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Advanced Cell Technology Develops In Vitro Way to Generate Hepatocytes from hESCs

Advanced Cell Technology has developed what it believes to be the first in vitro method for generating high-purity hepatocytes from human embryonic stem cells, the company announced this week.
The investigators found that in a mouse model of acute liver injury, the liver cells were able to repopulate the damaged liver. Because of this, the company believes the liver cells could be a valuable model as drug-discovery assays and in new drug metabolism and cytotoxicity screens, Advanced Cell Technology CSO Robert Lanza told Cell-Based Assay News this week. 
“If you talk to those in the stem-cell field, you’ll find that a lot of them feel that the significance of stem-cell research is not going to be in the field of regenerative medicine, but in the field of drug discovery and in using these cells for drug discovery assays and to understand the various mechanisms of human disease,” Lanza said.
He said that the ability to efficiently generate large numbers of hepatocytes from hESCs that are highly pure — in this case 70 percent — is the first important step.
Previous studies have examined the differentiation of hESCs to hepatocytes, and were able to establish the feasibility of the hepatic differentiation of hESCs and suggest induction strategies. These methods were inappropriate for large scale applications such as that suggested by Lanza and his team’s work, however.   
In those studies, inefficient differentiation resulted in heterogeneous cultures containing many cell types and low proportions of hepatocytes.
“Liver failure affects millions of Americans, and so hepatocytes could be of enormous value” as research tools, Lanza said. “We are also talking about an enormous scarcity of donor livers and hepatocytes. The ability to generate an unlimited number of these cells would be of great value not only for regenerative medicine, but also for drug discovery.”
In their study, which appeared online last week in the journal Stem Cells, the investigators differentiated hESCs into highly enriched populations of definitive endoderm, the embryonic germ layer that develops into the liver, lung, pancreas, thyroid, and intestines. They then generated hepatocytes by inducing the definitive endoderm to differentiate along the hepatic lineage.
According to the researchers, the hepatocytes exhibited functional hepatic characteristics such as the ability to store glycogen, uptake and release indocyanine green, and secrete albumin. 
The investigators found that in a mouse model of acute liver injury, the hESC-derived definitive endoderm differentiated into hepatocytes and repopulated the damaged liver.
‘Open-Ended Results’?
ACT’s work has its critics. “The only conclusion that I can draw based on their data is that, yes, they have hepatocyte-like cells, but more work would have to be done to show that they are as good a standard as primary hepatocytes,” Salman Khetani, a postdoctoral fellow in the Harvard-MIT division of health sciences and technology, told CBA News this week.
He added that until that happens, one “cannot even begin to compare toxicity and metabolism in the two models, because one has not even been tested for functionality against primary hepatocytes.”
Human ESC-derived hepatocytes would have to go through extensive characterization if people are going to trust them for toxicity testing, simply because toxicity testing is used to predict the outcomes of clinical trials, Khetani said.

“The only conclusion that I can draw based on their data is that, yes, they have hepatocyte-like cells, but more work would have to be done to show that they are as good a standard as primary hepatocytes.”

He pointed out that until hESC-derived hepatocytes have gone through rigorous testing, no one is going to use them for that purpose.
Primary hepatocytes from deceased donors are currently the gold standard, Khetani said. He also said that Lanza and the Advanced Cell Technologies team used the HepG2 carcinoma cell line as its positive control.
“I believe they should have used primary hepatocytes,” Khetani said. He mentioned that HepG2 cells are notoriously low in their liver-specific functions.
Khetani said that until he sees data comparing the hESC-derived hepatocytes to primary hepatocytes, it will be very hard for him to determine if Lanza’s cells are as functional as primary hepatocytes. “The paper sort of left that open-ended in this particular study.” 
To get people to trust the data generated by hESC-derived hepatocytes, the cells would have to go through the same rigorous characterization that was done for primary hepatocytes, Khetani said.
“I would say that we are probably several years away from that, and it would probably take at least six years or even more for the FDA to support the use of hESC-derived heaptocytes in ADME/tox testing,” said Khetani. He said that the FDA wants limited in vitro data sets but a lot of in vivo data. “So for them to accept hESC-derived hepatocytes as an alternative would be a challenge.“         
Finding a Better Way
By describing a method for generating human hepatocytes in vitro, the authors may enable government agencies and organizations to study the cells while reducing or eliminating the need for animal testing.
The study is timely: Earlier this month, three large US government agencies announced that they plan to establish a collaborative research program to overhaul how environmental chemicals are tested by shifting the focus away from animal testing and toward cell-based or biomolecular assays and computer models (see CBA News, 2/15/08).  
The program was spearheaded by the National Toxicology Program, the National Institutes of Health Chemical Genomics Center, and the Environmental Protection Agency’s National Center for Computational Toxicology.
Christopher Austin, director of the NCGC, told CBA News at the time that the project is expected to last between five and 10 years, and “depends on funding, to a great degree.”
The NTP-EPA-NCGC collaborative effort was closely preceded by the release of the Interagency Coordinating Committee on the Validation of Alternative Methods (or ICVAAM) — National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (or NICEATM) Five-Year Plan (see CBA News, 2/8/08).
The plan, available here, identifies priority areas for research, development, translation, and validation activities necessary to achieve regulatory acceptance of alternative in vitro testing methods, such as high-throughput screens, William Stokes, director of NICEATM, told CBA News recently.
An integral part of the plan is to work with the pharmaceutical and biotech companies and other US and international stakeholders to accept and use alternative methods, Marilyn Wind, ICCVAM chair and the deputy associate director of the Consumer Products Safety Commission, recently told CBA News.
Wind also said that ICCVAM wants to take advantage of new in vitro test methods and approaches currently in development.
“We recognize that some of these [methods] will require years to develop and validate, and others will be available for use more quickly,” she said. “To meet that challenge, we will be working with federal agencies and other stakeholders so that can we can link the research and development activities to the standardization and validation of alternative test methods.”
Another challenge is fostering the acceptance and use of these alternative methods by federal regulatory and research agencies, said Wind. Under the ICCVAM Authorization Act of 2000, the Committee has a responsibility to look at the validation status of new test methods. It is responsible for making recommendations to its member federal agencies, she said.
“We need to correlate the data generated by hESC-derived hepatocytes with that generated by in vivo animal tests,” said ACT’s Lanza.

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