Cell-secreted soluble factor signaling in a diffusion dominant microenvironment plays an important role on early stage differentiation of pluripotent stem cells in vivo. In this study, we utilized a membrane-based two-chambered microbioreactor (MB) to differentiate mouse embryonic stem cells (mESCs) in a diffusion dominant microenvironment of the top chamber while providing enough nutrient through the bottom chamber. Speculating that accumulated FGF4 in the small top chamber will augment neuronal differentiation in the MB culture, we first differentiated mESCs for 8 days by using a chemically optimized culture medium for neuronal induction. However, comparison of cellular morphology and expression of neuronal markers in the MB with that in the 6-well plate (6WP) indicated relatively lower neuronal differentiation in the MB culture. Therefore, to investigate whether microenvironment in the MB facilitates non-neuronal differentiation, we differentiated mESCs for 8 days by using chemically defined basal medium. In this case, differentiated cell morphology differed markedly between the MB and 6WP cultures: epithelial sheet-like morphology in the MB, whereas rosette morphology in the 6WP. Expression of markers from the three germ layers indicated lower neuronal but higher meso- and endo-dermal differentiation of mESCs in the MB than the 6WP culture. Moreover, among various cell-secreted soluble factors, BMP4 expression was remarkably upregulated in the MB culture. Inhibition of BMP4 signaling demonstrated that enhanced effect of upregulated BMP4 was responsible for the prominent meso- and endo-dermal differentiation in the MB. However, in the 6WP, downregulated BMP4 had a minimal influence on the differentiation behavior. Our study demonstrated utilization of a microbioreactor to modulate the effect of cell-secreted soluble factors by autoregulation and thereby inducing alternative self-capability of mESCs. Understanding and implementation of autoregulation of soluble factors similar to this study will lead to the development of robust culture systems to control ESC behavior.
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March 2012
Research Article|
March 07 2012
Induction of alternative fate other than default neuronal fate of embryonic stem cells in a membrane-based two-chambered microbioreactor by cell-secreted BMP4
Mohammad Mahfuz Chowdhury;
Mohammad Mahfuz Chowdhury
a)
Institute of Industrial Science, The University of Tokyo
, Tokyo, Japan
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Hiroshi Kimura;
Hiroshi Kimura
Institute of Industrial Science, The University of Tokyo
, Tokyo, Japan
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Teruo Fujii;
Teruo Fujii
Institute of Industrial Science, The University of Tokyo
, Tokyo, Japan
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Yasuyuki Sakai
Yasuyuki Sakai
Institute of Industrial Science, The University of Tokyo
, Tokyo, Japan
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a)
Author to whom correspondence should be addressed. Electronic mail: [email protected]. Telephone: +81-3-5452-6352. Fax: +81-3-5452-6353.
Biomicrofluidics 6, 014117 (2012)
Article history
Received:
December 11 2011
Accepted:
February 24 2012
Citation
Mohammad Mahfuz Chowdhury, Hiroshi Kimura, Teruo Fujii, Yasuyuki Sakai; Induction of alternative fate other than default neuronal fate of embryonic stem cells in a membrane-based two-chambered microbioreactor by cell-secreted BMP4. Biomicrofluidics 1 March 2012; 6 (1): 014117. https://doi.org/10.1063/1.3693590
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