Differentiation of human amniotic epithelial cells into cardiomyocyte-like cell

Abstract

AIM：To investigate the plasticity of human amniotic epithelial cells into cardiomyocyte-like cells through the experiment of induced differentiation in vitro.

METHODS：Experiments were conducted at the Key Laboratory of Cell Engineering of Guizhou Province from September 2005 to December 2006. ①Six placenta samples through cesarean delivery were collected aseptically with the informed consents of parturients and the experiment was approved by the Ethics Committee of the Affiliated Hospital of Zunyi Medical College. ②Human amnion was peeled from placenta by mechanism method. After being rinsed with D-Hank's solution, the tissue was cut into pieces with eye scissors and digested with 0.2 g/L ethylenediamine tetraacetic acid and 0.5 g/L trypsin. The digested mixture was centrifugated, digested, filtrated, and the fetal bovine serum was added into filter liquor to terminate digestion. Repeated the digestion procedure for twice and combined three digested cell suspension liquid. After being centrifuged, the cell deposition was resuspended and cultured in L-DMEM medium at density of 1.25×108 L-1. The culture medium was replaced at day 3 and the cells were digested with ethylenediamine tetraacetic acid plus trypsin when 80%~90% confluence was reached. After terminating of digestion, the mixture was centrifuged and the supernatant was discarded. Cell deposition was resuspended with culture medium and subcultivated at the density of 1×107 L-1. ③Phenotype of human amniotic epithelial cells was analyzed by flow cytometry. 10 μmol/L 5-azacytidine and 1 mmol/L ascorbic acid 2-phosphate were used to induce the 2nd generation of human amniotic epithelial cells. The expressions of desmin and α-actinin on induced cell were evaluated by immunofluorescent staining，and the levels of cardiac-specific transcription factors Nkx2.5, GATA-4 mRNA and alpha-myosin heavy chain mRNA were assayed by reverse transcription polymerase chain reaction.

RESULTS: ①Immunohistochemical properties of human amniotic epithelial cells: Cytokeratin 19 was expressed in human amniotic epithelial cells, while vimentin was negative and CD44 was nearly absent. ②The expression of α-actinin and desmin in human amniotic epithelial cells: The differentiated human amniotic epithelial cells were positive for myocyte-specific markers of desmin and α-actinin. ③The expression of Nkx2.5, GATA-4 and α-MHC mRNA in human amniotic epithelial cells: Cardiac-specific transcription factors of Nkx2.5 and GATA-4 mRNA were expressed in the differentiated human amniotic epithelial cells, while cardiac specific contractile protein of α- myosin heavy chain mRNA was absent.

CONCLUSION: Human amniotic epithelial cells acquired from trypsin digesting and separating possess the potential to differentiate into cardiomyocyte-like cells in vitro and human amniotic epithelial cells may be a candidate cells for cellular cardiomyoplasty.

Zhang L, Fang N, Chen DX, Liu ZL, Wan WH, Liu JW, Zhang T.Differentiation of human amniotic epithelial cells into cardiomyocyte-like cells.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(3):401-405(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-3/3k-401(ps).pdf]

摘要
目的：通过体外诱导分化实验，评价人羊膜上皮细胞向心肌样细胞分化的能力。
方法：实验于2005-09/2006-12在贵州省细胞工程重点实验室完成。①对象：经产妇知情同意，无菌采集健康足月剖宫产胎盘6份，实验经医院医学伦理委员会批准。②实验方法：采用机械法将羊膜从胎盘组织上剥离，D-Hank’s液冲洗后剪成碎片，加入含有0.2 g/L乙二胺四乙酸的0.5 g/L胰蛋白酶溶液，离心、消化、过滤，收集滤液，加入胎牛血清终止消化，重复消化2次。合并3次消化所得的细胞悬液，离心后将细胞沉淀悬浮于L-DMEM培养基中，按1.25×108 L-1密度接种，常规培养3 d后更换培养基，待细胞达80%~90%融合后用胰蛋白酶+乙二胺四乙酸联合消化，终止后离心弃上清，细胞沉淀用培养基重新悬浮，按1×107 L-1密度传代。③实验评估：用流式细胞仪鉴定人羊膜上皮细胞表型；使用10 μmol/L 5-氮杂胞苷和1 mmol/L抗坏血酸磷酸盐诱导第2代人羊膜上皮细胞，免疫荧光染色法检测诱导后细胞中特异蛋白结蛋白和 α-辅肌动蛋白的表达，RT-PCR检测心肌特异性转录因子Nkx2.5、GATA-4和心肌特异性收缩蛋白α-肌球蛋白重链mRNA的表达。
结果：①人羊膜上皮细胞的免疫组化特征：人羊膜上皮细胞几乎不表达CD44，不表达波形蛋白，表达角蛋白19。②人羊膜上皮细胞α-辅肌动蛋白和结蛋白的表达：人羊膜上皮细胞经诱导分化后，表达肌系细胞标志α-辅肌动蛋白和结蛋白。③人羊膜上皮细胞Nkx2.5、GATA-4和α-肌球蛋白重链mRNA的表达：人羊膜上皮细胞经诱导分化后，表达心肌特异性转录因子Nkx2.5 mRNA和GATA-4 mRNA，心肌特异的可收缩蛋白α-肌球蛋白重链mRNA未见表达。
结论：通过胰蛋白酶消化法分离获得的人羊膜上皮细胞具有向心肌样细胞分化的特性，可能成为细胞心肌成形术的候选供体细胞。
关键词：人羊膜上皮细胞；心肌样细胞；诱导分化

张路，方宁，陈代雄，刘祖林，万卫红，刘金伟，章涛.人羊膜上皮细胞有向心肌样细胞分化的特性[J].中国组织工程研究与临床康复，2008，12(3):401-405 [www.zglckf.com/zglckf/ejournal/upfiles/08-3/3k-401(ps).pdf]