Scale Up Manufacturing Approach for Production of Human Induced Pluripotent Stem Cell-Derived Islets Using Vertical Wheel®Bioreactors
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE266878 | GEO | 2025/05/30
REPOSITORIES: GEO
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Project description:We profiled the transcritpome and ATAC profiles of human pancreatic islets generated from pluripotent stem cells. Multiomic profiling was also performed on primary human islets and in vivo matured SC-islets for comparision. We catalogued the ATAC associated signatures for each cell types in SC-islets and compared them to their human primiary islet counterparts. In vivo maturation of SC-islets were also compared with in vitro SC-islets. In this study, we identified key regulators associated with islet identity during differentiation and maturation. Gene manipulation of CTCF affects differentiating SC-islet cell fate to enteroendocrine-like lineage. ARID1B knockdown caueses islet cells to present mature signatures. These gene altered SC-islets were also sequenced.
2023-03-25 | GSE199636 | GEO
Project description:Aims/Introduction: Metformin treatment for hyperglycemia in pregnancy (HIP) beneficially improves maternal glucose metabolism and reduces perinatal complications. However, metformin could impede pancreatic β cell development via impaired mitochondrial function. A new anti-diabetes drug imeglimin, developed based on metformin, improves mitochondrial function. Here we examine the effect of imeglimin on β cell differentiation using human induced pluripotent stem cell (iPSC)-derived pancreatic islet-like spheroid (SC-islet) models. Materials and Methods: Human iPSCs are differentiated into SC-islets by three-dimensional culture with and without imeglimin or metformin. Differentiation efficiencies of SC-islets were analyzed by flow cytometry, immunostaining, quantitative PCR, and insulin secretion assay. RNA sequencing and oxygen consumption rate were obtained for further characterization of SC-islets. SC-islets were cultured with proinflammatory cytokines, in part mimicking the uterus environment in HIP. Results: Metformin perturbed SC-islet differentiation while imeglimin did not alter it. Furthermore, imeglimin enhanced the gene expressions of β cell lineage markers. Maintenance of mitochondrial function and optimization of TGF-β and Wnt signaling were considered potential mechanisms for augmented β cell maturation by imeglimin. In the presence of proinflammatory cytokines, imeglimin ameliorated β cell differentiation impaired by cytokines and metformin. Conclusions: Imeglimin does not perturb differentiation of SC-islet cells and rather enhances gain in β cell identity gene sets in contrast to metformin. This may lead to the improvement of in vitro β cell differentiation protocols.
2025-01-20 | GSE242776 | GEO
Project description:Pluripotent stem cell-derived (SC) islets offer a renewable source for β cell replacement for type 1 diabetes, yet functional and metabolic immaturity may limit their long-term therapeutic efficacy. Here, we describe that limitations in mitochondrial transcriptional programming prevent the maturation of SC-β cells. We observe several alterations in SC-islet mitochondria compared to human islets, including reductions in mitochondrial respiration, expression of OXPHOS machinery, and mitochondrial lipid metabolism. Surprisingly, these effects were not related to reductions in mitochondrial mass, genome integrity, or structure. Transcriptomic profiling throughout differentiation to SC-islets revealed several unstudied candidates in β cell maturation that regulate programming of mitochondrial oxidative and fatty acid metabolism, including the nuclear receptors PPAR⍺ and PPARγ. Indeed, treatment of SC-islets with WY14643, a potent PPAR⍺ agonist with PPARγ activity, promoted expression of mitochondrial targets, improved insulin secretion, and increased the presence of β cells both in vitro and upon transplantation into immunodeficient mice. Mitochondria are vital to fuel β cell insulin release; however, our studies here revealed the potential for targeting mitochondrial programming to enhance the differentiation and metabolic maturation of SC-β cells.
2025-01-15 | GSE270220 | GEO
Project description:Islet transplantation for treatment of diabetes is limited by availability of donor islets and requirements for immunosuppression. Stem cell-derived islets might circumvent these issues. SC-islets effectively control glucose metabolism post transplantation, but do not yet achieve full function in vitro with currently published differentiation protocols. We aimed to identify markers of mature subpopulations of SC-β cells by studying transcriptional changes associated with in vivo maturation of SC-β cells using RNA-seq and co-expression network analysis. The β cell-specific hormone islet amyloid polypeptide (IAPP) emerged as the top candidate to be such a marker. IAPP+ cells had more mature β cell gene expression and higher cellular insulin content than IAPP- cells in vitro. IAPP+ INS+ cells were more stable in long-term culture than IAPP- INS+ cells and retained insulin expression after transplantation into mice. Finally, we conducted a small molecule screen to identify compounds that enhance IAPP expression. Aconitine up-regulated IAPP and could help to optimize differentiation protocols.
2024-04-15 | GSE263366 | GEO
Project description:Stem cell-derived islet (SC-islet) cell therapy holds immense potential for the treatment of Type 1 Diabetes. However, low oxygen supply leads to cell dysfunction post-transplantation, especially in subcutaneous spaces and encapsulation devices. The response of SC-islets to hypoxia and effective strategies to alleviate its detrimental effects remain poorly understood. This study investigates the impact of hypoxia on SC-islets and elucidate the dynamics of hypoxia-induced stress. We performed transcriptional profiling of human SC-islets exposed to hypoxic conditions, and drew the transcriptomic and epigenetic profiles of single cells. Our findings demonstrate that β cells within SC-islets gradually undergo a decline in cell identity and metabolic function in response to hypoxia. The loss of expression from immediate early genes, specifically EGR1, FOS, and JUN, results in the downregulation of key transcription factors (TFs) that are essential for maintaining SC-islet cell identity under hypoxic conditions. By comparing SC-islets under low and high oxygen conditions, we identified genes that play a role in maintaining the fitness of SC-islets in a low-oxygen environment. Notably, the expression of EDN3, a potent vasoconstrictor gene that is enriched in native pancreatic β cells, significantly aids in preserving β cell identity under hypoxic conditions. Elevated expression of EDN3 in SC-islets effectively mitigated the deleterious effects of hypoxia by modulating genes involved in SC-islet maturation including genes associated with glucose sensing and regulation in β cells. These insights improve the understanding of SC-islets under hypoxic conditions, offering a potential point of intervention for future clinical applications in the treatment of Type 1 Diabetes
2025-03-24 | GSE263130 | GEO
Project description:Stem cell-derived islet (SC-islet) organoids offer hope for cell replacement therapy in diabetes, but their immature function remains a challenge. Mature islet function requires the β-cell circadian clock, yet how the clock regulates maturation is unclear. Here, we show that a circadian transcription factor specific to maturing SC-β cells, DEC1, regulates insulin responsiveness to glucose. SC-islet organoids form normally from DEC1-ablated human pluripotent stem cells, but their insulin release capacity and glucose threshold fail to increase during in vitro culture and upon transplant. This deficit reflects downregulation of maturity-linked effectors of glucose utilization and insulin exocytosis, blunting glycolytic and oxidative metabolism, and is rescued by increasing metabolic flux. Moreover, DEC1 is needed to boost SC-islet maturity by synchronizing circadian glucose-responsive insulin secretion rhythms and clock machinery. Thus, DEC1 links circadian control to human β-cell maturation, highlighting its vitality to foster fully functional SC-islets.
2025-07-24 | GSE302939 | GEO
Project description:Using quantitative phosphoproteomic analysis of human islets and human stem-cell-derived islets (SC-islets), we show that mTORC1 inhibition with Torin-1 or rapamycin induces significant alterations in the phosphorylation profile of human islet cells.
2025-05-12 | PXD062389 | Pride
Project description:Background: Thioredoxin-interacting protein (TXNIP) regulates endoplasmic reticulum (ER) and oxidative stress, impairing glucose homeostasis in diabetes. However, it is unclear if TXNIP deficiency can improve differentiation or functionality of human stem cell-derived somatic metabolic cells. Methods: Using CRISPR-Cas12a genome editing, we generated TXNIP-deficient (TXNIP-/-) H1 human embryonic stem cells (H1-hESCs). These cells were differentiated into hepatocyte-like cells (HLCs) and stem-cell-derived insulin-producing islets (SC-islets). TXNIP-/- and TXNIP+/+ SC-islet maturation and functionality was assessed by implantation under the kidney capsule of NOD-SCID mice. Results: TXNIP deficiency significantly increased H1-hESC proliferation without affecting pluripotency, viability, or differentiation potential into HLCs and SC-islets. Bulk RNA-sequencing of thapsigargin-treated TXNIP-/- and TXNIP+/+ hESCs revealed differential expression of stress-responsive genes, with enriched apoptosis-related pathways in TXNIP+/+ cells but minimal transcriptional changes specific to TXNIP deficiency. In HLCs, TXNIP deletion reduced albumin secretion and insulin signalling, as indicated by decreased AKT phosphorylation, while showing no differences in glycolytic activity or lipid metabolism markers. Under thapsigargin-induced ER stress, TXNIP-/- HLCs exhibited transiently reduced eIF2α phosphorylation and lower BiP expression, suggesting compromised adaptive responses to prolonged stress. SC-islets derived from TXNIP-/- hESCs showed comparable viability, endocrine cell composition, and cytokine responses to TXNIP+/+ islets. Upon IFNα and IFNγ treatment, STAT1 phosphorylation was increased in TXNIP-/- SC-islets, indicating that IFN signalling remains active despite TXNIP deficiency. TXNIP-/- or TXNIP+/+ islets SC-islets implanted into NOD-SCID mice produced human C-peptide and responded to glucose stimulation. However, TXNIP-/- SC-islets showed no enhancement in glycaemic control or glucose-stimulated insulin secretion compared to controls. Conclusions: Our study demonstrates that TXNIP deficiency does not improve the differentiation of HLCs and SC-islets. We report the generation and characterization of TXNIP-/- and TXNIP+/+ H1-hESCs, HLCs and SC-islets as robust models for future studies of TXNIP’s role in metabolic cell biology.
2025-05-14 | GSE284753 | GEO
Project description:Stem cell-derived islets (SC-islets) hold significant promise for treating diabetes, but optimizing their functional maturity remains a challenge. CD49a, an integrin subunit involved in cell adhesion and signaling, has been identified as a potential marker associated with islet cell differentiation. In this study, we employed magnetic-activated cell sorting (MACS) to isolate CD49a-enriched and CD49a-depleted populations from SC-islets. These two distinct populations were evaluated for functional and molecular differences using single-cell RNA sequencing (scRNA-seq), in vitro glucose-stimulated insulin secretion (GSIS), and in vivo transplantation. Our scRNA-seq analysis revealed distinct transcriptional profiles between the CD49a-enriched and CD49a-depleted populations, with the CD49a-enriched population exhibiting higher expression of key β cell lineage markers, including insulin. Functional assays demonstrated that CD49a-enriched SC-islets had significantly improved GSIS, indicative of enhanced β cell functionality. Additionally, when transplanted into mice, the CD49a-enriched SC-islets exhibited superior graft performance, as evidenced by human C-peptide secretion. These findings support that CD49a enrichment through MACS represents a promising approach to enhance the functional maturity of SC-islets. Further investigations into the role of CD49a in islet cell differentiation and function may facilitate the development of more effective stem cell-based therapies for diabetes.
2025-01-28 | GSE278800 | GEO
Project description:Objective: The developmental effects of mutations in genes associated with monogenic diabetes on human pancreas development is not well understood. More specifically, if insulin gene recessive mutations influence the human endocrine lineage segregation still needs to be investigated. Methods: We generated a novel knock-in H2B-Cherry reporter human induced pluripotent stem cell (iPSCs) line expressing no insulin upon differentiation to stem cell-derived (SC-) β cells in vitro. This cell line enabled us to have a model mimicking the extremely reduced insulin levels in patients with recessive insulin mutations. We combined immunostaining, Western blotting and proteomics analysis to characterize the SC-islets from this iPSC line. Furthermore, we leveraged FACS analysis and imaging to explore the impact of insulin shortage on human endocrine cell induction, composition and proliferation. Results: We found that lack of insulin hampers insulin receptor (IR) signaling in SC-islets but increases the IR sensitivity. Furthermore, insulin deficiency showed no effects on human endocrine lineage induction. However, lack of insulin skewed the SC-islet cell composition. We found an increased in SC-β cell number at the expense of SC-α cell differentiation in the absence of insulin. Finally, insulin shortage reduced the rate of SC-β cell proliferation but had no impact of the expansion of SC-α cells. Conclusions: We provided evidence of the developmental impacts of reduced insulin levels on human β cell characteristics and endocrine lineage formation. These findings help to better understand the pathomechanisms of recessive insulin mutations during embryonic development and also shed some lights on the possible physiological function of this hormone coordinating human islet cell composition and architecture during endocrinogenesis.
2024-01-17 | PXD045366 | Pride