Colby C, Chang Q, Fuchimoto Y, Ferrara V, Murphy M, Sackstein R, Spitzer T R, White-Scharf M E, Sachs D H
Transplantation Biology Research Center, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston 02129, USA.
Transplantation. 2000 Jan 15;69(1):135-40. doi: 10.1097/00007890-200001150-00023.
Because of the relative ease of acquisition, increased yield, and improved engraftment characteristics, mobilized peripheral blood progenitor (stem) cells (PBSCs) have recently become the preferred source for hematopoietic stem cell transplantation. In our laboratory, procurement of a megadose of PBSCs is necessary for on-going studies evaluating non-myelosuppressive transplant regimens for the induction of mixed chimerism and allograft tolerance. To exploit hematopoietic growth factor synergy, we have sought to combine growth factors with proven utility to improve PBSC mobilization and maximize our PBSC procurement through an automated collection procedure.
Mobilization characteristics of PBSCs were determined in 2-5-month-old miniature swine. Animals received either swine recombinant stem cell factor (pSCF, 100 microg/kg) and swine recombinant interleukin 3 (pIL-3, 100 microg/kg), administered intramuscularly for 8 days, or pSCF, pIL-3, and human recombinant granulocyte-colony stimulating factor (hG-CSF), at 10 microg/kg. Leukapheresis was performed beginning on day 5 of cytokine treatment and continued daily for 3 days.
Collection of PBSCs from cytokine-mobilized animals via an automated leukapheresis procedure demonstrated a 10-fold increase in the number of total nucleated cells (TNC) (20-30 x 10(10) TNC) compared to bone marrow harvesting (2-3 x 10(10) total TNC). A more rapid rise in white blood cells (WBCs) was seen after administration of all three cytokines compared to pSCF and pIL-3 alone. An increase in colony-forming unit granulocyte-macrophage frequency measured daily from peripheral blood during cytokine treatment, was seen with the addition of hG-CSF to pSCF/pIL-3 correlating well with the rise in WBCs. Similarly, the addition of hG-CSF demonstrated a notable increase in the median progenitor cell yield from the 3-day leukapheresis procedure. Cytokine-mobilized PBSCs were capable of hematopoietic reconstitution. PBSCs mobilized with pSCF/pIL-3 were infused into an SLA-matched recipient conditioned with cyclophosphamide (50 mg/kg) and total body irradiation 1150 cGy. Neutrophil and platelet engraftment occurred on days 5 and 7, respectively, with minimal evidence of graft-versus-host disease. Complete donor chimerism has been demonstrated 331 days after transplant.
Our preliminary results show that in this well-defined miniature swine model, recombinant swine cytokine combinations (pSCF, pIL-3 with or without hG-CSF) successfully mobilize a high yield of progenitor cells for allogeneic transplantation. Furthermore, these cytokine-mobilized PBSCs demonstrate the potential to reconstitute hematopoiesis and provide long-term engraftment in miniature swine.
由于获取相对容易、产量增加以及植入特性改善，动员外周血祖（干）细胞（PBSCs）最近已成为造血干细胞移植的首选来源。在我们实验室，为正在进行的评估非骨髓抑制性移植方案以诱导混合嵌合体和同种异体移植耐受的研究，获取大剂量的PBSCs是必要的。为利用造血生长因子协同作用，我们试图将已证实有实用价值的生长因子联合起来，以改善PBSC动员，并通过自动化采集程序使我们的PBSC获取量最大化。
在2至5月龄的小型猪中测定PBSCs的动员特性。动物接受猪重组干细胞因子（pSCF，100μg/kg）和猪重组白细胞介素3（pIL-3，100μg/kg），肌肉注射8天，或接受pSCF、pIL-3和人重组粒细胞集落刺激因子（hG-CSF），剂量为10μg/kg。从细胞因子治疗第5天开始进行白细胞分离术，并持续3天每天进行。
通过自动化白细胞分离术从细胞因子动员的动物中采集PBSCs，与骨髓采集（2 - 3×10¹⁰个总有核细胞）相比，总核细胞（TNC）数量增加了10倍（20 - 30×10¹⁰个TNC）。与单独使用pSCF和pIL-3相比，给予所有三种细胞因子后白细胞（WBC）上升更快。在细胞因子治疗期间，每天从外周血中测量的集落形成单位粒细胞 - 巨噬细胞频率增加，在pSCF/pIL-3中添加hG-CSF与WBC上升密切相关。同样，添加hG-CSF显示3天白细胞分离术过程中祖细胞产量中位数显著增加。细胞因子动员的PBSCs能够进行造血重建。用pSCF/pIL-3动员的PBSCs被输注到用环磷酰胺（50mg/kg）和全身照射1150cGy预处理的SLA匹配受体中。中性粒细胞和血小板分别在第5天和第7天植入，移植物抗宿主病的证据极少。移植后331天已证明完全供体嵌合体。
我们的初步结果表明，在这个明确的小型猪模型中，重组猪细胞因子组合（pSCF、pIL-3加或不加hG-CSF）成功动员了高产量的祖细胞用于同种异体移植。此外，这些细胞因子动员的PBSCs显示出在小型猪中重建造血和提供长期植入的潜力。
