UNC School of Medicine scientists led by Scott Magness, PhD, sequenced the genes expressed in individual single cells from human digestive tracts to discover new cell-type characteristics and gain insights into important cell functions such as nutrient absorption and immune defense.
For the first time, the Magness lab used entire human GI tracts from three organ donors to show how cell types differ across all regions of the intestines, to shed light on cellular functions, and to show gene expression differences between these cells and between individuals.
This work, published in Cellular and Molecular Gastroenterology and Hepatology, opens the door to exploring the many facets of gut health in a much more precise manner at greater resolution than ever before.
“Our lab showed it’s possible to learn about each cell type’s function in important processes, such as nutrient absorption, protection from parasites, and the production of mucus and hormones that regulate eating behavior and gut motility,” said Magness, Associate Professor in the Joint UNC-NC State Department of Biomedical Engineering and senior author of the paper. “We also learned how the gut lining might interact with the environment through receptors and sensors, and how drugs could interact with different cell types.”
The Sensitive gut
For this research, the Magness lab focused on the epithelium: the single-cell thick layer separating the inside of the intestines and colon from everything else. Like other cell populations and the microbiota, the epithelium is incredibly important to human health, and for years scientists have been exploring it. But until now, researchers could only take tiny biopsies the size of grains of rice from a few parts of the digestive tract, usually from the colon or limited regions of the small intestine.
In the past, researchers would mash up those rice-sized biopsies to identify all epithelial cell types and learn some general features of these cells. Magness’s approach was to sample thousands of individual cells from every part of the lower digestive tract (small intestine and colon) to create an atlas and then study the potential roles of these cells through the genes that each cell expresses. Knowing all of this would deepen scientific knowledge about the gut epithelium and hopefully encourage other scientists to explore each cell’s function in biology, in disease, and in the unfortunate scenario of pharmaceutical side effects.
To do such a deep individual cell dive, Magness needed two things: better technology and the entire digestive tracts of humans.
RNA sequencing technology
UNC-Chapel Hill acquired state-of-the-art RNA sequencing technology several years ago for the creation of the Advanced Analytics Core Facility through the UNC Center for Gastrointestinal Disease and Biology, which developed the scientific and intellectual heft — research faculty, staff, postdocs, and students — to use state-of-the-art equipment.
Proteins CFTR (green) and FKBP1A (red) — a primary target of the drug Tacrolimus — in BEST4+ cells in small intestine.
The Magness Group acquired human digestive tracts through a research agreement with organ donor services at HonorBridge. When intestines are harvested for transplant and if they are not claimed by higher-priority groups, HonorBridge staff coordinates with the Magness Group to donate the transplant-grade organs for research.
Using sequencing technology to characterize gene expression, the Magness group first extracts RNA from each cell while keeping each cell separate, and then they run single-cell sequencing, which takes a snapshot of which genes each intestinal cell is expressing and how much.
A major problem with this kind of research is the sheer amount of data produced. The single cell sequencing picks up about 11,000 ‘reads’, or individual samples of gene products in just one cell, and in many thousands of individual cells, each with different combinations of the 20,000-plus human genes that are turned on or off. This creates almost 140,000,000 data points for all the 12,590 cells in the study that have to be put into a “visual” format so that scientists can make sense of the vast amount of information.
Researchers devised computational techniques to filter the data to produce a manageable data set that included cell populations from all portions of the GI tract. Then, based on what Magness and other researchers had already learned of each cell type, researchers could computationally identify each cell type from each region
