Two Proteins Safeguard Skin Stem Cell Function


Ever scraped your knees or elbows? It’s a good thing that they didn’t stay that way, since human skin readily renews, heals wounds, and regenerates the hair that covers it thanks to a resident population of stem cells. These cells continually produce new ones. Depending on someone’s age, the complete skin is renewed every 10-30 days.

A new study led by Salvador Aznar Benitah (Institute for Research in Biomedicine, Barcelona, Spain) has identified two proteins that are integral to the conservation of skin stem cells. Without these proteins these skin-based skin cells are lost.

The proteins identified, Dnmt3a and Dnmt3b, trigger the first step of the genetic program that leads to stem cell renewal and regeneration of the skin. “Without them (i.e. Dnmt3a & Dnmt3b), this program is not activated and the stem cells collapse and disappear from the tissue,” said Benitah.

Dnmt3a & 3b are enzymes that attach methyl groups (-CH3) to the cytosines in DNA molecules.  The full name of these enzymes, DNA (cytosine-5)-methyltransferase 3A, catalyze the transfer of methyl groups to specific CpG structures in DNA.  This process is known as “DNA methylation.”  These particular DNA methyltransferases participate in de novo DNA methylation.  They must be distinguished from so-called “maintenance DNA methylation,” which ensures the fidelity of replication of inherited epigenetic patterns.

Epigenetics refers to cellular and physiological trait variations that result from external or environmental factors that switch genes on and off and affect how cells express genes, but do not involve changes in nucleotide sequences, but in chemical modifications to DNA or higher-order structures of DNA.

DNMT3A forms part of the family of DNA methyltransferase enzymes that includes DNMT1, DNMT3A and DNMT3B.  While de novo DNA methylation modifies the information passed on by parents to their progeny, it enables key epigenetic modifications essential for processes such as cellular differentiation and embryonic development, transcriptional regulation, heterochromatin formation, X-inactivation, imprinting and genome stability.

Lorenzo Rinaldi, a graduate student in Benitah’s laboratory who was also the first author of this study, has mapped the regions of the genome that houses the genes that encodes these two proteins. Rinaldi and others have shown that Dnmt3a & 3b affect gene expression by methylating “gene enhancers” and “superenhancers.” Gene enhancers and superenhancers are sequences that tend to be far away from genes but still have the ability to increase the speed of gene expression up to 200-fold.

“It was surprising to see that two proteins that have always been associated with gene repression through DNA methylation are activated in the most transcriptionally active regions of stem cells. We had never observed this activity because we were unable to study the global distribution of Dnmt3a and Dnmt3b at the genomic level. Thanks to advances in sequencing techniques, more researchers are observing the very mechanism that we have described,” said Rinaldi.

Of the 12,000 gene enhancers in the genome, about 300 are superenhancers related to stem cell activity. Dnmt3a & 3b activate expression of the approximately 1,000 genes that are required for the self-renewing capacity of stem cells. By methylating the superenhancer, these proteins trigger the first step of the machinery that leads to the amplified expression of these essential genes for the stem cell.  Dnmt3a and Dnmt3b clearly associate with the most active enhancers in human epidermal SCs.

Graphical_Done

The expression of Dnmt3a & 3b is also altered in cancer cells. Cancer cells tend to show altered DNA methylation and altered gene enhancers that affect gene expression. The mass sequencing of tumor cell genomes has provided these observations. Dnmt3a and Dnmt3b activities are altered in many types of tumor, including leukemias, and cancers of the lung and the colon.

“Each of these three components is associated with the development of various kinds of cancer. Given that these proteins activate gene expression enhancers through DNA methylation, we believe that it would be of interest to study them in cancer cells in order to determine whether they participate in tumor development,” said Benitah.

This work appeared in: Lorenzo Rinaldi et al., “Dnmt3a and Dnmt3b Associate with Enhancers to Regulate Human Epidermal Stem Cell Homeostasis,” Cell Stem Cell, July 2016 DOI: 0.1016/j.stem.2016.06.020.

Mouse Study Suggests Stem Cells Can Ward Off Glaucoma


Regulating the internal pressure of the eyeball (known as the “intraocular pressure” or IOP) is crucial for the health of the eye.  Failure to maintain a healthy IOP can lead to vision loss in glaucoma.  However, a new set of experiments by Dr. Markus Kuehn and his colleagues at the Iowa City Veterans Affairs Medical Center and the University of Iowa has shown that infusions of stem cells could help restore proper drainage for plugged-up eyes that are at risk for glaucoma.

Kuehn and his coworkers injected stem cells into the eyes of laboratory mice suffering with glaucoma.  These infused cells regenerated the tiny, fragile patch of tissue known as the trabecular meshwork, which functions as a drain for the eyes.  When fluid accumulates in the eye, the increase in IOP can lead to glaucoma.  Glaucoma damages the optic nerve leads to blindness.

“We believe that replacement of damaged or lost trabecular meshwork cells with healthy cells can lead to functional restoration following transplantation into glaucoma eyes,” Kuehn wrote on his lab’s website.  One potential advantage of the approach is that induced pluripotent stem cells (iPSCs) could be created from cells harvested from a patient’s own skin. That gets around the ethical problems with using fetal stem cells.  It also lessens the chance of the patient’s body rejecting the transplanted cells.

In order to differentiate iPSCs into trabecular meshwork (TM) cells, Kuehn’s team cultured the iPSCs in medium that had previously been “conditioned” by actual human trabecular meshwork cells.  Injection of these TM cells into the eyes of laboratory rodents led to a proliferation of new endogenous cells within the trabecular meshwork.  The injected stem cells not only survived in the eyes of the animals, but also induced the eye into producing more of its own TM cells, thus multiplying the therapeutic effect.

Glaucoma has robbed some 120,000 Americans of their sight, according to data provided by the Glaucoma Research Foundation.  African-Americans are at especially high risk, as are people over age 60, those with diabetes, and those with a family history of the disease.  Glaucoma can be treated with medicines, but is not curable.  Management of the disease can delay or even prevent the eventual loss of vision. Among the treatments used are eye drops and laser or traditional surgery.

Kuehn and his team think that their findings show some promise for the most common form of glaucoma, known as primary open angle glaucoma.  It remains unclear if this mouse model is as relevant for other forms of the disease.  Another possible limitation of this research is that the new trabecular meshwork cells generated from the stem cell infusion eventually succumb to the same disease process that caused the breakdown in the first place.  This would require re-treatment and it is unclear whether an approach requiring multiple treatments over time would be viable. Kuehn and others to continue investigate this potentially fruitful approach.

This paper was published in the journal Proceedings of the National Academy of Sciences:  Wei Zhu et al., “Transplantation of iPSC-derived TM cells rescues glaucoma phenotypes in vivo,” Proceedings of the National Academy of Sciences, 2016; 113 (25): E3492 DOI: 10.1073/pnas.1604153113.

B-Complex Vitamins Promote Stem Cell Proliferation


B vitamins called folates can stimulate stem cell proliferation independently of their role as vitamins.  This is according to a collaborative study from the University of Georgia and Tufts University, which used a cell culture system in addition to a live animal model system to establish these results.

Folates, whether supplemental B vitamins or natural folates found in food, are an essential cofactor for single-carbon transfer reactions.  Folates are integral for the proper functioning of all cells in the body and are critical to prevent birth defects, in particular defects of the neural tube (spinal cord).

Folic-Acid
Folic acid

This study shows, for the first time, that an adult stem cell population is controlled by an external factor arising from outside the animal. The animal model system was the small, free-living roundworm, Caenorhabditis elegans.

Is this the case for human stem cell populations?  Difficult to say, but it surely can’t hurt to make sure that you are getting enough folate in your diet.

This work was published in this article: Snehal N. Chaudhari et al., “Bacterial Folates Provide an Exogenous Signal for C. elegans Germline Stem Cell Proliferation,” Developmental Cell, 2016; 38 (1):33 DOI: 10.1016/j.devcel.2016.06.013.

Gauging The Quality of Naive Stem Cells


Researchers from the Salk Institute for Biological Studies in La Jolla, CA and collaborators from Ecole Polytechnique Fédérale de Lausanne in Switzerland and Massachusetts Institute of Technology in Cambridge, Massachusetts have developed a new benchmark for generating the most primitive type of stem cell. These new molecular criteria can allow scientists know just how close laboratory-generated “naïve stem cells” mimic embryonic blastomeres that exist in the very earliest stages of human development.

Naïve stem cells potentially have a greater ability to differentiate into a wider variety of tissue types. They might have many different applications for research and regenerative medicine. Mature human bodies have their own adult stem cells populations. However, these stem cell populations have the capacity to differentiate into a subset of different cell types (multipotent), or only one cell type (unipotent). Stem cells derived from embryos are pluripotent, which means that they can differentiate into any cell type in the adult human body. Likewise, adult cells that have been subjected to particular genetic engineering and cell culture techniques can be reprogrammed into pluripotent stem cells known as induced pluripotent stem cells or iPSCs. These have many (although not all) of the characteristics of embryonic stem cells.

Several different research groups have developed cocktails of molecules that can de-differentiate pluripotent stem cells into cells that resemble cells from postimplantation embryos. Essentially, these protocols can effectively turn the clock back on pluripotent stem cells to make them resemble naïve stem cells, or those blastomeres that are found in preimplantation embryos only days after fertilization.

Naïve stem cells are “totipotent,” which means that they can differentiate into any cell type in either the adult body or in the embryo, including placenta. These cells constitute the primordial cells that produce all the cells of the human body and those that make up the placenta as well. Most of the published protocols to generate so-called naïve stem cells, however, are rather inefficient. They tend to produce cells that are very much like the starting pluripotent cells and produce few changes in gene expression.

The Salk team and their collaborators used a battery of molecular tests on these “primed” cells and embryonic stem cells (ESCs) that had been exposed to factors that are thought to induce the naïve state. Their experiments compared gene expression in ESCs with ESCs that had been subjected to the laboratory protocols to convert them into naïve stem cells, and blastomeres from early embryos. The discovered that three main tests were the most indicative of the differences between naïve stem cells and other stem cells.

First, they measured the expression levels of transposons, DNA sequences that can jump around the genome. It was clear that the expression of transposons provided a sensitive measure of the similarities pluripotent stem cells and early human development. In fact, naive human ESCs shared a unique transposon signature with cleavage-stage embryos, and the expression of certain transposons was indicative of naïve stem cells. Next, they found that the genomes of naïve embryonic stem cells have less methylation (the addition of methyl chemical groups or –CH3 groups – to the bases of DNA. They then examined the state of X chromosomes in naïve cells of female embryos’, which contain two active X chromosomes, unlike more mature embryonic cells that have silenced one of the X chromosomes. These three tests collectively include tens of thousands of genetic biomarkers to characterize the developmental state of stem cells.

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When current methods for generating naïve stem cells in the lab were judged using the three tests, each fell short of fully mimicking the naïve embryonic cells in different ways. One new technique, for instance, led to cells that had two active X chromosomes but didn’t match the exact methylation patterns desired. In fact, none of the current, published protocols lead to truly naïve stem cells.

These established guidelines may help researchers achieve that goal and eventually elucidate where the current methods fall short. Generating naïve stem cells would be a boon to both basic research and to medical applications of stem cells. The analysis provided in this paper is likely to become a gold standard for quality control of stem cells, including induced pluripotent stem cells, regardless of their use in research or in clinical applications.

See Rudolf Jaenisch et al., “Molecular Criteria for Defining the Naive Human Pluripotent State,” Cell Stem Cell, July 2016 DOI: 10.1016/j.stem.2016.06.011.

Weissman Laboratory Define Roadmap for Pluripotent Human Stem Cell Differentiation into Mesodermal Fates: Cells Rapidly Generate Bone, Heart Muscle


How do we get stem cells to differentiate into the cell types we want? Implanting undifferentiated stem cells into a living organism can sometimes result in cells that differentiate into unwanted cell types. Such a phenomenon is called heterotropic differentiation and it is a genuine concern of regenerative medicine. What is a clinical researcher to do? Answer: make a road map of the events that drive cells to differentiate into specific cell types and their respective precursors.

Researchers in the laboratory of Irving Weissman at Stanford University Researchers at the Stanford University School of Medicine have mapped out the bifurcating lineage choices that lead from pluripotency to 12 human mesodermal lineages, including bone, muscle, and heart. The experiments also defined the sets of biological and chemical signals necessary to quickly and efficiently direct pluripotent stem cells to differentiate into pure populations of any of 12 cell types. This is certainly a remarkable paper in many aspects, since Weissman and his group defined the extrinsic signals that control each binary lineage decision that occur during stem cell differentiation. This knowledge enables any lab to successfully block differentiation toward unwanted cell fates and rapidly steer pluripotent stem cells toward largely pure human mesodermal lineages at most of these differentiation branchpoints.

The ability to make pure populations of these cells within days rather than the weeks or months is one of the Holy Grails of regenerative medicine. Such abilities can, potentially, allow researchers and clinicians to make new beating heart cells to repair damage after a heart attack, or cartilage for osteoarthritic knees or hips, or bone to reinvigorate broken bones or malfunctioning joints, or heal from accidental or surgical trauma.

The Weissman study also highlights those key, but short-lived, patterns of gene expression that occur during human early embryonic segmentation. By mapping stepwise chromatin and single-cell gene expression changes during the somite segmentation stage of mesodermal development, the Weissman group discovered a previously unobservable human embryonic event transiently marked by expression of the HOPX gene. It turns out that these decisions made during human development rely on processes that are evolutionarily conserved among many animals. These insights may also lead to a better understanding of how congenital defects occur.

“Regenerative medicine relies on the ability to turn pluripotent human stem cells into specialized tissue stem cells that can engraft and function in patients,” said Irving Weissman of Stanford. “It took us years to be able to isolate blood-forming and brain-forming stem cells. Here we used our knowledge of the developmental biology of many other animal models to provide the positive and negative signaling factors to guide the developmental choices of these tissue and organ stem cells. Within five to nine days we can generate virtually all the pure cell populations that we need.”

All in all, this roadmap enables scientists to navigate mesodermal development to produce transplantable, human tissue progenitors, and uncover developmental processes.

This paper was published in the journal Cell: Irving L. Weissman et al., “Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types,” Cell, July 2016 DOI: 10.1016/j.cell.2016.06.011.

Patient-Specific Neurons Reveal Vital Clues About Autism


The brains of some people with autism spectrum disorder grow faster than usual early on in life, often before diagnosis. Now new research from scientists at the Salk Institute has used cutting-edge stem cell-based techniques to elucidate those mechanisms that drive excess brain growth, which affects as many as 30 percent of people with autism.

These findings show that it is possible to use stem cell reprogramming technologies to model the earliest stages of complex disorders and to evaluate potential therapeutic drugs. The Salk team, led by Alysson Muotri, discovered that stem cell-derived neurons, derived from stem cells that had been made from cells taken from autism patients, made fewer connections in culture compared to cells from healthy individuals. These same scientists also restored cell-cell communication between these cells by adding a growth factor called IGF-1 (insulin-like growth factor-1). IGF-1 is in the process of being evaluated in clinical trials of autism.

“This technology allows us to generate views of neuron development that have historically been intractable,” said senior investigator Fred H. Gage. “We’re excited by the possibility of using stem cell methods to unravel the biology of autism and to possibly screen for new drug treatments for this debilitating disorder.”

In the United States alone, autism affects approximately one out of every 68 children. Autistic children have problems communicating, show an inhibited ability to interact with others, and usually engage in repetitive behaviors. Mind you, the symptomatic manifestations in autistic children can vary dramatically in type and severity. Autism, to date, has no known, identified cause.

In 2010, Gage and collaborators recreated features of Rett syndrome (a rare disorder that shares features of autism but is caused by mutations in a single gene; MECP2) in a cell culture system. They extracted skin cells from Rett Syndrome patients and converted those cells into induced pluripotent stem cells (iPSCs). Then Gage and others differentiated those Rett-Syndrome-specific iPSCs into neurons, which they grew in culture. These neurons were then studied in detail in a neuron-specific culture system. “In that study, induced pluripotent stem cells gave us a window into the birth of a neuron that we would not otherwise have,” said Marchetto, the study’s first author. “Seeing features of Rett syndrome in a dish gave us the confidence to next study classical autism.”

In this new study, Gage and others created iPSCs from autism patients whose brains had grown up to 23 percent faster than usual during toddlerhood but had subsequently normalized. These iPSCs were then differentiated into neuron precursor cells (NPCs). Examinations of these NPCs revealed that the NPCs made from iPSCs derived from autism patients proliferated faster than those derived from typically developing individuals. This finding supports a theory advanced by some experts that brain enlargement is caused by disruptions to the cell’s normal cycle of division, according to Marchetto.

In addition, the neurons derived from autism-specific iPSCs behaved abnormally in culture. They fired less often compared with those cells derived from healthy people. The activity of these neurons, however, improved if they were treated with IGF-1. IGF-1 enhances the formation of cell-cell connections between neurons, and the establishment and stabilization of these connections seem to normalize neuronal function.

Muotri and Gage and others plan to use these patient-derived cells to elucidate the molecular mechanisms behind IGF-1’s effects. They will examine changes in gene expression and attempt to correlate them with changes in neuronal function. Although the newly derived cells are far from the patients’ brains, a brain cell by itself may, hopefully, reveal important clues about a person and their brain.

This work was published in the journal Molecular Psychiatry: M. C. Marchetto et al., “Altered proliferation and networks in neural cells derived from idiopathic autistic individuals,” Molecular Psychiatry, 2016; DOI: 10.1038/mp.2016.95.

Beta-Integrin Implicated In Slow Healing Of Aged Muscles


With age, the function and regenerative abilities of skeletal muscles decrease. Therefore, the elderly can find it difficult to recover from injury or surgery.

A new study from the laboratory of Chen-Ming Fan from Johns Hopkins University has shown that a protein called β1-integrin is crucial for muscle regeneration. β1-integrin seems to provide a promising target for therapeutic intervention to combat muscle aging or disease.

Muscle stem cells are the primary source of muscle regeneration after muscle injury from exercise, accidents, or surgery. These specialized adult stem cells lie dormant in the muscle tissue, and muscles even have them stored off to the side of the individual muscle fibers. Because of their location, these muscle stem cells are known as muscle “satellite cells.” After damage, these satellite cells awaken and proliferate, and go on to make new muscle fibers and restore muscle function. Some satellite cells return to dormancy, which allows the muscle to keep a reservoir of healing cells that can repair the muscle over and over again. Fan and her colleagues determined that proteins called integrins, and in particular, β1-integrin, are integral for maintaining the cycle of hibernation, activation, proliferation, and then return to hibernation, in muscle stem cells.

Integrins are cell surface proteins that provide tight connections between cells and the immediate external environment.

Integrin Dimer Structure: Globular domain structures of α and β subunits in a stable dimer. Ligand binding happens at the interface of the αI (left panel) or β-propeller (right panel) and the βI domain.
Integrin Dimer Structure: Globular domain structures of α and β subunits in a stable dimer. Ligand binding happens at the interface of the αI (left panel) or β-propeller (right panel) and the βI domain.

Without integrins, almost every stage of the regeneration is disrupted. Fan and her group predicted that defects in β1-integrin likely contribute to aging, which is associated with reduced muscle stem cell function and decreased quantities of muscle stem cells. This means that healing after injury or surgery is very slow, which can cause a long period of immobility and an accompanying loss of muscle mass. Inefficient muscular healing in the elderly is a significant clinical problem. Therapeutic approaches would be quite welcome by the aging population and their physicians. One way to improve muscle regeneration would be to stimulate muscle satellite cells in older individuals.

Fan and others determined that β1-integrin function is diminished in aged muscle stem cells. When they artificially activated integrins in aged mice, their regenerative abilities were restored to youthful levels. Improvement in regeneration, strength, and function were also seen when this treatment was applied to animals with muscular dystrophy, which underscores the potential importance of such an approach for the treatment of muscle disorders.

Muscle stem cells use β1-integrin to interact with many other proteins in the external environment of the muscle. Among this forest of proteins in the external environment of the muscle, Fan and her coworkers found one called fibronectin that might be the most relevant. They discovered that aged muscles contain substantially less fibronectin compared to young muscles. Like β1-integrin, eliminating fibronectin from young muscles makes them function as though they were old. However, restoring fibronectin to aged muscle tissue restores muscle regeneration to youthful levels. Fan’s group demonstrated a strong link between β1-integrin, fibronectin and muscle stem cell regeneration.

Taken together, the results show that aged muscle stem cells with compromised β1-integrin activity and aged muscles with insufficient amount of fibronectin both root causes of muscle aging. This makes β1-integrin and fibronectin very promising therapeutic targets.

This work appeared in the following journal: Michelle Rozo et al., “Targeting β1-integrin signaling enhances regeneration in aged and dystrophic muscle in mice,” Nature Medicine, 2016; DOI: 10.1038/nm.4116.