Pericellular matrix of decidua-derived mesenchymal cells: A potent human-derived substrate for the maintenance culture of human ES cells
Version of Record online: 21 APR 2009
Copyright © 2009 Wiley-Liss, Inc.
Volume 238, Issue 5, pages 1118–1130, May 2009
How to Cite
Nagase, T., Ueno, M., Matsumura, M., Muguruma, K., Ohgushi, M., Kondo, N., Kanematsu, D., Kanemura, Y. and Sasai, Y. (2009), Pericellular matrix of decidua-derived mesenchymal cells: A potent human-derived substrate for the maintenance culture of human ES cells. Dev. Dyn., 238: 1118–1130. doi: 10.1002/dvdy.21944
- Issue online: 21 APR 2009
- Version of Record online: 21 APR 2009
- Manuscript Accepted: 27 FEB 2009
- Kobe Cluster Project
- Leading Project
Additional Supporting Information may be found in the online version of this article.
|DVDY_21944_sm_SupFigS1.tif||766K||Supp. Fig. 1. Schematic of the preparation of decidua-derived mesenchymal cells and PCM-DM. Decidual tissues were separated manually from the fetal membrane and cut into small pieces with scissors. After enzymatic digestion, the dissociated cells were collected and seeded onto culture plates.|
|DVDY_21944_sm_SupFigS2.tif||6717K||Supp. Fig. 2. Decidua-derived mesenchymal cells (DMCs) can be used as a feeder layer for human ES cell maintenance culture. hES cells were maintained on mitomycin C-treated human DMCs (20,000 cells/cm2) for eight passages. A: Phase contrast view of hES cell colonies on DMCs. B: Growth curve for hES cells on DMCs. C–F: hES cells on DMCs expressed undifferentiated cell markers, SSEA4 (C), Oct3/4, (D), TRA-1-60 (E), and Nanog (F). Scale bar = 100 μm.|
|DVDY_21944_sm_SupFigS3.tif||567K||Supp. Fig. 3. Schematic of preparation of the pericellular matrix of decidua-derived mesenchymal cells (PCM-DM). A: Preparation of decidual mesenchymal cells and their pericellular matrix. B: Culture of hES cells on PCM-DM.|
|DVDY_21944_sm_SupFigS4.tif||4795K||Supp. Fig. 4. PCM-DM supports the maintenance of hES cells in chemically defined medium (StemPro) after long-term culture. hES cells cultured on PCM-DM in StemPro (A; a high-magnification view) expressed undifferentiated-state markers such as TRA-1-60 (B) and SSEA4 (C), but not the early differentiation marker SSEA1 (D; unlike mES cells, hES cells express SSEA1 after starting differentiation). Scale bar = 100 μm.|
|DVDY_21944_sm_SupFigS5.tif||10976K||Supp. Fig. 5. hES cells cultured on PCM-DM do not express most of the differentiated markers. hES colonies, cultured on PCM-DM in StemPro medium, were stained with antibodies for differentiated markers CD133 (A), N-cadherin (B), CK14 (C), Pax6 (G), Brachyury (H), and HNF3β (I). Differentiated cells from hES cells (H, I, and J) are used as positive controls to hES staining (D–F; see Experimental Procedures section for differentiation). Scale bar = 100 μm.|
|DVDY_21944_sm_SupFigS6.tif||724K||Supp. Fig. 6. FACS analysis showing hES cells cultured on PCM-DM keep undifferentiated state. Most of the cells expressed SSEA3, SSEA4, and Tra-1-60 (markers for the undifferentiated state) but not SSEA1 (marker for the differentiated state). Black lines in histogram plots represent staining without primary antibody (negative control).|
|DVDY_21944_sm_SupFigS7.tif||2588K||Supp. Fig. 7. PCM-DM contains a substantial amount of fibronectin and collagen IV and little laminin or heparan sulfate proteoglycan. Serial twofold dilutions of each protein were subjected to dot blot immunoassay. The starting mount of each protein (μg/dot) was indicated under the name of each applied sample. col IV, collagen IV; hLaminin, human laminin; mLaminin, mouse laminin; Heparan S, heparan sulfate proteoglycan.|
|DVDY_21944_sm_SupFigS8.tif||2869K||Supp. Fig. 8. hES cells were cultured on culture wells coated with gelatin, laminin (1 μg/cm2), PCM-DM, PCM-DM + laminin as in Figure 2A. No remarkable differences were seen between PCM-DM and PCM-DM + laminin.|
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