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
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摘要
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.
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.
引文
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[3] Xu MJ,Tsuji K,Ueda T,et al.Stimulation of mouse and human primitive hematopoiesis by murine embryonic aorta-gonad-mesonephros-derived stromal cell lines.Blood,1998,92(6):2032-2040.
[4] de Lima M,McNiece I,Robinson SN,et al.Cord-blood engraftment with ex vivo mesenchymal-cell coculture.N Engl J Med,2012,367(24):2305-2315.
[5] Agarwala S,Tamplin OJ,et al.Neural crossroads in the hematopoietic stem cell niche.Trends Cell Biol,2018,28(12):987-998.
[6] Li Z,Lan Y,He W,et al.Mouse embryonic head as a site for hematopoietic stem cell development.Cell Stem Cell,2012,11(5):663-675.
[7] Mao B,Huang S,Lu X,et al.Early development of definitive erythroblasts from human pluripotent stem cells defined by expression of glycophorin A/CD235a,CD34,and CD36.Stem Cell Reports,2016,7(5):869-883.
[8] 元力,潘旭,边国慧,等.不同体外诱导分化来源巨噬细胞表型分子分析及极化功能比较.国际输血及血液学杂志,2018,41( 2 ):121-132.
[9] Kfoury Y,Scadden DT.Mesenchymal cell contributions to the stem cell niche.Cell Stem Cell,2015,16(3):239-253.
[10] Dominici M,Le Blanc K,Mueller I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement.Cytotherapy,2006,8(4):315-317.
[11] Barberi T,Willis LM,Socci ND,et al.,Derivation of multipotent mesenchymal precursors from human embryonic stem cells.PLoS Med,2005,2(6):554-560.
[12] Chinzei R,Tanaka Y,Shimizu-Saito K,et al.,Embryoid-body cells derived from a mouse embryonic stem cell line show differentiation into functional hepatocytes.Hepatology,2002.36(1):22-29.
[13] Olivier EN,Rybicki AC,Bouhassira EE.Differentiation of human embryonic stem cells into bipotent mesenchymal stem cells.Stem Cells,2006,24(8):1914-1922.
[14] Leyendecker JA.TGF-beta inhibitor SB431542 promotes the differentiation of induced pluripotent stem cells and embryonic stem cells into mesenchymal-like cells.Stem Cells Int,2018,doi:10.1155/2018/7878201.
[15] Jiang B,Yan L,Wang X,et al.Concise review:mesenchymal stem cells derived from human pluripotent cells,an unlimited and quality-controllable source,for therapeutic applications.Stem Cells,2018,doi:10.1002/stem.2964.
[16] Angelopoulou M,Novelli E,Grove JE,et al.Cotransplantation of human mesenchymal stem cells enhances human myelopoiesis and megakaryocytopoiesis in NOD/SCID mice.Exp Hematol,2003,31(5):413-420.
[17] Moslem M,Eberle I,Weber I,et al.,Mesenchymal stem/stromal cells derived from induced pluripotent stem cells support CD34(pos) hematopoietic stem cell propagation and suppress inflammatory reaction.Stem Cells Int,2015,doi:10.1155/2015/843058.
[18] 郁金凤,孙文翠,路旭琳,等.人胚胎干细胞高效诱导产生巨噬细胞方法的建立.中国输血杂志,2015,28(5):482-487.
[19] Wynn TA,Chawla A,Pollard JW.Macrophage biology in development,homeostasis and disease.Nature,2013,496(7446):445-455.
[20] Sieweke MH,Allen JE.Beyond stem cells:self-renewal of differentiated macrophages.Science,2013,doi:10.1126/science.1242974.
[21] Hu Y,Qin C,Zheng G,Lai D,et al.,Mesenchymal stem cell-educated macrophages ameliorate LPS-induced systemic response.Mediators Inflamm,2016,doi:10.1115/2016/3735452.