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Additional Supporting Information may be found in the online version of this article.

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sc-12-0849_sm_SupplFigure1.pdf19KSupplementary Figure 1. Secretion of MMP from feeder cells. (A) Secreted protein levels of MMP2 in conditioned media of indicated cultures, analyzed by ELISA. Concentrations are normalized by final cell density. (B) Fraction of ESCs in the total cell population after two days of growth on a feeder layer in the presence or absence of Ro32. Data was obtained using two separate ESC cell lines with GFP conjugated to histone H2A or H2B (to ease cell counting). **=p<0.001,*=p<0.05.
sc-12-0849_sm_SupplFigure2.pdf20KSupplementary Figure 2. Additional marker expression with MMP addition. (A) mRNA expression levels in the indicated conditions in serum-containing media. (B) mRNA expression levels in the indicated conditions in serum-free media. * =p<0.05 and #=p<0.05 for all pairwise comparisons, all data represents averages of at least three independent experiments and error bars represent SD.
sc-12-0849_sm_SupplFigure3.pdf24KSupplementary Figure 3. Correlation between all genes expressed in the RNA-sequencing dataset in the indicated conditions.
sc-12-0849_sm_SupplFigure4.pdf42KSupplementary Figure 4. MMP1 overcomes RA differentiation stimulus. (A) Flow cytometry histogram of Sox2-GFP reporter mESCs fluorescent intensity after growth in the indicated conditions (RA = retinoic acid). (B) mRNA expression in the presence of RA with or without MMP1. #=p<0.05 for all pairwise comparisons, all data represents averages of at least three independent experiments and error bars represent SD.
sc-12-0849_sm_SupplFigure5.pdf41KSupplementary Figure 5. Images of representative morphology of cells grown in serum-free N2B27 media with the indicated additions. Labels at left indicate passage number. Scale bar = 200μm.
sc-12-0849_sm_SupplFigure6.pdf64KSupplementary Figure 6. EpiSCs cultured in the presence or absence of MMP1. (A) Representative images of EpiSCs seeded at equal density and cultured for three passages in N2B27 medium supplemented with various combinations of Fgf, Activin and MMP1. (B) Counts and representative images of EpiSCs seeded at equal density and cultured for two passages in EpiSC medium supplemented with the MMP inhibitor Ro32-555 or an equal volume of DMSO, n=2, **=p<0.001. (C) Counts and representative images of EpiSCs seeded at equal density and cultured for two passages in a 1:1 ratio of EpiSC medium and conditioned medium from ESCs treated with recombinant MMP1, n=2.
sc-12-0849_sm_SupplFigure7.pdf22KSupplementary Figure 7. mRNA expression level timecourse from embryoid bodies grown for the indicated number of days with no LIF or MMP1, after five passages in media supplemented with either LIF or MMP1. Image shows representative embryoid bodies from MMP1 condition on day 5. Scale bar = 400μm.
sc-12-0849_sm_SupplFigure8.pdf26KSupplementary Figure 8. Additional evidence regarding signaling pathways involved in MMPmediated self-renewal. (A) mRNA expression of differentiation and self-renewal markers in the presence or absence of SB431542, an Alk4/5/7 inhibitor of Activin/Nodal signaling. (B) mRNA expression levels of Wnt downstream target genes after short-term addition of MMP for the indicated number of hours. (C) mRNA expression levels of self-renewal and differentiation markers in cells grown in indicated conditions with a control shRNA or Stat3 shRNA, construct 1413. **=p<0.001,*=p<0.05.
sc-12-0849_sm_SupplFigure9.pdf24KSupplementary Figure 9. LIF-family ligands in MMP1-mediated self-renewal. (A) mRNA expression levels of self-renewal and differentiation markers after addition of a LIF-blocking antibody to cells with added LIF or added MMP1. (B) mRNA expression levels of a Stat3 target gene after 1 hour of indicated additions. (C) mRNA expression levels of Stat3 downstream target genes after 1 h in the indicated conditions. * =p<0.05; ##=p<0.001, #=p<0.05 for pairwise comparisons, all data represents averages of at least three independent experiments and error bars represent SD.
sc-12-0849_sm_SupplTable1.pdf23KSupplementary Table 1. Forward and reverse primers used for quantitative RT-PCR.

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