Endometrial stromal stem cells: in vitro isolation, culture and biological features

doi:10.3969/j.issn.2095-4344.2017.05.018

Abstract

Abstract:

BACKGROUND: Understanding and studying endometrial cells from the perspective of stem cells can provide a new therapeutic approach and research entry point for the clinical treatment of endometrial diseases.
OBJECTIVE: To compare the biological features of endometrial stromal stem cells isolated and cultured using different methods.
METHODS: Three commonly used cell separation methods, including trypsin, collagenase I and their combination, were used to isolate and culture endometrial stromal stem cells from the uterus after removal. Afterwards, passage 2 cells cultured for 5 days were subjected to trypan blue staining and living cell counting. Immunohistochemical staining for CD146 and CD90 and RT-PCR were used to identify harvested endometrial stromal stem cells. Logarithmically growing cells were cultured to draw cell growth curves.
RESULTS AND CONCLUSION: Harvested endometrial stromal stem cells presented with spindle, adherent cell growth, nonpolar arrangement, and fibroblast morphology. The DNA of endometrial stem cell marker CD146 and CD90 highly expressed in 500 bp and 150 bp respectively, but did not express in the mesometrium. S-shaped growth curve of cultured cells was found, and there was no difference in the cell cycle of cells cultured using three different methods. Among the three methods, collagenase I method could harvest the highest number of endometrial stromal stem cells that grew fastest. These findings indicate that endometrial stromal stem cells can be successfully isolated from the removed uterine tissues, and collagenase I method is an efficient, practical and stable method for in vitro culture of endometrial stromal stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Uterus, Cell Culture Techniques, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Bi Yu-hu, Teng Jun-ru. Endometrial stromal stem cells: in vitro isolation, culture and biological features[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(5): 760-765.

Figures/Tables 2

2.1 子宫内膜基质干细胞生长情况接种24-48 h后即可看到少量贴壁细胞，细胞形态不均一。培养三至四天细胞逐渐汇合形成大小不一的细胞集落，7 d时细胞集落明显增大，细胞多呈梭形或多角形。细胞呈漩涡状或辐射状生长，10 d左右细胞互相连接，铺满皿底(图1)。传代培养细胞生长方式与原代细胞相似，第2代细胞呈均一的成纤维状，融合后呈典型的极性。3种方法培养比较：胰蛋白酶法消化时间短，活细胞数量较少，贴壁时间较长，杂质多，胶原酶Ⅰ法消化时间长，但活细胞数量较多，贴壁时间较短，杂质少，胰蛋白酶联合胶原酶Ⅰ法介于两者之间。可见胶原酶Ⅰ法分离培养子宫内膜基质干细胞具有一定优势。 2.2 子宫内膜基质干细胞鉴定 2.2.1 流式细胞仪分析流式细胞仪检测发现子宫内膜基质干细胞阳性表达干细胞表面标志CD44、CD90，不表达造血细胞表面标志CD34、CD45(图2)。 2.2.2 免疫组化分析显微镜观察结果显示，CD90和CD146抗体主要表现为细胞核染色，部分为胞浆染色。染色阳性结果呈现淡黄色至深棕色，显色强度不明显为阴性(图3)。 2.2.3 RT-PCR分析结果在紫外线投射仪下观察电泳情况，分别比较目的基因CD90和CD146 mRNA的表达量[12]，标本选取子宫内膜与子宫肌层交界部位，以相近的子宫肌层组织作为对照，目的基因CD90、CD146在子宫肌层不表达，但是CD90的DNA在150 bp左右、基因CD146的DNA在500 bp左右高表达(图4)。 2.2.4 成骨成脂分化能力子宫内膜基质干细胞成脂诱导2周进行油红O染色，可见红色脂滴(图5A)；成骨诱导2周进行茜素红染色，可见钙盐被染为红色(图5B)。 2.3 不同分离方法下子宫内膜基质干细胞的生长曲线 3种分离方法所得单细胞悬液经细胞培养后，通过采用细胞计数法对第2代细胞进行计数，所得生长曲线均表现为S型，存在一定的滞留期、对数期和平台期，各组比较差异无显著性意义(图6)。 2.4 不同分离方法获得活细胞数比较 3种方法所获得活细胞数比较差异有显著性意义。胶原酶Ⅰ法所获得的活细胞数显著高于胰蛋白酶法和胰蛋白酶联合胶原酶Ⅰ法(P < 0.05，表1)。"

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