Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

In vitro Expansion of Hematopoietic Stem and Progenitor Cells Induces Tightly Regulated DNA-Hypermethylation

ABSTRACT: This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

ORGANISM(S): Homo sapiens

SUBMITTER: Wolfgang Wagner

PROVIDER: E-GEOD-40800 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Hematopoietic stem and progenitor cells acquire distinct DNA-hypermethylation during in vitro culture.

Weidner Carola Ingrid CI Walenda Thomas T Lin Qiong Q Wölfler Monika Martina MM Denecke Bernd B Costa Ivan Gesteira IG Zenke Martin M Wagner Wolfgang W

Scientific reports 20131128

Hematopoietic stem and progenitor cells (HPCs) can be maintained in vitro, but the vast majority of their progeny loses stemness during culture. In this study, we compared DNA-methylation (DNAm) profiles of freshly isolated and culture-expanded HPCs. Culture conditions of CD34(+) cells - either with or without mesenchymal stromal cells (MSCs) - had relatively little impact on DNAm, although proliferation is greatly increased by stromal support. However, all cultured HPCs - even those which remai ...[more]

PMID: 24284763

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Project description:Mononuclear cells were isolated from umbilical cord blood (UCB) using Lymphoprep sucrose-gradient centrifugation (1.077 g/ml, Nycomed Pharma, Oslo, Norway). Immunomagnetic cell separation, using magnetic beads coated with CD34 antibodies (Miltenyi Biotec, Gladbach, Germany), was performed to isolate CD34-positive hematopoietic progenitor cells (CB-CD34+ cells). To generate retrovirus, bicistronic retroviral DNA constructs were used expressing the ETV6-RUNX1 or AML1-ETO fusion gene and enhanced Green Fluorescent Protein (eGFP). As a control, a construct expressing only eGFP was used. Both vectors consisted of a pMSCV promoter region, an internal ribosomal entry site and an ampicillin resistance cassette. HEK293T cells were co-transfected with these constructs and second-generation retroviral packaging vectors using XtremeGENE 9 tranfection reagents (Roche, Basel, Switzerland). Viral particles were collected in IMDM 48 hours after transfection. CD34+ haematopoietic progenitors, pre-cultured overnight as described above, upon which cells were divided in two fractions. One fraction was transduced with ETV6-RUNX1-IRES-eGFP or AML1-ETO-IRES-eGFP, while the other fraction was transduced with control EV-IRES-eGFP. Transductions were performed with fresh retrovirus in retronectin (Takara, Otsu, Japan) coated wells. After sorting, DAPI- CD34+ GFP+ CB-CD34+ cells were lysed and RNA was extracted using Nucleospin RNA XS extraction columns according to manufacturer’s protocol (Macherey-Nagel, Düren, Germany). Quality of RNA was determined by on-chip-electrophoresis using a RNA Pico Chip according to manufacturer’s protocol (Agilent Technologies, Santa Clara, CA, USA). RNA Integrity scores (RIN) were higher than 8 for all samples. RNA was subsequently linearly amplified using the Nugen WT-Amplification™ pico system (Nugen, San Carlos, CA, USA). This system is based on RNA-dependent DNA polymerase activity and was previously reported to be most suitable for amplification and gene expression of picograms of input RNA.

Dataset Information

In vitro Expansion of Hematopoietic Stem and Progenitor Cells Induces Tightly Regulated DNA-Hypermethylation

Publications

Hematopoietic stem and progenitor cells acquire distinct DNA-hypermethylation during in vitro culture.

Similar Datasets

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