In Vivo Survival of Human Endometrial Mesenchymal Stem Cells Transplanted under the Kidney Capsule of Immunocompromised Mice

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% O₂ 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 (1 μM) for 7 days. 5 × 10⁵ 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.