Abstract
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.
Highlights
Human somatic cells can be reprogrammed into induced pluripotent stem cells by introduction of transcription factors such as Oct3/4, Sox2, Klf4 and c-Myc [1]
We examined whether hESF9 medium and each of three ECM -coated surfaces could be used for Induced Pluripotent Stem (iPS) cell derivation
Human induced pluripotent stem cells (iPSCs) generated under hESF9 and cultured in hESF9T (DP-F-iPS-CL8 passage 38, DP-F-iPS-CL4 passage 38, DP-F-iPSCL16 passage 33) were seeded in a 24-well plate coated with fibronectin and counted every 24 hr
Summary
Human somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by introduction of transcription factors such as Oct3/4, Sox, Klf and c-Myc [1]. Embryonic stem cells (ESCs) and human iPSCs (hiPSCs) can proliferate without limit and yet maintain the potential to generate derivatives of all three germ layers. These properties make them useful for understanding the basic biology of the human body, for drug discovery and testing, and for transplantation therapies. The inclusion of uncharacterized animal protein supplements makes culture conditions more complex with increased variability in nutrients and factors that contribute to cell growth and the maintenance of pluripotency. There is unavoidable variability in using live cells as feeders, which may affect reprogramming steps. For these reasons defined culture conditions without feeder cells are desirable. As we show below, hiPSCs cultured in serum-free and feeder-free conditions in the absence of exogenous TGF-b1 lose pluripotency with passaging over time
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