RESUMEN
Rabbit induced pluripotent stem cells (rbiPSCs) possess the characteristic features of primed pluripotency as defined in rodents and primates. In the present study, we reprogrammed rbiPSCs using human Krüppel-like factors (KLFs) 2 and 4 and cultured them in a medium supplemented with fetal calf serum and leukemia inhibitory factor. These cells (designated rbEKA) were propagated by enzymatic dissociation for at least 30 passages, during which they maintained a normal karyotype. This new culturing protocol resulted in transcriptional and epigenetic reconfiguration, as substantiated by the expression of transcription factors and the presence of histone modifications associated with naïve pluripotency. Furthermore, microarray analysis of rbiPSCs, rbEKA cells, rabbit ICM cells, and rabbit epiblast showed that the global gene expression profile of the reprogrammed rbiPSCs was more similar to that of rabbit ICM and epiblast cells. Injection of rbEKA cells into 8-cell stage rabbit embryos resulted in extensive colonization of ICM in 9% early-blastocysts (E3.5), epiblast in 10% mid-blastocysts (E4.5), and embryonic disk in 1.4% pre-gastrulae (E6). Thus, these results indicate that KLF2 and KLF4 triggered the conversion of rbiPSCs into epiblast-like, embryo colonization-competent PSCs. Our results highlight some of the requirements to achieve bona fide chimeric competency.
Asunto(s)
Reprogramación Celular , Estratos Germinativos/citología , Células Madre Pluripotentes Inducidas/citología , Factores de Transcripción de Tipo Kruppel/metabolismo , Animales , Blastocisto/citología , Blastocisto/metabolismo , Proliferación Celular , Supervivencia Celular , Quimera/metabolismo , Epigénesis Genética , Perfilación de la Expresión Génica , Humanos , Factor 4 Similar a Kruppel , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Conejos , Transducción de SeñalRESUMEN
Conventional rabbit embryonic stem cell (ESC) lines are derived from the inner cell mass (ICM) of pre-implantation embryos using methods and culture conditions that are established for primate ESCs. In this study, we explored the capacity of the rabbit ICM to give rise to ESC lines using conditions similar to those utilized to generate naive ESCs in mice. On single-cell dissociation and culture in fibroblast growth factor 2 (FGF2)-free, serum-supplemented medium, rabbit ICMs gave rise to ESC lines lacking the DNA-damage checkpoint in the G1 phase like mouse ESCs, and with a pluripotency gene expression profile closer to the rabbit ICM/epiblast profiles. These cell lines can be converted to FGF2-dependent ESCs after culture in conventional conditions. They can also colonize the rabbit pre-implantation embryo. These results indicate that rabbit epiblast cells can be coaxed toward different types of pluripotent stem cells and reveal the dynamics of pluripotent states in rabbit ESCs.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Animales , Biomarcadores , Blastocisto/citología , Blastocisto/metabolismo , Técnicas de Cultivo de Célula , Ciclo Celular , Diferenciación Celular/genética , Línea Celular , Autorrenovación de las Células/genética , Células Cultivadas , Biología Computacional/métodos , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Quinasas Janus/metabolismo , Factor Inhibidor de Leucemia/metabolismo , MicroARNs/genética , Conejos , Transducción de Señal , TranscriptomaRESUMEN
Leukemia inhibitory factor (LIF)/STAT3 signalling is a hallmark of naive pluripotency in rodent pluripotent stem cells (PSCs), whereas fibroblast growth factor (FGF)-2 and activin/nodal signalling is required to sustain self-renewal of human PSCs in a condition referred to as the primed state. It is unknown why LIF/STAT3 signalling alone fails to sustain pluripotency in human PSCs. Here we show that the forced expression of the hormone-dependent STAT3-ER (ER, ligand-binding domain of the human oestrogen receptor) in combination with 2i/LIF and tamoxifen allows human PSCs to escape from the primed state and enter a state characterized by the activation of STAT3 target genes and long-term self-renewal in FGF2- and feeder-free conditions. These cells acquire growth properties, a gene expression profile and an epigenetic landscape closer to those described in mouse naive PSCs. Together, these results show that temporarily increasing STAT3 activity is sufficient to reprogramme human PSCs to naive-like pluripotent cells.
Asunto(s)
Células Madre Embrionarias/fisiología , Regulación de la Expresión Génica/fisiología , Células Madre Pluripotentes/fisiología , Factor de Transcripción STAT3/metabolismo , Animales , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Nutrientes , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Factor Inhibidor de Leucemia/genética , Factor Inhibidor de Leucemia/metabolismo , Ratones , Análisis por Matrices de Proteínas , Factor de Transcripción STAT3/genética , Transducción de Señal , Tamoxifeno/farmacologíaRESUMEN
Krüppel-like factors (Klf) 4 and 5 are two closely related members of the Klf family, known to play key roles in cell cycle regulation, somatic cell reprogramming and pluripotency. Here we focus on the functional divergence between Klf4 and Klf5 in the inhibition of mouse embryonic stem (ES) cell differentiation. Using microarrays and chromatin immunoprecipitation coupled to ultra-high-throughput DNA sequencing, we show that Klf4 negatively regulates the expression of endodermal markers in the undifferentiated ES cells, including transcription factors involved in the commitment of pluripotent stem cells to endoderm differentiation. Knockdown of Klf4 enhances differentiation towards visceral and definitive endoderm. In contrast, Klf5 negatively regulates the expression of mesodermal markers, some of which control commitment to the mesoderm lineage, and knockdown of Klf5 specifically enhances differentiation towards mesoderm. We conclude that Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in murine ES cells.
