RESUMEN
Mature nephrons originate from a small population of uninduced nephrogenic progenitor cells (NPs) within the cap mesenchyme. These cells are characterized by the coexpression of SIX2 and CITED1. Many studies on mouse models as well as on human pluripotent stem cells have advanced our knowledge of NPs, but very little is known about this population in humans, since it is exhausted before birth and strategies for its direct isolation are still limited. Here we report an efficient protocol for direct isolation of human NPs without genetic manipulation or stepwise induction procedures. With the use of RNA-labeling probes, we isolated SIX2+ CITED1+ cells from human fetal kidney for the first time. We confirmed their nephrogenic state by gene profiling and evaluated their nephrogenic capabilities in giving rise to mature renal cells. We also evaluated the ability to culture these cells without complete loss of SIX2 and CITED1 expression over time. In addition to defining the gene profile of human NPs, this in vitro system facilitates studies of human renal development and provides a novel tool for renal regeneration and bioengineering purposes. Stem Cells Translational Medicine 2017;6:419-433.
Asunto(s)
Separación Celular/métodos , Nefronas/embriología , Células Madre/fisiología , Animales , Proteínas Reguladoras de la Apoptosis , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Morfogénesis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Células Madre/metabolismo , Factores de Tiempo , Transactivadores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
The outcome of tissue engineered organ transplants depends on the capacity of the biomaterial to promote a pro-healing response once implanted in vivo. Multiple studies, including ours, have demonstrated the possibility of using the extracellular matrix (ECM) of animal organs as platform for tissue engineering and more recently, discarded human organs have also been proposed as scaffold source. In contrast to artificial biomaterials, natural ECM has the advantage of undergoing continuous remodeling which allows adaptation to diverse conditions. It is known that natural matrices present diverse immune properties when compared to artificial biomaterials. However, how these properties compare between diseased and healthy ECM and artificial scaffolds has not yet been defined. To answer this question, we used decellularized renal ECM derived from WT mice and from mice affected by Alport Syndrome at different time-points of disease progression as a model of renal failure with extensive fibrosis. We characterized the morphology and composition of these ECMs and compared their in vitro effects on macrophage activation with that of synthetic scaffolds commonly used in the clinic (collagen type I and poly-L-(lactic) acid, PLLA). We showed that ECM derived from Alport kidneys differed in fibrous protein deposition and cytokine content when compared to ECM derived from WT kidneys. Yet, both WT and Alport renal ECM induced macrophage differentiation mainly towards a reparative (M2) phenotype, while artificial biomaterials towards an inflammatory (M1) phenotype. Anti-inflammatory properties of natural ECMs were lost when homogenized, hence three-dimensional structure of ECM seems crucial for generating an anti-inflammatory response. Together, these data support the notion that natural ECM, even if derived from diseased kidneys promote a M2 protolerogenic macrophage polarization, thus providing novel insights on the applicability of ECM obtained from discarded organs as ideal scaffold for tissue engineering.
Asunto(s)
Matriz Extracelular/química , Riñón/química , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Nefritis Hereditaria/inmunología , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Colágeno Tipo I/química , Colágeno Tipo I/farmacología , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Matriz Extracelular/inmunología , Matriz Extracelular/ultraestructura , Humanos , Inmunohistoquímica , Inmunofenotipificación , Riñón/inmunología , Macrófagos/clasificación , Macrófagos/citología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Nefritis Hereditaria/metabolismo , Nefritis Hereditaria/patología , Fenotipo , Poliésteres/química , Poliésteres/farmacología , Cultivo Primario de Células , Ingeniería de Tejidos/métodos , Andamios del TejidoRESUMEN
Over the past years, extracellular matrix (ECM) obtained from whole organ decellularization has been investigated as a platform for organ engineering. The ECM is composed of fibrous and nonfibrous molecules providing structural and biochemical support to the surrounding cells. Multiple decellularization techniques, including ours, have been optimized to maintain the composition, microstructure, and biomechanical properties of the native renal ECM that are difficult to obtain during the generation of synthetic substrates. There are evidences suggesting that in vivo implanted renal ECM has the capacity to induce formation of vasculature-like structures, but long-term in vivo transplantation and filtration activity by these tissue-engineered constructs have not been investigated or reported. Therefore, even if the process of renal decellularization is possible, the repopulation of the renal matrix with functional renal cell types is still very challenging. This review aims to summarize the current reports on kidney tissue engineering with the use of decellularized matrices and addresses the challenges in creating functional kidney units. Finally, this review discusses how future studies investigating cell-matrix interaction may aid the generation of a functional renal unit that would be transplantable into patients one day.
Asunto(s)
Riñón , Matriz Extracelular , Humanos , Medicina Regenerativa , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
Alveolar capillary dysplasia (ACD) is a congenital, lethal disorder of the pulmonary vasculature. Phosphatase and tensin homologue deleted from chromosome 10 (Pten) encodes a lipid phosphatase controlling key cellular functions, including stem/progenitor cell proliferation and differentiation; however, the role of PTEN in mesodermal lung cell lineage formation remains unexamined. To determine the role of mesodermal PTEN in the ontogeny of various mesenchymal cell lineages during lung development, we specifically deleted Pten in early embryonic lung mesenchyme in mice. Pups lacking Pten died at birth, with evidence of failure in blood oxygenation. Analysis at the cellular level showed defects in angioblast differentiation to endothelial cells and an accompanying accumulation of the angioblast cell population that was associated with disorganized capillary beds. We also found decreased expression of Forkhead box protein F1 (Foxf1), a gene associated with the ACD human phenotype. Analysis of human samples for ACD revealed a significant decrease in PTEN and increased activated protein kinase B (AKT). These studies demonstrate that mesodermal PTEN has a key role in controlling the amplification of angioblasts as well as their differentiation into endothelial cells, thereby directing the establishment of a functional gas exchange interface. Additionally, these mice could serve as a murine model of ACD.
Asunto(s)
Diferenciación Celular , Células Endoteliales/enzimología , Pulmón/embriología , Mesodermo/embriología , Fosfohidrolasa PTEN/metabolismo , Síndrome de Circulación Fetal Persistente/embriología , Animales , Linaje de la Célula , Células Endoteliales/patología , Activación Enzimática , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Pulmón/enzimología , Pulmón/patología , Mesodermo/enzimología , Mesodermo/patología , Ratones , Ratones Noqueados , Fosfohidrolasa PTEN/genética , Síndrome de Circulación Fetal Persistente/enzimología , Síndrome de Circulación Fetal Persistente/genética , Síndrome de Circulación Fetal Persistente/patología , Fenotipo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Alveolos Pulmonares/anomalías , Alveolos Pulmonares/embriología , Alveolos Pulmonares/enzimología , Alveolos Pulmonares/patologíaRESUMEN
Even though the role of the tyrosine phosphatase Pten as a tumor suppressor gene has been well established in thyroid cancer, its role during thyroid development is still elusive. We therefore targeted Pten deletion in the thyroid epithelium by crossing Pten(flox/flox) with a newly developed Nkx2.1-cre driver line in the BALB/c and C57BL/6 genetic backgrounds. C57BL/6 homozygous Pten mutant mice died around 2 weeks of age due to tracheal and esophageal compression by a hyperplasic thyroid. By contrast, BALB/c homozygous Pten mutant mice survived up to 2 years, but with a slightly increased thyroid volume. Characterization of the thyroid glands from C57BL/6 homozygous Pten mutant mice at postnatal day 14 (PN14) showed abnormally enlarged tissue with areas of cellular hyperplasia, disruption of the normal architecture, and follicular degeneration. In addition, differing degrees of hypothyroidism, thyroxine (T(4)) decrease, and thyroid-stimulating hormone elevation between the strains in the mutants and the heterozygous mutant were detected at PN14. Finally, C57BL/6 heterozygous Pten mutant mice developed thyroid tumors after 2 years of age. Our results indicate that Pten has a pivotal role in thyroid development and its deletion results in thyroid tumor formation, with the timing and severity of the tumor depending on the particular genetic background.