RESUMEN
Isolation of highly pure specific cell types is crucial for successful adult stem cell-based therapy. As the number of such cells in adult tissue is low, an extremely efficient method is needed for their isolation. Here, we describe cell-separation methodologies based on magnetic-affinity cell sorting (MACS) MicroBeads with monoclonal antibodies against specific membrane proteins conjugated to superparamagnetic particles. Cells labeled with MACS MicroBeads are retained in a magnetic field within a MACS column placed in a MACS separator, allowing fast and efficient separation. Both positively labeled and non-labeled fractions can be used directly for downstream applications as the separated cell fractions remain viable with no functional impairment. As immunomagnetic separation depends on the interaction between a cell's membrane and the magnetically labeled antibody, separation of specific cells originating from solid tissues is more complex and demands a cell-dissociating pretreatment. In this paper, we detail the use of immunomagnetic separation for the purpose of regenerating damaged salivary gland (SG) function in animal and human models of irradiated head and neck cancer. Each year 500,000 new cases of head and neck cancer occur worldwide. Most of these patients lose SG function following irradiation therapy. SGs contain integrin α6ß1-expressing epithelial stem cells. We hypothesized that these cells can be isolated, multiplied in culture and auto-implanted into the irradiated SGs to regenerate damaged SG function.
Asunto(s)
Células Madre Adultas/química , Separación Inmunomagnética/métodos , Integrina alfa6beta1/química , Marcadores de Afinidad/química , Animales , Anticuerpos Monoclonales/química , Membrana Celular/química , Supervivencia Celular , Citometría de Flujo , Neoplasias de Cabeza y Cuello/química , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Glándulas Salivales/química , Glándulas Salivales/patología , Sensibilidad y EspecificidadRESUMEN
INTRODUCTION: Cardiac troponin (cTn) is the biomarker of choice for detection of myocardial injury. There is a great need for simple point-of-care (POC) troponin testing among patients with chest pain, mainly in the prehospital setting. The purpose of this study was to evaluate the presence of cardiac troponin I (cTnI) in saliva of patients with myocardial injury using alpha-amylase depletion technique. METHODS: Saliva samples were collected from 40 patients with myocardial injury who were tested positive for conventional high-sensitivity cardiac troponin T (cTnT) blood tests, and from 66 healthy volunteers. Saliva samples were treated for the removal of salivary alpha-amylase. Treated and untreated samples were tested with blood cTnI Rapid Diagnostic Test. Salivary cTnI levels were compared to blood cTnT levels. RESULTS: Thirty-six of 40 patients with positive blood cTnT had positive salivary samples for cTnI following alpha-amylase depletion treatment (90.00% sensitivity). Moreover, three of the four negative saliva samples were obtained from patients with relatively low blood cTnT levels of 100 ng/L or less (96.88% sensitivity for 100 ng/L and above). The negative predictive value was 93.65% and rose up to 98.33% considering the 100 ng/L cutoff. Positive predictive values were 83.72% and 81.58%, respectively. Among 66 healthy volunteers and 7 samples yielded positive results (89.39% specificity). CONCLUSION: In this preliminary work, the presence of cTnI in saliva was demonstrated for the first time to be feasibly identified by a POC oriented assay. The specific salivary alpha-amylase depletion technique was shown to be crucial for the suggested assay.
Asunto(s)
alfa-Amilasas Salivales , Troponina I , Humanos , Estudios de Factibilidad , Saliva , Troponina T , Biomarcadores , Pruebas en el Punto de AtenciónRESUMEN
Impaired salivary gland (SG) function leading to oral diseases is relatively common with no adequate solution. Previously, tissue engineering of SG had been proposed to overcome this morbidity, however, not yet clinically available. Multiwall inorganic (tungsten disulfide [WS2]) nanotubes (INT-WS2) and fullerene-like nanoparticles (IF-WS2) have many potential medical applications. A yet unexplored venue application is their interaction with SG, and therefore, our aim was to test the biocompatibility of INT/IF-WS2 with the A5 and rat submandibular cells (RSC) SG cells. The cells were cultured and subjected after 1 day to different concentrations of INT-WS2 and were compared to control groups. Growth curves, trypan blue viability test, and carboxyfluorescein succinimidyl ester (CFSE) proliferation assay were obtained. Furthermore, cells morphology and interaction with the nanoparticles were observed by light microscopy, scanning electron microscopy and transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy. The results showed no significant differences in growth curves, proliferation kinetics, and viability between the groups compared. Moreover, no alterations were observed in the cell morphology. Interestingly, TEM images indicated that the nanoparticles are uptaken by the cells and accumulate in cytoplasmic vesicles. These results suggest promising future medical applications for these nanoparticles.
Asunto(s)
Materiales Biocompatibles/farmacología , Fulerenos/farmacología , Ensayo de Materiales/métodos , Nanopartículas/química , Nanotubos/química , Glándula Submandibular/citología , Sulfuros/farmacología , Compuestos de Tungsteno/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Nanopartículas/ultraestructura , Nanotubos/ultraestructura , Ratas , Glándula Submandibular/efectos de los fármacos , Glándula Submandibular/ultraestructuraRESUMEN
Head and neck cancer is the sixth most common cancer worldwide, resulting in ~ 640,000 cases. Most of these patients have irreversible damage to their salivary glands due to irradiation therapy, which typically leads to significant decrease in quality of life. In the last 2 decades, several strategies have been suggested to overcome this problem; however, no biologically based treatments are available. In the past few years, the authors of the present article and other researchers have focused on a new strategy of re-implantation of autologous salivary gland cells into the residual irradiated salivary glands. This article reviews the current prospective of the irradiation-induced salivary gland impairment mechanisms and the envisioned therapeutic modalities based on stem cell therapy.
Asunto(s)
Regeneración/fisiología , Glándulas Salivales/fisiología , Células Madre/fisiología , Autoinjertos/trasplante , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , Traumatismos por Radiación/etiología , Enfermedades de las Glándulas Salivales/etiología , Glándulas Salivales/efectos de la radiación , Trasplante de Células Madre/métodosRESUMEN
Irradiated head and neck cancer patients suffer from irreversible loss of salivary gland (SG) function, along with significant morbidity and compromised quality of life. To date there is no biologically-based treatment for this distress. Adult salivary gland stem cells are promising candidates for autologous transplantation therapy in the context of tissue-engineered artificial SGs or direct cell therapy. The major restrictions in handling such cells are their limited lifespan during in vitro cultivation, resulting in a narrow time-window for implantation and a risk of tumorigenic changes during culture. To overcome these difficulties, we tested in a rat model the possibility of establishing a personal/autologous SG stem cell bank. SG's integrin-α6ß1-expressing cells were shown to hold a subpopulation of SG-specific progenitor-cells. Explanted and cultured single cell-originated clones were cryopreserved for up to 3 years and shown to exhibit genetic and functional stability similar to noncryopreserved cells, as was emphasized by soft agar assay, division potential assessment, flow cytometric analysis, real-time reverse transcriptase-polymerase chain reaction, in vitro three-dimensional differentiation assay, and immunofluorescence confocal microscopy. Future integration of the novel strategies presented herein to a clinical therapeutic model will allow safe preservation until transplantation and repeated transplantation if needed. These tools open a new venue for adult autologous stem-cell transplantation-based SG regeneration.
Asunto(s)
Trasplante de Células/métodos , Neoplasias de Cabeza y Cuello/radioterapia , Glándulas Salivales/citología , Células Madre/citología , Células 3T3 , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Criopreservación , Neoplasias de Cabeza y Cuello/terapia , Humanos , Integrina alfa6beta1/biosíntesis , Masculino , Ratones , Microscopía Confocal/métodos , Reacción en Cadena de la Polimerasa/métodos , Ratas , Regeneración , Ingeniería de Tejidos/métodosRESUMEN
Salivary glands (SGs) are irreversibly damaged by irradiation (IR) treatment in head and neck cancer patients. Here, we used an animal irradiation model to investigate and define the molecular mechanisms affecting SGs following IR, focusing on saliva proteome and global transcription profile of submandibular salivary gland (SSG) tissue.We show that saliva secretion was gradually reduced to 50% of its initial level 12 weeks post-IR. Saliva protein composition was further examined by proteomic analysis following mass spectrometry (MS) analysis that revealed proteins with reduced expression originating from SSGs and proteins with increased expression derived from the serum, both indicating salivary tissue damage. To examine alterations in mRNA expression levels microarray analysis was performed. We found significant alterations in 95 genes, including cell-cycle arrest genes, SG functional genes and a DNA repair gene.Tissue damage was seen by confocal immunofluorescence of α-amylase and c-Kit that showed an increase and decrease, respectively, in protein expression. This was coherent with real-time PCR results.This data indicates that IR damages the SSG cells' ability to produce and secrete saliva and proteins, and maintain the physiological barrier between serum and saliva. The damage does not heal due to cell-cycle arrest, which prevents tissue regeneration. Taken together, our results reveal a new insight into IR pathobiology.
Asunto(s)
Proteoma/efectos de la radiación , Glándula Submandibular/metabolismo , Glándula Submandibular/efectos de la radiación , Transcriptoma/efectos de la radiación , Animales , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de la radiación , Ratas , Ratas Sprague-Dawley , Saliva/metabolismo , Factores de TiempoRESUMEN
Adult salivary gland stem cells are promising candidates for cell therapy and tissue regeneration in cases of irreversible damage to salivary glands in head and neck cancer patients undergoing irradiation therapy. At present, the major restriction in handling such cells is their relatively limited life span during in vitro cultivation, resulting in an inadequate experimental platform to explore the salivary gland-originated stem cells as candidates for future clinical application in therapy. We established a spontaneous immortal integrin α6ß1-expressing cell line of adult salivary progenitor cells from rats (rat salivary clone [RSC]) and investigated their ability to sustain cellular properties. This line was able to propagate for more than 400 doublings without loss of differentiation potential. RSC could differentiate in vitro to both acinar- and ductal-like structures and could be further manipulated upon culturing on a 3D scaffolds with different media supplements. Moreover, RSC expressed salivary-specific mRNAs and proteins as well as epithelial stem cell markers, and upon differentiation process their expression was changed. These results suggest RSC as a good model for further studies exploring cellular senescence, differentiation, and in vitro tissue engineering features as a crucial step toward reengineering irradiation-impaired salivary glands.