Brandt J E, Bartholomew A M, Fortman J D, Nelson M C, Bruno E, Chen L M, Turian J V, Davis T A, Chute J P, Hoffman R
Department of Medicine and the Biological Resources Laboratory, University of Illinois at Chicago, Chicago, IL 60607, USA.
Blood. 1999 Jul 1;94(1):106-13.
Hematopoietic stem cell (HSC) self-renewal in vitro has been reported to result in a diminished proliferative capacity or acquisition of a homing defect that might compromise marrow repopulation. Our group has demonstrated that human HSC expanded ex vivo in the presence of porcine microvascular endothelial cells (PMVEC) retain the capacity to competitively repopulate human bone fragments implanted in severe combined immunodeficiency (SCID) mice. To further test the marrow repopulating capacity of expanded stem cells, our laboratory has established a myeloablative, fractionated total body irradiation conditioning protocol for autologous marrow transplantation in baboons. A control animal, which received no transplant, as well as two animals, which received a suboptimal number of marrow mononuclear cells, died 37, 43, and 59 days postirradiation, respectively. Immunomagnetically selected CD34(+) marrow cells from two baboons were placed in PMVEC coculture with exogenous human cytokines. After 10 days of expansion, the grafts represented a 14-fold to 22-fold increase in cell number, a 4-fold to 5-fold expansion of CD34(+) cells, a 3-fold to 4-fold increase of colony-forming unit-granulocyte-macrophage (CFU-GM), and a 12-fold to 17-fold increase of cobblestone area-forming cells (CAFC) over input. Both baboons became transfusion independent by day 23 posttransplant and achieved absolute neutrophil count (ANC) >500/microL by day 25 +/- 1 and platelets >20,000/microL by day 29 +/- 2. This hematopoietic recovery was delayed in comparison to two animals that received either a graft consisting of freshly isolated, unexpanded CD34(+) cells or 175 x 10(6)/kg unfractionated marrow mononuclear cells. Analysis of the proliferative status of cells in PMVEC expansion cultures demonstrated that by 10 days, 99.8% of CD34(+) cells present in the cultures had undergone cycling, and that the population of cells expressing a CD34(+) CD38(-) phenotype in the cultures was also the result of active cell division. These data indicate that isolated bone marrow CD34(+) cells may undergo cell division during ex vivo expansion in the presence of endothelial cells to provide a graft capable of rescuing a myeloablated autologous host.
据报道，造血干细胞（HSC）在体外自我更新会导致增殖能力下降或出现归巢缺陷，这可能会影响骨髓再填充。我们的研究小组已经证明，在猪微血管内皮细胞（PMVEC）存在的情况下离体扩增的人HSC保留了竞争性再填充植入重症联合免疫缺陷（SCID）小鼠体内的人骨碎片的能力。为了进一步测试扩增干细胞的骨髓再填充能力，我们实验室建立了一种用于狒狒自体骨髓移植的清髓性、分次全身照射预处理方案。一只未接受移植的对照动物以及两只接受次优数量骨髓单个核细胞的动物分别在照射后37、43和59天死亡。从两只狒狒中免疫磁珠分选的CD34(+)骨髓细胞与外源性人细胞因子一起置于PMVEC共培养体系中。扩增10天后，移植物的细胞数量增加了14倍至22倍，CD34(+)细胞扩增了4倍至5倍，集落形成单位-粒细胞-巨噬细胞（CFU-GM）增加了3倍至4倍，鹅卵石区域形成细胞（CAFC）比输入细胞增加了12倍至17倍。两只狒狒在移植后第23天不再依赖输血，在第25±1天绝对中性粒细胞计数（ANC）>500/μL，在第29±2天血小板>20,000/μL。与接受由新鲜分离的、未扩增的CD34(+)细胞组成的移植物或175×10(6)/kg未分级骨髓单个核细胞的两只动物相比，这种造血恢复有所延迟。对PMVEC扩增培养物中细胞增殖状态的分析表明，到第10天时，培养物中存在的CD34(+)细胞中有99.8%经历了细胞周期循环，并且培养物中表达CD34(+)CD38(-)表型的细胞群体也是活跃细胞分裂的结果。这些数据表明，分离的骨髓CD34(+)细胞在体外与内皮细胞共培养扩增过程中可能会发生细胞分裂，从而提供一种能够挽救清髓性自体宿主的移植物。
