Nanog Gene Reverses Aging in Adult Stem Cells


Professor Stelios Andreadis from SUNY Buffalo and his colleagues have, in a series of elegant experiments, shown that the gene Nanog can stimulate dormant cellular processes that seem to be vital for preventing weak bones, clogged arteries and other telltale signs of aging. The findings might help counteract premature aging disorders such as Hutchinson-Gilford progeria syndrome.

“Our research into Nanog is helping us to better understand the process of aging and ultimately how to reverse it,” said Andreadis.

In order to delay or even reverse the ravages of aging, the human body holds a reservoir of nonspecialized progenitor cells that can regenerate organs. These cells are collectively called “adult stem cells,” and they are in every tissue of the body. Adult stem cells can rapidly respond to tissue damage to regenerate and heal organs and tissues. Unfortunately, as people age, fewer adult stem cells pare able to properly perform their function. This leads to the clinical scenarios associated with aging. Reversing the effects of aging in adult stem cells – re-booting them if you will – can potentially overcome this problem.

Andreadis and his coworkers have previously shown that the capacity of adult stem cells to form muscle and generate force declines with age. Specifically, Andreadis and others examined smooth muscle cells found in arteries, intestines and other tissues. In this new study, grad student Panagiotis Mistriotis introduced a gene called Nanog into aged stem cells. He found that Nanog activated two key cellular pathways that include Rho-associated protein kinase (ROCK) and Transforming growth factor beta (TGF-β). Activation of these two signaling pathways awakens dormant proteins like actin to build the new cytoskeletal networks that adult stem cells need to form contracting muscle cells. Force generated by these cells ultimately helps restore the regenerative properties that adult stem cells lose due to aging.

“Not only does Nanog have the capacity to delay aging, it has the potential in some cases to reverse it,” said Andreadis, who noted that introduction of the Nanog gene worked in three different models of aging: cells isolated from aged donors, cells aged in culture, and cells isolated from patients with Hutchinson-Gilford progeria syndrome.

Additionally, Andreadis and his group found that Nanog activated the central regulator of muscle formation, a signaling protein called serum response factor (SRF), which suggests that the same results may be applicable for skeletal, cardiac and other muscle types.

Andreadis and others are now examining potential drugs that can replace or mimic the effects of the Nanog gene. This will allow them to study the consequences of aging inside the body can also be reversed. This could have implications in an array of illnesses, everything from atherosclerosis, high blood pressure, and osteoporosis to Alzheimer’s disease.

This fascinating paper was published here: Panagiotis Mistriotis et al., “NANOG Reverses the Myogenic Differentiation Potential of Senescent Stem Cells by Restoring ACTIN Filamentous Organization and SRF-Dependent Gene Expression,” Stem Cells, 2016; DOI: 10.1002/stem.2452.

Computer Simulations of MSC-Heart Muscle Interactions Identify A Family of MSCs that Produce Few Side Effects


A research team at the Icahn School of Medicine at Mount Sinai has utilized a mathematical modeling to simulate the delivery of human mesenchymal stem cells to a damaged heart. In doing so, they found that a particular subset of harvested MSCs minimizes the risks associated with this therapy. This study represents a development that could lead to novel strategies to repair and regenerate heart muscle and might even improve stem cell treatments for heart attack patients.

In the United States alone, one person suffers a myocardial infarction or heart attack every 43 seconds (on the average). The urgency of this situation has motivated stem cells scientists and cardiologists to develop novel therapies to repair and regenerate heart muscle. One of these therapies includes the implantation of human mesenchymal stem cells (hMSCs). However, in clinical trials the benefits of hMSC implantation have often been modest and even transient. This might reflect our understanding of the mechanism by which hMSCs influence cardiac function.

Kevin D. Costa and his colleagues at the Icahn School of Medicine have used mathematical modeling to simulate the electrical interactions between implanted hMSCs and endogenous heart cells. They hoped to eventually understand the possible adverse effects of hMSC transplantation and new methods for reducing some potential risks of this therapy.

Implanted hMSCs can disrupt the electrical connections between heart muscle cells and can even cause the heart to beat irregularly; a condition called “arrhythmias.” One particular type of hMSCs, however, did not express an ion channel called EAG1 (which stands for “ether-a-go-go”). The EAG1-less hMSCs did not cause arrhythmias at nearly the rate as the EAG1-containing hMSC, in computer simulations run by Costa’s group.

These EAG1-less hMSCs, also known as “Type C” MSCs, minimized electrochemical disturbances in cardiac single-cell and tissue-level electrical activity. The benefits of these EAG1-less hMSCs may enhance the safety of hMSC treatments in heart attack patients who receive stem cell therapy. This advance could therefore lead to new clinical trials and future improvements in treatment of patients with heart failure.

Costa’s study might provide a template for future computational studies on mesenchymal stem cells. It also provides novel insights into hMSC-heart cell interactions that can guide future experimental studies to understand the mechanisms that underlie hMSC therapy for the heart.

This work was published in Joshua Mayourian et al., “Modeling Electrophysiological Coupling and Fusion between Human Mesenchymal Stem Cells and Cardiomyocytes, PLOS Computational Biology, 2016; 12 (7): e1005014. DOI: 10.1371/journal.pcbi.1005014.

Adjustable Gels Used to Determine Those Molecules That Drive Stem Cell Differentiation


Scientists have been very interested in the details of stem cell differentiation. To that end, several laboratories have designed hydrogels that mimic the stiffness of biological tissue in order to grow stem cells and study their differentiation.

In one enterprising laboratory, led by Rein Ulijn of the City University of New York and the University of Strathclyde, scientists have used a novel culture-based gel system to study mesenchymal stem cell differentiation and identify those metabolites used by stem cells when they select bone and cartilage cell fates. When these molecules are provided to standard stem cell cultures, these molecules can guide stem cells to generate desired cell types. This new study illustrates how new biomaterials can provide an exacting model system that can help scientists precisely determine those identifying factors that drive stem cell differentiation.

Stem-cell scientists have known that the rigidity of a hydrogel surface can instruct stem cells to differentiate. A rigid surface, as it turns out, can result in bone cell formation, whereas soft surfaces induce the differentiation of cells into neuron-like cells. With this information, Ulijn and others developed a protocol that generates gels by combining small building-block molecules that spontaneously form a network of nanosized fibers. Furthermore, by varying the concentration of these building blocks, the stiffness of these gels can be adjusted. By mimicking the stiffness of bone (40 kilopascal) or cartilage (15 kilopascal), the gel stimulates stem cells applied to its surface to differentiate accordingly.

“This paper is a great example of how chemistry can help make step changes in biology,” said Matthew Dalby of the University of Glasgow and co-senior author. “As a biologist, I needed simple yet tunable cell-culture gels that would give me a defined system to study metabolites in the laboratory. Rein had developed the chemistry to allow this to happen.”

The available gels for growing stem cells are typically derived from animal products. Unfortunately, this can affect the reproducibility of results, since different preparations of particular animal products can have rather different properties. Synthetic components usually require coatings or coupling of cell-adhesive ligands. However, the gel developed by Ulijn’s group is composed of two simple synthetic peptide derivatives. One component binds to copies of itself with high directional preference, which results in the spontaneous formation of nanoscale fibers when the molecules are dissolved in water. The second components consists of a surfactant-like molecule that binds to the fiber surface and presents simple, cell-compatible chemical groups to any cells.

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The components are held together by relatively weak and reversible interactions, e.g., hydrogen bonding and aromatic stacking. Interestingly, variants of these gels are commercially available through a spinoff company called Biogelx, Ltd., where Ulijn serves as chief scientific officer.

“We wanted a platform that provides nanofiber morphology and as-simple-as-possible chemistry and tunable stiffness to serve as a blank-slate background so that we could focus on changes in stem cell metabolism,” said Ulijn. “Matt and his team performed metabolomics analysis to find out how the key metabolites within a stem cell are used up during the differentiation process.”

Particular transcription factors are often the ingredients scientists use to induce stem cell fate in the case of induced pluripotent stem cells. However, Dalby and Ulijn think that certain metabolites might drive those pathways that cause the different intracellular concentrations of transcription factors that drive the various differentiation pathways.

One metabolite featured in the study is cholesterol sulfate. Cholesterol sulfate is used up during osteogenesis on a rigid matrix and can also be used to convert stem cells into bone-like cells in cell culture.

In their paper, Ulijn and his coworkers showed how small molecules, like cholesterol sulfate, can put into motion those cell-signaling pathways that culminate in the activation of the transcription factors that drive the transcription of major bone-related genes. The expression of these bone-specific genes drives bone formation, and this demonstrates a connection between the metabolites and the activation of transcription factors.

It must be noted that this gel does not precisely recapitulate the microenvironment inside the body. Therefore, it is unclear if the stem cells grown on it behave differently on the designed gel surfaces than they would in the body.

Although the full list of metabolites derived from the analysis is preliminary, “it could certainly point researchers in the right direction,” Ulijn said. “Our ambition is to simplify drug discovery by using the cell’s own metabolites as drug candidates,” Dalby said.

This paper was published here: Alakpa et al., “Tunable Supramolecular Hydrogels for Selection of Lineage Guiding Metabolites in Stem Cell Cultures,” Chem, 2016 DOI:10.1016/j.chempr.2016.07.001.

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.

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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.

Insulin-Producing Beta Cell Subtypes May Impact Diabetes Treatment


Researchers from the Oregon Stem Cell Center in Portland, Oregon have demonstrated the existence of at least four separate subtypes of human insulin-producing beta cells that may be important in the understanding and treatment of diabetes.

“This study marks the first description of several different kinds of human insulin producing beta cells,” said Markus Grompe of the Oregon Stem Cell Center at Oregon Health Science University. “Some of the cells are better at releasing insulin than others, whereas others may regenerate quicker. Therefore, it is possible that people with different percentages of the subtypes are more prone to diabetes. Further understanding of cell characteristics could be the key to uncovering new treatment options, as well as the reason why some people are diabetic and others are not.”

Diabetes mellitus affects more than 29 million people in the United States. There are two main types of diabetes mellitus, type I and type II. Type I diabetes mellitus is caused by insufficient production of insulin. Type II diabetes mellitus is caused by insulin resistance or the inability of the body to properly respond to the insulin produced by the body. Type I diabetes mellitus results from dysfunction or loss of insulin producing beta cells in the endocrine portion of the pancreas. Insulin is a hormone that helps the body keep normal blood sugar levels, and incorporate sugar in the bloodstream into cells to grow and repair tissues. Previously, only a single variety of beta cell was known to exist. However, using human pancreatic islets, or clusters of up to 4,000 cells, Grompe and colleagues identified a method to identify and isolate four distinct types of beta cells. They also found that hundreds of genes were differently expressed between cell subtypes and that these distinct beta cell subtypes produced different amounts of insulin.

All type 2 diabetics had abnormal percentages of the subtypes, suggesting a possible role in the disease process. Additional research is needed to determine how different forms of diabetes – and other diseases – affect the new cell subtypes, as well as how researchers may take advantage of these differences for medical treatment.

This work was published in: Craig Dorrell et al., “Human islets contain four distinct subtypes of β cells,” Nature Communications, 2016; 7: 11756 DOI: 10.1038/ ncomms11756.

New Study Validates Cellular Bone Allograft Technology


In a study in laboratory rats that had defects in their femurs (upper leg bone) showed new bone formation after they were treated with adipose-derived mesenchymal stromal cells that had been seeded on demineralized bone matrix and implanted.  Implanted stem cells were detected for up to 84 days in areas of new formation and had differentiated within the bony repair tissue.

According to AlloSource, the procedures used similar technology to the company’s AlloStem Cellular Bone Allograft process.

Surgical oncologist Nicole Ehrhart of Colorado State University presented these data at the State of Spine Surgery annual symposium and at the Korean American Spine Society meeting.

AlloStem is partially demineralized allograft bone combined with adipose-derived mesenchymal stem cells.  AlloStem is suitable for general bone grafting applications, and is similar to autograft bone because it provides the three key properties necessary for bone formation: osteoconduction, osteoinduction and osteogenesis.

Ehrhart’s study has been accepted for publication in Journal of Biomaterials and Tissue Engineering.

AlloSource provides 200 types of precise cartilage, cellular, bone, skin and soft-tissue allografts.

STEMTRA Trial Tests The Efficacy of Genetically-Modified SB623 Mesenchymal Stem Cells in Stroke Patients


SanBio, Inc., has announced the randomization of the first patient in their STEMTRA Phase 2 clinical trial study for traumatic brain injury. The STEMTRA trial is presently enrolling patients in both the United States and Japan, and the first patient was randomized at Emory University Hospital in Atlanta, Ga.

STEMTRA stands for “Stem cell therapy for traumatic brain injury,” and this trial will examine the effects of SB623 stem cells to treat patients with chronic motor deficits that result from traumatic brain injury (TBI).

SB623, a proprietary product of SanBio, are bone marrow-derived mesenchymal stem cells that have been genetically engineered to express the intracellular domain of Notch-1. When injected into neural tissue, SB623 cells seem to reverse neural damage. Since SB623 cells come from donors, a single donor’s cells can be used to treat thousands of patients. In cell culture and animal models, SB623 cells restore function to neurons damaged by strokes, spinal cord injury and Parkinson’s disease. There have been no serious adverse events attributable to the cell therapy product and patients benefit on all three stroke scales.

Traumatic brain injuries (TBIs) can be caused by a wide range of events, including falls, fights, car accidents, gunshot wounds to the head, blows to the head from falling objects, and battlefield injuries. These events often result in permanent damage, including significant motor deficits; leaving more than 5.3 million people living with disabilities in the United States alone.

Damien Bates of SanBio, said, “This modified stem cell treatment has improved outcomes in patients with persistent limb weakness secondary to ischemic stroke. Our preclinical data suggest it may also help TBI patients. For people suffering from the often debilitating effects of TBI, this milestone brings us one step closer to proving whether it’s an effective treatment option.”

The STEMTRA trial follows a Phase 1/2a clinical trial in patients afflicted with chronic motor deficit secondary as a result of an ischemic stroke were treated with SB623 cells. In this trial, SB623 cells statistically significantly improved motor function following implantation. The STEMTRA study will evaluate the tolerability, efficacy, and safety of the SB623 cell treatment and the administration process in those patients who have suffered a TBI.  As a Phase 2 trial, STEMTRA will evaluate the clinical efficacy and safety of intracranial administration of SB623 cells in patients with chronic motor deficit from TBI.

STEMTRA will be conducted across approximately 25 clinical trial sites throughout the United States and five sites in Japan. Total enrollment is expected to reach 52 patients in total, and all enrolled patients must have suffered their TBI at least 12 months ago.

Patient-Specific Heart Cells Made from Amniotic Fluid Cells Before a Baby is Born


The dream of cardiologists is to have stockpiles of cardiac progenitor cells that could be transplanted into a sick heart and regenerate it. Even more remarkable would be a source of heart cells for newborn babies with congenital heart problems. What about making these cells before they are born? Science fiction?

Probably not. Dr. Shaun M. Kunisaki from Mott Children’s Hospital and the University of Michigan School of Medicine and his colleagues made heart progenitor cells from Amniotic Fluid Cells. These cells were acquired from routine amniocentesis procedures, with proper institutional review board approval.

These amniotic fluid specimens (8–10 ml), which were taken from babies at 20 weeks gestation, were expanded in culture and then reprogrammed toward pluripotency using nonintegrating Sendai virus (SeV) vectors that expressed the four commonly-used reprogramming genes; OCT4, SOX2, cMYC, and KLF4. The resulted induced pluripotent stem cell (iPSC) lines were then exposed to cardiogenic differentiation conditions in order to generate spontaneously beating amniotic fluid-derived cardiomyocytes (AF-CMs). AF-CMs were formed with high efficiency.

After 6 weeks, Kunisaki and his team subjected their AF-CMs to a battery of quantitative gene expression experiments. They discovered that their AF-CMs expressed high levels of heart-specific genes (including MYH6, MYL7, TNNT2, TTN, and HCN4). However, Kunisaki and others also found that their AF-CMs consisted of a mixed population of differentiated atrial, ventricular, and nodal cells, as demonstrated by various genes expression profiles.

All AF-CMs were chromosomally normal and had no remnants of the SeV transgenes. Functional characterization of these AF-CMs showed a higher spontaneous beat frequency in comparison with heart cells made from dermal fibroblasts. The AF-CMs also showed normal calcium currents and appropriately responded to neurotransmitters that usually speed up the heart, like norepinephrine.

Collectively, these data suggest that human amniotic fluid-derived cells can be used to produce highly scalable sources of functional, transgene-free, autologous heart cells before child is born. Such an approach may be ideally suited for patients with prenatally diagnosed cardiac anomalies.

Key Molecules Tha Control Stem Cell Fate Identified


Adult stem cells, such as mesenchymal stem cells and blood-vessel-associated pericytes represent patient-specific stem cells that are excellent candidates for regenerative medicine. To that end being able to control the differentiation of these stem cells with drugs or small molecules is extremely desirable for eliciting targeted tissue and organ regeneration.

However, identifying these stem-cell-inducing molecules is time-consuming, expensive, and fraught with dead ends. Is there an easier way to control the behavior of stem cells in culture or in your own body?

Research from the City University of New York (CUNY) suggests that the answer to this question might be “yes.” According to Rein Ulijn from CUNY, “Simple small metabolites present in the body already can dictate cell behavior.”

In collaboration with Matthew Dalby from the University of Glasgow, Ulijn and his colleagues discovered that when they grew stem cells on a gel-like medium, the stiffness of which could be easily adjusted, they found molecules that could direct the differentiation of cultured stem cells. As an added bonus, they could direct the differentiation of cultured stem cells much more cheaply.

Ulijn and Dalby began their collaboration in 2011 after other laboratories had demonstrated that the stiffness of the medium could affect the differentiation of stem cells. “On a stiff gel you might get bone-like differentiation,” Ulijn explained. “On a softer gel differentiation into neurons is more likely.” They wanted to use such a system to identify small molecules that can control stem cell differentiation in culture. Such a finding could also “aid the discovery of natural metabolite-based drugs,” added Ulijn added. Such natural-based drugs could be used to, for example, reinforce bones in osteoporosis.

Dalby was interested in the role metabolites played in this stem cell differentiation. Unfortunately, these metabolites are present in fleetingly low concentrations. To complicate the picture, the different formulations of stiffer and floppier materials can mask subtle changes in metabolite concentration. Ulijn found a way around this problem by turning to the two-component peptide gels made by Biogelx (full disclosure: Ulijn serves as the chief scientific officer for Biogelx). Fine-tuning the concentration of the two different gel components changes the rigidity of the gel without changing any other components of the gel that might mask metabolite variation.

The researchers therefore studied concentration changes of hundreds of metabolites during stiffness-controlled stem cell differentiation of stem cells into bone or cartilage. Several metabolites that seemed to make a significant difference for stem cell differentiation were lysophosphatidic acid, which drove stem cells to form cartilage and cholesterol sulfate, which helped stem cells form bone. When Ulijn and his coworkers fed these metabolites to standard stem cell cultures, they differentiated into the desired cell type.

Helena Azevedo of Queen Mary University of London, said, “We will see, for sure, studies exploiting these metabolites for inducing controlled differentiation of stem cells.” She went on to called this study “highly innovative” and said that it might directly influence future stem cell differentiation experiments; particularly those that involve the formation of cartilage or bone.

The Founder Cell Identity Does Not Affect iPS Cell Differentiation to Hematopoietic Stem Cell Fate


Induced pluripotent stem cells (iPSCs) have many of the characteristics of embryonic stem cells, but are made from mature cells by means of a process called cell reprogramming. To reprogram cells, particular genes are delivered into mature cells, which are then cultured until they h:ave the growth properties of pluripotent cells. Further tests are required to demonstrate that the growing cells actually are iPSCs, but once they pass these tests, these cells can be grown in culture indefinitely and, ideally, differentiated into just about any cell type in our bodies (caveat: some iPSC lines can only differentiate into particular cell lineages). Theoretically, any cell type can be reprogrammed into iPSCs, but work from many laboratories has demonstrated that the identity of the founder cell influences the type of cell into which it can be reprogrammed.

Founder cells can be easily acquired from a donor and come in one of four types: fibroblasts (in skin), keratinocytes (also from skin), peripheral and umbilical cord blood, and dental pulp cells (from baby teeth). A variety of laboratories from around the world have made iPSC lines from a gaggle of different founder cells. Because of the significant influence of founder cells for iPSC characteristics, the use of iPSCs for regenerative medicine and other medical applications requires that the desired iPSC line should be selected based on the founder cell type and the characteristics of the iPSC line.

However, the founder cell identity is not the only factor that affects the characteristics of derived iPSC lines. The methods by which the founder cells are reprogrammed can also profoundly contribute to the differentiation efficiency of iPSC lines. According to Yoshinori Yoshida, Associate Professor at the Center for iPSC Research and Application (CiRA) at Kyoto University, the most commonly used methods of cell reprogramming utilize retroviruses, episomal/plasmids, and Sendai viruses to move genes into cells.

The cells found in blood represent a diverse group of cells that includes red blood cells that carry oxygen, platelets that heal wounds, and white blood cells that fight off infection. All the cells in blood are made by bone marrow-specific stem cells called “hematopoietic stem cells.” The production of clinical grade blood has remained a kind of “holy grail” for cellular reprogramming studies. Some scientists have argued that in order to make good-quality hematopoietic cells, the best founder cells are hematopoietic cells. Is this true? Yoshida and his colleagues examined a very large number of iPSC lines that were made from different founder cells and with differing reprogramming methods.  The results of these experiments were published in the journal Cell Stem Cell (doi:10.1016/j.stem.2016.06.019).

Remarkably, Yoshida and his crew discovered that neither of these factors has a significant effect. What did have a significant effect were the expression of certain genes and the position of particular DNA methylations. These two factors were better indicators of the efficiency at which an iPSC line could differentiate into the hematopoietic stem cells.

“We found the IGF2 (Insulin-like Growth Factor-2) gene marks the beginning of reprogramming to hematopoietic cells”, said Dr. Masatoshi Nishizawa, a hematologist who works in Yoshida’s lab and is the first author of this new study. Higher expression of the IGF2 gene is indicative of iPSCs initiating differentiation into hematopoietic cells. Even though IGF2 itself is not directly related to hematopoiesis, its uptake corresponded to an increase in the expression of those genes involved in directing differentiation into hematopoietic stem cells.

Although IGF2 marked the beginnings of differentiation to hematopoietic lineage, the completion of differentiation was marked by the methylation profiles of the iPS cell DNA. “DNA methylation has an effect on a cell staying pluripotent or differentiating,” explained Yoshida. Completion of the final stages of differentiation was highly correlated with less aberrant methylation during the reprogramming process. Blood founder cells showed a much lesser tendency to display aberrant DNA methylation patterns than did other iPSC lines made from other founder cells. This probably explains why past experiments seemed to indicate that the founder cell contributes to the effectiveness of differentiating iPS cells to the hematopoietic stem cell lineage.

These findings reveal molecular factors that can be used to evaluate the differentiation potential of different iPSC lines, which should, hopefully, expedite the progression of iPSCs to clinical use. Nishizawa expects this work to provide the basis for evaluating iPSC lines for the preparation of other cell types. “I think each cell type will have its own special patterns,” he said.

Breakthrough in scaling up life-changing stem cell production


Research teams at the University of Nottingham, Uppsala University and GE Healthcare in Sweden have discovered a new method that could solve the big problem of the large-scale stem cell production required to fully realize the potential of these remarkable cells for understanding and treating disease.

Human pluripotent stem cells are undifferentiated and possess the unique potential to differentiate into all the different cell types of the body. With applications in disease modeling, drug screening, regenerative medicine and tissue engineering, there is an enormous demand for these cells, which will only grow as clinical applications and the pharmaceutical industry increase the use of these cells.

However, large-scale production of stem cells is not currently feasible because available culture methods are either too expensive, or rely on materials that are not be safe for clinical use in humans, such as animal-based proteins.

In this new publication, which appeared on Wednesday July 13 2016 in Nature Communications, a collaborative team that consisted of researchers from The University of Nottingham’s Wolfson Centre for Stem Cells, Tissue Engineering and Modelling, Uppsala University and GE Healthcare have identified an improved method for human stem cell culture that, at least in principle, provide a faster and cheaper way for grow stem cells for large-scale industrial production.

The project had its genesis at Uppsala University in Sweden, and the first author, Dr Sara Pijuan-Galitó, is now continuing her work as a Swedish Research Council Research Fellow at Nottingham. Sara said: “By using a protein derived from human blood called Inter-alpha inhibitor, we have grown human pluripotent stem cells in a minimal medium without the need for costly and time-consuming biological substrates. Inter-alpha inhibitor is found in human blood at high concentrations, and is currently a by-product of standard drug purification schemes.

“The protein can make stem cells attach on unmodified tissue culture plastic, and improve survival of the stem cells in harsh conditions. It is the first stem cell culture method that does not require a pre-treated biological substrate for attachment, and therefore, is more cost and time-efficient and paves the way for easier and cheaper large-scale production.”

Lead supervisor Dr Cecilia Annerén, who has a joint position at Uppsala University and at GE Healthcare in Uppsala, said: “As coating is a time-consuming step and adds cost to human stem cell culture, this new method has the potential to save time and money in large-scale and high-throughput cultures, and be highly valuable for both basic research and commercial applications.”

Co-author on the paper Dr Cathy Merry added: “We now intend to combine Inter-alpha inhibitor protein with our innovative hydrogel technology to improve on current methods to control cell differentiation and apply it to disease modelling. This will help research into many diseases but our focus is on understanding rare conditions like Multiple Osteochondroma (an inherited disease associated with painful lumps developing on bones) at the cellular level. Our aim is to replicate the three-dimensional environments that cells experience in the body so that our lab-bench biology is more accurate in modelling diseases.”

Dr Sara Pijuan-Galitó’s next task is to combine the Inter-alpha inhibitor with improved synthetic polymers in collaboration with other regenerative medicine pioneers at the University, Professor Morgan Alexander and Professor Chris Denning. This team plans to further improve current human stem cell culture methods. Their goal is to design an economical and safe method that can be easily translated to large-scale production and deliver the billions of cells necessary to start taking cellular therapeutics to individual patients.