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Keywords:

  • Very small embryonic-like stem cells;
  • CXCR4;
  • stage-specific embryonic antigen-1;
  • Granulocyte colony-stimulating factor;
  • Mobilization

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Recently, we identified in murine adult tissues, including bone marrow, a population of very small embryonic-like (VSEL) stem cells. Here, we provide further evidence that under steady-state conditions these cells circulate at very low levels in peripheral blood (PB) (∼100–200 cells/ml) and could be additionally mobilized during pharmacological granulocyte-colony-stimulating factor-induced or stress-related mobilization, as demonstrated in a model of toxic liver or skeletal muscle damage induced by injection of carbon tetrachloride or cardiotoxin, respectively. The number of circulating VSEL stem cells under steady-state conditions in PB of 2-month-old animals was five times higher than that in 1-year-old mice. In conclusion, this study supports a hypothesis that VSEL stem cells are a mobile pool of primitive stem cells that could be released from the stem cell niches into PB. Further studies are needed, however, to see whether the level of these cells circulating in PB could become a prognostic indicator to assess the regenerative potential of an adult organism and/or clinical outcome from an injury.

Disclosure of potential conflicts of interest is found at the end of this article.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Author contributions: M.J.K.: collection and/or assembly of data, conception and design, financial support, manuscript co-writing; M.W., E.K.Z.-S., J.R.: collection and/or assembly of data; W.W.: provision of study material; M.Z.R.: conception and design, financial support, data analysis and, interpretation, manuscript writing, final approval of manuscript.

Circulating bone marrow (BM)-derived stem/progenitor cells could play an important role in turnover of stem cells in peripheral tissues. For example, hematopoietic stem cells (HSCs) continuously circulate at a very low level in peripheral blood (PB) to maintain a pool of HSCs in marrow tissue that is distributed in bones located in the distant part of the body. These HSCs are mobilized into PB, for example, during pharmacological mobilization induced by administration of granulocyte colony-stimulating factor (G-CSF) [1] or CXCR4 receptor antagonists (AMD3100 or T140) [2, [3]4]. They are also mobilized after BM damage as seen, for example, after partial BM irradiation [5] or administration of cytostatic agents (e.g., cyclophosphamide) [6, 7].

On the other hand, there is compelling evidence that BM contains a population of non-HSCs in addition to HSCs, which, similarly to HSCs, could be mobilized into PB during tissue/organ injuries. Accordingly, cells that express markers for mesenchymal stem cells [8], fibrocytes [9, 10], skeletal progenitors [11, 12], endothelial progenitors [13, [14]15], liver oval stem cells [16], kidney [17], and bronchial epithelium progenitors [18] were identified in PB in various tissue/organ injury models.

In accordance with these data we recently reported that during G-CSF-induced mobilization [19] as well as after heart infarct [20, 21] or stroke [22], the number of CXCR4+ mononuclear cells circulating in PB, enriched for se veral tissue-specific developmental markers, increases. Based on this finding, we postulated that a population of CXCR4+ tissue-committed stem cells (TCSCs) [19, 23] that respond robustly to a chemotactic gradient of stromal-derived factor-1 (SDF-1) resides in BM. Because SDF-1 is upregulated in a hypoxia-dependent manner in damaged organs [24, 25], TCSCs could be mobilized into PB in response to this chemotactic factor. We also suggested that, in addition to SDF-1, other chemoattractants such as hepatocyte growth factor/scatter factor (HGF/SF), leukemia inhibitory factor (LIF), and vascular endothelial growth factor (VEGF) [26, [27], [28]29] also direct mobilization of these cells. Thus, the concept emerged that chemotactic factors upregulated in damaged tissues may orchestrate release of non-HSCs from BM as well as from other organs into PB.

Furthermore, recent work from our group revealed that BM-derived CXCR4+ TCSCs (a) are small in size, (b) contain large nuclei with unorganized euchromatin, (c) express stage-specific embryonic antigen-1 (SSEA-1) on the surface and early embryonic transcription factors such as Oct-4 and Nanog in nuclei, and (d) show high activity of telomerase. Based on these observations and on the fact that these cells differentiate in vitro into cells from all three germ layers, we changed the name of these cells, which we initially described as TCSCs, to very small embryonic-like (VSEL) stem cells [30]. The fact that VSEL stem cells express mRNA for several TCSC markers could be explained by the open status of the chromatin in these cells that allows transcription of several genes, including those involved in tissue/organ development.

In this article we investigated whether cells that display the VSEL stem cell phenotype and morphology could be released into PB as a result of pharmacological G-CSF-induced and/or organ injury-directed mobilization. For the first time, by use of multiparameter analysis, we present evidence that VSEL stem cells could be detected in circulating PB as a population of small Sca-1+linCD45 cells that express SSEA-1 and Oct-4 at the protein level and that the number of these cells increases in stress situations. We also demonstrate that the number of these cells circulating in PB is reduced in older animals.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

The present study was performed in accordance with the guidelines of the Animal Care and Use Committee of the University of Louisville School of Medicine and with the Guide for the Care and Use of Laboratory Animals [31].

Mice and PB-Derived Cells

Murine mononuclear cells (MNCs) were isolated from PB of pathogen-free, 4- to 6-week-old and 12-month-old female C57BL/6, BALB/c, or C57BL/6-Tg(ACTB-EGFP)1Osb/J mice (Jackson Laboratory, Bar Harbor, ME, http://www.jax.org). Whole PB samples were lysed in BD lysing buffer (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) for 15 minutes in room temperature and washed twice in phosphate-buffered saline (PBS).

Mobilization of Mice

Mice were mobilized by subcutaneous injection of 250 μg/kg human G-CSF or 50 μg/kg human G-CSF (Amgen, Thousand Oaks, CA, http://www.amgen.com) daily for 6 days. Mice were bled from the retro-orbital plexus to obtain leukocyte counts using a MicroVette system for capillary blood collection (Sarstedt, Nümbrecht, Germany. http://www.sarstedt.com/php/main.php). Six hours after the last G-CSF injection, PB was obtained from the vena cava (with a 25-gauge needle and 1-ml syringe containing 250 U of heparin). In some mobilization protocols the specific blocking agent for CXCR4-T140-truncated polyphemusin analog (a gift from Dr. N. Fuji, Kyoto University, Kyoto, Japan) was injected intraperitoneally as indicated in the figure legends.

White Blood Cell Counts

Samples (50 μl) of PB were taken from the retro-orbital plexus of the mice and collected into MicroVette EDTA-coated tubes (Sarstedt Inc., Newton, NC). Samples were run within 2 hours of collection on a Hemavet 950 (Drew Scientific Inc., Oxford, CT, http://www.drew-scientific.com).

Staining of PB-Derived VSEL Stem Cells

To determine the amount of Sca-1+LinCD45 (VSEL) cells, flow cytometry analysis was performed. In brief, a single cell suspension was stained for lineage markers (CD45R/B220 clone RA3-6B2, Gr-1 clone RB6-8C5, TCRαβ clone H57-597, TCRγζ clone GL3, CD11b clone M1/70, and Ter-119 clone TER-119) conjugated with phycoerythrin (PE), CD45 (clone 30-F11) conjugated with PE-Cy5, and Sca-1 (clone D7) conjugated with fluorescein isothiocyanate (FITC) for 30 minutes on ice. After washing, samples were analyzed by fluorescence-activated cell sorting (FACS) (BD Biosciences). At least 106 events were acquired and analyzed by using Cell Quest software. Samples stained with appropriate isotype controls (BD Pharmingen, San Diego, CA, http://www.bdbiosciences.com/index_us.shtml) were examined in parallel.

Sorting of PB-Derived Cells

Lin/Sca-1+/CD45 (VSEL stem cells) and Lin/Sca-1+/CD45+ (HSCs) were isolated from a suspension of murine PB MNCs by multiparameter, live sterile cell sorting (MoFlo; Dako A/S, Fort Collins, CO, http://www.dako.com). Briefly, PB MNCs (100 × 106 cells/ml) were resuspended in cell sort medium (CSM), containing 1× Hanks' balanced salt solution without phenol red (Gibco, Grand Island, NY, http://www.invitrogen.com), 2% heat-inactivated fetal calf serum (Gibco), 10 mM HEPES buffer (Gibco), and 30 U/ml gentamicin (Gibco). The following monoclonal antibodies (mAbs) were used to stain these cells: biotin-conjugated rat anti-mouse Ly-6A/E (Sca-1) (clone E13-161.7), streptavidin-PE-Cy5 conjugate, anti-CD45-APC-Cy7 (clone 30-F11), anti-CD45R/B220-PE (clone RA3-6B2), anti-Gr-1-PE (clone RB6-8C5), anti-TCRαβ PE (clone H57-597), anti-TCRγζ PE (clone GL3), anti-CD11b PE (clone M1/70), and anti-Ter-119 PE (clone TER-119). All mAbs were added at saturating concentrations, and the cells were incubated for 30 minutes on ice and washed twice and then resuspended for sort in CSM at a concentration of 5 × 106 cells/ml.

Fluorescent Staining of PB-Derived VSEL Stem Cells

The expression of each antigen was examined in cells from four independent experiments. Sca-1+linCD45 or Sca-1+linCD45+ cells were stained with CD45 (clone 30-F11) conjugated with FITC (1:200; BD Biosciences), fixed in 3.5% paraformaldehyde for 20 minutes, permeabilized by 0.1% Triton X-100, washed in PBS, preblocked with 2% bovine serum albumin (BSA), and subsequently stained with antibodies to SSEA-1 (clone MC-480, 1:100, mouse monoclonal IgM; Chemicon, Temecula, CA, http://www.chemicon.com) and Oct-4 (clone 9E3, 1:100, mouse monoclonal IgG1; Chemicon). Appropriate secondary antibodies, Alexa Fluor 594 goat anti-mouse IgM and Alexa Fluor 594 goat anti-mouse IgG (1:400; Molecular Probes, Eugene, OR, http://probes.invitrogen.com), were used.

The expressions of α-sarcomeric actinin, troponin I, βIII tubulin, C-peptide, glial fibrillary acidic protein (GFAP), Olig1, and insulin were evaluated after 20 days of culture of green fluorescence protein-positive (GFP+) VSEL stem cells isolated from G-CSF-mobilized PB on the layer of BM stromal cells. The cells were fixed in 3.5% paraformaldehyde for 20 minutes, permeabilized by 0.1% Triton X-100, washed in PBS, preblocked with 2% BSA, and subsequently stained with α-sarcomeric actinin (1:100, mouse monoclonal IgM; Abcam, Cambridge, U.K., http://www.abcam.com), troponin I (1:200, mouse monoclonal IgG2b; Chemicon), βIII tubulin (1:100, rabbit polyclonal IgG; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), C-peptide (1:100, guinea pig IgG; Linco Research Inc., St. Charles, MO, http://www.lincoresearch.com), GFAP (1:200, mouse monoclonal IgG1; Chemicon), Olig1 (1:200, mouse monoclonal IgG2b; Millipore Corporation, Billerica, MA, http://www.millipore.com), and insulin (1:300, guinea pig polyclonal; Abcam). Appropriate secondary antibodies, Alexa Fluor 594 goat anti-mouse IgM, goat anti-mouse IgG, goat anti-rabbit IgG, and anti-guinea pig IgG (1;400; Molecular Probes), were used.

In control experiments, cells were stained with secondary antibodies only. The nuclei were labeled with 4,6-diamidino-2-phenylindole (Molecular Probes). The fluorescence images were collected with the TE-FM epifluorescence system attached to a Eclipse TE300 inverted microscope (Nikon, Tokyo, Japan, http://www.nikon.com) and captured by a Cool Snap HQ digital black and white CCD (Roper Scientific, Tucson, AZ, http://www.roperscientific.com) camera.

Reverse Transcriptase-Polymerase Chain Reaction

Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA, http://www1.qiagen.com). mRNA (10 ng) was reverse-transcribed with a One Step reverse transcriptase (RT)-polymerase chain reaction (PCR) (Qiagen) according to the manufacturer's instructions. The resulting cDNA fragments were amplified using HotStarTaq DNA polymerase (Qiagen). Primer sequences were as follows: for Oct-4, forward primer 5′-GGA GAG GTG AAA CCG TCC CTA GG-3′ and reverse primer 5′-AGA GGA GGT TCC CTC TGA GTT GC-3′; for Nanog, forward primer 5′-CTG GGA ACG CCT CAT CAA-3′ and reverse primer 5′-CAT CTT CTG CTT CCT GGC AA-3′; for Rex1, forward primer 5′-CCC CAA ATA CCA CTG ACC AAA A-3′ and reverse primer 5′-GGT TCG GAA AAC TCA CCT CGT A-3′; for Dppa3, forward primer 5′-ACC CAA TGA AGG ACC CTG AAA C-3′ and reverse primer 5′-GCT CAC TGT CCC GTT CAA ACT C-3′; and for Sox2, forward primer 5′-GTG GAA ACT TTT GTC CGA GAC-3′ and reverse primer 5′-TGG AGT GGG AGG AAG AGG TAA C-3′. The correct size of PCR products was confirmed by separation on agarose gel.

Real-Time RT-PCR (Quantitative RT-PCR)

For analysis of Oct-4, Nanog, Rex1, Dppa1, Rif1, Myf5, MyoD, Myogenin, GFAP, nestin, Nsx2.5/Csx, GATA-4, vascular epithelium (VE)-cadherin, Nkx 6.1, and Pdx1 mRNA levels, total mRNA was isolated from cells with an RNeasy Mini Kit. mRNA was reverse-transcribed with TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). Detection of Oct-4, Nanog, Rex1, Dppa1, Rif1, Myf5, MyoD, Myogenin, GFAP, nestin, Nsx2.5/Csx, GATA-4, VE-cadherin, Nkx 6.1, Pdx1, and β2-microglobulin mRNA levels was performed by real-time RT-PCR using an ABI PRISM 7000 sequence detection system (Applied Biosystems). A 25-μl reaction mixture contained 12.5 μl of SYBR Green PCR Master Mix, 10 ng of cDNA template, and forward and reverse primers. Primers were designed with Primer Express software (supplemental online data). The threshold cycle (Ct), that is, the cycle number at which the amount of amplified gene of interest reached a fixed threshold, was determined subsequently. Relative quantization of Oct-4, Nanog, Rex1, Dppa1, Rif1, Myf5, MyoD, Myogenin, GFAP, nestin, Nsx2.5/Csx, GATA-4, VE-cadherin, Nkx 6.1, and Pdx1 mRNA expression was calculated with the comparative Ct method. The relative quantization value of the target, normalized to an endogenous control β2-microglobulin gene and relative to a calibrator, is expressed as 2−ΔΔCt (fold difference), where ΔCt is Ct of target genes − Ct of the endogenous control gene, and ΔΔCt = ΔCt of samples for target gene − ΔCt of calibrator for the target gene.

To avoid the possibility of amplifying contaminating DNA (a) all of the primers for real-time RTR-PCR were designed with an intron sequence inside cDNA to be amplified, (b) reactions were performed with appropriate negative controls (template-free controls), (c) a uniform amplification of the products was rechecked by analyzing the melting curves of the amplified products (dissociation graphs), (4) the melting temperature (Tm) was 57–60°C, with the product Tm being at least 10°C higher than primer Tm, and, finally, (e) gel electrophoresis was performed to confirm the correct size of the amplification and the absence of unspecific bands.

Muscle Injury

Cardiotoxin (Naja mossambica mossambica, 100 μM; Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com) diluted in 100 μl of PBS was injected into the anterior tibialis muscle of BALB/c mice using a 27-gauge needle and a 1-ml syringe. The needle was inserted deep into the anterior tibialis muscle longitudinally toward the knee from the ankle. Cardiotoxin was injected along the length of the muscle. Mice in a control group were injected with 100 μl of PBS. Mice (treated and control groups) were sacrificed at 24, 48, and 72 hours after the cardiotoxin injection, and blood samples (1.0–1.5 ml from each mouse) were collected in heparin-rinsed syringes for the isolation of PB MNCs.

Liver Injury

A mouse hepatic cirrhosis model was induced by carbon tetrachloride (CCl4). On day 0 BALB/c mice were injected subcutaneously with 1 ml/kg CCl4 (Sigma-Aldrich) dissolved in corn oil (Sigma-Aldrich). Mice in a control group were injected with 100 μl of corn oil. Mice were sacrificed at 24, 48, and 72 hours after the injection, and blood samples (1.0–1.5 ml from each mouse) were collected in heparin-rinsed syringes for the isolation of PB MNCs.

Differentiation Assays

For coculture assays to prepare a supportive cell layer, 2 × 106 wild-type BM MNCs were plated in 23-mm Fluorodishes (World Precision Instruments, Sarasota, FL, http://www.wpiinc.com). At 72 hours, MNCs, purified by FACS GFP+ VSEL stem cells sorted from G-CSF-mobilized PB, were added as described below.

Neuronal Differentiation.

GFP+ VSEL stem cells (5 × 104) were cultured in NeuroCult Basal Medium (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) supplemented with 10 ng/ml recombinant human epidermal growth factor, 10 ng/ml fibroblast growth factor-2 (FGF-2), and 20 ng/ml nerve growth factor (all growth factors from R&D Systems, Inc., Minneapolis, MN, http://www.rndsystems.com). Growth factors were added every 24 hours and medium was replaced every 2–3 days. The cultures were examined after 23 days.

Pancreatic Differentiation.

G-CSF-mobilized GFP+ VSEL stem cells (5 × 104) were cultured on the layer of BM stromal cells in Dulbecco's modified Eagle's medium's medium (DMEM)/Ham's F-12 medium with 1% heat-inactivated FBS and 20 ng/ml of recombinant human Activin A (R&D Systems, Inc.). After 48 hours, the medium was changed, and differentiation was carried out in DMEM/Ham's F-12 medium with 5% heat-inactivated FBS in the presence of N2 supplement-A, B27 supplement, and 10 mM nicotinamide (Stem Cell Technologies). Medium was changed every second day. The cultures were examined after 20 days.

Cardiomyocyte Differentiation.

G-CSF-mobilized GFP+ VSEL stem cells (5 × 104) were plated on the layer of BM stromal cells in DMEM with 10% heat-inactivated FBS in the presence of 10 ng/ml basic FGF-2, 10 ng/ml VEGF, and 10 ng/ml transforming growth factor β1 (all growth factors from R&D Systems, Inc.). Growth factors were added every 24 hours. The cultures were examined after 25 days.

Statistical Analysis

Arithmetic means and SDs of our FACS data were calculated on a Macintosh PowerBase 180 computer, using Instat 1.14 software (GraphPad Software Inc., San Diego, CA, http://www.graphpad.com). Data were analyzed using the Student's t test for unpaired samples or analysis of variance for multiple comparisons. Statistical significance was defined as p < .05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Sca-1+LinCD45 Cells (VSEL Stem Cells) Circulate in PB

VSEL stem cells contain open type euchromatin and thus may transcribe several types of mRNA for early tissue-specific developmental markers similarly to embryonic stem cells [30]. In our previous studies, we demonstrated an increase in expression of mRNA for early skeletal muscles, neural tissue, and endothelium in mononuclear cells isolated from PB from a patient mobilized by G-CSF [19], partially irradiated mice [26], and mice after experimental acute heart infarct [20] and stroke [22]. However, direct evidence that this increase in mRNA for several tissue-specific developmental markers could be explained by an increased number of circulating VSEL stem cells in PB was missing.

Figure 1A and 1B shows direct FACS-based evidence that small cells that express VSEL stem cell markers (Sca-1+linCD45) are detectable at a very low level under steady-state conditions in murine PB (∼160 cells/ml; 0.0016% of white blood cells [WBCs]) and that their total number increases ∼5 times during G-CSF-induced mobilization (∼800 cells/ml; 0.0035% of WBCs). At the same time the number of WBCs increased ∼2 times. An increase in the number of these cells circulating in PB is further supported by an increase in the expression of mRNA for early developmental markers expressed in VSEL stem cells such as embryonic transcription factors Oct-4, Nanog, and Rex1 and expression of Rif1 and Dppa3 as shown by both real time (Fig. 1C) and normal RT-PCR (Fig. 1D). To exclude the possibility that expression of mRNA for these pluripotent genes could be from some other non-VSEL stem cells, in control experiments we evaluated expression of these genes in VSEL-depleted PB MNCs. As expected VSEL-depleted PB MNCs did not express these genes (supplemental online Fig. 1). Furthermore, supplemental online Table 1 demonstrates that at the same time they are mobilized into PB MNCs are highly enriched for mRNA for several early developmental tissue specific markers, a phenomenon that could be explained as mentioned above by open-type status of chromatin in these cells. Finally, we also found by using quantitative RT-PCR that the expression of Oct-4, Nanog, and Rex1 in VSEL stem cells is comparable to the expression of these genes in murine ES-D3 cells (supplemental online Fig. 2).

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Figure Figure 1.. VSEL stem cells circulate in PB under steady-state conditions and are mobilized into PB by G-CSF. C57BL/6 mice were mobilized for 6 days with G-CSF (250 μg/kg s.c. per day) (n = 10 animals per group). Data are pooled from four independent experiments. (A): Fluorescence-activated cell sorter analysis of murine PB MNCs. Erythrocytes were removed by hypotonic lysis, and PB MNCs were stained with lineage markers, CD45 and Sca-1. Upper left, dot plot of murine PB MNCs after hypotonic lysis. Upper right, histogram showing staining of cells from the R1 gate for lineage markers expression. R2 indicates lineage-negative PB MNCs. Cells from R1 and R2 were subsequently analyzed for expression of CD45 and coexpression of Sca-1 antigen in unmobilized PB MNCs (lower left panel) and in G-CSF-mobilized PB MNCs (lower right panel). VSEL stem cells are present in region R3. Hematopoietic stem cells are shown in R4. Representative data from four independent experiments are shown. (B): Left, number of circulating VSEL stem cells per 1 ml of PB (bars). Right, number of WBCs per microliter (♦). *p < .05. (C): Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the expression of mRNA for the markers of pluripotent stem cells. Markers were compared between the same number of unmobilized and G-CSF-mobilized PB MNCs. Data are mean ± SD and represent the average of four independent experiments. *p < .001. (D): Expression of mRNA for Oct-4, Nanog, Rex1, Dppa3, and Sox2 by unmobilized PB MNCs (lane 1), G-CSF-mobilized PB MNC (lane 3), and murine embryonic stem cells from the ES-D3 line (lane 5). RT-PCR was run for 30 cycles. Negative RT-PCR reactions indicate DNA instead of cDNA (lanes 2, 4, and 6). A representative result from three independent sorts is shown. Abbreviations: FL1-H, fluorescence 1st channel; FSC-H, forward scatter-height; G-CSF, granulocyte colony-stimulating factor; MNC, mononuclear cell; PB, peripheral blood; SSC-H, side scatter-height; VSEL, very small embryonic-like; WBC, white blood cell.

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Finally, we sorted these rare cells from murine PB by FACS for immunofluorescence staining and, as shown in Figure 2, we provide evidence that they express SSEA-1 on the surface (Fig. 2A) and Oct-4 in the nucleus (Fig. 2B). Figure 2 also demonstrates a side-by-side comparison of VSEL stem cells (3–4 μm in diameter) to murine Sca-1+linCD45+ cells (6–8 μm in diameter), which are a population of cells highly enriched in HSCs. Figure 2C shows that we detected Oct-4 in ∼20 and 50%, SSEA-1 in ∼15 and 38%, and both Oct-4 and SSEA-1 in ∼10 and 22% of VSEL stem cells sorted from nonmobilized and G-CSF-mobilized PB, respectively. This result demonstrates that the VSEL stem cell population isolated as very small Sca-1+linCD45 cells expresses Oct-4 and SSEA-1 at different levels.

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Figure Figure 2.. Immunohistochemical analysis of hematopoietic stem cells (HSCs) and VSEL stem cells mobilized into peripheral blood. Sca-1+linCD45+ (HSCs) and Sca-1+linCD45 (VSEL stem cells) isolated by fluorescence-activated cell sorting were evaluated for expression of SSEA-1 (red fluorescence in (A) and Oct-4 (red fluorescence in (B). Green fluorescence in (A) and (B) indicates expression of CD45 antigen. Nuclei were visualized after DAPI staining. All images were taken under a Plan Apo 60XA/1.40 oil objective (Nikon). Staining was performed on cells isolated from three independent sorts. Representative data are shown. Cells positive for Oct-4 and SSEA-1 were counted using an epifluorescence system attached to a Nikon Eclipse TE300 inverted microscope and are expressed as a percentage of sorted VSEL stem cells (C). Note that no Oct-4 or SSEA-1 expression was detected in sorted HSCs. Control staining data performed on ES-D3-derived embryoid bodies are shown in supplemental online Figure 2. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; G-CSF, granulocyte colony-stimulating factor; PE, phycoerythrin; SSEA-1, stage-specific embryonic antigen-1; VSEL, very small embryonic-like.

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Mobilized VSEL Stem Cells Differentiate in Vitro into Cardiomyocytes and Neural and Pancreatic Cells

To provide evidence that mobilized VSEL stem cells not only express pluripotent stem cell markers but also are able to differentiate into cells from all three germ layers, we performed differentiation studies in vitro. To provide such proof, VSEL stem cells were cultured in appropriate differentiation media on the layer of BM-derived stromal support. To demonstrate better tissue-specific contribution of VSEL stem cells in cocultures with stromal cells, VSEL stem cells were isolated from G-CSF-mobilized GFP+ mice. As expected and as shown in Figure 3, mobilized VSEL stem cells are able to differentiate into cardiomyocytes (Fig. 3A, 3B), neural cells (Fig. 3C, 3D, 3E) and pancreatic cell-like cluster (Fig. 3F, 3G). The analysis of DNA content in GFP+ cells isolated from the cocultures excluded the contribution of cell fusion to this effect. Thus, these experiments revealed the in vitro pluripotency of VSEL stem cells mobilized by G-CSF and circulating in PB by demonstrating their ability to differentiate into cells from all three germ layers.

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Figure Figure 3.. Differentiation of mobilized very small embryonic-like (VSEL) stem cells into cardiomyocytes and neural and pancreatic cells. Granulocyte colony-stimulating factor mobilized green fluorescence protein (GFP)-positive VSEL stem cells were cocultured with GFP-negative bone marrow stromal cells in cardiac (A,B), neural (C,D,E), and pancreatic (F,G) differentiation media. Expressions of α-sarcomeric actinin (A), Troponin I (B), Olig1 (C), β III tubulin (D), glial fibrillary acidic protein (E), C-peptide (F), and insulin (G) are shown. All proteins were detected by red fluorescence. Nuclei were identified by 4,6-diamidino-2-phenylindole. All images were taken under a Plan Fluor 20XA/0.50 objective (Nikon). Representative data are shown. In control experiments cells were stained with secondary antibodies only (supplemental online Fig. 3).

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Numbers of Circulating VSEL Stem Cells Are Reduced in Older Mice

In our previous work in which we compared 2-month-old and 1-year-old mice, we demonstrated that the numbers of VSEL stem cells residing in BM decrease (∼7 times) with age [30]. In the current study, we compared the numbers of circulating VSEL stem cells in PB between young (2-month-old) and old (1-year-old) mice and also evaluated changes in the numbers of these cells circulating in PB in response to G-CSF.

Figure 4A shows that the number of circulating VSEL stem cells in PB under steady-state conditions is reduced by ∼5 times in 1-year-old mice. Both young and old mice respond robustly in a similar manner to G-CSF-induced mobilization; however, the total number of mobilized VSEL stem cells in older mice was again ∼5 times lower compared with the number in young 2-month-old mice (Fig. 4A). This increase in the number of circulating VSEL stem cells was paralleled by an increase in the expression of mRNA for selected VSEL stem cell markers (Fig. 4B).

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Figure Figure 4.. The number of VSEL stem cells circulating in PB both under steady-state conditions and after G-CSF-induced mobilization is lower in older mice. C57BL/6 mice (2 months and 1 year old) were mobilized for 6 days with G-CSF (250 μg/kg s.c. per day) (n = 10 animals per group). Data from three independent experiments are pooled. (A): The number of circulating VSEL stem cells per 1 ml of PB. *p < .05. (B): Real-time reverse transcription-polymerase chain reaction for the expression of mRNA for the markers of pluripotent stem cells. Markers were compared between the same numbers of G-CSF-mobilized PB MNCs isolated from 2-month-old or 1-year-old C57BL/6 mice. Data are means ± SD and represent the average of three independent experiments. *p < .001. Abbreviations: G-CSF, granulocyte colony-stimulating factor; MNC, mononuclear cell; PB, peripheral blood; VSEL, very small embryonic-like.

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VSEL Stem Cells Are Mobilized at Similar Levels Both in Poor (C57BL/6) and Good (BALB/c) Mobilizing Murine Strains

It is well known that C57BL/6 mice are poor G-CSF mobilizers of HSCs compared with BALB/c mice, which are good G-CSF mobilizers [32]. Therefore, we compared the G-CSF-induced mobilization efficiency of VSEL stem cells in these two murine strains. To our surprise, the basic level of VSEL stem cells circulating under steady-state conditions was slightly higher in C57BL/6 mice compared with that in BALB/c mice. Furthermore, both strains responded similarly to G-CSF-induced mobilization (data not shown).

The mobilization of VSEL stem cells was in striking contrast to mobilization of colony-forming units granulocyte-macrophage that circulated in PB at higher levels in BALB/c mice under steady-state conditions and increased ∼3 times more compared with the levels in C57BL/6 animals (not shown). This finding suggests that different mobilization mechanisms may be involved in mobilization of HSCs and more primitive VSEL stem cells.

CXCR4 Antagonist T140 Enhances Mobilization of VSEL Stem Cells

Furthermore, because VSEL stem cells express CXCR4, we hypothesized that CXCR4 blockage by T140, which is a small molecular CXCR4 antagonist, should additionally increase mobilization of these cells. To evaluate the effect of T140 on VSEL stem cell mobilization, we used G-CSF at a low dose (Fig. 5). We found that T140-mediated blockage of CXCR4 enhanced G-CSF-induced mobilization of VSEL stem cells (∼2 times). However, at the same time T140, if used alone, did not mobilize either VSEL stem cells or HSCs (Fig. 5).

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Figure Figure 5.. T140 enhances G-CSF-induced mobilization of VSEL stem cells. C57BL/6 mice were mobilized for 6 days with a LD of G-CSF (50 μg/kg s.c. per day) (n = 10 animals per group). T140 (16 μg per mouse) was injected i.p. with a LD of G-CSF. Left, number of circulating VSEL stem cells per 1 ml of PB (bars). Right, number of circulating HSCs per 1 ml of PB (♦). Data represent the average of three independent experiments. *, **, p < .05 compared with immobilized animals. Abbreviations: G-CSF, granulocyte colony-stimulating factor; HSC, hematopoietic cell; LD, low dose; MNC, mononuclear cell; PB, peripheral blood; VSEL, very small embryonic-like.

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VSEL Stem Cells Are Mobilized into PB After Toxic Liver and Skeletal Muscle Injuries

Circulating stem cells have been demonstrated in PB in several models of tissue injury [11, 14, 17, 18, 25, 33, [34], [35], [36]37]. Thus, we became interested in whether VSEL stem cells could also be mobilized into PB and used as experimental models for toxic liver and skeletal muscle injury.

Figure 6A and 6B shows an increase in VSEL stem cell numbers circulating in PB in CCl4-induced liver damage. As is seen, the number of circulating VSEL stem cells increased up to 5 times in PB 48 hours after liver intoxication. These changes were paralleled by changes in expression of mRNA for selected VSEL stem cell markers (Fig. 6C). Similar changes were observed in cardiotoxin-induced skeletal muscle damage (Fig. 7A, 7B); however, the number of circulating VSEL stem cells in these animals was highest at 48 hours after injury and the number of circulating VSEL stem cells increased ∼2 times only.

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Figure Figure 6.. VSEL stem cells are mobilized in response to toxic liver injury. BALB/c mice were injected with CCl4 (1 ml/kg) (n = 5 animals per group). (A): Fluorescence activated cell sorter analysis of murine PB MNCs after removal of erythrocytes by hypotonic lysis. PB MNCs were stained 24, 48, and 72 h after injection of CCl4 with lineage markers, CD45 and Sca-1. Lin cells were analyzed for expression of CD45 and coexpression of Sca-1 antigen. VSEL stem cells are shown in R3 and HSCs in R5. Representative data for three independent experiments are shown. (B): PB MNCs were isolated 24, 48, and 72 h after injection. Left, number of circulating VSEL stem cells per 1 ml of PB (bars). *p < .05. Right, number of WBCs per microliter (♦). (C): Real-time reverse transcriptase-polymerase chain reaction for the expression of mRNA for the markers of PSCs. Markers were compared between the same number of PB MNCs from control animals and isolated 24, 48, and 72 h after injection of CCl4. Data are mean ± SD and represent the average of three independent experiments. *p < .00001 versus control PB MNCs. Abbreviations: CCl4, carbon tetrachloride; FL1-H, fluorescence 1st channel; FL3-H, fluorescence 3rd channel; h, hour; HSC, hematopoietic cell; MNC, mononuclear cell; PB, peripheral blood; PSC, pluripotent stem cell; VSEL, very small embryonic-like; WBC, white blood cells.

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Figure Figure 7.. VSEL stem cells are mobilized after skeletal muscle injury. BALB/c mice were injected with 100 μM cardiotoxin (n = 5 animals per group). (A): PB MNCs were isolated 24, 48, and 72 h after injection. Left, number of circulating VSEL stem cells per 1 ml of PB. *p < .05. Right, number of WBCs per microliter. Data are pooled from three independent experiments. (B): Real-time reverse transferase-polymerase chain reaction for the expression of mRNA for the markers of PSCs. Markers were compared between the same number of PB MNCs from control animals and isolated 24, 48, and 72 h after injection of cardiotoxin. Data are mean ± SD and represent the average of three independent experiments. *p < .00001 versus control PB MNCs. Abbreviations: CT, cardiotoxin; h, hour; MNC, mononuclear cell; PB, peripheral blood; PSC, pluripotent stem cell; VSEL, very small embryonic-like; WBC, white blood cells.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

PB could be envisioned as a “highway” by which stem cells are trafficking in the body to keep in balance a pool of stem cells located in different niches in peripheral tissues. In this context, BM has been proposed to be a main reservoir for these circulating cells [38]. To support this notion, BM develops by the end of the second trimester of gestation as a result of colonization by a mobile pool of stem cells that move from the fetal liver into the developing BM microenvironment [39, [40], [41]42].

It is well known that BM secretes several chemoattractants not only for HSCs but also for other non-HSCs (e.g., SDF-1, HGF/SF, LIF, and VEGF) [43, [44], [45]46]. Thus, stem cells that circulate in PB both during (a) fetal development or (b) postnatally while relocating between tissue specific niches may become chemoattracted from circulating PB to settle down in BM, where they find a permissive environment. This explains why in addition to HSCs, several other types of non-HSCs have been described in BM, for example, mesenchymal stem cells (MSCs) [47], multipotent adult progenitor cells (MAPCs) [48], marrow-isolated adult multilineage inducible cells (MIAMIs) [49], or multipotent adult stem cells (MASCs) [50].

We have recently purified from BM as well as other adult tissues and described at a single cell level a population of VSEL stem cells that contain large nuclei with unorganized euchromatin. These cells express on the surface SSEA-1 and in the nuclei early embryonic transcription factors such as Oct-4 and Nanog and display a high activity of telomerase. More importantly, VSEL stem cells differentiate in vitro in cells from all three germ layers [30]. The potential relationship of VSEL stem cells with MSCs, MAPCs, MIAMIs, and MASCs is not clear at this point, although it is possible that these could be overlapping populations of primitive cells that are identified by different isolation/expansion strategies.

In this article, we provide for the first time, direct experimental evidence that VSEL stem cells circulate at very low levels in murine PB and their number increases during G-CSF-induced mobilization as well during organ injuries as seen in models of toxic liver and skeletal muscle damage. The fact that VSEL stem cells circulate under steady-state conditions in PB supports our concept that VSEL stem cells are a most primitive population of mobile epiblast-derived pluripotent stem cells, which are deposited in developing organs and survive into adulthood and may play a role as a stem cell mother lineage for tissue-specific stem cells located in various organs [51, 52]. Thus, under steady-state conditions, circulating VSEL stem cells probably play an important role in maintaining the homeostasis of the stem cell compartment. These cells possess, similarly to epiblast-derived cells, high motility and respond to the same motto-morphogens (e.g., SDF-1, HGF/SF, and LIF) that orchestrate stem cell trafficking during both embryogenesis and regeneration. This explains why they mobilize robustly into PB during organ/tissue injury in an attempt to participate in repair of damaged organs.

In our studies, we directly enumerated circulating VSEL stem cells by using FACS-based multiparameter analysis. However, we are aware that in addition to VSEL stem cells, other types of stem cells, for example, MSCs as mentioned above [8], endothelial progenitors [14, 32, 53], liver oval stem cells [16, 54], skeletal muscle progenitors [11, 12], and kidney [55]-committed stem cells may circulate in PB in response to various stress situations. In accordance with these observations, we have reported in the past that mononuclear cells isolated from PB in patients mobilized by G-CSF [19] and mice after experimental heart infarct [20] and stroke [22] are enriched in mRNA for early developmental tissue-specific markers. Data presented in this article suggest that the expression of mRNA for these various tissues/organs detected by us in PB-mobilized mononuclear cells could be explained at least partly by mobilization of VSEL stem cells themselves that is due to the open type of the chromatin expressing mRNA for organ/tissue-committed stem cells.

We reported that ∼57 and ∼43% of VSEL stem cells isolated from BM express Oct-4 and SSEA-1, respectively, at the protein level [30]. In this study, we noted that the percentages of Oct-4+ and SSEA-1+ VSEL stem cells circulating in PB (non-mobilized and mobilized) were lower (Fig. 2C). This finding demonstrates that (a) VSEL stem cells express both of these genes at a different level and (b) some selection process of VSEL stem cells could be involved in mobilization. Thus, VSEL stem cells circulating under the steady-state condition in PB express Oct-4 and SSEA-1 protein and at a relatively low level; however, mobilization selects for cells with higher expression of both genes.

The lower number of circulating VSEL stem cells in older animals both under steady-state conditions and after response to G-CSF (Fig. 4) supports our concept that a decrease in a pool of these cells in the tissues may explain accelerated senescence with advancing age [30]. Furthermore, to our surprise we found that young C57BL/6 and BALB/c mice described as poor and good mobilizers of HSC, respectively [31], released a similar number of VSEL stem cells into PB. This suggests that VSEL stem cells are important players in the regeneration mechanism that operates at similar levels in both of these murine strains. In this context, further studies are needed to evaluate mobilization of VSEL stem cells in short-living DBA/2J mice, which, as we reported in the past, may have a reduced number of VSEL stem cells in their tissues [30].

Finally, we confirmed that the SDF-1-CXCR4 axis seems to play a pivotal role in regulating the trafficking of VSEL stem cells [19, 26, 56]. However, blockage of T140 did not increase the number of VSEL stem cells circulating in PB during steady-state conditions; rather, it enhanced a number of mobilized VSEL stem cells after administration of a suboptimal dose of G-CSF. Thus, CXCR4 antagonists could be used to mobilize these cells, for example, for potential clinical application.

Similarly, as we reported that the blockage of the third complement protein cleavage fragment receptor (C3aR) enhances G-CSF-induced mobilization of HSCs by modulating the SDF-1-CXCR4 axis [57], it would be important to evaluate the effectiveness of this strategy on VSEL stem cell mobilization. Further studies are also needed to evaluate mobilization of VSEL stem cells in other types of organ injury (e.g., heart infarct or stroke), after administration of other mobilizing agents or during systemic hypoxia. For example, as reported recently in rats, systemic hypoxia increases by ∼15 times the number of circulating MSCs in PB [8].

In conclusion, we provide for the first time direct evidence that murine VSEL stem cells circulate in PB under steady-state conditions and that their number increases during various stress situations. We hypothesize that VSEL stem cells are released mainly from the BM; however, we cannot exclude the possibility that, in addition to BM, other organs may also release these cells into PB. Thus, this mobile pool of early stem cells may play an underappreciated role in turnover of the tissue-specific stem cells located in various organs and can be directly involved in tissue/organ regeneration in response to injuries. Further studies are warranted to assess the role of these mobilized cells in tissue repair as well as to provide more evidence that a similar phenomenon also occurs in humans. To support this latter possibility, we recently described a population of human stem cells that corresponds to murine VSEL stem cells in human cord blood [58]. Finally, we envision that VSEL stem cells mobilized into PB, for example, after G-CSF administration in combination with a CXCR4 antagonist (e.g., T140 or AMD3100), could be harvested by leukapheresis as a potential source of stem cells for regenerative medicine.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

This work was supported by NIH Grants R01 CA106281-01 and NIH R01 DK074720 and Stella and Henry Endowment (to M.Z.R.) and NIH Grant P20RR018733 from the National Center for Research Resources (to M.K.).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information
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