Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, NC, USA.
Exp Hematol. 2011 Mar;39(3):375-83, 383.e1-4. doi: 10.1016/j.exphem.2010.12.007. Epub 2010 Dec 22.
Autologous bone marrow (BM) cells with a faulty gene corrected by gene targeting could provide a powerful therapeutic option for patients with genetic blood diseases. Achieving this goal is hindered by the low abundance of therapeutically useful BM cells and the difficulty maintaining them in tissue culture long enough to complete gene targeting without differentiating. Our objective was to devise a simple long-term culture system, using unfractioned BM cells, that maintains and expands therapeutically useful cells for ≥4 weeks.
From 2 to 60 million BM cells from wild-type (WT) mice or from mice carrying a truncated erythropoietin receptor transgene were plated with or without irradiated fetal-liver-derived AFT024 stromal cells in 25-cm(2) culture flasks. Four-week-cultured cells were analyzed and transplanted into sublethally irradiated thalassemic mice (1 million cells/mouse).
After 4 weeks, cultures with AFT024 cells had extensive "cobblestone" areas. Optimum expansion of Sca-1-positive cells was 5.5-fold with 20 × 10(6) WT cells/flask and 27-fold with 2 × 10(6) truncated erythropoietin receptor transgene cells. More than 85% of thalassemic mice transplanted with either type of cells had almost complete reversal of their thalassemic phenotype for at least 6 months, including blood smear dysmorphology, reticulocytosis, high ferritin plasma levels, and hepatic/renal hemosiderosis.
When plated at high cell densities on irradiated fetal-liver-derived stromal cells, BM cells from WT mice maintain their therapeutic potential for 4 weeks in culture, which is sufficient time for correction of a faulty gene by targeting.
通过基因靶向技术纠正有缺陷基因的自体骨髓（BM）细胞可为遗传性血液疾病患者提供一种强有力的治疗选择。然而，这一目标受到治疗性有用 BM 细胞数量稀少以及难以在组织培养中维持足够长时间以完成基因靶向而不分化的阻碍。我们的目标是设计一种简单的长期培养系统，使用未分离的 BM 细胞，维持和扩增治疗性有用细胞≥4 周。
从野生型（WT）小鼠或携带截断的促红细胞生成素受体转基因的小鼠中，将 2 至 6000 万个 BM 细胞与或不与辐照的胎肝衍生的 AFT024 基质细胞一起铺板于 25cm(2)培养瓶中。对培养 4 周的细胞进行分析，并移植到亚致死性照射的地中海贫血小鼠（100 万个细胞/只）中。
4 周后，AFT024 细胞培养物具有广泛的“鹅卵石”区域。用 20×10(6) WT 细胞/瓶培养物可使 Sca-1 阳性细胞最佳扩增 5.5 倍，用 2×10(6)截断的促红细胞生成素受体转基因细胞可扩增 27 倍。用这两种类型的细胞移植的地中海贫血小鼠中，超过 85%的小鼠的地中海贫血表型至少在 6 个月内几乎完全逆转，包括血液涂片形态异常、网织红细胞增多、高铁蛋白血浆水平升高和肝/肾含铁血黄素沉着症。
当在辐照的胎肝衍生基质细胞上以高细胞密度铺板时，WT 小鼠的 BM 细胞在培养中保持其治疗潜力 4 周，这足以完成基因靶向纠正有缺陷基因。
