胶原支架促进人类多能干细胞向红细胞的诱导分化

Biomimetic macroporous collagen hydrogels as 3D scaffolds promotes the in vitroproliferation of erythroblasts from human embryonic stem cell-derived definitive hematopoiesis

目的建立人胚胎干细胞（hESCs）来源的造血干/组细胞向红细胞分化的体外3D培养方法。方法hESCs与小鼠主动脉-性腺-中肾区（AGM-S3）基质细胞共培养14 d后获得CD34^＋CD45^＋造血干/祖细胞,对获得的CD34^＋CD45^＋细胞扩增5 d后,以相同初始接种数量分别接种于胶原水凝胶、透明质酸水凝胶、Ⅰ型胶原蛋白构建的三维（3D）支架材料（简称胶原支架）中,在体外模拟骨髓微环境,做红细胞定向分化,以普通液体悬浮培养（简称液体培养）为对照,通过MGG染色、流式细胞术、免疫染色、qRT-PCR等手段分别对收获的细胞做形态学、红系特异性表面标志表达情况、红系细胞成熟程度以及红系细胞发育过程中相关基因的表达情况分析。结果红系定向分化14 d后,胶原支架中收获的总细胞数量分别是液体培养、透明质酸水凝胶、胶原水凝胶的1.50、1.19、1.33倍,GPA^＋CD71^＋细胞数量分别是液体培养、透明质酸水凝胶、胶原水凝胶的1.55、1.25及1.48倍;GPA^＋CD36^＋细胞数量分别是液体培养、透明质酸水凝胶、胶原水凝胶的1.65、1.07、1.36倍。结论利用Ⅰ型胶原蛋白构建的3D支架材料可模拟骨髓微环境,促进红细胞分化。

Objective To establish anin vitrothree-dimensional（3D） microenvironment favoring the differentiationand proliferation of hematopoietic progenitors,derived from humanembryonicstemcells（hESCs）,towards erythroblasts with biomimetic macroporous hydrogel scaffolds mimicking the bone marrow architecture. Methods According to the methods previously reported,we co-cultured hESCs with the mice aorta-gonad-mesonephros（AGM-S3） stromal cell,allowing for efficient acquisition of CD34^＋ CD45^＋ hematopoietic progenitors. Hyaluronic acid hydrogel,type Ⅰ collagen hydrogel and type Ⅰ collagen hydrogel scaffold with microporous structures were synthesized to replicate the in vivo physical environment and functioned as the housing frame for the culture of these progenitors withconventional liquid culture being the control group. Erythroblasts were collected from these culture models andanalyzed by May-Grünwald-Giemsa staining,flow cytometry analysis and immunochemical staining.The expression of hematopoiesis-relatedgeneswas observed by qRT-PCR.Results After 14 days of culture,the total cell number harvested from thecollagen scaffold model was 1. 50 times,1. 19 times and 1. 33 times ofthe cell numbers of the liquid culture model,the hyaluronic acid hydrogel model and the collagen hydrogel model,respectively.The cell number of GPA^＋ CD71^＋ cells harvested from thecollagen scaffold model was 1. 55 times,1. 25 times and 1. 48 times of the cell numbersthe liquid culture model,the hyaluronic acid hydrogel model and the collagen hydrogel model,respectively.The cell number of GPA^＋ CD36^＋ cells harvested from the collagen scaffold model was 1. 65 times,1. 07 times and 1. 36 times of the cell numbers from the liquid culture model,the hyaluronic acid hydrogel model and the collagen hydrogel model,respectively. Conclusion we have successfully developed a microporous-scaffold culture modelthat partially mimicked the bone marrow microenvironment in vivo. This model can promote the proliferation of erythroblasts in vitro.