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

SC_11-0951_sm_supplFigure1.pdf109KFigure S1: Analysis of initial OCT4 and NANOG protein levels in the elutriated fractions. Western blot densitometry revealed indistinguishable levels of OCT4 protein across all isolated fractions and moderate, fraction-dependent variation in the protein levels of NANOG. OCT4 and NANOG levels were normalized to the levels measured for β-tubulin (loading control) and displayed as fold difference relative to non-elutriated cells (Bulk). N=2.
SC_11-0951_sm_supplFigure2.pdf73KFigure S2: Wide comparison of expression of pluripotency genes in cells from the ‘40’ and ‘80’ fractions. RT-qPCR measurement of Pluripotency associated genes revealed statistically indistinguishable mRNA levels in the ‘40’ and ‘80’ fractions. Expression is displayed relative to the average of all fractions in each experiment. N=3. n.s. - not significant.
SC_11-0951_sm_supplFigure3.pdf62KFigure S3: The ‘80’ fraction regenerates the original elutriation profile. Re-elutriation profile (A) and differentiation outcome (B, C) after repeated elutriation of cells from the ‘80’ fraction. (A) Elutriation profile of cells from the ‘80’ fraction after culturing for 3 days on Matrigel (with CM), followed by culturing on MEFs for 7 days and re-elutriation. (B, C) Expression of pluripotency (B) and differentiation genes (C) in re-elutriated cells from the ‘80’ and ‘40’ fractions after 3 more days of culture on Matrigel with CM. N=2.
SC_11-0951_sm_supplFigure4.pdf128KFigure S4: Rescue from differentiation in high density cultures of cells from the ‘40’ fraction does not reflect expansion of S-phase cells hESCs were treated for 1hr with BrdU prior to elutriation and analyzed for BrdU incorporation right after the elutriation (0hr) and following 72hrs of culture. BrdU+ cells represent cells that resided in S-phase at the time of elutriation. After 72 hrs, the fraction of these cells in high and low density cultures of the ‘40’ fraction was the same (bottom left), indicating that the rescue from differentiation in the high density culture is not due to preferential expansion of residual S-phase cells.
SC_11-0951_sm_supplFigure5.pdf619KFigure S5: Effect of 40/80 co-culture on colony formation of the individual fractions. Top: Colony formation assay for CFP-labeled cells from the ‘40’ fraction that were first cultured on Matrigel for 3 days with (right) or without (left) GFP-labeled cells from the ‘80’ fraction. The labeled cells were isolated from two consecutive elutriations. Following 3 days on Matrigel with conditioned medium (and bFGF), the elutriated cells were sorted by FACS and re-plated on MEFs for 5 days prior to the scoring of colonies. Bottom: Same as top for GFP-labeled cells from the ‘80’ fraction. Shown are representative images for each case. Scale bar = 40μm.
SC_11-0951_sm_supplTable1.pdf76KTable S1: Primers used in the various quantitative RT-qPCR measurements.
SC_11-0951_sm_supplTable2.pdf256KTable S2: Differential formation of cell doublets in cells from the ‘80’ versus the ‘40’ fraction.

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