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Pancreatic β-cells are responsible for maintaining glucose homeostasis. Therefore, their dysregulation leads to diabetes. Pancreas or islet transplants can be used to treat diabetes but these human tissues remain in short supply. Significant progress has now been made in differentiating human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) into pancreatic β-like cells for potential cell replacement therapy. Additionally, these hPSC-derived β-like cells represent a new invaluable model for studying diabetes disease mechanisms. Here, we review the use of hPSC-derived β-like cells as a platform to model various types of defects in human β-cells in diabetes, comparing them against existing animal models, ex vivo human islets and human β-cell line. We also discuss how hPSC-derived β-like cells are being used as a platform for screening novel therapeutic compounds. Last but not least, we evaluate the strengths and limitations of this human cell-based platform as an avenue to study and reveal new insights into human β-cell biology.

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-producing beta cells in the pancreas. Consequently, T1D patients produce little to no insulin, resulting in absolute insulin deficiency and an inability to regulate blood glucose levels effectively. Insulin replacement is the mainstream treatment for T1D, but it comes with a major drawback—the risk of hypoglycemia unawareness. As such, replenishment of glucose-sensing and insulin-producing beta cells via cell replacement therapy is believed to have great therapeutic value for T1D patients. Human pluripotent stem cells (hPSCs) are considered an attractive source for the derivation of insulin-producing beta-like cells for cell replacement therapy. However, several challenges need to be addressed before hPSC-derived beta-like cells can be used for T1D treatment. In this chapter, we provide a snapshot of the current progress of hPSC therapy with an emphasis on T1D, discuss some of the clinical considerations and technical strategies in the generation of current good manufacturing practice (cGMP)-compliant hPSC-derived beta-like cells and the future outlook for T1D hPSC therapy.

There is now a sizeable number of single cell transcriptomics studies performed on human and rodent pancreatic islets that have shed light on the unique gene signatures and level of heterogeneity within each individual islet cell type. Following closely from these studies, there is also rapidly-growing activity on characterizing islet-like cells derived from in vitro differentiation of human pluripotent stem cells (hPSCs) at the single cell level. The overall consensus across the studies so far suggests that the first few stages of differentiation are largely uniform, whereas during pancreatic endocrine commitment, cell trajectories start to diverge, resulting in multiple end-stage pancreatic cells that include progenitor-like, endocrine and non-endocrine cells. Comprehensive transcriptional profiling is important for understanding how and why islet cells, especially the insulin-secreting beta cells, exist in subpopulations that differ in maturity, proliferation rate, sensitivity to stress, and insulin secretion function. For hPSC-derived beta cells to be used confidently for cell therapy, optimal differentiation and thorough characterization is required. The key questions to address are—What is the trajectory of differentiation? Is heterogeneity a natural occurrence or is it a consequence of imperfect differentiation protocols? Can lessons be drawn from the extensive single cell transcriptomic data to help guide maturation of beta cells in vitro? This book chapter seeks to address some of these questions, and facilitate ongoing efforts in improving the beta cell differentiation pipeline or enriching for desired beta cell populations following differentiation, to make way for better mechanistic studies and future clinical translation.

Current complexities in human pluripotent stem cell (hPSC)-based studies. hPSC studies begin with the recruitment of patients harboring disease-associated gene variant(s) that may increase susceptibility to disease development. Somatic reprogramming is then performed to derive patient-specific human induced pluripotent stem cells (hiPSCs), followed by step-wise directed differentiation to a relevant cell type before qualitative/quantitative assays are performed to assess for phenotypic or gene expression differences between the healthy and diseased hiPSCs.

The differentiation of human pluripotent stem cells into pancreatic cells involves cellular proliferation and apoptosis during cell fate transitions. However, their implications for establishing cellular identity are unclear. Here, we profiled the expression of BCL-2 family of proteins during pancreatic specification and observed an upregulation of BCL-xL, downregulation of BAK and corresponding downregulation of cleaved CASP3 representative of apoptosis. Experimental inhibition of BCL-xL reciprocally increased apoptosis and resulted in a decreased gene expression of pancreatic markers despite a compensatory increase in anti-apoptotic protein BCL-2. RNA-Seq analyses then revealed a downregulation of multiple metabolic genes upon inhibition of BCL-xL. Follow-up bioenergetics assays revealed broad downregulation of both glycolysis and oxidative phosphorylation when BCL-xL was inhibited. Early perturbation of BCL-xL during pancreatic specification also had subsequent detrimental effects on the formation of INS+ pancreatic beta-like cells. In conclusion, the more differentiated pancreatic progenitors are dependent on anti-apoptotic BCL-xL for survival, whereas the less differentiated pancreatic progenitors that survived after WEHI-539 treatment would exhibit a more immature phenotype. Therefore, modulation of the expression level of BCL-xL can potentially increase the survival and robustness of pancreatic progenitors that ultimately define human pancreatic beta cell mass and function.

A comprehensive characterization of the molecular processes controlling cell fate decisions is essential to derive stable progenitors and terminally differentiated cells that are functional from human pluripotent stem cells (hPSCs). Here, we report the use of quantitative proteomics to describe early proteome adaptations during hPSC differentiation toward pancreatic progenitors. We report that the use of unbiased quantitative proteomics allows the simultaneous profiling of numerous proteins at multiple time points, and is a valuable tool to guide the discovery of signaling events and molecular signatures underlying cellular differentiation. We also monitored the activity level of pathways whose roles are pivotal in the early pancreas differentiation, including the Hippo signaling pathway. The quantitative proteomics data set provides insights into the dynamics of the global proteome during the transition of hPSCs from a pluripotent state toward pancreatic differentiation.

The differentiation of human pluripotent stem cells (hPSCs) into desired cell types such as the pancreatic cells involve cellular proliferation and apoptosis during cell fate transitions. Although different rates of cell growth and death are typically observed during differentiation, its implications for pancreatic tissue formation in humans remain unclear. Here, we profiled the expression of BCL-2 family of proteins during pancreatic specification from hPSCs and observed an upregulation of BCL- XL, downregulation of BAK and corresponding downregulation of cleaved CASP3 representative of apoptosis. The inhibition or downregulation of BCL-xL reciprocally increased apoptosis and resulted in a decreased gene expression of pancreatic markers despite a compensatory increase in companion anti-apoptotic protein BCL2. RNA-Seq analyses revealed a downregulation of multiple metabolic genes upon inhibition of BCL-xL function. Bioenergetics assays then revealed a broad downregulation of both glycolysis and oxidative phosphorylation when BCL-xL function was inhibited. In conclusion, we have identified anti-apoptotic protein BCL-xL to be necessary for suppressing the expression of pro-apoptotic protein BAK to facilitate proper pancreas formation in human cells. This is the first report that links a member of the BCL-2 family with pancreatic specification. Therefore, modulation of these two BCL-2 family of proteins can potentially increase the survival and robustness of pancreatic progenitors that ultimately defines human pancreatic beta cell mass and function. Disclosure L. Loo: None. A. Teo: None. S. Ghosh: None. A. Soetedjo: None. L. Nguyen: None. V.G. Krishnan: None. S. Hoon: None. Funding Agency for Science, Technology and Research; Institute of Molecular and Cell Biology

Summary Patients with an HNF1BS148L/+ mutation (MODY5) typically exhibit pancreatic hypoplasia. However, the molecular mechanisms are unknown due to inaccessibility of patient material and because mouse models do not fully recapitulate MODY5. Here, we differentiated MODY5 human-induced pluripotent stem cells (hiPSCs) into pancreatic progenitors, and show that the HNF1BS148L/+ mutation causes a compensatory increase in several pancreatic transcription factors, and surprisingly, a decrease in PAX6 pancreatic gene expression. The lack of suppression of PDX1, PTF1A, GATA4, and GATA6 indicates that MODY5-mediated pancreatic hypoplasia is mechanistically independent. Overexpression studies demonstrate that a compensatory increase in PDX1 gene expression is due to mutant HNF1BS148L/+ but not wild-type HNF1B or HNF1A. Furthermore, HNF1B does not appear to directly regulate PAX6 gene expression necessary for glucose tolerance. Our results demonstrate compensatory mechanisms in the pancreatic transcription factor network due to mutant HNF1BS148L/+ protein. Thus, patients typically develop MODY5 but not neonatal diabetes despite exhibiting pancreatic hypoplasia., Graphical Abstract, Highlights • HNF1BS148L/+ mutation elicits a compensatory increase in DE and pancreatic genes • MODY5-mediated pancreatic hypoplasia is independent of PDX1, PTF1A, GATA4, and GATA6 • HNF1BS148L mutation directly causes a compensatory increase in PDX1 gene expression • HNF1BS148L/+ mutation limits PAX6 expression and consequently leads to MODY5, Kulkarni, Teo, and colleagues differentiated MODY5-hiPSCs into pancreatic progenitors and demonstrated that HNF1BS148L/+ mutation elicits a compensatory mechanism in the pancreatic transcription factor network. They report that MODY5-mediated pancreatic hypoplasia is independent of PDX1, PTF1A, GATA4, and GATA6. In addition, a decrease in PAX6 gene expression may lead to MODY5 development.

Human embryonic stem cells are a type of pluripotent stem cells (hPSCs) that are used to investigate their differentiation into diverse mature cell types for molecular studies. The mechanisms underlying insulin receptor (IR)-mediated signaling in the maintenance of human pluripotent stem cell (hPSC) identity and cell fate specification are not fully understood. Here, we used two independent shRNAs to stably knock down IRs in two hPSC lines that represent pluripotent stem cells and explored the consequences on expression of key proteins in pathways linked to proliferation and differentiation. We consistently observed lowered pAKT in contrast to increased pERK1/2 and a concordant elevation in pluripotency gene expression. ERK2 chromatin immunoprecipitation, luciferase assays, and ERK1/2 inhibitors established direct causality between ERK1/2 and OCT4 expression. Of importance, RNA sequencing analyses indicated a dysregulation of genes involved in cell differentiation and organismal development. Mass spectrometry–based proteomic analyses further confirmed a global downregulation of extracellular matrix proteins. Subsequent differentiation toward the neural lineage reflected alterations in SOX1+PAX6+ neuroectoderm and FOXG1+ cortical neuron marker expression and protein localization. Collectively, our data underscore the role of IR-mediated signaling in maintaining pluripotency, the extracellular matrix necessary for the stem cell niche, and regulating cell fate specification including the neural lineage.

Summary Maturity-onset diabetes of the young 1 (MODY1) is a monogenic diabetes condition caused by heterozygous HNF4A mutations. We investigate how HNF4A haploinsufficiency from a MODY1/HNF4A mutation influences the development of foregut-derived liver and pancreatic cells through differentiation of human induced pluripotent stem cells from a MODY1 family down the foregut lineage. In MODY1-derived hepatopancreatic progenitors, which expressed reduced HNF4A levels and mislocalized HNF4A, foregut genes were downregulated, whereas hindgut-specifying HOX genes were upregulated. MODY1-derived hepatocyte-like cells were found to exhibit altered morphology. Hepatic and β cell gene signatures were also perturbed in MODY1-derived hepatocyte-like and β-like cells, respectively. As mutant HNF4A (p.Ile271fs) did not undergo complete nonsense-mediated decay or exert dominant negativity, HNF4A-mediated loss of function is likely due to impaired transcriptional activation of target genes. Our results suggest that in MODY1, liver and pancreas development is perturbed early on, contributing to altered hepatic proteins and β cell defects in patients., Graphical Abstract, Highlights • HNF4A is downregulated and predominantly mislocalized in the cytoplasm in MODY1 • Foregut markers, pancreatic and hepatic genes, were downregulated in MODY1-HPPs • A reciprocal upregulation of hindgut HOX genes was observed in MODY1-HPPs • Mutant HNF4A resulted in loss of transcriptional activation of target genes, Molecular Mechanism of Gene Regulation; Diabetology; Stem Cells Research; Developmental Biology

Type 1 and type 2 diabetes are caused by a destruction and decrease in the number of functional insulin-producing β cells, respectively; therefore, the generation of functional β cells from human embryonic stem cells and human induced pluripotent stem cells, collectively known as human pluripotent stem cells (hPSCs), for potential cell replacement therapy and disease modelling is an intensely investigated area. Recent scientific breakthroughs enabled derivation of large quantities of human pancreatic β-like cells in vitro, although with varied glucose-stimulated insulin secretion kinetics. In the present review, we comprehensively summarize, compare and critically analyze the intricacies of these developing technologies, including differentiation platforms, robustness of protocols, and methodologies used to characterize hPSC-derived β-like cells. We also discuss experimental issues that need to be resolved before these β-like cells can be used clinically.

Activin/Nodal signaling is necessary to maintain pluripotency of human embryonic stem cells (hESCs) and to induce their differentiation toward endoderm. However, the mechanisms by which Activin/Nodal signaling achieves these opposite functions remain unclear. To unravel these mechanisms, we examined the transcriptional network controlled in hESCs by Smad2 and Smad3, which represent the direct effectors of Activin/Nodal signaling. These analyses reveal that Smad2/3 participate in the control of the core transcriptional network characterizing pluripotency, which includes Oct-4, Nanog, FoxD3, Dppa4, Tert, Myc, and UTF1. In addition, similar experiments performed on endoderm cells confirm that a broad part of the transcriptional network directing differentiation is downstream of Smad2/3. Therefore, Activin/Nodal signaling appears to control divergent transcriptional networks in hESCs and in endoderm. Importantly, we observed an overlap between the transcriptional network downstream of Nanog and Smad2/3 in hESCs; whereas, functional studies showed that both factors cooperate to control the expression of pluripotency genes. Therefore, the effect of Activin/Nodal signaling on pluripotency and differentiation could be dictated by tissue specific Smad2/3 partners such as Nanog, explaining the mechanisms by which signaling pathways can orchestrate divergent cell fate decisions.

Summary Human embryonic stem cells (hESCs) rely on fibroblast growth factor and Activin-Nodal signaling to maintain their pluripotency. However, Activin-Nodal signaling is also known to induce mesendoderm differentiation. The mechanisms by which Activin-Nodal signaling can achieve these contradictory functions remain unknown. Here, we demonstrate that Smad-interacting protein 1 (SIP1) limits the mesendoderm-inducing effects of Activin-Nodal signaling without inhibiting the pluripotency-maintaining effects exerted by SMAD2/3. In turn, Activin-Nodal signaling cooperates with NANOG, OCT4, and SOX2 to control the expression of SIP1 in hESCs, thereby limiting the neuroectoderm-promoting effects of SIP1. Similar results were obtained with mouse epiblast stem cells, implying that these mechanisms are evolutionarily conserved and may operate in vivo during mammalian development. Overall, our results reveal the mechanisms by which Activin-Nodal signaling acts through SIP1 to regulate the cell-fate decision between neuroectoderm and mesendoderm in the progression from pluripotency to primary germ layer differentiation.

In the field of stem cell biology and diabetes, we and others seek to derive mature and functional human pancreatic β cells for disease modeling and cell replacement therapy. Traditionally, knowledge gathered from rodents is extended to human pancreas developmental biology research involving human pluripotent stem cells (hPSCs). While much has been learnt from rodent pancreas biology in the early steps toward Pdx1+ pancreatic progenitors, much less is known about the transition toward Ngn3+ pancreatic endocrine progenitors. Essentially, the later steps of pancreatic β cell development and maturation remain elusive to date. As a result, the most recent advances in the stem cell and diabetes field have relied upon combinatorial testing of numerous growth factors and chemical compounds in an arbitrary trial-and-error fashion to derive mature and functional human pancreatic β cells from hPSCs. Although this hit-or-miss approach appears to have made some headway in maturing human pancreatic β cells in vitro, its underlying biology is vaguely understood. Therefore, in this mini-review, we discuss some of these late-stage signaling pathways that are involved in human pancreatic β cell differentiation and highlight our current understanding of their relevance in rodent pancreas biology. Our efforts here unravel several novel signaling pathways that can be further studied to shed light on unexplored aspects of rodent pancreas biology. New investigations into these signaling pathways are expected to advance our knowledge in human pancreas developmental biology and to aid in the translation of stem cell biology in the context of diabetes treatments. Published version

The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts. Significance The efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells.

Background: Diabetes and metabolic syndromes are chronic, devastating diseases with increasing prevalence. Human pluripotent stem cells are gaining popularity in their usage for human in vitro disease modeling. With recent rapid advances in genome editing tools, these cells can now be genetically manipulated with relative ease to study how genes and gene variants contribute to diabetes and metabolic syndromes. Scope of review: We highlight the diabetes and metabolic genes and gene variants, which could potentially be studied, using two powerful technologies – human pluripotent stem cells (hPSCs) and genome editing tools – to aid the elucidation of yet elusive mechanisms underlying these complex diseases. Major conclusions: hPSCs and the advancing genome editing tools appear to be a timely and potent combination for probing molecular mechanism(s) underlying diseases such as diabetes and metabolic syndromes. The knowledge gained from these hiPSC-based disease modeling studies can potentially be translated into the clinics by guiding clinicians on the appropriate type of medication to use for each condition based on the mechanism of action of the disease. Published version

Summary There is considerable interest in differentiating human pluripotent stem cells (hPSCs) into definitive endoderm (DE) and pancreatic cells for in vitro disease modeling and cell replacement therapy. Numerous protocols use fetal bovine serum, which contains poorly defined factors to induce DE formation. Here, we compared Wnt and BMP in their ability to cooperate with Activin signaling to promote DE formation in a chemically defined medium. Varying concentrations of WNT3A, glycogen synthase kinase (GSK)-3 inhibitors CHIR99021 and 6-bromoindirubin-3′-oxime (BIO), and BMP4 could independently co-operate with Activin to effectively induce DE formation even in the absence of serum. Overall, CHIR99021 is favored due to its cost effectiveness. Surprisingly, WNT3A was ineffective in suppressing E-CADHERIN/CDH1 and pluripotency factor gene expression unlike GSK-3 inhibitors or BMP4. Our findings indicate that both Wnt and BMP effectively synergize with Activin signaling to generate DE from hPSCs, although WNT3A requires additional factors to suppress the pluripotency program inherent in hPSCs., Graphical Abstract, Highlights • High dose of WNT3A cooperates with Activin to induce DE without serum • GSK-3 inhibitors, CHIR99021 and BIO, work with Activin to induce DE without serum • Wnt and BMP signaling can induce DE with comparable efficiencies without serum • WNT3A is not as effective as BMP4, CHIR99021, or BIO in suppressing pluripotency, It is yet unclear how Wnt compares with BMP signaling in promoting Activin-induced definitive endoderm formation from human pluripotent stem cells in serum-free conditions. Kulkarni and colleagues demonstrate that Wnt or BMP signaling generates Activin-induced definitive endoderm comparably and that WNT3A is ineffective at suppressing E-CADHERIN and pluripotency.

The landmark discovery of induced pluripotent stem cells (iPSCs) by Shinya Yamanaka has transformed regenerative biology. Previously, insights into the pathogenesis of chronic human diseases have been hindered by the inaccessibility of patient samples. However, scientists are now able to convert patient fibroblasts into iPSCs and differentiate them into disease-relevant cell types. This ability opens new avenues for investigating disease pathogenesis and designing novel treatments. In this review, we highlight the uses of human iPSCs to uncover the underlying causes and pathological consequences of diabetes and metabolic syndromes, multi-factorial diseases whose etiologies have been difficult to unravel using traditional methodologies.

Maturity onset diabetes of the young (MODY) is an autosomal dominant disease. Despite extensive research, the mechanism by which a mutant MODY gene results in monogenic diabetes is not yet clear due to the inaccessibility of patient samples. Induced pluripotency and directed differentiation toward the pancreatic lineage are now viable and attractive methods to uncover the molecular mechanisms underlying MODY. Here we report, for the first time, the derivation of human induced pluripotent stem cells (hiPSCs) from patients with five types of MODY: MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY5 (HNF1B), and MODY8 (CEL) with a polycistronic lentiviral vector expressing a Cre-excisable human “stem cell cassette” containing the four reprogramming factors OCT4, KLF4, SOX2, and CMYC. These MODY-hiPSCs morphologically resemble human pluripotent stem cells (hPSCs), express pluripotency markers OCT4, SOX2, NANOG, SSEA-4, and TRA-1–60, give rise to derivatives of the three germ layers in a teratoma assay, and are karyotypically normal. Overall, our MODY-hiPSCs serve as invaluable tools to dissect the role of MODY genes in the development of pancreas and islet cells and to evaluate their significance in regulating beta cell function. This knowledge will aid future attempts aimed at deriving functional mature beta cells from hPSCs.

Stem cells represent a unique opportunity for regenerative medicine to cure a broad number of diseases for which current treatment only alleviates symptoms or retards further disease progression. However, the number of stem cells available has speedily increased these past 10 years and their diversity presents new challenges to clinicians and basic scientists who intend to use them in clinics or to study their unique properties. In addition, the recent possibility to derive pluripotent stem cells from somatic cells using epigenetic reprogramming has further increased the clinical interest of stem cells since induced pluripotent stem cells could render personalized cell-based therapy possible. The present review will attempt to summarize the advantages and challenges of each type of stem cell for current and future clinical applications using specific examples.

The pluripotent status of embryonic stem cells (ESCs) confers upon them the capacity to differentiate into the three primary germ layers, ectoderm,mesoderm and endoderm, from which all the cells of the adult body are derived. An understanding of the mechanisms controlling pluripotency is thus essential for driving the differentiation of human pluripotent cells into cell types useful for clinical applications. The Activin/Nodal signalling pathway is necessary to maintain pluripotency in human ESCs and in mouse epiblast stem cells (EpiSCs), but the molecular mechanisms by which it achieves this effect remain obscure. Here, we demonstrate that Activin/Nodal signalling controls expression of the key pluripotency factor Nanog in human ESCs and in mouse EpiSCs. Nanog in turn prevents neuroectoderm differentiation induced by FGF signalling and limits the transcriptional activity of the Smad2/3 cascade,blocking progression along the endoderm lineage. This negative-feedback loop imposes stasis in neuroectoderm and mesendoderm differentiation, thereby maintaining the pluripotent status of human ESCs and mouse EpiSCs.