FACS-purified myoblasts producing controlled VEGF levels induce safe and stable angiogenesis in chronic hind limb ischemia.
FACS-purified myoblasts producing controlled VEGF levels induce safe and stable angiogenesis in chronic hind limb ischemia.
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DOI：
10.1111/j.1582-4934.2011.01308.x
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发表时间：
2012-01
影响因子：
5.3
通讯作者：
Banfi A
中科院分区：
文献类型：
--
作者：
Wolff T;Mujagic E;Gianni-Barrera R;Fueglistaler P;Helmrich U;Misteli H;Gurke L;Heberer M;Banfi A
我们最近开发了一种方法来控制在体内分布的血管内皮生长因子（VEGF）的高通量流式细胞仪激活细胞分选（FACS）纯化转导的祖细胞，使他们均匀地表达特定的VEGF水平。在这里，我们研究了这种方法在裸大鼠慢性后肢缺血中的长期安全性。将原代成肌细胞转导以共表达大鼠VEGF-A164（rVEGF）和截短的大鼠CD 8a，后者用作FACS可定量的表面标志物。基于表达所需VEGF水平的参考克隆群体的CD 8荧光，从包含具有非常异质的VEGF水平的细胞的原代群体中分选产生相似VEGF水平的细胞。缺血诱导后1周，将12 × 106个细胞植入大腿肌肉中。未分选的成肌细胞引起血管瘤样结构，而纯化的细胞仅诱导3个月后稳定的正常毛细血管。移植区域的血管密度增加了一倍，但只有约0.1%的肌肉体积显示细胞移植，这解释了为什么没有观察到总血流量增加。总之，使用FACS纯化的成肌细胞可以逐个细胞控制VEGF的表达水平，这确保了在慢性缺血模型中的长期安全性。基于这些结果，在临床应用之前，需要确定达到疗效所需的植入细胞总数。
We recently developed a method to control the in vivo distribution of vascular endothelial growth factor (VEGF) by high throughput Fluorescence-Activated Cell Sorting (FACS) purification of transduced progenitors such that they homogeneously express specific VEGF levels. Here we investigated the long-term safety of this method in chronic hind limb ischemia in nude rats. Primary myoblasts were transduced to co-express rat VEGF-A164 (rVEGF) and truncated ratCD8a, the latter serving as a FACS-quantifiable surface marker. Based on the CD8 fluorescence of a reference clonal population, which expressed the desired VEGF level, cells producing similar VEGF levels were sorted from the primary population, which contained cells with very heterogeneous VEGF levels. One week after ischemia induction, 12 × 106 cells were implanted in the thigh muscles. Unsorted myoblasts caused angioma-like structures, whereas purified cells only induced normal capillaries that were stable after 3 months. Vessel density was doubled in engrafted areas, but only approximately 0.1% of muscle volume showed cell engraftment, explaining why no increase in total blood flow was observed. In conclusion, the use of FACS-purified myoblasts granted the cell-by-cell control of VEGF expression levels, which ensured long-term safety in a model of chronic ischemia. Based on these results, the total number of implanted cells required to achieve efficacy will need to be determined before a clinical application.
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