Modulating heterochromatin to improve beta cell differentiation from stem cells

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，并调节此类异染色质以从人胚胎干细胞中获得更成熟的 β 样细胞。 I 型糖尿病 (T1D) 是由于胰岛内 β 细胞的自身免疫性耗竭导致的，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 细胞去分化。 2000 年，《埃德蒙顿方案》证明，从尸体移植 β 细胞可以使严重 I 型糖尿病患者在无外源胰岛素的情况下存活。然而，成功通常需要来自两具尸体的胰岛，捐赠者是有限的，并且随着时间的推移，患者可能会回归胰岛素依赖。因此，对供体人类β细胞的医疗需求尚未得到满足。过去 20 年的广泛研究（包括我的实验室）已经确定了小鼠内胚层 β 细胞发育的不同阶段，并确定了许多信号传导效应器。这些信息已被用来指导人类胚胎干细胞（huESC）在体外分化出类β细胞。尽管取得了这些进展，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，我的实验室仅在来自 huESC 的有限数量的 β 类器官中观察到强大的 GSIS，而从诱导多能干细胞 (iPSC) 中生成具有 GSIS 能力的细胞是一个更大的挑战。 huESC 缺乏一致性，而 iPSC 则更困难，因为 iPSC 可以保留其原始细胞类型的表观遗传记忆，这表明存在表观遗传基础。虽然基于 H3K9me3 的异染色质基因沉默长期以来被认为可以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 细胞发育中高度动态，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激活的基因。事实上，我们提供的证据表明 H3K9me3 异染色质在源自 huESC 的 β 样细胞中没有得到适当的调节。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。 目标 2：在 huESC 分化为 β 样细胞的过程中，采用广泛且特异性的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。

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.