Gene Therapy Sprouts New Neurons in Alzheimer’s Patients’ Brains


The journal JAMA Neurology has published a new study that describes an experimental gene therapy that reduces the rate at which nerve cells in the brains of Alzheimer’s patients degenerate and die. See Tuszynski, M. H., et al. (2015). Nerve Growth Factor Gene Therapy: Activation of Neuronal Responses in Alzheimer Disease. JAMA Neurology, published online August 24, 2015. DOI: 10.1001/jamaneurol.2015.1807.

In this study, targeted injections of a growth factor called “nerve growth factor” or NGF into the brain of patients rescued dying cells around the injection site, enhanced the growth of these cells and induced them to sprout new nerve fibers. Surprisingly, in some cases, these beneficial effects persisted for 10 years after the therapy was first delivered.

Alzheimer’s disease (AD) is the world’s leading form of dementia. It affects approximately 47 million people worldwide, and this number is expected to almost double every 20 years. Despite the huge amounts of time, effort, and money devoted to developing an effective cure, the vast majority of new drugs for AD have failed in clinical trials.

While these new results are preliminary findings, they come from the first human trials designed to test the potential benefits of NGF gene therapy for AD patients.

NGF was discovered in the 1940s by Rita Levi-Montalcini, who demonstrated, quite convincingly, that a small protein that she had isolated and purified promoted the survival of certain sub-types of sensory neurons during development of the nervous system. Since that time, others have shown that it also promotes the survival of neurons that produce acetylcholine in the basal forebrain; these cells die off at an alarming rate in AD patients.

In 2001, Mark Tuszynski and his coworkers at the University of California, San Diego School of Medicine initiated a clinical trial based on these laboratory findings. This trial was the first of its kind, and it was designed to investigate the ability of NGF gene therapy to slow or prevent the neuronal degeneration and cell death characteristic of AD.

In phase I of this trial, eight patients with mild AD received so-called “ex vivo” therapy to deliver the NGF gene directly into the brain. This trial extracted skin fibroblasts from the skin on the patient’s backs, and then genetically engineered those cells to express the NGF genes. These NGF-expressing cells were then implanted into the patients’ basal forebrain. Since NGF is too large to cross the blood-brain barrier, it had to be administered directly into the brain. Also, outside the brain, exogenous NGF can stimulate other nerve cells can cause unwanted side-effects such as pain and weight loss.

One of these patients died just 5 weeks after receiving the therapy. Tuszynski’s team secured permission to perform an autopsy of this patient, and in 2005 they reported that the treatment led to robust growth responses, and did not cause any adverse effects (Tuszynski, M. H., et al. (2005). A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease. Nature Medicine, 11: 551 – 555).

The latest results come from postmortem examination of these patients’ brains, all of whom had also been recruited in a safety trial between March 2001 and October 2012. Additionally, two other were included who had received in vivo therapy that included injecting a modified virus that carried the NGF gene into the basal forebrain.

Some of the participants died about one year after undergoing therapy, and others survived for 10 years after the treatment. These autopsies showed that all of them had responded to the treatment.

Essentially, all the brain tissue samples taken from around the implantation sites contained diseased neurons, as expected, but the cells were overgrown, and had sprouted axonal fibers that had grown towards the region into which NGF had been delivered. In contrast, samples taken from the untreated side of the brain exhibited no such response.

This trial was conducted to test the safety of the treatment and it did confirm that none of the patients experienced long-term adverse effects from the treatment, even after long periods of time. These results also suggest that NGF is successfully taken up by nerve cells following targeted delivery. Also the cells synthesize NGF protein so that its concentration dramatically increases in and around the delivery site. Probably the most exciting part of these findings is that the responses to NGF can persist for many years after the gene has been delivered into the brain.

Cholinergic Neuronal Hypertrophy and Sprouting Shown is labeling for p75, a neurotrophin receptor expressed on cholinergic neurons of the nucleus basalis of Meynert. Images were obtained 3 years after adeno-associated viral vectors (serotype 2)–nerve growth factor (AAV2-NGF) delivery (A-C) and 7 years after ex vivo gene transfer (D-F). A-C, Cholinergic neurons are labeled for p75 within the zone of transduction (A), 3 mm from the zone of transduction (B), and in a control Alzheimer disease (AD) brain of the same Braak stage (C). Cells near the NGF transduction region appear larger. The inset shows higher-magnification views of a typical neuron from each region. D, Shown is a graft of fibroblasts transduced to secrete NGF (yellow arrowhead) adjacent to the nucleus basalis of Meynert (red arrowheads). E, The graft (G) at higher magnification is densely penetrated by p75-labeled axons arising from the nucleus basalis of Meynert. These axons are sprouting toward the graft, a classic trophic response. F, Shown are p75-labeled axons from the nucleus basalis of Meynert sprouting toward the graft. Individual axons coursing toward the graft are evident (arrowheads). The bar represents 125 µm in A through C, 500 µm in D, and 100 µm in E and F.
Cholinergic Neuronal Hypertrophy and Sprouting
Shown is labeling for p75, a neurotrophin receptor expressed on cholinergic neurons of the nucleus basalis of Meynert. Images were obtained 3 years after adeno-associated viral vectors (serotype 2)–nerve growth factor (AAV2-NGF) delivery (A-C) and 7 years after ex vivo gene transfer (D-F). A-C, Cholinergic neurons are labeled for p75 within the zone of transduction (A), 3 mm from the zone of transduction (B), and in a control Alzheimer disease (AD) brain of the same Braak stage (C). Cells near the NGF transduction region appear larger. The inset shows higher-magnification views of a typical neuron from each region. D, Shown is a graft of fibroblasts transduced to secrete NGF (yellow arrowhead) adjacent to the nucleus basalis of Meynert (red arrowheads). E, The graft (G) at higher magnification is densely penetrated by p75-labeled axons arising from the nucleus basalis of Meynert. These axons are sprouting toward the graft, a classic trophic response. F, Shown are p75-labeled axons from the nucleus basalis of Meynert sprouting toward the graft. Individual axons coursing toward the graft are evident (arrowheads). The bar represents 125 µm in A through C, 500 µm in D, and 100 µm in E and F.

Now, does the observed cellular response to NGF alleviate disease symptoms? Although phase II trials testing the efficacy of the treatment are ongoing, preliminary findings from the initial study suggest that the therapy did indeed slow the rate at which mental function declined in one of the patients involved. These new results indicate that gene therapy is a viable strategy for treating Alzheimer’s and other neurodegenerative diseases, and warrants further research and development.

Interleukin-17A Augments Mesenchymal Stem Cell Function


One of the biggest problems with organ transplantations is that the immune system can reject the transplanted tissue. Transplant rejection requires the patient to go through another traumatic surgery, and if another organ is not available, then they might very well die. Is there a way to reduce organ rejection?

Our bodies have on our cell surfaces a series of “bar codes” that are the result of “Major Histocompatibility Antigens” or MHC proteins. These proteins are encoded by genes that vary extensively. Therefore, each person has a distinct combination of cell surface proteins that decorate the outside of their cells. If your immune system finds cells that have a different set of MHCs that what you were born with, the immune system, which has been “taught” to accept your own proteins and reject different proteins, will attack and destroy the transplanted tissue. This is known as “Graft vs Host disease” or GvHD. To avoid GvHD, transplant physicians try to find organs that match your own tissue type as closely as possible. However, it is virtually impossible to find organs that perfectly match your own. Even if the patient is given anti-rejection drugs, sometimes the immune system begins to reject the transplanted organ. Is there a way around this?

Mesenchymal stem cells (MSCs) have the ability to suppress unwanted immune responses. If MSCs are pre-treated with cytokines, they can do this even better. Typically, MSCs are pre-treated with a molecule called interferon-γ. This molecule stimulates MSCs and causes then to suppress the immune response, but it also causes MSCs to express proteins that cause them to be rejected by the immune system. Thus interferon-γ seems to cause problems that prevent the MSCs from working properly.

In a really beautiful paper by Kisha Sivanathan in the Coates lab at the School of Medical Sciences, Adelaide, Australia and her colleagues, showed that pre-treating human MSCs with interleukin-17A did a better job at stimulating MSCs without sensitizing the cells to the immune system.

When Sivanathan and her co-workers treated human bone marrow-derived MSC with interleukin-17A, this molecule enhanced the ability of MSCs to suppress the immune response without making the MSCs subjects for rejection by the immune system.  These interleukin-17A-treate MSCs (MSC-17s) showed no induction or upregulation of molecules that the immune system reacts against (MHC class I, MHC class II, and T cell costimulatory molecule CD40), and the the interleukin-17A-treated MSCs maintained normal MSC morphology and made all the common MSC markers.

When MSC-17s were placed in culture with activated human T cells, the MSCs-17s potently suppressed T cell proliferation.  Additionally, MSC-17s prevented activated T-cells from making the whole cocktail of molecules they normally make once they are activated.

Not all T-cells are created equal. There is a group of T-cells called “regulator T-cells” or T-regs for short. T-regs tend to down regulate the immune response. It turns out that MSCs-17s turn on T-regs and stimulate them to potently suppress T-cell activation. They do this without inducing immunogenicity in the MSCs.

Thus, pre-treating MSCs with interleukin-17A represents a superior way for stimulate MSCs to suppress T cell activity in clinical situations.  Dr. Coates and his colleagues are hopeful that this protocol can be the subject of clinical trials in the near future.

Mesenchymal Stem Cells Heal Gastrointestinal Ulcers


Stomach ulcers are a complication of routine use of aspirin, Advil, or other non-steroidal anti-inflammatory drugs. Additionally, radiation therapy, or inflammatory bowel disease can also cause stomach ulcers, and these are painful and potentially dangerous for patients. Trying to get our heads around ulcers is not easy, but a new study by Manieri and colleagues have provided some understanding of ulcer formation and ways that mesenchymal stem cells (MSCs) might help heal these painful lesions.

Manieri and others used prostaglandin-deficient mice as a model system for ulcer formation. In these mice, their stomachs do not produce the prostaglandins that protect the layers of the stomach from being digested by its own acid and enzymes. Consequently, these mice are subject to so-called “penetrating ulcer formation,” or ulcers that penetrate the underlying muscular layer (muscularis propria). When Manieri and his colleagues took biopsies of the colon of these prostaglandin-deficient mice, they observed extensive necrosis of the upper and lower layers of the colon.

When these mice were treated with stable prostaglandin-I2 (PGI2) analogs, Manieri and others showed that they could ameliorate the damage to the colon. However, when this research group analyzed the ulcer beds in these mutant mice, they noticed that CD31+ endothelial cells, which form blood vessels, were found in very low numbers. This suggested that reduced blood vessel formation could be a key driver of penetrating ulcer formation. To confirm their hypothesis, the authors stained the wound sites for vascular endothelial growth factor (VEGF). They saw fewer VEGF+ cells in the mutant mice compared with wild-type animals, which suggests that impaired blood vessel production contributes to ulceration. To further test this hypothesis, Manieri and others treated wild-type mice with tivozanib (a VEGF receptor antagonist), which also caused smooth muscle necrosis in the colon.

Next Manieri and others injected MSCs from the colons of mice that showed increased expression of VEGF into the ulcerated colon of mutant mice. The MSCs dutifully migrated to the ulcer beds, and rescued the muscle necrosis phenotype. These results show that MSC administration can provide a soothing treatment prospect for patients who are dealing with gastrointestinal ulceration.

See N. A. Manieri et al., Mucosally transplanted mesenchymal stem cells stimulate intestinal healing by promoting angiogenesis. J. Clin. Invest. 10.1172/JCI81423 (2015).

So What About Three-Person Embryos?


In 2013, Deiter Egli’s group at Harvard University successfully transferred chromosomes that were in the process of dividing and segregating (known as an incompletely assembled spindle-chromosome complex) from one human egg into another egg whose nucleus had been removed (Nature 493, 632–637 (31 January 2013) doi:10.1038/nature11800). They prevented the eggs from prematurely re-entering meiosis by cooling the chromosome/spindle complex to room temperature. This allowed normal polar body extrusion, efficient development to the blastocyst stage, and, eventually, the derivation of normal stem cells.

3-person-ivf

Egli’s technique allows the genome of one egg to initiate development in the cytoplasm of another egg. Why is this significant? Because within out cells is a bean-shaped vesicle called a mitochondrion. Mitochondria make the energy for our cells. To do this, mitochondria use a variety of proteins encoded on genes found in the nuclear genome. However, mitochondria also have their own genome that encodes some crucial mitochondrial proteins and RNAs. The human mitochondrial genome is a small, circular DNA molecule that encodes 37 different genes.

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Mutations in genes encoded by the mitochondrial genome tend to have rather catastrophic consequences for the fertility of women. When the egg undergoes fertilization, the vast majority of the mitochondria of the sperm are degraded and their mitochondrial DNA is eliminated (Katsumi Kasashima, Yasumitsu Nagao, and Hitoshi Endo. Reprod Med Biol. 2014; 13(1): 11–20). Research has shown that the father’s mitochondrial genome can make some very small contribution to the embryo, a phenomenon known as “paternal leakage,” but it is usually pretty small (Kuijper B1, Lane N, Pomiankowski A. J Evol Biol. 2015 Feb;28(2):468-80). Therefore, if the mother carries a deleterious mutation in her mitochondrial DNA, her eggs will usually not be able to progress through fertilization successfully and support the growth and development of the embryo. Consequently, the mother will be infertile.

This new technique by Egli, however, allows mothers who are infertile because of mutations in their mitochondria DNA, to have children who are genetically related to them. All that is needed are eggs from a healthy donor, and a laboratory that has the know-how and will to do this procedure. The mother’s eggs are harvested by standard IVF technologies, fertilized by the father’s spermatozoa, and after fertilization has ended, the chromosome-spindle complex is lifted from the young embryos and transferred into enucleated donor eggs that contain mitochondria with normal genomes. Development will then ensue without a hitch. Right?

Well not so fast. As it turns out, this procedure has been carried out in several different animal species, and the results are decidedly mixed (see Reinhardt and others, Science 2013;341:1345).

If we begin with insects, we can move new mitochondrial genomes into embryos by standard genetic techniques. If we do so in the fruit fly, Drosophila melanogaster, such mitochondrial transfer produces fly embryos that develop normally, but the animals show altered juvenile viability, adult male animals show accelerated aging and reduced fertility. Genetically, it is clear that transferring new mitochondria into an egg messes up the expression of nuclear genes. Identical experiments in the seed beetle causes altered development and metabolic rates, reduced fertility in males and reduced survival in females. Similar studies in copepods (Tigriopus californicus) causes reduced juvenile viability, and reduced mitochondrial function and energy production in adults.

If mice are subjected to these same experiments, the animals develop normally and survive to adulthood, but these adult mice show reduced growth and exercise ability and reduced learning ability in males.

The above-mentioned experiments used standard genetic breeding techniques to generate animal strains that had a mismatch between the nuclear and mitochondrial genome.  Such techniques are demonstrably non-invasive.  However, the technology applied in Egli’s laboratory were invasive, and included removing chromosome/spindle complexes and transferring them to donor eggs that had been enucleated. Therefore, the effects of these invasive procedures had to be tested as well. If such invasive procedures were tested in cultured mouse cells, the hybrid cells showed altered cellular respiration and growth. In short, their mitochondria worked poorly inside their new homes.

If Egli’s technique was used in non-human primates, macaques in particular, the animals developed to the juvenile stage and appeared normal.

On the strength (or weakness) of these experiments, some reproductive specialists in countries where such techniques can be performed without fear of prosecution have used mitochondrial transfer in human embryos. Again the results are quite mixed. Healthy children have been born by this procedure, but several others have not. Helen Pearson reported in Nature News on the 14th of October, 2005 about two Chinese babies that were made with mitochondrial transfer that died in utero at 24 and 29 weeks. Other outcomes include a miscarriage, an abortion of a fetus that had Turner Syndrome, at least two children with mixed mitochondria that studies linked with cognitive dysfunction and obesity, and a child born with a severe developmental disorder. I do not call these hopeful results.

Another experiment that gives me pause was published in the journal Cell Reports in June of 2014 by Joerg Patrick Burgstaller and others. This paper showed that even small amounts of diseased mitochondrial DNA in an embryo would spread throughout the organism. The amount of spread is wide and varied, but even small amounts of variant mitochondrial DNA did spread. This significance of this is stark for this debate. You see, Egli’s original paper in Nature showed that very small amounts of the original mitochondrial DNA are transferred to the donor egg. Granted it below 1% of the total mitochondrial DNA in the embryo, but it is still detectable. Burgstaller and others have shown that even with this small amount of mitochondrial DNA, it will still spread throughout the developing baby and given them a body with some cells that have most the diseased mitochondrial DNA, and others that have the normal mitochondrial DNA, and other cells that have a mixture of the two. Therefore, Egli’s technique is NOT a cure for conditions linked to mitochondrial DNA mutations. Let me repeat this for every one – Egli’s technique is NOT a cure for conditions linked to mitochondrial DNA mutations.

No vertebrates have yet been studied who have gone through mitochondrial replacement and survived to reproductive age. Given the decidedly mixed record of this technology in a variety of animal models and the paucity of data so far, this technology is clearly not ready for use in humans.

However, that has not stopped scientists and politicians in the United Kingdom from pushing this technology forward as a fertility treatment for infertile women who harbor mitochondrial DNA mutations.  Some in the scientific community warned about the potential dangers of this technology.  Their concerns were largely ignored and in many cases severely criticized.  Even worse, some thought that three-person embryos could grease the slippery slope in which this technology or similar ones like cloning would be applied as generalized treatments for infertility.  That concern was labeled ridiculous. No longer.

Science magazine reported that cloning magnate Shoukhrat Mitalipov has formed a partnership with disgraced fraudster Woo Suk Hwang.  The two are teaming up to form a joint commercial venture to use Mitalipov’s cloning techniques as a way to treat infertility and perhaps other diseases.  Mitalipov’s commercial venture Mitogenome Therapuetics and Hwang along with the company BoyaLife, which will reportedly put up more than $90 million into the effort.  Mitalipov has also generated news reports by asking FDA approval to use so-called 3-person IVF “mitochondrial transfer” technology, which shares some technical elements with cloning, to treat infertility. This surprised some in the UK, including members of Parliament who were hoodwinked into voting to approve the three-person embryo procedure by being told that this technology would only be used to treat mitochondrial diseases.

The slippery slope is real and unless citizens rise up and make noise, we are going to be dragged where angels fear to tread by over-zealous scientists who are willing to sacrifice young children for the sake of their own fame and success.  This technology is not ready for use in humans.  The approval of this technology in the UK is a very bad idea.  It will also spread to the use of cloning in general as a treatment for diseases, and we will then move to fetus farming.  May God give us the strength to say enough is enough.

The United States FDA’s Cellular, Tissue and Gene Therapies Advisory Committee will be holding a public hearing to “discuss considerations for the design of early-phase clinical trials of cellular and gene therapy products” including the three-parent IVF method. The public has until October 15 to send in written comments. If you are interested in making your views known, go here.

Intravenous Administration of Lipitor-treated Stem Cells on the Heart


Hao Zhang and colleagues from the Chinese Academy of Medical Sciences and Peking Union Medical College have published a rather unusual experiment in the American Journal of Translational Research. This experiment, however, could have implications for stem cell therapy in heart attack patients.

When heart attack patients are treated with stem cells, they are either injected directly into the heart muscle or released into the heart through the coronary arteries by means of angioplasty. Injecting stem cells directly into the heart requires special equipment and training. Releasing cells into the coronary arteries causes most cells to end up in the lungs or other organs, and the retention of the stem cells is poor. Introducing cells by means intravenous administration would be supremely simple, but in animal experiments, intravenously administered stem cells almost never get to their target organ.

When the heart undergoes a heart attack, the damaged heart cells release a molecule called SDF1 or stromal cell-derived factor 1. SDF1 summons stem cells to the damaged areas by binding to the surfaces of stem cells and drawing them to the higher concentrations of SDF1. SDF1 binds to a receptor on the surfaces of stem cells called CXCR4. Unfortunately, when stem cells are administered intravenously to animals that have just experienced a heart attack, the stem cells do not have enough CXCR4 on their surfaces to properly respond to the SDF1 being secreted by the damaged heart.

Zhang and his colleagues capitalized on an observation made several years ago. When stem cells are exposed to statin drugs that are normally used to lower serum cholesterol levels, the stem cells increase the number of CXCR4 molecules on their surfaces. Statins have also been shown to increase stem cell survival once the cells get to the heart, but Zhang and his team wanted to know if pre-treating stem cells with statins could increase their migration to the damaged heart.

The Zhang group isolated mesenchymal stem cells from rat bone marrow and treated these cells with increasing concentrations of the drug Lipitor (atorvastatin). Indeed, increasing amounts of Lipitor increased the number of CXCR4 molecules on the surfaces of the mesenchymal stem cells (MSCs), This increase in CXCR4 molecules peaked at 24 hours, after which the number of receptors declined. These Lipitor-treated MSCs also migrated much more robustly in culture when treated with SDF1.

Next, Zhang’s group pre-treated MSCs with Lipitor and labeled them with an innocuous tracking molecule. 24 hours after giving some laboratory rats heart attacks, these MSCs were administered to the rats in their tail veins. Two other groups of similarly treated rats were given either MSCs that had not been pre-treated with Lipitor, or just buffer.

The Lipitor-treated MSCs were found in significantly higher quantities in the hearts of laboratory animals, relative to the other animals. Secondly, these Lipitor pre-treated MSCs cut the size of the heart scar in half, and there was also substantially less inflammation in hearts from animals treated with Lipitor pre-treated MSCs than the other groups. Heart function was also increased in the pre-treated group.

Live MSCs were observed in the hearts of the animals given Lipitor pre-treated MSCs. This is a remarkable finding, because most experiments have shown that MSCs administered to the heart after a heart attack ad usually dead within 21 day after administration. However the Lipitor pre-treated MSCs survive and flourish in the damaged heart, which suggests that SDF1 not only attracts stem cells but also increases their rates of survival.

This is a somewhat off-beat experiment at first glance, but if MSCs could be pre-treated with a drug like Lipitor and then administered to heart patients intravenously, they would survive in the heart, convey greater benefits, and their administration would be safer, and not require special equipment or training. With a little luck, this idea will reach human clinical trials in a few years; provided that further animal and cell culture studies confirm these results, elucidate the mechanism of SDF1-mediated survival, and show that such augmentation of function is also observed in human MSCs.

Rejuvenation Factor Discovered in Human Eggs


When the egg is fertilized by a sperm, it is transformed into a single-celled embryo or zygote that is metabolically active and driven to divide and develop. The egg, on the other hand, is a rather inert cell from a metabolic perspective. What is it in the egg that allows it to transform into something so remarkably different?

A new study by Swea-Ling Khaw and others in the laboratory of Ng Shyh-Chang at the Genome Institute of Singapore (GIS) has elucidated two main factors that help rejuvenate the egg and might also help reprogram adult cells into induced pluripotent stem cells (iPSCs).

Eggs express large amounts of a protein called Tcl1. Tcl1 suppresses the function of old, potentially malfunctioning mitochondria (the structure in cells that makes the energy for the cell). This suppression prevents damaged mitochondria from adversely affecting the egg’s transformation from into an embryo.

Remember also that if an adult cell is fused to an egg, it can cause the egg to divide and form an early embryo. Therefore, the egg cytoplasm is able to reprogram adult cells as well, and Tcl1 seems to play a role in this reprogramming capability as well.

In a screen for genes that are important to the reprogramming process, Shyh-Chang’s laboratory isolated two genes, Tcl1 and Tcl1b1. Further investigation of these two proteins showed that Tcl1 affects mitochondria by inhibiting a mitochondrial protein called polynucleotide phosphorylase (PNP). By locking PNP in the cytoplasm rather than the mitochondria, the growth and function of the mitochondria are inhibited. Tcl1b1 activates the Akt kinase, which stimulate cell growth, survival, and metabolism.

In a review article in the journal Stem Cells and Development, Anaïs Wanet and others explain that energy production in pluripotent stem cells is largely by means of glycolysis, which occurs in the cytoplasm. Mitochondria in pluripotent stem cells are immature subfunctional. When adult cells are reprogrammed into iPSCs, mitochondria function is shut down and energy production is largely derived from glycolysis. When the cells differentiate, the mitochondria are remodeled and become functional once again. Tcl1 is the protein that help shut down the mitochondria so that the pluripotent state can ensue and Tcf1b1 gears up the pluripotent stem cells to grow and divide at will.

Given this remarkable finding, can Tcf1 help make better iPSCs? Almost certainly, but how does one use this important factor to make better iPSCs?  That awaits further experimentation.  Additionally, this finding might also help aging and infertility issues as well. Hopefully this work by Shyh-Chang and her colleagues will lead to many more fruitful and exciting experiments.

Intravenous Bone Marrow For Stroke: Clinical Trial


Akihiko Taguchi from the Institute of Biomedical Research and Innovation in Kobe, Japan, in collaboration with a whole host of colleagues from various places treated stroke with their own bone marrow. This is a Phase 1/2 clinical trial but it is a very small trial that was neither blinded not placebo-controlled. Therefore, while this trial is useful, the results are of limited value.

In this clinical trial, 12 stroke patients were divided into two groups, one of which received 25 milliliters and the other of which received 50 milliliters of bone marrow cells 7-10 days after their strokes. The bone marrow cells were administered intravenously. To isolate bone marrow cells, the so-called “mononuclear fraction” was isolated from whole bone marrow samples that came from bone marrow aspirations. Patients were evaluated by means of brain imaging to measure blood flow in their brains, and a series of neurological tests. The National Institute of Health Stroke Scale or NIHSS scores were used to grade the neurological capabilities of each patient. Patients were examined 1 month and then 6 months after treatment.

All treated patients were compared with the records of other stroke patients in the past who were not treated with bone marrow cells. These comparisons showed that the bone marrow-treated patients showed a trend towards improved neurological outcomes. Statistically, the bone marrow-treated patients had significantly better blood flow and oxygen consumption in their brains 6 months after treatment compared to the historic controls. Also, the NIHSS scores of the bone marrow-treated patients were also significantly better than those of the historic controls. Patients who received the higher doses of bone marrow cells did better than those who received the lower doses.

There were also no apparent adverse effects to administering the bone marrow cells. One patient experienced pneumonia and sepsis 3 months after cell therapy, but data monitoring largely eliminated the cell therapy as being a contributing factor to this issue. Another patient experienced a seconded stroke that was detected the day after the cell therapy. Because the patient had shown signs of a stroke the day before treatment, the association between the cell therapy and the recurrent stroke is rather unclear. None of the other patients showed any worsening of their present strokes, seizures, or other complications.

All in aloe, it seems as though this procedure is safe, and there is a trend towards increased metabolic and neurological recovery. However, this is a very small study and these trends may not hold in a larger study. Secondly, these patients must be followed for an extended period of time in order to determine if these improvements are durable or transient. Finally, these improvements must be compared with a placebo if there are going to convince the FDA.

Bone marrow cells contain a variety of stem cells and other types of cells that may release cocktails of healing molecules that help cells survive, make new blood vessels, and tamp down inflammation. Additionally, bone marrow cells might stimulate resident populations of stem cells to proliferate and make new neurons and glial cells. Until these positive results can be reproduced in larger, better controlled studies, these results will remain interesting and hopeful, but ultimately inconclusive.

These results were published in Stem Cells and Development 2015 DOI: 10.1089/scd.2015.0160.

Fat-Derived Stem Cells Form Muscle in Muscular Dystrophy Mice


Stem cell therapy for Duchenne muscular dystrophy (DMD) has been plagued by poor cell engraftment into diseased muscles. Additionally, there are no reports to date describing the efficient generation of muscle progenitors from fat-derived stem cells (ADSCs) that can contribute to muscle regeneration.

A study by Cheng Zhang and others from Sun Yat-sen University in Guangzhou, China, Guangdong Province has examined the ability of progenitor cells differentiated from ADSCs using forskolin, basic fibroblast growth factor, the glycogen synthase kinase 3β inhibitor 6-bromoindirubin-3′-oxime as well as the supernatant of ADSC cultures to form workable muscle cells.

When these fat-derived stem cells were treated as described above, they formed a proliferative population of muscle progenitors from ADSCs that had characteristics similar to muscle satellite cells. Furthermore, in culture, these cells were capable of terminal differentiation into multinucleated myotubes.

When these fat-derived stem cells were transplanted into mice that had an inherited type of DMD, the progenitor cells successfully engrafted in skeletal muscle for up to 12 weeks, and generated new muscle fibers, restored dystrophin expression, and contributed to the satellite cell compartment.

These findings highlight the potential application of ADSCs for the treatment of muscular dystrophy. They also illustrate the ability of ADSCs to differentiate into functional skeletal muscle cells when treated properly in culture. These same cells might serve as a treatment for DMD patients.

This article was published in Hum. Mol. Genet. (2015) doi: 10.1093/hmg/ddv316.

Differential Immunogenicity of Cells Derived from Induced Pluripotent Stem Cells


Induced pluripotent stem cell (iPSC) technology has raised the possibility that patient-specific pluripotent stem cells may become a renewable source of a patient’s own cells for regenerative therapy without the concern of immune rejection. However, the immunogenicity of autologous human iPSC (hiPSC)-derived cells is not well understood.

Using a humanized mouse model (denoted Hu-mice) with a functional human immune system, Yang Xu and his colleagues from UC San Diego has shown that most teratomas or tumors formed by human iPSCs were readily recognized by immune cells and rejected. However, when these human iPSCs were differentiated into smooth muscle cells or retinal pigmented epithelial cells, the results were rather different. Human iPSC-derived smooth muscle cells appear to be highly immunogenic, but human iPSC-derived retinal pigment epithelial (RPE) cells are tolerated by the immune system, even when transplanted outside the eye.

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When Xu and others examined these results more closely, they discovered that this differential immunogenicity is due to the abnormal expression of cell surface proteins in hiPSC-derived Smooth Muscle Cells, but not in hiPSC-derived RPEs.

These findings support the feasibility of developing hiPSC-derived RPEs for treating macular degeneration. They also show that iPSC lines must be individually screened to determine if their derivatives are recognized by the patient’s immune system as foreign.

These results were published in Cell Stem Cell.

Gamida Cell Phase 3 Study Design Outline Approved by FDA and EMA


Gamida Cell, a cell therapy company based in Jerusalem, Israel, has reached agreements with the US Food and Drug Administration (USFDA) and the European Medicines Agency (EMA) with regards to a Phase III study design outline for testing their NiCord product. NiCord is a blood cancer treatment derived from a single umbilical cord blood until expanded in culture and enriched with stem cells by means of the company’s proprietary NAM technology.

Gamida Cell is moving forward now with plans to commence an international, multi-center, Phase III study of NiCord in 2016. Phase I/II data of 15 patients are expected in the fourth quarter of 2015. NiCord is in development as an experimental treatment for various types of blood cancers including Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Myelodysplastic Syndrome (MDS), and Chronic Myelogenous Leukemia (CML).

NiCord® is derived from a single cord blood unit which has been expanded in culture and enriched with stem cells using Gamida Cell’s proprietary NAM technology. NAM technology proceeds from the observation that nicotinamide, a form of vitamin B3, inhibits the loss of functionality that usually occurs during the culture process of umbilical cord blood stem cells, when added to the culture medium. Pre-clinical studies have also shown that the expanded cell grafts manufactured using NAM technology demonstrate improved functionality following infusion in a living animal. These stem cells show improved movement, home to the bone marrow, and show higher rates of engraftment, or durable retention in the bone marrow. Based on these results, Gamida Cell is currently testing in clinical trials (in patients) cells expanded in culture with the NAM platform to determine their safety and effectiveness as a treatment for blood cancers, sickle-cell anemia and thalassemia. NiCord is intended to fill the crucial clinical need for a treatment for the vast majority of blood cancer patients indicated for bone marrow transplantation, with insufficient treatment options. This segment has a multi-billion dollar market potential.

“The FDA and EMA feedback is a major regulatory milestone for NiCord. NiCord is a life-saving therapy intended to provide a successful treatment for the large number of blood cancer patients who do not have a family related matched donor. Gamida Cell is dedicated to changing the paradigm in transplantation by bringing this therapy to market as soon as possible,” said Dr. Yael Margolin, president and CEO of Gamida Cell.

“The positive regulatory feedback confirms that Gamida Cell’s NiCord program is on a clear path to approval both in the U.S. and EU. We look forward to continuing the development of this very important product in cooperation with sites of excellence in cord blood transplantation worldwide,” said Dr. David Snyder, V.P. of Clinical Development and Regulatory Affairs at Gamida Cell.

The Phase III study will be a randomized, controlled study of approximately 120 patients. It will compare the outcomes of patients transplanted with NiCord to those of patients transplanted with un-manipulated umbilical cord blood.

 

Regenerating Nerve Tissue in Spinal Cord Injuries


Severe injuries to the neck during recreational activities such as horseback riding or playing football can permanently alter someone’s life dramatically. With no options for the repair of spinal cord injuries, many are left with little hope for recovery.

New work by researchers at Rush University Medical Center (RUMC) in Chicago is investigating a new therapy that uses stem cells to treat spinal cord injuries within the first 14 to 30 days of injury. Rush is one of only two centers in the country currently studying this new approach.

“There are currently no therapies that successfully reverse the damage seen in the more than 12,000 individuals who suffer a spinal cord injury each year in the United States alone,” says Richard G. Fessler, MD, PhD, professor of neurological surgery at RUMC. An estimated 1.3 million Americans are living with a spinal cord injury.

“These injuries can be devastating, causing both emotional and physical distress, but there is now hope. This is a new era where we are now able to test whether a dose of stem cells delivered directly to the injured site can have an impact on motor or sensory function,” Fessler continued. “If we could generate even modest improvements in motor or sensory function, it would result in significant improvements in quality of life.”

Dr. Fessler is the principal investigator at RUMC of a clinical trial that involves progenitor cells that are likely to develop into a certain cell types. Specifically, this study is studying nerve cells known as oligodendrocyte progenitor cells, which potentially can make poorly functioning nerves function better. A San Francisco Bay-area biotechnology company known as Asterias Biotherapeutics, developed the cells and is sponsoring the trial.

This clinical trial is designed to assess the safety and efficacy of increasing doses of AST-OPC1 to treat individuals with a cervical spinal cord injury that resulted in tetraplegia, the partial or total paralysis of arms, legs and torso. As of mid-August, one individual has been enrolled in the study at Rush and there are high hopes that others will be enrolled as well in the near future.

Three escalating doses of AST-OPC1 will be examined in patients with subacute, neurologically complete injury to the cervical spinal cord (the spinal cord in the neck, specifically, the spinal nerves known as C5 to C7). These individuals essentially have lost all sensation and movement below their injury site and have severe paralysis of the upper and lower limbs.

In order for this therapy to work, the spinal cord must be continuous not severed. Patients must be able to begin treatment within 25 days of their injury.

Fessler and his group will administer AST-OPC1 between 14 to 30 days after sustaining the injury. Following the treatment, patients will receive frequent neurological exams and imaging in order to assess the efficacy of the treatment. Furthermore, patients will be followed for 15 years thereafter.

“If this treatment proves to be safe and effective, in the future, it also might be used for peripheral nerve injury or other conditions that affect the spinal cord, such as multiple sclerosis or ALS,” Fessler says.

The study is recruiting male and female patients ages 18 to 65 who have recently experienced a cervical spinal cord injury at the neck that resulted in partial or total paralysis of arms, legs and torso. All participants must be able to provide consent and commit to a long-term follow-up study.

Stem Cell Clinical Trials in 2014


Dr. Alexey Bersenev has done the stem cell community a great service by compiling the clinical trials that involved the used of stem cells for 2011-2014.

In 2014, there were 373 clinical trials registered in international databases that used stem cells.  36% of these trials were in the United States, 17% of them were in China, 8% in Japan, 5% in Spain, just under 5% were in India, 3.5 % were in South Korea and Iran, and 2% were in the UK.  To further break down these numbers according to geographical region, 36% were in the North America, 35% were in Asia, 19% were in Europe, 5% were in the Middle East, 3% were in Central and South America, and 2% were in Australia.

Of these clinical trials, 116 used mesenchymal stem cells, 81 used T-Cells, 31 used dendritic cells, 26 used mononuclear cells from bone marrow, 10 used Natural Killer cells, 22 used stromal vascular fraction (SVF) cells from fat, 16 used HSPCs (hematopoietic and progenitor cells) from bone marrow, and three were embryonic stem cell trials.

What were these trials trying to treat?  123 were for cancers of some sort, there were 51 trials examining neurological diseases and also 51 trials examining musculoskeletal disorders, 26 trials trying to help people with cardiovascular diseases, 17 attempting to treat skin diseases, 15 treating eye diseases, 8 that treated liver diseases, and 5 diabetes trials.

These are the rough trends.  As you can see, clinical trials that utilize adult and umbilical stem cell stem cells VASTLY outnumber those that use embryonic stem cells.

Bersenev Alexey. Trends in cell therapy clinical trials 2011 – 2014. CellTrials blog. February 14, 2015. Available: http://celltrials.info/2015/02/14/trends-2014/

Lab-Grown Muscle FIbers Aid in Studying Muscular Dystrophy


Skeletal muscle is the most abundant tissue in the human body, but, strangely, growing large quantities of it in the laboratory have proven rather challenging. While it is possible to reprogram other mature cells into heart muscle cells, or neurons, differentiating cells into skeletal muscle cells has simply not worked. So where do we go from here?

A new study from Brigham and Women’s Hospital (BWH) published in Nature Biotechnology has identified and even mimicked integral cues in the development of skeletal muscle. They used these cues to grow millimeter-long muscle fibers that are capable of contracting in the laboratory. This new method for growing functional muscle fibers in the laboratory potentially offer a better model for studying muscle diseases such as muscular dystrophy and for testing new treatments for these diseases.

Previous studies have used genetic modification techniques to grow small numbers of skeletal muscle cells in the laboratory. However, this new technique, which is the result of a collaboration between BWH and Harvard Stem Cell Institute, has produced a way to grow large numbers of skeletal muscle cells for use in clinical applications.

Olivier Pourquié of Harvard Medical School said, “We took the hard route: we wanted to recapitulate all of the early stages of muscle cell development that happen in the body and recreate that in a dish in the lab. We analyzed each stage of early development, and generated cell lines that glowed green when they reached a each stage. Going step by step, we managed to mimic each stage of development and coax cells toward muscle cell fate.”

The team found that a combination of secreted factors are important at the very early stages of embryonic development to stimulate muscle differentiation. By recapitulation this cocktail in the laboratory, Pourquié and his colleagues were able to mature muscle fibers in the laboratory from mouse or human pluripotent stem cells. Additionally, they produced muscle fibers in mice afflicted with muscular dystrophy by using muscle satellite cells. It is unknown if this method could help humans who suffer from muscular dystrophy, as more research is needed.

“This has been the missing piece: the ability to produce muscle cells in the lab could give us the ability to test out new treatments and tackle a spectrum of muscle diseases,” Pourquié said.

This new method also has the potential to help researchers study other muscle diseases, such as sarcopenia, or degenerative muscle loss and cachexia, the wasting away of muscle that typically occurs during severe illness.

Chemical-Only Cell Reprogramming Cocktails Direct Converts Skin Cells into Neurons


Two Chinese laboratories have independently transformed skin cells into neurons using only a cocktail of chemicals. One laboratory used skins cells from Alzheimer’s patients and the other used healthy laboratory mice, and therefore, the protocols developed by each laboratory differ. However, the success of these protocols suggests that it might be economically possible to use neurons made a patient’s own cells to test drug regiments for clinical purposes.

These two studies reinforce the idea that a purely chemical approach represents a promising way to scale up cell reprogramming research that might avoid the technical challenges and safety concerns associated with the more popular method of using transcription factors.

One of the challenges of reprogramming cells to change their identity is that you may end up with cells that look normal on the outside, but inside, many of their internal workings are quite different from the cell type you want to make. In these two papers, neurons made from chemically reprogrammed cells showed neuron-specific gene expression, action potentials, and synapse formation, which is strong evidence that these protocols produce fully operational neurons.

In both cases, the protocols employed decreased the expression of skin-specific genes and increased the expression of neuron-specific genes. These chemicals promoted neuronal cell fates by coordinating multiple signaling pathways that worked together to commit the cells to a neuronal fate. This direct reprogramming procedure bypasses the so-called “proliferative intermediate stage” that put cells under stress and increases the mutation rates. Therefore direct conversion protocols are inherently safer than other reprogramming protocols.

The paper from the laboratory of Jian Zhao (Cell Stem Cell 2015;17(2):204) designed a purely chemical protocol to convert skin cells from human Alzheimer’s disease patients into neurons. Direct reprogramming protocols are available for converting human skin cells into neurons, but these protocols require that cells be transfected with genes that encode transcription factors. Such manipulation requires that cells be treated with viruses or subjected to potentially stressful transfection conditions. This purely chemical protocol is a potentially welcome alternative that would be both safer and easier. The chemicals used in these procedures are easy to synthesize, stable, and standardization of the procedures would also be much easier.

The paper that uses a purely chemical protocol to directly reprogram mouse skin cells comes from the laboratory of Hongkui Deng (Cell Stem Cell 2015;17(2):195) is the culmination of four years of work. The main hurdle was suppressing skin-specific gene expression. Then Dong identified a compound called I-BET151 that suppressed skin cell-specific gene expression. This allowed Deng and his colleagues to successfully reprogram mouse cells with a purely chemical protocol.

The next step for both of these laboratories is to show that, in principle, these chemically reprogrammed cells can be used for therapeutic purposes. Such a proof-of-principle experiment will put direct reprogramming on the map for regenerative medicine in a powerful way.

3D Printed Sugar Network to feed Engineered Organs


A 3-D sugar network to feed bioengineered organs: moldable, biodegradable, and useable. In a word – Sweet!!

Leaders in Pharmaceutical Business Intelligence (LPBI) Group

3D Printed Sugar Network to feed Engineered Organs

Reporter: Irina Robu, PhD

“Tissue engineers have long dreamed of building an organ in a dish. But without vessels running through the tissue, cells in the centre starve and suffocate.

Now, US researchers can build vessels into a cell-containing gel – the beginnings of a thick tissue. Scientists form the gel around a lattice of printed sugar fibres. The fibres dissolve after the gel sets, leaving a network of channels that carry nutrients like blood vessels.

For the past decade, tissue engineers have looked for ways to build a 3D tissue in such a way that vessels are immediately available to feed growing cells. One way to create these vessels uses a tiny silicon template to pattern grooves in a sheet of cell-containing gel. Covering these cut outs with another sheet of engineered tissue creates enclosed channels. While these sheets can be…

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Gene Therapy for Blind Mice Might Lead to Human Trials


When a mouse sees an owl, it scurries for cover as fast as it can to escape the back-breaking talons of the swooping owl. If the mouse has defective vision and cannot properly see the owl, then the mouse is simply doomed. So ingrained is this escape response into the psyche of mice, that simply showing laboratory mice a video of a swooping owl will send them off into various directions for safety. Consequently, vision scientists can use this behavior to test treatments of blindness.

Rob Lucas of the University of Manchester, UK and his colleagues have played videos of swooping owls to normal and blind laboratory mice, and to blind mice that were treated with an experimental treatment for blindness. Lucas and others showed the owl video to mice that were so blind that they did not respond to the video. Then after giving these blind mice a treatment for blindness, those same mice were shown the same owl video, and they reacted as though they could see the swooping owl just fine. As Lucas explained: “You could say they were trying to escape, but we don’t know for sure. What we can say is that they react to the owl in the same way as sighted mice, whereas the untreated mice didn’t do anything.”

This best evidence acquired by Lucas and others to date that injecting the gene for a pigment that detects light into the eyes of blind mice can help them see again.

The gene therapy used by Lucas and others is meant to treat those types of blindness that are caused by damaged or missing photoreceptor, which are the cells in the neural retina that detect light. There are two types of photoreceptors in the retina: rods and cones. Rods and cones contain a pigment known as an “opsin,” which allows them to respond to light.  Opsin genes encode proteins that contain a vitamin A-based cofactor that helps it respond to light. The amino acid sequence of each opsin gene allows it to specifically respond to a range of frequencies of light. Different types of cones express specific opsins that allow them to specialize in the colors they can detect. Mutations in the opsin genes can cause blindness, and Lucas and his colleagues are interested in replacing the defective opsin genes in the retinas of laboratory mice. The majority of gene therapy experiments to treat blindness to date have concentrated on replacing faulty genes in rarer, specific forms of inherited blindness, such as

When a mouse sees an owl, it scurries for cover as fast as it can to escape the back-breaking talons of the swooping owl. If the mouse has defective vision and cannot properly see the owl, then the mouse is simply doomed. So ingrained is this escape response into the psyche of mice, that simply showing laboratory mice a video of a swooping owl will send them off into various directions for safety. Consequently, vision scientists can use this behavior to test treatments of blindness.

Rob Lucas of the University of Manchester, UK and his colleagues have played videos of swooping owls to normal and blind laboratory mice, and to blind mice that were treated with an experimental treatment for blindness. Lucas and others showed the owl video to mice that were so blind that they did not respond to the video. Then after giving these blind mice a treatment for blindness, those same mice were shown the same owl video, and they reacted as though they could see the swooping owl just fine. As Lucas explained: “You could say they were trying to escape, but we don’t know for sure. What we can say is that they react to the owl in the same way as sighted mice, whereas the untreated mice didn’t do anything.”

This best evidence acquired by Lucas and others to date that injecting the gene for a pigment that detects light into the eyes of blind mice can help them see again.

The gene therapy used by Lucas and others is meant to treat those types of blindness that are caused by damaged or missing photoreceptor, which are the cells in the neural retina that detect light. There are two types of photoreceptors in the retina: rods and cones. Rods and cones contain a pigment known as an “opsin,” which allows them to respond to light. Opsin genes encode proteins that contain a vitamin A-based cofactor that helps it respond to light. The amino acid sequence of each opsin gene allows it to specifically respond to a range of frequencies of light. Different types of cones express specific opsins that allow them to specialize in the colors they can detect. Mutations in the opsin genes can cause blindness, and Lucas and his colleagues are interested in replacing the defective opsin genes in the retinas of laboratory mice. The majority of gene therapy experiments to treat blindness to date have concentrated on replacing faulty genes in rarer, specific forms of inherited blindness, such as Leber congenital amaurosis.

Structures of opsins and of the chromophore retinal. (a) A model of the secondary structure of bovine rhodopsin. Amino-acid residues that are highly conserved in the whole opsin family are shown with a gray background. The retinal-binding site (K296) and the counterion position (E113) are marked with bold circles, as is E181, the counterion in opsins other than the vertebrate visual and non-visual ones. C110 and C187 form a disulfide bond. (b) The chemical structures of the 11-cis and all-trans forms of retinal. (c) The crystal structure of bovine rhodopsin (Protein DataBank ID: 1U19 [PDB:1U19]). The chromophore 11-cis-retinal, K296 and E113 are shown in stick representation in the ringed area. (d) The structure of the Schiff base linkage formed by retinal within the bovine opsin, together with the counterion that stabilizes it.
Structures of opsins and of the chromophore retinal. (a) A model of the secondary structure of bovine rhodopsin. Amino-acid residues that are highly conserved in the whole opsin family are shown with a gray background. The retinal-binding site (K296) and the counter ion position (E113) are marked with bold circles, as is E181, the counter ion in opsins other than the vertebrate visual and non-visual ones. C110 and C187 form a disulfide bond. (b) The chemical structures of the 11-cis and all-trans forms of retinal. (c) The crystal structure of bovine rhodopsin (Protein DataBank ID: 1U19 [PDB:1U19]). The chromophore 11-cis-retinal, K296 and E113 are shown in stick representation in the ringed area. (d) The structure of the Schiff base linkage formed by retinal within the bovine opsin, together with the counter ion that stabilizes it.  Taken from Terakita A, The opsins. Genome Biol 2005; 6(5):213.
The new treatment strategy employed by Lucas and others seek to enable other cells that lie just above the photoreceptors to capture light. Rod and cone cells normally detect light and convert it into an electrochemical signal that is sent to bipolar and then ganglion cells above them, which processing these signals and send them to the brain. By engineering bipolar or even ganglion cells to produce their own light-detecting pigment, they can to some extent compensate for the lost receptors, although the resolution of the vision is poor.

retina_schema

Lucas and others used the human gene for rhodopsin, the pigment used by rod cells to detect light and hooked this gene to a genetic “switch” that would only turn on the gene inside ganglion and bipolar cells. Then they inserted this DNA into a virus that infected the retinal cells of mice whose rods and cones had been destroyed.

After treatment, Lucas and his colleagues found that the mice could distinguish objects by their size quite well, but not as well as sighted mice. “The treated mice could discriminate black and white bars, but only ones that were 10 times thicker than what sighted mice could see,” says Lucas.

In earlier attempts, mice could only tell objects apart under extremely bright light. Therefore, this new finding is crucial. “Our mice could respond in ordinary light, the equivalent of looking at a computer monitor under ordinary office lighting,” says Lucas.

This is also the first time a human gene has been tested this way. The virus they used to deliver the gene therapy to mouse retinal cells has already been approved for use in humans, and Lucas says he hopes to begin trials of a human treatment in about five years.

“This is the most effective example yet of the use of genetic therapy to treat advanced retinal degeneration,” says Robin Ali, whose team at University College London has given gene therapy treatments of people with Leber congenital amaurosis.

But Robert Lanza, chief medical officer at Ocata Therapeutics in Marlborough, Massachusetts, warns that we don’t yet know how long the beneficial effects of the new treatment might last, since it seems that the sight in people with Leber congenital amaurosis who were treated with gene therapy between one and three years ago has begun to wane.

See Current Biology DOI: 10.1016/j.cub.2015.07.029.

Liver Cells from Circulating Blood Cells Under Clinically Safe Conditions


Can we convert circulating blood cells into working liver cells? Think of what this would mean for people who have liver problems. While is sounds like science fiction, the laboratory of James Ross at the University of Edinburgh, in collaboration with other scientists, has managed to do exactly that.

Ross and his colleagues developed an efficient method for converting circulating white blood cells into induced pluripotent stem cells (iPSCs). As previously mentioned on this blog, iPSCs are made from mature, adult cells by genetically engineering those cells with a cocktail of genes (in this case Oct4, Sox2, Klf4, L-Myc, and Lin28), and then culturing the cells in a special culture system that allows them to grow and become pluripotent stem cells that can theoretically differentiate into any of the 210 adult cell types in the human body.

Since the production of iPSCs from mature cells requires the insertion of particular genes into those cells, scientists typically use viruses or other vehicles to do this, which can introduce mutations into the genomes of the cells. Ross and his coworkers, however, used a non-integration method for reprogramming fresh or frozen white blood cells. They inserted small circles of DNA called “episomes” into these cells using a technique called “electroporation,” which binds the DNA to the surfaces of the cells and then subjects them to an electrical pulse that quickly moves the DNA into the cells without harming them. The genes on the episome are then expressed, but only transiently, which is all that is required to reprogram the adult cells into iPSCs. The cells were also cultured in a feeder-free system, which means that no animal products were involved in the production of these iPSC lines.  This constitutes, so-called “Good Manufacturing Practice” or GMP, which is required is a product is to be used for human patients.

Ross and others achieved a reprogramming efficiency of up to 0.033% (65 colonies from 2×105 seeded MNC), and when they used the same protocol to cord blood or fetal liver-derived blood-making (CD34+) cells, they achieved a reprogramming rate of 0.148% (148 iPSC colonies from 105 seeding cells). These iPSC lines were then used to make differentiated liver cells. This procedure tends to produce quasi-liver cells that do not have the characteristics of mature liver cells, but in this case, Ross and others derived cells that have proper drug metabolic function. This suggests that the iPSC-derived liver cells were at least mature enough to express many of the enzymes necessary to properly metabolize drugs. While these cells were probably not fully mature, they were a good deal further along than those derived in other experiments.

These experiments show that it is feasible to make liver cells for drug screen from circulating blood cells in a manner that is clinically safe. It is presently unclear if these cells can serve as material to heal a damaged liver, and that will take more work. Also, this procedure almost certainly would cost a good deal of money, and for that reason, banked iPSCs from white blood cells that have been fully tissue typed might be a better way to use cells made in this manner.

See Jing Liu, and others, Experimental Cell Research, 6 August 2015, Article ECR15383.

Cartilage Repair Using Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Embedded in Hyaluronic Acid Hydrogel in a Minipig Model


Cartilage shows lousy regenerative capabilities. Fortunately, it is possible to regenerate cartilage with human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) that have been embedded in a hyaluronic acid (HA) hydrogel composite. In fact, such a combination has shown remarkable results in rat and rabbit models.

In this present study, published in Stem Cells Translational Medicine, Yong-Geun Park and his colleagues from SungKyunKwan University School of Medicine, in Seoul, South Korea sought to confirm the efficacy of this protocol in a in a pig model using three different hUCB-MSC cell lines.

Park and his coworkers generated full-thickness cartilage injuries in the trochlear groove of each knee in 6 minipigs. Three weeks later, an even larger cartilage defect, 5 mm wide by 10 mm deep, was created, followed by an 8-mm-wide and 5-mm-deep boring. In short, the knee cartilages of these minipigs were very messed up.

Trochlear-groove

To these knee cartilages, a mixture (1.5 ml) of hUCB-MSCs (0.5 × 107 cells per milliliter) and 4% HA hydrogel composite were troweled into was then cartilage defects of the right knee. The left knee served as an untreated control. Each cell line was used in two minipigs.

Macroscopic findings of the osteochondral defects of the porcine knees. At 12 weeks postoperatively, the defects of both knees had produced regenerated tissues that were pearly white and firm. These new tissues, which resembled articular cartilage, appeared adherent to the adjacent cartilage and had restored the contour of the femoral condyles (smooth articular surfacewithout depression). The regenerated tissue of the control knee (left knee) looked fibrillated. Grossly, no differencewas seen in the quality of the repaired tissue in the transplanted knee (right knee) among the three groups with different cell lines. (A): Group A. (B): Group B. (C): Group C. Abbreviations: HA, hyaluronic acid; hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells.
Macroscopic findings of the osteochondral defects of the porcine knees. At 12 weeks postoperatively, the defects of both
knees had produced regenerated tissues that were pearly white and firm. These new tissues, which resembled articular cartilage, appeared adherent to the adjacent cartilage and had restored the contour of the femoral condyles (smooth articular surface without depression). The regenerated tissue of the control knee (left knee) looked fibrillated. Grossly, no difference was seen in the quality of the repaired tissue in the transplanted knee (right knee) among the three groups with different cell lines. (A): Group A. (B): Group B. (C): Group C. Abbreviations: HA, hyaluronic acid; hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells.

12 weeks after surgery, the pigs were sacrificed, and the degree of subsequent cartilage regeneration was evaluated by gross and more detailed microscopic analysis of the knee tissue. The transplanted knee showed superior and more complete joint-specific (hyaline) cartilage regeneration compared with the control knee. The microscopic characteristics of the knee cartilage showed that those animals that received the hUCB-MSCs had greater rates of cell proliferation and cells that differentiated into cartilage-making cells.

Microscopic findings of the regenerating osteochondral defects on porcine articular cartilage (safranin O and fast green staining). At 12 weeks postoperatively, the surface of the repairing tissue in the control knee (left knee) was poorly stained for glycosaminoglycan. In the transplanted knee (right knee), both the regenerated tissue and the adjacent cartilage to which it had become adherent exhibited the normal orthochromatic staining properties with safranin O. (A): Group A. (B): Group B. (C): Group C. Scale bars = 2 mm. Abbreviations: HA, hyaluronic acid; hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells.
Microscopic findings of the regenerating osteochondral defects on porcine articular cartilage (safranin O and fast green staining). At 12 weeks postoperatively, the surface of the repairing tissue in the control knee (left knee) was poorly stained for glycosaminoglycan. In the transplanted knee (right knee), both the regenerated tissue and the adjacent cartilage to which it had become adherent exhibited the normal orthochromatic staining properties with safranin O. (A): Group A. (B): Group B. (C): Group C. Scale bars = 2 mm. Abbreviations: HA, hyaluronic acid; hUCB-MSCs, human umbilical cord blood-derived mesenchymal stem cells.

These data show consistent cartilage regeneration using composites of hUCB-MSCs and HA hydrogel in a large animal model. These experiments could be a stepping stone to a human clinical trial in the future that treats osteoarthritis of the knees with hUCB-MSCs embedded in HA hydrogel.

Converting Immune Cell into Another Type of Immune Cell


What does it take to directly convert an antibody-producing B cell into a scavenging macrophage? The answer: one gene, according to a report in the July 30th issue of Stem Cell Reports. This directly reprogramming is transformation is possible because a transcription factor called C/EBPa can short-circuit the cells so that they re-express genes reserved for embryonic development.

Over the past 65 years, research teams in laboratories all over the world have shown that many different types of specialized cell types can be forcibly reprogrammed into another, but how this occurs is only recently been realized. These “transdifferentiations,” as they’re called, include reprogramming a skin cell into a muscle cell, or a muscle cell into a brown fat cell with the addition of just one or two transcription factors that bind to a cell’s DNA and induce the expression of other genes.

“For a long time it was unclear whether forcing cell fate decisions by expressing transcription factors in the wrong cell type could teach us something about what happens normally during physiological differentiation,” said senior study author Thomas Graf, Ph.D., group leader at the Centre for Genomic Regulation in Spain. “What we have now found is that the two processes are actually surprisingly similar.”

According to lead author of this study, Chris van Oevelen, Ph.D., B cell transdifferentiation occurs when C/EBPa binds to two regions of DNA that act as gene expression enhancers. One of these regions is typically active in immune cells, but the other is only activated when macrophage precursors are ready to differentiate. Thus, the synergism of these two enhancer pathways can cause the B cell to act like a macrophage precursor, which triggers B cell-to-macrophage transdifferentiation.

“This has taught us a great deal about how a transcription factor can activate a new gene expression program (in our case, that of macrophages) but has left us in the dark about the other part of the equation; namely, how the factor silences the B cell program, something that must happen if transdifferentiation is to work,” Dr. Graf said. “This is one of the questions we are focusing on now.”

Dr. Graf is interested in this pathway because of its potential therapeutic applications. As it turns out, C/EBPa-induced B cell-to-macrophage transdifferentiation can convert both human B cell lymphoma or leukemia cells into functional, non-cancerous macrophages. Graf believes that induced transdifferentiation could become therapeutically relevant if drug researchers can find a molecule that can replace C/EBPa. Additionally, understanding the mechanisms of this process would help labs worldwide who use transdifferentiation approach to generate cells for regenerative purposes.

First Clinical Trial for Genetically Engineered Stem Cell Treatment for Pulmonary Arterial Hypertension


A Canadian research team has published the results of the world’s first clinical trial of a genetically enhanced stem cell therapy for pulmonary arterial hypertension (PAH).

PAH is a rare and deadly disease that mainly affects young women, and is characterized by very high blood pressure in those arteries that supply blood to the lungs. Some cases of PAH are caused by mutations in the BMPR2 gene, but in many cases the cause remains unknown. Currently, PAH patients are treated with combination of various drug and oxygen. Drug treatments include blood vessel dilators, such as epoprosternol (Flolan or the inhaled form known as iloprost or Ventavis), endothelin receptor antagonists, such as bosentan (Tracleer) or ambrisentan (Letaris), sildenafil (Viagra) or tadalafil (Cialis), high doses of calcium channel blockers, anticoagulants, and diuretics. Such treatments can improve symptoms and exercise capacity (at best), but they cannot repair the blood vessel damage to the lungs or cure the disease.

This new study, entitled “Endothelial NO-Synthase Gene-Enhanced Progenitor Cell Therapy for Pulmonary Arterial Hypertension: the PHACeT Trial“ was published in the journal Circulation Research, and was coauthored lead investigator Duncan J. Stewart of the Ottawa Hospital Research Institute, and his collaborators.

The paper describes PAH as a progressive and eventually lethal disease that is characterized by eventual loss of functional lung microvasculature. This paper also argues that cell-based therapies offer the possibility of repairing and regenerating the lung microcirculation. The paper also reports that stem-cell therapy has shown promise in a pre-clinical evaluation that utilized experimental models of PAH.

This trial was a phase 1, dose-escalating clinical study whose goal was to test the tolerability, feasibility, and side-effects of a genetically-enhanced stem cell therapy to repair and regenerate lung blood vessels in PAH patients. Seven PAH patients who volunteered for this study underwent a blood cell selection process known as apheresis in order to harvest a certain population of white blood cells from their blood. These white blood cells were grown in the laboratory under special conditions that specifically selected for stem-like cells called endothelial progenitor cells (EPCs). These EPCs were genetically engineered to produce greater amounts of nitric oxide synthase, which makes the signaling molecule, nitric oxide (NO), a natural substance that widens blood vessels and is essential for efficient vascular repair and regeneration. These genetically enhanced cells were then injected directly into the lung circulation of the patient from whom there were originally harvested.

Of these seven patients, five were female and two were male, and all seven patients received treatment from December 2006 to March 2010. Continued observation and follow-up exams of these patients showed that the cell infusion procedure was well tolerated, and, on the whole, these patients showed a trend towards improvement in total pulmonary resistance (TPR) over the three-day delivery period. However, there was one serious adverse event (death) that occurred immediately after discharge in a patient who had severe, end-stage disease.

These investigators concluded that delivery of EPCs overexpressing eNOS was tolerated in PAH patients, and also produced evidence of short-term improvements, associated with long-term benefits in functional and quality-of-life assessments. However, they caution that future studies will be needed in order to further establish the efficacy of this therapy.

It must be noted that this study was not designed to rigorously assess the benefits the stem cell therapy versus a placebo. However, this research group observed improved blood flow in the lungs of patients during days following the therapy, and enhanced ability to exercise and better quality of life for up to six months after the therapy. Once again, I must provide the caveat that since this was not a double-blinded, placebo-controlled study, it is no possible to determine for sure if these observed effects were due to the cells or to psychological effects.

The therapy was generally well-tolerated, but one patient who had very severe and disease and signs of poor prognosis died one day after treatment. As unfortunate as this is, it is an expected outcome, given how sick the patient was and given their declining condition prior to treatment.

“Pulmonary arterial hypertension is a deadly and incurable disease that often strikes people in the prime of their life,” says the Circulation Research paper’s senior author Dr. Duncan Stewart, a practicing cardiologist and Executive Vice-President of Research at The Ottawa Hospital, and a professor of medicine at the University of Ottawa. “We desperately need new therapies for this disease, and regenerative medicine approaches have shown great promise in laboratory models and in clinical trials for other conditions.”

“This trial shows that genetically-enhanced stem cell therapy is a promising treatment approach for pulmonary arterial hypertension,” observes Dr. Stewart. “Although this is an important start, we will need to do larger studies to establish whether this therapy can produce important and durable benefits for people suffering from this challenging disease.”

Dr. Stewart is also the lead researcher of the first clinical trial in the world of a genetically-enhanced stem cell therapy for heart attack.