| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 1, 2002 as doi:10.1096/fj.01-0806fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Program in Tissue Engineering and Department of Parasitology,
* Third Department of Internal Medicine, Nara Medical University, Kashihara, Nara, Japan;
Second Department of Biochemistry,
Second Department of Anatomy, Osaka City University, Medical School, Osaka, Japan; and
Department of Anatomy and Physiology, Osaka Perfecture Collage of Health Sciences, Habikino, Osaka, Japan
2Correspondence: Program in Tissue Engineering and Department of Parasitology, Nara Medical University, 840 Shijo-Cho, Kashihara, Nara 634–8521, Japan. E-mail: ishizaka{at}naramed-u.ac.jp
SPECIFIC AIM
Cells differentiated from embryonic stem (ES) cells rather than tissue stem cells, have abundant proliferative capacity, will provide new opportunities for tissue transplantation. We have attempted to generate ES cell-derived hepatocytes expressing liver-specific functional properties by use of ES cell technology.
PRINCIPAL FINDINGS
1. Detection of albumin and glycogen in hepatocyte nuclear factor (HNF) -3ß-transfected ES(HTC) cells
HTC cells were cultured in 
-MEM (minimum essential medium) containing 10% fetal bovine serum (FBS), fibroblast growth factor 2 (FGF-2), dexamethasone, L-ascorbic-2-phosphate, and nicotinamide in 96-well round-bottomed plates for spheroid formation or plastic flasks containing chitin fibers. The liver-specific serum albumin production was detected in HTC spheroids at least 4 months after the initial culture with FGF-2 but not ES cells by immunostaining with anti-mouse albumin antibodies. The presence of glycogen was also observed in HTC spheroids but not in ES cells as positivity for -amylase-sensitive periodic acid Schiff staining.
2. Transcription of liver-enriched proteins in HTC cells
The expression of mRNA of albumin, complement C3, cytochrome P450 (P450), phosphoenolpyruvate carboxykinase (PEPCK), and peroxisomal membrane protein 1-like protein (PXMP1-L) as differentiated hepatocyte-enriched proteins was detected in HTC cells 1 and 4 months after the initial culture of HNF-3ß-transfected ES cells by RT-PCR (Fig. 1
).
|
3. Hepatic morphology of HTC cells
Cytokeratin (CK) 18 positive cells were observed on the outer layers of HTC spheroids. HTC cells were negative for anti-CK 19 antibodies as a biliary-type marker. Electron microscopy demonstrated that HTC cells possessed hepatocyte-like morphometric characterizations associated with a great number of mitochondria and highly organized rough endoplasmic reticulum, large quantities of glycogen granules, and abundant microvilli.
4. Urea and triacylglycerol synthesis in HTC cells
Ammonium chloride increased significantly in a dose-dependent manner the concentration of urea synthesized in HTC cells. Urea nitrogen in ES cell-cultured medium and control cell-free medium containing 20 mM ammonium chloride were undetectable (< 0.1 mg/dL) (Fig. 2
A). The urea synthesis rate in four clones of HTC cells cultured in plastic flasks containing chitin fibers was maintained constantly for at least 4 months in culture.
|
Triacylglycerol secretion in HTC cells was stimulated by insulin as a stimulator of hepatic lipogenesis in a dose-dependent manner, but was not observed in these cells cultured in the absence of insulin (Fig. 2B
). Insulin (4 U/mL) persistently induced the increased secretion of triacylglycerol from four clones of HTC cells for at least 1 month followed by 4 months. Cellular triacylglycerol content in HTC cells stimulated with insulin was measured after total lipid was extracted from the cell pellet. Insulin also augmented the storage of cellular triacylglycerol in HTC cells. A high dose of insulin induced a rapid secretion and synthesis of triacylglycerol and subsequently exhausted the storage of cellular triacylglycerol in HTC cells. It is proposed that the net de novo synthesis of triacylglycerol in HTC cells is markedly enhanced by insulin.
CONCLUSIONS AND SIGNIFICANCE
This study demonstrates that mouse ES cells were able to differentiate into hepatocytes with liver-specific metabolic functions when HNF-3ß-transfected ES cells were cultured in -MEM supplemented with FGF-2, nicotinamide, dexamethasone, L-ascorbic-2-phosphate, and 10% FBS in a 3-dimensional culture system to form spheroids. HTC cells formed spheroids when grown on 96-well round-bottomed spheroid plates or plastic flasks containing chitin fiber. The surface cells of HTC spheroids in long-term comfortable cultures maintained metabolic functions such as albumin production, whereas albumin synthesis in normal hepatocytes cultured in a monolayer was restrained a few days earlier. The cells of the inner layer in spheroid clusters are predicted to be functionally inactive because their cells are limited by diffusion of oxygen and nutrients into the spheroids; cell necrosis is observed > 150 µm from the spheroid surface.
Subcellular architecture of HTC cells subcultured in the maintenance medium containing insulin and glucose (4 g/L) possessed plentiful mitochondria and endoplasmic reticulum and glycogen granules as shown by electron microscopy. HTC cells rapidly promoted glucogenesis and lipogenic metabolic pathways by stimulation with 4 g/L of glucose and insulin, or oleate and insulin, respectively. Furthermore, HTC cells had the capacity to express mRNA of C3, P450, PEPCK, and PXMP1-L proteins as differentiated hepatocyte-enriched proteins.
Urea or triacylglycerol synthesis in HTC cells was readily induced by ammonium chloride or insulin, respectively. The urea and triacylglycerol synthesis rate in four clones of HTC cells cultured in flasks containing chitin fibers was kept constant for at least 4 months. These findings also suggested that hepatocytes in the 3-dimensional culture system could maintain liver-specific metabolic functions in a long-term culture. The present findings provide new evidence that mouse ES cells are able to differentiate into hepatocytes with liver-specific metabolic functions in long-term cultures by transfection of HNF-3ß gene, addition of FGF-2, and a 3-dimensional culture system (Fig. 3
). In vivo experiments are in progress to investigate the transplantation of HTC spheroids in liver of syngeneic mice treated with retrorsine and partial hepatectomy. Ongoing studies of hepatic differentiation are expected to provide further useful insights into current problems of cell transplantation, embryogenesis, and human ES cell technology.
|
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0806fje; to cite this article, use FASEB J. (July 1, 2002) 10.1096/fj.01-0806fje 
This article has been cited by other articles:
|
|
 |
|
  M. Inada, A. Follenzi, K. Cheng, M. Surana, B. Joseph, D. Benten, S. Bandi, H. Qian, and S. Gupta Phenotype reversion in fetal human liver epithelial cells identifies the role of an intermediate meso-endodermal stage before hepatic maturation J. Cell Sci., April 1, 2008; 121(7): 1002 - 1013. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H. Walkup and D. A. Gerber Hepatic Stem Cells: In Search of Stem Cells, August 1, 2006; 24(8): 1833 - 1840. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Bosch, S. L. Pratt, and S. L. Stice Isolation, Characterization, Gene Modification, and Nuclear Reprogramming of Porcine Mesenchymal Stem Cells Biol Reprod, January 1, 2006; 74(1): 46 - 57. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Eventov-Friedman, H. Katchman, E. Shezen, A. Aronovich, D. Tchorsh, B. Dekel, E. Freud, and Y. Reisner Embryonic pig liver, pancreas, and lung as a source for transplantation: Optimal organogenesis without teratoma depends on distinct time windows PNAS, February 22, 2005; 102(8): 2928 - 2933. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Lakshmipathy, B. Pelacho, K. Sudo, J. L. Linehan, E. Coucouvanis, D. S. Kaufman, and C. M. Verfaillie Efficient Transfection of Embryonic and Adult Stem Cells Stem Cells, July 1, 2004; 22(4): 531 - 543. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Davila, G. G. Cezar, M. Thiede, S. Strom, T. Miki, and J. Trosko Use and Application of Stem Cells in Toxicology Toxicol. Sci., June 1, 2004; 79(2): 214 - 223. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 10 (•), and 20 (
) mM ammonium chloride. The values represent the sum that integrates the quantity of urea in the medium at 5 min intervals. B) HTC cells and ES cells (
) or with 0.2 (•) or 4 (