Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions

ABSTRACT: To identify the molecular characterisitics of parallel lineage-biased MPP populations arising from hematopoietic stem cells (HSC) we conducted genome-wide analyses of hematopoietic stem, progenitor and mature myeloid cell populations using Affymetrix Gene ST1.0 arrays. Microarray analysis of 3-5 biological replicates of the indicated hematopoietic populations, isolated by FACS sorting from C57BL/6 mouse BM. Immunophenotypic definitions: Long-term HSC (HSCLT) (Lin-/cKit+/Sca1+/Flk2-/CD48-/CD150+); Short-term HSC (HSCST) (Lin-/cKit+/Sca1+/Flk2-/CD48-/CD150-); MPP2 (Lin-/cKit+/Sca1+/Flk2-/CD48+/CD150+); MPP3 (Lin-/cKit+/Sca1+/Flk2-/CD48+/CD150-); MPP4 (Lin-/cKit+/Sca1+/Flk2+); CMP (Lin-/cKit+/Fc?R-/CD34+); GMP (Lin-/cKit+/Fc?R+/CD34+); Pre-granulocyte (PreGr) (Mac1+/Gr1int); Granulocyte (Gr) (Mac1+/Gr1hi). HSC and GMP samples listed here were also used as controls for our related microarray study GSE48893.

ORGANISM(S): Mus musculus

SUBMITTER: Eric Pietras

PROVIDER: E-GEOD-68529 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We discovered that mice that lack Lsd1 in hematopoietic cells were exhibited increased frequencies of CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs, but completely lacked the lin- c-Kit+ Sca-1- myeloid progenitor compartment. To determine the genes altered by Lsd1-loss, CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs from Lsd1fl/fl and Lsd1fl/fl Mx1Cre mice were FACS-purified to be analyzed by gene expression profiling. Primary CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs were isolated by FACS-sorting, from the bone marrow of Lsd1fl/fl and Lsd1fl/fl Mx1Cre animals, one week after the final p(I:C) dose. Total RNA from three biological replicates per genotype was extracted with the RNeasy Micro Kit (QIAGEN), treated with DNaseI, reverse transcribed. RNA extracted from CD150+ CD48- lin- c-Kit+ Sca-1+ cells was amplified with the Ovation Pico WTA RNA Amplification System 2 (NuGEN Technologies). Single-stranded cDNA amplification products were purified using QIAquick PCR Purification Kit (QIAGEN) and labeled with the FL-Ovation cDNA Biotin Module V2 (NuGEN). The Biotin-labeled cDNA was used to hybridize to Affymetrix expression arrays using the Mouse Genome 430 2.0 array platform. Primary CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs were isolated by FACS-sorting, from the bone marrow of Lsd1fl/fl and Lsd1fl/fl Mx1Cre animals, one week after the final p(I:C) dose. Total RNA from three biological replicates per genotype was extracted with the RNeasy Micro Kit (QIAGEN), treated with DNaseI, reverse transcribed. RNA extracted from CD150+ CD48- lin- c-Kit+ Sca-1+ cells was amplified with the Ovation Pico WTA RNA Amplification System 2 (NuGEN Technologies). Single-stranded cDNA amplification products were purified using QIAquick PCR Purification Kit (QIAGEN) and labeled with the FL-Ovation cDNA Biotin Module V2 (NuGEN). The Biotin-labeled cDNA was used to hybridize to Affymetrix expression arrays using the Mouse Genome 430 2.0 array platform. Primary CD150+ CD48- lin- c-Kit+ Sca-1+ LT-HSCs were isolated by FACS-sorting, from the bone marrow of Lsd1fl/fl and Lsd1fl/fl Mx1Cre animals, one week after the final p(I:C) dose. Total RNA from three biological replicates per genotype was extracted with the RNeasy Micro Kit (QIAGEN), treated with DNaseI, reverse transcribed. RNA extracted from CD150+ CD48- lin- c-Kit+ Sca-1+ cells was amplified with the Ovation Pico WTA RNA Amplification System 2 (NuGEN Technologies). Single-stranded cDNA amplification products were purified using QIAquick PCR Purification Kit (QIAGEN) and labeled with the FL-Ovation cDNA Biotin Module V2 (NuGEN). The Biotin-labeled cDNA was used to hybridize to Affymetrix expression arrays using the Mouse Genome 430 2.0 array platform.

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Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions

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