Establishment of culturing system for ex-vivo expansion of angiogenic immature erythroid cells, and its application for treatment of patients with chronic severe lower limb ischemia.
Angiogenesis therapy by bone marrow-mononuclear cell implantation (BMI) has been utilized. We found that erythroid cells played an essential role in angiogenesis by BMI. We then tried to establish a novel cell therapy by implantation of ex vivo expanded immature erythroblasts cultured from hematopoietic stem/precursor cells. Immature to mature erythroblasts were purified from human bone marrow, and mRNA expression were analyzed. Strongly expressed VEGF and PLGF in immature erythroid cells decreased according to erythroid maturation. To expand very immature erythroid cells, we established a two-step culturing system, i.e., bone marrow cells were cultured in the presence of Flt-3L, SCF and TPO for 7 days, and the cells were further cultured in the presence of SCF, IGF-I and EPO for an additional 7 days. The in vivo angiogenic effects of implantation of the ex vivo expanded cells were stronger than that of BMI in mouse limb ischemia model. Three patients with severe chronic lower limb ischemia accompanied by Burger's disease or collagen arteritis were enrolled in a pilot clinical trial of the novel cell therapy by transplantation of ex-vivo expanded immature erythroid cells. In the clinical trial, most clinical symptoms such as rest pain and skin ulcers improved in 4 weeks, and did not recur in the one-year follow-up. No adverse events were observed in any of the patients. Moreover this novel cell therapy required only a small amount of bone marrow collection. Further enrollment of patients with chronic severe lower limb ischemia is necessary to confirm the efficacy and safety of this novel cell therapy, and to estimate the necessary amount of bone marrow aspirate.
Oda
Masato
M
First Department of Internal Medicine, Niigata University Medical and Dental Hospital, Niigata, Japan.
Toba
Ken
K
Ozawa
Takuya
T
Kato
Kiminori
K
Yanagawa
Takao
T
Ikarashi
Noboru
N
Takayama
Tsugumi
T
Suzuki
Tomoyasu
T
Hanawa
Haruo
H
Fuse
Ichiro
I
Nakata
Kou
K
Narita
Miwako
M
Takahashi
Masuhiro
M
Aizawa
Yoshifusa
Y
D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015153:Blotting, Western; D001853:Bone Marrow / Q000473:pathology; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D002908:Chronic Disease; D015672:Erythroid Precursor Cells / Q000166:cytology* / Q000637:transplantation*; D005240:Feasibility Studies; D005260:Female; D006614:Hindlimb / Q000098:blood supply* / Q000601:surgery; D006801:Humans; D007511:Ischemia / Q000473:pathology / Q000628:therapy*; D008180:Lupus Erythematosus, Systemic / Q000473:pathology / Q000628:therapy; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D008875:Middle Aged; D018919:Neovascularization, Physiologic; D000072483:Placenta Growth Factor; D011257:Pregnancy Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D033581:Stem Cell Transplantation / Q000379:methods*; D013919:Thromboangiitis Obliterans / Q000473:pathology / Q000628:therapy; D023822:Tissue Engineering / Q000379:methods*; D016896:Treatment Outcome; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
C000608036:PGF protein, human; C000608037:Pgf protein, mouse; D011257:Pregnancy Proteins; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D000072483:Placenta Growth Factor