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Frontiers of Materials Science

Front Mater Sci    2013, Vol. 7 Issue (1) : 51-61     DOI: 10.1007/s11706-013-0194-8
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Maintenance and induction of murine embryonic stem cell differentiation using E-cadherin-Fc substrata without colony formation
Qing-Yuan MENG1,2, Toshihiro AKAIKE2()
1. Department of Materials Science and Engineering, Tsinghua University, Beijing 100084, China; 2. Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
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Abstract

Induced embryonic stem (ES) cells are expected to be promising cell resources for the observation of the cell behaviors in developmental biology as well as the implantation in cell treatments in human diseases. A recombinant E-cadherin substratum was developed as a cell recognizable substratum to maintain the ES cells’ self-renewal and pluripotency at single cell level. Furthermore, the generation of various cell lineages in different germ layers, including hepatic or neural cells, was achieved on the chimeric protein layer precisely and effectively. The induction and isolation of specific cell population was carried out with the enhancing effect of other artificial extracellular matrices (ECMs) in enzyme-free process. The murine ES cell-derived cells showed highly morphological similarities and functional expressions to matured hepatocytes or neural progenitor cells.

Keywords cell adhesion molecule      embryonic stem cell differentiation      E-cadherin-Fc      single cell culture system     
Corresponding Authors: AKAIKE Toshihiro,Email:takaike@bio.titech.ac.jp   
Issue Date: 05 March 2013
 Cite this article:   
Qing-Yuan MENG,Toshihiro AKAIKE. Maintenance and induction of murine embryonic stem cell differentiation using E-cadherin-Fc substrata without colony formation[J]. Front Mater Sci, 2013, 7(1): 51-61.
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http://journal.hep.com.cn/foms/EN/10.1007/s11706-013-0194-8
http://journal.hep.com.cn/foms/EN/Y2013/V7/I1/51
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Fig.1  Production of E-cad-Fc chimeric protein. The chimera is constructed consisting of E-cadherin extracellular domain and IgG Fc region. And the ES cells cultured on the fusion protein show single cell morphology.
Fig.2  Adsorption behaviors of E-cad-Fc on polystyrene surface. The fusion protein adsorbed on PS using QCM method is revealed. The adsorption of E-cad-Fc on PS is shown using ELISA assay. The adsorption isotherm curve is described by Langmuir adsorption equation, in which the points (?) and the solid lines (–) represent the experimental results and the calculation models respectively.
Fig.3  ES cells maintain the undifferentiated phenotype on E-cad-Fc-coated plates. The ES cells cultured on E-cad-Fc and gelatin were stained with Oct3/4 antibody (Day5). The expression of Oct3/4, Rex1 and nanog was analyzed by RT-PCR with GAPDH as house-keeping gene. The proliferative capability of undifferentiated cells on gelatin and E-cad-Fc is evaluated, in which the number of adhered cells after 4 h on each matrix is considered as 1 × 10. Data are mean±SD, = 3. Scale bar: 50 μm. (Figs. 3(a) and 3(b): Reproduced with permission from Ref. [], Copyright 2006 Public Library of Science (PLoS); Fig. 3(c): Reproduced with permission from Ref. [], Copyright 2012 Elsevier)
Fig.4  Pluripotency of ES cells on E-cad-Fc-coated surface. H&E staining of teratomas showed the differentiation into ectoderm (epidermis), mesoderm (cartilage, and striated muscle cells) and endoderm (ciliated columnar epithelium, possibly bronchial epithelium). Differentiation into ectoderm was confirmed by specific staining for the neural markers βIII-tubulin, GFAP, Neurofilament-M and GAP-43. Scale bar: 50 μm. (Figs. 4(a) and 4(b): Reproduced with permission from Ref. [], Copyright 2006 Public Library of Science (PLoS))
Fig.5  The differentiation of mES cells toward hepaticyte-like cells on gelatin, E-cad-Fc, E-cad-Fc/PVLA hybrid matrix. E-cadherin and AFP signals are demonstrated in red and green, respectively on day 9 at single cell level using laser scanning confocal microscopy. The transcription levels of markers in hepatic progenitor cells (AFP, ALB, CK18 and HNF-4α) and E-cadherin are analyzed using RT-PCR with β-actin as the housekeeping gene. The immunofluorescence images show that the differentiated cells expressed high levels of Sox17 (red) and E-cadherin (red) on hybrid matrix on day 5. The immunofluorescence images of ALB (red) on day 16 are revealed on gelatin, E-cad-Fc and co-immobilized matrix. The immunofluorescence images reveal the ALB (red) and ASGPR (red) expression on day 24 in the differentiated cells cultured on different matrices. DAPI is used for nuclear staining (blue). The transcription markers of early hepatic stage (AFP, ALB, CK18 and HNF-4α) and final differentiating stage to hepatocytes (ASGPR and mTO) together with E-cadherin are analyzed using RT-PCR using β-actin as the house-keeping gene. Scale bar: 50 μm. (Fig. 5(a): Reproduced with permission from Ref. [], Copyright 2011 Elsevier; Figs. 5(b)-5(f): Reproduced with permission from Ref. [], Copyright 2012 Elsevier)
Fig.6  The enrichment of hepatocyte population after re-seeding onto PVLA matrix. The morphology of differentiated ES cells on gelatin and E-cad-Fc/PVLA hybrid matrix are compared with the differentiated cells that are re-seeded onto PVLA and maintained for three days. The morphology of primary hepatocytes cultured on gelatin, co-immobilized matrix and PVLA for three days are used as control. The transcript markers of differentiated cells on gelatin, E-cad-Fc and hybrid matrix on day 24 (marked as ‘before’) are compared with the differentiated cells (marked as ‘after’) that are re-seeded after 22 days of differentiation and maintained on PVLA for three days. The albumin secretion is measured for the differentiated cells on various matrices and re-seeded cells after three days incubation on PVLA with the primary hepatocytes on PVLA for three days as control. Relative glycogen storage amount in differentiated cells before and after re-seeding is compared with the one of the primary hepatocytes on PVLA for three days. Data are mean±SD, = 3. Abbreviations: P, PVLA; G, gelatin; E, E-cad-Fc; EP, E-cad-Fc/PVLA hybrid matrix; ES, mouse ES cells; PH, primary hepatocyte; D3, day 3; D22, day 22. Scale bar: 50 μm. (Figs. 6(a)-6(d): Reproduced with permission from Ref. [], Copyright 2012 Elsevier)
Fig.7  Differentiation of neural progenitor cells into βIII-tubulin expressing neural cells. mES and miPS cell-derived neural cells were stained with βIII-tubulin (red) after differentiation induction for 12 days. Two different culture conditions (low cell density and high cell density) were used. mES cells in high cell density showed confluent growth with elongated neurites covering all the surfaces coated with E-/N-cad-Fc. Cells on gelatin formed elongated neurites from the cluster. Scale bar: 200 μm. ( mES cells in low cell density on gelatin and cadherin-based co-immobilized substratum expressed βIII-tubulin. Scale bar: 50 μm. Transcript expression for Pax6, MAP2, TH, and GFAP was determined by RT-PCR in 8 and 10 days of differentiation. The expression level was normalized using house-keeping gene, β-actin. Abbreviations: microtubule associated protein 2, MAP2; tyrosine hydroxylase, TH; glial fibrillary acidic protein, GFAP; undifferentiated, UD. (Figs. 7(a)-7(c): Reproduced with permission from Ref. [], Copyright 2012 Elsevier)
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