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Deterministic direct reprogramming of somatic cells to pluripotency

A Corrigendum to this article was published on 01 April 2015

Abstract

Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.

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Figure 1: Boosting primed to naive pluripotency reversion.
Figure 2: Deterministic and synchronized iPS cell reprogramming.
Figure 3: Alleviating Mbd3 expression facilitates transition to pluripotency.
Figure 4: Numerical description of reprogramming after Mbd3 depletion.
Figure 5: Mechanisms for Mbd3 inhibitory effect on induced pluripotency.

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Data deposits

Chromatin immunoprecipitation data are available at the National Center for Biotechnology Information Gene Expression Omnibus database under the series accession number GSE49766. Microarray data are available at the National Center for Biotechnology Information Gene Expression Omnibus database under the series accession number GSE45352.

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Acknowledgements

J.H.H. is supported by a generous gift from I. and P. Mantoux; and grants from the Leona M. and Harry B. Helmsley Charitable Trust, ERC (StG-281906) grant, BIRAX initiative, Israel Science Foundation (BIKURA, ICORE and Regular programs), ICRF, Fritz Thyssen Stiftung, The Benoziyo Endowment fund, Alon Scholar Program, and the Clore research prize. I.A is supported by the HFSP Career Development Award, an ISF-Bikura and the ERC (StG-309788). A.A.M. is supported by a Weizmann Dean fellowship. We thank N. Barkai and her group, K. Saha, B. Hendrich, J. Nichols and A. Surani, for reagents and advice. We thank the Weizmann Institute management for providing critical financial and infrastructural support.

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Affiliations

Authors

Contributions

Y.R., A.Z., S.Ge., N.N. and J.H.H. conceived the idea for this project, designed and conducted experiments and wrote the manuscript. S.Ge. conducted protein biochemical analysis. A.Z. conducted numerical modelling analysis. O.G., L.W. and N.M. assisted in chromatin immunoprecipitation experiments. N.N. and A.Z. conducted bioinformatics analysis. Y.R. and A.Z. conducted live imaging experiments and analysis. S.V. engineered human stem cell lines. I.A., D.A.J., D.L.-A., S.Gi., D.A.-Z. and R.B.-G. assisted with ChIP-seq experiments. E.C., Z.S., Z.M. and A.T. conducted RRBS analysis. Y.R. and M.Z. conducted microinjections. Y.R. and A.A.M. conduced embryo staining. Y.R., S.Ge. and J.H.H. conducted reprogramming experiments with help from I.C., I.M., V.K., T.H. and D.B.

Corresponding authors

Correspondence to Noa Novershtern or Jacob H. Hanna.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Knockdown screen for epigenetic repressors in EpiSCs.

a, Knockdown efficiency of the indicated siRNA pools in EpiSCs measured by qRT–PCR. Expression values for each gene were normalized to those measured in control siRNA. Error bars indicated s.d. from average. Asterisks indicate t-test P value <0.05. b, Phase images of Mbd3+/+ and Mbd3fl/− EpiSC lines in this study. c, Oct4 immunostaining on EpiSC lines. d, RT–PCR expression level validation for pluripotency genes in naive V6.5 ES cells and primed Mbd3+/+ and Mbd3fl/− EpiSCs. In comparison to naive ES cells, primed EpiSCs downregulate naive pluripotency markers Nanog and Klf4, and upregulate FGF5 transcription (n = 3). e, EpiSC lines were pluripotent as evident by their ability to form mature differentiated teratomas. f, Representative agouti-coloured chimaeras obtained from reverted EpiSCs after Mbd3 depletion.

Extended Data Figure 2 Derivation of ES cells from

Mbd3−/− blastocysts. a, RT–PCR analysis for Oct4 and trophoblast marker expression of Mbd3+/+ and Mbd3−/− ES cells expanded either in FBS/LIF or 2i/LIF conditions. Only Mbd3−/− ES cells, and only in serum conditions, upregulate trophoblast differentiation markers. Error bars indicate s.d. from average (n = 3). b, Mbd3+/− heterozygous mice were mated, and Mdb3−/− ES cells were derived from blastocysts in naive defined 2i/LIF conditions. Western blot for pluripotency marker expression also indicated that the derived Mbd3/− ES cell lines adequately expressed all pluripotency factors tested. c, Transcriptional expression of Mbd3 and Nanog during pre-implantation development. RT–PCR analysis demonstrating the expression of Mbd3 during early mouse development, presented as a relative quantification column scheme. Error bars indicate s.d. from average (n = 3). Mbd3 transcript is detected at low levels in oocytes whereas Mbd3 protein is weakly detected by immunostaining in oocytes and zygotes (Fig. 1e), consistent with maternal inheritance. Mbd3 transcription becomes increased towards the end of pre-implantation development at the morula and blastocyst stages, consistent with strong re-expression of Mbd3 protein at the blastocyst stage (Fig. 1e). d, Immunostaining for Mbd3 and lineage markers in E5.5 post-implantation epiblast, indicating prominent expression (n = 3 embryos stained).

Extended Data Figure 3 Genetically engineered systems for deterministic reprogramming in mouse cells.

a, We established a reprogrammable mouse Mbd3+/+ and Mbd3fl/− iPS cell lines carrying (1) an Oct4–GFP reporter, (2) nuclear mCherry constitutively expressed marker, (3) m2RtTa transgene and (4) a TetO inducible STEMCCA-OKSM polycistronic cassette. These lines were injected into host blastocysts, and their differentiated derivatives were re-isolated in vitro. Subsequently, reprogramming efficiency and progression were analysed after doxycycline induction. b, Reprogramming efficiency after infection with indicated MEF lines with moloney retroviruses encoding individual factors. c, Reprogramming efficiency after infection with indicated MEF lines with polycistronic OKSM encoding lentivirus. d, Mbd3fl/− MEFs were infected with polycistronic OKSM vector in LIF-containing ES medium with or without the indicated exogenous supplements. Reprogramming efficiency was evaluated by Oct4–GFP levels on day 9 after transduction without cell splitting during the process. e, Mbd3+/+, Mbd3−/− and Mbd3fl/− MEFs, adult tail-tip-derived fibroblast (TTF) and neural precursor cells (NPC) were tested for iPS cell formation in 2i/LIF with or without OKSM lentiviral transduction. Our analysis indicates that OKSM is essential for iPS formation, and that Mbd3 depletion alone is not sufficient to reprogram any of these cells types to pluripotency (even after 30 days of follow up). f, Reprogramming efficiency of MEFs after transduction with the indicated combinations of reprogramming factors at day 10. Polycistronic lentiviral vectors were used for OSK and OSKM combinations. Asterisk indicates t-test P value <0.01 relative to Mbd3+/+ control. Error bars indicate s.d. from average (n = 4).

Extended Data Figure 4 Reprogramming kinetics on perturbation of Mbd3 expression.

a, Flow cytometry measurements of Oct4–GFP reactivation dynamics in 2i/LIF after doxycycline (OSKM) induction. Notably, wells at the indicated time points were collected for analysis without prior passaging and splitting during the reprogramming course. 1 out of 3 independent experiments is shown. FSC, forward scatter. b, Characterizing the effect for Mbd3 expression reconstitution during deterministic reprogramming of somatic cells to pluripotency. Scheme demonstrates experimental strategy for defining the temporal ability of Mbd3 during reprogramming to inhibit iPS formation. Secondary OSKM reprogrammable Mbd3fl/− MEFs were tested for their amenability to reprogramming after overexpression of Mbd3, Mbd2 or empty FUW lentiviruses at different time points during reprogramming. Mbd2 or mock-vector transfection did not result in a decrease in iPS cell reprogramming efficiency. Error bars indicate s.d. from average (n = 3). One out of two representative data sets is shown.

Extended Data Figure 5 Genetic and epigenetic changes during iPS cell reprogramming after Mbd3 depletion.

a, Hierarchical clustering was carried out on chromatin IP-seq measurements in fibroblasts before and after doxycycline induction. Clustering was calculated over concatenate vectors including z-scores of all histone marks (H3K4me3, H3K27me3 and H3K27ac) for each gene (n = 1,323 genes with differential gene expression between MEFs and ES cells). Spearman correlation was used as a distance metric and average linkage. b, Graph shows genome-wide methylation levels as measured by reduced representation bisulphite sequencing (RRBS). Results are averaged over all CpGs that were covered by five or more distinct sequencing reads (34,522 CpG sites in total). The average methylation level of low-passage Mbd3+/+ iPS cells is provided as a dashed line for reference. c, Hierarchical clustering for CpG methylation was made using Ward’s method and the Pearson correlation score as the similarity matrix. d, Single cell RT–PCR analysis for detection of pluripotency gene markers. Analysis was conducted on Mbd3+/+ and Mbd3fl/− MEFs before and 6 days after doxycycline induction. Undetected expression (marked by red boxes) indicates lack of amplification even after 50 amplification cycles are marked in red. Expressed genes are marked by green boxes. One biological replicate is shown of two performed.

Extended Data Figure 6 Depleting Mbd3 expression facilitates human iPS cell formation.

a, In vitro differentiated fibroblasts from MBD3WT and MBD3mut iPS cells carrying the doxycycline-inducible OKSM transgenes, were reprogrammed as indicated in Fig. 3f. Pluripotency of randomly selected iPS cell clones is shown as evident by teratoma. b, Secondary human reprogrammable C1 fibroblasts carrying doxycycline-inducible OSKM transgenes were subjected to the depicted reprogramming protocol. Knockdown of Mbd3 at days 2 and 4, but not with scrambled control siRNA, markedly increased the reprogramming efficiency as evaluated by formation of NANOG/SSEA4+ colonies. Pluripotency of a randomly selected iPS cell clone expanded and validated by in vivo teratoma formation. Western blot confirmed specific and significant decrease in MBD3 protein expression after MBD3 siRNA transfection. Error bars indicate s.d. from average (n = 3). One out of three representative experiments is shown. c, MBD3 siRNA treatment of human primary fibroblasts allows generation of iPS cells by only two rounds of reprogramming with mRNA transfection with OSKM and LIN28 (OSKML) factors. Representative human iPS cell clones are shown at different time points and passages (P indicates passage number). Pluripotency of randomly selected clones is shown by specific staining for OCT4 and SSEA4 pluripotency markers and teratoma formation. These results indicate that inhibition of MBD3 expression and/or function promotes iPS cell formation by transient mRNA or other transient transfection protocols for iPS cell reprogramming.

Extended Data Figure 7 Statistical analysis of iPS cell reprogramming after Mbd3 depletion.

a, Distribution of Nanog–GFP+ cells at initial time of detection, by quantifying the amount of Nanog–GFP+ cells detected above the 0.5% threshold. Graphs show box-plot medians and 25th/75th percentiles. b, Illustration of the first passage time model. In this model, we assume that reprogramming time depends on the first time in which some master regulator (that is, Nanog or Oct4) makes a transition from a low state to a high state of expression. c, Mbd3KD and Mbd3+/+ reprogramming dynamics were fit to Gaussian distribution. Figures show maximum likelihood estimates of mean and standard deviation, with 95% confidence intervals. d, Mbd3KD and Mbd3+/+ reprogramming dynamics were fit to multiple tandem rate-limiting step models, where convergence of adjusted R2 indicates the best fit (right panel). Results show that Mbd3+/+ (blue) fit best to a multi-phase process with one or two intermediate states, whereas Mbd3KD (red) fit best to a single exponential transition with no intermediate states.

Extended Data Figure 8 Effect of Mbd3 depletion on OSKM target genes.

a, Normalized single gene expression for selected group of genes in MEF and 8 days after doxycycline induction. Expression values represent distance from MEF expression values (set to 0) towards iPS values (set to 1), indicating absence of transcription in MEF and fast activation after doxycycline induction in Mbd3 depleted samples. b, Reprograming efficiency of Mbd3+/+ secondary MEFs after knockdown of Mbd3 or Chd4. Error bars indicate s.d. from average (n = 3). Asterisks indicate Student’s t-test P value <0.01. Western blot indicating protein depletion efficiency on siRNA transfection of either Mbd3 or Chd4 targeting siRNA pools. c, Distribution of gene expression fold-change relative to MEF, calculated over 2,928 genes bound by at least one of the OSKM factors20 and upregulated during reprogramming. Graphs show box-plot medians and 25th/75th percentiles, and P values by paired sample t-test. d, Distribution of histone marks and Oct4 binding levels in z-score values at day 4 after OSKM (doxycycline) induction, calculated over the same set of 2,928 genes described above. e, Histone mark z-score profiles for three representative OSKM target genes, calculated between 1 kb upstream to TSS and TES.

Extended Data Figure 9 Direct interaction of Mbd3 with OSKM pluripotency factors during reprogramming.

a, Overexpression of Flag-tagged Mbd3 simultaneously with OCT4, SOX2, KLF4, MYC or Nanog in HEK293 cells was followed by co-immunoprecipitation (co-IP) assay. Immunoblot analysis (IB) using antibodies against Oct4, Sox2, Klf4, Myc and Nanog showed specific binding between Mbd3 and the pluripotent factors except Nanog (n = 2). b, Co-immunoprecipitation assay of Chd4 (Mi2b), the core subunit of the NuRD complex, in secondary Mbd3+/+ fibroblasts 3 days after doxycycline induction. Co-immunoprecipitation for NuRD component, Chd4, followed by immunoblot analysis indicated specific pull-down of other Mbd3/NuRD components (Mbd3 and Mta2) and OSKM reprogramming factors (n = 3). c, Deletion mutations in the MBD site of Mbd3 was planned to find the binding region of Mbd3. Flag-tagged mutation constructs were co-transfected with Oct4, Sox2, Klf4 and Myc in HEK293T cells for 48 h followed by co-immunoprecipitation with anti-Flag beads and immunoblotted against OSKM. This analysis shows loss of binding and interaction between OSKM and selected Mbd3 mutants (n = 3).

Extended Data Figure 10 Pluripotency-promoting epigenetic activators are essential for both deterministic and stochastic iPS cell formation.

a, Requirement for doxycycline-mediated transgene induction during iPS cell reprogramming form Mbd3+/+ and Mbd3fl/− secondary MEFs. Percentage of Oct4–GFP colonies was quantified at final set time point on day 9. Similar time frame for minimal doxycycline induction was required for iPS cell formation in both cell samples (irrespective of the total iPS formation efficiency obtained). Representative data from one out of three biological replicates conducted. b, c, Specific knockdown of Utx and Wdr5 epigenetic regulators that are required for iPS cell formation significantly inhibited iPS cell formation in both Mbd3+/+ and Mbd3fl/− cells. Asterisks indicate t-test P value <0.01 in comparison to control siRNA sample. Error bars indicate s.d. from average (n = 3).

Supplementary information

Supplementary Information

This file contains supplementary discussions, a supplementary description for numerical modelling analysis and supplementary references. (PDF 420 kb)

Supplementary Dataset 1

This Supplementary Table shows Mbd3 localization following ChIP-Seq analysis. (i) Mbd3 bound regions in MEF cells and (ii) Mbd3 bound regions in MEF+OSKM samples, measured with ChIP-Seq and estimated with MACS software. Data appear in “bed” file format. (iii) Mbd3 bound target genes in MEF and (iv) Mbd3 bound target genes in MEF+OSKM cells. List generated by mapping of Mbd3 MACS peaks to an interval of 1Kb around the Transcription Start Sites of all mouse genes (Taken from USCS RefSeq known gene list). (XLSX 493 kb)

Highly enhanced ES-like colony formation by OSKM upon Mbd3 depletion

Live imaging of reprogramming in equivalent regions (5*6 mosaic) and phase contrast, after plating 150 cells per well. Video was prepared from time-lapse measurements taken every 8 hours for 6.5 days at 50X magnification (5X objective lens), time from DOX induction is indicated in the upper title. Note the accelerated ES-like colony formation in Mbd3flox/- cells in comparison to Mbd3+/+ donor MEFs. (n=4 independent experiments). (AVI 1468 kb)

Time-lapse microscopic imaging of deterministic reprogramming

Live imaging of Mbd3flox/- and control Mbd3+/+ full well mosaics with fluorescent mCherry and Oct4-GFP markers. Measurements were taken every 12 hours for 6 days at 50X magnification. In house automated segmentation protocol was run over time-lapse data tracking Oct4-GFP activation dynamic. Right upper image show Mbd3+/+ and left upper image show Mbd3flox/- full well mosaics. Time from DOX induction is given in the upper title. Lower left graph indicates cumulative Oct4-GFP+ colonies for Mbd3flox/- (red graph) and Mbd3+/+ (blue graph). Lower right graph indicates the average fraction of Oct4-GFP+ cells within single colonies. (n=4 independent experiments). (AVI 1136 kb)

Time-lapse imaging of reprogramming dynamics of Mbd3flox/- cells with single colony view

Live imaging of Mbd3flox/- full well mosaics with fluorescent mCherry and Oct4-GFP markers. Measurements were taken every 12 hours for 6 days at 50X magnification. For each time point full well mosaic (upper image) and up to 40 representative single colony images (two lower images) are shown. The two white rectangles on the full well mosaic represent the bounding box of the two single colonies that are shown in the lower images. In addition, lower left graph indicates cumulative Oct4-GFP+ colonies and lower right graph indicates the average fraction of Oct4-GFP+ cells within single colonies for Mbd3flox/-. (n=4 independent experiments). (AVI 5480 kb)

Time-lapse imaging of reprogramming dynamics of Mbd3+/+ cells with single colony view

As in supplementary Video 3, live imaging of Mbd3+/+ full well mosaics with fluorescent mCherry and Oct4-GFP markers, measurements were taken every 12h for 6 days at 50X magnification. (n=4 independent experiments). (AVI 2903 kb)

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Rais, Y., Zviran, A., Geula, S. et al. Deterministic direct reprogramming of somatic cells to pluripotency. Nature 502, 65–70 (2013). https://doi.org/10.1038/nature12587

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