scholarly journals Assessment of the in vitro function of human stem cell-derived β cells

2019 ◽  
Author(s):  
Arvind R. Srivatsava ◽  
Stefanie T. Shahan ◽  
Lisa C. Gutgesell ◽  
Leonardo Velazco-Cruz ◽  
Jeffrey R. Millman
Keyword(s):  
Stem Cell ◽  
Insulin Secretion ◽  
High Glucose ◽  
Embryonic Stem ◽  
Ion Concentration ◽  
Cellular Respiration ◽  
Second Phase ◽  
Cytoplasmic Calcium ◽  
Β Cells

AbstractInsulin-producing human embryonic stem cell-derived β (SC-β) cells are a promising cell source for diabetes cell replacement therapy. We have recently reported a differentiation strategy that produces SC-β cells in islet organoids that not only undergo glucose-stimulated insulin secretion but also have an islet-like dynamic insulin release profile, displaying both first and second phase insulin secretion. The goal of this study was to further characterize the functional profile of these SC-β cells in vitro. We utilized a Seahorse extracellular flux analyzer to measure mitochondrial respiration of SC-β cells at low and high glucose. We also used photolithography to fabricate a microfluidic device containing microwells to immobilize SC-β cells for perfusional analysis, monitoring cytoplasmic calcium using Fluo-4 AM at low and high glucose. Here we find that in addition to increased insulin secretion, SC-β cells have increased cellular respiration and cytoplasmic calcium ion concentration in response to a high glucose stimulation. Our results indicate that SC-β cells have similar function to that reported for islets, providing further performance characterization that could help with eventual development for diabetes cell therapy and drug screening.

Differential effects of microtubule-altering agents on beta-cells during development

1983 ◽  
Vol 245 (4) ◽  
pp. E391-E400
Author(s):  
R. S. Hill ◽  
W. B. Rhoten
Keyword(s):  
Insulin Secretion ◽  
Beta Cells ◽  
Insulin Release ◽  
High Glucose ◽  
Deuterium Oxide ◽  
Inhibitory Effect ◽  
Secretory Response ◽  
Second Phase ◽  
Insulin Secretory Response

The effect of microtubule-altering agents on the insulin secretory response to glucose during the perinatal period was investigated with an in vitro perifusion system. Rat pancreatic mince from day 17 of gestation (D17G) to day 6 postnatally (D6PN) were perifused for 60 min in basal glucose followed by 45 min with high glucose (3.5 mg/ml) or with high glucose plus 10 mM arginine (D17G). The two phases of insulin secretion in response to high glucose developed in an age-dependent and asynchronous manner. The first phase matured between D17G and D18G, and maturation of the second phase occurred subsequently. Vinblastine (VB) (20 or 100 microM) had a differential effect on the insulin secretory response. VB did not inhibit stimulated insulin release at D17G. This absence of an inhibitory effect of VB at D17G could not be explained by the absence of polymerized tubulin because microtubules were present in the control beta-cells and, in addition, VB treatment resulted in the formation of paracrystalline deposits. Subsequently in development, and with isolated islets of the adult, VB inhibited stimulated insulin release. Heavy water (deuterium oxide, D2O) inhibited stimulated insulin secretion at D17G but blocked completely insulin release from the near-term beta-cell. The inhibition of insulin secretion by D2O was rapidly reversed when water replaced D2O in the perifusion media. The results indicate that the maturation of the second phase of insulin secretion coincides with the ability of the microtubule-altering agents to modify the insulin secretory response. One possible explanation for these findings is that at D17G the microtubules are not coupled physicochemically to other molecules or structures necessary for their role in insulin secretion to be expressed fully.

scholarly journals MAFA and T3 Drive Maturation of Both Fetal Human Islets and Insulin-Producing Cells Differentiated From hESC

2015 ◽  
Vol 100 (10) ◽  
pp. 3651-3659 ◽  
Author(s):  
Cristina Aguayo-Mazzucato ◽  
Amanda DiIenno ◽  
Jennifer Hollister-Lock ◽  
Christopher Cahill ◽  
Arun Sharma ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Islet Cells ◽  
Embryonic Stem ◽  
Human Islets ◽  
Insulin Content ◽  
Β Cells ◽  
Β Cell ◽  
Functional Maturation ◽  
Glucose Responsiveness

Context: Human embryonic stem cells (hESCs) differentiated toward β-cells and fetal human pancreatic islet cells resemble each other transcriptionally and are characterized by immaturity with a lack of glucose responsiveness, low levels of insulin content, and impaired proinsulin-to-insulin processing. However, their response to stimuli that promote functionality have not been compared. Objective: The objective of the study was to evaluate the effects of our previous strategies for functional maturation developed in rodents in these two human models of β-cell immaturity and compare their responses. Design, Settings, Participants, and Interventions: In proof-of-principle experiments using either adenoviral-mediated overexpression of V-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA) or the physiologically driven path via thyroid hormone (T3) and human fetal islet-like cluster (ICC) functional maturity was evaluated. Then the effects of T3 were evaluated upon the functional maturation of hESCs differentiated toward β-cells. Main Outcome Measures: Functional maturation was evaluated by the following parameters: glucose responsiveness, insulin content, expression of the mature β-cell transcription factor MAFA, and proinsulin-to-insulin processing. Results: ICCs responded positively to MAFA overexpression and T3 treatment as assessed by two different maturation parameters: increased insulin secretion at 16.8 mM glucose and increased proinsulin-to-insulin processing. In hESCs differentiated toward β-cells, T3 enhanced MAFA expression, increased insulin content (probably mediated by the increased MAFA), and increased insulin secretion at 16.8 mM glucose. Conclusion: T3 is a useful in vitro stimulus to promote human β-cell maturation as shown in both human fetal ICCs and differentiated hESCs. The degree of maturation induced varied in the two models, possibly due to the different developmental status at the beginning of the study.

scholarly journals Hydrogel platform for in vitro three-dimensional assembly of human stem cell-derived β cells and endothelial cells

2019 ◽  
Author(s):  
Punn Augsornworawat ◽  
Leonardo Velazco-Cruz ◽  
Jiwon Song ◽  
Jeffrey R. Millman
Keyword(s):  
Endothelial Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Human Embryonic Stem Cell ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Cell Types ◽  
Diabetic Patients ◽  
Β Cells

AbstractDifferentiation of stem cells into functional replacement cells and tissues is a major goal of the regenerative medicine field. However, one limitation has been organization of differentiated cells into multi-cellular, three-dimensional assemblies. The islets of Langerhans contain many endocrine and non-endocrine cell types, such as insulin-producing β cells and endothelial cells. Transplantation of exogenous islets into diabetic patients can serve as a cell replacement therapy, replacing the need for patients to inject themselves with insulin, but the number of available islets from cadaveric donors is low. We have developed a strategy of assembling human embryonic stem cell-derived β cells with endothelial cells into three-dimensional aggregates on a hydrogel. The resulting islet organoids express β cell markers and are functional, capable of undergoing glucose-stimulated insulin secretion. These results provide a platform for evaluating the effects of the islet tissue microenvironment on human embryonic stem cell-derived β cells and other islet endocrine cells to develop tissue engineered islets.

scholarly journals Capsaicin Analogues Derived from n-3 Polyunsaturated Fatty Acids (PUFAs) Reduce Inflammatory Activity of Macrophages and Stimulate Insulin Secretion by β-Cells In Vitro

Nutrients ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 915 ◽  
Author(s):  
Erika Cione ◽  
Pierluigi Plastina ◽  
Attilio Pingitore ◽  
Mariarita Perri ◽  
Maria Cristina Caroleo ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Insulin Secretion ◽  
Polyunsaturated Fatty Acids ◽  
Second Phase ◽  
Β Cells ◽  
Monocyte Chemoattractant Protein 1 ◽  
Raw264.7 Macrophages ◽  
Inflammatory Protein ◽  
Capsaicin Analogues

In this study, two capsaicin analogues, N-eicosapentaenoyl vanillylamine (EPVA) and N-docosahexaenoyl vanillylamine (DHVA), were enzymatically synthesized from their corresponding n-3 long chain polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), both dietary relevant components. The compounds significantly reduced the production of some lipopolysaccharide (LPS)-induced inflammatory mediators, including nitric oxide (NO), macrophage-inflammatory protein-3α (CCL20) and monocyte chemoattractant protein-1 (MCP-1 or CCL2), by RAW264.7 macrophages. Next to this, only EPVA increased insulin secretion by pancreatic INS-1 832/13 β-cells, while raising intracellular Ca2+ and ATP concentrations. This suggests that the stimulation of insulin release occurs through an increase in the intracellular ATP/ADP ratio in the first phase, while is calcium-mediated in the second phase. Although it is not yet known whether EPVA is endogenously produced, its potential therapeutic value for diabetes treatment merits further investigation.

scholarly journals Stem Cell-Based Clinical Trials for Diabetes Mellitus

2021 ◽  
Vol 12 ◽  
Author(s):  
Eleonora de Klerk ◽  
Matthias Hebrok
Keyword(s):  
Clinical Trials ◽  
Stem Cell ◽  
Islet Transplantation ◽  
Type I Diabetes ◽  
Embryonic Stem ◽  
Insurance Companies ◽  
Type I ◽  
Β Cells ◽  
The Impact

Since its introduction more than twenty years ago, intraportal allogeneic cadaveric islet transplantation has been shown to be a promising therapy for patients with Type I Diabetes (T1D). Despite its positive outcome, the impact of islet transplantation has been limited due to a number of confounding issues, including the limited availability of cadaveric islets, the typically lifelong dependence of immunosuppressive drugs, and the lack of coverage of transplant costs by health insurance companies in some countries. Despite improvements in the immunosuppressive regimen, the number of required islets remains high, with two or more donors per patient often needed. Insulin independence is typically achieved upon islet transplantation, but on average just 25% of patients do not require exogenous insulin injections five years after. For these reasons, implementation of islet transplantation has been restricted almost exclusively to patients with brittle T1D who cannot avoid hypoglycemic events despite optimized insulin therapy. To improve C-peptide levels in patients with both T1 and T2 Diabetes, numerous clinical trials have explored the efficacy of mesenchymal stem cells (MSCs), both as supporting cells to protect existing β cells, and as source for newly generated β cells. Transplantation of MSCs is found to be effective for T2D patients, but its efficacy in T1D is controversial, as the ability of MSCs to differentiate into functional β cells in vitro is poor, and transdifferentiation in vivo does not seem to occur. Instead, to address limitations related to supply, human embryonic stem cell (hESC)-derived β cells are being explored as surrogates for cadaveric islets. Transplantation of allogeneic hESC-derived insulin-producing organoids has recently entered Phase I and Phase II clinical trials. Stem cell replacement therapies overcome the barrier of finite availability, but they still face immune rejection. Immune protective strategies, including coupling hESC-derived insulin-producing organoids with macroencapsulation devices and microencapsulation technologies, are being tested to balance the necessity of immune protection with the need for vascularization. Here, we compare the diverse human stem cell approaches and outcomes of recently completed and ongoing clinical trials, and discuss innovative strategies developed to overcome the most significant challenges remaining for transplanting stem cell-derived β cells.

scholarly journals HNF1A deficiency causes reduced calcium levels, accumulation of abnormal insulin granules and uncoupled insulin to C-peptide secretion in a stem cell model of MODY3

2021 ◽  
Author(s):  
Bryan Gonzalez ◽  
Haoquan Zhao ◽  
Jacqueline Niu ◽  
Damian Williams ◽  
Jaeyop Lee ◽  
...  
Keyword(s):  
Stem Cell ◽  
Insulin Secretion ◽  
Intracellular Calcium ◽  
Cell Model ◽  
Secretory Granules ◽  
Embryonic Stem ◽  
Loss Of Function ◽  
Β Cells ◽  
C Peptide ◽  
Cell Therapies

Abstract Mutations in HNF1A cause Maturity Onset Diabetes of the Young type 3 (MODY3), the most prevalent form of monogenic diabetes. Using stem cell-derived pancreatic endocrine cells from human embryonic stem cells (hESCs) with induced hypomorphic mutations in HNF1A, we show that HNF1A orchestrates a transcriptional program required for calcium-dependent insulin secretion. HNF1A-deficient β-cells display a reduction in CACNA1A and intracellular calcium levels, as well as impaired insulin granule exocytosis in association with SYT13 down-regulation. Knockout of CACNA1A and SYT13 reproduce the relevant phenotypes. Retention of insulin is associated with accumulation of enlarged secretory granules, and altered stoichiometry of secreted insulin to C-peptide. Glibenclamide, a sulfonylurea drug used in the treatment of MODY3 patients, increases intracellular calcium, and thereby restores C-peptide and insulin secretion to a normal ratio. While insulin secretion defects are constitutive in cells with complete HNF1A loss of function, β-cells from patients with heterozygous hypomorphic HNF1A mutations are initially normal, but lose the ability to secrete insulin and acquire abnormal stoichiometric secretion ratios, while gene corrected cells remain normal. Our studies provide the molecular basis for the treatment of MODY3 with sulfonylureas, and demonstrate promise for the use of cell therapies for MODY3.

scholarly journals The TGFβ Family in Human Placental Development at the Fetal-Maternal Interface

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 453
Author(s):  
Susana M. Chuva de Sousa Lopes ◽  
Marta S. Alexdottir ◽  
Gudrun Valdimarsdottir
Keyword(s):  
Stem Cell ◽  
Transforming Growth Factor Beta ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Cell Model ◽  
Embryonic Stem ◽  
Model Systems ◽  
Special Focus ◽  
Placental Development

Emerging data suggest that a trophoblast stem cell (TSC) population exists in the early human placenta. However, in vitro stem cell culture models are still in development and it remains under debate how well they reflect primary trophoblast (TB) cells. The absence of robust protocols to generate TSCs from humans has resulted in limited knowledge of the molecular mechanisms that regulate human placental development and TB lineage specification when compared to other human embryonic stem cells (hESCs). As placentation in mouse and human differ considerably, it is only with the development of human-based disease models using TSCs that we will be able to understand the various diseases caused by abnormal placentation in humans, such as preeclampsia. In this review, we summarize the knowledge on normal human placental development, the placental disease preeclampsia, and current stem cell model systems used to mimic TB differentiation. A special focus is given to the transforming growth factor-beta (TGFβ) family as it has been shown that the TGFβ family has an important role in human placental development and disease.

scholarly journals The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yick W Fong ◽  
Jaclyn J Ho ◽  
Carla Inouye ◽  
Robert Tjian
Keyword(s):  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
In Vitro Transcription ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Ips Cell ◽  
Ribonucleoprotein Complex ◽  
Transcription System

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.

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