SEARCH

SEARCH BY CITATION

Keywords:

  • pluripotency;
  • adult stem cells;
  • umbilical cord blood;
  • mesenchymal stem cell;
  • bone marrow

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Murine VSEL Cells
  5. Human UCB VSEL Cell Phenotype
  6. CD45LINCXCR4+ VSEL Cells from Human UCB Lack Stem Cell Activity
  7. Summary
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

In 2006, very small embryonic-like (VSEL) stem cells were described as a pluripotent population of prospectively isolated stem cells in adult murine bone marrow (mBM) and human umbilical cord blood (hUCB). While rigorous proof of pluripotency is still lacking, murine VSEL cells have been shown to overlap with an independently identified population of neural crest derived mesenchymal stem cells (MSC). The presence of primitive mesenchymal precursors within the VSEL cell population may partially explain the findings that have led to the concept of an “embryonic-like” stem cell in mBM. However, our own studies on human VSEL cells revealed very little similarity between murine VSEL cells and their reportedly equivalent population in hUCB. On the contrary, our data strongly suggest that human VSEL cells are an aberrant and inactive population that cannot expand in vitro and has neither embryonic nor adult stem cell like properties. Here we critically re-examine the data supporting stemness and pluripotency of murine and human VSEL cells, respectively. © 2012 International Society for Advancement of Cytometry


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Murine VSEL Cells
  5. Human UCB VSEL Cell Phenotype
  6. CD45LINCXCR4+ VSEL Cells from Human UCB Lack Stem Cell Activity
  7. Summary
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

The original description in 2006 of a cell population with embryonic stem cell-like characteristics in mouse bone marrow and human umbilical cord blood (hUCB) joins a number of reports over the years that have raised hopes of a non-embryonic source of pluripotent stem cells for autologous regenerative therapies (1, 2). However, unlike previous reports of extended regenerative potential arising under extended mesenchymal stem cell (MSC)-like culture conditions (3–5), the very small embryonic-like (VSEL) stem cells could be isolated prospectively from primary tissue using defined surface markers. Although the availability of well defined phenotypic markers should increase the reproducibility of identification and isolation between labs and make the detailed characterization of stem cell potential more straightforward, six years after the original publication there has been no independent confirmation of the data and no functional proof of the putative pluripotent nature of these cells (6). On the contrary, the evidence for pluripotency of adult cells has been repeatedly called into question (7–10). Therefore, it is time for a critical re-examination of the VSEL hypothesis and the data supporting stemness and pluripotency of the human and murine VSEL populations.

Murine VSEL Cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Murine VSEL Cells
  5. Human UCB VSEL Cell Phenotype
  6. CD45LINCXCR4+ VSEL Cells from Human UCB Lack Stem Cell Activity
  7. Summary
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

Murine VSEL cells were first described as a CD45LinSca-1+ population in adult murine bone marrow (mBM) that showed high transcript levels of pluripotency genes, a nuclear localization of both Oct 4 and Nanog proteins, a demethylated Oct4 promotor, an overall euchromatic nuclear organization, a quiescent status that is maintained by a unique methylation pattern of certain imprinting genes, and an in vitro differentiation potential into neuronal, pancreatic, and cardiomyocyte-like cells representative of ectoderm, endoderm, and mesoderm, respectively (2, 11). The putative pluripotency of murine VSEL cells remains based on these preliminary findings, there having been in the meantime no demonstration of teratoma formation, tissue contribution following blastocyst injection or multilineage differential at a clonal level. Thus, the gold standards of pluripotent stem cell function have still not been demonstrated, and the mere detection of pluripotency-associated markers at mRNA and protein level must be regarded as an insufficient basis from which to claim pluripotency, especially as the assays used to detect the markers are prone to artifacts such as false-positives (7, 8, 12). While the case for pluripotency and quiescence of VSEL cells has not been convincingly made, murine VSEL cells have indeed been shown to have proliferation capacity and differentiation potential in vitro (2, 13). Furthermore, murine VSEL cells are able to form bone tissue in vivo, and may contribute to heart regeneration (14, 15), although the same cells failed in hematopoietic reconstitution assays and in teratoma formation assays (2, 11). Most recently, a population similar to murine VSEL cells has been identified in rat bone marrow and has been shown both to expand and differentiate in vitro and to contribute to heart regeneration in vivo (16). These data are not definitive proof of a specific adult stem cell function and are not compatible with the pluripotent and quiescent status claimed for murine VSEL, but they would be consistent with a bone marrow VSEL population containing mesenchymal stem or progenitor cells with in vivo regenerative potential.

Interestingly, supporting evidence in this respect has been provided by an independent Japanese group with the identification of a mesenchymal precursor overlapping with the CD45LinSca-1+ VSEL cell population (Fig. 1) (17). The CD45Ter119Sca-1+PDGFR-α+ (PαS) cells give rise to multipotent mesenchymal stromal cells in vitro, and are able to engraft in bone marrow when transplanted i.v. into irradiated mice. Furthermore, it was shown that at least some of the PαS cells originate from the neural crest and can give rise to neuronal cell types in vitro, suggesting that there may be a mixed mesodermal/ectodermal potential in the PαS population (18). Consistent with this, it has been shown that Nestin, a marker previously associated with neuronal stem cells, specifies the entire mesenchymal potential of the CD45 bone marrow population (19, 20). The fact that mesenchymal stem cells with very similar properties have been independently isolated from PαS cells and from CD45Nestin+ cells suggests a degree of overlap also between these populations.

thumbnail image

Figure 1. Heterogeneity of the murine VSEL cell population. The gating of VSEL cells shows that ∼60% of the VSEL cells express PDGF-Rα, thus resembling the PαS phenotype. A: Gating of Lin mBM cells (CD5, CD45R, CD11b, Ly-6G/C, 7-4, Ter-119; lineage cell depletion kit, Miltenyi Biotec). B: Lin cells: Gating of CD45-Sca-1+ VSEL cells. C: LinCD45+ cells: gating of c-kit+Sca-1+ hematopoietic stem and progenitor cells (KSL). D: Expression of PDGF-Rα in KSL cells and VSEL cells. Further experimental details are provided as Supporting Information.

Download figure to PowerPoint

In summary, these examples demonstrate: (1) that mBM VSEL cells do contain multipotent mesenchymal stem cells, (2) that the mBM VSEL population is heterogeneous and currently insufficiently characterized, and (3) that an extra-mesenchymal differentiation potential of the VSEL population (into the neuronal lineage in this case) can be explained without invoking a pluripotent stem cell. The set of recently identified markers that allow a prospective isolation of mesenchymal stem cells from mBM should also help to clarify the true nature of the murine VSEL population.

Human UCB VSEL Cell Phenotype

  1. Top of page
  2. Abstract
  3. Introduction
  4. Murine VSEL Cells
  5. Human UCB VSEL Cell Phenotype
  6. CD45LINCXCR4+ VSEL Cells from Human UCB Lack Stem Cell Activity
  7. Summary
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

Clearly, the degree to which the perceived therapeutic potential of VSEL or related populations can be realized depends entirely on the presence of similar cells in human tissues. It was therefore encouraging that the description of VSEL in murine tissues was followed rapidly by a report from the same group of VSEL cells in hUCB (1). These data were fundamental to the definition of human VSEL cells as CD45LinCXCR4+ cells. The CD133 and CD34 markers were also described as being expressed by VSEL cells, but the data provided was not sufficient to clarify the relationship between the subsets of CXCR4+, CD133+, and CD34+ cells within the CD45Lin cell population. The transcript levels of the selected pluripotency genes including Oct4 and Nanog were indistinguishable between the CD45LinCXCR4+ and CD45LinCD133+ populations, but there was no indication that CD133 should be preferred in any way to CXCR4 as a positive marker for VSEL cells. Indeed, the CD45LinCXCR4+ cells were the only ones to be further characterized by the expression of Oct4 and Nanog on the protein level and by electron microscopy.

Following this initial definition of the human VSEL phenotype, alternative strategies for the isolation of VSEL from human UCB via magnetic enrichment of CD133+ cells have been published (21, 22). However, none of these reports provide functional indications of VSEL cell-like activity, let alone evidence of pluripotency, that would support superiority of CD133 over CD34 or CXCR4 for VSEL isolation. Until functional comparisons have been performed between the original CD45LinCXCR4+ and other populations such as the CD45LinCD133+, it seems sensible to base any comparison of data between different groups on the originally and more comprehensively described CD45LinCXCR4+ VSEL population.

CD45LINCXCR4+ VSEL Cells from Human UCB Lack Stem Cell Activity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Murine VSEL Cells
  5. Human UCB VSEL Cell Phenotype
  6. CD45LINCXCR4+ VSEL Cells from Human UCB Lack Stem Cell Activity
  7. Summary
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

With the aim of defining the true therapeutic potential of human VSEL cells, we have carried out an extensive characterization of purified populations of CD45LinCXCR4+ VSEL cells from hUCB (10). Consistent with the original description of these cells, our purified populations have a high density, are significantly smaller than hematopoietic stem cells, and appear to possess hardly any cytoplasm. However, a comprehensive flow cytometric screen for 22 surface markers typical of embryonic and adult stem cells revealed negligible expression on VSEL cells. Contrary to our expectations, purified VSEL cells also failed to expand in vitro under a range of culture conditions commonly used in embryonic or adult stem cell culture. Since our purification protocol excluded neither CD133+ nor CD34+ cells from the CD45LinCXCR4+ VSEL population, any stem cell activity arising from a CD133+ or CD34+ subpopulation should have been detected in our cell culture experiments.

In order to further extend our analysis and in the hope of gaining some indication of how to better culture and expand VSEL cells in vitro, we conducted a microarray analysis. The transcriptional profile of highly purified hUCB VSEL cells contradicted the previous report by Ratajczak's group and showed the VSEL cells to be clearly distinct both from the mature hematopoietic lineages, and from well-defined populations of pluripotent and adult stem cells (1). We further conducted a karyotypic analysis that revealed an aneuploid karyotype in the majority of purified VSEL cells by fluorescence in situ hybridization. Although aneuploidy has long been associated with cancer, it has recently been observed in cultured pluripotent and neuronal stem cells as well as normal neuronal progenitors and primary cells from blastocysts, so that the tendency to generate aneuploid cells may be a normal feature of regenerative systems (23–26). Even so, the aneuploid products themselves are unlikely to make a long-term contribution to regeneration, and it is unsurprising that these cells are unable to expand in vitro.

Summary

  1. Top of page
  2. Abstract
  3. Introduction
  4. Murine VSEL Cells
  5. Human UCB VSEL Cell Phenotype
  6. CD45LINCXCR4+ VSEL Cells from Human UCB Lack Stem Cell Activity
  7. Summary
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

There can be little doubt that the VSEL cells isolated from mBM contain very promising stem cell functions, including the capacity to generate mesenchymal stem cells with high efficiency (Table 1). However, in the continuing absence of functional data there is no basis in our view to assume pluripotency. It is to be hoped that MSC similar to those present in the mBM VSEL population are present in human tissues. However, our studies on the reportedly equivalent “VSEL” population derived from hUCB strongly suggest that these are an aberrant and inactive population that cannot expand in vitro and has neither an embryonic nor an adult stem cell like phenotype. There has been no confirmation by an independent group to date to support that human UCB-derived VSEL cells possess the functional hallmarks of stem cells, such as high proliferative capacity, in vitro differentiation potential, or the ability to self-renew in vivo. In the absence of such evidence and in the light of our own data, we currently see no justification for use of the term “stem cell” in reference to this population.

Table 1. Phenotype and differentiation potential of prospectively isolated VSEL and related adult stem cells
OriginPhenotypeExpansion in vitroDifferentiation potentialPluripotency markersElectron micrographyRef.
mBMCD45LinSca-1+YesNeuronal, pancreatic and  cardiomyocyte like cellsmRNA, proteinYes(2)
mBMCD45Ter119 Sca-1+PDGFRα+YesAdipocyte, chondrocyte, osteoblast,  endothelial cell; neuronal like cellsnot testedNo(17, 18)
mBMCD45nestin+YesAdipocyte, chondrocyte, osteoblastnot testedNo(19)
rat BMSSEA-1+(CD45Lin)YesCardiomyocyte, endothelial cellmRNA, proteinYes(16)
hUCBCD45LinCXCR4+NoNonemRNA, proteinYes(1)

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Murine VSEL Cells
  5. Human UCB VSEL Cell Phenotype
  6. CD45LINCXCR4+ VSEL Cells from Human UCB Lack Stem Cell Activity
  7. Summary
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

The authors declare the following conflicts of interest: Ralitza Danova-Alt was employed by Vita34 AG during the course of this project. Dietmar Egger is employed by Vita 34 AG. Rüdiger Alt received co-funding and cord blood samples from Vita 34 AG while working on this project at the Universität Leipzig and is now employed by Vita 34 AG.

Literature Cited

  1. Top of page
  2. Abstract
  3. Introduction
  4. Murine VSEL Cells
  5. Human UCB VSEL Cell Phenotype
  6. CD45LINCXCR4+ VSEL Cells from Human UCB Lack Stem Cell Activity
  7. Summary
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information
  • 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: 297303.
  • 2
    Kucia M, Reca R, Campbell FR, Zuba-Surma E, Majka M, Ratajczak J, Ratajczak MZ. A population of very small embryonic-like (VSEL) CXCR4(+)SSEA-1(+)Oct-4(+) stem cells identified in adult bone marrow. Leukemia 2006; 20: 857869.
  • 3
    Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002; 418: 4149.
  • 4
    D'Ippolito G, Diabira S, Howard GA, Menei P, Roos BA, Schiller PC. Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential. J Cell Sci 2004; 117: 29712981.
  • 5
    Kogler G, Sensken S, Airey JA, Trapp T, Muschen M, Feldhahn N, Liedtke S, Sorg RV, Fischer J, Rosenbaum C, et al. A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J Exp Med 2004; 200: 123135.
  • 6
    Zuba-Surma EK, Kucia M, Ratajczak J, Ratajczak MZ. “Small stem cells” in adult tissues: Very small embryonic-like stem cells stand up! Cytometry A 2009; 75A: 413.
  • 7
    Liedtke S, Enczmann J, Waclawczyk S, Wernet P, Kogler G. Oct4 and its pseudogenes confuse stem cell research. Cell Stem Cell 2007; 1: 364366.
  • 8
    Lengner CJ, Welstead GG, Jaenisch R. The pluripotency regulator Oct4: A role in somatic stem cells? Cell Cycle 2008; 7: 725728.
  • 9
    Ko K, Arauzo-Bravo MJ, Tapia N, Kim J, Lin Q, Bernemann C, Han DW, Gentile L, Reinhardt P, Greber B, et al. Human adult germline stem cells in question. Nature 2010; 465: E1; discussion E3.
  • 10
    Danova-Alt R, Heider A, Egger D, Cross M, Alt R. Very small embryonic-like stem cells purified from umbilical cord blood lack stem cell characteristics. PLoS One 2012; 7: e34899.
  • 11
    Shin DM, Zuba-Surma EK, Wu W, Ratajczak J, Wysoczynski M, Ratajczak MZ, Kucia M. Novel epigenetic mechanisms that control pluripotency and quiescence of adult bone marrow-derived Oct4(+) very small embryonic-like stem cells. Leukemia 2009; 23: 20422051.
  • 12
    Redshaw Z, Strain AJ. Human haematopoietic stem cells express Oct4 pseudogenes and lack the ability to initiate Oct4 promoter-driven gene expression. J Negat Results Biomed 2010; 9: 2.
  • 13
    Zuba-Surma EK, Guo Y, Taher H, Sanganalmath SK, Hunt G, Vincent RJ, Kucia M, Abdel-Latif A, Tang XL, Ratajczak MZ, et al. Transplantation of expanded bone marrow-derived very small embryonic-like stem cells (VSEL-SCs) improves left ventricular function and remodelling after myocardial infarction. J Cell Mol Med 2011; 15: 13191328.
  • 14
    Kucia M, Wysoczynski M, Ratajczak J, Ratajczak MZ. Identification of very small embryonic like (VSEL) stem cells in bone marrow. Cell Tissue Res 2008; 331: 125134.
  • 15
    Taichman RS, Wang Z, Shiozawa Y, Jung Y, Song J, Balduino A, Wang J, Patel LR, Havens AM, Kucia M, et al. Prospective identification and skeletal localization of cells capable of multilineage differentiation in vivo. Stem Cells Dev 2010; 19: 15571570.
  • 16
    Wu JH, Wang HJ, Tan YZ, Li ZH. Characterization of rat very small embryonic-like stem cells and cardiac repair after cell transplantation for myocardial infarction. Stem Cells Dev 2012; 21: 13671379.
  • 17
    Morikawa S, Mabuchi Y, Kubota Y, Nagai Y, Niibe K, Hiratsu E, Suzuki S, Miyauchi-Hara C, Nagoshi N, Sunabori T, et al. Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow. J Exp Med 2009; 206: 24832496.
  • 18
    Morikawa S, Mabuchi Y, Niibe K, Suzuki S, Nagoshi N, Sunabori T, Shimmura S, Nagai Y, Nakagawa T, Okano H, et al. Development of mesenchymal stem cells partially originate from the neural crest. Biochem Biophys Res Commun 2009; 379: 11141119.
  • 19
    Mendez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD, Lira SA, Scadden DT, Ma'ayan A, Enikolopov GN, Frenette PS. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 2010; 466: 829834.
  • 20
    Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein. Cell 1990; 60: 585595.
  • 21
    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: 3446.
  • 22
    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;Chapter 9:Unit9 29.
  • 23
    Peterson SE, Westra JW, Rehen SK, Young H, Bushman DM, Paczkowski CM, Yung YC, Lynch CL, Tran HT, Nickey KS, et al. Normal human pluripotent stem cell lines exhibit pervasive mosaic aneuploidy. PLoS One 2011; 6: e23018.
  • 24
    Peterson SE, Westra JW, Paczkowski CM, Chun J. Chromosomal mosaicism in neural stem cells. Methods Mol Biol 2008; 438: 197204.
  • 25
    Vanneste E, Voet T, Le Caignec C, Ampe M, Konings P, Melotte C, Debrock S, Amyere M, Vikkula M, Schuit F, et al. Chromosome instability is common in human cleavage-stage embryos. Nat Med 2009; 15: 577583.
  • 26
    Rehen SK, McConnell MJ, Kaushal D, Kingsbury MA, Yang AH, Chun J. Chromosomal variation in neurons of the developing and adult mammalian nervous system. Proc Natl Acad Sci USA 2001; 98: 1336113366.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Murine VSEL Cells
  5. Human UCB VSEL Cell Phenotype
  6. CD45LINCXCR4+ VSEL Cells from Human UCB Lack Stem Cell Activity
  7. Summary
  8. Acknowledgements
  9. Literature Cited
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
CYTO_22229_sm_SuppInfo.doc60KSupporting Information

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.