NKX3-1 is required for induced pluripotent stem cell reprogramming and can replace OCT4 in mouse and human iPSC induction

Abstract

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is now routinely accomplished by overexpression of the four Yamanaka factors (OCT4, SOX2, KLF4, MYC (or OSKM))1. These iPSCs can be derived from patients’ somatic cells and differentiated toward diverse fates, serving as a resource for basic and translational research. However, mechanistic insights into regulators and pathways that initiate the pluripotency network remain to be resolved. In particular, naturally occurring molecules that activate endogenous OCT4 and replace exogenous OCT4 in human iPSC reprogramming have yet to be found. Using a heterokaryon reprogramming system we identified NKX3-1 as an early and transiently expressed homeobox transcription factor. Following knockdown of NKX3-1, iPSC reprogramming is abrogated. NKX3-1 functions downstream of the IL-6–STAT3 regulatory network to activate endogenous OCT4. Importantly, NKX3-1 substitutes for exogenous OCT4 to reprogram both mouse and human fibroblasts at comparable efficiencies and generate fully pluripotent stem cells. Our findings establish an essential role for NKX3-1, a prostate-specific tumour suppressor, in iPSC reprogramming.

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Fig. 1: Nuclear reprogramming of human fibroblasts after fusion with mouse ESCs.
Fig. 2: Motifs at early accessible chromatin and gene expression dynamics identify NKX3-1.
Fig. 3: NKX3-1 induces Oct4 expression and is required for iPSC formation.
Fig. 4: NKX3-1 can substitute for OCT4 to generate pluripotent mouse and human iPSCs.
Fig. 5: NKX3-1 functions downstream of the IL-6–STAT3 cascade during iPSC induction.

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Acknowledgements

The authors apologize to those investigators whose important work we were unable to cite or describe due to space constraints. The authors thank D. Burns for critical discussions of the manuscript and P. Chu (Comparative Medicine, Stanford) for technical assistance. The authors acknowledge the Stanford Shared FACS Facility (SSFF) and FACS Core Facility in the Stanford Lokey Stem Cell Research Building for technical support. This work was supported by F32 GM112425-02, T32 HD007249 to T.M., Bio-X Graduate Research Fellowships to J.J.B., a NSF Graduate Research Fellowship to G.M., a GSK Sir James Black Program for Drug Discovery Postdoctoral Fellowship to A.P., and the Baxter Foundation, California Institute for Regenerative Medicine (CIRM) grant RB1-01292 and US National Institutes of Health (NIH) grants U01 HL100397, R01 AG009521 and R01 AG020961 to H.M.B.

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Contributions

The study was designed by T.M. and H.M.B. T.M. performed the majority of the experiments. G.M., J.J.B., A.P., H.Z. and V.S. also performed experiments. T.M. and G.M. performed data analysis. The manuscript was written by T.M., G.M. and H.M.B.

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Correspondence to Helen M. Blau.

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Integrated supplementary information

Supplementary Figure 1 Dynamic transcriptional kinetics during early heterokaryon reprogramming.

(a) Schematic diagram of the heterokaryon time course. Heterokaryons were generated by fusion of mouse ESCs and human MRC5 fibroblasts in a 3:1 ratio respectively. Cells were collected at the indicated time points for RNA-seq. Gene expression analysis was performed using only human transcripts. (a) FACS sorting gating strategy to isolate hetorkaryons. Hetorkaryons are detected as dsRed and GFP double positive cells and are sorted to >90% purity. (c) Line plots showing clusters of gene expression patterns during heterokaryon reprogramming. Individual gene expression pattern (gray), the median pattern (red), trajectory, and Gene Ontology/Gene Identity classes. Data represent 1 of 3 biological experiments with similar results.

Supplementary Figure 2 Differential gene expression during early heterokaryon reprogramming.

(a) Scatter plot of log(Fold Change) and mean expression of genes. Red dots signify a DE gene.

Supplementary Figure 3 Nkx3-1 knock-down does not affect MEF proliferation or survival.

(a) Line plot showing growth curves of MEFs transduced with OSKM and an shctrl or shNKX3-1 lentivirus (n = 3 biological replicates). Unpaired Student’s t-test was used and data represents mean ± s.d. (b) Histogram plot showing the percentage of live cells in MEFs transduced with OSKM and an shctrl or shNKX3-1 lentivirus. Live cells were determined as having no 7-AAD staining (n = 3 biological replicates). Unpaired Student’s t-test was used and data represents mean ± s.d. Statistical source data and exact P values for a and b can be found in Supplementary Table 5.

Supplementary Figure 4 NSK-derived iPSCs and OSK-derived have comparable levels of pluripotency gene expression.

(a) Histogram showing numbers of iPSC colonies number 21 days after lentivirus transduction of plasmids encoding NS, NK, and NKS. N = NKX3-1, S = SOX2, K = KLF4 (n = 2 biological replicates). (b) Human OCT4 and NKX3-1 expression in OSK-iPSCs and NSK-iPSCs. (n = 1 biological replicate) (c) Intracellular flow cytometry shows OCT4 protein expression in OSK-iPSCs and NSK-iPSCs. (Data represent 1 of 2 biological replicates with similar results). (d) Expression heatmap of pluripotency genes in MEFs, mouse NSK-iPSCs, mouse OSK-iPSCs and mouse ESCs. (e) Expression heatmap of pluripotency genes in human fibroblasts, human NSK-iPSCs, human OSK-iPSCs and human ESCs.

Supplementary Figure 5 IL6R and NKX3-1 expression kinetics during heterokaryon reprogramming.

(a) Line plot showing IL6R and NKX3-1 expression kinetics during heterokaryon reprogramming (n = 3 biological replicates). Data represents mean ± s.d. (b) Histogram plot showing expression of IL6R and NKX3-1 after transduction of an shIL6R lentivirus 2 h post-fusion (n = 3 biological replicates). Unpaired Student’s t-test was used and data represents mean ± s.d. (c) Histogram plot showing efficiency of shIL6R knock-down in mouse and human fibroblasts after infection with various targeted shRNAs. RT-qPCR was performed 5 days post transduction. (n = 3 technical replicates). (d) ATAC-seq tracks at the NKX3-1 locus during heterokaryon reprogramming. (Data represent 1 of 3 biological replicates with similar results). (e) Histogram plot showing expression of Nkx3-1 following transduction of OSKM alone and with Cre lentivirus (n = 3 biological replicates). Unpaired Student’s t-test was used and data represents mean ± s.d. (f) Histogram plot showing expression of NKX3-1 in human fibroblast after transduction of lentivirus encoding NKX3-1 (n = 3 biological replicates). Unpaired two-sided Student’s t-test was used. (g, h) Histogram plot showing expression of specific MET or EMT genes after transduction of NKX3-1 (n = 3 biological replicates). Unpaired Student’s t-test was used and data are shown as mean ± s.d. Statistical source data and exact P values for b, e, and f-h can be found in Supplementary Table 5.

Electronic supplementary material

Supplementary Information

Supplementary Figures 1–5 and Supplementary Table legends

Reporting Summary

Supplementary Table 1

Top 100 DE genes in each time point ordered by adjusted P-value

Supplementary Table 2

Human ESC signature gene expression in heterokaryon by 48h

Supplementary Table 3

Gene expression profile of IL-6 associated genes

Supplementary Table 4

Oligos

Supplementary Table 5

Statistics source data

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Mai, T., Markov, G.J., Brady, J.J. et al. NKX3-1 is required for induced pluripotent stem cell reprogramming and can replace OCT4 in mouse and human iPSC induction. Nat Cell Biol 20, 900–908 (2018). https://doi.org/10.1038/s41556-018-0136-x

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