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AML1-ETO transduced human cord blood cells, CD34 selected, compared to normal cord blood cells, CD34 selected

ABSTRACT: AML1-ETO expression in normal human umbilical cord blood CD34+ cells leads to long-term proliferation of an early self-renewing primitive progenitor cell with multilineage potential and stem cell ability, but these cells do not induce leukemia in immunodeficient mice. This comparative microarray study was initiated to determine the differences in the transcriptome of AML-ETO-expressing CD34+ cells after extended culture in vitro, using normal cord blood cells expanded for 6-8 weeks in vitro and subsequently purified for the CD34+ population as the control comparison. Keywords: Disease state analysis; comparison of changes in transcriptome due to long-term AML1-ETO expression in normal human hematopoietic CD34+ progenitor cells

ORGANISM(S): Homo sapiens

PROVIDER: GSE8023 | GEO | 2007/12/20

SECONDARY ACCESSION(S): PRJNA100827

REPOSITORIES: GEO

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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.

Project description:MLL-AF9 expression in normal human umbilical cord blood CD34+ cells leads to long-term proliferation of a myeloid progenitor cell with leukemogenic potential. Expression of a Core Binding Factor leukemia fusion (AML1-ETO or CBFbeta-SMMHC) in human CD34+ cells results in self-renewal of primitive progenitor cells with multilineage potential and stem cell ability, but these cells do not induce leukemia in immunodeficient mice. This comparative microarray study was initiated to determine how faithful these cell cultures are to the transcriptome of patient samples expressing each of these different fusion proteins, and to analyze the signaling pathways that are unique to CBF cultures and MLL-fusion cultures, with the hope of determining why the MLL-fusion cells are leukemogenic while the CBF cells are not. Experiment Overall Design: We have established a culture system whereby we retrovirally transduce human CD34+ cells, obtained from cord blood, with the leukemia fusion genes MLL-AF9 (MA9), AML1-ETO (AE) or CBFbetaMYH11 (CM). Cells expressing each of these fusion proteins are able to proliferate long-term in vitro in a cytokine dependent manner. These cultures resemble somewhat the blast samples from patients that contain these different fusion proteins, in that MA9 cells are predominantly monocytic (M5-like), AE cells have a large population of primitive CD34+ cells with continued abnormal differentiation (M2-like) and CM cells also contain a population of primitive CD34+ cells with continued abnormal differentiation but also have a significant population of abnormal eosinophils (M4Eo). We grow these cells in the presence of fetal bovine serum (FBS), and also grow them in serum-free conditions using the BIT supplement from Stem Cell Technologies. For the current experiments we used cell cultures that had been proliferating in vitro for 8-12 weeks, in either serum-free conditions or with FBS. Replicate samples were included in some instances, and the same clonal cultures that were grown under either BIT or FBS were also included.

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AML1-ETO transduced human cord blood cells, CD34 selected, compared to normal cord blood cells, CD34 selected

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