Miyoshi Hirotoshi, Sato Chiaki, Shimizu Yuichiro, Morita Misa
Department of Biomedical Engineering, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
Int J Artif Organs. 2019 Jul;42(7):374-379. doi: 10.1177/0391398819827596. Epub 2019 Feb 12.
With the aim of establishing an effective method to expand hematopoietic stem/progenitor cells for application in hematopoietic stem cell transplantation, we performed ex vivo expansion of hematopoietic stem/progenitor cells derived from mouse fetal liver cells in three-dimensional cocultures with stromal cells. In these cocultures, stromal cells were first cultured within three-dimensional scaffolds to form stromal layers and then fetal liver cells containing hematopoietic cells were seeded on these scaffolds to expand the hematopoietic cells over the 2 weeks of coculture in a serum-containing medium without the addition of cytokines. Prior to coculture, stromal cell growth was suppressed by treatment with the DNA synthesis inhibitor mitomycin C, and its effect on hematopoietic stem/progenitor cell expansion was compared with that in control cocultures in which fetal liver cells were cocultured with three-dimensional freeze-thawed stromal cells. After coculture with mitomycin C-treated stromal cells, we achieved a several-fold expansion of the primitive hematopoietic cells (c-kit hematopoietic progenitor cells >7.8-fold, and CD34 hematopoietic stem/progenitor cells >3.5-fold). Compared with control cocultures, expansion of hematopoietic stem/progenitor cells tended to be lower, although that of hematopoietic progenitor cells was comparable. Thus, our results suggest that three-dimensional freeze-thawed stromal cells have higher potential to expand hematopoietic stem/progenitor cells compared with mitomycin C-treated stromal cells.
为了建立一种有效的方法来扩增造血干/祖细胞以应用于造血干细胞移植，我们将源自小鼠胎肝细胞的造血干/祖细胞与基质细胞进行三维共培养，进行了体外扩增。在这些共培养中，首先将基质细胞培养在三维支架内以形成基质层，然后将含有造血细胞的胎肝细胞接种在这些支架上，在含血清的培养基中于不添加细胞因子的情况下共培养2周来扩增造血细胞。在共培养之前，用DNA合成抑制剂丝裂霉素C处理抑制基质细胞生长，并将其对造血干/祖细胞扩增的影响与对照共培养（其中胎肝细胞与三维冻融基质细胞共培养）中的影响进行比较。在用丝裂霉素C处理的基质细胞共培养后，我们实现了原始造血细胞的数倍扩增（c-kit造血祖细胞>7.8倍，CD34造血干/祖细胞>3.5倍）。与对照共培养相比，造血干/祖细胞的扩增虽然造血祖细胞的扩增相当，但往往较低。因此，我们的结果表明，与丝裂霉素C处理的基质细胞相比，三维冻融基质细胞具有更高的扩增造血干/祖细胞的潜力。
