DIRECTED DIFFERENTIATION OF ES AND IPS CELLS TO HEMATOPOIETIC STEM CELLS

ES 和 IPS 细胞定向分化为造血干细胞

• 批准号: 8293183
• 负责人: KYUNGHEE CHOI
• 金额: $ 19万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8293183
• 关键词: Affect; Applications Grants; Basic Science; Biological Models; Biology; Blood; Blood Cells; Cell Lineage; Cell Transplants; Cells; Development; Disease; Embryo; Endothelial Cells; Endothelium; Engraftment; Frequencies; Funding Mechanisms; Generations; Genes; Goals; Growth; Hematopoietic; Hematopoietic System; Hematopoietic stem cells; Human Development; In Vitro; Individual; Medicine; Mesoderm; Molecular; Mus; Natural regeneration; Nature; Outcome; Pluripotent Stem Cells; Regenerative Medicine; SLAM protein; Somatic Cell; Sorting - Cell Movement; Stem cells; Testing; embryonic stem cell; improved; in vivo; induced pluripotent stem cell; novel; progenitor; reconstitution; regenerative; stem

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.

描述(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为组成个体的所有体细胞的独特能力。在从多能干细胞中生成具有功能的造血干细胞(HSCs)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表达HOXB4(或CDX4-HOXB4)已被证明是从ES细胞(1-3)分化而来的功能性HSCs。尽管取得了这些重大进展，但在利用体外衍生的造血干细胞和祖细胞用于再生医学方面仍然存在重大挑战。例如，ES来源的造血祖细胞的造血植入一直保持在令人失望的低水平。即使有HOXB4的表达，在CDX4-HOXB4诱导的ES来源细胞中，能够重新填充受者造血系统的造血干细胞和祖细胞的频率估计最多为1/2-5×106(3)。移植细胞中结构性HOXB4的表达也可能是有问题的。总体目标是从ES和iPS细胞中强劲地产生具有功能的HSCs。为了实现这一目标，我们假设干细胞是由ES细胞产生的，遵循在体内发生的相同的分子和细胞规则。为此，我们证明了由体外分化的ES细胞分化而来的表达Flk-1的血管母细胞(Flk-1+)可以在培养中生成造血细胞(4，5)。此外，我们还证明了小鼠的原始和确定的造血系统都来自Flk-1+中胚层(6)。我们的初步研究表明，当Flk-1+血管母细胞在OP9细胞上被分选和进一步分化时，我们能够有效地产生CD41+cKit+CD150+细胞，这些细胞以前被鉴定为ES来源的HSC/前体细胞(3)。此外，ER71、GATA2和scl的时间共表达可从ES细胞中正向诱导CD41+cKit+CD150+细胞：1)强劲地诱导ES细胞形成Flk-1+成血管细胞；2)单独诱导CD41+cKit+CD150+细胞从Flk-1+成血管细胞分化为CD41+cKit+CD150+细胞。在此，我们验证了一种假设，即通过瞬时ER71、GATA2和scl的共同表达可以显著提高ES细胞产生功能性HSC的能力。我们将确定ES和iPS来源的CD41+cKit+CD150+细胞是否可以重建小鼠的造血系统，以及瞬时表达ER71、GATA2和scl在生成功能性HSCs方面是否优于HOXB4表达。有效地将ES和iPS细胞定向为HSCs的能力将提供一种新的分化途径，并将在再生生物学和医学领域产生重大的潜在影响。这一结果还可能彻底改变我们对细胞谱系发展和分化的设想，从而在基础研究领域也将产生重大的潜在影响。因此，潜在的影响很大。