Stem Cell Factor Delivery into Heart Muscle After Heart Attack May Enhance Cardiac Repair and Reverse Injury

Stem Cell Factor or SCF is a small peptide that circulates throughout the bloodstream and eventually finds its way to the bone marrow where it summons bone marrow-based stem cells to the sight of injury for tissue repair purposes. Unfortunately, it takes injured tissues time to express SCF at high enough levels to recruit bone marrow stem cells to come and accelerate tissue healing. This is particularly the case in the heart after a heart attack. For this reason, scientists are trying to find new and better ways to increase SCF production in the damaged heart.

To that end, cardiologists at the Icahn School of Medicine at Mount Sinai have discovered that delivering SCF directly to damaged heart muscle after a heart attack seems to augment heart muscle repair and regenerate injured tissue.

“Our discoveries offer insight into the power of stem cells to regenerate damaged muscle after a heart attack,” said lead study author Kenneth Fish, Director of the Cardiology Laboratory for Translational Research, Cardiovascular Research Center, Mount Sinai Heart, Icahn School of Medicine at Mount Sinai.

In this study, Fish and his colleagues used gene transfer to administer SCF to the heart shortly after inducing heart attacks in a pig model system in order to test its regenerative repair response. Fish and his coworkers developed a novel SCF gene transfer delivery system that stimulated the recruitment and expansion of adult cardiac stem cells directly to injury sites that reversed heart attack damage. In addition, the gene therapy improved cardiac function, decreased the death of heart muscle cells, increased regeneration of heart tissue blood vessels, and reduced the formation of heart tissue scarring.

“It is clear that the expression of the stem cell factor gene results in the generation of specific signals to neighboring cells in the damaged heart resulting in improved outcomes at the molecular, cellular, and organ level,” says Roger J. Haijar, senior study author and Director of the Cardiovascular Research Center at Mount Sinai. “Thus, while still in the early stages of investigation, there is evidence that recruiting this small group of stem cells to the heart could be the basis of novel therapies for halting the clinical deterioration in patients with advanced heart failure.”

The cell surface receptor for SCF is the c-Kit protein, and cells that possess the c-Kit protein are called c-Kit+ cells. c-Kit+ cells not only respond to SCF, but serve as resident cardiac stem cells that naturally increase in numbers after a heart attack and through cell proliferation are directly involved in cardiac repair.

To date, there is a great need for new interventional strategies for cardiomyopathy to prevent the progression of this disease to heart failure. Heart disease is the number one cause of death in the United States, with cardiomyopathy or an enlarged heart from heart attack or poor blood supply due to clogged arteries being the most common cause of the condition. Cardiomyopathy also causes a loss of heart muscle cells and changes in heart shape, which lead to the heart’s decreased pumping efficiency.

“Our study shows our SCF gene transfer strategy can mobilize a promising adult stem cell type to the damaged region of the heart to improve cardiac pumping function and reduce myocardial infarction sizes resulting in improved cardiac performance and potentially increase long-term survival and improve quality of life in patients at risk of progressing to heart failure,” says Dr. Fish.

“This study adds to the emerging evidence that a small population of adult stem cells can be recruited to the damaged areas of the heart and improve clinical outcomes,” says Dr. Hajjar.

Treating A Genetic Skin Disorder with Induced Pluripotent Stem Cells

Dystrophic epidermolysis bullosa (RDEB) is an inherited skin disease that causes fragile skin. RDEB is caused by mutations in the gene that encoded a protein called type VII collagen. Because collagen is a major structural component of skin, collagen mutations result in fragile skin and mucous membranes that blister easily if they are subjected to even slight mechanical stresses. There are no cures for such diseases, but skin creams and palliative care can decrease the severity of the symptoms.

Induced pluripotent stem cells (iPSCs) have the ability to treat such genetic diseases. In order to provide proof of principle of the applicability of iPSCs for the treatment of RDEB, Daniel Wenzel and his colleagues in the laboratory of Arabella Meixner from the Institute of Molecular Biotechnology of the Austrian Academy of Sciences in Vienna, Austria made iPSCs from mice that harbored mutations in the gene that encodes type VII collagen (Col7a1) and exhibited skin fragility and blistering. The symptoms displayed by these Col7a1-mutant mice resembled human RDEB.

Wenzel and his coworkers then genetically repaired the Col7a1 mutations in these iPSCs, and then differentiated these cells into functional fibroblasts that expressed and secreted normal type VII collagen. When implanted, the genetically-repaired iPSC–derived fibroblasts did not form tumors, and could be successfully traced up to 16 weeks after intradermal injection. Therapy with iPSC-derived fibroblasts also resulted in faithful and long-term restoration of type VII collagen deposition at the epidermal-dermal junction of Col7a1 mutant mice, and restored the resistance of the skin to mechanical stresses.

Thus, intradermal injection of genetically repaired iPSC-derived fibroblasts restored the mechanical resistance of the skin to blistering in RDEB mice. These data demonstrate that, at least in principle, RDEB skin can be effectively and safely repaired using a combination of gene therapy and iPSC-based cell therapy.

A similar study examined another type of epidermolysis bullosa.  Noriko Umegaki-Arao and her colleagues in the laboratory of Angela Christiano from Columbia University used iPSCs to treat mice with a distinct type of epidermolysis bullosa that resulted from mutations in COL17A1 gene, which encodes type XVII collagen (Col17).  In this case, however, the mutation has been observed to revert or fix itself in patients.  Patients tend to have patches of skin that are normal in a sea of abnormal skin.

Therefore, Umegaki-Arao and her coworkers derived iPSCs from Col17-mutant mice, differentiated them into skin cells (keratinocytes) and then cultured them, examining individual clones for reversion to normal Col17, which was fairly easy to do as it turns out.  Once revertant-iPSC keratinocytes were properly secured, and then used them to reconstitute human skin in mutant mice.  Thus, revertant keratinocytes can be a viable source of spontaneously gene-corrected cells for developing iPSC-based therapeutic approaches in the treatment of epidermolysis bullosa.

Young Blood Vessels Rejuvenate Aged Insulin-Producing Beta Cells

Professor Per-Olof Berggren Rolf Luft at the Research Center for Diabetes and Endocrinology at Karolinska Institutet has led an important study in collaboration with Alejandro Caicedo at University of Miami Miller School of Medicine and Hong Gil Nam at DGIST from the Republic of Korea that demonstrates that young capillary vessels can rejuvenate aged pancreatic islets. This study was published in the Proceedings of the National Academy of Sciences, USA and challenges prevailing views on the causes of age-dependent impaired glucose balance regulation, which often, in older patients, develops into type 2 diabetes mellitus.  These results suggest that treating inflammation and fibrosis in the small blood vessels of the pancreatic islets might provide a new way to treat age-dependent dysregulation of blood glucose levels.

“This is an unexpected but highly important finding, which we expect will have a significant impact on diabetes research in the future. The results indicate that beta cell function does not decline with age, and instead suggest that islet function is threatened by an age-dependent impairment of vessels that support them with oxygen and nutrients”, says Berggren.

Pancreatic beta cells are in the pancreatic islets and secrete the hormone insulin, which regulates blood glucose levels and also is one of the most important anabolic hormones of the human body.  Ageing may lead to a progressive decline in glucose regulation which may contribute to the onset of diabetes.  Generally, it has been assumed that this is due to reduced capacity of the beta cell to secrete insulin or multiply.

“In the study we challenged the view that the age-dependent impairment in glucose homeostasis is solely due to intrinsic, dysfunction of islet cells, and hypothesized that it is instead affected by systemic aging factors”, says first author Joana Almaça at the Diabetes Research Institute, University of Miami.

Even though the common wisdom in modern medicine is that insulin-producing pancreatic beta cell lose function through the constant demands on them,  Berggren and his collaborators showed that mouse and human beta cells are fully functional at advanced age.  When they replaced the islet vasculature in aged islet grafts with young capillaries, the investigators found that the islets were rejuvenated and glucose homeostasis fully restored.

“While expanding beta cell mass may still be desirable for future diabetes therapy, improving the local environment of the otherwise healthy aged beta cell could prevent age-associated deterioration in glucose homeostasis and thereby promote healthy ageing, which is conceptually novel and highly exciting”, says Per-Olof Berggren.

Mesenchymal Stem Cells Make Blood Vessel Cells and Improve Wound Healing

Mesenchymal stem cells from umbilical cord have the ability to differentiate into cartilage cells, fat cells, bone cells, and blood vessels cells. These cells also are poorly recognized by the immune system of the patient and are at a low risk of being rejected by the patient’s immune system.

Valeria Aguilera and her colleagues from the laboratory of Claudio AguayoWe at the University of Concepción, Chilee have evaluated the use of mesenchymal stem cells from umbilical cord in the formation of new blood vessels in damaged tissues. Wharton’s jelly mesenchymal stem cells of hWMSCs were used to potentially accelerate tissue repair in living animals.

Aguilera and her co-workers began by isolating mesenchymal stem cells from human Wharton’s jelly (a connective tissue in umbilical cord). Then they grew these cells in culture for 14 or 30 days. Interestingly, the longer the WMSCs grew in culture, the more they looked like blood vessel cells. They began to express blood vessel-specific genes and proteins. WMSCs cultured for 30 days were even more like blood vessels than those grown in culture for 14 days.

When these cells were injected in the mice with damaged skin, the results showed that the WMSCs cultured for 30 days significantly accelerated wound healing compared with animals injected with either undifferentiated hWMSCs or with no cells.

Effect of hWMSCs and endothelial-differentiated hWMSC transplantation in a wound-healing model. A) Representative images of wounds at day 1 (top panels) and 12 (lower panels) after injury and subcutaneous injection of hWMSCs, hWMSC trans-differentiated into endothelial cells for 14 days (hWMSC-End14d) or 30 days (hWMSC-End30d), or control (PBS). B) Wound healing quantified in PBS (○), hWMSC (•), hWMSC-End14d (□) or hWMSC-End30d (▪) treated mice (n = 5 independent experiments, in duplicate). Values are expressed as mean±S.E.M, +P<0.05 in hWMSC-End30d v/s hWMSC, hWMSC-End14d, at the corresponding time; **P<0.03 in hWMSC-End30d v/s PBS; *P<0.001 in hWMSC-End30d v/s PBS; # P<0.01 in hWMSC-End30d v/s PBS.
Effect of hWMSCs and endothelial-differentiated hWMSC transplantation in a wound-healing model.
A) Representative images of wounds at day 1 (top panels) and 12 (lower panels) after injury and subcutaneous injection of hWMSCs, hWMSC trans-differentiated into endothelial cells for 14 days (hWMSC-End14d) or 30 days (hWMSC-End30d), or control (PBS). B) Wound healing quantified in PBS (○), hWMSC (•), hWMSC-End14d (□) or hWMSC-End30d (▪) treated mice (n = 5 independent experiments, in duplicate). Values are expressed as mean±S.E.M, +P



The wounds of mice treated with the WMSCs cultured for 30 days looked healthier, but they had many more blood vessels.

Histologic analysis of wounds in the wound-healing model. A) Representative photographs of wounds (hematoxilin/eosin staining) 12 days after injury and subcutaneous injection of PBS, hWMSCs, hWMSC-End14d or hWMSC-End30d. Quantification of histological images, for blood vessels area (B) and histological score (C) for each group of mice. Values are mean ± S.E.M (n = 5 independent experiments, in duplicate), *P<0.001 in hWMSC-End30d or hWMSC-End14d v/s MSC; +P<0.05 in hWMSC-End30d or hWMSC-End14d v/s hWMSC. Magnification x40 (-). Ep, epidermis; D, dermis; H, hypodermis.
Histologic analysis of wounds in the wound-healing model.
A) Representative photographs of wounds (hematoxilin/eosin staining) 12 days after injury and subcutaneous injection of PBS, hWMSCs, hWMSC-End14d or hWMSC-End30d. Quantification of histological images, for blood vessels area (B) and histological score (C) for each group of mice. Values are mean ± S.E.M (n = 5 independent experiments, in duplicate), *P

When laboratory animals received the culture medium from the WMSCs cultured for 30-days also showed significant acceleration of their healing, which suggests that these cells secrete a host of healing molecules that induced the formation of new blood vessels.  One might also conclude that the implanted WMSCs did not contribute to the formation of new blood vessels, but simply directed the formation of new blood vessels by secreting healing molecules.  However, when WMSCs were detected in the healed tissue, they were predominantly found in the walls of new blood vessels.

Immunohistochemical detection of human mesenchymal cells in a wound-healing model. A. Immunohistochemical staining of human mitochondria was performed in permeabilized tissue sections obtained after 12 days of subcutaneous injection of PBS, hWMSCs, hWMSC-End14d or hWMSC-End30d in mice. Cell nuclei were stained with hematoxyline. In B. Number of positive cells per vessel. Representative images of 5 independent experiments, in duplicate. Magnification x40 and insert 100x. Bars 50 µm.
Immunohistochemical detection of human mesenchymal cells in a wound-healing model.
A. Immunohistochemical staining of human mitochondria was performed in permeabilized tissue sections obtained after 12 days of subcutaneous injection of PBS, hWMSCs, hWMSC-End14d or hWMSC-End30d in mice. Cell nuclei were stained with hematoxyline. In B. Number of positive cells per vessel. Representative images of 5 independent experiments, in duplicate. Magnification x40 and insert 100x. Bars 50 µm.

These results, which were published in PLOS ONE, demonstrate that mesenchymal stem cells isolated from umbilical cord connective tissue or Wharton’s jelly can be successfully grown in culture in the laboratory and trans-differentiated into blood vessels-forming cells (endothelial cells).  These differentiated hWMSC-derived endothelial cells seem to promote the formation of new networks of blood vessels, which augments tissue repair in laboratory animals through the secretion of soluble pro-blood vessel-making molecules and, occasionally, by contributing to the formation of new vessels, themselves.

Cells Made From Embryonic Stem Cells Derived from Cloned Embryos Are Rejected by the Immune System

Researchers from Stanford University have shown that genetic differences in mitochondria found in cells made from pluripotent stem cells that were originally derived from cloned embryos can prompt rejection by the immune system of the host animal from which they were made, at least in mice.

According to a study in mice by researchers at the Stanford University School of Medicine and colleagues in Germany, England and at MIT, cells and tissues in mice made from cloned embryos are rejected by the body because of a previously unknown immune response to the cell’s mitochondria. These findings reveal a likely hurdle if such therapies are ever used in humans.

Regenerative therapies that utilize stem cells have the potential to repair organs, replace dead or dying tissues, and treat severe diseases.  Many stem cell scientists think that pluripotent stem cells, which can differentiate into any kind of cell in the body, show the most promise for regenerative medical applications in the clinic.  One method for deriving pluripotent stem cells that have the same genetic composition as that of the patients is called somatic cell nuclear transfer (SCNT) or cloning.  This method takes the nucleus of an adult cell and injects it into an egg cell from which the nucleus has been removed.

SCNT can potentially make pluripotent stem cells that can repair a patient’s body. “One attraction of SCNT has always been that the genetic identity of the new pluripotent cell would be the same as the patient’s, since the transplanted nucleus carries the patient’s DNA,” said cardiothoracic surgeon Sonja Schrepfer, MD, PhD, who was the co-senior author of the study, which was published online Nov. 20 in Cell Stem Cell.

“The hope has been that this would eliminate the problem of the patient’s immune system attacking the pluripotent cells as foreign tissue, which is a problem with most organs and tissues when they are transplanted from one patient to another,” added Schrepfer, a visiting scholar at Stanford’s Cardiovascular Institute, and Heisenberg Professor of the German Research Foundation at the University Heart Center in Hamburg, and at the German Center for Cardiovascular Research.

Several years ago, Stanford University professor of pathology and developmental biology, Irving Weissman, MD, chaired a National Academies panel on SCNT cells.  At this time, he raised the possibility that the immune system of a patient who received the cells derived from stem cells made from cloned embryos might still generate an immune response against proteins from the cells’ mitochondria.  Mitochondria are the energy factories for cells, and they have their own genetic system (a DNA chromosome, protein-making structures called ribosomes, and enzymes for expressing and replicating DNA).  This reaction could occur because cells created through SCNT contain mitochondria from the egg donor and not from the patient, and therefore could still appear as foreign tissue to the recipient’s immune system.

There were other indications that Weisman was probably correct.  An experiment that was published in 2002 by William Rideout in the laboratory of Rudolf Jaenisch at the Whitehead Institute for Biological Research in the journal Cell derived embryonic stem cells from cloned mouse embryos and then differentiated those embryonic stem cells into bone marrow-based blood making stem cells. These blood making stem cells were then used to reconstitute the bone marrow of a mouse that had a mutation that prevented their bone marrow from forming normal types of disease-fighting white blood cells. However, even though the recipient mouse was genetically identical to the embryonic stem cells that had been used to derived the blood-making stem cells, the immune systems of the recipient mouse still rejected the implanted cells after a time.  Weissman, however, was not able to directly test this claim himself at that time.  Weissman directs the Stanford Institute for Stem Cell Biology and Regenerative Medicine, and now, in collaboration with Schrepfer and her colleagues, he was able to test this hypothesis.

“There was a thought that because the mitochondria were on the inside of the cell, they would not be exposed to the host’s immune system,” Schrepfer said. “We found out that this was not the case.”

Schrepfer, who heads the Transplant and Stem Cell Immunobiology Laboratory in Hamburg, used cells that were created by transferring the nuclei of adult mouse cells into enucleated eggs cells from genetically different mice. When transplanted back into the nucleus donor strain, the cells were rejected although there were only two single nucleotide substitutions in the mitochondrial DNA of these SCNT-derived cells compared to that of the nucleus donor. “We were surprised to find that just two small differences in the mitochondrial DNA was enough to cause an immune reaction,” she said.

“We didn’t do the experiment in humans, but we assume the same sort of reaction could occur,” Schrepfer added.

Until recently, researchers were able to perform SCNT in many species, but not in humans.  However, scientists at the Oregon Health and Science University announced the successful derivation of human embryonic  stem cells from cloned, human embryos.  This reignited interest in eventually using SCNT for human therapies. Although many stem cell researchers are focused on a different method of creating pluripotent stem cells, called induced pluripotent stem cells, some believe that there are some applications for which SCNT-derived pluripotent cells are better suited.

The immunological reactions reported in the new paper will be a consideration if clinicians ever use SCNT-derived stem cells in human therapy, but Weissman thinks that such reactions should not prevent their use.  “This research informs us of the margin of safety that would be required if, in the distant future, we need to use SCNT to create pluripotent cells to produce the tissue stem cells to treat someone,” he said. “In that case, clinicians would likely be able to handle the immunological reaction using the immunosuppression methods that are currently available.”  I find such a statement somewhat cavalier given that the nature of the immunological rejection might be robust enough to endanger the patient regardless of the anti-rejection drugs that are used.

In the future, scientists might also lessen the immune reaction by using eggs from someone who is genetically similar to the recipient, such as a mother or sister, Schrepfer added.  Except that now you have added the dangers of egg retrieval to this treatment regimen, which not only greatly jacks up the price of this type of treatment, but now endangers another person just to treat this one patient.  Add to that the fact that you are making a cloned human embryo (a very young person) for the sole purpose of dismembering it, and now you have added a degree of barbarism to this treatment as well.

So if we some SCNT-based treatments for patients we have an added danger for the patient (immunological rejection), danger for the egg donor, the homicide of the young embryo, and a prohibitively expensive procedure that no insurance company in their right mind would fund. I say we abandon this mode of treatment for the morally-bankrupt option that it is and pursue more ethical ways of treating patients.

A Faster Way to Make Blood Vessels

Suchitra Sumitran-Holgersson and Michael Olausson from the Sahlgrenska Academy have designed a new way to make blood vessels that takes only seven days and a few tablespoons of blood.

Thanks to their new procedure, the ability to make new tissues from stem cells has taken a huge stride forward. Three years ago, a patient at Sahlgrenska University Hospital received a blood vessel transplant grown from her own stem cells. Suchitra Sumitran-Holgersson, Professor of Transplantation Biology at Sahlgrenska Academy, and Michael Olausson, Surgeon/Medical Director of the Transplant Center and Professor at Sahlgrenska Academy, came up with the idea, planned and carried out the procedure.

Now Sumitran-Holgersson and Olausson have published a new study in the journal EBioMedicine based on two other transplants that were performed in 2012 at Sahlgrenska University Hospital. The patients in this procedure were two young children who were afflicted with the same condition as in the first patient. All three patients were missing the vein that goes from the gastrointestinal tract to the liver.

“Once again we used the stem cells of the patients to grow a new blood vessel that would permit the two organs to collaborate properly,” Professor Olausson says.  This time, however, Sumitran-Holgersson, found a way to extract stem cells that did not necessitate taking them from the bone marrow. “Drilling in the bone marrow is very painful,” she says. “It occurred to me that there must be a way to obtain the cells from the blood instead.”

The extreme youth of these patients motivated Sumitran-Holgersson find a new way to extract these stem cells. She came upon the extraction of stem cells from 25 millilitres (approximately 2 tablespoons) of blood, which is the minimum quantity of blood needed to obtain enough stem cells.

Then they used a novel technique to generate transplantable vascular grafts by using decellularized allogeneic vascular scaffolds that were then populated with peripheral whole blood and then grown in a bioreactor.  Circulating, VEGFR-2 +/CD45 + and a smaller fraction of VEGFR-2 +/CD14 + cells largely repopulated the graft to form new vessels for transplantation.

Fortunately, her idea worked out better than she could have ever expected, and worked perfectly the first time. “Not only that, but the blood itself accelerated growth of the new vein,” Professor Sumitran-Holgersson says. “The entire process took only a week, as opposed to a month in the first case. The blood contains substances that naturally promote growth.”

Olausson and Sumitran-Holgersson have treated three patients so far, two children and one adult. Two of the three patients have recovered well and have veins that are functioning normally. In the third case the child is under medical surveillance and the outcome is less certain.

The technology for creating new tissues from stem cells has taken a giant leap forward. Two tablespoons of blood are all that is needed to grow a brand new blood vessel in just seven days. This is shown in a new study from Sahlgrenska Acadedmy and Sahlgrenska University Hospital published in EBioMedicine.
The technology for creating new tissues from stem cells has taken a giant leap forward. Two tablespoons of blood are all that is needed to grow a brand new blood vessel in just seven days. This is shown in a new study from Sahlgrenska Acadedmy and Sahlgrenska University Hospital published in EBioMedicine.

These studies show that it is feasible to avoid taking painful blood marrow samples to extract stem cells for blood vessel production, and that it is equally feasible to produce those blood vessels in a matter of a week.

“We believe that this technological progress can lead to dissemination of the method for the benefit of additional groups of patients, such as those with varicose veins or myocardial infarction, who need new blood vessels,” Professor Holgersson says. “Our dream is to be able to grow complete organs as a way of overcoming the current shortage from donors.”

Does the Mother’s Diet Affect Her Offspring?

Can what a mother eats affect her baby? Claudia Buss of the Charité – Universitätsmedizin Berlin and the University of California, Irvine and her colleagues conducted a longitudinal study of mothers and their newborn babies, and discovered that increased production of the cytokine interleukin-6 (IL-6) in mothers can lead to alterations in the brain connectivity of her offspring.

Buss and coworkers took blood samples of pregnant women and measured levels of the cytokine IL-6 early in pregnancy, during the middle of the pregnancy, and near the end of their pregnancy. Shortly after the birth of their babies, Buss and others conducted MRI scans of the newborns. “This is the only way that we will be able to understand prenatal influences that are not confounded by post-natal influences,” Buss said at a November 17th press conference at the Society for Neuroscience (SfN) annual meeting in Washington, DC. In particular, Buss and her team looked for patterns of synchronized activity in the default mode network (DMN). The DMN is a network of brain regions that are active when the individual is not focused on the outside world and the brain is awake, but at rest. During goal-oriented activity, the DMN is deactivated and another network called the task-positive network (TPN) is activated. The DMN may correspond to task-independent introspection, or self-referential thought, while the TPN corresponds to action. Dysfunction of the DMN has been linked to psychiatric disorders.

The group found that the infant DMN “doesn’t look like adult network, but it’s emerging,” Buss said. “It’s there in an immature state.” More importantly, higher maternal gestational IL-6 concentration predicted reduced DFM connectivity. The infant brain was “less strongly connected under conditions of high maternal IL-6 concentrations,” Buss said.

In another study by neuroscientists at Duke University showed that the maternal diet of mice can cause inflammatory and behavior changes in offspring. Staci Bilbo of Duke University and her team found that a high-fat diet in the mother can lead to inflammation in the body’s fat tissue as well as immune changes in brain that may be linked to psychiatric disorders like anxiety and depression. The researchers fed mice either a low-fat diet or a high-fat diet, either enriched or not enriched for branched chain amino acids (BCAAs). Bilbo’s group examined the mothers’ brains midway through pregnancy and found increased expression of inflammatory cytokines in the hypothalamuses of mice fed a high-fat diet. These changes were also accompanied by postpartum increases in depressive-like behaviors in mice fed a BCAA-enriched diet and an increase in anxiety-like behaviors in mice fed a high-fat diet.

According to Bilbro, the offspring of these mothers showed “striking” differences in the expression of inflammatory cell types and in the behavior of the newborn pups. Infants born to moms fed a high-fat diet showed decreased expression of microglia markers and increased anxiety-like behaviors. However, mice born to moms on a high-fat, high-BCAA diet showed increased expression of a marker for astrocytes.

“Maternal diet does matter,” said Bilbo. “We believe [these changes] may be contributing to both metabolic changes as well as mood changes” in the moms and their offspring.

Society for Neuroscience Conference – More to Report

A very interesting poster at the SfN meeting described experiments with the antihypertensive medicine Telmisartan and its ability to protect brain cells from dying from an overdose of neurotransmitters.

During a stroke, dead or dying neurons tend to dump enormous quantities of neurotransmitters into their surrounding environment, and these excessive concentrations of neurotransmitters are deleterious for the surrounding neurons. This phenomenon is called “excitoxicity,” and it is an important killer of neurons in a stroke.

In this poster, a Chinese scientist used Telmisartan to pre-treat cultured neurons that were then given large quantities of the neurotransmitter glutamate. The drug protected the neurons from dying from the excessive concentrations of glutamate. Telmisartan also profected cells by binding to the AT[1A] receptor, and activating the PPAR[gamma] transcription factor. While these results may sound cryptic, PPAR[gamma] is a target for a group of anti-diabetic drugs called the triglitazones. By activating this transcription factor, telmisartan rescued these cultured neurons from certain death, and Dr. Wang (the poster presenter) suggested that Telmisartan could potentially be prescribed to delay the effects of stroke are even Alzheimer’s disease.

I also attended a series of short oral presentations at this meeting, and one symposium included modeling diseases with induced pluripotent stem cells. That was a fascinating symposium and I felt like a kid in a candy store. One Japanese researchers discussed his successes at using induced pluripotent stem cells (iPSCs) to make brain “organics.” These organoids contain multiple organ-specific cell types, recapitulate some function of the organ, and share at least some of the cellular organization of the organ. Brain organoids were made by deriving iPSCs from cells taken from human volunteers, which were ten grown in embryonic stem cell medium for one week to expand the cells. Then the cells were for about another week in Neural Induction Medium, and then shaken for four more weeks. The cells self-organized into minibrains that exhibited cortical organization with the layered structure of a brain that expressed many of the same genes as the layers of a developing brain. These minibrains also showed glutamate-induced calcium mobilization. Thus these minibrains qualify as a brain organoid.

Next, he used this same procedure to make minibrains from iPSCs derived from patients with fragile X syndrome, which, besides Down Syndrome, is the leading cause of mental retardation, globally speaking. Minibrains from these Fragile X Syndrome patients formed and looked normal. However, they showed abnormal connections between neurons. This tremendous model system can provide ways to study neurological diseases at very detailed levels.

The next talk was by Haruhisa Inoue from Kyoto University who examined the use of iPSCs as a way to treat neurological diseases. In particular, Dr. Inoue was interested in Amyotrophic Lateral Sclerosis or ALS. In the case of ALS, a cells called astrocytes are the problem. The astrocytes generate a foul environment that causes the neurons in the spinal cord to die off.

Dr. Inoue used iPSC technology to derive mature astrocytes from non-ALS and ALS patients. The two sets of astrocytes showed profound functional differences. When he transplanted normal astrocytes into the spinal cords of ALS mice, her also discovered that the mice showed rather significant functional improvements. Thus, Dr. Inoue thinks that transplantation of astrocytes made from iPSCs derived from the cells of healthy volunteers might provide an excellent way to delay or even reverse the effects of ALS.

Society for Neuroscience Conference 2014 Continued

Let me emphasize that the huge number of posters and talks at the SfN conference made it impossible to attend all of them, so my recollections here are some of the high points that I was able to take in. There is a lot of terrific science going on out there and these conferences are windows into it.

One poster described a feeding study in rats. One group of rats received a diet rich in omega-3 fatty acids, which are found in fish oils and soy. Another group was fed a standard laboratory diet that tends to skim on the omega-3 fatty acids. In the brains of the omega-3-fed rats, the expression off the gene that encodes Brain Derived Neurotropic Factor or BDNF increased significantly.

This is significant because BDNF promotes the survival of nerve cells (neurons) by playing a role in the growth, maturation (differentiation), and maintenance of these cells. In the brain, BDNF protein is active at the connections between nerve cells (synapses), where cell-to-cell communication occurs. The synapses can change and adapt over time in response to experience, a characteristic called synaptic plasticity, and BDNF regulates synaptic plasticity, which is important for learning and memory.

When these researchers examined why the BDNF gene was unregulated in rats fed the omega-3-rich diet, they discovered that the starting point of the gene, which is called the promoter was nice and clear. In the standard diet rats, the promoter of the BDNF gene was chemically modified with methyl (-CH3) groups. In the absence of the methyl groups, the transcription factor CTCF was able to bind and increase the rate of transcription. If the promoter was chemically modified with methyl groups, then a protein called MeCP2 bound to the promoter and prevented expression of BDNF.

This group looked further and discovered that the omega-3-rich diet seemed to influence the expression of BDNF by means of the balance of reduced and oxidized versions of electron carriers in cells, in particular, the ratio of NAD+ to NADH. NAD is a major electron carrier in cells and the ratio of NAD+, the oxidized version of this molecule, to the reduced version of this molecule, NADH, is a measure of the energy charge of the cell and how well-fed the individual is. More importantly, NAD is a substrate for another regulator of gene expression called Sirtuins.

Sirtuins are protein deacetylases, but they are unusual deacetylases since many of them they do not simply hydrolyze acetyl-lysine residues. Instead they couple lysine deacetylation to NAD hydrolysis. This hydrolysis produces O-acetyl-ADP-ribose, which is the deacetylated substrate and nicotinamide, which is an inhibitor of sirtuin activity. The dependence of sirtuins on NAD links their enzymatic activity directly to the energy status of the cell via the cellular NAD:NADH ratio.

The fact that a diet high in omega-3 fatty acids affects the NAD/NADH ratio is significant for Alzheimer’s disease because the sirtuin, SIRT1, deacetylates and coactivates the promoter for the gene that encodes the retinoic acid receptor beta gene, which subsequently upregulates the expression of alpha-secretase (ADAM10). Alpha-secretase is able to suppress beta-amyloid production. ADAM10 activation by SIRT1 also induces the Notch signaling pathway, which is known to repair neuronal damage in the brain. All of this begins with a dietary factor that actually protects the brain from Alzheimer’s disease by profound changes in gene expression.

Another poster from an Italian group used the 5XFAD mouse model of Alzheimer’s disease to test a growth factor called “painless Nerve Growth Factor” on mice with protein plaque formation in their brains. The growth factor was given by placing droplets of the growth factor in the noses of the mice while they were anesthetized. The results were stunning. Normally, 5XFAD mice get plaques quickly in their brains and lots of them. However, the growth factor was able to rescue the onset of behavioral deficits and reduces, although not eliminate, plaque formation. Other brain-specific pathologies found in these mice were reduced, such as astrocytosis. The wandering white cells in the brain known as microglia did a better job of gobbling up protein aggregates and clearing them from the brain, and the markers of inflammation were significantly reduced. I asked the investigator if there were plans to try to move this to clinical trials, and she said that she was unable to do so because of a lack of funding. Maybe someone will collaborate with this dear lady to make it so?

In another poster, the overexpression of an enzyme called heparanase in the brain decreased the burden of protein aggregates in the brains of mice with Alzheimer’s disease. I was not able to get into the details of this poster because of time.

In another poster, a very energetic young man told me about his very interesting work with a Parkinson’s disease model in rodents. If mice are administered a drug called MPTP (short for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), the dopamine-using neurons in the brain will specifically take up this drug in high concentrations and it will kill them. Therefore, this drug is an excellent model system to study Parkinson’s disease in mice.

Prokineticin-2 is a gene that is expressed in high quantities in the surviving dopamine-using neurons that came from the brains of Parkinson’s disease patients after their deaths. When Prokineticin-2 was overexpressed in cultured dopaminergic neurons, they unregulated a protein called Bcl-2. Bcl-2 is one of the group of proteins can protect cells from dying. Therefore, Prokineticin-2 is a prosurvival protein.

Next, this chap switched from a culture system to a “in a living animal” system or an in vivo system. By using genetically engineered viruses that overexpressed Prokineticin-2 in the brains of mice, he discovered that this viruses did not adversely affect the mice and he did in fact achieve high levels of Prokineticin-2 in the brains of mice with this recombinant viruses. The overexpression did not affect the mice in the least. When he did the same experiment with MPTP-treated mice – oh, just to be clear, he overexpressed Prokineticin-2 first and then administered the MPTP because it takes about 30 days for the viruses to properly upregulate Prokineticin-2 – he saw decreased inflammation in the brain, and increase in Bcl-2 and Pink1 expression in the brain (both of these genes are pro-survival genes), and the behavioral problems of the mice never emerged with the severity of the MPTP mice. When he examined TH – an enzyme that makes the neurotransmitter dopamine, he saw that levels of this enzyme were up too. This means that the dopamine-using neurons were surviving. Is this cool stuff or what?

That’s enough for now. More later.

Cardiac Stem Cells or their Exosomes Heal Heart Damage Caused by Duchenne Muscular Dystrophy

One of the research institutions that has been at the forefront of developing investigational stem cell treatments for heart attack patients is The Cedars-Sinai Heart Institute. Recently, a research team at Cedars-Sinai Heart Institute (CSHI) has injected cardiac stem cells into the hearts of laboratory mice afflicted with a rodent form of Duchenne muscular dystrophy. This disease can also adversely affect the heart, and these stem cell injections actually improved the heart function of these laboratory animals and resulted in greater survival rates for those mice. This work might provide the means to extend the lives and improve the quality of life of patients with this chronic muscle-wasting disease.

The CSHI team presented their results at the American Heart Association Scientific Sessions in Chicago. Their results clearly demonstrated that once laboratory mice with Duchenne muscular dystrophy were infused with cardiac stem cells, the animals showed progressive and significant improvements in heart function and increased exercise capacity.

Specifically, 78 lab mice that had been given laboratory-induced heart attacks were injected with their own cardiac stem cells, and over the next three months, these mice demonstrated improved pumping ability and exercise capacity in addition to a reduction in heart-specific inflammation. The CSHI team also discovered that the stem cells work indirectly, by secreting tiny vesicles called exosomes that are filled with molecules that induce tissue healing. When these exosomes were purified and administered alone, they reproduced the key benefits of the cardiac stem cells.

Apparently, this particular procedure could be ready for testing in human clinical studies as soon as next year.

Duchenne muscular dystrophy or DMD is a genetic disease that results from mutations in a gene found on the X chromosome in humans. DMD affects 1 in 3,600 boys and is a neuromuscular disease caused by abnormalities in a muscle protein called dystrophin.  Because dystrophin is an important structural protein for muscle that anchors muscle to other muscles and to the substratum, deficiencies for functional copies of the dystrophin protein cause progressive muscle wasting, destruction, and muscle weakness.

Dystrophin acts as an important link between the internal cytoskeleton and the extracellular matrix. Neuronal nitric oxide synthase (nNOS) binds to α-syntrophin but also has a binding site in repeat 17 of the rod domain of dystrophin (see Fig. 2A for details of dystrophin domains). αDG, α-dystroglycan; βDG, β-dystroglycan
Dystrophin acts as an important link between the internal cytoskeleton and the extracellular matrix. Neuronal nitric oxide synthase (nNOS) binds to α-syntrophin but also has a binding site in repeat 17 of the rod domain of dystrophin (see Fig. 2A for details of dystrophin domains). αDG, α-dystroglycan; βDG, β-dystroglycan.  See here

The majority of DMD patients lose their ability to walk by twelve years of age, although the severity of the disease varies from patient to patient. The average life expectancy is about 25, and the cause of death is usually heart failure. Dystrophin deficiency causes heart muscle weakness, and, ultimately, heart insufficiency, since the chronic weakness of the heart muscle prevents the heart from pumping enough blood to maintain a regular heart rhythm and provide for the needs of the rest of the body. Such a heart condition is called “cardiomyopathy.”

“Most research into treatments for Duchenne muscular dystrophy patients has focused on the skeletal muscle aspects of the disease, but more often than not, the cause of death has been the heart failure that affects Duchenne patients,” said Eduardo Marbán, MD, PhD, who is the director of the CSHI and the principal investigator of this particular study. “Currently, there is no treatment to address the loss of functional heart muscle in these patients.”

In 2009, Marbán and his team completed the world’s first procedure in which a patient’s own heart tissue was used to grow specialized heart stem cells. Stem cells from the heart were isolated, cultured, and then injected back into the patient’s heart in order to repair and regrow healthy heart muscle that had been injured by a heart attack. Results, Marbán and his colleagues published these results in The Lancet in 2012, and also demonstrated that one year after their patients had received the experimental stem cell treatment, they showed significant reductions in the size of the heart scar that had been produced by their heart attacks.

Earlier this year, CSHI researchers commenced a new clinical trial entitled “ALLSTAR,” which stands for Allogeneic Heart Stem Cells to Achieve Myocardial Regeneration (Clinical trial number NCT01458405). In this study, heart attack patients are given injections of stem cells from healthy donors, which should work better than the patient’s own stem cells, which were damaged by the heart attack.

CSHI has recently opened the nation’s first Regenerative Medicine Clinic, which is designed to match heart and vascular disease patients with the appropriate stem cell clinical trial being conducted at CSHI and other institutions.

“We are committed to thoroughly investigating whether stem cells could repair heart damage caused by Duchenne muscular dystrophy,” Marbán said.

The protocols for growing cardiac-derived stem cells were developed by Marbán when he was on the faculty of Johns Hopkins University. Johns Hopkins has filed for a patent on that intellectual property and has licensed it to Capricor, a company in which Cedars-Sinai and Marbán have a financial interest. Capricor is providing funds for the ALLSTAR clinical trial at Cedars-Sinai.

The Society for Neuroscience Meeting Continued

Glymphatics is a new subdiscipline in neuroscience that was essentially discovered by a Danish neuroscientist named Maiken Nedergaard. Dr. Nedergaard gave a fine seminar on this subject on Sunday.

Glymphatics consists of the system that removes waste products from the brain. Dr. Nedergaard showed movies that showed how the cerebrospinal fluid that bathes the periphery of the brain pulsates as it moves over the brain. When die molecules are injected into the cerebrospinal fluid, these dyes wend up in the blood system. How does this happen?

Nedergaard reasoned that diffusion of the fluid was far too slow for the dye to get to the blood system as fast as it does. Instead, she suspected that fluid moves by means of a “convection current.” How does this work? The blood vessels that feed the brain are surrounded by cells known as astrocytes. These astrocytes prevent molecules from entering the brain unless they can properly negotiate their way across these astrocytes, and this forms the basis for the blood-brain barrier. Cerebrospinal fluid moves across the cells of the brain and is removed by the astrocyte-surrounded vessels. This sink for the cerebrospinal fluid essentially pulls the cerebrospinal fluid across the brain cells and serves as the means by which the brain is cleansed of waste products.

This system, however, is subject to regulation, since the flow of fluid from the cerebrospinal fluid depends on the size of the spaces between brain cells. As it turns out, the spaces between brain cells in larger during sleep than when we are awake. Therefore, sleep seems to be the means by which our bodies clear the rubbish from our brains.

The molecule that controls the space between brain cells is norepinephrine. How it does that remains uncertain, but this is the molecule that is released during sleep to help clear out the garbage in the brain.

Since Alzheimer’s disease, Parkinson’s disease, other neurodegenerative diseases include the accumulation of protein aggregates in the brain, the removal of waste products in the brain would seem to be a rather important process. Also, when there is a head injury, surgeons sometimes leave the skull cap open while the brain heals. This, however, hamstrings the glymphatic system and surgeons should replace the skull cap so that the glymphatic system can do its job. Secondly, if norepinephrine can regulate this system, then this might be a way to increase clearance of waste products from the brain to reduce or delay the accumulation of protein aggregates in the brain.

Remarkable isn’t it?

Society for Neuroscience Meeting

I am in Washington DC at the Society for Neuroscience 2014 meeting. There is some incredible science here. Let me just share a few of the things I saw today:

The Thompson laboratory from UC Irvine (my alma mater – go anteaters!) made a model system for the blood-brain barrier from induced pluripotent stem cells. These scientists made iPSCs that had similar genetic defects to those observed in patients with Huntington’s disease. These iPSCs were then differentiated into blood-brain barrier cells and showed that these cells showed defects similar to those seen in patients with Huntington’s disease. The barrier leaked, which makes this a good model to study blood brain barrier defects in patients with neurological diseases.

Another poster described the use of vesicles from human fat-based stem cells to treat laboratory animals with a type of Huntington’s disease. These vesicles attenuated Huntington’s disease pathology and delayed its onset.

There were several other brilliant posters, and tomorrow, there will be even more. I will blog about those as time permits.

Growth Factor Delivery Stimulates Endogenous Heart Repair After Heart Attacks in Pigs

Steven Chamulean and his colleagues at the University Medical Center Utrecht in Holland have examined the use of growth factors to induce healing in the heart after a heart attack. Because simply applying growth factors to the heart will cause them to simply be washed out, Chamulean and his coworkers embedded the growth factors in a material called hydrogel. They were able to measure how long the implanted growth factors lasted. As it turns out, when the growth factors were embedded in the hydrogel, they lasted for four days, and the hydrogel caused the growth factors to spread out into heart tissue with a gradient with the highest concentration at the site of injection (see Bastings, et al., Advanced Healthcare Materials 2013 doi: 10.1002/adhm.201300076).

In his new publication in the Journal of Cardiovascular Translational Research, Chamulean and his group used a new hydrogen called UPy to into which they embedded their growth factors. UPy stands for ureido-pyrimidinone end-capped poly(ethylene glycol) polymer. At the pH of our bodies, UPy hydrogels form a gel-like material made of fibers. When the pH changes, the gel becomes liquid. They embedded the growth factors insulin-like growth factor-1 (IGF-1), and hepatocyte growth factor (HGF).

The experimental design of this paper used pigs that were given heart attacks and then reperfused 75 minutes later. One month later, the animals were broken into three groups: just hydrogel, hydrogel with growth factors embedded in it, and growth factors injected into other heart without hydrogel. One month later, the animals were examined for their heart function, and then the animals were sacrificed to examine their heart tissue.

In every case, the hearts treated with only the hydrogel did the poorest of the three groups. The animals injected with gel-less growth factors did better than the controls, but those animals treated with growth factors embedded in UPy hydrogel did the best. The physiological indicators of the hearts from the animals treated with UPy embedded with IGF-1 and HGF improved significantly more than the controls that were treated with only UPy hydrogel. The hearts from animals treated with IGF and HGF without hydrogels improved over controls, by not nearly as well as those treated with growth factor-embedded UPy hydrogels.

When the hearts were examined even more surprises were observed. The animals with hearts that had been treated with UPy + growth factors did not show the enlargement observed in the control hearts. This is significant, because enlargement of the heart is a side effect for a heart attack and is the sign of heart failure. The UPy + growth factor hearts also displayed many signs of dividing cells; far more than hearts from the other two groups. Since the heart has its own resident stem cell population, these growth factors stimulated these stem cells to divide and form new heart muscle, and new blood vessels. Blood vessel density was much higher in the UPy + growth factor group and the pressure against which blood flowed in these hearts was substantially less in this groups, demonstrating that not only was the blood vessel density higher, but blood flow through these vessel networks was much more efficient. There was also plentiful evidence of the formation of new muscle in the UPy + growth factor group. When these hearts were also stained for c-kit, which is a cell surface marker for cardiac stem cells, the UPy + growth factor hearts had lots of them – much more than the other two groups.

This paper reports significant findings because the resident stem cell population in the heart was actively mobilized without having to extract them by means of a biopsy. There is also evidence from Torella and others that IGF-1 and HGF can reactivate the sleeping cardiac stem cells of aged laboratory animals (Circulation Research 2004 94: 514-524). The UP{y hydrogels are well tolerated and are biodegradable. They provide a medium that stays in place and releases embedded growth factors in a sustained manner. The results in this paper provide the rationale to develop growth factor therapy for human patients.

Embryonic Stem Cells From Cloned Embryos Vs Induced Pluripotent Stem Cells: Let the Debate Begin

In May of 2013, Shoukhrat Mitalipov and his coworkers from the Oregon Health and Science University, reported the derivation of human embryonic stem cells from cloned human embryos. Other stem cell scientists have confirmed that Mitalipov’s protocol works as well as he says it does.

Mitalipov and others have also examined the genetic integrity of embryonic stem cells made from cloned human embryos and induced pluripotent stem cells made from mature adult cells through genetic engineering and cell culture techniques. This paper was published in Nature in June 2014 and used genetically matched sets of human Embryonic Stem cells made from embryos donated from in vitro fertilization clinics, induced Pluripotent Stem cells and nuclear transfer ES cells (NT-ES cells) derived by somatic cell nuclear transfer (SCNT). All three of these sets of stem cells were subjected to genome-wide analyses. These analyses sowed that both NT-ES cells and iPS cells derived from the same somatic cells contained comparable numbers of genetic variations. However, DNA methylation, a form of DNA modification for regulatory purposes and gene expression profiles of NT-ES cells corresponded closely to those of IVF ES cells. However, the gene expression provide of iPS cells differed from these other two cell types and iPS cells also retained residual DNA methylation patterns typical of the parental somatic cells. From this study, Mitalipov stated that “human somatic cells can be faithfully reprogrammed to pluripotency by SCNT (that means cloning) and are therefore ideal for cell replacement therapies.”

Now a new study by Dieter Egli of the New York Stem Cell Foundation (NYSCF) in New York City, which included Mitalipov as a collaborator, has failed to demonstrate significant genetic differences between iPS cells and NT-ES cells. This is significant because Eglin has long been a rather vigorous proponent of cloning to make patient-specific stem cells. Egli gave an oral preview of his forthcoming paper on October 22nd, at the NYSCF annual conference. Egli told his audience, “This means that all of you who are working on iPS cells are probably working with cells that are actually very good. So I have good news for you,” he told them, eliciting murmurs and chuckles. “What this exactly means for the SCNT program, I don’t know yet.”

Egli and colleagues used skin cells from two people—a newborn and an adult—to create both stem cells from cloned embryos (using donor eggs) and iPS cells. Then they compared the genomes of these two types of cell lines with the genomes of the original skin cells in terms of genetic mutations, changes in gene expression, and differences in DNA methylation. Both methods resulted in about 10 mutations compared with the average genome of the mature source cells. These changes didn’t necessarily happen during reprogramming, however, Egli says, since many of these mutations were likely present in the original skin cells, and some could have arisen during the handling of cells before they were reprogrammed.

Both types of stem cells also carried a similar amount of methylation changes. Overall, the method didn’t seem to matter, Egli and his team concluded. Because he is a longtime proponent of SCNT, Egli says it would have been “more attractive” to reveal significant differences between the two kinds of stem cells. “This is simply not what we found.”

Now it would be premature to conclude that iPS cells are as good as NT-ES cells for regenerative purposes, but this certainly seems to throw a monkey wrench in the cloning bandwagon. Cloning would be quite complicated and expensive and also requires young, fertile women to donate their eggs. These egg donors must undergo potentially risky procedures to donate their eggs. Jennifer Lahl’s documentary Eggsploitation provides just a few of some of the horror stories that some women experienced donating their eggs. The long-term effects of this procedure is simply not known and asking young women to do this and potentially compromise their health or future fertility seems beyond the pale to me.

Alternatively, iPS technology keeps improving and may come to the clinic sooner than we think. Also, is a cloned embryo essentially different from one made through IVF or “the old-fashioned way.?” This whole things seems to me to involved the creation of very young human beings just so that we can dismember them and use them as spare parts. Such a practice is barbaric in the extreme.

For those who are interested, please see chapters 18 and 19 of my book The Stem Cell Epistles to read more about this important topic.

A New Way to Mend Broken Hearts

Salk Institute researchers have discovered a way to heal injured hearts by reactivating long dormant molecular machinery found in the heart cells. This significant finding could open the door to new therapies for heart disorders in humans.

These new results were published in the November 6th, 2014 edition of the journal Cell Stem Cell. Although adult mammals don’t normally regenerate damaged tissue, they seem to retain a latent ability to do so. When the Salk team inhibited four different molecules that suppress genetic programs that lead to organ regeneration, they observed a dramatic improvement in heart regeneration and healing in laboratory mice.

These experiments provide proof-of-concept for a new type of clinical treatment in the fight against heart disease, which, according to the US Centers for Disease Control and Prevention, kills about 600,000 people each year in the United States alone.

“Organ regeneration is a fascinating phenomenon that seemingly recapitulates the processes observed during development. However, despite our current understanding of how embryogenesis and development proceeds, the mechanisms preventing regeneration in adult mammals have remained elusive,” says the study’s senior author Juan Carlos Izpisua Belmonte, holder of the Roger Guillemin Chair and primary investigator in the Gene Expression Laboratory and the Salk Institute.

We have within every cell of our bodies, the genes for organ regeneration. For several years, Izpisua Belmonte and his coworkers have attempted to clarify the genes that organism uses during embryonic development and during tissue healing highly regenerative organisms such as the zebrafish.

An injured zebrafish heart showing proliferating cells in the wounded area of the heart (red) and cardiac muscle cells (green).
An injured zebrafish heart showing proliferating cells in the wounded area of the heart (red) and cardiac muscle cells (green).

In 2003, Izpisua Belmonte’s laboratory first identified the signals that precede zebrafish heart regeneration, which they followed-up with a 2010 Nature paper, in which scientists from Izpisua Belmonte’s laboratory described how regeneration occurred in the zebrafish. Rather than stem cells invading injured heart tissue, the cardiac cells themselves reverted to a precursor-like state (a process called ‘dedifferentiation’). Dedifferentiation allowed the cells to proliferate within the damaged tissue.

n a dish, heart muscle cells return to a precursor-like state after pro-regenerative treatment with microRNA inhibitors. Green shows a disorganized cardiomyocyte cytoskeleton indicative of cell dedifferentiation; red shows mitochondrial organization.
In a dish, heart muscle cells return to a precursor-like state after pro-regenerative treatment with microRNA inhibitors. Green shows a disorganized cardiomyocyte cytoskeleton indicative of cell dedifferentiation; red shows mitochondrial organization.

They next determined if mammals retained any of the molecular players responsible for this kind of regenerative reprogramming. However, such an experiment came with some risks, recalls Ignacio Sancho-Martinez, a postdoctoral researcher in Izpisua Belmonte’s lab.

“When you speak about these things, the first thing that comes to peoples’ minds is that you’re crazy,” he says. “It’s a strange-sounding idea, since we associate regeneration with salamanders and fish, but not mammals.”

What are the things that cause a heart to regenerate in these smaller animals? Extensive work on the regenerating hearts of fish and salamanders failed to reveal anything concrete. Therefore, the laboratory changed its tack. “Instead, we thought, ‘If fish know how to do it, there must be something they can teach us about it,’” says the study’s first author Aitor Aguirre, a postdoctoral researcher in Izpisua Belmonte’s group.

The team focused on microRNAs, which control the expression of many genes. They used an extensive genetic screen for microRNAs that changed their expression levels during the healing of the zebrafish heart and that were found in the mammalian genome.

Their studies uncovered four molecules in particular–MiR-99, MiR-100, Let-7a and Let-7c–that fit their criteria. All were heavily repressed during heart injury in zebrafish and they were also present in rats, mice and humans.

However, in studies of mammalian cells in a culture dish and studies of living mice with heart damage, the group saw that the levels of these molecules were high in adults and failed to decline after the heart experienced injury. Therefore, Izpisua Belmonte’s team used adeno-associated viruses that could specifically infect the heart to target each of those four microRNAs and experimentally suppress their expressing levels.

Injecting these inhibitors into the hearts of mice that had suffered a heart attack triggered the regeneration of cardiac cells, and improved numerous physical and functional aspects of the heart, such as the thickness of its walls and its ability to pump blood. The scarring caused by the heart attack was significantly reduced with treatment compared to controls.

The improvements were still obvious three and six months after treatment–a long time in a mouse’s life. “The good thing is that the success was not limited to the short-term, which is quite common in cardiac regenerative biology,” Sancho-Martinez says.

The new study focused only on a handful of 70 some microRNA candidates that turned up in their initial screen. These other molecules might also play a part in heart cell proliferation, healing scars and promoting the formation of new blood vessels–all processes critical for heart repair, Sancho-Martinez says. The data are available so that other research groups can focus on molecules that interest them.

The next step for Izpisua Belmonte’s team is to move into larger animals and see whether “regenerative reprogramming” can work in larger hearts, and for extended periods after treatment, says Sancho-Martinez. And, although the virus packaging disappeared from the animals’ bodies by 2 weeks after treatment, the scientists are working on a new way to deliver the inhibitors to avoid the need for viruses altogether.

Human Amniotic Epithelial Cells Modulate Tooth Socket Restoration in Rats

Human amniotic epithelial cells have the capacity to differentiate into several different cell types. To that list, we can now add bone.

A study from Steve G.F. Shen at his colleagues at the Shanghai Jiao Tong University School of Medicine, Shanghai, China has used human amniotic epithelial cells to regenerate the tooth sockets in laboratory animals.

The first set of experiments examined the ability of human amniotic epithelial cells (hAECs) to form bone under controlled laboratory conditions. Then hAECs were loaded into artificial scaffolds that were then placed into the mouths of rats with tooth socket defects.

In culture, hAECs expressed bone-specific genes 10-14 days after induction. The cells also changed shape and made bone-specific proteins. When implanted into rat tooth sockets, the hAECs were embedded in a scaffold imbued with growth factors known to induce bone differentiation. These implants improved bone regeneration by directly participating in bone repair of the tooth socket defect. They also had an additional benefit in that they modulated the localized immune response against the implanted scaffolds. This immune response modulation augmented regeneration of the tooth sockets and allowed the implanted cells to get on with the job of fixing the surrounding bone without dealing with insults from the immune system.

This study has provided the first evidence that hAECs exhibit direct involvement in new bone regeneration and a localized modulatory influence on the early tissue remodeling process. These cells indirectly contributed to the bone-making process in the alveolar defect. Altogether, these results imply the potential clinical use of hAECs as an alternative stem cell-based for restoring tooth socket deformities.

Engineering Stem Cells to Fight Cancer

Advanced brain tumors are typically treated by surgical removal. However, it is difficult in the extreme to extirpate an entire tumor and therefore, tumor relapse is a perennial problem. A special group of small proteins known as ‘cytotoxic proteins” can target and destroy remaining cancer cells, but these proteins have short half-lives in the body and recent clinical trial called the PRECISE trial was not able to demonstrate that administered cytotoxic proteins had any efficacy against glioblastoma multiforme (GBM) brain tumors.

A new study, however, published online from the journal Stem Cells, a research group led by Khakid Shah from the Harvard Stem Cells Institute, have devised a new strategy designed around these engineered cytotoxic proteins has shown that neural stem cells (NSCs) can be genetically engineered to express these proteins and help treat GBM tumors.

So how did Shah and his colleagues design this novel strategy? They engineered NSCs to not only express specific cancer cell-killing toxins, but also have resistance to these toxins. Secondly, they designed cytotoxins that have the ability to enter cancer cells and target proteins known to be over-expressed by GBM tumors. Then these neural stem cells were encapsulated, they were transplanted into the space left after the bulk of the tumor was surgically removed.

In a mouse model of GMB, the implanted engineered stem cells survived and mediated an increase in long-term survival. This therapy was also effective against multiple patient-derived GBM cancer cell lines, which demonstrated their potential clinical relevance and applicability.

Shah and his coworkers want to bring these results to human trials within the next five years. They hope that this strategy can be successfully deployed in combination with surgical removal of the tumor mass. Shah also hopes that this approach can be adapted to treat other tumor types by using tissue-specific stem cells that express tumor-specific cytotoxins.

See Stuckey DW, Hingtgen SD, Karakas N, et al. Engineering toxin-resistant therapeutic stem cells to treat brain tumors. Stem Cells 2014.

Stem Cells in Breast Milk Might Help Baby Grow Up Strong

We have all probably heard about the benefits of breast milk for your baby as opposed to some other source of nutrition. This list of benefits is extensive: antibodies, better microfloral adaptation throughout the gastrointestinal tract, it helps you get your figure back, lower rates of illness in breast-fed babies, better for the environment, and so on. However, this long litany of benefits, and do not get me wrong, I am not knocking the benefits of breast-feeding, does not include one other benefit, and that includes a dose of breast-specific stem cells. Preliminary evidence has shown that mouse pups take in stem cells from their mother during breastfeeding, suggesting that the same thing might happen in humans.

Several years ago, it became clear that human breast milk contains a breast-specific kind of stem cells. This remarkable finding however did not answer the question of whether these cells coincidentally leaked into breast milk or they do anything useful with respect to the breast-feeding infant.

A presentation at the National Breastfeeding and Lactation Symposium in London last week presented data that suggests that, in mice at least, breast milk stem cells cross into the offspring’s blood from their stomach and play a functional role later in life.

Foteini Hassiotou from the University of Western Australia and her coworker used genetically modified mice whose cells contain a gene called tdTomato, which glows and intense shade of red under fluorescent light. The red-glowing females were mated and gave birth to mouse pups, but they were then presented with mouse pups from mothers who were genetically unmodified. Thus any red-glowing cells in these unmodified pups must have come to them from their mother’s milk.

When the these mouse pups that had been suckled by the tdTomato-expressing mothers grew to adulthood, assays of their tissues showed that red-glowing cells were found in their blood and the brain, thymus, pancreas, liver, spleen and kidneys. Hassiotou’s team also discovered that the breast-specific stem cells had differentiated into mature cells. Those red-glowing cells in the brain had the characteristic shape of neurons, those cells in the liver made the liver protein albumin, and those in the pancreas made insulin. According to Hassiotou, “They seem to integrate and become functional cells.”

What, precisely, is the role of these cells in the life of mice? Do they play a role in normal growth and development, or could they help to make the offspring tolerant to its mother’s cells and proteins, to reduce chances of an allergic reaction to her breast milk? “There must be some evolutionary advantage,” says Hassiotou.

According to Hassiotou, since her work and that of her colleagues clearly shows that these breast milk stem cells can differentiate into several different types of tissues makes it more likely they could be used for therapeutic applications. Chris Mason of University College London adds: “If these intriguing cells are functional, they could be a novel option for producing future cell therapies.”

Breast milk stem cells seem to have less capacity for unlimited cell division than embryonic stem cells. “But that’s actually a good thing,” says Hassiotou, because they do not form tumors when injected into mice. Therefore they may be less likely to trigger cancer if used to treat people.

Hassiotou points out that this kind of work cannot be done in humans, but she is planning to repeat it in a non-human primate species known as macaques.