Abstract
Purpose :
Human bone marrow (BM) contains CD34+ stem cells and mesenchymal stem cells (MSCs) that are being explored for tissue regeneration and revascularization. CD34+ cells, which include endothelial progenitor cells, are known to promote angiogenesis which may be impaired by the diabetic state. MSCs, which have paracrine effects via exosomes, reverse the impaired migration of diabetic CD34+ cells in vivo to enhance angiogenesis. This study used an in vitro tube formation assay (angiogenesis assay) to evaluate the angiogenic potential of human CD34+ cells and exosomes from MSCs, alone and in combination, to determine whether the exosomes can enhance the angiogenic potential of CD34+ cells.
Methods :
CD34+ cells were harvested from the mononuclear cell fraction of BM of a healthy or diabetic human donor. Exosomes were harvested from MSCs from a healthy BM donor cultured under hypoxic conditions. Tubule formation assay was conducted using HUVEC treated with PBS control, healthy CD34+ cells (50k cells), diabetic CD34+ cells (50k cells), or exosomes (0, 1, 10, and 100 μg) alone or in combination with CD34+ cells. Angiogenesis assay was imaged with ImageXpress Micro system and analyzed using built-in MetaXpress software for angiogenesis.
Results :
Healthy CD34+ cells significantly increased HUVECs’ tube length, area, and thickness compared to PBS (p ≤ 0.007). Diabetic CD34+ cells significantly increased tube area compared to PBS (p = 0.050), although no significant effect on tube length or thickness was noted. In addition, a significant increase in the number of tube segments, nodes, and branching points was observed in HUVEC treated with either healthy or diabetic CD34+ cells compared to PBS (p ≤ 0.003). Exosomes (100 ug only) significantly increased tube length, area, and the number of tube segments (p ≤ 0.033), but its combination with healthy or diabetic CD34+ cells had no additional effect .
Conclusions :
Human CD34+ cells from a healthy or diabetic donor both showed an angiogenic effect on HUVEC in vitro, but the angiogenic effect was less robust for diabetic CD34+ cells. Exosomes also had an angiogenic effect in vitro, significant only at the highest dose tested. No additional angiogenic effects were observed when exosomes were added to either healthy or diabetic CD34+ cells. Further studies are ongoing to evaluate the effects of higher doses of exosomes on angiogenesis of CD34+ cells.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.