Development of mesenchymal stem cell engineering for reconstructive therapy

脂肪组织是自体干细胞丰富的沉积位点，具有潜在的再生医学应用前景，而脂肪源性基质血管部分（SVF）是具有间充质、造血、神经源性、血管源性或上皮谱系分化潜力的自体干细胞的来源。通过在含成纤维细胞生长因子(FGF)-2的高、低血清培养基中培养SVF细胞，发现细胞在低血清培养基中生长更快，可无限传代。在低血清培养基中扩增的细胞表现出较高的间充质分化潜能。我们认为，由于血清中含有生长因子，高血清浓度不利于干细胞的自我更新。不同年龄患者的SVF细胞在高血清培养基中培养时，具有成骨潜能的干细胞在20岁以上就已经耗尽，而82岁患者的SVF细胞在低血清培养基中也可以扩增。由于人成人血清可以替代2%的胎牛血清，使人干细胞具有更好的增殖和分化能力，因此我们可以进行人源化体外培养，用于临床应用。通过我们的方法，从任何年龄的患者获得1 g皮下脂肪组织和200 ml血液，在3周内即可制备出10^9个细胞的自体干细胞群。

Adipose tissues are abundant deposition sites of autologous stem cells with potential application for regenerative medicine and adipose-derived stromal vascular fraction (SVF) is a source of autologous stem cells with differentiation potential along mesenchymal, hematopoietic, neurogenic, angiogenic or epithelial lineage. By culturing SVF cells in high- or low- serum medium containing fibroblast growth factor (FGF)-2, we found that the cells grew faster in low-serum medium and could be subcultured unlimitedly. Cells expanded in low-serum medium showed higher potential of mesenchymal differentiation. We suggest that high-serum concentration is unfavorable for self-renewing of stem cells due to growth factors contained in the serum. When SVF cells from patients at different ages were culture in high-serum medium, stem cells with osteogenic potential were depleted at ages above 20 years, whereas they could be expanded in low-serum medium even from 82 years old patient. Since human adult serum could substitute for 2% fetal bovine serum to give even better proliferation and differentiation, we could humanized ex vivo expansion culture of human stem cells for immediate clinical applications. By our method, autologous stem cell population corresponding to 10^9 cells can be prepared within 3 weeks after obtaining 1 g of subcutaneous adipose tissue and 200 ml of blood from patients at any age.