Optimized Isolation Of Porcine Bone Marrow Very Small Embryonic-like Stem Cells And Identification Of Its Biological Characteristics

Objective:To acquire porcine bone marrow very small embryonic-like stem cells by combined using of magnetic activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). And exploring the biological characteristics, the methods of culture and proliferation of porcine bone marrow VSELs in vitro.Methods:After getting porcine bone marrow by bone marrow puncture, we obtained porcine bone marrow mononuclear cells by using of red blood cell lysis buffer. Using the CD133 and Lin antibody coupled magnetic bead respectively, we obtained the CD133(+) and Lin(-) cells from mononuclear cells by MACS. After CD133(+) and Lin(-) cells respectively incubating with CD 133, CD45 and Lin fluorescein antibodies, VSELs were obtained by flow cytometry. The statistical method was used to test the sorting efficiency of two methods respectively and to discuss the value of combining the two methods. We use optical microscope and transmission electron microscope to observe the morphology and ultrastructure of VSELs. Then alkaline phosphatase staining and immunofluorescence staining were used to identify the marker expression of pluripotent stem cells. Porcine bone marrow VSELs were cultured and amplified in conditioned medium, at the same time, their proliferation and adherent rate were observed.Results:Magnetic activated cell sorting (MACS) combined with fluorescence-activated cell sorting (FACS) can fast and efficiently sort out porcine bone marrow VSELs and of which stained by CD133 was more efficient. But the combined method cost high price and is more likely to cause cell pollution. The number of porcine bone marrow VSELs is 0.01%-0.03% of the total mononuclear cell. These cells have a small size, uniform shape between every cell, diploid karyotype, big nucleus and little cytoplasm. They can express multiple antigen marker Oct-4、 Nanog and Sox2, and can be stained by alkaline phosphatase. We can obvious visible cells adherent after incubated in conditioned medium for 3-4 hours. But There is no apparent proliferation for 2-3 weeks.Conclusions:The combined use of magnetic activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) can quickly select large quantity, high purity of VSELs from porcine bone marrow. They can expression of embryonic like stem cells specific antigen, the biological characteristics of these cells are similar to embryonic stem cells (ESCs), and not easy to culture and reproduce in vitro.