RESUMO
Human embryonic stem cells (hESCs) can differentiate into any cell lineage (pluripotency potential) derived from the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is usually demonstrated in vitro by spontaneous differentiation of hESCs grown on a monolayer of feeder-cells using an embryoid bodies (EBs)-based method. However, currently hESCs are grown mostly using fully defined media in the absence of a feeder layer. Here we describe a EBs-based protocol that allows multilineage differentiation of hESCs and human induced pluripotent stem cells (hiPSCs) grown on feeder-free conditions.
Assuntos
Técnicas de Cultura de Células , Células-Tronco Embrionárias Humanas , Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Corpos Embrioides , Células Alimentadoras , HumanosRESUMO
Human induced pluripotent stem (hiPS) cell technology has already revolutionized some aspects of fundamental and applied research such as study of disease mechanisms and pharmacology screening. The first clinical trial using hiPS cell-derived cells began in Japan, only 10 years after the publication of the proof-of concept article. In this exciting context, strategies to generate hiPS cells have evolved quickly, tending towards non-invasive protocols to sample somatic cells combined with "safer" reprogramming strategies. In this unit, we describe a protocol combining both of these advantages to generate hiPS cells with episomal plasmid transfection from urine samples of individuals carrying the desired genotype. Based on previous published works, this simplified protocol requires minimal equipment and reagents, and is suitable both for scientists familiar with the hiPS cells technology and neophytes. HiPS cells displaying classical features of pluripotency and suitable for all desired downstream applications are generated rapidly (<10 weeks) and with high efficiency. © 2017 by John Wiley & Sons, Inc.
Assuntos
Separação Celular , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Urina/citologia , Animais , Técnicas de Cultura de Células , Células Alimentadoras , Feminino , Humanos , Masculino , Camundongos , Plasmídeos/genética , TransfecçãoRESUMO
The aim of the study has been to evaluate the morphology, proliferation, and pluripotency maintenance of mouse embryonic stem cells (mESCs) cultivated on poly(lactic-co-glycolic acid) scaffolds. The scaffolds were hydrolyzed with NaOH (treated) and nonhydrolyzed (untreated). Morphological and mechanical characterization of the scaffolds was performed. mESC were evaluated for cell viability, cytotoxicity, expression of pluripotency markers, colony morphology, and overall distribution. The treatment generated a reduction in the hydrophobic characteristics of the scaffolds, leading to a higher wettability compared to the untreated group. The viability, cytotoxicity, number of colonies, and the thickness of the cell layer presented similar results between the scaffold groups. The viability test showed that it was possible to cultivate the mESCs on the scaffolds. The cytotoxicity analysis showed that the PLGA scaffolds were not harmful for the cells. The cells maintained the expression of the pluripotency markers Oct4 and Sox2. The number of colonies and the thickness of the cell layer on the scaffold showed that they were not able to colonize the entire volume of the scaffolds. The area occupied by the mESCs was the same between the treated and untreated groups after 14 days in culture. It is possible to conclude that both conditions are equally suitable for maintaining mESC culture. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 424-432, 2017.