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. 2019 Dec 5;25(6):737-753.e4.
doi: 10.1016/j.stem.2019.10.002. Epub 2019 Nov 7.

Excluding Oct4 from Yamanaka Cocktail Unleashes the Developmental Potential of iPSCs

Sergiy Velychko  1 , Kenjiro Adachi  1 , Kee-Pyo Kim  1 , Yanlin Hou  1 , Caitlin M MacCarthy  1 , Guangming Wu  2 , Hans R Schöler  3
Affiliations

Excluding Oct4 from Yamanaka Cocktail Unleashes the Developmental Potential of iPSCs

Sergiy Velychko et al. Cell Stem Cell. .

Abstract

Oct4 is widely considered the most important among the four Yamanaka reprogramming factors. Here, we show that the combination of Sox2, Klf4, and cMyc (SKM) suffices for reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs). Simultaneous induction of Sox2 and cMyc in fibroblasts triggers immediate retroviral silencing, which explains the discrepancy with previous studies that attempted but failed to generate iPSCs without Oct4 using retroviral vectors. SKM induction could partially activate the pluripotency network, even in Oct4-knockout fibroblasts. Importantly, reprogramming in the absence of exogenous Oct4 results in greatly improved developmental potential of iPSCs, determined by their ability to give rise to all-iPSC mice in the tetraploid complementation assay. Our data suggest that overexpression of Oct4 during reprogramming leads to off-target gene activation during reprogramming and epigenetic aberrations in resulting iPSCs and thereby bear major implications for further development and application of iPSC technology.

Keywords: Klf4; Myc; Oct4; Sox2; Yamanaka cocktail; developmental potential; induced pluripotent stem cells; reprogramming; retroviral silencing; seesaw model of pluripotency; tetraploid complementation; transcription factor.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Sox2, Klf4, and cMyc Can Reprogram to Pluripotency in the Absence of Exogenous POU Factor (A and C) Scheme of KSM and SKM polycistronic vectors derived from OKSM (A) and OSKM (C) reprogramming cassettes. (B and D) Generation of iPSCs with tetO-KSM (B) and SKM (D) vectors, respectively. Phase-contrast and fluorescence microscopy images of primary colonies and passaged clonal lines of KSM and SKM iPSCs (scale bars represent 250 μm). (E) PCR genotyping verifying tetO-KSM and tetO-SKM transgenes in KSM and SKM iPSC lines, respectively, while confirming the absence of Oct4 or Brn4 integration. (F) Bisulfite sequencing analysis of DNA methylation in Oct4, Nanog, and Col1a1 promoters in MEFs and KSM and SKM iPSC lines. (G) H&E staining of teratoma sections with representation of three germ layers (ectoderm: keratinizing epithelium; mesoderm: smooth muscles; endoderm: cuboidal and respiratory epithelium). (H) An adult chimeric mouse generated from SKM iPSC line. (I) Bright-field and GFP merged images of the gonads from E13.5 KSM and SKM iPSC chimeric embryos. (J) Schematic representation of the time course reprogramming experiment. (K) Time course reprogramming experiment of Oct4-GFP MEFs using polycistronic vectors. 103 transduced MEFs were plated on feeders and induced with dox for the indicated number of days. GFP+ colonies were counted on 10 dpi. Error bars represent SD; n = 3. (L) Western blot analysis of MEFs after transduction of polycistronic vectors, 1 dpi.
Figure 2
Figure 2
Highly Proliferative Cells Can Be Reprogrammed by Sox2 and Klf4 Alone (A) Reprogramming Oct4-GFP MEFs with dissected KSM and SKM cassettes. Different levels of transgene expression were achieved by induction with three concentrations of dox: 1 μg/mL (H); 50 ng/mL (M); and 10 ng/mL (L; see the tetO-mCherry panel, right). GFP+ colonies were counted after 7 and 14 dpi. Error bars represent SD; n = 3; scale bars represent 250 μm. (B) Bright-field and Oct4-GFP merged overview images of Sox1-KM and Sox2-KM iPSCs after 14 dpi (scale bars represent 1 mm). (C) Reprogramming of Oct4-GFP MEFs with Sox2-KM or Sox1-KM polycistronic cassettes in combination with Oct4, Gata4, or Gata6. Different level of transgene expression was achieved by induction with 1 μg/mL or 50 ng/mL of dox for 12 days. GFP+ colonies were counted on D14. Error bars represent SD; n = 3. (D) Reprogramming of SV40LT-immortalized Oct4-GFP MEFs by ectopic expression of Sox2-Klf4 bicistronic cassette in combination with Oct4, cMyc, Gata4, or Gata6. Error bars represent SD; n = 3. Statistical significance was calculated with Student’s t test. (E) Cell proliferation assay. 2,000 MEFs were transduced with indicated constructs in 96-well plates. The cells were harvested and counted after 0, 2, 4, and 6 dpi. SK sample was used as control for calculation of statistical significance. Error bars represent SD; n = 3. Statistical significance was calculated with Student’s t test.
Figure 3
Figure 3
Omitting Oct4 in the Reprogramming Cocktail Increases the Quality of iPSCs (A) Schematic representation of tetraploid complementation experiment. (B) All-iPSC pups generated by tetraploid complementation assay with SKM#1 iPSC line. 23 aggregates were transferred to 2 pseudopregnant CD-1 (white) females. (C) The total ratios of 4N-on versus 4N-off iPSC and ESC lines generated in this study compared to published data. (D) Percentage of 4N-aggregated embryos derived from tetO-OSKM, SKM/KSM iPSCs or ESCs that gave rise to full-term pups, pups that initiated breathing, pups that survived foster-nursing for at least 48 h, and those survived to adulthood (at least 3 months). Bars are representing the mean between all tested lines. The statistical significance was determined by Mann-Whitney test. Error bars represent SEM. (E) 6-month-old all-iPSC mice generated by tetraploid complementation assay with SKM#1 iPSC line. (F) PCR genotyping of F1 offspring of SKM and KSM all-iPSC mice for SKM or KSM transgenes, respectively.
Figure 4
Figure 4
Co-expression of Sox2 and cMyc Leads to Immediate Retroviral Silencing in MEFs (A) Time course FACS of pMX-mCherry+ Oct4-GFP MEFs during OSKM and SKM reprogramming. (B) FACS analysis of pMX-mCherry+ MEFs expressing Oct4, Sox2, Klf4, cMyc, and combinations of the factors cloned into tet-inducible polycistronic vector with IRES-Puro on D3 of induction and puromycin selection. (C) Experimental design to follow the fate of cells that did or did not undergo retrovirus silencing by 3 dpi with OSKM or SKM. (D) Reprogramming of pMX-mCherry+ Oct4-GFP MEFs with OSKM or SKM. The cells were sorted for mCherry after 3 dpi and plated on a feeder layer. GFP+ colonies were counted after 7 and 14 dpi. Error bars represent SD; n = 3. Statistical significance was calculated with Student’s t test. (E) Experimental design of time course SKM versus OSKM reprogramming RNA-seq experiment. (F) Heatmap showing the Spearman correlation coefficient between time course reprogramming samples based on RNA-seq data. (G) Heatmap depicting the relative fold change (FC) of gene expression after induction with OSKM or SKM. The selected genes were reported as top 100 hits in systemic siRNA screen for reactivation of retrovirus in pluripotent stem cells (Yang et al., 2015). Hierarchical clustering was based on Euclidean distance.
Figure 5
Figure 5
SKM Can Activate Pluripotency Program in Oct4-KO MEFs (A and B) Time course expression plots of indicated fibroblast (A) and MET (B) genes during OSKM and SKM reprogramming. Only Epcam+ and GFP+ sorted samples are shown for d6 and d9, respectively. (C) Time course expression of MEF- and ESC-specific genes (Chronis et al., 2017). Only differentially expressed genes (DEGs) with FC ≥ 4 were plotted. (D) Time course expression plots of indicated genes during OSKM and SKM reprogramming. (E) Time course expression of Oct4, Nanog (ChIP-Atlas), and Sall4 (Lim et al., 2008) targets in ESCs. Only top peaks (macs score ≥ 200, on Chip-Atlas) within 5 kb of DEGs (FC ≥ 4 in iPSCs versus MEFs) were plotted. (F) qPCR gene expression analysis of Nr5a2, Nanog, and Sall4 after short hairpin RNA (shRNA)-driven KD during reprogramming after 2 dpi with OSKM. ActB was used as a reference gene. Error bars represent SD; n = 3. (G) Reprogramming of Oct4-GFP MEFs, expressing Nr5a2, Nanog, Sall4, or control shRNAs with OSKM or SKM. GFP+ colonies were counted after 7 and 14 dpi. Error bars represent SD; n = 3. Statistical significance was calculated with Student’s t test. (H) Strategy for generating Pou5f1 (Oct4)-KO MEFs. (I) PCR genotyping confirming homozygous deletion of Oct4 in 3 MEF lines. (J) Immunofluorescence imaging of Oct4F/F and Oct4Δ/Δ MEFs reprogrammed with SKM, on 7 dpi. (K) qPCR gene expression analysis of Epcam+ Oct4F/F and Oct4Δ/Δ MEFs reprogrammed with OSKM or SKM on 7 dpi. Error bars represent SD; n = 3.
Figure 6
Figure 6
Exogenous Oct4 Diverts the Cells from Direct Route to Pluripotency (A and B) PCA (A) and t-SNE (B) analysis of global gene expression in time course samples during OSKM and SKM reprogramming. (C) Heatmap depicting the relative FC of gene expression during the course of SKM and OSKM reprogramming based on RNA-seq. Hierarchical clustering was based on Euclidean distance. (D) Time course plots of representative GO terms of gene cluster characterized by specific kinetics (see Figure S5A). Gene set enrichment analysis was performed by DAVID (enrichment > 2; p Z score values of expression; shading depicts SD.
Figure 7
Figure 7
OSKM, but Not SKM, Reprogramming Leads to Loss of Imprinting (LOI) and Alterations of iPSC Differentiation during Embryo Development (A) Hierarchical clustering analysis of the iPSC and ESC lines based on global gene expression. Clustering was based on Euclidean distance. (B) MA plots displaying differentially expressed (DE) genes in 4 4N-off OSKM lines versus 15 4N-on iPSC lines and 6 low- versus 8 high-quality iPSCs (4N complementation efficiency of ≤2% and ≥30%, respectively). No DEG with p adj Gtl2 and Zrsr1 in representative iPSC lines validating the COBRA results from Figure S7D. (D) E9.5 embryos generated by 4N complementation with mCherry-labeled SKM and OSKM iPSCs (scale = 500 μm). (E) PCA analysis of bulk RNA-seq data of mCherry+ cells from E9.5 embryos generated by 4N-complementation with high- or low-quality iPSCs. (F and G) Venn diagrams showing the overlap between down- and upregulated DEGs in 3 differentiated 4N-off OSKM iPSCs at E9.5 (≥5 reads in any sample; FC > 1.2; p + or d9 Oct4-GFP+ OSKM versus SKM samples (≥5 reads in any sample; FC > 2; p < 0.05 by DESeq2). (I) Gene set enrichment analysis of overlapping genes from (H). The analysis was performed using Enrichr web server. The bars represent combined Enrichr scores. (J) Adapted Waddington’s epigenetic landscape model showing an epigenetic landscape with trajectories representing reprogramming from fibroblasts to pluripotency driven by OSKM or SKM cocktails.
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