脐血CD34^+细胞体外诱导分化为成熟巨核细胞及产出血小板(英文)

In vitro differentiation of umbilical cord blood CD34^+ cells into mature megakaryocytes and generation of platelets

背景:据作者查新检索,国外尚无通过体外诱导干细胞生成具有功能的血细胞并形成产品的报道。目的:体外诱导脐血CD34+细胞向成熟巨核细胞分化,并观察血小板产出情况。设计、时间及地点:细胞学体外观察,于2004/2006在湘雅医院及湘雅三医院中心实验室完成。材料:脐带来源于足月妊娠健康产妇,由湘雅医院提供。方法:免疫磁珠法分选脐血CD34+细胞,按5×107L-1密度接种于24孔培养板,加入含L-谷氨酰胺、铁饱和的人转铁蛋白、CaCl2、胰岛素、去离子牛血清白蛋白及重组人血小板生成素的StemPro-34无血清培养基,置于37℃、体积分数为0.05的CO2饱和湿度条件下向巨核细胞诱导培养14~21d。吸取细胞培养液,离心取上清,再次离心弃上清,余下物质即为细胞培养液中比重较小的血小板样颗粒。同法分离正常富血小板血浆中的血小板。主要观察指标:培养细胞与上清液中血小板样颗粒的形态变化、免疫组织化学染色结果、显微及超微结构观察,血小板聚集情况及CD41的表达。结果:培养第10天,巨核细胞诱导培养体系中出现丝状物质,并有血小板样颗粒产生,第16天达高峰;培养细胞强阳性表达血小板特异性抗原GPⅡbⅢa;光镜观察培养细胞呈成熟巨核细胞形态,但也可见幼稚巨核细胞样,巨核细胞旁可见血小板样颗粒;电镜观察培养细胞大多呈成熟巨核细胞形态,少量呈凋亡状态,上清液中血小板样颗粒与富血小板血浆中的血小板大小、超微结构基本一致,有的血小板表面光滑,有的则呈现不规则表面。上清液中血小板样颗粒与正常富血小板血浆中的血小板均能对凝血酶产生聚集反应,流式细胞仪检测两者具有同样的CD41高表达率。结论:脐血CD34+细胞能在体外诱导生成高纯度且成熟的巨核细胞,并产出血小板。

BACKGROUND： There still was not any report about inducing stem cells into matured cells to form products in vitro. OBJECTIVE： To induce CD34^＋ cells of umbilical cord blood to differentiate into mature megakaryocytes, and to investigate the mechanism of production of platelets. DESIGN, TIME AND SETTING： This cytology in vitro study was conducted at the Central Laboratory of Xiangya Hospital and Xiangya Third Hospital from 2004 to 2006. MATERIALS： Umbilical cord was collected from healthy full-term pregnant puerperants at the Xiangya Hospital. METHODS： The CD34^＋ cells were isolated from umbilical cord blood by magnetic activated cell sorting （MACS） and then cultured in 24-well culture plate at 5×10^7/L in StemPro-34 serum-free medium, supplemented with L-glutamine, saturated human transferrin, CaCl2, insulin, deionized bovine serum albumin and recombinant human thrombopoietin at 37 ℃, under 0.05 volume fraction CO2 saturated humidity to be differentiated into rnegakaryocytes for 14 21 days. Cell medium was absorbed, and centrifuged to obtain supernatant. Samples were centrifuged again, and then supernatant was removed. The remaining was platelet-like particles in cell culture plate. Platelet was isolated from normal platelet-rich plasma. MAIN OUTCOME MEASURES： The following parameters were measured： morphological changes in cultured cells and platelet-like particles in supernatant; results of immunohistochemistry; observation results under a microscope; platelet aggregation; CD41 expression. RESULTS： At day 10, silk-like substances were found in megakaryocyte culture medium, with the presence of platelet-sized particles. The production of platelet-sized particles reached a peal at day 16, Cultured cells were strongly positively for platelet-specific antigen GP Ⅱb Ⅲa. Under the optical microscope, mature megakaryocytes were detected, with the presence of some immature megakaryocytes, and platelet-sized particles were found surrounding megakaryocytes. Under the electron microscope, a majority of mature megakaryocytes and a few apoptotic megakaryocytes were detected, and platelet-sized particles in the supernatant had the same size and structure with the platelet in the platelet-rich plasma. Some platelet surfaces were smooth or irregular. Platelet-sized particles in the supernatant aggregated in response to thrombin as platelets in normal platelet-rich plasma. Flow cytometry demonstrated that the cultured platelets had the same high expression rate of CD41 as the platelets from platelet rich plasma. CONCLUSION： Umbilical cord blood CD34^＋ cells can be induced to differentiate into purified and mature megakaryocytes and platelets in vitro.