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: 10030974
• 负责人: Kenneth Zaret
• 金额: $ 40.5万
• 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/10030974
• 关键词: Address; Animals; Autoimmune Process; 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; Estrogen receptor positive; 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

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.

该提案的目标是量化基于H3K9me3的异染色质基因沉默在胰腺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获得更成熟的β样细胞。I型糖尿病（T1D）是由胰岛内β细胞的自身免疫性消耗引起的，导致胰岛素分泌不足、损害健康和生活方式的代谢失衡以及对外源性胰岛素的依赖。最近的研究表明，T1D还涉及β细胞去分化。在2000年，埃德蒙顿方案证明，从尸体β细胞移植可以引起严重的I型糖尿病患者的外源性无胰岛素生存。然而，成功通常需要来自两具尸体的胰岛，供体有限，患者可能会随着时间的推移而回归胰岛素依赖。因此，对于供体人辟田胞存在未满足的医学需求。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β细胞从内胚层发育的不同阶段，并确定了许多信号效应子。这些信息已被用于指导从人胚胎干细胞（huESC）体外分化β样细胞。尽管取得了这一进展，但大多数实验室在终末细胞产物中难以产生稳健的葡萄糖响应性胰岛素分泌（GSIS）。例如，我的实验室仅在huESC的有限数量的β样类器官中观察到强大的GSIS，而从诱导多能干细胞（iPSC）中产生GSIS感受态细胞是一个更大的挑战。huESC缺乏一致性，而iPSC更难，因为iPSC可以保留其原始细胞类型的表观遗传记忆，这表明了表观遗传基础。虽然基于H3K9me3的异染色质基因沉默长期以来被认为是组成性抑制基因组中的重复元件，但我的实验室最近发现，这种异染色质在肝脏和胰腺β细胞发育中具有高度动态性，并且这种异染色质还标记了在各种细胞重编程方法中最难以激活的基因。事实上，我们提供的证据表明，H3K9me3异染色质在来源于人胚胎干细胞的β样细胞中没有得到适当的调节。我们提出了两个目标，以利用这些见解：目标1：量化的作用H3K9me3异染色质在体内和体外β细胞成熟。 目标二：在huESC分化为β样细胞期间采用广泛和特异性异染色质调节剂的敲低筛选，以改善体外和移植动物中葡萄糖刺激的胰岛素分泌。