Unknown,Transcriptomics,Genomics,Proteomics

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Landscape of Hematopoiesis Described in Induced Pluripotent Stem Cells and Human Bone Marrow

ABSTRACT: Granulopoietic differentiation of myeloid progenitor cells derived from control iPSCs was performed in a two-step liquid culture. At the end of culture, stages of differentiation were identified by morphological analysis and submitted for RNA-sequencing analysis in order to provide insight into the genomic landscape of myeloid lineage hematopoiesis as modeled by the in vitro induced differentiation of iPSCs as compared to in vivo bone marrow-derived promyelocytes. Peripheral blood from healthy controls was obtained and iPSC were generated from peripheral blood mononuclear cells. Hematopoietic progenitors generated from control iPSCs when cultured in myeloid expansion medium containing 50ng/mL SCF, 10ng/mL IL-3 and 10ng/mL GM-CSF for 5 days at which point cells were stained for CD45-Pacific blue, CD34-PECy7, CD33-AP, CD11b-APC-Cy7, CD15-FITC. 7-AAD was used to eliminate the dead cells. The promyelocytic population (CD45+CD34-CD33+CD11b-CD15+/lo) was sorted and the RNA from control iPSC promyelocytes was isolated using QIAGEN RNAeasy mini kit. The RNA samples were processed for RNA-seq analyses using RNA-seq protocol from NuGEN and Illumina. The amplified products were sequenced to analyze the gene expression profile of each replicate sample. A total of 20 samples were used in this analysis to characterize and compare iPSC in vitro differentiated myeloid cells with those isolated from human bone marrow.

ORGANISM(S): Homo sapiens

SUBMITTER: Rebekah Karns

PROVIDER: E-GEOD-69622 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We performed a genome-wide analysis of gene expression in primary human CD151 myeloid progenitor cells. By using the serial analysis of gene expression (SAGE) technique, we obtained quantitative information for the expression of 37,519 unique SAGE-tag sequences. Of these unique tags, (i) 25% were detected at high and intermediate levels, whereas 75% were present as single copies, (ii) 53% of the tags matched known expressed sequences, 34% of which were matched to more than one known expressed sequence, and (iii) 47% of the tags had no matches and represent potentially novel genes. The correct genes were confirmed by application of the generation of longer cDNA fragments from SAGE tags for gene identification (GLGI) technique for high-copy tags with multiple matches. A set of genes known to be important in myeloid differentiation were expressed at various levels and used different spliced forms. This study provides a normal baseline for comparison of gene expression in myeloid diseases. The strategy of using SAGE and GLGI techniques in this study has broad applications to the genome-wide identification of expressed genes. (Lee S et al. The pattern of gene expression in human CD15+ myeloid progenitor cells. Proc Natl Acad Sci U S A. 98(6):3340-5, 2001; Lee S et al. Gene expression profiles in acute myeloid leukemia with common translocations using SAGE. Proc Natl Acad Sci U S A. 103(4):1030-5, 2006) Human bone-marrow mononuclear cells were isolated from bone marrow with FicollyPaque solution and stored in liquid nitrogen. Cells from three donors were thawed at 37°C, pooled, and used immediately for the isolation of myeloid progenitor cells with CD15 magnetic beads (Dynal, Oslo, Norway). The cells isolated by CD15 magnetic beads were lysed directly with TRIzol reagent for isolation of total RNA. mRNA was purified from 5 mg of total RNA with oligo(dT)25 beads. cDNA was synthesized with a cDNA synthesis kit, and SAGE was performed according to the SAGE protocol with the exceptions described in the original paper. SAGE-tag sequences were collected with the Big-Dye sequencing kit and ABI377 sequencer, and tag sequences were extracted with SAGE 300 software.

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Landscape of Hematopoiesis Described in Induced Pluripotent Stem Cells and Human Bone Marrow

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