Control of Developmental Regulators by Polycomb in Human Embryonic Stem Cells

Mapping RNA Polymerase II Occupancy in Embryonic Stem Cells

• Human ES Cells
• Growth Conditions
• Immunohistochemical Analysis of Pluripotency Markers
• Teratoma Formation
• Embryoid Bodies (EB)
• Technology and Protocols
• Antibodies
• Chromatin Immunoprecipitations
• Microarray Design

• Download Data
• Data Analysis

Human embryonic stem (ES) cells were obtained from WiCell (Madison, WI; NIH Code WA09) and grown according to the supplier’s recommendations. Briefly, passage 34 cells were grown in KO-DMEM medium supplemented with serum replacement, basic fibroblast growth factor (FGF), recombinant human leukemia inhibitory factor (LIF) and a human plasma protein fraction. Detailed protocol information on human ES cell growth conditions and culture reagents are available at http://www.mcb.harvard.edu/melton/hues.

In order to minimize any murine embryonic fibroblast (MEF) contribution in our analysis, H9 cells were cultured on a low density of irradiated MEFs (ICR MEFs) resulting in a ratio of approximately >8:1 H9 cell to MEF (Figure S1).

Figure S1. Human H9 ES cells cultured on a low density of irradiated murine embryonic fibroblasts. Bright-field image of H9 cell culture.

The culture of H9 on low-density MEFs had no adverse affects on cell morphology, growth rate, or undifferentiated status as determined by immunohistochemistry for pluripotency markers (e.g. Oct4, SSEA-3, Tra-1-60). In addition, H9 cells grown on a minimal feeder layer maintain the ability to generate derivates of ectoderm, mesoderm, and endoderm upon differentiation.