Abstract
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.
Highlights
Stem cell based therapy for ischemic diseases of the cardiovascular system has become an important area of stem cell research and translation
To remove the majority of the nonadherent blood cells, primary culture of bone marrow cells was performed by seeding the cells at 1.66104 cells/cm2 in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA) and 0.2% penicillin/streptomycin
To test its effect on bone marrow Endothelial progenitor cells (EPCs) survival and proliferation, rat bone marrow cells were first cultured in tissue culture dishes for 7 days to remove the majority of the non-adherent blood cells
Summary
Stem cell based therapy for ischemic diseases of the cardiovascular system has become an important area of stem cell research and translation. Endothelial progenitor cells (EPCs), which were first discovered in circulating blood [1], have been intensively investigated for their ability to enhance tissue angiogenesis and attenuate ischemic injury in both animal models and patients [2]. Efficient expansion of EPCs in culture becomes a prerequisite for their therapeutic application. High costs and safety concerns when using growth factors hinder the clinical application of EPC-based therapy. The establishment of an ideal culture method to expand EPCs without the need for growth factors is a critical goal to facilitate clinical translation
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