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Combined characterization of microRNA and mRNA profiles delineates early differentiation pathways of CD133+ and CD34+ hematopoietic stem and progenitor cells.

Bissels U, Wild S, Tomiuk S, Hafner M, Scheel H, Mihailovic A, Choi YH, Tuschl T, Bosio A - Stem Cells (2011)

Bottom Line: Using quantitative whole genome miRNA microarray and sequencing-based profiling, we found that between 109 (CD133(+) ) and 216 (CD34(-) CD133(-) ) miRNAs were expressed.In conclusion, the miRNAs expressed differentially between the CD133(+) and CD34(+) CD133(-) cells are involved in inhibition of differentiation, prevention of apoptosis, and cytoskeletal remodeling.These results are highly relevant for stem cell-based therapies with CD133(+) cells and delineate for the first time how the stem cell character of CD133(+) cells is defined by the expression of specific miRNAs.

View Article: PubMed Central - PubMed

Affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.

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Experimental procedure for miRNA expression analysis in hematopoietic stem cells and hematopoietic progenitor cells. (A): After separation of CD133+, CD34+CD133−, and CD34−CD133− cells, the RNA was isolated and further processed for miRNA or mRNA profiling. Hybridization was carried out using miRXplore microarrays for miRNA analysis and Agilent Whole Human Genome Oligo Microarrays for mRNA analysis. Subsequently, the miRNA and mRNA profiles were used to elucidate the biological function of the significantly differentially expressed miRNAs. (B): Detailed workflow for the combined miRNA and mRNA analysis. Abbreviations: miRNA, microRNAS.
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fig01: Experimental procedure for miRNA expression analysis in hematopoietic stem cells and hematopoietic progenitor cells. (A): After separation of CD133+, CD34+CD133−, and CD34−CD133− cells, the RNA was isolated and further processed for miRNA or mRNA profiling. Hybridization was carried out using miRXplore microarrays for miRNA analysis and Agilent Whole Human Genome Oligo Microarrays for mRNA analysis. Subsequently, the miRNA and mRNA profiles were used to elucidate the biological function of the significantly differentially expressed miRNAs. (B): Detailed workflow for the combined miRNA and mRNA analysis. Abbreviations: miRNA, microRNAS.

Mentions: CD133+ cells were magnetically isolated from bone marrow by MACS technology. Subsequently, CD34+CD133− cells were isolated using the negative fraction of the first separation (Fig. 1A). Besides the CD34+CD133− cell population, CD34−CD133− cells (the negative fraction of the second separation) were also analyzed and compared with CD133+ cells to get a general impression of miRNA expression in bone marrow cells. As the main cell type present in the CD34−CD133− fraction are erythrocytes, an additional analysis of CD34−CD133− cells after red blood cell (RBC) lysis was performed (Neg −R). Here, the main cell population is neutrophilic granulocytes [37].


Combined characterization of microRNA and mRNA profiles delineates early differentiation pathways of CD133+ and CD34+ hematopoietic stem and progenitor cells.

Bissels U, Wild S, Tomiuk S, Hafner M, Scheel H, Mihailovic A, Choi YH, Tuschl T, Bosio A - Stem Cells (2011)

Experimental procedure for miRNA expression analysis in hematopoietic stem cells and hematopoietic progenitor cells. (A): After separation of CD133+, CD34+CD133−, and CD34−CD133− cells, the RNA was isolated and further processed for miRNA or mRNA profiling. Hybridization was carried out using miRXplore microarrays for miRNA analysis and Agilent Whole Human Genome Oligo Microarrays for mRNA analysis. Subsequently, the miRNA and mRNA profiles were used to elucidate the biological function of the significantly differentially expressed miRNAs. (B): Detailed workflow for the combined miRNA and mRNA analysis. Abbreviations: miRNA, microRNAS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3116150&req=5

fig01: Experimental procedure for miRNA expression analysis in hematopoietic stem cells and hematopoietic progenitor cells. (A): After separation of CD133+, CD34+CD133−, and CD34−CD133− cells, the RNA was isolated and further processed for miRNA or mRNA profiling. Hybridization was carried out using miRXplore microarrays for miRNA analysis and Agilent Whole Human Genome Oligo Microarrays for mRNA analysis. Subsequently, the miRNA and mRNA profiles were used to elucidate the biological function of the significantly differentially expressed miRNAs. (B): Detailed workflow for the combined miRNA and mRNA analysis. Abbreviations: miRNA, microRNAS.
Mentions: CD133+ cells were magnetically isolated from bone marrow by MACS technology. Subsequently, CD34+CD133− cells were isolated using the negative fraction of the first separation (Fig. 1A). Besides the CD34+CD133− cell population, CD34−CD133− cells (the negative fraction of the second separation) were also analyzed and compared with CD133+ cells to get a general impression of miRNA expression in bone marrow cells. As the main cell type present in the CD34−CD133− fraction are erythrocytes, an additional analysis of CD34−CD133− cells after red blood cell (RBC) lysis was performed (Neg −R). Here, the main cell population is neutrophilic granulocytes [37].

Bottom Line: Using quantitative whole genome miRNA microarray and sequencing-based profiling, we found that between 109 (CD133(+) ) and 216 (CD34(-) CD133(-) ) miRNAs were expressed.In conclusion, the miRNAs expressed differentially between the CD133(+) and CD34(+) CD133(-) cells are involved in inhibition of differentiation, prevention of apoptosis, and cytoskeletal remodeling.These results are highly relevant for stem cell-based therapies with CD133(+) cells and delineate for the first time how the stem cell character of CD133(+) cells is defined by the expression of specific miRNAs.

View Article: PubMed Central - PubMed

Affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.

Show MeSH
Related in: MedlinePlus