RESUMEN
Insulin secretion from pancreatic ß cells is essential to the maintenance of glucose homeostasis. Defects in this process result in diabetes. Identifying genetic regulators that impair insulin secretion is crucial for the identification of novel therapeutic targets. Here, we show that reduction of ZNF148 in human islets, and its deletion in stem cell-derived ß cells (SC-ß cells), enhances insulin secretion. Transcriptomics of ZNF148-deficient SC-ß cells identifies increased expression of annexin and S100 genes whose proteins form tetrameric complexes involved in regulation of insulin vesicle trafficking and exocytosis. ZNF148 in SC-ß cells prevents translocation of annexin A2 from the nucleus to its functional place at the cell membrane via direct repression of S100A16 expression. These findings point to ZNF148 as a regulator of annexin-S100 complexes in human ß cells and suggest that suppression of ZNF148 may provide a novel therapeutic strategy to enhance insulin secretion.
Asunto(s)
Células Secretoras de Insulina , Humanos , Células Secretoras de Insulina/metabolismo , Secreción de Insulina , Glucosa/metabolismo , Insulina/metabolismo , Exocitosis , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Human embryonic stem cell-derived ß cells (SC-ß cells) hold great promise for treatment of diabetes, yet how to achieve functional maturation and protect them against metabolic stresses such as glucotoxicity and lipotoxicity remains elusive. Our single-cell RNA-seq analysis reveals that ZnT8 loss of function (LOF) accelerates the functional maturation of SC-ß cells. As a result, ZnT8 LOF improves glucose-stimulated insulin secretion (GSIS) by releasing the negative feedback of zinc inhibition on insulin secretion. Furthermore, we demonstrate that ZnT8 LOF mutations endow SC-ß cells with resistance to lipotoxicity/glucotoxicity-triggered cell death by alleviating endoplasmic reticulum (ER) stress through modulation of zinc levels. Importantly, transplantation of SC-ß cells with ZnT8 LOF into mice with preexisting diabetes significantly improves glycemia restoration and glucose tolerance. These findings highlight the beneficial effect of ZnT8 LOF on the functional maturation and survival of SC-ß cells that are useful as a potential source for cell replacement therapies.
Asunto(s)
Proteínas de Transporte de Catión , Diabetes Mellitus , Células Madre Embrionarias Humanas , Células Secretoras de Insulina , Animales , Proteínas de Transporte de Catión/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Glucosa/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ratones , Estrés Fisiológico , Zinc/metabolismoRESUMEN
The ever-increasing therapeutic and pharmaceutical demand for liver cells calls for systems that enable mass production of hepatic cells. Here we describe a large-scale suspension system that uses human endoderm stem cells (hEnSCs) as precursors to generate functional and transplantable hepatocytes (E-heps) or cholangiocytes (E-chos). hEnSC-derived hepatic populations are characterized by single-cell transcriptomic analyses and compared with hESC-derived counterparts, in-vitro-maintained or -expanded primary hepatocytes and adult cells, which reveals that hepatic differentiation of hEnSCs recapitulates in vivo development and that the heterogeneities of the resultant populations can be manipulated by regulating the EGF and MAPK signaling pathways. Functional assessments demonstrate that E-heps and E-chos possess properties comparable with adult counterparts and that, when transplanted intraperitoneally, encapsulated E-heps were able to rescue rats with acute liver failure. Our study lays the foundation for cell-based therapeutic agents and in vitro applications for liver diseases.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Endodermo/citología , Hepatocitos/citología , Células Madre Embrionarias Humanas/citología , Conductos Biliares/citología , Conductos Biliares/metabolismo , Diferenciación Celular/fisiología , Endodermo/metabolismo , Endodermo/trasplante , Células Epiteliales/citología , Células Epiteliales/metabolismo , Hepatocitos/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/trasplante , Humanos , Hígado/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/trasplanteRESUMEN
The pancreas of vertebrates is separately derived from both the dorsal and ventral endodermal domains. However, the difference between these two programs has been unclear. Here, using a pancreatic determination gene, Pdx1, driven GFP transgenic mouse strain, we identified Pdx1-GFP highly expressing cells (Pdx1high) and Pdx1-GFP lowly expressing cells (Pdx1low) in both embryonic dorsal Pdx1-expressing region (DPR) and ventral Pdx1-expressing region (VPR). We analyzed the transcriptomes of single Pdx1low and Pdx1high cells from the DPR and VPR. In the VPR, Pdx1low cells have an intermediate progenitor identity and can generate hepatoblasts, extrahepatobiliary cells, and Pdx1high pancreatic progenitor cells. In the DPR, Pdx1high cells are directly specified as pancreatic progenitors, whereas Pdx1low cells are precocious endocrine cells. Therefore, our study defines distinct road maps for dorsal and ventral pancreatic progenitor specification. The findings provide guidance for optimization of current ß-cell induction protocols by following the in vivo dorsal pancreatic specification program.
Asunto(s)
Proteínas de Homeodominio/genética , Páncreas/crecimiento & desarrollo , Células Madre/metabolismo , Transactivadores/genética , Transcriptoma/genética , Animales , Linaje de la Célula/genética , Regulación del Desarrollo de la Expresión Génica , Células Secretoras de Insulina/metabolismo , Ratones , Ratones Transgénicos/genética , Páncreas/embriología , Páncreas/metabolismo , Análisis de la Célula IndividualRESUMEN
Human induced pluripotent stem cells (hiPSCs) can proliferate infinitely. Their ability to differentiate into cardiomyocytes provides abundant sources for disease modeling, drug screening and regenerative medicine. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) display a low degree of maturation and fetal-like properties. Current in vitro differentiation methods do not mimic the structural, mechanical, or physiological properties of the cardiogenesis niche. Recently, we present an efficient cardiac maturation platform that combines hiPSCs monolayer cardiac differentiation with graphene substrate, which is a biocompatible and superconductive material. The hiPSCs lines were successfully maintained on the graphene sheets and were able to differentiate into functional cardiomyocytes. This strategy markedly increased the myofibril ultrastructural organization, elevated the conduction velocity, and enhanced both the Ca2+ handling and electrophysiological properties in the absence of electrical stimulation. On the graphene substrate, the expression of connexin 43 increased along with the conduction velocity. Interestingly, the bone morphogenetic proteins signaling was also significantly activated during early cardiogenesis, confirmed by RNA sequencing analysis. Here, we reasoned that graphene substrate as a conductive biomimetic surface could facilitate the intrinsic electrical propagation, mimicking the microenvironment of the native heart, to further promote the global maturation of hiPSC-CMs. Our findings highlight the capability of electrically active substrates to influence cardiomyocyte development. We believe that application of graphene sheets will be useful for simple, fast, and scalable maturation of regenerated cardiomyocytes.