体外诱导小鼠胚内生血内皮细胞向造血分化的不同培养体系的比较研究

A comparative study of different incubation culture systems for hematopoi⁃etic differentiation of mouse embryonic hemogenic endothelial cells in vitro

目的:比较二维平铺法和三维悬滴法两种培养体系对小鼠胚胎期生血内皮细胞体外诱导生成造血产物的支持作用。方法:分别利用二维平铺法和三维悬滴法将流式细胞术分选的造血干细胞潜能的生血内皮细胞PK44(CD41-CD43-CD45-CD31^(+)CD201^(+)Kit^(+)CD44^(+))群体与OP9-DL1-GFP基质细胞共孵育6 d后,在显微镜下观察两种培养体系产生造血产物的形态特点;利用流式细胞术分析两种培养体系孵育后的造血产物,并统计分析造血产物的比例和绝对数量;分别对两种培养体系共孵育后的造血产物进行造血细胞集落形成单位培养(colony-forming unit culture,CFU-C)实验,并对不同形态的细胞集落进行计数。结果:(1)利用二维平铺法形成的造血细胞平铺或悬浮于基质细胞上,而利用三维悬滴法共孵育后的产物形成了以基质细胞为中心,造血细胞黏附在周围的细胞聚集体。(2)二维平铺法产生更多CD45^(+)造血细胞;三维悬滴法对造血产物的分化具有谱系偏向性,更有利于肥大细胞的分化。(3)二维平铺法诱导生血内皮的分化产物在体外CFU-C实验中可以产生更多的造血集落。结论:二维平铺法更支持小鼠胚内生血内皮细胞体外诱导产生CD45^(+)的造血细胞以及有集落形成能力的造血干祖细胞。

AIM:To compare the supportive effects of two-dimension(2D)tiling and three-dimension(3D)hanging drop culture systems on the in vitro induction of hematopoiesis of mouse embryonic hemogenic endothelial cells.METHODS:The hematopoietic stem cell-competent hemogenic endothelial cell PK44(CD41-CD43-CD45-CD31^(+)CD201^(+)Kit^(+)CD44^(+))populations were sorted by flow cytometry and co-cultured with OP9-DL1-GFP stromal cells for 6 d using 2D tiling and 3D hanging drop culture systems,respectively,and the morphological characteristics of hematopoietic products were observed under the microscope.Hematopoietic products of the two induction systems were analyzed by flow cytometry,and the proportion and absolute number of hematopoietic products were counted and analyzed.Hematopoietic products after co-incubation with the two induction systems were subjected to colony-forming unit culture(CFU-C)assay and the different types of colonies were counted.RESULTS:(1)The hematopoietic products formed by incubation with the 2D tiling method laid or suspended on the stromal cells,whereas the products of co-incubation by the 3D hanging drop method formed an aggregate of cells centered on the stromal cells with hematopoietic cells adhering to the surrounding area.(2)The 2D tiling method produced more CD45^(+)hematopoietic cells;the 3D hanging drop method had a biased ef-fect on hematopoietic lineage differentiation,which was more conducive to the differentiation of mast cells.(3)The products from the 2D tiling method produced more hematopoietic colonies in the in vitro CFU-C assay.CONCLUSION:The 2D tiling method is more conducive to mouse embryonic hemogenic endothelial cells to produce CD45^(+)hematopoietic cells and hematopoietic stem/progenitor cells with colony-forming ability in the induction of hematopoiesis in vitro.