DIRECTED DIFFERENTIATION OF ES AND IPS CELLS TO HEMATOPOIETIC STEM CELLS

描述（由申请人提供）：胚胎干细胞（ES）在再生生物学和医学领域提供了一个令人兴奋的机会，因为它们具有在培养中分化成构成个体的所有体细胞的独特能力。从多能干细胞制备功能性造血干细胞（hsc）已经取得了重大进展。例如，在胚胎干细胞的体外分化过程中，HoxB4（或Cdx4-HoxB4）的表达已被证明可从胚胎干细胞中获得功能性造血干细胞（1-3）。尽管取得了这些重大进展，但在利用体外来源的造血干细胞和祖细胞进行再生医学方面仍然存在主要挑战。例如，胚胎干细胞衍生的造血祖细胞的造血植入率一直很低，令人失望。即使有HoxB4表达，在Cdx4-HoxB4诱导的ES衍生细胞中，能够重新填充受体造血系统的造血干细胞和祖细胞的频率估计最多为1 / 2-5x106(3)。移植细胞中的组成性HoxB4表达也可能存在问题。总体目标是从胚胎干细胞和iPS细胞中生成功能性造血干细胞。为了实现这一目标，我们假设造血干细胞是由胚胎干细胞产生的，遵循体内发生的相同的分子和细胞规则。为此，我们证明了Flk-1表达（Flk-1+）的成血管细胞，一种造血细胞和内皮细胞（或造血内皮）的共同祖细胞，来源于体外分化的胚胎干细胞，可以在培养中产生造血细胞（4,5）。此外，我们证明小鼠的原始和最终的造血系统都来源于Flk-1+中胚层(6)。我们的初步研究表明，当Flk-1+血管母细胞被分类并在OP9细胞上进一步分化时，我们能够有效地生成CD41+cKit+CD150+细胞，这些细胞之前被鉴定为ES来源的HSC/祖细胞(3)。此外，ER71、GATA2和Scl的时间共表达对ES细胞的CD41+cKit+CD150+细胞有正向诱导作用：1)通过强烈诱导ES细胞形成Flk-1+成血管细胞，2)通过独立诱导Flk-1+成血管细胞形成CD41+cKit+CD150+细胞。在此，我们验证了一个假设，即ER71、GATA2和Scl的时间共表达可以极大地促进ES细胞的功能性HSC生成。我们将确定ES和iPS衍生的CD41+cKit+CD150+细胞是否可以重建小鼠的造血系统，以及在生成功能性造血干细胞方面，时间ER71、GATA2和Scl的共表达是否优于HoxB4的表达。有效引导胚胎干细胞和iPS细胞分化为造血干细胞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。这一结果也可能会彻底改变我们对细胞谱系发育和分化的看法，因此也将在基础研究领域产生重大的潜在影响。因此，潜在的影响是很大的。

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.