Dear Editor,
The comments and concerns raised by the readers centre mainly around the claim that the population of CD45-Lin-CXCR4+ cells included in our analyses was not identical to the previously reported VSEL cell population.
We can neither accept nor understand this criticism, as the population of CD45-Lin-CXCR4+ cells was the one first described as VSEL cells:
The original paper by Kucia et al. first describing VSEL cells in human umbilical cord blood shows a detailed morphological and transcriptional analysis of CD45-Lin-CXCR4+ cells [1]. The data presented in Figure 3 of that paper explicitly shows CD45-Lin-CXCR4+ cells isolated by FACS as positive for SSEA-4, Oct-4 and Nanog on the protein level. Figure 4 of the same paper presents TEM images from CD45-Lin-CXCR4+ cells, the first and up to now the only ultra-structural images of VSEL cells. These data provide the basis both for the authors’ conclusion and title of the paper: CD45-Lin-CXCR4+ are very small embryonic-like (VSEL) cells.
The readers claim that we should have used CD133 rather than CXCR4 as a marker for VSEL cells. This, however, is also contradicted by the first description by Kucia et al. The authors show in Figure 2b of the above paper, that the transcript levels of the selected genes (incl. Oct4 and Nanog) are indistinguishable between the CD45-Lin-CXCR4+ and CD45-Lin-CD133+ populations. There is no indication here that CD133 should be preferred in any way to CXCR4 as a marker for VSEL cells.
The readers next cite papers describing “improved” isolation strategies for VSEL cells from human umbilical cord blood using CD133 either in combination with or instead of CXCR4 [2,3]. We are well aware of these papers. However, in our opinion they fail to contribute to the understanding of the VSEL cell type originally described, as none provide functional indications of VSEL cell-like activity, let alone evidence of pluripotency using widely accepted functional assays, such as differentiation potential into three germ layers in vitro or teratoma formation in vivo. The mere detection of pluripotency-associated markers at mRNA and protein level is insufficient to claim pluripotency, especially as the assays used to detect the markers are prone to false-positives [4,5]. Given the lack of functional assays for VSEL activity in these publications it is unclear how one can claim “improvement” of isolation strategies or interpret the suitability of CD133 or CD34 in comparison to to CXCR4 in this respect. The claim for a higher potential of one population over another (e.g. Lin-CD45-CD133+ vs. Lin-CD45-CXCR4+) needs to be based on functional data. Otherwise, the mode of enrichment or usage of markers is meaningless.
Having said this, we would like to point out that the population of Lin-CD45-CXCR4+ cells contains a very small number of CD133+ cells and that we did not (as the readers imply) actively exclude these from the assays. Had these cells possessed functional pluripotent properties, we should have detected this in our cell culture experiments.
Finally, it is important to note that we show the population of Lin-CD45-CXCR4 + cells to contain a high number of aneuploid (not polyploid) cells which we assume to be the products of defective cell division rather than malignant transformation. Aneuploid cells are very unlikely to expand and indeed failed to do so under any of our in vitro culture conditions. We therefore consider it very unlikely that these cells have any impact whatsoever (either positive or negative) in the setting of an umbilical cord blood transplant.
In summary, in the absence of any functional data concerning the superiority of either CD133 or CD34 over CXCR4 we see no compelling reason to deviate from the markers originally reported to isolate “pluripotent” VSEL cells from human umbilical cord blood. There is clearly controversy concerning the nature and properties of VSEL populations and this has not been helped by the description in the literature of marker profiles with no demonstration of functional relevance. In providing an independent, detailed and systematic analysis of that population for which VSEL phenotype has been claimed, we hope to have provided a reference point from which these controversies can be resolved.

Yours sincerely,
Rüdiger Alt & co-autors

1. Kucia M, Halasa M, Wysoczynski M, Baskiewicz-Masiuk M, Moldenhawer S, et al. (2007) Morphological and molecular characterization of novel population of CXCR4+ SSEA-4+ Oct-4+ very small embryonic-like cells purified from human cord blood: preliminary report. Leukemia 21: 297-303.
2. Zuba-Surma EK, Klich I, Greco N, Laughlin MJ, Ratajczak J, et al. (2010) Optimization of isolation and further characterization of umbilical-cord-blood-derived very small embryonic/ epiblast-like stem cells (VSELs). Eur J Haematol 84: 34-46.
3. Zuba-Surma EK, Ratajczak MZ (2010) Overview of very small embryonic-like stem cells (VSELs) and methodology of their identification and isolation by flow cytometric methods. Curr Protoc Cytom Chapter 9: Unit9 29.
4. Lengner CJ, Welstead GG, Jaenisch R (2008) The pluripotency regulator Oct4: a role in somatic stem cells? Cell Cycle 7: 725-728.
5. Liedtke S, Enczmann J, Waclawczyk S, Wernet P, Kogler G (2007) Oct4 and its pseudogenes confuse stem cell research. Cell Stem Cell 1: 364-366.