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2024. Organoid culture promotes dedifferentiation of mouse myoblasts into stem cells capable of complete muscle regeneration. Nature Biotechnology. DOI:https://doi.org/10.1038/s41587-024-02344-7 Price FD, Matyas MN, Gehrke AR, Chen W, Wolin EA, Chen W, Holton KM, Gibbs RM, Lee A, Singu P, Sakakeeny J,Poteracki JM, Goune K, Pfeiffer IT, Boswell SA, Sorger PK, Srivastava M, Pfaff KL, Gussoni E, Buchanan SM, Rubin LL. 2024. Organoid culture promotes dedifferentiation of mouse myoblasts into stem cells capable of complete muscle regeneration. Nature Biotechnology. DOI:https://doi.org/10.1038/s41587-024-02344-7 Experimental cell therapies for skeletal muscle conditions have shown little success, primarily because they use committed myogenic progenitors rather than true muscle stem cells, known as satellite cells. Here we present a method to generate in vitro-derived satellite cells (idSCs) from skeletal muscle tissue. When transplanted in small numbers into mouse muscle, mouse idSCs fuse into myofibers, repopulate the satellite cell niche, self-renew, support multiple rounds of muscle regeneration and improve force production on par with freshly isolated satellite cells in damaged skeletal muscle. We compared the epigenomic and transcriptional signatures between idSCs, myoblasts and satellite cells and used these signatures to identify core signaling pathways and genes that confer idSC functionality. Finally, from human muscle biopsies, we successfully generated satellite cell-like cells in vitro. After further development, idSCs may provide a scalable source of cells for the treatment of genetic muscle disorders, trauma-induced muscle damage and age-related muscle weakness. -
Watts ME, Giadone RM, Ordureau A, Holton KM, Harper JW, Rubin LL. 2023. Analyzing the ER stress response in ALS patient derived motor neurons identifies druggable neuroprotective targets. Frontiers in cellular neuroscience. 17:1327361. Pubmed: 38314348 DOI:10.3389/fncel.2023.1327361 Watts ME, Giadone RM, Ordureau A, Holton KM, Harper JW, Rubin LL. 2023. Analyzing the ER stress response in ALS patient derived motor neurons identifies druggable neuroprotective targets. Frontiers in cellular neuroscience. 17:1327361. Pubmed: 38314348 DOI:10.3389/fncel.2023.1327361 Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron (MN) disease with severely limited treatment options. Identification of effective treatments has been limited in part by the lack of predictive animal models for complex human disorders. Here, we utilized pharmacologic ER stressors to exacerbate underlying sensitivities conferred by ALS patient genetics in induced pluripotent stem cell (iPSC)-derived motor neurons (MNs). In doing so, we found that thapsigargin and tunicamycin exposure recapitulated ALS-associated degeneration, and that we could rescue this degeneration via MAP4K4 inhibition (MAP4K4i). We subsequently identified mechanisms underlying MAP4K4i-mediated protection by performing phosphoproteomics on iPSC-derived MNs treated with ER stressors ±MAP4K4i. Through these analyses, we found JNK, PKC, and BRAF to be differentially modulated in MAP4K4i-protected MNs, and that inhibitors to these proteins could also rescue MN toxicity. Collectively, this study highlights the value of utilizing ER stressors in ALS patient MNs to identify novel druggable targets.Copyright © 2024 Watts, Giadone, Ordureau, Holton, Harper and Rubin. -
Ximerakis M*, Holton KM*, Giadone RM, Ozek C, Saxena M, Santiago S, Adiconis X, Dionne D, Nguyen L, Shah KM, Goldstein JM, Gasperini C, Gampierakis IA, Lipnick SL, Simmons SK, Buchanan SM, Wagers AJ, Regev A, Levin JZ, Rubin LL. (* these authors contributed equally). 2023. Heterochronic parabiosis reprograms the mouse brain transcriptome by shifting aging signatures in multiple cell types. Nature Aging. 3. DOI:https://doi.org/10.1038/s43587-023-00373-6 Ximerakis M*, Holton KM*, Giadone RM, Ozek C, Saxena M, Santiago S, Adiconis X, Dionne D, Nguyen L, Shah KM, Goldstein JM, Gasperini C, Gampierakis IA, Lipnick SL, Simmons SK, Buchanan SM, Wagers AJ, Regev A, Levin JZ, Rubin LL. (* these authors contributed equally). 2023. Heterochronic parabiosis reprograms the mouse brain transcriptome by shifting aging signatures in multiple cell types. Nature Aging. 3. DOI:https://doi.org/10.1038/s43587-023-00373-6 Aging is a complex process involving transcriptomic changes associated with deterioration across multiple tissues and organs, including the brain. Recent studies using heterochronic parabiosis have shown that various aspects of aging-associated decline are modifiable or even reversible. To better understand how this occurs, we performed single-cell transcriptomic profiling of young and old mouse brains following parabiosis. For each cell type, we catalogued alterations in gene expression, molecular pathways, transcriptional networks, ligand-receptor interactions, and senescence status. Our analyses identified gene signatures demonstrating that heterochronic parabiosis regulates several hallmarks of aging in a cell-type-specific manner. Brain endothelial cells were found to be especially malleable to this intervention, exhibiting dynamic transcriptional changes that affect vascular structure and function. These findings suggest novel strategies for slowing deterioration and driving regeneration in the aging brain through approaches that do not rely on disease-specific mechanisms or actions of individual circulating factors. -
Rapino F, Natoli T, Limone F, O'Connor E, Blank J, Tegtmeyer M, Chen W, Norabuena E, Narula J, Hazelbaker D, Angelini G, Barrett L, O'Neil A, Beattie UK, Thanos JM, de Rivera H, Sheridan SD, Perlis RH, McCarroll SA, Stevens B, Subramanian A, Nehme R, Rubin LL. 2023. Small-molecule screen reveals pathways that regulate C4 secretion in stem cell-derived astrocytes. Stem cell reports. 18(1):237-253. Pubmed: 36563689 DOI:S2213-6711(22)00551-3 Rapino F, Natoli T, Limone F, O'Connor E, Blank J, Tegtmeyer M, Chen W, Norabuena E, Narula J, Hazelbaker D, Angelini G, Barrett L, O'Neil A, Beattie UK, Thanos JM, de Rivera H, Sheridan SD, Perlis RH, McCarroll SA, Stevens B, Subramanian A, Nehme R, Rubin LL. 2023. Small-molecule screen reveals pathways that regulate C4 secretion in stem cell-derived astrocytes. Stem cell reports. 18(1):237-253. Pubmed: 36563689 DOI:S2213-6711(22)00551-3 In the brain, the complement system plays a crucial role in the immune response and in synaptic elimination during normal development and disease. Here, we sought to identify pathways that modulate the production of complement component 4 (C4), recently associated with an increased risk of schizophrenia. To design a disease-relevant assay, we first developed a rapid and robust 3D protocol capable of producing large numbers of astrocytes from pluripotent cells. Transcriptional profiling of these astrocytes confirmed the homogeneity of this population of dorsal fetal-like astrocytes. Using a novel ELISA-based small-molecule screen, we identified epigenetic regulators, as well as inhibitors of intracellular signaling pathways, able to modulate C4 secretion from astrocytes. We then built a connectivity map to predict and validate additional key regulatory pathways, including one involving c-Jun-kinase. This work provides a foundation for developing therapies for CNS diseases involving the complement cascade.Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved. -
Paul KC, Krolewski RC, Lucumi Moreno E, Blank J, Holton KM, Ahfeldt T, Furlong M, Yu Y, Cockburn M, Thompson LK, Kreymerman A, Ricci-Blair EM, Li YJ, Patel HB, Lee RT, Bronstein J, Rubin LL, Khurana V, Ritz B. 2023. A pesticide and iPSC dopaminergic neuron screen identifies and classifies Parkinson-relevant pesticides. Nature communications. 14(1):2803. Pubmed: 37193692 DOI:10.1038/s41467-023-38215-z Paul KC, Krolewski RC, Lucumi Moreno E, Blank J, Holton KM, Ahfeldt T, Furlong M, Yu Y, Cockburn M, Thompson LK, Kreymerman A, Ricci-Blair EM, Li YJ, Patel HB, Lee RT, Bronstein J, Rubin LL, Khurana V, Ritz B. 2023. A pesticide and iPSC dopaminergic neuron screen identifies and classifies Parkinson-relevant pesticides. Nature communications. 14(1):2803. Pubmed: 37193692 DOI:10.1038/s41467-023-38215-z Parkinson's disease (PD) is a complex neurodegenerative disease with etiology rooted in genetic vulnerability and environmental factors. Here we combine quantitative epidemiologic study of pesticide exposures and PD with toxicity screening in dopaminergic neurons derived from PD patient induced pluripotent stem cells (iPSCs) to identify Parkinson's-relevant pesticides. Agricultural records enable investigation of 288 specific pesticides and PD risk in a comprehensive, pesticide-wide association study. We associate long-term exposure to 53 pesticides with PD and identify co-exposure profiles. We then employ a live-cell imaging screening paradigm exposing dopaminergic neurons to 39 PD-associated pesticides. We find that 10 pesticides are directly toxic to these neurons. Further, we analyze pesticides typically used in combinations in cotton farming, demonstrating that co-exposures result in greater toxicity than any single pesticide. We find trifluralin is a driver of toxicity to dopaminergic neurons and leads to mitochondrial dysfunction. Our paradigm may prove useful to mechanistically dissect pesticide exposures implicated in PD risk and guide agricultural policy.© 2023. The Author(s). -
Mayweather BA, Buchanan SM, Rubin LL. 2021. GDF11 expressed in the adult brain negatively regulates hippocampal neurogenesis. Molecular brain. 14(1):134. Pubmed: 34488822 DOI:10.1186/s13041-021-00845-z Mayweather BA, Buchanan SM, Rubin LL. 2021. GDF11 expressed in the adult brain negatively regulates hippocampal neurogenesis. Molecular brain. 14(1):134. Pubmed: 34488822 DOI:10.1186/s13041-021-00845-z Growth differentiation factor 11 (GDF11) is a transforming factor-β superfamily member that functions as a negative regulator of neurogenesis during embryonic development. However, when recombinant GDF11 (rGDF11) is administered systemically in aged mice, it promotes neurogenesis, the opposite of its role during development. The goal of the present study was to reconcile this apparent discrepancy by performing the first detailed investigation into the expression of endogenous GDF11 in the adult brain and its effects on neurogenesis. Using quantitative histological analysis, we observed that Gdf11 is most highly expressed in adult neurogenic niches and non-neurogenic regions within the hippocampus, choroid plexus, thalamus, habenula, and cerebellum. To investigate the role of endogenous GDF11 during adult hippocampal neurogenesis, we generated a tamoxifen inducible mouse that allowed us to reduce GDF11 levels. Depletion of Gdf11 during adulthood increased proliferation of neural progenitors and decreased the number of newborn neurons in the hippocampus, suggesting that endogenous GDF11 remains a negative regulator of hippocampal neurogenesis in adult mice. These findings further support the idea that circulating systemic GDF11 and endogenously expressed GDF11 in the adult brain have different target cells or mechanisms of action. Our data describe a role for GDF11-dependent signaling in adult neurogenesis that has implications for how GDF11 may be used to treat CNS disease.© 2021. The Author(s). -
Buchanan SM, Price FD, Castiglioni A, Gee AW, Schneider J, Matyas MN, Hayhurst M, Tabebordbar M, Wagers AJ, Rubin LL. 2020. Pro-myogenic small molecules revealed by a chemical screen on primary muscle stem cells. Skeletal muscle. 10(1):28. Pubmed: 33036659 DOI:10.1186/s13395-020-00248-z Buchanan SM, Price FD, Castiglioni A, Gee AW, Schneider J, Matyas MN, Hayhurst M, Tabebordbar M, Wagers AJ, Rubin LL. 2020. Pro-myogenic small molecules revealed by a chemical screen on primary muscle stem cells. Skeletal muscle. 10(1):28. Pubmed: 33036659 DOI:10.1186/s13395-020-00248-z Satellite cells are the canonical muscle stem cells that regenerate damaged skeletal muscle. Loss of function of these cells has been linked to reduced muscle repair capacity and compromised muscle health in acute muscle injury and congenital neuromuscular diseases. To identify new pathways that can prevent loss of skeletal muscle function or enhance regenerative potential, we established an imaging-based screen capable of identifying small molecules that promote the expansion of freshly isolated satellite cells. We found several classes of receptor tyrosine kinase (RTK) inhibitors that increased freshly isolated satellite cell numbers in vitro. Further exploration of one of these compounds, the RTK inhibitor CEP-701 (also known as lestaurtinib), revealed potent activity on mouse satellite cells both in vitro and in vivo. This expansion potential was not seen upon exposure of proliferating committed myoblasts or non-myogenic fibroblasts to CEP-701. When delivered subcutaneously to acutely injured animals, CEP-701 increased both the total number of satellite cells and the rate of muscle repair, as revealed by an increased cross-sectional area of regenerating fibers. Moreover, freshly isolated satellite cells expanded ex vivo in the presence of CEP-701 displayed enhanced muscle engraftment potential upon in vivo transplantation. We provide compelling evidence that certain RTKs, and in particular RET, regulate satellite cell expansion during muscle regeneration. This study demonstrates the power of small molecule screens of even rare adult stem cell populations for identifying stem cell-targeting compounds with therapeutic potential.