Controlling cells’ environments: A step toward building much-needed tissues and organs

Posted by Biomechanism 

With stem cells so fickle and indecisive that they make Shakespeare’s Hamlet pale by comparison, scientists today described an advance in encouraging stem cells to make decisions about their fate. The technology for doing so, reported here at the 242nd National Meeting & Exposition of the American Chemical Society (ACS), is an advance toward using stem cells in “regenerative medicine” — to grow from scratch organs for transplants and tissues for treating diseases.

Human embryonic stem cells offer the unique ability to not only renew themselves, but to also differentiate into any one of the more than 200 cell types found in the human body.

“Stem cells have great potential in regenerative medicine, in developing new drugs and in advancing biomedical research,” said Laura L. Kiessling, Ph.D., who presented the report. “To exploit that potential, we need two things: first, reproducible methods to grow human stem cells in the laboratory, and second, the ability to make stem cells grow into heart cells, brain cells or whatever kind of cell. Our technology takes a different approach to both of these problems, and the results are very encouraging.”

Biologically, so-called pluripotent human embryonic stem cells have not made up their minds about what to become. That’s essential because these cells, which are derived from embryos, have the agility to develop into the hundreds of different kinds of cells in a fully-formed human body. But controlling their differentiation has also stood as a major barrier to making the stem cell dream come true and using these all-purpose cells in medicine.

Past approaches to growing and scripting the fate of stem cells have involved adding growth-regulating and other substances to cultures of stem cells growing in the laboratory. These conditions left scientists guessing about exactly what wound up in the stem cells. Kiessling and colleagues are pioneering a new approach that involves using chemically controlled surfaces.

Kiessling previously developed chemically modified plastic and glass surfaces that take much of the guess work out of growing stem cells in laboratory cultures. In the past, scientists grew stem cells on surfaces that contained mouse cells. That left scientists with nagging questions about possible contamination of stem cells with disease-causing animal viruses — a stumbling block for using stem cells in potential medical applications. And that growth system was what scientists term “undefined.” There were variations from batch to batch of mouse cells, and scientists never really knew what the stem cells were coming into contact with and how it might be changing them. The synthetic, chemically-defined, surfaces ended that uncertainty. The approach was inexpensive, simple and a much-needed advance in producing stem cells, Kiessling explained.

With the ability to grow stem cells on the synthetic surfaces under chemically defined, or known, conditions, Kiessling’s group took an additional step in their latest research. It found that chemically defined surfaces can exert control over signaling pathways. “Signaling” is how molecules talk to one another and get things done inside a cell. It’s how an immune cell knows to fight an infection or how a pancreatic cell determines that more insulin is needed in the bloodstream, for example. By controlling how molecules inside a stem cell communicate, researchers could someday in the future nudge them to become one type of cell or tissue over another.

To see whether a new chemically defined surface could change signaling in a pilot experiment, Kiessling tested cancer cells. The research involved use of a signaling substance, transforming growth factor-beta (TGF-beta), which controls a range of activities, from cell growth to self-destruction.

“The new surfaces give scientists much more control over cells, opening up a wide range of possible future applications,” Kiessling explained. Building directly on the results of the pilot study, the surfaces could have applications in wound healing. TGF- beta can help wounds heal, but if it touches healthy skin, inflammation or even a cancerous tumor could develop. “We haven’t done this, but you could imagine a bandage that has a localized concentration of the special peptide surface that would recruit TGF-beta just to the wound site,” said Kiessling.

The surfaces also could make it easier to manufacture organs and tissues in the laboratory someday. “We think that this strategy, with different sets of peptides (building blocks of proteins) bound to the surface, could direct certain human embryonic stem cells on the surface to become one type of cell and other stem cells to become a second cell type, right next to each other. For the tissue engineering involved in growing replacement organs, you need to organize specialized cells in particular ways like this.”

Free radicals crucial to suppressing appetite

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Obesity is growing at alarming rates worldwide, and the biggest culprit is overeating. In a study of brain circuits that control hunger and satiety, Yale School of Medicine researchers have found that molecular mechanisms controlling free radicals—molecules tied to aging and tissue damage—are at the heart of increased appetite in diet-induced obesity.

Caption: This image shows satiety promoting melanocortin neurons (green) in the hypothalamus, some of which are activated (red nuclei) after treatment. Credit: Tamas Horvath, Yale University

Published Aug. 28 in the advanced online issue of Nature Medicine, the study found that elevating free radical levels in the hypothalamus directly or indirectly suppresses appetite in obese mice by activating satiety-promoting melanocortin neurons. Free radicals, however, are also thought to drive the aging process.

“It’s a catch-22,” said senior author Tamas Horvath, the Jean and David W. Wallace Professor of Biomedical Research, chair of comparative medicine and director of the Yale Program on Integrative Cell Signaling and Neurobiology of Metabolism. “On one hand, you must have these critical signaling molecules to stop eating. On the other hand, if exposed to them chronically, free radicals damage cells and promote aging.”

“That’s why, in response to continuous overeating, a cellular mechanism kicks in to suppress the generation of these free radicals,” added lead author Sabrina Diano, associate professor of Ob/Gyn, neurobiology and comparative medicine. “While this free radical-suppressing mechanism—promoted by growth of intracellular organelles, called peroxisomes—protects the cells from damage, this same process will decrease the ability to feel full after eating.”

After the mice ate, the team saw that the neurons responsible for stopping overeating had high levels of free radicals. This process is driven by the hormone leptin and glucose, which signal the brain to modulate food intake. When mice eat, leptin and glucose levels go up, as does free radical levels. However, in mice with diet-induced obesity, these same neurons display impaired firing and activity (leptin resistance); in these mice, levels of free radicals were buffered by peroxisomes, preventing the activation of these neurons and thus the ability to feel sated after eating.

According to Horvath and Diano, the crucial role of free radicals in promoting satiety as well as degenerative processes associated with aging may explain why it has been difficult to develop successful therapeutic strategies for obesity without major side effects. Current studies address the question of whether, under any circumstance, satiety could be promoted without sustained elevation of free radicals in the brain and periphery.

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Other authors on the study include Zhong-Wu Liu, Jin Kwoan Jeong, Marcelo O. Dietrich, Hai-Bin Ruan, Esther Kim, Shigetomo Suyama, Kaitlin Kelly, Erika Gyengesi, Jack L. Arbiser, Denise D. Belsham, David A. Sarruf, Michael W. Schwartz, Anton M. Bennett, Marya Shanabrough, Charles V. Mobbs, Xiaoyong Yang, and Xiao-Bing Gao.

The study was supported by grants form the National Institutes of Health and the American Diabetes Association.

Citation: Nature Medicine, DOI: 10.1038/nm.2421

Nano-thermometers show first temperature response differences within living cells

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Using a modern version of open-wide-and-keep-this-under-your-tongue, scientists today reported taking the temperature of individual cells in the human body, and finding for the first time that temperatures inside do not adhere to the familiar 98.6 degree Fahrenheit norm. They presented the research at the 242nd National Meeting & Exposition of the American Chemical Society (ACS), being held here this week.

Researchers are using quantum dots (shown in red) to take the temperature of living cells. Image Credit: Haw Yang, Ph.D.

Haw Yang and Liwei Lin, who collaborated on the research, did not use a familiar fever thermometer to check the temperature of cells, the 100 trillion or so microscopic packages of skin, nerve, heart, liver and other material that make up the human body. Cells are so small that almost 60,000 would fit on the head of a common pin. Yang is with Princeton University and Lin is with the University California-Berkeley.

“We used ‘nano-thermometers’,” Yang explained. “They are quantum dots, semiconductor crystals small enough to go right into an individual cell, where they change color as the temperature changes. We used quantum dots of cadmium and selenium that emit different colors (wavelengths) of light that correspond to temperature, and we can see that as a color change with our instruments.”

Yang said that information about the temperatures inside cells is important, but surprisingly lacking among the uncountable terabytes of scientific data available today.

“The inside of a cell is so complicated, and we know very little about it,” he pointed out. “When one thinks about chemistry, temperature is one of the most important physical factors that can change in a chemical reaction. So, we really wanted to know more about the chemistry inside a cell, which can tell us more about how the chemistry of life occurs.”

Scientists long have suspected that temperatures vary inside individual cells. Yang explained that thousands of biochemical reactions at the basis of life are constantly underway inside cells. Some of those reactions produce energy and heat. But some cells are more active than others, and the unused energy is discharged as heat. Parts of individual cells also may be warmer because they harbor biochemical power plants termed mitochondria for producing energy.

The researchers got that information by inserting the nano-thermometers into mouse cells growing in laboratory dishes. They found temperature differences of a few degrees Fahrenheit between one part of some cells and another, with parts of cells both warmer and cooler than others. Their temperature measurements are not yet accurate enough to give an exact numerical figure. Yang’s team also intentionally stimulated cells in ways that boosted the biochemical activity inside cells and observed temperature changes.

Yang says that those temperature changes may have body-wide impacts in determining health and disease. Increases in temperature inside a cell, for instance, may change the way that the genetic material called DNA works, and thus the way that the genes, which are made from DNA, work. Changing the temperature will also change how protein molecular machines operate. At higher temperatures, some proteins may become denatured, shutting down production.

“With these nano thermometer experiments, I believe we are the first to show that the temperature responses inside individual living cells are heterogeneous — or different,” said Yang. “This leads us to our next hypothesis, which is that cells may use differences in temperature as a way to communicate.”

Yang’s team is now conducting experiments to determine what regulates the temperature inside individual cells. One goal is to apply the information in improving prevention, diagnosis and treatment of diseases.