Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profile of murine bone marrow endosteal populations (2)

ABSTRACT: A subset of osteoblasts functions as a key component of the hematopoietic stem cell niche. But it is still unclear that the precise cellular and molecular contribution of endosteal cell populations on the HSC supportive microenvironment. We purified the endosteal cells and subdivided them into mesenchymal progenitor cells (MPCs: PDGF-Ra(+)Sca-1(+)) and two osteoblast populations (PDGF-Ra(-)Sca-1(-) and PDGF-Ra(+)Sca-1(-)). In this study, to clarify the characteristics of these fractionated cells in detail, gene expression analysis were performed using the GeneChip Mouse Genome Array 430 2.0 (Affymetrix). The bone fragments of femur and tibiae were treated with collagenase and endosteal cells were obtained. For enrichment of non-hematopoietic and non-endothelial population, CD45(-)CD31(-)Ter119(-) population was isolated and then the expressions of Sca-1 and PDGF-Ra was analyzed by FACS. FACS analysis showed that CD45(-)CD31(-)Ter119(-) cells were subdivided into PDGF-Ra(+)Sca-1(+), PDGF-Ra(-)Sca-1(-) and PDGF-Ra(+)Sca-1(-). These three populations were sorted and RNA samples were used for amplification, labeling, and hybridization to independent arrays.

ORGANISM(S): Mus musculus

SUBMITTER: Fumio Arai

PROVIDER: E-GEOD-17570 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Murine bronchioalveolar stem cells play a key role in pulmonary epithelial maintenance and repair but their molecular profile is poorly described so far. In this study, we used antibodies directed against Sca-1 and CD34, two markers originally ascribed to pulmonary cells harboring regenerative potential, to isolate single putative stem cells from murine lung tissue. The mean detection rate of positive cells was 8 per 106 lung cells. We then isolated and globally amplified the mRNA of positive cells to analyze gene expression in single cells. The resulting amplicons were then used for molecular profiling by transcript specific polymerase chain reaction (PCR) and global gene expression analysis using microarrays. Single marker-positive cells displayed a striking heterogeneity for the expression of epithelial and mesenchymal transcripts on the single cell level. Nevertheless, they could be subdivided into two cell populations: Sca-1+/CD34- and Sca-1+/CD34+ cells. In these subpopulations, transcripts of the epithelial marker Epcam (CD326) were exclusively detected in Sca-1+/CD34- cells (p = 0.03), whereas mRNA of the mesenchymal marker PdgfrM-NM-1 (CD140a) was detected in both subpopulations and more frequently in Sca-1+/CD34+ cells (p = 0.04). FACS analysis confirmed the existence of a PdgfrM-NM-1 positive subpopulation within Epcam+/Sca-1+/CD34- epithelial cells. Gene expression analysis by microarray hybridization identified transcripts differentially expressed between the two cell types as well as between epithelial reference cells and Sca-1+/CD34+ single cells, and selected transcripts were validated by quantitative PCR. Our results suggest a more mesenchymal commitment of Sca-1+/CD34+ cells and a more epithelial commitment of Sca-1+/CD34- cells. In summary, the study shows that single cell analysis enables the identification of novel molecular markers in yet poorly characterized populations of rare cells. Our results could further improve our understanding of Sca-1+/CD34+,- cells in the biology of the murine lung. Single cells of 10 Sca-1+/CD34+/CD31-/CD45-, 7 Sca-1+/CD34-/CD31-/CD45-, and 12 Sca-1-/CD34-/CD31-/CD45- were analyzed. Although the raw data are two channel only Cy5 signal of each file as analyzed. The Cy5 channel for each gene is normalized to the average Cy5 intensity of the gene across all samples.

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Gene expression profile of murine bone marrow endosteal populations (2)

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