人羊膜来源干细胞的性质及分化潜能研究
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摘要
背景基于干细胞研究的再生医学有望修复心肌损伤,是当前研究的热点。人羊膜来源干细胞具有多向分化潜能,且因其易于获得、不引起伦理学争议、干细胞含量丰富、免疫原性低等特点,有潜力成为未来再生医学临床应用的重要种子细胞来源。
目的
1.了解人羊膜不同部位(层面)来源干细胞的性质差异。
2.评价RCCS(旋转细胞培养系统)等培养方式在人羊膜来源干细胞培养中的价值。
3.比较不同方法诱导人羊膜来源干细胞向心肌细胞方向分化的效果。
方法与结果
1.羊膜上皮层来源的细胞表达各种干细胞标记物的水平普遍高于羊膜间质层来源的间充质干细胞;前者仅CD73表达水平低于后者。而羊膜上皮层内,近基底部的上皮细胞表达干细胞标记物的水平均高于近表层部(流式细胞学分析方法)。
2.RCCS培养较静止培养更利于维持各种干细胞标记物的表达水平(流式细胞学分析方法)。RCCS培养羊膜上皮细胞2周后,细胞倾向聚集生长,并有类胚体样细胞团形成,其中存在高表达胚胎干细胞标记物的细胞(免疫组化法、免疫荧光法)。
3.以心肌细胞裂解液、GDF-15、BMP2+FGF10分别对羊膜来源干细胞进行诱导,2周后各诱导组在蛋白水平及mRNA水平不同程度的表达心肌标记物(免疫组化法,RT-PCR法)。GDF-15诱导组表达MEF2C及GATA-4水平较高,BMP2+FGF10组表达HCN2水平较高,仅心肌细胞裂解液组明确表达KIR2.1。
4.羊膜来源干细胞具有不对称分裂的特点,以体积较大、形态较圆、胞内颗粒样物质较丰富明显的细胞表达干细胞标记物水平较高;而体积偏小、形态近圆或略不规则的细胞表达功能蛋白较强(免疫组化法、免疫荧光法)。
结论
1.羊膜来源干细胞的性质及分布存在不均一性,这为进一步筛选更具临床应用价值的细胞来源提供了依据。
2.RCCS培养有助于维持人羊膜来源干细胞的多向分化潜能。
3.诱导人羊膜来源干细胞向心肌细胞方向分化的过程中,GDF-15具有一定作用。干细胞向心肌细胞方向分化可能需要多种细胞因子的共同作用。
Background Regenerative medicine based on cytotherapy draws great interests as a potential solution of cardiac repair.Recent reports indicate that amnion-derived stem cells have pluripotency and differentiate toward all three germ layers.Besides,some unique properties such as the ease of availability without invasive procedures,not elicit ethical debate,abundant in cell reserve,and low immunogenicity,have made amnion a potential source of cells for regenerative medicine.
Objectives
1.Determine the characteristics of stem cells isolated from different regions of amnion.
2.Evaluate the application of RCCS(rotary cell culture system) in maintaining human amnion-derived cells.
3.Induce cardiogenic differentiation of human amnion-derived cells by different agents.
Methods and Results
1.By flow cytometry,it was revealed that human amniotic epithelial cells(hAEC) isolated from amniotic epithelial layer expressed stem cell markers generally at higher levels than human amniotic mesenchymal stromal cells(hAMSC) isolated from amniotic mesenchymal layer(except for CD73);besides,among hAEC,the basal group(digested later) expressed stem cell markers at higher levels than the superficial group(digested earlier).
2.By flow cytometry,RCCS showed positive effects in maintaining the expression levels of stem cell markers in hAECs.
3.After inducing cardiogenic differentiation of human amnion-derived cells by cardiac lysis,GDF-15,and BMP2+FGF10 respectively,the expressions of cardiac markers were detected by immunohistochemistry and RT-PCR in induced cells.
4.By Immunofluorescence and immunohistochemistry,uneven divisions of amion-derived stem cells were observed.
Conclusions
1.The characteristics of stem cells isolated from different regions of amnion were heterogeneous,which made possible further screening for cells holding greater therapeutic potential for transplantation and regenerative medicine.
2.RCCS showed positive effects in maintaining the pluripotency of human amnion-derived stem cells.
3.GDF-15 played a role in inducing cardiogenic differentiation of human amnion-derived stem cells.The cardiogenic differentiation might require the coordination of multiple cytokines.
目的
1.了解人羊膜不同部位(层面)来源干细胞的性质差异。
2.评价RCCS(旋转细胞培养系统)等培养方式在人羊膜来源干细胞培养中的价值。
3.比较不同方法诱导人羊膜来源干细胞向心肌细胞方向分化的效果。
方法与结果
1.羊膜上皮层来源的细胞表达各种干细胞标记物的水平普遍高于羊膜间质层来源的间充质干细胞;前者仅CD73表达水平低于后者。而羊膜上皮层内,近基底部的上皮细胞表达干细胞标记物的水平均高于近表层部(流式细胞学分析方法)。
2.RCCS培养较静止培养更利于维持各种干细胞标记物的表达水平(流式细胞学分析方法)。RCCS培养羊膜上皮细胞2周后,细胞倾向聚集生长,并有类胚体样细胞团形成,其中存在高表达胚胎干细胞标记物的细胞(免疫组化法、免疫荧光法)。
3.以心肌细胞裂解液、GDF-15、BMP2+FGF10分别对羊膜来源干细胞进行诱导,2周后各诱导组在蛋白水平及mRNA水平不同程度的表达心肌标记物(免疫组化法,RT-PCR法)。GDF-15诱导组表达MEF2C及GATA-4水平较高,BMP2+FGF10组表达HCN2水平较高,仅心肌细胞裂解液组明确表达KIR2.1。
4.羊膜来源干细胞具有不对称分裂的特点,以体积较大、形态较圆、胞内颗粒样物质较丰富明显的细胞表达干细胞标记物水平较高;而体积偏小、形态近圆或略不规则的细胞表达功能蛋白较强(免疫组化法、免疫荧光法)。
结论
1.羊膜来源干细胞的性质及分布存在不均一性,这为进一步筛选更具临床应用价值的细胞来源提供了依据。
2.RCCS培养有助于维持人羊膜来源干细胞的多向分化潜能。
3.诱导人羊膜来源干细胞向心肌细胞方向分化的过程中,GDF-15具有一定作用。干细胞向心肌细胞方向分化可能需要多种细胞因子的共同作用。
Background Regenerative medicine based on cytotherapy draws great interests as a potential solution of cardiac repair.Recent reports indicate that amnion-derived stem cells have pluripotency and differentiate toward all three germ layers.Besides,some unique properties such as the ease of availability without invasive procedures,not elicit ethical debate,abundant in cell reserve,and low immunogenicity,have made amnion a potential source of cells for regenerative medicine.
Objectives
1.Determine the characteristics of stem cells isolated from different regions of amnion.
2.Evaluate the application of RCCS(rotary cell culture system) in maintaining human amnion-derived cells.
3.Induce cardiogenic differentiation of human amnion-derived cells by different agents.
Methods and Results
1.By flow cytometry,it was revealed that human amniotic epithelial cells(hAEC) isolated from amniotic epithelial layer expressed stem cell markers generally at higher levels than human amniotic mesenchymal stromal cells(hAMSC) isolated from amniotic mesenchymal layer(except for CD73);besides,among hAEC,the basal group(digested later) expressed stem cell markers at higher levels than the superficial group(digested earlier).
2.By flow cytometry,RCCS showed positive effects in maintaining the expression levels of stem cell markers in hAECs.
3.After inducing cardiogenic differentiation of human amnion-derived cells by cardiac lysis,GDF-15,and BMP2+FGF10 respectively,the expressions of cardiac markers were detected by immunohistochemistry and RT-PCR in induced cells.
4.By Immunofluorescence and immunohistochemistry,uneven divisions of amion-derived stem cells were observed.
Conclusions
1.The characteristics of stem cells isolated from different regions of amnion were heterogeneous,which made possible further screening for cells holding greater therapeutic potential for transplantation and regenerative medicine.
2.RCCS showed positive effects in maintaining the pluripotency of human amnion-derived stem cells.
3.GDF-15 played a role in inducing cardiogenic differentiation of human amnion-derived stem cells.The cardiogenic differentiation might require the coordination of multiple cytokines.
引文
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3.Takahashi K,Tanabe K,Ohnuki M,Narita M,Ichisaka T,Tomoda K,Yamanaka S.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell.2007 Nov 30;131(5):861-872
4.Yu J,Vodyanik MA,Smuga-Otto K,Antosiewicz-Bourget J,Frane JL,Tian S,Nie J,Jonsdottir GA,Ruotti V,Stewart R,Slukvin Ⅱ,Thomson JA.Induced pluripotent stem cell lines derived from human somatic cells.Science.2007 Dec 21;318(5858):1917 -1920.
5.Toshio Miki,Thomas Lehmann,Hongbo Cai,Donna B.Stolz and Stephen C.Strom.Stem Cell Characteristics of Amniotic Epithelial Cells.Stem Cells 2005;23;1549-1559
6.Ayaka Toda,Motonori Okabe,Toshiko Yoshida,and Toshio Nikaido,The Potential of Amniotic Membrane/Amnion-Derived Cells for Regeneration of Various Tissues J Pharmacol Sci 2007;105:215 - 228
7.Miki T,Strom SC.Amnion-derived pluripotent/multipotent stem cells.Stem Cell Rev.2006;2(2):133-142.
8.Steffen Zeisberger,Andreas Zisch and Stephen C.Strom,et al.Concise Review:Isolation and Characterization of Cells from Human Term Placenta:Outcome of the First International Workshop on Placenta Derived Stem Cells.Stem Cells 2008;26;300-311
9.Ji Xiao-xin,Li Fei,Li Duo.Application of microgravity bioreactor in cell culture.Chinese Journal of Rehabilitation.June 10,2006,vol 10,No 21
10.袁岩,陈莲凤,张抒扬等.心肌细胞裂解液对骨髓间充质干细胞向心肌细胞分化诱导作用的研究.中华心血管病杂志,2005,Vol.33,No.2:169-173
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2.Odorico JS,Kaufman DS,Thomson JA Multilineage differentiation from human embryonic stem cell lines.Stem Cells.2001;19(3):193-204
3.Takahashi K,Tanabe K,Ohnuki M,Narita M,Ichisaka T,Tomoda K,Yamanaka S.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell.2007 Nov 30;131(5):861-872
4.Yu J,Vodyanik MA,Smuga-Otto K,Antosiewicz-Bourget J,Frane JL,Tian S,Nie J,Jonsdottir GA,Ruotti V,Stewart R,Slukvin Ⅱ,Thomson JA.Induced pluripotent stem cell lines derived from human somatic cells.Science.2007 Dec 21;318(5858):1917 -1920.
5.Toshio Miki,Thomas Lehmann,Hongbo Cai,Donna B.Stolz and Stephen C.Strom.Stem Cell Characteristics of Amniotic Epithelial Cells.Stem Cells 2005;23;1549-1559
6.Ayaka Toda,Motonori Okabe,Toshiko Yoshida,and Toshio Nikaido,The Potential of Amniotic Membrane/Amnion-Derived Cells for Regeneration of Various Tissues J Pharmacol Sci 2007;105:215 - 228
7.Miki T,Strom SC.Amnion-derived pluripotent/multipotent stem cells.Stem Cell Rev.2006;2(2):133-142.
8.Steffen Zeisberger,Andreas Zisch and Stephen C.Strom,et al.Concise Review:Isolation and Characterization of Cells from Human Term Placenta:Outcome of the First International Workshop on Placenta Derived Stem Cells.Stem Cells 2008;26;300-311
9.Ji Xiao-xin,Li Fei,Li Duo.Application of microgravity bioreactor in cell culture.Chinese Journal of Rehabilitation.June 10,2006,vol 10,No 21
10.袁岩,陈莲凤,张抒扬等.心肌细胞裂解液对骨髓间充质干细胞向心肌细胞分化诱导作用的研究.中华心血管病杂志,2005,Vol.33,No.2:169-173
11.Tamagawa T,Ishiwata I,Saito S.Establishment and characterization of a pluripotent stem cell line derived from human amniotic membranes and initiation of germ layers in vitro. Hum Cell. 2004 Sep;17(3): 125-130
12. Zhao P., Ise H, Hongo M, Ota M, Konishi I, Nikaido T. Human amniotic mesenchymal cells have some characteristics of cardiomyocytes. Transplantation. 2005 Mar 15;79(5):528-535
13. Pan G, Thomson JA.Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell Res. 2007 Jan;17(1):42-49
14. Dominici M,Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop Dj, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-317
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