Aging tends to rob us of our ability to concentrate, recall facts, and reason, and this decline seems to stem from the fact that older brains generate fewer neurons than they did when they were younger. However, German researchers have discovered a molecule that accumulates with age that inhibits the formation of new neurons. This finding might help scientists design therapies to prevent age-related mental decline.
This molecule, Dkk1 or Dickkopf-1, accumulated in the brains of aged mice. If Dkk1 production was blocked, neurons were born at much higher rates. Dr. Ana Martin-Villalba, the senior author of this work and a member of the German Cancer Research Center in Heidelberg. Said, “We released a brake on neuronal birth, thereby resetting performance in spatial memory tasks to levels observed in younger animals.”
Aged mice that lacked Dkk1 performed just as well in cognitive tests that included memory and recognition tests as younger mice because of the ability of their neural stem cells to self-renew and generate immature neurons.
Younger mice that lacked Dkk1 were less susceptible to developing acute stress-induced depression than normal mice. This seems to indicate that in addition to slowing memory loss during aging, neutralizing Dkk1 could be beneficial in counteracting symptoms of depression.
Martin-Villalba said that there are ongoing clinical trials to test inhibitors of Dkk1 for other medical purposes. “The design of inhibitors that reach the brain might enable the prevention of cognitive decline in the aging population and depression in the general population,” she said.
According to the American Lung Association and the National Institutes of Health, lung diseases such as asthma and chronic obstructive pulmonary disease (COPD) are on the rise. These are chronic ailments that affect the small airways of the lung. Asthma and COPD involve an injury-repair cycle that leads to the destruction of normal airway structure and function. Presently, drug treatments for COPD only treat the symptoms.
“A healthy lung has some capacity to regenerate itself like the liver,” noted Ed Morrisey, professor of Medicine and Cell and Developmental Biology and the scientific director of the Penn Institute for Regenerative Medicine in the Perelman School of Medicine, University of Pennsylvania. “In COPD, these reparative mechanisms fail.”
Morrisey and his colleagues are examining how epigenetic mechanisms control lung repair and regeneration. Epigenetics consists of chemical modifications to DNA and its supporting proteins that affect gene expression. Previous studies have discovered that smokers with COPD had the most significant decrease in one of the enzymes that controls these modifications, called HDAC2.
“HDAC therapies may be useful for COPD, as well as other airway diseases,” he explained. “The levels of HDAC2 expression and its activity are greatly reduced in COPD patients. We believe that decreased HDAC activity may impair the ability of the lung epithelium to regenerate.”
By using genetic and pharmacological approaches, Morrisey and others showed that the development of progenitor cells in the lung is specifically regulated by the combined function of two highly related HDACs, HDAC/1 and /2. Morrisey and his colleagues published their findings in the prestigious journal Developmental Cell.
By studying how HDAC activity and other epigenetic regulators control lung development and regeneration, they hope to develop new therapies to alleviate the unmet needs of patients with asthma and COPD.
HDAC1/2 deficiency leads to a loss of expression of the an essential transcription factor, a protein called Sox2, which in turn leads to disruption of airway epithelial cell development. This is affected in part by increasing the expression of two genes, Bmp4 and the tumor suppressor Rb1, both of which are inhibitors of cell proliferation including the proteins p16, and p21. This results in decreased epithelial proliferation in lung injury and inhibition of regeneration.
Together, these data support a critical role for HDAC-mediated mechanisms in regulating both development and regeneration of lung tissue. Since HDAC inhibitors and activators are currently in clinical trials for other diseases, including cancer, such compounds could be tested in the future for efficacy in COPD, acute lung injury and other lung diseases that involve defective repair and regeneration, said Morrisey.
ALS or amyotrophic lateral sclerosis is a disease that results in he death of motor neurons. Motor neurons enable skeletal muscles to contract, which drives movement. The death of motor neurons robs the patient of the ability to move and ALS patients suffer a relentless, progressive, and sad decline that culminates in death from asphyxiation. Treatments are largely palliative, but stem cells treatments might delay the onset of the disease, or even regenerate the dead neurons.
To this end a Mexican group from Monterrey has used a protocol to isolate white blood cells from the circulating blood of ALS patients, and differentiate a specific population of stem cells from peripheral blood into preneurons. Although these cells were not used to treat the patients in this study, such cells do show neuroprotective features and using them in a clinical study does seem to be the next step.
In this study, CD133 cells were isolated from peripheral blood and subjected to a special culture system called a neuroinduction system. After 2-48 hours in this system, the cells showed many features that were similar to those of neurons. The cells express a cadre of neural genes (beta-tubulin III, Oligo 2, Islet-2, Nkx6.1, and Hb9). Some of the ells also grew extensions that resemble the axons of true neurons.
Interestingly, the conversion of the CD133 cells into preneurons showed similar efficiency regardless of the age, sex, or health of the individual. Even those patients with more advanced ALS had CD133 cells that differentiated into preneurons with efficiencies equal to those of their healthier counterparts. While each patient showed variation with regards to the efficiency at which their CD133 cells differentiated into preneurons, these variations could not be correlated with the age, health or sex of the patient.
The fact that these preneurons expressed Oligo2, suggests that they could differentiate into motor neurons. Therefore, even though this study was small (13 patients), it certainly shows that cells that might provide treatment possibilities for ALS patients can be made from the patient’s own blood cells.
See Maria Teresa Gonzalez-Garza et al., Differentiation of CD133+ Stem Cells from Amyotrophic Lateral Sclerosis Patients into Preneuron Cells. Stem Cells Translational Medicine 2013;2:129-35.
Researchers at Johns Hopkins University have used experiments in mice and humans to determine to target of antidepressant drugs and electroconvulsive therapy. The results of these experiments explain how these therapies work to relieve depression. Apparently, antidepressant drugs and electroconvulsive therapy stimulate stem cells in the brain to grow and mature. These findings also provide a way to determine how well a patient might respond to anti-depression therapies, which will allow fine-tuning of these therapies.
Hongjun Song, Professor of neurology and director of the Stem Cell Program at the John Hopkins University School of Medicine’s Institute or Cell Engineering, main this comment: “Previous studies have shown that antidepressants and electroconvulsive therapy both activate neural stem cells in the adult brain to divide and form new neurons. What were missing were the specific molecules linking antidepressant treatment and stem cell activation.”
Song’s team made this link by assembling evidence from several different experiments. First, they examined gene expression profiles in the brains of mice that had and had not been treated with electroconvulsive therapy. They found that one gene in particular showed decreased expression in mice that had undergone electroconvulsive therapy (ECT), and that gene is called sFRP3 (secreted frizzled-related protein 3). Secreted frizzled-related protein 3 regulates the activity of secreted Wnt signaling proteins. Wnt proteins stimulate neural stem cells growth, but the presence of sFRP3 prevents Wnt proteins from doing so. Since ECT decreases sFRP3 production, Wnt proteins have a freer hand to stimulate neural stem cell proliferation.
A second experiment in mice confirmed that antidepressants targeted sFRP3. When knock-out mice were constructed that did not possess a functional copy of the sFRP3 gene, both sFRP3+ and sFRP3- mice were given antidepressants, but neither one showed any difference in behavior. These data strongly suggest that antidepressant treatments blocking the function of the sFRP3 protein, since in the absence of sFRP3, there is nothing to block and the drugs fail to elicit any changes in behavior.
Finally, Song and his collaborators examined sFRP3 genes from 541 patients diagnosed with clinical depression. The response of these patients to antidepressant drugs was tracked and correlated with their DNA sequences of their sFRP3 genes. They discovered that there were three variants of the sFRP3 gene that presaged a stronger response to antidepressant drugs.
A complicating factor is that the levels of sFRP3 are regulated by other factors such as exercise. According to Song, “This gene’s activity is very sensitive to the amount of activity in the brain so that sFRP3 seems to be a gatekeeper that links activity to new neuronal growth.”
This funding has two major near-term implications. First it could lead to genetic tests that enable doctors to predict a patient’s response to antidepressants. Second, it could provide new targets for potential new therapies for clinical depression.
See M-H Jang, Y Kitabatake, E Kang, H Jun, M V Pletnikov, K M Christian, R Hen, S Lucae, E B Binder, H Song and G-I Ming. Secreted frizzled-related protein 3 (sFRP3) regulates antidepressant responses in mice and humans. Molecular Psychiatry , (4 December 2012) | doi:10.1038/mp.2012.158.
Mi-Hyeon Jang, et al., Secreted Frizzled-Related Protein 3 Regulates Activity-Dependent Adult Hippocampal Neurogenesis. Cell Stem Cell, Volume 12, Issue 2, 215-223, 7 February 2013.
Congestive heart failure is the leading cause of morbidity and mortality worldwide. Implanting stem cells into the damaged heart to regenerate the dead heart cells is a potentially exciting prospect for regenerative medicine. Finding the right cell for the job is the greatest challenge, and to this end the heart itself may provide an interesting source of stem cells for regenerative medicine. This source of cells resides on the outside of the heart, a layer known as the epicardium.
After a heart attack, the cells of the epicardium differentiate into smooth muscle cells and heart-specific fibroblasts. They do not form heart muscle cells or blood vessels, but they do secrete a whole cadre of growth factors that encourage the heart to form blood vessels. In mice, preconditioning the heart with a protein called “thymosin beta4” induces the epicardial cells to migrate into the heart and form new heart muscle cells (Smart et al., Nature 2001 474: 640-4). Unfortunately, using thymosin beta4 in human patients who have had heart attacks fails to elicit appropriate changes in the myocardium (Zhou et al., J Mol Cell Cardiol 2012 52: 43-7).
Chong and his colleagues have discovered a new stem cell in the epicardium of mice that can grow for long periods of time in culture and are found near blood vessels. Chong and others call this new epicardial stem cell a “cardiac colony-forming-unit fibroblast” or cCFU-F for short (Chong et al., Cell Stem Cell 2011 9: 527-40).
These cCFU-Fs form from the epicardium, but they do not express heart-specific genes (e.g., c-Kit, CD31, Flk1, CD45, Nkx2-5, NG2). When the gene expression profile of cCFU-Fs was examined in some detail, they expressed the same clusters of genes as bone marrow stem cells (Pelekanos et al., Stem Cell Research 2012 8: 58-73). Are these two cell populations the same? Apparently not. Chong and his crew tried to reconstitute the cCFU-Fs in mice that had had their bone marrow completely replaced with green-glowing bone marrow stem cells. The glowing bone marrow stem cells never contributed to the cCFU-F population.
Therefore, can cCFU-Fs contribute to heart regeneration after a heart attack? Can they be primed to form heart muscle with thymosin beta4? Many questions abound, but these cCFU-Fs seem to represent an easily accessed and robust population for regenerative medicine for the heart after a heart attack.
Materials researchers at the University of Southampton, UK, have invented a new plastic that directs pluripotent stem cells to attach and differentiate into bone. This technology could lead to new therapies for people who suffer osteoporosis and osteoarthritis and need hip replacements.
Dr. Emmajayne Kingham has collaborated with University of Glasgow researchers to develop special plastics, grow embryonic stem cells on them, and assess the behavior of the cells on the plastic material. Normally, stem cells require chemicals in order to direct their differentiation. However, in this experiment, the only directing the cells was the microscopic topography of the plastic surface.
This plastic material, polycarbonate, is a very versatile material and is found in substances as hard an inflexible and bullet-proof glass to compact discs. This material is also rather inexpensive and because it can direct the differentiation of embryonic stem cells into bone tissue cells, it can make bone from pluripotent stem cells quite cheaply.
Professor Richard Oreffo, the leader of the Southampton research team said: “To generate bone cells for regenerative medicine and further medical research remains a significant challenge. However, we have found that by harnessing surface technologies that allow the generation and ultimately scale up of human embryonic stem cells to skeletal cells, we can aid the tissue engineering process. This is very exciting.”
Oreffo continued: “Our research may offer a whole new approach to skeletal regenerative medicine. The use of nontopographical patterns could enable new cell culture designs, new device designs, and could herald the development of new bone repair therapies as well as further human stem cell research.”
These data expand on previous work in which the Southampton research group teamed up with another research group from the University of Glasgow to show that plastic surfaces with embossed patterns encouraged the growth and spread of adult stem cells while preventing the stem cells from differentiating. Such a process uses inexpensive polycarbonate plastics that are inexpensive and relatively easy top manufacture.
“Our previous collaborative research showed exciting new ways to control mesenchymal stem cell – stem cells from bone marrow of adults – growth and differentiation on nanoscale patterns,: said Nikolaj Gadegaard from the University of Glasgow.
He continued, “This new Southampton discovery shows a totally different cell source, embryonic, also respond in a similar manner and this really starts to open this new field of discovery up. With more research impetus, it gives us the hope that we can go on to target a wider variety of degenerative conditions than we originally aspired to. This result is of fundamental significance.”
The Food and Drug Administration (FDA) of the United States has approved the first retinal implant for use in the United States. This approval is for Second Sight’s Argus II Retinal Prosthesis System, which provides limited sight to those patients blinded by a rare genetic eye condition called advanced retinitis pigmentosa. This condition damages the light-sensitive cells that line the outer layer of the retina and causes them to die. This severely reduces vision and eventually leads to blindness.
Second Sight has devoted more than 20 years of research and development to the development of the Argus II Retinal Prosthesis. It has succeeded in two clinical trials, and the funding for the development of this device – more than $200 million – came from the National Eye Institute, the Department of Energy and the National Science Foundation. The remaining money came from private investors. European regulators approved the Argus II for use in 2011 and it has been used in 30 patients in clinical-trial patients since 2007. The Ophthalmic Devices Advisory Panel of the FDA unanimously recommended approval for the Argus II in September 2012.
The Argus II includes a small video camera, a video processing unit and a 60-electrode implanted retinal prosthesis with a transmitter mounted on a pair of eyeglasses. This device replaces the function of degenerated cells in the retina. It must be stressed that the Argus II does not fully restore vision, but it can improve a patient’s ability to perceive images and movement. It uses the video processing unit to transform images from the video camera into electronic data that is wirelessly transmitted to the retinal prosthesis.
Retinitis pigmentosa affects about one in 4,000 people in the US and about 1.5 million people worldwide. It kills off the retina’s photoreceptors, which convert light into electrical signals that are transmitted by means of the optic nerve to the brain’s visual cortex for processing. Second Sight plans to adapt its technology to assist people afflicted with age-related macular degeneration, which is a similar but more common disease.
Second Sight has plans to make the Argus II available later this year in clinical centers throughout the US. They want to establish a network of surgeons who have the skills to implant the device and, eventually recruit hospitals to offer it.
The Argus II is not the only retinal implant under development. A medical start-up company called Retina Implant AG uses a different approach in its device. In this case, the prosthetic device, the Alpha IMS Implant, is inserted beneath a portion of the retina. The three- by three-millimeter microelectronic chip (0.1-millimeter thick) contains ~1,500 light-sensitive photodiodes, amplifiers and electrodes. The Alpha IMS Implant is surgically inserted beneath a portion of the retina known as the fovea (which contains a rich concentration of particular photoreceptors known as cone cells) in the retina’s macula region. The fovea enables the highest clarity of vision for people to read, watch TV and drive. This chip helps generate at least partial vision by stimulating intact nerve cells in the retina. The nerve impulses from these cells are then fed by means of the optic nerve to the visual cortex where they create impressions of sight. The power source for the chip is implanted under the skin behind the ear and connected by a thin cable to the chip. In May the company announced its first UK patients for its latest trial. To date surgeons have implanted the Alpha IMS Implant prosthetic in 36 patients through two clinical trials over six years.
Researchers from Stanford University researchers are developing self-powered retinal implants in which each pixel in the device is fitted with silicon photodiodes. These sensors detect light, and control the output of a pulsed electric current. Patients would be required to wear a set of goggles for these devices that emit near-infrared pulses that transmit power and data directly to the photodiodes. Inductive coils that must be surgically implanted in the patient’s head to power these other retinal prostheses. This design was reported in May 2012 issue of Nature Photonics, and in the article, they described in vitro electrical stimulation of healthy and degenerate rat retina by photodiodes powered by near-infrared light.
Other researchers are utilizing yet another design for retinal prosthesis design. Researchers from Weill Cornell Medical College in New York City have deciphered the neural codes that mouse and monkey retinas use to turn light patterns into patterns of electrical pulses that their brains translate into meaningful images. Next they programmed this information into an “encoder” chip that was combined with a mini-projector to create an implantable prosthetic. This chip converts images that come into the eye into a series of electrical impulses, and the mini-projector then converts the electrical impulses into light impulses that are sent to the brain. With this approach, instead of increasing the number of electrodes placed in an eye to capture more information and send signals to the brain, this approach increases the quality of the artificial signals themselves, which improves their ability to carry impulses to the brain.
Scientists at Queen’s University Belfast hope to design a new approach for treating the eyesight of diabetic patients by using adult stem cells.
Millions of diabetics every year are at risk for losing their eyesight due to diabetic retinopathy. When high blood sugar causes blood vessels in the eye to leak or become blocked, failed blood flow damages the retina and lead to vision impairment. If left untreated, diabetic retinopathy can lead to blindness.
The Queen’s University Belfast group have initiated the REDDSTAR study, which stands for Repair of Diabetic Damage by Stromal Cell Administration, and this study involves researchers from the Queen’s Center for Vision and Vascular Science in the School of Medicine, Dentistry and Biomedical Sciences. REDDSTAR begins with the isolation of stem cells from patients and expanding them in the laboratory. Then these patient-specific cells are delivered to the patient from whom they were originally drawn in order to repair the blood vessels in the eye. This blood vessel repair is especially useful in patients with diabetic retinopathy.
Presently, diabetic retinopathy is treated with laser ablation of new blood vessels that grow in response to damage. These new blood vessels become so dense that they obscure vision. However, presently, there are no treatments to control the progression of diabetic complications.
Alan Stitt, the director of the Centre for Vision and Vascular Science at Queen’s and lead scientist for the REDDSTAR study, said, “The Queen’s component of the REDDSTAR study involves investigating the potential of a unique stem cell population to promote repair of damaged blood vessels in the retina during diabetes.” Professor Stitt continued: “The impact could be profound for patients, because regeneration of damaged retina could prevent progression of diabetic retinopathy and reduce the risk of vision loss.”
“Treatments for diabetic retinopathy are not always satisfactory. They focus on end-stages and fail to address the root causes of the condition. A novel, alternative therapeutic approach is to harness adult stem cells to promote regeneration of the damaged retinal blood vessels and thereby prevent and/or reverse retinopathy.”
Stitt said the new research project is one of several regenerative medicine approaches ongoing at his research center. Their approach is to isolate a rather well-defined population of stem cells and then deliver those stem cells to sites in the body that have been ravaged by diabetes. In particular patients, these strategies have produced remarkable benefits from stem cell-mediated repair of their blood vessels. Treatments such as this one are simply the first step in the quest to develop exciting, effective and new therapies in an area of medicine where such therapies are desperately needed.
In the REDDSTAR study, stem cells from bone marrow are used and these stem cells are provided by Orbsen Therapuetics, which is a spin-off from the Science Foundation Ireland-funded Regenerative Medicine Institute (REMEDI) at NUI Galway.
This project will design protocols for growing these bone marrow-derived stem cells and they will be tested in several preclinical models of diabetes and diabetic complications at research centers in Belfast, Galway, Munich, Berlin, and Porto before human clinical trails take place in Denmark.
Queen’s Centre for Vision and Vascular Science is a key focus of the University’s ambitious 140-million pound “together we can go Beyond” fundraising campaign. This campaign is due to expand the Vision Science program further when the University’s new 32-million pound Wellcome-Wolfson Centre for Experimental Medicine opens in 2015. Along with vision, two new programs in Diabetes and Genomics will also be established in the new Center. These Center should stimulate further investment and global collaborations between biotech and health companies in Ireland.
Within human bone, cells called osteoblasts make new bone and without the constant activity of osteoblasts, bone becomes thin and fragile. Osteoblasts are derived from mesenchymal stem cells in the bone marrow. When bones break, orthopedic surgeons try to use growth factors to push more mesenchymal stems and their progeny to become osteoblasts. The growth factor in question is bone morphogen protein (BMP). BMP, however, does not work consistently, and it has some rather nasty side effects (cancer, The specific complications that are drawing the most concern include swelling in the neck and throat, radiating leg pain, and male sterility). Therefore, an alternative method for converting mesenchymal stem cells into osteoblasts is highly desirable.
Kurt Hankenson from the University of Pennsylvania School of Veterinary Medicine has worked on this very problem and described the situation this way, “In the field, we’re always searching for new ways for progenitor cells to become osteoblasts so we became interested in the Notch signaling pathway.” When it comes to BMP, Hankenson said, “it has become clear that BMPs have some issues with safety and efficacy.”
Is there a better way to make bone? There seems to be. A protein called Jagged-1 has been shown by Hankenson’s team to be highly expressed in bone. Jagged-1 is a component of the widely used Notch signaling pathway, which is found in the nervous system and in many other cells as well.
In mouse stem cells, introducing Jagged-1 blocks the progression of mesenchymal stem cells to osteoblasts. This finding has actually hampered osteoblast research for the last two years. Hankenson again, “That had been our operating dogma for a year or two.”
However, as is so often the case in science, you never truly know the result of an experiment until you actually do it. When Jagged-1 was added to human mesenchymal stem cells, the results were very different. Hankenson said, “It was remarkable to find that just putting the cells onto the Jagged-1 ligand seemed sufficient for driving the formation of bone-producing cells.”
From a developmental genetics perspective, this makes perfect sense, since mutations in the Jagged-1 gene cause an inherited disease known as Alagille syndrome which causes liver problems, abnormal metabolisms, and fragile bones that break easily. Also, genome-wide association studies have shown that particular versions of the Jagged-1 gene cause low bone density.
Hankenson and his collaborators are examining ways to manipulate the levels of the Jagged-1 protein in patients with bone problems. To that end, Hankenson is collaborating with Kathleen Loomes of Penn’s Perelman School of Medicine and the Children’s Hospital of Pennsylvania to study pediatric patients with Alagille syndrome to determine if bone abnormalities in these patients are indeed connected to Jagged-1 malfunctions.
Hankenson and his former graduate student Mike Dishowitz started a company called Skelegen through the University of Pennsylvania’s Center for Technology Transfer (CTT) UPstart program. The goal of Skelegen is to develop and improve a system for delivering Jagged-1 to sites that require new bone growth in the hopes of treating bone fractures and other skeletal problems.
See Fengchang Zhu et al., “Pkcdelta is required for Jagged-1 induction of hMSC osteogenic differentiation.” Stem Cells 2013; DOI 10.1002/stem.1353.
Induced pluripotent stem cells (iPSCs) are made from adult cells by means of genetic engineering techniques that introduce specific genes into adult cells. These genes express their proteins and they push the adult cell to de-differentiate into cells that, in many ways, resemble embryonic stem cells. These de-differentiated cells can form all the cell types, and they can potentially be used for regenerative medicine.
In particular, the heart can experience the death of heart muscle cells after a heart attack, and replacement of dead heart muscle cells can return the heart to its full potential. To this end, iPSCs and embryonic stem cells (ESCs) can be differentiated into heart muscle cells and transplanted into the damaged heart. Such experiments have been done and they do improve heart function, but what is the best way to apply the heart muscle cells? Should they be injected into the heart wall? Should they be applied on sheets?
Hidetoshi Matsumoto and colleagues in the laboratory of Jun Yamashita at Kyoto University has made mouse iPSCs, and differentiated them into heart muscle cells that were grown in sheets. These sheets were laid over the heart of athymic rats that had suffered a heart attack (athymic mice do not have a thymus gland and therefore are unable to reject transplanted, foreign tissue). The results were telling not for what they did to the heart, but how they did it.
Matsumoto made sheets of heart muscle, sheets of endothelial cells, which make blood vessels, and sheets of mural cells, which are the smooth muscle cells that control the diameter of blood vessels. He also made sheets that contained combinations of cells and sheets with all three cell types.
The sheets that contained all three cell types improved heart function, but after four weeks, no transplanted cells could be detected. How then did these sheets improve heart function? The answer was in a vast increase in the density of blood vessels. When the sheets were applied, they quickly showed a vast increase in the expression of those genes that induce the formation of blood vessels. Thus, even though the cells did not survive, the blood vessels they induced endured and improved heart function.
When cell sheets consisting only of endothelial cells or endothelial cells and mural cells were applied, only slight improvements in heart function were observed and the vast increase in blood vessel density did not ensue. Therefore, heart muscle cells are necessary to induce the formation of new blood vessels.
These results are very similar to those of Yoon et al, who used genetically engineered bone marrow cells to treat rodent hearts that had experienced a heart attack. When Yoon and others depleted these populations of either endothelial or mural cells, the implanted cells failed to improve heart function (Yoon et al., Circulation 2010 121: 2001-11).
Thus. iPSC-derived heart muscle sheets might very improve heart function after a heart attack, but they might do so without actually integrating into the heart.
On a closing note, it seems to me that preconditioning these cells to survive in the hostile environment of the infarcted heart might improve the survival of these cells, and therefore, and their ability to improve heart function. This might be an experiment for future researchers.
See Matsumoto et al, Stem Cells 2012 30:1196-1205.
My apologies to my readers for my inactivity. Many deadlines make for less blogging. Nevertheless, I hope to get back to a more regular blogging schedule once things quiet down a bit.
Today’s entry is about a fascinating group of cells found in the extraembryonic membranes of the fetus known as the amnion. The amniotic sac is a thin, transparent pair of membranes that is actually rather tough. This sac holds the fetus until shortly before birth. In inner membrane of the amnion sac contains the amniotic fluid and fetus and the outer membrane, the chorion, surrounds the amnion and is part of the placenta.
The amniotic membrane contains a remarkable cell type known as amniotic epithelial cells or hAECs (the “h” is for human). Upon isolation after birth, the amnion membrane and manually separated from the chorion membrane and washed in a saline (salt) solution in order to remove all the blood. Then the epithelial cells are liberated from the basement membrane upon which they sit by a product called TrypZean. TrypZean is a recombinant trypsin, which is very clean and devoid of animal products. Trypsin is one of the enzymes in your digestive system that degrades proteins. By expressing the human trypsin gene in bacteria and purifying the protein, Sigma-Aldrich corporation can sell it for a profit to scientists for various procedures.
A single amnion membrane can yield in the vicinity of 120 million viable hAECs, which can be maintained in serum-free culture conditions. After being grown for some time, hAECs will have normal chromosome compositions and will also maintain chromosomes that have nice, long ends (telomeres). This indicates that the cells are healthy and dying while they grow in culture (see Murphy et al., Current Protocols in Stem Cell Biology, 2010; Chapter 1: Unit 1E.6). .
In culture,. hAECs do not grow like weeds. Mesenchymal stem cells (MSCs) tend to grow better than their hAEC brethren, but hAECs possess a remarkable ability to differentiate into a wide variety of different cell types. Sivakami Ilancheran in the laboratory of Martin Pera at the University of Monash in Clayton, Australia showed that hAECs were able to differentiate into heart muscle, skeletal muscle, bone, fat cells, pancreatic cells, liver, and at least two kinds of nerve cells. Also, when injected into mice, hAECs never formed tumors (Ilancheran et al., Biology of Reproduction 77 (2007): 577-88). Murphy and others have also shown that hAECs can be isolated after collection and stored for clinical therapies.
Given that hAECs are accessible, what are they good for? When it comes to regenerative medicine, preclinical studies with hAECs have produced very solid results that may pave the way for other studies.
HAECs can differentiate into lung cells and this feature makes them an attractive candidate for lung diseases. Lung diseases cause inflammation of the lung and scarring that decreases overall lung capacity. Cystic fibrosis, acute respiratory distress syndrome, chronic obstructive pulmonary disease, pulmonary fibrosis, pulmonary edema, and pulmonary hypertension are all lung diseases that could potentially be treated with hAECs.
In animal models of lung disease, particular chemicals are given to the animal that damage the lung. The wounded lung tissue initiates inflammation that brings white blood cells into the lung that augment the lung damage, which results in lung scarring. If hAECs are given to mice whose lungs have been damaged by the anti-cancer drug bleomycin, the signs of inflammation and the genes normally expressed during inflammation fade away. There is also less scarring in the lungs and the functional recovery of these animals is significantly better than those animals that do not receive hAECs (Murphy et al., Cell Transplantation 2011 20(6): 909-23). In fact, hAECs can differentiate into lung cells and integrate into lung tissue. The significance of this is not lost on respiratory specialists who treat patients with cystic fibrosis. Cystic fibrosis patients lack a functional copy of a ion transport protein and poor ion transport cause the production of thick, sticky mucous that clogs up the lung pathways and causes patients to suffocate to death. However, hAECs can differentiate into lung cells that express this ion transporter. Therefore, hAECs could be a potential treatment for cystic fibrosis. Clearly hAECs have great potential for tissue engineering applications with lung disease.
Lungs are not the only organ that hAECs can help heal. These cells can also differentiate into pancreatic insulin-making cells. In the laboratory, Wei and coworkers succeeded in stimulating hAECs to secrete insulin and express the main sugar transport protein found in pancreatic insulin-secreting cells (Wei et al., Cell Transplantation 2003 12(5): 545-552). When transplanted into diabetic mice, hAECs normalize their blood sugar levels and their weights returned to normal. This shows that hAECs might represent a major breakthrough in the management of diabetes.
Clearly these cells, which come from a tissue that is normally thrown out after birth, are brimming with possibilities for regenerative medicine. Hopefully more research will produce even more possibilities.
Patients that suffer from an inherited condition known arrhythmogenic right ventricular dysplasia/cardiomyopathy or ARVD/C usually have no idea that they had a heart problem until they are in their 20s. The lack of symptoms at a younger age makes it virtually impossible for researchers to study this condition or to know how it develops. Fortunately, by making induced pluripotent stem cells (iPSCs) from patients with ARVD/C, researchers now have way to solve this very problem.
Small skin biopsies of ARVD/C patients can yield enough cellular material to make iPSCs. These iPSCs can be differentiated into heart muscle cells that are immature. These may not be terribly useful, since the goal is to model a disease that manifest itself during adulthood.
Researchers at Sanford-Burnham Medical Research Institute and Johns Hopkins University have created the first maturation-based “disease in a dish” model for ARVD/C. They created this model system by using iPSCs technology and employing a new method to mimic maturity that makes the metabolism of the hearts muscle cells more like those adult hearts. Thus, this model is likely more relevant to human ARVD/C than other models and therefore better suited for studying the disease and testing new treatments.
Huei-Sheng Vincent Chen, associate professor at Sanford-Burnham and the senior author of this study said, “It’s tough to demonstrate that a disease-in-a-dish model is clinically relevant for an adult-onset disease. But we made a key finding here – we can recapitulate the defects in this disease only when we induce adult-like metabolism. This is an important breakthrough considering that ARVD/C symptoms usually don’t arise until young adulthood. Yet the stem cells we’re working with are embryonic in nature.”
Daniel Judge, associate professor of medicine at Johns Hopkins University School of Medicine, said, “There is currently no treatment to prevent progression of ARVD/C, as rare disorder that preferentially affects athletes. With this new model, we hope we are not on a path to develop better therapies for this life-threatening disease.”
To make this model system, Cheng and his collaborators took skin samples from ARVD/C patients and converted various skin-based cells into iPSCs. After iPSC lines had been established, they differentiated them into heart muscle cells that had the characteristics of embryonic heart muscle cells. Unfortunately, these heart muscle cells showed no signs of ARVD/C, even when grown for over a year in culture.
What was the key? Metabolism. The young heart muscle cells primarily burn sugar for energy, but more mature heart muscle cells burn fat. Therefore, Chen’s group used a cocktail of molecules to get the heart muscle cells to preferentially burn fat.
As it turns out, metabolic malfunction is at the heart of ARVD/C. Chen and his group managed to track down the one piece that would get heart muscle cells made from patient-specific iPSCs to behave like sick ARVD/C heart muscle cells. The answer was the over-stimulation of a protein called PPAR-gamma.
PPAR-gamma plays an absolutely central role in type II diabetes. It regulates fatty acid storage and sugar metabolism. When PPAR-gamma activates genes, those genes stimulate lipid uptake and the production of fat in fat cells. If mice are made that do not have functional versions of PPAR-gamma, these mice fail to make fat, even when fed a high-fat diet.
PPAR stands for “peroxisome proliferator-activated receptor,” which is a subfamily of nuclear receptors. PPAR proteins bind DNA in combination with retinoid X receptors (RXRs), and these two proteins pair up to regulate transcription of various genes. There are three subtypes of PPARs: PPAR-alpha, PPAR-delta, and PPAR-gamma.
The fact that PPAR-gamma plays such a central role in the pathology of ARVD/C suggests a link between those mechanisms that cause type II diabetes and ARVD/C. According to Chen and Judge, ARVD/C heart muscle cells undergo exaggerated fat production, which leads to cell death. Because PPAR-gamma is a target for a group of drugs known as “glitazones,” perhaps these drugs can play a role in treating ARVD/C.
Acute lung injury and acute respiratory distress syndrome remain major causes of death and suffering despite advances in management of these conditions. The incidence of these conditions is expected to double in the next 25 years, and treatment for it is largely supportive.
Fortunately, mesenchymal stem cells (MSCs) from bone marrow have been used in experimental models to treat lung injury in rodents. MSCs can engraft into lung tissue and become lung tissue (or at least turn into cells that sure look a whole lot like lung tissue). MSCs can also suppress the types of immune responses that tend to really chew up lung tissue. Thus, MSC administration seems to improve the condition of lungs that have been attacked by infections or damaging agents.
However, the rates at which MSCs engraft into lung tissue is rather low; too low, in fact, to account for the benefit provided by MSCs. Therefore, MSCs appear to help repair lung tissue by means of “paracrine” mechanisms. This 50-cent word simply means that MSCs repair the lung by secreting molecules that promote lung healing.
To test this hypothesis, researchers in the laboratory of Bernard Thérband from the Ottawa Hospital Research Institute in Ottawa, Canada has grown MSCs in culture, and used the growth medium after the MSCs had been removed from it to treat mice that suffered from lung injuries.
To induce lung injury, mice were treated with isolated bits of bacterial cells that are known to promote acute lung injury. Then a group of these lung-injured mice were treated with conditioned medium from bone marrow MSCs that had been grown in culture dishes.
The MSC-conditioned medium decreased lung inflammation, and disruptions of the blood vessels in the lung normally observed during lung injury. Therefore, the lungs did not fill up with liquid and pus. However, the conditioned medium did not prevent the weight loss associated with lung injury. The overall tissue architecture of the lung tissue was much more normal in the mice treated with the conditioned medium from MSCs than in the untreated mice. Conditioned medium from other cultured cells had no such sanative effect.
MSC conditioned culture media also modified the activity of white blood cells in the lung. Instead of charging forward into lung tissue and damaging it in response to damage, the white blood cells (so-called “alveolar macrophages”) worked with the lung tissue to help heal it.
Finally, when Thébaud and his colleagues examined the molecules secreted into the medium by the MSCs, they discovered that the culture medium was filled with lots of interesting molecules, but one in particular caught their eye: Insulin-like growth factor-1 (IGF-1). This molecule has all kinds of healing properties, and it seemed to Thébaud and company that IGF-1 could be responsible for a good portion of the healing. Therefore, they infused the lung-injured mice with purified IGF-1, and, wouldn’t you know, the lungs showed rather robust healing after being damaged with bacterial bits.
Thus MSCs provide lung healing properties and they do so by means of the molecules they secrete. Many of these healing properties can be recapitulated by infusing IGF-1.
Such experiments provide hope that future clinical trials for such treatments are not far off.
Researchers from Harvard’s Department of Stem Cell and Regenerative Biology have succeeded in reprogramming one type of neuron into a different type of neurons in a living animals. Such an experiment has never been done before. These researchers, Paola Arlotta and Caroline Rouaux said that their work “tells you that maybe the brain is not as immutable as we always thought, because at least during an early window of time one can reprogram the identity of one neuronal class into another” Arlotta, an associate professor in Harvard’s Department of Stem Cell and Regenerative Biology (SCRB).
Direct lineage reprogramming of differentiated cells within the body was first proven by the SCRB co-chair and Harvard Stem Cell Institute (HSCI) co-director Doug Melton and colleagues five years ago. Workers in Melton’s lab succeeded in reprogramming exocrine pancreatic cells directly into insulin-producing beta cells. Now Arlotta and Rouaux now have shown that neurons can change too. Their work has been published in the journal Nature Cell Biology,
In their experiments, Arlotta and Rouaux targeted a group of neurons known as callosal projection neurons. Collosal projection neurons connect the two hemispheres of the brain. After specific treatments, the collosal projections neurons in this study were converted into corticofugal projection neurons. The significance of corticofugal projection neurons are not lost on Arlotta and Rouaux because they are a type of corticospinal motor neuron, which is one of two populations of neurons destroyed in Amyotrophic lateral sclerosis (ALS), or Lou Gehrig’s disease.
To achieve such reprogramming of neuronal identity, the researchers inserted a gene for a transcription factor known as Fezf2 into the collosal neurons. Fexf2 plays a central role in the development of corticospinal neurons in the embryo. The collosal neurons retracted their connects to the other hemisphere and made connections with neurons in the lower layers of the cerebral cortex.
Luci Bruijn, a neuroscientist who was not directly involved in this work noted, “This discovery tells us again that the brain is a somehow flexible system and gives us more evidence that reprogramming neurons to take on new identities and, perhaps, that new functions are possible. For those working to treat neurodegenerative diseases, that is reassuring.”
This work did not take take place in a culture dish in a laboratory. Instead it was done in the brains of living mice. The mice were young, so it is still not certain if such reprogramming could occur in older animals or even humans. If such reprogramming is possible, the implications for the treatment of neurodegenerative diseases could be enormous.
“Neurodegenerative diseases typically affect a specific population of neurons, leaving many others untouched. For example, in ALS it is corticospinal motor neurons in the brain and motor neurons in the spinal cord, among the many neurons of the nervous system, that selectively die,” Arlotta said. “What if one could take neurons that are spared in a given disease and turn them directly into the neurons that die off? In ALS, if you could generate even a small percentage of corticospinal motor neurons, it would likely be sufficient to recover basic functioning.”
Bruijn said of this work, “Understanding the constraints and possibilities of nervous system development allows us to consider new experiments and new strategies for therapy development. The most immediate importance of this finding is likely to be in the laboratory, where it will help us understand more about how the nervous system may respond when neurons are injured as they are in ALS.”
Benjamin Dekel, the head of the Pediatric Stem Cell Research Institute in Tel Aviv, Israel, and his team have isolated cancer stem cells from tumors found in the kidneys of some children. Wilms’ tumor is an inherited for of cancer that is found in the kidneys of particular children at a young age. Fortunately, these tumors are easily removed, but these children are at risk for other cancers throughout their lives.
Accord to Professor Dekel, “In earlier studies, cancer stem cells were isolation from adult cancers of the breast, pancreas, and brain, but so far much less is known about stem cells in pediatric cancers.” Professor Dekel continued, “Cancer stem cells contain the complete genetic machinery necessary to start, sustain and propagate tumor growth and they are often referred to as cancer initiating cells. As such, they not only represent a useful system to study cancer development but they also serve as a way to study new drug targets and potential treatments designed to stop the growth and spread of different types of cancer. We have demonstrated for the first time the presence of cancer stem cells in a type of tumor that is often found in children.”
Wilm’s tumors represent the most frequent type of kidney tumor found in children, and while children do usually respond well if the tumors are removed early surgically and if the patients are given chemotherapy, recurrences are possible and they can spread to other tissues.
Conventional chemotherapy is toxic to all cells in the body and if given to children may lead to the development of secondary cancers when they become adults. Thus, scientists would like to target tumor cells in as specific a manner as possible.
Researchers were able to remove parts of the tumors of cancer patients and graft them into mice. This procedure allowed researchers to test for the presence of cancer stem cells, since only the cancer stem cells could propagate the tumor from one animal to another. In the case of Wilms’ tumor, it was clear that cancer stem cells were present and could even be isolated from the rest of the tumor cells.
An international research team led by researchers at the University of California, San Diego School of Medicine has identified an enzyme that plays a key role in cancer stem cell reprogramming in a blood-based cancer known as chronic myeloid leukemia (CML).
CML treatment has received a tremendous boost by the discovery and development of chemotherapuetic agents known as tyrosine kinase inhibitors. Tyrosine kinase inhibitors attack a very specific group of signaling molecules that go awry in CML, and because of their high degree of specificity, these drugs are well tolerated and rather effective.
Tyrosine kinase inhibitors or TKIs block receptors called receptor tyrosine kinases that bind growth factors. Such receptors include molecules like Epidermal Growth Factor Receptor, which binds the growth factor EGF (Epidermal Growth Factor), Plate-Derived Growth Factor Receptor, which binds Platelet-Derived Growth Factor, and several others. Receptor tyrosine kinases are proteins that are embedded in the membrane of cells and when they are engaged by a specific growth factor, they pair up with another molecule and bind the growth factor tightly. Because this binding of their growth factor targets pairs two receptor tyrosine kinases together, the portion of the receptor protein that sticks toward the inside of the cell is activated. This internal portion of the receptor has “kinase” activity. Kinases are enzymes that stick phosphate groups on other molecules. Kinases place their phosphate groups on very specific targets. In the case of receptor tyrosine kinases, the target is the amino acid tyrosine. It just so happens that the internal piece of receptor tyrosine kinases contains several tyrosine residues and the paired receptors molecules tag each other with several phosphate groups on their tyrosines.
Phosphotyrosine acts as a signal to the inside of the cell, because specific protein contain pieces that can bind to phosphotyrosine. These phosphotyrosine-binding proteins (SH2-domain proteins for those who care about such things) drag powerful signaling molecules to the cell membrane. These signaling molecules are activated and the cell undergoes changes that cause it to move, growth, divide, or do other types of things.
In the case of blood cells, activation of particular receptor tyrosine kinases induces cells to grow and divide. Because there are exquisite controls on the signals set in motion by tyrosine, these signals cells divide and then stop. However, if the genes that encode these receptor tyrosine kinases undergo mutations that allow the receptors to pair up without binding growth factors, then the receptors will activate themselves at will without being dependent on the availability of growth factors. Cell will grow uncontrollably and fill up the bone marrow and blood.
At this point, we can see how TKIs work. These small molecules bind to the kinase part of receptor tyrosine kinases and gum them up. Because cells do not receive the signal to grow, they stop growing uncontrollably and this send the cancer into remission. TKIs include such famous drugs as Gleevec (imatinib), which was one of the first TKIs and has provent very successful against CML. However, after long periods of time on Gleevec, tumor cells can become resistant to it, and the physician must change drugs. Other TKIs include gefitinib (Iressa), and erlotinib (Tarceva), which inhibit Epidermal Growth Factor Receptor, Lapatinib (Tykerb), which is a dual inhibitor of EGFR and a subclass called Human EGFR type 2, and Sunitinib (Sutent) which is multi-targeted drug that inhibits Platelet-Derived Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor.
There are also other most specialized TKIs such as sorafenib (Nexavar), which targets a complex pathway that leads to a kinase signaling cascade, and nilotinib (Tasinga) which inhibits the fusion protein bcr-abl and is typically prescribed when a patient has shown resistance to imatinib (Gleevex).
Well, with all these new drugs, what’s the problem? The problem is that blood cancers, leukemias, can find ways around these treatments. Therefore, we must learn more these cancers in order to improve treatment of them. Leukemias are definitely tumors that emerge from cancer stem cells. Therefore, if you kill the cancer stem cells, you kill the tumor.
Principle investigator of this research, Catriona Jamieson, associate professor of medicine at UC San Diego, in collaboration with colleagues from Canada and Italy reported that inflammation, a phenomenon long associated with the development of cancer, increases the activity of the enzyme ADAR1 or adenosine deamiinase 1.
ADAR1 is expressed during embryonic development and it is essential in blood cell development. After embryonic development, ADAR1 switches off, but is reactivated by viral infections. Its role during viral infections is to protect blood cell-making stem cells from viral attacks. In leukemia stem cells, however, ADAR1 enhances the abnormal processing of RNA molecules. This causes enhanced cell renewal and resistance of malignant stem cells to chemotherapy.
Jamieson has already studied the link between cancer stem cell instability and abnormal RNA processing. She said, “People normally think about DNA instability in cancer, but in this case, it’s how the RNA is edited by enzymes that really matters in terms of cancer stem cell generation and resistance to conventional therapy.”
Because this RNA processing process is basic to cells and occurs in closely related organisms as well, studying it should be possible in model systems. It also represents a novel target for new therapies. According to Jamieson, inflammation is “an essential driver of cancer relapse and therapeutic resistance.”
Jamieson continued, “ADAR1 is an enzyme that we may be able to specifically target with a small molecule inhibitor, an approach we have already used effectively with other inhibitors. If we can block the capacity of leukemia stem cells to use ADAR1, if we can knock down that pathway, maybe we can put stem cells back on the right track and stop malignant cloning.”
The initiation of CML requires a mutation that fuses two genes together. One of these genes, BCR, fuses to a protein tyrosine kinase called ABL to generate the BCR-ABL gene that makes a fusion protein with uninhibited activity. The white blood cells that contain the BCR-ABL fusion protein expand slowly. The slowness with which this leukemia expands makes it difficult to diagnose early, and diagnosis is only possible once there are large numbers of precursors and malignant cells throughout the bloodstream and bone marrow. The median age at which CML is diagnosed is 66, and despite the advances in chemotherapeutic treatments, the vast majority of patients relapse if therapy is discontinued, since the cancer stem cells are dormant and resistant to treatment. If ADAR1 is addressed as a target, then perhaps treatments that target ADAR1 will overcome cancer stem cell resistance and prevent relapse.
In the United States alone, there are an estimated 70,00 people with CML and as the population ages, the prevalence of this disease is projected to jump to approximately 181,000 by 2050.
See “ADAR1 promotes malignant progenitor reprogramming in chronic myeloid leukemia.” Qingfei Jianga et al., PNAS DOI: 10.1073/pnas.2123021110.