Isolation And Comparison Of Mesenchymal Stem-like Cells From Human Gastric Cancer And Adjacent Non-cancerous Tissues

Objective To isolated mesenchymal stem cell-like cells from human gastric cancer (hGC-MSCs) and adjacent non-cancerous tissues (hGCN-MSCs), compared with their biological characteristics and the different expression genes between hGC-MSCs and hGCN-MSCs.Methods 120 human gastric cancer tissues and adjacent non-cancerous tissues were collected for cell culture using cell adherent method. The proper culture conditions for hGC-MSCs and hGCN-MSCs were established with the special stem cell medium: the completed serum culture medium (10% FBS DMEM); proliferation, morphology and molecular genetics of hGC-MSCs and hGCN-MSCs were analyzed by cell growth curve, cell cycle, surface antigens, genetics and DNA contents. The expression of stem cell-associated genes in hGC-MSCs and hGCN-MSCs were examined by reverse transcription-PCR (RT-PCR) and real-time PCR. Capabilities of differentiation of hGC-MSCs and hGCN-MSCs were analyzed by osteoblasts and adipocytes.α-SMA,CK18,PCNA,P53,ABCG2 and SALL4 expression difference in hGC-MSCs and hGCN-MSCs at the protein level were decected by immunohistochemistry (IHC). The transwell migration assay was used to study the migration ability of hGC-MSCs and hGCN-MSCs. hGC-MSCs and hGCN-MSCs cells were injected subcutaneously into the right or left back of the same BALB/c nude mouse to determine their tumorigenic capacity respectively.Results 35 hGC-MSCs and 24 hGCN-MSCs were successfully isolated from the 120 human gastric cancer tissues and adjacent non-cancerous tissues . The 11 paired hGC-MSCs and hGCN-MSCs from them were obtained. Our results showed hGC-MSCs and hGCN-MSCs possessed similar stem cell characteristics compare to bone marrows MSC (hBM-MSCs). cell growth curve and cell cycle results revealed that proliferation activity of hGC-MSCs was significantly higher than those in hGCN-MSCs. Flow cytometry showed that both hGC-MSCs and hGCN-MSCs were positive for CD29, HLA-1 and CD105 but negative for CD14, CD31, CD34 and CD71. hGC-MSCs expressed CD44 but not CD13 while hGCN-MSCs expressed these two antigens Contrarily. RT-PCR showed that stem cells-related genes such as Oct-4, Nanog, Bmi-1, Nucleostemin and ABCG2, mesenchyme lineage-related genes including CD44, CD73 andα-SMA, TGF-β, VEGF and IGF were expressed by both hGC-MSCs and hGCN-MSCs. hGC-MSCs and hGCN-MSCs were positive for N-cadherin but negative for E-cadherin. hGC-MSCs were positive for the proliferation-related genes MDM2, p21 and zinc finger transcriptional factor 4 (SALL4) while hGCN-MSCs did not express these genes. Karyotype, osteogenic or adipogenic differentiation and tumor formation assay revealed that the hGC-MSCs and hGCN-MSCs share almost all characteristics with normal hBM-MSCs.α-SMA, CK18, P53, ABCG2 and SALL4 express strong positive and PCNA express weakly positive in immunohistochemistry analysis. The transwell migration assay was used to study the migration ability of hGC-MSCs and hGCN-MSCs, Comparing to hGCN-MSCs, hGC-MSCs showed a higher migration ability.Conclusions hGC-MSCs and hGCN-MSCs were successfully isolated using cell adherent method. Our results indicated that hGC-MSCs and hGCN-MSCs possess the basic characteristcs of mesenchymal stem cells such as proliferation, morphology and molecular genetics. There were also many different between hGC-MSCs and hGCN-MSCs in growth curves, surface markers and gene expression. Our findings suggest that hGC-MSCs are components of the tumor microenvironment, The difference between hGC-MSCs and hGCN-MSCs may be related to interaction in cancer cells,stroma cells in gastric microenvironment. Our data provide a proof for the origin of carcinoma associated fibroblast (CAF), and therefore may potentially be used as a target for gastric cancer diagnosis and therapy.