Human peripheral blood mononuclear cells enriched in endothelial progenitor cells via quality and quantity controlled culture accelerate vascularization and wound healing in a porcine wound model

Abstract

The transplantation of endothelial progenitor cells (EPCs) is used to promote wound angiogenesis. In patients with chronic wounds and accompanying morbidities, EPCs are often compromised in number and function. To overcome these limitations, we previously developed a quality and quantity controlled (QQ) culture system to enrich peripheral blood mononuclear cells (PBMNCs) in EPCs. To evaluate the wound healing efficacy of mononuclear cells (MNCs) harvested after QQ culture (QQMNCs), preclinical studies were performed on large animals. MNCs harvested from the blood of healthy human subjects were cultured in the presence of angiogenic cytokines and growth factors in a serum-free medium for 7 days. A total of 5 × 106 QQMNCs per full-thickness skin defect or control saline was injected into wounds induced in cyclosporine-immunosuppressed pigs. EPC colony-forming assays revealed a significantly higher number of definitive (partially differentiated) EPC colony-forming units in QQMNCs. Flow cytometry evaluation of QQMNC surface markers showed enrichment of CD34+ and CD133+ stem cell populations, significant reduction in CCR2+ cell percentages, and a greater than 10-fold increase in the percentage of anti-inflammatory M2-type macrophages (CD206+ cells) compared with PBMNCs. Wounds treated with QQMNCs had a significantly higher closure rate. Wounds were harvested, frozen, and sectioned at day 21 postoperatively. Hematoxylin and eosin staining revealed that the epithelization of QQMNC-treated wounds was more advanced than in controls. Treated wounds developed granulation tissue with more mature collagen and larger capillary networks. CD31 and human mitochondrial co-staining confirmed the presence of differentiated human cells within newly formed vessels. Real-time polymerase chain reaction (PCR) showed upregulation of interleukin 6 (IL-6), IL-10, and IL-4 in the wound bed, suggesting paracrine activity of the transplanted QQMNCs. Our data demonstrate for the first time that QQ culture of MNCs obtained from a small amount of peripheral blood yields vasculogenic and therapeutic cells effective in wound healing.

Acceleration of Skin Wound-Healing Reactions by Autologous Micrograft Tissue Suspension

“…Granulation tissue is composed of fibroblasts and extracellular matrix including collagens. In the matrix, neovascularization takes place by angiogenesis and vasculogenesis [ 11 ]. These events finally terminate after full epidermal coverage of the wounded surface.…”

CCL2-Mediated Reversal of Impaired Skin Wound Healing in Diabetic Mice by Normalization of Neovascularization and Collagen Accumulation

“…Thus, the administration of CCL2 can promote neovascularization by affecting the migration of EPCs, as well as macrophages, which have crucial impacts on neovascularization. This was further supported by the observation that EPCs induced to migrate by the CCL2-CCR2 axis could contribute to neovascularization, an indispensable event sustaining newly formed granulation tissue (Kado et al, 2018;Spring et al, 2005). Moreover, VEGF also induced in vitro EPC migration as CCL2 did.…”

Premobilization of CD133+ cells by granulocyte colony- stimulating factor attenuates ischemic acute kidney injury induced by cardiopulmonary bypass

“…Accumulation of peripheral blood CD133+ cells in the renal tissue are likely to promote kidney regeneration. Moreover, in the rodent studies, CD133 ‘progenitor’ cells reported may not exist in the mouse kidney nor in the bone marrow, whether the therapeutic effects of the cells are due to autocrine, paracrine or endocrine effects still remain controversial 23,54–57 . Our results showed that premobilization of CD133+ cells prior to CPB significantly abated IAKI while mobilization of CD133+ cells after CPB procedure couldn’t prevent IAKI efficiently.…”