RESUMEN
The limited efficacy of cardiac cell-based therapy is thought to be due to poor cell retention within the myocardium. Hence, there is an urgent need for biomaterials that aid in long-term cell retention. This study describes the development of injectable microcapsules for the delivery of mesenchymal stem cells (MSCs) into the infarcted cardiac wall. These microcapsules comprise of low concentrations of agarose supplemented with extracellular matrix (ECM) proteins collagen and fibrin. Dextran sulfate, a negatively charged polycarbohydrate, was added to mimic glycosaminoglycans in the ECM. Cell viability assays showed that a combination of all components is necessary to support long-term survival and proliferation of MSCs within microcapsules. Following intramyocardial transplantation, microcapsules degraded slowly in vivo and did not induce a fibrotic foreign body response. Pre-labeling of encapsulated MSCs with iron oxide nanoparticles allowed continued cell-tracking by MRI over several weeks following transplantation into infarcted myocardium. In contrast, MSCs injected as cell suspension were only detectable for two days post transplantation by MRI. Histological analysis confirmed integration of transplanted cells at the infarct site. Therefore, microcapsules proved to be suitable for stem cell delivery into the infarcted myocardium and can overcome current limitations of poor cell retention in cardiac cell-based therapy.
Asunto(s)
Cápsulas , Proliferación Celular , Supervivencia Celular , Células Madre Mesenquimatosas/citología , Infarto del Miocardio/patología , Animales , Masculino , Ratas , Ratas WistarRESUMEN
The competition for access to space that arises between macromolecules is the basis of the macromolecular crowding phenomenon, known to modulate biochemical reactions in subtle ways. Crowding is a highly conserved physiological condition in and around cells in metazoans, and originates from a mixture of heterogeneous biomolecules. Here, using collagen fibrillogenesis as an experimental test platform and ideas from the theory of nonideal solutions, we show that an entropy-based synergy is created by a mixture of two different populations of artificial crowders, providing small crowders with extra volume occupancy when in the vicinity of bigger crowders. We present the physiological mechanism by which synergistic effects maximize volume exclusion with the minimum amount of heterogeneous crowders, demonstrating how the evolutionarily optimized crowded conditions found in vivo can be reproduced effectively in vitro.
Asunto(s)
Colágenos Fibrilares/química , Animales , Bovinos , Entropía , Cinética , Modelos Químicos , Piel , TemperaturaRESUMEN
Autologous cells hold great potential for personalized cell therapy, reducing immunological and risk of infections. However, low cell counts at harvest with subsequently long expansion times with associated cell function loss currently impede the advancement of autologous cell therapy approaches. Here, we aimed to source clinically relevant numbers of proangiogenic cells from an easy accessible cell source, namely peripheral blood. Using macromolecular crowding (MMC) as a biotechnological platform, we derived a novel cell type from peripheral blood that is generated within 5 days in large numbers (10-40 million cells per 100 ml of blood). This blood-derived angiogenic cell (BDAC) type is of monocytic origin, but exhibits pericyte markers PDGFR-ß and NG2 and demonstrates strong angiogenic activity, hitherto ascribed only to MSC-like pericytes. Our findings suggest that BDACs represent an alternative pericyte-like cell population of hematopoietic origin that is involved in promoting early stages of microvasculature formation. As a proof of principle of BDAC efficacy in an ischemic disease model, BDAC injection rescued affected tissues in a murine hind limb ischemia model by accelerating and enhancing revascularization. Derived from a renewable tissue that is easy to collect, BDACs overcome current short-comings of autologous cell therapy, in particular for tissue repair strategies.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Isquemia/terapia , Leucocitos Mononucleares/citología , Neovascularización Fisiológica , Pericitos/trasplante , Animales , Antígenos/genética , Antígenos/metabolismo , Biomarcadores/metabolismo , Adhesión Celular , Recuento de Células , Diferenciación Celular , Proliferación Celular , Expresión Génica , Miembro Posterior/irrigación sanguínea , Miembro Posterior/metabolismo , Miembro Posterior/patología , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Isquemia/metabolismo , Isquemia/patología , Leucocitos Mononucleares/fisiología , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Desnudos , Pericitos/citología , Pericitos/fisiología , Cultivo Primario de Células , Proteoglicanos/genética , Proteoglicanos/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
Macromolecular crowding is an optimal physiological feature in intracellular and extracellular spaces, and results from a variety of macromolecules occupying space and contributing to a fractional volume occupancy. Here, we show that soft collagen hydrogels assembled in nature-inspired crowded conditions feature enhanced biophysical properties. We demonstrate that crowding tunes the rate of collagen nucleation and fiber growth, affecting fiber diameter and organization. Adjustments of crowding levels during collagen assembly tune the gel pore size, protein permeability, transparency and resistance to enzymatic degradation. Furthermore, gels assembled in crowded conditions are twice as resistant to mechanical stress as the controls, inducing a 70% boost of proliferation of stem cells cultured on tuned hydrogels. Emulating the crowdedness of interstitial fluids therefore represents a way to optimize the properties of soft collagen gels, with promising applications in soft biomaterials design.
Asunto(s)
Colágeno Tipo I/química , Células Madre Mesenquimatosas/metabolismo , Células Cultivadas , Hidrogeles , Células Madre Mesenquimatosas/citología , PorosidadRESUMEN
Achondroplasia is the most common form of human dwarfism caused by a mutation in the fibroblast growth factor receptor 3 (FGFR3), resulting in abnormal endochondral bone formation. C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth and represents a potential therapy for achondroplasia. We have developed a novel, simple and cost effective method to produce a CNP analogue, PG-CNP37, at a large scale from Escherichia coli. A PG-CNP37 fusion protein was over-expressed as inclusion bodies in E. coli, which were purified then cleaved by formic acid to release the PG-CNP37 peptide. Approximately 0.5g of 95% pure, soluble and active PG-CNP37 peptide was produced from 1L of culture using this method and may represent a viable means for large-scale production of other therapeutic peptides.