The hESC line H1 (NIH code: WA01) was maintained on irradiated primary murine embryonic feeders (PMEF), and hEB were formed from hESC clumps in semisolid, serum-supplemented medium, exactly as previously described (Zambidis et al., 2005). Twenty-four hours after hESC plating, (Day 2) simple hEB were harvested, washed, re-suspended in PBS in 50-ml conical tubes, and collected by gentle gravity settling for 2-3 minutes. Formed hEB were re-suspended into 6-well ultra-non-adherent plates (Corning) at ~100-200 hEB/ml in SF liquid differentiation medium (SF LDM), consisting of serum-free expansion medium (SFEM, StemCell Technologies) supplemented with 50 ug/ml ascorbic acid (SIGMA), 1% EXCYTE, 0.5% insulin/transferrin/selenium (Invitrogen), 3% PFHM-II (Invitrogen), and penicillin/streptomycin (SIGMA). Various combinations of human growth factors (all from R&D Systems, Peprotech, or Invitrogen) were added directly into SF LDM at 2-20 days of hEB development: BMP4 (50 ng/ml), VEGF (50 ng/ml), FGF1 (50 ng/ml+ 5ug/ml heparin sulfate, SIGMA), FGF2 (50 ng/ml+ 5 ug/ml heparin sulfate), murine Nodal (50 ng/ml), IGF-II (50 ng/ml), murine wnt3a (50 ng/ml), and activin A (50 ng/ml). hEB cultures were fed by hemi-depletion and replacement of medium every 2-3 days with fresh SF LDM and GFs. To expand hEB-derived hematopoietic progenitors, enzymatically-digested (Accutase, SIGMA) day 9-10 hEB clumps, differentiated as above, were transferred into SF LDM and allowed to adhere to gelatinized tissue culture plates. SF cultures were supplemented with various hematopoietic inductive factors including: BMP4 (50 ng/ml), SCF (50 ng/ml), Flt3L (50 ng/ml), TPO (50 ng/ml), GM-CSF (50 ng/ml), G-CSF (50 ng/ml), IL-3 (20 ng/ml), IL-6 (20 ng/ml), VEGF (20 ng/ml), EPO (2 units/ml), FGF1 (10 ng/ml) and FGF2 (10 ng/ml).
Extracted molecule
total RNA
Extraction protocol
Total RNA are isolated from cell pellets using TriZol reagent(Invitrogen) followed by Rneasy mini kit (Qiagen) clean-up.
Label
Cy3
Label protocol
400 ng total Rna is amplified and labeled using Agilent low in put RNA amplification and labeling kit and Cy3-CTP (Perkin Elmer) following manufacturer's protocol.
Hybridization protocol
Using Agilent hybridization chambers and oven following manufacturer's protocol (65oC for 17 hours)
Scan protocol
Scanned on an Agilent G2565AA scanner.
Description
Individual sample
Data processing
Processed signal intensities are imported into GeneSpring Gx 7.3 and normalized to the median signal intensities of each arrays. Intensities of replicate probes are represented as single value of average.
