A team of British researchers from Durham University, the North East England Stem Cell Institute, and a startup called Reinnervate have developed two synthetic versions of retinoic acid that can be used to drive stem-cell differentiation.
The use of these molecules, dubbed EC23 and EC19, could ultimately reduce the number of animals used in laboratory research, Stefan Przyborski, a reader in the department of stem-cell biology at Durham University and chief scientific officer of Reinnervate, told CBA News this week.
In addition, these molecules have been found to be more effective than all-trans retinoic acid at driving the differentiation of human stem cells into particular types of tissue.
“Retinoic acid, the molecule known to cause stem cells to differentiate, is known to be unstable, so we had to work with the chemists to develop new compounds that were physically stable and did not degrade when used in normal laboratory conditions,” said Przyborski, whose research appeared in a paper in last week’s Organic and Biomolecular Chemistry.
When Przyborski and his colleagues tested the compounds, they found that they could get stem cells to differentiate into neurons in a more consistent fashion than with all-trans retinoic acid.
“In fact, the molecules that we came up with were actually more potent than the natural retinoid, and we found that we actually got more neurons,” he said. The neurons derived from these stem cells could then be used in drug discovery, for example to develop new compounds to treat neurodegenerative diseases.
For their cell-based assays, pharmaceutical companies require reagents that are consistent and work in a similar manner every time. “If we can offer [pharmaceutical companies] a suite of molecules that can be used to induce their cells to differentiate in a consistent manner, [such molecules are] of commercial value to us, and of value to them,” said Przyborski.
If more work can be done in the laboratory using human stem cell-derived neurons, he said, researchers would therefore require fewer animals.
“The other thing is that if you work in a human in vitro system, it is obviously more relevant to man, therefore the jump from species to species does not happen,” said Przyborski. However, he added, investigators still need to use an in vivo animal model at some point as well.
Przyborski said that the structure of EC19 is very closely related to EC23, but is subtly different. That small change to the structure has a significant effect on its ability to induce differentiation of the cells. “It appears that EC19 will induce the differentiation of epithelial cells, whereas EC23 promotes the differentiation of neurons,” he said.
The Path Less Traveled
The Durham University scientists are trying to develop a library of different molecules that selectively trigger the differentiation of particular cell types, for use by those wanting to grow specific cell types from stem cells and use them for ADME/tox or compound screening, said Andrew Whiting, a reader in the department of chemistry at Durham University and a collaborator on the project.
“We have been working for a while to develop small molecules that act as a library of compounds that we can use to control a whole range of different cellular processes,” said Whiting. He added that the researchers are also interested in designing molecules that selectively trigger development of different cell types.
“It appears that EC19 will induce the differentiation of epithelial cells, whereas EC23 promotes the differentiation of neurons.”
It is widely known that retinoic acid causes stem cells to differentiate down neural pathways, although it is unstable, very light-sensitive, and rapidly isomerizes to the 9 and 3 cis isomers, and will undergo oxidation and isomerization.
“When I started collaborating with [Przyborski], one of the things that we discussed was if he and his colleagues knew if the all-trans-retinoic acid they were using for differentiation purposes was pure, and if they ever had issues with reproducibility,” said Whiting.
Przyborski and his team did have issues regarding the reproducibility of their experiments, and he did not know the purity of the compounds that were used. “We realized that a need existed” for designing compounds that did the same thing as the natural compounds, but did it in such a way that they could not degrade, they could not isomerize, and they would give completely reproducible results, so that they could be used as a routine model for differentiation pathways and as probes for looking at biological mechanisms, Whiting said.
He added that this study is “really the core for this particular piece of work,” and he and Przyborski made a range of molecules that are similar to all-trans-retinoic acid but have built-in physical and chemical stability so they cannot isomerize. They also lack a molecular mechanism that enables them to change shape or undergo degradation or oxidation.
“We have continued to make a number of other compounds that are mimics of different isomers of the natural retinoic acids,” said Whiting. The researchers are controlling both the size and shape of the mimics, and looking at how those molecules behave. “We have also thought about using some of these molecules as markers, so we can use them in imaging techniques,” Whiting said.
“We are structurally changing some of these compounds so that we can determine, for example, where they go within cells, and determine the mechanisms by which they are actually interacting,” he said.
Whiting and Przyborski and their teams, who are all members of the NESCI, are also designing other classes of compounds that trigger differentiation down other pathways, and using those lead molecules as probes for investigating biological structure and mechanisms.
“We are at the beginning of quite a wide-ranging set of experiments to really look at greater and greater depth within the stem cells, as they are changing and developing and their morphology is changing,” Whiting said.
The molecules being developed by the Whiting and Przyborski research groups are currently being tested by undisclosed “leading global suppliers” of cell culture products.
Przyborski said that Reinnervate, the Durham University spin-out of which he is the chief scientific officer and director, has had EC23 mass produced. “We have hundreds of vials of this material now, and [Reinnervate is] going to start selling it through its website.” In addition, the recently published paper represents an important step, because it is a peer-reviewed article, and “once that goes out, people start to request the product.”
Separately this week, Reinnervate said it has secured £750,000 ($1.3 million) in a financing round led by VC shop NorthStar Equity Investors to help it continue developing a polystyrene foam scaffold that enables cells to grow in three dimensions in a similar way to how they grow in the human body.
Reinnervate received £550,000 through NSEI's Co-Investment Fund, while a further £200,000 was raised through Angel Investors. It has also received significant support from the Centre for Excellence in Life Sciences in the form of an undisclosed loan and consultancy.
Traditionally, cells have been grown in 2D single layers on a flat-polystyrene substrate, either in culture vessels or flat plates containing a number of wells. Studies suggest, however, that cells grown on the 3D scaffold behave more naturally, which for drug testing purposes is not only more cost effective but gives a truer reading of how the drug would perform within the body, Reinnervate said.