OBJECTIVE To aimed at the establishment of mouse stably knockout of MYSM1 mesenchymal stem cell(MSC) line C3H10T1/2, and to investigate its immunological capacity of MSC in vitro. METHODS To establish the stably transfected MSC cell line by using CRISPR-Cas9 technology. Then the Flow cytometry, quantitative PCR and Western blot were employed to detect whether the MYSM1 have been knockout yet. Furthermore, the immune modulatory effect of MYSM1-/- MSC was tested by addition of MYSM1-/- MSC supernatant into spleen lymphocyte and Foxp3 culture. The mRNA expression of inflammatory cytokines such as interleukin-4, interferon-γ and interleukin-17 were detected by quatitatine PCR. RESULTS The expression of MYSM1 was steadily knock out in MSC. In addition, MYSM1-/- MSC showed a stronger inhibitory effect on the expression of inflammatory cytokines. Therefore, the MYSM1 has been stably knocked out in C3H10T1/2. CONCLUSION The mouse stably knockout of MYSM1 mesenchymal stem cells has been successfully established, the knock-out of MYSM1 in MSC can induce more potent immunosuppressive effects on cellular immune reaction in vitro. Our data laid a foundation for the further MSC-based applications in immune related diseases.

Mesenchymal stem cells (MSCs) have been widely used in allogeneic stem cell transplantation. We compared immunologic and hematopoietic characteristics of MSCs derived from whole human umbilical cord (UC), as well as from different sections of UCs, including the amniotic membrane (AM), Wharton's jelly (WJ), and umbilical vessel (UV). Cell phenotypes were examined by flow cytometry. Lymphocyte transformation test and mixed lymphocyte reaction were performed to evaluate the immuno-modulatory activity of MSCs derived from UCs. The mRNA expression of cytokines was detected by real-time polymerase chain reaction. Hematopoietic function was studied by co-culturing MSCs with CD34(+) cells isolated from cord blood. Our results showed that MSCs separated from these four different sections including UC, WJ, UV, and AM had similar biological characteristics. All of the MSCs had multi-lineage differentiation ability and were able to differentiate into osteoblasts, adipocytes, and chondrocytes. The MSCs also inhibited the proliferation of allogeneic T cells in a dose-dependent manner. The relative mRNA expression of cytokines was examined, and the results showed that UCMSCs had higher interleukin-6 (IL6), IL11, stem cell factor, and FLT3 expression than MSCs derived from specific sections of UCs. CD34(+) cells had high propagation efficiencies when co-cultured with MSCs derived from different sections of UCs, among which UCMSCs are the most efficient feeding layer. Our study demonstrated that MSCs could be isolated from whole UC or specific sections of UC with similar immunomodulation and hematopoiesis supporting characteristics.