Single-cell transcriptome profiling reveals β cell maturation in stem cell-derived islets after transplantation

Human pluripotent stem cells differentiated to insulin-secreting β cells (SC-β cells) in islet organoids could provide an unlimited cell source for diabetes cell replacement therapy. However, current SC-β cells generated in vitro are transcriptionally and functionally immature compared to native adult β cells. Here, we use single-cell transcriptomic profiling to catalog changes that occur in transplanted SC-β cells. We find that transplanted SC-β cells exhibit drastic transcriptional changes and mature to more closely resemble adult β cells. Insulin and IAPP protein secretions increase upon transplantation, along with expression of maturation genes lacking with differentiation in vitro, including INS, MAFA, CHGB, and G6PC2. Other differentiated cell types, such as SC-α and SC-enterochromaffin (SC-EC) cells, also exhibit large transcriptional changes. This study provides a comprehensive resource for understanding human islet cell maturation and provides important insights into maturation of SC-β cells and other SC-islet cell types to enable future differentiation strategy improvements

SIX2 regulates human β cell differentiation from stem cells and functional maturation in vitro

Generation of insulin-secreting β cells in vitro is a promising approach for diabetes cell therapy. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are differentiated to β cells (SC-β cells) and mature to undergo glucose-stimulated insulin secretion, but molecular regulation of this defining β cell phenotype is unknown. Here, we show that maturation of SC-β cells is regulated by the transcription factor SIX2. Knockdown (KD) or knockout (KO) of SIX2 in SC-β cells drastically limits glucose-stimulated insulin secretion in both static and dynamic assays, along with the upstream processes of cytoplasmic calcium flux and mitochondrial respiration. Furthermore, SIX2 regulates the expression of genes associated with these key β cell processes, and its expression is restricted to endocrine cells. Our results demonstrate that expression of SIX2 influences the generation of human SC-β cells in vitro

Multidimensional analysis and therapeutic development using patient iPSC-derived disease models of Wolfram syndrome

Wolfram syndrome is a rare genetic disorder largely caused by pathogenic variants in the WFS1 gene and manifested by diabetes mellitus, optic nerve atrophy, and progressive neurodegeneration. Recent genetic and clinical findings have revealed Wolfram syndrome as a spectrum disorder. Therefore, a genotype-phenotype correlation analysis is needed for diagnosis and therapeutic development. Here, we focus on the WFS1 c.1672C\u3eT, p.R558C variant, which is highly prevalent in the Ashkenazi Jewish population. Clinical investigation indicated that patients carrying the homozygous WFS1 c.1672C\u3eT, p.R558C variant showed mild forms of Wolfram syndrome phenotypes. Expression of WFS1 p.R558C was more stable compared with the other known recessive pathogenic variants associated with Wolfram syndrome. Human induced pluripotent stem cell-derived (iPSC-derived) islets (SC-islets) homozygous for WFS1 c.1672C\u3eT variant recapitulated genotype-related Wolfram syndrome phenotypes. Enhancing residual WFS1 function through a combination treatment of chemical chaperones mitigated detrimental effects caused by the WFS1 c.1672C\u3eT, p.R558C variant and increased insulin secretion in SC-islets. Thus, the WFS1 c.1672C\u3eT, p.R558C variant causes a mild form of Wolfram syndrome phenotypes, which can be remitted with a combination treatment of chemical chaperones. We demonstrate that our patient iPSC-derived disease model provides a valuable platform for further genotype-phenotype analysis and therapeutic development for Wolfram syndrome

Current limiting and negative differential resistance in indium oxide based ceramics

Indium oxide based ceramics with bismuth oxide addition were sintered in air in the temperature range 800-1300 ºC. Current-voltage characteristics of In2O3-Bi2O3 ceramics sintered at different temperatures are weakly nonlinear. After an additional heat treatment in air at about 200 ºC samples sintered at a temperature within the narrow range of about 1050-1100 ºC exhibit a current-limiting effect accompanied by low-frequency current oscillations. It is shown that the observed electrical properties are controlled by the grain-boundary barriers and the heat treatment in air at 200 ºC leads to the decrease in the barrier height. Electrical measurements, scanning electron microscopy and X-ray photoelectron spectroscopy results suggest that the current-limiting effect observed in In2O3-Bi2O3 may be explained in terms of a modified barrier model; the observed current-limiting effect is the result of an increase of barrier height with increasing electric field, due to additional oxygen absorption. It is found that In2O3-Bi2O3-Co3O4-Cr2O3 ceramic exhibits current-voltage characteristics with negative differential resistance due to Joule micro heating.This study was performed in part in the frames of the project SEP-2003-C02-42821, CONACYT, México. Funding from the Royal Society, United Kingdom (2007R1/R26999) is gratefully acknowledged

Single-nucleus multi-omics of human stem cell-derived islets identifies deficiencies in lineage specification

Insulin-producing β cells created from human pluripotent stem cells have potential as a therapy for insulin-dependent diabetes, but human pluripotent stem cell-derived islets (SC-islets) still differ from their in vivo counterparts. To better understand the state of cell types within SC-islets and identify lineage specification deficiencies, we used single-nucleus multi-omic sequencing to analyse chromatin accessibility and transcriptional profiles of SC-islets and primary human islets. Here we provide an analysis that enabled the derivation of gene lists and activity for identifying each SC-islet cell type compared with primary islets. Within SC-islets, we found that the difference between β cells and awry enterochromaffin-like cells is a gradient of cell states rather than a stark difference in identity. Furthermore, transplantation of SC-islets in vivo improved cellular identities overtime, while long-term in vitro culture did not. Collectively, our results highlight the importance of chromatin and transcriptional landscapes during islet cell specification and maturation

Expression and regulation of type 2A protein phosphatases and alpha4 signalling in cardiac health and hypertrophy

Abstract Cardiac physiology and hypertrophy are regulated by the phosphorylation status of many proteins, which is partly controlled by a poorly defined type 2A protein phosphatase-alpha4 intracellular signalling axis. Quantitative PCR analysis revealed that mRNA levels of the type 2A catalytic subunits were differentially expressed in H9c2 cardiomyocytes (PP2ACb[PP2ACa[PP4C[PP6C), NRVM (PP2ACb[PP2ACa = PP4C = PP6C), and adult rat ventricular myocytes (PP2ACa[ PP2ACb[PP6C[PP4C). Western analysis confirmed that all type 2A catalytic subunits were expressed in H9c2 cardiomyocytes; however, PP4C protein was absent in adult myocytes and only detectable following 26S proteasome inhibition. Short-term knockdown of alpha4 protein expression attenuated expression of all type 2A catalytic subunits. Pressure overload-induced left ventricular (LV) hypertrophy was associated with an increase in both PP2AC and alpha4 protein expression. Although PP6C expression was unchanged, expression of PP6C regulatory subunits (1) Sit4-associated protein 1 (SAP1) and (2) ankyrin repeat domain (ANKRD) 28 and 44 proteins was elevated, whereas SAP2 expression was reduced in hypertrophied LV tissue. Co-immunoprecipitation studies demonstrated that the interaction between alpha4 and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Phosphorylation status of phospholemman (Ser63 and Ser68) was significantly increased by knockdown of PP2ACa, PP2ACb, or PP4C protein expression. DNA damage assessed by histone H2A.X phosphorylation (cH2A.X) in hypertrophied tissue remained unchanged. However, exposure of cardiomyocytes to H2O2 increased levels of cH2A.X which was unaffected by knockdown of PP6C expression, but was abolished by the short-term knockdown of alpha4 expression. This study illustrates the significance and altered activity of the type 2A protein phosphatase-alpha4 complex in healthy and hypertrophied myocardium