Insulin-producing cells derived from ‘induced pluripotent stem cells’ of patients with fulminant type 1 diabetes: vulnerability to cytokine insults and increased expression of apoptosis-related genes

Aims/Introduction: The present study was carried out to generate induced pluripotent stem cells (iPSCs) from patients with fulminant type 1 diabetes, and evaluate the cytokine‐induced apoptotic reactions of β‐like insulin‐producing cells differentiated from the iPSCs.Materials and Methods: iPSCs were generated from fibroblasts of patients with fulminant type 1 diabetes by inducing six reprogramming factors. Insulin‐producing cells were differentiated from the iPSCs in vitro. The proportion of cleaved caspase‐3‐positive or terminal deoxynucleotidyl transferase 2′‐deoxyuridine, 5′‐triphosphate nick end labeling‐positive cells among insulin (INS)‐positive cells derived from fulminant type 1 diabetes iPSC and control human iPSC lines was evaluated under treatment with tumor necrosis factor‐α, interleukin‐1β and interferon‐γ. Ribonucleic acid sequencing was carried out to compare gene expressions in INS‐positive cells derived from fulminant type 1 diabetes iPSC and control human iPSC lines.Results: Two iPSC clones were established from each of three patients with fulminant type 1 diabetes. The differentiation of insulin‐producing cells from fulminant type 1 diabetes iPSC was confirmed by immunofluorescence analysis and KCl‐induced C‐peptide secretion. After treatment with pro‐inflammatory cytokines, these INS‐positive cells showed higher expression of cleaved caspase‐3 than those derived from control human iPSCs. Altered expression levels of several apoptosis‐related genes were observed in INS‐positive cells derived from the fulminant type 1 diabetes iPSCs by ribonucleic acid sequencing.Conclusions: We generated iPSCs from patients with fulminant type 1 diabetes and differentiated them into insulin‐producing cells. This in vitro disease model can be used to elucidate the disease mechanisms of fulminant type 1 diabetes

Cell aggregation optimizes the differentiation of human ESCs and iPSCs into pancreatic bud-like progenitor cells

Embryonic pancreatic bud cells, the earliest pancreas-committed cells, generated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into mature pancreatic β-cells in vivo, indicating the feasibility of hESC/iPSC-based cell therapy for diabetes. However, the key factors required for the differentiation of these cells into pancreatic bud cells are incompletely understood. The purpose of this study was to establish culture conditions that efficiently induce PDX1+NKX6.1+ pancreatic bud cells from hESCs/iPSCs. We differentiated a hESC line, KhES-3, into pancreatic lineages with a stepwise protocol recapitulating developmental process. The induction rate of PDX1+NKX6.1+ cells was correlated with cell density in adherent cultures, and markedly improved with cell aggregation cultures. The positive effects of cell aggregation cultures on the differentiation of pancreatic bud cells were reproduced in multiple hESC/iPSC lines. The human PDX1+NKX6.1+ cells developed into pancreatic epithelia after implantation into immunocompromised mice. Moreover, human C-peptide secretion into mouse bloodstream was stimulated by glucose challenges after in vivo maturation. Taken together, these results suggest that cultures with high cell density are crucial for the differentiation of pancreas-committed progenitor cells from hESCs/iPSCs. Our findings may be applicable for the development of hESC/iPSC-based cell therapy for diabetes

Generation of Alveolar Epithelial Spheroids via Isolated Progenitor Cells from Human Pluripotent Stem Cells.

ヒトiPS細胞から肺胞上皮細胞を分化誘導し、単離する方法を確立 -肺の再生/創薬研究につながる大きな一歩-. 京都大学プレスリリース. 2014-08-22.No methods for isolating induced alveolar epithelial progenitor cells (AEPCs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been reported. Based on a study of the stepwise induction of alveolar epithelial cells (AECs), we identified carboxypeptidase M (CPM) as a surface marker of NKX2-1(+) "ventralized" anterior foregut endoderm cells (VAFECs) in vitro and in fetal human and murine lungs. Using SFTPC-GFP reporter hPSCs and a 3D coculture system with fetal human lung fibroblasts, we showed that CPM(+) cells isolated from VAFECs differentiate into AECs, demonstrating that CPM is a marker of AEPCs. Moreover, 3D coculture differentiation of CPM(+) cells formed spheroids with lamellar-body-like structures and an increased expression of surfactant proteins compared with 2D differentiation. Methods to induce and isolate AEPCs using CPM and consequently generate alveolar epithelial spheroids would aid human pulmonary disease modeling and regenerative medicine

Ablation of Central Serotonergic Neurons Decreased REM Sleep and Attenuated Arousal Response

Sleep/wake behavior is regulated by distinct groups of neurons, such as dopaminergic, noradrenergic, and orexinergic neurons. Although monoaminergic neurons are usually considered to be wake-promoting, the role of serotonergic neurons in sleep/wake behavior remains inconclusive because of the effect of serotonin (5-HT)-deficiency on brain development and the compensation for inborn 5-HT deficiency by other sleep/wake-regulating neurons. Here, we performed selective ablation of central 5-HT neurons in the newly developed Rosa-diphtheria toxin receptor (DTR)-tdTomato mouse line that was crossed with Pet1(Cre/+) mice to examine the role of 5-HT neurons in the sleep/wake behavior of adult mice. Intracerebroventricular administration of diphtheria toxin completely ablated tdTomato-positive cells in Pet1(Cre/+);Rosa-DTR-tdTomato mice. Electroencephalogram/electromyogram-based sleep/wake analysis demonstrated that central 5-HT neuron ablation in adult mice decreased the time spent in rapid eye movement (REM) sleep, which was associated with fewer transitions from non-REM (NREM) sleep to REM sleep than in control mice. Central 5-HT neuron-ablated mice showed attenuated wake response to a novel environment and increased theta power during wakefulness compared to control mice. The current findings indicated that adult 5-HT neurons work to support wakefulness and regulate REM sleep time through a biased transition from NREM sleep to REM sleep