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. 2009 Aug 4;106(31):12759-64.
doi: 10.1073/pnas.0904825106. Epub 2009 Jun 23.

Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation

Eirini P Papapetrou  1 , Mark J Tomishima, Stuart M Chambers, Yvonne Mica, Evan Reed, Jayanthi Menon, Viviane Tabar, Qianxing Mo, Lorenz Studer, Michel Sadelain
Affiliations

Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation

Eirini P Papapetrou et al. Proc Natl Acad Sci U S A. .

Abstract

Human-induced pluripotent stem cells (hiPSCs) are generated from somatic cells by ectopic expression of the 4 reprogramming factors (RFs) Oct-4, Sox2, Klf4, and c-Myc. To better define the stoichiometric requirements and dynamic expression patterns required for successful hiPSC induction, we generated 4 bicistronic lentiviral vectors encoding the 4 RFs co-expressed with discernable fluorescent proteins. Using this system, we define the optimal stoichiometry of RF expression to be highly sensitive to Oct4 dosage, and we demonstrate the impact that variations in the relative ratios of RF expression exert on the efficiency of hiPSC induction. Monitoring of expression of each individual RF in single cells during the course of reprogramming revealed that vector silencing follows acquisition of pluripotent cell markers. Pronounced lentiviral vector silencing was a characteristic of successfully reprogrammed hiPSC clones, but lack of complete silencing did not hinder hiPSC induction, maintenance, or directed differentiation. The vector system described here presents a powerful tool for mechanistic studies of reprogramming and the optimization of hiPSC generation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
hiPSCs derived from bicistronic vectors co-expressing each RF linked to a fluorescent protein. (A) Schematic representation of the vectors used in this study. LTR, long terminal repeat. (B) hiPSC colony at day 11 after transduction. (C) Hematoxylin and eosin staining of histological sections of a teratoma derived from line iPS-27. From Left to Right: Low power image demonstrating areas of heterogeneous differentiation: neuroectoderm (black arrow), smooth muscle (blank arrow), and primitive myxoid tissue (arrowhead), 4×. Pigmented epithelial tissue compatible with retinal neuroectoderm, 40×. Intestinal like epithelium including goblet cells (endoderm), 40×. Smooth muscle tissue, 20×. Inset demonstrates a high power image of immature mesenchymal tissue, potentially cartilage (40×).
Fig. 2.
Fig. 2.
Stoichiometric requirements of factor expression. (A) Representative flow cytometry analysis of MRC-5 fibroblasts on day 6 after transduction with the 4 vectors at increasing MOI as indicated. Numbers within plots denote percentage of cells in the respective quadrants. Dot plots in the Right are gated on vexGFP-Oct4+/mCerulean-cMyc+ double positive cells in the corresponding Left. The orange squares in the Right and the numbers above denote quadruple positive cells. (B) Representative flow cytometry analysis of MRC-5 fibroblasts on day 6 after transduction with titrated relative vector amounts (0.3, 1, 3), as indicated. UT, untransduced. (C) MRC-5 fibroblasts were transduced with relative vector amounts shown below the bars. Bars, mean ± SEM from 5 independent experiments. Absolute efficiency was calculated as the number of Tra-1–81+ colonies per number of plated cells divided by the fraction of quadruple positive cells estimated by flow cytometry analysis (as exemplified in A) at day 5 after transduction. Relative efficiency was calculated by normalizing absolute efficiency to the reference group (vector dose 1:1:1:1) to facilitate comparison across independent experiments. (D) Effect of relative Oct4 vector amount (x axis) on reprogramming efficiency (y axis), calculated as detailed in C. Bars, mean ± SEM from 3 independent experiments.
Fig. 3.
Fig. 3.
Silencing of ectopic factor expression in hiPSC lines. MFI of each co-expressed fluorescent protein, in 38 reprogrammed (Tra-1–81+, red circles) and 30 non-reprogrammed (Tra-1–81-, blue squares) clones. Error bars, SEM.
Fig. 4.
Fig. 4.
Differentiation of hiPSC clones with incomplete RF silencing. Left, flow cytometry analysis of hiPS clones 72, 71, 62 and 50. Right, in vitro differentiation of the same clones in endoderm, mesoderm and ectoderm, followed by staining for Sox17, Brachyury, and Pax6, respectively.
Fig. 5.
Fig. 5.
Real-time simultaneous monitoring of vector-encoded RF expression and pluripotency marker induction. (A) Vector expression in Tra-1–81+ versus HLA-ABChigh cells on day 12 (Left) and 20 (Right) after transduction. (B) Vector silencing in Tra-1–81+ cells over time. (Day 0 denotes time of transduction.) Fold difference was estimated as ratio of MFI in the Tra-1–81+ cells to the MFI in HLA-ABChigh cells. (C) Appearance of pluripotency marker Tra-1–81 and downregulation of HLA-ABC during reprogramming. Shown is 1 of 3 independent time course experiments.
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