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

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STEM_748_sm_supplFig1.pdf17KSupplemental Figure 1: Characterization of HSF1 control and PTEN KD hESCs. a) Bright field images of control and PTEN KD hESCs cultured on a mouse feeder layer. b) QPCR of PTEN levels in control and PTEN KD hESCs show an 80% knockdown of PTEN. c) Western analysis of PTEN, p-AKT, p-GSK3ß, and p-S6 protein levels in control and PTEN KD hESCs. d) FACS analysis showing the percentage of TRA-1-81 positive HSF1 hESCs that are positive for p-AKT. e) Immunofluorescent staining of ß-catenin (red) and OCT4 (green) in control and PTEN KD hESCs. Experiments were performed with control and PTEN KD passage 65-70 hESCs. Data shown are representative of three independent experiments. Error bars represent standard errors and asterisks denote p-value < 0.05.
STEM_748_sm_supplFig2.pdf16KSupplemental Figure 2: PTEN KD hESCs are less likely to differentiate spontaneously when cultured on Matrigel compared to control cells. Bright field images of H9 (passage 40) and HSF1 (passage 65) control and PTEN KD hESCs cultured on Matrigel for 5 days. Arrows denote fibroblasts and differentiated cells that surround control hESCs.
STEM_748_sm_supplFig3.pdf15KSupplemental Figure 3: PTEN KD hESCs have increased self-renewal over 5 passages. a) Control and PTEN KD H9 hESCs were plated as single cells, cultured under self-renewing conditions for 7 days, and stained for AP. Passage 5 colonies shown. b) Quantification of the number of AP+ colonies in (a) after 3 and 5 passages. c) Immunofluorescent staining of β-catenin (red) and OCT4 (green) of H9 control and PTEN KD hESCs after 5 passages under self-renewing conditions. Experiments were performed with control and PTEN KD passage 47-50 hESCs. Error bars represent standard errors and asterisks denote p-value < 0.05.
STEM_748_sm_supplFig4.pdf13KSupplemental Figure 4: HSF1 PTEN KD hESCs differentiate abnormally as embryoid bodies. a) QPCR of lineage markers representing all three germ layers in control and PTEN KD day 5 EBs. Graph shows control and PTEN KD levels in EBs relative to control hESCs. b) QPCR of NEUROD1 expression in control and PTEN KD day 5 EBs. c) Western analysis of PTEN, p-AKT and OCT4 in day 5 HSF1 EBs. Experiments were performed with passage 65-70 control and PTEN KD hESCs. Western blots are representative of three independent experiments. Error bars represent standard errors of three independent experiments and asterisks denote p-value < 0.05.
STEM_748_sm_supplFig5.pdf13KSupplemental Figure 5: H9 PTEN KD EBs maintain OCT4 and NANOG gene and protein levels compared to control EBs. After 5 days of EB differentiation, the levels of OCT4 and NANOG were evaluated using qPCR (a) and Western blot assays (b). Data shown are representative of three independent experiments. Error bars represent standard errors of three independent experiments and asterisks denote p-value < 0.05.
STEM_748_sm_supplFig6.pdf12KSupplemental Figure 6: H9 PTEN KD hESCs have aberrant late-stage neuroectoderm differentiation. Immunofluorescent staining of PAX6 (red), TUJ1 (green) and Hoechst (blue) after neural directed differentiation of control and PTEN KD differentiated cells. All experiments were performed using control and PTEN KD passage 37-45 hESCs. Immunofluorescent staining is representative of three independent experiments.
STEM_748_sm_supplFig7.pdf16KSupplemental Figure 7: HSF1 PTEN KD hESCs have increased pluripotency gene expression when cultured under self-renewing conditions. a) Quantification of FACS analysis showing the percentage of high TRA-1-81 positive cells in control and PTEN KD hESCs cultured under self-renewing conditions. b) FACS analysis of nuclear expression of OCT4 and NANOG in control and PTEN KD hESCs. Graph shows the percentage of TRA-1-81 positive hESCs that were positive for OCT4 or NANOG. c) Pluripotency marker gene expression was evaluated using qPCR. d) Expression of the early progenitor markers, PAX6 and GATA4, in control and PTEN KD hESCs. Graphs show PTEN KD fold over control. All experiments were performed using control and PTEN KD passage 68-72 hESCs. Error bars represent standard error of three independent experiments and asterisks denote p-values < 0.05.
STEM_748_sm_supplinfo.pdf84KSupplemental Data
STEM_748_sm_supptable.pdf16KSupplemental Table 1: List of primers used for qPCR.

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