Scientists at the University of Cambridge have successfully created 'mini-lungs' using stem cells derived from skin cells of patients with cystic fibrosis, and have shown that these can be used to test potential new drugs for this debilitating lung disease.

The research is one of a number of studies that have used stem cells -- the body's master cells -- to grow 'organoids', 3D clusters of cells that mimic the behaviour and function of specific organs within the body. Other recent examples have been 'mini-brains' to study Alzheimer's disease and 'mini-livers' to model liver disease. Scientists use the technique to model how diseases occur and to screen for potential drugs; they are an alternative to the use of animals in research.

Cystic fibrosis is a monogenic condition -- in other words, it is caused by a single genetic mutation in patients, though in some cases the mutation responsible may differ between patients. One of the main features of cystic fibrosis is the lungs become overwhelmed with thickened mucus causing difficulty breathing and increasing the incidence of respiratory infection. Although patients have a shorter than average lifespan, advances in treatment mean the outlook has improved significantly in recent years.

Researchers at the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute used skin cells from patients with the most common form of cystic fibrosis caused by a mutation in the CFTR gene referred to as the delta-F508 mutation. Approximately three in four cystic fibrosis patients in the UK have this particular mutation. They then reprogrammed the skin cells to an induced pluripotent state, the state at which the cells can develop into any type of cell within the body.

Using these cells -- known as induced pluripotent stem cells, or iPS cells -- the researchers were able to recreate embryonic lung development in the lab by activating a process known as gastrulation, in which the cells form distinct layers including the endoderm and then the foregut, from which the lung 'grows', and then pushed these cells further to develop into distal airway tissue. The distal airway is the part of the lung responsible for gas exchange and is often implicated in disease, such as cystic fibrosis, some forms of lung cancer and emphysema.

The results of the study are published in the journal Stem Cells and Development.

"In a sense, what we've created are 'mini-lungs'," explains Dr Nick Hannan, who led the study. "While they only represent the distal part of lung tissue, they are grown from human cells and so can be more reliable than using traditional animal models, such as mice. We can use them to learn more about key aspects of serious diseases -- in our case, cystic fibrosis."

The genetic mutation delta-F508 causes the CFTR protein found in distal airway tissue to misfold and malfunction, meaning it is not appropriately expressed on the surface of the cell, where its purpose is to facilitate the movement of chloride in and out of the cells. This in turn reduces the movement of water to the inside of the lung; as a consequence, the mucus becomes particular thick and prone to bacterial infection, which over time leads to scarring -- the 'fibrosis' in the disease's name.

Using a fluorescent dye that is sensitive to the presence of chloride, the researchers were able to see whether the 'mini-lungs' were functioning correctly. If they were, they would allow passage of the chloride and hence changes in fluorescence; malfunctioning cells from cystic fibrosis patients would not allow such passage and the fluorescence would not change. This technique allowed the researchers to show that the 'mini-lungs' could be used in principle to test potential new drugs: when a small molecule currently the subject of clinical trials was added to the cystic fibrosis 'mini lungs', the fluorescence changed -- a sign that the cells were now functioning when compared to the same cells not treated with the small molecule.

"We're confident this process could be scaled up to enable us to screen tens of thousands of compounds and develop mini-lungs with other diseases such as lung cancer and idiopathic pulmonary fibrosis," adds Dr Hannan. "This is far more practical, should provide more reliable data and is also more ethical than using large numbers of mice for such research."

Originally posted here:
Scientists grow 'mini-lungs' to aid study of cystic fibrosis

Scientists at the University of Cambridge have successfully created 'mini-lungs' using stem cells derived from skin cells of patients with cystic fibrosis, and have shown that these can be used to test potential new drugs for this debilitating lung disease.

The research is one of a number of studies that have used stem cells - the body's master cells - to grow 'organoids', 3D clusters of cells that mimic the behaviour and function of specific organs within the body. Other recent examples have been 'mini-brains' to study Alzheimer's disease and 'mini-livers' to model liver disease. Scientists use the technique to model how diseases occur and to screen for potential drugs; they are an alternative to the use of animals in research.

Cystic fibrosis is a monogenic condition - in other words, it is caused by a single genetic mutation in patients, though in some cases the mutation responsible may differ between patients. One of the main features of cystic fibrosis is the lungs become overwhelmed with thickened mucus causing difficulty breathing and increasing the incidence of respiratory infection. Although patients have a shorter than average lifespan, advances in treatment mean the outlook has improved significantly in recent years.

Researchers at the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute used skin cells from patients with the most common form of cystic fibrosis caused by a mutation in the CFTR gene referred to as the delta-F508 mutation. Approximately three in four cystic fibrosis patients in the UK have this particular mutation. They then reprogrammed the skin cells to an induced pluripotent state, the state at which the cells can develop into any type of cell within the body.

Using these cells - known as induced pluripotent stem cells, or iPS cells - the researchers were able to recreate embryonic lung development in the lab by activating a process known as gastrulation, in which the cells form distinct layers including the endoderm and then the foregut, from which the lung 'grows', and then pushed these cells further to develop into distal airway tissue. The distal airway is the part of the lung responsible for gas exchange and is often implicated in disease, such as cystic fibrosis, some forms of lung cancer and emphysema.

The results of the study are published in the journal Stem Cells and Development.

"In a sense, what we've created are 'mini-lungs'," explains Dr Nick Hannan, who led the study. "While they only represent the distal part of lung tissue, they are grown from human cells and so can be more reliable than using traditional animal models, such as mice. We can use them to learn more about key aspects of serious diseases - in our case, cystic fibrosis."

The genetic mutation delta-F508 causes the CFTR protein found in distal airway tissue to misfold and malfunction, meaning it is not appropriately expressed on the surface of the cell, where its purpose is to facilitate the movement of chloride in and out of the cells. This in turn reduces the movement of water to the inside of the lung; as a consequence, the mucus becomes particular thick and prone to bacterial infection, which over time leads to scarring - the 'fibrosis' in the disease's name.

Using a fluorescent dye that is sensitive to the presence of chloride, the researchers were able to see whether the 'mini-lungs' were functioning correctly. If they were, they would allow passage of the chloride and hence changes in fluorescence; malfunctioning cells from cystic fibrosis patients would not allow such passage and the fluorescence would not change. This technique allowed the researchers to show that the 'mini-lungs' could be used in principle to test potential new drugs: when a small molecule currently the subject of clinical trials was added to the cystic fibrosis 'mini lungs', the fluorescence changed - a sign that the cells were now functioning when compared to the same cells not treated with the small molecule.

"We're confident this process could be scaled up to enable us to screen tens of thousands of compounds and develop mini-lungs with other diseases such as lung cancer and idiopathic pulmonary fibrosis," adds Dr Hannan. "This is far more practical, should provide more reliable data and is also more ethical than using large numbers of mice for such research."

Continued here:
Scientists grow 'mini-lungs' to aid the study of cystic fibrosis

4 hours ago By labeling a piece of a stem cell superenhancer, which is a gene amplifying region, researchers made stem cells fluoresce in green in living mice. The cells are visible above lining the base of the two hair follicles. Although they do not contain stem cells, the hair shafts also fluoresce green.

Stem cells can have a strong sense of identity. Taken out of their home in the hair follicle, for example, and grown in culture, these cells remain true to themselves. After waiting in limbo, these cultured cells become capable of regenerating follicles and other skin structures once transplanted back into skin. It's not clear just how these stem cells and others elsewhere in the body retain their ability to produce new tissue and heal wounds, even under extraordinary conditions.

New research at Rockefeller University has identified a protein, Sox9, that takes the lead in controlling stem cell plasticity. In a paper published today (March 18) in Nature, the team describes Sox9 as a "pioneer factor" that breaks ground for the activation of genes associated with stem cell identity in the hair follicle.

"We found that in the hair follicle, Sox9 lays the foundation for stem cell plasticity. First, Sox9 makes the genes needed by stem cells accessible, so they can become active. Then, Sox9 recruits other proteins that work together to give these "stemness" genes a boost, amplifying their expression," says study author Elaine Fuchs, Rebecca C. Lancefield Professor, Robin Chemers Neustein Laboratory of Mammalian Cell Biology and Development. "Without Sox9, this process never happens, and hair follicle stem cells cannot survive."

Sox9 is a type of protein called a transcription factor, which can act like a volume dial for genes. When a transcription factor binds to a segment of DNA known as an enhancer, it cranks up the activity of the associated gene. Recently, scientists identified a less common, but more powerful version: the super-enhancer. Super-enhancers are much longer pieces of DNA, and host large numbers of cell type-specific transcription factors that bind cooperatively. Super-enhancers also containhistones, DNA-packagingproteins, that harbor specific chemical groups epigenetic marks that make genes they are associated with accessible so they can be expressed.

Using an epigenetic mark associated specifically with the histones of enhancers, first author Rene Adam, a graduate student in the lab, and colleagues, identified 377 of these high-powered gene-amplifying regions in hair follicle stem cells. The majority of these super-enhancers were bound by at least five transcription factors, often including Sox9. Then, they compared the stem cell super-enhancers to those of short-lived stem cell progeny, which have begun to choose a fate, and so lost the plasticity of stem cells. These two types of cells shared only 32 percent of their superenhancers, suggesting these regions played an important role in skin cell identity. By switching off super-enhancers associated with stem cell genes, these genes were silenced while new super-enhancers were being activated to turn on hair genes.

To better understand these dynamics, the researchers took a piece of a super-enhancer, which they called an "epicenter," where all the stem cell transcription factors bind, and they linked it to a gene thatglowed green whenever the transcription factors were present. In living mice, all the hair follicle stem cells glowed green, but surprisingly, the green gene turned off when the stem cells were taken from the follicle and placed in culture. When they put the cells back into living skin, the green glow returned.

Another clue came from experiments performed by Hanseul Yang, another student in the lab. By examining the new super-enhancers that were gained when the stem cells were cultured, they learned that these new super-enhancers bound transcription factors that were known to be activated during wound-repair. When they used one of these epicenters to drive the green gene, the green glow was seen in culture, but not in skin. When they wounded the skin, then the green glow switched on.

"We were learning that some super-enhancers are specifically activated in the stem cells within their native niche, while other super-enhancers specifically switch on during injury," explained Adam. "By shifting epicenters, you can shift from one cohort of transcription factors to another to adapt to different environments. But we still needed to determine what was controlling these shifts."

The culprit turned out to be Sox9, the only transcription factor expressed in both living tissue and culture. Further experiments confirmed Sox9's importance by showing, for example, that removing it spelled death for stem cells, while expressing it in the epidermis gave the skin cells features of hair follicle stem cells. These powers seemed to be special to Sox9, placing it atop the hierarchy of transcription factors in the stem cells. Sox9 is one of only a few pioneer factors known in biology which can initiate such dramatic changes in gene expression.

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Scientists pinpoint molecule that controls stem cell plasticity by boosting gene expression