Abstract
Studies have demonstrated that STAT3 is essential in maintaining self-renewal of embryonic stem cells (ESCs) and modulates ESC differentiation. However, there is still lack of direct evidence on STAT3 functions in ESCs and embryogenesis because constitutive STAT3 knockout (KO) mouse is embryonic lethal at E6.5–E7.5, prior to potential functional role in early development can be assessed. Therefore, in this study, two inducible STAT3 ESC lines were established, including the STAT3 knockout (InSTAT3 KO) and pSTAT3 overexpressed (InSTAT3 CA) using Tet-on inducible system in which STAT3 expression can be strictly controlled by doxycycline (Dox) stimulation. Through genotyping, deletion of STAT3 alleles was detected in InSTAT3 KO ESCs following 24 hours Dox stimulation. Western blot also showed that pSTAT3 and STAT3 protein levels were significantly reduced in InSTAT3 KO ESCs while dominantly elevated in InSTAT3 CA ECSs upon Dox stimulation. Likewise, it was found that STAT3-null ESCs would affect the differentiation of ESCs into mesoderm and cardiac lineage. Taken together, the findings of this study indicated that InSTAT3 KO and InSTAT3 CA ESCs could provide a new tool to clarify the direct targets of STAT3 and its role in ESC maintenance, which will facilitate the elaboration of the mechanisms whereby STAT3 maintains ESC pluripotency and regulates ESC differentiation during mammalian embryogenesis.
Highlights
Signal transducer and activator of transcription 3 (STAT3) belongs to the STAT family and is the only member essential for early embryo development since STAT3 null mice were found to be embryonic lethal at E6.5 to E7.5 [1]
Time course Quantitative Real-Time PCR (qRT-PCR) suggested that deletion of STAT3 resulted in decreased expression of mesodermal markers such as T-Brachyury, mesoderm posterior basic helix-loop-helix transcription factor 1 (Mesp1), Fgf5, and Hand1 during differentiation as compared to uninduced embryoid bodies (EBs) (Figure 1(c)). These results demonstrated that STAT3 is required for early embryonic stem cells (ESCs) differentiation into cardiaccommitted mesoderm
We have established an in vitro inducible STAT3 knockout and pSTAT3 overexpressed ESC system in which STAT3 expression can be strictly controlled by Dox stimulation, which facilitate the study of the direct functions or targets of STAT3 during modulation of ESC differentiation (Figure 2(a))
Summary
Signal transducer and activator of transcription 3 (STAT3) belongs to the STAT family and is the only member essential for early embryo development since STAT3 null mice were found to be embryonic lethal at E6.5 to E7.5 [1]. STAT3 is an essential mediator downstream of leukemia inhibitory factor (LIF) in maintaining ESC pluripotency. Studies have proved that STAT3 activation is essential to maintain ESC self-renewal [13,14,15]. Through a conditional active STAT3 system, it was found that STAT3 activation is adequate to maintain the self-renewal of ESCs [15] It is unclear how downstream targets of LIF/JAK/STAT3 signaling play a preeminent role in maintaining ESC pluripotency by interacting with the master regulators of pluripotency factors for ESCs, namely, Oct, Nanog, and Sox2 [16, 17]. STAT3 has been reported that it directly regulates Oct and Nanog transcription to maintain pluripotency of ESCs and iPSCs. Knockdown of STAT3 in ESCs leads to substantial reduction of Oct expression. Expression of STAT3 dominant-negative mutant using an inducible promoter in ESCs abolishes the self-renewal of ESCs as maintained by LIF and promotes differentiation [13, 14]
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