Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
Más filtros











Base de datos
Intervalo de año de publicación
1.
Nat Biomed Eng ; 2024 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-39187662

RESUMEN

Circularization can improve RNA persistence, yet simple and scalable approaches to achieve this are lacking. Here we report two methods that facilitate the pursuit of circular RNAs (cRNAs): cRNAs developed via in vitro circularization using group II introns, and cRNAs developed via in-cell circularization by the ubiquitously expressed RtcB protein. We also report simple purification protocols that enable high cRNA yields (40-75%) while maintaining low immune responses. These methods and protocols facilitate a broad range of applications in stem cell engineering as well as robust genome and epigenome targeting via zinc finger proteins and CRISPR-Cas9. Notably, cRNAs bearing the encephalomyocarditis internal ribosome entry enabled robust expression and persistence compared with linear capped RNAs in cardiomyocytes and neurons, which highlights the utility of cRNAs in these non-dividing cells. We also describe genome targeting via deimmunized Cas9 delivered as cRNA and a long-range multiplexed protein engineering methodology for the combinatorial screening of deimmunized protein variants that enables compatibility between persistence of expression and immunogenicity in cRNA-delivered proteins. The cRNA toolset will aid research and the development of therapeutics.

2.
bioRxiv ; 2024 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-38826258

RESUMEN

This article describes the Cell Maps for Artificial Intelligence (CM4AI) project and its goals, methods, standards, current datasets, software tools , status, and future directions. CM4AI is the Functional Genomics Data Generation Project in the U.S. National Institute of Health's (NIH) Bridge2AI program. Its overarching mission is to produce ethical, AI-ready datasets of cell architecture, inferred from multimodal data collected for human cell lines, to enable transformative biomedical AI research.

3.
iScience ; 24(10): 103149, 2021 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-34646987

RESUMEN

Deconstructing tissue-specific effects of genes and variants on proliferation is critical to understanding cellular transformation and systematically selecting cancer therapeutics. This requires scalable methods for multiplexed genetic screens tracking fitness across time, across lineages, and in a suitable niche, since physiological cues influence functional differences. Towards this, we present an approach, coupling single-cell cancer driver screens in teratomas with hit enrichment by serial teratoma reinjection, to simultaneously screen drivers across multiple lineages in vivo. Using this system, we analyzed population shifts and lineage-specific enrichment for 51 cancer associated genes and variants, profiling over 100,000 cells spanning over 20 lineages, across two rounds of serial reinjection. We confirmed that c-MYC alone or combined with myristoylated AKT1 potently drives proliferation in progenitor neural lineages, demonstrating signatures of malignancy. Additionally, mutant MEK1 S218D/S222D provides a proliferative advantage in mesenchymal lineages like fibroblasts. Our method provides a powerful platform for multi-lineage longitudinal study of oncogenesis.

4.
Stem Cell Reports ; 16(10): 2432-2441, 2021 10 12.
Artículo en Inglés | MEDLINE | ID: mdl-34559998

RESUMEN

Pluripotent stem cell-derived organoids have transformed our ability to recreate complex three-dimensional models of human tissue. However, the directed differentiation methods used to create them do not afford the ability to introduce cross-germ-layer cell types. Here, we present a bottom-up engineering approach to building vascularized human tissue by combining genetic reprogramming with chemically directed organoid differentiation. As a proof of concept, we created neuro-vascular and myo-vascular organoids via transcription factor overexpression in vascular organoids. We comprehensively characterized neuro-vascular organoids in terms of marker gene expression and composition, and demonstrated that the organoids maintain neural and vascular function for at least 45 days in culture. Finally, we demonstrated chronic electrical stimulation of myo-vascular organoid aggregates as a potential path toward engineering mature and large-scale vascularized skeletal muscle tissue from organoids. Our approach offers a roadmap to build diverse vascularized tissues of any type derived entirely from pluripotent stem cells.


Asunto(s)
Vasos Sanguíneos/citología , Organoides/irrigación sanguínea , Organoides/citología , Organoides/fisiología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Ingeniería de Tejidos/métodos , Vasos Sanguíneos/fisiología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Humanos , Neovascularización Fisiológica , Tejido Parenquimatoso/fisiología , Factores de Transcripción/metabolismo
5.
Sci Transl Med ; 13(584)2021 03 10.
Artículo en Inglés | MEDLINE | ID: mdl-33692134

RESUMEN

Current treatments for chronic pain rely largely on opioids despite their substantial side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing. In particular, a hereditary loss-of-function mutation in NaV1.7, a sodium channel protein associated with signaling in nociceptive sensory afferents, leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence and structural similarity between NaV subtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 in primary afferents via epigenome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and zinc finger proteins at the spinal level as a potential treatment for chronic pain. Toward this end, we first optimized the efficiency of NaV1.7 repression in vitro in Neuro2A cells and then, by the lumbar intrathecal route, delivered both epigenome engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain, and BzATP-induced pain. Our results show effective repression of NaV1.7 in lumbar dorsal root ganglia, reduced thermal hyperalgesia in the inflammatory state, decreased tactile allodynia in the neuropathic state, and no changes in normal motor function in mice. We anticipate that this long-lasting analgesia via targeted in vivo epigenetic repression of NaV1.7 methodology we dub pain LATER, might have therapeutic potential in management of persistent pain states.


Asunto(s)
Analgesia , Dolor Crónico , Neuralgia , Animales , Ganglios Espinales , Hiperalgesia , Ratones
6.
Cell ; 183(5): 1402-1419.e18, 2020 11 25.
Artículo en Inglés | MEDLINE | ID: mdl-33152263

RESUMEN

We propose that the teratoma, a recognized standard for validating pluripotency in stem cells, could be a promising platform for studying human developmental processes. Performing single-cell RNA sequencing (RNA-seq) of 179,632 cells across 23 teratomas from 4 cell lines, we found that teratomas reproducibly contain approximately 20 cell types across all 3 germ layers, that inter-teratoma cell type heterogeneity is comparable with organoid systems, and teratoma gut and brain cell types correspond well to similar fetal cell types. Furthermore, cellular barcoding confirmed that injected stem cells robustly engraft and contribute to all lineages. Using pooled CRISPR-Cas9 knockout screens, we showed that teratomas can enable simultaneous assaying of the effects of genetic perturbations across all germ layers. Additionally, we demonstrated that teratomas can be sculpted molecularly via microRNA (miRNA)-regulated suicide gene expression to enrich for specific tissues. Taken together, teratomas are a promising platform for modeling multi-lineage development, pan-tissue functional genetic screening, and tissue engineering.


Asunto(s)
Linaje de la Célula , Modelos Biológicos , Teratoma/patología , Animales , Células HEK293 , Humanos , Masculino , Ratones Endogámicos NOD , Ratones SCID , MicroARNs/genética , MicroARNs/metabolismo , Reproducibilidad de los Resultados , Teratoma/genética
7.
Adv Healthc Mater ; 7(23): e1800845, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30369101

RESUMEN

Recent advances in tissue engineering and 3D bioprinting have enabled construction of cell-laden scaffolds containing perfusable vascular networks. Although these methods partially address the nutrient-diffusion limitations present in engineered tissues, they are still restricted in both their viable vascular geometries and matrix material compatibility. To address this, tissue constructs are engineered via encapsulation of 3D printed, evacuable, free standing scaffolds of poly(vinyl alcohol) (PVA) in biologically derived matrices. The ease of printability and water-soluble nature of PVA grant compatibility with biologically relevant matrix materials and allow for easily repeatable generation of complex vascular patterns. This study confirms the ability of this approach to produce perfusable vascularized matrices capable of sustaining both cocultures of multiple cell types and excised tumor fragments ex vivo over multiple weeks. The study further demonstrates the ability of the approach to produce hybrid patterns allowing for coculture of vasculature and epithelial cell-lined lumens in close proximity, thereby enabling ex vivo recapitulation of gut-like systems. Taken together, the methodology is versatile, broadly applicable, and importantly, simple to use, enabling ready applicability in many research settings. It is believed that this technique has the potential to significantly accelerate progress in engineering and study of ex vivo organotypic tissue constructs.


Asunto(s)
Bioimpresión , Ingeniería de Tejidos , Animales , Línea Celular Tumoral , Células Endoteliales de la Vena Umbilical Humana , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias/metabolismo , Neoplasias/patología , Neovascularización Fisiológica , Alcohol Polivinílico/química , Impresión Tridimensional , Andamios del Tejido/química , Trasplante Heterólogo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA