Imaging of STAT3 Signaling Pathway During Mouse Embryonic Stem Cell Differentiation

Abstract

Signal transducers and activators of transcription 3 (STAT3) is a pleiotropic transcription factor involved in a variety of physiological processes. STAT3 acts as a key transcriptional determinant of mouse embryonic stem (ES) cell self-renewal and plays a pivotal function in early mammalian embryogenesis because the development of many organs requires STAT3 activation. However, little is known about the role of STAT3 function during ES cell differentiation. To answer this question, we built a lentiviral construct with 7-repeat STAT3-binding sequence (enhancer) and minimal TA (promoter) driving renilla luciferase and monomeric red fl uorescence protein (Rluc-mRFP), followed by a constitutive cytomegalovirus promoter driving green fl uorescent protein as a selection marker. The specifi city of our custom-designed 7-repeat STAT3 reporter construct was fi rst confi rmed by cotransfection with constitutively active version of STAT3 (STAT3C) into human embryonic kidney 293T cells. Next, a mouse ES cell line stably transduced with STAT3 reporter construct was isolated. This ES cell line showed a tight response in reporter gene expression with leukemia inhibitory factor (LIF) induction and was chosen as a developmental model for the STAT3 functional study. Using serial noninvasive bioluminescence imaging, we showed that the onset of embryoid body (EB) formation involved inhibition of STAT3 activity. However, during differentiation, STAT3 activity steadily increased from day 5 to 14 and was reduced by day 21. STAT3 activity was also confi rmed separately by Western blots. Finally, phosphorylation of STAT3 was also found to correspond with cardiomyocyte differentiation. In summary, this is the fi rst study to monitor real-time STAT3 activity during ES cell differentiation. This genetically modifi ed line can be used to study the biological role of STAT3 during ES cell differentiation into different derivatives.

STAT3 and cardiac remodeling

“…Controversially to these reported data, other studies in ES cells point to STAT3 as an important factor for cardiomyogenesis within murine ES cells [86][87][88][89][90]. Whether this is a universal mechanism varies between species or depends on different laboratory conditions remains to be elucidated.…”

Midazolam Inhibits the Apoptosis of Astrocytes Induced by Oxygen Glucose Deprivation via Targeting JAK2-STAT3 Signaling Pathway

“…And the effects of HIF-1α, EPO and VEGF on cell survival likely involve the expression of JAK2/STAT3 signal pathway, which plays a crucial role in regulation of inflammatory reaction [13], the growth and differentiation of cells [14], and cells apoptosis [15]. IL-6 is a member of the neuropoietin family of cytokines which includes leukemia inhibitory factor, ciliary neurotrophic factor, cardiotrophin oncostatin M, and IL-11 [16,17].…”

Caveolin-1 Plays an Important Role in the Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells into Cardiomyocytes

“…A further understanding of the biochemical pathways involved in signaling transduction will hopefully provide new insights for the improvement of stem-cell based therapies. A number of previous studies have demonstrated that the inhibition of STAT3 activation by specific inhibitors drastically suppresses the differentiation of mESCs into cardiomyocytes induced by the coapplication of leukemia inhibitory factor and bone morphogenic protein 2 [50,51]. The involvement of activated STAT3 in the differentiation of BMSCs seems contradictory.…”