Timecourse of in vitro erythroid differentiation of human adult-derived peripheral blood CD34+ cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE4655 | GEO | 2006/11/09
SECONDARY ACCESSION(S): PRJNA95299
REPOSITORIES: GEO
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Project description:The aim of the study is to develop a scalable continuous agitation suspension culture bioprocess for differentiation of hiPSCs towards erythroid cells. Using process optimization, we developed the entire process completely in agitation suspension culture condition starting from hiPSC expansion through to hematopoietic induction and erythroid differentiation. Transcriptome profiling was carried out to facilitate comparisons between erythroid cells differentiated from hiPSCs using this bioprocess, and erythroid cells differentiated from cord blood and adult CD34+ cells.
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Project description:The Affymetrix Human Gene 2.0 ST array was used to measure differential expression of RNA isolated from normal and Diamond Blackfan anemia (DBA) erythroid progenitors after ex vivo expansion of circulating, peripheral blood derived hematopoietic stem cells under erythroid growth conditions. The gene-level probe summaries reported in this series were computed using RMA as implemented in the Bioconductor package Oligo v1.36.1. Diamond Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by erythroid aplasia, usually without perturbation of other hematopoietic lineages. Approximately 65% of DBA patients with autosomal dominant inheritance have heterozygous mutations or deletions in ribosomal protein (RP) genes while <1% of patients with X-linked inheritance have been identified with mutations in the transcription factor, GATA1. Erythroid cells from patients with DBA have not been well characterized and the mechanisms underlying the erythroid specific effects of either RP or GATA1 associated DBA remain unclear. We have developed an in vitro culture system to expand peripheral blood CD34+ progenitor cells from patients with DBA and differentiate them into erythroid cells. Cells from patients with RP or GATA1 mutations showed decreased proliferation and delayed erythroid differentiation compared to controls. RNA transcript analyses of erythroid cells from controls and patients with RP or GATA1 mutations showed distinctive differences, with upregulation of heme biosynthesis genes prominently in RP-mediated DBA and failure to upregulate components of the translational apparatus in GATA1-mediated DBA. Our data show that dysregulation of translational function is a common feature of DBA caused by both RP and GATA1 mutations.
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Project description:Analysis of mobilized peripheral blood CD34+ cells from a healthy volunteer under erythroid differentiation conditions with and without stimulation to the BMP or Wnt signaling pathways. For erythroid differentiation, expanded CD34+ cells were placed in Stemspan SFEM medium supplemented with 2% pen/strep, 20ng/ml SCF, 1U/ml Epo, 5ng/ml IL3, 2uM dexamethasone, and 1uM beta-estradiol. Arrays were performed 2 hours after addition of cytokines. For signaling pathway stimulation, cells were exposed to 0.5uM BIO (a GSK3 inhibitor) for Wnt pathway activation, 25ng/ml rhBMP4 for BMP pathway activation, or vehicle control for 2 hours. Three biological replicates were performed per treatment group. We used microarrays to detail the global program of gene expression changes after Wnt or BMP pathway stimulation in human CD34+ hematopoietic progenitors under erythroid differentiation conditions. To investigate the changes to gene expression in human CD34+ hematopoietic progenitors following stimulation of the Wnt or BMP pathways during the early stages of erythroid differentiation. Three biological replicates were performed per treatment group.
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