Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro

Directed differentiation of human pluripotent stem cells into functional insulin-producing beta-like cells holds great promise for cell replacement therapy for patients suffering from diabetes. This approach also offers the unique opportunity to study otherwise inaccessible aspects of human beta cell development and function in vitro. Here, we show that current pancreatic progenitor differentiation protocols promote precocious endocrine commitment, ultimately resulting in the generation of non-functional polyhormonal cells. Omission of commonly used BMP inhibitors during pancreatic specification prevents precocious endocrine formation while treatment with retinoic acid followed by combined EGF/KGF efficiently generates both PDX1⁺ and subsequent PDX1⁺/NKX6.1⁺ pancreatic progenitor populations, respectively. Precise temporal activation of endocrine differentiation in PDX1⁺/NKX6.1⁺ progenitors produces glucose-responsive beta-like cells in vitro that exhibit key features of bona fide human beta cells, remain functional after short-term transplantation, and reduce blood glucose levels in diabetic mice. Thus, our simplified and scalable system accurately recapitulates key steps of human pancreas development and provides a fast and reproducible supply of functional human beta-like cells.     

Resveratrol Ameliorates the Maturation Process of β-Cell-Like Cells Obtained from an Optimized Differentiation Protocol of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic β-cells. For this particular lineage, although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucose-stimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulin-secreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1, SST, GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating β-cell-like cells and demonstrates that RSV improves the maturation process.     

Novel Pancreatic Endocrine Maturation Pathways Identified by Genomic Profiling and Causal Reasoning

We have used a previously unavailable model of pancreatic development, derived in vitro from human embryonic stem cells, to capture a time-course of gene, miRNA and histone modification levels in pancreatic endocrine cells. We investigated whether it is possible to better understand, and hence control, the biological pathways leading to pancreatic endocrine formation by analysing this information and combining it with the available scientific literature to generate models using a casual reasoning approach. We show that the embryonic stem cell differentiation protocol is highly reproducible in producing endocrine precursor cells and generates cells that recapitulate many aspects of human embryonic pancreas development, including maturation into functional endocrine cells when transplanted into recipient animals. The availability of whole genome gene and miRNA expression data from the early stages of human pancreatic development will be of great benefit to those in the fields of developmental biology and diabetes research. Our causal reasoning algorithm suggested the involvement of novel gene networks, such as NEUROG3/E2F1/KDM5B and SOCS3/STAT3/IL-6, in endocrine cell development We experimentally investigated the role of the top-ranked prediction by showing that addition of exogenous IL-6 could affect the expression of the endocrine progenitor genes NEUROG3 and NKX2.2.     

Adenoviral Vectors Stimulate Glucagon Transcription in Human Mesenchymal Stem Cells Expressing Pancreatic Transcription Factors

Viral gene carriers are being widely used as gene transfer systems in (trans)differentiation and reprogramming strategies. Forced expression of key regulators of pancreatic differentiation in stem cells, liver cells, pancreatic duct cells, or cells from the exocrine pancreas, can lead to the initiation of endocrine pancreatic differentiation. While several viral vector systems have been employed in such studies, the results reported with adenovirus vectors have been the most promising in vitro and in vivo. In this study, we examined whether the viral vector system itself could impact the differentiation capacity of human bone-marrow derived mesenchymal stem cells (hMSCs) toward the endocrine lineage. Lentivirus-mediated expression of Pdx-1, Ngn-3, and Maf-A alone or in combination does not lead to robust expression of any of the endocrine hormones (i.e. insulin, glucagon and somatostatin) in hMSCs. Remarkably, subsequent transduction of these genetically modified cells with an irrelevant early region 1 (E1)-deleted adenoviral vector potentiates the differentiation stimulus and promotes glucagon gene expression in hMSCs by affecting the chromatin structure. This adenovirus stimulation was observed upon infection with an E1-deleted adenovirus vector, but not after exposure to helper-dependent adenovirus vectors, pointing at the involvement of genes retained in the E1-deleted adenovirus vector in this phenomenon. Lentivirus mediated expression of the adenovirus E4-ORF3 mimics the adenovirus effect. From these data we conclude that E1-deleted adenoviral vectors are not inert gene-transfer vectors and contribute to the modulation of the cellular differentiation pathways.     

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications ¹. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages ², starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A ³ in combination with several growth factors ^4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine ⁸. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation ⁹. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation ^10,11.The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.     

Co-transplantation of mesenchymal stromal cells and cord blood cells in treatment of diabetes

Background aimsStem cells provide a promising source for treatment of type 1 diabetes, but the treatment strategy and mechanism remain unclear. The aims of this study were to investigate whether co-transplantation of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) and cord blood mononuclear cells (CB-MNCs) could reverse hyperglycemia in type 1 diabetic mice and to determine the appropriate ratio for co-transplantation. The treatment mechanism was also studied.MethodsA simple and efficient isolation method was developed to generate qualified UC-MSCs. UC-MSCs and CB-MNCs were then transplanted into type 1 diabetic mice at different ratios (UC-MSCs to CB-MNCs = 1:1, 1:4, 1:10) to observe the change in blood glucose concentration. Histology, immunohistochemistry, and human Alu polymerase chain reaction assay were performed to evaluate for the presence of donor-derived cells and the repair of endogenous islets. We also induced UC-MSCs into islet-like cells under specific culture conditions to determine their differentiate potential in vitro.ResultsCo-transplantation of UC-MSCs and CB-MNCs at a ratio of 1:4 effectively reversed hyperglycemia in diabetic mice. The detection of human Alu sequence indicated that the engraftment of donor-derived cells had homed into the recipient's pancreas and kidney. Although neither human insulin nor human nuclei antigen was detected in the regenerated pancreas, UC-MSCs could differentiate into insulin-secreted cells in vitro.ConclusionsCo-transplantation of UC-MSCs and CB-MNCs at a ratio of 1:4 could efficiently reverse hyperglycemia and repair pancreatic tissue.     

Insulin-Producing Cells Differentiated from Human Bone Marrow Mesenchymal Stem Cells In Vitro Ameliorate Streptozotocin-Induced Diabetic Hyperglycemia

Background

The two major obstacles in the successful transplantation of islets for diabetes treatment are inadequate supply of insulin-producing tissue and immune rejection. Induction of the differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) into insulin-producing cells (IPCs) for autologous transplantation may alleviate those limitations.

Methods

hMSCs were isolated and induced to differentiate into IPCs through a three-stage differentiation protocol in a defined media with high glucose, nicotinamide, and exendin-4. The physiological characteristics and functions of IPCs were then evaluated. Next, about 3 × 10⁶ differentiated cells were transplanted into the renal sub-capsular space of streptozotocin (STZ)-induced diabetic nude mice. Graft survival and function were assessed by immunohistochemistry, TUNEL staining and measurements of blood glucose levels in the mice.

Results

The differentiated IPCs were characterized by Dithizone (DTZ) positive staining, expression of pancreatic β-cell markers, and human insulin secretion in response to glucose stimulation. Moreover, 43% of the IPCs showed L-type Ca²⁺ channel activity and similar changes in intracellular Ca²⁺ in response to glucose stimulation as that seen in pancreatic β-cells in the process of glucose-stimulated insulin secretion. Transplantation of functional IPCs into the renal subcapsular space of STZ-induced diabetic nude mice ameliorated the hyperglycemia. Immunofluorescence staining revealed that transplanted IPCs sustainably expressed insulin, c-peptide, and PDX-1 without apparent apoptosis in vivo.

Conclusions

IPCs derived from hMSCs in vitro can ameliorate STZ-induced diabetic hyperglycemia, which indicates that these hMSCs may be a promising approach to overcome the limitations of islet transplantation.     

Activin, BMP and FGF pathways cooperate to promote endoderm and pancreatic lineage cell differentiation from human embryonic stem cells

The study of how human embryonic stem cells (hESCs) differentiate into insulin-producing beta cells has twofold significance: first, it provides an in vitro model system for the study of human pancreatic development, and second, it serves as a platform for the ultimate production of beta cells for transplantation into patients with diabetes. The delineation of growth factor interactions regulating pancreas specification from hESCs in vitro is critical to achieving these goals. In this study, we describe the roles of growth factors bFGF, BMP4 and Activin A in early hESC fate determination. The entire differentiation process is carried out in serum-free chemically-defined media (CDM) and results in reliable and robust induction of pancreatic endoderm cells, marked by PDX1, and cell clusters co-expressing markers characteristic of beta cells, including PDX1 and insulin/C-peptide. Varying the combinations of growth factors, we found that treatment of hESCs with bFGF, Activin A and BMP4 (FAB) together for 3–4 days resulted in strong induction of primitive-streak and definitive endoderm-associated genes, including MIXL1, GSC, SOX17 and FOXA2. Early proliferative foregut endoderm and pancreatic lineage cells marked by PDX1, FOXA2 and SOX9 expression are specified in EBs made from FAB-treated hESCs, but not from Activin A alone treated cells. Our results suggest that important tissue interactions occur in EB-based suspension culture that contribute to the complete induction of definitive endoderm and pancreas progenitors. Further differentiation occurs after EBs are embedded in Matrigel and cultured in serum-free media containing insulin, transferrin, selenium, FGF7, nicotinamide, islet neogenesis associated peptide (INGAP) and exendin-4, a long acting GLP-1 agonist. 21–28 days after embedding, PDX1 gene expression levels are comparable to those of human islets used for transplantation, and many PDX1⁺ clusters are formed. Almost all cells in PDX1⁺ clusters co-express FOXA2, HNF1ß, HNF6 and SOX9 proteins, and many cells also express CPA1, NKX6.1 and PTF1a. If cells are then switched to medium containing B27 and nicotinamide for 7–14 days, then the number of insulin⁺ cells increases markedly. Our study identifies a new chemically defined culture protocol for inducing endoderm- and pancreas-committed cells from hESCs and reveals an interplay between FGF, Activin A and BMP signaling in early hESC fate determination.     

Deconstructing Pancreas Developmental Biology

The relentless nature and increasing prevalence of human pancreatic diseases, in particular, diabetes mellitus and adenocarcinoma, has motivated further understanding of pancreas organogenesis. The pancreas is a multifunctional organ whose epithelial cells govern a diversity of physiologically vital endocrine and exocrine functions. The mechanisms governing the birth, differentiation, morphogenesis, growth, maturation, and maintenance of the endocrine and exocrine components in the pancreas have been discovered recently with increasing tempo. This includes recent studies unveiling mechanisms permitting unexpected flexibility in the developmental potential of immature and mature pancreatic cell subsets, including the ability to interconvert fates. In this article, we describe how classical cell biology, genetic analysis, lineage tracing, and embryological investigations are being complemented by powerful modern methods including epigenetic analysis, time-lapse imaging, and flow cytometry-based cell purification to dissect fundamental processes of pancreas development.