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 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.

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.

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.

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.

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.

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.

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.

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.

Recent progress in generation of human surrogate ß cells.

PURPOSE OF REVIEW: This review evaluates recent progress in several approaches aimed at developing human surrogate ß cells, and identifies gaps that need to be filled for bringing them closer to clinical application. RECENT FINDINGS: Cells expanded in vitro from human cadaver donor ß cells under conditions causing dedifferentiation have been shown to undergo redifferentiation following inhibition of the Notch pathway. Efforts for differentiation of insulin-producing cells from human pluripotent stem cells have focused on isolation and expansion of intermediate-stage cells. The role of mesenchyme in expansion of pancreas progenitors has been emphasized by mouse cell ablation, and co-culture of human embryonic stem cell-derived definitive endoderm with mesenchyme. Incomplete removal of Polycomb-mediated repression of endocrine genes in embryonic stem cell-derived insulin-producing cells generated in vitro has been suggested to be responsible for their immature phenotype. Induced pluripotent stem cells reprogrammed from ß cells have been shown to exhibit an enhanced differentiation capacity toward insulin-producing cells, compared with other pluripotent stem cells. A new approach for reprogramming non-ß into ß-like cells involving transcription factor gene ablation has been demonstrated in mouse enteroendocrine cells in vivo. SUMMARY: New insights into the stumbling blocks in expansion of human donor islet cells, differentiation of pluripotent stem cells, and reprogramming of non-ß cell types are shaping improved strategies, which are likely to bring us closer to the goal of generating abundant human surrogate ß cells.

Divalent metal transporter 1 regulates iron-mediated ROS and pancreatic ß cell fate in response to cytokines.

Reactive oxygen species (ROS) contribute to target-cell damage in inflammatory and iron-overload diseases. Little is known about iron transport regulation during inflammatory attack. Through a combination of in vitro and in vivo studies, we show that the proinflammatory cytokine IL-1ß induces divalent metal transporter 1 (DMT1) expression correlating with increased ß cell iron content and ROS production. Iron chelation and siRNA and genetic knockdown of DMT1 expression reduce cytokine-induced ROS formation and cell death. Glucose-stimulated insulin secretion in the absence of cytokines in Dmt1 knockout islets is defective, highlighting a physiological role of iron and ROS in the regulation of insulin secretion. Dmt1 knockout mice are protected against multiple low-dose streptozotocin and high-fat diet-induced glucose intolerance, models of type 1 and type 2 diabetes, respectively. Thus, ß cells become prone to ROS-mediated inflammatory damage via aberrant cellular iron metabolism, a finding with potential general cellular implications.

Making ß cells from adult tissues.

ß-Cell replacement represents an attractive prospect for diabetes therapy. Although much hope has been placed on derivation of insulin-producing cells from human pluripotent stem cells, this approach continues to face considerable challenges. Cells from adult human tissues, with both stem/progenitor and mature phenotypes, offer a possible alternative. This review summarizes recent progress in two major strategies based on this cell source, ex vivo expansion of human islet ß cells and conversion of non-ß cells into insulin-producing cells by nuclear reprogramming, and examines the obstacles that remain to be overcome for bringing these strategies closer to clinical application in diabetes therapy.

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.

Insulin-producing cells generated from dedifferentiated human pancreatic beta cells expanded in vitro.

BACKGROUND: Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD) cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT). Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells. METHODOLOGY/PRINCIPAL FINDING: Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2) using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug screening.

Epigenetic memory and preferential lineage-specific differentiation in induced pluripotent stem cells derived from human pancreatic islet beta cells.

Human induced pluripotent stem cells (HiPSCs) appear to be highly similar to human embryonic stem cells (HESCs). Using two genetic lineage-tracing systems, we demonstrate the generation of iPSC lines from human pancreatic islet beta cells. These reprogrammed cells acquired markers of pluripotent cells and differentiated into the three embryonic germ layers. However, the beta cell-derived iPSCs (BiPSCs) maintained open chromatin structure at key beta-cell genes, together with a unique DNA methylation signature that distinguishes them from other PSCs. BiPSCs also demonstrated an increased ability to differentiate into insulin-producing cells both in vitro and in vivo, compared with ESCs and isogenic non-beta iPSCs. Our results suggest that the epigenetic memory may predispose BiPSCs to differentiate more readily into insulin producing cells. These findings demonstrate that HiPSC phenotype may be influenced by their cells of origin, and suggest that their skewed differentiation potential may be advantageous for cell replacement therapy.

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.

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.

Epithelial-mesenchymal transition in cells expanded in vitro from lineage-traced adult human pancreatic beta cells.

BACKGROUND: In-vitro expansion of functional beta cells from adult human islets is an attractive approach for generating an abundant source of cells for beta-cell replacement therapy of diabetes. Using genetic cell-lineage tracing we have recently shown that beta cells cultured from adult human islets undergo rapid dedifferentiation and proliferate for up to 16 population doublings. These cells have raised interest as potential candidates for redifferentiation into functional insulin-producing cells. Previous work has associated dedifferentiation of cultured epithelial cells with epithelial-mesenchymal transition (EMT), and suggested that EMT generates cells with stem cell properties. Here we investigated the occurrence of EMT in these cultures and assessed their stem cell potential. METHODOLOGY/PRINCIPAL FINDINGS: Using cell-lineage tracing we provide direct evidence for occurrence of EMT in cells originating from beta cells in cultures of adult human islet cells. These cells express multiple mesenchymal markers, as well as markers associated with mesenchymal stem cells (MSC). However, we do not find evidence for the ability of such cells, nor of cells in these cultures derived from a non-beta-cell origin, to significantly differentiate into mesodermal cell types. CONCLUSIONS/SIGNIFICANCE: These findings constitute the first demonstration based on genetic lineage-tracing of EMT in cultured adult primary human cells, and show that EMT does not induce multipotency in cells derived from human beta cells.

Ex-vivo Expansion of Adult Human Pancreatic Beta-Cells.

Ex-vivo generation of human insulin-producing cells is considered a promising approach to providing an abundant source of cells for beta-cell replacement therapy in diabetes. Expansion of adult beta-cells from the limited number of islet donors is an attractive prospect. However, while evidence supports the replicative capacity of both rodent and human beta-cells in vivo, attempts at expanding these cells in tissue culture result in loss of beta-cell phenotype, making it difficult to track their fate during continuous propagation and raising doubts about their therapeutic potential. Recent lineage-tracing studies demonstrate the ability of human beta-cells to survive and replicate to a significant degree in vitro. Beta-cell delamination out of the normal epithelial structure, a process that results in dedifferentiation, seems to be required for significant in-vitro proliferation. Therefore, ways must be found of inducing redifferentiation of the expanded cells ex vivo, or of restoring their function upon transplantation. Elucidation of the signaling pathways altered during beta-cell adaptation to growth in culture may provide clues to cell redifferentiation. In a recent study, we found that human beta-cell dedifferentiation and entrance into the cell cycle in vitro correlated with activation of the Notch pathway and downregulation of the cell cycle inhibitor p57. Inhibition of the Notch downstream target HES1 using short hairpin RNA reduced beta-cell dedifferentiation and replication, suggesting a potential target for inducing cell redifferentiation following expansion in culture. This review critically discusses the potential for using ex-vivo beta-cell replication and redifferentiation in cell replacement therapy in diabetes.