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Damaged muscles don’t just die, they regenerate themselves — ScienceDaily

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While building a muscle damage model in a cultured system, a research collaboration between Kumamoto University and Nagasaki University in Japan has found that components leaking from broken muscle fibers activate “satellite” muscle stem cells. While attempting to identify the proteins that activate satellite cells, they found that metabolic enzymes, such as GAPDH, rapidly activated dormant satellite cells and accelerated muscle injury regeneration. This is a highly rational and efficient regeneration mechanism in which the damaged muscle itself activates the satellite cells that begin the regeneration process.

Skeletal muscle is made up of bundles of contracting muscle fibers and each muscle fiber is surrounded by satellite cells — muscle stem cells that can produce new muscle fibers. Thanks to the work of these satellite cells, muscle fibers can be regenerated even after being bruised or torn during intense exercise. Satellite cells also play essential roles in muscle growth during developmental stages and muscle hypertrophy during strength training. However, in refractory muscle diseases like muscular dystrophy and age-related muscular fragility (sarcopenia), the number and function of satellite cells decreases. It is therefore important to understand the regulatory mechanism of satellite cells in muscle regeneration therapy.

In mature skeletal muscle, satellite cells are usually present in a dormant state. Upon stimulation after muscle injury, satellite cells are rapidly activated and proliferate repeatedly. During the subsequent myogenesis, they differentiate and regenerate muscle fibers by fusing with existing muscle fibers or with together. Of these three steps (satellite cell activation, proliferation, and muscle differentiation), little is known about how the first step, activation, is induced.

Since satellite cells are activated when muscle fibers are damaged, researchers hypothesized that muscle damage itself could trigger activation. However, this is difficult to prove in animal models of muscle injury so they constructed a cell culture model in which single muscle fibers, isolated from mouse muscle tissue, were physically damaged and destroyed. Using this injury model, they found that components leaking from the injured muscle fibers activated satellite cells, and the activated cells entered the G1 preparatory phase of cell division. Further, the activated cells returned to a dormant state when the damaged components were removed, thereby suggesting that the damaged components act as the activation switch.

The research team named the leaking components “Damaged myofiber-derived factors” (DMDFs), after the broken muscle fibers, and identified them using mass spectrometry. Most of the identified proteins were metabolic enzymes, including glycolytic enzymes such as GAPDH, and muscle deviation enzymes that are used as biomarkers for muscle disorders and diseases. GAPDH is known as a “moonlighting protein” that has other roles in addition to its original function in glycolysis, such as cell death control and immune response mediation. The researchers therefore analyzed the effects of DMDFs, including GAPDH, on satellite cell activation and confirmed that exposure resulted in their entry into the G1 phase. Furthermore, the researchers injected GAPDH into mouse skeletal muscle and observed accelerated satellite cell proliferation after subsequent drug-induced muscle damage. These results suggest that DMDFs have the ability to activate dormant satellite cells and induce rapid muscle regeneration after injury. The mechanism by which broken muscle activates satellite cells is a highly effective and efficient tissue regeneration mechanism.

“In this study, we proposed a new muscle injury-regeneration model. However, the detailed molecular mechanism of how DMDFs activate satellite cells remains an unclear issue for future research. In addition to satellite cell activation, DMDF moonlighting functions are expected to be diverse,” said Associate Professor Yusuke Ono, leader of the study. “Recent studies have shown that skeletal muscle secretes various factors that affect other organs and tissues, such as the brain and fat, into the bloodstream, so it may be possible that DMDFs are involved in the linkage between injured muscle and other organs via blood circulation. We believe that further elucidation of the functions of DMDFs could clarify the pathologies of some muscle diseases and help in the development of new drugs.”

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Materials provided by Kumamoto University. Note: Content may be edited for style and length.

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The functional importance of estrogen receptor beta — ScienceDaily

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Researchers at Kumamoto University, Japan generated mice lacking the estrogen receptor beta (ERβ) gene, both fiber-specific and muscle stem cell-specific, which resulted in abnormalities in the growth and regeneration of skeletal muscle in female mice. This was not observed in male mice that lacked the ERβ gene, suggesting that estrogen and its downstream signals may be a female-specific mechanism for muscle growth and regeneration.

In humans, skeletal muscle mass generally peaks in the 20s with a gradual decline beginning in the 30s, but it is possible to maintain muscle mass through strength training and a healthy lifestyle. Skeletal muscle can be damaged through excessive exercise or bruising, but it has the ability to regenerate. The muscle stem cells that surround muscle fibers are essential for this regeneration; they also play a part in increasing muscle size (hypertrophy). Muscle stem cell dysfunction is thought to be associated with various muscle weakness, such as age-related sarcopenia and muscular dystrophy. Although basic research on skeletal muscle has progressed rapidly in recent years, most studies were conducted on male animals and gender differences were given much consideration.

Estrogen is a female hormone that maintains the homeostasis of various tissues and organs. A decrease in estrogen levels due to amenorrhea, menopause, or other factors can lead to a disturbance in biological homeostasis. When estrogen binds to estrogen receptors (ERs) in cells, it is transferred into the nucleus and binds to genomic DNA to induce the expression of specific genes as transcription factors. There are two types of ERs, ERα and ERβ. While both ERα and ERβ have high binding capacity to estrogen, their tissue distribution is different, they do not have a common DNA-binding domain, and they may act as antagonists to each other, suggesting that they have different roles. Furthermore, estrogen’s effects on cells can be both ER-mediated and non-ER-mediated.

An epidemiological study of pre and postmenopausal women in their 50s indicated an association between decreased blood estrogen levels and muscle weakness. A research group at Kumamoto University previously showed that estrogen is important for skeletal muscle development and regeneration using an ovariectomized estrogen deficiency mouse model (Kitajima and Ono, J Endocrinol 2016). They also examined the effectiveness of nutritional interventions in estrogen-deficient conditions (Kitajima et al., Nutrients 2017). However, whether estrogen acts directly on the ER of muscle fibers and muscle stem cells to regulate skeletal muscle growth and regeneration, or whether it acts indirectly through other tissues and organs was unclear. In this study, the researchers generated mice with either myofiber-specific or muscle stem cell-specific ERβ gene deletion and analyzed the function of ERβ in skeletal muscle.

To clarify the role of ERβ in the growth of skeletal muscle, researchers generated mice (mKO) in which the action of the ERβ gene could be turned off in myofibers with the administration of the drug doxycycline. ERβ deficiency was induced at 6 weeks of age, and muscle fiber area and strength of the tibialis anterior muscle was measured at 10-12 weeks. Compared to control mice, both indices were reduced in female mKO mice but not in male mice. Since there was no change in the expression of muscle atrophy-related genes, this reduced growth of female mice was not thought to be due to an increase in muscle atrophy. Ovariectomy-induced estrogen deficiency is known to be associated with muscle quality changes, such as a relative increase in the proportion of fast-type fibers (Kitajima and Ono, J Endocrinol 2016), but no such qualitative changes were observed in mKO mice. It was therefore suggested that, while it may have a direct effect on myofiber growth via ERβ (as expressed in myofibers), estrogen may also regulate the quality of myofibers in a non-ERβ-mediated manner.

To determine the function of ERβ in muscle stem cells, the researchers generated scKO mice in which the ERβ gene could be deleted in muscle stem cells with the administration of the drug tamoxifen. They then evaluated muscle regenerative capacity by locally inducing muscle damage. While muscle regeneration was efficient in control mice, the regenerated muscle tissue of female scKO mice showed thin regenerated muscle fibers, fibrosis caused by collagen deposition, and significantly reduced muscle regenerative capacity. Muscle regeneration in male scKO mice, however, was not impaired. Because impaired muscle regeneration in females was not exacerbated by ovariectomies that made them estrogen deficient, the researchers thus thought that estrogen regulates muscle regeneration via ERβ expressed by muscle stem cells.

To further investigate the cause of reduced muscle regenerative capacity, researchers isolated and cultured muscle stem cells for evaluation. ERβ in cells from scKO mice was evaluated in several experiments using siRNAs and inhibitors. ERβ was found to contribute to the promotion of muscle stem cell proliferation and the inhibition of cell death. Gene expression analysis (RNA-seq) of scKO muscle stem cells showed that the expression of “niche”-related genes, which are required for the maintenance of stem cell properties, was reduced in scKO muscle stem cells. Therefore, the researchers hypothesize that the inactivation of ERβ may have affected the proliferation and survival of muscle stem cells by inhibiting the formation of stem cell niches.

This study is thought to be the first to show that ERβ in genetic mouse models plays an important role in the growth and regeneration of skeletal muscle through its function in both muscle fibers and muscle stem cells. However, the role of ERβ in male mice has not yet been elucidated and remains to be addressed even though its expression in both male and female mice is comparable.

“Amenorrhea is induced in female athletes through rigorous training or excessive dieting and has become one of three major problems, together with low energy availability and osteoporosis, faced by female athletes worldwide,” said study leader Associate Professor Yusuke Ono. “Although the animal findings of this study cannot be directly applied to humans, they do suggest that decreased estrogen during amenorrhea may suppress ERβ activity in muscle fibers and muscle stem cells. For female athletes, this may lead to poor athletic performance and delayed recovery from injuries, and puts them at risk for adverse competitive conditions. Our plan is to continue investigating the pathogenesis of age-related sarcopenia and muscular dystrophy by targeting ERβ and its downstream signals with the goal of developing treatments.”

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Researchers make counterintuitive discoveries about immune-like characteristics of cells — ScienceDaily

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Vanderbilt University researchers have reported the counterintuitive discovery that certain chemotherapeutic agents used to treat tumors can have the opposite effect of tissue overgrowth in normal, intact mammary glands, epidermis and hair follicles. The researchers also are the first to report the discovery of an innate immune signaling pathway in fibroblasts — the spindle-shaped cells responsible for wound healing and collagen production — that causes cells to proliferate. Such signaling pathways previously were attributed only to immune cells.

The article describing the research, “DNA Damage Promotes Epithelial Hyperplasia and Fate Mis-specification via Fibroblast Inflammasome Activation,” was published in the journal Developmental Cell on Oct. 13.

The findings of this work, led by postdoctoral fellow Lindsey Seldin and Professor and Chair of the Department of Cell and Developmental Biology Ian Macara, have broad implications for diseases associated with the immune system like psoriasis, as well as cancer and stem cell research.

Understanding the functionality of stem cells and the way that their behavior is regulated has been a longstanding research interest for Seldin. “Normal stem cells have an amazing ability to continuously divide to maintain tissue function without forming tumors,” she explained. “We wanted to understand what happens to these cells in their native environment when subjected to damage, and if the response was connected to a specific tissue.”

By testing perturbations to the epidermis, mammary gland and hair follicles vis-à-vis mechanical damage or DNA damage through chemotherapeutic agents, the researchers saw a paradoxical response: Stem cells, which otherwise would divide slowly, instead divided rapidly, promoting tissue overgrowth.

When the tissues were subjected to DNA damage, their stem cells overly proliferated, giving rise to different cells than they normally would. “This was a very perplexing result,” said Seldin, the paper’s lead author. “We were determined to figure out if this was a direct response by the stem cells themselves or by inductive signals within their environment.” The key clue was that stem cells isolated from the body did not behave the same way as in intact tissue — an indication that the response must be provoked from signals being sent to the stem cells from other surrounding cell types.

The investigators turned their attention to fibroblasts, the predominant component of the tissue microenvironment. When fibroblasts in the epidermis were removed, the stem cell responsiveness to DNA damage was diminished, indicating that they played an important role. RNA sequencing revealed that fibroblasts can signal by way of inflammasomes — complexes within cells that help tissues respond to stress by clearing damaged cells or pathogens, which also in this case caused stem cells to divide. “This is an astounding discovery,” said Macara. “Inflammasome signaling has previously been attributed only to immune cells, but now it seems that fibroblasts can assume an immune-like nature.”

Seldin intends to replicate this work in the mammary gland to determine whether fibroblasts initiate the same innate immune response as in the epidermis, and more broadly how fibroblasts contribute to the development of cancer and other diseases associated with the immune system.

This work was supported by NCI/NIH grants R35CA132898, F32CA213794 and T32CA119925, as well as American Cancer Society grant PF-18-007-01-CCG.

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Materials provided by Vanderbilt University. Original written by Marissa Shapiro. Note: Content may be edited for style and length.

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Hair loss might be prevented by regulating stem cell metabolism — ScienceDaily

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Hair follicle stem cells, which promote hair growth, can prolong their life by switching their metabolic state. In experiments conducted with mice, a research group active in Helsinki and Cologne, Germany, has demonstrated that a protein called Rictor holds a key role in the process.

The study was published in the Cell Metabolism journal.

Mechanisms that regulate stem cells

Ultraviolet radiation and other environmental factors damage our skin and other tissues every day, with the body continuously removing and renewing the damaged tissue. On average, humans shed daily 500 million cells and a quantity of hairs weighing a total of 1.5 grams.

The dead material is replaced by specialised stem cells that promote tissue growth. Tissue function is dependent on the activity and health of these stem cells, as impaired activity results in the ageing of the tissues.

“Although the critical role of stem cells in ageing is established, little is known about the mechanisms that regulate the long-term maintenance of these important cells. The hair follicle with its well understood functions and clearly identifiable stem cells was a perfect model system to study this important question,” says Sara Wickstrom.

Reduced metabolic flexibility in stem cells underlying hair loss

At the end of hair follicles’ regenerative cycle, the moment a new hair is created, stem cells return to their specific location and resume a quiescent state. The key finding in the new study is that this return to the stem cell state requires a change in the cells’ metabolic state. They switch from glutamine-based metabolism and cellular respiration to glycolysis, a shift triggered by signalling induced by a protein called Rictor, in response to the low oxygen concentration in the tissue. Correspondingly, the present study demonstrated that the absence of the Rictor protein impaired the reversibility of the stem cells, initiating a slow exhaustion of the stem cells and hair loss caused by ageing.

The research group created a genetic mouse model to study the function of the Rictor protein, observing that hair follicle regeneration and cycle were significantly delayed in mice lacking the protein. Ageing mice suffering from Rictor deficiency showed a gradual decrease in their stem cell, resulting in loss of hair.

Precursors for developing hair loss drug therapies

Further research will now be conducted to investigate how these preclinical findings could be utilised in human stem cell biology and potentially also in drug therapies that would protect hair follicles from ageing. In other words, the mechanisms identified in the study could possibly be utilised in preventing hair loss.

“We are particularly excited about the observation that the application of a glutaminase inhibitor was able to restore stem cell function in the Rictor-deficient mice, proving the principle that modifying metabolic pathways could be a powerful way to boost the regenerative capacity of our tissues,” Wickstrom explains.

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Study finds that compressing cells, and crowding their contents, can coax them to grow and divide — ScienceDaily

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The closer people are physically to one another, the higher the chance for exchange, of things like ideas, information, and even infection. Now researchers at MIT and Boston Children’s Hospital have found that, even in the microscopic environment within a single cell, physical crowding increases the chance for interactions, in a way that can significantly alter a cell’s health and development.

In a paper published today in the journal Cell Stem Cell, the researchers have shown that physically squeezing cells, and crowding their contents, can trigger cells to grow and divide faster than they normally would.

While squeezing something to make it grow may sound counterintuitive, the team has an explanation: Squeezing acts to wring water out of a cell. With less water to swim in, proteins and other cell constituents are packed closer together. And when certain proteins are brought in close proximity, they can trigger cell signaling and activate genes within the cell.

In their new study, the scientists found that squeezing intestinal cells triggered proteins to cluster along a specific signaling pathway, which can help cells maintain their stem-cell state, an undifferentiated state in which can quickly grow and divide into more specialized cells. Ming Guo, associate professor of mechanical engineering at MIT, says that if cells can simply be squeezed to promote their “stemness,” they can then be directed to quickly build up miniature organs, such as artificial intestines or colons, which could then be used as platforms to understand organ function and test drug candidates for various diseases, and even as transplants for regenerative medicine.

Guo’s co-authors are lead author Yiwei Li, Jiliang Hu, and Qirong Lin from MIT, and Maorong Chen, Ren Sheng, and Xi He of Boston Children’s Hospital.

Packed in

To study squeezing’s effect on cells, the researchers mixed various cell types in solutions that solidified as rubbery slabs of hydrogel. To squeeze the cells, they placed weights on the hydrogel’s surface, in the form of either a quarter or a dime.

“We wanted to achieve a significant amount of cell size change, and those two weights can compress the cell by something like 10 to 30 percent of their total volume,” Guo explains.

The team used a confocal microscope to measure in 3D how individual cells’ shapes changed as each sample was compressed. As they expected, the cells shrank with pressure. But did squeezing also affect the cell’s contents? To answer this, the researchers first looked to see whether a cell’s water content changed. If squeezing acts to wring water out of a cell, the researchers reasoned that the cells should be less hydrated, and stiffer as a result.

They measured the stiffness of cells before and after weights were applied, using optical tweezers, a laser-based technique that Guo’s lab has employed for years to study interactions within cells, and found that indeed, cells stiffened with pressure. They also saw that there was less movement within cells that were squeezed, suggesting that their contents were more packed than usual.

Next, they looked at whether there were changes in the interactions between certain proteins in the cells, in response to cells being squeezed. They focused on several proteins that are known to trigger Wnt/?-catenin signaling, which is involved in cell growth and maintenance of “stemness.”

“In general, this pathway is known to make a cell more like a stem cell,” Guo says. “If you change this pathway’s activity, how cancer progresses and how embryos develop have been shown to be very different. So we thought we could use this pathway to demonstrate how cell crowding is important.”

A “refreshing” path

To see whether cell squeezing affects the Wnt pathway, and how fast a cell grows, the researchers grew small organoids — miniature organs, and in this case, clusters of cells that were collected from the intestines of mice.

“The Wnt pathway is particularly important in the colon,” Guo says, pointing out that the cells that line the human intestine are constantly being replenished. The Wnt pathway, he says, is essential for maintaining intestinal stem cells, generating new cells, and “refreshing” the intestinal lining.

He and his colleagues grew intestinal organoids, each measuring about half a millimeter, in several Petri dishes, then “squeezed” the organoids by infusing the dishes with polymers. This influx of polymers increased the osmotic pressure surrounding each organoid and forced water out of their cells. The team observed that as a result, specific proteins involved in activating the Wnt pathway were packed closer together, and were more likely to cluster to turn on the pathway and its growth-regulating genes.

The upshot: Those organoids that were squeezed actually grew larger and more quickly, with more stem cells on their surface than those that were not squeezed.

“The difference was very obvious,” Guo says. “Whenever you apply pressure, the organoids grow even bigger, with a lot more stem cells.”

He says the results demonstrate how squeezing can affect a organoid’s growth. The findings also show that a cell’s behavior can change depending on the amount of water that it contains.

“This is very general and broad, and the potential impact is profound, that cells can simply tune how much water they have to tune their biological consequences,” Guo says.

Going forward, he and his colleagues plan to explore cell squeezing as a way to speed up the growth of artificial organs that scientists may use to test new, personalized drugs.

“I could take my own cells and transfect them to make stem cells that can then be developed into a lung or intestinal organoid that would mimic my own organs,” Guo says. “I could then apply different pressures to make organoids of different size, then try different drugs. I imagine there would be a lot of possibilities.”

This research is supported, in part, by the National Cancer Institute and the Alfred P. Sloan Foundation.

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Human intestinal organoids grown from stem cells used to model congenital disorder in babies — ScienceDaily

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Scientists at Cincinnati Children’s used human intestinal organoids grown from stem cells to discover how our bodies control the absorption of nutrients from the food we eat. They further found that one hormone might be able to reverse a congenital disorder in babies who cannot adequately absorb nutrients and need intravenous feeding to survive.

Heather A. McCauley, PhD, a research associate at Cincinnati Children’s Hospital Medical Center, found that the hormone peptide YY, also called PYY, can reverse congenital malabsorption in mice. With a single PYY injection per day, 80% of the mice survived. Normally, only 20% to 30% survive.

This indicates PYY might be a possible therapeutic for people with severe malabsorption.

Poor absorption of macronutrients is a global health concern, underlying ailments such as malnutrition, intestinal infections and short-gut syndrome. So, identification of factors regulating nutrient absorption has significant therapeutic potential, the researchers noted.

McCauley was lead author of a manuscript published Sept. 22 in Nature Communications, which reported that the absorption of nutrients — in particular, carbohydrates and proteins — is controlled by enteroendocrine cells in the gastrointestinal tract.

Babies born without enteroendocrine cells — or whose enteroendocrine cells don’t function properly — have severe malabsorption and require IV nutrition.

“This study allowed us to understand how important this one rare cell type is in controlling how the intestine absorbs nutrients and functions on a daily basis,” McCauley said.

The Cincinnati Children’s study, “Enteroendocrine cells couple nutrient sensing to nutrient absorption by regulating ion transport,” was the first to describe a mechanism linking enteroendocrine cells to the absorption of macronutrients like carbohydrates and amino acids.

One key finding of the study is how these cells, upon sensing ingested nutrients, prepare the intestine to absorb nutrients by controlling the influx and outflux of electrolytes and water, the researchers stated. Absorption of carbohydrates and protein is then linked to the movement of ions in the intestine.

For this study, the scientists relied on human intestinal organoid models created in a lab, said James Wells, PhD, senior author of the study and chief scientific officer of the Center for Stem Cell and Organoid Medicine (CuSTOM) at Cincinnati Children’s.

Grown from stem cells, organoids are small formations of human organ that have an architecture and functions that are similar to their full-size counterparts.

Cincinnati Children’s launched efforts to make organoids from human pluripotent stem cells in 2006, said Wells, who is also director for basic research in the Division of Endocrinology at the medical center and an Allen Foundation Distinguished Investigator.

“What this study highlights is how decades of basic research into how organs are made and how they function is now leading to breakthroughs in identifying new therapeutics,” said Wells, who has led a team of investigators at Cincinnati Children’s who developed some of the first human organoid technologies that are now used globally.

The study on malabsorption used three different human small intestinal tissue models — all derived from pluripotent stem cells, which can form any kind of tissue in the body.

“The human organoids are essentially a much more realistic avatar to these patients with these rare mutations,” Wells said. “They allow us to model much more faithfully the human disease.”

McCauley and Wells conceived and initiated the recent study on malabsorption, designed the experiments and wrote the manuscript. Contributors to the study included intestinal physiology experts Marshall “Chip” Montrose, PhD, and Eitaro Aihara, PhD, of the University of Cincinnati.

The study was supported by grants from the National Institutes of Health (U19 AI116491, P01 HD093363, UG3 DK119982, U01 DK103117); S&R Foundation and American Physiological Society; the American Diabetes Association (1-17-PDF-102); the Shipley Foundation and the Allen Foundation. Support was also received from the Digestive Disease Research Center (P30 DK078392).

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Stem cell sheets harvested in just two days — ScienceDaily

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Stem cells are cell factories that constantly divide themselves to create new cells. Implanting stem cells in damaged organs can regenerate new tissues. Cell sheet engineering, which allows stem cells to be transplanted into damaged areas in the form of sheets made up of only cells, completely eliminates immune rejection caused by external substances and encourages tissue regeneration. A research team led by POSTECH recently succeeded in drastically reducing the harvest period of such stem cell sheets.

A joint research team comprised of Professor Dong Sung Kim and researcher Andrew Choi of POSTECH’s Department of Mechanical Engineering and Dr. InHyeok Rhyou and Dr. Ji-Ho Lee of the Department of Orthopedic Surgery at Pohang Semyung Christianity Hospital has significantly reduced the total harvest period of a stem cell sheet to two days. The nanotopography of poly(N-isopropylacrylamide) (PNIPAAm), which abruptly changes its roughness depending on temperature, allows harvesting of cell sheets that consist of mesenchymal stem cells derived from human bone marrow. Considering that it takes one week on average to make stem cells into sheets using the existing techniques developed so far, this is the shortest harvest time on record. These research findings were published as a cover paper in the latest issue of Biomaterials Science, an international journal in the biomaterials field.

Professor Kim’s research team focused on PNIPAAm, a polymer that either combines with water or averts it depending on the temperature. In previous studies, PNIPAAm has been introduced as a coating material for cell culture platform to harvest cell sheets, but the range of utilization had been hampered due to the limited types of cells that can be made into sheets. For the first time in 2019, the research team developed a technology of easily regulating the roughness of 3D bulk PNIPAAm and has stably produced various types of cells into sheets.

The study conducted this time focused on making stem cells — that are effective in tissue regeneration — into sheets in a short time in order to increase their direct utility. The team achieved this by applying an isotropic pattern of nanopores measuring 400 nanometers (nm, 1 billionth of a meter) on the surface of a 3D bulk PNIPAAm. As a result, not only did the formation and maturity of human bone marrow-derived mesenchymal stem cells on the nanotopography of bulk PNIPAAm accelerate, but the surface roughness of bulk PNIPAAm at room temperature below the lower critical solution temperature (LCST) was also rapidly increased, effectively inducing the detachment of cell sheets. This in turn enabled the rapid harvesting of human bone marrow-derived mesenchymal stem cell sheets.

“At least five days are needed to harvest stem cell sheets reported through previous researches,” commented Andrew Choi, the ” author of the paper. “We can now harvest them in just two days with the PNIPAAm nanotopography developed this time.”

“We have significantly shortened the harvest time by introducing nanotopography on the surface of the 3D bulk PNIPAAm to produce mature stem cell sheets for the first time in the world,” remarked Professor Dong Sung Kim who led the study. He added, “We have opened up the possibility of applying the sheets directly to patients in the future.”

The research was conducted with the support from Basic Research Program (Mid-career Researcher Program) and the Biomedical Technology Development Program of the National Research Foundation and the Ministry of Science and ICT of Korea.

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Coupling antibiotics with stem cells to fight off bone infections — ScienceDaily

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Bone infections caused by implants are difficult to treat and usually require a prolonged course of antibiotic treatment. In a new study, researchers from Kanazawa University discovered that implant-related bone infections can be effectively treated with a combinational treatment consisting of antibiotics and antibiotic-laden stem cells.

Bone fractures often require implants for stabilization and effective healing of the broken bone. However, implants can cause serious bone infections, such as osteomyelitis, that can only be managed with a prolonged antibiotic treatment. This in turn bears the risk of contributing to the development of antibiotic-resistant bacteria. While major efforts are currently underway to develop new antibiotics that cover these antibiotic-resistant bacteria, a different path has been to study the antibiotic effects of stem cells. One type is the so-called mesenchymal stem cells that naturally reside in the bone marrow and adipose tissue, among others, and that have been shown to possess antimicrobial properties.

“Adipose-derived stem cells, or ADSCs, have the distinct advantage of being abundant in subcutaneous adipose tissues and can thus be easily harvested,” says the corresponding author of the study Tamon Kabata. “The goal of our study was to investigate the therapeutic effects of ADSCs in combination with the antibiotic ciprofloxacin in an animal model of implant-related bone infection.”

To achieve their goal, the researchers first focused on the effects of ciprofloxacin on ADSCs and found an efficient, time-dependent loading of ADSCs with the antibiotic in the first 24 hours with no adverse effects of ciprofloxacin on the function or viability of the stem cells. The researchers then tested the antimicrobial activity of the antibiotic-loaded ADSCs in vitro (in a tube) and found that they effectively decreased the growth of the bacterium S. aureus, which is also the main microbe causing bone implant-related infections.

But could this novel approach also mitigate implant-related infection in a living organism? The researchers tested this on rats, who received bone implants using screws coated with S. aureus bacteria. The rats developed osteomyelitis 7 days after surgery. Then, the researchers administered one of the following to the animals: ADSCs loaded with ciprofloxacin, ADSCs alone, ciprofloxacin alone, or no treatment at all. Because osteomyelitis can lead to soft tissue swelling and abscess formation at the site of the infection, the researchers quantified the extent of the disease in the animals and found that only ADSCs loaded with ciprofloxacin presented as an effective treatment. Using the imaging modality micro-computed tomography to visualize the affected bones, the researchers further found that ADSC-loaded ciprofloxacin decreased the appearance of osteolysis, or bone degradation, which is not only important for bone health, but also for the stability of the implant.

“These are striking results that show how ADSCs can efficiently be loaded with antibiotics to exert a strong antimicrobial effect. Our findings suggest a potential novel therapy for implant-associated osteomyelitis, for which conventional treatment with only antibiotics is usually insufficient,” says Kabata.

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A small switch with a big impact — ScienceDaily

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T cells play a key role in the human immune system. They are capable of distinguishing diseased or foreign tissue from the body’s own, healthy tissue with great accuracy; they are capable of triggering the actions necessary to fight off the troublemakers. The details of this immune response are manifold and the individual steps are not yet fully understood.

Scientists of the universities of Würzburg and Mainz have now figured out new details of these processes, showing that tiny point mutations in a gene can modify T cells to be less aggressive. This could be an advantage after stem cell transplantation which includes T-cell transfusion in order to keep a number of severe side effects in check. The researchers have now published the results of their study in the Journal of Experimental Medicine. The study is led by Dr Friederike Berberich-Siebelt, head of the “Molecular and cellular immunology” research group at the Institute of Pathology of the University of Würzburg.

A protein family with multiple tasks

When T cells detect foreign or altered tissue, such as an infected or tumour tissue, this usually happens through the receptors on their cell surface. These T-cell receptors then send signals into the cell interior, initiating a response. In a first step, they activate a special family of transcription factors — scientifically called NFAT for nuclear factor of activated T-cells. The NFATs then bind to the DNA in the cell nucleus and trigger also the production of cytokines such as interleukin-2.

NFAT is composed of many family members which may have overlapping tasks or assume completely different functions. But that’s not all: Like many other proteins in the cell, they can still be modified after their synthesis to customize their function. The recently published study focuses on one specific modification of the NFATc1 “family member” which is called sumoylation.

Advantageous point mutations

“Sumoylation plays a role in different cellular processes such as nuclear transport, programmed cell death or as an antiviral mechanism,” Friederike Berberich-Siebelt explains. Sumoylation defects have also been observed in various diseases such as cancer and herpesvirus infections.

In the study now published, the scientists worked with laboratory animals that had two actually insignificant point mutations in the NFATc1 gene which, however, prevent sumoylation. This is not necessarily a disadvantage: “The offspring of these animals is perfectly healthy. The modified NFATc1 even mediates specific signals that reduce the clinical symptoms of multiple sclerosis at least in the animal model,” Berberich-Siebelt explains. When using T cells that carry these mutations in stem cell transplantation, they are much less aggressive against the tissues of the host animals than “normal” cells.

Fascinating fundamental research

This effect is due to an increase in interleukin-2 at the beginning of the immune response at the biomolecular level. Interleukin-2 counteracts the differentiation into inflammatory T-cell subtypes and at the same time supports so-called regulatory T cells according to the authors of the study. It is quite possible that this discovery will have consequences for future stem cell transplantation which includes T-cell infusion. When using T cells in which NFATc1 is not sumoylated, this might prevent severe side effects, making the point mutation “a small modification with a big impact” according to Berberich-Siebelt.

To investigate this in more detail, Berberich-Siebelt and her team will continue to research the possibilities of therapeutic implementation within the framework of the Collaborative Research Center/Transregio “Control of graft-versus-host and graft-versus-leukaemia immune responses after allogeneic stem cell transplantation” funded by the German Research Foundation (DFG). “We want to find out whether CRISPR/Cas9 gene editing can be applied to human T cells to exhibit just the right amount of activity during hematopoietic stem cell transplantation,” the scientist says.

But the new findings are also relevant independently of these potential consequences for therapeutic applications. “We are basically interested in understanding the fine regulation in cells, such as the T-cell receptor signalling and the function of NFAT family members and their isoforms in this context,” says Berberich-Siebelt who finds the newly published results “fascinating.” After all, the scientists did not have to switch off a gene or activate it excessively as is often the case in research. Instead, two actually harmless point mutations and subtle direct effects were sufficient to ultimately flip the switch from inflammation, autoimmunity and rejection to tolerance. A small shift of the focus at the beginning of the immune response had been sufficient to accomplish this.

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Materials provided by University of Würzburg. Original written by Gunnar Bartsch. Note: Content may be edited for style and length.

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Exosome treatment improves recovery from heart attacks in a preclinical study — ScienceDaily

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Science has long known that recovery from experimental heart attacks is improved by injection of a mixture of heart muscle cells, endothelial cells and smooth muscle cells, yet results have been limited by poor engraftment and retention, and researchers worry about potential tumorigenesis and heart arrhythmia.

Now research in pigs shows that using the exosomes naturally produced from that mixture of heart muscle cells, endothelial cells and smooth muscle cells — which were all derived from human induced pluripotent stem cells — yields regenerative benefits equivalent to the injected human induced pluripotent stem cell-cardiac cells, or hiPSC-CCs.

Exosomes are membrane-bound extracellular vesicles that contain biologically active proteins, RNAs and microRNAs. Exosomes are well known to participate in cell-to-cell communication, and they are actively studied as potential clinical therapies.

“The hiPSC-CC exosomes are acellular and, consequently, may enable physicians to exploit the cardioprotective and reparative properties of hiPSC-derived cells while avoiding the complexities associated with tumorigenic risks, cell storage, transportation and immune rejection,” said Ling Gao, Ph.D., and Jianyi “Jay” Zhang, M.D., Ph.D., University of Alabama at Birmingham corresponding authors of the study, published in Science Translational Medicine. “Thus, exosomes secreted by hiPSC-derived cardiac cells improved myocardial recovery without increasing the frequency of arrhythmogenic complications and may provide an acellular therapeutic option for myocardial injury.”

At UAB, Gao was a postdoctoral fellow in Biomedical Engineering, a joint department of the UAB School of Medicine and the UAB School of Engineering. Zhang is chair of the department.

Studies in large animals are necessary to identify, characterize and quantify responses to potential treatments. Prior to this current study, the feasibility of hiPSC-CC exosomes for cardiac therapy had been shown only in mouse models and in vitro work.

In the UAB experiments, juvenile pigs with experimental heart attacks had one of three treatments injected into the damaged myocardium: 1) a mixture of cardiomyocytes, endothelial cells and smooth muscle cells derived from human induced pluripotent stem cells, 2) exosomes extracted from the three cell types, or 3) homogenized fragments from the three cell types.

The researchers had two primary findings from the pig studies. First, they found that measurements of left-ventricle function, infarct size, wall stress, cardiac hypertrophy, apoptosis and angiogenesis in animals treated with hiPSC-CCs, hiPSC-CC fragments or hiPSC-CC exosomes were similar and significantly improved compared to animals that recovered without any of the three experimental treatments. Second, they found that exosome therapy did not increase the frequency of arrhythmia.

In experiments with cells or aortic rings grown in culture, they found that exosomes produced by hiPSC-CCs promoted blood vessel growth in cultured endothelial cells and isolated aortic rings. Furthermore, the exosomes protected cultured hiPSC-cardiomyocytes from the cytotoxic effects of serum-free low-oxygen media by reducing the programmed cell death called apoptosis and by maintaining intracellular calcium homeostasis, which has a direct beneficial effect on heart conductivity. The exosomes also increased cellular ATP content, which is beneficial since deficiencies in cellular ATP metabolism are believed to contribute to the progressive decline in heart function for patients with left ventricle hypertrophy and heart failure.

The researchers also found that some of these in vitro beneficial effects could also be mediated by synthetic mimics of the 15 most abundant microRNAs found in the hiPSC-CC exosomes. The researchers noted that knowledge of the potential role of microRNAs in clinical applications is still far from complete.

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Materials provided by University of Alabama at Birmingham. Original written by Jeff Hansen. Note: Content may be edited for style and length.

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