Scientists Pinpoint Molecule that Switches On Stem Cell Genes

Experiments placed Sox9 at the crux of a shift in gene expression associated with hair follicle stem cell identity

Friday, 20 March 2015

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, which takes the lead in controlling stem cell plasticity. In a paper published 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 contain histones, DNA-packaging proteins, which harbour 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 super-enhancers, 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, called an epicentre, where all the stem cell transcription factors bind, and they linked it to a gene that glowed 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 epicentres to drive the green gene, the green glow appeared in culture, but not in skin. When they wounded the skin, then the green glow switched on.

Researchers made stem cells fluoresce green (at

the base of hair follicles above) by labelling their

super-enhancers, regions of the genome bound

by gene-amplifying proteins. It appears one such

protein, Sox9, leads the activation of super-

enhancers that boost genes associated with stem

cell plasticity. Credit: Laboratory of

Mammalian Cell Biology and Development at

the Rockefeller University/Nature. 

"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 epicentres, 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.

"Importantly, we link this pioneer factor to super-enhancer dynamics, giving these domains a 'one-two punch' in governing cell identity. In the case of stem cell plasticity, Sox9 appears to be the lead factor that activates the super-enhancers that amplify genes associated with stemness," Fuchs says.

"These discoveries offer new insights into the way in which stem cells choose their fates and maintain plasticity while in transitional states, such as in culture or when repairing wounds."

Source: Rockefeller University

Contact: Wynne Parry

Reference:

Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice

Rene C. Adam, Hanseul Yang, Shira Rockowitz, Samantha B. Larsen, Maria Nikolova, Daniel S. Oristian, Lisa Polak, Meelis Kadaja, Amma Asare, Deyou Zheng, and Elaine Fuchs

Nature, March 18, 2015, doi:10.1038/nature14289

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Converting Adult Human Cells to Hair-follicle Generating Stem Cells

Implications for hair regeneration

Tuesday, 28 January 2014

If the content of many a situation comedy, not to mention late-night TV advertisements, is to be believed, there's an epidemic of balding men, and an intense desire to fix their follicular deficiencies.

This shows hair shafts (arrows) formed by

induced pluripotent stem cell-derived epithelial

stem cells. Credit: Ruifeng Yang, Perelman

School of Medicine, University of

Pennsylvania. 

One potential approach to reversing hair loss is to use stem cells to regenerate the missing or dying hair follicles. But it hasn't been possible to generate sufficient number of hair-follicle-generating stem cells – until now.

Xiaowei "George" Xu, MD, PhD, associate professor of Pathology and Laboratory Medicine and Dermatology at the Perelman School of Medicine, University of Pennsylvania, and colleagues published in Nature Communications a method for converting adult cells into epithelial stem cells (EpSCs), the first time anyone has achieved this in either humans or mice.

The epithelial stem cells, when implanted into immunocompromised mice, regenerated the different cell types of human skin and hair follicles, and even produced structurally recognizable hair shaft, raising the possibility that they may eventually enable hair regeneration in people.

Xu and his team, which includes researchers from Penn's departments of Dermatology and Biology, as well as the New Jersey Institute of Technology, started with human skin cells called dermal fibroblasts. By adding three genes, they converted those cells into induced pluripotent stem cells (iPSCs), which have the capability to differentiate into any cell types in the body. They then converted the iPS cells into epithelial stem cells, normally found at the bulge of hair follicles.

Starting with procedures other research teams had previously worked out to convert iPSCs into keratinocytes, Xu's team demonstrated that by carefully controlling the timing of the growth factors the cells received, they could force the iPSCs to generate large numbers of epithelial stem cells. In the Xu study, the team's protocol succeeded in turning over 25% of the iPSCs into epithelial stem cells in 18 days. Those cells were then purified using the proteins they expressed on their surfaces.

Comparison of the gene expression patterns of the human iPSC-derived epithelial stem cells with epithelial stem cells obtained from human hair follicles showed that the team had succeeded in producing the cells they set out to make in the first place. When they mixed those cells with mouse follicular inductive dermal cells and grafted them onto the skin of immunodeficient mice, they produced functional human epidermis (the outermost layers of skin cells) and follicles structurally similar to human hair follicles.

"This is the first time anyone has made scalable amounts of epithelial stem cells that are capable of generating the epithelial component of hair follicles," Xu says.

And those cells have many potential applications, he adds, including wound healing, cosmetics, and hair regeneration.

That said, iPSC-derived epithelial stem cells are not yet ready for use in human subjects, Xu adds. First, a hair follicle contains epithelial cells – a cell type that lines the body's vessels and cavities – as well as a specific kind of adult stem cell called dermal papillae. Xu and his team mixed iPSC-derived EpSCs and mouse dermal cells to generate hair follicles to achieve the growth of the follicles.

"When a person loses hair, they lose both types of cells." Xu explains.

"We have solved one major problem, the epithelial component of the hair follicle. We need to figure out a way to also make new dermal papillae cells, and no one has figured that part out yet."

What's more, the process Xu used to create iPSCs involves genetic modification of human cells with genes encoding oncogenic proteins and so needs more refinement. Still, he notes that stem-cell researchers are developing more workarounds, including strategies using only chemical agents.

Source: University of Pennsylvania School of Medicine 

Contact: Karen Kreeger

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Hair Regeneration Method is First to Induce New Human Hair Growth

Technique uses a patient's own cells to grow new hair

Tuesday, 22 October 2013

The source of new hair: For the first time,

researchers have been able to take human

dermal papilla cells (those inside the base

of human hair follicles) and use them to

create new hairs. Credit: Claire Higgins/

Christiano Lab at Columbia University

Medical Center.

Researchers at Columbia University Medical Center (CUMC) have devised a hair restoration method that can generate new human hair growth, rather than simply redistribute hair from one part of the scalp to another. The approach could significantly expand the use of hair transplantation to women with hair loss, who tend to have insufficient donor hair, as well as to men in early stages of baldness. The study was published today in the online edition of the Proceedings of the National Academy of Sciences (PNAS).

"About 90 percent of women with hair loss are not strong candidates for hair transplantation surgery because of insufficient donor hair," said co-study leader Angela M. Christiano, PhD, the Richard and Mildred Rhodebeck Professor of Dermatology and professor of genetics & development.

"This method offers the possibility of inducing large numbers of hair follicles or rejuvenating existing hair follicles, starting with cells grown from just a few hundred donor hairs. It could make hair transplantation available to individuals with a limited number of follicles, including those with female-pattern hair loss, scarring alopecia, and hair loss due to burns."

According to Dr. Christiano, such patients gain little benefit from existing hair-loss medications, which tend to slow the rate of hair loss but usually do not stimulate robust new hair growth.

"Dermal papilla cells give rise to hair follicles, and the notion of cloning hair follicles using inductive dermal papilla cells has been around for 40 years or so," said co-study leader Colin Jahoda, PhD, professor of stem cell sciences at Durham University, England, and co-director of North East England Stem Cell Institute, who is one of the early founders of the field.

Spheres of cultured papillae cells from human

hair follicles successfully produced new human

hair when transplanted between the dermis and

epidermis of human skin. Credit: Claire Higgins/

Christiano Lab at Columbia University Medical

Center.

"However, once the dermal papilla cells are put into conventional, two-dimensional tissue culture, they revert to basic skin cells and lose their ability to produce hair follicles. So we were faced with a Catch-22: how to expand a sufficiently large number of cells for hair regeneration while retaining their inductive properties."

The researchers found a clue to overcoming this barrier in their observations of rodent hair. Rodent papillae can be easily harvested, expanded, and successfully transplanted back into rodent skin, a method pioneered by Dr. Jahoda several years ago. The main reason that rodent hair is readily transplantable, the researchers suspected, is that their dermal papillae (unlike human papillae) tend to spontaneously aggregate, or form clumps, in tissue culture. The team reasoned that these aggregations must create their own extracellular environment, which allows the papillae to interact and release signals that ultimately reprogram the recipient skin to grow new follicles.

"This suggested that if we cultured human papillae in such a way as to encourage them to aggregate the way rodent cells do spontaneously, it could create the conditions needed to induce hair growth in human skin," said first author Claire A. Higgins, PhD, associate research scientist.

To test their hypothesis, the researchers harvested dermal papillae from seven human donors and cloned the cells in tissue culture; no additional growth factors were added to the cultures. After a few days, the cultured papillae were transplanted between the dermis and epidermis of human skin that had been grafted onto the backs of mice. In five of the seven tests, the transplants resulted in new hair growth that lasted at least six weeks. DNA analysis confirmed that the new hair follicles were human and genetically matched the donors.

"This approach has the potential to transform the medical treatment of hair loss," said Dr. Christiano.

"Current hair-loss medications tend to slow the loss of hair follicles or potentially stimulate the growth of existing hairs, but they do not create new hair follicles. Neither do conventional hair transplants, which relocate a set number of hairs from the back of the scalp to the front. Our method, in contrast, has the potential to actually grow new follicles using a patient's own cells. This could greatly expand the utility of hair restoration surgery to women and to younger patients — now it is largely restricted to the treatment of male-pattern baldness in patients with stable disease."

More work needs to be done before the method can be tested in humans, according to the researchers.

"We need to establish the origins of the critical intrinsic properties of the newly induced hairs, such as their hair cycle kinetics, colour, angle, positioning, and texture" said Dr. Jahoda.

"We also need to establish the role of the host epidermal cells that the dermal papilla cells interact with, to make the new structures."

The team is optimistic that clinical trials could begin in the near future.

"We also think that this study is an important step toward the goal of creating a replacement skin that contains hair follicles for use with, for example, burn patients," said Dr Jahoda.

The researchers used gene expression analyses to determine that the three-dimensional cultures restored 22 percent of the gene expression seen in normal hair follicles. "That's less than we expected, but it was sufficient for inducing the growth of new hair follicles," said Dr. Christiano.

In addition, using methods for the analysis of regulatory networks developed by the Califano lab in the Center for Computational Biology and Bioinformatics, Department of Systems Biology, the researchers identified a number of transcription factors (gene regulators) that have the potential to mimic the environmental signals that trigger papillae to induce new hair growth. This information could help researchers develop ways to restore the expression of more genes involved in hair growth and to increase the efficiency of the induction.

Source: Columbia University Medical Center 

Contact: Karin Eskenazi

Reference:

Microenvironmental reprogramming by three-dimensional culture enables dermal papilla cells to induce de novo human hair-follicle growth

Claire A. Higgins, James C. Chen, Jane E. Cerise, Colin A. B. Jahoda, and Angela M. Christiano

PNAS, October 21, 2013, doi:10.1073/pnas.1309970110

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New Skin Stem Cells Surprisingly Similar to Those Found in Embryos

New Skin Stem Cells Surprisingly Similar to Those Found in Embryos Tuesday, 08 December 2009 Scientists have discovered a new type of stem cell in the skin that acts surprisingly like certain stem cells found in embryos: both can generate fat, bone, cartilage, and even nerve cells. These newly described dermal stem cells may one day prove useful for treating neurological disorders and persistent wounds, such as diabetic ulcers, says Freda Miller, an HHMI international research scholar. Miller and her colleagues first saw the cells several years ago in both rodents and people, but only now confirmed that the cells are stem cells. Like other stem cells, these cell scan self-renew and, under the right conditions, they can grow into the cell types that constitute the skin's dermal layer, which lies under the surface epidermal layer. "We showed that these cells are, in fact, the real thing," says Miller, a professor at the University of Toronto and a senior scientist in the department of developmental biology at the Hospital for Sick Children in Toronto. The dermal stem cells also appear to help form the basis for hair growth. The new work was published December 4, 2009, in the journal Cell Stem Cell. Though this research focuses on the skin, Miller has spent her career searching for cures for neurological diseases such as Parkinson's. About a decade ago, she decided to find an easily accessible cell that could be coaxed into making nerves. Brain stem cells, some of which can grow into nerves, lie deep in the middle of the organ and are too difficult to reach if the scientists eventually wanted to cultivate the cells from individual patients. "I thought, 'This is blue sky stuff, but you never know.'" She searched the literature and found that amphibians can regenerate nerves in their skin. She also found published "hints" that mammalian nerve cells could do the same. Her team looked in the dermal layer of the skin in both mice and people. Hair follicles and sweat glands are rooted in the dermis, a thick layer of cells that also help support and nourish blood vessels and touch-perceiving nerves. In 2001, Miller's team hit pay dirt when they discovered cells that respond to the same growth factors that make brain stem cells differentiate. She named them skin-derived precursors (SKPs, or 'skips'). Miller soon discovered that the cells act like neural crest cells from embryos — stem cells that generate the entire peripheral nervous system and part of the head — in that they could turn into nerves, fat, bone, and cartilage. "That gave us the idea that these were some kind of embryonic-like precursor cell that migrated into the skin of the embryo," Miller said. "But instead of disappearing as the embryo develops, the cells survive into adulthood." Even though the SKPs acted like stem cells in Petri dishes, Miller didn't know if they behaved the same way in the body. "We were obviously very excited about these cells," she said. "The problem was, cells can do all kinds of weird things in culture dishes that look right but really aren't. We thought, 'Maybe we're being deceived.'" So lab member Jeffrey Biernaskie put the cells through their paces, performing a series of experiments to test whether the SKPs indeed acted like stem cells in the body. Earlier work in the lab had shown that the SKPs produce a transcription factor called SOX2, which is produced in many types of stem cells. The team used genetically engineered mice with SOX2 genes tagged with green fluorescent protein, which allowed them to track where SOX2 was expressed in the animals. They found that about 1% of skin cells from adult mice contained the SOX2-making cells, and they were concentrated in the bulb at the base of hair follicles. When the team cultured these cells, they began behaving like SKPs. Next, the scientists decided to see if the cells would not just settle at the base of hair follicles but grow new hair. They took the fluorescent cells, mixed them with epidermal cells — that makes up the majority of cells in a hair follicle — and transplanted the mixture under the skin of hairless mice. These mice began growing hair, and analysis showed the green cells migrated to their "home base" in the bulb of the new hair follicles. The team also transplanted rat SKP cells under the skin of mice. The cells behaved exactly like dermal stem cells – they spread out through the dermis and differentiated into various dermal cell types, including fat cells and dermal fibroblasts, which form the structural framework of the dermal layer. Intriguingly, the mice that carried transplanted rat SKPs also grew longer, thicker, rat-like hair, instead of short, thin mouse hair. "These cells are instructive, they tell the epidermal cells – which form the bulk of the hair follicle – to make bigger, rat-like hair follicles," Miller said. "There are a lot of jokes in my lab about bald men running around with rat hair on their heads." Finally, the team gave mice small puncture wounds and then transplanted their fluorescent SKPs next to the wound. Within a month, many transplanted cells appeared in the scar, showing they had contributed to wound healing. The SKPs were also found in new hair follicles in the healed skin. The cells behaviour both in wound healing and hair growth led the team to conclude that the SKPs are, in fact, dermal stem cells. Miller said the finding complements work by HHMI investigator Elaine Fuchs, who found epidermal stem cells, which help renew the top layer of skin. Combining the evidence from the two labs suggests a possible path to baldness treatments, Miller said — the dermal stem cells at the base of the hair follicle seem to be signalling the epidermal cells that form the shaft of the follicle to grow hair. But much about the signalling mechanism remains unknown. Miller wants to investigate less cosmetic applications, such as treating nerve and brain diseases. Experiments she published between 2005 and 2007 showed that SKPs can grow into nerves and help repair spinal cord damage in rats. Her lab is continuing to pursue that research. She is also searching for signals that could trigger the dermal stem cells to rev up their innate wound-healing ability. If such a signal can be found and mimicked, Miller can envision one day treating chronic wounds – such as diabetic ulcers – with a topical cream. Such a treatment is years or decades away, she said, but now researchers know which cell types to focus on. Another possibility: improving skin grafts, which today consist of only epidermal, not dermal, cells. While skin grafts can dramatically help burn victims, those grafts don't function like normal skin. "Stem cell researchers like to talk about building organs in a dish," said Miller. "You can imagine, if you have all the right players – dermal stem cells and epidermal stem cells – working together, you could do that with skin in a very real way." Reference: SKPs Derive from Hair Follicle Precursors and Exhibit Properties of Adult Dermal Stem Cells Jeffrey Biernaskie, Maryline Paris, Olena Morozova, B. Matthew Fagan, Marco Marra, Larysa Pevny and Freda D. Miller Cell Stem Cell, Volume 5, Issue 6, 610-623, 4 December 2009, doi:10.1016/j.stem.2009.10.019 ......... ZenMaster

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Stem Cell Therapy May Lead to Treatment for Deafness

A new study has successfully isolated human auditory stem cells from foetal cochleae Tuesday, 24 March 2009 Deafness affects more than 250 million people worldwide. It typically involves the loss of sensory receptors, called hair cells, for their "tufts" of hair-like protrusions, and their associated neurons. The transplantation of stem cells that are capable of producing functional cell types might be a promising treatment for hearing impairment, but no human candidate cell type has been available to develop this technology. A new study led by Dr. Marcelo N. Rivolta of the University of Sheffield has successfully isolated human auditory stem cells from foetal cochleae (the auditory portion of the inner ear) and found they had the capacity to differentiate into sensory hair cells and neurons. The study is published in the April issue of STEM CELLS. The researchers painstakingly dissected and cultured cochlear cells from 9-11 week-old human foetuses. The cells were expanded and maintained in vitro for up to one year, with continued division for the first 7 to 8 months and up to 30 population doublings. This is similar to other non-embryonic stem cell populations, such as bone marrow. Gene expression analysis showed that all cell lines expressed optic markers that lead to the development of the inner ear as well as markers expressed by pluripotent embryonic stem cells, from which all tissues and organs develop. They were able to formulate conditions that allowed for the progressive differentiation into neurons and hair cells with the same functional electrophysiological characteristics as cells seen in vivo. "The results are the first in vitro renewable stem cell system derived from the human auditory organ and have the potential for a variety of applications, such as studying the development of human cochlear neurons and hair cells, as models for drug screening and helping to develop cell-based therapies for deafness," say the authors. Although the hair cell-like cells did not show the typical formation of a hair bundle, the authors suggest that future studies will aim to improve the differentiation system. They are currently working on using the knowledge gleaned from this study to optimize the differentiation of human embryonic stem cells into ear cell types. "Although considerable information has been obtained about the embryology of the ear using animal models, the lack of a human system has impaired the validation of such information," the authors note. "Access to human cells that can differentiate should allow the exploration of features unique to humans that may not be applicable to animal models," says Donald G. Phinney, co-editor of the journal. The protocol they developed to expand and isolate human foetal auditory stem cells may be able to be adapted for deriving clinical-grade cells with potential therapeutic applications. Dr Ralph Holme, director of biomedical research for Royal National Institute for Deaf and Hard of Hearing People, said: "There are currently no treatments to restore permanent hearing loss so this has the potential to make a difference to millions of deaf people." Reference: Human Fetal Auditory Stem Cells (hFASCs) Can Be Expanded In Vitro And Differentiate Into Functional Auditory Neurons And Hair Cell-Like Cells Wei Chen, Stuart L. Johnson, Walter Marcotti, Peter W. Andrews, Harry D. Moore, Marcelo N. Rivolta STEM CELLS Express March 23, 2009, 10.1002/stem.62 ......... ZenMaster

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