Tisdale J F, Hanazono Y, Sellers S E, Agricola B A, Metzger M E, Donahue R E, Dunbar C E
Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, MD 20892-1652, USA.
Blood. 1998 Aug 15;92(4):1131-41.
The possibility of primitive hematopoietic cell ex vivo expansion is of interest for both gene therapy and transplantation applications. The engraftment of autologous rhesus peripheral blood (PB) progenitors expanded 10 to 14 days were tracked in vivo using genetic marking. Stem cell factor (SCF)/granulocyte colony-stimulating factor (G-CSF)-mobilized and CD34-enriched PB cells were divided into two equal aliquots and transduced with one of two retroviral vectors carrying the neomycin-resistance gene (neo) for 4 days in the presence of interleukin-3 (IL-3), IL-6, and SCF in the first 5 animals, IL-3/IL-6/SCF/Flt-3 ligand (FLT) in 2 subsequent animals, or IL-3/IL-6/SCF/FLT plus an autologous stromal monolayer (STR) in the final 2. At the end of transduction period, one aliquot (nonexpanded) from each animal was frozen, whereas the other was expanded under the same conditions but without vector for a total of 14 days before freezing. After total body irradiation, both the nonexpanded and expanded transduced cells were reinfused. Despite 5- to 13-fold higher cell and colony-forming unit (CFU) doses from the expanded fraction of marked cells, there was greater short- and long-term marking from the nonexpanded cells in all animals. In animals receiving cells transduced and expanded in the presence of IL-3/IL-6/SCF/FLT, engraftment by the marked expanded cells was further diminished. This discrepancy was even more pronounced in the animals who received cells transduced and expanded in the presence of FLT and autologous stroma, with no marking detectable from the expanded cells. Despite lack of evidence for expansion of engrafting cells, we found that the addition of FLT and especially STR during the initial brief transduction period increased engraftment with marked cells into a clinically relevant range. Levels of marked progeny cells originating from the nonexpanded aliqouts were significantly higher than that seen in previous 4 animals receiving cells transduced in the presence of IL-3/IL-6/SCF, with levels of 10% to 20% confirmed by Southern blotting from the nonexpanded IL-3/IL-6/SCF/FLT/STR graft compared with 0.01% in the original IL-3/IL-6/SCF cohort. These results suggest that, although expansion of PB progenitors is feasible ex vivo, their contribution towards both short- and long-term engraftment is markedly impaired. However, a brief transduction in the presence of specific cytokines and stromal support allows engraftment with an encouraging number of retrovirally modified cells.
原始造血细胞体外扩增的可能性在基因治疗和移植应用中都备受关注。利用基因标记在体内追踪了自体恒河猴外周血（PB）祖细胞在扩增10至14天后的植入情况。将干细胞因子（SCF）/粒细胞集落刺激因子（G-CSF）动员的且富含CD34的PB细胞分成两个相等的等分试样，并在白细胞介素-3（IL-3）、IL-6和SCF存在的情况下，用携带新霉素抗性基因（neo）的两种逆转录病毒载体之一转导4天，前5只动物如此处理；随后的2只动物在IL-3/IL-6/SCF/Flt-3配体（FLT）存在的情况下进行转导；最后2只动物在IL-3/IL-6/SCF/FLT加上自体基质单层（STR）存在的情况下进行转导。在转导期结束时，将每只动物的一个等分试样（未扩增）冷冻，而另一个在相同条件下但无载体的情况下扩增总共14天，然后冷冻。全身照射后，将未扩增和扩增的转导细胞重新注入。尽管标记细胞的扩增部分的细胞和集落形成单位（CFU）剂量高出5至13倍，但在所有动物中，未扩增细胞的短期和长期标记都更多。在接受在IL-3/IL-6/SCF/FLT存在下转导和扩增的细胞的动物中，标记的扩增细胞的植入进一步减少。这种差异在接受在FLT和自体基质存在下转导和扩增的细胞的动物中更为明显，从扩增细胞中未检测到标记。尽管缺乏植入细胞扩增的证据，但我们发现，在最初短暂的转导期添加FLT，尤其是STR，可使标记细胞的植入增加到临床相关范围。来自未扩增等分试样的标记后代细胞水平显著高于之前4只接受在IL-3/IL-6/SCF存在下转导的细胞的动物，通过Southern印迹法证实，未扩增的IL-3/IL-6/SCF/FLT/STR移植的水平为10%至20%，而原始IL-3/IL-6/SCF组为0.01%。这些结果表明，虽然PB祖细胞的扩增在体外是可行的，但其对短期和长期植入的贡献明显受损。然而，在特定细胞因子和基质支持存在下进行短暂转导可使大量逆转录病毒修饰的细胞实现植入。
