人诱导性多能干细胞来源的间充质干细胞促进成体造血发生研究

Human induced pluripotent stem cellsderived mesenchymal stem cells promote definitive hematopoiesis

目的探讨人诱导性多能干细胞(hiPSCs)在神经分化因子作用下生成的间充质干细胞(N-MSCs)对成体造血的影响。方法 1)与神经发育相关的MSCs的获得:hiPSCs在向前脑类器官培养过程中分离纯化出1个性质稳定的细胞系,命名为N-MSCs,通过形态学观察、流式检测和脂肪、成骨、软骨细胞分化实验对其进行鉴定。2)N-MSCs对成体造血的影响:人胚胎干细胞(hESCs)与鼠AGM-S3细胞共培养获得的CD34^+细胞分别与AGM-S3、N-MSCs(2×10~4个)共培养,单独悬浮培养为对照组(CD34^+组,1×10~4个),流式检测关于造血干/祖细胞(CD34^+CD45^+)和巨噬细胞(Mφ)表面标记的变化。3)N-MSCs对人脐带血来源的巨噬细胞(CB-Mφ)的影响:Mφ分别与脐带MSCs(UC-MSCs)、N-MSCs(2.5×10~5个)在transwell中共培养,Mφ单独培养为对照组(5×10~5个),再用IL-4对Mφ做刺激试验。用MGG染色、流式检测、qRT-PCR等方法分析Mφ形态学、M2型Mφ相关表面标记CD206和IL-10基因表达情况。结果 CD34^+组及其分别与AGM-S3、N-MSCs 2个共培养组培养10 d时收获的CD34^+CD45^+细胞数量(×10~3个)分别为:0.44±0.045 vs 0.63±0.170 vs 2.93±0.190(P<0.01);Mφ数量(×10~4个)分别为1.70±0.046 vs 1.49±0.057 vs 2.46±0.086(P<0.05)。Mφ与UC-MSCs共培养组及与N-MSCs共培养组CB-Mφ的CD206占比(%)为56.1±1.15 vs 60.0±1.76(P<0.05)、IL-10的相对表达量为6.8±0.748 vs 8.10±0.804(P<0.01)。结论 N-MSCs能促进成体造血和CB-Mφ向M2型Mφ的转变。

Objective To investigate the mesenchymal stem cells(N-MSCs) derived from neural-factor-conditioned human induced pluripotent stem cells(hiPSCs) and its influence on definitive hematopoiesis.Methods A stable cell line(N-MSCs) was obtained and purified in the course of hiPSCs′ differentiation to forebrain organoids. The cell line was characterized and confirmed by morphological observation, flow cytometry, adipogensis, osteogenesis and chondrogenesis. CD34+ cells were harvested from a human embryonic stem cell(hESC)/mouse aorta-gonad-mesonephros(AGM)-S3 cell co-culture system and further co-cultured with the AGM-S3 and the N-MSCs(2×10^4 cells), respectively. Additional suspending single-culture was performed as control(1×10^4 CD34+ cells). Flow cytometry was adopted to observe surface marker changes of the hematopoietic stem/progenitor cells(CD34+CD45+) and macrophages(Mφ). Umbilical cord blood macrophages(CB-Mφ) were co-cultured with Umbilical cord MSCs(UC-MSCs) and N-MSCs(2.5×10^5 cells) respectively in transwells with a Mφ single-culture as control(5×10^5 Mφ). The 3 Mφ cultures were then stimulated by IL-4 for 24 h. The Morphology, M2 subtype Mφ surface marker CD206 and IL-10 gene expression levels were analyzed by MGG staining, flow cytometry and qRT-PCR. Results After a ten-day co-culture period for the harvested CD34^+ cells, the CD34+ single-culture group yielded(×10^3 cells) 0.44±0.045 vs 0.63±0.170 vs 2.93±0.190 CD34+CD45^+ cells compared to the AGM-S3 co-culture group and the N-MSCs co-culture group. The number of Mφ(×10^4 cells) harvested in the CD34^+ single-culture group was 1.70±0.046 vs 1.49±0.057 vs 2.46±0.086 compared to the AGM-S3 co-culture group and the N-MSCs co-culture group. Genetically, the N-MSCs is capable of promoting the expression of CD206(%) 56.1±1.15 vs 60.0±1.76(P<0.05)and IL-10 6.8±0.748 vs 8.10±0.804(P<0.01)in CB-Mφ compared to the UC-MSCs. Conclusion N-MSCs cells promote definitive hematopoiesis and the transformation of CB-Mφ to the M2 subtype.