In vivo characterization of transplanted human embryonic stem cell‐derived pancreatic endocrine islet cells

Abstract. Objectives: Islet‐like clusters (ILCs), differentiated from human embryonic stem cells (hESCs), were characterized both before and after transplantation under the kidney capsule of streptozotocin‐induced diabetic immuno‐incompetent mice. Materials and methods: Multiple independent ILC preparations (n = 8) were characterized by immunohistochemistry, flow cytometry and cell insulin content, with six preparations transplanted into diabetic mice (n = 42), compared to controls, which were transplanted with either a human fibroblast cell line or undifferentiated hESCs (n = 28). Results: Prior to transplantation, ILCs were immunoreactive for the islet hormones insulin, C‐peptide and glucagon, and for the ductal epithelial marker cytokeratin‐19. ILCs also had cellular insulin contents similar to or higher than human foetal islets. Expression of islet and pancreas‐specific cell markers was maintained for 70 days post‐transplantation. The mean survival of recipients was increased by transplanted ILCs as compared to transplanted human fibroblast cells (P < 0.0001), or undifferentiated hESCs (P < 0.042). Graft function was confirmed by secretion of human C‐peptide in response to an oral bolus of glucose. Conclusions: hESC‐derived ILC grafts continued to contain cells that were positive for islet endocrine hormones and were shown to be functional by their ability to secrete human C‐peptide. Further enrichment and maturation of ILCs could lead to generation of a sufficient source of insulin‐producing cells for transplantation into patients with type 1 diabetes.     

Controlled aggregation of primary human pancreatic islet cells leads to glucose‐responsive pseudoislets comparable to native islets

Clinical islet transplantation is a promising treatment for patients with type 1 diabetes. However, pancreatic islets vary in size and shape affecting their survival and function after transplantation because of mass transport limitations. To reduce diffusion restrictions and improve islet cell survival, the generation of islets with optimal dimensions by dispersion followed by reassembly of islet cells, can help limit the length of diffusion pathways. This study describes a microwell platform that supports the controlled and reproducible production of three‐dimensional pancreatic cell clusters of human donor islets. We observed that primary human islet cell aggregates with a diameter of 100–150 μm consisting of about 1000 cells best resembled intact pancreatic islets as they showed low apoptotic cell death (<2%), comparable glucose‐responsiveness and increasing PDX1, MAFA and INSULIN gene expression with increasing aggregate size. The re‐associated human islet cells showed an a‐typical core shell configuration with beta cells predominantly on the outside unlike human islets, which became more randomized after implantation similar to native human islets. After transplantation of these islet cell aggregates under the kidney capsule of immunodeficient mice, human C‐peptide was detected in the serum indicating that beta cells retained their endocrine function similar to human islets. The agarose microwell platform was shown to be an easy and very reproducible method to aggregate pancreatic islet cells with high accuracy providing a reliable tool to study cell–cell interactions between insuloma and/or primary islet cells.     

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.     

Islet-like cell aggregates generated from human adipose tissue derived stem cells ameliorate experimental diabetes in mice

Background

Type 1 Diabetes Mellitus is caused by auto immune destruction of insulin producing beta cells in the pancreas. Currently available treatments include transplantation of isolated islets from donor pancreas to the patient. However, this method is limited by inadequate means of immuno-suppression to prevent islet rejection and importantly, limited supply of islets for transplantation. Autologous adult stem cells are now considered for cell replacement therapy in diabetes as it has the potential to generate neo-islets which are genetically part of the treated individual. Adopting methods of islet encapsulation in immuno-isolatory devices would eliminate the need for immuno-suppressants.

Methodology/Principal Findings

In the present study we explore the potential of human adipose tissue derived adult stem cells (h-ASCs) to differentiate into functional islet like cell aggregates (ICAs). Our stage specific differentiation protocol permit the conversion of mesodermic h-ASCs to definitive endoderm (Hnf3β, TCF2 and Sox17) and to PDX1, Ngn3, NeuroD, Pax4 positive pancreatic endoderm which further matures in vitro to secrete insulin. These ICAs are shown to produce human C-peptide in a glucose dependent manner exhibiting in-vitro functionality. Transplantation of mature ICAs, packed in immuno-isolatory biocompatible capsules to STZ induced diabetic mice restored near normoglycemia within 3–4 weeks. The detection of human C-peptide, 1155±165 pM in blood serum of experimental mice demonstrate the efficacy of our differentiation approach.

Conclusions

h-ASC is an ideal population of personal stem cells for cell replacement therapy, given that they are abundant, easily available and autologous in origin. Our findings present evidence that h-ASCs could be induced to differentiate into physiologically competent functional islet like cell aggregates, which may provide as a source of alternative islets for cell replacement therapy in type 1 diabetes.     

Selective destruction of mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 diabetic model

Type 1 diabetes (T1D) is caused by a T cell-mediated autoimmune response that leads to the loss of insulin-producing β cells. The optimal preclinical testing of promising therapies would be aided by a humanized immune-mediated T1D model. We develop this model in NOD-scid IL2rγ^null mice. The selective destruction of pancreatic islet β cells was mediated by human T lymphocytes after an initial trigger was supplied by the injection of irradiated spleen mononuclear cells (SMC) from diabetic nonobese diabetic (NOD) mice. This resulted in severe insulitis, a marked loss of total β-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the β cell-produced highly conserved chemokine stromal cell-derived factor 1 (SDF-1) and its receptor C-X-C chemokine receptor (CXCR) 4, as demonstrated by in vivo blocking experiments using antibody to CXCR4. The specificity of humanized T cell-mediated immune responses against islet β cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4⁺ T cell infiltration and clonal expansion, and the mouse islet β-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet β cells by a human T cell-mediated immune response in this humanized T1D model can mimic those observed in T1D patients. This model can provide a valuable tool for translational research into T1D.     

Gimmicks of gamma‐aminobutyric acid (GABA) in pancreatic β‐cell regeneration through transdifferentiation of pancreatic α‐ to β‐cells

In vivo regeneration of lost or dysfunctional islet β cells can fulfill the promise of improved therapy for diabetic patients. To achieve this, many mitogenic factors have been attempted, including gamma‐aminobutyric acid (GABA). GABA remarkably affects pancreatic islet cells’ (α cells and β cells) function through paracrine and/or autocrine binding to its membrane receptors on these cells. GABA has also been studied for promoting the transformation of α cells to β cells. Nonetheless, the gimmickry of GABA‐induced α‐cell transformation to β cells has two different perspectives. On the one hand, GABA was found to induce α‐cell transformation to β cells in vivo and insulin‐secreting β‐like cells in vitro. On the other hand, GABA treatment showed that it has no α‐ to β‐cell transformation response. Here, we will summarize the physiological effects of GABA on pancreatic islet β cells with an emphasis on its regenerative effects for transdifferentiation of islet α cells to β cells. We will also critically discuss the controversial results about GABA‐mediated transdifferentiation of α cells to β cells.     

Enhanced islet expansion by β‐cell proliferation in young diabetes‐prone rats fed a protective diet

Type 1 diabetes is inhibited in diabetes‐prone BioBreeding (BBdp) rats fed a low‐antigen hydrolyzed casein (HC) diet. In cereal‐fed BBdp rats, islet expansion is defective accompanied by a futile upregulation of islet neogenesis without increased islet mass, due to a subtle blockage in islet cell cycle. We hypothesized that islet growth is enhanced before insulitis in HC‐fed young BBdp rats and that islet neogenesis could be stimulated by a trophic factor, islet neogenesis‐associated protein (INGAP). β‐Cell homeostasis was analyzed using immunohistochemistry, morphometry, laser capture microdissection and RT‐PCR in BBdp rats fed HC or cereal diets. β‐cell proliferation in small and medium islets, and the number and area fraction of medium and large islets were increased in HC‐fed animals. In situ islet cell cycle analysis revealed an increased proportion of proliferating S + G2 cells in medium and large islets of 25–45 day HC‐fed rats. Expression of the cell cycle inhibitor, p16^INK4a correlated with islet size and the percentage of p16^INK4a+ β‐cells increased in HC‐fed BBdp rats, likely reflecting an increase in large islet area fraction. In HC‐fed rats, extra‐islet insulin⁺ clusters (EIC), insulin⁺ duct cells, large islet area fraction, and β‐cell mass were increased. Neurogenin‐3 and Pdx‐1, markers of β‐cell progenitors, were increased in EIC of weanling HC‐fed rats. Daily injection of INGAP (30–45 days) increased the number of small islets, total islets, and insulin⁺ cells in small ducts. Thus, in BBdp rats fed a protective HC diet, β‐cell expansion is enhanced through increased β‐cell proliferation and stimulation of islet neogenesis. J. Cell. Physiol. 224: 501–508, 2010. © 2010 Wiley‐Liss, Inc.     

Variation in plasma glucose and pancreatic β cells in the turtle, Lissemys punctata (order: Chelonia; family: Trionychidae)

The present study relates to the determination of the plasma glucose level and volumetric analysis of β cells in pancreatic islets of the soft‐shelled turtle Lissemys punctata during different phases of its reproductive cycle. Reproductive events play a vital role in influencing the plasma glucose level and β‐cell behaviour in the pancreatic islets. The colour of the pancreas is either yellowish or pinkish, depending on endocrine activity. Islets are present throughout the gland and range from individual cells to small or large clumps, depending on the seasonal cycle. Splenic islets are dense with more blood capillaries and nerve innervations irrespective of sex and season. The endocrine cell mass forms irregular patches without connective tissue capsule. β cells occupy the inner region of the islets, being surrounded by other cell types. Lissemys punctata exhibits higher β‐cell activity during hibernation. Most insulin‐secreting cells acquire a larger size during the regressive period. An analysis indicates that β cells outnumber the non‐β endocrine cell mass in both number and per cent volume. There is negative correlation between islet mass and animal weight. Between the periods of reproductive cycles, a difference exists with respect to fasting plasma glucose and β‐cell volume.     

Human pancreatic islets develop through fusion of distinct β and α/δ islets

Human pancreatic islets show unique architecture in which α and δ cells are mostly at the peripheral and perivascular areas. It has remained unknown how such prototype is realized in every islet. Here, I report that fetal islets develop first in two distinct types consisting of β or α/δ cells, respectively. The α/δ islets are variable in shape, composed of α and δ cells evenly intermixed. They are vascularized better but encapsulated poorer than β islets in general. During the development, the β and α/δ islets adjoin and fuse with each other in such a way that α and δ cells form a crescent on β cells and, then, progress to encompass and encroach into β cells. Most mature‐form islets appear to develop through the fusion. Islets at various stages of fusion are present concurrently until late gestation, suggesting that the islet fusion is an ongoing developmental process. The α/δ islets appear to play a primary role for the process, approaching toward the fusion partner actively. Direct connection is present between the α/δ islets and neural ganglia undergoing active neurogenesis, suggesting an organ‐wide neuroendocrine network development. The fusion of precursor islets appears to be a principle of human pancreatic development providing the prototype of mature islets. The complex development might be a reference for in vitro reproduction of biologically competent islets.     

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 low¹⁰; 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 tissue⁶. Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.     

Analysis of gene expressions of embryonic stem‐derived Pdx1‐expressing cells: Implications of genes involved in pancreas differentiation

We have recently reported the method by which embryonic stem (ES) cells were induced into Pdx1‐expressing cells. To gain insights into the ES cell‐derived Pdx1‐expressing cells, we examined gene expression profiles of the cells by microarray experiments. Microarray analyses followed by a comparison with the data of the cells in developing pancreatic and adult islet suggested that the ES cell‐derived Pdx1‐positive cells were immature pancreatic progenitor cells with endodermal characteristics. The analyses of the genes upregulated in the ES cell‐derived Pdx1‐positive cells would give us knowledge on early pancreatic development. Here, we first listed the genes and found that these contained not only those known to be expressed in the endoderm or pancreatic progenitor cells, but also those known to be involved in left–right axis formation. Second, we examined the gene expression patterns and found that several genes were expressed in the ventral foregut lip at the anterior intestinal portal in E8.5 embryo. Given that the Pdx1/GFP‐expressing cells are first observed in the same region at the anterior intestinal portal, these results suggest that the pancreatic progenitor cells first give rise at the ventral endoderm prior to the formation of dorsal and ventral pancreatic buds.     

The nucleosome binding protein HMGN3 is expressed in pancreatic α‐cells and affects plasma glucagon levels in mice

Glucose homeostasis requires the coordinated actions of various organs and is critically dependent on the proper functioning of the various cell types present in the pancreatic Langerhans islets. Here we report that chromatin architectural protein HMGN3 is highly expressed in all pancreatic endocrine islet cells, and that Hmgn3?/? mice which have a mild diabetic phenotype, have reduced glucagon levels in their blood. To elucidate the mechanism leading to altered glucagon secretion of Hmgn3?/? mice, we tested whether HMGN3 affect glucagon synthesis and secretion in αTC1‐9 cells, a glucagon secreting cell line that is used to study pancreatic α‐cell function. We find that in these cells deletion of either HMGN3 or other HMGN variants, does not significantly affect glucagon gene expression or glucagon secretion. Our studies demonstrate a link between HMGN3 and glucagon blood levels that is not directly dependent of the function of pancreatic α‐cells. J. Cell. Biochem. 109: 49–57, 2010. Published 2009 Wiley‐Liss, Inc.     

Development of endocrine pancreatic islets in embryos of the grass snake Natrix natrix (Lepidosauria,Serpentes)

Differentiation of the pancreatic islets in grass snake Natrix natrix embryos, was analyzed using light, transmission electron microscopy, and immuno-gold labeling. The study focuses on the origin of islets, mode of islet formation, and cell arrangement within islets. Two waves of pancreatic islet formation in grass snake embryos were described. The first wave begins just after egg laying when precursors of endocrine cells located within large cell agglomerates in the dorsal pancreatic bud differentiate. The large cell agglomerates were divided by mesenchymal cells thus forming the first islets. This mode of islet formation is described as fission. During the second wave of pancreatic islet formation which is related to the formation of the duct mantle, we observed four phases of islet formation: (a) differentiation of individual endocrine cells from the progenitor layer of duct walls (budding) and their incomplete delamination; (b) formation of two types of small groups of endocrine cells (A/D and B) in the wall of pancreatic ducts; (c) joining groups of cells emerging from neighboring ducts (fusion) and rearrangement of cells within islets; (d) differentiated pancreatic islets with characteristic arrangement of endocrine cells. Mature pancreatic islets of the grass snake contained mainly A endocrine cells. Single B and D or PP–cells were present at the periphery of the islets. This arrangement of endocrine cells within pancreatic islets of the grass snake differs from that reported from most others vertebrate species. Endocrine cells in the pancreas of grass snake embryos were also present in the walls of intralobular and intercalated ducts. At hatching, some endocrine cells were in contact with the lumen of the pancreatic ducts.     

Identification of microRNAs expressed highly in pancreatic islet‐like cell clusters differentiated from human embryonic stem cells

Type 1 diabetes is an autoimmune destruction of pancreatic islet beta cell disease, making it important to find a new alternative source of the islet beta cells to replace the damaged cells. hES (human embryonic stem) cells possess unlimited self‐renewal and pluripotency and thus have the potential to provide an unlimited supply of different cell types for tissue replacement. The hES‐T3 cells with normal female karyotype were first differentiated into EBs (embryoid bodies) and then induced to generate the T3pi (pancreatic islet‐like cell clusters derived from T3 cells), which expressed pancreatic islet cell‐specific markers of insulin, glucagon and somatostatin. The expression profiles of microRNAs and mRNAs from the T3pi were analysed and compared with those of undifferentiated hES‐T3 cells and differentiated EBs. MicroRNAs negatively regulate the expression of protein‐coding mRNAs. The T3pi showed very high expression of microRNAs, miR‐186, miR‐199a and miR‐339, which down‐regulated the expression of LIN28, PRDM1, CALB1, GCNT2, RBM47, PLEKHH1, RBPMS2 and PAK6. Therefore, these microRNAs and their target genes are very likely to play important regulatory roles in the development of pancreas and/or differentiation of islet cells, and they may be manipulated to increase the proportion of beta cells and insulin synthesis in the differentiated T3pi for cell therapy of type I diabetics.