Basic and clinical research for the application of "Ex vivo expanded erythroblast transplantation"

自体骨髓细胞移植(BMI)已被用于治疗肢体缺血，但其血管生成机制尚不清楚。治疗通常需要500到1000毫升的骨髓采集。本研究的目的是利用体外细胞培养技术开发一种复杂的细胞治疗方法。我们期望该疗法的血管生成与骨髓组织中的结构性血管生成相似。几种血管生成因子是由红细胞强烈产生的。在体外，HUVEC的血管生成是由红系集落进行的，而不是由人的血液干细胞制备的髓系集落进行的。在小鼠肢体缺血模型中，骨髓中的红系细胞对血管生成起着至关重要的作用。在体内模型中，同时给予促红细胞生成素和BMI可能通过对骨髓外红系细胞的存活作用来促进血管生成。其次，我们开发了一种从骨髓干细胞体外扩增红系细胞的系统。结果，只有1毫升的骨髓足以产生与500到1,000毫升新鲜采集的骨髓完全相容的血管生成因子产生细胞。体外扩增的红细胞在小鼠肢体缺血模型中也表现出较强的血管生成作用。“体外扩增的自体红细胞移植治疗肢体缺血患者(EVEETA)”的I/II期试验已获得伦理委员会和医院IRB的批准。临床使用的细胞培养将在医院GMP级细胞处理中心(CPC)实施。

Autologous bone marrow cell implantation (BMI) has been utilized for the treatment of limb ischemia, however the mechanism of angiogenesis by the therapy is not well known. As large as 500 to 1,000 mL of bone marrow collection is usually required for the treatment. The aim of this study was to develop a sophisticated cell therapy by using ex vivo cell culture technique.In the beginning, we studied the mechanism of angiogenesis by BMI. We expected that the angiogenesis by the therapy mimicked the constitutional angiogenesis in the bone marrow tissue. Several angiogenic factors were strongly produced by erythroblasts. In vitro angiogenesis of HUVEC was conducted by erythroid colonies but not by myeloid colonies prepared from human blood stem cells. Erythroid cells in bone marrow played essential roles for angiogenesis in mouse limb ischemia model. In the in vivo model, simultaneous administration of erythropoietin with BMI enhanced angiogenesis presumably through surviving effect on erythroid cells extramedullary.Next, we developed an ex vivo expansion system of erythroid cells from bone marrow stem cells. As the results, only 1 mL of bone marrow was enough to produce angiogenic factor producing cells that were fully compatible with 500 to 1,000 mL of freshly harvested bone marrow. The ex vivo expanded erythroblasts also expressed strong angiogenic effect in the mouse limb ischemic model.A phase I/II trial of "Ex vivo expanded autologous erythroblast transplantation for the treatment of the patients with limb ischemia (EVEETA)" has been approved by the ethical committee and the IRB in the hospital. Cell culture for the clinical use will be enforced in the GMP-grade Cell Processing Center (CPC) in the hospital.