Activation of Hes1 and Msx1 in Transgenic Mouse Embryonic Stem Cells Increases Differentiation into Neural Crest Derivatives

KARLA MÉNDEZ-MALDONADO; VEGA-LOPEZ, GUILLERMO A.; SARA CABALLERO; AYBAR, MANUEL J.; IVÁN VELASCO

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES

MOLECULAR DIVERSITY PRESERVATION INTERNATIONAL-MDPI

Lugar: Basel; Año: 2018 vol. 19 p. 1 - 1

1422-0067

Pluripotent cells have been used as a model to understand some critical factors that dictate cell fate. Embryonic stem cells (ESC) can be modified genetically to express different transcriptional regulators. The unstimulated human glucocorticoid receptor is located in the cytoplasm, but hormone binding causes its nuclear translocation. This feature is useful for designing inducible fusion proteins taking advantage of its glucocorticoid binding domain (GR). In this work we generated constructs that allow expression of chimeric proteins that includes the ligand binding domain of GR fused to either mouse Hes1 (HGR), mouse Msx1 (MGR), double transgenic for Hes1 and Msx1 (HGR+MGR) or GR only. We performed transient transfections of mouse fibroblasts with GR, HGR or MGR, and observed that the stimulation of GR with dexamethasone (Dex) caused the re-localization of the protein from the cytoplasm to the nucleus. Subsequently, stable mouse ESC lines containing the listed constructs were produced after antibiotic selection; these lines continue expressing pluripotency markers and were tested for Neural Crest (NC) differentiation. After differentiation for 10 days, all cell lines significantly decreased the expression of Sox2 and up-regulated Sox9, Snai1 and Msx1, indicating NC commitment. Addition of Dex to promote nuclear translocation of the different constructs during NC differentiation showed that Collagen IIa transcripts were increased in MGR cells; the co-activation in HGR+MGR cells by Dex caused a significant increase in Smooth Muscle Actin mRNA (α-Sma). Immunostaining of differentiated cells showed that in HGR+MGR cells Dex induced higher proportions of β-Tubulin III and α-Smooth Muscle Actin positive cells. These findings indicate that nuclear translocation of Hes1 and Msx1 increase smooth muscle differentiation during NC induction of pluripotent cells.