RESUMEN
Apolipoprotein L1 (APOL1) high-risk genotypes are associated with increased risk of chronic kidney disease (CKD) in people of West African ancestry. Given the importance of endothelial cells (ECs) in CKD, we hypothesized that APOL1 high-risk genotypes may contribute to disease via EC-intrinsic activation and dysfunction. Single cell RNA sequencing (scRNA-seq) analysis of the Kidney Precision Medicine Project dataset revealed APOL1 expression in ECs from various renal vascular compartments. Utilizing two public transcriptomic datasets of kidney tissue from African Americans with CKD and a dataset of APOL1-expressing transgenic mice, we identified an EC activation signature; specifically, increased intercellular adhesion molecule 1 (ICAM-1) expression and enrichment in leukocyte migration pathways. In vitro, APOL1 expression in ECs derived from genetically modified human induced pluripotent stem cells and glomerular ECs triggered changes in ICAM-1 and platelet endothelial cell adhesion molecule 1 (PECAM-1) leading to an increase in monocyte attachment. Overall, our data suggest the involvement of APOL1 as an inducer of EC activation in multiple renal vascular beds with potential effects beyond the glomerular vasculature.
RESUMEN
Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective.
Asunto(s)
Edición Génica/métodos , Hepatocitos/trasplante , Mutación , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/terapia , Ornitina Carbamoiltransferasa/genética , Adulto , Anciano , Amoníaco/metabolismo , Animales , Células Cultivadas , Niño , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Hepatocitos/química , Hepatocitos/citología , Humanos , Intrones , Masculino , Ratones , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/genética , Ácido Orótico/orina , Empalme del ARNRESUMEN
Development of physiologically relevant cellular models with strong translatability to human pathophysiology is critical for identification and validation of novel therapeutic targets. Herein we describe a detailed protocol for generation of an advanced 3-dimensional kidney cellular model using induced pluripotent stem cells, where differentiation and maturation of kidney progenitors and podocytes can be monitored in live cells due to CRISPR/Cas9-mediated fluorescent tagging of kidney lineage markers (SIX2 and NPHS1). Utilizing these cell lines, we have refined the previously published procedures to generate a new, higher throughput protocol suitable for drug discovery. Using paraffin-embedded sectioning and whole-mount immunostaining, we demonstrated that organoids grown in suspension culture express key markers of kidney biology (WT1, ECAD, LTL, nephrin) and vasculature (CD31) within renal cortical structures with microvilli, tight junctions and podocyte foot processes visualized by electron microscopy. Additionally, the organoids resemble the adult kidney transcriptomics profile, thereby strengthening the translatability of our in vitro model. Thus, development of human nephron-like structures in vitro fills a major gap in our ability to assess the effect of potential treatment on key kidney structures, opening up a wide range of possibilities to improve clinical translation.
Asunto(s)
Sistemas CRISPR-Cas , Descubrimiento de Drogas/métodos , Edición Génica/métodos , Células Madre Pluripotentes Inducidas/fisiología , Riñón/fisiología , Organoides/fisiología , Podocitos/fisiología , Biomarcadores/metabolismo , Diferenciación Celular , Línea Celular , Linaje de la Célula , Regulación de la Expresión Génica , Genotipo , Ensayos Analíticos de Alto Rendimiento , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/ultraestructura , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/ultraestructura , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Organoides/efectos de los fármacos , Organoides/metabolismo , Organoides/ultraestructura , Fenotipo , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Podocitos/ultraestructura , Factores de Tiempo , TranscriptomaRESUMEN
There is a need for physiologically relevant assay platforms to provide functionally relevant models of diabetes, to accelerate the discovery of new treatment options and boost developments in drug discovery. In this review, we compare several 3D-strategies that have been used to increase the functional relevance of ex vivo human primary pancreatic islets and developments into the generation of stem cell derived pancreatic beta-cells (ß-cells). Special attention will be given to recent approaches combining the use of extracellular matrix (ECM) scaffolds with pancreatic molecular memory, which can be used to improve yield and functionality of in vitro stem cell-derived pancreatic models. The ultimate goal is to develop scalable cell-based platforms for diabetes research and drug screening. This article will critically assess key aspects related to in vitro pancreatic 3D-ECM models and highlight the most promising approaches for future research.
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Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/citología , Células Madre/citología , Matriz Extracelular/metabolismo , Humanos , Islotes Pancreáticos/metabolismo , Células Madre/metabolismo , Andamios del TejidoRESUMEN
It has been suggested that extracellular vesicles (EVs) can mediate crosstalk between hormones and metabolites within pancreatic tissue. However, the possible effect of pancreatic EVs on stem cell differentiation into pancreatic lineages remains unknown. Herein, human islet-derived EVs (h-Islet-EVs) were isolated, characterized and subsequently added to human induced pluripotent stem cell (iPSC) clusters during pancreatic differentiation. The h-islet-EVs had a mean size of 117±7 nm and showed positive expression of CD63 and CD81 EV markers as measured by ELISA. The presence of key pancreatic transcription factor mRNA, such as NGN3, MAFA and PDX1, and pancreatic hormone proteins such as C-peptide and glucagon, were confirmed in h-Islet-EVs. iPSC clusters were differentiated in suspension and at the end stages of the differentiation protocol, the mRNA expression of the main pancreatic transcription factors and pancreatic hormones was increased. H-Islet-EVs were supplemented to the iPSC clusters in the later stages of differentiation. It was observed that h-Islet-EVs were able to up-regulate the intracellular levels of C-peptide in iPSC clusters in a concentration-dependent manner. The effect of h-Islet-EVs on the differentiation of iPSC clusters cultured in 3D-collagen hydrogels was also assessed. Although increased mRNA expression for pancreatic markers was observed when culturing the iPSC clusters in 3D-collagen hydrogels, delivery of EVs did not affect the insulin or C-peptide intracellular content. Our results provide new information on the role of h-Islet-EVs in the regulation of insulin expression in differentiating iPSC clusters, and are highly relevant for pancreatic tissue engineering applications.
Asunto(s)
Diferenciación Celular , Vesículas Extracelulares/metabolismo , Células Madre Pluripotentes Inducidas/citología , Insulina/metabolismo , Islotes Pancreáticos/citología , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Agregación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Colágeno/farmacología , Ensayo de Inmunoadsorción Enzimática , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/ultraestructura , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacología , Células Madre Pluripotentes Inducidas/ultraestructura , Suspensiones , Factores de TiempoRESUMEN
Extracellular vesicles (EVs) are small vesicles released by cells to aid cell-cell communication and tissue homeostasis. Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in pancreatic islets of patients with type 2 diabetes (T2D). IAPP is secreted in conjunction with insulin from pancreatic ß cells to regulate glucose metabolism. Here, using a combination of analytical and biophysical methods in vitro, we tested whether EVs isolated from pancreatic islets of healthy patients and patients with T2D modulate IAPP amyloid formation. We discovered that pancreatic EVs from healthy patients reduce IAPP amyloid formation by peptide scavenging, but T2D pancreatic and human serum EVs have no effect. In accordance with these differential effects, the insulin:C-peptide ratio and lipid composition differ between EVs from healthy pancreas and EVs from T2D pancreas and serum. It appears that healthy pancreatic EVs limit IAPP amyloid formation via direct binding as a tissue-specific control mechanism.
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Diabetes Mellitus Tipo 2/metabolismo , Vesículas Extracelulares/fisiología , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/metabolismo , Placa Amiloide/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Heat-producing beige/brite (brown-in-white) adipocytes in white adipose tissue have the potential to suppress metabolic disease in mice and hold great promise for the treatment of obesity and type 2 diabetes in humans. Here, we demonstrate that human adipose-derived stromal/progenitor cells (hASCs) from subcutaneous white adipose tissue can be efficiently converted into beige adipocytes. Upon pharmacological activation of peroxisome proliferator-activated receptor-γ, hASC-derived adipocytes activated beige fat-selective genes and a brown/beige fat-selective electron transport chain gene program. Importantly, hASC-derived beige fat cells displayed the bioenergetic characteristics of genuine brown fat cells, including a capacity for increased respiratory uncoupling in response to ß-adrenergic agonists. Furthermore, knock-down experiments reveal that the thermogenic capacity of human beige fat cells was entirely dependent on the presence of Uncoupling protein 1. In summary, this study reveals that hASCs can be readily differentiated into beige adipocytes that, upon activation, undergo uncoupling protein 1-dependent thermogenesis.
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Adipocitos Marrones/citología , Adipocitos Blancos/metabolismo , Metabolismo Energético/fisiología , Canales Iónicos/metabolismo , Proteínas Mitocondriales/metabolismo , Termogénesis/fisiología , Tejido Adiposo Blanco/citología , Diferenciación Celular , Células Cultivadas , Transporte de Electrón/genética , Transporte de Electrón/fisiología , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Activación Enzimática , Humanos , Canales Iónicos/genética , Células Madre Mesenquimatosas/citología , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Oxígeno/metabolismo , Consumo de Oxígeno/fisiología , PPAR gamma/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Proteína Desacopladora 1RESUMEN
With accelerating rates of obesity and type 2 diabetes world-wide, interest in studying the adipocyte and adipose tissue is increasing. Human adipose derived stem cells--differentiated to adipocytes in vitro--are frequently used as a model system for white adipocytes, as most of their pathways and functions resemble mature adipocytes in vivo. However, these cells are not completely like in vivo mature adipocytes. Hosting the cells in a more physiologically relevant environment compared to conventional two-dimensional cell culturing on plastic surfaces, can produce spatial cues that drive the cells towards a more mature state. We investigated the adipogenesis of adipose derived stem cells on electro spun polycaprolactone matrices and compared functionality to conventional two-dimensional cultures as well as to human primary mature adipocytes. To assess the degree of adipogenesis we measured cellular glucose-uptake and lipolysis and used a range of different methods to evaluate lipid accumulation. We compared the averaged results from a whole population with the single cell characteristics--studied by coherent anti-Stokes Raman scattering microscopy--to gain a comprehensive picture of the cell phenotypes. In adipose derived stem cells differentiated on a polycaprolactone-fiber matrix; an increased sensitivity in insulin-stimulated glucose uptake was detected when cells were grown on either aligned or random matrices. Furthermore, comparing differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrixes, to those differentiated in two-dimensional cultures showed, an increase in the cellular lipid accumulation, and hormone sensitive lipase content. In conclusion, we propose an adipocyte cell model created by differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrices which demonstrates increased maturity, compared to 2D cultured cells.
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Adipogénesis , Tejido Adiposo/citología , Poliésteres , Células Madre/citología , Adipocitos/citología , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Adulto , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/genética , Proliferación Celular/genética , Células Cultivadas , Femenino , Expresión Génica , Glucosa/metabolismo , Glucosa/farmacocinética , Humanos , Lípidos/análisis , Lipólisis , Masculino , Microscopía de Fluorescencia por Excitación Multifotónica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría Raman/métodos , Células Madre/metabolismo , Andamios del Tejido/química , Adulto JovenRESUMEN
Chemokines are key regulators of cell trafficking, and dipeptidyl peptidase IV/CD26 (CD26) inactivates chemokines. Here we show that the CD26-processed chemokines SDF1alpha/CXCL12 and RANTES/CCL5, in contrast to a control chemokine not processed by CD26, are potent inducers of cell surface expression of thrombospondin-1 (TSP-1) in T lymphocytes through a CD26-controlled mechanism and that TSP-1 stimulates expression of lipoprotein receptor related protein/CD91. Accordingly, intact TSP-1 and a peptide mimetic of a sequence in TSP-1 were sufficient to stimulate CD91 expression. The chemokine-induced expression of TSP-1 and CD91 was mimicked by inhibitors of CD26 and CXCL12 and CCL5 as well as inhibitors of CD26 stimulated polarized cytoplasmic spreading and migration through TSP-1. Silencing of CD26 using small interfering RNA or Ab-induced modulation of CD26 also increased TSP-1 expression and enhanced cytoplasmic spreading and T cell migration markedly. These results indicate that CD26 is an endogenous inhibitor of T cell motility through inhibition of TSP-1 expression and that chemokines stimulate cell polarity and migration through abrogation of the CD26-dependent inhibition. This suggests that T cell motility is regulated by a cascade of interacting cell surface molecules.
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Quimiocinas/metabolismo , Quimiotaxis de Leucocito , Dipeptidil Peptidasa 4/fisiología , Proteínas de la Membrana/metabolismo , Transducción de Señal/inmunología , Trombospondina 1/genética , Células Cultivadas , Quimiocina CCL5/metabolismo , Quimiocina CXCL12/metabolismo , Regulación de la Expresión Génica/inmunología , Humanos , Linfocitos T/fisiologíaRESUMEN
The mechanisms regulating T lymphocyte migration within the extracellular matrix are not understood. We show in this study that the thrombospondin-1 binding site of calreticulin, spanning aa 19-32, is a major triggering factor for T cell motility and migration within a three-dimensional collagen type 1 matrix, and that exogenous motogenic factors such as chemokines can stimulate migration via a calreticulin-thrombospondin-1 pathway. Endogenous calreticulin binding to the N-terminal domain of endogenous thrombospondin-1 elicited a motogenic signal to the T cells through the C-terminal domain of thrombospondin-1 and its cell surface receptor integrin-associated protein (CD47). Our data further revealed that thrombospondin-1 was expressed on the cell surface with a high turnover, and that PI3K and the Janus family of tyrosine kinases were required for T cell motility mediated through calreticulin, thrombospondin-1, and CD47. These results unveil an autocrine mechanism of calreticulin-thrombospondin-1-CD47 interaction for the control of T cell motility and migration within three-dimensional extracellular matrix substrata.