Enhanced engraftment of mesenchymal stem cells in a cutaneous wound model by culture in allogenic species-specific serum and administration in fibrin constructs
Enhanced engraftment of mesenchymal stem cells in a cutaneous wound model by culture in allogenic species-specific serum and administration in fibrin constructs
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DOI：
10.1634/stemcells.2005-0612
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发表时间：
2006-10-01
期刊：
影响因子：
5.2
通讯作者：
Spees, Jeffrey L.
中科院分区：
文献类型：
--
作者：
Gregory, Carl A.;Reyes, Emigdio;Spees, Jeffrey L.
人骨髓间充质干细胞(HMSCs)也被称为多能基质细胞，目前正被应用于骨病、移植物抗宿主病和心肌梗死的临床试验。然而，hMSCs的标准生长培养液中含有10%-20%的胎牛血清(FCS)，而且FCS对啮齿动物和人类都具有很强的免疫原性。此前，我们曾报道，通过一项基于荧光的灵敏分析，7-30毫克的内化FCS与10(8)hMSCs相关，这一剂量可能是大多数治疗所需要的。我们还发现，在含有20%自体成人血清(AHS)或自体10%AHS加生长因子(AHS(+))的培养液中短暂培养可使污染减少99.9%以上。我们现在已经对培养条件进行了广泛的鉴定，并表明使用异源20%AHS或异源10%AHS(+)扩增hMSC是可能的。FCS的摄取是一个活跃的过程，即使在低血清条件下(2%FCS)也能使污染集中在细胞内，但通过将细胞培养在含AHS的培养液中可以积极地转移污染。大鼠骨髓间充质干细胞(RMSCs)可在类似条件下用添加异种成年大鼠血清(ARS(+))进行扩增。在FCS中扩增后，再用ARS(+)培养8d，将rMSCs包裹在纤维蛋白中，然后皮下移植，显著提高了rMSCs在体内的活力。我们的结果有可能极大地改善使用hMSCs的细胞和遗传治疗。
Human mesenchymal stem cells (hMSCs), also referred to as multipotent stromal cells, are currently being applied in clinical trials for bone diseases, graft versus host disease, and myocardial infarction. However, the standard growth medium for hMSCs contains 10%-20% fetal calf serum (FCS), and FCS is strongly immunogenic in both rodents and humans. Previously, we reported that by a sensitive fluorescence-based assay, 7-30 mg of internalized FCS is associated with 10(8) hMSCs, a dosage that will probably be needed for most therapies. We also found that a brief culture in medium containing autologous 20% adult human serum (AHS) or autologous 10% AHS supplemented with growth factors (AHS(+)) reduced the contamination by more than 99.9%. We have now extensively characterized the culture conditions and shown that hMSC expansion is possible using heterologous 20% AHS or heterologous 10% AHS(+). The uptake of FCS is an active process that acts to concentrate contamination in the cells even under low serum conditions (2% FCS) but can be actively displaced by incubation of the cells in medium with AHS. Rat MSCs (rMSCs) can be expanded under similar conditions using supplemented heterologous adult rat serum (ARS(+)). After expansion in FCS, a further 8 days of culture with ARS(+) significantly improves the viability of the rMSCs in vivo after encapsulation in fibrin followed by subcutaneous implantation in rats. Our results have the potential to dramatically improve cellular and genetic therapies using hMSCs.