Modulating heterochromatin to improve beta cell differentiation from stem cells

调节异染色质以改善 β 细胞与干细胞的分化

基本信息

批准号：
10410417
负责人：
Kenneth Zaret
金额：
$ 40.63万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10410417
关键词：
Address Animals Autoimmune Beta Cell Biological Assay Birth Cadaver Cell Count Cell Differentiation process Cell Maturation Cell Transplantation Cell physiology Cells Dependence Elements Embryo Embryonic Development Endoderm Endoderm Cell Epigenetic Process Euchromatin Fibroblasts Gene Activation Gene Silencing Genes Genetic Genome Germ Layers Glucose Goals Hepatic Heterochromatin Human Impaired health In Vitro Insulin Insulin-Dependent Diabetes Mellitus Islets of Langerhans Knockout Mice Laboratories Life Style Liver Maps Medical Metabolic Metabolism Methods Methyltransferase Mouse Strains Mus Organoids Patients Physiological Process Proteins Proteomics Protocols documentation Public Health Recovery Research Role Signal Transduction Stem Cell Development Structure of beta Cell of islet Sucrose System Testing Time Tissues Transplantation Undifferentiated base cell type diabetic endocrine pancreas development epigenetic memory health goals human embryonic stem cell improved in vivo induced pluripotent stem cell insight insulin secretion islet knock-down laboratory experience mouse development stem cell differentiation stem cells success type I diabetic zinc finger nuclease

项目摘要

The goal of this proposal is to quantify the role of H3K9me3-based heterochromatin gene silencing in pancreatic beta cell development and to modulate such heterochromatin to obtain more mature beta-like cells from human embryonic stem cells. Type I diabetes (T1D) results from an autoimmune depletion of beta cells within pancreatic islets, resulting in a deficiency in insulin secretion, metabolic imbalances that impair health and lifestyle, and a dependency upon exogenous insulin. Recent studies indicate that T1D also involves beta cell de-differentiation. In 2000, the Edmonton Protocol demonstrated that beta cell transplants from cadavers could elicit exogenous insulin-free survival in severe type I diabetics. Yet success typically requires islets from two cadavers, donors are limiting, and patients can regress to insulin dependence over time. Thus, there is an unmet medical need for donor human beta cells. Extensive research over the past 20 years, including from my laboratory, has defined distinct stages of development of mouse beta cells from the endoderm germ layer and identified numerous signaling effectors. Such information has been used to guide beta-like cell differentiation in vitro from human embryonic stem cells (huESCs). Despite this progress, most laboratories experience difficulty generating robust glucose-responsive insulin secretion (GSIS) in terminal cell products. My lab, for instance, observes robust GSIS in only a limited number of beta-like organoids from huESCs, and generating GSIS-competent cells from induced pluripotent stem cells (iPSCs) is an even greater challenge. The lack of consistency from huESCs and the greater difficulty from iPSCs, which can retain an epigenetic memory of their originating cell type, suggests an epigenetic basis. While H3K9me3-based heterochromatic gene silencing has long been thought to constitutively suppress repeat elements in the genome, my laboratory has recently discovered that such heterochromatin is highly dynamic in liver and pancreatic beta cell development and that such heterochromatin also marks the genes that are the most difficult to activate in various cell reprogramming approaches. Indeed, we present evidence that H3K9me3 heterochromatin is not appropriately modulated in beta-like cells derived from huESCs. We propose two Aims to exploit these insights: Aim 1: Quantify the role of H3K9me3 heterochromatin during beta cell maturation in vivo and in vitro. Aim 2: Employ a knockdown screen of broad and specific heterochromatin modulators during huESC differentiation to beta-like cells to improve glucose-stimulated insulin secretion in vitro and in transplanted animals.

该建议的目的是量化基于H3K9me3的异染色质基因在胰腺β细胞发育中的作用，并调节这种异染色质从人类胚胎干细胞中获得更成熟的β样细胞。 I型糖尿病（T1D）是由胰岛中β细胞的自身免疫性耗竭，导致胰岛素分泌缺乏，损害健康和生活方式的代谢失衡以及对外源胰岛素的依赖性。最近的研究表明，T1D还涉及β细胞脱不同。在2000年，埃德蒙顿方案表明，来自尸体的β细胞移植物可能会在严重的I型糖尿病患者中引起无胰岛素的生存。然而，成功通常需要两个尸体的胰岛，供体有限，并且患者可以随着时间的流逝而回归胰岛素依赖性。因此，对供体人β细胞的医疗需求未满足。在过去的20年中，包括我的实验室在内的广泛研究定义了内胚层生殖层的小鼠β细胞开发的不同阶段，并确定了许多信号效应子。此类信息已用于指导人类胚胎干细胞（HUESCS）的体外β样细胞分化。尽管取得了这种进步，但大多数实验室在末端细胞产物中产生强大的葡萄糖胰岛素分泌（GSIS）遇到困难。例如，我的实验室仅在有限数量的HUESC中观察到鲁棒的GSI，并从诱导的多能干细胞（IPSC）中产生GSIS竞争性细胞是一个更大的挑战。 HUESC缺乏一致性和IPSC的更大难度，这可能保留了其原始细胞类型的表观遗传记忆，这表明了表观遗传基础。虽然长期以来人们认为基于H3K9ME3的异质基因沉默一直构成抑制基因组中的重复元素，但我的实验室最近发现，这种异染色质在肝脏和胰腺β细胞的发育中具有高度动态性，并且这种异染色质也标志着在各种细胞中都很难激活各种细胞的方法。确实，我们提供了证据表明，H3K9ME3异染色质在衍生自Huescs的β样细胞中没有适当调节。我们提出了两个旨在利用这些见解的目的：目标1：量化H3K9me3异染色质在体内和体外的β细胞成熟过程中的作用。 AIM 2：在HUESC分化为β样细胞期间，采用较宽的特定异染色质调节剂的敲低筛查，以改善体外和移植动物中葡萄糖刺激的胰岛素分泌。