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Comment on " 'Stemness': Transcriptional Profiling of Embryonic and Adult Stem Cells" and "A Stem Cell Molecular Signature" (I)

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Science  17 Oct 2003:
Vol. 302, Issue 5644, pp. 393
DOI: 10.1126/science.1086384

Ramalho-Santos et al. (1) and Ivanova et al. (2), comparing the same three “stem cells”— embryonic stem cells (ESCs); neural stem cells (NSCs), referred to as neural progenitor/stem cells (NPCs) in the present study; and hematopoietic stem cells (HSCs)—with their differentiated counterparts, each identified a list of commonly expressed “stemness” genes, proposed to be important for conferring the functional characteristics of stem cells. The ability to capture expression profiles of cells using microarrays offers the possibility of defining a stem cell by its constellation of active genes. An intriguing question, however, is whether the functional commonalities (self-renewal and pluripotency) (3) among stem cells can be defined at the genetic level. Do all stem cells express a similar set of “stemness” genes necessary for their unique properties, or do different stem cells express different sets of genes that confer stemness?

We have independently carried out gene expression profiling of three types of stem or immature progenitor cells: ESCs, NPCs, and retinal progenitor/stem cells, or RPCs (Fig. 1) (4, 5). The intersection of ESC-, NPC- and RPC-enriched genes defined a list of 385 genes that are collectively expressed by all three stem cells (6). It can be inferred that these genes may represent or include putative “stemness” genes. For the approach taken here to be able to define and support the notion of “stemness” genes, however, would also require that very similar sets of genes can be identified regardless of the type of stem cells used. To test the validity of this notion, we have collectively analyzed our results along with those from the studies of Ramalho-Santos et al. (1) and Ivanova et al. (2) (Figs. 2 and 3). To our surprise, a comparison of the three independently derived lists of “stemness” genes showed only one gene (integrin alpha-6) commonly identified in the three studies (Figs. 2A and 3A) (6). This finding raised serious concerns about the conclusions reported in (1) and (2), as was also critically highlighted by Burns and Zon (7).

Fig. 1.

(A) Hierarchical clustering of stem cells and their differentiated progenies (6), as identified in this study. The clustering of NPCs and RPCs with their differentiated progenies [tissues adjacent to lateral ventricles of adult brain (LVBr) and mature retina (Ret)] suggest that somatic stem cells are already primed to become specific lineages. (B) Venn diagram showing the number of genes enriched in ESCs, NPCs and RPCs, as identified in this study. The overlap of these genes identified a common list of 385 stemness genes enriched in all three “stem cells” (6).

Fig. 2.

Venn diagrams showing overlap of “stemness” genes and stem cell–enriched genes among studies by Ramalho-Santos et al. (1), Ivanova et al. (2), and Fortunel et al. (this study). Ivanova et al. used three different Affymetrix chips (U74v2 A, B, and C); Fortunel et al. and Ramalho-Santos et al. used only the U74v2 A chip (Fig. 3 shows same comparison, limiting Ivanova et al. results to the A chip). (A) “Stemness” genes found by the three groups overlap by only one gene. (B) ESC-enriched genes identified by each study overlap by 332 genes; the probability that such overlap occurs by chance is extremely low (P < 10–8). (C) NPC-enriched genes overlapping by 236 genes between the three groups (P < 10–8). (D) Overlap of “stemness” genes—two types of stem cell (ESC/NPC)-enriched genes—is limited to 10 genes. The probability of this number of genes overlapping by chance is greatly increased. P > 10–4 is not significant because there are more than 104 genes studied (8).

Fig. 3.

(A to D) Same as Fig. 2, but with data limited to the Affymetrix U74v2 A chip for all three groups. Although the total number of genes identified by Ivanova et al. (2) is reduced, the number of genes found in the intersecting regions among the three studies are not changed significantly: The same single “stemness” gene common among the three studies remains in (A); the number of ESC- and NPC-enriched genes in (B) and (C), though it has declined, remains in the same order of magnitude; and the number of ESC/NPC-enriched genes in (D) has declined by only one, from 10 to 9. The decrease in the number of genes found in the intersecting regions of ESC- and NPC-enriched genes (panels B and C) is due to the fact that probes for some genes are present in more than one chip and that some genes that were detected in chip A by Ramalho-Santos et al. (1) and Fortunel et al. (this study) were detected by Ivanova et al. in either chip B or chip C but not in chip A. The P values remain low in both Fig. 2 and Fig. 3.

We then examined whether the same extreme discrepancy was observed for the lists of genes expressed in one specific stem cell type. In marked contrast, there was a very significant overlap in the lists of stem cell–specific genes from the three studies. A total of 332 “ESC-enriched genes” (Fig. 2B) (6) and 236 “NPC-enriched genes” (Fig. 2C) (6) were identified by all three investigators. Statistical analysis (8) showed that these numbers are highly significant (P < 10–8). These results strongly go against major differences in cells and analytical methodologies between groups as an explanation for the lack of overlap in the three lists of “stemness” genes. However, when we computed for “stemness” genes, based on genes commonly expressed in two types of stem cells, we found that the three studies overlap by only 10 genes, with P = 1.4 × 10–4 (Fig. 2D). Therefore, as the number of stem cell types intersected to identify “stemness” genes is increased, the overlap between datasets from different investigators drops dramatically.

To examine why that may be the case, we plotted a comparison of significance scores (Fig. 4) for the different categories of genes derived by each group [fold change (FC) value or lower confidence bound (LCB) score] (9). It is quite apparent that as one increases the number of stem cell types for comparison, the genes that occur in the intersection are genes that show progressively lower differential expression between stem and differentiated cells (Fig. 4). For example, we found that the log of the significance scores for individual ESC or NPC-specific transcripts are typically in the range of 6 to 10 over differentiated progenies in all three groups. Transcripts of “stemness” genes from intersection of two or more stem cells, from all three groups, commonly showed log significance scores in the 2 to 4 level. In contrast to ESC and NPC, for which each study came up with overlapping sets of genes that are uniquely or highly expressed exclusively in each stem cell type, none of the putative “stemness” genes in the three studies were highly expressed genes compared with differentiated cells. These results indicate that the expression of “stemness” genes common to all stem cells, if such genes exist at all, is only relatively elevated compared with differentiated cells. This would create a significant variation in the genes identified by differential expression, amplified by subtle differences in experimental conditions between different studies. We propose this as the most important basis for the extreme discrepancies in the lists of putative “stemness” genes.

Fig. 4.

(A to C) Comparison of relative expression levels of”stemness“genes by fold changes. The graphs depict significance scores for log FC or log LCB changes in the expression levels of the top 80 genes from the list of ESC-enriched genes (blue), NPC-enriched genes (orange), ESC/NPC-enriched genes (red), and “stemness” genes (green). “Stemness” genes here are (A) the top 80 genes from the 385 genes identified in this study (Fig. 1), (B) the 230 genes identified in (1), and (C) the 283 genes identified in (2), as published; values in (B) and (C) were derived from published data. Fold changes are highest for genes enriched in a single stem cell type; with two stem cell types (NPC and ESC), the top 80 genes have much lower fold change compared to one stem cell type. With three stem cells, the fold changes compared with individual stem cells are markedly reduced.

Our observation does not rule out the possibility that genes unique to all stem cells which are expressed at low levels may exist. However, it is clear that no one single study can confidently identify the bona fide genes that specify “stemness,” and cross-validation of lists generated independently by different investigators is crucial. For instance, the total of 24 genes commonly identified in two of the studies (Figs. 2A and 3A) is statistically significant and warrants further investigation (6). It is possible also that there are “stemness” genes that have not yet been identified and are not represented in the chips used. Genes that may be important for stem cell functions such as self-renewal but that are also expressed in non-stem cells (for example, Stat3, gp130) are unlikely to be identified by a comparative microarray approach. Another possibility is that different stem cell types may use different gene networks to achieve self-renewal or multipotency (10). Finally, “stemness” genes may only be transiently expressed, so that they are easily missed by comparing two homeostatic states. Further efforts to identify these genes will require different strategies.

In summary, speculations made from independent studies (1, 2) about identity of stemness genes do not hold up when the studies are compared. Our explanation for this also demonstrates the inherent problem of testing the stemness hypothesis using a profiling approach.

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