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1.
Blood ; 140(16): 1774-1789, 2022 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-35714307

RESUMEN

Individuals with age-related clonal hematopoiesis (CH) are at greater risk for hematologic malignancies and cardiovascular diseases. However, predictive preclinical animal models to recapitulate the spectrum of human CH are lacking. Through error-corrected sequencing of 56 human CH/myeloid malignancy genes, we identified natural CH driver mutations in aged rhesus macaques matching genes somatically mutated in human CH, with DNMT3A mutations being the most frequent. A CH model in young adult macaques was generated via autologous transplantation of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated gene-edited hematopoietic stem and progenitor cells (HSPCs), targeting the top human CH genes with loss-of-function (LOF) mutations. Long-term follow-up revealed reproducible and significant expansion of multiple HSPC clones with heterozygous TET2 LOF mutations, compared with minimal expansion of clones bearing other mutations. Although the blood counts of these CH macaques were normal, their bone marrows were hypercellular and myeloid-predominant. TET2-disrupted myeloid colony-forming units isolated from these animals showed a distinct hyperinflammatory gene expression profile compared with wild type. In addition, mature macrophages purified from the CH macaques showed elevated NLRP3 inflammasome activity and increased interleukin-1ß (IL-1ß) and IL-6 production. The model was used to test the impact of IL-6 blockage by tocilizumab, documenting a slowing of TET2-mutated expansion, suggesting that interruption of the IL-6 axis may remove the selective advantage of mutant HSPCs. These findings provide a model for examining the pathophysiology of CH and give insights into potential therapeutic interventions.


Asunto(s)
Hematopoyesis Clonal , Dioxigenasas , Humanos , Adulto Joven , Animales , Anciano , Hematopoyesis Clonal/genética , Hematopoyesis/genética , Interleucina-1beta/genética , Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Macaca mulatta , Proteína 9 Asociada a CRISPR , Interleucina-6/genética , Células Clonales , Proteínas de Unión al ADN/genética , Dioxigenasas/genética
2.
Blood ; 129(14): 1940-1946, 2017 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-27903532

RESUMEN

Natural killer (NK) cells have long been considered short-lived effectors of innate immunity. However, recent animal models and human studies suggest that subsets of NK cells have adaptive features. We investigate clonal relationships of various NK-cell subsets, including the adaptive population, by taking advantage of naturally occurring X-linked somatic PIGA mutations in hematopoietic stem and progenitor cells (HSPCs) from patients with paroxysmal nocturnal hemoglobinuria (PNH). The affected HSPCs and their progeny lack expression of glycosylphosphatidylinositol (GPI) anchors on their cell surface, allowing quantification of PIGA-mutant (GPI-negative) HSPC-derived peripheral blood cell populations. The fraction of GPI-negative cells within the CD56dim NK cells was markedly lower than that of neutrophils and the CD56bright NK-cell compartments. This discrepancy was most prominent within the adaptive CD56dim NK-cell population lacking PLZF expression. The functional properties of these adaptive NK cells were similar in PNH patients and healthy individuals. Our findings support the existence of a long-lived, adaptive NK-cell population maintained independently from GPIposCD56dim.


Asunto(s)
Regulación de la Expresión Génica/inmunología , Células Madre Hematopoyéticas/inmunología , Hemoglobinuria Paroxística , Células Asesinas Naturales/inmunología , Proteínas de la Membrana , Adulto , Anciano , Antígeno CD56/genética , Antígeno CD56/inmunología , Femenino , Hemoglobinuria Paroxística/genética , Hemoglobinuria Paroxística/inmunología , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/inmunología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Persona de Mediana Edad , Oligosacáridos/genética , Oligosacáridos/inmunología , Proteína de la Leucemia Promielocítica con Dedos de Zinc
3.
Mol Ther ; 25(1): 44-53, 2017 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-28129126

RESUMEN

Nonhuman primate (NHP) induced pluripotent stem cells (iPSCs) offer the opportunity to investigate the safety, feasibility, and efficacy of proposed iPSC-derived cellular delivery in clinically relevant in vivo models. However, there is need for stable, robust, and safe labeling methods for NHP iPSCs and their differentiated lineages to study survival, proliferation, tissue integration, and biodistribution following transplantation. Here we investigate the utility of the adeno-associated virus integration site 1 (AAVS1) as a safe harbor for the addition of transgenes in our rhesus macaque iPSC (RhiPSC) model. A clinically relevant marker gene, human truncated CD19 (hΔCD19), or GFP was inserted into the AAVS1 site in RhiPSCs using the CRISPR/Cas9 system. Genetically modified RhiPSCs maintained normal karyotype and pluripotency, and these clones were able to further differentiate into all three germ layers in vitro and in vivo. In contrast to transgene delivery using randomly integrating viral vectors, AAVS1 targeting allowed stable transgene expression following differentiation. Off-target mutations were observed in some edited clones, highlighting the importance of careful characterization of these cells prior to downstream applications. Genetically marked RhiPSCs will be useful to further advance clinically relevant models for iPSC-based cell therapies.


Asunto(s)
Diferenciación Celular , Edición Génica , Expresión Génica , Estratos Germinativos/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Transgenes , Animales , Biomarcadores , Sistemas CRISPR-Cas , Reprogramación Celular , Marcación de Gen , Sitios Genéticos , Estratos Germinativos/embriología , Macaca mulatta , Especificidad de Órganos/genética
5.
Nat Med ; 13(11): 1349-58, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17982464

RESUMEN

Data providing direct evidence for a causative link between endothelial dysfunction, microvascular disease and diabetic end-organ damage are scarce. Here we show that activated protein C (APC) formation, which is regulated by endothelial thrombomodulin, is reduced in diabetic mice and causally linked to nephropathy. Thrombomodulin-dependent APC formation mediates cytoprotection in diabetic nephropathy by inhibiting glomerular apoptosis. APC prevents glucose-induced apoptosis in endothelial cells and podocytes, the cellular components of the glomerular filtration barrier. APC modulates the mitochondrial apoptosis pathway via the protease-activated receptor PAR-1 and the endothelial protein C receptor EPCR in glucose-stressed cells. These experiments establish a new pathway, in which hyperglycemia impairs endothelial thrombomodulin-dependent APC formation. Loss of thrombomodulin-dependent APC formation interrupts cross-talk between the vascular compartment and podocytes, causing glomerular apoptosis and diabetic nephropathy. Conversely, maintaining high APC levels during long-term diabetes protects against diabetic nephropathy.


Asunto(s)
Apoptosis , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/prevención & control , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/prevención & control , Endotelio Vascular/patología , Podocitos/patología , Proteína C/fisiología , Sustitución de Aminoácidos/genética , Animales , Apoptosis/genética , Línea Celular Transformada , Células Cultivadas , Citoprotección/genética , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/genética , Nefropatías Diabéticas/enzimología , Nefropatías Diabéticas/genética , Endotelio Vascular/enzimología , Activación Enzimática/genética , Humanos , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/enzimología , Glomérulos Renales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Microcirculación/enzimología , Microcirculación/patología , Podocitos/enzimología , Proteína C/biosíntesis , Proteína C/genética , Transducción de Señal/genética , Trombomodulina/fisiología
6.
ACS Appl Bio Mater ; 5(3): 1013-1024, 2022 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-35171572

RESUMEN

Clinically, bone tissue replacements and/or bone repair are challenging. Strategies based on well-defined combinations of osteoconductive materials and osteogenic cells are promising to improve bone regeneration but still require improvement. Herein, we combined polycaprolactone (PCL) fibers, carbon nanotubes (CNT), and hydroxyapatite (nHap) nanoparticles to develop the next generation of bone regeneration material. Fibers formed by rotary jet spinning (RJS) instead of traditional electrospinning (ES) with embedded bone marrow mesenchymal stem cells (BMMSCs) showed the best outcomes to repair rat calvarial defects after 6 weeks. To understand this, it was observed that different morphologies were formed depending on the manufacturing method used. RJS fibers presented a particular topography with rough fibers, which allowed for better cellular growth and cell spreading in vitro around and into a three-dimensional (3D) mesh, while fibers made by ES were more smooth and cellular growth was only measured on the 3D mesh surface. The fibers with incorporated nHap/CNT nanoparticles enhanced in vitro cell performance as indicated by more cellular proliferation, alkaline phosphatase activity, proliferation, and deposition of calcium. Greater bone neoformation occurred by combining three characteristics: the presence of nHap and CNT nanoparticles, the topography of the RJS fibers, and the addition of BMMSCs. RJS fibers with nanoparticles and seeded with BMMSCs showed 10 136 mm3 of bone neoformation, meaning a 10-fold increase compared to using RJS only and BMMSCs (0.853 mm3) and a 5-fold increase from using ES only (2054 mm3) after 6 weeks of implantation. Conversely, none of these approaches used individually showed any significant difference for in vivo bone neoformation, suggesting that their combination is essential for optimizing bone formation. In summary, our work generated a potential material composed of well-defined combinations of suitable scaffolds seeded with BMMSCs for enhancing numerous orthopedic tissue engineering applications.


Asunto(s)
Células Madre Mesenquimatosas , Nanotubos de Carbono , Animales , Huesos , Durapatita/farmacología , Poliésteres , Ratas , Andamios del Tejido
7.
Langmuir ; 26(23): 18308-14, 2010 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-20961085

RESUMEN

A method for the electrodeposition of hydroxyapatite films on superhydrophilic vertically aligned multiwalled carbon nanotubes is presented. The formation of a thin homogeneous film with high crystallinity was observed without any thermal treatment and with bioactivity properties that accelerate the in vitro biomineralization process and osteoblast adhesion.


Asunto(s)
Química/métodos , Durapatita/química , Nanotubos de Carbono/química , Osteoblastos/citología , Plaquetas/citología , Adhesión Celular , Línea Celular , Electroquímica/métodos , Humanos , Microscopía Electrónica de Rastreo/métodos , Microondas , Espectrometría Raman/métodos , Rayos X
8.
Mol Ther Methods Clin Dev ; 11: 143-154, 2018 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-30547048

RESUMEN

Gene therapies using integrating retrovirus vectors to modify hematopoietic stem and progenitor cells have shown great promise for the treatment of immune system and hematologic diseases. However, activation of proto-oncogenes via insertional mutagenesis has resulted in the development of leukemia. We have utilized cellular bar coding to investigate the impact of different vector designs on the clonal behavior of hematopoietic stem and progenitor cells (HSPCs) during in vivo expansion, as a quantitative surrogate assay for genotoxicity in a non-human primate model with high relevance for human biology. We transplanted two rhesus macaques with autologous CD34+ HSPCs transduced with three lentiviral vectors containing different promoters and/or enhancers of a predicted range of genotoxicities, each containing a high-diversity barcode library that uniquely tags each individual transduced HSPC. Analysis of clonal output from thousands of individual HSPCs transduced with these barcoded vectors revealed sustained clonal diversity, with no progressive dominance of clones containing any of the three vectors for up to almost 3 years post-transplantation. Our data support a low genotoxic risk for lentivirus vectors in HSPCs, even those containing strong promoters and/or enhancers. Additionally, this flexible system can be used for the testing of future vector designs.

9.
Cardiovasc Res ; 68(1): 87-97, 2005 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-15961069

RESUMEN

OBJECTIVE: We have previously reported that myocyte enhancer factor-2 (MEF2) transcription factors and c-Jun are rapidly activated by pressure overload and that these events are involved in the early activation of the myocardial hypertrophic genetic program. In this study, we investigated whether focal adhesion kinase (FAK) mediates the activation of MEF2 and c-Jun by mechanical stress in isolated neonatal rat ventricular myocytes (NRVMs). METHODS: NRVMs were subjected to cyclic stretch up to 4 h and studied by immunoblotting, reverse transcriptase-polymerase chain reaction, laser confocal analysis, and reporter gene and electrophoretic mobility shift assays. Analysis was extended to NRVMs transfected with FAK-antisense oligodeoxynucleotide, treated with FAK/Src inhibitor PP2 or JNK/c-Jun inhibitor SP600125. RESULTS: Cyclic stretch increased c-Jun expression, JNK/c-Jun phosphorylation, and MEF2-DNA binding activity in NRVMs. Reporter gene assays indicated that the MEF2 site is critical to c-jun transcription in stretched cells. FAK-antisense transfection abolished MEF2 and c-jun promoter activation, while either FAK-antisense or PP2 treatment inhibited the stretch-induced c-Jun expression and JNK/c-Jun phosphorylation. Finally, treatment of NRVMs with the specific JNK/c-Jun inhibitor SP600125 significantly reduced the stretch-induced increase of atrial natriuretic factor promoter activity. CONCLUSION: The present data indicate that FAK regulates the activation of MEF2 and JNK/c-Jun pathways, which in turn have a key role in the early activation of the hypertrophic genetic program by mechanical stress in cardiac myocytes.


Asunto(s)
Cardiomegalia/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Regulación de la Expresión Génica , Miocitos Cardíacos/metabolismo , Factores Reguladores Miogénicos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Animales , Cardiomegalia/genética , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Técnicas In Vitro , Factores de Transcripción MEF2 , Microscopía Confocal , Oligonucleótidos Antisentido/farmacología , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estrés Mecánico
10.
Am J Physiol Regul Integr Comp Physiol ; 289(3): R862-70, 2005 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15890789

RESUMEN

This study examined whether focal adhesion kinase (FAK) plays a role in the differentiation of C(2)C(12) myoblasts into myotubes. Differentiation of C(2)C(12) myoblasts induced by switch to differentiation culture medium was accompanied by a transient reduction of FAK phosphorylation at Tyr-397 (to approximately 50%, at 1 and 2 h), followed by an increase thereafter (to 240% up to 5 days), although FAK protein expression remained unchanged. FAK and phosphorylated FAK were found at the edge of lamellipodia in proliferating cells, whereas the later increase in FAK phosphorylation in differentiating cells was accompanied by its preferential location at the tip of well-organized actin stress fibers. Hyperexpression of FAK autophosphorylation site (Tyr-397) mutant (MT-FAK) reduced FAK phosphorylation at Tyr-397 in proliferating cells and was accompanied by reduction of cyclin D1 and increase of myogenin expression. These cells failed to progress to myotubes in differentiation medium. In contrast, hyperexpression of a wild-type FAK construction (WT-FAK) increased baseline and abolished the transient reduction of FAK phosphorylation at Tyr-397 in serum-starved C(2)C(12) cells. Cells transfected with WT-FAK failed to reduce cyclin D1 and to increase myogenin expression, as well as to progress to terminal differentiation in differentiation medium. These data indicate that FAK signaling plays a critical role in the control of cell cycle as well as in the progression of C(2)C(12) cells to terminal differentiation. Transient inhibition of FAK phosphorylation at Tyr-397 contributes to trigger the myogenic genetic program, but its later activation is also central to terminal differentiation into myotubes.


Asunto(s)
Diferenciación Celular/fisiología , Mioblastos/citología , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Proliferación Celular , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Ratones , Desarrollo de Músculos/fisiología , Mutación , Fosforilación , Proteínas Tirosina Quinasas/genética , Fracciones Subcelulares/metabolismo , Distribución Tisular
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