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.
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.
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DOI：
10.1016/j.yjmcc.2010.04.001
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发表时间：
2010-09
影响因子：
5
通讯作者：
M. Oda;K. Toba;T. Ozawa;Kiminori Kato;Takao Yanagawa;Noboru Ikarashi;Tsugumi Takayama;Tomoyasu Suzuki;H. Hanawa;I. Fuse;Kou Nakata;M. Narita;Masuhiro Takahashi;Y. Aizawa
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文献类型：
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作者：
M. Oda;K. Toba;T. Ozawa;Kiminori Kato;Takao Yanagawa;Noboru Ikarashi;Tsugumi Takayama;Tomoyasu Suzuki;H. Hanawa;I. Fuse;Kou Nakata;M. Narita;Masuhiro Takahashi;Y. Aizawa
通过骨髓单个核细胞植入（BMI）的血管生成疗法已经被利用。我们发现红系细胞在BMI引起的血管生成中起着重要作用。然后，我们试图建立一种新的细胞疗法，通过植入从造血干细胞/前体细胞培养的离体扩增的未成熟成红细胞。从人骨髓中纯化未成熟至成熟的成红细胞，并分析mRNA表达。未成熟红系细胞强表达的VEGF和PLGF随红系成熟而减少。为了扩增非常不成熟的红系细胞，我们建立了两步培养系统，即，在Flt-3L、SCF和TPO存在下培养骨髓细胞7天，并在SCF、IGF-1和EPO存在下再培养细胞7天。在小鼠肢体缺血模型中，体外扩增细胞移植的体内血管生成作用强于BMI。三名患有严重慢性下肢缺血并伴有Burger病或胶原性动脉炎的患者参加了通过移植离体扩增的未成熟红系细胞进行的新型细胞疗法的试点临床试验。在临床试验中，大多数临床症状如静息痛和皮肤溃疡在4周内得到改善，并且在一年的随访中没有复发。在任何患者中均未观察到不良事件。此外，这种新的细胞疗法只需要少量的骨髓收集。需要进一步招募慢性严重下肢缺血患者，以确认这种新型细胞疗法的疗效和安全性，并估计必要的骨髓穿刺量。
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.