RESUMO
Poly-(1,6)-beta-d-glucopyranosyl-(1,3)-beta-d-glucopyranose (PGG) beta-glucan is a soluble yeast-derived polysaccharide that has previously been shown to induce hematopoietic progenitor cell (HPC) mobilization. However, the mobilizing mechanism of action remains unknown. Here, we confirmed that PGG beta-glucan alone or in combination with granulocyte colony-stimulating factor (G-CSF) mobilizes HPC into the periphery. Optimal mobilizing effects were seen 24-48 hours after PGG beta-glucan doses of 4.8-9.6 mg/kg. Animals treated with G-CSF and PGG beta-glucan showed a collaborative effect in HPC mobilization compared with G-CSF treatment alone. Additional studies demonstrated that neither complement 3 nor complement receptor 3 played a role in this effect and that PGG beta-glucan treatment did not induce proinflammatory cytokine secretion. However, bone marrow cells from PGG beta-glucan-treated mice secreted abundant matrix metalloproteinase-9 (MMP-9), and PGG beta-glucan-induced HPC mobilization was abrogated in MMP-9 knockout mice. Moreover, we demonstrated that both hematopoietic and nonhematopoietic cells contributed to MMP-9 secretion upon PGG beta-glucan treatment. In addition, HPCs mobilized by PGG beta-glucan had similar levels of engraftment in host and lineage differentiation capability compared with those mobilized by G-CSF. Thus, PGG beta-glucan is an agent that enhances HPC mobilization and may improve the outcome of clinical stem cell transplantation.
Assuntos
Mobilização de Células-Tronco Hematopoéticas , Metaloproteinase 9 da Matriz/metabolismo , Saccharomyces cerevisiae/química , beta-Glucanas/farmacologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/enzimologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Quimiocina CXCL12/sangue , Ativação do Complemento/imunologia , Complemento C3/imunologia , Citocinas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Fator Estimulador de Colônias de Granulócitos/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/enzimologia , Humanos , Mediadores da Inflamação , Antígeno de Macrófago 1/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas RecombinantesRESUMO
BACKGROUND: Fms-related tyrosine kinase 3 (Flt3)-ligand (FL) promotes the proliferation, differentiation, development, and mobilization of hematopoietic cells. We previously found that FL-mobilized hematopoietic stem cells (HSC) engraft efficiently, whereas FL-expanded bone marrow HSC do not. The function of FL-mobilized c-Kit(+) Sca-1(+)Lin(-)(KSL) subpopulations has not been systematically evaluated. A precise definition of the repopulating ability is needed to define which HSC subpopulations are critical for long-term chimerism and tolerance induction. FL significantly mobilized c-Kit(hi) and c-Kit(lo) Sca-1(+)Lin(-) cells into peripheral blood (PB). Here, we evaluated the influence of Flt3 expression on long-term repopulating ability of HSC subpopulations. METHODS: c-Kit(hi) or c-Kit(lo) KSL cells were sorted from PB of FL-treated green fluorescent protein-positive donors. The function of these cells was evaluated using competitive reconstitution assays, colony-forming units spleen, and colony forming cell assays. The function of c-Kit(hi) CD34(-)Flt3(-) KSL, c-Kit CD34(+)Flt3(-) KSL, c-Kit(hi) CD34(+)Flt3(+) KSL were investigated in an in vivo transplantation model. RESULTS: Only FL-mobilized PB c-Kit(hi) KSL cells exhibited high spleen colony-forming unit activity, generated high numbers of both lymphoid and myeloid colonies in vitro, and rescued ablated recipients. FL-mobilization expanded both c-Kit(hi) CD34(+)Flt3(-) cells (short-term HSC) and c-Kit(hi) CD34(-)Flt3(-) KSL cells (long-term HSC). There was a significant decrease in c-Kit CD34Flt3 KSL late multipotent progenitors in PB. A combination of c-Kit(hi) CD34Flt3 and c-Kit CD34(+)Flt3(-) KSL cells offered the most effective rescue of ablated recipients. CONCLUSIONS: These data suggest that engraftment of purified HSC is influenced by both short- and long-term repopulating populations and that Flt3 expression may be useful for selecting the most critical HSC subpopulations for transplantation.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Tirosina Quinase 3 Semelhante a fms/fisiologia , Animais , Antígenos CD34/análise , Diferenciação Celular , Mobilização de Células-Tronco Hematopoéticas , Masculino , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Complement (C) and innate immunity emerge as important and underappreciated modulators of mobilization of hematopoietic stem/progenitor cells (HSPC). We reported that (a) C becomes activated in bone marrow (BM) during granulocyte-colony-stimulating factor (G-CSF)-induced mobilization by the classic immunoglobulin (Ig)-dependent pathway and that (b) C3 cleavage fragments increase the responsiveness of HSPC to a stromal derived factor-1 gradient. Since patients suffering from severe combined immunodeficiency (SCID) mobilize poorly, we hypothesized that this could be directly linked to the lack of C activating Ig in these patients. In the current study to better elucidate the role of C activation in HSPC mobilization, we mobilized mice that lack Ig (RAG2, SCID, and Jh) by G-CSF or zymosan, compounds that activate C by the classic Ig-dependent and the alternative Ig-independent pathways, respectively. In addition, we evaluated mobilization in C5-deficient animals. Mobilization was evaluated by measuring the number of colony-forming unit-granulocyte macrophage and leukocytes circulating in peripheral blood. We found that (a) G-CSF- but not zymosan-induced mobilization was severely reduced in RAG2, SCID, and Jh mice; (b) impaired G-CSF-induced mobilization was restored after infusion of purified wild-type Ig; and (c) mobilization was severely reduced in C5-deficient mice. These data provide strong evidence that the C system plays a pivotal role in mobilization of HSPC and that egress of HSPC from BM occurs as part of an immune response. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Movimento Celular/imunologia , Ativação do Complemento/genética , Ativação do Complemento/imunologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Mobilização de Células-Tronco Hematopoéticas , Imunoglobulinas/deficiência , Síndromes de Imunodeficiência/genética , Animais , Movimento Celular/genética , Feminino , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/imunologia , Imunoglobulinas/genética , Imunoglobulinas/fisiologia , Síndromes de Imunodeficiência/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCIDRESUMO
Complement cascade (CC) and innate immunity emerge as important and underappreciated modulators of trafficking of hematopoietic stem/progenitor cells (HSPC). Accordingly, we reported that (i) C becomes activated in bone marrow (BM) during G-CSF-induced mobilization by the classical immunoglobulin (Ig)-dependent pathway, and that (ii) C3 cleavage fragments increase the responsiveness of HSPC to an stromal derived factor-1 (SDF-1) gradient. Furthermore, our recent data in immunodeficient mice support the concept that the CC is a major factor modulating egress of HSPC from bone marrow (BM) into peripheral blood (PB). Thus, in light of these findings, mobilization of HSPC could be envisioned as part of an immune response that requires CC activation by the classical Ig-dependent and/or Ig-independent pathways. Hence modulation of CC activation could allow for the development of more efficient mobilization strategies in patients who are poor mobilizers of HSPC.
Assuntos
Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/imunologia , Animais , Medula Óssea/imunologia , Ativação do Complemento/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos SCID , Modelos BiológicosRESUMO
Several organs including hematopoietic ones may regenerate by attracting stem cells that are mobilized from their niches in response to stress related to tissue/organ damage and after mobilization circulate in the peripheral blood. The trafficking of these cells is regulated by alpha-chemokine stromal derived factor-1 (SDF-1) that is upregulated in damaged organs and binds to seven-transmembrane-span G-protein-coupled CXCR4 receptor that is expressed on circulating stem cells. In parallel, evidence has accumulated that the complement (C) system, which is part of innate immunity, may also orchestrate regeneration. C becomes activated with the release of the third complement component (C3) cleavage fragments (e.g., C3a, desArgC3a, and iC3b) during tissue/organ injury. Our recent work demonstrated that these fragments modulate responsiveness of CXCR4+ stem cells to an SDF-1 gradient. Thus the high concentration of both SDF-1 and C3 cleavage fragments in damaged organs results in the formation of an optimal gradient for chemoattracting circulating CXCR4+ stem cells. In this review we will focus on interactions between the SDF-1-CXCR4 axis and the C3 cleavage fragments in a model of mobilization, trafficking, and homing of hematopoietic stem/progenitor cells (HSPC).
Assuntos
Quimiocinas CXC/fisiologia , Complemento C3/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Receptores CXCR4/fisiologia , Animais , Células da Medula Óssea/fisiologia , Movimento Celular , Quimiocina CXCL12 , Hematopoese , Transplante de Células-Tronco Hematopoéticas , HumanosRESUMO
PURPOSE: To characterize chemoattractants expressed by the retinal pigment epithelium (RPE) after sodium iodate (NaIO3)-induced damage and to investigate whether ocular-committed stem cells preexist in the bone marrow (BM) and migrate in response to the chemoattractive signals expressed by the damaged RPE. METHODS: C57/BL6 mice were treated with a single intravenous injection of NaIO3 (50 mg/kg) to create RPE damage. At different time points real-time RT-PCR, ELISA, and immunohistochemistry were used to identify chemoattractants secreted in the subretinal space. Conditioned medium from NaIO3-treated mouse RPE was used in an in vitro assay to assess chemotaxis of stem cell antigen-1 positive (Sca-1+) BM mononuclear cells (MNCs). The expression of early ocular markers (MITF, Pax-6, Six-3, Otx) in migrated cells and in MNCs isolated from granulocyte colony-stimulating factor (G-CSF) and Flt3 ligand (FL)-mobilized and nonmobilized peripheral blood (PB) was analyzed by real-time RT-PCR. RESULTS: mRNA for stromal cell-derived factor-1 (SDF-1), C3, hepatocyte growth factor (HGF), and leukemia inhibitory factor (LIF) was significantly increased, and higher SDF-1 and C3 protein secretion from the RPE was found after NaIO3 treatment. A higher number of BMMNCs expressing early ocular markers migrated to conditioned medium from damaged retina. There was also increased expression of early ocular markers in PBMNCs after mobilization. CONCLUSIONS: Damaged RPE secretes cytokines that have been shown to serve as chemoattractants for BM-derived stem cells (BMSCs). Retina-committed stem cells appear to reside in the BM and can be mobilized into the PB by G-CSF and FL. These stem cells may have the potential to serve as an endogenous source for tissue regeneration after RPE damage.
Assuntos
Fatores Quimiotáticos/metabolismo , Quimiotaxia de Leucócito/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Iodatos/toxicidade , Epitélio Pigmentado Ocular/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Fatores Quimiotáticos/genética , Ensaio de Imunoadsorção Enzimática , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Injeções Intravenosas , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Homeobox SIX3RESUMO
INTRODUCTION: Recently we identified in bone marrow (BM) by employing chemotactic isolation to SDF-1 gradient combined with real time RT-PCR analysis a mobile population of CXCR4+ BM mononuclear cells that express mRNA for various markers of early tissue-committed stem cells (TCSCs). In this study we evaluated whether TCSCs respond to other motomorphogens, such as hepatocyte growth factor (HGF) and leukemia inhibitory factor (LIF). MATERIALS AND METHODS: We again employed chemotactic isolation combined with real-time RT-PCR analysis to assess whether murine and human BM contain TCSCs that respond to HGF and LIF gradients. We also evaluated expressions of HGF and LIF in damaged organs. RESULTS: We noted that the number of TCSCs is highest in BM from young (1- to 2-month-old) mice and decreases in 1-year-old animals. Murine and human TCSCs 1) respond to HGF and LIF gradients in addition to an SDF-1 gradient, 2) reside in populations of BM-derived non-hematopoietic CD45-cells, and 3) are released (mobilized) from BM into the peripheral blood (PB) during tissue injury (e.g. after partial body irradiation). CONCLUSIONS: These findings further support our theory of the BM as a "hideout" for TCSCs and we suggest that their presence in BM tissue should be considered before experimental evidence is interpreted simply as transdifferentiation/plasticity of hematopoietic stem cells. Since we demonstrated that not only SDF-1, but also HGF and LIF are upregulated in damaged tissues, we postulate that CXCR4+ c-Met+ LIF-R+ TCSC could be mobilized from the BM into the PB, from which they are subsequently chemoattracted to damaged organs, where they play a role in tissue repair/regeneration.
Assuntos
Células da Medula Óssea/fisiologia , Movimento Celular , Quimiocinas CXC/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Interleucina-6/metabolismo , Células-Tronco/fisiologia , Fatores Etários , Animais , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Quimiocina CXCL12 , Quimiocinas CXC/fisiologia , Feminino , Regulação da Expressão Gênica , Fator de Crescimento de Hepatócito/fisiologia , Humanos , Interleucina-6/fisiologia , Fator Inibidor de Leucemia , Subunidade alfa de Receptor de Fator Inibidor de Leucemia , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores CXCR4/metabolismo , Receptores de Citocinas/metabolismo , Receptores de OSM-LIF , Células-Tronco/metabolismo , Distribuição TecidualRESUMO
Rhabdomyosarcomas (RMSs) are frequently characterized by bone marrow involvement. Recently, we reported that human RMS cells express the CXC chemokine receptor-4 (CXCR4) and postulated a role for the CXCR4 stromal-derived factor (SDF)-1 axis in the metastasis of RMS cells to bone marrow. Because RMS cells also express the tyrosine kinase receptor c-MET, the specific ligand hepatocyte growth factor (HGF) that is secreted in bone marrow and lymph node stroma, we hypothesized that the c-MET-HGF axis modulates the metastatic behavior of RMS cells as well. Supporting this concept is our observation that conditioned media harvested from expanded ex vivo human bone marrow fibroblasts chemoattracted RMS cells in an HGF- and SDF-1-dependent manner. Six human alveolar and three embryonal RMS cell lines were examined. We found that although HGF, similar to SDF-1, did not affect the proliferation of RMS cells, it induced in several of them: (a) locomotion; (b) stress fiber formation; (c) chemotaxis; (d) adhesion to human umbilical vein endothelial cells; (e) trans-Matrigel invasion and matrix metalloproteinase secretion; and (f) phosphorylation of mitogen-activated protein kinase p42/44 and AKT. Moreover HGF, but not SDF-1, increased the survival of RMS cells exposed to radio- and chemotherapy. We also found that the more aggressive alveolar RMS cells express higher levels of c-MET than embryonal RMS cell lines and "home/seed" better into bone marrow after i.v. injection into immunocompromised mice. Because we could not find any activating mutations in the kinase region of c-MET or any evidence for HGF autocrine stimulation, we suggest that the increased response of RMS cell lines depends on overexpression of functional c-MET. We conclude that HGF regulates the metastatic behavior of c-MET-positive RMS cells, directing them to the bone marrow and lymph nodes. Signaling from the c-MET receptor may also contribute to the resistance of RMS cells to conventional treatment modalities.
Assuntos
Quimiocinas CXC/farmacologia , Fator de Crescimento de Hepatócito/farmacologia , Proteínas Serina-Treonina Quinases , Rabdomiossarcoma/patologia , Actinas/metabolismo , Neoplasias da Medula Óssea/patologia , Neoplasias da Medula Óssea/secundário , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Quimiocina CXCL12 , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Citoesqueleto/metabolismo , Resistencia a Medicamentos Antineoplásicos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-met/biossíntese , Tolerância a Radiação , Receptores CXCR4/biossíntese , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/radioterapiaRESUMO
OBJECTIVE: Peripheral blood platelet-derived microparticles (PMPs) circulate in blood and may interact directly with target cells affecting their various biological functions. METHODS: To investigate the effect of human PMPs on hematopoiesis, we first phenotyped them for expression of various surface molecules and subsequently studied various biological responses of normal stem/progenitor (CD34(+)) and more differentiated precursor cells as well as several leukemic cell lines to PMPs. RESULTS: We found that, in addition to platelet-endothelium attachment receptors (CD41, CD61 and CD62), PMPs express G-protein-coupled seven transmembrane-span receptors such as CXCR4 and PAR-1; cytokine receptors including TNF-RI, TNF-RII, and CD95; and ligands such as CD40L and PF-4. Moreover, we found that several of these receptors could be transferred by PMPs to the membranes of normal as well as malignant cells and observed that PMPs: 1) chemoattract these cells, 2) increase their adhesion, proliferation, and survival, and 3) activate in these cells various intracellular signaling cascades including MAPK p42/44, PI-3K-AKT, and STAT proteins. The biological effects of PMPs were only partly reduced by heat inactivation or trypsin digest, indicating that, in addition to the protein components of PMPs, lipid components are also responsible for their biological activity. CONCLUSIONS: We conclude that PMPs modulate biological functions of hematopoietic cells and postulate that they play an important but as yet not fully understood role in intercellular cross-talk in hematopoiesis. Further studies, however, are needed to identify the PMP components that exert specific biological effects.
Assuntos
Plaquetas/fisiologia , Adesão Celular/fisiologia , Estruturas Celulares/fisiologia , Quimiotaxia/fisiologia , Células-Tronco Hematopoéticas/citologia , Antígenos CD34/análise , Biomarcadores/análise , Células da Medula Óssea/citologia , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Eritroblastos/citologia , Eritroblastos/fisiologia , Células HL-60 , Células-Tronco Hematopoéticas/fisiologia , Humanos , Fosfoproteínas/metabolismo , Fosforilação , Células Tumorais CultivadasRESUMO
Chemokines, small pro-inflammatory chemoattractant cytokines, that bind to specific G-protein-coupled seven-span transmembrane receptors present on plasma membranes of target cells are the major regulators of cell trafficking. In addition some chemokines have been reported to modulate cell survival and growth. Moreover, compelling evidence is accumulating that cancer cells may employ several mechanisms involving chemokine-chemokine receptor axes during their metastasis that also regulate the trafficking of normal cells. Of all the chemokines, stromal-derived factor-1 (SDF-1), an alpha-chemokine that binds to G-protein-coupled CXCR4, plays an important and unique role in the regulation of stem/progenitor cell trafficking. First, SDF-1 regulates the trafficking of CXCR4+ haemato/lymphopoietic cells, their homing/retention in major haemato/lymphopoietic organs and accumulation of CXCR4+ immune cells in tissues affected by inflammation. Second, CXCR4 plays an essential role in the trafficking of other tissue/organ specific stem/progenitor cells expressing CXCR4 on their surface, e.g., during embryo/organogenesis and tissue/organ regeneration. Third, since CXCR4 is expressed on several tumour cells, these CXCR4 positive tumour cells may metastasize to the organs that secrete/express SDF-1 (e.g., bones, lymph nodes, lung and liver). SDF-1 exerts pleiotropic effects regulating processes essential to tumour metastasis such as locomotion of malignant cells, their chemoattraction and adhesion, as well as plays an important role in tumour vascularization. This implies that new therapeutic strategies aimed at blocking the SDF-1-CXCR4 axis could have important applications in the clinic by modulating the trafficking of haemato/lymphopoietic cells and inhibiting the metastatic behaviour of tumour cells as well. In this review, we focus on a role of the SDF-1-CXCR4 axis in regulating the metastatic behaviour of tumour cells and discuss the molecular mechanisms that are essential to this process.
Assuntos
Movimento Celular/fisiologia , Quimiocinas CXC/metabolismo , Quimiotaxia/fisiologia , Neoplasias/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais , Animais , Adesão Celular/fisiologia , Quimiocina CXCL12 , Humanos , Metástase Neoplásica/patologia , Metástase Neoplásica/fisiopatologia , Neoplasias/patologiaRESUMO
Evidence is accumulating that autocrine/paracrine regulatory mechanisms play an important role in regulating normal hematopoiesis. To support this, various growth factors, cytokines and chemokines are expressed and secreted by normal early and differentiated hematopoietic cells. In this review, we summarize recent advances in the identification and understanding of the role of autocrine/paracrine axes in normal human erythropoiesis. We will also address a biological significance of the secretion of (i) metalloproteinases which in addition to growth factors and cytokines are secreted by normal erythroid cells and (ii) membrane-derived microvesicles (MV), that are shed from the surface of maturating erythroblasts/reticulocytes, and as we postulate may also play a role in intercellular communication. We hypothesize that all these factors together play an important role in a crosstalk between erythroid cells and their environment. A better understanding of intercellular crosstalk operating in normal erythropoiesis and of the mechanisms regulating synthesis of these endogenously produced factors may allow us to develop more efficient therapeutic strategies to treat various erythropoietic disorders.
Assuntos
Eritrócitos/metabolismo , Eritropoese , Substâncias de Crescimento/fisiologia , Comunicação Celular , Substâncias de Crescimento/metabolismo , Células-Tronco Hematopoéticas/metabolismo , HumanosRESUMO
Accumulated evidence suggests that in addition to hematopoietic stem cells (HSC), bone marrow (BM) also harbors endothelial stem cells (ESC), mesenchymal stem cells (MSC), multipotential adult progenitor cells (MAPC), pluripotent stem cells (PCS) as well as tissue committed stem cells (TCSC) recently identified by us. In this review we discuss the similarities and differences between these cell populations. Furthermore, we will present the hypothesis that all of these versatile BM derived stem cells are in fact different subpopulations of TCSC. These cells accumulate in bone marrow during ontogenesis and being a mobile population of cells are released from BM into peripheral blood after tissue injury to regenerate damaged organs. Furthermore, since BM is a "hideout" for TCSC, their presence in preparations of bone marrow derived mononuclear cells should be considered before experimental evidence is interpreted simply as trans-differentiation or plasticity of HSC. Finally, our observation that the number of TCSC accumulate in the bone marrow of young animals and their numbers decrease during senescence provides a new insight into aging and may explain why the regeneration processes becomes less effective in older individuals.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco/citologia , Adulto , Envelhecimento , Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Multipotentes/citologia , Células-Tronco Pluripotentes/citologia , Receptores CXCR4/sangue , Receptores CXCR4/isolamento & purificação , RegeneraçãoAssuntos
Plaquetas/metabolismo , Complemento C3/imunologia , Trombopoese/fisiologia , Animais , Adesão Celular , Movimento Celular , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Ativação do Complemento , Complemento C3a/metabolismo , Células Endoteliais/fisiologia , Humanos , Microdomínios da Membrana/metabolismo , Osteoblastos/fisiologia , Transdução de Sinais/fisiologiaAssuntos
Medula Óssea , Movimento Celular/fisiologia , Complemento C3/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco/fisiologia , Animais , Transplante de Células , Ativação do Complemento , Complemento C3/genética , Humanos , Fatores Imunológicos/genética , Fatores Imunológicos/metabolismo , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Knockout , Receptores CXCR4/imunologia , Condicionamento Pré-TransplanteRESUMO
Rhabdomyosarcomas (RMS) express CXCR4 and CXCR7 receptors that bind prometastatic alpha-chemokine stromal-derived factor-1 (SDF-1). In this report, we analyzed the activity of both promoters in a model of less metastatic human embryonal-RMS cell line (RD) and more metastatic alveolar-like RMS (RD cells transduced with paired box gene 3/forkhead homologue; PAX3-FKHR fusion gene). First, CXCR4 is barely detectable in RD and becomes upregulated in RD/PAX3-FKHR cells. In contrast, CXCR7 highly expressed in RD becomes downregulated in RD/PAX3-FKHR cells. Next, promoter deletion and mutation studies revealed that whereas (a) expression of CXCR4 in RD and RD/PAX3-FKHR cells required nuclear respiratory factor-1 (NRF-1) binding site and (b) was additionally upregulated by direct interaction of NRF-1 with PAX3-FKHR, CXCR7 promoter activity required a proximal nuclear factor-kappaB-binding motif. The requirement of these factors for CXCR4 and CXCR7 promoter activities was additionally supported after blocking NRF-1 and nuclear factor-kappaB. Furthermore, CXCR4 expression in PAX3-FKHR(+) RMS cells seems to be enhanced because of the interaction of PAX3-FKHR and NRF-1 proteins in the proximal part of the promoter that prevents access of the negative regulator of transcription YY1 to its binding site. Finally, although hypoxia enhances CXCR4 and CXCR7 promoter activity and receptor expression in RD cells, it inhibits CXCR7 expression in RD/PAX3-FKHR cells. In conclusion, SDF-1 binding receptors CXCR4 and CXCR7 are differently regulated in RMS cells. The upregulation of CXCR4 and downregulation of CXCR7 expression by PAX3-FKHR or hypoxia may give SDF-1 an advantage to better engage the CXCR4 receptor, thus increasing RMS motility.
Assuntos
Receptores CXCR4/genética , Receptores CXCR/genética , Receptores de Quimiocinas/genética , Rabdomiossarcoma/genética , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/genética , Quimiocina CXCL12/metabolismo , Clonagem Molecular , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Fator 1 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , NF-kappa B/fisiologia , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição Box Pareados/fisiologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Receptores CXCR/metabolismo , Receptores CXCR4/metabolismo , Receptores de Quimiocinas/metabolismo , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , TransfecçãoRESUMO
The alpha-chemokine stromal-derived factor (SDF)-1 and the G-protein-coupled seven-span transmembrane receptor CXCR4 axis regulates the trafficking of various cell types. In this review, we present the concept that the SDF-1-CXCR4 axis is a master regulator of trafficking of both normal and cancer stem cells. Supporting this is growing evidence that SDF-1 plays a pivotal role in the regulation of trafficking of normal hematopoietic stem cells (HSCs) and their homing/retention in bone marrow. Moreover, functional CXCR4 is also expressed on nonhematopoietic tissue-committed stem/progenitor cells (TCSCs); hence, the SDF-1-CXCR4 axis emerges as a pivotal regulator of trafficking of various types of stem cells in the body. Furthermore, because most if not all malignancies originate in the stem/progenitor cell compartment, cancer stem cells also express CXCR4 on their surface and, as a result, the SDF-1-CXCR4 axis is also involved in directing their trafficking/metastasis to organs that highly express SDF-1 (e.g., lymph nodes, lungs, liver, and bones). Hence, we postulate that the metastasis of cancer stem cells and trafficking of normal stem cells involve similar mechanisms, and we discuss here the common molecular mechanisms involved in these processes. Finally, the responsiveness of CXCR4+ normal and malignant stem cells to an SDF-1 gradient may be regulated positively/primed by several small molecules related to inflammation which enhance incorporation of CXCR4 into membrane lipid rafts, or may be inhibited/blocked by small CXCR4 antagonist peptides. Consequently, strategies aimed at modulating the SDF-1-CXCR4 axis could have important clinical applications both in regenerative medicine to deliver normal stem cells to the tissues/organs and in clinical hematology/oncology to inhibit metastasis of cancer stem cells.
Assuntos
Quimiocinas CXC/metabolismo , Neoplasias/patologia , Receptores CXCR4/metabolismo , Células-Tronco/citologia , Animais , Medula Óssea/patologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Quimiocina CXCL12 , Quimiotaxia , Humanos , Inflamação , Ligantes , Microdomínios da Membrana , Modelos Biológicos , Metástase Neoplásica , Neoplasias/metabolismo , Transdução de Sinais , Distribuição TecidualRESUMO
We found that supernatants of leukapheresis products (SLPs) of patients mobilized with granulocyte-colony-stimulating factor (G-CSF) or the various components of SLPs (fibrinogen, fibronectin, soluble vascular cell adhesion molecule-1 [VCAM-1], intercellular adhesion molecule-1 [ICAM-1], and urokinase plasminogen activator receptor [uPAR]) increase the chemotactic responses of hematopoietic stem/progenitor cells (HSPCs) to stromal-derived factor-1 (SDF-1). However, alone they do not chemoattract HSPCs, but they do increase or prime the cells' chemotactic responses to a low or threshold dose of SDF-1. We observed that SLPs increased calcium flux, phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT, secretion of matrix metalloproteinases, and adhesion to endothelium in CD34+ cells. Furthermore, SLPs increased SDF-dependent actin polymerization and significantly enhanced the homing of human cord blood (CB)- and bone marrow (BM)-derived CD34+ cells in a NOD/SCID mouse transplantation model. Moreover, the sensitization or priming of cell chemotaxis to an SDF-1 gradient was dependent on cholesterol content in the cell membrane and on the incorporation of the SDF-1 binding receptor CXCR4 and the small GTPase Rac-1 into membrane lipid rafts. This colocalization of CXCR4 and Rac-1 in lipid rafts facilitated guanosine triphosphate (GTP) binding/activation of Rac-1. Hence, we postulate that CXCR4 could be primed by various factors related to leukapheresis and mobilization that increase its association with membrane lipid rafts, allowing the HSPCs to better sense the SDF-1 gradient. This may partially explain why HSPCs from mobilized peripheral blood leukapheresis products engraft more quickly in patients than do those from BM or CB. Based on our findings, we suggest that the homing of HSPCs is optimal when CXCR4 is incorporated in membrane lipid rafts and that ex vivo priming of HSPCs with some of the SLP-related molecules before transplantation could increase their engraftment.
Assuntos
Quimiocinas CXC/farmacologia , Quimiotaxia/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Receptores CXCR4/metabolismo , Actinas/metabolismo , Animais , Antígenos CD34/metabolismo , Medula Óssea/metabolismo , Cálcio/metabolismo , Adesão Celular , Células Cultivadas , Quimiocina CXCL12 , Colágeno , Combinação de Medicamentos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Laminina , Leucaférese , Metaloproteinases da Matriz/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteoglicanas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
In this study, quiescent bone marrow-derived CD34+ erythroid burst-forming units (BFU-E) were found to be resistant to the inhibitory effects of tumour necrosis factor (TNF)-alpha and -beta as well as interferon (IFN)-alpha, -beta and -gamma, in contrast to those stimulated by a combination of erytrhropoietin (Epo) plus kit ligand (KL). Unexpectedly, we found that TNF-alpha also inhibited the apoptosis of quiescent normal human CD34+ BFU-E cells. Accordingly, TNF-alpha added to CD34+ cells cultured for 2 d in serum-free medium protected clonogeneic BFU-E from undergoing serum deprivation-mediated apoptosis. Furthermore, the prosurvival effect of TNF-alpha in quiescent CD34+ cells was consistent with its ability to induce phosphorylation of mitogen-activated protein kinase (MAPK) p42/44. However, when added to CD34+ cells that were stimulated by Epo + KL, TNF-alpha induced apoptosis and inhibited proliferation of BFU-E. To explain this intriguing differential sensitivity between unstimulated CD34+ cells versus those stimulated by Epo + KL, we examined the expression of apoptosis-regulating genes (FLIP, BCL-2, BCL-XL, BAD and BAX) in these cells. Of all the genes tested, FLIP became rapidly downregulated in CD34+ cells 24 h after stimulation with Epo + KL, suggesting that it may protect quiescent CD34+ BFU-E progenitors residing in the bone marrow from the inhibitory effects of inflammatory cytokines. Thus, we hypothesize that cycling cells may become more sensitive to proapoptotic stimuli (e.g. chemotherapy, inhibitory cytokines) than quiescent ones because of the downregulation of protective FLIP.
Assuntos
Antígenos CD34/imunologia , Citocinas/imunologia , Proteínas de Ligação a DNA , Células Precursoras Eritroides/imunologia , Regulação da Expressão Gênica , Fatores de Transcrição/genética , Apoptose/genética , Western Blotting/métodos , Células Cultivadas , Eritropoetina/farmacologia , Humanos , Ativação Linfocitária , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Células-Tronco/farmacologia , Fator de Necrose Tumoral alfa/imunologia , Fatores Estimuladores UpstreamRESUMO
Several reports imply that bone marrow hematopoietic stem cells transdifferentiate into tissue-specific stem cells; however, the possibility of committed tissue-specific stem cells pre-existing in the bone marrow has not been dealt with adequately. We present here an alternative explanation of the so-called phenomenon of stem cell transdifferentiation. First, we postulate that tissue-committed stem/progenitor cells circulate in the peripheral blood and compete for tissue-specific niches. The circulation of these cells plays an important physiological role in maintaining a pool of stem cells in distant parts of the body and the number of these cells in peripheral blood can be increased by the administration of agents similar to those used for mobilization of hematopoietic stem cells. Second, we postulate that bone marrow tissue is a source of various stem-cell chemoattractants and survival factors and provides an environment that chemoattracts tissue-specific circulating stem/progenitor cells. In this context, we envision bone marrow as a "home" or "hide-out place" not only of hematopoietic stem cells but also of already differentiated circulating tissue-specific stem/progenitors. In support of this concept, we report here that mRNA of several early markers for muscle (Myf-5, Myo-D), neural (GFAP, nestin) and liver (CK19, fetoprotein) is detectable in circulating (adherent cell-depleted) peripheral blood mononuclear cells. Moreover, using real-time RT-PCR, we found that the level of expression of these markers increases in the peripheral blood of humans and mice after mobilization by G-CSF. Furthermore, using stromal-derived factor-1 (SDF-1) chemotaxis and real-time RT-PCR analysis, we present evidence that early tissue-specific stem cells reside in normal human and murine bone marrow, express the CXCR4 receptor on their surface and can be highly enriched (in humans and mice) after chemotaxis to SDF-1 gradient. All our experiments were performed on freshly isolated cells to exclude the potential contribution of transdifferentiated hematopoietic stem or mesenchymal cells in the culture. We maintain that any transdifferentiation studies employing populations of bone marrow cells should rule out the possibility that the apparently pure hematopoietic stem cell population could in fact contain pre-existing tissue-specific stem/progenitors.
Assuntos
Células da Medula Óssea/citologia , Quimiocinas CXC/fisiologia , Quimiotaxia , Células-Tronco/citologia , Animais , Biomarcadores/sangue , Circulação Sanguínea , Células da Medula Óssea/fisiologia , Sistema Nervoso Central/citologia , Quimiocina CXCL12 , Quimiocinas CXC/farmacologia , Quimiotaxia/efeitos dos fármacos , Humanos , Fígado/citologia , Camundongos , Modelos Biológicos , Músculos/citologia , Especificidade de Órgãos , RNA Mensageiro/sangue , Estresse Fisiológico/patologiaRESUMO
Complement has recently been implicated in developmental pathways and noninflammatory processes. The expression of various complement components and receptors has been shown in a wide range of circulating myeloid and lymphoid cells, but their role in normal hematopoiesis and stem cell homing has not yet been investigated. We report that normal human CD34(+) cells and lineage-differentiated hematopoietic progenitors express the complement anaphylatoxin C3a receptor (C3aR) and respond to C3a. Moreover, C3a, but not the biologically inactive desArg-C3a, induces calcium flux in these cells. Furthermore, we found that C3 is secreted by bone marrow stroma and that, although C3a does not influence directly the proliferation/survival of hematopoietic progenitors, it (1) potentiates the stromal cell-derived factor 1 (SDF-1)-dependent chemotaxis of human CD34(+) cells and lineage-committed myeloid, erythroid, and megakaryocytic progenitors; (2) primes SDF-1-dependent trans-Matrigel migration; and (3) stimulates matrix metalloproteinase-9 secretion and very late antigen 4 (VLA-4)-mediated adhesion to vascular cell adhesion molecule 1 (VCAM-1). Furthermore, we found that murine Sca-1(+) cells primed by C3a engrafted faster in lethally irradiated animals. These results indicate that normal human hematopoietic stem and progenitor cells express functional C3aR and that the C3aR-C3a axis sensitizes the responses of these cells to SDF-1 and thus may be involved in promoting their homing into the bone marrow via cross talk with the SDF-CXC chemokine receptor-4 (CXCR4) signaling axis. C3a is the first positive regulator of this axis to be identified.