After a few days or weeks, you test each well to see which compounds are the most promising for further testing. If you tried to repeat the same massive screening process with pipette-wielding lab workers, it might take years to complete.
Alzheimer’s researchers want to use the same high-throughput screening technique to test thousands of potentially life-saving compounds on brain cells. For that to work, though, they first need to find a reliably homogeneous supply of identical brain cells to run through the screening process.
But unlike human cancer cells, which are easily cloned and grown in the lab, human brain cells don’t survive in a dish. Instead, researchers have attempted to grow their own brain cells from induced pluripotent stem cells, which are skin or blood cells that have been reprogramed to become undifferentiated, blank-slate stem cells.
That’s easier said than done, though.
To grow a brain cell from a stem cell, you need to mimic the natural biological differentiation process that results in a specific type of brain cell. That requires a cocktail of precisely-timed doses of chemicals called growth factors that activate and deactivate different genes in the growing cell.
The trouble with most lab-grown brain cells is they’re highly heterogeneous by nature, meaning that different subtypes of neurons and glia — a “helper” cell in the central nervous system — emerge from the same differentiation process and then need to be sorted out. This multi-step process is expensive and time-consuming, especially for researchers who want to generate millions of identical brain cells for high-throughput screening.
The good news is a far cheaper and much faster system may be on the way. A team of Alzheimer’s researchers from the Gladstone Institutes has developed a simple, two-step process for growing thousands of identical neurons from a single stem cell, according to a paper published in the journal Stem Cell Reports.
Li Gan is an associate director and senior investigator at the Gladstone Institute of Neurological Disease and a professor of neurology at the University of California, San Francisco.
Like many recent breakthroughs in biotech, Gan told Seeker, this one started with gene-editing. Using a CRISPR-like genome-editor called TALEN, the Gladstone researchers programmed their stem cells to overexpress a certain gene that’s responsible for differentiating cells into neurons. But the gene would only be activated when the stem cell was exposed to a common antibiotic called doxycycline.
The gene-editing program itself wasn’t new, Gan explained but had previously been installed in stem cells by viruses, which led to unpredictable results. Some stem cells would take up more of the virus than others, ultimately resulting in a mix of neurons and glia, not a pure sample of neurons. With TALEN, Gan and her team could precision edit one stem cell and clone it as many times as they wanted — creating “tens of millions” of identically pre-programed stem cells.
After just three days of exposure to doxycycline, the stem cells transformed into precursor neurons. And not just any old neuron, but a specific subtype of neuron that’s of particular interest to Alzheimer’s researchers. Most importantly, every single precursor neuron was exactly the same.
“The key to our system is that we allow our cells to be completely synchronized,” said Gan. “They turn into the same type of neurons at exactly the same time, because they express this particular gene exactly the same.”
When the cells were removed from the doxycycline solution, the gene immediately turned off, ensuring that none of the cells continued to differentiate into another neuron subtype or glial cell. The second step in the two-step process was to culture the cells for four weeks until they developed into functionally active neurons.
The reason Gan and her team chose this particular neuron subtype was that it contained tau, the naturally occurring brain protein that can form tangles that choke off and kill brain cells in Alzheimer’s and other neurodegenerative diseases. Abnormally high levels of tau are strongly associated with Alzheimer’s and other forms of dementia, and previous studies had shown that lowering tau levels in mice with Alzheimer’s can recover some memory deficits.
If the Gladstone team could find a new compound that lowers tau levels in human brain cells, it would be an excellent proof of concept that the high-throughput screening system worked.
Robotic arms quickly filled each of the 394 wells on a large plastic tray with 2,000 of Gan’s engineered neurons. To test the broadest sample of treatments possible, the Gladstone team ran the screening against 1,280 different bioactive molecules contained in the Library of Pharmaceutically Active Compounds. The top two tau-lowering candidates were both from the same family of compounds known as AR agonists.
What’s exciting to Gan, beyond her own Alzheimer’s research, is that her lab’s low-cost, two-step technique for growing countless numbers of identical brain cells is available for any researcher to use.
“We’ve shipped the programmed stem cells to many people,” said Gan, including labs with limited cell-culturing experience. “They can use the stem cells to differentiate into neurons, just like you would grow cancer cells. They just need to add this little inducer and put them into media that’s suitable for neurons and that’s it.”
Armed with a stockpile of inexpensive and identical human brain cells, researchers can reliably repeat each other’s experiments, and make apples-to-apples comparisons between different treatments — and potentially cures — for Alzheimer’s and other neurodegenerative diseases.