人脐带华通氏胶间充质干细胞的生物学特性及其向肌源性细胞分化的能力
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摘要
目的探讨人脐带华通氏胶间充质干细胞(human umbilical cord Wharton′s Jelly mesenchymal stem cells,hUCMSCs)的生物学特性及其向肌源性细胞分化的能力。方法收集健康足月产胎儿脐带组织,采用酶消化法从华通氏胶中分离hUCMSCs,选取第3代对数生长期细胞,显微镜观察细胞形态,流式细胞术检测细胞免疫表型分子的表达,免疫荧光染色检测干细胞标记物及其体外向肌源性细胞的分化,荧光显微镜观察及流式细胞术检测携带绿色荧光蛋白(green fluorescent protein,GFP)的慢病毒转染hUCMSCs后GFP的表达。结果采用酶消化法获得可贴壁生长的大量hUCMSCs,其能在体外成功进行扩增培养。hUCMSCs可高表达CD29、CD44、CD73、CD90和CD105,而极低表达CD14、CD34、CD45和HLA-Ⅱ;能表达干细胞标记物Oct4和Sox2,且在体外特殊培养条件下表达骨骼肌干细胞标记物Pax7和Myo D;转染的hUCMSCs可稳定表达GFP,转染率高达50%~70%。结论人脐带华通氏胶组织存在间充质干细胞,且其具有向肌源性细胞分化的潜能。
Objective To investigate the biological characteristics of human umbilical cord Whalton′ s jelly mesenchymal stem cells(hUCMSCs)as well as their ability in differentiation to myogenic cells. Methods The umbilical cord tissue of healthy full-term fetuses,from which hUCMSCs were isolated by digestion with collagenase Ⅱ. The hUCMSCs of passage 3 in logarithmic growth period were observed for morphology by microscopy, analyzed for expression of cellular immunophenotypic molecules by flow cytometry,and for stem cell markers and the differentiation of the cells to myogenic cells in vitro by IFA. The expression of green fluorescent protein(GFP) in hUCMSCs transfected with lentivirus carrying GFP gene was observed by IFA and flow cytometry. Results A large quantity of adherent hUCMSCs were obtained,which could be cultured and proliferated in vitro. CD29,CD44,CD73,CD90 and CD105 were highly expressed,while CD14, CD34, CD45 and HLA-Ⅱ were lowly expressed, in hUCMSCs. Stem cell markers Oct4 and Sox2 were expressed,moreover,skeletal muscular stem cell markers Pax7 and MyoD were expressed under special culture condition in vitro,in hUCMSCs. GFP was stably expressed in hUCMSCs transfected with lentivirus,while the transfection rate was50%. Conclusion Mesenchymal stem cells existed in human umbilical cord Wharton′s Jelly, which showed the capacity of differentiation to myogenic cells.
Objective To investigate the biological characteristics of human umbilical cord Whalton′ s jelly mesenchymal stem cells(hUCMSCs)as well as their ability in differentiation to myogenic cells. Methods The umbilical cord tissue of healthy full-term fetuses,from which hUCMSCs were isolated by digestion with collagenase Ⅱ. The hUCMSCs of passage 3 in logarithmic growth period were observed for morphology by microscopy, analyzed for expression of cellular immunophenotypic molecules by flow cytometry,and for stem cell markers and the differentiation of the cells to myogenic cells in vitro by IFA. The expression of green fluorescent protein(GFP) in hUCMSCs transfected with lentivirus carrying GFP gene was observed by IFA and flow cytometry. Results A large quantity of adherent hUCMSCs were obtained,which could be cultured and proliferated in vitro. CD29,CD44,CD73,CD90 and CD105 were highly expressed,while CD14, CD34, CD45 and HLA-Ⅱ were lowly expressed, in hUCMSCs. Stem cell markers Oct4 and Sox2 were expressed,moreover,skeletal muscular stem cell markers Pax7 and MyoD were expressed under special culture condition in vitro,in hUCMSCs. GFP was stably expressed in hUCMSCs transfected with lentivirus,while the transfection rate was50%. Conclusion Mesenchymal stem cells existed in human umbilical cord Wharton′s Jelly, which showed the capacity of differentiation to myogenic cells.
引文
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[19]WEINTRAUB H,TAPSCOTT S J,DAVIS R L,et al.Activation of muscle-specific genes in pigment,nerve,fat,liver,and fibroblast cell lines by forced expression of Myo D[J].Proc Natl Acad Sci USA,1989,86(14):5434-5438.
[20]KUANG S,CHARGS B,SEALE P,et al.Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis[J].J Cell Biol,2006,172(1):103-113.
[21]LEPPER C,CONWAY S J,FAN C M.Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements[J].Nature,2009,460(7255):627-631.
[22]NINAGAWA N T,ISOBE E,HIRAYAMA Y,et al.Transplantated mesenchymal stem cells derived from embryonic stem cells promote muscle regeneration and accelerate functional recovery of injured skeletal muscle[J].Biores Open Access,2013,2(4):295-306.
[23]JOE A W,YI L,NATARAJAN A,et al.Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis[J].Nat Cell Biol,2010,12(2):153-163.
[24]TANG L,CHANG J.Effect of GFP-containing lentivirus infection on the expression of octamer transcription factor 4 in human umbilical cord mesenchymal stem cells[J].Chin J Cell Mol Immunol,2013,29(3):292-296.(in Chinese)唐莉,常静.携带GFP的慢病毒感染人脐带间充质干细胞及其对Oct4表达的影响[J].细胞与分子免疫学杂志,2013,29(3):292-296.
[2]KONALA V B,MAMIDI M K,BHONDE R,et al.The current landscape of the mesenchymal stromal cell secretome:A new paradigm for cell-free regeneration[J].Cytotherapy,2016,18(1):13-24.
[3]HUNT C J.Cryopreservation of human stem cells for clinical application:a review[J].Transfus Med Hemother,2011,38(2):107-123.
[4]HE B,YANG X Q,ZHANG Y Q.Biological characteristics and their latest research progress of human umbilical cord Wharton′s Jelly-derived mesenchymal stem cells[J].J Int Obstel Gynecol,2016,43(1):94-98.(in Chinese)何蓓,杨晓清,张玉泉.人脐带华通胶间充质干细胞的生物学特性及研究新进展[J].国际妇产科学杂志,2016,43(1):94-98.
[5]SUBRAMANIAN A,FONG C Y,BISWAS A,et al.Comparative characterization of cells from the various compartments of the human umbilical cord shows that the Wharton′s Jelly compartment provides the best source of clinically utilizable mesenchymal stem cells[J].PLo S One,2015,10(6):e0127992.doi:10.1371/journal.pone.0127992.
[6]TAGHIZADEH R R,CETRULO K J,CETRULO C L.Wharton′s Jelly stem cells:future clinical applications[J].Placenta,2011,32(Suppl 4):S311-S315.
[7]CONCONI M T,BURRA P,DI LIDDO R,et al.CD105(+)cells from Wharton′s jelly show in vitro and in vivo myogenic differentiative potential[J].Int J Mol Med,2006,18(6):1089-1096.
[8]MCELREAVEY K D,IRVINE A I,ENNIS K T,et al.Isolation,culture and characterisation of fibroblast-like cells derived from the Wharton′s jelly portion of human umbilical cord[J].Biochem Soc Trans,1991,19(1):29S.
[9]LU L L,LIU Y J,YANG S G,et al.Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials[J].Haematologica,2006,91(8):1017-1026.
[10]CHEN Y,ZHANG N K,YANG M,et al.Isolation and identification of mesenchymal stem cells from human umbilical cord Wharton′s Jelly[J].China J Mod Med,2010,20(16):2412-2415,2420.(in Chinese)陈宇,张宁坤,杨明,等.脐带华通胶间充质干细胞的分离培养及鉴定[J].中国现代医学杂志,2010,20(16):2412-2415,2420.
[11]NEJAD N A,AMIDI F,HOSEINI M A,et al.Male germ-like cell differentiation potential of human umbilical cord Wharton′s jelly-derived mesenchymal stem cells in co-culture with human placenta cells in presence of BMP4 and retinoic acid[J].Iran J Basic Med Sci,2015,18(4):325-333.
[12]KALASZCZYNSKA I,FERDYN K.Wharton′s jelly derived mesenchymal stem cells:future of regenerative medicine?Recent findings and clinical significance[J].Biomed Res Int,2015,2015:430847.DOI:10.1155/2015/430847.
[13]LA ROCCA G,LO IACONO M,CORSELLO T,et al.Human Wharton′s jelly mesenchymal stem cells maintain the expression of key immunomodulatory molecules when subjected to osteogenic,adipogenic and chondrogenic differentiation in vitro:new perspectives for cellular therapy[J].Curr Stem Cell Res Ther,2013,8(1):100-113.
[14]ZHAO G,LIU F,LAN S,et al.Large-scale expansion of Wharton′s jelly-derived mesenchymal stem cells on gelatin microbeads,with retention of self-renewal and multipotency characteristics and the capacity for enhancing skin wound healing[J].Stem Cell Res Ther,2015,6:38.
[15]ATLASI Y,MOWLA S J,ZIAEE S A,et al.OCT-4,an embryonic stem cell marker,is highly expressed in bladder cancer[J].Int J Cancer,2007,120(7):1598-1602.
[16]PIERANTOZZI E,GAVA B,MANINI I,et al.Pluripotency regulators in human mesenchymal stem cells:expression of NANOG but not of OCT-4 and SOX-2[J].Stem Cells Dev,2011,20(5):915-923.
[17]TAPSCOTT S J.The circuitry of a master switch:Myod and the regulation of skeletal muscle gene transcription[J].Development,2005,132(12):2685-2695.
[18]KIM J A,SHON Y H,LIM J O,et al.MYOD mediates skeletal myogenic differentiation of human amniotic fluid stem cells and regeneration of muscle injury[J].Stem Cell Res Ther,2013,4(6):147.
[19]WEINTRAUB H,TAPSCOTT S J,DAVIS R L,et al.Activation of muscle-specific genes in pigment,nerve,fat,liver,and fibroblast cell lines by forced expression of Myo D[J].Proc Natl Acad Sci USA,1989,86(14):5434-5438.
[20]KUANG S,CHARGS B,SEALE P,et al.Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis[J].J Cell Biol,2006,172(1):103-113.
[21]LEPPER C,CONWAY S J,FAN C M.Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements[J].Nature,2009,460(7255):627-631.
[22]NINAGAWA N T,ISOBE E,HIRAYAMA Y,et al.Transplantated mesenchymal stem cells derived from embryonic stem cells promote muscle regeneration and accelerate functional recovery of injured skeletal muscle[J].Biores Open Access,2013,2(4):295-306.
[23]JOE A W,YI L,NATARAJAN A,et al.Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis[J].Nat Cell Biol,2010,12(2):153-163.
[24]TANG L,CHANG J.Effect of GFP-containing lentivirus infection on the expression of octamer transcription factor 4 in human umbilical cord mesenchymal stem cells[J].Chin J Cell Mol Immunol,2013,29(3):292-296.(in Chinese)唐莉,常静.携带GFP的慢病毒感染人脐带间充质干细胞及其对Oct4表达的影响[J].细胞与分子免疫学杂志,2013,29(3):292-296.