RESUMEN
Mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome, is a rare, lysosomal disorder caused by mutations in a gene encoding iduronate-2-sulfatase (IDS). IDS deficiency results in an accumulation of glycosaminoglycans (GAGs) and secondary accumulations of other lipids in lysosomes. Symptoms of MPS II include a variety of soft and hard tissue problems, developmental delay, and deterioration of multiple organs. Enzyme replacement therapy is an approved treatment for MPS II, but fails to improve neuronal symptoms. Cell-based neuronal models of MPS II disease are needed for compound screening and drug development for the treatment of the neuronal symptoms in MPS II. In this study, three induced pluripotent stem cell (iPSC) lines were generated from three MPS II patient-derived dermal fibroblast cell lines that were differentiated into neural stem cells and neurons. The disease phenotypes were measured using immunofluorescence staining and Nile red dye staining. In addition, the therapeutic effects of recombinant human IDS enzyme, delta-tocopherol (DT), and hydroxypropyl-beta-cyclodextrin (HPBCD) were determined in the MPS II disease cells. Finally, the neural stem cells from two of the MPS II iPSC lines exhibited typical disease features including a deficiency of IDS activity, abnormal glycosaminoglycan storage, and secondary lipid accumulation. Enzyme replacement therapy partially rescued the disease phenotypes in these cells. DT showed a significant effect in reducing the secondary accumulation of lipids in the MPS II neural stem cells. In contrast, HPBCD displayed limited or no effect in these cells. Our data indicate that these MPS II cells can be used as a cell-based disease model to study disease pathogenesis, evaluate drug efficacy, and screen compounds for drug development.
Asunto(s)
Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Mucopolisacaridosis II/tratamiento farmacológico , Mucopolisacaridosis II/metabolismo , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , 2-Hidroxipropil-beta-Ciclodextrina/uso terapéutico , Línea Celular , Terapia de Reemplazo Enzimático , Glicosaminoglicanos/metabolismo , Humanos , Iduronato Sulfatasa/uso terapéutico , Células Madre Pluripotentes Inducidas/patología , Metabolismo de los Lípidos/efectos de los fármacos , Modelos Neurológicos , Mucopolisacaridosis II/patología , Células-Madre Neurales/patología , Fenotipo , Proteínas Recombinantes/uso terapéutico , Tocoferoles/uso terapéuticoRESUMEN
Alagille syndrome (ALGS) is an autosomal dominant, multisystemic disorder due to haploinsufficiency in JAG1 or less frequently, mutations in NOTCH2. The disease has been difficult to diagnose and treat due to variable expression. The generation of this iPSC line (TRNDi036-A) carrying a heterozygous mutation (p.Cys693*) in the JAG1 gene provides a means of studying the disease and developing novel therapeutics towards patient treatment.
Asunto(s)
Síndrome de Alagille , Heterocigoto , Células Madre Pluripotentes Inducidas , Proteína Jagged-1 , Mutación , Síndrome de Alagille/genética , Humanos , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Línea Celular , Masculino , FemeninoRESUMEN
Expanded human lymphoblast cells from three different aged healthy individuals, 8-year-old male, 0-year-old newborn (NB) male, and 26-year-old female, were used to generate induced pluripotent stem cell (iPSC) lines TRNDi033-A, TRNDi034-A and TRNDi035-A, respectively, by exogenous expression of five reprogramming factors, human OCT4, SOX2, KLF4, L-MYC and LIN28. The authenticity of established iPSC lines was confirmed by the expressions of stem cell markers, karyotype analysis, embryoid body formation, and scorecard analysis. These iPSC lines could serve as healthy donor controls that are age and sex matched for the studies involving patient-specific iPSCs.
Asunto(s)
Células Madre Pluripotentes Inducidas , Femenino , Recién Nacido , Humanos , Masculino , Anciano , Niño , Células Madre Pluripotentes Inducidas/metabolismo , Diferenciación Celular , Factor 4 Similar a Kruppel , Linfocitos , Cariotipificación , Reprogramación CelularRESUMEN
Alagille syndrome (ALGS) is an autosomal dominant, multisystemic disorder due to haploinsufficiency in either the JAG1 gene (ALGS type 1) or the NOTCH2 gene (ALGS type 2). The disease has been difficult to diagnose and treat due to its muti-system clinical presentation, variable expressivity, and prenatal onset for some of the features. The generation of this iPSC line (TRNDi032-A) carrying a heterozygous mutation, p.Cys682Leufs*7 (c.2044dup), in the JAG1 gene provides a means of studying the disease and developing novel therapeutics towards patient treatment.
Asunto(s)
Síndrome de Alagille , Células Madre Pluripotentes Inducidas , Humanos , Síndrome de Alagille/genética , Síndrome de Alagille/diagnóstico , Síndrome de Alagille/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Mutación/genéticaRESUMEN
Human induced pluripotent stem cells (hiPSCs) are characterized by unlimited self-renewal and the capability to differentiate into all three germ layers, with the potential to further differentiate into all types of cells and tissues. Human iPSCs retain all genetic information from their original donors and can be developed into disease models to study disease pathophysiology, identify disease phenotypes and biomarkers, and evaluate therapeutic efficacy and toxicity for drug development. Human iPSCs can also be used to develop cell therapies and regenerative medicine. In the last decade, the technologies for hiPSC generation and differentiation have advanced rapidly. Human iPSC culture and propagation are tedious and require careful handling. High-quality hiPSCs are necessary for downstream applications. The methods, techniques, and skills for hiPSC maintenance and characterization are very different from those for immortalized cell lines. It can be a challenge for new laboratory staff, and sometimes even for experienced staff, to properly culture and maintain the high quality of these cells. Here, we describe a comprehensive set of protocols for hiPSC propagation under chemically defined and feeder-free culture conditions. These step-by-step protocols describe in detail all the reagents and experimental procedures needed to culture hiPSCs. The protocols also describe experimental methods for hiPSC characterization, including immunofluorescence staining and flow cytometric analysis with a panel of pluripotency markers, a teratoma formation assay for validation of in vivo pluripotency, and detection of Sendai virus to ensure elimination of the viral vectors. These protocols have been successfully used in our laboratory for hiPSC expansion and propagation, and this article provide a useful reference guide for laboratory staff to work on hiPSC culture. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Propagation and cryopreservation of hiPSC cultures Basic Protocol 2: Recovery of cryopreserved hiPSCs Basic Protocol 3: Validation of pluripotency markers via immunocytochemical analysis Alternate Protocol: Determination of the expression of pluripotency markers via flow cytometry analysis Basic Protocol 4: Assessment of pluripotency via in vivo teratoma formation assay Basic Protocol 5: Confirmation of Sendai viral vector clearance via RT-PCR.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Bioensayo , Diferenciación Celular , Línea Celular , Tratamiento Basado en Trasplante de Células y TejidosRESUMEN
RATIONALE: Arginine methylation by protein N-arginine methyltransferases (PRMTs) is an important posttranslational modification in the regulation of protein signaling. PRMT2 contains a highly conserved catalytic Ado-Met binding domain, but the enzymatic function of PRMT2 with respect to methylation is unknown. The JAK-STAT pathway is proposed to be regulated through direct arginine methylation of STAT transcription factors, and STAT3 signaling is known to be required for leptin regulation of energy balance. OBJECTIVE: To identify the potential role of STAT3 arginine methylation by PRMT2 in the regulation of leptin signaling and energy homeostasis. METHODS AND RESULTS: We identified that PRMT2(-/-) mice are hypophagic, lean, and have significantly reduced serum leptin levels. This lean phenotype is accompanied by resistance to food-dependent obesity and an increased sensitivity to exogenous leptin administration. PRMT2 colocalizes with STAT3 in hypothalamic nuclei, where it binds and methylates STAT3 through its Ado-Met binding domain. In vitro studies further clarified that the Ado-Met binding domain of PRMT2 induces STAT3 methylation at the Arg31 residue. Absence of PRMT2 results in decreased methylation and prolonged tyrosine phosphorylation of hypothalamic STAT3, which was associated with increased expression of hypothalamic proopiomelanocortin following leptin stimulation. CONCLUSIONS: These data elucidate a molecular pathway that directly links arginine methylation of STAT3 by PRMT2 to the regulation of leptin signaling, suggesting a potential role for PRMT2 antagonism in the treatment of obesity and obesity-related syndromes.
Asunto(s)
Metabolismo Energético/fisiología , Leptina/metabolismo , Metiltransferasas/metabolismo , Obesidad/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/fisiología , Animales , Núcleo Arqueado del Hipotálamo/metabolismo , Peso Corporal/fisiología , Ingestión de Alimentos/fisiología , Glucógeno/metabolismo , Hígado/metabolismo , Metilación , Metiltransferasas/genética , Ratones , Ratones Mutantes , Obesidad/fisiopatología , Fosforilación/fisiología , Estructura Terciaria de Proteína , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genéticaRESUMEN
We have successfully generated induced pluripotent stem cells (iPSC) from dermal fibroblasts and peripheral blood mononuclear cells from patients with a homozygous missense mutation in the gene encoding PSMB8. Biallelic loss of function mutations in this gene are responsible for the PSMB8 deficiency termed Chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE). The iPSC carrying the homozygous PSMB8 gene mutation (c.224C > T, T75M) are phenotypically normal and have the capacity to differentiate toward the three germ layers. These iPSC have great potential to study the role of PMSB8 in the regulation of immune responses and other cellular pathways.
Asunto(s)
Células Madre Pluripotentes Inducidas , Lipodistrofia , Enfermedad Crónica , Eritema Nudoso , Fiebre , Dedos/anomalías , Humanos , Síndromes de Inmunodeficiencia , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Lipodistrofia/genética , Lipodistrofia/metabolismo , Mutación , SíndromeRESUMEN
We have successfully generated induced pluripotent stem cells (iPSC) from dermal fibroblasts of the patient with a germline mutation in the coding region of the LYN kinase gene. This gain of function (GOF) mutation eliminates the inhibitory tyrosine (Y) at the position p.Y508, with an unknown established disease etiology. The iPSC carrying germline mutation in LYN are phenotypically normal, and they have capacity to differentiate toward the three germ layers. These iPSCs are critical for studying this unknown disease etiology and to the further understand the role of Lyn kinases in autoimmune disease.
Asunto(s)
Células Madre Pluripotentes Inducidas , Familia-src Quinasas , Humanos , Homocigoto , Mutación/genética , Tirosina/genética , Familia-src Quinasas/genéticaRESUMEN
Genetic studies show that BLOC1S1 modulates mitochondrial and endosome-lysosome function (Wu et al., 2021a). Furthermore, Bloc1s1 mutations are linked to leukodystrophy (Bertoli-Avella et al., 2021). The Vanderver laboratory identified additional individuals with leukodystrophy that harbored either complex heterozygous (Bloc1s1 c.206A > C and c.359G > A), or homozygous (Bloc1s1 c.185 T > C) point mutations. We generated induced pluripotential stem cell (iPSC) lines from these subjects, from parents of the complex heterozygous mutations patient, and from CRISPR isogenic (c.206A > C and c.359G > A) corrected iPSC-line. These complex heterozygous, homozygous, and isogenic-corrected Bloc1s1 lines were phenotypically normal and were capable of differentiation towards the three germ layers.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Homocigoto , Células Madre Pluripotentes Inducidas/metabolismo , Heterocigoto , Mutación/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteínas del Tejido Nervioso/metabolismoRESUMEN
We have successfully created induced pluripotent stem cells (iPSC) from patients carrying a heterozygous mutation in the gene encoding STING. The gain-of-function mutation leads to constitutive activation of STING which leads to the development of the disease STING-associated vasculopathy with onset in infancy (SAVI). The iPSC lines derived from the SAVI patitents are shown to be morphologically and phenotypically normal and have the potential to self renew and differentiate into the three germ layers. These iPSC provide a powerful tools to investigate the role of STING in the regulation of immune responses and vascular renegeration.
Asunto(s)
Inmunidad , Células Madre Pluripotentes Inducidas , Enfermedades Vasculares , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/patología , Mutación con Ganancia de Función , Enfermedades Vasculares/genética , Enfermedades Vasculares/inmunologíaRESUMEN
OBJECTIVE: Children with Hutchinson-Gilford progeria syndrome (HGPS) exhibit dramatically accelerated cardiovascular disease (CVD), causing death from myocardial infarction or stroke between the ages of 7 and 20 years. We undertook the first histological comparative evaluation between genetically confirmed HGPS and the CVD of aging. METHODS AND RESULTS: We present structural and immunohistological analysis of cardiovascular tissues from 2 children with HGPS who died of myocardial infarction. Both had features classically associated with the atherosclerosis of aging, as well as arteriolosclerosis of small vessels. In addition, vessels exhibited prominent adventitial fibrosis, a previously undescribed feature of HGPS. Importantly, although progerin was detected at higher rates in the HGPS coronary arteries, it was also present in non-HGPS individuals. Between the ages of 1 month and 97 years, progerin staining increased an average of 3.34% per year (P<0.0001) in coronary arteries. CONCLUSIONS: We find concordance among many aspects of cardiovascular pathology in both HGPS and geriatric patients. HGPS generates a more prominent adventitial fibrosis than typical CVD. Vascular progerin generation in young non-HGPS individuals, which significantly increases throughout life, strongly suggests that progerin has a role in cardiovascular aging of the general population.
Asunto(s)
Aterosclerosis/patología , Enfermedad de la Arteria Coronaria/patología , Progeria/patología , Adolescente , Envejecimiento/patología , Enfermedades Cardiovasculares/patología , Niño , Femenino , Humanos , Lamina Tipo A/análisis , Masculino , Infarto del Miocardio/etiología , Progeria/complicacionesRESUMEN
Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder of progressive ossification of skeletal muscle, fascia, tendons, and ligaments. Most FOP cases are caused by a heterozygous c. 617G > A mutation in the ACVR1 gene which encodes a gain-of-function of bone morphogenetic protein type I receptor. A human induced pluripotent stem cell (iPSC) line was generated from the dermal skin fibroblasts of a FOP patient who carries the c. 617G > A mutation in the ACVR1 gene. This iPSC line provides an attractive resource for FOP disease modeling.
Asunto(s)
Células Madre Pluripotentes Inducidas , Miositis Osificante , Receptores de Activinas Tipo I/genética , Heterocigoto , Humanos , Mutación , Miositis Osificante/genéticaRESUMEN
Alagille syndrome (ALGS) is a multisystem autosomal dominant disorder caused by defects in the Notch signaling pathway, including the mutation in JAGGED1 (JAG1) (ALGS type 1) or NOTCH2 (ALGS type 2). An induced pluripotent stem cell (iPSC) line was generated from the dermal fibroblasts of a 3-month-old patient with heterozygous mutation at JAG1 splicing site (Chr20: 10,629,709C>A) before exon 11. This iPSC model offers a useful resource for disease modeling to study the disease pathophysiology and to develop therapeutics for treatment of ALGS.
Asunto(s)
Síndrome de Alagille , Células Madre Pluripotentes Inducidas , Síndrome de Alagille/genética , Exones/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Lactante , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , MutaciónRESUMEN
Farber disease is an ultra-rare lysosomal storage disease. Mutations in the N-acylsphingosine amidohydrolase (ASAH1) gene, which encodes for the enzyme acid ceramidase (ACDase), cause ceramides to accumulate in the body. A human induced pluripotent stem cell (iPSC) line TRNDi030-A was generated from fibroblasts of a male patient with a homozygous p. Y36C (c.107 A>G) variant in the second exon of the ASAH1 producing the alpha subunit of ACDase. This Farber disease iPSC line is a useful resource to study disease pathophysiology and to develop therapeutics for treatment of patients with Farber disease.
Asunto(s)
Lipogranulomatosis de Farber , Células Madre Pluripotentes Inducidas , Ceramidasa Ácida/genética , Homocigoto , Humanos , Masculino , Mutación/genéticaRESUMEN
NGLY1 deficiency is a rare disorder caused by mutations in the NGLY1 gene which codes for the highly conserved N-glycanase1 (NGLY1). This enzyme functions in cytosolic deglycosylation of N- linked glycoproteins. An induced pluripotent stem cell (iPSC) line was generated from the dermal fibroblasts of a 2-year-old patient carrying compound heterozygous mutations, p.R390P and p.L318P in the NGLY1 gene. This cell-based iPSC disease model provides a resource to study disease pathophysiology and to develop a cell-based disease model for drug development for NGLY1 patients.
Asunto(s)
Células Madre Pluripotentes Inducidas , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Línea Celular , Preescolar , Glicoproteínas , Heterocigoto , Humanos , MutaciónRESUMEN
NGLY1 deficiency is a rare recessive genetic disease caused by mutations in the NGLY1 gene which codes for N-glycanase 1 (NGLY1). Here, we report the generation of two gene corrected iPSC lines using a patient-derived iPSC line (NCATS-CL6103) that carried a homozygous p.R401X mutation in the NGLY1 gene. These lines contain either one (NCATS-CL6104) or two (NCATS-CL6105) CRISPR/Cas9 corrected alleles of NGLY1. This pair of NGLY1 mutation corrected iPSC lines can be used as a control for the NCATS-CL6103 which serves as a cell-based NGLY1 disease model for the study of the disease pathophysiology and evaluation of therapeutics under development.
Asunto(s)
Trastornos Congénitos de Glicosilación , Células Madre Pluripotentes Inducidas , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/deficiencia , Sistemas CRISPR-Cas/genética , Trastornos Congénitos de Glicosilación/genética , Homocigoto , Humanos , Mutación/genética , National Center for Advancing Translational Sciences (U.S.) , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Estados UnidosRESUMEN
Alagille syndrome (ALGS) is a rare autosomal dominant disorder caused by disruption of the Notch signaling pathway due to mutations in either JAGGED1 (JAG1) (ALGS type 1) or NOTCH2 (ALGS type 2). Loss of this signaling interferes with the development of many organs, but especially the liver. A human induced pluripotent stem cell (iPSC) line was generated from the fibroblasts of a patient with a p. C312X (c. 936 T > A) variant in JAG1. This iPSC line offers a valuable resource to study the disease pathophysiology and develop therapeutics to treat patients with ALGS.
Asunto(s)
Síndrome de Alagille , Células Madre Pluripotentes Inducidas , Síndrome de Alagille/genética , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Mutación/genéticaRESUMEN
BACKGROUND: In murine embryonic stem cells, the onset of vascular endothelial growth factor receptor 2 (VEGFR-2) expression identifies endothelial precursors. Undifferentiated human embryonic stem cells express VEGFR-2, and VEGFR-2 expression persists on differentiation. The objective of our study was to identify a single population of endothelial precursors with common identifying features from both human and murine embryonic stem cells. METHODS AND RESULTS: We report that expression of the VEGF coreceptor neuropilin-1 (NRP-1) coincides with expression of Brachyury and VEGFR-2 and identifies endothelial precursors in murine and human embryonic stem cells before CD31 or CD34 expression. When sorted and differentiated, VEGFR-2(+)NRP-1(+) cells form endothelial-like colonies that express CD31 and CD34 7-fold more efficiently than NRP-1 cells. Finally, antagonism of both the VEGF and Semaphorin binding functions of NRP-1 impairs the differentiation of vascular precursors to endothelial cells. CONCLUSIONS: The onset of NRP-1 expression identifies endothelial precursors in murine and human stem cells. The findings define the origin of a single population of endothelial precursors from human and murine stem cells to endothelial cells. Additionally, the function of both the VEGF and Semaphorin binding activities of NRP-1 has important roles in the differentiation of stem cells to endothelial cells, providing novel insights into the role of NRP-1 in a model of vasculogenesis.
Asunto(s)
Antígenos CD34/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Neovascularización Fisiológica/fisiología , Neuropilina-1/genética , Animales , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular/fisiología , Línea Celular , Medio de Cultivo Libre de Suero/farmacología , Células Endoteliales/citología , Células Endoteliales/fisiología , Proteínas Fetales/genética , Proteínas Fetales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Técnicas In Vitro , Ratones , Neuropilina-1/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Semaforinas/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Human induced pluripotent stem cells (iPSCs) that express stable and robust fluorescent proteins have proven to be indispensable in basic and translational research. These reporter iPSC lines can greatly facilitate cell imaging, sorting, and tracking in vitro and in vivo studies. Here, we document two reporter human iPSC lines generated by gene-editing technologies that precisely integrated one-copy of a tdTomato transgene driven by strong CAG promoter into the AAVS1 human safe harbor locus.