Comment[ArrayExpressAccession] E-GEOD-44136 MAGE-TAB Version 1.1 Public Release Date 2013-12-31 Investigation Title 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 Comment[Submitted Name] 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 Experiment Description The supply of red blood cells (RBCs) is not sufficient in many developing countries or in developed countries for patients who need chronic transfusion from best-matched donors. Ex vivo expansion and maturation of human erythroid precursor cells (erythroblasts) could represent a potential solution. Proliferating erythroblasts can be expanded from human umbilical cord blood mononuclear cells (CB MNCs) ex vivo for 10^6-10^7 fold (in ~50 days) before undergoing senescence. Here, we report that ectopic expression of three to four genetic factors that have been used for iPS cell derivation enables CB-derived erythroblasts to undergo extended ex vivo expansion (M-bM-^IM-%10^51 fold in ~9 months) in a defined suspension culture condition without change of cell identity or function. These vastly expanding erythroblasts maintain homogeneously immature erythroblast phenotypes, a normal diploid karyotype and dependence on specific combination of cytokines and hormone for survival and proliferation throughout the continuous expansion period. When switched to a culture condition for terminal maturation, these immortalized erythroblasts gradually exit cell cycle, decrease cell size, accumulate hemoglobin, condense nuclei and eventually give rise to enucleated hemoglobin-containing erythrocytes. Our result may ultimately lead to the development of unlimited sources of cultured RBCs for optimally-matched or personalized transfusion medicine. We compared the global gene expression profiles of different human cell types: iE: immortalized erythroblasts generated by genetic reprogramming from pCBE; pCBE: primary cord blood-derived erythroblasts; CD34+: CD34+ purified hematopoietic stem/progenitor cells from adult blood or fetal liver; TF-1: a human erythroleukemia cell line; ESC: human embryonic stem cells; iPSCs: human induced pluripotent stem cells. We want to see the relationship among these cell types. We included multiple samples (biological replicates) for most cell types. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Huang Huang Talbot Jr. Tsang Person First Name Xiaosong Xiaosong Conover Kitman Person Email xhuang18@jhmi.edu Person Affiliation Johns Hopkins University Person Address Medicine/Hematology, Johns Hopkins University, 733 North Broadway, Rm780, Baltimore, Maryland, USA Person Roles submitter Protocol Name P-GSE44136-1 P-GSE44136-5 P-GSE44136-6 P-GSE44136-2 P-GSE44136-3 P-GSE44136-4 P-GSE44136-7 Protocol Description Image analysis used the Affymetrix Command Console version 3 (AGCC v3.0) software, processed with the Expression Console PLIER algorithm and the manufacturerM-bM-^@M-^Ys 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. ID_REF = VALUE = RMA Log2 values One round amplification protocol for total RNA following AffymetrixM-bM-^@M-^Y specifications using T7 promoter engineered random primers for cDNA synthesis, and AmbionM-. WT Expression Kit (www.affymetrix.com). 16hrs at 45M-0 C with rotation (60rpm) as described by Affymetrix in their GeneChipM-. Expression Wash, Stain and Scan User Manual (www.affymetrix.com). no treatment pCBE19: After thawing, frozen CB MNCs (5x106/ml) were cultivated in serum-free medium (SFM; 50% IMDM with 50% HamM-bM-^@M-^Ys 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 M-5M; Sigma), insulin-like growth factor 1 (IGF-1; 40 ng/mL), stem cell factor (SCF; 100 ng/mL), and holo-transferrin (100 M-5g/ml; Sigma), termed M-bM-^@M-^\erythroblast expansion mediumM-bM-^@M-^] 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 M-CM-^W 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. RNeasy according to manufacture's protocol Using AffymetrixM-bM-^@M-^Y GeneChip Scanner 3000 7G and default parameters described by the manufacturer in their GeneChip Expression Analysis Technical Manual. Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name GENETIC FACTORS EXPANSION DAYS TISSUE OF ORIGIN Experimental Factor Type genetic factors expansion days tissue of origin Comment[SecondaryAccession] GSE44136 Comment[GEOReleaseDate] 2013-12-31 Comment[ArrayExpressSubmissionDate] 2013-02-07 Comment[GEOLastUpdateDate] 2014-01-01 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-44136.sdrf.txt