DIRECTED DIFFERENTIATION OF ES AND IPS CELLS TO HEMATOPOIETIC STEM CELLS

描述（由申请人提供）：胚胎干（ES）细胞在再生生物学和医学领域提供了一个令人兴奋的机会，因为它们具有独特的能力，可以在培养中分化成构成个体的所有体细胞。在从多能干细胞产生功能性造血干细胞（HSC）方面已经做出了重大努力。例如，在ES细胞的体外分化过程中HoxB 4（或Cdx 4-HoxB 4）的表达已显示从ES细胞衍生功能性HSC（1-3）。尽管取得了这些重大进展，但在利用体外衍生的HSC和祖细胞用于再生医学方面仍然存在重大挑战。例如，ES衍生的造血祖细胞的造血植入始终保持低水平。即使在HoxB 4表达的情况下，在Cdx 4-HoxB 4诱导的ES衍生细胞中，可以重新填充受体造血系统的HSC和祖细胞的频率估计最多为2- 5x 106中的1个（3）。移植细胞中的组成型HoxB 4表达也可能是有问题的。总体目标是从ES和iPS细胞稳健地产生功能性HSC。在实现这一目标的过程中，我们认为HSC是由ES细胞产生的，遵循体内发生的相同分子和细胞规则。为此，我们证明了Flk-1表达（Flk-1+）成血管细胞，造血和内皮细胞（或生血内皮）的共同祖细胞，衍生自体外分化的ES细胞，可以在培养物中产生造血细胞（4，5）。此外，我们证明了小鼠的原始和永久造血系统均来源于Flk-1+中胚层（6）。我们的初步研究表明，当Flk-1+成血管细胞被分选并在OP 9细胞上进一步分化时，我们能够有效地产生CD 41 +cKit+ CD 150+细胞，其先前已被鉴定为ES衍生的HSC/祖细胞（3）。此外，ER 71、GATA 2和Scl的时间共表达正诱导来自ES细胞的CD 41 +cKit+ CD 150+细胞：1）通过强烈诱导来自ES细胞的Flk-1+成血管细胞形成和2）通过独立诱导来自Flk-1+成血管细胞的CD 41 +cKit+ CD 150+细胞。在此，我们测试了一个假设，即ES细胞的功能性HSC生成可以通过时间ER 71、GATA 2和Scl共表达而大大改善。我们将确定ES和iPS衍生的CD 41 +cKit+ CD 150+细胞是否可以重建小鼠的造血系统，以及在产生功能性HSC中，时间ER 71、GATA 2和Scl共表达是否上级HoxB 4表达。有效地将ES和iPS细胞定向为HSC的能力将提供一种新的分化手段，并将在再生生物学和医学领域产生重大的潜在影响。这一结果也将可能彻底改变我们对细胞谱系发育和分化的设想，因此也将在基础研究领域产生重大的潜在影响。因此，潜在影响很大。

DESCRIPTION (provided by applicant): Embryonic stem (ES) cells provide an exciting opportunity in the field of regenerative biology and medicine, as they have the unique capacity to differentiate in culture into all somatic cells that make up an individual. Significant efforts have been made in generating functional hematopoietic stem cells (HSCs) from pluripotent stem cells. For instance, expression of HoxB4 (or Cdx4-HoxB4) during in vitro differentiation of ES cells has been shown to derive functional HSCs from ES cells (1-3). Despite these significant advancements, major challenges remain in utilizing in vitro derived HSCs and progenitors for regenerative medicine. For example, hematopoietic engraftment by ES derived hematopoietic progenitors remains consistently and disappointingly low. Even with HoxB4 expression, the frequency of HSCs and progenitors that can repopulate the recipient's hematopoietic system is estimated at best 1 in 2-5x106 among Cdx4-HoxB4 induced ES derived cells (3). Constitutive HoxB4 expression in transplanted cells could also be problematic. The overall objective is to robustly generate functional HSCs from ES and iPS cells. In achieving this goal, we posit that HSCs are generated from ES cells following the same molecular and cellular rules occurring in vivo. To this end, we demonstrated Flk-1 expressing (Flk-1+) hemangioblast, a common progenitor of hematopoietic and endothelial cells (or hemogenic endothelium), derived from in vitro differentiated ES cells can generate hematopoietic cells in culture (4, 5). In addition, we demonstrated that both primitive and definitive hematopoietic systems of the mouse are derived from the Flk-1+ mesoderm (6). Our preliminary studies indicate that when Flk-1+ hemangioblasts were sorted and further differentiated on OP9 cells, we were able to efficiently generate CD41+cKit+CD150+ cells, which have been previously identified as ES derived HSC/progenitors (3). Moreover, temporal co-expression of ER71, GATA2 and Scl positively induced CD41+cKit+CD150+ cells from ES cells: 1) by robustly inducing Flk-1+ hemangioblast formation from ES cells and 2) by independently inducing CD41+cKit+CD150+ cells from Flk-1+ hemangioblasts. Herein, we test a hypothesis that functional HSC generation from ES cells can be greatly improved by temporal ER71, GATA2 and Scl co-expression. We will determine if ES and iPS derived CD41+cKit+CD150+ cells could reconstitute hematopoietic system of the mouse and if temporal ER71, GATA2 and Scl co-expression would be superior to HoxB4 expression in generating functional HSCs. The ability to effectively direct ES and iPS cells to HSCs will provide a novel means of differentiation and will have a major potential impact in the field of regenerative biology and medicine. The outcome will also likely revolutionize the way we envision the cell lineage development and differentiation, thus will also have a major potential impact in the basic research field. Thus, the potential impact is high.