RESUMO
Stage-specific embryonic antigen-4 (SSEA-4) is a glycosphingolipid, which is overexpressed in some cancers and has been linked to disease progression. However, little is known about the functions of SSEA-4 and the characteristics of SSEA-4 expressing tumor cells. Our studies identified SSEA-4 expression on a subpopulation of cells in many solid tumor cell lines but not in leukemic cell lines. Fluorescence-activated cell sorting-sorted SSEA-4(+) prostate cancer cells formed fibroblast-like colonies with limited cell-cell contacts, whereas SSEA-4(-) cells formed cobblestone-like epithelial colonies. Only colonies derived from SSEA-4(+) cells were enriched for pluripotent embryonic stem cell markers. Moreover, major epithelial cell-associated markers Claudin-7, E-cadherin, ESRP1 and GRHL2 were down-regulated in the SSEA-4(+) fraction of DU145 and HCT-116 cells. Similar to cell lines, SSEA-4(+) primary prostate tumor cells also showed down-regulation of epithelial cell-associated markers. In addition, they showed up-regulation of epithelial-to-mesenchymal transition as well as mesenchymal markers. Furthermore, SSEA-4(+) cells escape from adhesive colonies spontaneously and form invadopodia-like migratory structures, in which SSEA-4, cortactin as well as active pPI3K, pAkt and pSrc are enriched and colocalized. Finally, SSEA-4(+) cells displayed strong tumorigenic ability and stable knockdown of SSEA-4 synthesis resulted in decreased cellular adhesion to different extracellular matrices. In conclusion, we introduce SSEA-4 as a novel marker to identify heterogeneous, invasive subpopulations of tumor cells. Moreover, increased cell-surface SSEA-4 expression is associated with the loss of cell-cell interactions and the gain of a migratory phenotype, suggesting an important role of SSEA-4 in cancer invasion by influencing cellular adhesion to the extracellular matrix.
Assuntos
Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Próstata/metabolismo , Antígenos Embrionários Estágio-Específicos/genética , Caderinas/genética , Caderinas/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Claudinas/genética , Claudinas/metabolismo , Cortactina/genética , Cortactina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/patologia , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Humanos , Masculino , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Antígenos Embrionários Estágio-Específicos/química , Antígenos Embrionários Estágio-Específicos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Nerves and blood vessels are present in most organs and are indispensable for their function and homeostasis. Within these organs, neurovascular (NV) tissue forms congruent patterns and establishes vital interactions. Several human pathologies, including diabetes type II, produce NV disruptions with serious consequences that are complicated to study using animal models. Complex in vitro organ platforms, with neural and vascular supply, allow the investigation of such interactions, whether in a normal or pathological context, in an affordable, simple, and direct manner. To date, a few in vitro models contain NV tissue, and most strategies report models with nonbiomimetic representations of the native environment. To this end, we have established here an NV platform that contains mature vasculature and neural tissue, composed of human microvascular endothelial cells (HMVECs), induced pluripotent stem cell (iPSCs)-derived sensory neurons, and primary rat Schwann cells (SCs) within a fibrin-embedded polymeric scaffold. First, we show that SCs can induce the formation of and stabilize vascular networks to the same degree as the traditional and more thoroughly studied human dermal fibroblasts (HDFs). We also show that through SC prepatterning, we are able to control vessel orientation. Using our NV platform, we demonstrate the concomitant formation of three-dimensional neural and vascular tissue, and the influence of different medium formulations and cell types on the NV tissue outcome. Finally, we propose a protocol to form mature NV tissue, via the integration of independent neural and vascular constituents. The platform described here provides a versatile and advanced model for in vitro research of the NV axis.
Assuntos
Células-Tronco Pluripotentes Induzidas , Tecido Nervoso , Animais , Biomimética , Células Endoteliais/metabolismo , Humanos , Ratos , Células de Schwann/metabolismoRESUMO
Functional humanizedin vitronerve models are coveted as an alternative to animal models due to their ease of access, lower cost, clinical relevance and no need for recurrent animal sacrifice. To this end, we developed a sensory nerve model using induced pluripotent stem cells-derived nociceptors that are electrically active and exhibit a functional response to noxious stimuli. The differentiated neurons were co-cultured with primary Schwann cells on an aligned microfibrous scaffold to produce biomimetic peripheral nerve tissue. Compared to glass coverslips, our scaffold enhances tissue development and stabilization. Using this model, we demonstrate that myelin damage can be induced from hyperglycemia exposure (glucose at 45 mM) and mitigated by epalrestat (1µM) supplementation. Through fibrin embedding of the platform, we were able to create 3D anisotropic myelinated tissue, reaching over 6.5 mm in length. Finally, as a proof-of-concept, we incorporated pancreatic pseudoislets and endometrial organoids into our nerve platform, to demonstrate the potential in generating nociceptor innervation models. In summary, we propose here an improved tool for neurobiology research with potential applications in pathology modeling, drug screening and target tissue innervation.
Assuntos
Células-Tronco Pluripotentes Induzidas , Nociceptores , Animais , Diferenciação Celular , Humanos , Bainha de Mielina , Nociceptores/fisiologia , Nervos Periféricos , Células de SchwannRESUMO
The use of hydrogels derived from the extracellular matrix (ECM) in tissue engineering applications aims to overcome the conundrum of mimicking the complexity of ECM composition in vitro. In this chapter, we describe a method of decellularization and subsequent formation of an ECM-based hydrogel using porcine heart tissue. These decellularized ECM hydrogels could be used to create semi-interpenetrating networks or as bioinks in bioprinting applications to further enhance the bioactivity and increase the biomimicry degree of the biological cardiac constructs.
Assuntos
Matriz Extracelular , Ventrículos do Coração/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Materiais Biomiméticos , Humanos , Hidrogéis , Infarto do Miocárdio/terapia , SuínosAssuntos
Anticorpos Anti-Idiotípicos/imunologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Animais , Reações Cruzadas , Cães , Citometria de Fluxo , Humanos , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/terapia , Dor Lombar/patologia , Dor Lombar/terapia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologiaRESUMO
BACKGROUND: Connective tissue progenitors (CTPs) from native bone marrow (BM) or their culture-expanded progeny, often referred to as mesenchymal stem/stromal cells, represents a promising strategy for treatment of cartilage injuries. But the cartilage regeneration capacity of these cells remains unpredictable because of cell heterogeneity. HYPOTHESIS: The harvest technique of BM may highly influence stem cell heterogeneity and, thus, cartilage formation because these cells have distinct spatial localization within BM from the same bone. STUDY DESIGN: Controlled laboratory study. METHODS: CTPs obtained from the femur of patients undergoing total hip replacement by 2 harvest techniques-BM aspiration and BM collection-after bone rasping were immunophenotyped by flow cytometry and evaluated for chondrogenic ability. The spatial localization of different CTP subsets in BM was verified by immunohistochemistry. RESULTS: Cells from the BM after rasping were significantly more chondrogenic than the donor-matched aspirate, whereas no notable difference in their osteogenic or adipogenic potential was observed. The authors then assessed whether distinct immunophenotypically defined CTP subsets were responsible for the different chondrogenic capacity. Cells directly isolated from BM after rasping contained a higher percentage (mean, 7.2-fold) of CD45-CD271+CD56+ CTPs as compared with BM aspirates. The presence of this subset in the harvested BM strongly correlated with chondrogenic ability, showing that CD271+CD56+ cells are enriched in chondroprogenitors. Furthermore, evaluation of these CTP subsets in BM revealed that CD271+CD56+ cells were localized in the bone-lining regions whereas CD271+CD56- cells were found in the perivascular regions. Since the iliac crest remains a frequent site of BM harvest for musculoskeletal regeneration, the authors also compared the spatial distribution of these subsets in trabeculae of femoral head and iliac crest and found CD271+CD56+ bone-lining cells in both tissues. CONCLUSION: Chondrogenically distinct CTP subsets have distinct spatial localization in BM; hence, the harvest technique of BM determines the efficiency of cartilage formation. CLINICAL RELEVANCE: The harvest technique of BM may be of major importance in determining the clinical success of BM mesenchymal stem/stromal cells in cartilage repair.
Assuntos
Cartilagem/fisiologia , Regeneração/fisiologia , Coleta de Tecidos e Órgãos/métodos , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/fisiologia , Células da Medula Óssea/fisiologia , Transplante de Medula Óssea , Cartilagem/lesões , Doenças das Cartilagens , Contagem de Células , Diferenciação Celular , Condrogênese/fisiologia , Feminino , Citometria de Fluxo , Humanos , Ílio/cirurgia , Antígenos Comuns de Leucócito/fisiologia , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Osteogênese/fisiologia , Receptores de Fator de Crescimento Neural/metabolismo , Células-Tronco/fisiologiaRESUMO
In-depth understanding of human spermatogenesis requires studying specific molecular signatures and interactions of spermatogonia with other testicular cell populations, for which isolation of pure populations of different cell types is crucial. Here, we describe a technique to simultaneously enrich pure, multiple testicular cell populations, including spermatogonia, endothelial (TECs), and perivascular mesenchymal stem/stromal cells (TMSCs), from testicular tissue by flow cytometry using a combination of defined markers. Immunohistochemical studies, multicolor staining, and cell sorting followed by multiplex quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that spermatogonia were highly enriched in the CD49f+CD49a-HLA-ABC-SSEA-4+ fraction of primary testicular cells. In contrast to spermatogonia, TMSCs and TECs were highly enriched in the CD49f+CD49a+HLA-ABC+CD144- and CD49f+CD49a+HLA-ABC+CD144+subsets, respectively. The delineation was confirmed by the expression of specific stromal and endothelial key markers as well as by the differentiation and angiogenic capacity of the sorted populations. In this article, for the first time, we performed transcriptome profiling of highly enriched, freshly isolated human spermatogonia and compared their expression profile with that of TMSCs. Our RNA sequencing data favor the hypothesis that TMSCs are candidate niche components for spermatogonia. The composite genotype and phenotype of defined testicular cell populations combined with a robust isolation procedure from small biopsies contributes to a better understanding of cellular interactions and for the establishment of efficient culture techniques to maintain spermatogonial progenitors.
Assuntos
Compartimento Celular , Perfilação da Expressão Gênica , Espermatogônias/citologia , Espermatogônias/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Adulto , Idoso , Antígenos CD/metabolismo , Diferenciação Celular , Separação Celular , Análise por Conglomerados , Células Endoteliais/citologia , Regulação da Expressão Gênica , Genoma Humano , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Fisiológica , Fenótipo , Antígenos Embrionários Estágio-Específicos/metabolismo , Células Estromais/metabolismo , Testículo/citologiaRESUMO
Several strategies have been developed to facilitate the prospective isolation of bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) based on the selective expression or absence of surface markers. Recently, we described the monoclonal antibodies W3D5 and W5C5, which selectively react with BM-MSCs, but not with hematopoietic cells. Both antibodies showed an identical reactivity pattern, indicating that they may recognize the same molecule. To identify the cognate antigen, cultured MSCs were sorted for cells expressing either very high levels of W5C5/W3D5 antigen or for cells which were negative for this antigen. Further processing of these cells for microarray analysis revealed a 20-fold enrichment of the type 1 integral membrane protein Sushi domain containing 2 (SUSD2) in the in W5C5(+) subset. To confirm the identity of the W5C5/W3D5 antigen to SUSD2, HEK293 cells were transfected with the full-length coding sequence of human SUSD2 followed by reactivity analysis of W5C5 and W3D5 antibodies with the transfected line. Flow cytometric analysis showed that both antibodies selectively recognized HEK293/huSUSD2 cells, but not the parental cell line. In line with this, SUSD2 siRNA treatment of SUSD2(+) WERI-RB-1 retinoblastoma cells reduced the expression levels of W3D5 and W5C5 antigens to ~39% and 37%, respectively. Finally, FACSorting and colony assays revealed that only SUSD2(+), but not SUSD2(-) BM cells give rise to colony-forming units-fibroblasts and are able to differentiate into osteoblasts, adipocytes, and chondrocytes. In conclusion, we identified SUSD2 as a novel and specific marker for the prospective isolation of BM-MSCs.
Assuntos
Anticorpos Monoclonais , Células da Medula Óssea/citologia , Glicoproteínas de Membrana/imunologia , Células-Tronco Mesenquimais/citologia , Adipócitos/citologia , Células da Medula Óssea/imunologia , Diferenciação Celular , Linhagem da Célula , Condrócitos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Células-Tronco Hematopoéticas/imunologia , Humanos , Glicoproteínas de Membrana/biossíntese , Células-Tronco Mesenquimais/imunologia , Osteoblastos/citologiaRESUMO
Bone marrow-derived mesenchymal stromal/stem cells (MSCs) are nonhematopoietic cells that are able to differentiate into osteoblasts, adipocytes, and chondrocytes. In addition, they are known to participate in niche formation for hematopoietic stem cells and to display immunomodulatory properties. Conventionally, these cells are functionally isolated from tissue based on their capacity to adhere to the surface of culture flasks. This isolation procedure is hampered by the unpredictable influence of secreted molecules, the interactions between cocultured hematopoietic and other unrelated cells, and by the arbitrarily selected removal time of nonadherent cells before the expansion of MSCs. Finally, functionally isolated cells do not provide biological information about the starting population. To circumvent these limitations, several strategies have been developed to facilitate the prospective isolation of MSCs based on the selective expression, or absence, of surface markers. In this report, we summarize the most frequently used markers and introduce new targets for antibody-based isolation procedures of primary bone marrow- and amnion-derived MSCs.
Assuntos
Âmnio/citologia , Células da Medula Óssea/classificação , Células-Tronco Mesenquimais/classificação , Âmnio/metabolismo , Anticorpos Monoclonais , Antígenos CD/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Diferenciação Celular , Separação Celular , Ensaio de Unidades Formadoras de Colônias , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Fenótipo , Gravidez , Nicho de Células-TroncoRESUMO
Conventionally, mesenchymal/stromal stem cells (MSC) are functionally isolated from primary tissue based on their capacity to adhere to the plastic surface. This isolation procedure is hampered by the unpredictable influence of secreted molecules or interactions with co-cultured hematopoietic and other unrelated cells as well as by the arbitrarily selected removal time of non-adherent cells prior to expansion of MSC. Early removal of non-adherent cells may result in the elimination of a late adhering MSC subsets and late removal increases the influence of undesired cells on the growth and differentiation of MSC. Finally, in conventional protocols MSC are co-expanded together with macrophages, endothelial cells and other adherent cells. To circumvent these limitations, several strategies have been developed to facilitate the prospective isolation of MSC based on the selective expression or absence of surface markers. Here we summarize the most frequently used markers and introduce new targets for antibody-based isolation procedures of primary bone marrow-derived MSC.