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Paper Information

Journal:   CELL JOURNAL (YAKHTEH)   2008 , Volume 10 , Number SUPPLEMENT 1; Page(s) 65 To 65.
 
Paper: 

DIFFERENTIATION AND ENRICHMENT OF HEPATOCYPTE- LIKE CELLS FROM HUMAN EMBRYONIC STEM CELLS IN VITRO AND IN VIVO

 
 
Author(s):  ZERN MARK A.*
 
* UC DAVIS MEDICAL CENTER, IM: GASTROENTEROLOGY, SACRAMENTO USA
 
Abstract: 

Objective: Human embryonic stem cells (hESC) may provide a cell source of functional hepatocytes for therapeutic use. The aim of this study is to develop viable hepatocytes from hESC that can be used for cell-based therapies.
Materials and Methods: Using a combinatorial approach, we developed culture conditions that differentiated a percentage of hESC along a hepatocyte lineage. The differentiated hESC were further enriched by transducing with a lentiviral vector containing the human a1- antitrypsin (
a1-AT) promoter driving the GFP gene. The GFP+ hESC were then purified by laser microdissection and pressure catapulting.
Results: After transduction with a lentivirus containing α1-AT promoter, differentiated GFP+ hESC expressed a large series of liver proteins: -fetoprotein (AFP), albumin (ALB), α1-AT, CK18, transferrin (TF), tyrosin aminotransferase (TAT), arginase (ARG), glucose-6- phosphatase, CYP1A1, CYP2B6, CYP1B1, CYP2E1, CYP2C9, and CYP3A. AFP expression decreased with time. Differentiated hESC also had liver-specific functions. Differentiated hESC also had liver-specific functions. They accumulated glycogen, showed the cellular uptake of indocyanine green, and expressed high levels of CYP1A2 activity as well as urea synthesis. Quantitative RT-PCR revealed that the expression level of purified hESC over time was comparable to primary human hepatoctes: 24 to 35% for ALB, 44 to 57% for α1-AT, 73 to 91% for TF, 13 to 61% for TAT, and 128 to 230% for ARG. When NOD-SCID mice were transplanted with the differentiated hESC transduced with a lentiviral triple fusion vector, positive luciferase signals were obtained by a charged coupled device camera over time. The differentiated hESC survived and engrafted in mouse livers, and human mRNA and protein species (ALB,
a 1-AT, TF, CYP1A1 and GAPDH) were expressed in the transplanted mouse liver at three weeks after transplantation, and human albumin was detected in the mouse serum. Of note, human AFP expression was not found, indicating that the cells had expression profiles consistent with mature human hepatocytes.
Conclusion: Our results demonstrated for the first time that purified differentiated hESC expressed near physiological levels of liver-specific genes and had liverspecific functions that are comparable to primary human hepatocytes. In addition, this represents the first successful transplantation of differentiated hESC into an animal liver, and the first bioluminescence imaging of hESC in the liver.

 
Keyword(s): HEPATOCYPTE, GUMAN EMBRYONIC STEM CELLS, LENTIVIRUS
 
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