Expansion of Endothelial Progenitor Cells in High Density Dot Culture of Rat Bone Marrow Cells

<div><p>In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2×10⁵ cells/cm²) by seeding total 9×10⁵ cells into six high density dots or cultured in regular density (1.6×10⁴ cells/cm²) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells.</p></div

Engraftment and endothelial differentiation of high density cultured cells in ischemic limbs.

<p>A and B, Confocal images revealed that some of high density cultured cells (CM-DiI⁺, white arrows) participated in neovascularization by direct differentiation into endothelial cells (ILB4⁺). C, CM-DiI⁺ cells (white arrows) close to capillaries, but negative for ILB4 staining, might support vessel formation via paracrine effect of released pro-angiogenic growth factors. D, The percentages of endothelial and non-endothelial differentiation of injected cells, and the percentage of vessels derived from donor and recipient (n = 3). *p<0.05.</p

Up-regulation of cell adhesion molecules and pro-angiogenic growth factors in high density cultured cells.

<p>A, Pathway analysis of microarray data. Genes involved in focal adhesion and ECM-receptor interactions were highly expressed in high density cultured cells compared with those in regular density cultured cells. B, Expression of integrin family genes validated by qRT-PCR analysis. Data are presented as the fold increase of gene expression in high density (HD) cultured cells compared with that in regular density (RD) cultured cells (n = 3). *p<0.05. C, Expression of growth factors validated by qRT-PCR analysis. Data are presented as the fold increase of gene expression in high density cultured cells compared with that in regular density cultured cells. (n = 3). *p<0.05. D, ELISA analysis of VEGF, PDGF, TGF-β, HGF, bFGF and SDF-1α in the supernatants of cultured cells. (n = 3). *p<0.05. E, Tube formation of HUVECs on Matrigel induced by conditioned medium from regular (CM-RD) or high density (CM-HD) cultures. Cells incubated in DMEM with 10% FBS served as a control medium (Ctrl-M). The number of branch points per field was counted after 12 hours of network formation (n = 9). *p<0.05. F, Growth factor expression of CD45⁺ and CD45⁻ cells sorted from high density culture validated by qRT-PCR analysis. (n = 3). *p<0.05.</p

Pro-angiogenic potential of high density cultured cells in vitro and in vivo.

<p>A, In vitro tube formation assay with Matrigel for the cells derived from high density (HD) and regular density (RD) groups with quantification of the number of branch points per field (n = 9). *p<0.05, Scale bars, 100 µm. B, An in vivo angiogenic assay was performed by transplantation of expanded cells into ischemic hind limbs of nude mice. Representative views of ischemic left hind limbs at 3 weeks after treatment with PBS, regular density cultured cells or high density cultured cells and percent distribution of outcomes after treatment (n = 10). C, Laser Doppler images of blood perfusion with mice in a supine position at day 1 after femoral artery ligation and at day 21 after cell transplantation or PBS injection. Restoration of blood flow was calculated by comparing the laser Doppler image data of ischemic left limbs with that of non-ischemic right limbs at day 21. A significant improvement of blood supply was achieved in the group treated with high density cultured cells (n = 4). *p<0.05. D, Blood vessels in hind limb adductor muscles were identified by ILB4 staining. A higher number of capillaries were observed in the group treated with high density cultured cells (n = 4). *p<0.05, Scale bars, 100 µm. E, Donor-derived cells were detected by CM-DiI labeling of hind limb adductor muscles. A higher number of CM-DiI⁺ cells were observed in the muscles treated with high density cultured cells (n = 5). *p<0.05, Scale bars, 100 µm.</p

Bone marrow cells in high density culture.

<p>A, Small bright cells were observed in high density culture (2×10⁵ cells/cm²) of rat bone marrow cells. Cells were incubated with DiI-ac-LDL and stained with FITC-conjugated UEA lectin and DAPI. Small bright cells were double-positive for DiI-ac-LDL and UEA lectin with counterstained DAPI. B, Spindle-shaped cells were observed in regular density culture (1.6×10⁴ cells/cm²). The majority of cells were negative for DiI-ac-LDL uptake and UEA lectin binding. C, Bone marrow cells were seeded at different densities. After 3 days of culture, fluorescence microscopic observation and flow cytometric analysis revealed an increase of DiI-ac-LDL-positive cells with the increase of cell seeding density (n = 3). Scale bars, 100 µm.</p

Endothelial precursors are enriched in the CD45⁺ cell population.

<p>A, Flow cytometric analysis showed that the majority of cells in regular density (RD) culture became larger after their expansion, whereas the cells in high density (HD) culture maintained the small size of their parental cells (P0). B, Cells from high density culture were analyzed further by gating based on different cell sizes. The small cells showed higher expression levels of EPC related markers than those of the large cells. C, In high density culture, the majority of cells positive for KDR, CD34 and DiI were also positive for CD45. D, In vitro angiogenesis assay of CD45⁺ and CD45⁻ cells sorted from high density culture. E, CD45⁺, but not CD45⁻, cells from high density culture differentiated into endothelial cells that were able to uptake DiI-ac-LDL and express vWF and eNOs. Scale bars, 100 µm. F, Percentages of endothelial cell marker-positive cells in CD45⁺ and CD45⁻ populations after 14 days of induction (n = 3). *p<0.05.</p

Characterization of bone marrow cells cultured in high density dots.

<p>A, Diagram of the high density dot culture system. Adherent cells from primary cultures (P0) of rat bone marrow aspirates were subcultured at a regular density (RD) or in high density dots (HD). B, Typical morphologies of primary cells (P0), the cells in RD- and HD-culture at day 15, and the proliferation of cells in RD- and HD-culture (n = 3). Scale bars, 100 µm. C, Representative histograms of cell surface marker expression analyzed by flow cytometer. D, Quantitative analysis of the flow cytometric data showed higher expression levels of EPC related markers in high density cultured cells (n = 3). *p<0.05.</p