人胎盘来源的造血干细胞和间充质干细胞的分离及鉴定
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摘要
目的建立人胎盘来源的造血干细胞(placenta hematopoietic stem cells,hP-HSCs)和间充质干细胞(placentaderived mesenchymal stem cells,hP-MSCs)的分离方法,并进行鉴定和组分分析。方法选取10份新生健康婴儿胎盘组织,采用机械破碎法联合磁珠分选法分离h P-HSCs,胎盘绒毛膜组织块贴壁法分离hP-MSCs,利用形态学观察、集落培养、流式细胞术等进行鉴定。结果 hP-HSCs:分离后有核细胞数(total nucleated cell number,TNC)为(11. 82±2. 46)×10~8个,TNC回收率≥80%;细胞活性为(99. 7±0. 3)%;细胞表面抗体CD34~+CD45dim表达率为(8. 69±0. 36)%,CD34~+总数为(108. 0±6. 48)×10~6个;集落形成总数为(1. 88±1. 07)×10~6。hP-MSCs:冻存细胞总数为(40. 78±9. 35)×10~7个;细胞活性为(99. 0±1. 5)%;细胞表面抗体CD34~+CD45~+表达率为(0. 1±0. 1)%,CD44~+CD105~+为(99. 6±0. 2)%,CD14~+CD19~+为(0. 1±0. 1)%,CD90~+CD73+为(98. 9±0. 2)%;且具有良好成脂、成骨分化潜能。结论成功建立了hP-HSCs和hP-MSCs体外分离培养方法,为胎盘的临床应用奠定了基础,并提供了细胞种子资源。
Objective To develop a method for isolation of human placenta-derived hematopoietic stem cells and mesenchymal stem cells,identify the isolated cells and analyze their components. Methods Ten placental tissues of healthy newborns were collected,from which placenta hematopoietic stem cells(hP-HSCs) were isolated by mechanical disruption combined with magnetic bead sorting,while placenta-derived mesenchymal stem cells(hP-MSCs)by placental chorionic membrane tissue block adherence method,observed for morphology,and identified by colony culture and flow cytometry. Results The total nucleated cell(TNC) number of hP-HSCs was(11. 82 ± 2. 46) × 10~8,while the TNC recovery was not less than 80%,the cell viability was(99. 7 ± 0. 3)%,the proportion of CD34~+CD45 dim cells was(8. 69 ± 0. 36)%,the total CD34~+ cell number was(108. 0 ± 6. 48)× 10~6,and the total number of colonies formed was(1. 88 ± 1. 07) × 10~6. However,the total number of frozen hP-MSCs was(40. 78 ± 9. 35) × 10~7,while the cell viability was(99. 0 ± 1. 5)%,and the proportions of CD34~+CD45~+,CD45~+CD105~+,CD14~+CD19~+ and CD90~+CD73~+ cells were(0. 1 ± 0. 1)%,(99. 6 ± 0. 2)%,(0. 1 ± 0. 1)% and(98. 9 ± 0. 2)%,respectively. The hP-MSCs showed potential in osteogenic and adipogenic differentiations. Conclusion The methods were isolation and culture of h P-hSCs and hP-MSCs were successfully developed,which laid a foundation of clinical application of placenta and provided a resource of cell seeds.
Objective To develop a method for isolation of human placenta-derived hematopoietic stem cells and mesenchymal stem cells,identify the isolated cells and analyze their components. Methods Ten placental tissues of healthy newborns were collected,from which placenta hematopoietic stem cells(hP-HSCs) were isolated by mechanical disruption combined with magnetic bead sorting,while placenta-derived mesenchymal stem cells(hP-MSCs)by placental chorionic membrane tissue block adherence method,observed for morphology,and identified by colony culture and flow cytometry. Results The total nucleated cell(TNC) number of hP-HSCs was(11. 82 ± 2. 46) × 10~8,while the TNC recovery was not less than 80%,the cell viability was(99. 7 ± 0. 3)%,the proportion of CD34~+CD45 dim cells was(8. 69 ± 0. 36)%,the total CD34~+ cell number was(108. 0 ± 6. 48)× 10~6,and the total number of colonies formed was(1. 88 ± 1. 07) × 10~6. However,the total number of frozen hP-MSCs was(40. 78 ± 9. 35) × 10~7,while the cell viability was(99. 0 ± 1. 5)%,and the proportions of CD34~+CD45~+,CD45~+CD105~+,CD14~+CD19~+ and CD90~+CD73~+ cells were(0. 1 ± 0. 1)%,(99. 6 ± 0. 2)%,(0. 1 ± 0. 1)% and(98. 9 ± 0. 2)%,respectively. The hP-MSCs showed potential in osteogenic and adipogenic differentiations. Conclusion The methods were isolation and culture of h P-hSCs and hP-MSCs were successfully developed,which laid a foundation of clinical application of placenta and provided a resource of cell seeds.
引文
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[11]ZIADLOU R,SHAHHOSEININ M,SAFARI F,et al.Comparative analysis of neural differentiation potential in human mesenchymal stem cells derived from chorion and adult bone marrow[J].Cell Tissue Res,2015,362(2):367-377.
[12]KIL K,CHOI M Y,KONG J S,et al.Regenerative efficacy of mesenchymal stromal cells from human placenta in sensorineural hearing loss[J].Int J Pediatr Otorhinolaryngol,2016,91(1):72-81.
[13]BANA N,SANOOGHI D,SOLEIMANI M,et al.A comparative study to evaluate myogenic differentiation potential of human chorion versus umbilical cord blood-derived mesenchymal stem cells[J].Tissue Cell,2017,49(4):495-502.
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[15]FAGHIHI F,MIRZAEI E,AI J,et al.Differentiation potential of human chorion-derived mesenchymal stem cells into motor neuron-like cells in two-and three-dimensional culture systems[J].Mol Neurobiol,2016,53(3):1862-1872.
[16]JIN Y L,WU J Y,LU Y,et al.Isolation and cultivation of human placental chorionic-derived mesenchymal stem cells:optimization of the tissue explants method[J].Chin J Tissue Engi Res,2017,21(21):3388-3393.(in Chinese)金宇林,吴洁莹,陆琰,等.人胎盘绒毛膜间充质干细胞分离培养:组织块法的优化[J].中国组织工程研究,2017,21(21):3388-3393.
[17]LIU F,JIANG X L,LIU M M.Different culture methods for human umbilical cord mesenchymal stem cells[J].Chin JTissue Engi Res,2016,20(28):4136-4141.(in Chinese)刘奉,蒋祥林,刘明明.不同培养方法对人脐带间充质干细胞的影响[J].中国组织工程研究,2016,20(28):4136-4141.
[18]LI S P,WANG Z,WANG C,et al.Culture of mesenchymal stem cells derived from human umbilical cord without enzymatic treatment[J].Chin J Blood Transfusion,2017,30(8):885-889.(in Chinese)李素萍,王震,王超,等.人脐带非酶解法培养MSCs的方法建立[J].中国输血杂志,2017,30(8):885-889.
[19]LU H Y,DU W J,LIU C X,et al.Efficient generation of human umbilical cord derived mensenchymal stem cells by explant re-culture method[J].Chin J Biologicals,2017,30(6):649-654.(in Chinese)芦慧颖,杜文敬,刘春香,等.二次贴壁法高效制备人脐带间充质干细胞[J].中国生物制品学杂志,2017,30(6):649-654.
[2]ROBIN C,BOLLEROT K,MENDES S,et al.Human placenta is a potent hematopoietic niche containing hematopoietic stem and progenitor cells throughout development[J].Cell Stem Cell,2009,5(4):385-395.
[3]KURTZBERG J,LAUGHLIN M,GRAHAM M L,et al.Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients[J].N Engl J Med,1996,335(3):157-166.
[4]ZHAO G,LIU W W,GAO W W,et al.Comparison on the capacity of osteogenesis of mesenchymal stem cells from three different tissues[J].Basic&Clin Med,2017,37(10):1417-1423.(in Chinese)赵刚,刘薇薇,高伟玮,等.3种组织来源的间充质干细胞体外成骨能力的比较[J].基础医学与临床,2017,37(10):1417-1423.
[5]LI M.Isolation,extraction and in vitro amplification of placental derived hematopoietic cells[D].Beijing:Academy of Military Medical Sciences,2017.(in Chinese)李猛.胎盘来源造血干细胞分离提取和体外扩增研究[D].北京中国人民解放军军事医学科学院,2017.
[6]时庆,侯怀水,鞠秀丽,等.深低温冻存十六年后脐血造血干细胞活性的检测[J].中华血液学杂志,2008,29(8):555-556.
[7]ZHOU S L,ZHENG C,SU J Q,et al.Isolation and identification of human umbilical cord and placenta-derived stem cells and their component analysis[J].J Exp Hematol,2015,23(6):1684-1691.(in Chinese)周胜利,郑超,苏建强,等.人脐带和胎盘来源的干细胞分离鉴定及其组份分析[J].中国实验血液学杂志,2015,23(6):1684-1691.
[8]LI J,WEI Y,YAN L,et al.Multiplacenta derived stem cell/cytokine treatment increases survival time in a mouse model with radiation-induced bone marrow damage[J].Cytotechnology,2016,68(6):2677-2686.
[9]KATSIANI E,GARAS A,SKENTOU C,et al.Chorionic villi derived mesenchymal like stem cells and expression of embryonic stem cells markers during long-term culturing[J].Cell Tissue Bank,2016,17(3):517-529.
[10]QIN S Q,KUSUMA G D,AL-SOWaYAN B,et al.Establishment and characterization of fetal and maternal mesenchymal stem/stromal cell lines from the human term placenta[J].Placenta,2016,39(1):134-146.
[11]ZIADLOU R,SHAHHOSEININ M,SAFARI F,et al.Comparative analysis of neural differentiation potential in human mesenchymal stem cells derived from chorion and adult bone marrow[J].Cell Tissue Res,2015,362(2):367-377.
[12]KIL K,CHOI M Y,KONG J S,et al.Regenerative efficacy of mesenchymal stromal cells from human placenta in sensorineural hearing loss[J].Int J Pediatr Otorhinolaryngol,2016,91(1):72-81.
[13]BANA N,SANOOGHI D,SOLEIMANI M,et al.A comparative study to evaluate myogenic differentiation potential of human chorion versus umbilical cord blood-derived mesenchymal stem cells[J].Tissue Cell,2017,49(4):495-502.
[14]FAGHIHI F,MIRZAEI E,AI J,et al.Erratum to:differentiation potential of human chorion-derived mesenchymal stem cells into motor neuron-like cells in two-and three-dimensional culture systems[J].Mol Neurobiol,2016,53(3):1873.
[15]FAGHIHI F,MIRZAEI E,AI J,et al.Differentiation potential of human chorion-derived mesenchymal stem cells into motor neuron-like cells in two-and three-dimensional culture systems[J].Mol Neurobiol,2016,53(3):1862-1872.
[16]JIN Y L,WU J Y,LU Y,et al.Isolation and cultivation of human placental chorionic-derived mesenchymal stem cells:optimization of the tissue explants method[J].Chin J Tissue Engi Res,2017,21(21):3388-3393.(in Chinese)金宇林,吴洁莹,陆琰,等.人胎盘绒毛膜间充质干细胞分离培养:组织块法的优化[J].中国组织工程研究,2017,21(21):3388-3393.
[17]LIU F,JIANG X L,LIU M M.Different culture methods for human umbilical cord mesenchymal stem cells[J].Chin JTissue Engi Res,2016,20(28):4136-4141.(in Chinese)刘奉,蒋祥林,刘明明.不同培养方法对人脐带间充质干细胞的影响[J].中国组织工程研究,2016,20(28):4136-4141.
[18]LI S P,WANG Z,WANG C,et al.Culture of mesenchymal stem cells derived from human umbilical cord without enzymatic treatment[J].Chin J Blood Transfusion,2017,30(8):885-889.(in Chinese)李素萍,王震,王超,等.人脐带非酶解法培养MSCs的方法建立[J].中国输血杂志,2017,30(8):885-889.
[19]LU H Y,DU W J,LIU C X,et al.Efficient generation of human umbilical cord derived mensenchymal stem cells by explant re-culture method[J].Chin J Biologicals,2017,30(6):649-654.(in Chinese)芦慧颖,杜文敬,刘春香,等.二次贴壁法高效制备人脐带间充质干细胞[J].中国生物制品学杂志,2017,30(6):649-654.