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Isolation and characterization of endothelial progenitor cells from Rhesus monkeys.

Sun W, Zheng L, Han P, Kang YJ - Regen Med Res (2014)

Bottom Line: The results showed that nonselective mononuclear EPCs were a better choice for high yield of the target cells.The cells grew in M 200 better than in EGM-2, and supplementation with fetal bovine serum promoted cell proliferation; but serum level at 7.5% was better than at 10%.This procedure would help using these valuable cells for regenerative medicine research.

View Article: PubMed Central - PubMed

Affiliation: Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041 China.

ABSTRACT

Background: Endothelial progenitor cells (EPCs) are increasingly becoming a major focus of regenerative medicine research and practice. The present study was undertaken to establish an appropriate procedure for isolation and characterization of EPCs from Rhesus monkeys for regenerative medicine research.

Result: Selective CD34+ and nonselective mononuclear EPCs were isolated from bone marrow and cultured under varying conditions. The results showed that nonselective mononuclear EPCs were a better choice for high yield of the target cells. The cells grew in M 200 better than in EGM-2, and supplementation with fetal bovine serum promoted cell proliferation; but serum level at 7.5% was better than at 10%. In addition, surface coating of the culture dishes with human fibronectin significantly improved the proliferation and ontogeny of the isolated EPCs. Immunocytochemistry including detection of markers CD34, CD133 and CD31 and double-staining for Ac-LDL and lectin verified the purity of the cultured mononuclear EPCs.

Conclusion: By a thorough analysis, we established a practical procedure for isolation and propagation of EPCs from Rhesus monkeys. This procedure would help using these valuable cells for regenerative medicine research.

No MeSH data available.


Related in: MedlinePlus

Characterization of cells in cultures by staining cells with FITC-lectin and examing uptake of Dil AcLDL. Unselected mononuclear EPCs were cultured on FN coated dishes and in M 200 or EGM-2 medium, and characterization was proceeded by uptake of Dil AcLDL (left, red) and FITC-lectin staining (middle, green). Double positive cells (right, merge) were observed in both M 200 (A-C) and EGM-2 (D-F) on the 19th day after plating, but the number of double positive cells in EGM-2 was much more than in M 200 (G). *, significantly different (P <0.05). Bar =100 μm.
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Fig4: Characterization of cells in cultures by staining cells with FITC-lectin and examing uptake of Dil AcLDL. Unselected mononuclear EPCs were cultured on FN coated dishes and in M 200 or EGM-2 medium, and characterization was proceeded by uptake of Dil AcLDL (left, red) and FITC-lectin staining (middle, green). Double positive cells (right, merge) were observed in both M 200 (A-C) and EGM-2 (D-F) on the 19th day after plating, but the number of double positive cells in EGM-2 was much more than in M 200 (G). *, significantly different (P <0.05). Bar =100 μm.

Mentions: Unselected mononuclear EPCs were cultured on human FN coated culture dishes and in M 200 or EGM-2 without FBS supplementation. Under this condition, cells were characterized by surface markers CD34, CD133, CD31, by FITC-lectin staining, and by the uptake of Dil AcLDL. FITC-lectin staining and Dil AcLDL uptake are classical methods for EPCs characterization; binding of lectin is specific for human endothelial cells, and uptake of Dil AcLDL is a function associated with endothelial cells [3, 26, 27]. There were no differences in the expression of CD34, CD133, and CD31 between the two different media (data not shown). The data presented in Figure 4 show that almost all the cells were bound with FITC-lectin, but uptake of Dil AcLDL was only observed in a portion of cell population. Importantly, there was much more double-positive staining of FITC-lectin and Dil AcLDL in cells cultured in EGM-2 than those in M 200.Figure 4


Isolation and characterization of endothelial progenitor cells from Rhesus monkeys.

Sun W, Zheng L, Han P, Kang YJ - Regen Med Res (2014)

Characterization of cells in cultures by staining cells with FITC-lectin and examing uptake of Dil AcLDL. Unselected mononuclear EPCs were cultured on FN coated dishes and in M 200 or EGM-2 medium, and characterization was proceeded by uptake of Dil AcLDL (left, red) and FITC-lectin staining (middle, green). Double positive cells (right, merge) were observed in both M 200 (A-C) and EGM-2 (D-F) on the 19th day after plating, but the number of double positive cells in EGM-2 was much more than in M 200 (G). *, significantly different (P <0.05). Bar =100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4389970&req=5

Fig4: Characterization of cells in cultures by staining cells with FITC-lectin and examing uptake of Dil AcLDL. Unselected mononuclear EPCs were cultured on FN coated dishes and in M 200 or EGM-2 medium, and characterization was proceeded by uptake of Dil AcLDL (left, red) and FITC-lectin staining (middle, green). Double positive cells (right, merge) were observed in both M 200 (A-C) and EGM-2 (D-F) on the 19th day after plating, but the number of double positive cells in EGM-2 was much more than in M 200 (G). *, significantly different (P <0.05). Bar =100 μm.
Mentions: Unselected mononuclear EPCs were cultured on human FN coated culture dishes and in M 200 or EGM-2 without FBS supplementation. Under this condition, cells were characterized by surface markers CD34, CD133, CD31, by FITC-lectin staining, and by the uptake of Dil AcLDL. FITC-lectin staining and Dil AcLDL uptake are classical methods for EPCs characterization; binding of lectin is specific for human endothelial cells, and uptake of Dil AcLDL is a function associated with endothelial cells [3, 26, 27]. There were no differences in the expression of CD34, CD133, and CD31 between the two different media (data not shown). The data presented in Figure 4 show that almost all the cells were bound with FITC-lectin, but uptake of Dil AcLDL was only observed in a portion of cell population. Importantly, there was much more double-positive staining of FITC-lectin and Dil AcLDL in cells cultured in EGM-2 than those in M 200.Figure 4

Bottom Line: The results showed that nonselective mononuclear EPCs were a better choice for high yield of the target cells.The cells grew in M 200 better than in EGM-2, and supplementation with fetal bovine serum promoted cell proliferation; but serum level at 7.5% was better than at 10%.This procedure would help using these valuable cells for regenerative medicine research.

View Article: PubMed Central - PubMed

Affiliation: Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041 China.

ABSTRACT

Background: Endothelial progenitor cells (EPCs) are increasingly becoming a major focus of regenerative medicine research and practice. The present study was undertaken to establish an appropriate procedure for isolation and characterization of EPCs from Rhesus monkeys for regenerative medicine research.

Result: Selective CD34+ and nonselective mononuclear EPCs were isolated from bone marrow and cultured under varying conditions. The results showed that nonselective mononuclear EPCs were a better choice for high yield of the target cells. The cells grew in M 200 better than in EGM-2, and supplementation with fetal bovine serum promoted cell proliferation; but serum level at 7.5% was better than at 10%. In addition, surface coating of the culture dishes with human fibronectin significantly improved the proliferation and ontogeny of the isolated EPCs. Immunocytochemistry including detection of markers CD34, CD133 and CD31 and double-staining for Ac-LDL and lectin verified the purity of the cultured mononuclear EPCs.

Conclusion: By a thorough analysis, we established a practical procedure for isolation and propagation of EPCs from Rhesus monkeys. This procedure would help using these valuable cells for regenerative medicine research.

No MeSH data available.


Related in: MedlinePlus