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For VSEL studying group: Mariusz Z. Ratajczak, Ewa K. Zuba-Surma, Magda Kucia, Dong-Myung Shin, Wojciech Wojakowski

Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, Louisville KY, USA


Dear Editor,

We read with interest a recent paper "Very small embryonic-like stem cells purified from umbilical cord blood lack stem cell characteristics" published in PloS ONE (2012; 7(4): e34899) by Danova-Alt R, Heider A, Egger D, Cross M, and Alt R.

However, we are concerned that this manuscript is based on a misunderstanding of the phenotype and isolation protocol for very small embryonic-like stem cells (VSELs). The authors investigated a population of cells that is CD133– and made conclusions about CD133+ VSELs that we believe are too far-reaching and unsupported by their results. In studying rare populations of cells, one needs to compare apples to apples, which unfortunately was not done in this case. The cell population that was actually sorted does not contain CD133+ cells, and thus cannot contain VSELs. In a short preliminary report published 5 years ago [1], we showed that human umbilical cord blood (UCB) contains CXCR4+CD34+CD133+Lin–CD45– cells that are enriched for very rare, small Oct-4+ and SSEA-4+ cells corresponding to murine VSELs. We have since worked for several years with CD133+Lin–CD45– cells and have demonstrated that they are highly enriched for VSELs [2,3]. However, we are aware that even in this population not every single cell is a VSEL and we are still looking for more specific and unique markers, in addition to CD133, with which to sort these cells.

Instead, the authors sorted and analyzed CD45–lin–CXCR4+ cells that turned out, for unknown reasons, to be CD133–. According to the authors, only a very few cells expressed this marker, but it is these cells that should be analyzed because they are highly enriched for VSELs. Unfortunately, these very rare CD133+Lin–CD45– cells were omitted from the sorting protocol and studies were performed on a different (CD133–) population of Lin–CD45+ cells. Thus, conclusions about VSELs based on their gene-array studies are misleading.

Furthermore, the authors claim that all CXCR4+ CD45–lin– cells are polyploid. If this were true, then all small mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and fibrocytes circulating in cord blood should be polyploid as well, with an unacceptable risk of malignant transformation to patients transplanted with UCB cells. In fact, UCB may contain some extremely rare polyploid cells that are shed from the placenta, and the polyploid cells they displayed on cyto-spin slides could well be placenta-derived. Moreover, in our recently published paper we clearly demonstrated that CD133+Lin–CD45– cells have the normal number of chromosomes [4].

To summarize, this paper reports results that should not be interpreted as a molecular analysis of VSELs, because the isolation protocol was inappropriate and the authors analyzed a different population of cells. Therefore, their conclusions about VSELs are not justified. The detailed step-by-step protocol for isolation of these cells was published two years ago in Current Protocols of Cytometry [5] and Figure 1. It is possible that these authors have not had access to this publication, and we would be happy to provide it to them as well as to guide them on how to isolate these rare cells. Then they will be able to compare apples to apples (and not apples to oranges) and draw valid conclusions.


Figure 1. Standardized gating strategy for sorting of cord blood- derived VSELs (CB-VSELs) by FACS. VSELs were isolated from fraction of CB total nucleated cells by multiparameter fluorescence- activating cell sorting employing MoFlo XDP cell sorter (Beckman Coulter). Panel A: Objects ranging from 2 - 15 µm are included into gate R1 after comparison with six differently sized bead particles with standard diameters of 1, 2, 4, 6, 10, and 15 µm to avoid VSEL loss. Panel B: CB nucleated cells are visualized on dot plot representing forward scatter (FSC) vs. side scatter (SSC) signals, which are related to the cellular size and granularity/complexity, respectively. Panel C represents cells from region R1 following staining with 7-aminoactinomycin D (7-AAD) and only 7-AAD-negative objects (including all viable cells) from region R2 are shown in Panel D that visualizes their expression of hematopoietic lineages markers (Lin). Lin-negative cells (region R3) are further plotted on dot-plot (Panel E) visualizing their expression of CD45 and CD133/1 antigens. Panel E: CD133+/Lin-/CD45- cells (CB-VSELs) are sorted as objects enclosed in logic gate including regions R1, R2, R3 and R4, while CD133+/Lin-/CD45+ hematopoietic stem/ progenitor cells (HSCs) from the logic gate including regions R1, R2, R3 and R5. Percentages show the approximate average content of each cellular subpopulation in total CB nucleated cells.


References:

1. Kucia M, Halasa M, Wysoczynski M, Baskiewicz-Masiuk M, Moldenhawer S, Zuba-Surma E, Czajka R, Wojakowski W, Machalinski B, Ratajczak MZ. 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 2007;21:297-303.

2. Zuba-Surma EK, Klich I, Greco N, Laughlin MJ, Ratajczak J, Ratajczak MZ. Optimization of isolation and further characterization of umbilical-cord-blood-derived very small embryonic/ epiblast-like stem cells (VSELs). Eur J Haematol 2010;84:34-46.

3. Ratajczak J, Zuba-Surma E, Klich I, Liu R, Wysoczynski M, Greco N, Kucia M, Laughlin MJ, Ratajczak MZ. Hematopoietic differentiation of umbilical cord blood-derived very small embryonic/epiblast-like stem cells. Leukemia 2011;25:1278-85.

4. Zuba-Surma EK, Ratajczak MZ. Overview of very small embryonic-like stem cells (VSELs) and methodology of their identification and isolation by flow cytometric methods. Curr Protoc Cytom. 2010 Jan; Chapter 9: Unit 9.29

5. Ratajczak MZ, Shin DM, Liu R, Mierzejewska K, Ratajczak J, Kucia M, Zuba-Surma EK. Very small embryonic/epiblast-like stem cells (VSELs) and their potential role in aging and organ rejuvenation - an update and comparison to other primitive small stem cells isolated from adult tissues. Aging (Albany NY) 2012 Apr 7. [Epub ahead of print]