Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 12 de 12
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Antioxidants (Basel) ; 13(2)2024 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-38397799

RESUMO

Retinitis pigmentosa is a common cause of inherited blindness in adults, which in many cases is associated with an increase in the formation of reactive oxygen species (ROS) that induces DNA damage, triggering Poly-ADP-Ribose Polymerase 1 (PARP1) activation and leading to parthanatos-mediated cell death. Previous studies have shown that resveratrol (RSV) is a promising molecule that can mitigate PARP1 overactivity, but its low bioavailability is a limitation for medical use. This study examined the impact of a synthesized new acylated RSV prodrug, piceid octanoate (PIC-OCT), in the 661W cell line against H2O2 oxidative stress and in rd10 mice. PIC-OCT possesses a better ADME profile than RSV. In response to H2O2, 661W cells pretreated with PIC-OCT preserved cell viability in more than 38% of cells by significantly promoting SIRT1 nuclear translocation, preserving NAD+/NADH ratio, and suppressing intracellular ROS formation. These effects result from expressing antioxidant genes, maintaining mitochondrial function, reducing PARP1 nuclear expression, and preventing AIF nuclear translocation. In rd10 mice, PIC-OCT inhibited PAR-polymer formation, increased SIRT1 expression, significantly reduced TUNEL-positive cells in the retinal outer nuclear layer, preserved ERGs, and enhanced light chamber activity (all p values < 0.05). Our findings corroborate that PIC-OCT protects photoreceptors by modulating the SIRT1/PARP1 axis in models of retinal degeneration.

2.
Micromachines (Basel) ; 12(12)2021 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-34945319

RESUMO

Printed circuit board (PCB) technology is well known, reliable, and low-cost, and its application to biomedicine, which implies the integration of microfluidics and electronics, has led to Lab-on-PCB. However, the biocompatibility of the involved materials has to be examined if they are in contact with biological elements. In this paper, the solder mask (PSR-2000 CD02G/CA-25 CD01, Taiyo Ink (Suzhou) Co., Ltd., Suzhou, China) of a commercial PCB has been studied for retinal cultures. For this purpose, retinal explants have been cultured over this substrate, both on open and closed systems, with successful results. Cell viability data shows that the solder mask has no cytotoxic effect on the culture allowing the application of PCB as the substrate of customized microelectrode arrays (MEAs). Finally, a comparative study of the biocompatibility of the 3D printer Uniz zSG amber resin has also been carried out.

3.
Nat Commun ; 11(1): 1609, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-32231223

RESUMO

In vitro differentiation of human pluripotent stem cells into functional retinal pigment epithelial (RPE) cells provides a potentially unlimited source for cell based reparative therapy of age-related macular degeneration. Although the inherent pigmentation of the RPE cells have been useful to grossly evaluate differentiation efficiency and allowed manual isolation of pigmented structures, accurate quantification and automated isolation has been challenging. To address this issue, here we perform a comprehensive antibody screening and identify cell surface markers for RPE cells. We show that these markers can be used to isolate RPE cells during in vitro differentiation and to track, quantify and improve differentiation efficiency. Finally, these surface markers aided to develop a robust, direct and scalable monolayer differentiation protocol on human recombinant laminin-111 and -521 without the need for manual isolation.


Assuntos
Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Células Epiteliais/metabolismo , Neurônios/metabolismo , Pigmentos da Retina/metabolismo , Animais , Antígeno CD56 , Células-Tronco Embrionárias , Humanos , Laminina/genética , Degeneração Macular/metabolismo , Coelhos , Epitélio Pigmentado da Retina/metabolismo
4.
Stem Cells Transl Med ; 9(8): 936-953, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32319201

RESUMO

As pluripotent stem cell (PSC)-based reparative cell therapies are reaching the bedside, there is a growing need for the standardization of studies concerning safety of the derived products. Clinical trials using these promising strategies are in development, and treatment for age-related macular degeneration is one of the first that has reached patients. We have previously established a xeno-free and defined differentiation protocol to generate functional human embryonic stem cells (hESCs)-derived retinal pigment epithelial (RPE) cells. In this study, we perform preclinical safety studies including karyotype and whole-genome sequencing (WGS) to assess genome stability, single-cell RNA sequencing to ensure cell purity, and biodistribution and tumorigenicity analysis to rule out potential migratory or tumorigenic properties of these cells. WGS analysis illustrates that existing germline variants load is higher than the introduced variants acquired through in vitro culture or differentiation, and enforces the importance to examine the genome integrity at a deeper level than just karyotype. Altogether, we provide a strategy for preclinical evaluation of PSC-based therapies and the data support safety of the hESC-RPE cells generated through our in vitro differentiation methodology.


Assuntos
Células-Tronco Embrionárias Humanas/metabolismo , Degeneração Macular/terapia , Células-Tronco Pluripotentes/metabolismo , Idoso , Diferenciação Celular , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes/citologia
5.
Stem Cell Reports ; 14(4): 648-662, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32197113

RESUMO

Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells could serve as a replacement therapy in advanced stages of age-related macular degeneration. However, allogenic hESC-RPE transplants trigger immune rejection, supporting a strategy to evade their immune recognition. We established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. Activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. After transplantation of SKO-B2M, SKO-CIITA, or DKO hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a large-eyed animal model.


Assuntos
Células Epiteliais/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Epitélio Pigmentado da Retina/citologia , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Citotoxicidade Imunológica , Xenoenxertos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Imunomodulação , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Linfócitos T/metabolismo , Transativadores/metabolismo , Microglobulina beta-2/metabolismo
6.
Cell Stem Cell ; 23(2): 155-157, 2018 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-30075125

RESUMO

Although endonuclease-mediated genome editing techniques offer significant improvement over traditional methods, they are still ineffective for introduction of large DNA sequences. Recently in Nature Biotechnology, Gu et al. (2018) developed a CRISPR-Cas strategy termed 2C-HR-CRISPR that generates fluorescent reporter tagging of genes with up to 95% knockin efficiency in mouse embryos.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Camundongos , Microinjeções
7.
Curr Opin Genet Dev ; 52: 1-6, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29729430

RESUMO

Understanding the genetic underpinning of early human development is of great interest not only for basic developmental and stem cell biology but also for regenerative medicine, infertility treatments, and better understanding the causes of congenital disease. Our current knowledge has mainly been generated with the use of laboratory animals, especially the mouse. While human and mouse early development present morphological resemblance, we know that the timing of the events as well as the cellular and genetic mechanisms that control fundamental processes are distinct between the species. The rapid technological development of single-cell sequencing and genome editing together with novel stem cell models of the early human embryo has made it feasible and relevant to perform functional genetic studies directly in human cells and embryos. In this review we will discuss these latest advances where combined transcriptional analysis and genome engineering has begun to shed new insights into the key processes of zygotic genome activation, lineage specification, X-chromosome inactivation and postimplantation development including primordial germ cell specification in the human embryo.


Assuntos
Diferenciação Celular/genética , Desenvolvimento Embrionário/genética , Genoma Humano/genética , Inativação do Cromossomo X/genética , Embrião de Mamíferos , Células Germinativas/crescimento & desenvolvimento , Humanos , Zigoto
8.
Cell Stem Cell ; 20(6): 874-890.e7, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28343983

RESUMO

Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through primed-to-naive resetting, but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific, but not primed-specific, proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus, identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting.


Assuntos
Antígenos de Diferenciação/biossíntese , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/biossíntese , Células-Tronco Pluripotentes/metabolismo , Linhagem Celular , Humanos , Células-Tronco Pluripotentes/citologia
9.
Development ; 144(1): 3-7, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28049687

RESUMO

Developmental biologists have become increasingly aware that the wealth of knowledge generated through genetic studies of pre-implantation mouse development might not easily be translated to the human embryo. Comparative studies have been fueled by recent technological advances in single-cell analysis, allowing in-depth analysis of the human embryo. This field could shortly gain more momentum as novel genome editing technologies might, for the first time, also allow functional genetic studies in the human embryo. In this Spotlight article, we summarize the CRISPR-Cas9 genome editing system and discuss its potential applications and limitations in human pre-implantation embryos, and the ethical considerations thereof.


Assuntos
Sistemas CRISPR-Cas/genética , Pesquisas com Embriões , Desenvolvimento Embrionário/genética , Edição de Genes/ética , Edição de Genes/estatística & dados numéricos , Edição de Genes/tendências , Animais , Pesquisas com Embriões/ética , Embrião de Mamíferos , Humanos
10.
Development ; 143(19): 3459-3469, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27578796

RESUMO

Leucine twenty homeobox (LEUTX) is a paired (PRD)-like homeobox gene that is expressed almost exclusively in human embryos during preimplantation development. We previously identified a novel transcription start site for the predicted human LEUTX gene based on the transcriptional analysis of human preimplantation embryos. The novel variant encodes a protein with a complete homeodomain. Here, we provide a detailed description of the molecular cloning of the complete homeodomain-containing LEUTX Using a human embryonic stem cell overexpression model we show that the complete homeodomain isoform is functional and sufficient to activate the transcription of a large proportion of the genes that are upregulated in human embryo genome activation (EGA), whereas the previously predicted partial homeodomain isoform is largely inactive. Another PRD-like transcription factor, DPRX, is then upregulated as a powerful repressor of transcription. We propose a two-stage model of human EGA in which LEUTX acts as a transcriptional activator at the 4-cell stage, and DPRX as a balancing repressor at the 8-cell stage. We conclude that LEUTX is a candidate regulator of human EGA.


Assuntos
Blastocisto/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas de Homeodomínio/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Reação em Cadeia da Polimerase , Isoformas de Proteínas/genética
11.
Cell ; 165(4): 1012-26, 2016 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-27062923

RESUMO

Mouse studies have been instrumental in forming our current understanding of early cell-lineage decisions; however, similar insights into the early human development are severely limited. Here, we present a comprehensive transcriptional map of human embryo development, including the sequenced transcriptomes of 1,529 individual cells from 88 human preimplantation embryos. These data show that cells undergo an intermediate state of co-expression of lineage-specific genes, followed by a concurrent establishment of the trophectoderm, epiblast, and primitive endoderm lineages, which coincide with blastocyst formation. Female cells of all three lineages achieve dosage compensation of X chromosome RNA levels prior to implantation. However, in contrast to the mouse, XIST is transcribed from both alleles throughout the progression of this expression dampening, and X chromosome genes maintain biallelic expression while dosage compensation proceeds. We envision broad utility of this transcriptional atlas in future studies on human development as well as in stem cell research.


Assuntos
Blastocisto/metabolismo , Cromossomos Humanos X , Análise de Célula Única , Massa Celular Interna do Blastocisto/metabolismo , Mecanismo Genético de Compensação de Dose , Feminino , Humanos , Masculino , RNA Longo não Codificante/genética , Análise de Sequência de RNA , Caracteres Sexuais , Transcriptoma
12.
Stem Cell Reports ; 6(1): 9-17, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26724907

RESUMO

Human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model.


Assuntos
Diferenciação Celular/fisiologia , Atrofia Geográfica/fisiopatologia , Células-Tronco Embrionárias Humanas/fisiologia , Epitélio Pigmentado da Retina/fisiologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Meios de Cultura/química , Meios de Cultura/farmacologia , Modelos Animais de Doenças , Atrofia Geográfica/terapia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/transplante , Humanos , Laminina/metabolismo , Microscopia Confocal , Coelhos , Epitélio Pigmentado da Retina/citologia , Transplante de Células-Tronco/métodos , Imagem com Lapso de Tempo , Transplante Heterólogo , Xenobióticos/química , Xenobióticos/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...