Towards an Optimized Culture Medium for the Generation of Mouse Induced Pluripotent Stem Cells

Abstract

Generation of induced pluripotent stem cells from somatic cells using defined factors has potential relevant applications in regenerative medicine and biology. However, this promising technology remains inefficient and time consuming. We have devised a serum free culture medium termed iSF1 that facilitates the generation of mouse induced pluripotent stem cells. This optimization of the culture medium is sensitive to the presence of Myc in the reprogramming factors. Moreover, we could reprogram meningeal cells using only two factors Oct4/Klf4. Therefore, iSF1 represents a basal medium that may be used for mechanistic studies and testing new reprogramming approaches.The discovery that four transcription factors Oct4, Sox2, Klf4, and Myc can revert differentiated somatic cells to a pluripotent state resembling embryonic stem cells (ESCs) 3 (1-4) not only demonstrates the remarkable plasticity of the mammalian genome, but also offers a unique opportunity to investigate the mechanisms associated with cell fate determination at the molecular level (5, 6). Besides, human induced pluripotent stem cells (iPSCs) overcome ethical issues associated with human ESCs and the risk of immune rejection (7,8). However, the reprogramming process remains largely an art form, with low efficiency and inconsistency among different experimental settings (1,3,4,6). Nowadays, the mouse system remains the model in which many mechanistic/technical breakthroughs are achieved. Therefore, albeit more robust than the human setting, it is important to improve mouse iPSC generation to achieve a better understanding of nuclear reprogramming. Here, we report an optimized method to generate mouse iPSCs consistently with high efficiency that is potentially useful for multiple applications including screening for small molecules, dissection of molecular mechanisms, and testing of new methods. EXPERIMENTAL PROCEDURESCell Culture-Mouse embryonic fibroblasts (MEFs) were derived from day 13.5 embryos (e13.5) hemizygous for the Oct4-GFP transgenic allele (9, 10) and Rosa26 allele and were maintained in fibroblast medium: DMEM supplemented with 10% fetal bovine serum (FBS), L-glutamine, and non-essential amino acid. iPSCs and ESCs were routinely expanded on MEF feeder layers (MEFs inactivated with mitomycin C) in both FBS-containing medium (mES) or KSR medium. mES medium consisted of DMEM supplemented with 15% FBS, L-glutamine, NEAA, sodium pyruvate, penicillin/streptomycin, ␤-mercaptoethanol, and 1000 units/ml leukemia inhibitory factor (Millipore). KSR medium consisted of knock-out DMEM supplemented with 15% knock-out Serum Replacement (SR), L-glutamine, NEAA, penicillin/streptomycin, ␤-mercaptoethanol, and 1000 units/ml LIF. fSF1 consisted of DMEM/F12 supplemented with 10% SR, 1/200 N2, L-glutamine, NEAA, penicillin/streptomycin, 1000 units/ml LIF, and 5 ng/ml basic FGF. iSF1 was like fSF1 but contained high glucose DMEM. Unless otherwise indicated, all reagents were purchased from Invitrogen. The FBS lot number was 709778, which was used in our previo...

BMPs functionally replace Klf4 and support efficient reprogramming of mouse fibroblasts by Oct4 alone

“…By contrast, OS-mediated reprogramming was reported only with p53-null fibroblasts [14]. We previously reported that OKS-mediated reprogramming can be enhanced by the optimized serum-free medium iSF1 [19]. Further optimization led to improved efficiency of OKS-mediated reprogramming and we found that iPSCs can be generated by either OK or OS with very low efficiency (Supplementary information, Figure S1A) in a newly formulated medium iCD1.…”

“…However, the function of each factor employed in reprogramming is poorly understood and at least two factors are required (including ectopic and endogenous expression) in the literature [7,[12][13][14][15][16]. Among the four Yamanaka factors, c-Myc was proven dispensable and can be replaced by various methods [17][18][19][20]. Oct4, Sox2 and Klf4 are currently considered as indispensable factors, as replacement of them would greatly reduce reprogramming efficiency.…”

NuRD Blocks Reprogramming of Mouse Somatic Cells into Pluripotent Stem Cells

“…iPSC formation requires Sox2 both in ESC culture medium and in iSF1 medium containing vitamin C and bFGF [17,18]. Knockdown of Mbd3 in iSF1 medium did not only enhance OSKM-mediated iPSC formation, but also mediated the generation of Oct4-GFP-positive clones in the absence of Sox2 (Fig.…”

Generation of induced pluripotent mouse stem cells in an indirect co-culture system

“…Two micrograms of total RNA was used to synthesize first-strand cDNA using the M-MLV reverse transcriptase (Promega, Madison, WI, USA) and oligo dT(18). Conventional PCRs were performed with the primer sequences shown in Table 1 (Chen et al, 2009(Chen et al, , 2010. The expression levels of Nanog and SSEA-1 were assessed using FastStart Universal SYBR Green Master (ROX; Roche Diagnostics, Rotkreuz, Switzerland) and analyzed with an ABI7300 fluorescence quantitative PCR instrument.…”

“…The cells were cultured initially in MEF medium. After infection, the medium was gradually replaced by ESC medium supplemented with 50 μg/mL vitamin C (Sigma-Aldrich Corporation, St. Louis, MO, USA) using a 3-day stepwise process to enhance induction efficiency (Chen et al, 2010;Esteban et al, 2010).…”