Dissecting mechanisms of human islet differentiation and maturation through epigenome profiling

SUMMARYInvestigating pancreatic islet differentiation from human stem cells in vitro provides a unique opportunity to dissect mechanisms that operate during human development in utero. We developed methods to profile DNA methylation, chromatin accessibility, and histone modifications from pluripotent stem cells to mature pancreatic islet cells, uncovering widespread epigenome remodeling upon endocrine commitment. Key lineage-defining loci are epigenetically primed before activation, foreshadowing cell fate commitment, and we show that priming of α-cell-specific enhancers steers polyhormonal cells toward an α-cell fate. We further dissect pioneer factors and core regulatory circuits across islet cell differentiation and maturation stages, which identify LMX1B as a key regulator of in vitro-derived endocrine progenitors. Finally, by contrasting maturing stem cell-derived to natural β-cells, we discover that circadian metabolic cycles trigger rhythmic control of insulin synthesis and release and promote mature insulin responsiveness via an increased glucose threshold. These findings form a basis for understanding mechanisms orchestrating human islet cell specification and maturation.

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Recent progress in pancreatic islet cell therapy

Inflammation and Regeneration ◽

10.1186/s41232-020-00152-5 ◽

2021 ◽

Vol 41 (1) ◽

Author(s):

Erinn Zixuan Sim ◽

Nobuaki Shiraki ◽

Shoen Kume

Keyword(s):

Stem Cells ◽

Pancreatic Islet ◽

Islet Cell ◽

Recent Progress ◽

Disease Modeling ◽

Islet Cells ◽

Pancreatic Islet Cells ◽

Pancreatic Islet Cell

AbstractHuman pluripotent stem cells (PSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising cell sources in regenerating pancreatic islets through in vitro directed differentiation. Recent progress in this research field has made it possible to generate glucose-responsive pancreatic islet cells from PSCs. Single-cell RNA sequencing techniques have been applied to analyze PSC-derived endocrine beta-cells, which are then compared with human islets. This has led to the identification of novel signaling pathways and molecules involved in lineage commitment during pancreatic differentiation and maturation processes. Single-cell transcriptomics are also used to construct a detailed map of in vivo endocrine differentiation of developing mouse embryos to study pancreatic islet development. Mimicking those occurring in vivo, it was reported that differentiating PSCs can generate similar islet cell structures, while metabolomics analysis highlighted key components involved in PSC-derived pancreatic islet cell function, providing information for the improvement of in vitro pancreatic maturation procedures. In addition, cell transplantation into diabetic animal models, together with the cell delivery system, is studied to ensure the therapeutic potentials of PSC-derived pancreatic islet cells. Combined with gene-editing technology, the engineered mutation-corrected PSC lines originated from diabetes patients could be differentiated into functional pancreatic islet cells, suggesting possible autologous cell therapy in the future. These PSC-derived pancreatic islet cells are a potential tool for studies of disease modeling and drug testing. Herein, we outlined the directed differentiation procedures of PSC-derived pancreatic islet cells, novel findings through transcriptome and metabolome studies, and recent progress in disease modeling.

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Co-localization of nestin and insulin and expression of islet cell markers in long-term human pancreatic nestin-positive cell cultures

Journal of Endocrinology ◽

10.1677/joe.1.05703 ◽

2004 ◽

Vol 183 (3) ◽

pp. 455-467 ◽

Cited By ~ 26

Author(s):

Silvya Stuchi Maria-Engler ◽

Maria Lúcia C Corrêa-Giannella ◽

Letícia Labriola ◽

Karin Krogh ◽

Christian Colin ◽

...

Keyword(s):

Cell Cultures ◽

Islet Cell ◽

Islet Cells ◽

Human Islet ◽

Β Cells ◽

Short Term ◽

Cell Markers ◽

Insulin Expression

Strategies to differentiate progenitor cells into β cells in vitro have been considered as an alternative to increase β cell availability prior to transplantation. It has recently been suggested that nestin-positive cells could be multipotential stem cells capable of expressing endocrine markers upon specific stimulation; however, this issue still remains controversial. Here, we characterized short- and long-term islet cell cultures derived from three different human islet preparations, with respect to expression of nestin and islet cell markers, using confocal microscopy and semi-quantitative RT-PCR. The number of nestin-positive cells was found to be strikingly high in long-term cultures. In addition, a large proportion (49.7%) of these nestin-positive cells, present in long-term culture, are shown to be proliferative, as judged by BrdU incorporation. The proportion of insulin-positive cells was found to be high in short-term (up to 28 days) cultures and declined thereafter, when cells were maintained in the presence of 10% serum, concomitantly with the decrease in insulin and PDX-1 expression. Interestingly, insulin and nestin co-expression was observed as a rare event in a small proportion of cells present in freshly isolated human islets as well as in purified islet cells cultured in vitro for long periods of time. In addition, upon long-term subculturing of nestin-positive cells in 10% serum, we observed reappearance of insulin expression at the mRNA level; when these cultures were shifted to 1% serum for a month, expression of insulin, glucagon and somatostatin was also detected, indicating that manipulating the culture conditions can be used to modulate the nestin-positive cell’s fate. Attempts to induce cell differentiation by plating nestin-positive cells onto Matrigel revealed that these cells tend to aggregate to form islet-like clusters, but this is not sufficient to increase insulin expression upon short-term culture. Our data corroborate previous findings indicating that, at least in vitro, nestin-positive cells may undergo the early stages of differentiation to an islet cell phenotype and that long-term cultures of nestin-positive human islet cells may be considered as a potential source of precursor cells to generate fully differentiated/ functional β cells.

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Overexpression of the nuclear factor-κB subunit c-Rel protects against human islet cell death in vitro

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00212.2009 ◽

2009 ◽

Vol 297 (5) ◽

pp. E1067-E1077 ◽

Cited By ~ 22

Author(s):

Dariush Mokhtari ◽

Andreea Barbu ◽

Ilir Mehmeti ◽

Chantal Vercamer ◽

Nils Welsh

Keyword(s):

Cell Death ◽

Islet Cell ◽

Nuclear Translocation ◽

Islet Cells ◽

Human Islet ◽

Nuclear Fraction ◽

Nuclear Factor ◽

Subunit C

The transcription factor nuclear factor (NF)-κB is known to modulate rates of apoptosis and may therefore play a role in the increased β-cell death that occurs in type 1 and type 2 diabetes. The aim of the present investigation was to study the expression of NF-κB subunits in human islet cells and whether overexpression of the NF-κB subunit c-Rel affects islet cell survival. We detected expression of p65, Rel-B, p50, p105, p52, and the ribosomal protein S3 (rpS3) in human islet cells. Among these, only p65 and rpS3 were translocated from the cytosolic to the nuclear fraction in response to cytokines. Interestingly, rpS3 participated in p65 binding to the κB-element in gel shift analysis experiments. We observed cytoplasmic c-Rel expression in vivo in 6J mice, and signs of nuclear translocation in β-cells of infiltrated nonobese diabetic islets. Human islet cells were also dispersed by trypsin treatment and transduced with a c-Rel adenoviral vector. This resulted in increased expression of c-Rel and inhibitory factor κB, increased κB-binding activity, and augmented protein levels of Bcl-XL, c-IAP2, and heat shock protein 72. c-Rel expression in human islet cells protected against cytokine-induced caspase 3 activation and cell death. c-Rel protected also against streptozotocin- and H2O2-induced cell death, in both intact rat islets and human islet cells. We conclude that rpS3 participates in NF-κB signaling and that a genetic increase in the activity of the NF-κB subunit c-Rel results in protection against cell death in human islets.

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Intrapancreatic MSC transplantation facilitates pancreatic islet regeneration

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02173-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Rahul Khatri ◽

Sebastian Friedrich Petry ◽

Thomas Linn

Keyword(s):

Stem Cells ◽

Growth Factor ◽

Pancreatic Islet ◽

Nude Mice ◽

Direct Contact ◽

Islet Cells ◽

Physical Contact ◽

Min6 Cells

Abstract Background Type 1 diabetes mellitus (T1D) is characterized by the autoimmune destruction of the pancreatic β cells. The transplantation of mesenchymal stromal/stem cells (MSC) was reported to rescue the damaged pancreatic niche. However, there is an ongoing discussion on whether direct physical contact between MSC and pancreatic islets results in a superior outcome as opposed to indirect effects of soluble factors released from the MSC entrapped in the lung microvasculature after systemic administration. Hence, MSC were studied in direct contact (DC) and indirect contact (IDC) with murine pancreatic β cell line MIN6-cells damaged by nitrosourea derivative streptozotocin (STZ) in vitro. Further, the protective and antidiabetic outcome of MSC transplantation was evaluated through the intrapancreatic route (IPR) and intravenous route (IVR) in STZ-induced diabetic NMRI nude mice. Methods MSC were investigated in culture with STZ-damaged MIN6-cells, either under direct contact (DC) or separated through a semi-permeable membrane (IDC). Moreover, multiple low doses of STZ were administered to NMRI nude mice for the induction of hyperglycemia. 0.5 × 106 adipose-derived mesenchymal stem cells (ADMSC) were transferred through direct injection into the pancreas (IPR) or the tail vein (IVR), respectively. Bromodeoxyuridine (BrdU) was injected for the detection of proliferating islet cells in vivo, and real-time polymerase chain reaction (RT-PCR) was employed for the measurement of the expression of growth factor and immunomodulatory genes in the murine pancreas and human MSC. Phosphorylation of AKT and ERK was analyzed with Western blotting. Results The administration of MSC through IPR ameliorated hyperglycemia in contrast to IVR, STZ, and non-diabetic control in a 30-day window. IPR resulted in a higher number of replicating islet cells, number of islets, islet area, growth factor (EGF), and balancing of the Th1/Th2 response in vivo. Physical contact also provided a superior protection to MIN6-cells from STZ through the AKT and ERK pathway in vitro in comparison with IDC. Conclusion Our study suggests that the physical contact between MSC and pancreatic islet cells is required to fully unfold their protective potential.

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Nkx2.2 Activates the Ghrelin Promoter in Pancreatic Islet Cells

Molecular Endocrinology ◽

10.1210/me.2009-0360 ◽

2010 ◽

Vol 24 (2) ◽

pp. 381-390 ◽

Cited By ~ 10

Author(s):

Jonathon T. Hill ◽

Christina S. Chao ◽

Keith R. Anderson ◽

Fernanda Kaufman ◽

Christopher W. Johnson ◽

...

Keyword(s):

Cell Fate ◽

Islet Cell ◽

Islet Cells ◽

Cell Fate Decisions ◽

Endocrine Differentiation ◽

Embryonic Pancreas ◽

Translational Start ◽

Transcriptional Start Sites

Abstract Nkx2.2 is an essential regulator of pancreatic endocrine differentiation. Nkx2.2-null mice are completely devoid of β-ells and have a large reduction of α- and PP cells. In the place of these islet populations, there is a corresponding increase in the ghrelin-positive ε-cells. Molecular studies have indicated that Nkx2.2 functions as an activator and repressor to regulate islet cell fate decisions. To determine whether Nkx2.2 is solely important for islet cell fate decisions or also has the capability to control ghrelin at the promoter level, we studied the transcriptional regulation of the ghrelin promoter within the pancreas, in vitro and in vivo. These studies demonstrate that both of the previously identified transcriptional start sites in the ghrelin promoter are active within the embryonic pancreas; however, the long transcript is preferentially up-regulated in the Nkx2.2-null pancreas. We also show that the promoter region between −619 and −488 bp upstream of the translational start site is necessary for repression of ghrelin in αTC1 and βTC6 cells. Surprisingly, we also show that Nkx2.2 is able to bind to and activate the ghrelin promoter in several cell lines that do or do not express endogenous ghrelin. Together, these results suggest that the up-regulation of ghrelin expression in the Nkx2.2-null mice is not due to loss of repression of the ghrelin promoter in the nonghrelin islet populations. Furthermore, Nkx2.2 may contribute to the activation of ghrelin in mature islet ε-cells.

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A Modified Method of Insulin Producing Cells’ Generation from Bone Marrow-Derived Mesenchymal Stem Cells

Journal of Diabetes Research ◽

10.1155/2014/628591 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 14

Author(s):

Paweł Czubak ◽

Agnieszka Bojarska-Junak ◽

Jacek Tabarkiewicz ◽

Lechosław Putowski

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Growth Factors ◽

Pancreatic Islet ◽

Islet Cells ◽

Insulin Injection ◽

Modified Method ◽

Insulin Producing Cells

Type 1 diabetes mellitus is a result of autoimmune destruction of pancreatic insulin producingβ-cells and so far it can be cured only by insulin injection, by pancreas transplantation, or by pancreatic islet cells’ transplantation. The methods are, however, imperfect and have a lot of disadvantages. Therefore new solutions are needed. The best one would be the use of differentiated mesenchymal stem cells (MSCs). In the present study, we investigated the potential of the bone marrow-derived MSCs line forin vitrodifferentiation into insulin producing cells (IPSs). We applied an 18-day protocol to differentiate MSCs. Differentiating cells formed cell clusters some of which resembled pancreatic islet-like cells. Using dithizone we confirmed the presence of insulin in the cells. What is more, the expression of proinsulin C-peptide in differentiated IPCs was analyzed by flow cytometry. For the first time, we investigated the influence of growth factors’ concentration on IPCs differentiation efficiency. We have found that an increase in the concentration of growth factors up to 60 ng/mL ofβ-FGF/EGF and 30 ng/mL of activin A/β-cellulin increases the percentage of IPCs. Further increase of growth factors does not show any increase of the percentage of differentiated cells. Our findings suggest that the presented protocol can be adapted for differentiation of insulin producing cells from stem cells.

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