RESUMEN
Acute kidney injury (AKI) is one of the most relevant health issues, leading to millions of deaths. The magnitude of the phenomenon remarks the urgent need for innovative and effective therapeutic approaches. Cell-based therapy with renal progenitor cells (RPCs) has been proposed as a possible strategy. Studies have shown the feasibility of directing embryonic stem cells or induced Pluripotent Stem Cells (iPSCs) towards nephrogenic intermediate mesoderm and metanephric mesenchyme (MM). However, the functional activity of iPSC-derived RPCs has not been tested in animal models of kidney disease. Here, through an efficient inductive protocol, we directed human iPSCs towards RPCs that robustly engrafted into damaged tubuli and restored renal function and structure in cisplatin-mice with AKI. These results demonstrate that iPSCs are a valuable source of engraftable cells with regenerative activity for kidney disease and create the basis for future applications in stem cell-based therapy.
Asunto(s)
Lesión Renal Aguda/terapia , Células Madre Pluripotentes Inducidas/citología , Riñón/citología , Trasplante de Células Madre , Células Madre/citología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
The application of cell-based therapies in regenerative medicine is gaining recognition. Here, we show that human bone marrow stromal cells (BMSCs), also known as bone-marrow-derived mesenchymal cells, can be reprogrammed into renal proximal tubular-like epithelial cells using cell-free extracts. Streptolysin-O-permeabilized BMSCs exposed to HK2-cell extracts underwent morphological changes-formation of "domes" and tubule-like structures-and acquired epithelial functional properties such as transepithelial-resistance, albumin-binding, and uptake and specific markers E-cadherin and aquaporin-1. Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts. RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway. Reprogrammed BMSCs integrated into self-forming kidney tissue and formed tubular structures. Reprogrammed BMSCs infused in immunodeficient mice with cisplatin-induced acute kidney injury engrafted into proximal tubuli, reduced renal injury and improved function. Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy.
Asunto(s)
Diferenciación Celular , Reprogramación Celular , Riñón/citología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Animales , Línea Celular Transformada , Sistema Libre de Células , Femenino , Perfilación de la Expresión Génica , Humanos , Técnicas In Vitro , Túbulos Renales Proximales/citología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/ultraestructura , Ratones , Ratones Endogámicos NOD , Ratones SCID , TranscriptomaRESUMEN
For chronic kidney diseases, there is little chance that the vast majority of world's population will have access to renal replacement therapy with dialysis or transplantation. Tissue engineering would help to address this shortcoming by regeneration of damaged kidney using naturally occurring scaffolds seeded with precursor renal cells. The aims of the present study were to optimize the production of three-dimensional (3D) rat whole-kidney scaffolds by shortening the duration of organ decellularization process using detergents that avoid nonionic compounds, to investigate integrity of extracellular matrix (ECM) structure and to enhance the efficacy of scaffold cellularization using physiological perfusion method. Intact rat kidneys were successfully decellularized after 17 h perfusion with sodium dodecyl sulfate. The whole-kidney scaffolds preserved the 3D architecture of blood vessels, glomeruli, and tubuli as shown by transmission and scanning electron microscopy. Micro-computerized tomography (micro-CT) scan confirmed integrity, patency, and connection of the vascular network. Collagen IV, laminin, and fibronectin staining of decellularized scaffolds were similar to those of native kidney tissues. After infusion of whole-kidney scaffolds with murine embryonic stem (mES) cells through the renal artery, and pressure-controlled perfusion with recirculating cell medium for 24 and 72 h, seeded cells were almost completely retained into the organ and uniformly distributed in the vascular network and glomerular capillaries without major signs of apoptosis. Occasionally, mES cells reached peritubular capillary and tubular compartment. We observed the loss of cell pluripotency and the start of differentiation toward meso-endodermal lineage. Our findings indicate that, with the proposed optimized protocol, rat kidneys can be efficiently decellularized to produce renal ECM scaffolds in a relatively short time, and rapid recellularization of vascular structures and glomeruli. This experimental setup may open the possibility to obtain differentiation of stem cells with long lasting in vitro perfusion.
Asunto(s)
Órganos Bioartificiales , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Regeneración Tisular Dirigida/instrumentación , Riñón/química , Riñón/crecimiento & desarrollo , Andamios del Tejido , Animales , Diferenciación Celular , Sistema Libre de Células/química , Células Cultivadas , Análisis de Falla de Equipo , Riñón/citología , Masculino , Técnicas de Cultivo de Órganos/instrumentación , Preservación de Órganos/métodos , Diseño de Prótesis , Ratas , Ratas Sprague-DawleyRESUMEN
SHC genes codify for a family of adaptor molecules comprising four genes. Previous data have implicated the Shc(s) molecules in stem cell division and differentiation. Specifically, the p66(ShcA) isoform has been found to contribute to longevity and resistance from oxidative stress. Here we report that p66(ShcA) is up-regulated during in vitro neural induction in embryonic stem cells. p66(ShcA) over-expression in ES cells reduces GSK-3beta kinase activation and increases beta-catenin stabilization and its transcriptional activity. p66(ShcA) over-expression results in ES cells undergoing an anticipated neural induction and accelerated neuronal differentiation. Similar effects are obtained in human ES cells over-expressing p66(ShcA). This study reveals a role for p66(ShcA) in the modulation of Wnt/beta-catenin pathway and in ES cell neuralization which is consistent between mouse and human.
Asunto(s)
Diferenciación Celular/fisiología , Inducción Embrionaria/fisiología , Células Madre Embrionarias/fisiología , Neuronas/fisiología , Isoformas de Proteínas/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Animales , Células Cultivadas , Células Madre Embrionarias/citología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Ratones , Neuronas/citología , Isoformas de Proteínas/genética , Proteínas Adaptadoras de la Señalización Shc/genética , Transducción de Señal/fisiología , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
Novel mono- and diacylated spermines, readily obtained using isolable succinimidyl active esters of acidic retinoids for the selective acylation of free spermine or in situ activated acidic retinoids for acylating selectively protected spermine followed by deprotection, were shown to inhibit the ribozyme ribonuclease P more strongly than the parent retinoids.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Ribonucleasa P/antagonistas & inhibidores , Espermina/química , Tretinoina/farmacología , Animales , Dictyostelium/citología , Dictyostelium/enzimología , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Estructura Molecular , Ribonucleasa P/aislamiento & purificación , Estereoisomerismo , Tretinoina/análogos & derivados , Tretinoina/químicaRESUMEN
Neural stem cells are cells that can self-renew ad infinitum and have the potential to generate immature precursors and mature cells of both neuronal and glial lineages. During recent years, neural stem cells gained attention as major candidates for regenerative and cell replacement therapies in various pathological brain conditions; however, they have recently revealed unforeseen valuable characters. Here the authors review on the state of the field, with a particular focus on the most recent results on the optimisation of neural stem cells isolation/derivation and homogeneous long-term expansion, highlighting advantages of this resource and outlining their potential applications. Bearing in mind that stem-cell based therapies for the diseased and injured brain are still far from being a reality, these cells have been recently opened up to valuable exploitation for disease modelling, drug discovery and toxicology tests as short-term applications.
Asunto(s)
Enfermedades del Sistema Nervioso Central/terapia , Neurobiología/tendencias , Neuronas/fisiología , Células Madre/fisiología , Animales , Diferenciación Celular , Humanos , Neuronas/citología , Células Madre/citologíaRESUMEN
Huntingtin is a protein that is mutated in Huntington's disease (HD), a dominant inherited neurodegenerative disorder. We previously proposed that, in addition to the gained toxic activity of the mutant protein, selective molecular dysfunctions in HD may represent the consequences of the loss of wild-type protein activity. We first reported that wild-type huntingtin positively affects the transcription of the brain-derived neurotrophic factor (BDNF) gene, a cortically derived survival factor for the striatal neurons that are mainly affected in the disease. Mutation in huntingtin decreases BDNF gene transcription. One mechanism involves the activation of repressor element 1/neuron-restrictive silencer element (RE1/NRSE) located within the BDNF promoter. We now show that increased binding of the RE1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) repressor occurs at multiple genomic RE1/NRSE loci in HD cells, in animal models, and in postmortem brains, resulting in a decrease of RE1/NRSE-mediated gene transcription. The same molecular phenotype is produced in cells and brain tissue depleted of endogenous huntingtin, thereby directly validating the loss-of-function hypothesis of HD. Through a ChIP (chromatin immunoprecipitation)-on-chip approach, we examined occupancy of multiple REST/NRSF target genes in the postmortem HD brain, providing the first example of the application of this technology to neurodegenerative diseases. Finally, we show that attenuation of REST/NRSF binding restores BDNF levels, suggesting that relief of REST/NRSF mediated repression can restore aberrant neuronal gene transcription in HD.
Asunto(s)
Química Encefálica/genética , Regulación de la Expresión Génica/genética , Predisposición Genética a la Enfermedad/genética , Enfermedad de Huntington/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Línea Celular Transformada , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Cuerpo Estriado/fisiopatología , Regulación hacia Abajo/genética , Humanos , Proteína Huntingtina , Inmunoprecipitación/métodos , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Neuronas/patología , Proteínas Nucleares/genética , Unión Proteica/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The transcriptional mechanisms underlying lineage specification and differentiation of embryonic stem (ES) cells remain elusive. Oct-3/4 (POU5f1) is one of the earliest transcription factors expressed in the embryo. Both the pluripotency and the fate of ES cells depend upon a tight control of Oct-3/4 expression. We report that transgene- or TGFbeta-induced increase in Oct-3/4 mRNA and protein levels in undifferentiated ES cells and at early stages of differentiation triggers expression of mesodermal and cardiac specific genes through Smad2/4. cDNA antisense- and siRNA-mediated inhibition of upregulation of Oct-3/4 in ES cells prevent their specification toward the mesoderm and their differentiation into cardiomyocytes. Similarly, Oct-3/4 siRNA injected in the inner cell mass of blastocysts impairs cardiogenesis in early embryos. Thus, quantitative Oct-3/4 expression is regulated by a morphogen, pointing to a pivotal and physiological function of the POU factor in mesodermal and cardiac commitments of ES cells and of the epiblast.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Corazón/crecimiento & desarrollo , Miocitos Cardíacos/citología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre/citología , Células Madre/fisiología , Animales , Blastocisto/citología , Blastocisto/metabolismo , Linaje de la Célula , Inmunohistoquímica , Hibridación in Situ , Ratones , Microinyecciones , Miocitos Cardíacos/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Técnicas de Cultivo de Órganos , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/metabolismoRESUMEN
Embryonic stem (ES) cells represent a source for cell-based regenerative therapies of heart failure. The pluripotency and the plasticity of ES cells allow them to be committed to a cardiac lineage following treatment with growth factors of the transforming growth factor (TGF)-beta superfamily. We describe a protocol designed to turn on expression of cardiac-specific genes in undifferentiated murine ES cells stimulated with BMP2 and/or TGF-beta. Cell commitment results in a significant improvement in spontaneous cardiac differentiation of ES cells both in vitro and in vivo.
Asunto(s)
Proteínas Morfogenéticas Óseas/farmacología , Miocardio/metabolismo , Miocitos Cardíacos/citología , Células Madre/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/metabolismo , Línea Celular , Linaje de la Célula , Células Cultivadas , Cricetinae , Miocardio/citología , Reacción en Cadena de la Polimerasa , Trasplante de Células Madre , Células Madre/citología , Células Madre/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The molecular mechanisms governing early cardiogenesis are still largely unknown. Interestingly, the retinoblastoma protein (Rb), a regulator of cell cycle, has recently emerged as a new candidate regulating cell differentiation. Rb-/- mice die at midgestation and mice lacking E2f1/E2f3, downstream components of the Rb-dependent transcriptional pathway, die of heart failure. To gain insight into the function of Rb pathway in early cardiogenesis, we used Rb-/- embryonic stem (ES) cells differentiating into cardiomyocytes. Rb-/- cells displayed a dramatic delay in expression of cardiac-specific transcription factors and in turn in the whole process of cardiac differentiation. The phenotype of Rb-/- ES cell-derived cardiomyocytes was rescued by reintroducing Rb in cardiac progenitors, by stimulating the BMP-dependent cardiogenic pathway or by overexpression of Nkx2.5. ES cells deficient in the recently identified factor LEK1, a murine homolog of the cardiomyogenic factor 1, or specific disruption of Rb-LEK1 interaction into the nucleus of differentiating ES cells recapitulated the delay in cardiac differentiation of Rb-/- ES cells. Thus, we provide evidence for a novel Rb/LEK1-dependent and BMP-independent transcriptional program, which plays a pivotal role in priming ES cells toward a cardiac fate.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Miocardio/citología , Miocitos Cardíacos/citología , Proteína de Retinoblastoma/fisiología , Células Madre/fisiología , Animales , Linaje de la Célula/fisiología , Proteínas Cromosómicas no Histona/deficiencia , Ratones , Proteínas de Microfilamentos , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Células 3T3 NIH , Proteína de Retinoblastoma/deficiencia , Proteína de Retinoblastoma/metabolismo , Células Madre/citologíaRESUMEN
Ribonuclease P (RNase P) is a ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5'-end. We have investigated the effect of synthetic rertinoids (all-trans retinoic acid, acitretin) and arotinoids (Ro 13-7410, Ro 15-0778, Ro, 13-6298 and Ro 15-1570) on RNase P activity isolated for the first time from normal human epidermal keratinocytes (NHEK). All tested compounds but one (Ro 15-1570) revealed a dose-dependent inhibition of RNase P activity, indicating that they may have a direct effect on tRNA biogenesis. Detailed kinetic analysis showed that all retinoids behave as classic competitive inhibitors. On the basis of the Ki values Ro 13-7410 was found to be the strongest inhibitor among all compounds tested.