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Overexpression of bone morphogenetic protein 4 in STO fibroblast feeder cells represses the proliferation of mouse embryonic stem cells in 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 I?B (rAd-dnI?B). This blockage of the NF-?B signal pathway in STO cells led to a significant decrease in [(3)H]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 I?B (rAd-dnI?B). These results suggested that the NF-?B signaling pathway represses expression of BMP4 in STO feeder cells. Conditioned medium from the rAd-dnI?B-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.

SUBMITTER: Kim GH

PROVIDER: S-EPMC3406291 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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

Kim Gu-Hee GH Lee Gong-Rak GR Choi Hyung-Im HI Park Neung-Hwa NH Chung Hun Taeg HT Han In-Seob IS

Experimental & molecular medicine 20120701 7

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 [(3)H]thymidi ...[more]

PMID: 22595769

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Project description:Successful realization of the enormous potential of pluripotent stem cells in regenerative medicine demands the development of well-defined culture conditions. Maintenance of embryonic stem cells (ESCs) typically requires co-culture with feeder layer cells, generally mouse embryonic fibroblasts (MEFs). Concerns about xenogeneic pathogen contamination and immune reaction to feeder cells underlie the need for ensuring the safety and efficacy of future stem cell-based products through the development of a controlled culture environment. To gain insight into the effectiveness of MEF layers, here we have developed a biomimetic synthetic feeder layer (BSFL) that is acellular and replicates the stiffness and topography of MEFs. The mechanical properties of MEFs were measured using atomic force microscopy. The average Young's modulus of the MEF monolayers was replicated using tunable polyacrylamide (PA) gels. BSFLs replicated topographical features of the MEFs, including cellular, subcellular, and cytoskeletal features. On BSFLs, mouse ESCs adhered and formed compact round colonies; similar to on MEF controls but not on Flat PA. ESCs on BSFLs maintained their pluripotency and self-renewal across passages, formed embryoid bodies and differentiated into progenitors of the three germ layers. This acellular biomimetic synthetic feeder layer supports stem cell culture without requiring co-culture of live xenogeneic feeder cells, and provides a versatile, tailorable platform for investigating stem cell growth.Embryonic stem cells have enormous potential to aid therapeutics, because they can renew themselves and become different cell types. This study addresses a key challenge for ESC use - growing them safely for human patients. ESCs typically grow with a feeder layer of mouse fibroblasts. Since patients have a risk of immune response to feeder layer cells, we have developed a material to mimic the feeder layer and eliminate this risk. We investigated the influence of feeder layer topography and stiffness on mouse ESCs. While the biomimetic synthetic feeder layer contains no live cells, it replicates the stiffness and topography of feeder layer cells. Significantly, ESCs grown on BSFLs retain their abilities to grow and become multiple cell types.

Dataset Information

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

Publications

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

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