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.

Keywords: Chronic wounds; endothelial progenitor cells; peripheral blood mononuclear cells; porcine wound model; vasculogenesis; wound healing.

Figure 1.

Porcine wound model and time schedule for wound observation. (a) Six full-thickness skin defects (measured 2.5 × 2.5 cm²) were induced on the back of each pig. Wounds were separated from each other and the backbone by 3 cm. QQMNCs were injected intramuscularly into five spots within each wound at 1 × 10⁶ cells/spot. The remaining three wounds were injected with saline as control. (b) After wound induction and injection of QQMNCs, wounds were photographed at the indicated time points. On day 21, the wounds were excised, frozen, and sectioned. QQMNC: Mononuclear cells harvested after quality and quantity controlled culture.

Figure 2.

Characterization of untreated MNCs (PBMNCs) and QQ culture MNCs (QQMNCs). (a) PBMNC and QQMNC counts per 100 mL of peripheral blood. PBMNC n = 10, QQMNC n = 10. (b) Example of pEPC-CFU (left) and dEPC-CFU (right). 40× magnification FOV. (c) Quantification of total EPC-CFUs including pEPC-CFUs and dEPC-CFUs generated from PBMNCs or QQMNCs plated at 2 × 10⁵ cells/dish. Each bar represents mean ± SD, PBMNC n = 7, QQMNC n = 4. *p < 0.05. PBMNC total CFU versus QQMNC total CFU. (d) Percentage differentiation into pEPC-CFUs and dEPC-CFUs based on their respective average counts (from Figure 2(c) and included inside the bars). dEPC-CFU: definitive endothelial progenitor cells colony-forming units; PBMNC: peripheral blood mononuclear cells; pEPC-CFU: primitive endothelial progenitor cells colony-forming units; QQMNC: mononuclear cells harvested after quality and quantity controlled culture.

Figure 3.

Comparison of wound closure rates in QQMNC-treated and saline-treated wounds. (a) Wounds were photographed at the indicated time points. Wounds tended to epithelialize faster in the QQMNC-transplanted group than in the saline-treated control group. (b) Wounds were photographed on days 0, 3, 7, 10, 14, 17, and 21. Wound area was measured photogrammetrically, and the percentage of wound closure was calculated as [1 − (current wound area/original wound area)] × 100%. Each bar represents mean ± SD, saline n = 6, QQMNC n = 9 wounds. QQMNC: mononuclear cells harvested after quality and quantity controlled culture.

Figure 4.

Wound epithelialization. Hematoxylin and eosin (H-E) staining was performed to evaluate epithelialization. (a) Microscopic images of the H-E stained wounds. (b) Quantitation of the non-epithelialized areas of the wounds. Each bar represents mean ± SD, saline n = 8, QQMNC n = 7, 40× magnification FOV per wound. QQMNC: mononuclear cells harvested after quality and quantity controlled culture.

Figure 5.

Visualization and quantitation of mature collagen by van Gieson staining. (a) Representative microphotographs of wound sections after van Gieson staining. High-density, thick, and strongly stained collagen fibrils are seen in the QQMNC-treated group on day 21 after treatment. (b) Percentage of the area stained strongly with van Gieson staining. Percentage of mature collagen was calculated by measuring the red pixelated area. Each bar represents mean ± SD, saline n = 4, QQMNC n = 4, 200× magnification FOV. QQMNC: mononuclear cells harvested after quality and quantity controlled culture.

Figure 6.

Visualization and quantitation of capillaries in QQMNC-treated and control wounds by staining with the anti-CD31 antibody. (a) Representative microphotograph of granulation tissue in wound sections of control (top) and QQMNC-treated (bottom) wounds stained with the anti-CD31 antibody; 200× magnification. (b) Quantification of the number of capillaries. Significantly more CD31-positive vessels were present in QQMNC-treated wounds compared with saline-injected wounds. Each bar represents mean ± SD, saline n = 4, QQMNC n = 4, 200× magnification FOV. (c) Assessment of inclusion of human cells into newly formed capillaries in the muscular layer 1 cm from the edge of wounds injected with PKH26-QQMNC. 1: DAPI staining. 2: CD31 staining. Endothelial cells are stained in green. 3: Human mitochondria staining. The PKH26-QQMNC red signal is enhanced by HMA staining. 4: Merged microphotographs 1, 2, and 3. White arrows point to overlapping CD31 and HMA staining (orange) suggesting that QQMNC differentiated into endothelial cells.

Figure 7.

Gene expression evaluation of growth factors and cytokines related to wound healing and vasculogenesis in the wound bed. As demonstrated through real-time PCR analysis, expression of IL-6, IL-4, and IL-10 was significantly increased in QQMNC-treated wound tissues compared with saline-treated controls. Each bar represents mean ± SD; TGFβ: QQMNC/saline n = 10/6, FGF: QQMNC /saline n = 5/5, IL-6: QQMNC /saline n = 6/5, IL-4: QQMNC /saline n = 6/4, IL-10: QQMNC /saline n = 6/5.