RESUMO
Human culture-expanded mesenchymal stromal cells (MSC) are being considered for multiple therapeutic applications because of their regenerative and anti-inflammatory properties. Although a large number of MSC can be propagated from a small initial sample, several lines of evidence indicate that MSC lose their immunosuppressive and regenerative potency aftaer multiple passages. In this report, we use the FACSCAP Lyoplate proteomic analysis system to detect changes in cell surface protein expression of CD45- /CD31- /CD34- /CD73+ /CD105+ stromal cells in unpassaged bone marrow (BM) and through 10 serial culture passages. We provide for the first time a detailed characterization of native unpassaged BM MSC (0.08% of BM mononuclear cells) as well as the changes that occur during the initial expansion. Adipogenic and osteogenic differentiative potential was determined though the serial passages and correlated with immunophenotypic changes and senescence. Among the most prominent were striking decreases in Fas ligand, CD98, CD205, and CD106, accompanied by a gain in the expression of CD49c, CD63, CD98, and class 1 and class 2 major histocompatibility complex (MHC) molecules. Other molecules that are down-modulated with later passage include CD24, CD54, CD59, CD243/P-glycoprotein, and CD273/PD-L2. Early senescence, as defined by the loss of replicative capacity occurring with the loss of differentiative capacity, increase in CDKN2A p16, and increased time to confluence, was accompanied by loss of the motility-associated metalloproteinase CD10 and the proliferation-associated transferrin receptor CD71. Among the strongest statistical associations were loss of MAC-inhibitory protein/CD59, loss of ICAM-1/CD54, and increase in CDKN2A as a function of increasing passage, as well as increased CD10 expression with adipogenic and osteogenic capacities. The data provide a clear set of markers that can be used to assess MSC quality. We suggest that clinically relevant numbers of highly functional low passage MSC can be manufactured starting with large quantities of BM, which are readily available from cadaveric organ donors.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Proteoma/metabolismo , Células Estromais/metabolismo , Adipogenia/fisiologia , Antígenos CD/metabolismo , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Humanos , Osteogênese/fisiologia , Proteômica/métodosRESUMO
Flow cytometry is often performed on adherent cells or solid tissues that have been released from their growth substrate or disaggregated by enzymatic digestion. Although detection of strongly expressed cell surface proteins following such procedures indicates that many survive treatment with proteolytic enzymes, applications such as cell surface proteomics involve assessment of the expression of more than 200 proteins and it is important to know how to interpret negative results. To address this problem, we performed flow cytometry-based cell surface proteomic analysis on two non-adherent cell lines, THP1 and K562, after mock and authentic trypsin treatment, according to a widely used protocol to remove adherent cells (0.25% trypsin, 2.21 mM EDTA, 37°C, 5 min). In a single screening experiment, we examined the effect of treatment on mean fluorescence intensity and on the percent of positive cells and determined the false negative rate. Of 164 determinations that were ≥20% positive after mock treatment, 13 (7.9%) were <20% positive after trypsin treatment. Four proteins were chosen for time-course studies (performed in triplicate), confirming initial sensitivity results but revealing significant variability in the magnitude of the trypsin effect. When trypsin sensitivity of individual proteins was examined as a function of the number of predicted high probability extracellular trypsin cleavage sites, we found that the markers that yielded false negatives all had high numbers of sites (>30), but even so, the majority of proteins with high numbers of trypsin sites could still be detected after mild trypsin treatment. We conclude that the great majority of cell surface proteins can be detected after mild trypsin treatment, but that negative results should not be over-interpreted, due to the possibility of false negatives.
Assuntos
Citometria de Fluxo , Proteínas de Membrana/isolamento & purificação , Proteoma/genética , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana/genética , Proteômica/métodos , Tripsina/farmacologiaRESUMO
Various mesenchymal cell types have been identified as critical components of the hematopoietic stem/progenitor cell (HSPC) niche. Although several groups have described the generation of mesenchyme from human pluripotent stem cells (hPSCs), the capacity of such cells to support hematopoiesis has not been reported. Here, we demonstrate that distinct mesenchymal subpopulations co-emerge from mesoderm during hPSC differentiation. Despite co-expression of common mesenchymal markers (CD73, CD105, CD90, and PDGFRß), a subset of cells defined as CD146hiCD73hi expressed genes associated with the HSPC niche and supported the maintenance of functional HSPCs ex vivo, while CD146loCD73lo cells supported differentiation. Stromal support of HSPCs was contact dependent and mediated in part through high JAG1 expression and low WNT signaling. Molecular profiling revealed significant transcriptional similarity between hPSC-derived CD146++ and primary human CD146++ perivascular cells. The derivation of functionally diverse types of mesenchyme from hPSCs opens potential avenues to model the HSPC niche and develop PSC-based therapies.
Assuntos
Diferenciação Celular/genética , Células-Tronco Mesenquimais/citologia , Mesoderma/citologia , Células-Tronco Pluripotentes/citologia , Antígeno CD146/genética , Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Hematopoese/genética , Humanos , Células-Tronco Mesenquimais/metabolismo , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Células-Tronco Pluripotentes/metabolismo , Nicho de Células-Tronco/genética , Via de Sinalização Wnt/genéticaRESUMO
Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31- /CD45- /CD34+ /CD146- cells (adventitial stromal/stem cells [ASCs]) and CD31- /CD45- /CD34- /CD146+ cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDHbr ASC (most primitive); (b) ALDHdim ASC; (c) ALDHbr PC; (d) ALDHdim PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression networks. Stem Cells 2017;35:1273-1289.
Assuntos
Tecido Adiposo/citologia , Linhagem da Célula , Redes Reguladoras de Genes , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Aldeído Desidrogenase/metabolismo , Diferenciação Celular/genética , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Pericitos/citologia , Análise de Célula ÚnicaRESUMO
Perivascular mesenchymal precursor cells (i.e., pericytes) reside in skeletal muscle where they contribute to myofiber regeneration; however, the existence of similar microvessel-associated regenerative precursor cells in cardiac muscle has not yet been documented. We tested whether microvascular pericytes within human myocardium exhibit phenotypes and multipotency similar to their anatomically and developmentally distinct counterparts. Fetal and adult human heart pericytes (hHPs) express canonical pericyte markers in situ, including CD146, NG2, platelet-derived growth factor receptor (PDGFR) ß, PDGFRα, alpha-smooth muscle actin, and smooth muscle myosin heavy chain, but not CD117, CD133, and desmin, nor endothelial cell (EC) markers. hHPs were prospectively purified to homogeneity from ventricular myocardium by flow cytometry, based on a combination of positive- (CD146) and negative-selection (CD34, CD45, CD56, and CD117) cell lineage markers. Purified hHPs expanded in vitro were phenotypically similar to human skeletal muscle-derived pericytes (hSkMPs). hHPs express mesenchymal stem/stromal cell markers in situ and exhibited osteo-, chondro-, and adipogenic potentials but, importantly, no ability for skeletal myogenesis, diverging from pericytes of all other origins. hHPs supported network formation with/without ECs in Matrigel cultures; hHPs further stimulated angiogenic responses under hypoxia, markedly different from hSkMPs. The cardiomyogenic potential of hHPs was examined following 5-azacytidine treatment and neonatal cardiomyocyte coculture in vitro, and intramyocardial transplantation in vivo. Results indicated cardiomyocytic differentiation in a small fraction of hHPs. In conclusion, human myocardial pericytes share certain phenotypic and developmental similarities with their skeletal muscle homologs, yet exhibit different antigenic, myogenic, and angiogenic properties. This is the first example of an anatomical restriction in the developmental potential of pericytes as native mesenchymal stem cells.
Assuntos
Antígenos de Diferenciação/biossíntese , Células-Tronco Multipotentes/metabolismo , Miocárdio/metabolismo , Pericitos/metabolismo , Células Cultivadas , Feminino , Humanos , Masculino , Células-Tronco Multipotentes/citologia , Miocárdio/citologia , Especificidade de Órgãos/fisiologia , Pericitos/citologiaRESUMO
Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.
Assuntos
Túnica Adventícia/citologia , Células Endoteliais/citologia , Células-Tronco Mesenquimais/citologia , Músculo Esquelético/irrigação sanguínea , Pericitos/citologia , Túnica Íntima/citologia , HumanosRESUMO
Mesenchymal stem/stromal cells (MSCs) are adult multipotent progenitors of great promise for cell therapy. MSCs can mediate tissue regeneration, immunomodulation, and hematopoiesis support. Despite the unique properties of MSCs and their broad range of potential clinical applications, the very nature of these cells has been uncertain. Furthermore, MSCs are heterogeneous and only defined subpopulations of these are endowed with the particular abilities to sustain hematopoietic stem cells, regulate immune responses, or differentiate into mesodermal cell lineages. It is becoming evident that current criteria used to define cultured polyclonal MSCs (expression of nonspecific markers and in vitro mesodermal differentiation) are not sufficient to fully understand and exploit the potential of these cells. Here, we describe how flow cytometry has been used to reveal a perivascular origin of MSCs. As a result, the prospective purification of MSCs and specialized subsets thereof is now possible, and the clinical use of purified autologous MSCs is now within reach.
Assuntos
Citometria de Fluxo , Células-Tronco Mesenquimais/metabolismo , Tecido Adiposo Branco/citologia , Animais , Antígenos CD/metabolismo , Vasos Sanguíneos/citologia , Separação Celular , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Medicina RegenerativaRESUMO
Lysophosphatidic acid (LPA) is a pleiotropic phospholipid present in the blood and certain tissues at high concentrations; its diverse effects are mediated through differential, tissue specific expression of LPA receptors. Our goal was to determine if LPA exerts lineage-specific effects during normal human hematopoiesis. In vitro stimulation of CD34+ human hematopoietic progenitors by LPA induced myeloid differentiation but had no effect on lymphoid differentiation. LPA receptors were expressed at significantly higher levels on Common Myeloid Progenitors (CMP) than either multipotent Hematopoietic Stem/Progenitor Cells (HSPC) or Common Lymphoid Progenitors (CLP) suggesting that LPA acts on committed myeloid progenitors. Functional studies demonstrated that LPA enhanced migration, induced cell proliferation and reduced apoptosis of isolated CMP, but had no effect on either HSPC or CLP. Analysis of adult and fetal human bone marrow sections showed that PPAP2A, (the enzyme which degrades LPA) was highly expressed in the osteoblastic niche but not in the perivascular regions, whereas Autotaxin (the enzyme that synthesizes LPA) was expressed in perivascular regions of the marrow. We propose that a gradient of LPA with the highest levels in peri-sinusoidal regions and lowest near the endosteal zone, regulates the localization, proliferation and differentiation of myeloid progenitors within the bone marrow marrow.
Assuntos
Lisofosfolipídeos/farmacologia , Antígenos CD34/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/efeitos dos fármacosRESUMO
Hematopoietic stem and progenitor cells (HSPCs) emerge and develop adjacent to blood vessel walls in the yolk sac, aorta-gonad-mesonephros region, embryonic liver, and fetal bone marrow. In adult mouse bone marrow, perivascular cells shape a "niche" for HSPCs. Mesenchymal stem/stromal cells (MSCs), which support hematopoiesis in culture, are themselves derived in part from perivascular cells. In order to define their direct role in hematopoiesis, we tested the ability of purified human CD146(+) perivascular cells, as compared with unfractionated MSCs and CD146(-) cells, to sustain human HSPCs in coculture. CD146(+) perivascular cells support the long-term persistence, through cell-to-cell contact and at least partly via Notch activation, of human myelolymphoid HSPCs able to engraft primary and secondary immunodeficient mice. Conversely, unfractionated MSCs and CD146(-) cells induce differentiation and compromise ex vivo maintenance of HSPCs. Moreover, CD146(+) perivascular cells express, natively and in culture, molecular markers of the vascular hematopoietic niche. Unexpectedly, this dramatic, previously undocumented ability to support hematopoietic stem cells is present in CD146(+) perivascular cells extracted from the nonhematopoietic adipose tissue.
Assuntos
Vasos Sanguíneos/fisiologia , Antígeno CD146/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Adulto , Animais , Antígenos CD34/metabolismo , Vasos Sanguíneos/citologia , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Comunicação Celular , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Técnicas de Cocultura , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Receptores Notch/genética , Receptores Notch/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Serrate-JaggedRESUMO
The interaction of tumor cells with the tumor vasculature is mainly studied for its role in tumor angiogenesis and intravascular metastasis of circulating tumor cells. In addition, a specific interaction of tumor cells with the abluminal surfaces of vessels, or angiotropism, may promote the migration of angiotropic tumor cells along the abluminal vascular surfaces in a pericytic location. This process has been termed extravascular migratory metastasis. The abluminal vascular surface may also provide a vascular niche inducing or sustaining stemness to angiotropic tumor cells. This pilot study investigated if angiotropic melanoma cells might represent a subset population with pericytic and embryonic or stem cell properties. Through microarray analysis, we showed that the interaction between melanoma cells and the abluminal surface of endothelial cells triggers significant differential expression of several genes. The most significantly differentially expressed genes have demonstrated properties linked to cancer cell migration (CCL2, ICAM1 and IL6), cancer progression (CCL2, ICAM1, SELE, TRAF1, IL6, SERPINB2 and CXCL6), epithelial to mesenchymal transition (CCL2 and IL6), embryonic/stem cell properties (CCL2, PDGFB, EVX1 and CFDP1) and pericytic recruitment (PDGFB). In addition, bioinformatics-based analysis of the differentially expressed genes has shown that the most significantly enriched functional groups included development, cell movement, cancer, and embryonic development. Finally, the investigation of pericyte/mesenchymal stem cells markers via immunostaining of human melanoma samples revealed expression of PDGFRB, NG2 and CD146 by angiotropic melanoma cells. Taken together, these preliminary data are supportive of the "pericytic mimicry" by angiotropic melanoma cells, and suggest that the interaction between melanoma cells and the abluminal vascular surface induce differential expression of genes linked to cancer migration and embryonic/stem cell properties.
RESUMO
Human microvascular pericytes (CD146(+)/34(-)/45(-)/56(-)) contain multipotent precursors and repair/regenerate defective tissues, notably skeletal muscle. However, their ability to repair the ischemic heart remains unknown. We investigated the therapeutic potential of human pericytes, purified from skeletal muscle, for treating ischemic heart disease and mediating associated repair mechanisms in mice. Echocardiography revealed that pericyte transplantation attenuated left ventricular dilatation and significantly improved cardiac contractility, superior to CD56+ myogenic progenitor transplantation, in acutely infarcted mouse hearts. Pericyte treatment substantially reduced myocardial fibrosis and significantly diminished infiltration of host inflammatory cells at the infarct site. Hypoxic pericyte-conditioned medium suppressed murine fibroblast proliferation and inhibited macrophage proliferation in vitro. High expression by pericytes of immunoregulatory molecules, including interleukin-6, leukemia inhibitory factor, cyclooxygenase-2, and heme oxygenase-1, was sustained under hypoxia, except for monocyte chemotactic protein-1. Host angiogenesis was significantly increased. Pericytes supported microvascular structures in vivo and formed capillary-like networks with/without endothelial cells in three-dimensional cocultures. Under hypoxia, pericytes dramatically increased expression of vascular endothelial growth factor-A, platelet-derived growth factor-ß, transforming growth factor-ß1 and corresponding receptors while expression of basic fibroblast growth factor, hepatocyte growth factor, epidermal growth factor, and angiopoietin-1 was repressed. The capacity of pericytes to differentiate into and/or fuse with cardiac cells was revealed by green fluorescence protein labeling, although to a minor extent. In conclusion, intramyocardial transplantation of purified human pericytes promotes functional and structural recovery, attributable to multiple mechanisms involving paracrine effects and cellular interactions.
Assuntos
Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Miocárdio/patologia , Pericitos/transplante , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Fibrose/prevenção & controle , Expressão Gênica , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/genética , Miocárdio/metabolismo , Neovascularização Fisiológica , Pericitos/fisiologia , Proteínas Proto-Oncogênicas c-sis/genética , Proteínas Proto-Oncogênicas c-sis/metabolismo , Regeneração/fisiologia , Transplante Heterólogo , Ultrassonografia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Adipose tissue is an ideal source of mesenchymal stem cells for bone tissue engineering: it is largely dispensable and readily accessible with minimal morbidity. However, the stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population, which leads to unreliable bone formation. In the present study, we prospectively purified human perivascular stem cells (PSCs) from adipose tissue and compared their bone-forming capacity with that of traditionally derived SVF. PSCs are a population (sorted by fluorescence-activated cell sorting) of pericytes (CD146+CD34-CD45-) and adventitial cells (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. Here, we found that PSCs underwent osteogenic differentiation in vitro and formed bone after intramuscular implantation without the need for predifferentiation. We next sought to optimize PSCs for in vivo bone formation, adopting a demineralized bone matrix for osteoinduction and tricalcium phosphate particle formulation for protein release. Patient-matched, purified PSCs formed significantly more bone in comparison with traditionally derived SVF by all parameters. Recombinant bone morphogenetic protein 2 increased in vivo bone formation but with a massive adipogenic response. In contrast, recombinant Nel-like molecule 1 (NELL-1; a novel osteoinductive growth factor) selectively enhanced bone formation. These studies suggest that adipose-derived human PSCs are a new cell source for future efforts in skeletal regenerative medicine. Moreover, PSCs are a stem cell-based therapeutic that is readily approvable by the U.S. Food and Drug Administration, with potentially increased safety, purity, identity, potency, and efficacy. Finally, NELL-1 is a candidate growth factor able to induce human PSC osteogenesis.
Assuntos
Regeneração Óssea , Células-Tronco Mesenquimais/citologia , Osteogênese , Pericitos/citologia , Adipogenia , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/metabolismo , Animais , Antígenos CD34/metabolismo , Matriz Óssea/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 2/farmacologia , Antígeno CD146/metabolismo , Fosfatos de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio , Técnicas de Cultura de Células , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Antígenos Comuns de Leucócito/metabolismo , Lipectomia , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos SCID , Proteínas do Tecido Nervoso/metabolismo , Pericitos/efeitos dos fármacos , Estudos Prospectivos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Medicina Regenerativa/métodos , Alicerces Teciduais , Microtomografia por Raio-XRESUMO
Adipose tissue is an ideal mesenchymal stem cell (MSC) source, as it is dispensable and accessible with minimal morbidity. However, the stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population, which has disadvantages for tissue regeneration. In the present study, we prospectively purified human perivascular stem cells (PSCs) from n = 60 samples of human lipoaspirate and documented their frequency, viability, and variation with patient demographics. PSCs are a fluorescence-activated cell sorting-sorted population composed of pericytes (CD45-, CD146+, CD34-) and adventitial cells (CD45-, CD146-, CD34+), each of which we have previously reported to have properties of MSCs. Here, we found that PSCs make up, on average, 43.2% of SVF from human lipoaspirate (19.5% pericytes and 23.8% adventitial cells). These numbers were minimally changed by age, gender, or body mass index of the patient or by length of refrigerated storage time between liposuction and processing. In a previous publication, we observed that human PSCs (hPSCs) formed significantly more bone in vivo in comparison with unsorted human SVF (hSVF) in an intramuscular implantation model. We now extend this finding to a bone injury model, observing that purified hPSCs led to significantly greater healing of mouse critical-size calvarial defects than hSVF (60.9% healing as opposed to 15.4% healing at 2 weeks postoperative by microcomputed tomography analysis). These studies suggest that adipose-derived hPSCs are a new cell source for future efforts in skeletal regenerative medicine. Moreover, hPSCs are a stem cell-based therapeutic that is readily approvable by the U.S. Food and Drug Administration, with potentially increased safety, purity, identity, potency, and efficacy.
Assuntos
Regeneração Óssea , Osso e Ossos , Células-Tronco Mesenquimais/metabolismo , Engenharia Tecidual , Tecido Adiposo/citologia , Túnica Adventícia , Animais , Antígenos CD34/análise , Antígeno CD146/análise , Separação Celular , Humanos , Antígenos Comuns de Leucócito/análise , Camundongos , Pericitos , Alicerces Teciduais , Cicatrização , Ferimentos e Lesões/terapiaRESUMO
Mesenchymal stem/stromal cells (MSC) are currently the best candidate therapeutic cells for regenerative medicine related to osteoarticular, muscular, vascular and inflammatory diseases, although these cells remain heterogeneous and necessitate a better biological characterization. We and others recently described that MSC originate from two types of perivascular cells, namely pericytes and adventitial cells and contain the in situ counterpart of MSC in developing and adult human organs, which can be prospectively purified using well defined cell surface markers. Pericytes encircle endothelial cells of capillaries and microvessels and express the adhesion molecule CD146 and the PDGFRß, but lack endothelial and haematopoietic markers such as CD34, CD31, vWF (von Willebrand factor), the ligand for Ulex europaeus 1 (UEA1) and CD45 respectively. The proteoglycan NG2 is a pericyte marker exclusively associated with the arterial system. Besides its expression in smooth muscle cells, smooth muscle actin (αSMA) is also detected in subsets of pericytes. Adventitial cells surround the largest vessels and, opposite to pericytes, are not closely associated to endothelial cells. Adventitial cells express CD34 and lack αSMA and all endothelial and haematopoietic cell markers, as for pericytes. Altogether, pericytes and adventitial perivascular cells express in situ and in culture markers of MSC and display capacities to differentiate towards osteogenic, adipogenic and chondrogenic cell lineages. Importantly, adventitial cells can differentiate into pericyte-like cells under inductive conditions in vitro. Altogether, using purified perivascular cells instead of MSC may bring higher benefits to regenerative medicine, including the possibility, for the first time, to use these cells uncultured.
Assuntos
Tecido Adiposo/citologia , Túnica Adventícia/citologia , Células-Tronco Mesenquimais/fisiologia , Pericitos/fisiologia , Medicina Regenerativa , Túnica Adventícia/fisiologia , Antígenos/metabolismo , Biomarcadores/metabolismo , Antígeno CD146/metabolismo , Diferenciação Celular , Linhagem da Célula , Transplante de Células , Células Cultivadas , Humanos , Proteínas de Membrana , Pericitos/transplante , Proteoglicanas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
A theoretical inverse relationship exists between osteogenic (bone forming) and adipogenic (fat forming) mesenchymal stem cell (MSC) differentiation. This inverse relationship in theory partially underlies the clinical entity of osteoporosis, in which marrow MSCs have a preference for adipose differentiation that increases with age. Two pro-osteogenic cytokines have been recently studied that each also possesses antiadipogenic properties: Sonic Hedgehog (SHH) and NELL-1 proteins. In the present study, we assayed the potential additive effects of the biologically active N-terminus of SHH (SHH-N) and NELL-1 protein on osteogenic and adipogenic differentiation of human primary adipose-derived stromal cell (hASCs). We observed that both recombinant SHH-N and NELL-1 protein significantly enhanced osteogenic differentiation and reduced adipose differentiation across all markers examined (alkaline phosphatase, Alizarin red and Oil red O staining, and osteogenic gene expression). Moreover, SHH-N and NELL-1 directed signaling produced additive effects on the pro-osteogenic and antiadipogenic differentiation of hASCs. NELL-1 treatment increased Hedgehog signaling pathway expression; coapplication of the Smoothened antagonist Cyclopamine reversed the pro-osteogenic effect of NELL-1. In summary, Hedgehog and Nell-1 signaling exert additive effects on the pro-osteogenic and antiadipogenic differentiation of ASCs. These studies suggest that the combination cytokines SHH-N+NELL-1 may represent a viable future technique for inducing the osteogenic differentiation of MSCs.
Assuntos
Adipogenia , Tecido Adiposo/citologia , Células-Tronco Adultas/fisiologia , Proteínas Hedgehog/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Osteogênese , Adulto , Células-Tronco Adultas/enzimologia , Células-Tronco Adultas/metabolismo , Fosfatase Alcalina/metabolismo , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Proteínas de Ligação ao Cálcio , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Proteínas Hedgehog/farmacologia , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/farmacologia , Fenótipo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Transdução de Sinais , Receptor Smoothened , Alcaloides de Veratrum/farmacologiaRESUMO
We previously demonstrated that human pericytes, which encircle capillaries and microvessels, give rise in culture to genuine mesenchymal stem cells (MSCs). This raised the question as to whether all MSC are derived from pericytes. Pericytes and other cells defined on differential expression of CD34, CD31, and CD146 were sorted from the stromal vascular fraction of human white adipose tissue. Besides pericytes, CD34+ CD31- CD146- CD45- cells, which reside in the outmost layer of blood vessels, the tunica adventitia, natively expressed MSC markers and gave rise in culture to clonogenic multipotent progenitors identical to standard bone marrow-derived MSC. Despite common MSC features and developmental properties, adventitial cells and pericytes retain distinct phenotypes and genotypes through culture. However, in the presence of growth factors involved in vascular remodeling, adventitial cells acquire a pericytes-like phenotype. In conclusion, we demonstrate the co-existence of 2 separate perivascular MSC progenitors: pericytes in capillaries and microvessels and adventitial cells around larger vessels.
Assuntos
Artérias/citologia , Células-Tronco Mesenquimais/citologia , Veias/citologia , Tecido Adiposo/citologia , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Antígeno CD146/metabolismo , Separação Celular , Células Cultivadas , Células Clonais , Feminino , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Pericitos/citologia , Pericitos/efeitos dos fármacos , Pericitos/metabolismoRESUMO
The search for novel sources of stem cells other than bone marrow mesenchymal stem cells (MSCs) for bone regeneration and repair has been a critical endeavor. We previously established an effective protocol to homogeneously purify human pericytes from multiple fetal and adult tissues, including adipose, bone marrow, skeletal muscle, and pancreas, and identified pericytes as a primitive origin of human MSCs. In the present study, we further characterized the osteogenic potential of purified human pericytes combined with a novel osteoinductive growth factor, Nell-1. Purified pericytes grown on either standard culture ware or human cancellous bone chip (hCBC) scaffolds exhibited robust osteogenic differentiation in vitro. Using a nude mouse muscle pouch model, pericytes formed significant new bone in vivo as compared to scaffold alone (hCBC). Moreover, Nell-1 significantly increased pericyte osteogenic differentiation, both in vitro and in vivo. Interestingly, Nell-1 significantly induced pericyte proliferation and was observed to have pro-angiogenic effects, both in vitro and in vivo. These studies suggest that pericytes are a potential new cell source for future efforts in skeletal regenerative medicine, and that Nell-1 is a candidate growth factor able to induce pericyte osteogenic differentiation.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Osteogênese/efeitos dos fármacos , Pericitos/citologia , Pericitos/efeitos dos fármacos , Animais , Regeneração Óssea/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Proteínas de Ligação ao Cálcio , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Separação Celular , Feto/citologia , Membro Posterior/diagnóstico por imagem , Membro Posterior/efeitos dos fármacos , Membro Posterior/patologia , Membro Posterior/cirurgia , Humanos , Imuno-Histoquímica , Implantes Experimentais , Camundongos , Camundongos SCID , Neovascularização Fisiológica/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Pâncreas/embriologia , Pericitos/metabolismo , Pericitos/transplante , Implantação de Prótese , Alicerces Teciduais , Microtomografia por Raio-XRESUMO
Estrogens regulate osteoblast differentiation and mineralization. We identified GATA4 as a transcription factor expressed in osteoblasts and directly regulated by 17ß-estradiol in this cell type but not in breast cancer cells, another estrogen-responsive tissue. Chromatin immunoprecipitation sequencing (chromatin immunoprecipitation sequencing) reveals that estrogen receptor α (ERα) binds to chromatin near GATA4 at five different enhancers. GATA4 and ERα are both recruited to ERα binding sites near genes that are specifically expressed in osteoblasts and control osteoblast differentiation. Maximal binding of GATA4 precedes ERα binding, and GATA4 is necessary for histone 3 lysine 4 dimethylation at ERα binding sites, suggesting that GATA4 is a pioneer factor for ERα. As such, knockdown of GATA4 reduced recruitment of ERα to DNA. Our study illustrates that GATA4 is a pioneer factor for ERα recruitment to osteoblast-specific enhancers.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Osteoblastos/metabolismo , Transcrição Gênica , Fosfatase Alcalina/genética , Animais , Células da Medula Óssea/citologia , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Imunoprecipitação da Cromatina , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Elementos Facilitadores Genéticos , Receptor alfa de Estrogênio/genética , Estrogênios/farmacologia , Estrogênios/fisiologia , Proteína Ligante Fas/genética , Fêmur/citologia , Fêmur/metabolismo , Fator de Transcrição GATA4/genética , Estudo de Associação Genômica Ampla , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Ligação Proteica , Crânio/citologia , Crânio/metabolismo , Técnicas de Cultura de TecidosRESUMO
Independent studies by numerous investigators have shown that it is possible to harvest multipotent progenitor cells from diverse dissociated and cultured fetal, perinatal, and principally adult developed tissues. Despite the increasingly recognized medical value of these progenitor cells, the archetype of which remains the mesenchymal stem cell, this indirect extraction method has precluded the understanding of their native identity, tissue distribution, and frequency. Consistent with other researchers, we have hypothesized that blood vessels in virtually all organs harbor ubiquitous stem cells. We have identified, marked, and sorted to homogeneity by flow cytometry endothelial and perivascular cells in a large selection of human fetal, perinatal, and adult organs. Perivascular cells, including pericytes in the smallest blood vessels and adventitial cells around larger ones, natively express mesenchymal stem cell markers and produce in culture a long-lasting progeny of multilineage mesodermal progenitor cells. Herein, we review results from our and other laboratories that suggest a perivascular origin for mesenchymal stem cells and other adult progenitor cells. Recent experiments illustrate the therapeutic potential of human pericytes to regenerate skeletal muscle and promote functional recovery in the diseased heart and kidney.
Assuntos
Células-Tronco Adultas/fisiologia , Diferenciação Celular , Linhagem da Célula , Células-Tronco Mesenquimais/fisiologia , Células-Tronco Multipotentes/fisiologia , Pericitos/fisiologia , Células Estromais/fisiologia , Adulto , Células-Tronco Adultas/transplante , Animais , Biomarcadores/metabolismo , Proliferação de Células , Células-Tronco Fetais/fisiologia , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Multipotentes/transplante , Neovascularização Fisiológica , RegeneraçãoRESUMO
Two putative types of circulating endothelial progenitor cells have been recently identified in vitro: (1) endothelial colony-forming cell (ECFC) and (2) colony-forming unit-endothelial cell (CFU-EC). Only the former is now recognized to belong to endothelial lineage. We have used the ECFC and CFU-EC assays to readdress the issue of the clonal relation between endothelial progenitor cells and hematopoietic stem cells in patients with Philadelphia-positive and Philadelphia-negative chronic myeloproliferative disorders. Both ECFCs and CFU-ECs were cultured from peripheral blood mononuclear cells, and either BCR-ABL rearrangement or JAK2-V617F mutation were assessed in both types of endothelial colonies. We found that ECFCs lack the disease-specific markers, which are otherwise present in CFU-ECs, thus reinforcing the concept that the latter belongs to the hematopoietic lineage, and showing that in chronic myeloproliferative disorders the cell that gives rise to circulating ECFC has a distinct origin from the cell of the hematopoietic malignant clone.