Β-cell Differentiation Research Articles

Enhancing differentiation and functionality of insulin-producing cells derived from iPSCs using esterified collagen hydrogel for cell therapy in diabetes mellitus

BackgroundIslet transplantation is a recommended treatment for type 1 diabetes but is limited by donor organ shortage. This study introduces an innovative approach for improving the differentiation and functionality of insulin-producing cells (IPCs) from iPSCs using 3D spheroid formation and hydrogel matrix as an alternative pancreatic islet source. The extracellular matrix (ECM) is crucial for pancreatic islet functionality, but finding the ideal matrix for β-cell differentiation has been challenging. We aimed to advance IPC differentiation and maturation through an esterified collagen hydrogel, comparing its effectiveness with conventional basement membrane extract (BME) hydrogels.MethodsiPSCs were differentiated into IPCs using a small molecule-based sequential protocol, followed by spheroid formation in concave microwells. Rheological analysis, scanning electron microscopy, and proteomic profiling were used to characterize the chemical and physical properties of each matrix. IPCs, both in single-cell form and as spheroids, were embedded in either ionized collagen or BME hydrogels, which was followed by assessments of morphological changes, pancreatic islet-related gene expression, insulin secretion, and pathway activation using comprehensive analytical techniques.ResultsEsterified collagen hydrogels markedly improved the structural integrity, insulin expression, and cell-cell interactions in IPC spheroids, forming densely packed insulin-expressing clusters, in contrast to the dispersed cells observed in BME cultures. Collagen hydrogel significantly enhanced the mRNA expression of crucial endocrine markers and maturation factors, with IPC spheroids showing accelerated differentiation from day 5, suggesting a faster differentiation compared to single cells in hydrogel encapsulation. Insulin secretion in response to glucose in collagen environments, with a GSIS index of 2.46 ± 0.05, exceeded those in 2D and BME, demonstrating superior pancreatic islet functionality. Pathway analysis highlighted enhanced insulin secretion capabilities, evidenced by the upregulation of genes like Secretogranin III and Chromogranin A in collagen cultures. In vivo transplantation results showed that collagen hydrogel enhanced cluster integrity, tissue integration, and insulin secretion compared to non-embedded IPCs and BME groups.ConclusionEsterified collagen hydrogels demonstrated superior efficacy over 2D and BME in promoting IPC differentiation and maturation, possibly through upregulation of the expression of key secretion pathway genes. Our findings suggest that using collagen hydrogels presents a promising approach to enhance insulin secretion efficiency in differentiating pancreatic β-cells, advancing cell therapy in diabetes cell therapy.

A stepwise approach to deriving functional β-cells from human embryonic or induced pluripotent stem cells

Abstract Our understanding of β-cell differentiation from pluripotent stem cells (PSCs) is rapidly evolving. Although progress has been made, challenges remain, particularly in achieving glucose-stimulated insulin secretion (GSIS). Human embryonic stem cells (hESCs) are valuable due to their pluripotent ability. A fixed protocol targeting master regulatory genes initiates stem cells into pancreatic lineage commitment. Due to the observations that a single stem cell can differentiate into multiple cell types depending on various factors and conditions, non-linear differentiation pathways exist. Co-expression of key factors remains essential for successful β-cell differentiation. The mature β-cell marker MAFA plays a critical role in maintaining the differentiation state and preventing dedifferentiation. Recapitulating pancreatic islet clustering enhances physiological responses, offering potential avenues for diabetes treatment. On the other hand, several enhanced differentiation protocols from induced pluripotent stem cells (iPSCs) have improved the functional insulin producing β-cells generated. These findings, with their potential to revolutionize diabetes treatment, highlight the complexity of β-cell differentiation and guide further advancements in regenerative medicine.

REDOX SIGNALLING IN THE PANCREAS IN HEALTH AND DISEASE.

This review addresses oxidative stress and redox signaling in the pancreas under physiological conditions as well as in acute pancreatitis, chronic pancreatitis, pancreatic cancer, and diabetes. Physiological redox homeodynamics is maintained mainly by NRF2/KEAP1, NF-κB, protein tyrosine phosphatases, peroxisome proliferator-activated receptor-γ co-activator 1α (PGC1α), and normal autophagy. Depletion of reduced glutathione in the pancreas is a hallmark of acute pancreatitis and is initially accompanied by disulfide stress, which is characterized by protein cysteinylation without increased glutathione oxidation. A cross-talk between oxidative stress, MAPKs, and NF-κB amplifies the inflammatory cascade, acting PP2A and PGC1α as key redox regulatory nodes. In acute pancreatitis, nitration of cystathionine-β synthase causes blockade of the trans-sulfuration pathway leading to increased homocysteine levels, whereas p53 triggers necroptosis in the pancreas through downregulation of sulfiredoxin, PGC1α, and peroxiredoxin 3. Chronic pancreatitis exhibits oxidative distress mediated by NADPH oxidase 1 and/or CYP2E1, which promotes cell death, fibrosis, and inflammation. Oxidative stress cooperates with mutant KRAS to initiate and promote pancreatic adenocarcinoma. Mutant KRAS increases mitochondrial ROS, which trigger acinar-to-ductal metaplasia and progression to PanIN. ROS are maintained at sufficient level to promote cell proliferation, whilst avoiding cell death or senescence through formation of NADPH and GSH, and activation of NRF-2, HIF-1/2α, and CREB. Redox signalling also plays a fundamental role in differentiation, proliferation, and insulin secretion of β-cells. However, ROS overproduction promotes β-cell dysfunction and apoptosis in type 1 and type 2 diabetes.

Features of beta cell differentiation during the development of type 2 diabetes mellitus

Type 2 diabetes mellitus is characterized by a mild inflammatory reaction in the pancreas, which affects the structure and function of the pancreatic islets: the number of β-cells decreases and the number of α-cells increases. The work examined the features of β-cell differentiation in the development of experimental type 2 diabetes mellitus and while reducing the inflammatory process. Biochemical, histological methods, enzyme-linked immunosorbent assay, immunohistochemical methods were used using primary antibodies to insulin, glucagon, proliferation marker Ki-67 and secondary antibodies labeled with fluorescent dyes. Streptozotocin and nicotinamide were used to model type 2 diabetes mellitus, and the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione was used to reduce the inflammatory response. Previous studies have shown that it changes the macrophage phenotype from proinflammatory M1 to anti-inflammatory M2. In type 2 diabetes mellitus, against the background of a decrease in the number of macrophages with the CD163 marker and the concentration of the cytokine TGF-β1, which have an anti-inflammatory effect, in the pancreatic islets, a decrease in the number of β-cells and their functional activity was observed, while the content of α-cells synthesizing glucagon increased. After administration of the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione, the opposite picture was observed in the pancreatic islets: against the background of an increase in the number of CD163+ macrophages and the content of TGF-β1, the number of β cells increased and the number of α cells decreased-cells. The increase in the number of insulin-synthesizing cells was not accompanied by their mitotic activity. It is likely that a decrease in the number of CD163+ macrophages and the level of the antiinflammatory cytokine TGF-β1 in the islets are factors contributing to changes in the cell microenvironment and, as a consequence, the differentiation of β-cells into α-cells. On the contrary, an increase in the number of CD163+ macrophages and TGF-β1 against the background of administration of the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione presumably promotes reverse differentiation of α-cells into β-cells and restoration of insulin synthesis pancreas. Targeted effects on the microenvironment of cells in the pancreatic islet in type 2 diabetes mellitus may be a new approach to treating the disease.

MODY Only Monogenic? A Narrative Review of the Novel Rare and Low-Penetrant Variants.

Maturity-onset diabetes of the young (MODY) represents the most frequent form of monogenic diabetes mellitus (DM), currently classified in 14 distinct subtypes according to single gene mutations involved in the differentiation and function of pancreatic β-cells. A significant proportion of MODY has unknown etiology, suggesting that the genetic landscape is still to be explored. Recently, novel potentially MODY-causal genes, involved in the differentiation and function of β-cells, have been identified, such as RFX6, NKX2.2, NKX6.1, WFS1, PCBD1, MTOR, TBC1D4, CACNA1E, MNX1, AKT2, NEUROG3, EIF2AK3, GLIS3, HADH, and PTF1A. Genetic and clinical features of MODY variants remain highly heterogeneous, with no direct genotype-phenotype correlation, especially in the low-penetrant subtypes. This is a narrative review of the literature aimed at describing the current state-of-the-art of the novel likely MODY-associated variants. For a deeper understanding of MODY complexity, we also report some related controversies concerning the etiological role of some of the well-known pathological genes and MODY inheritance pattern, as well as the rare association of MODY with autoimmune diabetes. Due to the limited data available, the assessment of MODY-related genes pathogenicity remains challenging, especially in the setting of rare and low-penetrant subtypes. In consideration of the crucial importance of an accurate diagnosis, prognosis and management of MODY, more studies are warranted to further investigate its genetic landscape and the genotype-phenotype correlation, as well as the pathogenetic contribution of the nongenetic modifiers in this cohort of patients.

Harnessing androgen receptor: Revolutionizing diabetes treatment in men with selective androgen receptor modulators

Diabetes is a chronic metabolic disorder. Among different types, type 2 diabetes mellitus (T2DM) is the most prevalent accounting for 90 % to 95 % of all reported cases. T2DM is more prevalent in men compared to women. According to several literature reports, men with low testosterone levels and/or decreased androgen receptor (AR) expression are prone to T2DM. Consistently, androgen therapy was effective in treating T2DM in several preclinical and clinical studies. Activation of AR has been shown to induce the gene expression of NGN3 and NEUROD1 which helps in pancreatic β-cell differentiation, regeneration, and proliferation. AR activation can also increase the gene expression of glucose transporter (GLUT4) in the hepatocytes, skeletal muscle fibers, and adipocytes. An increase in GLUT4 expression could augment the activity of insulin and could help to reduce the insulin dose. However, testosterone therapy even for the short term was found to produce significant adverse effects. The rate-limiting being the increase in the hematocrit value. Hence, to minimize or avoid the serious adverse effects associated with androgen therapy, selective androgen receptor modulators (SARMs) could be employed to harness the goodness of the AR. A preliminary study based on the network analysis of the genes associated with T2DM has also pointed towards the importance of AR in T2DM.

Gene expression patterns and DNA methylation of neuron and pancreatic β-cell developments in zebrafish embryos treated with bisphenol F and AF

Bisphenol F (BPF) and bisphenol AF (BPAF) are structural analogues of bisphenol A (BPA) that are used in the manufacture of a myriad of BPA-free products; however, there is a paucity of information regarding their developmental effects. The present study investigates the effects of BPF and BPAF on neurodevelopment and pancreatic β-cell differentiation via altering DNA methylation and gene expression patterns using the zebrafish model. BPF and BPAF induced behavioral perturbations: increased average speed, increased maximum acceleration, increased mania time and decreased static time, in 0.3 and 1.0 μM groups in zebrafish embryos. Glucose level was significantly increased in 1.0 μM BPF (28 %); while a monotonic increase of 29 %, 55 %, and 74 % were observed in 0.1, 0.3, and 1.0 μM BPAF, respectively. Consistent with a decreased insulin mRNA level, the expression of two critical transcription factors (pdx-1 and foxa2) essential for the development and functioning of beta-cells decreased following the bisphenols exposure. In addition, embryonic exposure to BPF and BPAF upregulated the transcription of developmental genes (vegfa, wnt8a, and mstn1) and neuron-related genes (mbp, elavl3, gap43, gfap). Also, the expressions of DNA methyltransferases (dnmt1, dnmt3, dnmt4, dnmt5, dnmt6, dnmt7, and dnmt8) were significantly aberrant compared with the control group. The Bisulfite PCR results indicate increased DNA methylation at promoter regions of pdx-1 in BPF (8.2 %) and BPAF (7.6 %); α1-tubulin in BPF (5.3 %) and in BPAF (4.1 %), congruous with the increased dnmt1 and dnmt3 transcription, at early stage of zebrafish development. The present study indicates that zebrafish embryonic exposure to BPF and BPAF elicits islet dysfunction and neuron perturbations resulting in increased DNA methylation levels.

Developments in endoderm and pancreatic β-cell differentiation from human pluripotent stem cells

Human pluripotent stem cells (hPSCs) can differentiate into any cell type in the body, including cells or tissues from ectodermal, mesodermal, or endodermal lineages. Type 1 diabetes is characterized by the selective destruction of β cells in the islets of Langerhans within the pancreas, a process driven by autoimmunity. This destruction leads to the inability to produce and secrete insulin, resulting in poor blood glucose control. Patients with type 1 diabetes require exogenous insulin and regular blood glucose monitoring. However, achieving strict glycemic control solely through this approach is challenging, and there is a risk of side effects such as hypoglycemia when insulin levels rise following exogenous insulin administration. Thus, there is a pressing need for treatments that can overcome these limitations. Stem cell-derived pancreatic β cells are produced through a complex multi-step differentiation process, which involves the application of various growth factors and small molecules to precisely control intracellular signaling pathways at specific times. Our review primarily focused on significant studies concerning the differentiation of hPSCs into highly functional pancreatic β cells, covering key stages of pancreatic development such as definitive endoderm, primitive gut tube, posterior foregut/pancreatic progenitor cells, and pancreatic endocrine cells. Generating insulin-producing functional pancreatic β cells from hPSCs 77could be a viable and renewable cell source for alternative cell therapy to treat type 1 diabetes.

Effect of Akkermansia muciniphila on pancreatic islet β-cell function in rats with prediabetes mellitus induced by a high-fat diet

Prediabetes is an important stage in the development of diabetes. It is necessary to find a safe, effective and sustainable way to delay and reverse the progression of prediabetes. Akkermansia muciniphila (A. muciniphila) is one of the key bacteria associated with glucose metabolism. Recent studies mainly focus on the effect of A. muciniphila on obesity and insulin resistance, but there is no research on the effect of A. muciniphila on pancreatic β-cell function and its mechanism in prediabetes. In this study, we investigated the effects of A. muciniphila on β-cell function, apoptosis and differentiation, as well as its effects on the gut microbiome, intestinal barrier, metaflammation and the expression of Toll-like receptors (TLRs) in a high-fat diet (HFD)-induced prediabetic rat model. The effect of A. muciniphila was compared with dietary intervention. The results showed both A. muciniphila treatment and dietary intervention can reduce metaflammation by repairing the intestinal barrier in rats with prediabetes induced by an HFD and improve β-cell secretory function, apoptosis and differentiation through signaling pathways mediated by TLR2 and TLR4. Additionally, A. muciniphila can further elevate β-cell secretion, attenuate apoptosis and improve differentiation and the TLR signaling pathway on the basis of diet.

New Mechanisms to Prevent Heart Failure with Preserved Ejection Fraction Using Glucagon-like Peptide-1 Receptor Agonism (GLP-1 RA) in Metabolic Syndrome and in Type 2 Diabetes: A Review.

This review examines the impact of obesity on the pathophysiology of heart failure with preserved ejection fraction (HFpEF) and focuses on novel mechanisms for HFpEF prevention using a glucagon-like peptide-1 receptor agonism (GLP-1 RA). Obesity can lead to HFpEF through various mechanisms, including low-grade systemic inflammation, adipocyte dysfunction, accumulation of visceral adipose tissue, and increased pericardial/epicardial adipose tissue (contributing to an increase in myocardial fat content and interstitial fibrosis). Glucagon-like peptide 1 (GLP-1) is an incretin hormone that is released from the enteroendocrine L-cells in the gut. GLP-1 reduces blood glucose levels by stimulating insulin synthesis, suppressing islet α-cell function, and promoting the proliferation and differentiation of β-cells. GLP-1 regulates gastric emptying and appetite, and GLP-1 RA is currently indicated for treating type 2 diabetes (T2D), obesity, and metabolic syndrome (MS). Recent evidence indicates that GLP-1 RA may play a significant role in preventing HFpEF in patients with obesity, MS, or obese T2D. This effect may be due to activating cardioprotective mechanisms (the endogenous counter-regulatory renin angiotensin system and the AMPK/mTOR pathway) and by inhibiting deleterious remodeling mechanisms (the PKA/RhoA/ROCK pathway, aldosterone levels, and microinflammation). However, there is still a need for further research to validate the impact of these mechanisms on humans.

Abstract 4683: Suppression of ARL6ip1 inhibits malignant phenotypes of human colorectal cancer cells in vivo and > in situ

Abstract Introduction: Conophylline is an alkaloid isolated from the leaves of the Thai plant, Ervatamia microphylla. We isolated conophylline as an inhibitor of K-Ras functions and an activator of pancreatic β-cell differentiation. It ameliorates various disease models in animals, including cancer, diabetes mellitus, NASH, and hepatic cirrhosis. On the other hand, its molecular target was determined to be ADP-ribosylation factor-like 6-interacting protein 1 (ARL6ip1) using a conophylline-biotin conjugate. ARL6ip1 is located in the endoplasmic reticulum (ER) membrane, and conophylline binds to the cytoplasmic portion of ARL6ip1. Known functions of ARL6ip1 include inhibition of apoptosis, inhibition of glutamate transporter, and modulation of the ER structure. Previously, we knocked out ARL6ip1 in human colon carcinoma cells by CRISPR-Cas9 and found that either deletion of ARL6ip1 or addition of conophylline inhibited migration, invasion and tumorigenicity in cultured cancer cells. In the present research we have studied the in vivo anticancer effect of conophylline and ARL6ip1 knockout, and the mechanism of anticancer activity in cultured cells. Materials and Methods: Conophylline was isolated from the leaves of Ervatamia microphylla. We employed HCT116 and DLD1 cell lines as human colorectal cancer cells. Cellular migration was measured by a wound-healing assay. The activity of Akt was measured by the phospho-Akt antibody. BALB/cSlc-nu/nu mice were used for the animal experiment. Results: Colon adenocarcinoma tissue microarray comparing tumor and adjacent normal tissues revealed that ARL6ip1 expression was significantly higher in cancer than in normal tissues. Intraperitoneal administration of conophylline and knockout of ARL6ip1 both inhibited the growth of HCT116 in mice. Neither influenced the bodyweight of animals. Conophylline and deletion of ARL6ip1 both inhibited the migration. Rescue of ARL6ip1 canceled the effect of ARL6ip1 deletion on migration in HCT116 and DLD1 cells. As the mechanism, either conophylline or ARL6ip1 knockout inhibited the Akt activity, which was canceled by the rescue of ARL6ip1 in both cell lines. Conclusion: ARL6ip1 was found to be highly expressed in the tumor tissue in colorectal cancer. CRISPR-Cas9 knockout of ARL6ip1 showed a similar anticancer activity as treatment with conophylline in the animal experiment. It is likely that the anticancer activity of conophylline would be mediated by Akt. Thus, ARL6ip1 would be a useful molecular target for the treatment of cancer. Citation Format: Yinzhi Lin, Sivasundaram Karnan, Hideaki Ito, Kazuo Umezawa. Suppression of ARL6ip1 inhibits malignant phenotypes of human colorectal cancer cells in vivo and > in situ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4683.

Evaluation of Pancreatic β-cell Differentiation Efficiency of Human iPSC Lines for Clinical Use

Transplantation of pancreatic β-cells generated from human induced pluripotent stem cells (hiPSCs) has great potential as a root treatment for type 1 diabetes. However, their current level of efficiency to differentiate into β-cells is still not at par for clinical use. Previous research has shown that differentiation efficiency varies among human embryonic stem cells and mouse-induced pluripotent stem cell lines. Therefore, selecting a suitable cell line for efficient induction into desired tissues and organs is crucial. In this study, we have evaluated the efficiency of 15 hiPSC lines available for clinical use to differentiate into pancreatic β-cells. Our investigation has revealed induction efficiency to differ among the hiPSC lines, even when derived from the same donor. Among the hiPSC lines tested, the 16A01 cell line exhibited the highest Insulin expression and low Glucagon expression, suggesting that this cell line is suitable for differentiation into β-cells. Our study has demonstrated the importance of selecting a suitable hiPSC line for effective differentiation into β-cells.

Development, regeneration, and physiological expansion of functional β-cells: Cellular sources and regulators.

Pancreatic regeneration is a complex process observed in both normal and pathological conditions. The aim of this review is to provide a comprehensive understanding of the emergence of a functionally active population of insulin-secreting β-cells in the adult pancreas. The renewal of β-cells is governed by a multifaceted interaction between cellular sources of genetic and epigenetic factors. Understanding the development and heterogeneity of β-cell populations is crucial for functional β-cell regeneration. The functional mass of pancreatic β-cells increases in situations such as pregnancy and obesity. However, the specific markers of mature β-cell populations and postnatal pancreatic progenitors capable of increasing self-reproduction in these conditions remain to be elucidated. The capacity to regenerate the β-cell population through various pathways, including the proliferation of pre-existing β-cells, β-cell neogenesis, differentiation of β-cells from a population of progenitor cells, and transdifferentiation of non-β-cells into β-cells, reveals crucial molecular mechanisms for identifying cellular sources and inducers of functional cell renewal. This provides an opportunity to identify specific cellular sources and mechanisms of regeneration, which could have clinical applications in treating various pathologies, including in vitro cell-based technologies, and deepen our understanding of regeneration in different physiological conditions.

Downregulation of Sirt3 contributes to β-cell dedifferentiation via FoxO1 in type 2 diabetic mellitus.

FoxO1 is an important factor in the β-cell differentiation in type 2 diabetes mellitus (T2DM). Sirt3 is found to be involved in FoxO1 function. This study investigated the role of Sirt3 in the β-cell dedifferentiation and its mechanism. Twelve-week-old db/db mice and INS1 cells transfected with Sirt3-specific short hairpin RNA (shSirt3) were used to evaluate the dedifferentiation of β-cell. Insulin levels were measured by enzyme linked immunosorbent assay. The proteins of Sirt3, T-FoxO1, Ac-FoxO1 and differentiation indexes such as NGN3, OCT4, MAFA were determined by western blot or immunofluorescence staining. The combination of Sirt3 and FoxO1 was determined by the co-immunoprecipitation assay. The transcriptional activity of FoxO1 was detected by dual luciferase reporter assay. Both the in vivo and in vitro results showed that Sirt3 was decreased along with β-cell dedifferentiation and decreased function of insulin secretion under high glucose conditions. When Sirt3 was knocked down in INS1 cells, increased β-cell dedifferentiation and lowered insulin secretion were observed. This effect was closely related to the amount loss and the decreased deacetylation of FoxO1, which resulted in a reduction in transcriptional activity. Downregulation of Sirt3 contributes to β-cell dedifferentiation in high glucose via FoxO1. Intervention of Sirt3 may be an effective approach to prevent β-cell failure in T2DM.

In Silico Functional and Structural Analysis of Non-synonymous Single Nucleotide Polymorphisms (nsSNPs) in Human Paired Box 4 Gene.

In human genome, members of Paired box (PAX) transcription factor family are highly sequence-specific DNA-binding proteins. Among PAX gene family members, PAX4 gene has significant role in growth, proliferation, differentiation, and insulin secretion of pancreatic β-cells. Single nucleotide polymorphisms (SNPs) in PAX4 gene progress in the pathogenesis of various human diseases. Hence, the molecular mechanism of how these SNPs in PAX4 gene significantly progress diseases pathogenesis needs to be elucidated. For the reason, a series of bioinformatic analyzes were done to identify the SNPs of PAX4 gene that contribute in diseases pathogenesis. From the analyzes, 4145 SNPs (rsIDs) in PAX4 gene were obtained, where, 362 missense (8.73%), 169 synonymous (4.08%), and 2323 intron variants (56.04%). The rest SNPs were unspecified. Among the 362 missense variants, 118 nsSNPs were found as deleterious in SIFT analysis. Among those, 25 nsSNPs were most probably damaging and 23 were deleterious as observed in PolyPhen-2 and PROVEAN analyzes, respectively. Following all analyzes, 14 nsSNPs (rs149708455, rs115887120, rs147279315, rs35155575, rs370095957, rs373939873, rs145468905, rs121917718, rs2233580, rs3824004, rs372751660, rs369459316, rs375472849, rs372497946) were common and observed as deleterious, probably damaging, affective and diseases associated. Following structural analyzes, 11 nsSNPs guided proteins were found as most unstable and highly conserved. Among these, R20W, R39Q, R45Q, R60H, G65D, and A223D mutated proteins were highly harmful. Hence, the results from above-mentioned integrated comprehensive bioinformatic analyzes guide how different nsSNPs in PAX4 gene alter structural and functional characteristics of the protein that might progress diseases pathogenesis in human including type 2 diabetes.

LncRNA Malat1 regulates iPSC-derived β-cell differentiation by targeting the miR-15b-5p/Ihh axis

BackgroundDifferentiation of induced pluripotent stem cells (iPSCs)-derived β-like cells is a novel strategy for treatment of type 1 diabetes. Elucidation of the regulatory mechanisms of long noncoding RNAs (lncRNAs) in β-like cells derived from iPSCs is important for understanding the development of the pancreas and pancreatic β-cells and may improve the quality of β-like cells for stem cell therapy. Methodsβ-like cells were derived from iPSCs in a three-step protocol. RNA sequencing and bioinformatics analysis were carried out to screen the differentially expressed lncRNAs and identify the putative target genes separately. LncRNA Malat1 was chosen for further research. Series of loss and gain of functions experiments were performed to study the biological function of LncRNA Malat1. Quantitative real-time PCR (qRT-PCR), Western blot (WB) analysis and immunofluorescence (IF) staining were carried out to separately detect the functions of pancreatic β-cells at the mRNA and protein levels. Cytoplasmic and nuclear RNA fractionation and fluorescence in situ hybridization (FISH) were used to determine the subcellar location of lncRNA Malat1 in β-like cells. Enzyme-linked immunosorbent assays (ELISAs) were performed to examine the differentiation and insulin secretion of β-like cells after stimulation with different glucose concentrations. Structural interactions between lncRNA Malat1 and miR-15b-5p and between miR-15b-5p/Ihh were detected by dual luciferase reporter assays (LRAs). ResultsWe found that the expression of lncRNA Malat1 declined during differentiation, and overexpression (OE) of lncRNA Malat1 notably impaired the differentiation and maturation of β-like cells derived from iPSCs in vitro and in vivo. Most importantly, lncRNA Malat1 could function as a competing endogenous RNA (ceRNA) of miR-15b-5p to regulate the expression of Ihh according to bioinformatics prediction, mechanistic analysis and downstream experiments. ConclusionThis study established an unreported regulatory network of lncRNA Malat1 and the miR-15b-5p/Ihh axis during the differentiation of iPSCs into β-like cells. In addition to acting as an oncogene promoting tumorigenesis, lncRNA Malat1 may be an effective and novel target for treatment of diabetes in the future.

85-OR: Swi/Snf Controls Transcriptional Programs Required for ß-Cell Development and Postnatal Islet Function

A key feature of diabetes is insufficient functional β-cell mass. Strategies to augment functional β-cell mass include directed differentiation of stem cells towards a β-cell fate, which requires extensive knowledge of transcriptional programs governing differentiation in vivo. Pdx1, an essential transcription factor involved in β-cell differentiation, dynamically recruits coregulators to drive different gene expression programs. A recent study identified the ATP-dependent Swi/Snf chromatin remodeling complex as an important Pdx1-interacting partner. In the developing pancreas, conditional removal of the Brg Swi/Snf ATPase subunit compromised final pancreas mass, and loss of both ATPase subunits, Brg1 and Brm, led to glucose dyshomeostasis. While Swi/Snf is essential during early pancreas development and mature β-cell function, the role of Swi/Snf during endocrine cell development has yet to be explored. Here, we generated a conditional knockout of either Brg1 (Brg1ΔendoBrm+/-), Brm (Brg1Δendo/+Brm-/-), or both subunits (DKOΔendo) during endocrine development using Neurogenin 3 Cre. Four-week-old Brg1ΔendoBrm+/- mice are glucose intolerant, hyperglycemic, and hypoinsulinemic, with no phenotype observed in Brg1Δendo/+Brm-/-mice. Impaired glucose homeostasis of Brg1ΔendoBrm+/- mice was due in part to reduced islet number, impaired insulin secretion, and altered gene expression programs. DKOΔendo mice have not been found at weaning; however, postnatal day 6 DKOΔendo mice are severely hyperglycemic with reduced plasma insulin levels. Single-cell RNA-Seq of embryonic day 15.5 lineage-labeled cells revealed reduced insulin and glucagon transcript in the β-cell and α-cell clusters, respectively, in both Brg1ΔendoBrm+/- and DKOΔendo mice and reduced Neurogenin 3­ transcript in DKOΔendo endocrine progenitor clusters. These data suggest Swi/Snf plays a critical role in governing gene-expression programs essential for endocrine cell development. Disclosure R.K.Davidson: Employee; Eli Lilly and Company. S.Kanojia: None. M.E.Osmulski: None. J.Spaeth: None. Funding National Institutes of Health (R01DK129287, R03DK127129)

86-OR: Self-Organized Insulin-Producing ß-Cells Differentiated from Human Omentum-Derived Stem Cells

Human omentum-derived mesenchymal stem cells (hO-MSCs) possess a great potential to differentiate into multiple lineages and have self-renewal capacities, allowing them to be utilized as patient-specific cell-based therapeutics. Although various stem cell-derived insulin-producing β-cells have been proposed for the treatment of diabetes mellitus, developing an efficient method to establish highly functionalized insulin-producing β-cells remains a challenging task. Here, we aimed to develop a novel cell culture platform that could regulate important cell-cell/cell-matrix interactions by introducing a fibroblast growth factor 2 (FGF2)-immobilized matrix that can support cell adhesion, proliferation, and differentiation of hO-MSCs into insulin-producing β-cells. Our initial findings demonstrated that cells cultured on an FGF2-immobilized matrix were able to self-organize into insulin-producing β-cell-like progenitors, as evident from the upregulation of pancreatic β-cell-specific markers such as PDX-1, Insulin, and Glut-2. Furthermore, heparan sulfate proteoglycan, gap junction proteins including Cx36 and Cx43, and cell adhesion molecules such as E-cadherin and Ncam1 were significantly upregulated, leading to cell maturation and insulin secretion in the pancreatic niche environment. These results suggest that our novel platform could promote β-cell differentiation and glucose-stimulated insulin secretion from hO-MSCs by enhancing cell-cell junction proteins such as connexins (Cx36, Cx43), E-cadherin, and N-cam. Such a cell culture platform can offer novel strategies to obtain functional pancreatic β-cells from a patient-specific cell sources, ultimately enabling better treatments for diabetes mellitus. Disclosure Y.Hwang: None. Funding National Research Foundation of Korea (2019R1A5A8083404)

Molecular characterization and re-interpretation of HNF1A variants identified in Indian MODY subjects towards precision medicine.

HNF1A is an essential component of the transcription factor network that controls pancreatic β-cell differentiation, maintenance, and glucose stimulated insulin secretion (GSIS). A continuum of protein malfunction is caused by variations in the HNF1A gene, from severe loss-of-function (LOF) variants that cause the highly penetrant Maturity Onset Diabetes of the Young (MODY) to milder LOF variants that are far less penetrant but impart a population-wide risk of type 2 diabetes that is up to five times higher. Before classifying and reporting the discovered variations as relevant in clinical diagnosis, a critical review is required. Functional investigations offer substantial support for classifying a variant as pathogenic, or otherwise as advised by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) ACMG/AMP criteria for variant interpretation. To determine the molecular basis for the variations in the HNF1A gene found in patients with monogenic diabetes in India. We performed functional protein analyses such as transactivation, protein expression, DNA binding, nuclear localization, and glucose stimulated insulin secretion (GSIS) assay, along with structural prediction analysis for 14 HNF1A variants found in 20 patients with monogenic diabetes. Of the 14 variants, 4 (28.6%) were interpreted as pathogenic, 6 (42.8%) as likely pathogenic, 3 (21.4%) as variants of uncertain significance, and 1 (7.14%) as benign. Patients harboring the pathogenic/likely pathogenic variants were able to successfully switch from insulin to sulfonylureas (SU) making these variants clinically actionable. Our findings are the first to show the need of using additive scores during molecular characterization for accurate pathogenicity evaluations of HNF1A variants in precision medicine.

Heterogeneity of Islet Cells during Embryogenesis and Differentiation.

Diabetes is caused by insufficient insulin secretion due to β-cell dysfunction and/or β-cell loss. Therefore, the restoration of functional β-cells by the induction of β-cell differentiation from embryonic stem (ES) and induced-pluripotent stem (iPS) cells, or from somatic non-β-cells, may be a promising curative therapy. To establish an efficient and feasible method for generating functional insulin-producing cells, comprehensive knowledge of pancreas development and β-cell differentiation, including the mechanisms driving cell fate decisions and endocrine cell maturation is crucial. Recent advances in single-cell RNA sequencing (scRNA-seq) technologies have opened a new era in pancreas development and diabetes research, leading to clarification of the detailed transcriptomes of individual insulin-producing cells. Such extensive high-resolution data enables the inference of developmental trajectories during cell transitions and gene regulatory networks. Additionally, advancements in stem cell research have not only enabled their immediate clinical application, but also has made it possible to observe the genetic dynamics of human cell development and maturation in a dish. In this review, we provide an overview of the heterogeneity of islet cells during embryogenesis and differentiation as demonstrated by scRNA-seq studies on the developing and adult pancreata, with implications for the future application of regenerative medicine for diabetes.