RESUMO
Proteins of the CLCA gene family including the human ClCa1 (hClCa1) have been suggested to constitute a new family of chloride channels mediating Ca(2+)-dependent Cl- currents. The present study examines the relationship between the hClCa1 protein and Ca(2+)-dependent Cl- currents using heterologous expression of hClCa1 in HEK293 and NCIH522 cell lines and whole cell recordings. By contrast to previous reports claiming the absence of Cl- currents in HEK293 cells, we find that HEK293 and NCIH522 cell lines express constitutive Ca(2+)-dependent Cl- currents and show that hClCa1 increases the amplitude of Ca(2+)-dependent Cl- currents in those cells. We further show that hClCa1 does not modify the permeability sequence but increases the Cl- conductance while decreasing the G(SCN-)/G(Cl-) conductance ratio from approximately 2-3 to approximately 1. We use an Eyring rate theory (two barriers, one site channel) model and show that the effect of hClCa1 on the anionic channel can be simulated by its action on lowering the first and the second energy barriers. We conclude that hClCa1 does not form Ca(2+)-dependent Cl- channels per se or enhance the trafficking/insertion of constitutive channels in the HEK293 and NCIH522 expression systems. Rather, hClCa1 elevates the single channel conductance of endogenous Ca(2+)-dependent Cl- channels by lowering the energy barriers for ion translocation through the pore.
Assuntos
Canais de Cloreto/fisiologia , Fenômenos Eletrofisiológicos/fisiologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Cálcio/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Canais de Cloreto/antagonistas & inibidores , Cloretos/metabolismo , Estimulação Elétrica , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Expressão Gênica/genética , Gluconatos/farmacologia , Humanos , Potenciais da Membrana/fisiologia , Modelos Moleculares , Ácido Niflúmico/farmacologia , Permeabilidade , Termodinâmica , Tiocianatos/farmacologia , TransfecçãoRESUMO
Planar array electrophysiology techniques were applied to assays for modulators of recombinant hIK and hSK3 Ca2+-activated K+ channels. In CHO-hIK-expressing cells, under asymmetric K+ gradients, small-molecule channel activators evoked time- and voltage-independent currents characteristic of those previously described by classical patch clamp electrophysiology methods. In single-hole (cell) experiments, the large cell-to-cell heterogeneity in channel expression rendered it difficult to generate activator concentration-response curves. However, in population patch clamp mode, in which signals are averaged from up to 64 cells, well-to-well variation was substantially reduced such that concentration-response curves could be easily constructed. The absolute EC50 values and rank order of potency for a range of activators, including 1-EBIO and DC-EBIO, corresponded well with conventional patch clamp data. Activator responses of hIK and hSK3 channels could be fully and specifically blocked by the selective inhibitors TRAM-34 and apamin, with IC50 values of 0.31 microM and 3 nM, respectively. To demonstrate assay precision and robustness, a test set of 704 compounds was screened in a 384-well format of the hIK assay. All plates had Z' values greater than 0.6, and the statistical cutoff for activity was 8%. Eleven hits (1.6%) were identified from this set, in addition to the randomly spiked wells with known activators. Overall, our findings demonstrate that population patch clamp is a powerful and enabling method for screening Ca2+-activated K+ channels and provides significant advantages over single-cell electrophysiology (IonWorks(HT)) and other previously published approaches. Moreover, this work demonstrates for the 1st time the utility of population patch clamp for ion channel activator assays and for non-voltage-gated ion channels.
Assuntos
Eletrofisiologia/métodos , Técnicas de Patch-Clamp/métodos , Canais de Potássio Cálcio-Ativados/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Humanos , Moduladores de Transporte de Membrana/farmacologia , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Reprodutibilidade dos TestesRESUMO
Patients with laryngeal disorders may have severe morbidity relating to swallowing, vocalization, and respiratory function, for which conventional therapies are suboptimal. A tissue-engineered approach would aim to restore the vocal folds and maintain respiratory function while limiting the extent of scarring in the regenerated tissue. Under Good Laboratory Practice conditions, we decellularized porcine larynges, using detergents and enzymes under negative pressure to produce an acellular scaffold comprising cartilage, muscle, and mucosa. To assess safety and functionality before clinical trials, a decellularized hemilarynx seeded with human bone marrow-derived mesenchymal stem cells and a tissue-engineered oral mucosal sheet was implanted orthotopically into six pigs. The seeded grafts were left in situ for 6 months and assessed using computed tomography imaging, bronchoscopy, and mucosal brushings, together with vocal recording and histological analysis on explantation. The graft caused no adverse respiratory function, nor did it impact swallowing or vocalization. Rudimentary vocal folds covered by contiguous epithelium were easily identifiable. In conclusion, the proposed tissue-engineered approach represents a viable alternative treatment for laryngeal defects. Stem Cells Translational Medicine 2017;6:677-687.
Assuntos
Laringe/transplante , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Regeneração , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Fenômenos Biomecânicos , Broncoscopia , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Laringe/diagnóstico por imagem , Laringe/patologia , Laringe/fisiopatologia , Fonação , Recuperação de Função Fisiológica , Sus scrofa , Fatores de Tempo , Tomografia Computadorizada por Raios X , Vocalização AnimalRESUMO
Patients with large tracheal lesions unsuitable for conventional endoscopic or open operations may require a tracheal replacement but there is no present consensus of how this may be achieved. Tissue engineering using decellularized or synthetic tracheal scaffolds offers a new avenue for airway reconstruction. Decellularized human donor tracheal scaffolds have been applied in compassionate-use clinical cases but naturally derived extracellular matrix (ECM) scaffolds demand lengthy preparation times. Here, we compare a clinically applied detergent-enzymatic method (DEM) with an accelerated vacuum-assisted decellularization (VAD) protocol. We examined the histological appearance, DNA content and extracellular matrix composition of human donor tracheae decellularized using these techniques. Further, we performed scanning electron microscopy (SEM) and biomechanical testing to analyze decellularization performance. To assess the biocompatibility of scaffolds generated using VAD, we seeded scaffolds with primary human airway epithelial cells in vitro and performed in vivo chick chorioallantoic membrane (CAM) and subcutaneous implantation assays. Both DEM and VAD protocols produced well-decellularized tracheal scaffolds with no adverse mechanical effects and scaffolds retained the capacity for in vitro and in vivo cellular integration. We conclude that the substantial reduction in time required to produce scaffolds using VAD compared to DEM (approximately 9 days vs. 3-8 weeks) does not compromise the quality of human tracheal scaffold generated. These findings might inform clinical decellularization techniques as VAD offers accelerated scaffold production and reduces the associated costs.
Assuntos
Sistema Livre de Células/química , Matriz Extracelular/química , Matriz Extracelular/ultraestrutura , Engenharia Tecidual/instrumentação , Alicerces Teciduais , Traqueia/citologia , Traqueia/crescimento & desenvolvimento , Fracionamento Celular/instrumentação , Fracionamento Celular/métodos , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Engenharia Tecidual/métodos , Traqueia/ultraestrutura , VácuoRESUMO
Tracheal replacement for the treatment of end-stage airway disease remains an elusive goal. The use of tissue-engineered tracheae in compassionate use cases suggests that such an approach is a viable option. Here, a stem cell-seeded, decellularized tissue-engineered tracheal graft was used on a compassionate basis for a girl with critical tracheal stenosis after conventional reconstructive techniques failed. The graft represents the first cell-seeded tracheal graft manufactured to full good manufacturing practice (GMP) standards. We report important preclinical and clinical data from the case, which ended in the death of the recipient. Early results were encouraging, but an acute event, hypothesized to be an intrathoracic bleed, caused sudden airway obstruction 3 weeks post-transplantation, resulting in her death. We detail the clinical events and identify areas of priority to improve future grafts. In particular, we advocate the use of stents during the first few months post-implantation. The negative outcome of this case highlights the inherent difficulties in clinical translation where preclinical in vivo models cannot replicate complex clinical scenarios that are encountered. The practical difficulties in delivering GMP grafts underscore the need to refine protocols for phase I clinical trials. Stem Cells Translational Medicine 2017;6:1458-1464.
Assuntos
Órgãos Bioartificiais/efeitos adversos , Transplante de Órgãos/métodos , Complicações Pós-Operatórias/etiologia , Engenharia Tecidual/métodos , Traqueia/transplante , Estenose Traqueal/cirurgia , Adolescente , Células Cultivadas , Feminino , Humanos , Transplante de Órgãos/efeitos adversos , Transplante de Órgãos/instrumentação , Células-Tronco/citologia , Alicerces Teciduais/normasRESUMO
Stem cell-based tissue-engineered tracheas are at an early stage in their product development cycle. Tens of patients have been treated worldwide in predominantly compassionate use settings, demonstrating significant promise. This potentially life-saving treatment is complex, and the cost and its implications for such treatments are yet to be fully understood. The costs are compounded by varying strategies for graft preparation and transplant, resulting in differing clinical and laboratory costs from different research groups. In this study, we present a detailed breakdown of the clinical and manufacturing costs for three of the United Kingdom (UK) patients treated with such transplants. All three patients were treated under Compassionate Use legislation, within the UK National Health Service (NHS) hospital setting. The total costs for the three UK patients treated ranged from $174,420 to $740,500. All three patients were in a state of poor health at time of treatment and had a number of complexities in addition to the restricted airway. This is the first time a cost analysis has been made for a tissue-engineered organ and provides a benchmark for future studies, as well as comparative data for use in reimbursement considerations.