Mesenchymal stem/stromal cells in post-menopausal endometrium.
Mesenchymal stem/stromal cells in post-menopausal endometrium.
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DOI：
10.1093/humrep/deu159
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发表时间：
2014-09
影响因子：
6.1
通讯作者：
D. Ulrich;D. Ulrich;K. S. Tan;James A. Deane;K. E. Schwab;Amy Cheong;Anna Rosamilia;C. Gargett-C.-Garg
中科院分区：
文献类型：
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作者：
D. Ulrich;D. Ulrich;K. S. Tan;James A. Deane;K. E. Schwab;Amy Cheong;Anna Rosamilia;C. Gargett-C.-Garg
研究问题 绝经后子宫内膜是否含有具有成体干细胞特性并且可以通过活检前瞻性分离的间充质干细胞/基质细胞 (MSC)？摘要答案 从绝经后子宫内膜活检中分离的血管周围 W5C5(+) 细胞显示出克隆性、多能性和表面表型等特征性 MSC 特性，无论女性是否接受过雌激素预处理以再生子宫内膜。已知信息 最近，MSC 在人类绝经前子宫内膜中被发现，并且可以使用单一标记物 W5C5/SUSD2 进行前瞻性分离。研究设计、大小、持续时间 通过为期 2 年的前瞻性 IV 期临床试验，收集了 17 名绝经前 (MP 前) 女性、19 名未接受激素治疗的绝经后 (MP 后) 女性和 15 名接受雌激素替代治疗 (MP+ E2 后) 的绝经后女性的功能层和基底层子宫内膜组织。参与者/材料、环境、方法 65 岁以下的绝经后妇女在组织收集前接受或不接受 E2 治疗 6-8 周。通过雌二醇免疫酶法测定血清E2水平。子宫内膜组织是在子宫切除术之前通过活检（刮宫术）从女性身上获得的。使用图像分析确定 E2 对子宫内膜厚度以及腺体和管腔上皮高度的影响。将子宫内膜组织解离成单细胞悬液，并在新鲜分离和短期培养、磁珠纯化的 W5C5(+) 细胞中检查 MSC 特性。使用克隆形成、连续克隆、脂肪形成、软骨形成、成骨和肌原性诱导培养基中的中胚层分化以及通过流式细胞术进行表面表型分析来评估MSC特性。使用双色免疫荧光检查 W5C5(+) 细胞中雌激素受体 α 的表达。使用 CD34 和 α 平滑肌肌动蛋白免疫染色以及随后的图像分析来分析血管。主要结果和机会的作用 使用 W5C5 抗体从绝经后子宫内膜中纯化出一小群具有 MSC 特性的基质细胞，无论是因低循环雌激素而萎缩，还是因全身雌激素治疗而再生，与绝经前子宫内膜相似。源自经或未经E2处理的绝经后子宫内膜的MSC满足最低的MSC标准：克隆性、表面表型（CD29（+）、CD44（+）、CD73（+）、CD105（+）、CD140b（+）、CD146（+））和多能性。绝经后子宫内膜 MSC 在体外自我更新和 W5C5 表达方面也表现出与绝经前 eMSC 相当的特性。 W5C5(+)细胞如预期位于血管周围并且不表达雌激素受体α。局限性和注意理由 源自绝经后子宫内膜的 MSC 的特性是在体外进行评估的，其体内组织重建能力尚未像绝经前子宫内膜 MSC 那样确定。研究结果的更广泛意义 子宫内膜是 MSC 的一个可获取来源，其发病率最低，可作为基于细胞的疗法用于未来的临床应用。这项研究表明，更年期妇女可以通过简单的活检获得子宫内膜间充质干细胞，用于自体治疗，特别是如果她们的子宫内膜已通过短期 E2 治疗再生，前提是她们拥有完整的子宫并且没有短期 E2 治疗的禁忌。绝经后妇女的子宫内膜间充质干细胞具有关键的间充质干细胞特性，是一种有前途的间充质干细胞来源，与女性的年龄无关。研究经费/竞争利益 这项研究得到了澳大利亚国家健康和医学研究委员会 (NHMRC) 拨款 (1021126) (C.E.G., A.R.) 和高级研究奖学金 (1042298) (C.E.G.)、澳大利亚妇科内窥镜协会拨款 (A.R.)、莫纳什国际研究生研究奖学金 (DU)、澳大利亚干细胞中心、墨尔本东南部的支持再生疗法联盟和澳大利亚干细胞中心提供奖学金 (DU) 和维多利亚州政府的运营基础设施支持计划。利益竞争：AR 从 AMS 收取导师费、顾问委员会费和安斯泰来资助的研究，并为其他项目进行由 AMS 和波士顿科学资助的研究者主导的研究。试用注册号 CTNRN12610000563066。
STUDY QUESTION Does post-menopausal endometrium contain mesenchymal stem/stromal cells (MSC) that have adult stem cell properties and can be prospectively isolated from a biopsy? SUMMARY ANSWER Perivascular W5C5(+) cells isolated from post-menopausal endometrial biopsies displayed characteristic MSC properties of clonogenicity, multipotency and surface phenotype irrespective of whether the women were or were not pre-treated with estrogen to regenerate the endometrium. WHAT IS KNOWN ALREADY Recently MSCs have been identified in human premenopausal endometrium, and can be prospectively isolated using a single marker, W5C5/SUSD2. STUDY DESIGN, SIZE, DURATION Endometrial tissue of both the functional and basal layers, from 17 premenopausal (pre-MP) women, 19 post-menopausal (post-MP) women without hormonal treatment and 15 post-menopausal women on estrogen replacement therapy (post-MP+ E2), was collected through a prospective phase IV clinical trial over 2 years. PARTICIPANTS/MATERIALS, SETTING, METHODS Post-menopausal women <65 years of age were treated with or without E2 for 6-8 weeks prior to tissue collection. Serum E2 levels were determined by estradiol immunoenzymatic assay. Endometrial tissue was obtained from women by biopsy (curettage) just prior to the hysterectomy. The effect of E2 on endometrial thickness and glandular and luminal epithelial height was determined using image analysis. Endometrial tissue was dissociated into single cell suspensions and MSC properties were examined in freshly isolated and short-term cultured, magnetic bead-purified W5C5(+) cells. MSC properties were assessed using clonogenicity, serial cloning, mesodermal differentiation in adipogenic, chondrogenic, osteogenic and myogenic induction culture media, and surface phenotype analysis by flow cytometry. Estrogen receptor α expression in W5C5(+) cells was examined using dual colour immunofluorescence. Vascularity was analysed using CD34 and alpha smooth muscle actin immunostaining and subsequent image analysis. MAIN RESULTS AND THE ROLE OF CHANCE A small population of stromal cells with MSC properties was purified with the W5C5 antibody from post-menopausal endometrium, whether atrophic from low circulating estrogen or regenerated from systemic estrogen treatment, similar to premenopausal endometrium. The MSC derived from post-menopausal endometrium treated with or without E2 fulfilled the minimum MSC criteria: clonogenicity, surface phenotype (CD29(+), CD44(+), CD73(+), CD105(+), CD140b(+), CD146(+)) and multipotency. The post-menopausal endometrial MSCs also showed comparable properties to premenopausal eMSC with respect to self-renewal in vitro and W5C5 expression. The W5C5(+) cells were located perivascularly as expected and did not express estrogen receptor α. LIMITATIONS, REASONS FOR CAUTION The properties of the MSC derived from post-menopausal endometrium were evaluated in vitro and their in vivo tissue reconstitution capacity has not been established as it has for premenopausal endometrial MSC. WIDER IMPLICATIONS OF THE FINDINGS The endometrium is an accessible source of MSC obtainable with minimum morbidity that could be used for future clinical applications as a cell-based therapy. This study shows that menopausal women can access their endometrial MSC by a simple biopsy for use in autologous therapies, particularly if their endometrium has been regenerated by short-term E2 treatment, provided they have an intact uterus and are not contraindicated for short-term E2 treatment. Endometrial MSC in post-menopausal women possess key MSC properties and are a promising source of MSC independent of a woman's age. STUDY FUNDING/COMPETING INTERESTS This study was supported by the National Health and Medical Research Council (NHMRC) of Australia grant (1021126) (C.E.G., A.R.) and Senior Research Fellowship (1042298) (C.E.G.), Australian Gynaecological Endoscopic Society grant (A.R.) , Monash International Postgraduate Research Scholarship (DU), Australian Stem Cell Centre, South East Melbourne Alliance for Regenerative Therapies and Australian Stem Cell Centre top up scholarships (DU) and Victorian Government's Operational Infrastructure Support Program. Competing interests: AR receives Preceptorship fees from AMS, advisory board fees and sponsored study from Astellas, and conducts investigator led studies sponsored by AMS and Boston Scientific for other projects. TRIAL REGISTRATION NUMBER CTNRN12610000563066.