Overexpression of bone morphogenetic protein 4 in STO fibroblast feeder cells represses the proliferation of mouse embryonic stem cellsin vitro

Abstract

Embryonic stem cells (ESCs) can be propagated in vitro on feeder layers of mouse STO fibroblast cells. The STO cells secrete several cytokines that are essential for ESCs to maintain their undifferentiated state. In this study, we found significant growth inhibition of mouse ESCs (mESCs) cultured on STO cells infected with adenovirus containing a dominant-negative mutant form of IκB (rAd-dnIκB). This blockage of the NF-κB signal pathway in STO cells led to a significant decrease in [3H]thymidine incorporation and colony formation of mESCs. Expression profile of cytokines secreted from the STO cells revealed an increase in the bone morphogenetic protein4 (BMP4) transcript level in the STO cells infected with adenoviral vector encoding dominant negative IκB (rAd-dnIκB). These results suggested that the NF-κB signaling pathway represses expression of BMP4 in STO feeder cells. Conditioned medium from the rAd-dnIκB-infected STO cells also significantly reduced the colony size of mESCs. Addition of BMP4 prevented colony formation of mESCs cultured in the conditioned medium. Our finding suggested that an excess of BMP4 in the conditioned medium also inhibits proliferation of mESCs.

Growth inhibition of mouse embryonic stem (ES) cells on the feeders from domestic animals

“…The current system for mESCs culture uses mouse embryonic fibroblasts as a feeder supplemented with LIF, because LIF is an interleukin 6 class cytokine that affects cell growth by inhibiting differentiation (Ma et al, 2012). In addition, mESCs can be propagated in vitro on feeders of mouse STO cells, because the STO cells secrete several cytokines that are essential for mESCs to maintain their undifferentiated state, but after STO cells were infected with adenovirus containing a mutant form and overexpressed, the cytokine-bone morphogenetic protein 4 (BMP 4), overexpression of BMP 4 in STO feeder cells repressed the proliferation of mESCs in vitro (Kim et al, 2012). In this experiment, the feeders from goat, porcine and bovine embryonic fibroblasts inhibited the proliferation of mESCs, therefore, the results suggest that it probably provides a model for insight into BMP 4related cytokine signal pathway in goat, porcine and bovine embryonic fibroblasts.…”

“…The primary embryonic fibroblasts from 75-day-old and 6 cm long goat fetuses, four-month-old and 20 cm long bovine fetuses, 30-day-old and 8 cm long porcine fetuses, and 12.5-day-old and 5 mm long mouse fetuses were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) (Hyclone, China) with 10% fetal bovine serum (FBS) (Hyclone, Australia), respectively. After covering the dishes, the embryonic fibroblasts were treated for 2.5 h with 10 μg/ml mitomycin C (Sigma, America), digested with trypsin solution (Gibico, Australia), seeded at 1.2 × 10 5 cells/cm 2 in dishes treated with 0.1% gelatin, and cultured at 37.5°C (Kim et al, 2012;Ma et al, 2012). 29th generation mESCs from male R1 cell line were seeded at 1 × 10 5 cells/dish on the feeders from goat, bovine, porcine and mouse embryonic fibroblasts with stem cell medium consisting of 1000 U/ml LIF (Chemicon, America), 15% FBS and high glucose DMEM respectively, and cultured at 37.5°C.…”

The Role of Genetically Modified Human Feeder Cells in Maintaining the Integrity of Primary Cultured Human Deciduous Dental Pulp Cells

“…In contrast to MEFs, STO cells can continue to proliferate in vitro; however, their proliferation can be inhibited by MMC or gamma irradiation [ 17 ]. Moreover, feeder cells can be engineered to express and secrete growth factors, such as leukemia inhibitory factor (LIF), bone morphogenetic protein-4 (BMP4), and basic fibroblast growth factor (bFGF; also known as FGF-2), which are important for establishing and maintaining mouse and human ESCs [ 22 , 23 , 24 , 25 , 26 , 27 ]. These findings indicate that the addition of recombinant growth factors to the medium is unnecessary when ESCs are co-cultured with genetically engineered feeder cells capable of producing growth factors.…”