Cell and Tissue Research

, Volume 331, Issue 1, pp 125–134

Identification of very small embryonic like (VSEL) stem cells in bone marrow

Authors

  • M. Kucia
    • Stem Cell Institute at James Graham Brown Cancer CenterUniversity of Louisville
    • Department of PhysiopathologyPomeranian Medical University
  • M. Wysoczynski
    • Stem Cell Institute at James Graham Brown Cancer CenterUniversity of Louisville
    • Department of PhysiopathologyPomeranian Medical University
  • J. Ratajczak
    • Stem Cell Institute at James Graham Brown Cancer CenterUniversity of Louisville
    • Department of PhysiopathologyPomeranian Medical University
    • Stem Cell Institute at James Graham Brown Cancer CenterUniversity of Louisville
    • Department of PhysiopathologyPomeranian Medical University
Review

DOI: 10.1007/s00441-007-0485-4

Cite this article as:
Kucia, M., Wysoczynski, M., Ratajczak, J. et al. Cell Tissue Res (2008) 331: 125. doi:10.1007/s00441-007-0485-4

Abstract

Bone marrow (BM) develops in mammals by the end of the second/beginning of the third trimester of gestation and becomes a major hematopoietic organ in postnatal life. The α-chemokine stromal derived factor-1 (SDF-1) to CXCR4 (\( G_{{\alpha i}} \)-protein-coupled seven transmembrane-spanning chemokine receptor) axis plays a major role in BM colonization by stem cells. By the end of the second trimester of gestation, BM becomes colonized by hematopoietic stem cells (HSC), which are chemoattracted from the fetal liver in a CXCR4-SDF-1-dependent manner. Whereas CXCR4 is expressed on HSC, SDF-1 is secreted by BM stroma and osteoblasts that line BM cavities. Mounting evidence indicates that BM also contains rare CXCR4+ pluripotent stem cells (PSC). Recently, our group has identified a population of CXCR4+ very small embryonic like stem cells in murine BM and human cord blood. We hypothesize that these cells are deposited during development in BM as a mobile pool of circulating PSC that play a pivotal role in postnatal tissue turnover, both of non-hematopoietic and hematopoietic tissues.

Keywords

Oct-4NanogStage-specific embryonic antigen\( G_{{\alpha i}} \)-protein-coupled seven transmembrane-spanning chemokine receptorVery small embryonic like stem cellsEmbryonic stem cells

Introduction

Bone marrow (BM) was for many years primarily envisioned as the “home organ” of hematopoietic stem cells (HSC). Recent evidence, however, indicates that BM additionally contains a heterogeneous population of non-hematopoietic stem cells, which have been variously described in the literature as endothelial progenitor cells (Asahara et al. 1997; Shi et al. 1998), mesenchymal stem cells (MSC; Prockop 1997; Peister et al. 2004), multipotent adult progenitor cells (MAPC; Jiang et al. 2002), or marrow-isolated adult multilineage inducible (MIAMI) cells (D’Ippolito et al. 2004). Unexpectedly, BM has recently also been identified as a potential source of precursors of germ cells (oocytes and spermatogonial cells; Johnson et al. 2005; Nayernia et al. 2006). In some cases, similar or overlapping populations of primitive stem cells in the BM have probably been detected by using different experimental strategies and hence have been assigned different names.

The presence of these various populations of stem cells in the BM is hypothesized to be the result of the “developmental migration” of stem cells during ontogenesis. These cells are chemoattracted, during development, to the BM in which they find a permissive microenvironment. The most important chemotactic factors responsible for the accumulation of these cells in BM, a step that takes place by the end of the second and the beginning of the third trimester of gestation, are stromal derived factor-1 (SDF-1) or hepatocyte growth facto/scatter factor (HGF/SF; Nagasawa 2000; Taichman et al. 2001). On the other hand, the corresponding receptors for these factors, α-chemokine Gαi protein-coupled seven transmembrane-spanning receptor (CXCR4) and the tyrosine kinase receptor c-met are expressed on migrating stem cells. Mounting evidence indicates that, indeed, HSC and other non-hematopoietic stem cells are chemoattracted into BM in an SDF-1-CXCR4- and HGF/SF-c-met-dependent manner. Moreover, it is widely accepted that BM-derived stem cells, if needed, could be released/mobilized from the BM into circulation during tissue injury and stress, facilitating the regeneration of damaged organs (LaBarge and Blau 2002; Abbott et al. 2004; Kucia et al. 2004; Wojakowski et al. 2004; Gomperts et al. 2006).

Several attempts have been made in the past few years to purify, from BM, a pluripotent stem cell (PSC) that, in vitro, could give rise to cells from all three germ layers (meso-, ecto-, and endoderm) and, in vivo, after injection into the developing blastocyst, could contribute to the development of multiple organs and tissues. In contrast to positive data in vitro, this latter criterion for pluripotentiality in vivo for several potential candidates for PSC has not been demonstrated so far in a reproducible manner with any stem cell type isolated from adult tissues.

In this review, we focus on recent data from our laboratory identifying a population of CXCR4+, stage-specific embryonic antigen (SSEA)+, Oct-4+, Nanog+ stem cells in murine BM and human cord blood (CB). We have named these cells “very small embryonic like (VSEL) stem cells” (Kucia et al. 2006b, 2007). We have suggested that VSEL cells are deposited early in the development in marrow tissue and are descendants of epiblast-derived stem cells (EPSC) (Ratajczak et al. 2007) including some primordial germ cells (PGC; Kucia et al. 2006a).

We further hypothesize that VSEL cells are a dormant quiescent population of PSC that (1) are deposited during development and reside in BM, (2) may contribute to long-term hematopoiesis, (3) after being mobilized from BM into peripheral blood, may participate in the turnover of other tissue-specific (monopotent) stem cells that are located in peripheral niches, and (4) may play a role in tissue organ regeneration as seen, for example, during stress situation/organ injury (Kucia et al. 2004, 2006c). On the other hand, since “a thin red line” exists between regeneration and tumor formation, we consider it likely that this population of primitive stem cells may give rise to some types of cancer (e.g., pediatric sarcomas, teratomas, germinal tumors) if exposed to mutagens.

Identification of VSEL stem cells in BM

A few years ago, we proposed an alternative explanation of stem cell plasticity and postulated that BM may contain some rare non-hematopoietic stem cells that are committed to various tissues. We enriched murine and human BM-derived cells for this particular population of stem cells after chemotaxis to SDF-1 and described them as tissue committed stem cells (Ratajczak et al. 2004). However, we soon realized that these cells also express mRNA for several embryonic transcription factors, such as Oct-4, Nanog, and Rex-1 (Kucia et al. 2006b). This indicated the potential relationship of these cells to putative BM-residing PSC.

Based on this observation, our team began “a hunt to purify” such cells from BM. In order to isolate these potential PSC, we have assumed that they (1) will express CXCR4 receptor (mice and human) and Sca-1+ (mice), (2) will be non-hematopoietic (CD45), and (3) will be very small in size. This will be justified below.

The assumed expression of CXCR4 on these cells was based on our data showing that we can enrich murine BM-derived mononuclear cells (BMMNC) for this population of cells after chemotactic isolation to an SDF-1 gradient (Fig. 1a; Ratajczak et al. 2004). We noticed that murine BMMNC collected from the lower chamber of the transwell system after chemotaxis to SDF-1 were enriched in mRNA for several early developmental markers. If we subsequently sorted cells that responded to an SDF-1 gradient (CXCR4+ BMMNC), by employing a fluorescence-activated cell sorter, into populations of CD45+ and CD45 cells, the latter population was more enriched for mRNA for non-hematopoietic stem cells including several PSC markers (Fig. 1b). Finally, direct microscopic analysis of murine BMMNC isolated by chemotactic gradient to SDF-1 revealed that this population contained rare small cells (2–4 μm in diameter) that stained positively, for example, for Oct-4 or Nanog. We also noticed that these cells could be enriched and sorted from murine BM by fluorescence-activated cell sorting (FACS) to give a population of Sca-1+ cells; this indicated that Sca-1 antigen, instead of CXCR4, could be employed as a surrogate marker to isolate these cells by FACS (Kucia et al. 2006b).
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Fig. 1

Strategy to isolate non-hematopoietic stem cells enriched in very small embryonic like (VSEL) cells from bone-marrow-derived mononuclear cells (BMMNC) based on chemotactic isolation combined with fluorescence-activated cell sorting (FACS). a BMMNC freshly isolated from bone marrow were loaded into the upper chamber of transwells and allowed to undergo chemotaxis for 1–3 h to an SDF-1 gradient or control medium (Step I). b BMMNC that responded to the SDF-1 gradient (expressing functional CXCR4) were subsequently sorted by FACS into populations of CD45+ and CD45 cells (Step II). CXCR4+CD45 cells in contrast to CXCR4+CD45+ are highly enriched in non-hematopoietic developmental markers, including markers of VSEL cells

Based on these observations, we hypothesized that the potential PSC would reside in murine BM as a population of very small CXCR4+ Sca-1+ CD45 lin cells. Figure 2 shows a general strategy to sort a population of very small (2–4 μm) Sca-1+ CD45 lin cells from murine BM. These cells express (as determined by real-time quantitative polymerase chain reaction and immunhistochemistry) SSEA-1, Oct-4, Nanog, and Rex-1 and Rif-1 telomerase protein, do not express MHC-I and MHC-II (HLA-DR) antigens, and are CD90 CD105 CD29 (Fig. 3). Direct electron-microscopic analysis has revealed that they display several features typical of embryonic stem cells such as a large nucleus surrounded by a narrow rim of cytoplasm and open-type chromatin (euchromatin). Despite their small size, they posses diploid DNA, high telomerase activity, and numerous mitochondria (Fig. 4). Based on all these criteria, we have named these cells VSEL stem cells. Figure 4 shows a direct size comparison of murine VSEL cells with BM-derived murine HSC.
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Fig. 2

Multicolor FACS of VSEL cells from murine BMMNC. VSEL cells were sorted from BMMNC derived from 1-month-old mice. Cells were sorted by employing a MoFlo sorter (DAKO) after immunostaining for murine Sca-1, CD45, and lineage markers with specific antibodies conjugated with the fluorchoromes PE-Cy5, APC-Cy7, and PE, respectively (BD Pharmingen). Cells were sorted based on size, which was estimated by using beads (Flow Cytometry Size Calibration Kit, Invitrogen). a Region R1 contains cells with a size between 2 and 6 μm. b Dot-plots show cells from R1. VSEL cells were isolated from a fraction of cells that were negative for CD45 and did not express lineage markers (region R4, left upper quadrant). c Sca-1+ lin CD45 cells (VSEL cells) are shown in R9 (0.01% of murine BMMNC) and Sca-1+ lin CD45+ cells (HSC) are shown in R8. A representative sort is shown

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Fig. 3

Phenotype of murine VSEL cells. a Murine BMMNC were gated for small Sca-1+linCD45 cells (R1). b Subsequently, CD45lin cells (R2) were evaluated for expression of Sca-1 and MHC-1, MHC-II (HLA-DR), CD29, CD90, and CD105. In brief, BMMNCs were flushed from the femura of pathogen-free 1-month-old female C57Bl/6 (Jackson Laboratory, Bar Harbor, Me., USA). Erythrocytes were removed by a hypotonic solution (Lysing Buffer, BD Biosciences, San Jose, Calif., USA). BMMNCs were re-suspended in staining medium, containing 1× Hanks’ balanced salt solution without phenol red, 2% heat-inactivated fetal calf serum, 10 mM HEPES buffer, and 30 U/ml Gentamicin (all from GIBCO, Grand Island, N.Y., USA). The following monoclonal antibodies were employed to detect Sca-1+linCD45 VSEL cells: biotin-conjugated rat anti-mouse Ly-6A/E (Sca-1; clone E13-161.7), streptavidin-APC conjugate, anti-CD45-PE-Cy5 (clone 30-F11), anti-CD45R/B220-PE (clone RA3-6B2), anti-Gr-1-PE (clone RB6-8C5), anti-TCR alpha/beta PE (clone H57-597), anti-TCR gamma/delta PE (clone GL3), anti-CD11b PE (clone M1/70), and anti-Ter-119 PE (clone TER-119). c–g The following antibodies were employed to phenotype VSEL cells: anti-MHC-I fluorescein isothiocyanate (FITC; clone H-2Db; c), anti-HLA DR FITC (d), anti-CD90 FITC (clone HIS51; e), anti-CD29 FITC (clone HMβ1-1; f), anti-CD105 FITC (clone MJ7/18; g). All monoclonal antibodies were added at saturating concentrations. The cells were incubated for 30 min on ice, washed twice, and then re-suspended in staining solution; 106 events were collected. Representative FACS analyses are shown

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Fig. 4

Transmission election microscopy (TEM) of murine Sca-1+linCD45 VSEL stem cell and murine Sca-1+linCD45+ hematopoietic stem cells (HSC). For TEM analysis, the murine Sca-1+linCD45 (VSEL) and murine Sca-1+linCD45 (primary HSC) cells were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 10 h at 4°C, post-fixed in osmium tetroxide and dehydrated. Fixed cells were subsequently embedded in LX112 and sectioned at 800 Å, stained with uranyl acetate and lead citrate, and viewed on a Philips CM10 electron microscope operating at 60 kV. a VSEL stem cells are small and measure 2–4 μm in diameter. They possess a large nucleus surrounded by a narrow rim of cytoplasm. At the ultrastructural level, the narrow rim of cytoplasm possesses a few mitochondria, scattered ribosomes, small profiles of endoplasmic reticulum, and a few vesicles. The nucleus is contained within a nuclear envelope with nuclear pores. Chromatin is loosely packed and consists of euchromatin. b In contrast, Sca-1+linCD45+ hematopoietic stem cells display a heterogenous morphology and are larger. They measure on average 8–10 μm in diameter and possess scattered chromatin and prominent nucleoli. Representative cells are shown side by side to show the relative difference in their sizes

Interestingly 5%–10% of purified murine VSEL cells, if plated over a C2C12 murine myoblast cell feeder layer, are able to form spheres that resemble embryoid bodies (Fig. 5a) and stain positively for the fetal isoform of alkaline phosphatase (Fig. 5b). Furthermore, the appearance of green fluorescence protein (GFP)+ spheres after plating VSEL stem cells isolated from GFP+ mice and the diploid content of the DNA in these spheres excludes the possibility that these spheres are derived from the supportive C2C12 cell line (Fig. 2c) or are a result of cell fusion between VSEL and C2C12 cells.
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Fig. 5

Murine VSEL cells form spheres (VSEL-DS) in co-cultures over C2C12 cells. Isolated by FACS from murine BM Sca-1+linCD45 VSEL cells are plated over exponentially growing murine C2C12 myoblastic cells line in Dulbecco’s minimal essential medium supplemented with 2% heat-inactivated fetal bovine serum. In these co-culture conditions, by day 7–10, 5%–10% of plated VSEL cells form spheres (VSEL-DS) that consist of primitive stem cells that express Oct-4, Nanog, and SSEA-1 antigen. a Typical formation of sphere by murine VSEL cells. b Murine VSEL-DS stain positively for fetal alkaline phosphatase (red). c VSEL cells sorted from GFP+ mice form GFP+ VSEL-DS (green) in co-cultures over C2C12 cells. Representative images are shown

We found that the cells in murine VSEL-derived spheres (VSEL-DS) are immature with large nuclei containing euchromatin and, like purified VSEL cells, are CXCR4+SSEA-1+Oct-4+. Furthermore, cells from VSEL-DS, after being re-plated over C2C12 cells, may again (up to 5–7 passages) grow new spheres or, if plated into cultures promoting tissue differentiation, expand into cells from all three germ-cell layers. Similar spheres are also formed by VSEL cells isolated from murine fetal liver, spleen, and thymus. Unfortunately, we cannot reproduce this phenomenon so far with human VSEL cells; this suggests an involvement of species-specific factors.

Interestingly, the formation of VSEL-DS is associated with a young age in mice, and no VSEL-DS have been observed in cells isolated from old mice (~2 years old). The finding that VSEL cells express several markers of the germ cell line (fetal-type alkaline phosphatase, Oct-4, SSEA-1, CXCR4, Mvh, Stella, Fragilis, Nobox, Hdac6) indicates that they are probably closely related developmentally to a population of EPSC and PGC (Ratajczak et al. 2007). We will discuss this potential developmental link below.

We have noticed that VSEL cells are mobile and respond robustly to an SDF-1 gradient, adhere to fibronectin and fibrinogen, and may interact with BM-derived stromal fibroblasts. Confocal microscopy and time-lapse studies have revealed that these cells attach rapidly to, undergo emperipolesis, or migrate beneath BM-derived fibroblasts (Fig. 6; Kucia et al. 2005). This strong interaction of VSEL cells with BM-derived fibroblasts has an important implication, namely, populations of BM-adherent fibroblastic cells described in the literature (e.g., MSC, MAPC, or MIAMI) may “be contaminated” by these tiny rare cells that “hide out” among fibroblasts.
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Fig. 6

VSEL cells “hiding” in bone marrow fibroblasts. Sca-1+linCD45 cells sorted from BMMNC were plated on murine BM-derived stromal cells. Images of interaction were recorded and analyzed with computer-aided methods. Observations were carried out under a Nicon Inverted Microscope Eclipse TE300 with an oil immersion ×60 objective lens. All of the images were captured by a cool snap HQ digital B/W CCD (Roper Scientific) camera. Time-lapsing of the cells was carried out for 20 min. a VSEL cell approaches/attaches to bone marrow fibroblast (arrow). b Fibroblast stretches out a protrusion that captures VSEL cell (white arrow). c, d VSEL cell is dragged by protrusion from fibroblast and placed below the fibroblast or undergoes emperipolesis. Based on this, we postulate that trans-dedifferentiation experiments performed on BM-derived adherent fibroblast cell-like populations of cells (MSC, MAPC, MIAMI, or unrestricted somatic stem cells) must exclude “cross contamination” of these adherent fibroblastic cells by VSEL cells

To support this further, recent evidence indicates that MSC, MAPC, and MIAMI do indeed contain cells that express SSEA-1, Oct-4, Rex-1, or Nanog. For example, a subpopulation of undifferentiated MSC isolated from BM adherent cells has been found to express Oct-4, Rex1, and Nanog (Lamoury et al. 2006). Furthermore, a population of SSEA-1+ Oct-4+ Nanog+ Sca-1+ lin CD45 cells that is similar to our VSEL cells was recently isolated from the BM by another team with the suggestion that these cells could be associated with very early MSC (Anjos-Afonso and Bonnet 2007). Similarly, serum deprivation of human MSC cultures selects for the expansion of so-called serum-deprived MSC, which express mRNA for embryonic markers, e.g., Oct-4 (Pochampally et al. 2004). Moreover, another population of BM-derived adherent cells, described as MAPC, has recently been further enriched for small cells that are embryonic-like and express SSEA, Oct-4, and Nanog (Zeng et al. 2006). Furthermore, the population of MIAMI cells, which are isolated from human adult BM by culturing BM mononuclear cells in low-oxygen-tension conditions on fibronectin, express the embryonic stem cell markers Oct-4 and Rex-1 and differentiate into cells of multiple germ layers (D’Ippolito et al. 2004). Finally, BM has also been identified as a source of Oct-4+ oocyte-like (Johnson et al. 2005) and spermatogonia-like (Nayernia et al. 2006) cells and of Oct3/4+ cells, which may differentiate in vitro into cardiomyocytes (Pallante et al. 2007).

The potential relationship of all these versatile Oct-4+ cells to Oct-4+ VSEL cells described by us is not clear at this point, although these are overlapping populations of cells, which are identified by slightly different isolation/expansion strategies.

Developmental origin of VSEL cells

An important problem to be solved is the developmental origin of VSEL cells. We suggest that these cells are specified early during development in the primitive ectoderm (epiblast) at the stage of the so-called “cylinder embryo”. We also envision that VSEL cells are direct descendants from the germ lineage, which is the most important cell lineage in the body from a developmental standpoint. This will be justified below.

It is widely accepted that, from a developmental and evolutionary point of view, the main goal of the multicellular organism is to pass genes to the next generations; this process is orchestrated by the appropriate interplay between germ and somatic cell lines. The germ line carries the genome (nuclear and mitochondrial DNA) from one generation to the next and is the only cell lineage that retains true developmental totipotency. In this context, we can envision that all somatic cells are descendants of the germ line and help germ cells to accomplish this mission effectively (Fig. 7).
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Fig. 7

Hypothetical developmental origin of VSEL cells. From a developmental and evolutionary perspective, the germ line (red) carries the genome (nuclear and mitochondrial DNA) from one generation to the next, and all somatic cell lines (gray) “bud out” from the germ line during ontogenesis in order that the germ cells successfully acheive their goal. The germ potential is established in the fertilized oocyte (zygote) and is subsequently retained in the morula, inner cell mass, EPSC, PGC, and mature germ cells (oocytes and sperm). The first cells that “bud out” from the germ lineage are trophoectodermal cells, which give rise to the placenta. Subsequently, during gastrulation, EPSC are a source of pluripotent stem cells for all three germ layers (meso-, ecto-, and endoderm) and PGC. We propose that, at this stage, some EPSC might be deposited as Oct-4+ PSC in the peripheral tissues/organs (red dotted circles). Similarly, some migrating PGC might wander from their major migratory route to the genital ridges and also become deposited. Furthermore, we hypothesize that, like the PGC, other EPSC deposited in the developing tissues undergo erasure of their somatic imprint (yellow line). This mechanism of erasure of methylation of somatic imprinted genes protects the developing organism from the possibility of teratoma formation. However, it will affect some of the aspects of the pluripotentiality of these cells (e.g., potential of these cells to contribute to blastocyst development or teratoma formation after transplantation into immunodeficient mice)

To support this concept, we can envision a zygote, which is derived directly during conception from the fusion of two germ cells (female oocyte and male sperm), as the most primitive totipotent germ stem cell able to form both embryo and extra-embryonal tissues (placenta). During fertilization, the haploid DNA derived from an oocyte is combined with haploid DNA of a sperm, and thus the zygote can be envisioned as a mother cell to the germ lineage, which “down the road” will give rise to (1) more differentiated cells from the germ lineage, viz., gametes (to ensure transfer of the genome to the next generation, and (2) other somatic lineages that will provide the soma to fulfill this mission. These somatic cells are derived/“bud out” from the germ lineage during embryogenesis. Figure 7 presents this concept showing “the circle of reproductive life”: it begins with the establishment of the most primitive germ line cell (zygote), which gives rise to somatic lineages (meso-, ecto-, and endoderm) and, most importantly, the germ cells (oocytes or sperm), which ensure transfer of DNA to the next generation. In this context, the germ lineage, in order to pass genes to the progeny, must establish an adult organism that will provide a “vehicle” soma/body to fulfill this aim.

Figure 7 also shows that the germ potential (marked as red) of the zygote is retained in the first blastomers, cells that are present in the center of a developing morula, and subsequently in the cells from the inner cell mass (ICM) of a blastocyst. At this time of development, however, some level of specification occurs, and the trophoectoderm “buds out” from the germ line (Fig. 7). The trophoectoderm forms the placenta and the remaining part of the blastocyst, whereas the ICM will give rise to the epiblast (Yamanaka et al. 2006).

EPSC, as demonstrated experimentally, are also pluripotent and also retain germ lineage potential (Martin 1981; Amit et al. 2000). EPSC express SSEA-1 (mice), SSEA-3/4 (human), Oct-4, and Nanog. They will give rise to all three germ layers, viz., the ecto-, meso-, and endoderm including PGC. Thus, the epiblast, through the process of gastrulation, is the source of all stem cells for all the germ layers; the stem cells in these layers will give rise to all of the tissues and organs in the embryo. Thus, EPSC can be envisioned as a founder population of PSC for multipotent stem cells for ecto-, endo-, or mesoderm and, later in development, will give rise to unipotent stem cells that will develop given cell lineages. These Oct-4+ EPSC may survive in the tissues and organs into adulthood as a founder population of stem cells. Furthermore, additional evidence exists that some of the epiblast-derived PGC themselves go astry during their migration through the embryo proper on their way to the genital ridges and seed into peripheral tissues (Jordan 1917; Upadhyay and Zamboni 1982; Francavilla and Zamboni 1985).

In conclusion, we hypothesize that the cells identified in adult tissues as expressing ICM/epiblast/PGC markers, such as SSEA-1 (mice), SSEA-3/4 (human), Oct-4, and Nanog, are populations of PSC that are deposited in these tissues early during gastrulation/embryogenesis and are derived mostly from EPSC and to some extent from some rare migrating PGC that drift from their major migratory route to the genital ridges (Fig. 7).

Regulation of pluripotency in the germ line by status of somatic imprint: implications for other PSC in adult organs/tissues?

As mentioned in the Introduction, the most valuable assay to demonstrate that a given stem cell is truly pluripotent is one based on the developmental complementation of the blastocyst. In this assay, candidate PSC are injected into the developing blastocyst, and subsequently the embryo or the neonatal mouse is examined to determine whether the potential candidate cell has contributed to the development of the organs and tissues belonging to all three germ layers. Indeed, this had not as yet been shown in a reproducible manner for any type of stem cell isolated from adult tissues.

We suggest that the problem of providing such evidence arises because all PSC that reside in adult tissues probably erase their somatic imprint, viz., the differential methylation of some maternally and paternally inherited genes (e.g., H19, Igf-2, Igf-2R, Snrpn; Mann 2001; Yamazaki et al. 2003). For example, Igf-2 is expressed from the paternal and H19 from the maternal chromosome. The erasure of the somatic imprint on these genes by demethylation protects, on one hand, the developing organism from parthenogenesis and teratoma formation and, on the other, prevents PSC surviving into adulthood with the ability to complete blastocyst development. The phenomenon of the erasure of the somatic imprint has been studied extensively in the case of epiblast-derived PGC and will be discussed below.

Shortly before the epiblast is about to give rise to all three germ layers (ectoderm, mesoderm, and endoderm), the first morphologically identifiable precursors of PGC in mice become specified, at 6.0–6.5 days post conception (dpc) in the proximal part of the epiblast, as the first population of stem cells in the embryo at the beginning of gastrulation (McLaren 1992, 2003). PGC in mice subsequently move for a short period of time, first to the basis of alantois, which is located in the extra-embryonic mesoderm, and then migrate into the embryo proper toward the genital ridges in which they finally undergo developmental differentiation into oocytes or sperm, respectively (Molyneaux and Wylie 2004).

Interestingly, the migrating PGC, in contrast to EPSC, lose their pluripotency. Namely, migrating PGC freshly isolated from embryos proliferate in vitro for a few days only and then disappear, either because they differentiate or because they die (De Felici and McLaren 1982). Furthermore, whereas the nuclei of even terminally differentiated somatic cells (e.g., fibrocytes, lymphocytes) can be successfully used as donors for nuclear transfer and may give rise to the clonote, nuclei from PGC at 11.5 dpc and later are incompetent to support full-term development of the clonote (Yamazaki et al. 2003). This is intriguing, taking into consideration that PGC are the population of stem cells that carries “developmental totipotency” for oocytes and sperm.

However, on the other hand, when PGC are cultured over murine embryonic fibroblasts and exposed ex vivo to three growth factors, viz., kit ligand (KL), leukemia inhibitory factor (LIF), and basic fibroblast growth factor (FGF-2), they continue to proliferate and form large colonies of embryonic germ (EG) cells, which like embryonic stem cells (ESC) can be expanded indefinitely (Matsui et al. 1992; Shamblott et al. 1998; Turnpenny et al. 2003). Such EG cells have been derived from pre- and post-migratory and from migratory PGC in both mice and humans and are pluripotent (Matsui et al. 1992; Shamblott et al. 1998). Thus, EG cells in contrast to PGC fully contribute to all three germ layers in the blastocyst complementation assay giving rise, in the developing embryo, to all somatic lineages and germ cells.

As an explanation of this phenomenon at the molecular level, PGC until 9.5 dpc presumably display the proper somatic imprint (paternal and maternal pattern of methylation) of the H19, Igf-2, Igf-2R, and Snrpn genes crucial for maintaining their pluripotency (Mann 2001). The somatic imprint, however, is erased in PGC by demethylation while these cells migrate toward the genital ridges at approximately 10.5 dpc (Sato et al. 2003). The erasure of the methylation (imprint) of the H19, Igf-2, Igf-2R, and Snrpn genes in early PGC could make these cells resistant to parthenogenesis or the formation of teratomas, as mentioned above, but shuts down PGC developmental pluripotency (Macchiarini and Ostertag 2004; Oosterhuis and Looijenga 2005). A proper somatic imprint is however subsequently re-established in developing gonads in haploid gametes (sperm and oocytes), so that a fertilized egg (zygote) expresses a developmentally proper somatic imprint of these crucial genes.

We hypothesize that a similar mechanism of somatic imprint erasure takes place during development, not only in PGC, but also in other EPSC-derived PSC that are deposited in the developing organs (Fig. 7, yellow line). Because of the erasure of the somatic imprint, deposited PSC lose the ability to complete blastocyst development. However, as PGC-derived EG cells can regain their pluripoteniality, the erasure of the somatic imprint is reversible, and the re-establishment of a proper somatic imprint is possible, for example, in vitro under appropriate culture conditions (in the case of PGC, with murine embryonic fibroblasts as the feeder layer and in the presence of KL, LIF, and FGF-2). Moreover, like PGC, other PSC that reside in adult tissues may also regain a proper somatic imprint under certain circumstances (e.g., tissue/organ injury). This may occur through epigenetic changes. The identification of genes/proteins involved in this phenomenon will no doubt be crucial for manipulating the pluripotentiality of somatic stem cells.

VSEL-like stem cells in other tissues

A population of stem cells that express markers of ESC/epiblast/PGC cells has recently been described in several non-hematopoietic organs, e.g., in epidermis (Dyce et al. 2004, 2006; Yu et al. 2006), bronchial epithelium (Ling et al. 2006), myocardium (Mendez-Ferrer et al. (2006), pancreas (Kruse et al. 2006; Danner et al. 2007), testes (Kanatsu-Shinohara et al. 2004; Guan et al. 2006), retina (Koso et al. 2006), and amniotic fluid (De Coppi et al. 2007). These cells residing in different tissues, depending on the niche in which they are located, may slightly differ in size or panel of surface markers. Their closer analysis by in vitro assays, however, has demonstrated that they are able to differentiate in a similar way as their counterparts isolated from the BM or CB into cells belonging to the various germ layers. These data support an overall concept of developmental deposition of Oct-4+ EPSC in developing organs (Fig. 7). We also envision that these cells circulate in the peripheral blood and shuttle between BM and other organs, as seen for example during tissue organ injury (Kucia et al. 2004, 2006c; Eghbali-Fatourechi et al. 2005; Palermo et al. 2005; Togel et al. 2005; Gomperts et al. 2006).

Future implications

The “positive” data supporting the plasticity of BM-derived stem cells can thus be re-interpreted, at least partially, with respect to the finding that BM stem cells are heterogeneous and that BM tissue contains various types of stem cells, including a rare population of pluripotent VSEL cells. The question remains, however, as to whether these PSC can continuously contribute in adult life to the renewal of other stem cells, including HSC, which are the most numerous population of stem cells in BM.

Several answers can be given to this provocative question, which is especially timely in view of the current widely performed clinical trails with BM-derived stem cells in cardiology and neurology, and these answers should be considered before these cells find potential application in regenerative medicine. First, there is the obvious problem of isolating a sufficient number of VSEL cells from the BM. The number of these cells among murine BM MNC is extremely low, as VSEL cells represent 1 cell in 104–105 of BM MNC (Kucia et al. 2006b). Furthermore, our data show that these cells are enriched in the BM of young mammals and their number decreases with age (Kucia et al. 2006b). VSEL cells, even if they are released from the BM and are able to home toward the areas of tissue/organ injury, probably play a role only in the regeneration of minor tissue injuries. Heart infarct or stroke, on the other hand, involves severe tissue damage possibly beyond the capacity of these rare cells to repair effectively. Second, the allocation of these cells to the damaged areas depends on homing signals that may be inefficient in the presence of proteolytic enzymes released from leukocytes and macrophages associated with damaged tissue. For example, matrix metalloproteinases released from inflammatory cells may degrade SDF-1 locally and thus perturb the homing of CXCR4+ stem cells. Therefore, VSEL cells may circulate as a “homeless” population of stem cells in peripheral blood and return to BM or home to other organs. Third, in order to reveal their full regenerative potential, these cells have to be fully functional. We cannot exclude the possibility that VSEL cells, while residing/being “trapped” in the BM, are not fully functional but remain “locked” into a dormant state and need the appropriate activation signals by unidentified factors. As mentioned above, their somatic imprint is most probably erased, which may limit their pluripotentiality. Finally, we have not so far identified an efficient combination of growth factors/adhesion molecules that allows the efficient differentiation of VSEL cells without supportive feeder layer cells (e.g., C2C12, BM stroma fibroblasts). However, cells isolated from VSEL-DS may differentiate into all the germ layers, although, as mentioned above, they show some level of differentiation and limited self-renewal.

In conclusion, our data indicate that VSEL cells potentially provide a real therapeutic alternative to the controversial use of human ES cells and therapeutic cloning. Hence, while the ethical debate concerning the application of ES cells in therapy continues, the potential of VSEL cells is ripe for exploration. Researchers must determine whether these cells can be efficiently employed in the clinic, or whether they are merely developmental remnants that are found in the BM but that cannot be harnessed effectively for regeneration. The coming years will bring important answers to these questions.

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© Springer-Verlag 2007