Clinical trial for the treatment of severe limb ischaemia by implantation of cultured immature red cells developed from small amount of bone marrow of the patient

INTERVENTION: 1. Autologous Bone Marrow Collection: 20 ml of bone marrow is collected from iliac crest under local anaesthesia 14 days before the implantation. 2. Ex Vivo Expansion Culture: All procedures are enforced by exclusive technical exparts along the approved protocols in GMP‐grade Cell Processing Room established in Bioscience Medical Research Center, Niigata University Medical and Dental Hospital. Mononuclear cells separated from the bone marrow are incubated in a suspension culture in the presence of rh Flt‐ligand, rh SCF, rh Thrombopoietin, and culture supplements for 7 days to expand myeloid progenitors. Harvested and washed cells are further cultured in the presence of rh SCF, rh IGF‐I, rh Erythropoietin, and culture supplements for additional 7 days to expand immature erythroblasts and macrophages. 3. Preparation for implant: Cultured cells are harvested and washed. The cells are resuspended in 50 ml of platelet concentrate supplemented with 6,000 IU of erythropoietin. 4. Treatment: Aliquots of the 50 ml of cell suspension are intramuscularly injected in 100 points of the ischaemic limb. Daily intramuscular injection of rh erythropoietin in the same loci follows from the next day for consecutive 4 days. CONDITION: Severe peripheral arterial disease including arteriosclerosis obliterans, Buerger disease and arteritis ; Circulatory System ; Peripheral arterial disease PRIMARY OUTCOME: Efficacy evaluation: improvement in:; 1. Ankle‐Brachial Index (ABI) (1 month of implantation); 2. Transcutaneous Oxygen pressure (TcO2) (1 month); 3. Rest pain; ; Safety evaluation: ; 1. Adverse effects caused by the implanted cells including teratoma, ossification, etc.; 2. Adverse effects caysed by erythropoietin including polycythemia, hypertension, pure red cell aplasia, thrombosis in heart/lung/brain, etc.; 3. Adverse effects caused by possible residual components used as supplements for cell culture including allergy, serum sickness, infections, etc.; 4. Common adverse effects caused by drug administration stated in National Cancer Institute Common Toxicity Criteria (NCI‐CTC) SECONDARY OUTCOME: 1. Improvement in ABI (6, 12, 18 and 24 month of implantation) and Transcutaneous Oxygen pressure (TcO2) (6, 12, 18 and 24 months); 2. Improvement in subjective symptoms: Visual Analogue Scale (VAS) in 1, 6, 12, 18 and 24 months of implantation ; 3. Improvement in limb ulceration in 1, 6, 12, 18 and 24 months of implantation; 4. Elongation in range of pain‐free walking in 1, 6, 12, 18 and 24 months of implantation; 5. The requirement of analgesic in 1, 6, 12, 18 and 24 months of implantation; 6. Manifestation of new collaterals evaluated by angiography in 1 month of implantation INCLUSION CRITERIA: 1. Peripheral Arterial Disease (PAD) including Arteriosclerosis Obliterans (ASO), Buerger disease, and arteritis associated with collagen diseases 2. Fontaines stage: IIb, III, and IV 3. Chronic limb ischaemia, including rest pain, non‐healing ischaemic ulcers, or both, and were not candidates for non‐surgical or surgical revascularisation 4. Age: greater than 20 or less than 80 years 5. World Health Organization (WHO) performance status: 0 to 3