骨髓间充质干细胞与造血干细胞共培养对脐血造血干细胞扩增的作用

Effects of bone marrow-derived mesenchymal stem cell layer on expansion and differentiation of hematopoietic stem cells from cord blood.

脐带血造血干细胞的异体移植对高剂量化疗的患者恢复造血功能是一种有效的细胞学疗法.脐带血造血干细胞的体外扩增能提高有效植入的造血干/祖细胞的数量.将扩增10 d的造血干/祖细胞移植给高剂量化疗的成人患者,患者平均约在移植后的第26天(第15～45天)达到了嗜中性粒细胞的植入.为了加快有核细胞数量的扩增和嗜中性粒细胞的成熟.在以骨髓间充质干细胞为培养基质细胞层,采取阶段扩增方法培养单个核细胞和分选的CD34 细胞,并与无骨髓间充质干细胞的培养体系进行对照.从冻存的脐带血中分离的单个核细胞和CD34 细胞分别在含有骨髓间充质干细胞的100 mL培养袋中用50 mL ADM培养液培养7 d,7 d后收获细胞并移入含有1L ADM培养液的1 L培养袋中继续培养7 d.每天计数细胞量.14d后收获细胞,并进行相应的细胞分析.扩增的结果表明,在有骨髓间充质干细胞的共培养体系中,单个核细胞培养的有核细胞扩增了346倍,CD34 细胞培养的有核细胞扩增了674倍.无骨髓间充质干细胞的非共培养体系中,两者分别扩增了152倍和377倍.两种培养体系都获得了定向祖细胞(GM—CFC)和原始祖细胞(HPP—CFC)的扩增.有骨髓间充质干细胞的共培养体系获得了更多的嗜中性粒细胞.

Allogcncic cord blood (CB) products as a source of cellular support for patient receiving high-dose chemotherapy were used for pediatric patients. Ex vivo expansion of CB cells was proposed as a method to increase the number of hematopoietic stem / progenitor cells available for transplantation. Following high-dose chemotherapy, we transplanted adult patients with the CB nucleated cells expanded for 10 days. Patients achieved neutrophil engraftment in a median of 26 days (range 15 to 45). In an attempt to increase expansion of total nucleated cells and further maturation of neutrophil precursors, we developed a co-culture system with human bone marrow-derived mesenchymal stem cells (MSCs) as a trophoblastic layer.Low-density mononuclear cells and CD34+ cells isolated from the frozen CB were cultured for 7 days at 37 C in 100 mL Teflon culture bags with MSC layer and 50 mL of ADM containing SCF, G-CSF and MGDF (100 ng/ mL). The cells harvested from these bags after 7 days were transferred to 1 L Teflon bags with MSC layer and 1 L of ADM pus SCF, G-CSF, and MGDF. After a second culture period of incubation, the cells were harvested and assayed for phenotypes of expanded cells and mature of committed progenitor cells (GM-CFC) and primitive progenitor cells (HPP-CFC).The cultures with MSCs result in the median total nucleated cell expansions of 346- and 674-folds for the low-density mononuclear cell culture and the CD34 ' cell culture; the cultures without MSCs result in the median total nucleated cell expansions of 152- and 377-folds for the low-density mononuclear cell culture and the CD34+ cell culture. The cultures with MSCs lead to expansions of GM-CFC and HPP-CFC. We propose that the availability of increased numbers of expanded CB cells by the culture with MSCs may result in more rapid engraftment of neu-trophils following infusion to transplant recipients.