人胚胎干细胞诱导分化为具有完全骨髓定植能力的CD34+造血祖细胞的机制研究

CD34 is one of human hematopoietic-endothelial markers, and is used to quantify hematopoietic stem cells in clinical bone marrow transplantation. Human embryonic stem cell-derived CD34+ cells are able to differentiate into various mature blood cell in vitro. However, they were demonstrated to behave more towards endothelial differentiation and fail to engraft into adult bone marrow in animal models, indicating that current hESC differentiating system is unable to differentiate hESCs into hematopoietic progenitor cells with mature engrafting capability. We thus hypothesized that the levels of cellular differentiation between hESC-derived CD34+ cells and adult bone marrow might not be identical. We found in our previous study that, compared with cord blood (CB)-derived CD34+ cells, hESC-CD34+ cells expressed higher level of Nanog, indicating that hESC-CD34+ cells are less differentiated. Furthermore,expressions of Cadherins and CXCR4 on hESC-CD34+ cells, the regulating cellular engraftment into hematopietic tissues, were different from those on CB-CD34+ cells. In this study, we will investigate the regulating mechanism of Cadherins and CXCR4 expression on hESC-CD34+ cells during their differentiation. In addition, we will monitor the capability of hESC-CD34+ cells migration to and engraftment into adult bone marrow by using confocol system and in vivo flow cytometry system which we developped before.The clarification of molecular mechanism regulating engraftment of hESC-CD34+ cells into adult hematopoietic tissue will shed lights on their translational application.

CD34作为人造血-内皮祖细胞的表面标记，是临床进行骨髓移植时计数造血祖细胞的依据。人胚胎干细胞（hESC）来源的CD34+细胞虽能在体外分化为各种成熟的血细胞，但更倾向于内皮系分化，不能在骨髓等成体造血组织长期定植，我们推测这可能是现有方法无法诱导hESC分化为具有完全定植能力的造血祖细胞、细胞分化程度与成体骨髓微环境不吻合所致。我们以脐血为对照，发现hESC-CD34+细胞的分化成熟度不如脐血CD34+细胞，Cadherin、CXCR4等影响细胞迁移定植力的表面分子表达也与脐血CD34+细胞存在差异，调控细胞表型转化的转录因子表达也比脐血低。本课题拟进一步研究影响hESC-CD34+细胞表型转化和Cadherin、CXCR4等表面分子表达的相关分子机制，利用小鼠模型在体、定量、动态观测hESC-CD34+细胞在成体骨髓中的归巢定植能力及调控因素，为hESC在血液领域的转化应用提供依据。

人胚胎干细胞（hESC）来源的CD34+细胞虽能在体外分化为各种成熟的血细胞，但不能在骨髓等成体造血组织长期定植，更倾向于内皮系分化，推测这可能是现有方法无法诱导hESC分化为具有完全定植能力的造血祖细胞、细胞分化程度与成体骨髓微环境不吻合所致。在本课题研究中，我们将hESC来源CD34+细胞进一步进行造血系再诱导，发现hESC分化后的CD34+细胞在不同的细胞因子诱导下可以分化成造血系祖细胞或内皮系祖细胞。在造血因子SCF和Flt-3L的作用下，hESC来源CD34+细胞可以进一步分化为表达CD45的造血细胞。再通过离心EB的改良诱导方法，可以获得较大量的hESC来源CD34+CD45+细胞。我们比较了未分化hPSC、hPSC来源CD34+细胞和脐血CD34+细胞的影响细胞迁移的上皮间质转化（EMT）相关基因表达，发现人多能干细胞来源CD34+细胞分化成熟度不如脐血来源CD34+细胞。其中Cadherin、CXCR4 等影响细胞迁移定植力的表面分子表达、EMT相关转录因子表达与脐血CD34+细胞存在差异，抑制EMT相关转录因子Zeb的miRNA-200家族表达也存在差异。造血生长因子BMP4可以调控hPSC来源CD34+细胞EMT相关转录因子的表达水平，EMT相关转录因子Zeb2敲除的iPSC在向造血干细胞分化过程中存在缺陷，说明对Zeb2进行调控有望可增强hPSC来源CD34+细胞造血系分化成熟度，增强向骨髓迁移定植的能力。影响造血干细胞定植骨髓的另一重要因素是成体的骨髓微环境，其中骨髓间充质干细胞（BMSC）是最重要的骨髓微环境间质细胞，BMSC的成脂分化异常影响对造血干细胞的支持，我们发现AML患者的BMSC成脂分化提前，免疫介导骨髓衰竭的再生障碍性贫血患者的BMSC成脂分化能力也显著增强，相应的小鼠模型实验结果证明活化的T细胞导致小鼠BMSC成脂分化基因表达增高。本课题研究中我们成功建立了实时在体观测活体小鼠骨髓微环境的模型平台，观测到尾静脉移植小鼠骨髓细胞后，荧光标记的骨髓细胞迁移到颅骨骨髓腔早期归巢的时间-空间进程，该平台可用于观测在体定量动态观测hESC-CD34+细胞在成体骨髓中的迁移归巢定植研究。