Generation of Human Induced Pluripotent Stem (iPS) Cells in Serum- and Feeder-Free Defined Culture and TGF-β1 Regulation of Pluripotency

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.

Induced Pleuripotent Stem Cells: A Pioneer in Therapy

“…Together with mTeSR™1 or TeSR™2, mFreSR™ provides researchers with complete media solutions for maintaining high quality pluripotent cultures and eliminates the use of feeders and serum to reduce variability in experiments. 8 ES cells cryopreserved in mFreSR™ have thawing efficiencies 5 to 10 fold higher than conventional thawing methods using serum.…”

Fetal bovine serum (FBS): Past – present – future

“…Both hESCs and hiPSCs are generally maintained on inactivated mouse or human embryonic fibroblasts (feeder cells) or under feeder-free conditions (on extracellular matrices) in media supplemented with proprietary replacements as alternatives to FBS, in an effort to better control the phenotype of the cells (i.e., stem-ness maintenance, pluripotency, and differentiation towards a desired cell lineage). Indeed, given the presence of undefined or unknown components in FBS, serum-free culture systems now have been optimized to avoid the possible effects elicited by undefined differentiating growth factors and the risk of contaminations from pathogens potentially present in animal sera (e.g., mycoplasma, viruses, and prions) (Pistollato et al, 2012;Yamasaki et al, 2014).…”

Generation of a MLL-AF9-specific stem cell model of acute monocytic leukemia

“…AML-M5-iPSCs were maintained in human embryonic stem cell (hESC) medium on irradiated feeder cells as previously described. [10] For feeder dependent culture, AML-M5-iPSCs were maintained in feeder-free culture for at least three passages prior to three germ layers differentiation assay. For feeder-independent iPSC cell culture, plates were coated with Geltrex matrix (Invitrogen) and mTeSR TM 1 medium (Stemcell Technologies, Vancouver, Canada) was used.…”

“…[7] IPSC has been generated from many cell types, including cancer cells. [8][9][10] A reprogramed human pancreatic carcinoma cells has demonstrated the ability to study cancer progression using live cells. [11] This cancer-derived iPSC has provided precious disease models that mocks the diseases exactly and could be used for studying cancer development, elucidating oncogenes-associated mechanisms and potential drug discovery.…”