Directed hematopoietic differentiation of human pluripotent stem cells

人多能干细胞定向造血分化

• 批准号: 8009282
• 负责人: Linzhao Cheng
• 金额: $ 45.8万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8009282
• 关键词: Adult; BMP4; Blood; Blood Cell Count; Blood Cells; Bone Marrow; Bone Marrow Cells; CD34 gene; Cell Culture Techniques; Cell Line; Cell surface; Cells; Characteristics; Clonal Expansion; Clonal Hematopoietic Stem Cell; DNA Transposons; Data; Derivation procedure; Development; Disease; ES Cell Line; Embryo; Exhibits; Family suidae; Fibroblasts; Functional disorder; Funding; Gene Mutation; Genes; Genetic Models; Hematological Disease; Hematopoietic; Hematopoietic stem cells; Human; Inherited; Investigation; Laboratories; Lead; Leukocytes; Link; Marrow; Mesenchymal Stem Cells; Methodology; Methods; Modeling; Molecular Profiling; Mutate; Mutation; PTPRC gene; Paper; Patients; Phosphatidylinositols; Pluripotent Stem Cells; Process; Proteins; Protocols documentation; Publishing; Reporting; Research; Retroviral Vector; Role; Sickle Cell Anemia; Signal Pathway; Somatic Cell; Somatic Mutation; Stromal Cells; System; Testing; Tissues; Umbilical Cord Blood; Virus; base; blastocyst; cell type; clinical application; developmental genetics; embryonic stem cell; human embryonic stem cell; human stem cells; improved; induced pluripotent stem cell; notch protein; novel; peripheral blood; pluripotency; postnatal human; prospective; public health relevance; rat Piga protein; stem; success; vector

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.

描述（由申请人提供）：在过去的两年中，我们和其他人通过使用一些确定的转录因子，成功地将人类体细胞（例如成纤维细胞）重编程为诱导多能干（iPS）细胞。最近，我们和其他人还将人类出生后血细胞重新编程为功能性 iPS 细胞。尽管成功重编程的源自不同成体组织的 iPS 细胞在整体水平上表现出多能性特征和独特的分子特征，与 ES 细胞非常相似，但越来越多的证据也揭示了成体细胞衍生的 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人 ES (hES) 细胞造血分化中的 Notch/HES1 信号通路。我们计划继续这一研究方向，以改善 hES 和 iPS 细胞的定向造血分化。此外，我们将应用该系统来建立仅限于血细胞的体细胞突变引起的人类血液疾病的发育/遗传模型。基于我们最近将人类血液和骨髓 CD34+ 细胞重编程为 iPS 细胞的成功，我们将首先开发一种有效的方法，通过无病毒方法重编程未分级的成年白细胞（目标 1）。我们还将从同一成年捐赠者的 CD45+ 白细胞和成纤维细胞中衍生出配对的 iPS 细胞系。这种方法将使我们能够比较它们在造血分化方面的潜力，以进行如下所述的机制研究。在目标 2 中，我们将从血液来源和成纤维细胞来源的人类 iPS 细胞中进行定向造血分化，并与囊胚来源的 hES 细胞进行比较。为此，我们还将阐明和调节 Notch/HES1 信号通路在人类 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，我们将衍生自阵发性睡眠性血红蛋白尿症 (PNH) 患者的 iPS 细胞并对其进行造血分化，PNH 是一种与造血干细胞 (HSC) 中 X 连锁 PIG-A 基因突变相关的克隆性躯体疾病。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，从表型可区分的 GPI-AP-（PIG-A null）和 GPI-AP+（PIG-A 野生型）血细胞以及同一 PNH 患者的骨髓成纤维细胞（PIG-A 野生型）中衍生出 iPS 细胞系。然后，我们将采用目标 2 中建立的最佳方法，从 PNH 患者衍生的 iPS 中进行定向造血分化 具有或不具有 PIG-A 和 GPI-AP 缺陷的细胞。这项研究将帮助我们更好地了解 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。 公共健康相关性：本研究重点关注人类多能干细胞的造血分化。我们将利用人类干细胞建立一个新颖的前瞻性模型，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方法来研究数十种其他躯体血液疾病以及镰状细胞性贫血等遗传性血液疾病。