Comment on " 'Stemness': transcriptional profiling of embryonic and adult stem cells" and "a stem cell molecular signature".

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-, NPCand 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). 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 ). 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