DEV021121 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Characterization of ΔNβ-cateninER-expressing hES cells. (A) G-banding chromosome analysis of ΔNβ-cateninER-expressing hES cells. The transgenic cell lines derived from different ES cell lines are shown (K1βcatER derived from KhES-1 cells, K3βcatER derived from KhES-3 cells). No abnormalities were apparent in these transgenic cell lines. (B) FACS data for stem cell antigens expressed in the undifferentiated parental and ΔNβ-cateninER-expressing hES cells. Fluorescence histograms are shown from flowcytometry of undifferentiated hES cell lines, KhES-1 and KhES-3 cells cultured with or without 4OHT (100 nM) for 3 days, and undifferentiated hES cells stably expressing ΔNβ-cateninER. Cells were immunostained for each of the antibodies: SSEA3, SSEA4, TRA-1-60, TRA-1-81. (C) Expression of Oct4 and Nanog protein in parental and ΔNβ-cateninER-expressing hES cells. Cells were immunostained for indicated antibodies. Nuclei were counterstained by DAPI. Scale bar: 100 µm. (D) The qPCR analysis on RNA isolated from parental and ΔNβ-cateninER-expressing cells cultured with or without 4OHT for 3 days was performed using specific primers for indicated genes. K1, KhES-1 cells; K3, KhES-3 cells; K1βcat, ΔNβ-cateninER-expressing KhES-1 cells; K3βcat, ΔNβ-cateninER-expressing KhES-3 cells; v, vehicle control; OHT, 4OHT-treated.

• Supplemental Figure S2 -

  Fig. S2. The effect of various 4OHT and Noggin conditions on cell fate determination. (A) Dose-dependent effect of Noggin on the cell fate change from mesoderm to endoderm progenitors. Cells expressing ΔNβ-cateninER were treated with Noggin (0-500 ng/ml) in the presence or absence of 4OHT for 3 days, and then analyzed by qPCR. (B) Effect of 4OHT exposure duration on cell fate determination. Cells expressing ΔNβ-cateninER were treated with 4OHT (O) for the first day (1), the first 2 days (2) or for 3 consecutive days (3) in the presence or absence of Noggin (N), and then analyzed by qPCR at day 3. v, vehicle control. (C) Effect of the Noggin exposure duration on cell fate determination. Cells expressing ΔNβ-cateninER were treated with Noggin (N, 250 ng/ml) for the final day (1), the final 2 days (2) or for 3 consecutive 3 days (3) in the presence or absence of 4OHT (OHT), and then analyzed by qPCR at day 3.

• Supplemental Figure S3 -

  Fig. S3. Dose-dependent effects of BIO and 4OHT on the cell fate change from undifferentiated hES to the PS. (A) The hES cell lines, KhES-1 and KhES-3 cells, were treated with BIO-Acetoxime at different concentrations (0-10 µM) for 3 days, and then analyzed by qPCR using specific primers for the genes indicated. The relative gene expression was normalized to the first sample in the data set (see Materials and methods). ES, undifferentiated ES cells. (B) Cells expressing ΔNβ-cateninER were treated with 4OHT at different concentrations (0-500 nM) for 3 days and then analyzed as described in A.