RESUMEN
Transactivation systems are a promising application based on the CRISPR/Cas9 system and allow targeted control of gene expression levels in cell culture. However, their performance has been reported to vary considerably depending on the cell type and the activator system. Three activator systems (dCas9-VP160, dCas9-SunTag, and dCas9-VPR) were compared for the efficiency of activating expression of OCT4, NANOG, PDX1, FOXA2, NKX2-2, and NKX6-1 in an immortalized human skin fibroblast line. The activation efficiency was found to depend on the activation system type; the extent of activation depended on the system run time.
Asunto(s)
Activación Transcripcional , HumanosRESUMEN
Induced pluripotent stem cells (iPS cells) are a prospective resource for regenerative biomedicine. iPS cells can differentiate into any type of stem, progenitor and somatic cells to help replace structures within damaged organs or tissues. However, iPS cells themselves, can produce malignant tumors if they are injected into the body of an immunocompatible or immunodeficient recipient. Thus, it is necessary to detect any residual iPS cells content in biomedical cell products obtained from iPS cells and destined for transplantation. In this article we describe searches for iPS cells in heterogeneous cell mixtures, using two different methods-quantitative RT-PCR and droplet digital PCR (ddPCR). In experiments with various heterogeneous mixtures, including mixtures with neural stem cells, we found that the OCT4, TDGF1 and LIN28 genes are the best markers for such a search, and droplet digital PCR provides the greatest measurement accuracy, which is 0.002%. Thus, we have confirmed the advantage of using droplet digital PCR in the search for pluripotent stem cells in heterogeneous cell mixtures. We hope that this data can be useful for biosafety control in regenerative biomedicine.
Asunto(s)
Marcadores Genéticos , Células Madre Embrionarias Humanas/citología , Células Madre Pluripotentes Inducidas/citología , Línea Celular , Técnicas de Cocultivo , Contención de Riesgos Biológicos , Proteínas Ligadas a GPI/genética , Células Madre Embrionarias Humanas/química , Humanos , Células Madre Pluripotentes Inducidas/química , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de Neoplasias/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Proteínas de Unión al ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Medicina RegenerativaRESUMEN
AIM: to evaluate the role of laboratory biomarkers in monitoring effectiveness of rituximab (RTM) biosimilar therapy in a total dose of 1200 mg. MATERIALS AND METHODS: 20 patients (pts) with rheumatoid arthritis (RA) (18 woman, mean age 61.5(54-66.5) years, mean disease duration 39.5(20-84) months, mean DAS28 5.6(4.9-6.8)) received two intravenous RTM biosimilar infusions (600 mg â2) in combination with DMARDs and glucocorticoids. Laboratory biomarkers were assessed at baseline and weeks 12 and 24 after the first infusion of RTX. RESULTS: RTM biosimilar induced decreases in DAS28, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) at week 12 and 24, p.
Asunto(s)
Antirreumáticos , Artritis Reumatoide , Biosimilares Farmacéuticos , Anciano , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Biomarcadores , Biosimilares Farmacéuticos/uso terapéutico , Sedimentación Sanguínea , Proteína C-Reactiva , Femenino , Humanos , Persona de Mediana Edad , Rituximab/uso terapéutico , Resultado del TratamientoRESUMEN
Dermal papilla (DP) cells are unique regional stem cells of the skin that induce formation of a hair follicle and its regeneration cycle. DP are multipotent stem cells; therefore we supposed that the efficiency of DPC reprogramming could exceed that of dermal fibroblasts reprogramming. We generated induced pluripotent stem cells from human DP cells using lentiviral transfection with Oct4, Sox2, Klf4, and c-Myc, and cultivation of cells both in a medium supplemented with valproic acid and at a physiological level of oxygen (5%). The efficiency of DP cells reprogramming was ~0.03%, while the efficiency of dermal fibroblast reprogramming under the same conditions was ~0.01%. Therefore, we demonstrated the suitability of DP cells as an alternative source of iPS cells.