Beta-Cell Dedifferentiation in Type 2 Diabetes: Concise Review.

Type 2 diabetes (T2D) is caused by an inherited predisposition to pancreatic islet ß-cell failure, which is manifested under cellular stress induced by metabolic overload. The decrease in the functional ß-cell mass associated with T2D has been attributed primarily to ß-cell death; however, studies in recent years suggested that ß-cell dedifferentiation may contribute to this decline. The mechanisms linking genetic factors and cellular stress to ß-cell dedifferentiation remain largely unknown. This study evaluated the evidence for ß-cell dedifferentiation in T2D, and T2D and examined experimental systems in which its mechanisms may be studied. Understanding these mechanisms may allow prevention of ß-cell dedifferentiation or induction of cell redifferentiation for restoration of the functional ß-cell mass. Stem Cells 2019;37:1267-1272.

Redifferentiation of expanded human pancreatic ß-cell-derived cells by inhibition of the NOTCH pathway.

In vitro expansion of ß-cells from adult human pancreatic islets would overcome donor ß-cell shortage for cell replacement therapy for diabetes. Using a ß-cell-specific labeling system we have shown that ß-cell expansion is accompanied by dedifferentiation resembling epithelial-mesenchymal transition and loss of insulin expression. Epigenetic analyses indicate that key ß-cell genes maintain open chromatin structure in expanded ß-cell-derived (BCD) cells, although they are not transcribed. In the developing pancreas important cell-fate decisions are regulated by NOTCH receptors, which signal through the Hairy and Enhancer of Split 1 (HES1) transcription regulator. We have reported that BCD cell dedifferentiation and proliferation in vitro correlate with reactivation of the NOTCH pathway. Inhibition of HES1 expression using shRNA during culture initiation results in reduced ß-cell replication and dedifferentiation, suggesting that HES1 inhibition may also affect BCD cell redifferentiation following expansion. Here, we used HES1 shRNA to down-regulate HES1 expression in expanded human BCD cells, showing that HES1 inhibition is sufficient to induce BCD cell redifferentiation, as manifested by a significant increase in insulin expression. Combined treatment with HES1 shRNA, cell aggregation in serum-free medium, and a mixture of soluble factors further stimulated the redifferentiation of BCD cells. In vivo analyses demonstrated the ability of the redifferentiated cells to replace ß-cell function in hyperglycemic immunodeficient mice. These findings demonstrate the redifferentiation potential of ex vivo expanded BCD cells and the reproducible differentiating effect of HES1 inhibition in these cells.

Adenovirus E3 MHC inhibitory genes but not TNF/Fas apoptotic inhibitory genes expressed in beta cells prevent autoimmune diabetes.

To mimic events and molecules involved in type 1 insulin-dependent diabetes mellitus (T1D), we previously designed a transgenic (tg) mouse model where the viral nucleoprotein (NP) gene of lymphocytic choriomeningitis virus (LCMV) was expressed in the thymus to delete high affinity antiself (virus) T cells and in insulin-producing beta cells of the islets of Langerhans. Such tg mice, termed RIP-LCMV, fail to spontaneously develop diabetes. In contrast, when these mice are challenged with LCMV, they develop diabetes as they display hyperglycemia, low to absent levels of pancreatic insulin, and abundant mononuclear cell infiltrates in the islets. However, expressing the adenovirus early region (E3) gene in beta cells along with the LCMV transgene aborted the T1D. The present study utilizes this combined tg model (RIP LCMV x RIP E3) to define the requirement(s) of either pro-apoptotic TNF and Fas pathways or MHC class I up-regulation on beta cells for virus-induced T1D. Inhibitors to either pathway (TNF/Fas or MHC class I) are encoded in the E3 gene complex. To accomplish this task either the E3 region encoding the inhibitors of TNF and Fas pathways or the region encoding gp-19, a protein that inhibits transport of MHC class I molecules out of the endoplasmic reticulum were deleted in the RIP LCMV x RIP E3 model. Thus only the gp-19 is required to abort the virus-induced T1D. In contrast, removal of TNF- and Fas-pathway inhibitory genes had no effect on E3-mediated prevention of T1D.

Development of human insulin-producing cells for cell therapy of diabetes.

Diabetes mellitus is characterized by the loss of insulin-producing beta cells. While conventional treatment results in severe long-term complications, cell replacement therapy is a promising approach for the cure of this disease. However, its application is severally limited by the shortage of donor tissue. Hence, great research efforts concentrate on the development of an abundant cell source of functional beta-like cells, by pursuing three main strategies: Expansion of human donor beta cells in vitro, reprogramming of other cell types, and directed differentiation of pluripotent stem cells, both embryonic and patient-derived. The goal of all these approaches has been the generation of cells with properties that closely resemble the beta-cell phenotype, in particular production and storage of adequate amounts of mature insulin, and its regulated release in response to physiological signals. Here we review recent progress in all three approaches and discuss their advantages as well as remaining challenges.

Epigenetic Memory: Lessons From iPS Cells Derived From Human ß Cells.

Incomplete reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) may be responsible for the heterogeneity in differentiation capacity observed among iPSC lines. It remains unclear whether it results from stochastic reprogramming events, or reflects consistent genetic or cell-of-origin differences. Some evidence suggests that epigenetic memory predisposes iPSCs to enhanced differentiation into the parental cell type. We investigated iPSCs reprogrammed from human pancreatic islet ß cells (BiPSCs), as a step in development of a robust differentiation protocol for generation of ß-like cells. BiPSCs derived from multiple human donors manifested enhanced and reproducible spontaneous and induced differentiation towards insulin-producing cells, compared with iPSCs derived from isogenic non-ß-cell types and fibroblast-derived iPSCs (FiPSCs). Genome-wide analyses of open chromatin in BiPSCs and FiPSCs identified thousands of differential open chromatin sites (DOCs) between the two iPSC types. DOCs more open in BiPSCs (Bi-DOCs) were significantly enriched for known regulators of endodermal development, including bivalent and weak enhancers, and FOXA2 binding sites. Bi-DOCs were associated with genes related to pancreas development and ß-cell function. These studies provide evidence for reproducible epigenetic memory in BiPSCs. Bi-DOCs may provide clues to genes and pathways involved in the differentiation process, which could be manipulated to increase the efficiency and reproducibility of differentiation of pluripotent stem cells from non-ß-cell sources.

Beta-cell replacement for insulin-dependent diabetes mellitus.

Beta-cell replacement is considered the optimal treatment for type 1 diabetes, however, it is hindered by a shortage of human organ donors. Given the difficulty of expanding adult beta cells in vitro, stem/progenitor cells, which can be expanded in tissue culture and induced to differentiate into multiple cell types, represent an attractive source for generation of cells with beta-cell properties. In the absence of well-characterized human pancreas progenitor cells, investigators are exploring the use of embryonic stem cells and stem/progenitor cells from other tissues. Once abundant surrogate beta cells are available, the challenge will be to protect them from recurring autoimmunity.

Generation of insulin-producing cells from human bone marrow mesenchymal stem cells by genetic manipulation.

Beta cell replacement is a promising approach for treatment of type 1 diabetes; however, it is limited by a shortage of pancreas donors. The pluripotent MSC in adult bone marrow (BM) offer an attractive source of stem cells for generation of surrogate beta cells. BM-MSC can be obtained with relative ease from each patient, allowing potential circumvention of allograft rejection. Here, we report a procedure for expansion of BM-MSC in vitro and their differentiation into insulin-producing cells. The pancreatic duodenal homeobox 1 (Pdx1) gene was expressed in BM-MSC from 14 human donors, and the extent of differentiation of these cells toward the beta-cell phenotype was evaluated. RNA and protein analyses documented the activation of expression of all four islet hormones. However, the cells lacked expression of NEUROD1, a key transcription factor in differentiated beta cells. A significant insulin content, as well as glucose-stimulated insulin release, were demonstrated in vitro. Cell transplantation into streptozotocin-diabetic immunodeficient mice resulted in further differentiation, including induction of NEUROD1, and reduction of hyperglycemia. These findings were reproducible in BM-MSC from 9 of 14 donors of both sexes, ages 19-62. These results suggest a therapeutic potential for PDX1-expressing BM-MSC in beta-cell replacement in patients with type 1 diabetes.

Genes Associated with Pancreas Development and Function Maintain Open Chromatin in iPSCs Generated from Human Pancreatic Beta Cells.

Current in vitro islet differentiation protocols suffer from heterogeneity and low efficiency. Induced pluripotent stem cells (iPSCs) derived from pancreatic beta cells (BiPSCs) preferentially differentiate toward endocrine pancreas-like cells versus those from fibroblasts (FiPSCs). We interrogated genome-wide open chromatin in BiPSCs and FiPSCs via ATAC-seq and identified ∼8.3k significant, differential open chromatin sites (DOCS) between the two iPSC subtypes (false discovery rate [FDR] < 0.05). DOCS where chromatin was more accessible in BiPSCs (Bi-DOCS) were significantly enriched for known regulators of endodermal development, including bivalent and weak enhancers, and FOXA2 binding sites (FDR < 0.05). Bi-DOCS were associated with genes related to pancreas development and beta-cell function, including transcription factors mutated in monogenic diabetes (PDX1, NKX2-2, HNF1A; FDR < 0.05). Moreover, Bi-DOCS correlated with enhanced gene expression in BiPSC-derived definitive endoderm and pancreatic progenitor cells. Bi-DOCS therefore highlight genes and pathways governing islet-lineage commitment, which can be exploited for differentiation protocol optimization, diabetes disease modeling, and therapeutic purposes.

Differentiation of human liver-derived, insulin-producing cells toward the beta-cell phenotype.

beta-Cell transplantation is viewed as a cure for type 1 diabetes; however, it is limited by the number of pancreas donors. Human stem cells offer the promise of an abundant source of insulin-producing cells, given the existence of methods for manipulating their differentiation. We have previously demonstrated that the expression of the beta-cell transcription factor pancreatic duodenal homeobox 1 (PDX-1) in human fetal liver cells activates multiple aspects of the beta-cell phenotype. These cells, termed FH-B-TPN cells, produce insulin, release insulin in response to physiological glucose levels, and replace beta-cell function in diabetic immunodeficient mice. However, they deviate from the normal beta-cell phenotype by the lack of expression of a number of beta-cell genes, the expression of non-beta-cell genes, and a lower insulin content. Here we aimed to promote differentiation of FH-B-TPN cells toward the beta-cell phenotype using soluble factors. Cells cultured with activin A in serum-free medium upregulated expression of NeuroD and Nkx2.2 and downregulated paired box homeotic gene 6 (PAX-6). Glucokinase and prohormone convertase 1/3 were also upregulated, whereas pancreatic polypeptide and glucagon as well as liver markers were downregulated. Insulin content was increased by up to 33-fold, to approximately 60% of the insulin content of normal beta-cells. The cells were shown to contain human C-peptide and release insulin in response to physiological glucose levels. Cell transplantation into immunodeficient diabetic mice resulted in the restoration of stable euglycemia. The cells continued to express insulin in vivo, and no cell replication was detected. Thus, the manipulation of culture conditions induced a significant and stable differentiation of FH-B-TPN cells toward the beta-cell phenotype, making them excellent candidates for beta-cell replacement in type 1 diabetes.

Redifferentiation of expanded human islet ß cells by inhibition of ARX.

Ex-vivo expansion of adult human islet ß cells has been evaluated for generation of abundant insulin-producing cells for transplantation; however, lineage-tracing has demonstrated that this process results in ß-cell dedifferentiation. Redifferentiation of ß-cell-derived (BCD) cells can be achieved using a combination of soluble factors termed Redifferentiation Cocktail (RC); however, this treatment leads to redifferentiation of only a fraction of BCD cells. This study aimed at improving redifferentiation efficiency by affecting the balance of islet progenitor-cell transcription factors activated by RC treatment. Specifically, RC treatment induces the transcription factors PAX4 and ARX, which play key roles in directing pancreas endocrine progenitor cells into the ß/δ or α/PP developmental pathways, respectively. Misactivation of ARX in RC-treated BCD cells may inhibit their redifferentiation into ß cells. Blocking ARX expression by shRNA elevated insulin mRNA levels 12.8-fold, and more than doubled the number of insulin-positive BCD cells. ARX inhibition in expanded α-cell-derived cells treated with RC did not cause their transdifferentiation into insulin-producing cells. The combination of RC and ARX shRNA treatment may facilitate the generation of abundant insulin-producing cells for transplantation into patients with type 1 diabetes.

Adenovirus early region 3 antiapoptotic 10.4K, 14.5K, and 14.7K genes decrease the incidence of autoimmune diabetes in NOD mice.

Genes in the early region 3 (E3) of the adenovirus genome allow the virus to evade host immune responses by interfering with major histocompatibility (MHC) class I-mediated antigen presentation and tumor necrosis factor-alpha (TNF-alpha)- or Fas-induced apoptosis of infected cells. Autoimmune type 1 diabetes (T1D) is inhibited in NOD mice transgenically expressing all E3 genes under control of a rat insulin promoter (RIPE3/NOD). For dissecting the protective mechanisms afforded by various E3 genes, they were subdivided into RIP-driven transgene constructs. Strong T1D protection mediated at the beta-cell level characterized DL704/NOD mice lacking the E3 gp19K gene suppressing MHC class I expression but retaining the 10.4K, 14.5K, and 14.7K genes inhibiting Fas- or TNF-alpha-induced apoptosis and TNF-alpha-induced NF-kB activation. Much weaker protection characterized DL309/NOD mice expressing the gp19K but not the 10.4K, 14.5K, and 14.7K genes. While RIPE3/NOD splenocytes had an unexpected decrease in ability to adoptively transfer T1D, splenocytes from both the DL704 and DL309 stocks efficiently did so. These findings indicate that all E3 genes must be expressed to inhibit the diabetogenic potential of NOD immune cells. They also demonstrate that the antiapoptotic E3 genes most effectively protect pancreatic beta-cells from diabetogenic immune responses.

Cell replacement therapy for type 1 diabetes.

Replacement of the insulin-producing pancreatic islet beta cells represents the ultimate treatment for type 1 diabetes. Recent advances in islet transplantation underscore the urgent need for developing alternatives to human tissue donors, which are scarce. Two possible approaches are the expansion of differentiated beta cells by reversible immortalization and the generation of insulin-producing cells from embryonic or adult stem cells. It is possible that new insights into endocrine pancreas development will ultimately lead to manipulation of progenitor-cell fate towards the beta-cell phenotype of insulin production, storage and regulated secretion. Both allogeneic and autologous surrogate beta cells are likely to require protection from recurring autoimmunity. This protection might take the form of tolerization, cell encapsulation, or cell engineering with immunoprotective genes. If successful, these approaches could lead to widespread cell replacement therapy for type 1 diabetes.

TGFß Pathway Inhibition Redifferentiates Human Pancreatic Islet ß Cells Expanded In Vitro.

In-vitro expansion of insulin-producing cells from adult human pancreatic islets could provide an abundant cell source for diabetes therapy. However, proliferation of ß-cell-derived (BCD) cells is associated with loss of phenotype and epithelial-mesenchymal transition (EMT). Nevertheless, BCD cells maintain open chromatin structure at ß-cell genes, suggesting that they could be readily redifferentiated. The transforming growth factor ß (TGFß) pathway has been implicated in EMT in a range of cell types. Here we show that human islet cell expansion in vitro involves upregulation of the TGFß pathway. Blocking TGFß pathway activation using short hairpin RNA (shRNA) against TGFß Receptor 1 (TGFBR1, ALK5) transcripts inhibits BCD cell proliferation and dedifferentiation. Treatment of expanded BCD cells with ALK5 shRNA results in their redifferentiation, as judged by expression of ß-cell genes and decreased cell proliferation. These effects, which are reproducible in cells from multiple human donors, are mediated, at least in part, by AKT-FOXO1 signaling. ALK5 inhibition synergizes with a soluble factor cocktail to promote BCD cell redifferentiation. The combined treatment may offer a therapeutically applicable way for generating an abundant source of functional insulin-producing cells following ex-vivo expansion.

MiR-375 promotes redifferentiation of adult human ß cells expanded in vitro.

In-vitro expansion of ß cells from adult human pancreatic islets could provide abundant cells for cell replacement therapy of diabetes. However, proliferation of ß-cell-derived (BCD) cells is associated with dedifferentiation. Here we analyzed changes in microRNAs (miRNAs) during BCD cell dedifferentiation and identified miR-375 as one of the miRNAs greatly downregulated. We hypothesized that restoration of miR-375 expression in expanded BCD cells may contribute to their redifferentiation. Our findings demonstrate that overexpression of miR-375 alone leads to activation of ß-cell gene expression, reduced cell proliferation, and a switch from N-cadherin to E-cadherin expression, which characterizes mesenchymal-epithelial transition. These effects, which are reproducible in cells derived from multiple human donors, are likely mediated by repression of PDPK1 transcripts and indirect downregulation of GSK3 activity. These findings support an important role of miR-375 in regulation of human ß-cell phenotype, and suggest that miR-375 upregulation may facilitate the generation of functional insulin-producing cells following ex-vivo expansion of human islet cells.

Inhibition of ZEB1 expression induces redifferentiation of adult human ß cells expanded in vitro.

In-vitro expansion of functional adult human ß-cells is an attractive approach for generating insulin-producing cells for transplantation. However, human islet cell expansion in culture results in loss of ß-cell phenotype and epithelial-mesenchymal transition (EMT). This process activates expression of ZEB1 and ZEB2, two members of the zinc-finger homeobox family of E-cadherin repressors, which play key roles in EMT. Downregulation of ZEB1 using shRNA in expanded ß-cell-derived (BCD) cells induced mesenchymal-epithelial transition (MET), ß-cell gene expression, and proliferation attenuation. In addition, inhibition of ZEB1 expression potentiated redifferentiation induced by a combination of soluble factors, as judged by an improved response to glucose stimulation and a 3-fold increase in the fraction of C-peptide-positive cells to 60% of BCD cells. Furthermore, ZEB1 shRNA led to increased insulin secretion in cells transplanted in vivo. Our findings suggest that the effects of ZEB1 inhibition are mediated by attenuation of the miR-200c target genes SOX6 and SOX2. These findings, which were reproducible in cells derived from multiple human donors, emphasize the key role of ZEB1 in EMT in cultured BCD cells and support the value of ZEB1 inhibition for BCD cell redifferentiation and generation of functional human ß-like cells for cell therapy of diabetes.

Preventing type 1 diabetes mellitus: the promise of gene therapy.

Type 1 (insulin-dependent) diabetes mellitus is an autoimmune disease that has no cure. Closed-loop insulin administration strategies and approaches for replacement of the insulin-producing beta cells may offer improved treatments, which could delay or prevent diabetes complications. In the long run, however, prevention of type 1 diabetes in susceptible individuals represents the best chance for reducing the toll of the disease. Prevention of type 1 diabetes will require reliable methods for early diagnosis of predisposition to the disease, using improved genetic and serological screening on a wide scale. Identification of the primary antigenic target(s) for autoimmunity will allow intervention in prediabetes stages aimed at the induction of antigen-specific tolerance. In addition to manipulation of the immune system, the susceptibility of beta cells to autoimmunity could be reduced. A number of genes have been shown to increase beta-cell resistance to immune effector molecules in animal models and cultured beta-cell lines. These genes could be used for preventive gene therapy of type 1 diabetes mellitus if expressed in beta cells prior to the onset of autoimmune destruction. This prospect depends on the development of safe and efficient vectors, and approaches for cell-specific targeting of these vectors to beta cells in vivo.

Regulation of insulin secretion: insights from engineered beta-cell lines.

Abnormalities in insulin secretion are involved in a number of diseases, including type 1 and type 2 diabetes, persistent hyperinsulinemic hypoglycemia of infancy, and insulinoma. Understanding the mechanisms that regulate insulin secretion may allow the development of new therapies for these diseases as well as contribute to our ability to engineer insulin-producing cells for cell replacement therapy of type 1 diabetes. Glucose phosphorylation in beta-cells has been viewed as a key regulatory event in coupling insulin secretion to extracellular glucose concentrations. Work with transformed rodent beta-cell lines as well as recent findings from human progenitor cells induced to differentiate into insulin-producing cells has provided new insights into the role of glucose phosphorylating enzymes in the regulation of insulin secretion.

Generation of insulin-producing cells from stem cells for cell replacement therapy of type 1 diabetes.

Type 1 diabetes mellitus is caused by an autoimmune destruction of pancreatic islet beta cells, leading to insulin deficiency. Beta-cell replacement is considered the optimal treatment for type 1 diabetes, however it is severely limited by the shortage of human organ donors. An effective cell-replacement strategy depends on the development of an abundant supply of beta cells and their protection from recurring immune destruction. Stem/progenitor cells, which can be expanded in tissue culture and induced to differentiate into multiple cell types, represent an attractive source for generation of cells with beta-cell properties: insulin biosynthesis, storage, and regulated secretion in response to physiologic signals. Embryonic stem cells have been shown to spontaneously differentiate into insulin-producing cells at a low frequency, and this capacity could be further enhanced by tissue culture conditions, soluble agents, and expression of dominant transcription factor genes. Progenitor cells from fetal and adult tissues, such as liver and bone marrow, have also been shown capable of differentiation towards the beta-cell phenotype in vivo, or following expression of dominant transcription factors in vitro. These approaches offer novel ways for generation of cells for transplantation into patients with type 1 diabetes.

The NOTCH pathway in ß-cell growth and differentiation.

Beta-cell replacement represents the optimal therapy for type 1 diabetes. Efforts to manipulate ß-cell proliferation and differentiation could be advanced by a better understanding of the normal pathways regulating ß-cell development and renewal. NOTCH signaling is a highly conserved pathway which plays a central role in pancreas development. Cell-lineage tracing has revealed the reactivation of the NOTCH pathway in adult human ß cells cultured under conditions which induce cell proliferation and dedifferentiation. Inhibition of NOTCH signaling in dedifferentiated cells following ex vivo expansion has been shown to promote restoration of the ß-cell phenotype. This approach may increase the availability of functional ß cells for transplantation.

Redifferentiation of adult human ß cells expanded in vitro by inhibition of the WNT pathway.

In vitro expansion of adult human islet ß cells is an attractive solution for the shortage of tissue for cell replacement therapy of type 1 diabetes. Using a lineage tracing approach we have demonstrated that ß-cell-derived (BCD) cells rapidly dedifferentiate in culture and can proliferate for up to 16 population doublings. Dedifferentiation is associated with changes resembling epithelial-mesenchymal transition (EMT). The WNT pathway has been shown to induce EMT and plays key roles in regulating replication and differentiation in many cell types. Here we show that BCD cell dedifferentiation is associated with ß-catenin translocation into the nucleus and activation of the WNT pathway. Inhibition of ß-catenin expression in expanded BCD cells using short hairpin RNA resulted in growth arrest, mesenchymal-epithelial transition, and redifferentiation, as judged by activation of ß-cell gene expression. Furthermore, inhibition of ß-catenin expression synergized with redifferentiation induced by a combination of soluble factors, as judged by an increase in the number of C-peptide-positive cells. Simultaneous inhibition of the WNT and NOTCH pathways also resulted in a synergistic effect on redifferentiation. These findings, which were reproducible in cells derived from multiple human donors, suggest that inhibition of the WNT pathway may contribute to a therapeutically applicable way for generation of functional insulin-producing cells following ex-vivo expansion.