Directed hematopoietic differentiation of human pluripotent stem cells

描述（由申请人提供）：在过去的2年中，我们和其他人通过使用一些定义的转录因子成功地将人体细胞（如成人成纤维细胞）重编程为诱导多能干细胞（iPS）。最近，我们和其他人还将人类出生后的血细胞重新编程为功能性iPS细胞。尽管成功重编程的来自不同成体组织的iPS细胞在整体水平上表现出与ES细胞非常相似的多能性特征和独特的分子特征，但越来越多的证据也揭示了成体细胞来源的iPS和ES细胞系之间的差异。iPS和ES细胞之间以及来自不同成体细胞类型的各种iPS细胞系之间的分化潜力的相似性和差异性仍有待确定。在过去的资助周期中，我们投入了大量精力来阐明和调节Notch/HES 1信号通路在人ES（hES）细胞造血分化中的作用。我们计划继续这一系列的研究，以提高定向造血分化从hES和iPS细胞。此外，我们将应用该系统建立发育/遗传模型的人类血液疾病所造成的体细胞突变，仅限于血细胞。基于我们最近在将人血液和骨髓CD 34+细胞重编程为iPS细胞方面的成功，我们将首先开发一种通过无病毒方法重编程未分级分离的成人白细胞的有效方法（目的1）。我们还将从来自相同成年供体的CD 45+白细胞和成纤维细胞获得配对的iPS细胞系。这种方法将使我们能够比较它们在造血分化中的潜力，用于如下所述的机制研究。在目标2中，我们将从血液来源的和成纤维细胞来源的人iPS细胞进行定向造血分化，与囊胚来源的hES细胞进行比较。为此，我们还将阐明和调节Notch/HES 1信号通路在人iPS细胞造血分化中的作用，以便更好地理解和增强这一过程。在目标3中，我们将获得并进行来自阵发性睡眠性血红蛋白尿症（PNH）患者的iPS细胞的造血分化，PNH是一种与造血干细胞（HSC）中的X连锁PIG-A基因突变相关的克隆性体细胞疾病。在PNH患者中，PIG-A突变的HSC成为克隆优势，并产生大量缺乏糖基磷脂酰肌醇锚定蛋白（GPI-AP）的血细胞。我们将使用目标1中开发的最佳方法，从表型上可区分的GPI-AP-（PIG-A无效）和GPI-AP+（PIG-A野生型）血细胞以及相同PNH患者的骨髓成纤维细胞（PIG-A野生型）获得iPS细胞系。然后，我们将采取目标2中建立的最佳方法，从PNH患者来源的iPS细胞进行定向造血分化，无论是否缺乏PIG-A和GPI-AP。这项研究将有助于我们更好地了解PIG-A突变的作用，并确定其他可能导致PNH中HSC克隆优势的基因突变。 公共卫生相关性：这项研究的重点是从人类多能干细胞造血分化。我们将利用人类干细胞建立一种新型的前瞻性模型，以更好地了解阵发性睡眠性血红蛋白尿症（PNH）的克隆优势和其他令人困惑的病理生理学，现有模型尚未充分回答这些问题。这项研究也将帮助我们建立一个通用的方法来研究其他几十种体细胞血液疾病以及遗传性血液疾病，如镰状细胞贫血。

DESCRIPTION (provided by applicant): In the past 2 years, we and others have successfully reprogrammed human somatic cells such as adult fibroblasts into induced pluripotent stem (iPS) cells by using a few defined transcriptional factors. More recently, we and others also reprogrammed human postnatal blood cells into functional iPS cells. Although successfully reprogrammed iPS cells derived from different adult tissues exhibit pluripotency characteristics and unique molecular signatures at a global level remarkably similar to ES cells, increasing evidence also reveals differences between adult cell-derived iPS and ES cell lines. Similarities and differences in the differentiation potential between iPS and ES cells, and between various iPS cell lines from different adult cell types, remain to be determined. In the past funding cycle, we have devoted significant efforts to elucidate and modulate the Notch/HES1 signaling pathways in hematopoietic differentiation from human ES (hES) cells. We plan to continue this line of investigation to improve directed hematopoietic differentiation from both hES and iPS cells. In addition, we will apply this system to establish developmental/genetic models for human blood diseases resulting from somatic mutations that are restricted to blood cells. Based on our recent success in reprogramming human blood and marrow CD34+ cells to iPS cells, we will first develop an efficient method to reprogram un-fractionated adult leukocytes by a virus-free method (Aim 1). We will also derive paired iPS cell lines from CD45+ leukocytes and fibroblasts from the same adult donors. This approach would allow us to compare their potential in hematopoietic differentiation for mechanistic studies as outlined below. In Aim 2, we will conduct directed hematopoietic differentiation from blood-derived and fibroblast-derived human iPS cells, in comparison with that of blastocyst-derived hES cells. In this aim, we will also elucidate and modulate the role of Notch/HES1 signaling pathways in the hematopoietic differentiation from human iPS cells in order to better understand and enhance the process. In Aim 3, we will derive and conduct hematopoietic differentiation of iPS cells derived from patients with paroxysmal nocturnal hemoglobinuria (PNH), a clonal somatic disease related to the X-linked PIG-A gene mutation in hematopoietic stem cells (HSCs). In PNH patients, a PIG-A mutated HSC becomes clonally dominant and gives rise to large numbers of blood cells lacking glycosyl-phosphatidyl- inositol anchored proteins (GPI-APs). We will derive iPS cell lines from phenotypically distinguishable GPI-AP- (PIG-A null) and GPI-AP+ (PIG-A wildtype) blood cells and from marrow fibroblasts (of PIG-A wildtype) of the same PNH patients, using the best method developed in Aim 1. Then we will take the best approach established in Aim 2 to conduct directed hematopoietic differentiation from the PNH patient-derived iPS cells with or without the deficiency of PIG-A and GPI-APs. This study will help us to better understand the role of the PIG-A mutation and to identify other possible genetic mutations that may lead to HSC clonal dominance in PNH. PUBLIC HEALTH RELEVANCE: This study focuses on hematopoietic differentiation from human pluripotent stem cells. We will establish a novel prospective model using human stem cells to better understand clonal dominance and other puzzling pathophysiology of paroxysmal nocturnal hemoglobinuria (PNH), which has not been adequately answered by existing models. This study will also help us to establish a universal method to study dozens of other somatic blood diseases as well as inherited blood diseases such as sickle cell anemia.