Cultivation And Proliferation-promoting Cytokines Of HPP-EPC From Murine Bone Marrow

Objective: The present study was designed to culture and purify high proliferative potential endothelial progenitor cells (HPP-EPC) from murine bone marrow and their progeny cells using a culture system containing the murine bone marrow endothelial cell line-conditioned medium (BMEC-CM), in order that a new method for expansion of these cells was developed. The expression levels of VEGF, bFGF and IGF-1 mRNAs in the cells of bone marrow endothelial cell line were detected, so as to gain insight into the melocular mechanisms underlying promoted proliferation of EPC by BMEC-CM.Methods and results: This experiment was divided into the following three parts:1. Total EPC colony-forming assaySingle cells in murine bone marrow suspension were prepared and preplated for 6 hours to allow the preferential attachment of non-EPC to tissue culture plates. The unattached cells were repreplated in the culture system containing BMEC-CM. After five days, the adhered cells were trypsinized, washed and counted. The trypsinized cells were plated in 1 cell/mm~2 in the above-mentioned culture system. The adherent cell colonies were observed in cultures under an inverted microscope. The colonies were enumerated, and the colony-forming efficiency was calculated as the number of colonies formed per 10~6 nucleated bone marrow cells (NBMCs). The cell numbers of colonies were counted on digital micrographs. The growth cueves were plotted and the population doubling times (DTs) were calculated by using the mean cell number of colonies.EPC clusters appeared in cultures in 9～12 days, then EPC colonies, consisting of oval-, round- and spindle-shape cells, were formed in 2～3 days. The colony-forming efficiency was 600.0±52.4 per 10~6 NBMCs. Most colonies grew rapidly within 1 week and then slowly down. The average cell number of colonies got up 504.6±126.0 at the end of a 30-day culture. DTs of the cells were 58.0-, 74.4-, 131.2- and 358.2-hours respectively in periods of days 10-13, 14-17, 18-21 and 22-25 during cultivation.2. HPP-EPC colony-forming assay, and in vitro expansion and characterization of single colony-derived cellsThe above-mentioned trypsinized cells were plated in 50 cells/mm~2 in the culture system containing BMEC-CM, and the medium was changed every week. The adherent cell colonies were observed in cultures under an inverted microscope. The colonies containing more than 5000 cells were enumerated, and the colony-forming efficiency was calculated as number of colonies formed per 10~6 NBMCs. The culture expansion of single colony-derived cells was performed. The expanded cells were counted every 4 days. The growth cueves of cells were plotted and DTs were calculated based on individual colonies. Expanded cells were tested by immunofluorescent staining for CD31 and vWF, FITC-UEA-1 binding and Dil-Ac-LDL uptake.HPP-EPC colonies, most cells of them showing oval-, and round-shape, were formed in 28～35 days of cultivation. The colony-forming efficiency was 3.0±0.6 per 10~6 NBMCs. Single colony-derived cells can form the cord-like and network-like structures in subcultures at low cell density, and shown a cobblestone-like appearance at high cell density. The shortest TD of the expanded cells was 51.2 hours and observed in the period of days 50-53 during cultivation. TD was prolonged to 324.2 hours in the period of days 74-77. A HPP-EPC was able to yield about 8×10~6 progeny cells over the 80-day-culture period, and these cells were positive for CD31 and vWF immunofluorescent staining, taken up LDL and combined UEA.3. Quantifying mRNA expression of three cytokines in mBMECsTotal RNA was isolated from cells of the murine bone marrowendothelial cell line (mBMECs) using Trizol reagent. The expressionlevels of VEGF, bFGF and IGF-1 mRNAs were measured by Q-realtime RT-PCR.PCR results showed that mRNAs of VEGF and bFGF wereexpressed at high levels and IGF-1 mRNA was expressed at lowerlevels in mBMECs. Conclusions: The culture system containing BMEC-CM is very suitable for EPC growth, so that their progeny cells can be obtained with high yield by using the single colony-derived cell culture in this culture system. The high-level expression of VEGF and bFGF in mBMECs may contribute considerably to the molecular mechanisms of BMEC-CM promoted proliferation of EPC. These results also imply the feedback regulation between EPCs and endothelial cells.