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Volume 9, Pages 884-890
Short Communication

Hepatocyte Differentiation from Human ES Cells using the Simple Embryoid Body Formation Method and the Staged-Additional Cocktail

1Department of Histology and Embryology, Shinshu University, School of Medicine, 3-1-1 Asahi Matsumoto, Nagano 390-8621, Japan
2Bourbon Corporation, 44-2-14 Matsunami, Kashiwazaki City, Niigata 945-8611, Japan
3NOF Corporation, 5-10 Tokodai, Tsukuba, Ibaraki 300-2635, Japan
4Nissui Pharmaceutical Co., Ltd, 1075-2 Hokunanmoro, Yuki-shi, Ibaraki 307-0036, Japan

Received 3 June 2009; Revised 22 July 2009; Accepted 3 August 2009

Academic Editor: Margaret Tzaphlidou

Copyright © 2009 Katsunori Sasaki et al.


To induce hepatocytes from human embryonic stem (hES) cells easily and effectively, a simple suspension culture method that separates ES colonies with a scraper and transfers them into newly developed, nonadherent MPC (2-methacryloyloxyethyl phosphorylcholine) plates, and the staged-additional cocktail method, including growth factors, cytokines, and Lanford serum-free medium, were developed and evaluated mainly by morphological analysis. The formed embryoid bodies (EBs) showed compact cellular agglomeration until day 4 and later formed coeloms in their interior. RT-PCR (reverse transcriptase-polymerase chain reaction) analysis showed that they are gene markers of the three germ layers. Mesenchymal cells with rough endoplasmic reticulum (rER) and extracellular matrix (ECM), and without junctions, were recognized in the interior of the EBs by transmission electron microscopy (TEM) in addition to epithelial cells. When they were stimulated by the staged-additional cocktail, they expressed albumin-positive immunoreactivity, indocyanine green (ICG) uptake, and typical ultrastructures of the hepatocytes, including bile canaliculi. These results indicate that these combined methods promote EB formation and hepatocyte differentiation from hES cells.