A team of American, British, and Canadian researchers announced this week that they have used human embryonic stem cell differentiation cultures to generate a KDRlow/C-KIT(CD117)neg population that displays cardiac, endothelial, and vascular smooth muscle potential in vitro and, after transplantation, in vivo.
An official from VistaGen Therapeutics, which participated in the research, told CBA News this week that the company plans to apply the results of this work to further develop commercial applications for ESC-derived cardiomyocyte assays in drug screening and predictive toxicology.
“It is the electrophysiological characterization [of these KDRlow/C-KITneg-derived cells] that is getting to be an exciting validation of the system as a better in vitro tool for understanding cardiovascular biology, and the response to drugs by human CV cells,” said VistaGen CEO Ralph Snodgrass of the paper, which appeared online recently in the journal Nature.
VistaGen uses ESCs to customize and develop in vitro assays for drug discovery and predictive toxicology. Snodgrass said in an interview this week with CBA News that “those in drug discovery are beginning to appreciate that these assays have significant value. Many groups are beginning to talk about the value that ESCs have for developing more clinically relevant drug-discovery tools.”
As an example, Snodgrass cited an undisclosed pharmaceutical company that “has licensed three of our assays for diabetes research and diabetes drug screening,” and said that VistaGen is planning to announce more about this agreement “in a few weeks.”
Heart of the Matter
In the Nature paper, when the researchers plated the KDRlow/C-KITneg cells in monolayer culture, they found that the cells differentiated into populations comprising more than 50 percent contracting cardiomyocytes. When plated in methylcellulose, populations of cells derived from the KDRlow/C-KITneg fraction gave rise to colonies comprising cardiac cells, endothelial cells, and vascular smooth muscle cell lineages.
The investigators ran cell-mixing experiments and limiting dilution studies, the results of which supported their belief that the colonies grown in methylcellulose were derived from a clone, rather than from a cell aggregation.
John Cashman, director of the Human Biomolecular Research Institute, advised caution, however. “Before extensive animal toxicity studies are done on a new chemical entity, relatively inexpensive tests for cardiac cell toxicity should be accomplished,” he told CBA News in an e-mail.
Last year, the HBRI was awarded a $714,654 grant from the California Institute of Regenerative Medicine to support a project that aims to treat heart disease by using drug-like molecules that act as cardiomyocyte differentiation agents (see CBA News, 2/23/07).
“It is the electrophysiological characterization [of these cells] that is getting to be an exciting validation of the system as a better in vitro tool for understanding cardiovascular biology, and the response to drugs by human CV cells.”
According to Cashman, it is important to study the effects of new drug candidates on human Ether-a-go-go Related Gene (or hERG) function, because a number of notable examples of cardiovascular toxicity have been reported for certain new drugs. He added that the hERG channel is subject to blockade by drugs, and several approved drugs have been removed from the market because of adverse drug interactions with this target.
Researchers can currently perform a rapid and inexpensive screen for possible hERG toxicity using planar patch clamp techniques, said Cashman. But in the Nature paper, “the hERG channel was not fully characterized,” he said. “This needs to be done before anything can be said about the utility of the cells in ADME/Tox studies.”
To be sure, the authors have done “a nice job with certain aspects of the cardiomyoctes,” but further studies are needed to characterize the hERG channel before the cells can be used in cardiovascular toxicity studies, said Cashman.
“It would also be well to address other aspects of metabolism capacity with these cells, before they could be widely used for ADME/Tox studies. However, the authors appear to be making good progress,” he added.
Kyle Kolaja, director of discovery and investigative safety at Roche Palo Alto, said this week that the Nature paper is “fascinating for a few reasons. One, the time it takes to differentiate the cells [14 to 16 days] is much shorter than others have been able to demonstrate. This addresses one of the key issues with hESC-derived cardiac tissue, which is getting an adequate number of cells to be able to run experiments.”
The Swiss drug and diagnostics giant has some experience with this. In March, Cellular Dynamics International announced that it will use its cell-based assays to screen certain Roche Palo Alto drug candidates for cardiotoxicity (see CBA News, 3/7/08). Under the terms of the agreement, Roche will supply CDI with two sets of 25 well-characterized kinase inhibitors to validate CDI’s current crop of toxicology products and services, Kolaja told CBA News at the time. CDI would test those compounds using human cardiomyocytes derived from human embryonic stem cells.
Kolaja added that “it is also interesting that the authors [of the Nature paper] have identified how to create a pluripotent stem cell in the heart that can create various types of cardiomyocytes and endothelial cells. When you combine this type of fundamental understanding of cardiac cell biology with the revolutionary breakthrough of inducible pluripotent stem cells, where one can create stem cells from adult epithelial cells, it is apparent that hESC-derived models will likely become the dominant cellular system.”
Kolaja agreed with Cashman that although these findings are exciting, further studies are necessary. “New cell systems not only need to be extensively characterized for structural, functional, and biochemical endpoints, but then need to be tested for their performance as a predictive toxicology tool,” Kolaja said.
He added that he felt this was particularly relevant for iPS-derived systems where “genetic manipulation is needed to dedifferentiate the cells without really knowing what effect that may have on the tissue that these cells ultimately differentiate back into.”
In the Nature paper, the authors show that these cells lose certain stem-cell markers and gain cardiac ones, and begin to gain functional channel activities indicative of the human heart — at least in juvenile or neonatal versions. “In that respect, I would anticipate these cells to respond in a similar manner to cardiomyocytes derived from other means, such as primary, iPS, other hESC methods, et cetera, but more data is definitely needed,” said Kolaja.
For its part, VistaGen plans to do further studies, said Snodgrass, adding that the next step for the company is to format the cells in in vitro analytical tests.
“That allows us to efficiently evaluate drugs for their electrophysiological effects on the heart, and identify those that hit a variety of ion channels in the heart or are known to be toxic to various biological aspects of the heart, and start to validate those [cells] as a formal screening system for predicting cardiotoxicity,” he said.
VistaGen will be talking to various pharma companies that are interested in using this as an assay for cardiac toxicity prediction, said Snodgrass. He declined to elaborate.
“We are not in the process of developing kits [with these cells] right now, however,” said Snodgrass. He said VistaGen believes that the best way to start using a technology effectively is “through strategic collaboration, or by providing money and screening services.”