We hypothesized that the CXC chemokine receptor-4 (CXCR4)-stromal-derived factor-1 (SDF-1) axis may be involved in metastasis of CXCR4 ؉ tumor cells into the bone marrow and lymph nodes, which secrete the ␣-chemokine SDF-1. To explore this hypothesis, we phenotyped by fluorescence-activated cell sorter analysis various human tumor cell lines for expression of CXCR4 and found that it was highly expressed on several rhabdomyosarcoma (RMS) cell lines. We also observed that cell lines derived from alveolar RMS, which is characterized by recurrent PAX3-and PAX7-FKHR gene fusions and is associated with a poor prognosis, expressed higher levels of CXCR4 than lines derived from embryonal RMS. Furthermore, transfer of a PAX3-FKHR gene into embryonal RMS cell activates CXCR4 expression. Because alveolar RMS frequently metastasizes to the bone marrow and lymph nodes, it seems that the CXCR4-SDF-1 axis could play an important role in this process. These findings prompted us to determine whether SDF-1 regulates the metastatic behavior of RMS cells. Accordingly, we found that, although SDF-1 did not affect proliferation or survival of these cell lines, it induced in several of them (1)

Recently, we purified from adult murine bone marrow (BM) a population of CXCR4À very small embryonic-like (VSEL) stem cells and hypothesized that similar cells could be also present in human cord blood (CB). Here, we report that by employing a novel two-step isolation procedure -removal of erythrocytes by hypotonic lysis combined with multiparameter sorting -we could isolate from CB a population of human cells that are similar to murine BM-derived VSELs, described previously by us. These CBisolated VSELs (CB-VSEL) are very small (3-5 lm) and highly enriched in a population of CXCR4À CB mononuclear cells, possess large nuclei containing unorganized euchromatin and express nuclear embryonic transcription factors Oct-4 and Nanog and surface embryonic antigen SSEA-4. Further studies are needed to see if human CB-isolated VSELs similar to their murine BM-derived counterparts are endowed with pluripotent stem cell properties.

In patients with AMI and impaired LVEF, treatment with BM cells does not lead to a significant improvement of LVEF or volumes. There was however a trend in favour of cell therapy in patients with most severely impaired LVEF and longer delay between the symptoms and revascularization.

This study was designed to determine the characteristics of tumour cell-derived microvesicles (TMV) and their interactions with human monocytes. TMV were shed spontaneously by three different human cancer cell lines but their release was significantly increased upon activation of the cells with phorbol 12-myristate 13-acetate (PMA). TMV showed the presence of several surface determinants of tumour cells, e.g. HLA class I, CD29, CD44v7/8, CD51, chemokine receptors (CCR6, CX3CR1), extracellular matrix metalloproteinase inducer (EMMPRIN), epithelial cell adhesion molecule (EpCAM), but their level of expression differed from that on cells they originated from. TMV also carried mRNA for growth factors: vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), interleukin-8 (IL-8) and surface determinants (CD44H). TMV were localized at the monocytes surface following their short exposure to TMV, while at later times intracellularly. TMV transferred CCR6 and CD44v7/8 to monocytes, exerted antiapoptotic effect on monocytes and activated AKT kinase (Protein Kinase B). Thus, TMV interact with monocytes, alter their immunophenotype and biological activity. This implicates the novel mechanism by which tumour infiltrating macrophages may be affected by tumour cells not only by a direct cell to cell contact, soluble factors but also by TMV.

Background— Adult stem cells can contribute to myocardial regeneration after ischemic injury. Bone marrow and skeletal muscles contain a population of CXCR4 + cells expressing genes specific for muscle progenitor cells that can be mobilized into the peripheral blood. The aims of the study were (1) to confirm the presence of early tissue-committed cells expressing cardiac, muscle, and endothelial markers in populations of mononuclear cells in peripheral blood and (2) to assess the dynamics and magnitude of the mobilization of CD34 + , CD117 + , CXCR4 + , c-met + , CD34/CD117 + , and CD34/CXCR4 + stem cells into peripheral blood in relation to inflammatory and hematopoietic cytokines in patients with ST-segment–elevation acute myocardial infarction (STEMI). Methods and Results— Fifty-six patients with STEMI (<12 hours), 39 with stable angina, and 20 healthy control subjects were enrolled. Real-time reverse transcription–polymerase chain reaction (RT-PCR) was used for detection of tissue-specific markers. The number of the cells was assessed by use of a flow cytometer on admission, after 24 hours, and after 7 days. RT-PCR revealed increased expression of mRNA (up to 3.5-fold increase) for specific cardiac (GATA4, MEF2C, Nkx2.5/Csx), muscle (Myf5, Myogenin, MyoD), and endothelial (VE-cadherin, von Willebrand factor) markers in peripheral blood mononuclear cells. The number of CD34/CXCR4 + and CD34/CD117 + and c-met + stem cells in peripheral blood was significantly higher in STEMI patients than in stable angina and healthy subjects, peaking on admission, without further significant increase after 24 hours and 7 days. Conclusions— The study demonstrates in the setting of STEMI a marked mobilization of mononuclear cells expressing specific cardiac, muscle, and endothelial markers as well as CD34/CXCR4 + and CD34/CD117 + and c-met + stem cells and shows that stromal cell–derived factor-1 is an important factor influencing the mobilization.

We cloned the cDNA for stem cell tyrosine kinase 1 (STK-1), the human homolog of murine Flk-2/Flt-3, from a CD34+ hematopoietic stem cell-enriched library and investigated its expression in subsets of normal human bone marrow. The cDNA encodes a protein of 993 aa with 85% identity and 92% si to Flk-2/Flt-3. STK-1 is a member ofthe pe HI receptor tyrosine kinase family that includes KIT (steel factor receptor), FMS (colony-stimulating factor 1R), and platelet-derived growth factor receptor. STK-1 expression in human blood and marrow is restricted to CD34+ cells, a population greatly enriched for stem/progenitor cells. Anti-STK-1 antiserum recognizes polypeptides of 160 and 130 kDa in several STK-l-expressing cell lines and in 3T3 cells transfected with a STK-1 expression vector. Antisense oligonucleotides directed against STK-1 sequences inhibited hematopoietic colony formation, most strongly in long-term bone marrow cultures. These data suggest that STK-1 may function as a growth factor receptor on hematopoietic stem and/or progenitor cells. (17,18).Cloning of STK-1. RNA was isolated by the guanidium thiocyanate method (19). First-strand cDNA, generated from 1 &g of CD34+ total RNA with priming by random hexamers and reverse transcription by Moloney murine leukemia virus reverse transcriptase (M-MLV-RT) (GIBCO/BRL), was used as template. Degenerate oligonucleotide primers (designated HHSC-PTK1 and HHSC-PTK2), corresponding to the consensus sequences of the VIb and IX subdomains of TKs, were used for PCR amplification (11). A DNA fragment (HHSC-PTK.2A) was isolated with predicted amino acid identity of 97% when compared to the murine Flk-2/Flt-3. A CD34+ cDNA library was constructed using the Superscript plasmid system (GIBCO/BRL) and screened with this fragment. Positive clones were isolated and after sequencing (20) 5' rapid amplification of cDNA ends (21,22) was used to complete the missing 5' end of the cDNA.Reverse Transcriptase PCR (RT-PCR). Equal amounts (usually 1 ,ug) of total RNA were reverse transcribed with M-MLV-RT using random hexamers or oligo(dt)15 (Boehringer Mannheim) as primers. Aliquots of this material were used with specific primer pairs for PCR amplification. Primer pairs used for STK-1 amplification included nt 91-117 and 324-304 or 878-895 and 1557-1540. Amplification consisted of 95°C for 5 min before adding Taq polymerase (New England Biolabs), followed by cycles of 95°C for 1 min, 45°C for 1 min, and 72°C for 2 min, repeated for 35 cycles. The PCR products were electrophoresed through 1% agarose gels in TAE buffer and transferred overnight to nitrocellulose (23), processed, and probed (24).

We found that the murine cell lines C2C12 and G7 derived from muscle satellite cells, which are essential for muscle regeneration, express the functional CXCR4 receptor on their surface and that the specific ligand for this receptor, α α-chemokine stromal-derived factor 1 (SDF-1), is secreted in muscle tissue. These cell lines responded to SDF-1 stimulation by chemotaxis, phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT serine-threonine kinase, and calcium flux, confirming the functionality of the CXCR4 receptor. Moreover, supernatants derived from muscle fibroblasts chemoattracted both satellite cells and human CD34 + hematopoietic stem/progenitor cells. In a similar set of experiments, supernatants from bone marrow fibroblasts were found to chemoattract CXCR4 + satellite cells just as they chemoattract CD34 + cells.Moreover, preincubation of both muscle satellite cells and hematopoietic stem/progenitor CD34 + cells before chemotaxis with T140, a specific CXCR4 inhibitor, resulted in a significantly lower chemotaxis to media conditioned by either muscle-or bone marrow-derived fibroblasts. Based on these observations, we postulate that the SDF-1-CXCR4 axis is involved in chemoattracting circulating CXCR4 + muscle stem/progenitor and circulating CXCR4 + hematopoietic CD34 + cells to both muscle and bone marrow tissues. Thus, it appears that tissue-specific stem cells circulating in peripheral blood could compete for SDF-1 + niches, and this would explain, without invoking the concept of stem cell plasticity, why hematopoietic colonies can be cultured from muscles and early muscle progenitors can be cultured from bone marrow.

The mechanisms of hematopoietic progenitor cell egress and clinical mobilization are not fully understood. Herein, we report that in vivo desensitization of Sphingosine-1-phosphate (S1P) receptors by FTY720 as well as disruption of S1P gradient toward the blood, reduced steady state egress of immature progenitors and primitive Sca-1 ؉ /c-Kit ؉ /Lin ؊ (SKL) cells via inhibition of SDF-1 release. Administration of AMD3100 or G-CSF to mice with deficiencies in either S1P production or its receptor S1P 1 , or pretreated with FTY720, also resulted in reduced stem and progenitor cell mobilization. Mice injected with AMD3100 or G-CSF demonstrated transient increased S1P levels in the blood mediated via mTOR signaling, as well as an elevated rate of immature c-Kit ؉ /Lin ؊ cells expressing surface S1P 1 in the bone marrow (BM). Importantly, we found that S1P induced SDF-1 secretion from BM stromal cells including Nestin ؉ mesenchymal stem cells via reactive oxygen species (ROS) signaling. Moreover, elevated ROS production by hematopoietic progenitor cells is also regulated by S1P. Our findings reveal that the S1P/S1P 1 axis regulates progenitor cell egress and mobilization via activation of ROS signaling on both hematopoietic progenitors and BM stromal cells, and SDF-1 release. The dynamic cross-talk between S1P and SDF-1 integrates BM stromal cells and hematopoeitic progenitor cell motility. (Blood. 2012;119(11):2478-2488) IntroductionMotility is a key feature of hematopoietic stem and progenitor cells (HSPCs). These cells are continuously released at basal levels from the bone marrow (BM) reservoir to the circulation during steady state homeostasis together with maturing leukocytes, and at increased rates on stress situations, such as bleeding or inflammation. 1,2 The complex process of HSPC trafficking is orchestrated by various cytokines, chemokines, proteolytic enzymes, and adhesion molecules 3-5 through a dynamic interplay between the immune and nervous systems within the bone microenvironment. 1,2,6-8 HSPC mobilization can be clinically induced by a variety of cytokines and drugs, such as granulocyte colony stimulating factor (G-CSF, the most commonly used agent), 9,10 sulfated polysaccharides, 11,12 and recently also by AMD3100. 13,14 Repetitive G-CSF administrations cause mobilization by inducing proliferation and differentiation of HSPC, thus increasing their pool size, accompanied by reduced retention in the BM microenvironment. 15 The chemokine stromal cell-derived factor-1 (SDF-1, also termed CXCL12), which is the most powerful chemoattractant of both human and murine HSPCs, 16,17 and its major receptor CXCR4 are key players in HSPC mobilization. 10,12,13,[18][19][20][21] SDF-1 is transiently increased in the murine BM during G-CSF stimulation followed by its downregulation at both protein 18,22 and mRNA 23 levels, reaching a nadir at the peak of HSPC mobilization. 18 The intensified SDF-1/CXCR4 interactions induce enhanced production of reactive oxygen species (ROS) through activation of the HGF/c-Met path...