Generation of Human Induced Pluripotent Stem (Ips) Cells in Serum- and Feeder-Free Defined Culture and TGF-Β1 Regulation of Pluripotency

Human Embryonic Stem cells (hESCs) and human induced Pluripotent Stem cells (hiPSCs) are commonly maintained on inactivated mouse embryonic fibroblast as feeder cells in medium supplemented with FBS or proprietary replacements. Use of culture medium containing undefined or unknown components has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. In addition, there is no consensus as to the optimal formulation, or the nature of the cytokine requirements of the cells to promote their self-renewal and inhibit their differentiation. In this study, we successfully generated hiPSCs from human dental pulp cells (DPCs) using Yamanaka's factors (Oct3/4, Sox2, Klf4, and c-Myc) with retroviral vectors in serum- and feeder-free defined culture conditions. These hiPSCs retained the property of self-renewal as evaluated by the expression of self-renewal marker genes and proteins, morphology, cell growth rates, and pluripotency evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo. In this study, we found that TGF-β1 increased the expression levels of pluripotency markers in a dose-dependent manner. However, increasing doses of TGF-β1 suppressed the growth rate of hiPSCs cultured under the defined conditions. Furthermore, over short time periods the hiPSCs cultured in hESF9 or hESF9T exhibited similar morphology, but hiPSCs maintained in hESF9 could not survive beyond 30 passages. This result clearly confirmed that hiPSCs cultured in hESF9 medium absolutely required TGF-β1 to maintain pluripotency. This simple serum-free adherent monoculture system will allow us to elucidate the cell responses to growth factors under defined conditions and can eliminate the risk might be brought by undefined pathogens.

人类胚胎干细胞（Human Embryonic Stem cells, hESCs）与人类诱导多能干细胞（human induced Pluripotent Stem cells, hiPSCs）通常以灭活的小鼠胚胎成纤维细胞作为饲养层细胞，在添加胎牛血清（FBS）或专有替代物的培养基中进行培养。使用含有未定义或未知成分的培养基，因对细胞响应分化性生长因子的机制认知相对匮乏，限制了多能细胞应用的发展。此外，在促进细胞自我更新、抑制分化所需的最优配方，以及细胞因子需求的本质方面，目前尚未达成共识。

本研究在无血清、无饲养层的限定性培养条件下，利用逆转录病毒载体递送山中伸弥因子（Oct3/4、Sox2、Klf4及c-Myc），成功从人类牙髓细胞（human dental pulp cells, DPCs）中诱导生成了hiPSCs。通过自我更新标志物基因与蛋白的表达、细胞形态、生长速率对其自我更新特性进行评估；通过体外及体内分化为三个原始胚层的衍生物对其多能性进行验证，结果显示这些hiPSCs保留了多能干细胞的特性。

本研究发现，转化生长因子-β1（TGF-β1）以剂量依赖方式上调多能性标志物的表达水平。但随着TGF-β1剂量升高，限定培养条件下培养的hiPSCs生长速率受到抑制。此外，短期培养时，在hESF9或hESF9T培养基中培养的hiPSCs形态相似，但在hESF9中维持培养的hiPSCs无法传代超过30次。该结果明确证实：在hESF9培养基中培养的hiPSCs绝对需要TGF-β1以维持多能性。

这一简便的无血清贴壁单层培养系统，将有助于我们在限定条件下阐明细胞对生长因子的响应，同时可消除未定义病原体可能带来的潜在风险。