Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells
Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells
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DOI：
10.1016/s0301-472x(00)00551-8
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发表时间：
2000-12-01
影响因子：
2.6
通讯作者：
Flake, AW
中科院分区：
文献类型：
--
作者：
Kitano, Y;Radu, A;Flake, AW
Objective.由于骨髓间充质祖细胞（MPCs）中含有造血细胞，因此很难对MPCs进行鉴定。我们利用重新铺板后MPC的快速增殖，通过用携带LacZ和选择性新霉素抗性（neoR）基因的逆转录病毒载体转染来富集鼠MPC。将新鲜收获的小鼠骨髓细胞与由嗜异性psi-CRIP或嗜异性psi-CRE生产细胞产生的BAG逆转录病毒载体孵育48小时，并在G418存在下生长。在psi-CRIP上清液中孵育的细胞形成由不含CD 45（+）细胞的大的同质细胞组成的集落，但在psi-CRE上清液中孵育的细胞不形成基质细胞集落。在未分化状态下，细胞显示成纤维细胞样表型，碱性磷酸酶活性低。然而，在用地塞米松或5-氮杂胞苷处理后，逆转录病毒转导的细胞分化成油红O阳性脂肪细胞和成骨细胞，产生von Kossa阳性骨结节。由β-甘油磷酸盐、地塞米松和抗坏血酸组成的成骨补充剂诱导碱性磷酸酶活性增加和与早期细胞脱离相关的急性成骨。将逆转录病毒转导的细胞皮下注射到同一品系的第1天新生小鼠中，产生了被X-gal（+）细胞包围的异位钙沉积。逆转录病毒选择循环贴壁细胞是一种有效的方法来富集MPC。(C)2000年国际实验血液学学会。出版社：Elsevier Science Inc.
Objective. It has been difficult to characterize murine bone marrow (BM)-derived mesenchymal progenitor cells (MPCs) because of contamination with hematopoietic cells. We took advantage of the rapid proliferation of MPCs after replating to enrich murine MPCs by transfection with a retroviral vector carrying both LacZ and the selective neomycin resistance (neoR) gene.Materials and Methods. Freshly harvested BM cells from mice were incubated with BAG retroviral vector produced by amphotropic psi -CRIP or ecotropic psi -CRE producer cells for 48 hours and grown in the presence of G418.Results. Cells incubated in psi -CRIP supernatant formed colonies composed of large homogeneous cells that were free of CD45(+) cells, but cells incubated in psi -CRE supernatant did not form stromal cell colonies. In the undifferentiated state, the cells displayed a fibroblast-like phenotype with low alkaline phosphatase activity. However, upon treatment with dexamethasone or 5-azacytidine, the retrovirally transduced cells differentiated into oil-red-O-positive adipocytic cells and osteogenic cells generating von Kossa-positive bone nodules. Osteogenic supplements composed of beta -glycerophosphate, dexamethasone, and ascorbic acid induced an increase in alkaline phosphatase activity and acute osteogenesis associated with early cell detachment. Subcutaneous injection with retrovirally transduced cells into day 1 newborn mice of the same strain produced ectopic calcium depositions surrounded by X-gal(+) cells,Conclusions. Retroviral selection of cycling adherent cells is an effective approach for enrichment of MPCs. (C) 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.