Transplantation Of Canine Amniotic Mesenchymal Stem Cells Into Thrombocytopenia Mice

Recent years, people are paying more and more attention to amniotic membrane tissue which has a rich resource. Compared with stem cells derived from other tissues, amniotic mesenchymal stem cells have the advantages of non ethical limitation, non invasion, wide source, low immunogenicity and so on. In recent years, there have been some studies on the cell therapy using canine stem cells, but the source of stem cells is relatively single, most of them are derived from bone marrow, umbilical cord blood or adipose tissue. Therefore, it is needed to find a new source of canine stem cells. High dose chemotherapy drugs have the function of killing tumor cells, and also cause severe bone marrow suppression. Thrombocytopenia is an obvious characteristic. In addition to the use of drug therapy, stem cell transplantation is gradually used in the treatment of bone marrow injury.Canine amniotic mesenchymal stem cells (cAMSCs) were collected by enzyme digestion method from canine amniotic membrane tissues. The cell morphology was continuously observed at different passages and cell growth curves of passage 3 and passage 9 were drawn. Stem cell related proteins and genes were identified by immunofluorescence and RT-PCR. Expression of cell surface markers on cAMSCs were detected by flow cytometry. The multiple differentiation potential of cAMSCs was identified by osteogenesis, adipogenesis, neurogenesis and insulinogenesis. Then cyclophosphamide was used to induce thrombocytopenia in order to observe the effect of transplanted cAMSCs on the recovery of platelets in mice. This study showed that the cAMSCs was long fusiform and could be continuously subcultured in vitro. The immunofluorescence results showed that Vimentin and SSEA-4 were positively expressed. Flow cytometry analysis suggested that CD29, CD49d and CD73 were positively expressed, but CD34, CD45 and HLA-DR were negatively expressed on cAMSCs. The RT-PCR results showed that stem cell related genes OCT4, SOX2 and NANOG were positively expressed too. The cAMSCs could be induced to osteoblasts, adipocytes, neurocytes and pancreatic β islets in vitro. The markers associated with induced cells, such as COL1A1, LPL, MAP2 and INS, were positively expressed after differentiation. The cAMSCs can make the number of platelets return to normal levels four days earlier in thrombocytopenia mice. The results showed that the cAMSCs could be successfully isolated and cultured in vitro. Stem cell related proteins and genes were positively expressed. The cAMSCs has the multiple differentiation potential and can promote the recovery of platelets.