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有效且可重复地将诱导多能干细胞（IPS）分化为造血干细胞（HSC）的方法。本项目的目的一是确定iPS细胞向中胚层分化和HSC生成的最佳条件。这一目标将检验工程化的人基质细胞系提供可靠地将IPS细胞分化为造血前体的最佳方法的假设。实验将比较使用胚状体（EB）培养物和基质细胞系分化IPS细胞以支持IPS细胞分化成HSC的方法。 将研究这两种分化方法的各种要素，以确定促进中胚层分化和HSC产生的最佳方法。第二个目的是确定从iPS细胞产生的HSC的存活、生长和扩增的最佳细胞因子条件。这一目的将检验从IPS产生的HSC在其对胎儿HSC的细胞因子应答方面比成人HSC更相似的假设。HSC在培养物中的积累需要有利于其存活并使其分化为定向祖细胞的条件最小化。将联合测试已知在造血早期阶段发挥作用的各种细胞因子，以比较从iPS细胞产生的HSC与从胎儿组织、脐带血和成人外周血分离的HSC的相似性。这些实验将优化用于生产HSC的培养条件。第三个目标是确定不同IPS细胞系分化为HSC的能力的可变性。这一目标将检验不同的iPS细胞系类似地能够形成HSC的假设，所述HSC可以在免疫缺陷小鼠中产生长期的多谱系重建。这一目标将测试来自多种IPS细胞系的分离的HSC在没有畸胎瘤或白血病形成、染色体异常或其他明显功能缺陷的情况下提供长期重建的能力。实验的结果将是开发从遗传校正的IPS细胞有效产生可移植HSC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.