RESUMO
Cardiomyocytes (CMs) derived from human-induced pluripotent stem cells (hiPSCs) are considered a promising platform for multiple applications, including disease modeling, regenerative medicine, screening of drug toxicity and investigation of cardiomyogenesis. Despite remarkable improvement in methodology enabling differentiation of hiPSCs into CMs, applied protocols generate heterogeneous cell populations composed of CMs along with differentiated non-cardiac cell-types and undifferentiated hiPSCs. Here we describea procedure of automated Magnetic-Activated Cell Sorting (autoMACS) for the purification of hiPSCs-derived CMs under sterile culture conditions. We illustrate that this approach led to a robust depletion of non-cardiac cells and enrichment of CMs, a result particularly crucial for hiPSC lines with poor cardiac differentiation efficiencies.
Assuntos
Diferenciação Celular , Citometria de Fluxo , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Citometria de Fluxo/métodos , Separação Celular/métodos , Técnicas de Cultura de Células/métodos , Separação Imunomagnética/métodos , Células CultivadasRESUMO
Cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) offer an attractive platform to evaluate the mechanisms of cardiovascular-related incidents and to develop and test new drugs for heart diseases. This work focuses on the comparison of two hiPSC-CM differentiation protocols: the GiWi method based on temporal modulation of the Wnt/ß-catenin pathway and the commercially available PSC Cardiomyocyte Differentiation Kit. We underlined the need to optimize several parameters such as cell density or small molecule concentration (CHIR-99021, IWR-1) to obtain functional hiPSC-CMs. Both protocols yield a similar differentiation efficiency; therefore, the choice of a particular procedure may depend on the preferences of the experimenter.
Assuntos
Técnicas de Cultura de Células , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Técnicas de Cultura de Células/métodos , Via de Sinalização Wnt , Células CultivadasRESUMO
Slow-cycling cancer cells (SCC) contribute to the aggressiveness of many cancers, and their invasiveness and chemoresistance pose a great therapeutic challenge. However, in melanoma, their tumor-initiating abilities are not fully understood. In this study, we used the syngeneic transplantation assay to investigate the tumor-initiating properties of melanoma SCC in the physiologically relevant in vivo settings. For this we used B16-F10 murine melanoma cell line where we identified a small fraction of SCC. We found that, unlike human melanoma, the murine melanoma SCC were not marked by the high expression of the epigenetic enzyme Jarid1b. At the same time, their slow-cycling phenotype was a temporary state, similar to what was described in human melanoma. Progeny of SCC had slightly increased doxorubicin resistance and altered expression of melanogenesis genes, independent of the expression of cancer stem cell markers. Single-cell expansion of SCC revealed delayed growth and reduced clone formation when compared to non-SCC, which was further confirmed by an in vitro limiting dilution assay. Finally, syngeneic transplantation of 10 cells in vivo established that SCC were able to initiate growth in primary recipients and continue growth in the serial transplantation assay, suggesting their self-renewal nature. Together, our study highlights the high plasticity and tumorigenicity of murine melanoma SCC and suggests their role in melanoma aggressiveness.
Assuntos
Melanoma Experimental , Humanos , Animais , Camundongos , Transplante Isogênico , Melanoma Experimental/genética , Melanoma Experimental/tratamento farmacológico , Linhagem Celular , Proliferação de CélulasRESUMO
Psoriasis (Ps) is a chronic, inflammatory skin disease characterized by thickened, red and scaly plaques. Systemic inflammation associated with psoriasis results in an increased risk of death due to the development of psoriasis-associated comorbidities such as cardiovascular disease (CVD) and metabolic syndrome. Although the cardiometabolic features in psoriasis are clinically well described, the underlying molecular mechanisms linking these comorbidities remain poorly understood. Generation of induced pluripotent stem cells (hiPSCs) from peripheral blood mononuclear cells (PBMCs) and skin fibroblasts (SFs) of psoriatic patients provides a novel approach to investigate the pathway by which cutaneous inflammation promotes CV complications in this disorder.
Assuntos
Células-Tronco Pluripotentes Induzidas , Psoríase , Humanos , Leucócitos Mononucleares , Comorbidade , Psoríase/complicações , Psoríase/epidemiologia , InflamaçãoRESUMO
Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects joints but should be considered as a syndrome that also includes extra-articular manifestations and comorbidities. Human-derived induced pluripotent stem cells (hiPSCs) and their differentiated derivatives may be of special interest in the investigation of complex pathophysiology of RA. In this study, we demonstrate and compare the generation of hiPSC from peripheral blood mononuclear cells (PBMC) and fibroblast-like synoviocytes (FLS) from RA patients. Application of three-dimensional cardiac microtissues constructed from RA specific iPSC-derivatives may be a useful approach to investigate RA comorbidities and cardiac protection or toxicity of anti-rheumatic drugs.