Biochemists have utilized a new cell culture medium to ask how critical genes are to the survival and reproduction of human cells under different growth conditions. The technique could have important ramifications for the treatment of human diseases.
Cell culture media are designed to provide a sheltered and nutritive environment to support cell survival and rapid growth, but until now, these media have not closely resembled the biochemical conditions encountered by cells in the human body. Researchers previously designed a new medium that more closely reflects the nutrient composition of human blood, Human Plasma-like Medium (HPLM).
In a paper published online March 1 in the journal Cell Metabolism, researchers at UW-Madison, the Morgridge Institute for Research, and the Whitehead Institute used HPLM to show that medium composition can affect gene essentiality, or in other words, the extent to which a gene contributes to cell fitness, a characteristic that is critical to many human diseases.
Researchers used CRISPR technology to perform forward genetic screens in human blood cancer cell lines. This approach can be used to identify genes that drive a particular phenotype. Ultimately, the researchers wanted to ask how one such phenotype (cell growth) might change in a more human-like medium.
“With forward genetic screening,” said Nicholas Rossiter, a technician in the Cantor Lab and first author on the paper, “we can consider a phenotype, introduce a perturbation at the genome level, and then evaluate if there’s any change in that phenotype of interest.”
By using the CRISPR/Cas9 technology to systematically knock out each of the nearly 20,000 genes encoded by the human genome in a pooled format, they went on to identify hundreds of genes that were conditionally essential in traditional culture media versus in HPLM. These sets of genes are those that effectively become more important, or make greater contributions to cell fitness, in one growth condition versus another.
“The really core essential genes,” said Rossiter, “are almost universally important for growth across all human cell lines tested in any condition. When you knock them out, cells aren’t going to grow. For instance, growth conditions aren’t necessarily going to influence the fact that cells won’t proliferate if they can’t produce ribosomes or can’t perform DNA replication.”
But by leveraging CRISPR-based screens of cancer cells in different media, the researchers were also able to identify conditionally essential genes, and in specific cases highlighted in follow-up work, trace such effects to the availability of components that are uniquely defined in HPLM versus traditional media.
Jason Cantor, now assistant professor of biochemistry at UW-Madison and investigator at Morgridge Institute for Research, was previously a postdoc in the Sabitini Lab at Whitehead Institute, where HPLM was first developed and where this study was initiated.
“This shows,” said Cantor, “that there are aspects of the environment that we can tinker with to make a gene become more or less critical for cell growth. Certainly, there are also implications here in terms of how to potentially increase the fidelity of what we see in the lab and what might happen in the body.”
These implications could be far-reaching. Use of HPLM may allow researchers to conduct experiments in the lab that are more directly relevant to human disease. If, for instance, scientists can alter the importance of a specific gene for cancer cell growth, then its encoded protein could become a more promising target for treatment. And as researchers understand more about the target’s response to various perturbations, smarter therapeutic approaches can be developed.
“There are plenty of components that we can play around with just in HPLM,” said Cantor. “You can manipulate components of the media, or potentially, in the blood, to make an encoded protein become more or less essential for cell proliferation. From a therapeutic angle, down the line, this could perhaps allow for new treatment approaches, where targeted therapies are combined with this type of [environmental] manipulation of circulating metabolites.”
The Cantor lab is jointly affiliated with the Department of Biochemistry at the University of Wisconsin-Madison and the Morgridge Institute for Research. Their goal is to understand how environmental factors influence basic cell physiology and drug activity in the context of human cancers and normal cellular components of the immune system.
As an independent research organization, the Morgridge Institute for Research explores uncharted scientific territory to discover tomorrow’s cures. In affiliation with the University of Wisconsin-Madison, they support researchers who take a fearless approach to advancing human health in emerging fields such as regenerative biology, metabolism, virology and medical engineering. Through public programming, we work to inspire scientific curiosity in everyday life. Learn more at: www.morgridge.org
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