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Objective: Local intramuscular transplantation of granulocyte colony-stimulating factor (G-CSF)-mobilised peripheral blood mononuclear cells (PB-MNC) has been shown to be effective for treating patients with no-option critical limb ischaemia (CLI) who are not considered suitable to undergo surgical bypass or percutaneous transluminal angioplasty. The aim of this study was to investigate the effectiveness and safety of PB-MNCs as a treatment for no-option CLI patients. Method: This prospective cohort study was conducted between April 2013 and December 2017. Patients with no-option CLI were treated with G-CSF 5–10 µg/kg/day for 3 days. PB-MNCs (7.1±2.2×10 10 ) with CD34+ cells (2.1±1.2×10 8 ) were collected by blood cell separator and then injected into the calf or thigh of ischaemic limbs. Ankle–brachial index, toe–brachial index and transcutaneous oxygen tension were recorded at 1 and 3 months after injection. The amputation rate and the wound healing rate were also recorded. Results: Eight patients took part in the study. Two patients experienced rest pain relief 1 month after PB-MNC therapy. Five patients had healed ulcer at 6 months after PB-MNC therapy. Limb ischaemia did not improve after PB-MNC therapy in one patient. Below-knee amputation was performed in that patient due to extension of gangrene. Two patients required reinjection of PB-MNCs because of recurrence of ischaemic ulcer. The limb salvage rate after 1 year was 87.5%. Conclusion: Local intramuscular transplantation of G-CSF-mobilised PB-MNCs might be a safe and effective treatment for no-option CLI patients.

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Background Quality and Quantity culture media (QQ culture media) was reported to enhance vasculogenesis and angiogenesis function of mononuclear cells (MNCs) from healthy volunteers. In this study, MNCs from chronic limb-threatening ischemia (CLTI) patients were cultured in QQ culture media, and then investigated for angiogenesis-related phenotype and function. Methods Patients aged ≥ 18 years with CLTI caused by atherosclerosis of the lower extremities were prospectively recruited at Siriraj Hospital (Bangkok, Thailand) during July 2017–December 2018. Peripheral blood mononuclear cells (PBMNCs) were isolated from peripheral blood. PBMNCs were cultured in either QQ culture media or standard culture media. The number of CD34+CD133+ cells, CD206+ cells, CD4+CD25+CD127+ cells, colony formation assay, and human umbilical vein endothelial cell (HUVEC) tube formation assay in MNCs were compared between those cultured in QQ culture media and those cultured in standard culture media. Results Thirty-nine patients were included with a mean age of 69 ± 11 years. Diabetes mellitus was found in 25 (64%) patients. The percentage of CD34+CD133+ progenitor cells in MNCs cultured in QQ culture media and in MNCs cultured in standard culture media was 4.91 ± 5.30% and 0.40 ± 0.46%, respectively (p < 0.0001). The percentage of CD206+ cells in MNCs cultured in QQ culture media and in MNCs cultured in standard culture media was 19.31 ± 11.42% and 4.40 ± 2.54%, respectively (p < 0.0001). The percentage of inactive population of T regulatory cells (CD4+CD25+CD127+ cells) in MNCs cultured in standard culture media and in MNCs cultured in QQ culture media was 14.5 ± 10.68% and 1.84 ± 1.37%, respectively (p < 0.0001). The total number of colony-forming units from MNCs cultured in QQ culture media and in MNCs cultured in standard culture media was 8.86 ± 8.35 of 2 × 105 cells/dish, and 0.58 ± 1.05 of 2 × 105 cells/dish, respectively (p < 0.0001). The mean intensity of Dil-Ac-LDL uptake that incorporated into the HUVEC forming tube was 1.37 ± 0.88 in MNCs cultured in QQ culture media, and 0.78 ± 0.41 in MNCs cultured in standard culture media. (p < 0.0003). Conclusions MNCs from CLTI patients that were cultured in QQ culture media had a significantly higher number of CD34+CD133+ cells and anti-inflammatory cells, and higher angiogenesis-related function compared to MNCs cultured in standard culture media.

Background: Quality and Quantity (QQ) culture media was shown a promising effect in enhancing the vasculogenesis of mononuclear cells (MNCs) of healthy volunteers and chronic limb-threatening ischemia (CLTI) patients. In this study, the MNCs from CLTI patients were further investigated based of their risk factors. Methods: In this study, MNCs from chronic limb-threatening ischemia (CLTI) patients with coexisting diabetes mellitus (DM), hypertension (HT), current smoker status, or chronic kidney disease (CKD) stage 3 or above were cultured in QQ culture media, and then investigated for angiogenesis-related phenotype and function. CLTI patients with DM, HT, current smoker status, or CKD were prospectively recruited. Forty-eight patients (mean age: 67.5±8.0 years) were included. DM, HT, current smoker status, and CKD was found in 34 (71.0%), 39 (81.0%), 27(56.3%), and 32 (66.7%) patients, respectively. Results: In CLI patients with coexisting diseases, the percentages of CD34+, CD133+, CD34+ CD133+ progenitor cells; CD 206+ cells; colony forming cells; and, tube formation were significantly higher in the PBMNCs cultured in QQ media than in the PBMNCs cultured in standard culture media. However, the percentage of CD4+ CD25+ CD127+ cells was significantly lower in PBMNCs cultured in QQ culture media compared to the percentage in PBMNCs cultured in standard culture media. Conclusions: Quality and Quantity (QQ)culture media was shown to effectively restore the number of vascular progenitor cells and the vasculogenic function of mononuclear cells from chronic limb-threatening ischemia patients with coexisting diabetes mellitus, hypertension, current smoker status, or chronic kidney disease stage 3 or above.

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