In order to provide the basis for developing practical mouse embryonic stem cells (mESCs) culture method, how the endogenous level of self-renewal-stimulating factor genes was altered in the mESCs by different extracellular signaling was investigated in this study. For different extracellular signaling, mESCs were cultured in 2 dimension (D), 3D and integrin-stimulating 3D culture system in the presence or absence of leukemia inhibitory factor (LIF) and transcriptional level of Lif, Bmp4 and Wnt3a was evaluated in the mESCs cultured in each system. The expression of three genes was significantly increased in 3D system relative to 2D system under LIF-containing condition, while only Wnt3a expression was increased by 3D culture under LIF-free condition. Stimulation of integrin signaling in mESCs within 3D system with exogenous LIF significantly up-regulated transcriptional level of Bmp4, but did not induce transcriptional regulation of Lif and Wnt3a. In the absence of LIF inside 3D system, the expression of Lif and Bmp4 was significantly increased by integrin signaling, while it significantly decreased Wnt3a expression. Finally, the signal from exogenous LIF significantly caused increased expression of Lif in 2D system, decreased expression of Bmp4 in both 2D and 3D system, and decreased expression of Wnt3a in integrin-stimulating 3D system. From these results, we identified that endogenous expression level of self-renewal-stimulating factor genes in mESCs could be effectively regulated through artificial and proper manipulation of extracellular signaling. Moreover, synthetic 3D niche stimulating endogenous secretion of self-renewal-stimulating factors will be able to help develop growth factor-free maintenance system of mESCs.

ABSTRAC   INTRODUCTION   MATERIALS AND METHODS    Culture of Mouse Embryonic Stem Cells (mESCs)    Adhesion Peptide Synthesis and Purification    Formation of Poly(Ethylene Glycol) (PEG)-Based Hydrogeland Conjugation of Adhesion Peptides    Incorporation of mESCs into PEG-hydrogel and Culture    Analysis of Relative Gene Expression    Statistical Analysis   RESULTS    Effects of 3D or 2D Culture System on the Endogenous Expression of Self-Renewal-Stimulating Factor Genes in mESCs    Effects of Integrin Signaling inside 3D Culture System on the Endogenous Expression of Self-Renewal-StimulatingFactor Genes in mESCs    Effects of Stat3 Signaling under 2D, 3D and Integrin-Stimulating 3D Culture System on the Endogenous Expressionof Self-Renewal-Stimulating Factor Genes in mESCs   DISCUSSION   REFERENCES

• Seung Pyo Gong(Department of Marine Biomaterials and Aquaculture, Pukyong National University, Busan 608-737, Korea)
• Seung Tae Lee(Department of Animal Biotechnology, Kangwon National University, Chuncheon 200-701, Korea, Institute of Animal Resources, Kangwon National University, Chuncheon 200-701, Korea, Stem Cell Institute, Kangwon National University, Chuncheon 200-701, Korea) Corresponding author