In Vivo Survival of Human Endometrial Mesenchymal Stem Cells Transplanted Under the Kidney Capsule of Immunocompromised Mice
In Vivo Survival of Human Endometrial Mesenchymal Stem Cells Transplanted Under the Kidney Capsule of Immunocompromised Mice
复制标题
DOI：
10.1089/scd.2017.0177
复制
发表时间：
2017-12-05
影响因子：
4
通讯作者：
Gargett, Caroline E.
中科院分区：
文献类型：
--
作者：
Gurung, Shanti;Deane, James A.;Gargett, Caroline E.
人子宫内膜间充质干细胞(EMSCs)是一种具有良好特性的成体干细胞类型，具有用于再生医学或细胞治疗的潜力。作为原理的证明，我们在皮下创伤修复的大鼠模型中证明了eMSCs通过旁分泌作用减少炎症反应，从而促进了伤口的愈合。然而，缺乏一种在未分化状态下培养eMSCs的有效方案和一种可靠的标记细胞跟踪的方法。在本研究中，我们研究了利用含有mCherry荧光报告基因的慢病毒载体在含有A83-01的培养液中体外培养的eMSCs进行转导和标记，以及免疫低下的NSG小鼠肾被膜下移植后标记细胞的不同追踪方法。从人子宫内膜分离出血管周围的SUSD2(+)eMSCs。用携带mCherry荧光报告基因的慢病毒转导第1代eMSCs，用荧光激活细胞分选法分离mCherry(+)细胞，在含成纤维细胞生长因子2(FGF2)和表皮生长因子(EGF)的无血清培养液中培养至第6代。随后将细胞分成两个烧瓶，分别用0.01%二甲基亚砜或A83-01(1M)处理7天。将5×10(5)对照细胞或A83-01预处理细胞包裹于纤维蛋白凝胶中，移植到NSG小鼠的肾被膜下。分别于移植后7、14、30天进行组织学分析。MCherry基因可有效转导人eMSCs。它们在5代中增殖并保持高表达。用聚合酶链式反应、流式细胞仪和免疫荧光对移植的肾脏进行分析表明，这两种细胞类型都至少存活了30天。使用慢病毒载体对eMSCs进行高效标记，并将其培养在保持未分化状态的环境中，可以在临床前动物模型中进行可靠的检测，并强调需要产生纯的未分化MSCs群体，以便在体内长期存活，以延长其治疗效果。
Human endometrial mesenchymal stem cells (eMSCs) are a well-characterized adult stem cell type with potential for use in regenerative medicine or cell therapy. As a proof of principle, we demonstrated that eMSCs promoted wound healing by reducing the inflammatory response through a paracrine action in a subcutaneous rat model of wound repair. However, an efficient protocol for culturing eMSCs in the undifferentiated state and a reliable method of labeling them for cell tracking were lacking. In this study, we investigated the use of a lentiviral vector containing the mCherry fluorescent reporter gene to transduce and label eMSCs following in vitro culturing in A83-01 containing medium, and different methods of tracing the labeled cells following transplantation under the kidney capsule of immunocompromised NSG mice. Perivascular SUSD2(+) eMSCs were isolated from human endometrium. Passage 1 eMSCs were transduced by lentiviruses with mCherry fluorescent reporter gene; mCherry(+) cells were isolated by fluorescence-activated cell sorting and cultured until passage 6 in 5% O-2 in serum-free medium with fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF). The cells were subsequently divided into two flasks and treated with either dimethyl sulfoxide (0.01%) or A83-01 (1M) for 7 days. 5x10(5) control or A83-01 pretreated cells were encapsulated into a fibrin gel and transplanted under the subrenal capsules of NSG mice. Tissues were analyzed at 7, 14, and 30 days posttransplantation. Human eMSCs were efficiently transduced with mCherry gene. They proliferated and maintained high mCherry expression over five passages. Analyzing transplanted kidneys using polymerase chain reaction, flow cytometry, and immunofluorescence showed that both cell types survived at least 30 days. Efficient labeling of eMSCs using a lentiviral vector and culturing them in an environment maintaining them in an undifferentiated state enable reliable detection in preclinical animal models and highlight the need for generating a pure population of undifferentiated MSCs for long-term survival in vivo to prolong their treatment effect.