Researchers generate functioning, three-dimensional tissue to study what goes wrong - and right - in development
by Nick Miller
As organs go, the stomach does not get much respect.
It takes in food and fluids, helps turn them into energy, and then begins converting it all into substances too often the subject of tasteless humor. Unlike the brain, heart or lungs, the stomach is not one of science’s “glam” organs. Nor is it the subject of extensive developmental research literature - at least not yet.
Kyle McCracken, an MD/PhD student in the Developmental Biology laboratory of Jim Wells, PhD, hopes to change this. Wells is an expert in gastrointestinal development, diabetes research and stem cell technologies. When his team set out to see if they could use pluripotent stem cells (PSCs) to grow human stomachs in the laboratory to model and study human disease, McCracken started by looking for studies on basic stomach development.
“With organs like the pancreas, liver and the lungs, there are hundreds of publications about what controls their development,” McCracken says. “But we went to the stomach and there is nothing. We think we are addressing some of these gaps in knowledge with the in vitro modeling system we’ve developed.”
Stomachs In A Dish
Wells and McCracken successfully led a team of scientists to figure out how to generate in a petri dish a functioning, three-dimensional and critically important region of the human stomach called the antrum. They did this by using PSCs, some of them induced pluripotent stem cells (iPSCs). The iPSCs are made from human skin cells transformed with biochemical solutions to take on embryonic-like characteristics.
Like human embryonic stem cells (hESCs), iPSCs have the ability to become any cell or tissue type of the human body.
In early 2011, Wells’ laboratory published a paper in Nature on using iPSCs to generate functioning, three-dimensional intestinal tissue in a petri dish. That study — the first time any research team had generated functioning, three-dimensional intestinal organoids from iPSCs — helped start a new chapter in life sciences research for studying diseases and therapeutic solutions.
Unlike generating intestine — where the literature gave researchers some clues on where to begin — the stomach project forced the scientists to start from scratch. It was a tedious and time-consuming process of testing different genes and biochemical combinations to get PSCs to form stomach tissue.
“Not only were we trying to generate gastric organoids for research and therapeutic purposes, we were actually using the new in vitro system as a primary research and discovery tool to determine what makes stomach to begin with because so little was known,” Wells explains.
Recipe For Growth
To grow distal stomach through what is called directed differentiation, the team used a precise combination of signaling by important developmental pathways — including FGF (fibroblast growth factor), Wnt (protein signaling pathway), and BMP (bone morphogenetic protein). This allowed the scientists to mimic the normal steps of development that occur in an embryo. Importantly, during this phase the researchers discovered that BMP needed to be repressed. Through the carefully timed manipulation of these and other molecular components, the researchers coaxed two-dimensional cultures of PSCs into becoming three-dimensional foregut tube structures — an embryonic starting point for stomach.
To get foregut tissues to become gastric tissue corresponding to the antrum in the distal region of the stomach, the scientists manipulated other cellular processes by stimulating the signaling of retinoic acid and epidermal growth factor. Over the course of a month, these steps resulted in the formation of 3D gastric tissues that grew into large organoids similar to the antrum in the distal stomach.
Replicating A Variety Of Conditions
The new modeling system provides significant advantages for studying human disease, according to Wells. For example, the body’s response to food intake starts in the stomach. Controlling this response could hold the keys to preventing obesity and diabetes — a growing health epidemic. The distal portion of the stomach generated by the Wells lab houses the body’s satiety or “hunger” response. Wells says a breakdown in the hunger response is linked to obesity and resulting metabolic diseases like diabetes.
Stomach research may help explain why some gastric bypass surgery patients become diabetes-free even before they lose significant weight. And the new modeling system is already being used to study peptic ulcers and gastric cancers in unprecedented detail.
Wells, who has a dual appointment in the Division of Endocrinology, is part of a new research consortium at Cincinnati Children’s formed to study the endocrine system. The effort is designed to bring a multi-laboratory and multi-disciplinary focus to studying the endocrine system — and the new iPSC modeling technology will be central to that effort.
Mouse studies — long a backbone of life sciences and disease research — are poorly suited for studying diseases of the stomach. For one, Wells says the stomach is one of the least evolutionarily conserved organs among mammals, so structural development differs between mice and humans. One possible reason for this may be the wide dietary differences between species. Pathogens that run amok in human stomachs will not, in many cases, infect the stomachs of mice.
Because the gastric organoids are derived from human cells, McCracken says they will allow scientists to study the biology of human stomach tissue.
The new 3D organoids replicate in a laboratory what actually happens in a person far more precisely than do flat cell cultures.
“We haven’t just made a bunch of flat cells in a dish,” Wells says. “Embryos aren’t flat and we’ve figured out, at least partly, how the embryonic gastrointestinal system transitions from two-dimensional into three-dimensional, and then generated three-dimensional organ tissues with a fair level of complexity.”
Study of H. pylori is first to use 3D gastric model
Although the digestive system is full of helpful bacteria, Helicobacter pylori are not among them.
H. pylori are perhaps the worst bacterial villains to afflict the stomach, and the chief culprit behind peptic ulcers and gastric cancers.
In collaboration with Yana Zavros, PhD, a researcher at the University of Cincinnati’s Department of Molecular and Cellular Physiology who studies gastric cancer, one of the first studies using the new 3D gastric model is how H. pylori bacteria infect the human stomach.
An estimated 10 percent of the world’s population suffers from gastric diseases, largely because of H. pylori, says Kyle McCracken. During the study, the researchers were amazed to observe how quickly and efficiently H. pylori infected their 3D human gastric organoids.
“We didn’t model cancer, but we did infect the organoids and observe them for 24 hours,” McCracken explains. “We saw profound effects in that short period of time. H. pylori can cause cells to start dividing faster than they normally would and can activate other proteins in the cell that are known to drive cancer — all within a very short time.”
||Image shows gastric organoid before and after (white mass) infection with H. Pylori.