异体人脐带华通氏胶干细胞在体外和体内均可使人类脐血 CD34+细胞的扩增增加几倍。

BACKGROUND: The transplantation of human umbilical cord blood (UCB) CD34+ cells has been successfully used to treat hematological disorders but one major limitation has been the low cell numbers available. Mesenchymal stem cells (MSCs) lying within the bone marrow in vivo behave like a scaffold on which CD34+ cells interact and proliferate. We therefore evaluated the use of allogeneic MSCs from the human UC Wharton's jelly (hWJSCs) as stromal support for the ex vivo expansion of CD34+ cells. METHODS: We performed an in-depth evaluation of the primitiveness, migration, adhesion, maturation, mitochondrial behavior, and pathway mechanisms of this platform using conventional assays followed by the evaluation of engraftment potential of the expanded CD34+ cells in an in vivo murine model. RESULTS: We demonstrate that hWJSCs and its conditioned medium (hWJSC-CM) support the production of significantly high fold changes of CD34+, CD34+CD133+, CD34+CD90+, CD34+ALDH+, CD34+CD45+, and CD34+CD49f+ cells after 7 days of interaction when compared to controls. In the presence of hWJSCs or hWJSC-CM, the CD34+ cells produced significantly more primitive CFU-GEMM colonies, HoxB4, and HoxA9 gene expression and lower percentages of CD34+CXCR4+ cells. There were also significantly higher N-cadherin+ cell numbers and increased cell migration in transwell migration assays. The CD34+ cells expanded with hWJSCs had significantly lower mitochondrial mass, mitochondrial membrane potential, and oxidative stress. Green Mitotracker-tagged mitochondria from CD34+ cells were observed lying within red CellTracker-tagged hWJSCs under confocal microscopy indicating mitochondrial transfer via tunneling nanotubes. CD34+ cells expanded with hWJSCs and hWJSC-CM showed significantly reduced oxidative phosphorylation (ATP6VIH and NDUFA10) and increased glycolytic (HIF-1a and HK-1) pathway-related gene expression. CD34+ cells expanded with hWJSCs for 7 days showed significant greater CD45+ cell chimerism in the bone marrow of primary and secondary irradiated mice when transplanted intravenously. CONCLUSIONS: In this report, we confirmed that allogeneic hWJSCs provide an attractive platform for the ex vivo expansion of high fold numbers of UCB CD34+ cells while keeping them primitive. Allogeneic hWJSCs are readily available in abundance from discarded UCs, can be easily frozen in cord blood banks, thawed, and then used as a platform for UCB-HSC expansion if numbers are inadequate.

背景：人脐带血（UCB）CD34+细胞的移植已成功用于治疗血液系统疾病，但一个主要的限制是可用细胞数量低。体内骨髓中的间充质干细胞（MSCs）作为支架，CD34+细胞在其上相互作用和增殖。因此，我们评估了异体人 UC 沃顿胶（hWJSCs）MSC 作为体外扩增 CD34+细胞的基质支持物的用途。

方法：我们使用常规检测方法对这个平台的原始状态、迁移、黏附、成熟、线粒体行为和途径机制进行了深入评估，然后在体内小鼠模型中评估了扩增 CD34+细胞的植入潜力。

结果：我们证明 hWJSCs 及其条件培养基（hWJSC-CM）支持在 7 天的相互作用后，CD34+、CD34+CD133+、CD34+CD90+、CD34+ALDH+、CD34+CD45+和 CD34+CD49f+细胞的显著高倍变化。与对照组相比，在存在 hWJSCs 或 hWJSC-CM 的情况下，CD34+细胞产生的原始 CFU-GEMM 集落、HoxB4 和 HoxA9 基因表达以及 CD34+CXCR4+细胞的百分比显著降低。在 Transwell 迁移实验中，N-钙黏蛋白+细胞数量也显著增加，细胞迁移增加。用 hWJSCs 扩增的 CD34+细胞的线粒体质量、线粒体膜电位和氧化应激显著降低。共聚焦显微镜下观察到用绿色 Mitotracker 标记的 CD34+细胞内的红色 CellTracker 标记的 hWJSCs 中的线粒体，表明通过隧穿纳米管进行线粒体转移。用 hWJSCs 和 hWJSC-CM 扩增的 CD34+细胞表现出氧化磷酸化（ATP6VIH 和 NDUFA10）显著降低和糖酵解（HIF-1a 和 HK-1）途径相关基因表达增加。用 hWJSCs 扩增 7 天的 CD34+细胞在静脉移植到原发性和继发性照射小鼠的骨髓中显示出显著更高的 CD45+细胞嵌合体。

结论：在本报告中，我们证实同种异体 hWJSCs 为体外扩增高倍数 UCB CD34+细胞提供了一个有吸引力的平台，同时保持其原始状态。同种异体 hWJSCs 可从废弃的 UC 中大量获得，可在脐带血库中方便地冷冻，解冻后可作为 UCB-HSC 扩增的平台使用，如果数量不足。