sRNA-seq analysis of human embryonic stem cells and definitive endoderm reveals differentially expressed microRNAs and novel IsomiRs with distinct targets.

MicroRNAs (miRNAs) are noncoding, regulatory RNAs expressed dynamically during differentiation of human embryonic stem cells (hESCs) into defined lineages. Mapping developmental expression of miRNAs during transition from pluripotency to definitive endoderm (DE) should help to elucidate the mechanisms underlying lineage specification and ultimately enhance differentiation protocols. In this report, next generation sequencing was used to build upon our previous analysis of miRNA expression in human hESCs and DE. From millions of sequencing reads, 747 and 734 annotated miRNAs were identified in pluripotent and DE cells, respectively, including 77 differentially expressed miRNAs. Among these, four of the top five upregulated miRNAs were previously undetected in DE. Furthermore, the stem-loop for miR-302a, an important miRNA for both hESCs self-renewal and endoderm specification, produced several highly expressed miRNA species (isomiRs). Overall, isomiRs represented >10% of sequencing reads in >40% of all detected stem-loop arms, suggesting that the impact of these abundant miRNA species may have been overlooked in previous studies. Because of their relative abundance, the role of differential isomiR targeting was studied using the miR-302 cluster as a model system. A miRNA mimetic for miR-302a-5p, but not miR-302a-5p(+3), decreased expression of orthodenticle homeobox 2 (OTX2). Conversely, isomiR 302a-5p(+3) selectively decreased expression of tuberous sclerosis protein 1, but not OTX2, indicating nonoverlapping specificity of miRNA processing variants. Taken together, our characterization of miRNA expression, which includes novel miRNAs and isomiRs, helps establish a foundation for understanding the role of miRNAs in DE formation and selective targeting by isomiRs.

Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules.

Using a high-throughput chemical screen, we identified two small molecules that enhance the survival of human embryonic stem cells (hESCs). By characterizing their mechanisms of action, we discovered an essential role of E-cadherin signaling for ESC survival. Specifically, we showed that the primary cause of hESC death following enzymatic dissociation comes from an irreparable disruption of E-cadherin signaling, which then leads to a fatal perturbation of integrin signaling. Furthermore, we found that stability of E-cadherin and the resulting survival of ESCs were controlled by specific growth factor signaling. Finally, we generated mESC-like hESCs by culturing them in mESC conditions. And these converted hESCs rely more on E-cadherin signaling and significantly less on integrin signaling. Our data suggest that differential usage of cell adhesion systems by ESCs to maintain self-renewal may explain their profound differences in terms of morphology, growth factor requirement, and sensitivity to enzymatic cell dissociation.

Inhibitors of Src and focal adhesion kinase promote endocrine specification: impact on the derivation of ß-cells from human pluripotent stem cells.

Stepwise approaches for the derivation of ß-cells from human embryonic stem cells have been described. However, low levels of endocrine specification limit the final yield of insulin-producing ß-cells. In this study, we show that the pyrrolo-pyrimidine Src family kinase (SFK) inhibitor PP2 effectively promotes the endocrine specification of human embryonic stem cell derivatives based on its capacity to induce the expression of proendocrine transcription factors (NGN3, NEUROD1, NKX2.2, and PAX4) and to significantly increase the final yield of insulin-positive cells. We further demonstrate that PP2 inhibits the activation of focal adhesion kinase (FAK), and selective inhibition of this kinase is also sufficient to induce early endocrine commitment based on increased expression of NGN3, NEUROD1, and NKX2.2. Additional studies using dominant negative constructs and isolated human fetal pancreata suggest that c-Src is at least partially responsible for inhibiting early endocrine specification. Mechanistically, we propose that inhibition of SFK/FAK signaling can promote endocrine specification by limiting activation of the TGFßR/Smad2/3 pathway. Moreover, we show that inhibition of SFK/FAK signaling suppresses cell growth, increases the expression of the ß-cell-associated cyclin-dependent kinase inhibitor p57kip2, and simultaneously suppresses the expression of Id1 and Id2. This study has important implications for the derivation of ß-cells for the cell-based therapy of diabetes and sheds new light on the signaling events that regulate early endocrine specification.

A chemical platform for improved induction of human iPSCs.

The slow kinetics and low efficiency of reprogramming methods to generate human induced pluripotent stem cells (iPSCs) impose major limitations on their utility in biomedical applications. Here we describe a chemical approach that dramatically improves (200-fold) the efficiency of iPSC generation from human fibroblasts, within seven days of treatment. This will provide a basis for developing safer, more efficient, nonviral methods for reprogramming human somatic cells.

Chemical combinations potentiate human pluripotent stem cell-derived 3D pancreatic progenitor clusters toward functional ß cells.

Human pluripotent stem cell (hPSC)-derived pancreatic ß cells are an attractive cell source for treating diabetes. However, current derivation methods remain inefficient, heterogeneous, and cell line dependent. To address these issues, we first devised a strategy to efficiently cluster hPSC-derived pancreatic progenitors into 3D structures. Through a systematic study, we discovered 10 chemicals that not only retain the pancreatic progenitors in 3D clusters but also enhance their potentiality towards NKX6.1+/INS+ ß cells. We further systematically screened signaling pathway modulators in the three steps from pancreatic progenitors toward ß cells. The implementation of all these strategies and chemical combinations resulted in generating ß cells from different sources of hPSCs with high efficiency. The derived ß cells are functional and can reverse hyperglycemia in mice within two weeks. Our protocol provides a robust platform for studying human ß cells and developing hPSC-derived ß cells for cell replacement therapy.

Impact of integrin-matrix interaction and signaling on insulin gene expression and the mesenchymal transition of human beta-cells.

A critical shortage of donor pancreata currently prevents the development of a universal cell-based therapy for type I diabetes. The ex vivo expansion of insulin-producing beta-cells offers a potential solution but is problematic due to the inherent tendency of these cells to transition into mesenchymal-like cells that are devoid of function. Here, we demonstrate for the first time that exposure to elements of the extracellular matrix (ECM) directly potentiates the mesenchymal transition of cultured fetal beta-cells and causes associated declines in insulin gene expression. Individual ECM constituents varied in their ability to induce such responses, with collagen-IV (C-IV) and fibronectin inducing strong responses, whereas laminin-1 had no significant effect. Mesenchymal transition and concomitant losses in insulin gene expression observed on C-IV were found to be dependent on beta(1)-integrin ligation and were augmented in the presence of hepatocyte growth factor. Importantly, selective inhibition of c-Src, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) prior to exposure to C-IV prevented mesenchymal transition and effectively preserved insulin expression. Fetal beta-cells undergoing mesenchymal transition were found to acquire alpha(1)beta(1) expression, and ligation of this integrin then promotes declines in insulin gene expression and a marked increase in beta-cell motility. Inhibition of Src-, ERK-, or JNK-dependent signaling combined with the selective regulation of matrix exposure may ultimately facilitate the development of more effective beta-cell expansion protocols.

A novel approach for the derivation of putative primordial germ cells and sertoli cells from human embryonic stem cells.

Using human embryonic stem cells (hESCs), we describe a novel method for the rapid derivation and enrichment of cells that are comparable to primordial germ cells (PGCs) and Sertoli cells. The methodology described is based on modest changes to the growth conditions commonly used to expand hESCs and does not require genetic manipulation or complex three-dimensional culture. Remarkably, we have determined that simply reducing the size of cultured ESC colonies and manipulating the number of feeding cycles, results in the rapid emergence of cells that are comparable to migratory PGCs. Importantly, these cells can be monitored and purified on the basis of the expression of the chemokine receptor CXCR4. Under more stringent differentiating conditions these cells mature and upregulate the expression of specific germ cell markers. Importantly, this process is accompanied by the development of Sertoli-like support cells. Such cells normally provide trophic support and immunoprotection to developing germ cells and may have significant clinical utility in the prevention of graft rejection. The putative Sertoli-germ cell cocultures generated in this study may ultimately be developed to study and manipulate interactions and processes involved in human gametogenesis.

Generation of human-induced pluripotent stem cells in the absence of exogenous Sox2.

Induced pluripotent stem cell technology has attracted enormous interest for potential application in regenerative medicine. Here, we report that a specific glycogen synthase kinase 3 (GSK-3) inhibitor, CHIR99021, can induce the reprogramming of mouse embryonic fibroblasts transduced by only two factors, Oct4 and Klf4. When combined with Parnate (also named tranylcypromine), an inhibitor of lysine-specific demethylase 1, CHIR99021 can cause the reprogramming of human primary keratinocyte transduced with the two factors, Oct4 and Klf4. To our knowledge, this is the first time that human iPS cells have been generated from somatic cells without exogenous Sox2 expression. Our studies suggest that the GSK-3 inhibitor might have a general application to replace transcription factors in both mouse and human reprogramming.

Limited capacity of human adult islets expanded in vitro to redifferentiate into insulin-producing beta-cells.

Limited organ availability is an obstacle to the widespread use of islet transplantation in type 1 diabetic patients. To address this problem, many studies have explored methods for expanding functional human islets in vitro for diabetes cell therapy. We previously showed that islet cells replicate after monolayer formation under the influence of hepatocyte growth factor and selected extracellular matrices. However, under these conditions, senescence and loss of insulin expression occur after >15 doublings. In contrast, other groups have reported that islet cells expanded in monolayers for months progressed through a reversible epithelial-to-mesenchymal transition, and that on removal of serum from the cultures, islet-like structures producing insulin were formed (1). The aim of the current study was to compare the two methods for islet expansion using immunostaining, real-time quantitative PCR, and microarrays at the following time points: on arrival, after monolayer expansion, and after 1 week in serum-free media. At this time, cell aliquots were grafted into nude mice to study in vivo function. The two methods showed similar results in islet cell expansion. Attempts at cell differentiation after expansion by both methods failed to consistently recover a beta-cell phenotype. Redifferentiation of beta-cells after expansion is still a challenge in need of a solution.

Brief review: cell replacement therapies to treat type 1 diabetes mellitus.

Human embryonic stem cells (hESCs) and induced pluripotent cells (iPSCs) have the potential to differentiate into any somatic cell, making them ideal candidates for cell replacement therapies to treat a number of human diseases and regenerate damaged or non-functional tissues and organs. Key to the promise of regenerative medicine is developing standardized protocols that can safely be applied in patients. Progress towards this goal has occurred in a number of fields, including type 1 diabetes mellitus (T1D). During the past 10 years, significant technological advances in hESC/iPSC biochemistry have provided a roadmap to generate sufficient quantities of glucose-responsive, insulin-producing cells capable of eliminating diabetes in rodents. Although many of the molecular mechanisms underlying the genesis of these cells remain to be elucidated, the field of cell-based therapeutics to treat T1D has advanced to the point where the first Phase I/II trials in humans have begun. Here, we provide a concise review of the history of cell replacement therapies to treat T1D from islet transplantations and xenotranplantation, to current work in hESC/iPSC. We also highlight the latest advances in efforts to employ insulin-producing, glucose-responsive ß-like cells derived from hESC as therapeutics.

Characterization and isolation of promoter-defined nestin-positive cells from the human fetal pancreas.

Studies using adult human islets and mouse embryonic stem cells have suggested that the neurepithelial precursor cell marker nestin also identifies and can be used to purify beta-cell precursors. To determine whether nestin can be used to identify beta-cell progenitors in the developing human pancreas, we characterized nestin expression from 12 to 24 gestational weeks, purified nestin+ cells using an enhancer/promoter-driven selection plasmid, and determined whether nestin+ cells can differentiate into beta-cells. Nestin was visualized in the platelet endothelial cell adhesion molecule and alpha smooth muscle actin-positive blood vessels and colocalized with vimentin in the interstitium. Nestin was not observed in pan cytokeratin (pCK)-positive ductal epithelium or insulin cells. Purified nestin+ cells also coexpressed vimentin and lacked pCK immunoreactivity. Purified adult and fetal pancreatic fibroblasts also expressed nestin. The nestin enhancer/promoter used in the selection plasmid was sufficient to drive reporter gene expression, green fluorescent protein, in human fetal pancreatic tissue. Exposure of selected nestin+ cells to nicotinamide, hepatocyte growth factor/scatter factor, betacellulin, activin A, or exendin-4 failed to induce pancreatic and duodenal homeobox gene-1 or insulin message as determined by RT-PCR. Transplantation of nestin+ cells and fetal pancreatic fibroblasts into athymic mice also failed to result in the development of beta-cells, whereas nestin- fetal pancreatic epithelial cells gave rise to functional insulin-secreting beta-cells. We conclude that nestin is not a specific marker of beta-cell precursors in the developing human pancreas.

A novel approach to increase human islet cell mass while preserving beta-cell function.

Human islet expansion in monolayer culture leads to loss of function and senescence. By maintaining the 3-D configuration of islets in fibrin gels, it is feasible to expand beta-cells in response to hepatocyte growth factor (HGF) while preserving physiologic glucose responsiveness both in vitro and in vivo after transplantation into nude mice. Islets were cultured free floating with or without growth factors and nicotinamide and in fibrin gels with the same conditions. Proliferation was observed only in islets cultured in fibrin gels and the cocktail; total insulin increased by threefold, with a concomitant increase in beta-cell mass by morphometry. Insulin release after glucose challenge was also preserved. Islets in fibrin gels gave rise in vivo to large grafts rich in insulin and glucagon, and grafts from free-floating islets were smaller with fewer endocrine cells. Circulating human C-peptide levels were higher than in the mice receiving free-floating islets. In summary, fibrin allows for HGF-mediated cell proliferation while preserving glucose responsiveness in an environment that preserves cell-cell contacts. Limited islet ex vivo expansion under these conditions may improve recipient-donor tissue ratios to equal the functional results of whole-organ transplants.

Exendin 4 up-regulates expression of PDX 1 and hastens differentiation and maturation of human fetal pancreatic cells.

In addition to stimulating insulin secretion, glucagon-like peptide and its long-acting analog exendin 4 have been reported to increase beta-cell mass by both differentiation/neogenesis of precursor cells and enhanced replication of existing beta-cells. Here, we investigated the effect of exendin 4 in the growth and differentiation of beta-cells from undifferentiated precursors in islet-like cell clusters (ICCs) derived from human fetal pancreases. Our results show that the addition of exendin 4 to the culture media stimulates PDX 1 expression in ICCs as shown by immunofluorescence staining. The up-regulation of PDX 1 was not accompanied by changes in insulin expression because we did not find a significant difference in the number of insulin-positive cells in the exendin 4-treated ICCs, compared with controls. We also tested the effects of exendin 4 in the glucose-induced insulin secretion of human ICCs transplanted under the kidney capsule of athymic rats. In the exendin 4-treated rats (given ip during 10 d) 8 wk after the beginning of the treatment, insulin was released in response to glucose as detected by the measurement of circulating human C-peptide. In control (saline-treated) rats, the basal levels of human C-peptide did not change significantly after glucose stimulation. Thus, exendin 4 induces functional maturation of fetal beta-cells in response to glucose. In these rats, serial sections of the kidney-bearing grafts were examined histologically for insulin containing cells. We found a significant increase in beta-cell number, compared with the control rats. Overall, these results show that in vivo exendin 4 causes growth and differentiation of human fetal beta-cells from undifferentiated precursor cells. It also accelerates the functional maturation of fetal beta-cells as evidenced by their glucose-stimulated insulin secretion.

Isolation, culture, and imaging of human fetal pancreatic cell clusters.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation(1-9). However in vitro, genesis of insulin producing cells from human fetal ICCs is low(10); results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust(11-17). A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro(11-22), far fewer exist for ICCs(10,23,24). Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue(6). Although the examples presented here focus upon the pancreatic endoderm proliferation and ß cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

Is stage-specific embryonic antigen 4 a marker for human ductal stem/progenitor cells?

The presence of pancreatic stem cells (PnSCs) has not been firmly demonstrated in the human or animal pancreas. Previous reports have suggested that ductal and acinar structures in the exocrine pancreas can be a potential source of progenitor cells. More recently, immature insulin precursors in the periphery of human islets have been found to self-replicate and differentiate to endocrine cells in vitro. Transplantation of these cells under the kidney capsule improves the diabetic state in mice. The controversy surrounding where PnSCs reside could be resolved if a specific marker were to be found that allowed their identification, purification, and directed differentiation to endocrine cells. We have identified in human pancreas cells positive for the stage-specific embryonic antigen 4 (SSEA4), a stem cell marker. These cells also express ductal, pancreatic progenitor, and stem cell protein markers. Interestingly, some of the SSEA4(+) cells scattered in the ducts do not show a ductal cell phenotype. SSEA4(+)-sorted cells formed aggregate-like spheres in culture and robustly differentiated to pancreatic hormone-expressing cells in conditions of high glucose concentration and B27 supplementation. We hypothesize that SSEA4(+) cells or a subpopulation of those cells residing in the pancreatic ducts may be the elusive PnSCs, and in this case, SSEA4 may represent a potential surface antigen marker for human PnSCs. The discovery of specific markers for the identification and purification of human PnSCs would greatly facilitate studies aimed at the expansion of these cells and the development of targeting tools for their potential induction to insulin-producing cells.

The SDF-1α/CXCR4 axis is required for proliferation and maturation of human fetal pancreatic endocrine progenitor cells.

The chemokine receptor CXCR4 and ligand SDF-1α are expressed in fetal and adult mouse islets. Neutralization of CXCR4 has previously been shown to diminish ductal cell proliferation and increase apoptosis in the IFNγ transgenic mouse model in which the adult mouse pancreas displays islet regeneration. Here, we demonstrate that CXCR4 and SDF-1α are expressed in the human fetal pancreas and that during early gestation, CXCR4 colocalizes with neurogenin 3 (ngn3), a key transcription factor for endocrine specification in the pancreas. Treatment of islet like clusters (ICCs) derived from human fetal pancreas with SDF-1α resulted in increased proliferation of epithelial cells in ICCs without a concomitant increase in total insulin expression. Exposure of ICCs in vitro to AMD3100, a pharmacological inhibitor of CXCR4, did not alter expression of endocrine hormones insulin and glucagon, or the pancreatic endocrine transcription factors PDX1, Nkx6.1, Ngn3 and PAX4. However, a strong inhibition of ß cell genesis was observed when in vitro AMD3100 treatment of ICCs was followed by two weeks of in vivo treatment with AMD3100 after ICC transplantation into mice. Analysis of the grafts for human C-peptide found that inhibition of CXCR4 activity profoundly inhibits islet development. Subsequently, a model pancreatic epithelial cell system (CFPAC-1) was employed to study the signals that regulate proliferation and apoptosis by the SDF-1α/CXCR4 axis. From a selected panel of inhibitors tested, both the PI 3-kinase and MAPK pathways were identified as critical regulators of CFPAC-1 proliferation. SDF-1α stimulated Akt phosphorylation, but failed to increase phosphorylation of Erk above the high basal levels observed. Taken together, these results indicate that SDF-1α/CXCR4 axis plays a critical regulatory role in the genesis of human islets.

A distinct microRNA signature for definitive endoderm derived from human embryonic stem cells.

Human embryonic stem cells (hESCs) have the potential to differentiate into many adult cell types, and they are being explored as a resource for cell replacement therapies for multiple diseases. In order to optimize in vitro differentiation protocols, it will be necessary to elucidate regulatory mechanisms that contribute to lineage specification. MicroRNAs (miRNAs) are emerging as key regulators of hESC differentiation and embryonic development. In this study, we compare miRNA expression profiles between pluripotent hESCs and definitive endoderm (DE), an early step in the pathway toward the pancreatic lineage. Results from microarray analysis showed that DE can be distinguished by its unique miRNA profile, which consists of 37 significantly down-regulated and 17 up-regulated miRNAs in 2 different cell lines and in the presence/absence of feeder layers. Comparison to other hESC-derived lineages showed that most of the highly up-regulated miRNAs are specific to endoderm in early development. Notably, miR-375, which was previously implicated in regulating development and function of later stages of pancreatic development, is highly and specifically up-regulated during DE formation, suggesting that it may have a distinct role very early in development. Examination of potential mRNA targets showed that TIMM8A is repressed by ectopic miR-375 expression in pluripotent hESCs.