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

• 批准号: 10646396
• 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
• 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/10646396
• 关键词: Address; Animals; Autoimmune; Beta Cell; Biological Assay; Birth; Cadaver; Cell Count; Cell Differentiation process; Cell Maturation; Cell Reprogramming; 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; Pluripotent Stem Cells; 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; cell dedifferentiation; cell type; diabetic; embryonic stem cell; 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

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.

这项建议的目的是量化基于H3K9me3的异染色质基因沉默在胰岛β细胞发育中的作用，并调节这种异染色质以从人胚胎干细胞中获得更成熟的贝塔样细胞。I型糖尿病(T1D)是由于胰岛内β细胞的自身免疫耗竭，导致胰岛素分泌不足，代谢失衡损害健康和生活方式，以及对外源性胰岛素的依赖。最近的研究表明，T1D还涉及β细胞的去分化。2000年，埃德蒙顿协议证明，从身体中移植β细胞可以在重症I型糖尿病患者中诱导外源性无胰岛素生存。然而，成功通常需要两具身体的胰岛，捐赠者是有限的，随着时间的推移，患者可能会回归对胰岛素的依赖。因此，对捐赠者人类β细胞的医学需求尚未得到满足。在过去的20年里，包括我的实验室在内的广泛研究已经确定了小鼠内胚层β细胞发育的不同阶段，并确定了许多信号效应器。这些信息已经被用来指导从人类胚胎干细胞(HuESCs)体外分化为类似于β的细胞。尽管取得了这一进展，但大多数实验室在终末细胞产品中难以产生强大的葡萄糖反应性胰岛素分泌(GSIS)。例如，我的实验室只从人胚胎干细胞中观察到有限数量的类贝塔类有机物质中的GSIS，而从诱导的多能干细胞(IPSCs)中产生具有GSIS能力的细胞是一个更大的挑战。HuESCs缺乏一致性，而iPSCs的难度更大，可以保留其原始细胞类型的表观遗传记忆，这表明存在表观遗传学基础。虽然长期以来，基于H3K9me3的异染色质基因沉默一直被认为是对基因组中重复元件的结构性抑制，但我的实验室最近发现，这种异染色质在肝脏和胰腺的β细胞发育中具有高度的动态，而且这种异染色质还标记了在各种细胞重编程方法中最难激活的基因。事实上，我们提出的证据表明，H3K9me3异染色质在来源于HuESCs的β样细胞中没有适当的调节。我们提出了两个目标来利用这些见解：目标1：量化H3K9me3异染色质在体内和体外β细胞成熟过程中的作用。目的2：在体外和移植动物中，在huESC向β样细胞分化的过程中，使用广泛和特定的异染色质调节剂进行敲除筛选，以促进葡萄糖刺激的胰岛素分泌。