tissue of origin: adult blood expansion days: passage 36 genetic factors: OCT4, SOX2, KLF4, C-MYC, LIN28, Large T antigen
Treatment protocol
no treatment
Growth protocol
pCBE19: After thawing, frozen CB MNCs (5x106/ml) were cultivated in serum-free medium (SFM; 50% IMDM with 50% Ham’s F12 (Invitrogen), synthetic lipids (Invitrogen), insulin-transferrin-selenium supplement (Invitrogen) and 5 mg/ml BSA (Sigma), 50 ug/ml of ascorbic acid (Sigma) and 2 mM glutamax ) supplemented with Epo (2 U/mL; R&D systems), Dexamethasone (1 µM; Sigma), insulin-like growth factor 1 (IGF-1; 40 ng/mL), stem cell factor (SCF; 100 ng/mL), and holo-transferrin (100 µg/ml; Sigma), termed “erythroblast expansion medium” in gelatin-coated wells. Primary cultures of erythroblasts established after several days were kept less than 1x106 cells/ml by daily cell counting and partial medium changes. iE2: Grow in the erythroblast expansion medium. After the culture cells become proliferating constantly, only partial medium change and cell passaging every 2 or 3 days are performed to keep cell density at or below 5 × 105 cells/ml. CD34+: primary cells isolated by magenetic beads binding to CD34 cell surface marker. TF-1: RPMI-1640+10% FBS+ 3U/ml hEPO. iPSC and ESC: standard ESC medium and on MEF feeder.
Extracted molecule
total RNA
Extraction protocol
RNeasy according to manufacture's protocol
Label
biotin
Label protocol
One round amplification protocol for total RNA following Affymetrix’ specifications using T7 promoter engineered random primers for cDNA synthesis, and Ambion® WT Expression Kit (www.affymetrix.com).
Hybridization protocol
16hrs at 45° C with rotation (60rpm) as described by Affymetrix in their GeneChip® Expression Wash, Stain and Scan User Manual (www.affymetrix.com).
Scan protocol
Using Affymetrix’ GeneChip Scanner 3000 7G and default parameters described by the manufacturer in their GeneChip Expression Analysis Technical Manual.
Description
Human induced pluripotent stem cells derived from adult blood cells iPSC2
Data processing
Image analysis used the Affymetrix Command Console version 3 (AGCC v3.0) software, processed with the Expression Console PLIER algorithm and the manufacturer’s specifications. probe group file: HuGene-1_0-st-v1.r4.pgf meta-probeset file: HuGene-1_0-st-v1.na33.1.hg19.probeset.csv, HuGene-1_0-st-v1.na33.1.hg19.transcript.csv RMA Log2 values from the Partek Genomics Suite, using all probesets, were summarized for gene-level analysis.
Expression data from primary CB erythroblasts, immortalized/induced erythroblasts, Fetal liver CD34+ blood stem cells, adult CD34+ blood stem cells, erythroleukemia cell line (TF-1) and human ESC and iPSCs
