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.

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)

Encapsulated Human Islet Cells Halt Diabetes for 6 Months in Mice


Researchers from the Massachusetts Institute of Technology (MIT), Boston’s Children Hospital, and several other institutions have successfully used human pancreatic islet cells encased in a porous capsule to halt Type 1 diabetes in mice without causing an adverse immune response for six months. These experiments been reported in two separate scientific journals.

The first study utilized a modified alginate material to encapsulate the pancreatic islet cells. Alginate is a material that originally was derived from brown algae, and has been used to encapsulate cells without harming them or preventing them from sensing and responding to biochemical signals.

Despite all these advantages to alginate, nonspecific kept theior immune responses against it eventually result in the build up of scar tissue around alginate capsules that renders any implanted cells placed inside them ineffective.

The MIT group tested modified alginate derivatives that would not elicit this nonspecific immune response. From a library of over 800 alginate derivatives, the group came upon one particular alginate derivative called triazolethiomorpholine dioxide (TMTD).

To test TMTD capsules in diabetic mice, The MIT group teamed up with Harvard researcher Doug Melton, who supplied human pancreatic beta cells derived from human embryonic stem cells. Once the TMTD-encased beta cells were implanted into mice suffering from type 1 diabetes, the cells immediately began producing insulin in response to increases in blood glucose levels. Diabetic, laboratory mice with these islet cell TMTD-encased implants kept their blood glucose levels within a healthy range for 174 days, which was the whole length of the study.

“Encapsulation therapies have the potential to be groundbreaking for people with Type 1 Diabetes. These treatments aim to effectively establish long-term insulin independence and eliminate the daily burden of managing the disease for months, possibly years, at a time without the need for immune suppression,” said Julia Greenstein, an executive with the Juvenile Diabetes Research Foundation, who funded these two studies.

See here and here.

Stem Cell-Derived Retinal Grafts Integrate into Damaged Monkey Retinas


Retinal degenerations are the leading cause of blindness and fixing a defective retina is not an easy task.

Fortunately, a model system in nonhuman primates that has been used to test retinal replacement with stem cell-derived retinal cells has seen some success. In several experiment in small animals, retinal transplantations helped blind animals regain their sight. However, small laboratory rodents are not terribly good model systems for human eye problems.

To address the clinical relevancy of this transplantation system, Shirai and colleagues confirmed in rats and in macaques that transplantion of human embryonic stem cell (hESC)–derived retinas integrate into the already-existing retina and develop as fully mature retinal grafts.

In this paper, Shirai and others established the developmental stage at which embryonic stem cell-derived retinal cells could integrate into the retina and replace damaged cells. By transplanting cells into nude rats that do not have the ability to reject transplanted tissue, they refined their cell-based technique to heal damaged retinas. Then they took their refined technique into macques to treat two newly established monkey models of retinal degeneration.

In the first model system, Shirai et al. exposed one group monkeys to retina-damaging chemicals, and the other group had their retinas damaged by lasers. In both cases, the result was photoreceptor degeneration. Anywhere from 46 to 109 days after injury, the human embryonic stem cell-derived retinal sheets were implanted into the damaged retinas.

The retinal grafts integrated into the primate eyes and continued to differentiate into cone and rod cells, which are the two types of photoreceptor cells in the retina. Functional studies are still being conducted, but if vision can be improved, but these new macaque models confirm the clinical potential of stem cell–derived grafts for retinal blindness that results from photoreceptor degeneration.

See H. Shirai et al., Transplantation of human embryonic stem cell-derived retinal tissue in two primate models of retinal degeneration. Proc. Natl. Acad. Sci. U.S.A. 113, E81–E90 (2015).

Alveolar Macrophages Derived from Stem Cells Help Lung-Damaged Mice Recover and Survive from Airway Disease


Within the tiny alveolar sacs of our lungs is an immune cell that surveys and directs the immune response within the lung. This immune cell is called an “alveolar macrophage,” and this cell is an actively phagocytic cell. It gobbles up invading bacteria and foreign material in order to keep the lungs clean. When these cells work normally, they help our lungs function properly. When, however, they go rogue, they can fill the lungs with cells that clog the lungs and prevent you from breathing.

Alveolar Macrophages
Alveolar Macrophages

Certain diseases like chronic obstructive pulmonary disease, asthma, and lung fibrosis, have abnormal alveolar macrophages and no specific treatments can appropriately compensate for these abnormalities.

Since alveolar macrophages (AMs) can be made from pluripotent stem cells, perhaps transplanting exogenous AMs derived from pluripotent stem cells can clean up messy lungs.

Martin Post, from the University of Toronto, in Ontario, Canada, and his colleagues tested this very hypothesis in mice. Post and his coworkers differentiated mouse embryonic stem cells by using factor-defined media in order to generate embryonic macrophages that could be grown in culture. Then they conditioned their cells into an alveolar-like phenotype by treating them with the cytokine GM-CSF. The cells were surprisingly like normal AMs, at least in culture.

To test these cells in mice, Post and his group created mice that lacked the ADA (adenine deaminase) gene and these mice lacked proper AM activity and suffered chronic lung damage.

Next, Post’s team transplanted their embryonic stem cell-derived alveolar-like macrophages into the tracheas (windpipes) of these injured animals in order to view their therapeutic potential.

What Post and others saw truly amazed them. Not only was their differentiation protocol wonderfully efficient and adaptable to human pluripotent stem cells, but their PSC-derived macrophages essentially “walked and talked” like regular, normal AMs. These cells made all the right cell surface proteins to be identified as AMs and they engulfed bacteria and dying cells. In fact, they were better phagocytes than bone marrow-derived macrophages.

The implanted macrophages stayed in the airways of the recipient mice for at least 4 weeks, and were able to gobble up other types of rogue white blood cells (i.e., neutrophils) during acute lung injury. Thus, the implanted cells were able to protect the lung from further damage under conditions of lung injury. Additionally, the implanted AMs enhanced tissue repair in the lungs and promoted survival of these mice. Interestingly, the mice did not develop abnormal pathology or teratomas as a result of the implanted macrophages.

Thus, this work from Post and his colleagues shows that pluripotent stem cells are a viable source of therapeutically effective alveolar-like macrophages that can be implanted into the lungs and treat airway diseases. Further experiments in larger animals should prepare this strategy for clinical trials.

This study was published in the American Journal of Respiratory and Critical Care Medicine. published online 05 Jan 2016 as DOI: 10.1164/rccm.201509-1838OC.

Pluripotent Stem Cells Used to Make a Functional Thyroid


The thyroid gland sits over the main cartilage of the larynx and produces thyroid hormone (thyroxine); a hormone that regulates the basal metabolic rate. When the thyroid slows down and fails to make sufficient amounts of thyroid hormone, the result is a condition called hypothyroidism. The symptoms of hypothyroidism are fatigue, weakness, weight gain or increased difficulty losing weight, coarse, dry hair, dry, rough pale skin, hair loss, cold intolerance, muscle cramps and frequent muscle aches, constipation, depression, irritability, memory loss, abnormal menstrual cycles and decreased libido.

If someone has any evidence of a thyroid tumor, then the thyroid is removed, and the patient must take oral thyroid hormone. Because getting the dose right can be difficult, we might ask, “Can we replace the thyroid with stem cell treatments?”

Human pluripotent stem cells can differentiate into balls of cells that are mini-organs called “organoids.”  Unfortunately, if left to themselves, the formation of these organoids is rather haphazard and the cells tend to differentiate into a whole host of different cell types. This is not fatal, however, since the differentiation of these stem cells can be orchestrated by using growth factors or certain culture conditions. Can we use such innovations to make a minithyroid?

Darrell Kotton and his group at Boston University School of Medicine Pulmonary, Allergy, Sleep and Critical Care Medicine have spent their time tweaking the conditions to drive human pluripotent stem cells to form thyroid cells. A new study of theirs that appears in the journal Cell Stem Cell details how the use of two growth factors, BMP4 and FGF2 can drive pluripotent stem cells to commit to thyroid cell fates.

In order to make thyroid cells from embryonic stem cells (ESCs), Kotton and his group had to make endodermal progenitors from them first. Fortunately, a study from Kotton’s own laboratory that was published in 2012 employed a technique used in several other papers that grew ESCs in a serum-free medium with a growth factor called activin. Christodoulou, C., and others (J. Clin. Invest. 121, 2313–2325) showed that over 80 percent of the ESCs grown under these conditions differentiated into endodermal progenitors.  When Kotton and his colleagues cultured these endodermal progenitors in BMP4 and FGF2, some of them differentiated into thyroid progenitor cells. Interestingly, this mechanism by which thyroid-specific cell fates are specified is conserved in creatures as disparate as frogs and mice.

To make mature thyroid cells from these progenitors, a three-dimensional culture system was used in combination with thyroid stimulation hormone and dexamethasone. Under these conditions, the cells formed spherical thyroid follicles that secreted thyroid hormone. To perfect their protocols, Kotton’s group used mouse ESCs, but they additionally showed that this same strategy can make mature thyroid cells from human induced pluripotent stem cells (iPSCs).

Thyroid specification

The appearance of cells in a culture system that look like mature thyroid follicles and express many of the same iodine-metabolizing enzymes as mature thyroid cells is exciting, but can such cells stand in for thyroid tissue in an animal that lacks sufficient thyroid tissue?

Kotton’s laboratory took this to the next step by transplanting their cultured thyroid follicles into laboratory mice that lacked a functional thyroid. These transplants were not inserted into the neck of the animal, but instead were place underneath the kidney, which is area rich in blood vessels. Interestingly, the implanted thyroid “organoids” or little organs did not fall apart upon transplantation. Instead they retained their characteristic structure. More interestingly, these organoids kept expressing iodine-metabolizing enzymes and made thyroid hormone. The synthesis and release of thyroid hormone was also regulated by the hypothalamic hormone thyroid stimulating hormone (TSH). TSH is made and released in response to insufficient thyroid hormone levels. The thyroid responds to TSH by making a releasing more thyroid hormone, which causes a feed-back inhibition of the release of TSH. The fact that these implanted organoids were properly regulated by TSH bespeaks of the maturity of these cells. Also, significantly, none of the laboratory animals showed any signs that the implanted cells had formed any tumors.

Kotton and his coworkers were also able to used human ESCs and human iPSCs to make thyroid organoids. Human iPSCs-derived thyroid organoids were made from human patients with normal thyroid function and from hypothyroid children who carry a loss-of-function mutation in the NKX2-1 gene. This show that Kotton’s system can be used as a model to study inherited thyroid deficiencies. However, there is even more excitement that this system or something similar to it might be useful to safely treat thyroid loss in patients who have lost their thyroid as a result of cancer, or injury.

International Stem Cell Corp’s Parthenogenetic Stem Cells to Be Used in A Clinical Trial to Treat Parkinson’s Disease Patients


The Australian government has recently given its approval for a clinical trial of what is almost certainly a medical first. The Carlsbad-based stem cell company, International Stem Cell Corp. (ISCO), a publicly traded biotechnology company, has developed a unique stem cell technology to address particular conditions.

The clinical trial that has been approved will examine the use the ISCO’s unique stem cell products in the treatment of Parkinson’s disease. Twelve Parkinson’s patients will receive implantations of these cells sometime in the first quarter of 2016, according to Russell Kern, ISCO’s chief scientific officer. The implanted cells will be neural precursor cells, which are slightly immature neurons that will complete their maturation in the brain, hopefully into dopamingergic neurons, which are the precise kind of neurons that die off in patients with Parkinson’s disease.

Parkinson’s disease (PD) is a progressive disorder of the nervous system that affects voluntary movement. PD develops gradually and sometimes begins with a slight tremor in only one hand, but PD may also cause stiffness or slowing of movement. PD worsens over time.

PD patients suffer from tremor, or shaking of the limbs, particularly when it is relaxed and at rest. Over time, PD reduces the ability to move and slows movement (bradykinesis) which makes simple tasks difficult and time-consuming. Muscle stiffness may occur and this limits the range of motion and causes pain. PD patients also suffer from stooping posture and balance problems and a decreased ability to perform unconscious movements. For example, they have trouble swinging their arms while they walk, blinking, or smiling. They might also experience speech problems that can range from slurring of the speech to monotone speech devoid of inflexions, or softer speech with hesitations before speaking. Writing might also become problematic.

PD is caused by the gradual death of neurons in the midbrain that produce a chemical messenger called dopamine. The drop in dopamine levels in the system of the brain that controls voluntary movement leading to the signs and symptoms of Parkinson’s disease.

Several different animal experiments with a variety different cell types have established that transplantation to dopamine-making neuronal precursors into the midbrains of laboratory animals with artificially-induced PD can reverse the symptoms of PD. Dopaminergic neurons can be derived from embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), umbilical cord blood hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs), and NSCs (see Petit G. H., Olsson T. T., Brundin P. Neuropathology and Applied Neurobiology. 2014;40(1):60–67). Also, since the 1980s, various cell sources have been tested, including autografts of adrenal medulla, sympathetic ganglion, carotid body-derived cells, xenografts of fetal porcine ventral mesencephalon, and allografts of human fetal ventral mesencephalon (fVM) tissues have been implanted into the midbrains of PD patients (Buttery PC, Barker RA. J Comp Neurol. 2014 Aug 15;522(12):2802-16). While the results of these trials were varied and not terribly reproducible, these studies did show that the signs and symptoms of PD could be reversed, in some people, by implanting dopamine-making neurons into the midbrains of PD patients.

ISCO has derived neural precursor cells from a completely new source. ISCO scientists have taken unfertilized eggs from human egg donors and artificially activated them so that they self-fertilize, and then begin dividing until they form a blastocyst-stage embryo from which stem cells are derived. This new class of stem cells, which were pioneered by ISCO, human parthenogenetic stem cells (hpSCs) have the best characteristics of each of the other classes of stem cells. Since these stem cells are created by chemically stimulating the oocytes (eggs) to begin division, the oocytes are not fertilized and no viable embryo is created or destroyed. This process is called parthenogenesis and parthenogenetic stem cells derived from the parthenogenetically-activated oocytes, are produced from unfertilized human egg cells.

The stem cells are created by chemically stimulating the oocytes (eggs) to begin division.  The oocytes are not fertilized and no viable embryo is created or destroyed.
The stem cells are created by chemically stimulating the oocytes (eggs) to begin division. The oocytes are not fertilized and no viable embryo is created or destroyed.

Why did ISCO decide to do this trial in Australia? According to Kern, ISCO chose to conduct their clinical trial in Australia because its clinical trial system is more “interactive,” which allows for better collaboration with Australia’s Therapeutic Goods Administration on trial design. This clinical trial, in fact, is the first stem cell trial for PD according to the clinical trial tracking site clinicaltrials.gov. The test will be conducted by ISCO’s Australian subsidiary, Cyto Therapeutics.

The approach pioneered in this clinical trial might cure or even provide an extended period of relief from the symptoms of PD. If this clinical trial succeeds, the stem cell clinical trial dam might very well break and we will see proposed clinical trials that test stem cell-based treatments for other neurodegenerative diseases such as Huntington’s disease, Lou Gehrig’s disease (ALS), frontotemporal dementia, or even Alzheimer’s disease.

ISCO has spent many years developing their parthenogenetic technology with meager financing. However the company’s total market value amounts to something close to $11.1 million, presently.

hpSCs are pluripotent like embryonic stem cells. Because they are being used in the brain, they will not be exposed to the immune system. Therefore an exact tissue type match is not necessary for this type of transplantation. In their publications, ISCO scientists have found their cells to be quite stable, but other research groups who have worked with stem cells derived from parthenogenetically-activated embryos have found such cells to be less stable than other types of pluripotent stem cells. The stability of the ISCO hpSCs remains an open question. The lack of a paternal genome might pose a safety challenge for the use of hpSCs.

Rita Vassena and her colleagues in the laboratory of Juan Carlos Izpisua Belmonte at the Salk Institute for Biological Studies in La Jolla, CA examined the gene expression patterns of mesenchymal stem cells derived from hpSCs and found that the overall gene expression patterns were similar to MSCs made from embryonic stem cells or induced pluripotent stem cells. However, upon further differentiation and manipulation, the gene expression patterns of the cells began to show more variability and further depart from normal gene expression patterns (Vassena R, et al Human Molecular Genetics 2012; 21(15): 3366-3373). Therefore, the derivatives of hpSCs might not be as stable as cellular derivatives from other types of stem cells. The good news about hpSCs established from parthenogenetic ESCs were reported to be morphologically indistinguishable from embryonic stem cells derived from fertilized embryos, and seem to show normal gene expression or even correct genomic imprinting in chimeras, when pESCs were used in tissue contribution (T.Horii, et al Stem Cells, vol. 26, no. 1, pp. 79–88, 2008).

For those of us who view the early embryo as the youngest members of the human community who have the right not to be harmed, hpSCs made by ISCO remove this objection, since their derivation does not involve the death of any embryos.

The ISCO approach to Parkinson’s is similar to that of a San Diego group called Summit for Stem Cell, which is going to use induced pluripotent stem cell derivatives. This nonprofit organization is presently raising money for a clinical trial to test the efficacy of their treatment.

Both groups intend to transplant the cells while they are still slightly immature, so that they can complete their development in the brain. Animal studies suggest that implanting immature precursors are better than transplanting mature dopaminergic neurons into the midbrain. The precursors then differentiate into dopamine-making neurons, and other cells differentiate into supportive glial cells, which support the dopamine-making neurons.

“It’s a dual action,” Kern said. “Also, neural stem cells reduce inflammation, and inflammation is huge in Parkinson’s.”

Summit 4 Stem Cell will also take a similar approach, according to stem cell scientist Jeanne Loring, a leader of the Summit 4 Stem Cell project. The cells make proper connections with the brain better when they are still maturing, said Loring, who’s also head of the regenerative medicine program at The Scripps Research Institute in La Jolla. This is all provided that Summit 4 Stem Cell can raise the millions of dollars required for the clinical trial and secure the required approvals from the U.S. Food and Drug Administration.

Loring said she views ISCO as a partner in fighting Parkinson’s. One of her former students is working for the company, she said. “The whole idea is to treat patients by whatever means possible,” Loring said.

ISCO’s choice of Australia for its streamlined regulatory process makes sense, Loring said. Her team, with U.S.-based academics and medical professionals, doesn’t have the same flexibility as ISCO in looking for clinical trial locations, she said.

Researchers Grow Retinal Ganglion Cells in the Laboratory


Researchers from laboratory of Donald Zack at The Johns Hopkins University in Baltimore, Maryland have used genome editing methods to efficiently differentiate human pluripotent stem cells into retinal ganglion cells. Retinal ganglion cells are found in the retina that and helps transmit visual signals from the eye to the brain. Abnormalities or death of ganglion cells can cause vision loss, and conditions such as glaucoma and multiple sclerosis can wreak havoc on ganglion cells.

“Our work could lead not only to a better understanding of the biology of the optic nerve, but also to a cell-based human model that could be used to discover drugs that stop or treat blinding conditions,” said Zack, who is the Guerrieri Family Professor of Ophthalmology at the Johns Hopkins University School of Medicine. “And, eventually it could lead to the development of cell transplant therapies that restore vision in patients with glaucoma and MS.”

Published in the journal Scientific Reports, Zack and his team genetically modified a line of human embryonic stem cells so that they would fluoresce once they differentiated into retinal ganglion cells. Then they used these cells to develop new differentiation methods and characterize the resulting cells.

To genetically modify their cells, Zack and others used the CRISPR-Cas9 system. CRISPR stands for “clustered regularly interspaced short palindromic repeats” and these are short segments DNA, which are found in bacteria, contain short repeated sequences. Following each repeated sequence is a short spacer that usually comes from previous exposures to a bacterial virus or plasmid. Bacteria use the CRISPR/Cas system as a kind of immune system that prevents cells from being invaded by foreign DNA. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaeal genomes.

When bacteria are invaded by a virus, the particular Cas nucleases capture the viral DNA, cut it and insert it into the CRISPR array. When the bacterial cell is infected by a virus, an RNA is transcribed from the CRISPR array called the crRNA. This crRNA then hybridizes with the invading DNA or RNA and the double-stranded RNA or DNA/RNA hybrid is degraded by Cas proteins.

The CRISPR/Cas system is a useful laboratory tool for gene editing or adding, disrupting or changing the sequences of particular genes. If Cas9 and the appropriate crRNA are delivered into cells, you can cut a genome almost anywhere. CRISPR has a huge number of potential applications.

Zack and his group used the CRISPR/Cas system to insert a fluorescent protein gene into the DNA of their stem cells line. This red fluorescent protein would be expressed if a gene called BRN3B (POU4F2) was also expressed. BRN3B is expressed by mature retinal ganglion cells. Therefore, once these cells differentiated into retinal ganglion cells, they would glow red when viewed with a fluorescence microscope.

After differentiating their cells, Zack and his coworkers used a technique called fluorescence-activated cell sorting to isolate fully differentiated cells from other cells. The pure cell culture contained cells that displayed the biological and physical properties observed in retinal ganglion cells produced naturally, according to Zack.

As an added bonus, Valentin Sluch, a former graduate student in Zack’s laboratory, and her colleagues discovered that soaking the pluripotent stem cells in a chemical called “forskolin” at the commencement of the differentiation protocol significantly improved the efficiency of differentiation. Forskolin is a labdane diterpene found in the roots of the Indian Coleus plant (Coleus forskohlii), which belongs to the mint family.  It is used by some people as a weight loss supplement by some people.

“By the 30th day of culture, there were obvious clumps of fluorescent cells visible under the microscope,” said Sluch, who is now a postdoctoral scholar working at Novartis. Sluch continued, “I was very excited when it first worked. I just jumped up from the microscope and ran [to get a colleague]. It seems we can now isolate the cells and study them in a pure culture, which is something that wasn’t possible before.”

“We really see this as just the beginning,” adds Zack. In follow-up studies using CRISPR, his lab is looking to find other genes that are important for ganglion cell survival and function. “We hope that these cells can eventually lead to new treatments for glaucoma and other forms of optic nerve disease.”

To use these cells to develop new treatments for Multiple Sclerosis, Zack is collaborating with Dr. Peter Calabresi, professor of neurology and director of the Johns Hopkins Multiple Sclerosis Center.

Mesoderm Progenitor Cells With Reduced Tumor-Causing Potential Derived from Human Pluripotent Stem Cells


Karl Willert, PhD, associate professor in the Department of Cellular and Molecular Medicine at the University of California, San Diego and his colleagues have generated a new cell line in his laboratory that can potentially all the tissues in our bodies that are generated from mesoderm.

During embryonic development, 14 days after fertilization, the embryo is transformed from a single-cell thick sheet to a three-layered structure by a process called gastrulation. Gastrulation forms an outer layer of cells known as the ectoderm, which forms the skin and the nervous system, a middle layer of cells known as the mesoderm, which forms the muscles, heart, blood vessels, kidneys, gonads, dermis, adrenal glands, bones, and several other important tissues, and an innermost layer of cells called the endoderm, which forms the gastrointestinal tract as its associated structures. These three layers, the ectoderm, mesoderm, and the endoderm, are collectively known as the “primary germ layers” and they are formed at gastrulation.

Willert, in collaboration with co-corresponding author David Brafman from Arizona State University, used a high-throughput screening platform that had been previously developed in Brafman’s laboratory to define the exact cellular microenvironment that would drive pluripotent stem cells efficiently differentiate into mesodermal progenitor cells. Such cells could theoretically differentiate into any of the derivatives of the mesodermal germ layer, and these cells would also show a greatly reduced capacity to form tumors, since they are no longer pluripotent, but only multipotent.

After using their screening platform to differentiate human embryonic stem cells into cells that expressed mesodermal-specific genes, Willert and his team settled upon a microenvironment that differentiated these stem cells into intermediate mesodermal progenitor (IMP) cells that could be propagated in culture. Interestingly, these IMP cells had the ability to differentiate into mature kidney cells, without the risk of forming tumors. Oddly, these cells were not able to differentiate into other types of mesodermal derivatives.

“This work nicely complements recent advances in tissue engineering and the goal of rebuilding or recreating functional organs, such as what we’ve seen with the creation of ‘mini-kidneys’,” said Willert. “It represents a novel source of cells.” This study was published November 10, 2015 in the online journal eLIFE.

Extensive analyses showed that their IMP cells lacked tumor-forming potential. However, they retained the ability to differentiate into cells that compose the adult kidney. The ability to generate expandable populations of IMPs cells with limited differentiation have several advantages over pluripotent human stem cell cultures. First, pluripotent stem cell cultures can be differentiate into specific cell types but even under the best of conditions, such cell preparations can harbor undifferentiated cells that retain the potential to seed tumor growth. Secondly, it is much easier to manipulate and differentiate IMP cells than pluripotent stem cells. That simplifies the protocols for handling these cells, which also decreases the time and expense required to make anything from these cells.  Third, since IMP cells have limited differentiation capabilities, they are less likely than pluripotent stem cells to differentiate into unwanted cell types.

“Our cells can serve as building blocks to generate kidneys that may one day be suitable for cell replacement and transplantation,” said Willert. “I think such a therapeutic application is still a few years in the future, but engineered kidney tissue can serve as a powerful model system to study how the human kidney interacts with and filters drugs. Such an application would be of tremendous value to the pharmaceutical industry.”

Even though Willert’s IMP cells differentiated into kidney cells, Willert is optimistic that they are capable of differentiating into other mesodermal-derived cell types, like gonads. “We have only characterized their potential to differentiate into cells that contribute to the kidney. We are now investigating to what extent these cells can generate other tissues and organs that derive from intermediate mesoderm, including reproductive organs.”

Willert and his colleagues are using the same protocol to generate other expandable progenitor cell lines from pluripotent stem cells derived from other germ layers, such as ectoderm and endoderm.

Cardiac Muscle Cells Work as Well as Cardiac Progenitor Cells to Repair the Heart


Cell therapies for the heart after a heart attack provide some healing, but the success of these treatments in inconsistent and the majority of the improvements are modest. Whole bone marrow or even bone marrow stem cells can promote the growth of new blood vessels in the heart after a heart attack (Zhou Y, et al., Ann Thorac Surg. 2011 Apr;91(4):1206-12). The treatment of the heart after a heart attack, can also stimulate the regeneration of new heart muscle, but such new muscle comes from endogenous stem cells populations that are induced by the implanted stem cells (Hatzistergos KE, et al., Circ Res. 2010 Oct 1;107(7):913-22).

Nevertheless, the clinical trials with bone marrow cells have produced mixed results. Bone marrow implants work well in some patients and hardly at all in others. The quality of the patient’s bone marrow might be part of the reason for the disparate findings of these trials, but the fact remains, that using cells that can replace dead heart muscle can potentially treat a damaged heart better than bone marrow stem cells.

Pluripotent stem cells, either embryonic stem cells or induced pluripotent stem cells (iPSCs) can efficiently differentiate into heart muscle cells, but a debate remains as to which cell does a better job for healing the heart: Should young heart muscle cells called progenitor cells be used, or can mature heart muscle cells do the job just as well?

Charles Murray from the University of Washington, who has pioneered the use of stem cells to treat the hearts of laboratory animals, and his colleagues tested the ability of heart progenitor cells to repair the heart versus mature heart muscle cells. Both of these cell types were tested against bone marrow stem cells as a control.

Murray and his colleagues used heart muscle cells made from human embryonic stem cells and heart progenitor cells made from the same human embryonic stem cell line to treat the hearts of laboratory rats. These rats were given heart attacks and then the cells were injected directly into the walls of the heart. Injections were given four days after the heart attacks were induced. Each treatment group contained ten rats, including a control group that received injections of cells that are known to possess no healing capabilities.

Measurements of heart function four weeks after treatment showed that both heart progenitor cells and mature heart muscle cells improved the heart equally well and both cells improved heart significantly better than bone marrow stem cells.

Murray said, “There’s no reason to go back to more primitive cells, because they don’t seem to have a practical advantage over more definitive cells types in which the risk for tumor formation is lower.”

In the future, Murry would like to determine if these same cells work in a larger animal model system and then, eventually start clinical trials in human heart attack patients.

Fernandes and Chong et al., Stem Cell Reports, October 2015 DOI: 10.1016/jstemcr.2015.09.011.

Thyroid Organoids Made from Stem Cells Treat Thyroid-Deficient Mice


Darrell Kotton and his research team from Beth Deaconess Medical Center, in collaboration with researchers from the Boston University School of Medicine have devised a workable protocol for differentiating Human pluripotent stem cells into functional thyroid gland cells.

Every year, many people are diagnosed with an underactive thyroid and many others lose their thyroid as a result of thyroid cancer. Designing treatments that can help replace lost thyroid tissue would certainly be a welcome thing for these patients.

By working with mouse embryonic stem cells, Kotton and his colleagues showed that two growth factors, BMP4 and FGF2, and induce foregut endodermal cells to differentiate into thyroid cells. This simple signaling pathway not only efficiently generates thyroid tissue from endoderm, but this pathway turns out to be commonly used in species as diverse as frogs, mice and humans.

The BMP4/FGF2-treated foregut cells differentiated into small thyroid organics that Kotton and his team were able to transplant into thyroid-deficient mice. These transplantations restored normal thyroid function to these mice.

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While mice cells are a fine model system for human diseases, they are not exactly the same. Can this procedure work with human cells? To answer that question, Kotton and his coworkers used human induced pluripotent stem cells (iPSCs) and subjected them to the same BMP4/FGF2 protocol after they had first differentiated the cells into endoderm. In addition, Kotton and his team made thyroid cells from iPSCs derived from cells taken from patients with a specific type of hypothyroidism (interactive thyroid). These patients lack a gene called NKX2-1, and suffer from congenital hypothyroidism.

The thyroid is responsible for your basal metabolic rate. Hypothyroidism or an interactive thyroid can cause patients to gain weight, feel tired constantly, have trouble concentrating, and have a slow heart rate. Hypothyroidism is usually treated with synthetic thyroid hormones that are taken orally. However, restoring a patient’s own thyroid tissue or even replacing defective thyroid tissue with repaired thyroid tissue would be a huge boon to thyroid patients.

This work has discovered the regulatory mechanisms that drive the establishment of the thyroid. It also provides a significant step toward cell-based regenerative therapy for hypothyroidism and the replacement of the thyroid after thyroid cancer treatments.

These results were published in the journal Cell Stem Cell, October 2015 DOI:10.1016/j.stem.2015.09.004.

Protein Regulates Heart Muscle Development


Scientists from the Center for Genomic. Regulation in Barcelona, Spain have discovered a genetic regulatory network that revolves around a protein called Mel18. This regulatory network acts as a genetic switch during the differentiation of embryonic stem cells into heart muscle cells.

Mel18 acts in combination with a vitally important set of proteins called the “Polycomb Regulatory Complexes” or PRCs. PRCs are probably one of the major repressors of genes in adult and embryonic stem cells, and in this paper, Luciano De Croce and his colleagues showed that Mel18 acts with the PRCs to suppress gene expression.

Beyond that, however, once differentiation occurs, Mel18 combines with other proteins to continue to shut off the expression of unnecessary genes, but during early cardiac development, Mel18 completely shifts and becomes a driver of gene expression. It shifts its function by forming new complexes with other proteins that regulate gene expression in various ways.

Graphical abstract_5x5

Thus Mel18 acts as a genetic switch that guides stem cells into the cardiac fate and eventually into the heart muscle cell lineage.

This fascinating work, which was published in the journal Cell Stem Cell, can help stem cell scientists grow better heart muscle from induced pluripotent stem cells in the laboratory. It could also elucidate the underlying causes of heart defects in congenital heart disease. They may also lead to new ways of controlling stem cells in the laboratory to grow cellular repair kits and patches for patients with damaged or sick hearts.

Rejection of Induced Pluripotent Stem Cell Derivatives By the Immune System is a Function of Where They are Transplanted


Induced pluripotent stem cells (iPSCs) are made from mature, adult cells by a combination of genetic engineering and cell culture techniques. Master genes are transfected into mature cells, which are then cultured as they grow and revert to more immature states. Eventually, a population of cells grow in culture that have some, though not all of the characteristics, of embryonic stem cells. Because these cells are pluripotent, they should, theoretically have the ability to differentiate into any adult cell type. Also, since they are derived from a patient’s own cells, they should be tolerated by the patient’s immune system and should not experience tissue rejection.I

Or should they? Experiments with cells derived from iPSCs have generated mixed results. If C57BL/6 (B6) mice are transplanted with iPSC-derived cells, such cells show some levels of recognition by the immune system. However, another study has concluded that various lineages of B6 iPSC-derived cells are not recognized by the immune system when transplanted under the kidney capsule of B6 mice. Why the contradiction?

Yang Xu and his colleagues at the University of California, San Diego have attempted to resolve this controversy by utilizing a mouse model system. Xu and his colleagues used the same B6 transplantation model and transplanted a variety of different cells derived from iPSCs that were made from cells that came from the same laboratory mice.

Xu and others showed that iPSC-derived and embryonic stem cell (ESC)-derived cells are either tolerated or rejected, depending upon WHERE they are transplanted. You see the immune system depends upon a network of cells called “dendritic cells” to sample the fluids that circulate throughout the body and identify foreign substances. Some locations in our bodies are chock-full of dendritic cells, while other locations have a paucity of dendritic cells. When iPSC or ESC-derived cells are transplanted under the kidney capsule, they survive and thrive. The kidney capsule has a distinct lack of dendritic cells. However, if these same cells, which were so nicely tolerated under the kidney capsule, are transplanted under the skin or injected into muscles, they were rejected by the immune system. Why? These two sites are loaded with dendritic cells.

Therefore, the rejection of iPSC-derived cells by the patient’s body is more of a function of where the cells are transplanted than the cells themselves. Mind you, poor quality iPSCs can produce derivatives that are rejected by the immune system, but high-quality iPSCs can differentiate into cells that are accepted by the immune system, but it is wholly dependent on where they are transplanted.

Perhaps, transplanted IPSC derivatives will need the immune system suppressed for a short period of time and after they become integrated into the patient’s body, the immune suppression can be lifted. Alternatively it might be possible to induce tolerance to the transplanted cells with immunological tricks. Either way, understanding why iPSCs-derived cells are rejected or accepted by the patient’s immune system is the next step to using these amazing cells for regenerative medicine.

Xu’s paper appeared in the journal Stem Cells – DOI: 10.1002/stem.2227.

Elabela, A New Human Embryonic Stem Cell Growth Factor


When embryonic stem cell lines are made, they are traditionally grown on a layer of “feeder cells” that secrete growth factors that keep the embryonic stem cells (ESCs) from differentiating and drive them to grow. These feeder cells are usually irradiated mouse fibroblasts that coat the culture dish, but do not divide. Mouse ESCs can be grown without feeder cells if the growth factor LIF is provided in the medium. LIF, however, is not the growth factor required by human ESCs, and therefore, designing culture media for human ESCs to help them grow without feeder cells has proven more difficult.

Having said that, several laboratories have designed media that can be used to derive human embryonic stem cells without feeder cells. Such a procedure is very important if such cells are to be used for therapeutic purposes, since animal cells can harbor difficult to detect viruses and unusual sugars on their cell surfaces that can also be transferred to human ESCs in culture. These unusual sugars can elicit a strong immune response against them, and for this reason, ESCs must be cultivated or derived under cell-free conditions. However, to design good cell-free culture media, we must know more about the growth factors required by ESCs.

To that end, Bruno Reversade from The Institute of Molecular and Cell Biology in Singapore and others have identified a new growth factor that human ESCs secrete themselves. This protein, ELABELA (ELA), was first identified as a signal for heart development. However, Reversade’s laboratory has discovered that ELA is also abundantly secreted by human ESCs and is required for human ESCs to maintain their ability to self-renew.

Reversade and others deleted the ELA gene with the CRISPR/Cas9 system, and they also knocked the expression of this gene down in other cells with small interfering RNAs. Alternatively, they also incubated human ESCs with antibodies against ELA, which neutralized ELA and prevented it from binding to the cell surface. However Ela was inhibited, the results were the same; reduced ESC growth, increased amounts of cell death, and loss of pluripotency.

How does ELA signal to cells to grow? Global signaling studies of growing human ESCs showed that ELA activates the PI3K/AKT/mTORC1 signaling pathway, which has been show in other work to be required for cell survival. By activating this pathway, ELA drives human ESCs through the cell-cycle progression, activates protein synthesis, and inhibits stress-induced apoptosis.

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Interestingly, INSULIN and ELA have partially overlapping functions in human ESC culture medium, but only ELA seems to prime human ESCs toward the endoderm lineage. In the heart, ELA binds to the Apelin receptor APLNR. This receptor, however, is not expressed in human ESCs, which suggests that another receptor, whose identity remains unknown at the moment, binds ELA in human ESCs.

Thus ELA seems to act through an alternate cell-surface receptor, is an endogenous secreted growth factor in human

This paper was published in the journal Cell Stem Cell.