人脑类器官来源间充质干细胞促进脐带血造血干祖细胞向巨噬细胞分化和功能极化

Mesenchymal stem cells from human pluripotent stem cell-derived brain-specific organoids promote the differentiation and polarization of macrophages from umbilical cord blood HSPCs

本文探讨了人脑类器官来源神经源性间充质干细胞(neural mesenchymal stem cells,nMSCs)对脐带血来源CD34(umbilical cord blood CD34,UCB-CD34)细胞向各系分化的作用,尤其是对巨噬细胞(macrophage,Mφ)诱导分化和极化的影响。本文通过UCB-CD34细胞与H1-nMSCs或hi PSC-nMSCs贴壁共培养,无MSCs组作为对照(control),计数共培养组和对照组造血细胞集落数、对集落细胞进行MGG染色、流式检测Mφ相关表面分子、转录组测序检测共培养7 d后CD34细胞红系和髓系基因表达的差异。将nMSCs通过trans-well与UCB-CD34来源的Mφ(UCB-Mφ)共培养,经IL-4刺激后,流式检测Mφ表面分子CD206的表达情况,并用U-PLEX平台测定培养基上清中IL-10的浓度。相较于对照组,UCB-CD34细胞分别与H1-nMSCs、hi PSC-nMSCs共培养时可获得更多的造血细胞集落(P<0.05)以及更多的M集落(P<0.01);UCB-CD34细胞分别与H1-nMSCs、hi PSC-nMSCs共培养第7天,可产生更多GMP细胞,而CFU-E产量减少。UCB-CD34细胞分别与H1-nMSCs、hi PSC-nMSCs在Mφ诱导分化培养基或无细胞因子分化培养基中共培养21 d后,均可获得更高比例和数量的Mφ。共培养7 d后CD34细胞转录组测序显示,与H1-nMSCs、hi PSC-nMSCs共培养组高表达GMP相关基因:NAPSB、HCK、FCGR1A、MS4A6A、PRTN3等和Mφ相关基因CD14、CSF1R、TRIB1和APP等;与nMSCs共培养组的UCB-Mφ在IL-4刺激时,高表达CD206表面标记分子,且分泌更多IL-10,即向M2型极化。本研究初步证明,H1-nMSCs、hi PSC-nMSCs能较好地促进UCB-CD34向Mφ分化及向M2型极化。

This work was aimed to investigate the effect of human brain-specific organoids derived neural mesenchymal stem cells(nMSCs) on the differentiation of umbilical cord blood CD34(UCB-CD34),particularly the differentiation and polarization of macrophages, UCB-CD34cells were co-cultured with H1-nMSCs or hiPSC-nMSCs. The culture of UCB-CD34without MSCs was used as the control group. The number of co-culture group derived and control group derived hematopoietic colonies were counted, colony cells were detected by MGG staining, and the surface markers of macrophages were detected by flow cytometry. We detected the erythroid and myeloid gene expression in CD34cells by transcriptome sequencing. UCB derived macrophages with H1-nMSCs or hiPSC-nMSCs in trans-well were stimulated with IL-4. We detected the expression of CD206 in macrophages by flow cytometry and measured the expression of IL-10 in the supernatant of culture medium by the U-PLEX platform. Compared with control group, UCB-CD34cells could generate more hematopoietic colonies(P<0.05) when co-cultured with H1-nMSCs or hiPSCs-nMSCs, respectively;and more M colonies(P<0.01). UCB-CD34cells co-cultured with H1-nMSCs or hiPSC-nMSCs for 7 days could produce more GMP cells, while CFU-E cells decreased. Then, UCB-CD34cells were co-cultured with H1-nMSCs or hiPSC-nMSCs in macrophage inducing or cytokine-free differentiation medium for 21 days, respectively. The results showed a significantly higher number and proportion of macrophages, as compared with the control group. The transcriptome sequencing analysis of CD34cells at day-7 co-culture, either with H1-nMSCs or hiPSC-nMSCs, showed high expression of GMPrelated genes(NAPSB, HCK, FCGRLA, MS4A6A, PRTN3) and macrophage related genes(CD14, CSFLR,TRIBL, and APP). Upon stimulation by IL-4, UCB-Mφ from trans-well cultured with nMSCs upregulated the expression of CD206 and produced more IL-10, indicating their polarization towards M2 type. This study preliminarily proved that co-culture with H1-nMSCs or hi PSC-nMSCs can better promote the differentiation of UCB-CD34HSPCs into functionally mature macrophages and favor the polarization towards M2 type.