Phase I/IIa Feasibility Trial of Autologous Quality- and Quantity-Cultured Peripheral Blood Mononuclear Cell Therapy for Non-Healing Extremity Ulcers.

Non-healing wounds are among the main causes of morbidity and mortality. We recently described a novel, serum-free ex vivo expansion system, the quantity and quality culture system (QQc), which uses peripheral blood mononuclear cells (PBMNCs) for effective and noninvasive regeneration of tissue and vasculature in murine and porcine models. In this prospective clinical study, we investigated the safety and efficacy of QQ-cultured peripheral blood mononuclear cell (MNC-QQ) therapy for chronic non-healing ischemic extremity wounds. Peripheral blood was collected from 9 patients with 10 chronic (>1 month) non-healing wounds (8 males, 1 female; 64-74 years) corresponding to ischemic extremity ulcers. PBMNCs were isolated and cultured using QQc. Within a 20-cm area surrounding the ulcer, 2 × 107 cells were injected under local anesthesia. Wound healing was monitored photometrically every 2 weeks. The primary endpoint was safety, whereas the secondary endpoint was efficacy at 12-week post-injection. All patients remained ambulant, and no deaths, other serious adverse events, or major amputations were observed for 12 weeks after cell transplantation. Six of the 10 cases showed complete wound closure with an average wound closure rate of 73.2% ± 40.1% at 12 weeks. MNC-QQ therapy increased vascular perfusion, skin perfusion pressure, and decreased pain intensity in all patients. These results indicate the feasibility and safety of MNC-QQ therapy in patients with chronic non-healing ischemic extremity wounds. As the therapy involves transplanting highly vasculogenic cells obtained from a small blood sample, it may be an effective and highly vasculogenic strategy for limb salvage.

MMP9 secreted from mononuclear cell quality and quantity culture mediates STAT3 phosphorylation and fibroblast migration in wounds.

INTRODUCTION: Intractable ulcers may ultimately lead to amputation. To promote wound healing, researchers developed a serum-free ex vivo peripheral blood mononuclear cell quality and quantity culture (MNC-QQc) as a source for cell therapy. In mice, pigs, and even humans, cell therapy with MNC-QQc reportedly yields a high regenerative efficacy. However, the mechanism of wound healing by MNC-QQc cells remains largely unknown. Hence, using an in vitro wound healing model, this study aimed to investigate MNC-QQc cells and the migratory potential of dermal fibroblasts. METHODS: After separation from a 50 mL blood sample from healthy individuals, mononuclear cells were cultured for 7 days in a serum-free ex vivo expansion system with five different cytokines (MNC-QQc method). The effects of MNC-QQc cells on human dermal fibroblast migration were observed by scratch assay. An angiogenesis array screened the MNC-QQc cell supernatant for proteins related to wound healing. Finally, fibroblast migration was confirmed by observing the intracellular signal transduction pathways via Western blot. RESULTS: The migration of fibroblasts co-cultured with MNC-QQc cells increased by matrix metallopeptidase-9 (MMP9) secretion, as suggested by the angiogenesis array. Furthermore, the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in fibroblast/MNC-QQc cell co-culture and fibroblast culture with added recombinant human MMP9 protein increased. When fibroblasts were cultured with either an MMP9 inhibitor or a STAT3 inhibitor, both fibroblast migration and STAT3 phosphorylation were significantly suppressed. CONCLUSIONS: MNC-QQc cells promote wound healing by the secretion of MMP9, which induces fibroblast migration via the STAT3 signaling pathway.

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.

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.

Quality-Quantity Control Culture Enhances Vasculogenesis and Wound Healing Efficacy of Human Diabetic Peripheral Blood CD34+ Cells.

Autologous endothelial progenitor cell (EPC) therapy is commonly used to stimulate angiogenesis in ischemic repair and wound healing. However, low total numbers and functional deficits of EPCs make autologous EPC therapy ineffective in diabetes. Currently, no known ex vivo culture techniques can expand and/or ameliorate the functional deficits of EPCs for clinical usage. Recently, we showed that a quality-quantity culture (QQc) system restores the vasculogenic and wound-healing efficacy of murine diabetic EPCs. To validate these results and elucidate the mechanism in a translational study, we evaluated the efficacy of this QQc system to restore the vasculogenic potential of diabetic human peripheral blood (PB) CD34+ cells. CD34+ cells purified from PB of diabetic and healthy patients were subjected to QQc. Gene expression, vascular regeneration, and expression of cytokines and paracrine mediators were analyzed. Pre- or post-QQc diabetic human PB-CD34+ cells were transplanted into wounded BALB/c nude mice and streptozotocin-induced diabetic mice to assess functional efficacy. Post-QQc diabetic human PB-CD34+ cell therapy significantly accelerated wound closure, re-epithelialization, and angiogenesis. The higher therapeutic efficacy of post-QQc diabetic human PB-CD34+ cells was attributed to increased differentiation ability of diabetic CD34+ cells, direct vasculogenesis, and enhanced expression of angiogenic factors and wound-healing genes. Thus, QQc can significantly enhance the therapeutic efficacy of human PB-CD34+ cells in diabetic wounds, overcoming the inherent limitation of autologous cell therapy in diabetic patients, and could be useful for treatment of not only wounds but also other ischemic diseases. Stem Cells Translational Medicine 2018;7:428-438.

Successful Treatment of Infantile Hemangiomas With Propranolol in Low-Birth-Weight Infants.

BACKGROUND: Infantile hemangioma (IH) is a benign neoplasm that causes scarring and cosmetic problems after spontaneous regression. Therefore, aggressive treatments such as laser irradiation and corticosteroid have been used; however, recently, the effect of propranolol has been widely noticed. In this study, the authors applied propranolol to low-birth-weight infants with IHs and evaluated its effect. METHODS: Four low-birth-weight infants having IH were selected, with birth weights ranging from 582 to 814 g (average 703 g). The administration of propranolol was started within 4 days of hospitalization. The dosage of propranolol was increased from 0.5 to 2.0 mg/kg/day step by step. Vital signs and blood sugar level were checked prior to every administration of the drug. Continuous monitoring of electrocardiography and arterial oxygen saturation were performed during entire hospitalization.The outcomes were assessed by the patient's family and 2 board-certified plastic surgeons based on 5 parameters pertaining to clinical findings, using a scale of 1 to 10. Reduction rate of the hemangioma was calculated at the end of treatment and compared with the size prior to treatment. RESULTS: The authors could administrate propranolol without any severe side effects in all patients. Infantile hemangiomas gradually shrank soon after the authors started the treatment. Reduction ratios were 22.1% to 100% (average 48.72%), and the comprehensive evaluation of treatment was 7.5 to 10 (average 8.55) on a 10-point scale. CONCLUSIONS: With careful monitoring of their vital signs, propranolol could be a good treatment option even for IH in low-birth-weight infants whose birth weights were less than 1000 g.

Interleukin-6 stimulates Akt and p38 MAPK phosphorylation and fibroblast migration in non-diabetic but not diabetic mice.

Persistent inflammatory environment and abnormal macrophage activation are characteristics of chronic diabetic wounds. Here, we attempted to characterize the differences in macrophage activation and temporal variations in cytokine expression in diabetic and non-diabetic wounds, with a focus on interleukin (IL)-6 mRNA expression and the p38 MAPK and PI3K/Akt signaling pathways. Cutaneous wound closure, CD68- and arginase-1 (Arg-1)-expressing macrophages, and cytokine mRNA expression were examined in non-diabetic and streptozotocin-induced type 1 diabetic mice at different time points after injury. The effect of IL-6 on p38 MAPK and Akt phosphorylation was investigated, and an in vitro scratch assay was performed to determine the role of IL-6 in primary skin fibroblast migration. Before injury, mRNA expression levels of the inflammatory markers iNOS, IL-6, and TNF-α were higher in diabetic mice; however, IL-6 expression was significantly lower 6 h post injury in diabetic wounds than that in non-diabetic wounds. Non-diabetic wounds exhibited increased p38 MAPK and Akt phosphorylation; however, no such increase was found in diabetic wounds. In fibroblasts from non-diabetic mice, IL-6 increased the phosphorylation of p38 MAPK and levels of its downstream factor CREB, and also significantly increased Akt phosphorylation and levels of its upstream factor P13K. These effects of IL-6 were not detected in fibroblasts derived from the diabetic mice. In scratch assays, IL-6 stimulated the migration of primary cultured skin fibroblasts from the non-diabetic mice, and the inhibition of p38 MAPK was found to markedly suppress IL-6-stimulated fibroblast migration. These findings underscore the critical differences between diabetic and non-diabetic wounds in terms of macrophage activation, cytokine mRNA expression profile, and involvement of the IL-6-stimulated p38 MAPK-Akt signaling pathway. Aberrant macrophage activation and abnormalities in the cytokine mRNA expression profile during different phases of wound healing should be addressed when designing effective therapeutic modalities for refractory diabetic wounds.