Differentiation of two types of mobilized peripheral blood stem cells by microRNA and cDNA expression analysis

Abstract

Background: Mobilized-peripheral blood hematopoietic stem cells (HSCs) have been used for transplantation, immunotherapy, and cardiovascular regenerative medicine. Agents used for HSC mobilization include G-CSF and the CXCR4 inhibitor AMD3100 (plerixafor). The HSCs cells mobilized by each agent may contain different subtypes and have different functions. To characterize mobilized HSCs used for clinical applications, microRNA (miRNA) profiling and gene expression profiling were used to compare AMD3100-mobilized CD133+ cells from 4 subjects, AMD3100 plus G-CSF-mobilized CD133+ cells from 4 subjects and G-CSF-mobilized CD34+ cells from 5 subjects. The HSCs were compared to peripheral blood leukocytes (PBLs) from 7 subjects.

Results: Hierarchical clustering of miRNAs separated HSCs from PBLs. miRNAs up-regulated in all HSCs included hematopoiesis-associated miRNA; miR-126, miR-10a, miR-221 and miR-17-92 cluster. miRNAs up-regulated in PBLs included miR-142-3p, -218, -21, and -379. Hierarchical clustering analysis of miRNA expression separated the AMD3100-mobilized CD133+ cells from G-CSF-mobilized CD34+ cells. Gene expression analysis of the HSCs naturally segregated samples according to mobilization and isolation protocol and cell differentiation status.

Conclusion: HSCs and PBLs have unique miRNA and gene expression profiles. miRNA and gene expression microarrays maybe useful for assessing differences in HSCs.

Figure 1

MicroRNA (miRNA) expression profiles of hematopoietic stem cells (HSCs) and peripheral blood leukocytes (PBLs). RNA was isolated from T cells, B cells, monocytes and NK cells from 7 subjects, G-CSF-mobilized CD34+ cells from 5 subjects, AMD3100 (A)-mobilized CD133+ cells from 4 subjects, and AMD3100 plus G-CSF (A+G)-mobilized CD133+ cells from 4 subjects and miRNA expression was analyzed using an expression array with 457 human miRNAs. A) Unsupervised hierarchical clustering of Eisen was used to analyze the 148 miRNAs that remained after filtering (miRNA expressed in ≥ 80% of samples). B) Signature miRNAs whose expression was markedly up-regulated in HSCs or PBLs.

Figure 2

Gene expression profiles of hematopoietic stem cells (HSCs) and peripheral blood leukocytes (PBLs). cDNA was isolated from T cells, B cells, monocytes and NK cells from 7 subjects, G-CSF (G)-mobilized CD34+ cells from 5 subjects, AMD3100 (A)-mobilized CD133+ cells from 4 subjects and AMD3100 plus G-CSF (A+G)-mobilized CD133+ cells from 4 subjects. cDNA expression was analyzed using an expression microarray with 17,500 cDNA. Unsupervised hierarchical clustering of Eisen was used to analyze the 11,023 genes that remained after filtering (cDNA expressed in ≥ 80% of samples).