Cell Therapy Unit, Hematology Department, University Hospital of Salamanca, IBSAL, University of Salamanca, Paseo de San Vicente 58-182, 37007, Salamanca, Spain.
RETIC TerCel, ISCIII, Madrid, Spain.
Stem Cell Res Ther. 2021 Dec 7;12(1):601. doi: 10.1186/s13287-021-02669-z.
Poor graft function or graft failure after allogeneic stem cell transplantation is an unmet medical need, in which mesenchymal stromal cells (MSC) constitute an attractive potential therapeutic approach. Hypoxia-inducible factor-1α (HIF-1α) overexpression in MSC (HIF-MSC) potentiates the angiogenic and immunomodulatory properties of these cells, so we hypothesized that co-transplantation of MSC-HIF with CD34 human cord blood cells would also enhance hematopoietic stem cell engraftment and function both in vitro and in vivo.
Human MSC were obtained from dental pulp. Lentiviral overexpression of HIF-1α was performed transducing cells with pWPI-green fluorescent protein (GFP) (MSC WT) or pWPI-HIF-1α-GFP (HIF-MSC) expression vectors. Human cord blood CD34 cells were co-cultured with MSC WT or HIF-MSC (4:1) for 72 h. Then, viability (Annexin V and 7-AAD), cell cycle, ROS expression and immunophenotyping of key molecules involved in engraftment (CXCR4, CD34, ITGA4, c-KIT) were evaluated by flow cytometry in CD34 cells. In addition, CD34 cells clonal expansion was analyzed by clonogenic assays. Finally, in vivo engraftment was measured by flow cytometry 4-weeks after CD34 cell transplantation with or without intrabone MSC WT or HIF-MSC in NOD/SCID mice.
We did not observe significant differences in viability, cell cycle and ROS expression between CD34 cells co-cultured with MSC WT or HIF-MSC. Nevertheless, a significant increase in CD34, CXCR4 and ITGA4 expression (p = 0.009; p = 0.001; p = 0.013, respectively) was observed in CD34 cells co-cultured with HIF-MSC compared to MSC WT. In addition, CD34 cells cultured with HIF-MSC displayed a higher CFU-GM clonogenic potential than those cultured with MSC WT (p = 0.048). We also observed a significant increase in CD34 cells engraftment ability when they were co-transplanted with HIF-MSC compared to CD34 co-transplanted with MSC WT (p = 0.016) or alone (p = 0.015) in both the injected and contralateral femurs (p = 0.024, p = 0.008 respectively).
Co-transplantation of human CD34 cells with HIF-MSC enhances cell engraftment in vivo. This is probably due to the ability of HIF-MSC to increase clonogenic capacity of hematopoietic cells and to induce the expression of adhesion molecules involved in graft survival in the hematopoietic niche.
同种异体干细胞移植后移植物功能不良或移植物衰竭是一种未满足的医疗需求，间充质基质细胞（MSC）构成了一种有吸引力的潜在治疗方法。MSC 中缺氧诱导因子-1α（HIF-1α）的过表达（HIF-MSC）增强了这些细胞的血管生成和免疫调节特性，因此我们假设 MSC-HIF 与 CD34 人脐血细胞共移植也将增强造血干细胞在体外和体内的植入和功能。
从牙髓中获得人 MSC。通过转导细胞用 pWPI-绿色荧光蛋白（GFP）（MSC WT）或 pWPI-HIF-1α-GFP（HIF-MSC）表达载体进行慢病毒过表达 HIF-1α。将人脐血 CD34 细胞与 MSC WT 或 HIF-MSC（4:1）共培养 72 小时。然后，通过流式细胞术评估 CD34 细胞中与植入相关的关键分子（CXCR4、CD34、ITGA4、c-KIT）的活力（Annexin V 和 7-AAD）、细胞周期、ROS 表达和免疫表型。此外，通过克隆形成试验分析 CD34 细胞的克隆扩增。最后，通过流式细胞术在 NOD/SCID 小鼠中移植 CD34 细胞 4 周后，测量 MSC WT 或 HIF-MSC 骨髓内共移植后 CD34 细胞的体内植入情况。
我们没有观察到与 MSC WT 共培养的 CD34 细胞与 HIF-MSC 共培养的 CD34 细胞之间的活力、细胞周期和 ROS 表达有显著差异。然而，与 MSC WT 相比，与 HIF-MSC 共培养的 CD34 细胞中 CD34、CXCR4 和 ITGA4 的表达显著增加（p=0.009；p=0.001；p=0.013，分别）。此外，与 MSC WT 相比，与 HIF-MSC 共培养的 CD34 细胞具有更高的 CFU-GM 集落形成潜力（p=0.048）。我们还观察到，与 MSC WT 共移植的 CD34 细胞相比，与 HIF-MSC 共移植的 CD34 细胞的植入能力显著增加（p=0.016），或单独移植的 CD34 细胞（p=0.015），无论是在注射侧还是对侧股骨（p=0.024，p=0.008）。
与 HIF-MSC 共移植人 CD34 细胞可增强体内细胞植入。这可能是由于 HIF-MSC 能够增加造血细胞的集落形成能力，并诱导造血龛中与移植物存活相关的粘附分子的表达。
