Stem Cell-Derived Smooth Muscle Cells Help Restructure Urethral Sphincter Muscles in Rats


Stress urinary incontinence affects 25%-50% of the female population and is defined as the leakage of the bladder upon exertion. The exertions that can cause the bladder to leak can be as simple as laughing, coughing, sneezing, hiccups, yelling, or even jumping up and down. Stress urinary incontinence costs Americans some $12 billion a year and also causes a good deal of embarrassment and compromises quality of life. Unsurprisingly, stress urinary incontinence also is associated with an increased incidence of anxiety, stress, and depression.

In most cases of stress urinary incontinence, injury to the internal sphincter muscles of the urethra or to the nerves that innervate these muscles (both smooth and voluntary muscles) significantly contribute to the condition. Conservative management of stress urinary incontinence can work at first, but can fail later on. The other option is corrective surgery that reconstructs the urethral sphincter and increases urethral support. However, even though such surgeries can and often do work, recurrence of the incontinence is rather common. Is there a better way?

Yan Wen from Stanford University School of Medicine and colleagues and collaborators from College of Medicine of Case Western Reserve in Cleveland, Ohio, Southern Medical University in Guangzhou, China, and Montana State University have used a novel stem cell-based technique to treat laboratory Rowett nude rats that had a surgically-induced form of stress urinary incontinence. While the results are not overwhelming, they suggest that a stem cell-based approach might be a step in the right direction.

Wen and others used a human embryonic stem cell line called H9 and two different types of induced pluripotent stem cell lines to make, in culture, human smooth muscle progenitor cells (pSMCs). Fortunately, protocols for differentiating pluripotent stem cells into smooth muscle cells is well worked out and rather well understood. These pSMCs were also tagged with a firefly luciferase gene that allowed visualization of the cells after implantation.

Six groups of rats were treated in various ways. The first group had stress urinary incontinence and were only treated with saline solutions. The second group of animals also had stress urinary incontinence and were treated with cultured human pSMCs that were derived from human bladders. The third group of animals also had stress urinary incontinence and were treated with pSMCs made from H9 human embryonic stem cells. The next two groups also had stress urinary incontinence and were treated with two different induced pluripotent stem cell lines; one of which was induced with a retroviral vector and the second of which was made with episomal DNA. Both lines were originally derived from dermal fibroblasts. The final group of rats did not have stress urinary incontinence and were used as a control group.

The cells were introduced into the mice by means of injections into the urethra under anesthesia. Two million cells were introduced in each case, three weeks after the induction of stress urinary incontinence. All animals were examined five weeks after the cells were injected into the animals.

Because the cells were tagged with firefly luciferase, the animals could be given an injection of luciferin, which is the substrate for luciferase. Luciferase catalyzes a reaction with luciferin, and the cells glow. This glow is easily detected by means of a machine called the Xenogen Imaging System. Such experiments showed that the injected cells did not survive terribly well, and by 9 days after the injections, they were usually not detectable. Two rats that had been injected with retrovirally-induced induced pluripotent stem cell-derived pSMCs lasted until 35 days after injection, but these rats were the exception and not the rule.

Did the cells integrate into the urethral sphincter by the signal is too low to be detected using luciferase? The answer to this question was certainly yes, but the amount of integration was nothing to write home about. Small patches of cells showed up in the urethra sphincters that expressed human gene products, and therefore, had to be derived from the injected cells.

In vivo survival of transplanted pSMCs in RNU rats. (A): The RV-iPSC pSMCs were periurethrally injected into the rats and monitored with BLI. (B): At day 12, a small number of the transplanted cells were detected in the proximal rat urethra. The transplanted human cells were determined by positive staining of HuNuclei and smoothelin. (C): Gene expression of human ERV-3 in rat urethras 5 weeks after cell transplantation. Y-axis on left shows the scale for ERV-3 copy numbers from tissue samples. Each human cell contains one copy of ERV-3 transcript; hence, the number of copies is equal to the number of cells. Y-axis on right shows the ERV-3 copy numbers of the standard cell samples. The cell numbers in the standard graph are 0.5 × 103, 1 × 103, 2 × 103, 4 × 103, 8 × 103, and 10 × 103 (red dots). ERV-3 amplifications in all pSMC-treated groups were very low. Abbreviations: BLI, bioluminescent imaging; bSMC, bladder smooth muscle cell; DAPI, 4′,6-diamidino-2-phenylindole; Epi, episomal plasmid; ERV-3, endogenous retrovirus group 3; H&E, hematoxylin and eosin; Hu, human; HuNuclei, human nuclei; iPSC, induced pluripotent stem cell; pSMCs, smooth muscle progenitor cells; RNU, Rowett Nude; RV, retrovirus vector.
In vivo survival of transplanted pSMCs in RNU rats. (A): The RV-iPSC pSMCs were periurethrally injected into the rats and monitored with BLI. (B): At day 12, a small number of the transplanted cells were detected in the proximal rat urethra. The transplanted human cells were determined by positive staining of HuNuclei and smoothelin. (C): Gene expression of human ERV-3 in rat urethras 5 weeks after cell transplantation. Y-axis on left shows the scale for ERV-3 copy numbers from tissue samples. Each human cell contains one copy of ERV-3 transcript; hence, the number of copies is equal to the number of cells. Y-axis on right shows the ERV-3 copy numbers of the standard cell samples. The cell numbers in the standard graph are 0.5 × 103, 1 × 103, 2 × 103, 4 × 103, 8 × 103, and 10 × 103 (red dots). ERV-3 amplifications in all pSMC-treated groups were very low. Abbreviations: BLI, bioluminescent imaging; bSMC, bladder smooth muscle cell; DAPI, 4′,6-diamidino-2-phenylindole; Epi, episomal plasmid; ERV-3, endogenous retrovirus group 3; H&E, hematoxylin and eosin; Hu, human; HuNuclei, human nuclei; iPSC, induced pluripotent stem cell; pSMCs, smooth muscle progenitor cells; RNU, Rowett Nude; RV, retrovirus vector.

The exciting part about these results, however, was that when Wen and others examined the rat urethral sphincters for the presence of things like elastin and other proteins that make for a healthy urethral sphincter, there was a good deal of elastin, but it was not human elastin but rat elastin. Therefore, this elastin synthesis was INDUCED by the implanted cells even though it was not made by the implanted cells. Instead, the implanted cells seemed to signal to the native cells to beef up their own production of sphincter-specific gene products, which made from a better sphincter. This was not the case in animals that received injections of human pSMCs derived from human bladders.

Assessment of elastin fibers in the proximal urethra of the rat. (A): Representative images of cross-section of proximal urethra with Weigert’s Resorcin-Fuchsin’s elastin and van Gieson’s collagen staining. Elastic fiber shown as dark blue, collagen as red pink, and other tissue elements as yellow. Scale bars = 100 µm. (B): Quantification of elastin fibers was assessed using Image-Pro Plus software and expressed as a percentage of the arbitrary ROIs. Each bar represents the mean value ± SEM. Abbreviations: bSMC, human bladder smooth muscle cells; H9-pSMC, surgery plus H9-pSMC injection; Hu-bSMC, surgery plus injection of human bladder smooth muscle cells; pSMCs, smooth muscle progenitor cells; Pure control, no surgery and no treatment; ROIs, regions of interest; Sham saline, surgery plus saline injection.
Assessment of elastin fibers in the proximal urethra of the rat. (A): Representative images of cross-section of proximal urethra with Weigert’s Resorcin-Fuchsin’s elastin and van Gieson’s collagen staining. Elastic fiber shown as dark blue, collagen as red pink, and other tissue elements as yellow. Scale bars = 100 µm. (B): Quantification of elastin fibers was assessed using Image-Pro Plus software and expressed as a percentage of the arbitrary ROIs. Each bar represents the mean value ± SEM. Abbreviations: bSMC, human bladder smooth muscle cells; H9-pSMC, surgery plus H9-pSMC injection; Hu-bSMC, surgery plus injection of human bladder smooth muscle cells; pSMCs, smooth muscle progenitor cells; Pure control, no surgery and no treatment; ROIs, regions of interest; Sham saline, surgery plus saline injection.

Because these mice were sacrificed five weeks after the injections, Wen and others could not assess the urethral function of these animals. Therefore, it is uncertain if the improved tissue architecture of the urethral sphincter properly translated into improved function even though it is reasonable to assume that it would. Having said that, it is possible that the experiments that detected the presence of increased amounts of elastin and collagen in the sphincters of these rats was complicated by the presence of bladder tissue in the preparations. Since bladder tissue was included in all trials of this experiment, it is unlikely that bladder tissue is the sole cause of increase elastin and collagen in the stem cell-treated rats. Secondly, rat regenerative properties may not properly match the regenerative properties in older human patients. Here again, unless such an experiment is attempted in larger animal models and then in human patients, we will never know if this procedure is viable for regenerative treatments in the future.

For now, it is an interesting observation, and perhaps a promising start to might someday become a viable regenerative treatment for human patients.

This paper appeared in Stem Cells Translational Medicine, vol 5, number 12, December 2016, pp. 1719-1729.

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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.

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.

Large Screening and Analyses of Established Induced Pluripotent Stem Cell Lines Finds Rogue Lines


Induced pluripotent stem cells (iPSCs) have come a long way since the first lines were made by Shinya Yamanaka and his colleagues in 2006. Initial successes of iPSCs in animal models generated a good deal of hope that iPSCs might find a place in the annals of regenerative medicine. However, since that time, further work has created doubts about the safety of these cells, since some, though admittedly not all, iPSC lines show some genetic abnormalities. However, as screening techniques have become better and have increased in sensitivity, the possibility of accurately ascertaining the quality of iPSC lines draws closer and closer.

A new paper that appeared in the June 9 edition of the journal Stem Cell Reports by Carolyn Lutzko and others from a multi-institutional research group known as the Progenitor Cell Biology Consortium, have used these new screening technologies to screen large numbers of established iPSC lines. The results were somewhat sobering; about 30 percent of iPSC lines analyzed from 10 research institutions were genetically unstable and not safe for clinical use.

This work comprehensively characterized of a large collection of iPSC lines. The technology to produce safe and effective iPSCs exists. Nevertheless, this does not mean that all iPSC lines were produced safely and effectively. In this paper, Lutzko and her colleagues discovered that some iPSC lines that were made with inferior protocols. Some iPSC lines were contaminated with bacteria or carried mutations associated with cancer.

“It was very surprising to us the high number of unstable cell lines identified in the study, which highlights the importance of setting safety standards for stem cell therapies,” said Carolyn Lutzko, PhD, senior author and director of translational development in the Translational Core Laboratories at Cincinnati Children’s Hospital Medical Center. “A good number of the cell lines we studied met quality standards, although the unexpected number of lines that did not meet these standards could not be used for clinical therapies.”

In this paper, Lutzko and her collaborators compared 58 different iPSC lines that had been submitted by various research institutions. The cells were generated with a variety of genes, methods and cells of origin that ranged from skin fibroblasts to infant cord blood cells. All iPSC lines were analyzed for genetic stability, degree of pluripotency, and several other scientific criteria.

In order for an iPSC line to be considered for clinical work, they must exhibit a high degree of genetic stability. Genetically unstable iPSC lines run the risk of form derivatives that can become cancerous, show poor survival, or differentiate into unwanted cell types upon transplantation. It also is essential that iPSC lines exhibit the ability to continuously renew and expand without losing pluripotency or introducing new genetic mutations.

All iPSC lines were also compared to human embryonic stem cell lines in order to compare them to an outside standard.

How did these 58 iPSC lines fare in this rather exacting gauntlet of tests? It depended on several factors. First of all the cell of origin was very important. Skin fibroblasts tended to make rather low-quality iPSC lines, on the average, but cord blood stem cells usually made rather high-quality iPSC lines. Additionally, the specific reprogramming method employed also made a difference. Some of the iPSC lines included in the test were reprogrammed by means of viruses that integrate into the genome of the host cell (24%). Others were reprogrammed with plasmids (64%), which do not integrate into the host cell genome and are lost soon after reprogramming and growth occurs. Others were reprogrammed with modified RNAs (7%), and a few others (5%) were reprogrammed with other types of viruses that do not integrate into the genome of the host cell (Sendai virus). In all cases, the iPSC lines were made by introducing genes into a mature cell that drove that cell to de-differentiate and grow. Slightly different cocktails of genes were used, but the results were largely the same – the induction of pluripotency.  On the average, non-integrating methods of introducing reprogramming genes into cells resulted in higher-quality iPSC lines, with a few notable exceptions.

Pluripotency for each iPSC line was tested by means of implanting undifferentiated iPSCs into nude mice and observing the cells form differentiated tumors called “teratomas.” Teratomas contain tissues derived from all three primary germ layers; endoderm (gut region), ectoderm (epidermis, nerve tissue, etc.) and mesoderm (muscles, blood cells, etc.).

Prior to this study, the prevailing view was that low-quality iPSC lines were not pluripotent and could not form proper teratomas. This hypothesis had not been tested because of the expense of implanting all these iPSC lines into nude mice. To test this hypothesis, Lutzko and her colleagues tested if all iPSC lines, both high and low quality lines, could generate teratomas. Their tests showed that both genetically stable and unstable iPSC lines formed teratomas with cells from all three germ layers. Although genetically unstable iPSC lines demonstrated pluripotency, the concern in a clinical context would be that they also could result in cancer – again emphasizing the need for safe reprogramming methods, according to study authors.

The enormous amount of data generated by these experiments required sophisticated computing for high-level computational analyses. First author, Nathan Salomonis, PhD, a researcher in the Division of Biomedical Informatics at Cincinnati Children’s. Salomonis used computational approaches to collate, examine, and analyze the data and produce large data sets that can compare the different methods of cell programming, the differences in gene regulation between lines, and the functional quality of each iPSC line.

According to Salomonis, his robust data sets uncovered those iPSC lines that had lost their ability to differentiate into particular adult cell types. This massive collection of raw processed data is available through the online web database.

Salomonis said that, in the future, members of this research consortium will test the ability of each iPSCs line to differentiate into specific cell types – such as brain, heart, lung and other cells in the human body. After these data are verified and published, this information will be added to the online database as a public resource.

Antiaging Glycoprotein Quadruples Viability of Stem Cells in Retina


When pluripotent stem cells are differentiated into photoreceptor cells, and then implanted into the retina at the back of the eye of a laboratory animal, they do not always survive.  However, pre-treatment of those cells with an antiaging glycoprotein (AAGP), made by ProtoKinetix, causes those transplanted cells to be 300 times more viable than cells not treated with this protein according to a study recently accepted for publication.

AAGP was invented by Dr. Geraldine-Castelot-Deliencourt and developed in partnership with the Institute for Scientific Application (INSA) of France. For her work in this area Dr. Castelot-Deliencourt was honored with France’s highest award for scientific accomplishment, the Francinov Award, in 2006.

ProtoKinetix, Incorporated said that a paper submitted by Kevin Gregory-Evans on the company’s AAGP was accepted for publication by the Journal of Tissue Engineering and Regenerative Medicine for publication.

AAGP significantly improves the viable yield of stem cells transplanted in retinal tissue, according to experiments conducted at the University of British Columbia in the laboratory of Dr. Kevin Gregory-Evans.

AAGP seems to protect cells from inflammation-induced cell death. This is based on experiments in which cultured cells that were treated with AAGP were significantly more resistant to hydrogen peroxide, ultraviolet A (wavelengths of 320-400 nanometers), and ultraviolet C (shorter than 290 nm). In addition, when exposed to an inflammatory mediator, interleukin β (ILβ), AAGP exposure reduced COX-2 expression three-fold. COX-2 is an enzyme that is induced by the various stimuli that stimulate Inflammation. It is, therefore, an excellent read-out of the degree to which inflammation has been induced. The fact that AAGP prevented the induction of COX-2 shows that this protein can inhibit the induction of inflammation. These data suggest that AAGP™ may not just be usable in cell and organ storage but also in pharmacological treatments.

Pluripotent Stem Cells Actively Regulate The Openness of their Heterochromatin


Packaging DNA into a small area like the nucleus of the cell does not occur unless that DNA is tightly wound into compact structures collectively known as chromatin. However, not all regions of the genome show the same degree of compaction. Highly-expressed regions of the genome tend to be less highly compacted and regions of the genes that are not expressed to any degree tend to be squirreled away into tight chromatin.

Pluripotent stem cells tend to have an open and decondensed chromatin organization. In fact, this open and decondensed chromatin configuration is a defining property of pluripotent cells in general. The connection between pluripotency and the is open chromatin organization and the mediators of this chromatin configuration remain shrouded in uncertainty.

A new study from the laboratory of Peter J Rugg-Gunn at the Babraham Institute, in collaboration with scientists from Canada, the United Kingdom, and Japan, has identified two proteins, Nanog and Sall1 that participate in the chromatin structure of pluripotent stem cells. Such an understanding can contribute to making better pluripotent stem cells.

Cells tend to possess regions of the genome that are tightly wrapped into tight heterochomatin. These genomic regions are usually structural in nature and are, typically, not expressed. These include centromeric DNA and pericentromeric DNA, which plays a role in spindle attachment during cell division. These regions are collectively known as “constitutive heterochromatin.” However, previous research has demonstrated that this constitutive heterchromatin is maintained in an open and uncompacted conformation.

Clara Lopes Novo, in Rugg-Gunn’s laboratory and her colleagues discovered that transcription factor NANOG acts as an integral regulator of the conformation of constitutive heterochromatin in mouse embryonic stem cells. When Lopes Novo and others deleted the Nanog genes in mouse embryonic stem cells, the constitutive heterochromatin was remodeled in a manner that led to more intensive chromatin compaction. However, when Lopes Novo and her coworkers forced the expression of the Nanog gene in mouse embryonic stem cells, leading to spikes in the levels of NANOG proteim, the heterochromatin domains showed distinct decompaction.

When Lopes Novo and others determined where NANOG spent its time, they discovered that it was bound to heterochromatin. In particular, NANOG associated with satellite repeats within heterochromatin domains. Heterochromatin that was associated with NANOG had highly dispersed chromatin fibers, low levels of modified histone proteins that are usually associated with chromatin compaction (i.e. H3K9me3), and high levels of transcription.

The second heterochromatin-associated protein, SALL1, seems to work in cahoots with NANOG. In fact, when Lopes Novo and others deleted the Sall1 gene from mouse embryonic stem cells, the Sall1-/- cells recapitulate the Nanog -/- phenotype. However, further work showed that the loss of Sall1 can be rescued by forcing the recruitment of the NANOG to major portions of the heterochromatin (by over-expressing the NANOG protein).

These results demonstrate the connection between pluripotency and chromatin organization. This work seems to say, “embryonic stem cells actively maintain an open heterochromatin architecture.” They do this to stabilize their pluritotency.

Loss of heterochromatin regulation has potential consequences for the long-term genetic stability of stem cells, and the ability of stem cells, and the ability of stem cells to differentiate and mature into specialized cell types.

This work was published in the journal Genes and Development (http://www.genesdev.org/cgi/doi/10.1101/gad.275685.115)