RESUMO
Cellular stress can influence efficiency of iPSCs generation and their differentiation. However, the role of intracellular cytoprotective factors in these processes is still not well known. Therefore, we investigated the effect of HO-1 (Hmox1) or Nrf2 (Nfe2l2), two major cytoprotective genes. Hmox1-/- fibroblasts demonstrated decreased reprogramming efficiency in comparison to Hmox1+/+ cells. Reversely, pharmacological enhancement of HO-1 resulted in higher number of iPSCs colonies. Importantly, elevated level of both p53 and p53-regulated miR-34a and 14-3-3σ was observed in HO-1-deficient fibroblasts whereas downregulation of p53 in these cells markedly increased their reprogramming efficiency. In human fibroblasts HO-1 silencing also induced p53 expression and affected reprogramming outcome. Hmox1+/+ and Hmox1-/- iPSCs similarly differentiated in vitro to cells originating from three germ layers, however, lower number of contracting cells was observed during this process in HO-1-deficient cells indicating attenuated cardiac differentiation. Importantly, silencing of Hmox1 in murine ESC using CRISPR/Cas-9 editing also impaired their spontaneous cardiac differentiation. Decreased reprogramming efficiency was also observed in Nrf2-lacking fibroblasts. Reversely, sulforaphane, a Nrf2 activator, increased the number of iPSCs colonies. However, both Nfe2l2+/+ and Nfe2l2-/- iPSCs showed similar pluripotency and differentiation capacity. These results indicate that regulation of HO-1 expression can further optimize generation and cardiac differentiation of iPSCs. © 2018 IUBMB Life, 70(2):129-142, 2018.
Assuntos
Diferenciação Celular/fisiologia , Técnicas de Reprogramação Celular/métodos , Heme Oxigenase-1/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas de Membrana/metabolismo , Animais , Ciclo Celular/fisiologia , Fibroblastos , Heme Oxigenase-1/genética , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Recent advances in the stem cell field allow to obtain many human tissues in vitro. However, hepatic differentiation of induced pluripotent stem cells (iPSCs) still remains challenging. Hepatocyte-like cells (HLCs) obtained after differentiation resemble more fetal liver hepatocytes. MicroRNAs (miRNA) play an important role in the differentiation process. Here, we analysed noncoding RNA profiles from the last stages of differentiation and compare them to hepatocytes. Our results show that HLCs maintain an epithelial character and express miRNA which can block hepatocyte maturation by inhibiting the epithelial-mesenchymal transition (EMT). Additionally, we identified differentially expressed small nucleolar RNAs (snoRNAs) and discovered novel noncoding RNA (ncRNA) genes.
RESUMO
Fibroblasts from the foreskin of a 6-year-old healthy boy was used to generate induced pluripotent stem cell line (ULEIi001-A) by lipotransfection with episomal vectors. Pluripotency and differentiation potential of the cell line was confirmed in vitro. Additionally hepatic differentiation potential of the cell line was investigated.