Whereas hUCB-MSCs with low-angiogenic capacity did not cause any significant increase in total length or branching formation of HUVEC. sensitivity to hypoxic conditions is different between cells originating from different donors, and this difference affects the contribution to angiogenesis. The bioinformatics analysis of different donors under hypoxic culture conditions identified intrinsic variability in gene expression patterns and suggests alternative potential genetic factors ANGPTL4, ADM, SLC2A3, and CDON as guaranteed general indicators for further stem cell therapy. Introduction Peripheral artery disease (PAD) remains a leading cause of limb disability and loss, Flavopiridol HCl which is caused by crucial limb ischemia1. Although the disease severely diminishes Flavopiridol HCl quality of life and has a great risk of amputation, there are currently only a few treatment options. Recently, several types of research in cell therapy reported that cells have the potential to re-vascularize the ischemic limb2. Preclinical cell therapy studies have exhibited the improved regeneration of the vascular system in different experimental models with several types of cell applications through various injection routes3,4. However, in clinical trials, the cell therapies showed varied outcomes; some of them improved in revascularization and led to less amputation, while many other trials did not show any clinical benefits5. Mesenchymal stem cells (MSCs), a promising candidate source for cell transplantation therapies for PAD, are well-known for their unique qualities, such as immunomodulation6, maintaining endogenous stem cell niches7 and their potential to stimulate angiogenesis8. Additionally, they have been reported to migrate and proliferate in response to the cytokines or chemokines released from the ischemic site9. Recent studies have focused on modifying MSCs to improve revascularization and understand the cells biological role and mode of action in angiogenesis10. Despite these efforts and accomplishments, the results of current preclinical studies and clinical trials suggest that a better alleviation strategy with MSC therapy is still needed. One strongly suggested element is that there are individual differences in MSCs based on the variability from donor to donor11. To verify MSCs as a reliable cell source and establish MSC cell therapy for PAD, the strikingly variable behaviors among MSCs isolated from different donors must be comprehended. Recent studies addressing this issue have compared bone marrow MSCs from various donors and found significant differences in cell growth rates and alkaline phosphatase enzyme activity12. Differentiation capacity also showed contrasting results between cells from different donors, with distinguished osteogenic differentiation ability with different gene levels, and the adipocyte-specific gene expression varied as well13. In this study, we examined the angiogenesis capacity of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in vitro and in vivo. We focused on comparing hUCB-MSCs isolated from different donors, and examined the capability for therapeutic efficacy for PAD. To spotlight the fact that individual differences based on donor-specific cellular properties is crucial in the application of the cells, we optimized Rabbit Polyclonal to PDGFRb the culture conditions of hUCB-MSCs by incubating in hypoxic conditions for 1 day or 2 weeks and analyzed the change in revascularization. Moreover, genome-wide analysis of hUCB-MSCs between different donors exhibited Flavopiridol HCl different therapeutic efficacy through genetic profiling. Materials and methods Isolation and culture of hUCB-MSCs Entire experimental procedures involving hUCB-MSCs were conducted under approval of the Boramae Hospital Institutional Review Board (IRB) and the Seoul National University IRB (IRB No. 1608/001-021). Isolation and culture of hUCB-MSCs were previously described7. In brief, human cord blood samples were incubated with HetaSep answer (Stem Cell Technologies, Vancouver, Canada) at a ratio of 5:1 to remove red blood cells. Then, the supernatant was collected with Ficoll, and mononuclear cells were separated after centrifugation at 2500?r.p.m. for 20?min. The cells were washed twice in phosphate-buffered saline (PBS). Cell pellets were reconstituted and seeded in KSB-3 Complete media (Kangstem Biotech, Seoul, Republic of Korea) made up of 10% fetal bovine serum (Gibco BRL, NY, USA) and antibiotics. After 3 days of stabilization, unattached cells were washed out, and isolated stem cells were maintained at 5% CO2 Flavopiridol HCl and 21% O2 for normoxic Flavopiridol HCl condition. For hypoxic culture, hUCB-MSCs were transferred to a hypoxic chamber made up of 5% CO2 and 1% O2 gases for 24?h or 2 weeks. Immunocytochemistry Cells cultured under normoxic and hypoxic conditions were washed in PBS and fixed with 4% paraformaldehyde (PFA) at room heat for 10?min. The cells were permeabilized with 0.05% Triton X-100 solution at room temperature for 10?min and blocked with 5% normal goat.