Expansion Of Bone Marrow Proangiogenic Cells By High Density Dot Culture And The Related Mechanisms

Background—Adult stem cells are distributed in various tissues and their stemness is maintained by the stem cell?niche?.Mimicking the niche in vitro remains a great challenge.Growing evidence suggests that cell-cell interactions play an important role in the niche.We hypothesized that seeding cells at high density to physically maintain cell-cell interactions in culture would be beneficial for stem cell expansion in vitro.Methods—Rat bone marrow cells were extracted from the femurs and tibias of4-week-old male Wistar rats.Primary culture of bone marrow cells was carried out by seeding the cells at 1.6×10~4 cells/cm~2 in DMEM.For regular density culture,9×10~5primary cultured cells(P0)were seeded evenly at a density of 1.6×10~4 cells/cm~2 in a10-cm diameter tissue culture dish,for high density dot culture,9×10~5 primary cells were suspended in 300?l of culture medium,and then six drops(50?l each)of cell suspension were dot-seeded separately onto a 10-cm diameter culture dish.The average diameter of each dot was about 1 cm,resulting in a final local cell density of2×10~5 cells/cm~2.After 15 days,flow cytometric analyses were used to test the angiogenic markers,tube formation assay and hindlimb ischemia model were used to test the angiogenic ability of those cells in regular density culture or high density dot culture.At last,we carried out microarray analysis to compare global gene expression patterns between high density and regular density cultured cells.Results—After 15 days,flow cytometric analyses showed that cells in high density culture expressed higher levels of angiogenic markers,like CD14,CD133,CD45,KDR,CD144,CD31?CD34,than those of cells in regular density culture.In tube formation assays,the number of branch points per field,which were formed by cells from the high density culture,was significantly higher than those formed by cells from the regular density culture.In addition a significant difference in the physiological outcomes of ischemic limbs was observed.Injection of high density cultured cells achieved limb salvage in seven out of 10 mice(70%),whereas only one out of 10(10%)mice were fully protected by injection of regular density cultured cells,and limb salvage was not achieved in the PBS-treated group.Laser Doppler image analysis revealed that blood flow was restored to 80%in the high density group,whereas blood flow was restored to 55%and 40%in regular density and PBS groups,and histological analysis confirmed that the number of microvessels in ischemic muscles was significantly higher in the group treated with high density cultured cells compared with that in the group treated with regular density cultured cells or PBS.Global gene expression analyses revealed that high density cultured cells represented a population that highly expressed focal adhesion,ECM-receptor interaction and proangiogenic genes.Conclusions—This study establishes a novel and simple cell culture method that can mimic the bone marrow niche to maintain and expand endothelial progenitor cells without additional growth factors.Cells from this type of culture can serve as a new therapeutic cell source for treating ischemic diseases.