Human Primary Bone Marrow Mesenchymal Stromal Cells and Their in vitro Progenies Display Distinct Transcriptional Profile Signatures

Abstract

Bone marrow mesenchymal stromal cells (BM-MSCs) are a rare population of cells that gives rise to skeletal tissues and the hematopoietic stroma in vivo. Recently, we have demonstrated that BM-MSCs fulfill stringent in vivo stem cell criteria when propagated as non-adherent mesenspheres but not as adherent-cultured cells. Motivated by these profound functional differences, the current study aimed to identify potential important MSC regulators by investigating global gene expression profiles of adherent and non-adherent culture-derived BM-MSCs in comparison with primary BM-MSCs. A substantial number of genes were differentially expressed between primary and culture-expanded cells already early upon culture, and numerous genes were found to be different when comparing adherent and non-adherent BM-MSCs. Cluster analysis identified 16 sets of genes of which two displayed comparable gene expression levels in primary and non-adherent cultured cells, but not in adherent cultured cells. This pattern suggested that these clusters contained candidate regulators of BM-MSCs. Gene expression differences were confirmed for selected genes and BM-MSC transcription factors by protein analysis and RT-PCR, respectively. Taken together, these data demonstrated profound gene expression changes upon culture of primary BM-MSCs. Moreover, gene cluster differences provide the basis to uncover the regulatory mechanisms that control primary and cultured BM-MSCs.

Figure 1

FACS gating strategy and experimental design of the microarray analysis. a. Freshly isolated, lineage-depleted bone marrow mononuclear cells were stained with antibodies against CD45, CD31, CD71, CD235a and CD271 as described. Following forward/side scatter gating and dead cell exclusion, CD45⁻/CD31⁻/CD71⁻/CD235a⁻ cells were sorted by gating on the CD271⁺ population. A representative set of FACS plots is presented. b. Schematic overview of the experimental workflow. From each donor (n = 4), primary cells were sorted into lysis buffer for gene expression analysis, and for culture in mesensphere and standard MSC medium, respectively. Ex-vivo cultured adherent BM-MSCs and mesenspheres were harvested in passages 0 and 3, and prepared for microarray analysis.

Figure 2

Gene expression and cluster analysis of primary, adherent and non-adherent culture-expanded BM-MSCs. (a) Heatmap of significantly differentially expressed genes in primary, adherent and sphere cultured-derived lin⁻/CD45⁻/CD31⁻/CD71⁻/CD235a⁻/CD271⁺ cells at passages 0 and 3. (b) Cluster analysis of differentially expressed genes. The first data points from the left in each plot (1–3, light blue) represent primary cells, the next 8 boxes in orange represent adherent cultured cells (4–7: passage 0; 8–11: passage 3) and the last 8 boxes in dark blue represent the results for sphere cultured cells (12–15: passage 0, 16–19: passage 3).

Figure 3

Primary and sphere-cultured cells but not adherent cultured cells show similar expression patterns in clusters 3 and 5. (a) Biological process annotations with false discovery rate (FDR) ≤ 0.1% in clusters 3 and 5 were identified using the DAVID Bioinformatics Resources 6.8. The x-axis indicates the number of genes included in each functional group. Cluster 3 shows the genes that were up-regulated in primary and sphere-cultured cells, but down-regulated in adherent-cultured cells. Cluster 5 contains genes that were down-regulated in primary and sphere-cultured cells but up-regulated in adherent-cultured cells. The names of the genes in clusters 3 and 5 are listed in Supplementary Table S1. (b) Gene expression analysis of cluster 3 genes relative to the RPL11 reference gene (n = 3). Significant differences in gene expression between primary and cultured cells (P0 and P3) are indicated as: **p < 0.01, ***p < 0.001, ****p ≤ 0.0001 (2-way ANOVA, multiple comparisons test). (c) Percentage of positive cells for each surface marker in primary, adherent and sphere cultured BM-MSCs (n = 3, 2-way ANOVA, p = 0.0005), and (d) Mean fluorescence intensity (geometric means) of each surface marker minus their respective isotype (n = 3, 2-way ANOVA p = 0.55, cell group variation p = 0.0149, surface marker variation p = 0.1027). Bars show mean ± SD. SPH: sphere cultured cells, ADH: adherent cultured cells.