Isolation, Purification And Primary Identification Of Biological Characteristics Of Rat Bone Marrow Mesenchymal Stem Cells

Objective: Two methods were tried to compare the effects of isolation and purification in vitroof mesenchymal stem cells (MSCs) from rat bone marrow. Some biological characteristics ofMSCs were identified for further study in the mechanism of differentiated regulation andapplication of MSCs.Methods: MSCs from rat bone marrow were isolated and purified using the centrifugation ofgradient and density with human lymphocyte isolation liquid before culture or culturing directwith all bone marrow cells respectively, and the effects of these methods were observed andcompared. The MSCs from rat bone marrow marked with green fluorescent protein (GFP) werecompared with those marked with Hoechst 33342. The surface antigens CD45, CD44, CD29,cell cycle, karyotype of MSCs from rat bone marrow of different passages (subculture under 10times and beyond passage number 50) were compared. The MSCs of different passages wereinduced in vitro with adipogenic and osteogenic inductive medium. The changes of CD antigenswere measured and the cells were stained for identifying the primary biological characteristicsof MSCs. The marked rat bone marrow mesenchymal stem cells were induced with adipogenicand osteogenic inductive medium to observe whether the marking affects the differentiation ofMSCs. In addition, MSCs from rat bone marrow marked with Hoechst 33342 were injected intocaudal vein, abdominal cavity and inguen of rats for observing the safety of rat bone marrowmesenchymal stem cells in vivo transplant.Results: Compared with centrifugation of gradient and density, more MSCs, faster growth,simply operation and less time were found by culturing direct with all bone marrow cells. Themarked rate of rat bone marrow mesenchymal stem cells with the vector of pEGFP-N1 wasonly 20%, and the intensity of fluorescence didn't become weaken with time increase, but themarked MSCs stopped proliferatation and differentiation after G418 screening. While themarked rate of rat bone marrow mesenchymal stem cells with Hoechst 33342 was 100%, andthe marked MSCs maintained proliferatation, but the intensity of fluorescence became weakenwith the increasing of passages. In the rat bone marrow mesenchymal stem cells of subcultureunder 10 times, 6.86 % of the cells showed CD45~+, 14.91% showed CD44~+ and 97.89% showed CD29~+. The cell cycle analysis by measuring DNA content by flow cytometry revealed that76.41% of the cells were at G0/G1, 13.27 % at S, and 10.33 % at G2/M phases of the cycle.The chromosome number of the MSCs was 2n=42, G zone of the chromosome displayedclearly. In the rat bone marrow mesenchymal stem cells of highly subcultivated (over 50) cells,4.35 % of the cells showed CD45~+, 59.65% showed CD44~+ and 99.18% showed CD29~+. Thecell cycle analysis displayed that 57.86% of the cells were at G0/G1, 23.81% at S and 18.33 %at G2/M phases of the cycle. The chromosome number of the MSCs was 43, a excessivechromosome was observed. G zone of the chromosome displayed clearly. The adipogenie dropswere observed in the MSCs from rat bone marrow of different passages and marked withHoechst 33342 when they were induced with adipogenic and osteogenic inductive medium.Adipogenic drops became red after stained with oil red. Cytoplasm displayed violet and blueafter stained with BCIP/NBT. The positive rate of CD44 in rat bone marrow mesenchymal stemcells increased after induction in both low and highly subcultivated cells. The positive rates ofCD45 and CD29 in rat bone marrow mesenchymal stem cells of less than 10 passages displayeda slight increase after induction. No immunological rejection was observed in the rats whichhad been accepted the rat bone marrow mesenchymal stem cells marked with Hoechst 33342.No tumor, dermoid tumor or cells of marked with Hoechst 33342 were detected after transplantof MSCs in vivo.Conclusion: The cells isolated and purified in this experiment are considered as rat bonemarrow mesenchymal stem cells; MSCs that marked with Hoechst 33342 don't effectproliferatation and differentiation; Tumor and dermoid tumor can't be detected after in vivotransplant of MSCs, and the transplant of MSCs seems safe. These evidences support thefurther application research of MSCs.