Deterministic direct reprogramming of somatic cells to pluripotency

Journal name:
Nature
Volume:
502,
Pages:
65–70
Date published:
DOI:
doi:10.1038/nature12587
Received
Accepted
Published online

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.

At a glance

Figures

  1. Boosting primed to naive pluripotency reversion.
    Figure 1: Boosting primed to naive pluripotency reversion.

    a, An siRNA screen for factors that can boost epigenetic reversion of primed EpiSCs into naive ES cells. Percentage of naive Nanog–GFP+ cells detected by flow cytometry is indicated (n = 3). b, Single-cell reprogramming efficiency and quantification for EpiSC reprogramming from different mutant lines. The pBRY-Mbd3 rescue construct was stably expressed in the indicated lines (n = 4). c, Western blot analysis for Mbd3 expression in ES cells. d, Mbd3−/− ES cell derivation from blastocysts in 2i/LIF. e, Representative confocal immunostaining images for temporal Mbd3 expression in developing mouse embryos. Arrows indicate polar body (n = 15 embryos stained per stage). Scale bar, 25μm. f, Mbd3fl/− and Mbd3+/+ ES cell lines (with or without pBRY-Mbd3 overexpression allele) were targeted with an Oct4–GFP reporter and a constitutively expressed mCherry reporter, and injected into host chimaeras. Embryonic day (E)8.5 primordial germ cells (PGC) were sorted into defined conditions and evaluated for efficiency to generate EG cells (n = 6). Asterisk indicates t-test P value <0.01 in comparison to Mbd3+/+. All error bars indicate s.d. from average.

  2. Deterministic and synchronized iPS cell reprogramming.
    Figure 2: Deterministic and synchronized iPS cell reprogramming.

    a, Mbd3 wild-type and depleted (Mbd3fl/− or Mbd3−/−) MEFs were directly infected with lentiviruses expressing a polycistronic OKSM cassette. Reprogramming efficiency (Oct4–GFP) was measured by FACS at day 10. Asterisk indicates t-test P value <0.01 in comparison to Mbd3+/+ samples. Error bars indicate s.d. from average (n = 5). b, Secondary reprogrammable fibroblasts carrying an Oct4–GFP reporter and an mCherry constitutively expressed marker were single-cell-seeded and subjected to doxycycline-induced reprogramming. Reprogramming efficiency at day 8 was calculated by dividing the number of Oct4–GFP+ wells by mCherry+ wells (n = 3 per clone, ± indicates s.d. from average). c, Top: immunostaining of representative iPS cell clones for pluripotency markers (original magnification, ×100). Bottom: agouti-coat-coloured chimaera and germline transmission from Mbd3-depleted iPS cells. d, The indicated somatic cell types from NGFP1-control or NGFP1-Mbd3KD adult chimaeras were isolated and subjected to single-cell reprogramming and evaluation of Nanog–GFP expression after 8days of doxycycline (n = 4). CMP, common myeloid progenitor; HSC, haematopoietic stem cell; NPC, neural precursor. e, Full-well mosaic images of mCherry, Oct4–GFP and combined channels, shown for Mbd3fl/− and Mbd3+/+ at day 6. Scale bar, 3mm. f, Characterization of Oct4–GFP+ dynamic for Mbd3fl/− (red plot) and Mbd3+/+ (blue plot) based on live imaging. Top graph indicates cumulative Oct4–GFP+ colonies; bottom graph indicates fraction of Oct4–GFP+ cells within colonies. Dox, doxycycline. Graphs show means and s.d. of all tracked colonies in three biological replicates (one out of four biological data sets is shown).

  3. Alleviating Mbd3 expression facilitates transition to pluripotency.
    Figure 3: Alleviating Mbd3 expression facilitates transition to pluripotency.

    a, Spearman correlation matrix between the indicated mouse samples, measured over gene expression levels of all 16,620 expressed genes. The matrix is clustered with hierarchical clustering. b, Monoclonal lines established from Mbd3+/+ and Mbd3fl/− secondary cells and reprogrammed in 2i/LIF plus doxycycline. Fraction of pre-iPS clones that did not reactivate Oct4/Nanog GFP markers (either SSEA1 positive or negative) is shown. c, Representative partially reprogrammed cell line containing OSKM transgenes, and that did not reactivate GFP reporter, was subjected to the indicated manipulations and analysed for completion of reprogramming as assayed by FACS. FUW indicates lentiviral backbone vector used. d, Targeting strategy for human MBD3 locus. e, Biallelically targeted MBD3mut clone displayed ~90% reduction in MBD3 protein expression levels. f, MBD3WT and MBD3mut iPS cells carrying doxycycline-inducible OKSM transgenes were labelled with constitutively expressed mCherry and targeted with an OCT4-GFP knock-in allele. ROCKi, Rho-associated protein kinase inhibitor. In vitro differentiated fibroblasts from the latter lines were reprogrammed as indicated in the scheme. Error bars indicate s.d. of average (n = 6). Asterisks indicate t-test P value <0.01 in comparison to MBD3WT.

  4. Numerical description of reprogramming after Mbd3 depletion.
    Figure 4: Numerical description of reprogramming after Mbd3 depletion.

    a, Scheme demonstrating the monoclonal and polyclonal follow-ups for Nanog–GFP reactivation. KD, knockdown; OE, overexpression. b, Cumulative percentage of Nanog–GFP+ wells versus time on doxycycline, measured for various clonal B-cell-derived populations. c, Goodness of fit plots for the fitting of deterministic step function model to the observed reprogramming latency. d, Comparison of the calculated variation of Mbd3KD and control (dark red and blue colours, respectively), and the cell cycle variation of each sample (light red and blue colours, respectively). Sample variation was calculated using coefficient of variation (left panel) and dynamic variation (right panel) (Extended Data Fig. 7b). Graphs show maximum likelihood estimations. Error bars indicate 95% confidence intervals of maximal likelihood value (Mbd3KD reprogramming n = 7; control reprogramming n = 13; cell cycle for control and Mbd3KD n = 20). e, Goodness of fit plots for the fitting of cell-cycle time distribution to the observed reprogramming latency.

  5. Mechanisms for Mbd3 inhibitory effect on induced pluripotency.
    Figure 5: Mechanisms for Mbd3 inhibitory effect on induced pluripotency.

    a, Constructs encoding Flag-tagged OCT4, SOX2, KLF4, MYC, Nanog or HDAC1 were transfected into HEK293T cells in combination with Mbd3. The cell lysates were immunoprecipitated (IP) with an anti-Flag antibody (or anti-IgG as control), followed by an immunoblot analysis (IB) (n = 3 biological replicates). b, Functional enrichment of Mbd3 and Mi2β (Chd4) direct targets, measured in MEFs before and after OSKM induction. Colour levels indicate enrichment P values (by Fisher’s exact test) that pass the false discovery rate (FDR) threshold of 0.0001%. c, Distribution of gene expression fold change relative to MEFs of Mbd3+/+ (blue) and Mbd3fl/− (red) samples throughout 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. Graphs show box-plot medians and 25th/75th percentiles, and P-values by paired sample t-test. e, Reprogramming efficiency of Mbd3fl/− MEFs after infection with lentiviruses encoding wild type and different mutant Mbd3 inserts. Error bars indicate s.d. from average (n = 6). Asterisk in e indicates t-test P value <0.01. f, Mechanistic model scheme.

  6. Knockdown screen for epigenetic repressors in EpiSCs.
    Extended Data Fig. 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.

  7. Derivation of ES cells from
    Extended Data Fig. 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).

  8. Genetically engineered systems for deterministic reprogramming in mouse cells.
    Extended Data Fig. 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 30days 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).

  9. Reprogramming kinetics on perturbation of Mbd3 expression.
    Extended Data Fig. 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.

  10. Genetic and epigenetic changes during iPS cell reprogramming after Mbd3 depletion.
    Extended Data Fig. 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 6days 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.

  11. Depleting Mbd3 expression facilitates human iPS cell formation.
    Extended Data Fig. 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.

  12. Statistical analysis of iPS cell reprogramming after Mbd3 depletion.
    Extended Data Fig. 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.

  13. Effect of Mbd3 depletion on OSKM target genes.
    Extended Data Fig. 8: Effect of Mbd3 depletion on OSKM target genes.

    a, Normalized single gene expression for selected group of genes in MEF and 8days 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 1kb upstream to TSS and TES.

  14. Direct interaction of Mbd3 with OSKM pluripotency factors during reprogramming.
    Extended Data Fig. 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 3days 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 48h 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).

  15. Pluripotency-promoting epigenetic activators are essential for both deterministic and stochastic iPS cell formation.
    Extended Data Fig. 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).

Videos

  1. Highly enhanced ES-like colony formation by OSKM upon Mbd3 depletion
    Video 1: 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).
  2. Time-lapse microscopic imaging of deterministic reprogramming
    Video 2: 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).
  3. Time-lapse imaging of reprogramming dynamics of Mbd3flox/- cells with single colony view
    Video 3: 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).
  4. Time-lapse imaging of reprogramming dynamics of Mbd3+/+ cells with single colony view
    Video 4: 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).

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Author information

  1. These authors contributed equally to this work.

    • Yoach Rais,
    • Asaf Zviran &
    • Shay Geula

Affiliations

  1. The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

    • Yoach Rais,
    • Asaf Zviran,
    • Shay Geula,
    • Ohad Gafni,
    • Elad Chomsky,
    • Sergey Viukov,
    • Abed AlFatah Mansour,
    • Inbal Caspi,
    • Vladislav Krupalnik,
    • Mirie Zerbib,
    • Itay Maza,
    • Nofar Mor,
    • Dror Baran,
    • Leehee Weinberger,
    • Noa Novershtern &
    • Jacob H. Hanna
  2. The Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel

    • Diego A. Jaitin,
    • David Lara-Astiaso,
    • Ronnie Blecher-Gonen &
    • Ido Amit
  3. The Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel

    • Zohar Shipony,
    • Zohar Mukamel &
    • Amos Tanay
  4. The Department of Computer Science, Weizmann Institute of Science, Rehovot 76100, Israel

    • Zohar Shipony,
    • Zohar Mukamel &
    • Amos Tanay
  5. The Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel

    • Tzachi Hagai
  6. The Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot 76100, Israel

    • Shlomit Gilad &
    • Daniela Amann-Zalcenstein

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

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.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Knockdown screen for epigenetic repressors in EpiSCs. (506 KB)

    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.

  2. Extended Data Figure 2: Derivation of ES cells from (518 KB)

    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).

  3. Extended Data Figure 3: Genetically engineered systems for deterministic reprogramming in mouse cells. (413 KB)

    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 30days 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).

  4. Extended Data Figure 4: Reprogramming kinetics on perturbation of Mbd3 expression. (280 KB)

    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.

  5. Extended Data Figure 5: Genetic and epigenetic changes during iPS cell reprogramming after Mbd3 depletion. (479 KB)

    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 6days 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.

  6. Extended Data Figure 6: Depleting Mbd3 expression facilitates human iPS cell formation. (1,030 KB)

    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.

  7. Extended Data Figure 7: Statistical analysis of iPS cell reprogramming after Mbd3 depletion. (355 KB)

    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.

  8. Extended Data Figure 8: Effect of Mbd3 depletion on OSKM target genes. (511 KB)

    a, Normalized single gene expression for selected group of genes in MEF and 8days 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 1kb upstream to TSS and TES.

  9. Extended Data Figure 9: Direct interaction of Mbd3 with OSKM pluripotency factors during reprogramming. (336 KB)

    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 3days 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 48h 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).

  10. Extended Data Figure 10: Pluripotency-promoting epigenetic activators are essential for both deterministic and stochastic iPS cell formation. (181 KB)

    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

Video

  1. Video 1: Highly enhanced ES-like colony formation by OSKM upon Mbd3 depletion (1.43 MB, Download)
    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).
  2. Video 2: Time-lapse microscopic imaging of deterministic reprogramming (1.1 MB, Download)
    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).
  3. Video 3: Time-lapse imaging of reprogramming dynamics of Mbd3flox/- cells with single colony view (5.35 MB, Download)
    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).
  4. Video 4: Time-lapse imaging of reprogramming dynamics of Mbd3+/+ cells with single colony view (2.83 MB, Download)
    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).

PDF files

  1. Supplementary Information (420 KB)

    This file contains supplementary discussions, a supplementary description for numerical modelling analysis and supplementary references.

Excel files

  1. Supplementary Dataset 1 (493 KB)

    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).

Comments

  1. Report this comment #62955

    jacob hanna said:

    Detailed protocols, FAQs and comments are available at: http://www.weizmann.ac.il/molgen/Hanna/Protocols.html

  2. Report this comment #64027

    jacob hanna said:

    Further details on the experimental settings used to generate the data under GSE49766 have been added on the GEO website. It now states that in Fig. 3a and Extended Data Figure 5, the selected Mbd3+/+ clonal series carry deltaPE-Oct4-GFP transgenic reporter, and Mbd3flox/- and Mbd3-/- cells carry Oct4-GFP transgenic reporter (complete enhancer region with DE and PE elements), which can be identified when analyzing the Oct4 locus in genomic DNA input datasets. The difference in transgene reporters does not influence interpretation of our genomic analysis data, because we do not use Oct4-GFP or any selection for these experiments. Also note that the endogenous Nanog and Oct4 loci are not manipulated and identical between all cell lines used in genomic studies (Oct4-GFP reporter was introduced via random transgenesis and validated for specificity). Further, as we reprogram in 2i/LIF conditions, the PE element is not functionally relevant and thus they cannot differentially influence the molecular outcome of this experiment. Importantly, for all mouse iPSC efficiency quantitative results presented in our paper (e.g. Fig.1, Fig. 2, Supplementary Movies 1-4, Rais et al. Nature 2013) all lines carried matched Nanog-GFP knock- in reporter, or Oct4-GFP transgenic reporter (containing both DE and PE elements as delineated in Extended Data Figure 3a, Rais et al. Nature 2013). Thus, all iPSC efficiency and kinetic comparisons were conducted by using matched reporters systems.

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Additional data