Generation of Hematopoietic Stem Cells from Induced Pluripotent Stem Cells

项目总结(见说明)： 项目3的总体目标是开发一种高效且可重复地将诱导多能干细胞(IPS)分化为造血干细胞(HSCs)的方法。本项目的目标1将确定iPS细胞中胚层分化和HSCs生成的最佳条件。这一目标将检验这样一种假设，即工程化的人类基质细胞系提供了可靠地将IPS细胞分化为造血祖细胞的最佳方法。实验将比较使用类胚体(EB)培养和基质细胞系分化IPS细胞的方法，以支持IPS细胞分化为HSCs。 将对这两种分化方法的各种因素进行研究，以确定促进中胚层分化和HSC生成的最佳方法。第二个目的是确定iPS细胞产生的HSCs存活、生长和扩增的最佳细胞因子条件。这一目标将检验这样一种假设，即从IPS产生的HSCs对胎儿HSCs的细胞因子反应比成年HSCs更相似。HSCs在培养中的积累需要有利于其生存的条件，并将其分化为承诺的祖细胞的程度降至最低。已知在早期造血阶段发挥作用的各种细胞因子将被联合测试，以比较iPS细胞产生的HSCs与从胎儿组织、脐带血和成人外周血中分离出来的HSCs的相似性。这些实验将优化HSCs生产的培养条件。第三个目标将确定不同IPS细胞系分化为HSC的能力的差异性。这一目标将检验这样一种假设，即不同的iPS细胞系类似地能够形成能够在免疫缺陷小鼠中产生长期多谱系重建的HSC。这一目标将测试来自多个IPS细胞系的分离的HSCs提供长期重建的能力，而不会形成畸胎瘤或白血病、染色体异常或其他明显的功能缺陷。这些实验的结果将是开发出从经过基因修正的IPS细胞高效地产生可移植的造血干细胞的技术。

PROJECT SUMMARY (See instructions): The overall goal of Project 3 is to develop a method to efficiently and reproducibly differentiate induced pluripotent stem (IPS) cells into hematopoietic stem cells (HSCs). Aim 1 of this project will determine the best conditions for the mesodermal differentiation of iPS cells and the generation of HSCs. This aim will test the hypothesis that an engineered human stromal cell line offers the best method to reliably differentiate IPS cells into hematopoietic precursors. Experiments will compare methods of differentiating IPS cells using embryoid bodies (EB) cultures and stromal cell lines to support the differentiation of IPS cells into HSCs. Various elements of these two differentiation methods will be studied to determine the best method to promote mesodermal differentiation and HSC creation. The second aim is to determine the optimal cytokine conditions for the survival, growth and expansion of HSCs generated from iPS cells. This aim will test the hypothesis that HSCs generated from IPS are more similar in their cytokine responses to fetal HSCs than adult HSCs. Accumulation of HSCs in culture requires conditions that favor their survival and minimize their differentiation into committed progenitors. Various cytokines known to play a role in the early stages of hematopoiesis will be tested in combination to compare the similarity of HSCs generated from iPS cells to those isolated from fetal tissues, umbilical cord blood and adult peripheral blood. These experiments will optimize culture conditions for the production of HSCs. The third aim will determine the variability in the capacity of different IPS cell lines to differentiate into HSCs. This aim will test the hypothesis that different iPS cells lines are similarly capable to form HSCs that can generate long-term multilineage reconstitution in immunodeficient mice. This aim will test isolated HSCs derived from multiple IPS cell lines for their capacity to provide long-term reconstitution without teratoma or leukemia formation, chromosomal abnormalities or other obvious functional deficiencies. The outcome of the experiments will be development of technology to efficiently generate transplantable HSCs from genetically corrected IPS cells.