Insulin-expressing cells that have been differentiated from human pluripotent stem cells in vitro lack the glucose responsiveness characteristic of mature beta cells. Beta-cell maturation in mice was studied to find genetic markers that enable screens for factors that induce bona fide beta cells in vitro. We find that functional beta-cell maturation is marked by an increase in the glucose threshold for insulin secretion and by expression of the gene urocortin 3.
Studying the response of islet cells to glucose stimulation is important for understanding cell function in healthy and disease states. Most functional assays are performed on whole islets or cell populations, resulting in averaged observations and loss of information at the single cell level. We demonstrate methods to examine calcium fluxing in individual cells of intact islets in response to multiple glucose challenges. Wild-type mouse islets predominantly contained cells that responded to three (out of three) sequential high glucose challenges, whereas cells of diabetic islets (db/db or NOD) responded less frequently or not at all. Imaged islets were also immunostained for endocrine markers to associate the calcium flux profile of individual cells with gene expression. Wild-type mouse islet cells that robustly fluxed calcium expressed β cell markers (INS/NKX6.1), whereas islet cells that inversely fluxed at low glucose expressed α cell markers (GCG). Diabetic mouse islets showed a higher proportion of dysfunctional β cells that responded poorly to glucose challenges. Most of the failed calcium influx responses in β cells were observed in the second and third high glucose challenges, emphasizing the importance of multiple sequential glucose challenges for assessing the full function of islet cells. Human islet cells were also assessed and showed functional α and β cells. This approach to analyze islet responses to multiple glucose challenges in correlation with gene expression assays expands the understanding of β cell function and the diseased state.
Driving human pluripotent stem cells (hPSCs) into specific lineages is an inefficient and challenging process. We show that a potent Src inhibitor, PP1, regulates expression of genes involved in the G1 to S phase transition of the cell cycle, activates proteins in the retinoblastoma family, and subsequently increases the differentiation propensities of hPSCs into all three germ layers. We further demonstrate that genetic suppression of Src regulates the activity of the retinoblastoma protein and enhances the differentiation potential of hPSCs across all germ layers. These positive effects extend beyond the initial germ layer specification and enable efficient differentiation at subsequent stages of differentiation.
Transcriptional profiling is a key technique in the study of cell biology that is limited by the availability of reagents to uniquely identify specific cell types and isolate high quality RNA from them. We report a Method for Analyzing RNA following Intracellular Sorting (MARIS) that generates high quality RNA for transcriptome profiling following cellular fixation, intracellular immunofluorescent staining and FACS. MARIS can therefore be used to isolate high quality RNA from many otherwise inaccessible cell types simply based on immunofluorescent tagging of unique intracellular proteins. As proof of principle, we isolate RNA from sorted human embryonic stem cell-derived insulin-expressing cells as well as adult human β cells. MARIS is a basic molecular biology technique that could be used across several biological disciplines.
One goal of regenerative medicine, to use stem cells to replace cells lost by injury or disease, depends on producing an excess of the relevant cell for study or transplantation. To this end, the stepwise differentiation of stem cells into specialized derivatives has been successful for some cell types1, 2, 3, but a major problem remains the inefficient conversion of cells from one stage of differentiation to the next. If specialized cells are to be produced in large numbers it will be necessary to expand progenitor cells, without differentiation, at some steps of the process. Using the pancreatic lineage as a model for embryonic-stem-cell differentiation, we demonstrate that this is a solvable problem. Co-culture with organ-matched mesenchyme permits proliferation and self-renewal of progenitors, without differentiation, and enables an expansion of more than a million-fold for human endodermal cells with full retention of their developmental potential. This effect is specific both to the mesenchymal cell and to the progenitor being amplified. Progenitors that have been serially expanded on mesenchyme give rise to glucose-sensing, insulin-secreting cells when transplanted in vivo. Theoretically, the identification of stage-specific renewal signals can be incorporated into any scheme for the efficient production of large numbers of differentiated cells from stem cells and may therefore have wide application in regenerative biology.
Understanding the genes and signaling pathways that determine the differentiation and fate of a cell is a central goal of developmental biology. Using that information to gain mastery over the fates of cells presents new approaches to cell transplantation and drug discovery for human diseases including diabetes.
One goal of regenerative medicine is to instructively convert adult cells into other cell types for tissue repair and regeneration. Although isolated examples of adult cell reprogramming are known, there is no general understanding of how to turn one cell type into another in a controlled manner. Here, using a strategy of re-expressing key developmental regulators in vivo, we identify a specific combination of three transcription factors (Ngn3 (also known as Neurog3) Pdx1 and Mafa) that reprograms differentiated pancreatic exocrine cells in adult mice into cells that closely resemble β-cells. The induced β-cells are indistinguishable from endogenous islet β-cells in size, shape and ultrastructure. They express genes essential for β-cell function and can ameliorate hyperglycaemia by remodelling local vasculature and secreting insulin. This study provides an example of cellular reprogramming using defined factors in an adult organ and suggests a general paradigm for directing cell reprogramming without reversion to a pluripotent stem cell state.