Patient-Specific Heart Muscle Cells Before the Baby Is Born


Prenatal ultrasound scans can detect congenital heart defects (CHDs) before birth. Some 1% of all children born per year have some kind of CHD. Most of these children will require some kind of rather invasive, albeit life-saving surgery but an estimated 25% of these children will die before their first birthday. This underscores the need for netter therapies of children with CHDs.

To that end, Shaun Kunisaka from C.S. Mott Children’s Hospital in Ann Arbor, Michigan and his colleagues have used induced pluripotent stem cell (iPSC) technology to make patient-specific heart muscle cells in culture from the baby’s amniotic fluid cells. Because these cells can be generated in less than 16 weeks, and because the amniotic fluid can be harvested at about 20-weeks gestation, this procedure can potentially provide large quantities of heart muscle cells before the baby is born.

In this paper, which was published in Stem Cells Translational Medicine, Kunisaki and others collected 8-10 milliliter samples of amniotic fluid at 20 weeks gestation from two pregnant women who provided written consent for their amniocentesis procedures. The amniotic fluid cells from these small samples were expanded in culture, and between passages 3 and 5, cells were selected for mesenchymal stem cell properties. These amniotic fluid mesenchymal stem cells were then infected with genetically engineered non-integrating Sendai viruses that caused transient expression of the Oct4, Sox2, Klf4, and c-Myc genes in these cells. The transient expression of these four genes drove the cells to dedifferentiate into iPSCs that were then grown and then differentiated into heart muscle cells, using well-worked out protocols that have become rather standard in the field.

Not only were the amniotic fluid mesenchymal stem cells very well reprogrammed into iPSCs, but these iPSCs also could be reliably differentiated into cardiomyocytes (heart muscle cells, that is) that had no detectable signs of the transgenes that were used to reprogram them, and, also, had normal karyotypes. Karyotypes are spreads of a cell’s chromosomes, and the chromosome spreads of these reprogrammed cells were normal.

As to what kinds of heart muscle cells were made, these cells showed the usual types of calcium cycling common to heart muscle cells. These cells also beat faster when they were stimulated with epinephrine-like molecules (isoproterenol in this case). Interestingly, the heart muscle cells were a mixed population of ventricular cells that form the large, lower chambers of the heart, atrial cells, that form the small, upper chambers of the heart, and pacemaker cells that spontaneously form their own signals to beat.

This paper demonstrated that second-trimester human amniotic fluid cells can be reliably reprogrammed into iPSCs that can be reliably differentiated into heart muscle cells that are free of reprogramming factors. This approach does have the potential to produce patient-specific, therapeutic-grade heart muscle cells for treatment before the child is even born.

Some caveats do exist. The use of the Sendai virus means that cells have to be passaged several times to rid them of the viral DNA sequences. Also, to make these clinical-grade cells, all animal produces in their production must be removed. Tremendous advances have been made in this regard to date, but those advancements would have to be applied to this procedure in order to make cells under Good Manufacturing Practices (GMP) standards that are required for clinical-grade materials. Finally, neither of these mothers had children who were diagnosed with a CHD. Deriving heart muscle cells from children diagnosed with a CHD and showing that such cells had the ability to improve the function of the heart of such children is the true test of whether or not this procedure might work in the clinic.

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.

Cynata’s MSC Technology Produces Significant Relief of Asthma in Preclinical Study


An Australian stem cell company called Cynata Therapeutics Limited is in the process of developing a therapeutic stem cell platform technology that they called “Cymerus.” The idea for Cymerus originated at the University of Wisconsin-Madison, but Cymerus would generate a protocol by which clinical laboratories could produce very immature mesenchymal stem cells from induced pluripotent stem cells. Such cells would be personalized for patients and their needs, and Cynata’s goal is to produce a platform that is economically feasible and relatively fast so that patients can receive infusions of the cells they so badly need in a timely fashion. These are very ambitious goals to say the least, but Cynata has been hacking away at this problem for some time, and we certainly wish them the best.

Cynata has recently released some very encouraging data in which their personalized mesenchymal stem cells were used to treat laboratory animals with a laboratory-induces form of asthma. Briefly, female mice (BALB/c mice for those who are interested) were injected with a yolk-protein called “ovalbumin.” Ovalbumin is a protein found in egg whites, and because it is an egg-specific protein, mice do not have it and their immune systems have never seen it before. Such an injection causes the mice to mount an immune response to the ovalbumin, and these mice are then administered aerosolized ovalbumin by means of a nebulizer. This causes the animals to develop a rather severe asthmatic attack against ovalbumin.

In this study, Cynata scientists and their collaborators used 48 mice that were divided into six different groups. The first group was untreated animals that did not suffer from ovalbumin asthma. The second group contained eight animals that had no asthma but were treated intravenously with one million mesenchymal stem cells. The third group also had no asthma, but were treated with an intranasal infusions of one million mesenchymal stem cells. The fourth group contain eight asthmatic animals that were untreated during the course of the experiment. The fifth group contain eight asthmatic animals that were treated intravenously with one million mesenchymal stem cells. The final group contained eight asthmatic animals that were treated with intranasal infusions of one million mesenchymal stem cells. As a note, all animals that were treated mesenchymal stem cells were treated three times. So-called airway hyperresponsiveness (AHR) is a measure of the sensitivity and irritability of the bronchial tissues. AHR is an important measure of the tendency of the lungs to undergo constriction during an asthma attack and AHR is usually measured by administering a drug that can cause bronchoconstriction. The greater the degree of bronchoconstriction in such an experiment is indicative of great AHR. The successful treatment of asthma results in reduction in AHR.

The results of this experiment were wonderfully successful. Exposing mice to the ovalbumin caused them to exhibit significantly increased AHR. However, intravenous administration of Cynata’s MSCs in asthmatic animals caused a statistically significant (60-70%) decrease in AHR compared to untreated, sensitized animals. Additionally, intranasal administration of Cynata’s MSCs completely normalized AHR. The AHR in these asthmatic mice was brought down to a level that was largely the same as the non-asthmatic mice. Also, importantly, no adverse side effects were observed during the study.

This study was conducted under the supervision of Associate Professor Chrishan Samuel and Dr. Simon Royce from the Department of Pharmacology at Monash University, Melbourne, Australia. Because the features of this model asthma system closely resemble the clinical manifestations of asthma in humans, these results provide excellent evidence that such a treatment stands a chance of working in human patients.

“We are very excited by these results, which indicate that Cymerus™ MSCs could have a profound effect in the treatment of asthma. This is a debilitating condition, which affects about 10% of the population, resulting in close to 40,000 hospitalizations and several hundred deaths each year, in Australia alone,” said Cynata Vice President of Product Development, Dr. Kilian Kelly. “Although a number of drugs are approved for the treatment of asthma, studies have shown that conventional treatments result in as few as 5% of asthma patients achieving full control of their condition. Consequently, there is a widely recognized need for novel treatments that address – and potentially eliminate – the underlying disease”, added Dr. Kelly.

“This study has clearly demonstrated that Cynata’s MSCs have a dramatic effect on AHR in our model, particularly when directly administered into the allergic lung. We look forward to continuing our analysis of the effects of these unique cells on markers of inflammation and airway remodeling, and we are optimistic of building on the very positive data we have generated so far,” said Associate Professor Samuel.

Asthma is a condition characterized by the inflammation, narrowing, and swelling of the airways, accompanied by excessive mucous production that makes it difficult to breathe. According to the Global Asthma Network, asthma affects over 330 million people globally. Cynata had partnered with Monash University to examine the potential of its Cymerus technology as an alternate treatment for asthma sufferers.

Cymerus™ makes us of induced pluripotent stem cells (iPSCs) that are then differentiated into a specific type of mesenchymal stem cell precursor known as a “mesenchymoangioblast” or MCA. Cymerus potentially provides a source of MSCs that can be made for so-called “off-the-shelf” therapeutic uses.

Induced Pluripotent Stem Cell-Based Model System of Hypertrophic Cardiomyopathy Provides Unique Insights into Disease Pathology


A research team at the Icahn School of Medicine at Mount Sinai led by Bruce Gelb created a model of hypertrophic cardiomyopathy (HCM) by using human induced pluripotent stem cells.

Patients who suffer from an extreme thickening of the walls of the heart exhibit HCM. This excessive heart thickening is associated with a several rare and common illnesses. There is a strong genetic component to the risk for developing HCM. Can stem cell-based model system be used to study the genetics of HCM?

The answer to this question seems to be yes, since laboratory-generated induced pluripotent stem cells lines that have been differentiated into heart cells that, in many cases, closely resemble human heart tissue. Studies with such stem cell-based model systems have reaped useful insights into disease mechanisms (see F Kamdar, et al., J Card Fail. 2015 Sep;21(9):761-70; Lee YK, Ng KM, Tse HF. J Biomed Nanotechnol. 2014 Oct;10(10):2562-85).

In this paper, Bruce Gelb and his colleagues examined a genetic disorder called cardiofaciocutaneous syndrome (CFC). CFC is caused by mutations in a gene called BRAF. It is a rare condition that affects fewer than 300 people worldwide, and causes head, face, skin, and muscular abnormalities, including abnormalities of the heart.

Gelb and his coworkers isolated skin cells from three CFC patients and reprogrammed them into induced pluripotent stem cells, which were then differentiated into heart cells. In this disease model system, the heart muscle cells enlarged, but this seemed to be due to the interaction of the heart muscle cells with heart-specific fibroblasts. Fibroblasts constitute a significant portion of total heart tissue, even though the heart muscle cells are responsible for the actual pumping activity of the heart. In their model system, Gelb and others observed that these fibroblast-like cells produce an excess of a protein growth factor called TGF-beta, which causes the cardiomyocytes to undergo hypertrophy or abnormal enlargement.

This model system has relevance for research on several related and more common genetic disorders, including Noonan syndrome, which is characterized by unusual facial features, short stature, heart defects, and skeletal malformations.

There is no cure for HCM in patients with these related genetic conditions, but if these findings are correct, then scientists might be able to treat HCM by blocking specific cell signals. This is something that scientists already know how to do. Approximately 40 percent of patients with CFC suffer from HCM (two of the three participants in this study had HCM). This suggests a pathogenic connection, though the link has never been adequately researched.

“We believe this is the first time the phenomenon has been observed using a human induced pluripotent stem cell model of the disease,” said Bruce Gelb.

Please see Rebecca Josowitz et al., “Autonomous and Non-Autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC -Derived Cardiomyocytes,” Stem Cell Reports, 2016; DOI: 10.1016/j.stemcr.2016.07.018.

Functional, Though Not Completely Structurally Normal Tissue-Engineered Livers Made from Adult Liver Cells


Tracy C. Grikscheit and her research team from the Saban Research Institute at the Children’s Hospital Los Angeles have produced functional, tissue-engineered human and mouse liver from adult stem and progenitor cells.

The largest organ in our bodies, the liver executes many vital functions. It is located in the upper right portion of the abdomen protected by the rib cage. The liver has two main lobes that are divided into many tiny lobules.

Liver cells are supplied by two different sources of blood. The hepatic artery provides oxygen-rich blood from the heart and the portal vein supplies nutrients from the intestine and the spleen. Normally, veins return blood from the body to the heart, but the portal vein allows chemicals from the digestive tract to enter the liver for “detoxification” and filtering prior to entering the general circulation. The portal vein also delivers the precursors liver cells need to produce the proteins, cholesterol, and glycogen required for normal body activities.

The liver also makes bile. Bile is a mixture of water, bile acids (made from stored cholesterol in the liver), and other sundry chemicals. Bile made by the liver is then stored in the gallbladder. When food enters the duodenum (the uppermost part of the small intestine), the gallbladder contracts and secretes bile is secreted into the duodenum, to aid in the digestion of fats in food.

The liver also stores extra sugar in the form of glycogen, which is converted back into glucose when the body needs it for energy. It also produces blood clotting factors, processes and stores iron for red blood cell production, converts toxic nitrogenous wastes (usually in the form of ammonium) into urea, which is excreted in urine. Finally, the liver also metabolizes foreign substances, like drugs into substances that can effectively excreted by the kidneys.

Both adults and children are affected by various types of liver disease. Liver can be caused by infectious hepatitis, which is caused by a variety of viruses, chronic alcoholism, inherited liver abnormalities (e.g., Wilson’s disease, hemochromatosis, Gilbert’s disease) or various types of liver cancer. One in ten people in the United States suffer from liver cancer and need a liver transplant. Liver transplantation is the only effective treatment for end-stage liver disease, but the scarcity of liver donors and the necessity of life-long immunosuppressive therapy limit treatment options. In some cases (such as inborn errors of metabolism or acute bouts of liver insufficiency), patients may be effectively treated by transplanting small quantities of functional liver tissue.

Alternate approaches that have been investigated, but these protocols have significant limitations. For example, “hepatocyte transplantation” involves the infusion of liver cells from a donated liver. This protocol, however, wastes many cells that do not integrate into the existing liver and such a treatment is usually little more than a stop-gap solution, since most patients require a liver transplant within a year of this treatment.

Human-induced pluripotent stem (iPS) cells are another possibility but, so far, iPS cells differentiate into immature rather than mature, functional, proliferative hepatocytes.

A need remains for a robust treatment that can eliminate the need for immunosuppressive theory. “We hypothesized that by modifying the protocol used to generate intestine, we would be able to develop liver organoid units that could generate functional tissue-engineered liver when transplanted,” said Dr. Grikscheit.

Grikscheit and her co-workers extracted hardy, multicellular clusters of liver cells known as liver organoid units (LOUs) from resected human and mouse livers. These LOUs were loaded onto scaffolds made from nonwoven polyglycolic acid fibers. These scaffolds are completely biodegradable and they provide a structure upon which the LOUs can grow, fuse, and form a structure that resembles a liver.

After transplantation of the LOU/scaffold combinations, they generated tissue-engineered livers or TELis. Tissue-engineered livers developed from the human and mouse LOUs and possessed a variety of key liver-specific cell types that are required for normal hepatic function. However, the cellular organization of these TELis did differ from native liver tissue.

The tissue-engineered livers (TELis) made by Grikscheit’s laboratory contained normal liver components such as hepatocytes that properly expressed the liver-specific protein albumin, CK19-expressing bile ducts, vascular structures surrounded by smooth muscles that expressed smooth muscle-specific actin, desmin-expressing stellate cells, and CD31-expressing endothelial cells. The production of albumin by the TELi hepatocytes indicated that these cells were executing their normal secretory function. In a mouse model of liver failure, their tissue-engineered liver provided some hepatic function. In addition, the hepatocytes proliferated in the tissue-engineered liver.

A cellular therapy for liver disease that utilizes technologies like this would completely change the treatment options for many patients. In particular, children with metabolic disorders and require a new liver to survive might see particular benefits if such a treatment can come to the clinic. By generating functional hepatocytes comparable to those in native liver, establishing that these cells are functional and proliferative, Grikscheit and her colleagues have moved one step closer to that goal.

To access this paper, please see: Nirmala Mavila et al., “Functional Human and Murine Tissue-Engineered Liver Is Generated From Adult Stem/Progenitor Cells,” Stem Cells Translational Medicine, August 2016 DOI: 10.5966/sctm.2016-0205.

Mouse Study Suggests Stem Cells Can Ward Off Glaucoma


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

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

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

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

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

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

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

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.