Directed hematopoietic differentiation of human pluripotent stem cells

描述(申请人提供)：在过去的两年里，我们和其他人通过使用一些已定义的转录因子，成功地将成人成纤维细胞等人类体细胞重新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具有功能的iPS细胞。尽管来自不同成体组织的iPS细胞成功重编程，在全球水平上表现出与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不同iPS细胞系之间的分化潜力的相似和差异仍有待确定。在过去的资金周期中，我们致力于阐明和调节人类胚胎干细胞(HES)向造血分化过程中的Notch/HES1信号通路。我们计划继续这一研究，以改善HES和iPS细胞的定向造血分化。此外，我们将应用这一系统来建立人类血液疾病的发育/遗传模型，这些疾病是由限于血细胞的体细胞突变引起的。在我们最近成功地将人类血液和骨髓CD34+细胞重新编程为iPS细胞的基础上，我们将首先开发一种有效的方法，通过无病毒方法重新编程未分离的成人白细胞(目标1)。我们还将从相同的成人捐赠者的CD45+白细胞和成纤维细胞中获得成对的iPS细胞系。这种方法将使我们能够比较它们在造血分化机制研究中的潜力，如下所述。在目标2中，我们将进行血液来源和成纤维细胞来源的人iPS细胞的定向造血细胞分化，并与胚泡来源的HES细胞进行比较。为此，我们还将阐明和调节Notch/HES1信号通路在人iPS细胞向造血细胞分化过程中的作用，以更好地理解和促进这一过程。在目标3中，我们将从阵发性睡眠性血红蛋白尿症(PNH)患者中分离并进行iPS细胞的造血分化。PNH是一种克隆性躯体疾病，与造血干细胞(HSCs)中X连锁的PIG-A基因突变有关。在PNH患者中，PIG-A突变的HSC成为克隆性优势，并导致大量缺乏糖基磷脂酰肌醇锚定蛋白(GPI-APs)的血细胞。我们将使用目标1中开发的最佳方法，从相同PNH患者的表型可区分的GPI-AP-(PIG-A空)和GPI-AP+(PIG-A野生型)血细胞以及PIG-A野生型的骨髓成纤维细胞中分离出iPS细胞系。然后，我们将采用目标2中建立的最佳方法，在PNH患者来源的iPS细胞中进行定向造血分化，无论是否存在PIG-A和GPI-AP缺陷。这项研究将有助于我们更好地了解PIG-A突变的作用，并识别可能导致PNH中HSC克隆性优势的其他可能的基因突变。 公共卫生相关性：这项研究的重点是从人类多能干细胞分化为造血细胞。我们将使用人类干细胞建立一个新的前瞻性模型，以更好地了解阵发性睡眠性血红蛋白尿(PNH)的克隆显性和其他令人费解的病理生理学，现有模型尚未充分回答这一问题。这项研究还将帮助我们建立一种通用的方法来研究数十种其他躯体血液疾病以及遗传性血液疾病，如镰状细胞性贫血。

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.