The impact of cell source, culture methodology, culture location, and individual donors on gene expression profiles of bone marrow-derived and adipose-derived stromal cells

Abstract

Bone marrow (BM) stromal cells (MSCs), also known as mesenchymal stem cells, display a high degree of heterogeneity. To shed light on the causes of this heterogeneity, MSCs were collected from either human BM (n=5) or adipose tissue (AT) (n=5), and expanded using 2 different culture methods: one based on fetal calf serum, and one based on human platelet lysate. After initial expansion, MSCs were frozen, and the vials were transported to 3 different laboratories and grown for 1 passage using the same brand of culture plastic, medium, and supplements. Subsequently, the cells were harvested and assayed for their gene expression profile using the Affymetrix exon microarray platform. Based on gene expression profiles, the most discriminative feature was the anatomical harvesting site, followed by culture methodology. Remarkably, genes in the WNT pathway were expressed at higher levels in BM-derived MSCs than in AT-derived MSCs. Although differences were found between laboratories, cell culture location only slightly affects heterogeneity. Furthermore, individual donors contributed marginally to the observed differences in transcriptomes. Finally, BM-derived MSCs displayed the highest level of similarity, irrespective their culture conditions, when compared to AT-derived cells.

FIG. 1.

Hierarchical clustering of gene expression data. Two-dimensional representation of the unsupervised hierarchical clustering of standardized log2 gene expression values for 64 cell culture samples. Each row represents a sample and each column a gene. The 785 genes that are represented are differentially regulated (Benjamini Hochberg-corrected P<0.05, fold change >2) in at least one of the factors, cell cultures, media, or locations. The relative gene expression is visualized by a color gradient as shown by the color bar; blue means that the gene is downregulated; gray means that there is no change in expression, and red indicates upregulation. Color images available online at www.liebertpub.com/scd

FIG. 2.

PCA of MSCs cultured in FCS. BM-derived MSCs (red filled circles) cluster better than AT-derived MSCs (blue filled circles). MSC, marrow stromal cell; BM, bone marrow; FCS, fetal calf serum; PCA, principal component analysis. Color images available online at www.liebertpub.com/scd

FIG. 3.

PCA of MSCs cultured in hPL. BM-derived MSCs (red filled circles) and AT-derived MSCs (blue filled circles). hPL, human platelet lysate. Color images available online at www.liebertpub.com/scd

FIG. 4.

Pearson correlation matrix. Depicted are the Pearson correlation coefficients for every combination of samples. The color gradient indicates the relative height of the correlation. Relative low correlations are colored blue, whereas very high correlations are shown in red. Note that the BM samples correlate higher with each other than with the other samples. RN, AT-derived MSCs; ARH, samples cultured in FCS; Nijm, Nijmegen; Utre, Utrecht; Leid, Leiden. Color images available online at www.liebertpub.com/scd

FIG. 5.

Genome-wide locations of the 1134 differentially expressed genes between BM-MSCs and AT-derived MSCs. Chromosome overview showing the location and fold change of the 1134 differentially expressed genes between BM and AT. Each dot represents one gene. The vertical axes represent the base pair position of each gene on the respective chromosome. Fold changes are represented by both the position of the dot on the horizontal axes of the chromosome and a color gradient: a left alignment and blue color indicate downregulated genes; a position close to the middle and gray color are indicative for small changes; and an alignment right from the middle and red color represent upregulation of the gene. RN, AT-derived MSCs. Color images available online at www.liebertpub.com/scd