Neural progenitor cells in canonical rosette formations expressing Nestin (green), Pax6 (red), and DAPI (blue). Image courtesy of Michael Wells, postdoctoral fellow in the Eggan Lab.
All Publications
2020
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2020. Absence of Survival and Motor Deficits in 500 Repeat C9ORF72 BAC Mice. Neuron. 108(4):775-783.e4. Pubmed: 33022228 DOI:10.1016/j.neuron.2020.08.009 Mordes DA, Morrison BM, Ament XH, Cantrell C, Mok J, Eggan P, Xue C, Wang JY, Eggan K, Rothstein JD. 2020. Absence of Survival and Motor Deficits in 500 Repeat C9ORF72 BAC Mice. Neuron. 108(4):775-783.e4. Pubmed: 33022228 DOI:10.1016/j.neuron.2020.08.009 A hexanucleotide repeat expansion at C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Initial studies of bacterial artificial chromosome (BAC) transgenic mice harboring this expansion described an absence of motor and survival phenotypes. However, a recent study by Liu and colleagues described transgenic mice harboring a large repeat expansion (C9-500) and reported decreased survival and progressive motor phenotypes. To determine the utility of the C9-500 animals for understanding degenerative mechanisms, we validated and established two independent colonies of transgene carriers. However, extended studies of these animals for up to 1 year revealed no reproducible abnormalities in survival, motor function, or neurodegeneration. Here, we propose several potential explanations for the disparate nature of our findings from those of Liu and colleagues. Resolving the discrepancies we identify will be essential to settle the translational utility of C9-500 mice. This Matters Arising paper is in response to Liu et al. (2016), published in Neuron. See also the response by Nguyen et al. (2020), published in this issue.Copyright © 2020 Elsevier Inc. All rights reserved. -
Burberry A, Wells MF, Limone F, Couto A, Smith KS, Keaney J, Gillet G, van Gastel N, Wang JY, Pietilainen O, Qian M, Eggan P, Cantrell C, Mok J, Kadiu I, Scadden DT, Eggan K. 2020. C9orf72 suppresses systemic and neural inflammation induced by gut bacteria. Nature. 582(7810):89-94. Pubmed: 32483373 DOI:10.1038/s41586-020-2288-7 Burberry A, Wells MF, Limone F, Couto A, Smith KS, Keaney J, Gillet G, van Gastel N, Wang JY, Pietilainen O, Qian M, Eggan P, Cantrell C, Mok J, Kadiu I, Scadden DT, Eggan K. 2020. C9orf72 suppresses systemic and neural inflammation induced by gut bacteria. Nature. 582(7810):89-94. Pubmed: 32483373 DOI:10.1038/s41586-020-2288-7 A hexanucleotide-repeat expansion in C9ORF72 is the most common genetic variant that contributes to amyotrophic lateral sclerosis and frontotemporal dementia. The C9ORF72 mutation acts through gain- and loss-of-function mechanisms to induce pathways that are implicated in neural degeneration. The expansion is transcribed into a long repetitive RNA, which negatively sequesters RNA-binding proteins before its non-canonical translation into neural-toxic dipeptide proteins. The failure of RNA polymerase to read through the mutation also reduces the abundance of the endogenous C9ORF72 gene product, which functions in endolysosomal pathways and suppresses systemic and neural inflammation. Notably, the effects of the repeat expansion act with incomplete penetrance in families with a high prevalence of amyotrophic lateral sclerosis or frontotemporal dementia, indicating that either genetic or environmental factors modify the risk of disease for each individual. Identifying disease modifiers is of considerable translational interest, as it could suggest strategies to diminish the risk of developing amyotrophic lateral sclerosis or frontotemporal dementia, or to slow progression. Here we report that an environment with reduced abundance of immune-stimulating bacteria protects C9orf72-mutant mice from premature mortality and significantly ameliorates their underlying systemic inflammation and autoimmunity. Consistent with C9orf72 functioning to prevent microbiota from inducing a pathological inflammatory response, we found that reducing the microbial burden in mutant mice with broad spectrum antibiotics-as well as transplanting gut microflora from a protective environment-attenuated inflammatory phenotypes, even after their onset. Our studies provide further evidence that the microbial composition of our gut has an important role in brain health and can interact in surprising ways with well-known genetic risk factors for disorders of the nervous system. -
Hazelbaker DZ, Beccard A, Angelini G, Mazzucato P, Messana A, Lam D, Eggan K, Barrett LE. 2020. A multiplexed gRNA piggyBac transposon system facilitates efficient induction of CRISPRi and CRISPRa in human pluripotent stem cells. Scientific reports. 10(1):635. Pubmed: 31959800 DOI:10.1038/s41598-020-57500-1 Hazelbaker DZ, Beccard A, Angelini G, Mazzucato P, Messana A, Lam D, Eggan K, Barrett LE. 2020. A multiplexed gRNA piggyBac transposon system facilitates efficient induction of CRISPRi and CRISPRa in human pluripotent stem cells. Scientific reports. 10(1):635. Pubmed: 31959800 DOI:10.1038/s41598-020-57500-1 CRISPR-Cas9-mediated gene interference (CRISPRi) and activation (CRISPRa) approaches hold promise for functional gene studies and genome-wide screens in human pluripotent stem cells (hPSCs). However, in contrast to CRISPR-Cas9 nuclease approaches, the efficiency of CRISPRi/a depends on continued expression of the dead Cas9 (dCas9) effector and guide RNA (gRNA), which can vary substantially depending on transgene design and delivery. Here, we design and generate new fluorescently labeled piggyBac (PB) vectors to deliver uniform and sustained expression of multiplexed gRNAs. In addition, we generate hPSC lines harboring AAVS1-integrated, inducible and fluorescent dCas9-KRAB and dCas9-VPR transgenes to allow for accurate quantification and tracking of cells that express both the dCas9 effectors and gRNAs. We then employ these systems to target the TCF4 gene in hPSCs and assess expression levels of the dCas9 effectors, individual gRNAs and targeted gene. We also assess the performance of our PB system for single gRNA delivery, confirming its utility for library format applications. Collectively, our results provide proof-of-principle application of a stable, multiplexed PB gRNA delivery system that can be widely exploited to further enable genome engineering studies in hPSCs. Paired with diverse CRISPR tools including our dual fluorescence CRISPRi/a cell lines, this system can facilitate functional dissection of individual genes and pathways as well as larger-scale screens for studies of development and disease. -
Farhan SMK, Howrigan DP, Abbott LE, Klim JR, Topp SD, Byrnes AE, Churchhouse C, Phatnani H, Smith BN, Rampersaud E, Wu G, Wuu J, Shatunov A, Iacoangeli A, Khleifat AA, Mordes DA, Ghosh S, Eggan K, Rademakers R, McCauley JL, Schüle R, Züchner S, Benatar M, Taylor JP, Nalls M, Gotkine M, Shaw PJ, Morrison KE, Al-Chalabi A, Traynor B, Shaw CE, Goldstein DB, Harms MB, Daly MJ, Neale BM. 2020. Publisher Correction: Exome sequencing in amyotrophic lateral sclerosis implicates a novel gene, DNAJC7, encoding a heat-shock protein. Nature neuroscience. 23(2):295. Pubmed: 31857710 DOI:10.1038/s41593-019-0570-5 Farhan SMK, Howrigan DP, Abbott LE, Klim JR, Topp SD, Byrnes AE, Churchhouse C, Phatnani H, Smith BN, Rampersaud E, Wu G, Wuu J, Shatunov A, Iacoangeli A, Khleifat AA, Mordes DA, Ghosh S, Eggan K, Rademakers R, McCauley JL, Schüle R, Züchner S, Benatar M, Taylor JP, Nalls M, Gotkine M, Shaw PJ, Morrison KE, Al-Chalabi A, Traynor B, Shaw CE, Goldstein DB, Harms MB, Daly MJ, Neale BM. 2020. Publisher Correction: Exome sequencing in amyotrophic lateral sclerosis implicates a novel gene, DNAJC7, encoding a heat-shock protein. Nature neuroscience. 23(2):295. Pubmed: 31857710 DOI:10.1038/s41593-019-0570-5 An amendment to this paper has been published and can be accessed via a link at the top of the paper. 2019
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Farhan SMK, Howrigan DP, Abbott LE, Klim JR, Topp SD, Byrnes AE, Churchhouse C, Phatnani H, Smith BN, Rampersaud E, Wu G, Wuu J, Shatunov A, Iacoangeli A, Al Khleifat A, Mordes DA, Ghosh S, Eggan K, Rademakers R, McCauley JL, Schüle R, Züchner S, Benatar M, Taylor JP, Nalls M, Gotkine M, Shaw PJ, Morrison KE, Al-Chalabi A, Traynor B, Shaw CE, Goldstein DB, Harms MB, Daly MJ, Neale BM. 2019. Exome sequencing in amyotrophic lateral sclerosis implicates a novel gene, DNAJC7, encoding a heat-shock protein. Nature neuroscience. 22(12):1966-1974. Pubmed: 31768050 DOI:10.1038/s41593-019-0530-0 Farhan SMK, Howrigan DP, Abbott LE, Klim JR, Topp SD, Byrnes AE, Churchhouse C, Phatnani H, Smith BN, Rampersaud E, Wu G, Wuu J, Shatunov A, Iacoangeli A, Al Khleifat A, Mordes DA, Ghosh S, Eggan K, Rademakers R, McCauley JL, Schüle R, Züchner S, Benatar M, Taylor JP, Nalls M, Gotkine M, Shaw PJ, Morrison KE, Al-Chalabi A, Traynor B, Shaw CE, Goldstein DB, Harms MB, Daly MJ, Neale BM. 2019. Exome sequencing in amyotrophic lateral sclerosis implicates a novel gene, DNAJC7, encoding a heat-shock protein. Nature neuroscience. 22(12):1966-1974. Pubmed: 31768050 DOI:10.1038/s41593-019-0530-0 To discover novel genes underlying amyotrophic lateral sclerosis (ALS), we aggregated exomes from 3,864 cases and 7,839 ancestry-matched controls. We observed a significant excess of rare protein-truncating variants among ALS cases, and these variants were concentrated in constrained genes. Through gene level analyses, we replicated known ALS genes including SOD1, NEK1 and FUS. We also observed multiple distinct protein-truncating variants in a highly constrained gene, DNAJC7. The signal in DNAJC7 exceeded genome-wide significance, and immunoblotting assays showed depletion of DNAJC7 protein in fibroblasts in a patient with ALS carrying the p.Arg156Ter variant. DNAJC7 encodes a member of the heat-shock protein family, HSP40, which, along with HSP70 proteins, facilitates protein homeostasis, including folding of newly synthesized polypeptides and clearance of degraded proteins. When these processes are not regulated, misfolding and accumulation of aberrant proteins can occur and lead to protein aggregation, which is a pathological hallmark of neurodegeneration. Our results highlight DNAJC7 as a novel gene for ALS. -
Groff AF, Resetkova N, DiDomenico F, Sakkas D, Penzias A, Rinn JL, Eggan K. 2019. RNA-seq as a tool for evaluating human embryo competence. Genome research. 29(10):1705-1718. Pubmed: 31548358 DOI:10.1101/gr.252981.119 Groff AF, Resetkova N, DiDomenico F, Sakkas D, Penzias A, Rinn JL, Eggan K. 2019. RNA-seq as a tool for evaluating human embryo competence. Genome research. 29(10):1705-1718. Pubmed: 31548358 DOI:10.1101/gr.252981.119 The majority of embryos created through in vitro fertilization (IVF) do not implant. It seems plausible that rates of implantation would improve if we had a better understanding of molecular factors affecting embryo competence. Currently, the process of selecting an embryo for uterine transfer uses an ad hoc combination of morphological criteria, the kinetics of development, and genetic testing for aneuploidy. However, no single criterion can ensure selection of a viable embryo. In contrast, RNA-sequencing (RNA-seq) of embryos could yield high-dimensional data, which may provide additional insight and illuminate the discrepancies among current selection criteria. Recent advances enabling the production of RNA-seq libraries from single cells have facilitated the application of this technique to the study of transcriptional events in early human development. However, these studies have not assessed the quality of their constituent embryos relative to commonly used embryological criteria. Here, we perform proof-of-principle advancement to embryo selection procedures by generating RNA-seq libraries from a trophectoderm biopsy as well as the remaining whole embryo. We combine state-of-the-art embryological methods with low-input RNA-seq to develop the first transcriptome-wide approach for assessing embryo competence. Specifically, we show the capacity of RNA-seq as a promising tool in preimplantation screening by showing that biopsies of an embryo can capture valuable information available in the whole embryo from which they are derived. Furthermore, we show that this technique can be used to generate a RNA-based digital karyotype and to identify candidate competence-associated genes. Together, these data establish the foundation for a future RNA-based diagnostic in IVF.© 2019 Groff et al.; Published by Cold Spring Harbor Laboratory Press. -
Thams S, Lowry ER, Larraufie MH, Spiller KJ, Li H, Williams DJ, Hoang P, Jiang E, Williams LA, Sandoe J, Eggan K, Lieberam I, Kanning KC, Stockwell BR, Henderson CE, Wichterle H. 2019. A Stem Cell-Based Screening Platform Identifies Compounds that Desensitize Motor Neurons to Endoplasmic Reticulum Stress. Molecular therapy : the journal of the American Society of Gene Therapy. 27(1):87-101. Pubmed: 30446391 DOI:10.1016/j.ymthe.2018.10.010 Thams S, Lowry ER, Larraufie MH, Spiller KJ, Li H, Williams DJ, Hoang P, Jiang E, Williams LA, Sandoe J, Eggan K, Lieberam I, Kanning KC, Stockwell BR, Henderson CE, Wichterle H. 2019. A Stem Cell-Based Screening Platform Identifies Compounds that Desensitize Motor Neurons to Endoplasmic Reticulum Stress. Molecular therapy : the journal of the American Society of Gene Therapy. 27(1):87-101. Pubmed: 30446391 DOI:10.1016/j.ymthe.2018.10.010 Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease selectively targeting motor neurons in the brain and spinal cord. The reasons for differential motor neuron susceptibility remain elusive. We developed a stem cell-based motor neuron assay to study cell-autonomous mechanisms causing motor neuron degeneration, with implications for ALS. A small-molecule screen identified cyclopiazonic acid (CPA) as a stressor to which stem cell-derived motor neurons were more sensitive than interneurons. CPA induced endoplasmic reticulum stress and the unfolded protein response. Furthermore, CPA resulted in an accelerated degeneration of motor neurons expressing human superoxide dismutase 1 (hSOD1) carrying the ALS-causing G93A mutation, compared to motor neurons expressing wild-type hSOD1. A secondary screen identified compounds that alleviated CPA-mediated motor neuron degeneration: three kinase inhibitors and tauroursodeoxycholic acid (TUDCA), a bile acid derivative. The neuroprotective effects of these compounds were validated in human stem cell-derived motor neurons carrying a mutated SOD1 allele (hSOD1). Moreover, we found that the administration of TUDCA in an hSOD1 mouse model of ALS reduced muscle denervation. Jointly, these results provide insights into the mechanisms contributing to the preferential susceptibility of ALS motor neurons, and they demonstrate the utility of stem cell-derived motor neurons for the discovery of new neuroprotective compounds.Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved. -
Mukherjee-Clavin B, Mi R, Kern B, Choi IY, Lim H, Oh Y, Lannon B, Kim KJ, Bell S, Hur JK, Hwang W, Che YH, Habib O, Baloh RH, Eggan K, Brandacher G, Hoke A, Studer L, Kim YJ, Lee G. 2019. Comparison of three congruent patient-specific cell types for the modelling of a human genetic Schwann-cell disorder. Nature biomedical engineering. 3(7):571-582. Pubmed: 30962586 DOI:10.1038/s41551-019-0381-8 Mukherjee-Clavin B, Mi R, Kern B, Choi IY, Lim H, Oh Y, Lannon B, Kim KJ, Bell S, Hur JK, Hwang W, Che YH, Habib O, Baloh RH, Eggan K, Brandacher G, Hoke A, Studer L, Kim YJ, Lee G. 2019. Comparison of three congruent patient-specific cell types for the modelling of a human genetic Schwann-cell disorder. Nature biomedical engineering. 3(7):571-582. Pubmed: 30962586 DOI:10.1038/s41551-019-0381-8 Patient-specific human-induced pluripotent stem cells (hiPSCs) hold great promise for the modelling of genetic disorders. However, these cells display wide intra- and interindividual variations in gene expression, which makes distinguishing true-positive and false-positive phenotypes challenging. Data from hiPSC phenotypes and human embryonic stem cells (hESCs) harbouring the same disease mutation are also lacking. Here, we report a comparison of the molecular, cellular and functional characteristics of three congruent patient-specific cell types-hiPSCs, hESCs and direct-lineage-converted cells-derived from currently available differentiation and direct-reprogramming technologies for use in the modelling of Charcot-Marie-Tooth 1A, a human genetic Schwann-cell disorder featuring a 1.4 Mb chromosomal duplication. We find that the chemokines C-X-C motif ligand chemokine-1 (CXCL1) and macrophage chemoattractant protein-1 (MCP1) are commonly upregulated in all three congruent models and in clinical patient samples. The development of congruent models of a single genetic disease using somatic cells from a common patient will facilitate the search for convergent phenotypes. -
Klim JR, Williams LA, Limone F, Guerra San Juan I, Davis-Dusenbery BN, Mordes DA, Burberry A, Steinbaugh MJ, Gamage KK, Kirchner R, Moccia R, Cassel SH, Chen K, Wainger BJ, Woolf CJ, Eggan K. 2019. ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair. Nature neuroscience. 22(2):167-179. Pubmed: 30643292 DOI:10.1038/s41593-018-0300-4 Klim JR, Williams LA, Limone F, Guerra San Juan I, Davis-Dusenbery BN, Mordes DA, Burberry A, Steinbaugh MJ, Gamage KK, Kirchner R, Moccia R, Cassel SH, Chen K, Wainger BJ, Woolf CJ, Eggan K. 2019. ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair. Nature neuroscience. 22(2):167-179. Pubmed: 30643292 DOI:10.1038/s41593-018-0300-4 The findings that amyotrophic lateral sclerosis (ALS) patients almost universally display pathological mislocalization of the RNA-binding protein TDP-43 and that mutations in its gene cause familial ALS have nominated altered RNA metabolism as a disease mechanism. However, the RNAs regulated by TDP-43 in motor neurons and their connection to neuropathy remain to be identified. Here we report transcripts whose abundances in human motor neurons are sensitive to TDP-43 depletion. Notably, expression of STMN2, which encodes a microtubule regulator, declined after TDP-43 knockdown and TDP-43 mislocalization as well as in patient-specific motor neurons and postmortem patient spinal cord. STMN2 loss upon reduced TDP-43 function was due to altered splicing, which is functionally important, as we show STMN2 is necessary for normal axonal outgrowth and regeneration. Notably, post-translational stabilization of STMN2 rescued neurite outgrowth and axon regeneration deficits induced by TDP-43 depletion. We propose that restoring STMN2 expression warrants examination as a therapeutic strategy for ALS. -
Shao Z, Noh H, Bin Kim W, Ni P, Nguyen C, Cote SE, Noyes E, Zhao J, Parsons T, Park JM, Zheng K, Park JJ, Coyle JT, Weinberger DR, Straub RE, Berman KF, Apud J, Ongur D, Cohen BM, McPhie DL, Rapoport JL, Perlis RH, Lanz TA, Xi HS, Yin C, Huang W, Hirayama T, Fukuda E, Yagi T, Ghosh S, Eggan KC, Kim HY, Eisenberg LM, Moghadam AA, Stanton PK, Cho JH, Chung S. 2019. Dysregulated protocadherin-pathway activity as an intrinsic defect in induced pluripotent stem cell-derived cortical interneurons from subjects with schizophrenia. Nature neuroscience. 22(2):229-242. Pubmed: 30664768 DOI:10.1038/s41593-018-0313-z Shao Z, Noh H, Bin Kim W, Ni P, Nguyen C, Cote SE, Noyes E, Zhao J, Parsons T, Park JM, Zheng K, Park JJ, Coyle JT, Weinberger DR, Straub RE, Berman KF, Apud J, Ongur D, Cohen BM, McPhie DL, Rapoport JL, Perlis RH, Lanz TA, Xi HS, Yin C, Huang W, Hirayama T, Fukuda E, Yagi T, Ghosh S, Eggan KC, Kim HY, Eisenberg LM, Moghadam AA, Stanton PK, Cho JH, Chung S. 2019. Dysregulated protocadherin-pathway activity as an intrinsic defect in induced pluripotent stem cell-derived cortical interneurons from subjects with schizophrenia. Nature neuroscience. 22(2):229-242. Pubmed: 30664768 DOI:10.1038/s41593-018-0313-z We generated cortical interneurons (cINs) from induced pluripotent stem cells derived from 14 healthy controls and 14 subjects with schizophrenia. Both healthy control cINs and schizophrenia cINs were authentic, fired spontaneously, received functional excitatory inputs from host neurons, and induced GABA-mediated inhibition in host neurons in vivo. However, schizophrenia cINs had dysregulated expression of protocadherin genes, which lie within documented schizophrenia loci. Mice lacking protocadherin-α showed defective arborization and synaptic density of prefrontal cortex cINs and behavioral abnormalities. Schizophrenia cINs similarly showed defects in synaptic density and arborization that were reversed by inhibitors of protein kinase C, a downstream kinase in the protocadherin pathway. These findings reveal an intrinsic abnormality in schizophrenia cINs in the absence of any circuit-driven pathology. They also demonstrate the utility of homogenous and functional populations of a relevant neuronal subtype for probing pathogenesis mechanisms during development. -
Avior Y, Eggan K, Benvenisty N. 2019. Cancer-Related Mutations Identified in Primed and Naive Human Pluripotent Stem Cells. Cell stem cell. 25(4):456-461. Pubmed: 31543368 DOI:10.1016/j.stem.2019.09.001 Avior Y, Eggan K, Benvenisty N. 2019. Cancer-Related Mutations Identified in Primed and Naive Human Pluripotent Stem Cells. Cell stem cell. 25(4):456-461. Pubmed: 31543368 DOI:10.1016/j.stem.2019.09.001 Human pluripotent stem cells (hPSCs) are known to harbor chromosomal aberrations, affecting their tumorigenic potential. We established a strategy to identify cancer-related point mutations in hPSCs, detecting recurrent mutations in over 20 genes, alongside those previously detected in p53. Importantly, naive hPSCs harbor, on average, four times more mutations than their primed counterparts, which appear primarily in pathways inhibited during naive conversion. Such cancer-related mutations should be taken into consideration in future applications, especially in clinical contexts.Copyright © 2019 Elsevier Inc. All rights reserved. -
Oh HS, Neuhausser WM, Eggan P, Angelova M, Kirchner R, Eggan KC, Knipe DM. 2019. Herpesviral lytic gene functions render the viral genome susceptible to novel editing by CRISPR/Cas9. eLife. 8. Pubmed: 31789594 DOI:e51662 Oh HS, Neuhausser WM, Eggan P, Angelova M, Kirchner R, Eggan KC, Knipe DM. 2019. Herpesviral lytic gene functions render the viral genome susceptible to novel editing by CRISPR/Cas9. eLife. 8. Pubmed: 31789594 DOI:e51662 Herpes simplex virus (HSV) establishes lifelong latent infection and can cause serious human disease, but current antiviral therapies target lytic but not latent infection. We screened for sgRNAs that cleave HSV-1 DNA sequences efficiently in vitro and used these sgRNAs to observe the first editing of quiescent HSV-1 DNA. The sgRNAs targeted lytic replicating viral DNA genomes more efficiently than quiescent genomes, consistent with the open structure of lytic chromatin. Editing of latent genomes caused short indels while editing of replicating genomes produced indels, linear molecules, and large genomic sequence loss around the gRNA target site. The HSV ICP0 protein and viral DNA replication increased the loss of DNA sequences around the gRNA target site. We conclude that HSV, by promoting open chromatin needed for viral gene expression and by inhibiting the DNA damage response, makes the genome vulnerable to a novel form of editing by CRISPR-Cas9 during lytic replication.© 2019, Oh et al. -
Shlevkov E, Basu H, Bray MA, Sun Z, Wei W, Apaydin K, Karhohs K, Chen PF, Smith JLM, Wiskow O, Roet K, Huang X, Eggan K, Carpenter AE, Kleiman RJ, Schwarz TL. 2019. A High-Content Screen Identifies TPP1 and Aurora B as Regulators of Axonal Mitochondrial Transport. Cell reports. 28(12):3224-3237.e5. Pubmed: 31533043 DOI:10.1016/j.celrep.2019.08.035 Shlevkov E, Basu H, Bray MA, Sun Z, Wei W, Apaydin K, Karhohs K, Chen PF, Smith JLM, Wiskow O, Roet K, Huang X, Eggan K, Carpenter AE, Kleiman RJ, Schwarz TL. 2019. A High-Content Screen Identifies TPP1 and Aurora B as Regulators of Axonal Mitochondrial Transport. Cell reports. 28(12):3224-3237.e5. Pubmed: 31533043 DOI:10.1016/j.celrep.2019.08.035 Dysregulated axonal trafficking of mitochondria is linked to neurodegenerative disorders. We report a high-content screen for small-molecule regulators of the axonal transport of mitochondria. Six compounds enhanced mitochondrial transport in the sub-micromolar range, acting via three cellular targets: F-actin, Tripeptidyl peptidase 1 (TPP1), or Aurora Kinase B (AurKB). Pharmacological inhibition or small hairpin RNA (shRNA) knockdown of each target promotes mitochondrial axonal transport in rat hippocampal neurons and induced pluripotent stem cell (iPSC)-derived human cortical neurons and enhances mitochondrial transport in iPSC-derived motor neurons from an amyotrophic lateral sclerosis (ALS) patient bearing one copy of SOD1 mutation. Our work identifies druggable regulators of axonal transport of mitochondria, provides broadly applicable methods for similar image-based screens, and suggests that restoration of proper axonal trafficking of mitochondria can be achieved in human ALS neurons.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved. 2018
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Ichida JK, Staats KA, Davis-Dusenbery BN, Clement K, Galloway KE, Babos KN, Shi Y, Son EY, Kiskinis E, Atwater N, Gu H, Gnirke A, Meissner A, Eggan K. 2018. Comparative genomic analysis of embryonic, lineage-converted and stem cell-derived motor neurons. Development (Cambridge, England). 145(22). Pubmed: 30337375 DOI:10.1242/dev.168617 Ichida JK, Staats KA, Davis-Dusenbery BN, Clement K, Galloway KE, Babos KN, Shi Y, Son EY, Kiskinis E, Atwater N, Gu H, Gnirke A, Meissner A, Eggan K. 2018. Comparative genomic analysis of embryonic, lineage-converted and stem cell-derived motor neurons. Development (Cambridge, England). 145(22). Pubmed: 30337375 DOI:10.1242/dev.168617 Advances in stem cell science allow the production of different cell types either through the recapitulation of developmental processes, often termed 'directed differentiation', or the forced expression of lineage-specific transcription factors. Although cells produced by both approaches are increasingly used in translational applications, their quantitative similarity to their primary counterparts remains largely unresolved. To investigate the similarity between -derived and primary cell types, we harvested and purified mouse spinal motor neurons and compared them with motor neurons produced by transcription factor-mediated lineage conversion of fibroblasts or directed differentiation of pluripotent stem cells. To enable unbiased analysis of these motor neuron types and their cells of origin, we then subjected them to whole transcriptome and DNA methylome analysis by RNA sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS). Despite major differences in methodology, lineage conversion and directed differentiation both produce cells that closely approximate the primary motor neuron state. However, we identify differences in Fas signaling, the Hox code and synaptic gene expression between lineage-converted and directed differentiation motor neurons that affect their utility in translational studies.© 2018. Published by The Company of Biologists Ltd. -
McPhie DL, Nehme R, Ravichandran C, Babb SM, Ghosh SD, Staskus A, Kalinowski A, Kaur R, Douvaras P, Du F, Ongur D, Fossati V, Eggan K, Cohen BM. 2018. Oligodendrocyte differentiation of induced pluripotent stem cells derived from subjects with schizophrenias implicate abnormalities in development. Translational psychiatry. 8(1):230. Pubmed: 30352993 DOI:10.1038/s41398-018-0284-6 McPhie DL, Nehme R, Ravichandran C, Babb SM, Ghosh SD, Staskus A, Kalinowski A, Kaur R, Douvaras P, Du F, Ongur D, Fossati V, Eggan K, Cohen BM. 2018. Oligodendrocyte differentiation of induced pluripotent stem cells derived from subjects with schizophrenias implicate abnormalities in development. Translational psychiatry. 8(1):230. Pubmed: 30352993 DOI:10.1038/s41398-018-0284-6 Abnormalities of brain connectivity and signal transduction are consistently observed in individuals with schizophrenias (SZ). Underlying these anomalies, convergent in vivo, post mortem, and genomic evidence suggest abnormal oligodendrocyte (OL) development and function and lower myelination in SZ. Our primary hypothesis was that there would be abnormalities in the number of induced pluripotent stem (iPS) cell-derived OLs from subjects with SZ. Our secondary hypothesis was that these in vitro abnormalities would correlate with measures of white matter (WM) integrity and myelination in the same subjects in vivo, estimated from magnetic resonance imaging. Six healthy control (HC) and six SZ iPS cell lines, derived from skin fibroblasts from well-characterized subjects, were differentiated into OLs. FACS analysis of the oligodendrocyte-specific surface, glycoprotein O4, was performed at three time points of development (days 65, 75, and 85) to quantify the number of late oligodendrocyte progenitor cells (OPCs) and OLs in each line. Significantly fewer O4-positive cells developed from SZ versus HC lines (95% CI 1.0: 8.6, F = 8.06, p = 0.02). The difference was greater when corrected for age (95% CI 5.4:10.4, F = 53.6, p < 0.001). A correlation between myelin content in WM in vivo, estimated by magnetization transfer ratio (MTR) and number of O4-positive cells in vitro was also observed across all time points (F = 4.3, p = 0.07), reaching significance for mature OLs at day 85 in culture (r = 0.70, p < 0.02). Low production of OPCs may be a contributing mechanism underlying WM reduction in SZ. -
Vogt MA, Ehsaei Z, Knuckles P, Higginbottom A, Helmbrecht MS, Kunath T, Eggan K, Williams LA, Shaw PJ, Wurst W, Floss T, Huber AB, Taylor V. 2018. TDP-43 induces p53-mediated cell death of cortical progenitors and immature neurons. Scientific reports. 8(1):8097. Pubmed: 29802307 DOI:10.1038/s41598-018-26397-2 Vogt MA, Ehsaei Z, Knuckles P, Higginbottom A, Helmbrecht MS, Kunath T, Eggan K, Williams LA, Shaw PJ, Wurst W, Floss T, Huber AB, Taylor V. 2018. TDP-43 induces p53-mediated cell death of cortical progenitors and immature neurons. Scientific reports. 8(1):8097. Pubmed: 29802307 DOI:10.1038/s41598-018-26397-2 TAR DNA-binding protein 43 (TDP-43) is a key player in neurodegenerative diseases including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Accumulation of TDP-43 is associated with neuronal death in the brain. How increased and disease-causing mutant forms of TDP-43 induce cell death remains unclear. Here we addressed the role of TDP-43 during neural development and show that reduced TDP-43 causes defects in neural stem/progenitor cell proliferation but not cell death. However, overexpression of wild type and TDP-43 proteins induce p53-dependent apoptosis of neural stem/progenitors and human induced pluripotent cell (iPS)-derived immature cortical neurons. We show that TDP-43 induces expression of the proapoptotic BH3-only genes Bbc3 and Bax, and that p53 inhibition rescues TDP-43 induced cell death of embryonic mouse, and human cortical neurons, including those derived from TDP-43 ALS patient iPS cells. Hence, an increase in wild type and mutant TDP-43 induces p53-dependent cell death in neural progenitors developing neurons and this can be rescued. These findings may have important implications for accumulated or mutant TDP-43 induced neurodegenerative diseases. -
Zhang Y, Burberry A, Wang JY, Sandoe J, Ghosh S, Udeshi ND, Svinkina T, Mordes DA, Mok J, Charlton M, Li QZ, Carr SA, Eggan K. 2018. The C9orf72-interacting protein Smcr8 is a negative regulator of autoimmunity and lysosomal exocytosis. Genes & development. 32(13-14):929-943. Pubmed: 29950492 DOI:10.1101/gad.313932.118 Zhang Y, Burberry A, Wang JY, Sandoe J, Ghosh S, Udeshi ND, Svinkina T, Mordes DA, Mok J, Charlton M, Li QZ, Carr SA, Eggan K. 2018. The C9orf72-interacting protein Smcr8 is a negative regulator of autoimmunity and lysosomal exocytosis. Genes & development. 32(13-14):929-943. Pubmed: 29950492 DOI:10.1101/gad.313932.118 While a mutation in is the most common genetic contributor to amyotrophic lateral sclerosis (ALS), much remains to be learned concerning the function of the protein normally encoded at this locus. To elaborate further on functions for C9ORF72, we used quantitative mass spectrometry-based proteomics to identify interacting proteins in motor neurons and found that its long isoform complexes with and stabilizes SMCR8, which further enables interaction with WDR41. To study the organismal and cellular functions for this tripartite complex, we generated loss-of-function mutant mice and found that they developed phenotypes also observed in loss-of-function animals, including autoimmunity. Along with a loss of tolerance for many nervous system autoantigens, we found increased lysosomal exocytosis in mutant macrophages. In addition to elevated surface Lamp1 (lysosome-associated membrane protein 1) expression, we also observed enhanced secretion of lysosomal components-phenotypes that we subsequently observed in loss-of-function macrophages. Overall, our findings demonstrate that C9ORF72 and SMCR8 have interdependent functions in suppressing autoimmunity as well as negatively regulating lysosomal exocytosis-processes of potential importance to ALS.© 2018 Zhang et al.; Published by Cold Spring Harbor Laboratory Press. -
Sanders SJ, Neale BM, Huang H, Werling DM, An JY, Dong S, Abecasis G, Arguello PA, Blangero J, Boehnke M, Daly MJ, Eggan K, Geschwind DH, Glahn DC, Goldstein DB, Gur RE, Handsaker RE, McCarroll SA, Ophoff RA, Palotie A, Pato CN, Sabatti C, State MW, Willsey AJ, Hyman SE, Addington AM, Lehner T, Freimer NB. 2018. Publisher Correction: Whole genome sequencing in psychiatric disorders: the WGSPD consortium. Nature neuroscience. 21(7):1017. Pubmed: 29549319 DOI:10.1038/s41593-018-0102-8 Sanders SJ, Neale BM, Huang H, Werling DM, An JY, Dong S, Abecasis G, Arguello PA, Blangero J, Boehnke M, Daly MJ, Eggan K, Geschwind DH, Glahn DC, Goldstein DB, Gur RE, Handsaker RE, McCarroll SA, Ophoff RA, Palotie A, Pato CN, Sabatti C, State MW, Willsey AJ, Hyman SE, Addington AM, Lehner T, Freimer NB. 2018. Publisher Correction: Whole genome sequencing in psychiatric disorders: the WGSPD consortium. Nature neuroscience. 21(7):1017. Pubmed: 29549319 DOI:10.1038/s41593-018-0102-8 In the version of this article initially published, the consortium authorship and corresponding authors were not presented correctly. In the PDF and print versions, the Whole Genome Sequencing for Psychiatric Disorders (WGSPD) consortium was missing from the author list at the beginning of the paper, where it should have appeared as the seventh author; it was present in the author list at the end of the paper, but the footnote directing readers to the Supplementary Note for a list of members was missing. In the HTML version, the consortium was listed as the last author instead of as the seventh, and the line directing readers to the Supplementary Note for a list of members appeared at the end of the paper under Author Information but not in association with the consortium name itself. Also, this line stated that both member names and affiliations could be found in the Supplementary Note; in fact, only names are given. In all versions of the paper, the corresponding author symbols were attached to A. Jeremy Willsey, Steven E. Hyman, Anjene M. Addington and Thomas Lehner; they should have been attached, respectively, to Steven E. Hyman, Anjene M. Addington, Thomas Lehner and Nelson B. Freimer. As a result of this shift, the respective contact links in the HTML version did not lead to the indicated individuals. The errors have been corrected in the HTML and PDF versions of the article. -
Mordes DA, Prudencio M, Goodman LD, Klim JR, Moccia R, Limone F, Pietilainen O, Chowdhary K, Dickson DW, Rademakers R, Bonini NM, Petrucelli L, Eggan K. 2018. Dipeptide repeat proteins activate a heat shock response found in C9ORF72-ALS/FTLD patients. Acta neuropathologica communications. 6(1):55. Pubmed: 29973287 DOI:10.1186/s40478-018-0555-8 Mordes DA, Prudencio M, Goodman LD, Klim JR, Moccia R, Limone F, Pietilainen O, Chowdhary K, Dickson DW, Rademakers R, Bonini NM, Petrucelli L, Eggan K. 2018. Dipeptide repeat proteins activate a heat shock response found in C9ORF72-ALS/FTLD patients. Acta neuropathologica communications. 6(1):55. Pubmed: 29973287 DOI:10.1186/s40478-018-0555-8 A hexanucleotide (GGGGCC) repeat expansion in C9ORF72 is the most common genetic contributor to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Reduced expression of the C9ORF72 gene product has been proposed as a potential contributor to disease pathogenesis. Additionally, repetitive RNAs and dipeptide repeat proteins (DPRs), such as poly-GR, can be produced by this hexanucleotide expansion that disrupt a number of cellular processes, potentially contributing to neural degeneration. To better discern which of these mechanisms leads to disease-associated changes in patient brains, we analyzed gene expression data generated from the cortex and cerebellum. We found that transcripts encoding heat shock proteins (HSPs) regulated by the HSF1 transcription factor were significantly induced in C9ORF72-ALS/FTLD patients relative to both sporadic ALS/FTLD cases and controls. Treatment of human neurons with chemically synthesized DPRs was sufficient to activate a similar transcriptional response. Expression of GGGGCC repeats and also poly-GR in the brains of Drosophila lead to the upregulation of HSF1 and the same highly-conserved HSPs. Additionally, HSF1 was a modifier of poly-GR toxicity in Drosophila. Our results suggest that the expression of DPRs are associated with upregulation of HSF1 and activation of a heat shock response in C9ORF72-ALS/FTLD. -
Maas K, Mirabal S, Penzias A, Sweetnam PM, Eggan KC, Sakkas D. 2018. Hippo signaling in the ovary and polycystic ovarian syndrome. Journal of assisted reproduction and genetics. 35(10):1763-1771. Pubmed: 30120633 DOI:10.1007/s10815-018-1235-0 Maas K, Mirabal S, Penzias A, Sweetnam PM, Eggan KC, Sakkas D. 2018. Hippo signaling in the ovary and polycystic ovarian syndrome. Journal of assisted reproduction and genetics. 35(10):1763-1771. Pubmed: 30120633 DOI:10.1007/s10815-018-1235-0 Array -
Fan LZ, Nehme R, Adam Y, Jung ES, Wu H, Eggan K, Arnold DB, Cohen AE. 2018. All-optical synaptic electrophysiology probes mechanism of ketamine-induced disinhibition. Nature methods. 15(10):823-831. Pubmed: 30275587 DOI:10.1038/s41592-018-0142-8 Fan LZ, Nehme R, Adam Y, Jung ES, Wu H, Eggan K, Arnold DB, Cohen AE. 2018. All-optical synaptic electrophysiology probes mechanism of ketamine-induced disinhibition. Nature methods. 15(10):823-831. Pubmed: 30275587 DOI:10.1038/s41592-018-0142-8 Optical assays of synaptic strength could facilitate studies of neuronal transmission and its dysregulation in disease. Here we introduce a genetic toolbox for all-optical interrogation of synaptic electrophysiology (synOptopatch) via mutually exclusive expression of a channelrhodopsin actuator and an archaerhodopsin-derived voltage indicator. Optically induced activity in the channelrhodopsin-expressing neurons generated excitatory and inhibitory postsynaptic potentials that we optically resolved in reporter-expressing neurons. We further developed a yellow spine-targeted Ca indicator to localize optogenetically triggered synaptic inputs. We demonstrated synOptopatch recordings in cultured rodent neurons and in acute rodent brain slice. In synOptopatch measurements of primary rodent cultures, acute ketamine administration suppressed disynaptic inhibitory feedbacks, mimicking the effect of this drug on network function in both rodents and humans. We localized this action of ketamine to excitatory synapses onto interneurons. These results establish an in vitro all-optical model of disynaptic disinhibition, a synaptic defect hypothesized in schizophrenia-associated psychosis. -
Nehme R, Zuccaro E, Ghosh SD, Li C, Sherwood JL, Pietilainen O, Barrett LE, Limone F, Worringer KA, Kommineni S, Zang Y, Cacchiarelli D, Meissner A, Adolfsson R, Haggarty S, Madison J, Muller M, Arlotta P, Fu Z, Feng G, Eggan K. 2018. Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission. Cell reports. 23(8):2509-2523. Pubmed: 29791859 DOI:10.1016/j.celrep.2018.04.066 Nehme R, Zuccaro E, Ghosh SD, Li C, Sherwood JL, Pietilainen O, Barrett LE, Limone F, Worringer KA, Kommineni S, Zang Y, Cacchiarelli D, Meissner A, Adolfsson R, Haggarty S, Madison J, Muller M, Arlotta P, Fu Z, Feng G, Eggan K. 2018. Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission. Cell reports. 23(8):2509-2523. Pubmed: 29791859 DOI:10.1016/j.celrep.2018.04.066 Transcription factor programming of pluripotent stem cells (PSCs) has emerged as an approach to generate human neurons for disease modeling. However, programming schemes produce a variety of cell types, and those neurons that are made often retain an immature phenotype, which limits their utility in modeling neuronal processes, including synaptic transmission. We report that combining NGN2 programming with SMAD and WNT inhibition generates human patterned induced neurons (hpiNs). Single-cell analyses showed that hpiN cultures contained cells along a developmental continuum, ranging from poorly differentiated neuronal progenitors to well-differentiated, excitatory glutamatergic neurons. The most differentiated neurons could be identified using a CAMK2A::GFP reporter gene and exhibited greater functionality, including NMDAR-mediated synaptic transmission. We conclude that utilizing single-cell and reporter gene approaches for selecting successfully programmed cells for study will greatly enhance the utility of hpiNs and other programmed neuronal populations in the modeling of nervous system disorders.Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved. -
Kiskinis E, Kralj JM, Zou P, Weinstein EN, Zhang H, Tsioras K, Wiskow O, Ortega JA, Eggan K, Cohen AE. 2018. All-Optical Electrophysiology for High-Throughput Functional Characterization of a Human iPSC-Derived Motor Neuron Model of ALS. Stem cell reports. 10(6):1991-2004. Pubmed: 29779896 DOI:10.1016/j.stemcr.2018.04.020 Kiskinis E, Kralj JM, Zou P, Weinstein EN, Zhang H, Tsioras K, Wiskow O, Ortega JA, Eggan K, Cohen AE. 2018. All-Optical Electrophysiology for High-Throughput Functional Characterization of a Human iPSC-Derived Motor Neuron Model of ALS. Stem cell reports. 10(6):1991-2004. Pubmed: 29779896 DOI:10.1016/j.stemcr.2018.04.020 Human induced pluripotent stem cell (iPSC)-derived neurons are an attractive substrate for modeling disease, yet the heterogeneity of these cultures presents a challenge for functional characterization by manual patch-clamp electrophysiology. Here, we describe an optimized all-optical electrophysiology, "Optopatch," pipeline for high-throughput functional characterization of human iPSC-derived neuronal cultures. We demonstrate the method in a human iPSC-derived motor neuron (iPSC-MN) model of amyotrophic lateral sclerosis (ALS). In a comparison of iPSC-MNs with an ALS-causing mutation (SOD1 A4V) with their genome-corrected controls, the mutants showed elevated spike rates under weak or no stimulus and greater likelihood of entering depolarization block under strong optogenetic stimulus. We compared these results with numerical simulations of simple conductance-based neuronal models and with literature results in this and other iPSC-based models of ALS. Our data and simulations suggest that deficits in slowly activating potassium channels may underlie the changes in electrophysiology in the SOD1 A4V mutation.Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved. -
Srikanth P, Lagomarsino VN, Pearse RV, Liao M, Ghosh S, Nehme R, Seyfried N, Eggan K, Young-Pearse TL. 2018. Convergence of independent DISC1 mutations on impaired neurite growth via decreased UNC5D expression. Translational psychiatry. 8(1):245. Pubmed: 30410030 DOI:10.1038/s41398-018-0281-9 Srikanth P, Lagomarsino VN, Pearse RV, Liao M, Ghosh S, Nehme R, Seyfried N, Eggan K, Young-Pearse TL. 2018. Convergence of independent DISC1 mutations on impaired neurite growth via decreased UNC5D expression. Translational psychiatry. 8(1):245. Pubmed: 30410030 DOI:10.1038/s41398-018-0281-9 The identification of convergent phenotypes in different models of psychiatric illness highlights robust phenotypes that are more likely to be implicated in disease pathophysiology. Here, we utilize human iPSCs harboring distinct mutations in DISC1 that have been found in families with major mental illness. One mutation was engineered to mimic the consequences on DISC1 protein of a balanced translocation linked to mental illness in a Scottish pedigree; the other mutation was identified in an American pedigree with a high incidence of mental illness. Directed differentiation of these iPSCs using NGN2 expression shows rapid conversion to a homogenous population of mature excitatory neurons. Both DISC1 mutations result in reduced DISC1 protein expression, and show subtle effects on certain presynaptic proteins. In addition, RNA sequencing and qPCR showed decreased expression of UNC5D, DPP10, PCDHA6, and ZNF506 in neurons with both DISC1 mutations. Longitudinal analysis of neurite outgrowth revealed decreased neurite outgrowth in neurons with each DISC1 mutation, which was mimicked by UNC5D knockdown and rescued by transient upregulation of endogenous UNC5D. This study shows a narrow range of convergent phenotypes of two mutations found in families with major mental illness, and implicates dysregulated netrin signaling in DISC1 biology. 2017
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Sanders SJ, Neale BM, Huang H, Werling DM, An JY, Dong S, Abecasis G, Arguello PA, Blangero J, Boehnke M, Daly MJ, Eggan K, Geschwind DH, Glahn DC, Goldstein DB, Gur RE, Handsaker RE, McCarroll SA, Ophoff RA, Palotie A, Pato CN, Sabatti C, State MW, Willsey AJ, Hyman SE, Addington AM, Lehner T, Freimer NB. 2017. Whole genome sequencing in psychiatric disorders: the WGSPD consortium. Nature neuroscience. 20(12):1661-1668. Pubmed: 29184211 DOI:10.1038/s41593-017-0017-9 Sanders SJ, Neale BM, Huang H, Werling DM, An JY, Dong S, Abecasis G, Arguello PA, Blangero J, Boehnke M, Daly MJ, Eggan K, Geschwind DH, Glahn DC, Goldstein DB, Gur RE, Handsaker RE, McCarroll SA, Ophoff RA, Palotie A, Pato CN, Sabatti C, State MW, Willsey AJ, Hyman SE, Addington AM, Lehner T, Freimer NB. 2017. Whole genome sequencing in psychiatric disorders: the WGSPD consortium. Nature neuroscience. 20(12):1661-1668. Pubmed: 29184211 DOI:10.1038/s41593-017-0017-9 -
Hazelbaker DZ, Beccard A, Bara AM, Dabkowski N, Messana A, Mazzucato P, Lam D, Manning D, Eggan K, Barrett LE. 2017. A Scaled Framework for CRISPR Editing of Human Pluripotent Stem Cells to Study Psychiatric Disease. Stem cell reports. 9(4):1315-1327. Pubmed: 29020615 DOI:10.1016/j.stemcr.2017.09.006 Hazelbaker DZ, Beccard A, Bara AM, Dabkowski N, Messana A, Mazzucato P, Lam D, Manning D, Eggan K, Barrett LE. 2017. A Scaled Framework for CRISPR Editing of Human Pluripotent Stem Cells to Study Psychiatric Disease. Stem cell reports. 9(4):1315-1327. Pubmed: 29020615 DOI:10.1016/j.stemcr.2017.09.006 Scaling of CRISPR-Cas9 technology in human pluripotent stem cells (hPSCs) represents an important step for modeling complex disease and developing drug screens in human cells. However, variables affecting the scaling efficiency of gene editing in hPSCs remain poorly understood. Here, we report a standardized CRISPR-Cas9 approach, with robust benchmarking at each step, to successfully target and genotype a set of psychiatric disease-implicated genes in hPSCs and provide a resource of edited hPSC lines for six of these genes. We found that transcriptional state and nucleosome positioning around targeted loci was not correlated with editing efficiency. However, editing frequencies varied between different hPSC lines and correlated with genomic stability, underscoring the need for careful cell line selection and unbiased assessments of genomic integrity. Together, our step-by-step quantification and in-depth analyses provide an experimental roadmap for scaling Cas9-mediated editing in hPSCs to study psychiatric disease, with broader applicability for other polygenic diseases.Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved. -
Kawada J, Kaneda S, Kirihara T, Maroof A, Levi T, Eggan K, Fujii T, Ikeuchi Y. 2017. Generation of a Motor Nerve Organoid with Human Stem Cell-Derived Neurons. Stem cell reports. 9(5):1441-1449. Pubmed: 29107592 DOI:10.1016/j.stemcr.2017.09.021 Kawada J, Kaneda S, Kirihara T, Maroof A, Levi T, Eggan K, Fujii T, Ikeuchi Y. 2017. Generation of a Motor Nerve Organoid with Human Stem Cell-Derived Neurons. Stem cell reports. 9(5):1441-1449. Pubmed: 29107592 DOI:10.1016/j.stemcr.2017.09.021 During development, axons spontaneously assemble into a fascicle to form nerves and tracts in the nervous system as they extend within a spatially constrained path. However, understanding of the axonal fascicle has been hampered by lack of an in vitro model system. Here, we report generation of a nerve organoid composed of a robust fascicle of axons extended from a spheroid of human stem cell-derived motor neurons within our custom-designed microdevice. The device is equipped with a narrow channel providing a microenvironment that facilitates the growing axons to spontaneously assemble into a unidirectional fascicle. The fascicle was specifically made with axons. We found that it was electrically active and elastic and could serve as a model to evaluate degeneration of axons in vitro. This nerve organoid model should facilitate future studies on the development of the axonal fascicle and drug screening for diseases affecting axon fascicles.Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved. -
Salick MR, Wells MF, Eggan K, Kaykas A. 2017. Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids. Journal of visualized experiments : JoVE. Pubmed: 28994790 DOI:10.3791/56404 Salick MR, Wells MF, Eggan K, Kaykas A. 2017. Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids. Journal of visualized experiments : JoVE. Pubmed: 28994790 DOI:10.3791/56404 The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain. Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models. -
Andrews PW, Ben-David U, Benvenisty N, Coffey P, Eggan K, Knowles BB, Nagy A, Pera M, Reubinoff B, Rugg-Gunn PJ, Stacey GN. 2017. Assessing the Safety of Human Pluripotent Stem Cells and Their Derivatives for Clinical Applications. Stem cell reports. 9(1):1-4. Pubmed: 28700896 DOI:10.1016/j.stemcr.2017.05.029 Andrews PW, Ben-David U, Benvenisty N, Coffey P, Eggan K, Knowles BB, Nagy A, Pera M, Reubinoff B, Rugg-Gunn PJ, Stacey GN. 2017. Assessing the Safety of Human Pluripotent Stem Cells and Their Derivatives for Clinical Applications. Stem cell reports. 9(1):1-4. Pubmed: 28700896 DOI:10.1016/j.stemcr.2017.05.029 Pluripotent stem cells may acquire genetic and epigenetic variants during culture following their derivation. At a conference organized by the International Stem Cell Initiative, and held at The Jackson Laboratory, Bar Harbor, Maine, October 2016, participants discussed how the appearance of such variants can be monitored and minimized and, crucially, how their significance for the safety of therapeutic applications of these cells can be assessed. A strong recommendation from the meeting was that an international advisory group should be set up to review the genetic and epigenetic changes observed in human pluripotent stem cell lines and establish a framework for evaluating the risks that they may pose for clinical use.Copyright © 2017. -
Tripathi P, Rodriguez-Muela N, Klim JR, de Boer AS, Agrawal S, Sandoe J, Lopes CS, Ogliari KS, Williams LA, Shear M, Rubin LL, Eggan K, Zhou Q. 2017. Reactive Astrocytes Promote ALS-like Degeneration and Intracellular Protein Aggregation in Human Motor Neurons by Disrupting Autophagy through TGF-β1. Stem cell reports. 9(2):667-680. Pubmed: 28712846 DOI:10.1016/j.stemcr.2017.06.008 Tripathi P, Rodriguez-Muela N, Klim JR, de Boer AS, Agrawal S, Sandoe J, Lopes CS, Ogliari KS, Williams LA, Shear M, Rubin LL, Eggan K, Zhou Q. 2017. Reactive Astrocytes Promote ALS-like Degeneration and Intracellular Protein Aggregation in Human Motor Neurons by Disrupting Autophagy through TGF-β1. Stem cell reports. 9(2):667-680. Pubmed: 28712846 DOI:10.1016/j.stemcr.2017.06.008 Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing motor neuron disease. Astrocytic factors are known to contribute to motor neuron degeneration and death in ALS. However, the role of astrocyte in promoting motor neuron protein aggregation, a disease hallmark of ALS, remains largely unclear. Here, using culture models of human motor neurons and primary astrocytes of different genotypes (wild-type or SOD1 mutant) and reactive states (non-reactive or reactive), we show that reactive astrocytes, regardless of their genotypes, reduce motor neuron health and lead to moderate neuronal loss. After prolonged co-cultures of up to 2 months, motor neurons show increased axonal and cytoplasmic protein inclusions characteristic of ALS. Reactive astrocytes induce protein aggregation in part by releasing transforming growth factor β1 (TGF-β1), which disrupts motor neuron autophagy through the mTOR pathway. These results reveal the important contribution of reactive astrocytes in promoting aspects of ALS pathology independent of genetic influences.Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved. -
Merkle FT, Ghosh S, Kamitaki N, Mitchell J, Avior Y, Mello C, Kashin S, Mekhoubad S, Ilic D, Charlton M, Saphier G, Handsaker RE, Genovese G, Bar S, Benvenisty N, McCarroll SA, Eggan K. 2017. Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations. Nature. 545(7653):229-233. Pubmed: 28445466 DOI:10.1038/nature22312 Merkle FT, Ghosh S, Kamitaki N, Mitchell J, Avior Y, Mello C, Kashin S, Mekhoubad S, Ilic D, Charlton M, Saphier G, Handsaker RE, Genovese G, Bar S, Benvenisty N, McCarroll SA, Eggan K. 2017. Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations. Nature. 545(7653):229-233. Pubmed: 28445466 DOI:10.1038/nature22312 Human pluripotent stem cells (hPS cells) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with the acquisition of large copy number variants that provide mutated cells with a growth advantage in culture. The nature, extent and functional effects of other acquired genome sequence mutations in cultured hPS cells are not known. Here we sequence the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hES cell) lines, including 26 lines prepared for potential clinical use. We then apply computational strategies for identifying mutations present in a subset of cells in each hES cell line. Although such mosaic mutations were generally rare, we identified five unrelated hES cell lines that carried six mutations in the TP53 gene that encodes the tumour suppressor P53. The TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We found that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that the P53 mutations confer selective advantage. We then mined published RNA sequencing data from 117 hPS cell lines, and observed another nine TP53 mutations, all resulting in coding changes in the DNA-binding domain of P53. In three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from the loss of heterozygosity at the TP53 locus. As the acquisition and expansion of cancer-associated mutations in hPS cells may go unnoticed during most applications, we suggest that careful genetic characterization of hPS cells and their differentiated derivatives be carried out before clinical use. 2016
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Sances S, Bruijn LI, Chandran S, Eggan K, Ho R, Klim JR, Livesey MR, Lowry E, Macklis JD, Rushton D, Sadegh C, Sareen D, Wichterle H, Zhang SC, Svendsen CN. 2016. Modeling ALS with motor neurons derived from human induced pluripotent stem cells. Nature neuroscience. 19(4):542-53. Pubmed: 27021939 DOI:10.1038/nn.4273 Sances S, Bruijn LI, Chandran S, Eggan K, Ho R, Klim JR, Livesey MR, Lowry E, Macklis JD, Rushton D, Sadegh C, Sareen D, Wichterle H, Zhang SC, Svendsen CN. 2016. Modeling ALS with motor neurons derived from human induced pluripotent stem cells. Nature neuroscience. 19(4):542-53. Pubmed: 27021939 DOI:10.1038/nn.4273 Directing the differentiation of induced pluripotent stem cells into motor neurons has allowed investigators to develop new models of amyotrophic lateral sclerosis (ALS). However, techniques vary between laboratories and the cells do not appear to mature into fully functional adult motor neurons. Here we discuss common developmental principles of both lower and upper motor neuron development that have led to specific derivation techniques. We then suggest how these motor neurons may be matured further either through direct expression or administration of specific factors or coculture approaches with other tissues. Ultimately, through a greater understanding of motor neuron biology, it will be possible to establish more reliable models of ALS. These in turn will have a greater chance of validating new drugs that may be effective for the disease. -
Wells MF, Salick MR, Wiskow O, Ho DJ, Worringer KA, Ihry RJ, Kommineni S, Bilican B, Klim JR, Hill EJ, Kane LT, Ye C, Kaykas A, Eggan K. 2016. Genetic Ablation of AXL Does Not Protect Human Neural Progenitor Cells and Cerebral Organoids from Zika Virus Infection. Cell stem cell. 19(6):703-708. Pubmed: 27912091 DOI:10.1016/j.stem.2016.11.011 Wells MF, Salick MR, Wiskow O, Ho DJ, Worringer KA, Ihry RJ, Kommineni S, Bilican B, Klim JR, Hill EJ, Kane LT, Ye C, Kaykas A, Eggan K. 2016. Genetic Ablation of AXL Does Not Protect Human Neural Progenitor Cells and Cerebral Organoids from Zika Virus Infection. Cell stem cell. 19(6):703-708. Pubmed: 27912091 DOI:10.1016/j.stem.2016.11.011 Zika virus (ZIKV) can cross the placental barrier, resulting in infection of the fetal brain and neurological defects including microcephaly. The cellular tropism of ZIKV and the identity of attachment factors used by the virus to gain access to key cell types involved in pathogenesis are under intense investigation. Initial studies suggested that ZIKV preferentially targets neural progenitor cells (NPCs), providing an explanation for the developmental phenotypes observed in some pregnancies. The AXL protein has been nominated as a key attachment factor for ZIKV in several cell types including NPCs. However, here we show that genetic ablation of AXL has no effect on ZIKV entry or ZIKV-mediated cell death in human induced pluripotent stem cell (iPSC)-derived NPCs or cerebral organoids. These findings call into question the utility of AXL inhibitors for preventing birth defects after infection and suggest that further studies of viral attachment factors in NPCs are needed.Copyright © 2016 Elsevier Inc. All rights reserved. -
Hill SJ, Mordes DA, Cameron LA, Neuberg DS, Landini S, Eggan K, Livingston DM. 2016. Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage. Proceedings of the National Academy of Sciences of the United States of America. 113(48):E7701-E7709. Pubmed: 27849576 Hill SJ, Mordes DA, Cameron LA, Neuberg DS, Landini S, Eggan K, Livingston DM. 2016. Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage. Proceedings of the National Academy of Sciences of the United States of America. 113(48):E7701-E7709. Pubmed: 27849576 Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis. -
Tian F, Yang W, Mordes DA, Wang JY, Salameh JS, Mok J, Chew J, Sharma A, Leno-Duran E, Suzuki-Uematsu S, Suzuki N, Han SS, Lu FK, Ji M, Zhang R, Liu Y, Strominger J, Shneider NA, Petrucelli L, Xie XS, Eggan K. 2016. Monitoring peripheral nerve degeneration in ALS by label-free stimulated Raman scattering imaging. Nature communications. 7:13283. Pubmed: 27796305 DOI:10.1038/ncomms13283 Tian F, Yang W, Mordes DA, Wang JY, Salameh JS, Mok J, Chew J, Sharma A, Leno-Duran E, Suzuki-Uematsu S, Suzuki N, Han SS, Lu FK, Ji M, Zhang R, Liu Y, Strominger J, Shneider NA, Petrucelli L, Xie XS, Eggan K. 2016. Monitoring peripheral nerve degeneration in ALS by label-free stimulated Raman scattering imaging. Nature communications. 7:13283. Pubmed: 27796305 DOI:10.1038/ncomms13283 The study of amyotrophic lateral sclerosis (ALS) and potential interventions would be facilitated if motor axon degeneration could be more readily visualized. Here we demonstrate that stimulated Raman scattering (SRS) microscopy could be used to sensitively monitor peripheral nerve degeneration in ALS mouse models and ALS autopsy materials. Three-dimensional imaging of pre-symptomatic SOD1 mouse models and data processing by a correlation-based algorithm revealed that significant degeneration of peripheral nerves could be detected coincidentally with the earliest detectable signs of muscle denervation and preceded physiologically measurable motor function decline. We also found that peripheral degeneration was an early event in FUS as well as C9ORF72 repeat expansion models of ALS, and that serial imaging allowed long-term observation of disease progression and drug effects in living animals. Our study demonstrates that SRS imaging is a sensitive and quantitative means of measuring disease progression, greatly facilitating future studies of disease mechanisms and candidate therapeutics. -
Herring A, Messana A, Bara AM, Hazelbaker DZ, Eggan K, Barrett LE. 2016. Generation of a TLE1 homozygous knockout human embryonic stem cell line using CRISPR-Cas9. Stem cell research. 17(2):430-432. Pubmed: 27879218 DOI:10.1016/j.scr.2016.09.009 Herring A, Messana A, Bara AM, Hazelbaker DZ, Eggan K, Barrett LE. 2016. Generation of a TLE1 homozygous knockout human embryonic stem cell line using CRISPR-Cas9. Stem cell research. 17(2):430-432. Pubmed: 27879218 DOI:10.1016/j.scr.2016.09.009 Here, we generated a biallelic mutation in the TLE1 (Transducin Like Enhancer of Split 1) gene using CRISPR-Cas9 editing in the human embryonic stem cell (hESC) line WA01. The homozygous knockout cell line, TLE1-464-G04, displays loss of TLE1 protein expression while maintaining pluripotency, differentiation potential and genomic integrity.Copyright © 2016 Helmholtz Zentrum München. Published by Elsevier B.V. All rights reserved. -
Bara AM, Messana A, Herring A, Hazelbaker DZ, Eggan K, Barrett LE. 2016. Generation of a TLE3 heterozygous knockout human embryonic stem cell line using CRISPR-Cas9. Stem cell research. 17(2):441-443. Pubmed: 27879221 DOI:10.1016/j.scr.2016.09.008 Bara AM, Messana A, Herring A, Hazelbaker DZ, Eggan K, Barrett LE. 2016. Generation of a TLE3 heterozygous knockout human embryonic stem cell line using CRISPR-Cas9. Stem cell research. 17(2):441-443. Pubmed: 27879221 DOI:10.1016/j.scr.2016.09.008 Here, we generated a monoallelic mutation in the TLE3 (Transducin Like Enhancer of Split 3) gene using CRISPR-Cas9 editing in the human embryonic stem cell (hESC) line WA01. The heterozygous knockout cell line, TLE3-447-D08-A01, displays partial loss of TLE3 protein expression while maintaining pluripotency, differentiation potential and genomic integrity.Copyright © 2016 Helmholtz Zentrum München. Published by Elsevier B.V. All rights reserved. -
Santos DP, Kiskinis E, Eggan K, Merkle FT. 2016. Comprehensive Protocols for CRISPR/Cas9-based Gene Editing in Human Pluripotent Stem Cells. Current protocols in stem cell biology. 38:5B.6.1-5B.6.60. Pubmed: 27532820 DOI:10.1002/cpsc.15 Santos DP, Kiskinis E, Eggan K, Merkle FT. 2016. Comprehensive Protocols for CRISPR/Cas9-based Gene Editing in Human Pluripotent Stem Cells. Current protocols in stem cell biology. 38:5B.6.1-5B.6.60. Pubmed: 27532820 DOI:10.1002/cpsc.15 Genome editing of human pluripotent stem cells (hPSCs) with the CRISPR/Cas9 system has the potential to revolutionize hPSC-based disease modeling, drug screening, and transplantation therapy. Here, we aim to provide a single resource to enable groups, even those with limited experience with hPSC culture or the CRISPR/Cas9 system, to successfully perform genome editing. The methods are presented in detail and are supported by a theoretical framework to allow for the incorporation of inevitable improvements in the rapidly evolving gene-editing field. We describe protocols to generate hPSC lines with gene-specific knock-outs, small targeted mutations, or knock-in reporters. © 2016 by John Wiley & Sons, Inc.Copyright © 2016 John Wiley & Sons, Inc. -
Burberry A, Suzuki N, Wang JY, Moccia R, Mordes DA, Stewart MH, Suzuki-Uematsu S, Ghosh S, Singh A, Merkle FT, Koszka K, Li QZ, Zon L, Rossi DJ, Trowbridge JJ, Notarangelo LD, Eggan K. 2016. Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease. Science translational medicine. 8(347):347ra93. Pubmed: 27412785 DOI:10.1126/scitranslmed.aaf6038 Burberry A, Suzuki N, Wang JY, Moccia R, Mordes DA, Stewart MH, Suzuki-Uematsu S, Ghosh S, Singh A, Merkle FT, Koszka K, Li QZ, Zon L, Rossi DJ, Trowbridge JJ, Notarangelo LD, Eggan K. 2016. Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease. Science translational medicine. 8(347):347ra93. Pubmed: 27412785 DOI:10.1126/scitranslmed.aaf6038 C9ORF72 mutations are found in a significant fraction of patients suffering from amyotrophic lateral sclerosis and frontotemporal dementia, yet the function of the C9ORF72 gene product remains poorly understood. We show that mice harboring loss-of-function mutations in the ortholog of C9ORF72 develop splenomegaly, neutrophilia, thrombocytopenia, increased expression of inflammatory cytokines, and severe autoimmunity, ultimately leading to a high mortality rate. Transplantation of mutant mouse bone marrow into wild-type recipients was sufficient to recapitulate the phenotypes observed in the mutant animals, including autoimmunity and premature mortality. Reciprocally, transplantation of wild-type mouse bone marrow into mutant mice improved their phenotype. We conclude that C9ORF72 serves an important function within the hematopoietic system to restrict inflammation and the development of autoimmunity.Copyright © 2016, American Association for the Advancement of Science. -
Ray MK, Wiskow O, King MJ, Ismail N, Ergun A, Wang Y, Plys AJ, Davis CP, Kathrein K, Sadreyev R, Borowsky ML, Eggan K, Zon L, Galloway JL, Kingston RE. 2016. CAT7 and cat7l Long Non-coding RNAs Tune Polycomb Repressive Complex 1 Function during Human and Zebrafish Development. The Journal of biological chemistry. 291(37):19558-72. Pubmed: 27405765 DOI:10.1074/jbc.M116.730853 Ray MK, Wiskow O, King MJ, Ismail N, Ergun A, Wang Y, Plys AJ, Davis CP, Kathrein K, Sadreyev R, Borowsky ML, Eggan K, Zon L, Galloway JL, Kingston RE. 2016. CAT7 and cat7l Long Non-coding RNAs Tune Polycomb Repressive Complex 1 Function during Human and Zebrafish Development. The Journal of biological chemistry. 291(37):19558-72. Pubmed: 27405765 DOI:10.1074/jbc.M116.730853 The essential functions of polycomb repressive complex 1 (PRC1) in development and gene silencing are thought to involve long non-coding RNAs (lncRNAs), but few specific lncRNAs that guide PRC1 activity are known. We screened for lncRNAs, which co-precipitate with PRC1 from chromatin and found candidates that impact polycomb group protein (PcG)-regulated gene expression in vivo A novel lncRNA from this screen, CAT7, regulates expression and polycomb group binding at the MNX1 locus during early neuronal differentiation. CAT7 contains a unique tandem repeat domain that shares high sequence similarity to a non-syntenic zebrafish analog, cat7l Defects caused by interference of cat7l RNA during zebrafish embryogenesis were rescued by human CAT7 RNA, enhanced by interference of a PRC1 component, and suppressed by interference of a known PRC1 target gene, demonstrating cat7l genetically interacts with a PRC1. We propose a model whereby PRC1 acts in concert with specific lncRNAs and that CAT7/cat7l represents convergent lncRNAs that independently evolved to tune PRC1 repression at individual loci.© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. -
Intoh A, Suzuki N, Koszka K, Eggan K. 2016. SLC52A3, A Brown-Vialetto-van Laere syndrome candidate gene is essential for mouse development, but dispensable for motor neuron differentiation. Human molecular genetics. 25(9):1814-23. Pubmed: 26976849 DOI:10.1093/hmg/ddw053 Intoh A, Suzuki N, Koszka K, Eggan K. 2016. SLC52A3, A Brown-Vialetto-van Laere syndrome candidate gene is essential for mouse development, but dispensable for motor neuron differentiation. Human molecular genetics. 25(9):1814-23. Pubmed: 26976849 DOI:10.1093/hmg/ddw053 Riboflavin, also known as vitamin B2, is essential for cellular reduction-oxidation reactions, but is not readily synthesized by mammalian cells. It has been proposed that riboflavin absorption occurs through solute carrier family 52 members (SLC52) A1, A2 and A3. These transporters are also candidate genes for the childhood onset-neural degenerative syndrome Brown-Vialetto-Van Laere (BVVL). Although riboflavin is an essential nutrient, why mutations in its transporters result in a neural cell-specific disorder remains unclear. Here, we provide evidence that Slc52a3 is the mouse ortholog of SLC52A3 and show that Slc52a3 deficiency results in early embryonic lethality. Loss of mutant embryos was associated with both defects in placental formation and increased rates of apoptosis in embryonic cells. In contrast, Slc52a3 -/- embryonic stem cell lines could be readily established and differentiated into motor neurons, suggesting that this transporter is dispensable for neural differentiation and short-term maintenance. Consistent with this finding, examination of Slc52a3 gene products in adult tissues revealed expression in the testis and intestine but little or none in the brain and spinal cord. Our results suggest that BVVL patients with SCL52A3 mutations may be good candidates for riboflavin replacement therapy and suggests that either the mutations these individuals carry are hypomorphic, or that in these cases alternative transporters act during human embryogenesis to allow full-term development.© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com. 2015
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Wainger BJ, Buttermore ED, Oliveira JT, Mellin C, Lee S, Saber WA, Wang AJ, Ichida JK, Chiu IM, Barrett L, Huebner EA, Bilgin C, Tsujimoto N, Brenneis C, Kapur K, Rubin LL, Eggan K, Woolf CJ. 2015. Modeling pain in vitro using nociceptor neurons reprogrammed from fibroblasts. Nature neuroscience. 18(1):17-24. Pubmed: 25420066 DOI:10.1038/nn.3886 Wainger BJ, Buttermore ED, Oliveira JT, Mellin C, Lee S, Saber WA, Wang AJ, Ichida JK, Chiu IM, Barrett L, Huebner EA, Bilgin C, Tsujimoto N, Brenneis C, Kapur K, Rubin LL, Eggan K, Woolf CJ. 2015. Modeling pain in vitro using nociceptor neurons reprogrammed from fibroblasts. Nature neuroscience. 18(1):17-24. Pubmed: 25420066 DOI:10.1038/nn.3886 Reprogramming somatic cells from one cell fate to another can generate specific neurons suitable for disease modeling. To maximize the utility of patient-derived neurons, they must model not only disease-relevant cell classes, but also the diversity of neuronal subtypes found in vivo and the pathophysiological changes that underlie specific clinical diseases. We identified five transcription factors that reprogram mouse and human fibroblasts into noxious stimulus-detecting (nociceptor) neurons. These recapitulated the expression of quintessential nociceptor-specific functional receptors and channels found in adult mouse nociceptor neurons, as well as native subtype diversity. Moreover, the derived nociceptor neurons exhibited TrpV1 sensitization to the inflammatory mediator prostaglandin E2 and the chemotherapeutic drug oxaliplatin, modeling the inherent mechanisms underlying inflammatory pain hypersensitivity and painful chemotherapy-induced neuropathy. Using fibroblasts from patients with familial dysautonomia (hereditary sensory and autonomic neuropathy type III), we found that the technique was able to reveal previously unknown aspects of human disease phenotypes in vitro. -
McNeish J, Gardner JP, Wainger BJ, Woolf CJ, Eggan K. 2015. From Dish to Bedside: Lessons Learned While Translating Findings from a Stem Cell Model of Disease to a Clinical Trial. Cell stem cell. 17(1):8-10. Pubmed: 26140603 DOI:S1934-5909(15)00273-8 McNeish J, Gardner JP, Wainger BJ, Woolf CJ, Eggan K. 2015. From Dish to Bedside: Lessons Learned While Translating Findings from a Stem Cell Model of Disease to a Clinical Trial. Cell stem cell. 17(1):8-10. Pubmed: 26140603 DOI:S1934-5909(15)00273-8 While iPSCs have created unprecedented opportunities for drug discovery, there remains uncertainty concerning the path to the clinic for candidate therapeutics discovered with their use. Here we share lessons that we learned, and believe are generalizable to similar efforts, while taking a discovery made using iPSCs into a clinical trial.Copyright © 2015 Elsevier Inc. All rights reserved. -
Merkle FT, Neuhausser WM, Santos D, Valen E, Gagnon JA, Maas K, Sandoe J, Schier AF, Eggan K. 2015. Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus. Cell reports. 11(6):875-883. Pubmed: 25937281 DOI:10.1016/j.celrep.2015.04.007 Merkle FT, Neuhausser WM, Santos D, Valen E, Gagnon JA, Maas K, Sandoe J, Schier AF, Eggan K. 2015. Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus. Cell reports. 11(6):875-883. Pubmed: 25937281 DOI:10.1016/j.celrep.2015.04.007 The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus.Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved. -
Schwarz-Romond T, Kiskinis E, Eggan K. 2015. Focus on induced pluripotency and cellular reprogramming. The EMBO journal. 34(11):1435. Pubmed: 25916828 DOI:10.15252/embj.201591615 Schwarz-Romond T, Kiskinis E, Eggan K. 2015. Focus on induced pluripotency and cellular reprogramming. The EMBO journal. 34(11):1435. Pubmed: 25916828 DOI:10.15252/embj.201591615 -
de Boer AS, Eggan K. 2015. A perspective on stem cell modeling of amyotrophic lateral sclerosis. Cell cycle (Georgetown, Tex.). 14(23):3679-88. Pubmed: 26505672 DOI:10.1080/15384101.2015.1093712 de Boer AS, Eggan K. 2015. A perspective on stem cell modeling of amyotrophic lateral sclerosis. Cell cycle (Georgetown, Tex.). 14(23):3679-88. Pubmed: 26505672 DOI:10.1080/15384101.2015.1093712 Amyotrophic lateral sclerosis is a complex neurodegenerative disease. Limitations in animal models have impeded progress in studying disease pathology and potential drug discovery. Here, we will review recent advances in the development of stem cell models for the study of ALS. Additionally, we will discuss the progress toward therapeutic development derived from these stem cell based assays. -
Sun S, Ling SC, Qiu J, Albuquerque CP, Zhou Y, Tokunaga S, Li H, Qiu H, Bui A, Yeo GW, Huang EJ, Eggan K, Zhou H, Fu XD, Lagier-Tourenne C, Cleveland DW. 2015. ALS-causative mutations in FUS/TLS confer gain and loss of function by altered association with SMN and U1-snRNP. Nature communications. 6:6171. Pubmed: 25625564 DOI:10.1038/ncomms7171 Sun S, Ling SC, Qiu J, Albuquerque CP, Zhou Y, Tokunaga S, Li H, Qiu H, Bui A, Yeo GW, Huang EJ, Eggan K, Zhou H, Fu XD, Lagier-Tourenne C, Cleveland DW. 2015. ALS-causative mutations in FUS/TLS confer gain and loss of function by altered association with SMN and U1-snRNP. Nature communications. 6:6171. Pubmed: 25625564 DOI:10.1038/ncomms7171 The RNA-binding protein FUS/TLS, mutation in which is causative of the fatal motor neuron disease amyotrophic lateral sclerosis (ALS), is demonstrated to directly bind to the U1-snRNP and SMN complexes. ALS-causative mutations in FUS/TLS are shown to abnormally enhance their interaction with SMN and dysregulate its function, including loss of Gems and altered levels of small nuclear RNAs. The same mutants are found to have reduced association with U1-snRNP. Correspondingly, global RNA analysis reveals a mutant-dependent loss of splicing activity, with ALS-linked mutants failing to reverse changes caused by loss of wild-type FUS/TLS. Furthermore, a common FUS/TLS mutant-associated RNA splicing signature is identified in ALS patient fibroblasts. Taken together, these studies establish potentially converging disease mechanisms in ALS and spinal muscular atrophy, with ALS-causative mutants acquiring properties representing both gain (dysregulation of SMN) and loss (reduced RNA processing mediated by U1-snRNP) of function. -
Toma JS, Shettar BC, Chipman PH, Pinto DM, Borowska JP, Ichida JK, Fawcett JP, Zhang Y, Eggan K, Rafuse VF. 2015. Motoneurons derived from induced pluripotent stem cells develop mature phenotypes typical of endogenous spinal motoneurons. The Journal of neuroscience : the official journal of the Society for Neuroscience. 35(3):1291-306. Pubmed: 25609642 DOI:10.1523/JNEUROSCI.2126-14.2015 Toma JS, Shettar BC, Chipman PH, Pinto DM, Borowska JP, Ichida JK, Fawcett JP, Zhang Y, Eggan K, Rafuse VF. 2015. Motoneurons derived from induced pluripotent stem cells develop mature phenotypes typical of endogenous spinal motoneurons. The Journal of neuroscience : the official journal of the Society for Neuroscience. 35(3):1291-306. Pubmed: 25609642 DOI:10.1523/JNEUROSCI.2126-14.2015 Induced pluripotent cell-derived motoneurons (iPSCMNs) are sought for use in cell replacement therapies and treatment strategies for motoneuron diseases such as amyotrophic lateral sclerosis (ALS). However, much remains unknown about the physiological properties of iPSCMNs and how they compare with endogenous spinal motoneurons or embryonic stem cell-derived motoneurons (ESCMNs). In the present study, we first used a proteomic approach and compared protein expression profiles between iPSCMNs and ESCMNs to show that <4% of the proteins identified were differentially regulated. Like ESCs, we found that mouse iPSCs treated with retinoic acid and a smoothened agonist differentiated into motoneurons expressing the LIM homeodomain protein Lhx3. When transplanted into the neural tube of developing chick embryos, iPSCMNs selectively targeted muscles normally innervated by Lhx3 motoneurons. In vitro studies showed that iPSCMNs form anatomically mature and functional neuromuscular junctions (NMJs) when cocultured with chick myofibers for several weeks. Electrophysiologically, iPSCMNs developed passive membrane and firing characteristic typical of postnatal motoneurons after several weeks in culture. Finally, iPSCMNs grafted into transected mouse tibial nerve projected axons to denervated gastrocnemius muscle fibers, where they formed functional NMJs, restored contractile force. and attenuated denervation atrophy. Together, iPSCMNs possess many of the same cellular and physiological characteristics as ESCMNs and endogenous spinal motoneurons. These results further justify using iPSCMNs as a source of motoneurons for cell replacement therapies and to study motoneuron diseases such as ALS.Copyright © 2015 the authors 0270-6474/15/351291-16$15.00/0. -
Paull D, Sevilla A, Zhou H, Hahn AK, Kim H, Napolitano C, Tsankov A, Shang L, Krumholz K, Jagadeesan P, Woodard CM, Sun B, Vilboux T, Zimmer M, Forero E, Moroziewicz DN, Martinez H, Malicdan MC, Weiss KA, Vensand LB, Dusenberry CR, Polus H, Sy KT, Kahler DJ, Gahl WA, Solomon SL, Chang S, Meissner A, Eggan K, Noggle SA. 2015. Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells. Nature methods. 12(9):885-92. Pubmed: 26237226 DOI:10.1038/nmeth.3507 Paull D, Sevilla A, Zhou H, Hahn AK, Kim H, Napolitano C, Tsankov A, Shang L, Krumholz K, Jagadeesan P, Woodard CM, Sun B, Vilboux T, Zimmer M, Forero E, Moroziewicz DN, Martinez H, Malicdan MC, Weiss KA, Vensand LB, Dusenberry CR, Polus H, Sy KT, Kahler DJ, Gahl WA, Solomon SL, Chang S, Meissner A, Eggan K, Noggle SA. 2015. Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells. Nature methods. 12(9):885-92. Pubmed: 26237226 DOI:10.1038/nmeth.3507 Induced pluripotent stem cells (iPSCs) are an essential tool for modeling how causal genetic variants impact cellular function in disease, as well as an emerging source of tissue for regenerative medicine. The preparation of somatic cells, their reprogramming and the subsequent verification of iPSC pluripotency are laborious, manual processes limiting the scale and reproducibility of this technology. Here we describe a modular, robotic platform for iPSC reprogramming enabling automated, high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention. We demonstrate that automated reprogramming and the pooled selection of polyclonal pluripotent cells results in high-quality, stable iPSCs. These lines display less line-to-line variation than either manually produced lines or lines produced through automation followed by single-colony subcloning. The robotic platform we describe will enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized medicines. -
Merkle FT, Maroof A, Wataya T, Sasai Y, Studer L, Eggan K, Schier AF. 2015. Generation of neuropeptidergic hypothalamic neurons from human pluripotent stem cells. Development (Cambridge, England). 142(4):633-43. Pubmed: 25670790 DOI:10.1242/dev.117978 Merkle FT, Maroof A, Wataya T, Sasai Y, Studer L, Eggan K, Schier AF. 2015. Generation of neuropeptidergic hypothalamic neurons from human pluripotent stem cells. Development (Cambridge, England). 142(4):633-43. Pubmed: 25670790 DOI:10.1242/dev.117978 Hypothalamic neurons orchestrate many essential physiological and behavioral processes via secreted neuropeptides, and are relevant to human diseases such as obesity, narcolepsy and infertility. We report the differentiation of human pluripotent stem cells into many of the major types of neuropeptidergic hypothalamic neurons, including those producing pro-opiolemelanocortin, agouti-related peptide, hypocretin/orexin, melanin-concentrating hormone, oxytocin, arginine vasopressin, corticotropin-releasing hormone (CRH) or thyrotropin-releasing hormone. Hypothalamic neurons can be generated using a 'self-patterning' strategy that yields a broad array of cell types, or via a more reproducible directed differentiation approach. Stem cell-derived human hypothalamic neurons share characteristic morphological properties and gene expression patterns with their counterparts in vivo, and are able to integrate into the mouse brain. These neurons could form the basis of cellular models, chemical screens or cellular therapies to study and treat common human diseases.© 2015. Published by The Company of Biologists Ltd. 2014
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Carter AC, Davis-Dusenbery BN, Koszka K, Ichida JK, Eggan K. 2014. Nanog-independent reprogramming to iPSCs with canonical factors. Stem cell reports. 2(2):119-26. Pubmed: 24527385 DOI:10.1016/j.stemcr.2013.12.010 Carter AC, Davis-Dusenbery BN, Koszka K, Ichida JK, Eggan K. 2014. Nanog-independent reprogramming to iPSCs with canonical factors. Stem cell reports. 2(2):119-26. Pubmed: 24527385 DOI:10.1016/j.stemcr.2013.12.010 It has been suggested that the transcription factor Nanog is essential for the establishment of pluripotency during the derivation of embryonic stem cells and induced pluripotent stem cells (iPSCs). However, successful reprogramming to pluripotency with a growing list of divergent transcription factors, at ever-increasing efficiencies, suggests that there may be many distinct routes to a pluripotent state. Here, we have investigated whether Nanog is necessary for reprogramming murine fibroblasts under highly efficient conditions using the canonical-reprogramming factors Oct4, Sox2, Klf4, and cMyc. In agreement with prior results, the efficiency of reprogramming Nanog (-/-) fibroblasts was significantly lower than that of control fibroblasts. However, in contrast to previous findings, we were able to reproducibly generate iPSCs from Nanog (-/-) fibroblasts that effectively contributed to the germline of chimeric mice. Thus, whereas Nanog may be an important mediator of reprogramming, it is not required for establishing pluripotency in the mouse, even under standard conditions. -
Alami NH, Smith RB, Carrasco MA, Williams LA, Winborn CS, Han SSW, Kiskinis E, Winborn B, Freibaum BD, Kanagaraj A, Clare AJ, Badders NM, Bilican B, Chaum E, Chandran S, Shaw CE, Eggan KC, Maniatis T, Taylor JP. 2014. Axonal transport of TDP-43 mRNA granules is impaired by ALS-causing mutations. Neuron. 81(3):536-543. Pubmed: 24507191 DOI:10.1016/j.neuron.2013.12.018 Alami NH, Smith RB, Carrasco MA, Williams LA, Winborn CS, Han SSW, Kiskinis E, Winborn B, Freibaum BD, Kanagaraj A, Clare AJ, Badders NM, Bilican B, Chaum E, Chandran S, Shaw CE, Eggan KC, Maniatis T, Taylor JP. 2014. Axonal transport of TDP-43 mRNA granules is impaired by ALS-causing mutations. Neuron. 81(3):536-543. Pubmed: 24507191 DOI:10.1016/j.neuron.2013.12.018 The RNA-binding protein TDP-43 regulates RNA metabolism at multiple levels, including transcription, RNA splicing, and mRNA stability. TDP-43 is a major component of the cytoplasmic inclusions characteristic of amyotrophic lateral sclerosis and some types of frontotemporal lobar degeneration. The importance of TDP-43 in disease is underscored by the fact that dominant missense mutations are sufficient to cause disease, although the role of TDP-43 in pathogenesis is unknown. Here we show that TDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons in vitro and in vivo and facilitate delivery of target mRNA to distal neuronal compartments. TDP-43 mutations impair this mRNA transport function in vivo and in vitro, including in stem cell-derived motor neurons from ALS patients bearing any one of three different TDP-43 ALS-causing mutations. Thus, TDP-43 mutations that cause ALS lead to partial loss of a novel cytoplasmic function of TDP-43.Copyright © 2014 Elsevier Inc. All rights reserved. -
Davis-Dusenbery BN, Williams LA, Klim JR, Eggan K. 2014. How to make spinal motor neurons. Development (Cambridge, England). 141(3):491-501. Pubmed: 24449832 DOI:10.1242/dev.097410 Davis-Dusenbery BN, Williams LA, Klim JR, Eggan K. 2014. How to make spinal motor neurons. Development (Cambridge, England). 141(3):491-501. Pubmed: 24449832 DOI:10.1242/dev.097410 All muscle movements, including breathing, walking, and fine motor skills rely on the function of the spinal motor neuron to transmit signals from the brain to individual muscle groups. Loss of spinal motor neuron function underlies several neurological disorders for which treatment has been hampered by the inability to obtain sufficient quantities of primary motor neurons to perform mechanistic studies or drug screens. Progress towards overcoming this challenge has been achieved through the synthesis of developmental biology paradigms and advances in stem cell and reprogramming technology, which allow the production of motor neurons in vitro. In this Primer, we discuss how the logic of spinal motor neuron development has been applied to allow generation of motor neurons either from pluripotent stem cells by directed differentiation and transcriptional programming, or from somatic cells by direct lineage conversion. Finally, we discuss methods to evaluate the molecular and functional properties of motor neurons generated through each of these techniques. -
Koren A, Handsaker RE, Kamitaki N, Karlić R, Ghosh S, Polak P, Eggan K, McCarroll SA. 2014. Genetic variation in human DNA replication timing. Cell. 159(5):1015-1026. Pubmed: 25416942 DOI:10.1016/j.cell.2014.10.025 Koren A, Handsaker RE, Kamitaki N, Karlić R, Ghosh S, Polak P, Eggan K, McCarroll SA. 2014. Genetic variation in human DNA replication timing. Cell. 159(5):1015-1026. Pubmed: 25416942 DOI:10.1016/j.cell.2014.10.025 Genomic DNA replicates in a choreographed temporal order that impacts the distribution of mutations along the genome. We show here that DNA replication timing is shaped by genetic polymorphisms that act in cis upon megabase-scale DNA segments. In genome sequences from proliferating cells, read depth along chromosomes reflected DNA replication activity in those cells. We used this relationship to analyze variation in replication timing among 161 individuals sequenced by the 1000 Genomes Project. Genome-wide association of replication timing with genetic variation identified 16 loci at which inherited alleles associate with replication timing. We call these "replication timing quantitative trait loci" (rtQTLs). rtQTLs involved the differential use of replication origins, exhibited allele-specific effects on replication timing, and associated with gene expression variation at megabase scales. Our results show replication timing to be shaped by genetic polymorphism and identify a means by which inherited polymorphism regulates the mutability of nearby sequences. -
Haidet-Phillips AM, Roybon L, Gross SK, Tuteja A, Donnelly CJ, Richard JP, Ko M, Sherman A, Eggan K, Henderson CE, Maragakis NJ. 2014. Gene profiling of human induced pluripotent stem cell-derived astrocyte progenitors following spinal cord engraftment. Stem cells translational medicine. 3(5):575-85. Pubmed: 24604284 DOI:10.5966/sctm.2013-0153 Haidet-Phillips AM, Roybon L, Gross SK, Tuteja A, Donnelly CJ, Richard JP, Ko M, Sherman A, Eggan K, Henderson CE, Maragakis NJ. 2014. Gene profiling of human induced pluripotent stem cell-derived astrocyte progenitors following spinal cord engraftment. Stem cells translational medicine. 3(5):575-85. Pubmed: 24604284 DOI:10.5966/sctm.2013-0153 The generation of human induced pluripotent stem cells (hiPSCs) represents an exciting advancement with promise for stem cell transplantation therapies as well as for neurological disease modeling. Based on the emerging roles for astrocytes in neurological disorders, we investigated whether hiPSC-derived astrocyte progenitors could be engrafted to the rodent spinal cord and how the characteristics of these cells changed between in vitro culture and after transplantation to the in vivo spinal cord environment. Our results show that human embryonic stem cell- and hiPSC-derived astrocyte progenitors survive long-term after spinal cord engraftment and differentiate to astrocytes in vivo with few cells from other lineages present. Gene profiling of the transplanted cells demonstrates the astrocyte progenitors continue to mature in vivo and upregulate a variety of astrocyte-specific genes. Given this mature astrocyte gene profile, this work highlights hiPSCs as a tool to investigate disease-related astrocyte biology using in vivo disease modeling with significant implications for human neurological diseases currently lacking animal models. -
Ichida JK, Tcw J, Williams LA, Carter AC, Shi Y, Moura MT, Ziller M, Singh S, Amabile G, Bock C, Umezawa A, Rubin LL, Bradner JE, Akutsu H, Meissner A, Eggan K. 2014. Notch inhibition allows oncogene-independent generation of iPS cells. Nature chemical biology. 10(8):632-639. Pubmed: 24952596 DOI:10.1038/nchembio.1552 Ichida JK, Tcw J, Williams LA, Carter AC, Shi Y, Moura MT, Ziller M, Singh S, Amabile G, Bock C, Umezawa A, Rubin LL, Bradner JE, Akutsu H, Meissner A, Eggan K. 2014. Notch inhibition allows oncogene-independent generation of iPS cells. Nature chemical biology. 10(8):632-639. Pubmed: 24952596 DOI:10.1038/nchembio.1552 The reprogramming of somatic cells to pluripotency using defined transcription factors holds great promise for biomedicine. However, human reprogramming remains inefficient and relies either on the use of the potentially dangerous oncogenes KLF4 and CMYC or the genetic inhibition of the tumor suppressor gene p53. We hypothesized that inhibition of signal transduction pathways that promote differentiation of the target somatic cells during development might relieve the requirement for non-core pluripotency factors during induced pluripotent stem cell (iPSC) reprogramming. Here, we show that inhibition of Notch greatly improves the efficiency of iPSC generation from mouse and human keratinocytes by suppressing p21 in a p53-independent manner and thereby enriching for undifferentiated cells capable of long-term self-renewal. Pharmacological inhibition of Notch enabled routine production of human iPSCs without KLF4 and CMYC while leaving p53 activity intact. Thus, restricting the development of somatic cells by altering intercellular communication enables the production of safer human iPSCs. -
Wainger BJ, Kiskinis E, Mellin C, Wiskow O, Han SS, Sandoe J, Perez NP, Williams LA, Lee S, Boulting G, Berry JD, Brown RH, Cudkowicz ME, Bean BP, Eggan K, Woolf CJ. 2014. Intrinsic membrane hyperexcitability of amyotrophic lateral sclerosis patient-derived motor neurons. Cell reports. 7(1):1-11. Pubmed: 24703839 DOI:S2211-1247(14)00197-1 Wainger BJ, Kiskinis E, Mellin C, Wiskow O, Han SS, Sandoe J, Perez NP, Williams LA, Lee S, Boulting G, Berry JD, Brown RH, Cudkowicz ME, Bean BP, Eggan K, Woolf CJ. 2014. Intrinsic membrane hyperexcitability of amyotrophic lateral sclerosis patient-derived motor neurons. Cell reports. 7(1):1-11. Pubmed: 24703839 DOI:S2211-1247(14)00197-1 Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor nervous system. We show using multielectrode array and patch-clamp recordings that hyperexcitability detected by clinical neurophysiological studies of ALS patients is recapitulated in induced pluripotent stem cell-derived motor neurons from ALS patients harboring superoxide dismutase 1 (SOD1), C9orf72, and fused-in-sarcoma mutations. Motor neurons produced from a genetically corrected but otherwise isogenic SOD1(+/+) stem cell line do not display the hyperexcitability phenotype. SOD1(A4V/+) ALS patient-derived motor neurons have reduced delayed-rectifier potassium current amplitudes relative to control-derived motor neurons, a deficit that may underlie their hyperexcitability. The Kv7 channel activator retigabine both blocks the hyperexcitability and improves motor neuron survival in vitro when tested in SOD1 mutant ALS cases. Therefore, electrophysiological characterization of human stem cell-derived neurons can reveal disease-related mechanisms and identify therapeutic candidates.Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved. -
Kiskinis E, Sandoe J, Williams LA, Boulting GL, Moccia R, Wainger BJ, Han S, Peng T, Thams S, Mikkilineni S, Mellin C, Merkle FT, Davis-Dusenbery BN, Ziller M, Oakley D, Ichida J, Di Costanzo S, Atwater N, Maeder ML, Goodwin MJ, Nemesh J, Handsaker RE, Paull D, Noggle S, McCarroll SA, Joung JK, Woolf CJ, Brown RH, Eggan K. 2014. Pathways disrupted in human ALS motor neurons identified through genetic correction of mutant SOD1. Cell stem cell. 14(6):781-95. Pubmed: 24704492 DOI:S1934-5909(14)00098-8 Kiskinis E, Sandoe J, Williams LA, Boulting GL, Moccia R, Wainger BJ, Han S, Peng T, Thams S, Mikkilineni S, Mellin C, Merkle FT, Davis-Dusenbery BN, Ziller M, Oakley D, Ichida J, Di Costanzo S, Atwater N, Maeder ML, Goodwin MJ, Nemesh J, Handsaker RE, Paull D, Noggle S, McCarroll SA, Joung JK, Woolf CJ, Brown RH, Eggan K. 2014. Pathways disrupted in human ALS motor neurons identified through genetic correction of mutant SOD1. Cell stem cell. 14(6):781-95. Pubmed: 24704492 DOI:S1934-5909(14)00098-8 Although many distinct mutations in a variety of genes are known to cause Amyotrophic Lateral Sclerosis (ALS), it remains poorly understood how they selectively impact motor neuron biology and whether they converge on common pathways to cause neuronal degeneration. Here, we have combined reprogramming and stem cell differentiation approaches with genome engineering and RNA sequencing to define the transcriptional and functional changes that are induced in human motor neurons by mutant SOD1. Mutant SOD1 protein induced a transcriptional signature indicative of increased oxidative stress, reduced mitochondrial function, altered subcellular transport, and activation of the ER stress and unfolded protein response pathways. Functional studies demonstrated that these pathways were perturbed in a manner dependent on the SOD1 mutation. Finally, interrogation of stem-cell-derived motor neurons produced from ALS patients harboring a repeat expansion in C9orf72 indicates that at least a subset of these changes are more broadly conserved in ALS.Copyright © 2014 Elsevier Inc. All rights reserved. -
Fukuda A, Tomikawa J, Miura T, Hata K, Nakabayashi K, Eggan K, Akutsu H, Umezawa A. 2014. The role of maternal-specific H3K9me3 modification in establishing imprinted X-chromosome inactivation and embryogenesis in mice. Nature communications. 5:5464. Pubmed: 25394724 DOI:10.1038/ncomms6464 Fukuda A, Tomikawa J, Miura T, Hata K, Nakabayashi K, Eggan K, Akutsu H, Umezawa A. 2014. The role of maternal-specific H3K9me3 modification in establishing imprinted X-chromosome inactivation and embryogenesis in mice. Nature communications. 5:5464. Pubmed: 25394724 DOI:10.1038/ncomms6464 Maintaining a single active X-chromosome by repressing Xist is crucial for embryonic development in mice. Although the Xist activator RNF12/RLIM is present as a maternal factor, maternal Xist (Xm-Xist) is repressed during preimplantation phases to establish imprinted X-chromosome inactivation (XCI). Here we show, using a highly reproducible chromatin immunoprecipitation method that facilitates chromatin analysis of preimplantation embryos, that H3K9me3 is enriched at the Xist promoter region, preventing Xm-Xist activation by RNF12. The high levels of H3K9me3 at the Xist promoter region are lost in embryonic stem (ES) cells, and ES-cloned embryos show RNF12-dependent Xist expression. Moreover, lack of Xm-XCI in the trophectoderm, rather than loss of paternally expressed imprinted genes, is the primary cause of embryonic lethality in 70-80% of parthenogenotes immediately after implantation. This study reveals that H3K9me3 is involved in the imprinting that silences Xm-Xist. Our findings highlight the role of maternal-specific H3K9me3 modification in embryo development. -
de Boer AS, Koszka K, Kiskinis E, Suzuki N, Davis-Dusenbery BN, Eggan K. 2014. Genetic validation of a therapeutic target in a mouse model of ALS. Science translational medicine. 6(248):248ra104. Pubmed: 25100738 DOI:10.1126/scitranslmed.3009351 de Boer AS, Koszka K, Kiskinis E, Suzuki N, Davis-Dusenbery BN, Eggan K. 2014. Genetic validation of a therapeutic target in a mouse model of ALS. Science translational medicine. 6(248):248ra104. Pubmed: 25100738 DOI:10.1126/scitranslmed.3009351 Neurons produced from stem cells have emerged as a tool to identify new therapeutic targets for neurological diseases such as amyotrophic lateral sclerosis (ALS). However, it remains unclear to what extent these new mechanistic insights will translate to animal models, an important step in the validation of new targets. Previously, we found that glia from mice carrying the SOD1G93A mutation, a model of ALS, were toxic to stem cell-derived human motor neurons. We use pharmacological and genetic approaches to demonstrate that the prostanoid receptor DP1 mediates this glial toxicity. Furthermore, we validate the importance of this mechanism for neural degeneration in vivo. Genetic ablation of DP1 in SOD1G93A mice extended life span, decreased microglial activation, and reduced motor neuron loss. Our findings suggest that blocking DP1 may be a therapeutic strategy in ALS and demonstrate that discoveries from stem cell models of disease can be corroborated in vivo.Copyright © 2014, American Association for the Advancement of Science. -
Smith ZD, Chan MM, Humm KC, Karnik R, Mekhoubad S, Regev A, Eggan K, Meissner A. 2014. DNA methylation dynamics of the human preimplantation embryo. Nature. 511(7511):611-5. Pubmed: 25079558 DOI:10.1038/nature13581 Smith ZD, Chan MM, Humm KC, Karnik R, Mekhoubad S, Regev A, Eggan K, Meissner A. 2014. DNA methylation dynamics of the human preimplantation embryo. Nature. 511(7511):611-5. Pubmed: 25079558 DOI:10.1038/nature13581 In mammals, cytosine methylation is predominantly restricted to CpG dinucleotides and stably distributed across the genome, with local, cell-type-specific regulation directed by DNA binding factors. This comparatively static landscape is in marked contrast with the events of fertilization, during which the paternal genome is globally reprogrammed. Paternal genome demethylation includes the majority of CpGs, although methylation remains detectable at several notable features. These dynamics have been extensively characterized in the mouse, with only limited observations available in other mammals, and direct measurements are required to understand the extent to which early embryonic landscapes are conserved. We present genome-scale DNA methylation maps of human preimplantation development and embryonic stem cell derivation, confirming a transient state of global hypomethylation that includes most CpGs, while sites of residual maintenance are primarily restricted to gene bodies. Although most features share similar dynamics to those in mouse, maternally contributed methylation is divergently targeted to species-specific sets of CpG island promoters that extend beyond known imprint control regions. Retrotransposon regulation is also highly diverse, and transitions from maternally to embryonically expressed elements. Together, our data confirm that paternal genome demethylation is a general attribute of early mammalian development that is characterized by distinct modes of epigenetic regulation. -
Schwartz JC, Podell ER, Han SS, Berry JD, Eggan KC, Cech TR. 2014. FUS is sequestered in nuclear aggregates in ALS patient fibroblasts. Molecular biology of the cell. 25(17):2571-8. Pubmed: 25009283 DOI:10.1091/mbc.E14-05-1007 Schwartz JC, Podell ER, Han SS, Berry JD, Eggan KC, Cech TR. 2014. FUS is sequestered in nuclear aggregates in ALS patient fibroblasts. Molecular biology of the cell. 25(17):2571-8. Pubmed: 25009283 DOI:10.1091/mbc.E14-05-1007 Mutations in the RNA-binding protein FUS have been shown to cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We investigate whether mutant FUS protein in ALS patient-derived fibroblasts affects normal FUS functions in the nucleus. We investigated fibroblasts from two ALS patients possessing different FUS mutations and a normal control. Fibroblasts from these patients have their nuclear FUS protein trapped in SDS-resistant aggregates. Genome-wide analysis reveals an inappropriate accumulation of Ser-2 phosphorylation on RNA polymerase II (RNA Pol II) near the transcription start sites of 625 genes for ALS patient cells and after small interfering RNA (siRNA) knockdown of FUS in normal fibroblasts. Furthermore, both the presence of mutant FUS protein and siRNA knockdown of wild-type FUS correlate with altered distribution of RNA Pol II within fibroblast nuclei. A loss of FUS function in orchestrating Ser-2 phosphorylation of the CTD of RNA Pol II is detectable in ALS patient-derived fibroblasts expressing mutant FUS protein, even when the FUS protein remains largely nuclear. A likely explanation for this loss of function is the aggregation of FUS protein in nuclei. Thus our results suggest a specific mechanism by which mutant FUS can have biological consequences other than by the formation of cytoplasmic aggregates.© 2014 Schwartz et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). -
Woodard CM, Campos BA, Kuo SH, Nirenberg MJ, Nestor MW, Zimmer M, Mosharov EV, Sulzer D, Zhou H, Paull D, Clark L, Schadt EE, Sardi SP, Rubin L, Eggan K, Brock M, Lipnick S, Rao M, Chang S, Li A, Noggle SA. 2014. iPSC-derived dopamine neurons reveal differences between monozygotic twins discordant for Parkinson's disease. Cell reports. 9(4):1173-82. Pubmed: 25456120 DOI:S2211-1247(14)00877-8 Woodard CM, Campos BA, Kuo SH, Nirenberg MJ, Nestor MW, Zimmer M, Mosharov EV, Sulzer D, Zhou H, Paull D, Clark L, Schadt EE, Sardi SP, Rubin L, Eggan K, Brock M, Lipnick S, Rao M, Chang S, Li A, Noggle SA. 2014. iPSC-derived dopamine neurons reveal differences between monozygotic twins discordant for Parkinson's disease. Cell reports. 9(4):1173-82. Pubmed: 25456120 DOI:S2211-1247(14)00877-8 Parkinson's disease (PD) has been attributed to a combination of genetic and nongenetic factors. We studied a set of monozygotic twins harboring the heterozygous glucocerebrosidase mutation (GBA N370S) but clinically discordant for PD. We applied induced pluripotent stem cell (iPSC) technology for PD disease modeling using the twins' fibroblasts to evaluate and dissect the genetic and nongenetic contributions. Utilizing fluorescence-activated cell sorting, we obtained a homogenous population of "footprint-free" iPSC-derived midbrain dopaminergic (mDA) neurons. The mDA neurons from both twins had ∼50% GBA enzymatic activity, ∼3-fold elevated α-synuclein protein levels, and a reduced capacity to synthesize and release dopamine. Interestingly, the affected twin's neurons showed an even lower dopamine level, increased monoamine oxidase B (MAO-B) expression, and impaired intrinsic network activity. Overexpression of wild-type GBA and treatment with MAO-B inhibitors normalized α-synuclein and dopamine levels, suggesting a combination therapy for the affected twin.Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved. 2013
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Maroof AM, Keros S, Tyson JA, Ying SW, Ganat YM, Merkle FT, Liu B, Goulburn A, Stanley EG, Elefanty AG, Widmer HR, Eggan K, Goldstein PA, Anderson SA, Studer L. 2013. Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell stem cell. 12(5):559-72. Pubmed: 23642365 DOI:S1934-5909(13)00144-6 Maroof AM, Keros S, Tyson JA, Ying SW, Ganat YM, Merkle FT, Liu B, Goulburn A, Stanley EG, Elefanty AG, Widmer HR, Eggan K, Goldstein PA, Anderson SA, Studer L. 2013. Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell stem cell. 12(5):559-72. Pubmed: 23642365 DOI:S1934-5909(13)00144-6 Human pluripotent stem cells are a powerful tool for modeling brain development and disease. The human cortex is composed of two major neuronal populations: projection neurons and local interneurons. Cortical interneurons comprise a diverse class of cell types expressing the neurotransmitter GABA. Dysfunction of cortical interneurons has been implicated in neuropsychiatric diseases, including schizophrenia, autism, and epilepsy. Here, we demonstrate the highly efficient derivation of human cortical interneurons in an NKX2.1::GFP human embryonic stem cell reporter line. Manipulating the timing of SHH activation yields three distinct GFP+ populations with specific transcriptional profiles, neurotransmitter phenotypes, and migratory behaviors. Further differentiation in a murine cortical environment yields parvalbumin- and somatostatin-expressing neurons that exhibit synaptic inputs and electrophysiological properties of cortical interneurons. Our study defines the signals sufficient for modeling human ventral forebrain development in vitro and lays the foundation for studying cortical interneuron involvement in human disease pathology.Copyright © 2013 Elsevier Inc. All rights reserved. -
Hanada T, Weitzer S, Mair B, Bernreuther C, Wainger BJ, Ichida J, Hanada R, Orthofer M, Cronin SJ, Komnenovic V, Minis A, Sato F, Mimata H, Yoshimura A, Tamir I, Rainer J, Kofler R, Yaron A, Eggan KC, Woolf CJ, Glatzel M, Herbst R, Martinez J, Penninger JM. 2013. CLP1 links tRNA metabolism to progressive motor-neuron loss. Nature. 495(7442):474-80. Pubmed: 23474986 DOI:10.1038/nature11923 Hanada T, Weitzer S, Mair B, Bernreuther C, Wainger BJ, Ichida J, Hanada R, Orthofer M, Cronin SJ, Komnenovic V, Minis A, Sato F, Mimata H, Yoshimura A, Tamir I, Rainer J, Kofler R, Yaron A, Eggan KC, Woolf CJ, Glatzel M, Herbst R, Martinez J, Penninger JM. 2013. CLP1 links tRNA metabolism to progressive motor-neuron loss. Nature. 495(7442):474-80. Pubmed: 23474986 DOI:10.1038/nature11923 CLP1 was the first mammalian RNA kinase to be identified. However, determining its in vivo function has been elusive. Here we generated kinase-dead Clp1 (Clp1(K/K)) mice that show a progressive loss of spinal motor neurons associated with axonal degeneration in the peripheral nerves and denervation of neuromuscular junctions, resulting in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of a novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-transfer RNA. These tRNA fragments sensitize cells to oxidative-stress-induced p53 (also known as TRP53) activation and p53-dependent cell death. Genetic inactivation of p53 rescues Clp1(K/K) mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53. -
Niakan KK, Eggan K. 2013. Analysis of human embryos from zygote to blastocyst reveals distinct gene expression patterns relative to the mouse. Developmental biology. 375(1):54-64. Pubmed: 23261930 DOI:S0012-1606(12)00674-4 Niakan KK, Eggan K. 2013. Analysis of human embryos from zygote to blastocyst reveals distinct gene expression patterns relative to the mouse. Developmental biology. 375(1):54-64. Pubmed: 23261930 DOI:S0012-1606(12)00674-4 Early mammalian embryogenesis is controlled by mechanisms governing the balance between pluripotency and differentiation. The expression of early lineage-specific genes can vary significantly between species, with implications for developmental control and stem cell derivation. However, the mechanisms involved in patterning the human embryo are still unclear. We analyzed the appearance and localization of lineage-specific transcription factors in staged preimplantation human embryos from the zygote until the blastocyst. We observed that the pluripotency-associated transcription factor OCT4 was initially expressed in 8-cell embryos at 3 days post-fertilization (dpf), and restricted to the inner cell mass (ICM) in 128-256 cell blastocysts (6dpf), approximately 2 days later than the mouse. The trophectoderm (TE)-associated transcription factor CDX2 was upregulated in 5dpf blastocysts and initially coincident with OCT4, indicating a lag in CDX2 initiation in the TE lineage, relative to the mouse. Once established, the TE expressed intracellular and cell-surface proteins cytokeratin-7 (CK7) and fibroblast growth factor receptor-1 (FGFR1), which are thought to be specific to post-implantation human trophoblast progenitor cells. The primitive endoderm (PE)-associated transcription factor SOX17 was initially heterogeneously expressed in the ICM where it co-localized with a sub-set of OCT4 expressing cells at 4-5dpf. SOX17 was progressively restricted to the PE adjacent to the blastocoel cavity together with the transcription factor GATA6 by 6dpf. We observed low levels of Laminin expression in the human PE, though this basement membrane component is thought to play an important role in mouse PE cell sorting, suggesting divergence in differentiation mechanisms between species. Additionally, while stem cell lines representing the three distinct cell types that comprise a mouse blastocyst have been established, the identity of cell types that emerge during early human embryonic stem cell derivation is unclear. We observed that derivation from plating intact human blastocysts resulted predominantly in the outgrowth of TE-like cells, which impairs human embryonic stem cell derivation. Altogether, our findings provide important insight into developmental patterning of preimplantation human embryos with potential consequences for stem cell derivation.Copyright © 2012 Elsevier Inc. All rights reserved. -
Tabansky I, Lenarcic A, Draft RW, Loulier K, Keskin DB, Rosains J, Rivera-Feliciano J, Lichtman JW, Livet J, Stern JN, Sanes JR, Eggan K. 2013. Developmental bias in cleavage-stage mouse blastomeres. Current biology : CB. 23(1):21-31. Pubmed: 23177476 DOI:S0960-9822(12)01309-7 Tabansky I, Lenarcic A, Draft RW, Loulier K, Keskin DB, Rosains J, Rivera-Feliciano J, Lichtman JW, Livet J, Stern JN, Sanes JR, Eggan K. 2013. Developmental bias in cleavage-stage mouse blastomeres. Current biology : CB. 23(1):21-31. Pubmed: 23177476 DOI:S0960-9822(12)01309-7 ArrayCopyright © 2013 Elsevier Ltd. All rights reserved. -
Merkle FT, Eggan K. 2013. Modeling human disease with pluripotent stem cells: from genome association to function. Cell stem cell. 12(6):656-68. Pubmed: 23746975 DOI:S1934-5909(13)00209-9 Merkle FT, Eggan K. 2013. Modeling human disease with pluripotent stem cells: from genome association to function. Cell stem cell. 12(6):656-68. Pubmed: 23746975 DOI:S1934-5909(13)00209-9 Mechanistic insights into human disease may enable the development of treatments that are effective in broad patient populations. The confluence of gene-editing technologies, induced pluripotent stem cells, and genome-wide association as well as DNA sequencing studies is enabling new approaches for illuminating the molecular basis of human disease. We discuss the opportunities and challenges of combining these technologies and provide a workflow for interrogating the contribution of disease-associated candidate genetic variants to disease-relevant phenotypes. Finally, we discuss the potential utility of human pluripotent stem cells for placing disease-associated genetic variants into molecular pathways.Copyright © 2013 Elsevier Inc. All rights reserved. -
Eggan K. 2013. Picking the lock on pluripotency. The New England journal of medicine. 369(22):2150-1. Pubmed: 24283230 DOI:10.1056/NEJMcibr1311880 Eggan K. 2013. Picking the lock on pluripotency. The New England journal of medicine. 369(22):2150-1. Pubmed: 24283230 DOI:10.1056/NEJMcibr1311880 -
Sandoe J, Eggan K. 2013. Opportunities and challenges of pluripotent stem cell neurodegenerative disease models. Nature neuroscience. 16(7):780-9. Pubmed: 23799470 DOI:10.1038/nn.3425 Sandoe J, Eggan K. 2013. Opportunities and challenges of pluripotent stem cell neurodegenerative disease models. Nature neuroscience. 16(7):780-9. Pubmed: 23799470 DOI:10.1038/nn.3425 Human neurodegenerative disorders are among the most difficult to study. In particular, the inability to readily obtain the faulty cell types most relevant to these diseases has impeded progress for decades. Recent advances in pluripotent stem cell technology now grant access to substantial quantities of disease-pertinent neurons both with and without predisposing mutations. While this suite of technologies has revolutionized the field of 'in vitro disease modeling', great care must be taken in their deployment if robust, durable discoveries are to be made. Here we review what we perceive to be several of the stumbling blocks in the use of stem cells for the study of neurological disease and offer strategies to overcome them. -
Suzuki N, Maroof AM, Merkle FT, Koszka K, Intoh A, Armstrong I, Moccia R, Davis-Dusenbery BN, Eggan K. 2013. The mouse C9ORF72 ortholog is enriched in neurons known to degenerate in ALS and FTD. Nature neuroscience. 16(12):1725-7. Pubmed: 24185425 DOI:10.1038/nn.3566 Suzuki N, Maroof AM, Merkle FT, Koszka K, Intoh A, Armstrong I, Moccia R, Davis-Dusenbery BN, Eggan K. 2013. The mouse C9ORF72 ortholog is enriched in neurons known to degenerate in ALS and FTD. Nature neuroscience. 16(12):1725-7. Pubmed: 24185425 DOI:10.1038/nn.3566 Using transgenic mice harboring a targeted LacZ insertion, we studied the expression pattern of the C9ORF72 mouse ortholog (3110043O21Rik). Unlike most genes that are mutated in amyotrophic lateral sclerosis (ALS), which are ubiquitously expressed, the C9ORF72 ortholog was most highly transcribed in the neuronal populations that are sensitive to degeneration in ALS and frontotemporal dementia. Thus, our results provide a potential explanation for the cell type specificity of neuronal degeneration caused by C9ORF72 mutations. -
Navara CS, Hornecker J, Grow D, Chaudhari S, Hornsby PJ, Ichida JK, Eggan K, McCarrey JR. 2013. Derivation of induced pluripotent stem cells from the baboon: a nonhuman primate model for preclinical testing of stem cell therapies. Cellular reprogramming. 15(6):495-502. Pubmed: 24182315 DOI:10.1089/cell.2012.0093 Navara CS, Hornecker J, Grow D, Chaudhari S, Hornsby PJ, Ichida JK, Eggan K, McCarrey JR. 2013. Derivation of induced pluripotent stem cells from the baboon: a nonhuman primate model for preclinical testing of stem cell therapies. Cellular reprogramming. 15(6):495-502. Pubmed: 24182315 DOI:10.1089/cell.2012.0093 Development of effective pluripotent stem cell-based therapies will require safety and efficacy testing in a clinically relevant preclinical model such as nonhuman primates (NHPs). Baboons and macaques are equally similar to humans genetically and both have been extensively used for biomedical research. Macaques are preferred for human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) research whereas baboons are preferred for transplantation studies because of the greater similarity of their anatomy and immunogenetic system to those of humans. We generated four induced pluripotent stem cell (iPSC) lines from skin cells of the olive baboon (Papio anubis). Each line shows the distinct morphology of primate pluripotent stem cells, including flat colonies with well-defined borders and a high nuclear/cytoplasm ratio. Each is positive for the pluripotency markers OCT4, SOX2, NANOG, and SSEA4. Pluripotency was confirmed in two lines by teratoma formation with representative tissues from each germ layer, whereas a third produced cells from all three germ layers following embryoid body differentiation. Three lines have a normal male karyotype and the fourth is missing the short arm of one copy of chromosome 18. This may serve as an in vitro model for the human developmental disorder 18p-, which impacts 1 in 50,000 births/year. These iPSC lines represent the first step toward establishing the baboon as a NHP model for developing stem cell-based therapies. 2012
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Mekhoubad S, Bock C, de Boer AS, Kiskinis E, Meissner A, Eggan K. 2012. Erosion of dosage compensation impacts human iPSC disease modeling. Cell stem cell. 10(5):595-609. Pubmed: 22560080 DOI:10.1016/j.stem.2012.02.014 Mekhoubad S, Bock C, de Boer AS, Kiskinis E, Meissner A, Eggan K. 2012. Erosion of dosage compensation impacts human iPSC disease modeling. Cell stem cell. 10(5):595-609. Pubmed: 22560080 DOI:10.1016/j.stem.2012.02.014 Although distinct human induced pluripotent stem cell (hiPSC) lines can display considerable epigenetic variation, it has been unclear whether such variability impacts their utility for disease modeling. Here, we show that although low-passage female hiPSCs retain the inactive X chromosome of the somatic cell they are derived from, over time in culture they undergo an "erosion" of X chromosome inactivation (XCI). This erosion of XCI is characterized by loss of XIST expression and foci of H3-K27-trimethylation, as well as transcriptional derepression of genes on the inactive X that cannot be reversed by either differentiation or further reprogramming. We specifically demonstrate that erosion of XCI has a significant impact on the use of female hiPSCs for modeling Lesch-Nyhan syndrome. However, our finding that most genes subject to XCI are derepressed by this erosion of XCI suggests that it should be a significant consideration when selecting hiPSC lines for modeling any disease.Copyright © 2012 Elsevier Inc. All rights reserved. -
Williams LA, Davis-Dusenbery BN, Eggan KC. 2012. SnapShot: directed differentiation of pluripotent stem cells. Cell. 149(5):1174-1174.e1. Pubmed: 22632979 DOI:10.1016/j.cell.2012.05.015 Williams LA, Davis-Dusenbery BN, Eggan KC. 2012. SnapShot: directed differentiation of pluripotent stem cells. Cell. 149(5):1174-1174.e1. Pubmed: 22632979 DOI:10.1016/j.cell.2012.05.015 2011
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Egli D, Chen AE, Saphier G, Powers D, Alper M, Katz K, Berger B, Goland R, Leibel RL, Melton DA, Eggan K. 2011. Impracticality of egg donor recruitment in the absence of compensation. Cell stem cell. 9(4):293-4. Pubmed: 21982228 DOI:10.1016/j.stem.2011.08.002 Egli D, Chen AE, Saphier G, Powers D, Alper M, Katz K, Berger B, Goland R, Leibel RL, Melton DA, Eggan K. 2011. Impracticality of egg donor recruitment in the absence of compensation. Cell stem cell. 9(4):293-4. Pubmed: 21982228 DOI:10.1016/j.stem.2011.08.002 -
Boulting GL, Kiskinis E, Croft GF, Amoroso MW, Oakley DH, Wainger BJ, Williams DJ, Kahler DJ, Yamaki M, Davidow L, Rodolfa CT, Dimos JT, Mikkilineni S, MacDermott AB, Woolf CJ, Henderson CE, Wichterle H, Eggan K. 2011. A functionally characterized test set of human induced pluripotent stem cells. Nature biotechnology. 29(3):279-86. Pubmed: 21293464 DOI:10.1038/nbt.1783 Boulting GL, Kiskinis E, Croft GF, Amoroso MW, Oakley DH, Wainger BJ, Williams DJ, Kahler DJ, Yamaki M, Davidow L, Rodolfa CT, Dimos JT, Mikkilineni S, MacDermott AB, Woolf CJ, Henderson CE, Wichterle H, Eggan K. 2011. A functionally characterized test set of human induced pluripotent stem cells. Nature biotechnology. 29(3):279-86. Pubmed: 21293464 DOI:10.1038/nbt.1783 Human induced pluripotent stem cells (iPSCs) present exciting opportunities for studying development and for in vitro disease modeling. However, reported variability in the behavior of iPSCs has called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate. Under standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to that of human embryonic stem cells (ESCs). Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all 16 iPSC lines passed a stringent test of differentiation capacity despite variations in karyotype and in the expression of early pluripotency markers and transgenes. This iPSC and ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications. -
Bock C, Kiskinis E, Verstappen G, Gu H, Boulting G, Smith ZD, Ziller M, Croft GF, Amoroso MW, Oakley DH, Gnirke A, Eggan K, Meissner A. 2011. Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. Cell. 144(3):439-52. Pubmed: 21295703 DOI:10.1016/j.cell.2010.12.032 Bock C, Kiskinis E, Verstappen G, Gu H, Boulting G, Smith ZD, Ziller M, Croft GF, Amoroso MW, Oakley DH, Gnirke A, Eggan K, Meissner A. 2011. Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. Cell. 144(3):439-52. Pubmed: 21295703 DOI:10.1016/j.cell.2010.12.032 The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines.Copyright © 2011 Elsevier Inc. All rights reserved. -
Gore A, Li Z, Fung HL, Young JE, Agarwal S, Antosiewicz-Bourget J, Canto I, Giorgetti A, Israel MA, Kiskinis E, Lee JH, Loh YH, Manos PD, Montserrat N, Panopoulos AD, Ruiz S, Wilbert ML, Yu J, Kirkness EF, Izpisua Belmonte JC, Rossi DJ, Thomson JA, Eggan K, Daley GQ, Goldstein LS, Zhang K. 2011. Somatic coding mutations in human induced pluripotent stem cells. Nature. 471(7336):63-7. Pubmed: 21368825 DOI:10.1038/nature09805 Gore A, Li Z, Fung HL, Young JE, Agarwal S, Antosiewicz-Bourget J, Canto I, Giorgetti A, Israel MA, Kiskinis E, Lee JH, Loh YH, Manos PD, Montserrat N, Panopoulos AD, Ruiz S, Wilbert ML, Yu J, Kirkness EF, Izpisua Belmonte JC, Rossi DJ, Thomson JA, Eggan K, Daley GQ, Goldstein LS, Zhang K. 2011. Somatic coding mutations in human induced pluripotent stem cells. Nature. 471(7336):63-7. Pubmed: 21368825 DOI:10.1038/nature09805 Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use. -
Han SS, Williams LA, Eggan KC. 2011. Constructing and deconstructing stem cell models of neurological disease. Neuron. 70(4):626-44. Pubmed: 21609821 DOI:10.1016/j.neuron.2011.05.003 Han SS, Williams LA, Eggan KC. 2011. Constructing and deconstructing stem cell models of neurological disease. Neuron. 70(4):626-44. Pubmed: 21609821 DOI:10.1016/j.neuron.2011.05.003 Among the disciplines of medicine, the study of neurological disorders is particularly challenging. The fundamental inaccessibility of the human neural types affected by disease prevents their isolation for in vitro studies of degenerative mechanisms or for drug screening efforts. However, the ability to reprogram readily accessible tissue from patients into pluripotent stem (iPS) cells may now provide a general solution to this shortage of human neurons. Gradually improving methods for directing the differentiation of patient-specific stem cells has enabled the production of several neural cell types affected by disease. Furthermore, initial studies with stem cell lines derived from individuals with pediatric, monogenic disorders have validated the stem cell approach to disease modeling, allowing relevant neural phenotypes to be observed and studied. Whether iPS cell-derived neurons will always faithfully recapitulate the same degenerative processes observed in patients and serve as platforms for drug discovery relevant to common late-onset diseases remains to be determined.Copyright © 2011 Elsevier Inc. All rights reserved. -
Son EY, Ichida JK, Wainger BJ, Toma JS, Rafuse VF, Woolf CJ, Eggan K. 2011. Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell stem cell. 9(3):205-18. Pubmed: 21852222 DOI:10.1016/j.stem.2011.07.014 Son EY, Ichida JK, Wainger BJ, Toma JS, Rafuse VF, Woolf CJ, Eggan K. 2011. Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell stem cell. 9(3):205-18. Pubmed: 21852222 DOI:10.1016/j.stem.2011.07.014 The mammalian nervous system comprises many distinct neuronal subtypes, each with its own phenotype and differential sensitivity to degenerative disease. Although specific neuronal types can be isolated from rodent embryos or engineered from stem cells for translational studies, transcription factor-mediated reprogramming might provide a more direct route to their generation. Here we report that the forced expression of select transcription factors is sufficient to convert mouse and human fibroblasts into induced motor neurons (iMNs). iMNs displayed a morphology, gene expression signature, electrophysiology, synaptic functionality, in vivo engraftment capacity, and sensitivity to degenerative stimuli similar to those of embryo-derived motor neurons. We show that the converting fibroblasts do not transit through a proliferative neural progenitor state, and thus form bona fide motor neurons via a route distinct from embryonic development. Our findings demonstrate that fibroblasts can be converted directly into a specific differentiated and functional neural subtype, the spinal motor neuron.Copyright © 2011 Elsevier Inc. All rights reserved. -
Egli D, Chen AE, Saphier G, Ichida J, Fitzgerald C, Go KJ, Acevedo N, Patel J, Baetscher M, Kearns WG, Goland R, Leibel RL, Melton DA, Eggan K. 2011. Reprogramming within hours following nuclear transfer into mouse but not human zygotes. Nature communications. 2:488. Pubmed: 21971503 DOI:10.1038/ncomms1503 Egli D, Chen AE, Saphier G, Ichida J, Fitzgerald C, Go KJ, Acevedo N, Patel J, Baetscher M, Kearns WG, Goland R, Leibel RL, Melton DA, Eggan K. 2011. Reprogramming within hours following nuclear transfer into mouse but not human zygotes. Nature communications. 2:488. Pubmed: 21971503 DOI:10.1038/ncomms1503 Fertilized mouse zygotes can reprogram somatic cells to a pluripotent state. Human zygotes might therefore be useful for producing patient-derived pluripotent stem cells. However, logistical, legal and social considerations have limited the availability of human eggs for research. Here we show that a significant number of normal fertilized eggs (zygotes) can be obtained for reprogramming studies. Using these zygotes, we found that when the zygotic genome was replaced with that of a somatic cell, development progressed normally throughout the cleavage stages, but then arrested before the morula stage. This arrest was associated with a failure to activate transcription in the transferred somatic genome. In contrast to human zygotes, mouse zygotes reprogrammed the somatic cell genome to a pluripotent state within hours after transfer. Our results suggest that there may be a previously unappreciated barrier to successful human nuclear transfer, and that future studies could focus on the requirements for genome activation. 2010
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Egli D, Eggan K. 2010. Recipient cell nuclear factors are required for reprogramming by nuclear transfer. Development (Cambridge, England). 137(12):1953-63. Pubmed: 20463036 DOI:10.1242/dev.046151 Egli D, Eggan K. 2010. Recipient cell nuclear factors are required for reprogramming by nuclear transfer. Development (Cambridge, England). 137(12):1953-63. Pubmed: 20463036 DOI:10.1242/dev.046151 Nuclear transfer allows the reprogramming of somatic cells to totipotency. The cell cycle state of the donor and recipient cells, as well as their extent of differentiation, have each been cited as important determinants of reprogramming success. Here, we have used donor and recipient cells at various cell cycle and developmental stages to investigate the importance of these parameters. We found that many stages of the cell cycle were compatible with reprogramming as long as a sufficient supply of essential nuclear factors, such as Brg1, were retained in the recipient cell following enucleation. Consistent with this conclusion, the increased efficiency of reprogramming when using donor nuclei from embryonic cells could be explained, at least in part, by reintroduction of embryonic nuclear factors along with the donor nucleus. By contrast, cell cycle synchrony between the donor nucleus and the recipient cell was not required at the time of transfer, as long as synchrony was reached by the first mitosis. Our findings demonstrate the remarkable flexibility of the reprogramming process and support the importance of nuclear transcriptional regulators in mediating reprogramming. -
Niakan KK, Ji H, Maehr R, Vokes SA, Rodolfa KT, Sherwood RI, Yamaki M, Dimos JT, Chen AE, Melton DA, McMahon AP, Eggan K. 2010. Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal. Genes & development. 24(3):312-26. Pubmed: 20123909 DOI:10.1101/gad.1833510 Niakan KK, Ji H, Maehr R, Vokes SA, Rodolfa KT, Sherwood RI, Yamaki M, Dimos JT, Chen AE, Melton DA, McMahon AP, Eggan K. 2010. Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal. Genes & development. 24(3):312-26. Pubmed: 20123909 DOI:10.1101/gad.1833510 In embryonic stem (ES) cells, a well-characterized transcriptional network promotes pluripotency and represses gene expression required for differentiation. In comparison, the transcriptional networks that promote differentiation of ES cells and the blastocyst inner cell mass are poorly understood. Here, we show that Sox17 is a transcriptional regulator of differentiation in these pluripotent cells. ES cells deficient in Sox17 fail to differentiate into extraembryonic cell types and maintain expression of pluripotency-associated transcription factors, including Oct4, Nanog, and Sox2. In contrast, forced expression of Sox17 down-regulates ES cell-associated gene expression and directly activates genes functioning in differentiation toward an extraembryonic endoderm cell fate. We show these effects of Sox17 on ES cell gene expression are mediated at least in part through a competition between Sox17 and Nanog for common DNA-binding sites. By elaborating the function of Sox17, our results provide insight into how the transcriptional network promoting ES cell self-renewal is interrupted, allowing cellular differentiation. -
Ichida JK, Kiskinis E, Eggan K. 2010. Shushing down the epigenetic landscape towards stem cell differentiation. Development (Cambridge, England). 137(15):2455-60. Pubmed: 20554719 DOI:10.1242/dev.049130 Ichida JK, Kiskinis E, Eggan K. 2010. Shushing down the epigenetic landscape towards stem cell differentiation. Development (Cambridge, England). 137(15):2455-60. Pubmed: 20554719 DOI:10.1242/dev.049130 In February 2010, researchers interested in stem cell biology gathered in Keystone, Colorado, USA to discuss their findings on the origins and behaviors of pluripotent and multipotent stem cells, and their therapeutic potential. Here, we review the presentations at that meeting and the questions that emerged concerning how a stem cell ;decides' to self-renew or differentiate, what their distinct properties are and how this information can be used to develop novel therapies. -
Kiskinis E, Eggan K. 2010. Progress toward the clinical application of patient-specific pluripotent stem cells. The Journal of clinical investigation. 120(1):51-9. Pubmed: 20051636 DOI:40553 Kiskinis E, Eggan K. 2010. Progress toward the clinical application of patient-specific pluripotent stem cells. The Journal of clinical investigation. 120(1):51-9. Pubmed: 20051636 DOI:40553 Induced pluripotent stem (iPS) cells are generated by epigenetic reprogramming of somatic cells through the exogenous expression of transcription factors. These cells, just like embryonic stem cells, are likely to have a major impact on regenerative medicine, because they self-renew and retain the potential to be differentiated into all cell types of the human body. In this Review, we describe the current state of iPS cell technology, including approaches by which they are generated and what is known about their biology, and discuss the potential applications of these cells for disease modeling, drug discovery, and, eventually, cell replacement therapy. 2009
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Egli D, Sandler VM, Shinohara ML, Cantor H, Eggan K. 2009. Reprogramming after chromosome transfer into mouse blastomeres. Current biology : CB. 19(16):1403-9. Pubmed: 19682906 DOI:10.1016/j.cub.2009.06.065 Egli D, Sandler VM, Shinohara ML, Cantor H, Eggan K. 2009. Reprogramming after chromosome transfer into mouse blastomeres. Current biology : CB. 19(16):1403-9. Pubmed: 19682906 DOI:10.1016/j.cub.2009.06.065 It is well known that oocytes can reprogram differentiated cells, allowing animal cloning by nuclear transfer. We have recently shown that fertilized zygotes retain reprogramming activities, suggesting that such activities might also persist in cleavage-stage embryos. Here, we used chromosome transplantation techniques to investigate whether the blastomeres of two-cell-stage mouse embryos can reprogram more differentiated cells. When chromosomes from one of the two blastomeres were replaced with the chromosomes of an embryonic or CD4(+) T lymphocyte donor cell, we observed nuclear reprogramming and efficient contribution of the manipulated cell to the developing blastocyst. Embryos produced by this method could be used to derive stem cell lines and also developed to term, generating mosaic "cloned" animals. These results demonstrate that blastomeres retain reprogramming activities and support the notion that discarded human preimplantation embryos may be useful recipients for the production of genetically tailored human embryonic stem cell lines. -
Deng J, Shoemaker R, Xie B, Gore A, LeProust EM, Antosiewicz-Bourget J, Egli D, Maherali N, Park IH, Yu J, Daley GQ, Eggan K, Hochedlinger K, Thomson J, Wang W, Gao Y, Zhang K. 2009. Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nature biotechnology. 27(4):353-60. Pubmed: 19330000 DOI:10.1038/nbt.1530 Deng J, Shoemaker R, Xie B, Gore A, LeProust EM, Antosiewicz-Bourget J, Egli D, Maherali N, Park IH, Yu J, Daley GQ, Eggan K, Hochedlinger K, Thomson J, Wang W, Gao Y, Zhang K. 2009. Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nature biotechnology. 27(4):353-60. Pubmed: 19330000 DOI:10.1038/nbt.1530 Current DNA methylation assays are limited in the flexibility and efficiency of characterizing a large number of genomic targets. We report a method to specifically capture an arbitrary subset of genomic targets for single-molecule bisulfite sequencing for digital quantification of DNA methylation at single-nucleotide resolution. A set of ~30,000 padlock probes was designed to assess methylation of ~66,000 CpG sites within 2,020 CpG islands on human chromosome 12, chromosome 20, and 34 selected regions. To investigate epigenetic differences associated with dedifferentiation, we compared methylation in three human fibroblast lines and eight human pluripotent stem cell lines. Chromosome-wide methylation patterns were similar among all lines studied, but cytosine methylation was slightly more prevalent in the pluripotent cells than in the fibroblasts. Induced pluripotent stem (iPS) cells appeared to display more methylation than embryonic stem cells. We found 288 regions methylated differently in fibroblasts and pluripotent cells. This targeted approach should be particularly useful for analyzing DNA methylation in large genomes. -
Ichida JK, Blanchard J, Lam K, Son EY, Chung JE, Egli D, Loh KM, Carter AC, Di Giorgio FP, Koszka K, Huangfu D, Akutsu H, Liu DR, Rubin LL, Eggan K. 2009. A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog. Cell stem cell. 5(5):491-503. Pubmed: 19818703 DOI:10.1016/j.stem.2009.09.012 Ichida JK, Blanchard J, Lam K, Son EY, Chung JE, Egli D, Loh KM, Carter AC, Di Giorgio FP, Koszka K, Huangfu D, Akutsu H, Liu DR, Rubin LL, Eggan K. 2009. A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog. Cell stem cell. 5(5):491-503. Pubmed: 19818703 DOI:10.1016/j.stem.2009.09.012 The combined activity of three transcription factors can reprogram adult cells into induced pluripotent stem cells (iPSCs). However, the transgenic methods used for delivering reprogramming factors have raised concerns regarding the future utility of the resulting stem cells. These uncertainties could be overcome if each transgenic factor were replaced with a small molecule that either directly activated its expression from the somatic genome or in some way compensated for its activity. To this end, we have used high-content chemical screening to identify small molecules that can replace Sox2 in reprogramming. We show that one of these molecules functions in reprogramming by inhibiting Tgf-beta signaling in a stable and trapped intermediate cell type that forms during the process. We find that this inhibition promotes the completion of reprogramming through induction of the transcription factor Nanog. -
Chen AE, Egli D, Niakan K, Deng J, Akutsu H, Yamaki M, Cowan C, Fitz-Gerald C, Zhang K, Melton DA, Eggan K. 2009. Optimal timing of inner cell mass isolation increases the efficiency of human embryonic stem cell derivation and allows generation of sibling cell lines. Cell stem cell. 4(2):103-6. Pubmed: 19200798 DOI:10.1016/j.stem.2008.12.001 Chen AE, Egli D, Niakan K, Deng J, Akutsu H, Yamaki M, Cowan C, Fitz-Gerald C, Zhang K, Melton DA, Eggan K. 2009. Optimal timing of inner cell mass isolation increases the efficiency of human embryonic stem cell derivation and allows generation of sibling cell lines. Cell stem cell. 4(2):103-6. Pubmed: 19200798 DOI:10.1016/j.stem.2008.12.001 -
Zhang K, Li JB, Gao Y, Egli D, Xie B, Deng J, Li Z, Lee JH, Aach J, Leproust EM, Eggan K, Church GM. 2009. Digital RNA allelotyping reveals tissue-specific and allele-specific gene expression in human. Nature methods. 6(8):613-8. Pubmed: 19620972 DOI:10.1038/nmeth.1357 Zhang K, Li JB, Gao Y, Egli D, Xie B, Deng J, Li Z, Lee JH, Aach J, Leproust EM, Eggan K, Church GM. 2009. Digital RNA allelotyping reveals tissue-specific and allele-specific gene expression in human. Nature methods. 6(8):613-8. Pubmed: 19620972 DOI:10.1038/nmeth.1357 We developed a digital RNA allelotyping method for quantitatively interrogating allele-specific gene expression. This method involves ultra-deep sequencing of padlock-captured single-nucleotide polymorphisms (SNPs) from the transcriptome. We characterized four cell lines established from two human subjects in the Personal Genome Project. Approximately 11-22% of the heterozygous mRNA-associated SNPs showed allele-specific expression in each cell line and 4.3-8.5% were tissue-specific, suggesting the presence of tissue-specific cis regulation. When we applied allelotyping to two pairs of sibling human embryonic stem cell lines, the sibling lines were more similar in allele-specific expression than were the genetically unrelated lines. We found that the variation of allelic ratios in gene expression among different cell lines was primarily explained by genetic variations, much more so than by specific tissue types or growth conditions. Comparison of expressed SNPs on the sense and antisense transcripts suggested that allelic ratios are primarily determined by cis-regulatory mechanisms on the sense transcripts. 2008
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Eggan K. 2008. Using stem cells and reprogramming to understand disease. Regenerative medicine. 3(6):799-801. Pubmed: 18947303 DOI:10.2217/17460751.3.6.799 Eggan K. 2008. Using stem cells and reprogramming to understand disease. Regenerative medicine. 3(6):799-801. Pubmed: 18947303 DOI:10.2217/17460751.3.6.799 Kevin Eggan received his PhD in Biology from the Massachusetts Institute of Technology in February 2003. He is currently an Assistant Professor of Stem Cell and Regenerative Biology at Harvard University. Dr Eggan has devoted the last 7 years to performing stem cell research. He is currently leading a research group that is investigating the mechanisms regulating epigenetic reprogramming after somatic cell nuclear transfer and using nuclear transfer techniques to derive disease-specific human embryonic stem cell lines from diabetic and Parkinson's disease patients. His accomplishments include cloning mice from olfactory sensory neurons, deriving embryonic germ cells and male gametes from embryonic stem cells and characterizing the abnormalities that sometimes arise as a result of nuclear transfer. -
Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF, Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K. 2008. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science (New York, N.Y.). 321(5893):1218-21. Pubmed: 18669821 DOI:10.1126/science.1158799 Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF, Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K. 2008. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science (New York, N.Y.). 321(5893):1218-21. Pubmed: 18669821 DOI:10.1126/science.1158799 The generation of pluripotent stem cells from an individual patient would enable the large-scale production of the cell types affected by that patient's disease. These cells could in turn be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. Although recent studies have demonstrated the reprogramming of human fibroblasts to a pluripotent state, it remains unclear whether these induced pluripotent stem (iPS) cells can be produced directly from elderly patients with chronic disease. We have generated iPS cells from an 82-year-old woman diagnosed with a familial form of amyotrophic lateral sclerosis (ALS). These patient-specific iPS cells possess properties of embryonic stem cells and were successfully directed to differentiate into motor neurons, the cell type destroyed in ALS. -
Egli D, Birkhoff G, Eggan K. 2008. Mediators of reprogramming: transcription factors and transitions through mitosis. Nature reviews. Molecular cell biology. 9(7):505-16. Pubmed: 18568039 DOI:10.1038/nrm2439 Egli D, Birkhoff G, Eggan K. 2008. Mediators of reprogramming: transcription factors and transitions through mitosis. Nature reviews. Molecular cell biology. 9(7):505-16. Pubmed: 18568039 DOI:10.1038/nrm2439 It is thought that most cell types of the human body share the same genetic information as that contained in the zygote from which they originate. Consistent with this view, animal cloning studies demonstrated that the intact genome of a differentiated cell can be reprogrammed to support the development of an entire organism and allow the production of pluripotent stem cells. Recent progress in reprogramming research now points to an important role for transcription factors in the establishment and the maintenance of cellular phenotypes, and to cell division as a mediator of transitions between different states of gene expression. -
Di Giorgio FP, Boulting GL, Bobrowicz S, Eggan KC. 2008. Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation. Cell stem cell. 3(6):637-48. Pubmed: 19041780 DOI:10.1016/j.stem.2008.09.017 Di Giorgio FP, Boulting GL, Bobrowicz S, Eggan KC. 2008. Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation. Cell stem cell. 3(6):637-48. Pubmed: 19041780 DOI:10.1016/j.stem.2008.09.017 It has been proposed that human embryonic stem cells could be used to provide an inexhaustible supply of differentiated cell types for the study of disease processes. Although methods for differentiating embryonic stem cells into specific cell types have become increasingly sophisticated, the utility of the resulting cells for modeling disease has not been determined. We have asked whether specific neuronal subtypes produced from human embryonic stem cells can be used to investigate the mechanisms leading to neural degeneration in amyotrophic lateral sclerosis (ALS). We show that human spinal motor neurons, but not interneurons, are selectively sensitive to the toxic effect of glial cells carrying an ALS-causing mutation in the SOD1 gene. Our findings demonstrate the relevance of these non-cell-autonomous effects to human motor neurons and more broadly demonstrate the utility of human embryonic stem cells for studying disease and identifying potential therapeutics. 2007
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Eggan K. 2007. Short-circuiting epiblast development. Cell stem cell. 1(2):131-2. Pubmed: 18371343 DOI:10.1016/j.stem.2007.07.018 Eggan K. 2007. Short-circuiting epiblast development. Cell stem cell. 1(2):131-2. Pubmed: 18371343 DOI:10.1016/j.stem.2007.07.018 Although human and mouse embryonic stem cells share many characteristics, their behaviors are not identical. Two recent papers describe the derivation of stem cell lines from mouse epiblast that may provide an explanation for the unique character of human embryonic stem cells. -
Eggan K. 2007. Dolly's legacy: human nuclear transplantation and better medicines for our children. Cloning and stem cells. 9(1):21-5. Pubmed: 17386009 Eggan K. 2007. Dolly's legacy: human nuclear transplantation and better medicines for our children. Cloning and stem cells. 9(1):21-5. Pubmed: 17386009 -
Di Giorgio FP, Carrasco MA, Siao MC, Maniatis T, Eggan K. 2007. Non-cell autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model. Nature neuroscience. 10(5):608-14. Pubmed: 17435754 Di Giorgio FP, Carrasco MA, Siao MC, Maniatis T, Eggan K. 2007. Non-cell autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model. Nature neuroscience. 10(5):608-14. Pubmed: 17435754 Here we report an in vitro model system for studying the molecular and cellular mechanisms that underlie the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Embryonic stem cells (ESCs) derived from mice carrying normal or mutant transgenic alleles of the human SOD1 gene were used to generate motor neurons by in vitro differentiation. These motor neurons could be maintained in long-term coculture either with additional cells that arose during differentiation or with primary glial cells. Motor neurons carrying either the nonpathological human SOD1 transgene or the mutant SOD1(G93A) allele showed neurodegenerative properties when cocultured with SOD1(G93A) glial cells. Thus, our studies demonstrate that glial cells carrying a human SOD1(G93A) mutation have a direct, non-cell autonomous effect on motor neuron survival. More generally, our results show that ESC-based models of disease provide a powerful tool for studying the mechanisms of neural degeneration. These phenotypes displayed in culture could provide cell-based assays for the identification of new ALS drugs. -
Egli D, Rosains J, Birkhoff G, Eggan K. 2007. Developmental reprogramming after chromosome transfer into mitotic mouse zygotes. Nature. 447(7145):679-85. Pubmed: 17554301 Egli D, Rosains J, Birkhoff G, Eggan K. 2007. Developmental reprogramming after chromosome transfer into mitotic mouse zygotes. Nature. 447(7145):679-85. Pubmed: 17554301 Until now, animal cloning and the production of embryonic stem cell lines by somatic cell nuclear transfer have relied on introducing nuclei into meiotic oocytes. In contrast, attempts at somatic cell nuclear transfer into fertilized interphase zygotes have failed. As a result, it has generally been assumed that unfertilized human oocytes will be required for the generation of tailored human embryonic stem cell lines from patients by somatic cell nuclear transfer. Here we report, however, that, unlike interphase zygotes, mouse zygotes temporarily arrested in mitosis can support somatic cell reprogramming, the production of embryonic stem cell lines and the full-term development of cloned animals. Thus, human zygotes and perhaps human embryonic blastomeres may be useful supplements to human oocytes for the creation of patient-derived human embryonic stem cells. 2006
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Yang X, Eggan K, Seidel G, Jaenisch R, Melton D. 2006. A simple system of checks and balances to cut fraud. Nature. 439(7078):782. Pubmed: 16482128 Yang X, Eggan K, Seidel G, Jaenisch R, Melton D. 2006. A simple system of checks and balances to cut fraud. Nature. 439(7078):782. Pubmed: 16482128 -
Rodolfa KT, Eggan K. 2006. A transcriptional logic for nuclear reprogramming. Cell. 126(4):652-5. Pubmed: 16923385 Rodolfa KT, Eggan K. 2006. A transcriptional logic for nuclear reprogramming. Cell. 126(4):652-5. Pubmed: 16923385 Limitations on a differentiated cell's pluripotency can be erased by nuclear transfer or by fusion with embryonic stem cells, but attempts to recapitulate this process of nuclear reprogramming by molecular means have failed. In this issue of Cell, Takahashi and Yamanaka (2006) take a rational approach to identifying a suite of embryonic transcription factors whose overexpression restores pluripotency to adult somatic cells. -
Sullivan S, Eggan K. 2006. The potential of cell fusion for human therapy. Stem cell reviews. 2(4):341-9. Pubmed: 17848721 Sullivan S, Eggan K. 2006. The potential of cell fusion for human therapy. Stem cell reviews. 2(4):341-9. Pubmed: 17848721 As donor organs and tissues for transplantation medicine are scarce, alternative methods for replacing damaged cells or restoring organ function are highly needed. Here, we consider the therapeutic potential of cell fusion. After highlighting the various contexts in which cells are known to fuse during mammalian development, we discuss the implications of the observation that cell fusion can occur with restorative effects following tissue damage or cell transplantation. There are still, however, many challenges facing those who wish to implement cell fusion as a therapeutic tool. These include identifying the best cells to use for reparative fusion, determining the best route of introducing these cells into the desired tissue, discovering methods to increase the incidence of cell fusion, and ensuring the functionality of the resulting fusion products. If these difficulties can be overcome, cell fusion might have therapeutic potential as highlighted by several recent transplantation studies. -
Trish E, Dimos J, Eggan K. 2006. Passaging HuES human embryonic stem cell-lines with trypsin. Journal of visualized experiments : JoVE. Pubmed: 18704181 DOI:10.3791/49 Trish E, Dimos J, Eggan K. 2006. Passaging HuES human embryonic stem cell-lines with trypsin. Journal of visualized experiments : JoVE. Pubmed: 18704181 DOI:10.3791/49 In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings. Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture. First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA. Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette. The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation. The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media. An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells. The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter. It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities. -
Eggan K, Jurga S, Gosden R, Min IM, Wagers AJ. 2006. Ovulated oocytes in adult mice derive from non-circulating germ cells. Nature. 441(7097):1109-14. Pubmed: 16799565 Eggan K, Jurga S, Gosden R, Min IM, Wagers AJ. 2006. Ovulated oocytes in adult mice derive from non-circulating germ cells. Nature. 441(7097):1109-14. Pubmed: 16799565 Decades of research in reproductive biology have led to the generally accepted belief that in female mammals, all surviving germ cells enter meiosis at the end of fetal development and as a result, the postnatal ovary harbours a limited supply of oocytes that cannot be replenished or regenerated if lost to injury or disease. However, recent reports have challenged this view, suggesting instead that oocyte production is maintained through continual seeding of the ovary by circulating, bone-marrow-derived germ cells. To test directly the physiological relevance of circulating cells for female fertility, we established transplantation and parabiotic mouse models to assess the capacity of circulating bone marrow cells to generate ovulated oocytes, both in the steady state and after induced damage. Our studies showed no evidence that bone marrow cells, or any other normally circulating cells, contribute to the formation of mature, ovulated oocytes. Instead, cells that travelled to the ovary through the bloodstream exhibited properties characteristic of committed blood leukocytes. -
Trish E, Dimos J, Eggan K. 2006. Freezing human ES cells. Journal of visualized experiments : JoVE. Pubmed: 18704182 DOI:10.3791/50 Trish E, Dimos J, Eggan K. 2006. Freezing human ES cells. Journal of visualized experiments : JoVE. Pubmed: 18704182 DOI:10.3791/50 Here we demonstrate how our lab freezes HuES human embryonic stem cell lines. A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction. Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature. During this time cells can be observed rounding, and colonies lifting off the plate surface. Gentle repeated pipetting will remove cells and colonies from the plate surface. Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun. Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10 cm plate. After cells are adequately resuspended, ice cold freezing media is added at equal volume. Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80 C over 24 hours. Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months. -
Trish E, Dimos J, Eggan K. 2006. Human ES cells: starting culture from frozen cells. Journal of visualized experiments : JoVE. Pubmed: 18704174 DOI:10.3791/86 Trish E, Dimos J, Eggan K. 2006. Human ES cells: starting culture from frozen cells. Journal of visualized experiments : JoVE. Pubmed: 18704174 DOI:10.3791/86 Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock. First, a one to two day old ten cm plate of approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator. A frozen vial of cells from long term liquid nitrogen storage or a -80 C freezer is sourced and quickly submerged in a 37 C water bath for quick thawing. Cells in freezing media are then removed from the vial and placed in a large volume of HuES media. The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate. Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate. It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate. The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter. -
Egli D, Eggan K. 2006. Nuclear transfer into mouse oocytes. Journal of visualized experiments : JoVE. Pubmed: 18704178 DOI:10.3791/116 Egli D, Eggan K. 2006. Nuclear transfer into mouse oocytes. Journal of visualized experiments : JoVE. Pubmed: 18704178 DOI:10.3791/116 Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998). 2005
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Cowan CA, Atienza J, Melton DA, Eggan K. 2005. Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science (New York, N.Y.). 309(5739):1369-73. Pubmed: 16123299 Cowan CA, Atienza J, Melton DA, Eggan K. 2005. Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science (New York, N.Y.). 309(5739):1369-73. Pubmed: 16123299 We have explored the use of embryonic stem cells as an alternative to oocytes for reprogramming human somatic nuclei. Human embryonic stem (hES) cells were fused with human fibroblasts, resulting in hybrid cells that maintain a stable tetraploid DNA content and have morphology, growth rate, and antigen expression patterns characteristic of hES cells. Differentiation of hybrid cells in vitro and in vivo yielded cell types from each embryonic germ layer. Analysis of genome-wide transcriptional activity, reporter gene activation, allele-specific gene expression, and DNA methylation showed that the somatic genome was reprogrammed to an embryonic state. These results establish that hES cells can reprogram the transcriptional state of somatic nuclei and provide a system for investigating the underlying mechanisms. -
Jaenisch R, Hochedlinger K, Eggan K. 2005. Nuclear cloning, epigenetic reprogramming and cellular differentiation. Novartis Foundation symposium. 265:107-18; discussion 118-28. Pubmed: 16050253 Jaenisch R, Hochedlinger K, Eggan K. 2005. Nuclear cloning, epigenetic reprogramming and cellular differentiation. Novartis Foundation symposium. 265:107-18; discussion 118-28. Pubmed: 16050253 The full-term development of sheep, cows, goats, pigs and mice has been achieved through the transfer of somatic cell nuclei into enucleated oocytes. Despite these successes, mammalian cloning remains an inefficient process, with a preponderance of reconstructed embryos failing at early- to mid-gestation stages of development. The small percentage of conceptuses that survive to term are characterized by a high mortality rate and frequently display grossly increased placental and birth weights. It is likely that inappropriate expression of key developmental genes may contribute to lethality of cloned embryos. One of the most interesting issues of nuclear cloning is the question of genomic reprogramming, i.e. whether successful cloning requires the resetting of epigenetic modifications which are characteristic of the adult donor nucleus. Processes such as X-inactivation and genomic imprinting are known to depend on epigenetic modifications of the genome. The classical nuclear transfer experiments with frogs have suggested that the source of the donor nucleus affects the phenotype of the clone. We have, using expression profiling, compared gene expression in clones derived from embryonic stem (ES) cells and from somatic donor cell nuclei and find substantial gene dysregulation. Our results suggest that faulty reprogramming is caused by the nuclear cloning procedure itself. In addition, the type of donor nucleus contributes to the abnormal expression pattern seen in cloned animals. One of the major unresolved issues has been whether nuclei of terminally differentiated cells can be reprogrammed by transfer into the oocyte. To address this question we have derived monoclonal mice from B and T cells and used the genetic rearrangements of the immunoglobulin and T cell receptor genes to retrospectively verify the differentiation state of the donor nucleus. Finally, we discuss our recent studies on the reprogramming of nuclei from terminally differentiated neurons and from cancer cells. 2004
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Eggan K, Baldwin K, Tackett M, Osborne J, Gogos J, Chess A, Axel R, Jaenisch R. 2004. Mice cloned from olfactory sensory neurons. Nature. 428(6978):44-9. Pubmed: 14990966 Eggan K, Baldwin K, Tackett M, Osborne J, Gogos J, Chess A, Axel R, Jaenisch R. 2004. Mice cloned from olfactory sensory neurons. Nature. 428(6978):44-9. Pubmed: 14990966 Cloning by nuclear transplantation has been successfully carried out in various mammals, including mice. Until now mice have not been cloned from post-mitotic cells such as neurons. Here, we have generated fertile mouse clones derived by transferring the nuclei of post-mitotic, olfactory sensory neurons into oocytes. These results indicate that the genome of a post-mitotic, terminally differentiated neuron can re-enter the cell cycle and be reprogrammed to a state of totipotency after nuclear transfer. Moreover, the pattern of odorant receptor gene expression and the organization of odorant receptor genes in cloned mice was indistinguishable from wild-type animals, indicating that irreversible changes to the DNA of olfactory neurons do not accompany receptor gene choice. -
Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ. 2004. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature. 427(6970):148-54. Pubmed: 14668819 Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ. 2004. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature. 427(6970):148-54. Pubmed: 14668819 Egg and sperm cells (gametes) of the mouse are derived from a founder population of primordial germ cells that are set aside early in embryogenesis. Primordial germ cells arise from the proximal epiblast, a region of the early mouse embryo that also contributes to the first blood lineages of the embryonic yolk sac. Embryonic stem cells differentiate in vitro into cystic structures called embryoid bodies consisting of tissue lineages typical of the early mouse embryo. Because embryoid bodies sustain blood development, we reasoned that they might also support primordial germ cell formation. Here we isolate primordial germ cells from embryoid bodies, and derive continuously growing lines of embryonic germ cells. Embryonic germ cells show erasure of the methylation markers (imprints) of the Igf2r and H19 genes, a property characteristic of the germ lineage. We show that embryoid bodies support maturation of the primordial germ cells into haploid male gametes, which when injected into oocytes restore the somatic diploid chromosome complement and develop into blastocysts. Our ability to derive germ cells from embryonic stem cells provides an accessible in vitro model system for studies of germline epigenetic modification and mammalian gametogenesis. -
Jaenisch R, Hochedlinger K, Blelloch R, Yamada Y, Baldwin K, Eggan K. 2004. Nuclear cloning, epigenetic reprogramming, and cellular differentiation. Cold Spring Harbor symposia on quantitative biology. 69:19-27. Pubmed: 16117629 Jaenisch R, Hochedlinger K, Blelloch R, Yamada Y, Baldwin K, Eggan K. 2004. Nuclear cloning, epigenetic reprogramming, and cellular differentiation. Cold Spring Harbor symposia on quantitative biology. 69:19-27. Pubmed: 16117629 2003
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Eggan K, Jaenisch R. 2003. Differentiation of F1 embryonic stem cells into viable male and female mice by tetraploid embryo complementation. Methods in enzymology. 365:25-39. Pubmed: 14696335 Eggan K, Jaenisch R. 2003. Differentiation of F1 embryonic stem cells into viable male and female mice by tetraploid embryo complementation. Methods in enzymology. 365:25-39. Pubmed: 14696335 -
Bortvin A, Eggan K, Skaletsky H, Akutsu H, Berry DL, Yanagimachi R, Page DC, Jaenisch R. 2003. Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei. Development (Cambridge, England). 130(8):1673-80. Pubmed: 12620990 Bortvin A, Eggan K, Skaletsky H, Akutsu H, Berry DL, Yanagimachi R, Page DC, Jaenisch R. 2003. Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei. Development (Cambridge, England). 130(8):1673-80. Pubmed: 12620990 The majority of cloned animals derived by nuclear transfer from somatic cell nuclei develop to the blastocyst stage but die after implantation. Mouse embryos that lack an Oct4 gene, which plays an essential role in control of developmental pluripotency, develop to the blastocyst stage and also die after implantation, because they lack pluripotent embryonic cells. Based on this similarity, we posited that cloned embryos derived from differentiated cell nuclei fail to establish a population of truly pluripotent embryonic cells because of faulty reactivation of key embryonic genes such as Oct4. To explore this hypothesis, we used an in silico approach to identify a set of Oct4-related genes whose developmental expression pattern is similar to that of Oct4. When expression of Oct4 and 10 Oct4-related genes was analyzed in individual cumulus cell-derived cloned blastocysts, only 62% correctly expressed all tested genes. In contrast to this incomplete reactivation of Oct4-related genes in somatic clones, ES cell-derived cloned blastocysts and normal control embryos expressed these genes normally. Notably, the contrast between expression patterns of the Oct4-related genes correlated with efficiency of embryonic development of somatic and ES cell-derived cloned blastocysts to term. These observations suggest that failure to reactivate the full spectrum of these Oct4-related genes may contribute to embryonic lethality in somatic-cell clones. -
Eggan K, Jaenisch R. 2003. Micromanipulating dosage compensation: understanding X-chromosome inactivation through nuclear transplantation. Seminars in cell & developmental biology. 14(6):349-58. Pubmed: 15015742 Eggan K, Jaenisch R. 2003. Micromanipulating dosage compensation: understanding X-chromosome inactivation through nuclear transplantation. Seminars in cell & developmental biology. 14(6):349-58. Pubmed: 15015742 Nuclear transfer (NT) studies have provided insight into the functional importance of epigenetic alteration of the X chromosomes during X-inactivation. Uniparental embryos created by NT have been informative as to the time and location at which the imprint controlling extraembryonic X-inactivation is established. Experiments with female somatic cells, have demonstrated that the inactive X chromosome (Xi) is reactivated after NT, leading to random X-inactivation in the embryonic lineages of cloned embryos. However, in the extraembryonic lineages of clones, epigenetic information from the donor cell nucleus persists, leading to preferential inactivation of the donor cell's inactive X in the placenta of cloned animals. These results suggest epigenetic information established during embryonic X-inactivation is functionally equivalent to the gametic imprint. 2002
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Jeanisch R, Eggan K, Humpherys D, Rideout W, Hochedlinger K. 2002. Nuclear cloning, stem cells, and genomic reprogramming. Cloning and stem cells. 4(4):389-96. Pubmed: 12630413 Jeanisch R, Eggan K, Humpherys D, Rideout W, Hochedlinger K. 2002. Nuclear cloning, stem cells, and genomic reprogramming. Cloning and stem cells. 4(4):389-96. Pubmed: 12630413 The generation of adult animals by nuclear cloning from adult donor cells is extremely inefficient, with most clones dying soon after implantation. In contrast, cloning from embryonic stem cell donor nuclei is significanty more efficient than from adult donor cells. However, regardless of donor cell type, all clones that survive to birth and beyond suffer serious phenotypic and gene expression abnormalities. All available evidence is consistent with the notion that the anomalous phenotypes of cloned animals are caused by faulty epigenetic reprogramming of the donor nucleus. Faulty reprogramming appears to be caused by the cloning process itself as well as by the epigenetic state of the donor nucleus. In contrast to reproductive cloning, faulty reprogramming of the donor nucleus does not tend to interfere with the application of nuclear transfer technology for therapeutic purposes (therapeutic cloning). -
Possemato R, Eggan K, Moeller BJ, Jaenisch R, Jackson-Grusby L. 2002. Flp recombinase regulated lacZ expression at the ROSA26 locus. Genesis (New York, N.Y. : 2000). 32(2):184-6. Pubmed: 11857816 Possemato R, Eggan K, Moeller BJ, Jaenisch R, Jackson-Grusby L. 2002. Flp recombinase regulated lacZ expression at the ROSA26 locus. Genesis (New York, N.Y. : 2000). 32(2):184-6. Pubmed: 11857816 -
Biniszkiewicz D, Gribnau J, Ramsahoye B, Gaudet F, Eggan K, Humpherys D, Mastrangelo MA, Jun Z, Walter J, Jaenisch R. 2002. Dnmt1 overexpression causes genomic hypermethylation, loss of imprinting, and embryonic lethality. Molecular and cellular biology. 22(7):2124-35. Pubmed: 11884600 Biniszkiewicz D, Gribnau J, Ramsahoye B, Gaudet F, Eggan K, Humpherys D, Mastrangelo MA, Jun Z, Walter J, Jaenisch R. 2002. Dnmt1 overexpression causes genomic hypermethylation, loss of imprinting, and embryonic lethality. Molecular and cellular biology. 22(7):2124-35. Pubmed: 11884600 Biallelic expression of Igf2 is frequently seen in cancers because Igf2 functions as a survival factor. In many tumors the activation of Igf2 expression has been correlated with de novo methylation of the imprinted region. We have compared the intrinsic susceptibilities of the imprinted region of Igf2 and H19, other imprinted genes, bulk genomic DNA, and repetitive retroviral sequences to Dnmt1 overexpression. At low Dnmt1 methyltransferase levels repetitive retroviral elements were methylated and silenced. The nonmethylated imprinted region of Igf2 and H19 was resistant to methylation at low Dnmt1 levels but became fully methylated when Dnmt1 was overexpressed from a bacterial artificial chromosome transgene. Methylation caused the activation of the silent Igf2 allele in wild-type and Dnmt1 knockout cells, leading to biallelic Igf2 expression. In contrast, the imprinted genes Igf2r, Peg3, Snrpn, and Grf1 were completely resistant to de novo methylation, even when Dnmt1 was overexpressed. Therefore, the intrinsic difference between the imprinted region of Igf2 and H19 and of other imprinted genes to postzygotic de novo methylation may be the molecular basis for the frequently observed de novo methylation and upregulation of Igf2 in neoplastic cells and tumors. Injection of Dnmt1-overexpressing embryonic stem cells in diploid or tetraploid blastocysts resulted in lethality of the embryo, which resembled embryonic lethality caused by Dnmt1 deficiency. -
Humpherys D, Eggan K, Akutsu H, Friedman A, Hochedlinger K, Yanagimachi R, Lander ES, Golub TR, Jaenisch R. 2002. Abnormal gene expression in cloned mice derived from embryonic stem cell and cumulus cell nuclei. Proceedings of the National Academy of Sciences of the United States of America. 99(20):12889-94. Pubmed: 12235366 Humpherys D, Eggan K, Akutsu H, Friedman A, Hochedlinger K, Yanagimachi R, Lander ES, Golub TR, Jaenisch R. 2002. Abnormal gene expression in cloned mice derived from embryonic stem cell and cumulus cell nuclei. Proceedings of the National Academy of Sciences of the United States of America. 99(20):12889-94. Pubmed: 12235366 To assess the extent of abnormal gene expression in clones, we assessed global gene expression by microarray analysis on RNA from the placentas and livers of neonatal cloned mice derived by nuclear transfer (NT) from both cultured embryonic stem cells and freshly isolated cumulus cells. Direct comparison of gene expression profiles of more than 10,000 genes showed that for both donor cell types approximately 4% of the expressed genes in the NT placentas differed dramatically in expression levels from those in controls and that the majority of abnormally expressed genes were common to both types of clones. Importantly, however, the expression of a smaller set of genes differed between the embryonic stem cell- and cumulus cell-derived clones. The livers of the cloned mice also showed abnormal gene expression, although to a lesser extent, and with a different set of affected genes, than seen in the placentas. Our results demonstrate frequent abnormal gene expression in clones, in which most expression abnormalities appear common to the NT procedure whereas others appear to reflect the particular donor nucleus. -
Eggan K, Rode A, Jentsch I, Samuel C, Hennek T, Tintrup H, Zevnik B, Erwin J, Loring J, Jackson-Grusby L, Speicher MR, Kuehn R, Jaenisch R. 2002. Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complementation. Nature biotechnology. 20(5):455-9. Pubmed: 11981557 Eggan K, Rode A, Jentsch I, Samuel C, Hennek T, Tintrup H, Zevnik B, Erwin J, Loring J, Jackson-Grusby L, Speicher MR, Kuehn R, Jaenisch R. 2002. Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complementation. Nature biotechnology. 20(5):455-9. Pubmed: 11981557 We have devised a general strategy for producing female mice from 39,X0 embryonic stem (ES) cells derived from male cell lines carrying a targeted mutation of interest. We show that the Y chromosome is lost in 2% of subclones from 40,XY ES cell lines, making the identification of targeted 39,X0 subclones a routine procedure. After gene targeting, male and female mice carrying the mutation can be generated by tetraploid embryo complementation from the 40,XY ES cell line and its 39,X0 derivatives. A single intercross then produces homozygous mutant offspring. Because this strategy avoids outcrossing and therefore segregation of mutant alleles introduced into the ES cells, the time and expense required for production of experimental mutant animals from a targeted ES cell clone are substantially reduced. Our data also indicate that ES cells have inherently unstable karyotypes, but this instability does not interfere with production of adult ES cell tetraploid mice. 2001
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Humpherys D, Eggan K, Akutsu H, Hochedlinger K, Rideout WM, Biniszkiewicz D, Yanagimachi R, Jaenisch R. 2001. Epigenetic instability in ES cells and cloned mice. Science (New York, N.Y.). 293(5527):95-7. Pubmed: 11441181 Humpherys D, Eggan K, Akutsu H, Hochedlinger K, Rideout WM, Biniszkiewicz D, Yanagimachi R, Jaenisch R. 2001. Epigenetic instability in ES cells and cloned mice. Science (New York, N.Y.). 293(5527):95-7. Pubmed: 11441181 Cloning by nuclear transfer (NT) is an inefficient process in which most clones die before birth and survivors often display growth abnormalities. In an effort to correlate gene expression with survival and fetal overgrowth, we have examined imprinted gene expression in both mice cloned by nuclear transfer and in the embryonic stem (ES) cell donor populations from which they were derived. The epigenetic state of the ES cell genome was found to be extremely unstable. Similarly, variation in imprinted gene expression was observed in most cloned mice, even in those derived from ES cells of the same subclone. Many of the animals survived to adulthood despite widespread gene dysregulation, indicating that mammalian development may be rather tolerant to epigenetic aberrations of the genome. These data imply that even apparently normal cloned animals may have subtle abnormalities in gene expression. -
Eggan K, Akutsu H, Loring J, Jackson-Grusby L, Klemm M, Rideout WM, Yanagimachi R, Jaenisch R. 2001. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proceedings of the National Academy of Sciences of the United States of America. 98(11):6209-14. Pubmed: 11331774 Eggan K, Akutsu H, Loring J, Jackson-Grusby L, Klemm M, Rideout WM, Yanagimachi R, Jaenisch R. 2001. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proceedings of the National Academy of Sciences of the United States of America. 98(11):6209-14. Pubmed: 11331774 To assess whether heterozygosity of the donor cell genome was a general parameter crucial for long-term survival of cloned animals, we tested the ability of embryonic stem (ES) cells with either an inbred or F(1) genetic background to generate cloned mice by nuclear transfer. Most clones derived from five F(1) ES cell lines survived to adulthood. In contrast, clones from three inbred ES cell lines invariably died shortly after birth due to respiratory failure. Comparison of mice derived from nuclear cloning, in which a complete blastocyst is derived from a single ES cell, and tetraploid blastocyst complementation, in which only the inner cell mass is formed from a few injected ES cells, allows us to determine which phenotypes depend on the technique or on the characteristics of the ES cell line. Neonatal lethality also has been reported in mice entirely derived from inbred ES cells that had been injected into tetraploid blastocysts (ES cell-tetraploids). Like inbred clones, ES cell-tetraploid pups derived from inbred ES cell lines died shortly after delivery with signs of respiratory distress. In contrast, most ES cell-tetraploid neonates, derived from six F(1) ES cell lines, developed into fertile adults. Cloned pups obtained from both inbred and F(1) ES cell nuclei frequently displayed increased placental and birth weights whereas ES cell-tetraploid pups were of normal weight. The potency of F(1) ES cells to generate live, fertile adults was not lost after either long-term in vitro culture or serial gene targeting events. We conclude that genetic heterozygosity is a crucial parameter for postnatal survival of mice that are entirely derived from ES cells by either nuclear cloning or tetraploid embryo complementation. In addition, our results demonstrate that tetraploid embryo complementation using F(1) ES cells represents a simple, efficient procedure for deriving animals with complex genetic alterations without the need for a chimeric intermediate. -
Rideout WM, Eggan K, Jaenisch R. 2001. Nuclear cloning and epigenetic reprogramming of the genome. Science (New York, N.Y.). 293(5532):1093-8. Pubmed: 11498580 Rideout WM, Eggan K, Jaenisch R. 2001. Nuclear cloning and epigenetic reprogramming of the genome. Science (New York, N.Y.). 293(5532):1093-8. Pubmed: 11498580 Cloning of mammals by nuclear transfer (NT) results in gestational or neonatal failure with at most a few percent of manipulated embryos resulting in live births. Many of those that survive to term succumb to a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. Cloned embryos derived from donors, such as embryonic stem cells, that may require little or no reprogramming of early developmental genes develop substantially better beyond implantation than NT clones derived from somatic cells. Although recent experiments have demonstrated normal reprogramming of telomere length and X chromosome inactivation, epigenetic information established during gametogenesis, such as gametic imprints, cannot be restored after nuclear transfer. Survival of cloned animals to birth and beyond, despite substantial transcriptional dysregulation, is consistent with mammalian development being rather tolerant to epigenetic abnormalities, with lethality resulting only beyond a threshold of faulty gene reprogramming encompassing multiple loci. 2000
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Eggan K, Akutsu H, Hochedlinger K, Rideout W, Yanagimachi R, Jaenisch R. 2000. X-Chromosome inactivation in cloned mouse embryos. Science (New York, N.Y.). 290(5496):1578-81. Pubmed: 11090356 Eggan K, Akutsu H, Hochedlinger K, Rideout W, Yanagimachi R, Jaenisch R. 2000. X-Chromosome inactivation in cloned mouse embryos. Science (New York, N.Y.). 290(5496):1578-81. Pubmed: 11090356 To study whether cloning resets the epigenetic differences between the two X chromosomes of a somatic female nucleus, we monitored X inactivation in cloned mouse embryos. Both X chromosomes were active during cleavage of cloned embryos, followed by random X inactivation in the embryo proper. In the trophectoderm (TE), X inactivation was nonrandom with the inactivated X of the somatic donor being chosen for inactivation. When female embryonic stem cells with two active X chromosomes were used as donors, random X inactivation was seen in the TE and embryo. These results demonstrate that epigenetic marks can be removed and reestablished on either X chromosome during cloning. Our results also suggest that the epigenetic marks imposed on the X chromosomes during gametogenesis, responsible for normal imprinted X inactivation in the TE, are functionally equivalent to the marks imposed on the chromosomes during somatic X inactivation. -
Rideout WM, Wakayama T, Wutz A, Eggan K, Jackson-Grusby L, Dausman J, Yanagimachi R, Jaenisch R. 2000. Generation of mice from wild-type and targeted ES cells by nuclear cloning. Nature genetics. 24(2):109-10. Pubmed: 10655052 Rideout WM, Wakayama T, Wutz A, Eggan K, Jackson-Grusby L, Dausman J, Yanagimachi R, Jaenisch R. 2000. Generation of mice from wild-type and targeted ES cells by nuclear cloning. Nature genetics. 24(2):109-10. Pubmed: 10655052 -
Akeju O, Davis-Dusenbery BN, Cassel SH, Ichida JK, Eggan K. Ketamine exposure in early development impairs specification of the primary germ cell layers. Neurotoxicology and teratology. 43:59-68. Pubmed: 24746641 DOI:S0892-0362(14)00040-3 Akeju O, Davis-Dusenbery BN, Cassel SH, Ichida JK, Eggan K. Ketamine exposure in early development impairs specification of the primary germ cell layers. Neurotoxicology and teratology. 43:59-68. Pubmed: 24746641 DOI:S0892-0362(14)00040-3 Preclinical and clinical evidence implicates N-methyl-d-aspartate receptor (NMDAr) signaling in early embryological development. However, the role of NMDAr signaling in early development has not been well studied. Here, we use a mouse embryonic stem cell model to perform a step-wise exploration of the effects of NMDAr signaling on early cell fate specification. We found that antagonism of the NMDAr impaired specification into the neuroectodermal and mesoendodermal cell lineages, with little or no effect on specification of the extraembryonic endoderm cell lineage. Consistent with these findings, exogenous NMDA promoted neuroectodermal differentiation. Finally, NMDAr antagonism modified expression of several key targets of TGF-β superfamily signaling, suggesting a mechanism for these findings. In summary, this study shows that NMDAr antagonism interferes with the normal developmental pathways of embryogenesis, and suggests that interference is most pronounced prior to neuroectodermal and mesoendodermal cell fate specification.Copyright © 2014 Elsevier Inc. All rights reserved.