Comparative transcriptome analysis of embryonic and adult stem cells with extended and limited differentiation capacity

Abstract

Background: Recently, several populations of postnatal stem cells, such as multipotent adult progenitor cells (MAPCs), have been described that have broader differentiation ability than classical adult stem cells. Here we compare the transcriptome of pluripotent embryonic stem cells (ESCs), MAPCs, and lineage-restricted mesenchymal stem cells (MSCs) to determine their relationship.

Results: Applying principal component analysis, non-negative matrix factorization and k-means clustering algorithms to the gene-expression data, we identified a unique gene-expression profile for MAPCs. Apart from the ESC-specific transcription factor Oct4 and other ESC transcripts, some of them associated with maintaining ESC pluripotency, MAPCs also express transcripts characteristic of early endoderm and mesoderm. MAPCs do not, however, express Nanog or Sox2, two other key transcription factors involved in maintaining ESC properties. This unique molecular signature was seen irrespective of the microarray platform used and was very similar for both mouse and rat MAPCs. As MSC-like cells isolated under MAPC conditions are virtually identical to MSCs, and MSCs cultured in MAPC conditions do not upregulate MAPC-expressed transcripts, the MAPC signature is cell-type specific and not merely the result of differing culture conditions.

Conclusion: Multivariate analysis techniques clustered stem cells on the basis of their expressed gene profile, and the genes determining this clustering reflected the stem cells' differentiation potential in vitro. This comparative transcriptome analysis should significantly aid the isolation and culture of MAPCs and MAPC-like cells, and form the basis for studies to gain insights into genes that confer on these cells their greater developmental potency.

Figure 1

Oct4 expression and endothelial-like and hepatocyte-like differentiation for mMAPC-1, mMAPC-2, mClone-3, rMAPC, and rClone-2. (a) The levels of Oct4 (Pou5f1) mRNA in mouse (m, left) and rat (r, right) clones compared with those of Gapdh mRNA. The mouse clones are also compared with mESCs. ΔCT is difference in threshold cycles calculated as Oct4 CT - Gapdh CT. ND, not detected. (b) Endothelial-like differentiation. mRNA levels of endothelial markers in mouse (left panel) and rat (right panel) clones before and after differentiation, measured at day 9 in two independent differentiations of each clone. Levels are compared with those in universal mouse RNA and rat spleen RNA, respectively. Left panel: blue diamonds, Pecam (×10) (values shown were scaled by the factors in brackets); pink squares, Lyve1; orange triangles, vWF. Right panel: blue diamonds, VE-Cad (×100); pink squares, Flt-1; orange triangles, Flk-1; turquoise crosses, vWF (×100). (c) Hepatocyte-like differentiation. mRNA levels of hepatocyte markers in mouse and rat clones before and after differentiation. Levels are compared with levels in mouse hepatocytes and rat liver, respectively. Two representative differentiations measured at day 18 are shown. Left panel: blue diamonds, F2 (×100); pink squares, Tat, (×10⁵); green triangles, Afp (×10^-1); turquoise crosses, Ttr (×10³). Right panel: blue diamonds, Afp (×100^-1); pink squares, Alb, (×10³); orange triangles, Tat (×100); turquoise crosses, Ttr (×10³). See text for abbreviations.

Figure 2

Cell-surface phenotype and Oct4 protein expression of mouse and rat clones evaluated by flow cytometry. (a) Flow cytometry results for mouse clones. From left to right: MSCs; mMAPC-1; mMAPC-2; mClone-3; and MSCs cultured in MAPC conditions. The histogram in each panel plots the number of cells (as a percentage of the maximum) on the vertical axis against the fluorescence intensity of the labeled antibody bound to the indicated protein (horizontal axis). The horizontal line on each histogram indicates the fluorescence range that contains the indicated percentage of cells positive for that protein. (b) Flow cytometry results for the rat clones rMAPC-1 and rClone-2.

Figure 3

PCA and NMF analysis of the gene-expression data of mouse MSC, MAPC-1, MAPC-2 and ESC. (a) Percentage variation captured in each principal component (PC). PCs are ordered from 1 to 4 according to the percentage of the total variance they capture. (b) Samples plotted in the first two components' space. Distance of samples in the component space is indicative of similarity in expression profiles. (c) Consensus matrix from NMF. Model selection in NMF is based on a consensus matrix that contains the probability that a pair of samples is assigned to the same group. Probability values correspond to the colors in the key. M-1, MAPC-1; M-2, MAPC-2; (d) Metagene profiles from NMF plotted as logarithm of the probe set intensity.

Figure 4

Genes for cell-surface and extracellular space proteins and transcriptional regulators clustered according to their expression profiles in the mouse MAPC/ESC/MSC comparison and in rat rMAPC-1 and rClone-2. (a, b) Genes expressed in mMAPCs at levels at least twofold higher than in ESCs and MSCs. (c, d) Genes expressed in mMAPC/ESCs at levels at least twofold higher than in MSCs. (e, f) Genes expressed in mMAPC/MSCs at levels at least twofold higher than in ESCs. (g, h) Genes expressed at levels at least twofold higher in rMAPC-1 than in rClone-2. (i, j) Genes expressed in rClone-2 at levels at least twofold higher than in rMAPC-1. The yellow bars indicate genes for proteins localized to the extracellular space; the blue bars indicate genes for transcriptional regulators. Red and green indicate higher and lower levels of expression, respectively.

Figure 5

Q-RT-PCR validation of differentially expressed genes. (a) Comparison of relative expression levels of mRNA for the genes indicated by Q-RT-PCR on unamplified RNA (black bars) and Affymetrix microarray data (gray bars) (n = 3). Samples were normalized using Gapdh as the housekeeping gene and percent expression level was calculated with respect to the highest-expressing sample. The asterisk (*) indicates genes evaluated in mMSCs grown in the MAPC condition. (b) Relative mRNA expression levels of Ecats and other ESC-expressed transcripts in mMAPCs (black bars) and ESCs (gray bars) quantified using Q-RT-PCR. Three independent isolations of mMAPCs and ESC RNA for Q-RT-PCR were used to confirm microarray mRNA expression results. Samples were normalized to ESC expression levels and Gapdh mRNA levels were used for internal normalization.

Figure 6

Genes for proteins localized to the extracellular space and for transcriptional regulators clustered according to expression profiles in MAPC/ESC/NS comparison. (a, b) Genes expressed at levels at least twofold higher in both MAPCs and ESCs than in NS (k-means clusters 2 and 4). (c, d) Genes expressed in MAPCs at levels at least twofold higher than in both ESCs and NS (k-means cluster 3). The yellow bars indicate genes whose products are localized to the extracellular space; the blue bars indicate genes for transcriptional regulators. Red and green indicate higher and lower levels of expression, respectively.

Figure 7

PCA and NMF analysis on the gene-expression data of MSC, MSC-like (MSC-l, mClone-3), MAPC-1 (M-1), MAPC-2 (M-2) and ESC. (a) PCA on all differentially expressed genes with samples plotted in the first two components' space. (b) Consensus matrix from NMF on all differentially expressed genes. See Figure 3 for an explanation of the key in (b).