Institut de Recherche en Hématologie et Transplantation, Mulhouse, France.
Exp Hematol. 2011 Apr;39(4):495-505. doi: 10.1016/j.exphem.2011.01.003. Epub 2011 Jan 14.
Recently, we demonstrated that normal human bone marrow (hBM)-derived CD34(+) cells, released into the peripheral blood after granulocyte colony-stimulating factor mobilization, contain cell subpopulations committed along endothelial and cardiac differentiation pathways. These subpopulations could play a key role in the regeneration of post-ischemic myocardial lesion after their direct intracardiac delivery. We hypothesized that these relevant cells might be issued from very small embryonic-like stem cells deposited in the BM during ontogenesis and reside lifelong in the adult BM, and that they could be mobilized into peripheral blood by granulocyte colony-stimulating factor.
Samples of normal hBM and leukapheresis products harvested from cancer patients after granulocyte colony-stimulating factor mobilization were analyzed and sorted by multiparameter flow cytometry strategy. Immunofluorescence and reverse transcription quantitative polymerase chain reaction assays were performed to analyze the expression of typical pluripotent stem cells markers.
A population of CD34(+)/CD133(+)/CXCR4(+)/Lin(-) CD45(-) immature cells was first isolated from the hBM or from leukapheresis products. Among this population, very small (2-5 μm) cells expressing Oct-4, Nanog, and stage-specific embryonic antigen-4 at protein and messenger RNA levels were identified.
Our study supports the hypothesis that very small embryonic-like stem cells constitute a "mobile" pool of primitive/pluripotent stem cells that could be released from the BM into the peripheral blood under the influence of various physiological or pathological stimuli. In order to fully support that hBM- and leukapheresis product-derived very small embryonic-like stem cells are actually pluripotent, we are currently testing their ability to differentiate in vitro into cells from all three germ layers.
最近，我们证明了经过粒细胞集落刺激因子动员后释放到外周血中的正常人骨髓（hBM）来源的 CD34（+）细胞中含有沿内皮和心脏分化途径分化的细胞亚群。这些亚群可在直接心脏内给药后，在缺血性心肌损伤后再生中发挥关键作用。我们假设这些相关细胞可能来自于在个体发生过程中沉积在 BM 中的非常小的胚胎样干细胞，并在成年 BM 中终身存在，并且它们可以通过粒细胞集落刺激因子动员到外周血中。
分析和通过多参数流式细胞术策略对来自癌症患者在粒细胞集落刺激因子动员后收获的正常 hBM 和白细胞分离产物进行分选。进行免疫荧光和逆转录定量聚合酶链反应检测以分析典型多能干细胞标志物的表达。
首先从 hBM 或白细胞分离产物中分离出 CD34（+）/CD133（+）/CXCR4（+）/Lin（-）CD45（-）幼稚细胞群。在该群体中，鉴定出在蛋白质和信使 RNA 水平上表达 Oct-4、Nanog 和阶段特异性胚胎抗原-4 的非常小（2-5μm）细胞。
我们的研究支持了这样的假说，即非常小的胚胎样干细胞构成了一个“可移动”的原始/多能干细胞池，可在各种生理或病理刺激的影响下从 BM 释放到外周血中。为了充分支持 hBM 和白细胞分离产物来源的非常小的胚胎样干细胞实际上是多能的，我们目前正在测试它们在体外分化为来自所有三个胚层的细胞的能力。
