Abstract
Objective: This research is designed to explore the methods of isolation and culture for endothelial progenitor cells from bovine bone marrow, characteristic, induced differentiative capacity in vitro . Material and methods: Main experimental reagents contain DMEM/F12, fetal bovine serum, percoll lymphocyte separating, Trypsin 1: 250, VEGF, bFGF, GF-1, EDTA and so on. Cultivation system is DMEM/F12 with 10% FBS and VEGF 10 ng/mL, cultured under 37°C, 5% CO 2 , saturated humidity. Cell viability is measured by trypan blue solution exclusion test. Immunofluorescent detection is used to detected cell surface markers and double swallows, while bovine chromosome is analyzed by karyotyping. Results: We find that the majority of bovine endothelial progenitor cells (EPCs) are fibrous shaped. Frozen survival of bovine EPCs before and after cryopreservation is 95.2±0.14% and 80.9±0.30% respectively; cryopreservation affects little on the viability of bovine EPCs. Immunofluorescent detection of the cell surface markers CD34, CD133 and flk present positive, which can confirm that the cell cultured in vitro are EPCs. Then Dil-ac-LDL and FITC-UAE-1 uptake assays are carried out. Eventually, bovine EPCs are induced to differentiate into endothelial cells and smooth muscle cells respectively, demonstrating the multi-lineage differentiation potential of bovine EPCs in vitro . Conclusion: EPCs can be got with proper culture system. The little cell cryopreservation effect and stronger induced differentiation potential in vitro imply that EPCs can be applied in genetic resources conservation and reuse. http://doi.org/10.5455/javar.2017.d196
Highlights
Endothelial progenitor cells (EPCs) are known as precursor cells used in the treatment of atherosclerosis, which can differentiate into mature endothelial cells that participate in the repair of endothelial injury (Talbot and Blombergie, 2008; Spadaccio et al, 2010)
endothelial progenitor cells (EPCs) are passaged and we find that they develop fibrosis gradually (Figure 1C)
We find that EPCs are usually isolated from umbilical cord blood or peripheral blood in previous reports, 3% of EPCs can be found in bone marrow mononuclear cells approximately (Boyer et al, 2005; Rotmans et al, 2006) and there are fewer researches paying close attention to it
Summary
Endothelial progenitor cells (EPCs) are known as precursor cells used in the treatment of atherosclerosis, which can differentiate into mature endothelial cells that participate in the repair of endothelial injury (Talbot and Blombergie, 2008; Spadaccio et al, 2010). Later (Asahara et al, 1997; Kawamura et al, 2006), scientists isolated EPC from umbilical cord blood, liver, marrow, arterial outer membrane and fat tissue (Eggermann et al, 2003; Bonello et al, 2006). They all can directionally home to the ischemic area and differentiate into mature endothelial cells with delayed high proliferation potential, can they participate in the embryonic development of angiogenesis, and promote restoration of the damaged endothelial capillaries (Peichev et al, 2000; De Falco et al, 2009), apply to endangered species resources conservation. This research will explore the methods of isolation and culture, characterization and induced differentiation of EPC from bovine bone marrow
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