Rapid ex vivo expansion of human umbilical cord hematopoietic progenitors using a novel culture system
Rapid ex vivo expansion of human umbilical cord hematopoietic progenitors using a novel culture system
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DOI：
10.1016/s0301-472x(99)00012-0
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发表时间：
1999-05-01
影响因子：
2.6
通讯作者：
Hotta, T
中科院分区：
文献类型：
--
作者：
Kawada, H;Ando, K;Hotta, T
细胞数量限制了脐带血（CB）在成人基因治疗和骨髓替代中的广泛临床应用；需要一种简单有效的方法来离体扩增 CB 原始祖细胞 (PPC)。最近，据报道，血小板生成素 (TPO) 和 Flk-2/Flt-3 配体 (FL-2) 的组合可支持 CB-PPC 在无基质液体培养物中缓慢增殖。我们建立了一种新型培养系统，其中鼠基质细胞系 HESS-5 与 TPO/FL-2 协同作用，可显着支持冷冻保存的 CB-PPC 的快速扩增。此外，虽然 HESS-5 细胞在培养过程中直接粘附于人类祖细胞，但培养的人类细胞可以轻松收获，而不会被 HESS-5 细胞污染。培养 7 天内，含血清培养物中 CD34(bright)/CD38(dim) 细胞数量增加了 100 倍。当 HESS-5 细胞在 TPO/FL-2 存在下与人祖细胞物理分离时，协同作用被阻断，表明 HESS-5 细胞通过直接细胞间相互作用支持 PPC 的增殖。然后在非常短期（5天）的无血清培养，通过添加干细胞因子（SCF）或白介素-3（IL-3）进一步增强扩增，结果是，CD34（亮）/CD38（暗）细胞增加了50至100倍，培养物中的集落形成单位（CFU-C）和混合集落（CFU-GER IRI）增强了10至30倍，并且分别增加了 10 至 20 倍，此外，从 CD34(bright)/CD38(dim) 细胞产生的长期培养起始细胞 (LTC-IC) 扩增了 25 倍。严重联合免疫缺陷 (SCID) 小鼠再生细胞 (SRC) 测定证实了扩增细胞重建长期造血的广泛能力。这些结果表明，这种异种共培养系统与人类细胞因子结合，可以从冷冻保存的 CB 中快速生成 PPC，(C) 1999 国际实验血液学学会。由爱思唯尔科学公司出版
Cell numbers limit the widespread clinical use of cord blood (CB) for gene therapy and marrow replacement in adults; a simple and effective method for ex vivo expansion of CB primitive progenitor cells (PPC) is required, Recently, the combination of thrombopoietin (TPO) and Flk-2/Flt-3 ligand (FL-2) was reported to support slow proliferation of CB-PPC in stroma-free liquid culture. We established a novel culture system in which the murine stromal cell line HESS-5 dramatically supports the rapid expansion of cryopreserved CB-PPC in synergy with TPO/FL-2, Furthermore, while HESS-5 cells directly adhered to human progenitors during culture, the cultured human cells could easily be harvested without contamination by HESS-5 cells. Within 7 days of culture, a 100-fold increase in CD34(bright)/CD38(dim) cells was obtained in serum-containing culture, When HESS-5 cells were physically separated from human progenitor cells in the presence of TPO/FL-2, synergy was blocked, suggesting that HESS-5 cells support proliferation of PPC by direct cell-to-cell interaction, The hematopoietic-supportive effects of this xenogeneic coculture system were then assessed in a very short-term (5 days) serum-free culture, Expansion was further enhanced by addition of stem cell factor (SCF) or interleukin-3 (IL-3), As a result, a 50- to 100-fold increase in CD34(bright)/CD38(dim) cells was noted, Colony-forming units in culture (CFU-C) and mixed colonies (CFU-GER IRI) were enhanced by 10- to 30-fold and 10- to 20-fold, respectively, Moreover, generation of long-term-culture-initiating cells (LTC-IC) from CD34(bright)/CD38(dim) cells was amplified by 25-fold, The severe-combined immunodeficient (SCID) mouse-repopulating cell (SRC) assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. These results indicate that this xenogeneic coculture system, in combination with human cytokines, can rapidly generate PPC from cryopreserved CB, (C) 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc.