Effect of Gsk3 inhibitor CHIR99021 on aneuploidy levels in rat embryonic stem cells

Abstract

Germline competent embryonic stem (ES) cells can serve as a tool to create genetically engineered rat strains used to elucidate gene function or provide disease models. In optimum culture conditions, ES cells are able to retain their pluripotent state. The type of components present and their concentration in ES cell culture media greatly influences characteristics of ES cells including the ability to maintain the cells in a pluripotent state. We routinely use 2i media containing inhibitors CHIR99021 and PD0325901 to culture rat ES cells. CHIR99021 specifically inhibits the Gsk3β pathway. We have found that the vendor source of CHIR99021 has a measurable influence on the level of aneuploidy seen over time as rat ES cells are passaged. Karyotyping of three different rat ES cell lines passaged multiple times showed increased aneuploidy when CHIR99021 from source B was used. Mass spectrometry analysis of this inhibitor showed the presence of unexpected synthetic small molecules, which might directly or indirectly cause increases in chromosome instability. Identifying these molecules could further understanding of their influence on chromosome stability and indicate how to improve synthesis of this media component to prevent deleterious effects in culture.

Dynamic stem cell states: naïve to primed pluripotency in rodents and humans

“…With the development of naïve conditions and efforts to endow these cells with more features of naivety, one question that emerges is "how much naivety is needed and is there a dark side for permanently maintaining PSCs under naive conditions?" Rodent ESCs expanded in 2i/LIF have an increased tendency for acquiring genomic abnormalities 41 , and it remains unclear whether these alterations occur as a by-product of nonspecific activity of small molecule inhibitors used 95,116 , or as a direct result of intrinsic molecular features of naïve pluripotency (e.g. increased endogenous retroviral elements (ERVs) activity, reduction in epigenetic repressive marks).…”

A novel matrine derivate inhibits differentiated human hepatoma cells and hepatic cancer stem-like cells by suppressing PI3K/AKT signaling pathways

“…Accordingly, MASM treatment increased phosphorylation of β-catenin (Ser33/Ser37/Thr41) and decreased β-catenin protein levels, while phosphorylation of β-catenin (Ser675) remained unchanged ( Figure 6B). Conversely, CHIR [21] (a GSK3β inhibi-tor) increased phosphorylation of GSK3β (Ser9) and suppressed phosphorylation of β-catenin (Ser33/Ser37/Thr41) but not of β-catenin (Ser675); β-catenin protein levels increased. When co-treated with CHIR, the effects of MASM on GSK3β phosphorylation, β-catenin phosphorylation (Ser33/Ser37/ Thr41), and β-catenin accumulation were attenuated ( Figure 6B and 6D).…”

Cyclin G2 Suppresses Glomerulosclerosis by Regulating Canonical Wnt Signalling

“…To determine the regulatory mechanisms for cyclin G2 in renal glomerulosclerosis, we activated the canonical Wnt signalling pathway using the GSK3 β inhibitor CHIR99021 [ 24 ]. In the absence of CHIR99021, cyclin G2 overexpression inhibited the expression of proteins associated with canonical Wnt signalling and glomerulosclerosis.…”