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As a potential method for producing cells to repair failing systems in human bodies, many scientists are looking to stem cells ­- cells that have the power to differentiate or transform into many different cell types. Scientists already know how to extract stem cells from adult human fat and hope they will someday be able to take a person's own cells and develop the tissues they need.
But there is a major constraint to this plan - out of all the cells drawn from adult fat, only a small percentage can successfully turn into the desired cell type. Some could even turn into harmful cell types. To tackle this challenge, Eric Darling, assistant professor of biology, and his lab are working to produce two methods to sort the useable cells from the chaos.
"You're reliant on these cells," said Hetal Desai GS, lead researcher on one of the projects. "If you're trying to grow a bone, you're reliant on how well the cell's going to respond to (the chemical stimulus), how well they're going to accomplish turning into (something like) bone. So if you have a bunch of cells that are essentially going to just hang out and not do anything, that's bad. If you can weed those out and keep the ones that are optimal - that's where these techniques really have a lot of power."
Desai's research, published Sept. 5, focused on developing a probe that would light up when stem cells were transforming into the correct cell type. The lab's other recent project in the area took a different tack - measuring the physical properties of cells to see their potential to turn into bone, cartilage or fat.

Glowing beacons
When a stem cell is differentiating, it sends messenger RNA signals that produce specific proteins and help it transform. Darling's team created a probe that binds to specific RNA and lights up - showing when specific cells are beginning to differentiate specifically into bone.
"We were basically able to quantify, in living cells and in real time, how many of these cells are expressing the genes at different stages of turning into bone," Desai said. She started work on the project while she was on rotation in Darling's lab and stayed on to make it a main research focus.
The probe, which was developed in 1996 and can be designed to respond to any specific gene, was dispatched into two groups of stem cells derived from fat - one which had been treated by a chemical to induce differentiation into bone and one that had not. Looking at the cells, an observer could immediately tell by the glow which cells were responding positively to the chemical signals. Over the course of three weeks, the group watched the waves of fluorescence mark the different stages in the cells' transformations.
Desai said the hardest part of the study was designing the probe. "We needed it to be a fly on the wall of the process," she said. "We didn't want it to interfere with the cell, but we wanted to make sure we had a really clear signal that we could assess really easily."
The team designed the probe to only interface with the specific RNA that indicated differentiation, and they ran several experiments to ensure the probe was not blocking the cells from using the RNA normally.
Eventually, the researchers hope the probe can be used to pick out cells responding well to the chemical signals in a clinical setting, providing a mechanism for sorting between cells. Richard Freiman, associate professor of biology at Brown who was not involved in the study, called this the most exciting aspect of the research.
"In thinking about therapy, measuring changes on those cells before you actually put them back into a patient is absolutely essential," he said. "It's rare to be able to do that with living cells - if one can, it's very powerful."

A predictive approach
In addition to developing the molecular probe, Darling's lab tackled the sorting from a predictive angle in a study published this May. The idea came to Darling during his post-doctorate at Duke University, when he used an atomic force microscope to examine the physical properties of cells. Darling said he found that stem cells had a wide variation and wondered whether that variation could predict the cell's ability to turn into different kinds of tissue.
Darling took that question to Brown, where he also took on research assistants Rafael Gonzalez-Cruz GS and Vera Fonseca. He said he thought that the physical properties of a stem cell pulled from fat might reflect internal features that would make the cells more amenable to differentiation into specific types.
"Right now, when people do enrichment of stem cells, they often look at surface markers that are associated with certain lineages," Darling said. "It works great for identifying very specific populations, but stem cells sometimes share markers with other types of cells." The worry is that these stem cells would be discarded, even if they could still be useable.
 "We're looking at trying to use more of the cells that we have," Darling added. "Some of the cells might only be able to become bone. So they truly aren't stem cells, but if you're trying to create bone tissue, you can still use them."
The lab used cells from a set of donors that had been cultivated into a large population of potential stem cells. Ultimately, they were able to get 36 individual cells to grow into stem cell populations. The idea was, even if the original stem cells came in many physical forms, each population grown from a single cell would have a common structure.
Next, they used an atomic force microscope to poke at individuals from the different populations, determining how stiff they were, their size and their internal consistencies. They separated the populations into groups and gave them the chemical signals to differentiate - measuring how well each population turned into bone, fat and cartilage cells.
Sure enough, a trend was revealed. Stiff cells were best at differentiating into bone, soft and large cells were best at differentiating into fat, and viscous cells were best at differentiating into cartilage.
Attributing differentiation capabilities to mechanical properties of these stem cells is a unique idea, Freiman said. "This study sits at the intersect between biology and engineering," he said. "Darling's looking at stem cell replacement therapies using theories from the discipline of engineering - something that many biologists never consider thinking about."
But a lot of work is needed before this research can be applied clinically.
"For a clinical application we would need to sort large numbers of cells that could be useful for therapy or tissue engineering applications," said Gonzalez-Cruz, the study's head author. "We could use the properties that we found here to measure large numbers of cells and see if the trends still hold with these large numbers." The cells the team analyzed were all drawn from a relatively similar population, but they need to see if variations are still predictive for more diverse stem cells. This could potentially allow s
cientists to sort any population of fat-derived cells to function most effectively.
"Actually doing the sorting - that's going to be the hard part," Darling said.
 
Future plans
Since both of these approaches have worked in the lab, a next step is to turn them into tools that can be used with patients. A sorting method that combines multiple approaches could be tremendously helpful.
"You can enrich cells based on gene expressions, and you can enrich them based on mechanical properties, and hopefully down the road we can merge these two techniques and sort cells based both on mechanical properties and on gene expressions in a very high-throughout way," Desai said. "We're working towards a situation where a patient is in the hospital, we can isolate their cells, quickly assess them and place them right back into the patient for therapy. That would be the real goal of all of this."




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