Original Manuscript

Leukemia (2004) 18, 29–40. doi:10.1038/sj.leu.2403184 Published online 30 October 2003

Stem cell plasticity revisited: CXCR4-positive cells expressing mRNA for early muscle, liver and neural cells 'hide out' in the bone marrow

This paper was presented as an oral presentation at the 32nd Annual Meeting of the International Society for Experimental Hematology, Paris, France, July 5–8, 2003

M Z Ratajczak1,2, M Kucia1,2, R Reca1, M Majka2, A Janowska-Wieczorek3 and J Ratajczak1

  1. 1Stem Cell Biology Program at James Graham Brown Cancer Center and Department of Medicine, University of Louisville, Louisville, KY, USA
  2. 2European Stem Cell Therapeutic Excellence Center, Medical College, Jagiellonian University, Cracow, Poland
  3. 3University of Alberta and Canadian Blood Services, Edmonton, Canada

Correspondence: Dr MZ Ratajczak, Stem Cell Biology Program at James Graham Brown Cancer Center, University of Louisville, 529 South Jackson Street, Louisville, KY 40202, USA

Received 13 August 2003; Accepted 24 September 2003; Published online 30 October 2003.

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Abstract

It has been suggested that bone marrow (BM)-derived hematopoietic stem cells transdifferentiate into tissue-specific stem cells (the so-called phenomenon of stem cell plasticity), but the possibility of committed tissue-specific stem cells pre-existing in BM has not been given sufficient consideration. We hypothesized that (i) tissue-committed stem cells circulate at a low level in the peripheral blood (PB) under normal steady-state conditions, maintaining a pool of stem cells in peripheral tissues, and their levels increase in PB during stress/tissue injury, and (ii) they could be chemoattracted to the BM where they find a supportive environment and that the SDF-1–CXCR4 axis plays a prominent role in the homing/retention of these cells to BM niches. We performed all experiments using freshly isolated cells to exclude the potential for 'transdifferentiation' of hematopoietic stem or mesenchymal cells associated with in vitro culture systems. We detected mRNA for various early markers for muscle (Myf-5, Myo-D), neural (GFAP, nestin) and liver (CK19, fetoprotein) cells in circulating (adherent cell-depleted) PB mononuclear cells (MNC) and increased levels of expression of these markers in PB after mobilization by G-CSF (as measured using real-time RT-PCR). Furthermore, SDF-1 chemotaxis combined with real-time RT-PCR analysis revealed that (i) these early tissue-specific cells reside in normal murine BM, (ii) express CXCR4 on their surface and (iii) can be enriched (up to 60 times) after chemotaxis to an SDF-1 gradient. These cells were also highly enriched within purified populations of murine Sca-1+ BM MNC as well as of human CD34+-, AC133+- and CXCR4-positive cells. We also found that the expression of mRNA for SDF-1 is upregulated in damaged heart, kidney and liver. Hence our data provide a new perspective on BM not only as a home for hematopoietic stem cells but also a 'hideout' for already differentiated CXCR4-positive tissue-committed stem/progenitor cells that follow an SDF-1 gradient, could be mobilized into PB, and subsequently take part in organ/tissue regeneration.

Keywords:

CXCR4, SDF-1, stem cell plasticity, stem cell mobilization, stem cell homing

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Introduction

Bone marrow (BM) has been traditionally envisioned as a 'home' of hematopoietic stem cells. It is also known to contain mesenchymal stem cells that give rise to the various mesodermal tissues (eg smooth muscles, chondrocytes, osteocytes).1,2,3 Surprisingly, BM hematopoietic stem cells were also recently reported to be able to 'transdifferentiate' into cells that express early heart,4,5 skeletal muscle,6,7 neural,8 liver9 or pancreatic cell10,11 markers. This was supported in humans by the observations that transplantation of peripheral blood (PB) stem cells expressing early hematopoietic CD34+ antigen led to the appearance of donor-derived hepatocytes,12 epithelial cells12 and neurons.13 Similarly, human BM-derived cells contributed to the regeneration of infarcted myocardium.14 These findings have been interpreted as evidence for the existence of the phenomenon of transdifferentiation or plasticity of adult stem cells.

However, the concept of transdifferentiation of adult tissue-specific stem cells has recently been called into question.15,16 Studies aimed at reproducing these experiments with neural stem cells have failed.17 Similarly, it has been shown that hematopoietic stem/progenitor cells (HSPC) found in muscle tissue are in fact of BM origin.18,19,20 Recent studies on chimeric animals involving the transplantation of a single hematopoietic stem cell marked with a green fluorescent protein into lethally irradiated nontransgenic mice demonstrated that so-called transdifferentiation or plasticity of circulating HPSC and/or their progeny is an extremely rare event, if it occurs at all.21

Several potential alternative explanations for the phenomenon of apparent stem cell plasticity should be taken into consideration when evaluating reported data. First, some of the results could be explained by the phenomenon of cell fusion.22,23,24 The significant role of cell fusion has recently been demonstrated during liver regeneration in mice transplanted with BM cells.25 Next, it is likely that in certain circumstances cells may undergo epigenetic changes caused by external stimuli that affect/change gene expression.26 Supporting this are observations showing that, during animal cloning employing nuclear transfer, the nuclei isolated from differentiated somatic cells may be reprogrammed and de-differentiated when injected into the cytoplasm of enucleated embryonic stem cells or oocytes.27,28 It is likely that a similar 'reprogramming effect' may also occur in cells isolated from their physiological environment and exposed in vitro to stress factors related to culture conditions. Finally, in our opinion, there is the explanation, which we believe to be the most likely, that committed tissue-specific stem cells pre-exist in various organs/tissues.29 We present evidence for this explanation and discuss it with respect to BM tissue.

There is compelling evidence that the alpha-chemokine stromal derived factor (SDF)-1, which is secreted by BM stroma, plays an essential role not only in the homing of HSPC30 but also in chemoattracting to the BM other CXCR4-positive cells.31,32,33,34,35 Several pediatric tumors metastasizing to the BM such as rhabdomyosarcoma, neuroblastoma, nephroblastoma, hepatoblastoma and retinoblastoma express functional CXCR4 on their surface and follow an SDF-1 gradient.31,32,33,34,35 All these tumors derive from early muscle, neural, kidney, liver and retina pigment epithelial cells, respectively, which are also CXCR4-positive.31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46 Hence it seems likely that the CXCR4–SDF-1 axis plays an essential role in the chemoattraction/retention in BM not only of CXCR4-positive tumor cells but also CXCR4-positive tissue-committed stem/progenitor cells.43 Therefore the major aim of this study was to determine whether cells expressing mRNA for early muscle, neural and liver tissue-committed stem/progenitors reside under steady-state conditions in normal BM. We envision that these cells could circulate between BM and peripheral tissues and if needed could take part in the regeneration of damaged organs.

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Materials and methods

Murine cells

Murine mononuclear cells (MNC) were isolated from BM flushed from the femurs of pathogen-free, 4- to 6-week-old female Balb-C mice (The Jackson Laboratory, Bar Harbor, ME, USA) enriched for light-density MNC by Ficoll–Paque centrifugation. Sca-1+ cells were isolated by employing paramagnetic mini-beads (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer's protocol. The study was approved by the IACUC at the University of Louisville.

Human cells

Light-density BM MNC were obtained from four cadaveric BM donors and, if necessary, depleted of adherent cells and T lymphocytes (A-T- MNC) as described.47 Samples were obtained from six consenting patients who had been mobilized with G-CSF (Filgastrim; Amgen, Thousand Oaks, CA, USA) and had undergone leukapheresis using the Cobe Spectra Apheresis System (COBE, Lakewood, CO, USA). All the studies were approved by the human ethics committees of the Universities of Louisville and Alberta. Cells were enriched for CD34+ or AC133+ cells by immunoaffinity selection with MiniMacs paramagnetic beads (Miltenyj Biotec), according to the manufacturer's protocol and as described by us in detail.47,48 The purity of isolated CD34+ and AC133+ cells was determined as >98% by FACS analysis. For some experiments, we isolated CXCR4+, CD34+/CXCR4+ and CD34+/AC133+ BM MNC by employing FITC-alpha-CD34, PE-alpha-CXCR4 or PE-alpha-AC133 MoAb (Pharmingen, Palo Alto, CA, USA) and MoFlo cell sorter (Cytomation, Dako, CA, USA) as described.48 Briefly, cells were stained for 30 min at 4°C, washed twice, sorted, and spun down immediately after sorting to isolate RNA using the Qiagen RNA isolation kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's protocol.

Chemotactic isolation

We devised a new method for chemotactic isolation. Briefly, after the isolation of murine BM-, PB- and spleen-derived MNC, the cells were resuspended in serum-free medium and equilibrated for 10 min at 37°C. The lower chambers of the Costar Transwell 24-well plates, 6.5-mm diameter, 5-muM pore filter (Costar Corning, Cambridge, MA, USA), were filled with 650 mul of serum-free medium and 0.5% BSA containing SDF-1 (200 ng/ml) or with media alone (control). In all, 100 mul aliquots of cell suspension were added to the upper chambers. The plates were incubated at 37°C, 95% humidity, 5% CO2 for 5 h and evaluated under an inverted microscope. Cells from the lower chamber were collected and their numbers scored by FACS analysis (FACScan Becton Dickinson) as described.48

Models of organ injury

Excessive bleeding model: Female BALB/c mice (4–6 weeks old) were bled from the retro-orbital plexus (10 drops or approx500 mul of blood). Four animals in each group were killed at various times after bleeding (2, 12, 24 and 48 h), and subsequently mRNA was isolated from their heart muscles.

Irradiation model: Female BALB/c mice (4–6 weeks old) were irradiated with a lethal dose of italic gamma-irradiation (750 cGy). Four animals in each group were killed 24 and 48 h after irradiation, and subsequently mRNA was isolated from their heart muscles.

CCL4 exposure model: Acute CCL4 liver and kidney injury was induced in BalbC mice by the intraperitoneal injection of a single dose of carbon tetrachloride (CCL4; 0.8 ml/kg) diluted to 100 mul with corn oil (Sigma, St Louis, MO, USA). Four animals in each group were killed at various times after exposure (6, 12, 24, 48 and 72 h), and subsequently mRNA was isolated from kidney and liver.

Mobilization of mice

Mice were mobilized by subcutaneous (s.c.) injection of 250 mug/kg human G-CSF (Amgen) daily for 6 days. At 6 h 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 heparin) and enriched for light-density MNC as described. In some mobilization protocols, the specific CXCR4 antagonist T140 (16 mug/kg) was injected intraperitoneally (i.p.) after the last dose of G-CSF.

RT-PCR

Total RNA was isolated using the RNeasy Mini Kit (Quiagen Inc.). mRNA (0.5 mug) was reverse-transcribed with 500 U of Moloney murine leukemia virus reverse transcriptase (MoMLV-RT). The resulting cDNA fragments were amplified using 5 U of Thermus aquaticus (Taq) polymerase. Primer sequences for human MyoD were forward primer 5'-CGG CGG CGG AAC TGC TAC GAA-3' and reverse primer 5'-GGG GCG GGG GCG GAA ACT T-3', for human myogenin were forward primer 5'-AGC GCC CCC TCG TGT ATG-3' and reverse primer 5'-TGT CCC CGG CAA CTT CAG C-3', for human c-met were forward primer 5'-GGG TCG CTT CAT GCA GGT TGT GGT-3' and reverse primer 5'-ATG GTC AGC CTT GTC CCT CCT TCA-3', for human GFAP were forward primer 5'-GTG GGC AGG TGG GAG CTT GAT TCT-3' and reverse primer 5'-CTG GGG CGG CCT GGT ATG ACA-3'.

Real-time RT-PCR

For analysis of Myf5, MyoD, myogenin, GFAP, nestin, alpha-fetoprotein and CK19 mRNA levels, total mRNA was isolated from cells with the RNeasy Mini Kit (Quiagen Inc., Valencia, CA, USA). mRNA was reverse-transcribed with TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA). Detection of Myf5, MyoD, myogenin GFAP, nestin, alpha-fetoprotein, CK19 and beta-actin mRNA levels was performed by real-time RT-PCR using an ABI PRISM® 7000 Sequence Detection System (ABI, Foster City, CA, USA). A 25 mul reaction mixture contains 12.5 mul SYBR Green PCR Master Mix, 10 ng of cDNA template, 5'-ACC ATG GAT CGG CGG AAG G-3' forward and 5'-AAT CGG TGC TGG CAA CTG GAG-3' reverse primers for human Myf5, 5'-CTA GGA GGG CGT CCT TCA TG-3' forward and 5'-CAC GTA TTC TGC CCA GCT TTT-3' reverse primers for murine Myf5, 5'-CGG CGG CGG AAC TGC TAC GAA-3' forward and 5'-GGG GCG GGG GCG GAA ACT T-3' reverse primers for human MyoD, 5'-GGA CAG CCG GTG TGC ATT-3' forward and 5'-CAC TCC GGA ACC CCA ACA G-3' reverse primers for murine MyoD, 5'-AGC GCC CCC TCG TGT ATG-3' forward and 5'-TGT CCC CGG CAA CTT CAG C-3' reverse primers for human myogenin, 5'-GGA GAA GCG CAG GCT CAA G-3' forward and 5'-TTG AGC AGG GTG CTC CTC TT-3' reverse primers for murine myogenin, 5'-GTG GGC AGG TGG GAG CTT GAT TCT-3' forward and 5'-CTG GGG CGG CCT GGT ATG ACA-3' reverse primers for GFAP, 5'-GCA GGC CAC TGA AAA GTT CC-3' forward and 5'-TTC TCC TGC TCC AGG GCT T-3' reverse primers for human and murine nestine, 5'-CTG GAT GTG GCC CAT GTA CA-3' forward and 5'-ATC CAG CAC ATC TCC TCT GCA-3' reverse primers for human and murine alpha-fetoprotein, 5'-CAT GCG AAG CCA ATA TGA GGT-3' forward and 5'-TCA GCA TCC TTC CGG TTC TG-3' reverse primers for CK19, 5'-GGA TGC AGA AGG AGA TCA CTG-3' forward and 5'-CGA TCC ACA CGG AGT ACT TG-3' reverse primers for human and murine beta-actin, 5'-CGT GAG GCC AGG GAA GAG T-3' forward and 5'-TGA TGA GCA TGG TGG GTT GA-3' reverse primers for murine SDF-1. Primers were designed with Primer Express software. 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 quantitation of Myf5, MyoD, myogenin, GFAP, nestin, alpha-fetoprotein and CK19 mRNA expression was calculated with the comparative Ct method. The relative quantitation value of target, normalized to an endogenous control beta-actin gene and relative to a calibrator, is expressed as 2-DeltaDeltaCt (fold difference), where DeltaCt=Ct of target genes (Myf5, MyoD, myogenin, GFAP, nestin, alpha-fetoprotein, CK19)-Ct of endogenous control gene (beta-actin), and DeltaDeltaCt=DeltaCt of samples for target gene-DeltaCt of calibrator for the target gene.

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

Fluorescent staining of the muscle and nerve markers

For the visualization of the muscle and nerve markers, BM MNC were fixed after chemotactic isolation in 3.7%. paraformaldehyde/Ca- and Mg-free PBS for 15 min, and permeabilized by 0.1% Triton X-100 in PBS for 5 min at RT. The primary antibody used for this study was rabbit polyclonal beta-tubulin (1:400), and mouse monoclonal antibody alpha-Myf5 (1:400) was employed as well (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After rinsing in PBS, the sections were incubated with FITC polyclonal anti-rabbit Ig (PharMingen, Lexington, KY, USA) and Alexa Fluor 568 goat anti-mouse IgG (Molecular Probes, Eugene, OR, USA) for 45 min. The stained cells were examined using a BX51 fluorescence microscope (Olympus America, Melville, NY, USA) equipped with a charge-coupled device camera (Olympus America). Each staining was repeated three times on separate samples.

Statistical analysis

Arithmetic means and standard deviations of our FACS data were calculated on a Macintosh computer PowerBase 180, using Instat 1.14 (GraphPad, San Diego, CA, USA) software. Data were analyzed using the Student's t-test for unpaired samples. Statistical significance was defined as P<0.05.

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Results

Cells expressing early markers for muscle, nerve and liver circulate in the PB and their numbers increase during G-CSF mobilization

The presence of circulating stem cells in the PB is a well-known phenomenon described for HSPC and endothelial stem/progenitors.1,29,43 We hypothesized that in addition to HSPC and endothelial stem/progenitors other tissue-specific stem cells also circulate in the PB, maintaining a pool of stem cells for different organs/tissues.

We were able to detect mRNA for markers of early muscle (myogenin) and neural (GFAP) progenitors in MNC isolated from human PB (Figure 1a and Table 1). The number of these circulating tissue-committed stem cells increased after G-CSF mobilization (Figure 1b and Table 1). In mobilized PB MNC, messages for these markers were already easily detectable after 25 cycles of RT-PCR (Figure 1b) compared to nonmobilized blood MNC where 40 cycles were needed to amplify faint signals for myogenin and GFAP (Figure 1a). Furthermore, we were able to detect mRNA for MyoD, an early marker of proliferating muscle stem/progenitor cells, in the PB of 80% of mobilized patients (Table 1). To investigate this phenomenon further and compare the expression of mRNA for early muscle, neural and liver markers between MNC isolated from normal and mobilized PB, we employed real-time RT-PCR analysis. Figure 2a shows that the expression of mRNA for all of these markers was upregulated in MNC isolated from the PB of G-CSF-mobilized patients.

Figure 1.
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RT-PCR analysis of MNC from PB. Expression of mRNA for MyoD (lane 1), myogenin (lane 2), c-met (lane 3) and GFAP (lane 4) was evaluated by RT-PCR in MNC from PB isolated from normal donor (a) or patients mobilized by G-CSF (b). RT-PCR was run for 40 cycles (a) or 25 cycles (b). (c) Negative RT-PCR reactions – DNA instead of cDNA.

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Figure 2.
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Real-time RT-PCR analysis of expression of mRNA for early muscle, neural and liver markers in normal and G-CSF-mobilized human and murine peripheral blood (hPB) MNC. (a) PB MNC from normal human donors (n=3) as well as MNC isolated from patients mobilized with G-CSF (n=4) were compared by real-time RT-PCR for the expression of mRNA of early muscle (Myf5, MyoD and myogenin), early neural (GFAP, nestin) and early liver oval (fetoprotein, CK19) stem/progenitor cells. The data are shown as fold increase in the mRNA level of hPB MNC as compared to nonmobilized PB MNC. (b) PB MNC from normal Balb C mice (n=12) before mobilization as well as MNC isolated from these animals mobilized with G-CSF alone (n=8) and G-CSF + T140 (n=4) were compared by real-time RT-PCR for the expression of mRNA of early muscle (Myf5, MyoD and myogenin), early neural (GFAP, nestin) and early liver oval (fetoprotein, CK19) stem/progenitor cells. The data are shown as fold increase in the mRNA level of mobilized PB MNC compared to nonmobilized PB MNC. *P<0.00001 compared to nonmobilized MNC.

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Similarly, mRNA for all these markers was upregulated in a fraction of MNC isolated from PB of mice mobilized by G-CSF (Figure 2b). Moreover, the expression of mRNA for early muscle, neural and liver cells was additionally enhanced if mice were mobilized with G-CSF+T140, a small molecular inhibitor of the CXCR4 receptor.49 Since increases in the number of these cells paralleled the changes in the number of mobilized HSPC in the PB, we asked whether cells expressing these early markers could reside in the BM and be mobilized along with HSPC into the PB.

Murine BM MNC chemoattracted to an SDF-1 gradient are highly enriched in cells expressing mRNA for early muscle, liver and neural cell markers

We hypothesized that SDF-1, which is secreted at high levels by BM stroma cells,50 may chemoattract other CXCR4-positive cells such as muscle satellite,43,44 liver oval45,51 and neural46,52 stem/progenitor cells besides CXCR4-positive hematopoietic stem cells. To demonstrate this, we assumed that these cells would show chemotaxis to an SDF-1 gradient, which would allow us to isolate them from a suspension of BM MNC using a simple chemotaxis assay (chemotactic isolation). As expected, 18plusminus7% and 22plusminus9% of MNC from BM and spleen showed chemotaxis to SDF-1.

Figure 3 supports our concept. Furthermore, SDF-1 chemotactic isolation studies combined with real-time RT-PCR analysis revealed that CXCR4-positive cells that express mRNA for early tissue-specific cells for muscle, liver and neural tissue reside in normal murine BM and can be highly enriched after chemotaxis to an SDF-1 gradient. In particular, we were able to demonstrate an increase in mRNA for the markers MyoD and fetoprotein of early proliferating muscle and oval liver progenitor cells, respectively. In a direct histochemical analysis (Figure 4) we observed using available antibodies that the percentage of cells expressing Myf5 (an early muscle cell marker) and beta-tubulin (an early neural cell marker) increased after chemotaxis to SDF-1 from approx1 to approx4% and from 0 to approx1%, respectively. Since these studies were performed on freshly isolated cells, the potential contribution of transdifferentiated hematopoietic stem or mesenchymal cells to the phenomenon observed was ruled out.

Figure 3.
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CXCR4-positive cells that migrate to an SDF-1 gradient express markers of muscle and liver progenitor cells and are present in BM. BM cells were isolated from the lower transwell chambers after chemotaxis to SDF-1 (chemotactic isolation), and expressions of mRNA for early muscle (MyoD, myogenin) (a), neural (GFAP, nestin) (b) and liver (fetoprotein, CK19) (c) stem/progenitor markers were compared between the same number of cells from the input and lower chamber by employing real-time RT-PCR. The data are pooled together from three independent experiments. *P<0.00001 as compared to input cells.

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Figure 4.
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CXCR4-positive cells that express markers of muscle and neural progenitor cells are present in BM. BM MNC isolated after chemotaxis to SDF-1 that express Myf-5 (upper panel) and beta-tubulin (lower panel). Left part of the figure – Nomarski's optics; right part of the figure – immunohistochemical staining (times 400). Negative staining controls are not shown.

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Similarly, we found that murine MNC, isolated from spleen and enriched by chemotaxis to SDF-1, highly express mRNA for markers of proliferating muscle stem/progenitors and early liver precursor cells, MyoD and CK19, respectively. Accordingly, mRNA for MyoD and CK19 in spleen-derived MNC cells separated by an SDF-1 gradient was expressed 25plusminus3 times and 70plusminus6 times more, respectively, than control input spleen-derived MNC. Thus, our data suggest that, in addition to BM, murine spleen is an important source or 'hideout' of early CXCR4-positive tissue-committed precursors. Since expression of mRNA for fetoprotein was low in CXCR4-positive cells isolated from spleen, we assumed that this organ contains a different, most likely a more differentiated, population of potential liver precursors, which highly express CK19.

Finally, to exclude the possibility that SDF-1 itself could induce changes in the expression of genes encoding early muscle, neural and liver markers, we employed in our experiments BM MNC and BM CD34+ cells that had been stimulated for 5 h with a chemotactic dose of SDF-1. No changes in the expression of these genes were observed (data not shown).

Real-time RT-PCR reveals that human CD34+ AC133+ and CD34+ CXCR4+ BM MNC as well as murine Sca-1+ cells are highly enriched in mRNA for early muscle, neural and liver markers

Since cells enriched for early hematopoietic stem cell markers such as human CD34 and AC133 as well as murine Sca-1 antigen were reported to contribute to organ tissue regeneration,53,54,55,56,57 we sought to determine whether these cells are also enriched in early committed tissue-specific stem/progenitors.

To address this question we employed real-time RT-PCR to compare the expression of messages for early muscle, liver and neural markers between different subsets of cells isolated from human and murine BM. Figure 5 shows that mRNA for these markers was highly enriched (up to 400 times) in a fraction of human adherent-depleted BM MNC (A- BM MNC) compared with adherent BM MNC. This observation further supports our chemotactic isolation data suggesting that cells expressing these markers can be found in the fraction of the small nonadherent BM MNC that respond to an SDF-1 gradient. Furthermore, since cells expressing mRNA for early tissue stem/progenitors are nonadherent, they do not overlap with the population of adherent mesenchymal stem cells. Figure 6 shows that cells expressing mRNA for early muscle, neural and liver stem/progenitor cells are enriched in a fraction of purified CD34+ and AC133+ and, as was predicted from chemotactic studies, also in CXCR4-positive BM MNC.

Figure 5.
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Adherent cell depleted human BM MNC are highly enriched in mRNA for early muscle, neural and liver stem/progenitor cell markers. Human BM mononuclear adherent and adherent cell depleted fractions were compared for the expression of mRNA for early muscle (MyoD, myogenin) (a), neural (GFAP, nestin) (b) and liver (fetoprotein, CK19) (c) stem/progenitor markers. The data are pooled together from three independent experiments. *P<0.00001 as compared to control.

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Figure 6.
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Human CD34+, AC133+ and CXCR4+ BM MNC are highly enriched in mRNA for early muscle, neural and liver stem/progenitor cell markers. Human BM mononuclear CD34+- and CD34+-depleted cells, AC133+- and ACC133+-depleted cells as well as CXCR4+- and CXCR4+-depleted cells were compared for the expression of mRNA for early muscle (MyoD, myogenin) (a), neural (GFAP, nestin) (b) and liver (fetoprotein, CK19) (c) stem/progenitor markers. The data are pooled together from three independent experiments. *P<0.00001 as compared to control or negative fractions of cells.

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To further explore which cells are enriched in potential early tissue-committed HSPC, fractions of CD34+ CXCR4-, CD34+ CXCR4+ and CD34+ AC133+ BM MNC were sorted by FACS (Figure 7a) and evaluated by real-time RT-PCR for expression of tissue-committed stem/progenitor cell markers. Figure 7b shows that tissue-committed progenitor cells reside in the fraction of CD34+ CXCR4+ and CD34+ AC133+ BM MNC. Furthermore, the number of these cells was significantly lower in CD34+ CXCR4- BM MNC. These data together demonstrate that cells expressing markers of early muscle, neural and liver cells are highly enriched in a population of CXCR4-positive cells that co-express CD34 and AC133 antigens.

Figure 7.
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Sorted by FACS human CD34+/AC133+ and CD34+/CXCR4+ BM MNC are highly enriched in mRNA for early muscle, neural and liver stem/progenitor cell markers. (a) FACS histogram of BM MNC double labeled for expression of early stem cell markers. Left panel – cells labeled for expression of CD34 antigen (FITC) and CXCR4 (PE). Right panel – cells labeled for expression of CD34 antigen (FITC) and AC133 (PE). Cells shown in region R8 were sorted by FACS and evaluated by real-time RT-PCR for expression of markers of early tissue-committed stem/progenitor cells. Representative sort is shown. (b) Double positive CD34+/AC133+ and CD34+/CXCR4+ BM MNC sorted by FACS (Figure 8 – R8) were compared with unsorted BM MNC for expression of markers of early tissue-committed stem/progenitor cells. The data are pooled together from three independent experiments. *P<0.00001 as compared to unpurified BM MNC.

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Similar experiments performed on murine BM MNC revealed that murine Sca-1+cells are also highly enriched in cells expressing markers for early murine muscle, neural and liver stem/progenitor cells. Accordingly, we found an increase in the expression of mRNA for MyoD (113plusminus23 times), Myf-5 (11plusminus3 times), myogenin (10plusminus3 times), c-met (9plusminus2 times), GFAP (9plusminus3 times), nestin (14plusminus23), alpha-fetoprotein (29plusminus5 times) and CK19 (121plusminus14 times), compared to the control unpurified BM MNC.

SDF-1 mRNA is upregulated in damaged organs

To test the hypothesis that CXCR4-positive tissue-committed stem/progenitor cells could be released/mobilized from BM into PB during organ injury and chemoattracted to the damaged peripheral tissues by an SDF-1 gradient, we evaluated the expression of mRNA for SDF-1 in hypoxic and italic gamma-irradiated myocardium as well as kidney and liver damaged by CCL4. Figure 8 shows that SDF-1 mRNA is significantly upregulated in response to injury in all these tissues. However, to demonstrate fully the contribution of tissue-specific stem/progenitor cells mobilized into PB from the BM by tissue injury to organ regeneration, more extensive functional studies are needed. Such studies are currently under way in our laboratory.

Figure 8.
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Expression of SDF-1 is upregulated in murine hypoxic and irradiated myocardium and CCl4 damaged kidney and liver. Changes in the expression of mRNA for SDF-1 between normal and damaged tissues after heart irradiation and heart hypoxia (a) and CCl4 damaged kidney (b) and liver (c) evaluated by real-time RT-PCR. The data are pooled together from four independent experiments. *P<0.00001 as compared to intact tissues.

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Discussion

Using our new strategy of chemotactic separation combined with real-time RT-PCR, we demonstrated that the BM harbors a small population of nonadherent CXCR4-positive cells that express mRNA for early muscle-, neural- and liver-tissue-committed stem/progenitor cells. Furthermore, we found that these cells were enriched in populations of purified human CXCR4+, CD34+ and AC133+ and murine Sca-1+ cells. We hypothesized that these tissue-specific cells circulate in PB under normal steady-state conditions at low but detectable levels and they can be mobilized into the bloodstream during G-CSF mobilization or tissue stress/injury. In fact, we were able to detect increases in mRNA for early tissue-committed stem/progenitor cell markers in samples from human and murine PB mobilized by G-CSF. At the same time, we found that mRNA for SDF-1 is upregulated in damaged organs including hypoxic myocardium and liver and kidney exposed to CCl4.

These observations are important in two respects. First, they provide the basis for a new understanding of the processes related to tissue/organ regeneration. We envision a sequence of events where (i) the expression of SDF-1 is upregulated in damaged organs/tissues (Figure 8), (ii) the number of CXCR4-positive tissue-specific stem cells circulating in the PB increases, and (iii) the SDF-1–CXCR4 axis comes into play through chemoattraction of these cells to the damaged tissues/organs as the first step in the regeneration process. Second, we present evidence that CXCR4-positive tissue-committed cells circulate in PB and suggest how the phenomenon of the so-called stem cell de-differentiation/plasticity could be explained differently. This alternative concept, depicted in Figure 9, is based on four principles: (i) tissue-specific stem cells circulate in the PB, (ii) CXCR4 is a marker of various stem/progenitor cells (eg hematopoietic, endothelial, neural, muscle and liver stem cells), (iii) SDF-1, the ligand for CXCR4, is secreted in various organs to which circulating stem cells are chemoattracted and 'home/reside', and (iv) circulating stem cells may compete for common tissue-specific niches with the result that stem cells committed to other tissues may be detected in various organs.

Figure 9.
Figure 9 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Our hypothesis postulating circulation of tissue-committed CXCR4+ cells. Hematopoietic, muscle satellite, liver oval, neural and other tissue-specific stem/progenitor cells (eg for retina or renal tubular epithelium) express CXCR4 and circulate at low levels in the PB to maintain stem cell pools in distant parts of the body and compete for SDF-1-positive niches in various organs. The number of these cells in the PB increases during tissue damage or pharmacological mobilization. The identity of chemoattractants released from damaged tissues, besides SDF-1, requires further study. BM as a source of many stem cell chemoattractants and survival factors provides an environment that encourages circulating CXCR4+ tissue-committed stem/progenitor cells to home to it. In this context, BM tissue becomes a 'hideout' not only of hematopoietic stem cells but also of already differentiated circulating CXCR4+ tissue-specific stem/progenitor cells. These cells reside in BM and we hypothesize that they could play an important role in tissue/organ repair as a mobile reserve pool of tissue-committed progenitors. Whether these cells also originate in BM requires further study.

Full figure and legend (244K)

In our proposal of the circulation of tissue-specific stem cells, BM tissue takes center stage (Figure 9). The BM's well-developed vessel system as well as its secretion of SDF-1 and several other chemoattractants and cell survival factors provides a unique microenvironment for chemoattracting circulating cells. As a result, BM is the preferred site to which other cell types such as breast and prostate cancers, rhabdomyosarcomas and neuroblastomas metastasize.31,32,33,34,35,36,37,38,39,40,41,42 To explain this, we and others have suggested that SDF-1 secreted by BM stroma chemoattracts CXCR4-positive tumor cells to the BM.31,32,33,34,35

Here we postulate that an analogous mechanism operates at the level of normal circulating CXCR4-positive tissue-committed stem/progenitor cells whereby SDF-1 secreted by BM stroma may chemoattract these cells to the BM. In fact, compelling evidence has been accumulated that CXCR4 is a marker of various stem cells. Consistent with this, we recently observed that murine embryonic stem cells express functional CXCR4 (manuscript in preparation). Moreover, functional CXCR4 is expressed on skeletal muscle stem/progenitor satellite cells,43,44 primordial germ cells,39,40 neural stem cells41 and retinal pigment epithelial stem cells as well as liver oval stem cells.42,45 Hence all these tissue-specific stem cells could potentially be chemoattracted by SDF-1 secreted in BM.

We are aware, however, that before final conclusions can be drawn from our data, some issues require further clarification. First, we do not have final proof of the origin of the cells in the BM that are expressing markers for early tissue-committed stem/progenitor cells. These cells could be chemoattracted to the BM from the peripheral tissues or, alternatively, could originate locally from a population of early BM-residing stem cells that theoretically could be precursors both for hematopoietic and other tissue-committed unipotent stem cells. Second, assuming that all or even a part of early CXCR4-positive tissue-committed stem/progenitor cells home from the circulation into BM, we do not know much about the final fate of these cells after they home to the hematopoietic microenvironment. Various scenarios are possible here. These cells, for example, could remain in the BM as a quiescent mobile reserve population for circulating tissue/organ precursors, or they may even expand, for example, during peripheral tissue injury/organ damage. Third, the relationship of these cells to mesenchymal stem cells is not clear. Based on our observations that these tissue-specific stem/progenitor cells reside in a population of nonadherent BM MNC (Figure 5) and express markers not directly related to the mesenchymal phenotype (eg CD34 antigen), we believe that they constitute a different, nonoverlapping subset of cells. Fourth, we cannot rule out the possibility that, as has been shown for early hematopoietic stem cells, which express mRNA for multiple lineages before being locked into a myeloid vs a lymphoid fate,58 the population of BM-residing early stem cells that we isolated by means of an SDF-1-gradient expresses mRNA for multiple lineages before being 'developmentally locked' into the various cell lineages. Finally, we are aware that, because we worked with a population of FACS-sorted cells, further studies on better purified cell populations expressing tissue lineage-specific markers and studies at the single cell level are needed to better characterize these CXCR4-positive cells. Experiments are under way in our laboratory to address these questions.

The concept presented in this paper is based on the assumption that tissue-committed stem/progenitors circulate in the PB (Figure 9). In fact, PB has been shown to contain, in addition to HSPC, circulating endothelial progenitor cells,59 stromal cells60 and a poorly defined population of fibrocytic cells.61 Here we provide new evidence that PB, in addition to HSPC and endothelial progenitors, contains other circulating tissue-committed stem/progenitors. Although the percentage of early cells for muscle, liver or neural tissue circulating in PB is much lower than that of early HSPC, these mobilized circulating tissue-specific cells may play an important role in tissue repair following injury. We postulate that mobilization of these cells occurs during tissue damage/injury (eg heart infarct, stroke, toxic liver damage). Since we found that the number of these cells increases during G-CSF-induced mobilization, these cells could be enriched in PB for potential therapeutic purposes by employing mobilization protocols similar to those used for the mobilization of HSPC. Figure 2b suggests that antagonists of CXCR4 may increase the efficacy of such mobilization.

We hypothesize that CXCR4-positive cells after mobilization into PB may be subsequently chemoattracted to the damaged tissues. Compelling evidence from other laboratories37,38,45,62 and our own data presented here (Figure 8) show that expression of SDF-1 is upregulated in damaged tissues during toxic liver and kidney damage, or hypoxia and irradiation of myocardium. Thus, evidence is accumulating that SDF-1 may chemoattract circulating tissue-committed CXCR4-positive stem cells for tissue and organ repair/regeneration. However, the full identification of factors secreted by the damaged organs involved in chemoattracting circulating tissue-committed stem cells requires further investigation.

Another important outcome of our studies is the finding that cells enriched for markers for early muscle, neural and liver differentiation are present among human CXCR4-positive BM MNC co-expressing the CD34 and AC133 antigens. This may explain the reported data showing the contribution of BM- or PB-derived CD34+ cells to the regeneration of heart muscle.63 We suggest, however, a different explanation: that heart muscle regeneration was due to the proliferation of the early muscle CD34+/AC133+/CXCR4+ stem/progenitors originating from BM or mobilized PB rather than to the transdifferentiation/plasticity of CD34+ HSPC.

Thus, our data as a whole provide a new perspective on the BM as a 'hideout' or 'home' not only of HSPC but also of already differentiated tissue-committed stem/progenitor cells that could be mobilized into PB and play an important role in tissue/organ regeneration. This hypothesis, however, requires further studies using appropriate tissue/organ damage/regeneration models. Our data also explain why it is possible to grow early muscle, neural, liver, renal tubular and even pancreatic cells from BM MNC (isolated, for example, by FACS as a side population).64,65,66 Our findings suggest that the BM is a 'hideout' of tissue-committed progenitor cells, and the implications of this should be considered before the experimental evidence is interpreted as transdifferentiation/plasticity.

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Note

We recently obtained evidence that in addition to early skeletal, liver and neural tissue committed cells, normal bone marrow harbors cells expressing markers for early cardiac stem/progenitors (Nkx2.5/Csx, GATA-4 and MEF2C).

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Acknowledgements

Supported by NIH grant R01 HL61796-01, EU grant QLK3-CT-2002-30307 and KBN grant PBZ-501/Z/B/1/2002 to MZR.

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