From University of Washington, Press Release
Robots grow mini-organs from human stem cells
Bird's eye view of a microwell plate containing kidney organoids, generated by liquid handling robots from human stem cells. Yellow boxed region is shown at higher magnification. Red, green, and yellow colors mark distinct segments of the kidney.
An automated system that uses robots has been designed to rapidly produce human mini-organs derived from stem cells. Researchers at the University of Washington School of Medicine in Seattle developed the new system.
The advance promises to greatly expand the use of mini-organs in basic research and drug discovery, according to Benjamin Freedman, assistant professor of medicine, Division of Nephrology, at the UW School of Medicine, who led the research effort.
"This is a new 'secret weapon' in our fight against disease,' said Freedman, who is a scientist at the UW Institute for Stem Cell and Regenerative Medicine, as well as at the Kidney Research Institute, a collaboration between the Northwest Kidney Centers and UW Medicine.
A report describing the new technique will be published online May 17 in the journal Cell Stem Cell. The lead authors were research scientists Stefan Czerniecki, and Nelly Cruz from the Freedman lab, and Dr. Jennifer Harder, assistant professor of internal medicine, Division of Nephrology at the University of Michigan School of Medicine, where she is a kidney disease specialist.
The traditional way to grow cells for biomedical research, Freedman explained, is to culture them as flat, two-dimensional sheets, which are overly simplistic. In recent years, researchers have been increasingly successful in growing stem cells into more complex, three-dimensional structures called mini-organs or organoids. These resemble rudimentary organs and in many ways behave similarly. While these properties make organoids ideal for biomedical research, they also pose a challenge for mass production. The ability to mass produce organoids is the most exciting potential applications of the new robotic technology, according to the developers.
In the new study, the researchers used a robotic system to automate the procedure for growing stem cells into organoids. Although similar approaches have been successful with adult stem cells, this is the first report of successfully automating the manufacture of organoids from pluripotent stem cells. That cell type is versatile and capable of becoming any type of organ.
In this process, the liquid-handling robots introduced the stem cells into plates that contained as many as 384 miniature wells each, and then coaxed them to turn into kidney organoids over 21 days. Each little microwell typically contained ten or more organoids, and each plate contained thousands of organoids. With a speed that would have impressed Henry Ford's car assembly line, the robots could produce many plates in a fraction of the time.
"Ordinarily, just setting up an experiment of this magnitude would take a researcher all day, while the robot can do it in 20 minutes," said Freedman.
"On top of that, the robot doesn't get tired and make mistakes," he added. "There's no question. For repetitive, tedious tasks like this, robots do a better job than humans."
The researchers further trained robots to process and analyze the organoids they produced. Harder and her colleagues at the University of Michigan Kidney Center used an automated, cutting-edge technique called single cell RNA sequencing to identify all the different types of cells found in the organoids.
"We established that these organoids do resemble developing kidneys, but also that they contain non-kidney cells that had not previously been characterized in these cultures," said Harder.
"These findings give us a better idea of the nature of these organoids and provide a baseline from which we can make improvements," Freedman said. "The value of this high-throughput platform is that we can now alter our procedure at any point, in many different ways, and quickly see which of these changes produces a better result."
Demonstrating this, the researchers discovered a way to greatly expand the number of blood vessel cells in their organoids to make them more like real kidneys.
The researchers also used their new technique to search for drugs that could affect disease. In one of these experiments, they produced organoids with mutations that cause polycystic kidney disease, a common, inherited condition that affects one in 600 people worldwide and often leads to kidney failure.
In this disease, tiny tubes in the kidneys and other organs swell like balloons and form expanding cysts that crowd out the healthy tissue.
In their experiment, the researchers exposed the polycystic kidney disease organoids to a number of substances. They found that one, a factor called blebbistatin that blocks a protein called myosin, led to a significant increase in the number and size of cysts.
"This was unexpected, since myosin was not known to be involved in PKD," Freedman said. Myosin, which is better known for its role in muscle contraction, may allow kidney tubules to expand and contract. If it is not functioning properly it might lead to cysts, Freedman explained.
"It's definitely a pathway we will be looking at," he said.
The title of the research paper in Cell Stem Cell is, "High-throughput screening enhances kidney organoid differentiation from human pluripotent stem cells and enables automated multidimensional phenotyping."
The research was supported by the American Society of Nephrology, PKD Foundation, National Kidney Foundation, Northwest Kidney Centers, Howard Hughes Medical Institute and the U.S. National Institutes of Health. The NIH grant numbers are: K01DK102826, UH3TR000504, UG3TR002158, K08DK089119, U54DK083912, R00DK094873, and R01DK097598.
From Eureka Alert
Scientists develop method to tweak tiny 'antenna' on cells
IMAGE: THESE ARE PRIMARY CILIA IN A MOUSE EMBRYONIC NODE. view more
CREDIT: SHINOHARA KYOSUKE, TOKYO UNIVERSITY OF AGRICULTURE AND TECHNOLOGY
A report of their findings appeared online April 30 in Nature Communications.
With few exceptions, most cells in the body have cilia or can grow them. The tiny antenna sense chemicals such as hormones and growth factors, which regulate cell health and growth. Cilia also detect mechanical and physical cues in the body, such as light, gravity, sound and the flow of blood and urine.
When cilia malfunction, a range of human diseases and conditions can occur. For example, problems with cilia in kidney cells can cause polycystic kidney disease, an incurable condition in which fluid-filled cysts interfere with kidney function and which is conventionally treated with dialysis.
Because cilia are so small -- 10,000 times smaller than a cell -- scientists have long found it challenging to squeeze their tools into such tight spaces to study them.
"When I was a postdoc, a colleague in a neighboring laboratory was studying cilia, and I hoped that by combining his knowledge of the biology of cilia with my expertise in cellular engineering, we could figure out how to manipulate cilia within their tiny spaces," says Takanari Inoue, Ph.D., professor of cell biology at the Johns Hopkins University School of Medicine and an author of the new report.
After years of work, he says they figured out a way to manipulate a chemical signaling pathway within cilia that controls how molecules are shuttled up and down the length of the tiny structure.
To do it, Inoue and his colleagues in Taiwan used a tool called chemically inducible dimerization, which they say is faster than efforts to manipulate the pathway by rewriting the cilia's genetic code. The tool, essentially, is a matchmaker -- it helps to mesh two specific chemicals together at specific sites within a living cell.
For the new study, Inoue and his colleagues added a protein called FRB to cells from mice grown in the laboratory. The FRB protein is capable of glomming onto a rigid structure within cilia, called a microtubule, which acts as a railway, shuttling proteins up and down the length of cilia.
Then they added a molecule called FKBP to the cells, which is attached to an enzyme that acts as an eraser for a chemical modification in the cilia called glutamylation. The FKBP and enzyme pair floats around the cell until scientists add a chemical called rapamycin, which causes FKBP to get trapped at FRB molecules within the cilia.
Once inside the cilia, the enzyme attached to the FKBP molecule selectively erases the glutamylation modification inside the cilia. It also ignores other signaling pathways.
The scientists call their molecule matchmaking STRIP, for spatiotemporal rewriting intraciliary post-translational modifications.
As a result of rapidly removing glutamylation in cilia, the scientists found that molecules flowed up the cilia, toward the tip, more slowly -- about .3 micrometers per second -- compared with .4 micrometers per second, using a dead enzyme that doesn't affect glutamylation.
"We think our technique is faster than existing means of tracking cilia activity and enables scientists to access cilia parts faster and dive into specific chemical modifications for certain amounts of time," says Inoue.
"Our STRIP system offers a new strategy for precisely controlling microtubule modifications in living cells. With this approach, it becomes possible to understand how microtubules regulate cellular functions and may also serve as a new way to treat human diseases in the future," says Yu-Chun Lin, Ph.D., an assistant professor at the Institute of Molecular Medicine at the National Tsing Hua University in Taiwan.
Other diseases affected by flawed cilia include a brain disorder called Joubert syndrome, a kidney disorder called nephronophthisis, retinitis pigmentosa and a rare disorder called situs inversus, in which the internal organs of the body are in the reverse location of their normal position.
The scientists also found that microtubules in the mouse cells that are not located inside cilia were not affected when they tinkered with glutamylation.
Inoue and his colleagues also found that the genetic output of a developmental pathway called Hedgehog (which is connected to glutamylation) is decreased in cells treated with STRIP compared with their controls.
Inoue and his colleagues say they now plan to apply STRIP to human cells and look more closely at the molecular process of glutamylation in cilia. They may also use STRIP to control other chemical modifications within cilia.
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