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Category Archives: Stem Cell Human Trials

Stem Cell FAQ

Posted: October 19, 2015 at 6:41 am

Some of the promise of stem cell therapy has been realized. A prime example is bone marrow transplantation. Even here, however, manyproblems remain to be solved.

Challenges facing stem cell therapy include the following:

Adult stem cells Tissue-specific stem cells in adult individuals tend to be rare. Furthermore, while they can regenerate themselves in an animal or person they are generally very difficult to grow and to expand in the laboratory. Because of this, it is difficult to obtain sufficient numbers of many adult stem cell types for study and clinical use. Hematopoietic or blood-forming stem cells in the bone marrow, for example, only make up one in a hundred thousand cells of the bone marrow. They can be isolated, but can only be expanded a very limited amount in the laboratory. Fortunately, large numbers of whole bone marrow cells can be isolated and administered for the treatment for a variety of diseases of the blood. Skin stem cells can be expanded however, and are used to treat burns. For other types of stem cells, such as mesenchymal stem cells, some success has been achieved in expanding the cellsin vitro, but application in animals has been difficult. One major problem is the mode of administration. Bone marrow cells can be infused in the blood stream, and will find their way to the bone marrow. For other stem cells, such as muscle stem cells, mesenchymal stem cells and neural stem cells, the route of administration in humans is more problematic. It is believed, however, that once healthy stem cells find their niche, they will start repairing the tissue. In another approach, attempts are made to differentiate stem cells into functional tissue, which is then transplanted. A final problem is rejection. If stem cells from the patients are used, rejection by the immune system is not a problem. However, with donor stem cells, the immune system of the recipient will reject the cells, unless the immune system is suppressed by drugs. In the case of bone marrow transplantation, another problem arises. The bone marrow contains immune cells from the donor. These will attack the tissues of the recipient, causing the sometimes deadly graft-versus-host disease.

Pluripotent stem cells All embryonic stem cell lines are derived from very early stage embryos, and will therefore be genetically different from any patient. Hence, immune rejection will be major issue. For this reason, iPS cells, which are generated from the cells of the patient through a process of reprogramming, are a major breakthrough, since these will not be rejected. A problem however is that many iPS cell lines are generated by insertion of genes using viruses, carrying the risk of transformation into cancer cells. Furthermore, undifferentiated embryonic stem cells or iPS cells form tumors when transplanted into mice. Therefore, cells derived from embryonic stem cells or iPS cells have to be devoid of the original stem cells to avoid tumor formation. This is a major safety concern.

A second major challenge is differentiation of pluripotent cells into cells or tissues that are functional in an adult patient and that meet the standards that are required for ‘transplantation grade’ tissues and cells.

A major advantage of pluripotent cells is that they can be grown and expanded indefinitely in the laboratory. Therefore, in contrast to adult stem cells, cell number will be less of a limiting factor. Another advantage is that given their very broad potential, several cell types that are present in an organ might be generated. Sophisticated tissue engineering approaches are therefore being developed to reconstruct organs in the lab.

While results from animal models are promising, the research on stem cells and their applications to treat various human diseases is still at a preliminary stage. As with any medical treatment, a rigorous research and testing process must be followed to ensure long-term efficacy and safety.

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Stem Cell FAQ

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Ready or Not: Stem Cell Therapies Poised to Enter Trials …

Posted: October 12, 2015 at 11:44 am

20 Nov 2014

Stem cells have been hailed, and hailed some more, as a breakthrough technology. All the same, they have been slow to make real inroads in the understanding and treatment of Alzheimers disease. That is about to change, according to scientists who spoke at Accelerating the Cure for Alzheimers Disease through Regenerative Medicine. Held November 6 at Duke University, Durham, North Carolina, the symposium was co-chaired by Murali Doraiswamy and Joanne Kurtzberg. Kurtzberg is a pediatrician and cell therapy expert atDuke.

The first clinical trials of stem cells for AD are expected to begin in 2015, speakers said. Some cautioned that many questions remain about how stem cells affect the Alzheimers brain. They debated whether the move into the clinic is premature, noting the need for more research into where in the brain stem cells go and how long they last. On this, attendees were intrigued by some success tracking injected cells with MRI. In addition to therapeutic applications, induced stem cells made from patients with AD and related disorders are helping shed light on disease mechanisms and enabling screens for potentially therapeutic compounds. Research on stem cells remains limited, however, in part because it is largely supported by state initiatives, such as the California Institute for Regenerative Medicine, and private foundations, such as the New York Stem CellFoundation.

Can Stem Cells Treat Alzheimers? Therapies based on stem cells have had some success in other diseases. Transplants of cord blood, for example, are approved by the Food and Drug Administration to treat leukemia and inborn errors of metabolism, Kurtzberg said in a talk in which she described treatments pioneered by the Duke Stem Cell and Regenerative Medicine Program. Clinicians irradiate the patients bone marrow before administering cord blood, then stem cells in the donated blood engraft and replace unhealthy cells. In the last 25 years, more than 35,000 such life-saving transplants have taken place, Kurtzbergnoted.

In Alzheimers disease, however, neurons die off in massive numbers throughout the brain, making cell replacement impractical. This has caused many researchers to overlook stem cell therapy as an option for AD, Mahendra Rao said in his keynote address. Rao leads regenerative medicine at the New York Stem Cell Foundation Research Institute, New York City. Nonetheless, stem cells have demonstrated the ability to improve cognition in animal models. Rather than replacing neurons, they may benefit the brain in other ways, such as by modulating inflammation, stimulating remyelination, and supplying trophic support. This may enhance the life of dying neurons, Rao said. Would injected cells succumb to the surrounding disease, as has been found to happen with fetal neuron grafts in Parkinsons patients (see Apr 2008 news story;Jun 2014 news story)?Quite the opposite, Rao believes. Injected stem cells, which often mature into glia, may help modify the brain environment, thus lowering itstoxicity.

One example of how stem cells can promote neuron health came some time ago from Frank LaFerla of the University of California, Irvine. LaFerla injected mouse neural stem cells into the brains of animals that modeled hippocampal sclerosis and Alzheimers disease. Treated mice improved cognitively but, in synch with what Rao said, injected cells neither became neurons nor lowered A or tau pathology. Instead, they promoted formation of synapses in the CA1 region of the hippocampus, an effect LaFerla traced to stem cells releasing brain-derived neurotrophic factor (BDNF). Knocking down this growth factor in the cells before injecting them abrogated the treatment benefit (see Jul 2009 news story).Could stem cells slow disease progression? When LaFerla modified neural stem cells to express the A-degrading enzyme neprilysin, treatment with these cells lowered A deposits throughout mouse brain (see Aug 2010 conference story).It is not clear if this step improves cognition further, LaFerla said in answer to an audiencequestion.

Stem cells may also replace sick glia. Kurtzberg described the use of cord blood stem cells to treat Krabbes disease in children. This genetic disorder is caused by the lack of the enzyme galactosylceramidase, which helps maintain the myelin wrapped around axons. Babies born with this condition develop muscle weakness and seizures, and most die before they are 2 years old. However, those who receive cord blood transplants around one month of age, before symptoms appear, survive and generally thrive, with only mild development delays, Kurtzberg said (see Escolar et al., 2005).

Most of the stem cells that engraft in the brains of these children become glia. Studying engrafted cells in postmortem samples, Kurtzbergs group found that they shared properties of microglia and oligodendrocytes. In mouse models, these cells secrete soluble anti-inflammatory factors, stimulate neurogenesis, and promote remyelination. The researchers are now generating these cells in vitro and are about to begin a Phase 1 trial to test whether delivering them into cerebrospinal fluid, in addition to the standard cord blood transplant, is safe in children with Krabbes disease. The differentiated cells should engraft more quickly than do stem cells, which take months to provide the full benefit, and thus may improve outcomes, Kurtzbergnoted.

Amyloid deposits (green) in cortex and hippocampus of mice treated with human mesenchymal stem cells (right) are reduced by one-third compared to untreated mice (left). [Image courtesy of Alexei Lukashev and Tristan Bolmont, StemedicaInternational.]

In other cases, it is not yet clear what the stem cells do. Researchers led by Alexei Lukashev of the biotech company Stemedica International, Epalinges, Switzerland, presented a poster on the use of human adult mesenchymal stem cells to combat Alzheimers disease. First author Tristan Bolmont injected the cells into the bloodstream of 15-month-old APP/PS1 miceonce per week for 10 weeks. The amyloid load in the hippocampus of treated mice dropped by one-third compared with untreated controls. Meanwhile, more microglia clustered around plaques, while the number of pro-inflammatory microglia shrank, suggesting that the stem cells somehow influence this balance. In ongoing work, the authors are characterizing this, as well as testing behavior in treated mice. The cells appeared safe, with no increase in vascular amyloid ormicrohemorrhage.

Human mesenchymal stem cells are currently in a Phase 1/2 clinical trialfor stroke. Lukashev said he is applying for FDA approval for a Phase 2 Alzheimers study to start next year. Similar approaches by other groups are in trials for multiple sclerosis and amyotrophic lateral sclerosis (ALS) (see Oct 2010 news story).

Other stem cell-based Alzheimers treatments are also on the threshold of the clinic. Ellen Feigal, who leads research and development at the California Institute for Regenerative Medicine (CIRM),noted that her organization has given out 17 awards for Alzheimers projects; three of those are now applying for FDA approval for trials. Two academic studies identified small neuroprotective molecules through screens of stem cells; the third, led by Alexandra Capela at the biotech company StemCells Inc., Newark, California, proposes to transplant neural stem cells into AD patients (see CIRM award).

Conference attendees disagree whether the technology is ready for human study. We are rushing too fast to the clinic, LaFerla cautioned, noting that many questions of basic science remain
ed to be answered. Also, researchers do not yet know how many cells to deliver, where, and how often in order to optimize the response, he said. Bolmont conceded that questions remain, but said he felt many of them can be answeredonlyby trials. We could do two more years of mouse studies, and we would still have the same question about whether these treatments will work in humans, he told Alzforum. Feigal urged that trials and research run in parallel. We dont need to paralyze trials while answering basic questions. Clinical trials can inform research. It is a two-way street, shesaid.

Thomas Finn from the FDA spelled out potential safety concerns for which his agency will watch. They include whether intravenous injections of stem cells might block capillaries, causing embolisms and damaging brain tissue. Another question is whether stem cells might give rise to tumors, or make the wrong kind of cells or connections in the brain, leading to side effects like chronicpain.

Stem cells labeled with the MRI agent SPIO spread through one brain hemisphere after injection into a carotid artery. [Image courtesy of Piotr Walczak, Miroslaw Janowski, Jeff W.M. Bulte, etal.]

Tracking Cells in Vivo To allay these concerns, researchers want to be able to follow injected stem cells to see where they go and what kind of cells they become. In his talk, Jeff Bulte, a radiologist at Johns Hopkins University, Baltimore, discussed ways to label stem cells with contrast agents so that MRI can tracked them (see Ahrens and Bulte, 2013). One such agent, superparamagnetic iron oxide (SPIO), is sensitive and appears safe, having been used in several clinical trials. In a human case study, SPIO maintained a signal for more than four months (see Janowski et al., 2014). However, this compound has been pulled from the market for economic reasons, Bulte toldAlzforum.

Probes containing the stable isotope fluorine-19 (19F) emit a bright signal but are less sensitive than SPIO (see Ruiz-Cabello et al., 2008). Celsense Inc. of Pittsburgh markets these probes, which are in use in at least one clinical trial. Bultes own company, SenCEST LLC, Fulton, Maryland, develops chemical exchange saturation transfer (CEST) agents. Instead of using metal, the exchange of a proton between the agent and surrounding water creates the signal. All three approaches demonstrate potential for human use at this time, Bultesaid.

With all these labels, however, the signal fades over time. Bulte is experimenting with reporter genes that could give long-term information on injected cells, with the added advantage of turning off when cells die. As an example, he said that stem cells can be engineered to express a thymidine kinase from herpes simplex virus, which is then detected by a thymidine analogue probe using CEST (see Bar-Shir et al., 2013;Bar-Shir et al., 2013). Bulte also found that stem cells can be targeted to areas of inflammation by making them express the docking protein VLA-4 on their cell surface. This molecule binds to VCAM-1, which is expressed by inflamed endothelial cells. This way, stem cells penetrate into the brain three- to fourfold better, Bulte claimed (see Gorelik et al., 2012).

Such tracking technologies provide a glimpse into the life of injected cells. Using MRI labels, Bulte found that stem cells injected into carotid arteries fanned out across the brain more broadly than cells injected into the brains ventricles or parenchyma (see image above). In an animal study, injection of large cells by this route did not disrupt the integrity of blood vessels in the brain, Bultesaid.

Modeling Disease: What if Craig Had an Alzheimer Mutation? Short of treating disease, researchers hope that stem cells will at least model human disease more faithfully than do animals or cell lines. Lawrence Goldstein of the University of California, San Diego, previously reported that induced neurons generated from people with APP mutations produced loads of A40 and phosphorylated tau (see Jan 2012 news story).At Duke, Goldstein noted that genetic variability between individual people poses a problem for modeling disease with induced pluripotent stem (iPS) cells. To address this, he generated iPS cells from a person whose genome was fully sequenced: genetics pioneer Craig Venter (see Nov 2008 news story).Then he introduced mutations in either APP or presenilin and compared the results. To his surprise, presenilin mutations did not boost phosphorylated tau, while APP mutations did. Why this difference? Goldstein does not know yet. Currently, he is culturing induced Venter neurons with astrocytes to see if cellular interactions might influence tauprocessing.

Neurons in iPS cells can model other neurodegenerative diseases as well. Chris Henderson of Columbia University, New York City, generated induced corticospinal motor neurons, which selectively degenerate in ALS. Henderson compared their gene expression profile to that of oculomotor neurons, which remain intact throughout the disease, to learn what makes spinal motoneurons so vulnerable. He turned up 15 candidate susceptibility genes and four potential resistance genes. When he knocked out one of the susceptibility genes, matrix metalloproteinase-9 (MMP-9), from ALS mouse models, they lived 80 days longer than littermate controls, extending their lifespan by about one-quarter. MMP-9 is expressed almost exclusively by motor neurons and might make an attractive therapeutic target, Henderson suggested (see Kaplan et al., 2014).

Researchers also use induced neurons to screen drugs. Goldstein screened a library of 3,000 compounds on induced neurons from AD patients. He found four classes of drug that lowered A, and said he is working on advancing some of them to the clinic. Henderson screened 50,000 compounds on induced motor neurons to find some that stimulated axon growth in an inhibitory environment. Surprisingly, statins boosted axon extension the most, leading to a 30-fold increase in growth. Henderson believes that the drugs are acting via a mechanism other than cholesterol. Statins themselves would not make good ALS therapeutics because they barely enter the brain and have systemic side effects, he noted. He is looking for other ways to stimulate the samepathway.

Attendees debated whether stem cells will lead to personalized medicine. In theory, clinicians could use iPS cells to generate replacement cells that contain a patients own DNA. By studying iPS cells from multiple donors, researchers could find specific genetic factors that predict whether a given patient will respond to a particular therapy. Clinicians want these options, speakers said, but pharmaceutical companies protest that such approaches would not be commercially viable. In practice, stem cell therapies will not attempt genetic matching in the foreseeable future, clinicians agreed. The question of whether autologous injected stem cells will last for a lifetime without inflaming the immune system will likely be answered sooner.Madolyn BowmanRogers

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Cell Trials

Posted: October 1, 2015 at 3:42 pm

Starting from 2014, I was trying to capture results of clinical studies in cell therapy. Today, Id like to share some results of this attempt. I decided to narrow down my analysis to regenerative medicine, since most of cell-based therapies with published results belong to this category.

Inclusion criteria and definitions

Exclusion criteria

Search strategy I set very loose filter for search of clinical studies results by using PubMed database with query stem cell. I receive notifications about new publications from this query via RSS feed. Stem cell query captures about 50-100 publications daily, 90% of clinical studies.

Total number of studies and data capture

Demographics A great variety of countries published results of clinical studies in 2014. The biggest number of reports came from China. US was the second biggest contributor. I summarized major contributing countries (more than 4 reports) in this figure:

Cell types As one may predict, Mesenchymal Stromal Cells (MSC) was the major cell type (35%) in cell-based regenerative medicine clinical studies. Mobilized hematopoietic stem/progenitor cells (HPC-A) and bone marrow mononuclear cells (BM MNC) were another popular type of cells for tissue regeneration. Interestingly, 2 reports were about results of studies, involved embryonic stem cell-based (ESC) products. Few studies used 2 different types of cells cell simultaneously or concurrently.

Results interpretation I loosely judged all results of clinical studies as (1) positive, (2) mixed/ inconclusive and (3) failed. Positive are the results, reported no safety and feasibility concerns and/or provided at least some evidence of efficacy. Mixed/ inconclusive results included:

Failed studies were considered, based on termination of studies due to safety issues, lack of feasibility and/ or lack of efficacy. Failed efficacy usually reported by authors as lack of difference between control group (example: placebo) and experimental (cells) group. Few trials were not described as failed by authors, but judged as such by me, based on lack of significance between groups and missed end points.

The first figure shows total number of positive, mixed/ inconclusive and failed studies from analysis of all reported studies (116):

Next, I looked at only registered clinical trials and broke it down by phases 1, 1/2, 2, 2/3 and 3:

Only one trial is failed in phase 1 and almost 90% of them reported as positive. The only trial, which failed in phase 1 was designed with efficacy end points. There were no trials, which failed safety. A number of mixed/inconclusive and failed trials were increased from phase 1 to 2/3. Because efficacy of therapy is usually assessed in phase 2 trials, studies labeled as phase 1/2 are not necessarily included efficacy end points. Despite lack of any controls many studies concluded by authors as positive or promising. Placebo control was very rare. Most controls include: (i) historic, (ii) baseline and (iii) standard therapy.

Limitations of analysis

It was a snapshot of some data that I was able to capture in 2014. Please feel free to give me feedback and discuss these data in comments. Im open to suggestions and collaboration.

How to cite: Bersenev Alexey. Results of regenerative medicine clinical studies from 2014. CellTrials blog. March 1, 2015. Available:

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Cell Trials

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UK scientists start stem cell trial of potential blindness …

Posted: September 29, 2015 at 1:41 am

LONDON The first patient has been treated in Britain in a pioneering trial of a new treatment co-developed by Pfizer and derived from embryonic stem cells designed for patients with a condition that can cause blindness.

Specialists at London’s Moorfields Eye Hospital said the operation, described as “successful”, was the first of 10 planned for participants in a trial of the treatment for a disease called ‘wet’ age-related macular degeneration (AMD).

The trial will test the safety and efficacy of transplanting eye cells known as retinal pigment epithelium, which have been derived from embryonic stem cells.

Stem cells are the body’s master cells, the source of all other cells. Scientists who support the use of embryonic stem cells say they could transform medicine, providing treatments for blindness, juvenile diabetes or severe injuries. But critics object to them because they are harvested from human embryos.

This trial involves surgeons inserting a specially engineered patch behind the retina to deliver the treatment cells to replace diseased cells at the back of the eye.

The first surgery was successfully performed on a patient last month, Moorfields said in a statement on Tuesday, and “there have been no complications to date”.

“The patient wishes to remain anonymous, but the team hope to determine her outcome in terms of initial visual recovery by early December,” it added.

Retinal surgeon Lyndon Da Cruz, who is performing the operations, said he hoped many patients “will benefit in the future from transplantation of these cells.”

Macular degeneration accounts for almost 50 percent of all cases of blindness or vision loss in the developed world. It usually affects people over 50 and comes in two forms, wet and dry. Wet AMD, which is less common than dry AMD, is generally caused by abnormal blood vessels that leak fluid or blood into a region in center of the retina.

This trial is part of The London Project to Cure Blindness – a partnership between Moorfields, University College London’s (UCL) Institute of Ophthalmology, and Britain’s National Institute for Health Research. The U.S. pharmaceutical giant Pfizer joined in 2009.

Chris Mason, a professor of regenerative medicine at UCL, said the trial is important both as potential step towards curing a major cause of blindness, and as a way of deepening understanding of the use of embryonic stem cells in treatments.

“If the AMD trials are successful, then by using embryonic stem cells as the starting material, the therapy can then be affordably manufactured at large scale,” he said.

(Editing by David Evans)

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All Things Stem Cell Visual Stem Cell Glossary

Posted: September 27, 2015 at 1:44 am

Stem cells: Cells that are able to (1) self-renew (can create more stem cells indefinitely) and (2) differentiate into (become) specialized, mature cell types.

Embryonic stem cells: Stem cells that are harvested from a blastocyst. These cells are pluripotent, meaning they can differentiate into cells from all three germ layers.

Embryonic stem cells are isolated from cells in a blastocyst, a very early stage embryo. Once isolated from the blastocyst, these cells form colonies in culture (closely packed groups of cells) and can become cells of the three germ layers, which later make up the adult body.

Adult stem cells (or Somatic Stem Cell): Stem cells that are harvested from tissues in an adult body. These cells are usually multipotent, meaning they can differentiate into cells from some, but not all, of the three germ layers. They are thought to act to repair and regenerate the tissue in which they are found in, but usually they can differentiate into cells of completely different tissue types.

Adult stem cells can be found in a wide variety of tissues throughout the body; shown here are only a few examples.

The Three Germ Layers: These are three different tissue types that exist during development in the embryo and that, together, will later make up the body. These layers include the mesoderm, endoderm, and ectoderm.

The three germ layers form during the gastrula stage of development. The layers are determined by their physical position in the gastrula. This stage follows the zygote and blastocyst stages; the gastrula forms when the embryo is approximately 14-16 days old in humans.

Endoderm: One of the three germ layers. Specifically, this is the inner layer of cells in the embryo and it will develop into lungs, digestive organs, the liver, the pancreas, and other organs.

Mesoderm: One of the three germ layers. Specifically, this is the middle layer of cells in the embryo and it will develop into muscle, bone, blood, kidneys, connective tissue, and related structures.

Ectoderm: One of the three germ layers. Specifically, this is the outer layer of cells in the embryo and it will develop into skin, the nervous system, sensory organs, tooth enamel, eye lens, and other structures.

Differentiation, Differentiated: The process by which a stem cell turns into a different, mature cell. When a stem cell has become the mature cell type, it is called differentiated and has lost the ability to turn into multiple different cell types; it is also no longer undifferentiated.

Directed differentiation: To tightly control a stem cell to become a specific mature cell type. This can be done by regulating the conditions the cell is exposed to (i.e. specific media supplemented with different factors can be used).

The differentiation of stem cells can be controlled by exposing the cells to specific conditions. This regulation can cause the cells to become a specific, desired mature cell type, such as neurons in this example.

Undifferentiated: A stem cell that has not become a specific mature cell type. The stem cell holds the potential to differentiate, to become different cell types.

Potential, potency: The number of different kinds of mature cells a given stem cell can become, or differentiate into.

Totipotent: The ability to turn into all the mature cell types of the body as well as embryonic components that are required for development but do not become tissues of the adult body (i.e. the placenta).

A totipotent cell has the ability to become all the cells in the adult body; such cells could theoretically create a complete embryo, such as is shown here in the early stages.

Pluripotent: The ability to turn into all the mature cell types of the body. This is shown by differentiating these stem cells into cell types of the three different germ layers.

Embryonic stem cells are pluripotent cells isolated from an early stage embryo, called the blastocyst. These isolated cells can turn into cells representative of the three germ layers, all the mature cell types of the body.

Multipotent: The ability to turn into more than one mature cell type of the body, usually a restricted and related group of different cell types.

Mesenchymal stem cells are an example of multipotent stem cells; these stem cells can become a wide variety, but related group, of mature cell types (bone, cartilage, connective tissue, adipose tissue, and others).

Unipotent: The ability to give rise to a single mature cell type of the body.

Tissue Type: A group of cells that are similar in morphology and function, and function together as a unit.

Mesenchyme Tissue: Connective tissue from all three germ layers in the embryo. This tissue can become cells that make up connective tissue, cartilage, adipose tissue, the lymphatic system, and bone in the adult body.

Mesenchyme tissue can come from all three of the germ layers (ectoderm, mesoderm, and endoderm) in the developing embryo, shown here at the gastrula stage. The mesenchyme can become bone, cartilage, connective tissue, adipose tissue, and other components of the adult body.

Hematopoietic Stem Cells: Stem cells that can create all the blood cells (red blood cells, white blood cells, and platelets). These stem cells reside within bone marrow in adults and different organs in the fetus.

Hematopoietic stem cells can become, or differentiate into, all the different blood cell types. This process is referred to as hematopoiesis.

Bone marrow: Tissue within the hollow inside of bones that contains hematopoietic stem cells and mesenchymal stem cells.

Development: The process by which a fertilized egg (from the union of a sperm and egg) becomes an adult organism.

Zygote: The single cell that results from a sperm and egg uniting during fertilization. The zygote undergoes several rounds of cell division before it becomes an embryo (after about four days in humans).

When an egg is fertilized by a sperm, the resultant single cell is referred to as a zygote.

Blastocyst: A very early embryo (containing approximately 150 cells) that has not yet implanted into the uterus. The blastocyst is a fluid-filled sphere that contains a group of cells inside it (called the inner cell mass) and is surrounded by an outer layer of cells (the trophoblast, which forms the placenta).

The blastocyst contains three primary components: the inner cell mass, which can become the adult organism, the trophoblast, which becomes the placenta, and the blastocoele, which is a fluid-filled space. The blastocyst develops into the gastrula, a later stage embryo.

Inner Cell Mass: A small group of cells that are attached inside the blastocyst. Human embryonic stem cells are created from these cells in blastocysts that are four or five days post-fertilization. The
cells from the inner cell mass have the potential to develop into an embryo, then later the fetus, and eventually the entire body of the adult organism.

Cells taken from the inner cell mass of the blastocyst (a very early stage embryo) can become embryonic stem cells.

Embryo: The developing organism from the end of the zygote stage (after about four days in humans) until it becomes a fetus (until 7 to 8 weeks after conception in humans).

Models: A biological system that is easy to study and similar enough to another, more complex system of interest so that knowledge of the model system can be used to better understand the more complex system. Such systems can include cells and whole organisms.

Model organism: An organism that is easy to study and manipulate and is similar enough to another organism of interest so that by understanding the model organism, a greater understanding of the other organism may be gained. For example, rats and mice can be used as model organisms to better understand humans.

Shown are several different model organisms frequently used in laboratory studies.

Severe Combined Immune-Deficient (SCID) mouse: A mouse lacking a functional immune system, specifically lacking or abnormal T and B lymphocytes. This is due to inbreeding or genetic engineering. They are extensively used for tissue transplants, because they lack an immune system to reject foreign substances, and for studying an immunocompromised system.

Cellular models: A cell system that can be used to understand normal, or diseased, functions that the cell has within the body. By taking cells from the body and studying them outside of the body, in culture, different conditions can be manipulated and the results studied, whereas this can be much more difficult, or impossible, to do within the body.

Stem cells obtained from different tissues of the body can be used as models to study normal, or diseased, cells in these tissues.

Cell Types:

Somatic Cell: Any cell in the body, developing or adult, other than the germline cells (the gametes, or sperm and eggs).

Gametes: The cells in the body that carry the genetic information that will be passed to the offspring. In other words, these are the germline cells: an egg (for females) or sperm (for males) cell.

Other terms:

Regenerative Medicine: A field of research that investigates how to repair or replace damaged tissues, usually by using stem cells. In this manner, stem cells may be differentiated into, or made to become, the type of cell that is damaged and then used in transplants. Also see clinical trials.

Clinical trials: A controlled test of a new drug or treatment on human subjects, normally performed after successful trials with model organisms. lists many stem cell clinical trials.

Stem cells have great potential to treat a wide variety of human diseases and medical conditions.

Cell Surface Marker proteins, or simply Cell Markers: A protein on the surface of a cell that identifies the cell as a certain cell type.

Somatic Cell Nuclear Transfer (SCNT): A technique that uses an egg and a somatic cell (a non-germline cell). The nucleus, which contains the genetic material, is removed from the egg and the nucleus from the somatic cell is removed and combined with the egg. The resultant cell contains the genetic material of the nucleus donor, and is turned into a totipotent state by the egg. This cell has the potential to develop into an organism, a clone of the nucleus donor.

Dolly the sheep was cloned through somatic cell nuclear transfer (SCNT). An adult cell from the mammary gland of a Finn-Dorset ewe acted as the nuclear donor; it was fused with an enucleated egg from a Scottish Blackface ewe, which acted as the cytoplasmic (or egg) donor. An electrical pulse acted to fuse the cells and activate the oocyte after injection into the surrogate mother ewe. A successfully implanted oocyte developed into the lamb Dolly, a clone of the nuclear donor, the Finn-Dorset ewe.

Clone: A genetic, identical copy of an individual organism through asexual methods. A clone can be created through somatic cell nuclear transfer.

Other stem cell glossaries:

Image credits Images of Endoderm, Mesoderm, Ectoderm, Bone Marrow, Neurons, Cartilage, Hand Skeleton, Connective and Adipose Tissue, Gastrula, Clinical Trials, Mouse, Rat, Drosophila, C. Elegans, Arabidopsis, Sea Urchin, Xenopus, Somatic Cell Nuclear Transfer to Create Dolly and other images were taken from the Wikimedia Commons and redistributed and altered freely as they are all in the public domain. The image of Hematopoiesis was also taken from the Wikimedia Commons and redistributed according to the GNU Free Documentation License.

2009. Teisha Rowland. All rights reserved.

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Stem Cell Basics: Potential uses of human stem cells

Posted: September 17, 2015 at 6:41 am

There are many ways in which human stem cells can be used in research and the clinic. Studies of human embryonic stem cells will yield information about the complex events that occur during human development. A primary goal of this work is to identify how undifferentiated stem cells become the differentiated cells that form the tissues and organs. Scientists know that turning genes on and off is central to this process. Some of the most serious medical conditions, such as cancer and birth defects, are due to abnormal cell division and differentiation. A more complete understanding of the genetic and molecular controls of these processes may yield information about how such diseases arise and suggest new strategies for therapy. Predictably controlling cell proliferation and differentiation requires additional basic research on the molecular and genetic signals that regulate cell division and specialization. While recent developments with iPS cells suggest some of the specific factors that may be involved, techniques must be devised to introduce these factors safely into the cells and control the processes that are induced by these factors.

Human stem cells are currently being used to test new drugs. New medications are tested for safety on differentiated cells generated from human pluripotent cell lines. Other kinds of cell lines have a long history of being used in this way. Cancer cell lines, for example, are used to screen potential anti-tumor drugs. The availability of pluripotent stem cells would allow drug testing in a wider range of cell types. However, to screen drugs effectively, the conditions must be identical when comparing different drugs. Therefore, scientists must be able to precisely control the differentiation of stem cells into the specific cell type on which drugs will be tested. For some cell types and tissues, current knowledge of the signals controlling differentiation falls short of being able to mimic these conditions precisely to generate pure populations of differentiated cells for each drug being tested.

Perhaps the most important potential application of human stem cells is the generation of cells and tissues that could be used for cell-based therapies. Today, donated organs and tissues are often used to replace ailing or destroyed tissue, but the need for transplantable tissues and organs far outweighs the available supply. Stem cells, directed to differentiate into specific cell types, offer the possibility of a renewable source of replacement cells and tissues to treat diseases including maculardegeneration, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, and rheumatoid arthritis.

Figure 3. Strategies to repair heart muscle with adult stem cells. Click here for larger image.

2008 Terese Winslow

For example, it may become possible to generate healthy heart muscle cells in the laboratory and then transplant those cells into patients with chronic heart disease. Preliminary research in mice and other animals indicates that bone marrow stromal cells, transplanted into a damaged heart, can have beneficial effects. Whether these cells can generate heart muscle cells or stimulate the growth of new blood vessels that repopulate the heart tissue, or help via some other mechanism is actively under investigation. For example, injected cells may accomplish repair by secreting growth factors, rather than actually incorporating into the heart. Promising results from animal studies have served as the basis for a small number of exploratory studies in humans (for discussion, see call-out box, “Can Stem Cells Mend a Broken Heart?”). Other recent studies in cell culture systems indicate that it may be possible to direct the differentiation of embryonic stem cells or adult bone marrow cells into heart muscle cells (Figure 3).

Cardiovascular disease (CVD), which includes hypertension, coronary heart disease, stroke, and congestive heart failure, has ranked as the number one cause of death in the United States every year since 1900 except 1918, when the nation struggled with an influenza epidemic. Nearly 2,600 Americans die of CVD each day, roughly one person every 34 seconds. Given the aging of the population and the relatively dramatic recent increases in the prevalence of cardiovascular risk factors such as obesity and type 2 diabetes, CVD will be a significant health concern well into the 21st century.

Cardiovascular disease can deprive heart tissue of oxygen, thereby killing cardiac muscle cells (cardiomyocytes). This loss triggers a cascade of detrimental events, including formation of scar tissue, an overload of blood flow and pressure capacity, the overstretching of viable cardiac cells attempting to sustain cardiac output, leading to heart failure, and eventual death. Restoring damaged heart muscle tissue, through repair or regeneration, is therefore a potentially new strategy to treat heart failure.

The use of embryonic and adult-derived stem cells for cardiac repair is an active area of research. A number of stem cell types, including embryonic stem (ES) cells, cardiac stem cells that naturally reside within the heart, myoblasts (muscle stem cells), adult bone marrow-derived cells including mesenchymal cells (bone marrow-derived cells that give rise to tissues such as muscle, bone, tendons, ligaments, and adipose tissue), endothelial progenitor cells (cells that give rise to the endothelium, the interior lining of blood vessels), and umbilical cord blood cells, have been investigated as possible sources for regenerating damaged heart tissue. All have been explored in mouse or rat models, and some have been tested in larger animal models, such as pigs.

A few small studies have also been carried out in humans, usually in patients who are undergoing open-heart surgery. Several of these have demonstrated that stem cells that are injected into the circulation or directly into the injured heart tissue appear to improve cardiac function and/or induce the formation of new capillaries. The mechanism for this repair remains controversial, and the stem cells likely regenerate heart tissue through several pathways. However, the stem cell populations that have been tested in these experiments vary widely, as do the conditions of their purification and application. Although much more research is needed to assess the safety and improve the efficacy of this approach, these preliminary clinical experiments show how stem cells may one day be used to repair damaged heart tissue, thereby reducing the burden of cardiovascular disease.

In people who suffer from type1 diabetes, the cells of the pancreas that normally produce insulin are destroyed by the patient’s own immune system. New studies indicate that it may be possible to direct the differentiation of human embryonic stem cells in cell culture to form insulin-producing cells that eventually could be used in transplantation therapy for persons with diabetes.

To realize the promise of novel cell-based therapies for such pervasive and debilitating diseases, scientists must be able to manipulate stem cells so that they possess the necessary characteristics for successful differentiation, transplantation, and engraftment. The following is a list of steps in successful cell-based treatments that scientists will have to learn to control to bring such treatments to the clinic. To be useful for transplant purposes, stem cells must be reproducibly made to:

Also, to avoid the problem of immune rejectio
n, scientists are experimenting with different research strategies to generate tissues that will not be rejected.

To summarize, stem cells offer exciting promise for future therapies, but significant technical hurdles remain that will only be overcome through years of intensive research.

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Stem Cell Basics: Potential uses of human stem cells

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Human neural stem cell transplantation in ALS: initial …

Posted: September 13, 2015 at 8:46 pm

Abstract Background

We report the initial results from a phase I clinical trial for ALS. We transplanted GMP-grade, fetal human neural stem cells from natural in utero death (hNSCs) into the anterior horns of the spinal cord to test for the safety of both cells and neurosurgical procedures in these patients. The trial was approved by the Istituto Superiore di Sanit and the competent Ethics Committees and was monitored by an external Safety Board.

Six non-ambulatory patients were treated. Three of them received 3 unilateral hNSCs microinjections into the lumbar cord tract, while the remaining ones received bilateral (n=3+3) microinjections. None manifested severe adverse events related to the treatment, even though nearly 5 times more cells were injected in the patients receiving bilateral implants and a much milder immune-suppression regimen was used as compared to previous trials.

No increase of disease progression due to the treatment was observed for up to18 months after surgery. Rather, two patients showed a transitory improvement of the subscore ambulation on the ALS-FRS-R scale (from 1 to 2). A third patient showed improvement of the MRC score for tibialis anterior, which persisted for as long as 7months. The latter and two additional patients refused PEG and invasive ventilation and died 8months after surgery due to the progression of respiratory failure. The autopsies confirmed that this was related to the evolution of the disease.

We describe a safe cell therapy approach that will allow for the treatment of larger pools of patients for later-phase ALS clinical trials, while warranting good reproducibility. These can now be carried out under more standardized conditions, based on a more homogenous repertoire of clinical grade hNSCs. The use of brain tissue from natural miscarriages eliminates the ethical concerns that may arise from the use of fetal material.

EudraCT:2009-014484-39 webcite.

Amyotrophic Lateral Sclerosis (ALS) is a devastating, incurable neurodegenerative disease that targets motor neurons (MNs) in the primary motor cortex, brainstem, and spinal cord. Cell therapy is emerging as a potential therapeutic option in ALS, although numerous scientific, technical, ethical and regulatory issues remain to be overcome. One critical issue pertains to the type of stem cells to be used in cell therapy for ALS. Donor cells must not only survive in the human central nervous system (CNS), but ought to be capable of improving the tissue pathophysiological condition, possibly by modulating local inflammatory and immune reactions and by antagonizing toxic phenomena [1],[2]. Notwithstanding, the first critical requirement that stem cells must meet in clinical applications concerns their safety. At the same time, a pressing need exists for them to be easily isolated and reproducibly and stably expanded ex vivo[3]. Thus, both hematopoietic and mesenchymal stem cells (MSCs) have now been transplanted into the spinal cord of ALS patients, in the absence of long-term negative effects [4]-[6]. Others and we have previously documented the integration capacity and prospective therapeutic efficacy of human neural stem cells (hNSC) in preclinical rodent models of neurological diseases [7]-[10]. Of interest, we showed that, when implanted either intravenously or intrathecally, NSC ameliorate the pathophysiological and neurological traits in an experimental model of autoimmune encephalomyelitis, both in rodents [11] and non-human primates [12]. A key conclusion from these studies was that implanted NSCs would integrate and survive predominantly as astroglial cells. These would likely elicited their effects through the release of growth factors and immunomodulatory molecules [12].

The injection of candidate therapeutic cells in the proximity of the degeneration site(s) may better suit the key requirements in the neural transplantation context. In ALS, engraftment of donor cells close to the dying MNs might favour the diffusion of trophic and immunomodulatory factors to both the MNs themselves and the surrounding glial cells, thereby enhancing the likelihood of accomplishing therapeutic effects. Accordingly, a meta-analysis of 11 independent studies demonstrated that, when implanted close to the dying MNs, NSCs may slow both the onset and the progression of clinical signs and can prolong overall survival in ALS mice [13]. Furthermore, it has recently been shown that transplantation of human neural progenitor cells into the ventral cervical spinal cords of SOD1G93A rats can slow the death of phrenic motor neuron and increase activity in spared phrenic MNs [14].

A technique for the focal delivery of donor cells in the proximity of ventral MNs has recently been established using a stabilized, stereotaxic frame [15]. This system, previously standardized in animal models [16],[17], has now been employed in the first FDA-approved cell-therapy trial for ALS, implanting human neural progenitor cells [18],[19].

Here we expand on these studies and report the preliminary results from a first group of six ALS patients in an ongoing Phase I trial, in which bona fide, multipotent hNSCs were isolated and reproducibly expanded from human fetal tissue. These cells were obtained from spontaneous miscarriages [8] and implanted using a stereotaxic/surgical apparatus and injection procedures similar to those used by Riley and colleagues [17].

The study was performed as a prospective, open pilot study. The trial was approved and monitored by the competent Italian authorities, i.e. the Istituto Superiore di Sanit (ISS), Agenzia Italiana del Farmaco (AIFA, member of the European Medicine Agency) and by the Ethics Committees of the Umbria Region, of the Maggiore della Carit and of the San Antonio Hospitals. It goes by the European Clinical Trials Database (EudraCT) identification number 2009-014484-39. All patients provided written, informed consent. All the recorded data for the recruited patients concerning the whole follow-up period were registered in the Database For Clinical Studies With Gene And Somatic Therapy of the ISS. The same data were also communicated to an independent Safety Monitoring Board of multidisciplinary experts, whom controlled strict compliance with the protocol and monitored possible adverse events.

Patients were deemed eligible when they had definite or probable sporadic ALS, according to the El Escorial Revised Criteria [20]. Given the high iatrogenic risk, patients in this initial study were selected whom suffered from severe functional impairment in their lower limbs. The detailed list of inclusion and exclusion criteria is reported in Table1. All patients received ordinary medical treatment, including riluzole.

Table 1. Inclusion and exclusion criteria

We expected a large impact on the mass media and inferred that this kind of trial would raise significant expectations in a large population of patients. Hence, in order to deal with the likely rather large number of possible applications, a toll-free phone number and a web-based portal were set up. Thorough this, patients could apply for admission to the trial by filling out a form and by sending a standardized set of clinical information for initial screening. The neurologists of the st
aff reviewed these submissions and eligible patients were summoned for a visit at the recruitment centres, until the maximum number of patients allotted by the recruitment criteria was reached.

Case-reports for eligible patients were sent to the ISS, whose experts verified the appropriate adherence to the protocol and authorized the treatment. At this point the patients were informed of their recruitment into the study.

The informed consent was structured as an interview, which openly and clearly stated the experimental and preliminary nature of our clinical study and the risks associated with the procedure. The neurologist discussed each question with the patients and their relatives. This procedure was in agreement with the recommendations of the International Society for Stem Cell Research [21]. Subjects were made aware that their participation in this study was entirely voluntary and that participation or non-participation would not interfere with their ongoing clinical care. Before signing their consent, patients and close relatives were offered the possibility of meeting separately with their family physician, a neurosurgeon and a consultant neurologist, whom were in no manner involved in our trial, in order to discuss all pending issues. A psychologist closely examined the patients before and immediately after recruitment. A clinical interview and the MMPI-2 test were used to ensure that the participants fully comprehended that this was a safety trial and all of the risks associated with the procedure.

Patients were enrolled and evaluated at the Tertiary ALS Centres in Novara and Padova, Italy. All surgical procedures were performed at the Santa Maria Hospital in Terni, Italy, in close proximity to the cell factory that prepared and released the GMP-grade hNSCs upon the formal authorization granted by AIFA (protocol number aM 02/2014).

The primary outcome measure was the immediate and long-term safety profile. Mortality from any cause and serious adverse effects were the primary occurrences that were assessed. Patients were closely monitored for immediate adverse events, including allergic reactions (tachycardia, fever), respiratory failure, local complications (intraparenchymal hematoma, local infection at the site of surgery), systemic complications (systemic infections), paralysis or sensory loss below the level of the injection site. Potential, delayed adverse events included intraspinal tumor formation, aberrant connections (spinal myoclonus), persistent sensory loss or paralysis not related to the progression of the disease.

The secondary outcome measures were the difference in functional outcomes measured by the ALS-FRS-R scale and forced vital capacity (FVC).

To estimate the rate of disease progression, patients were monitored monthly for 1year and then every three months, until death. After enrollment, but prior to surgery, each patient was subjected to a three-month observation period. Clinical progression was assessed using the ALS-FRS-R, Ashworth Spasticity Scale and the Medical Research Council (MRC) scale of 34 muscle groups of the upper and lower limbs and FVC. At each examination a clinical psychologist also evaluated the patients. Profile of Mood State (POMS) [22] and SEIQoL-DW [23] questionnaires were provided to patients to assess the mood state and the quality of life.

Brain and spinal cord (SC) MRIs were obtained using a 1.5-T imaging system (Achieva Intera, Philips). Spinal cord MRIs were performed pre-operatively and at 21days and at 3, 6, 9 and 12months after surgery. Brain MRIs were performed at the time of entry in the study and 12months after surgery. In addition to the full conventional MRI, the spinal cord was imaged by a diffusion tensor imaging (DTI) pulse sequence in the axial plane, with 64 directions, in order to identify and quantitatively characterize the tissue. Using the fiber-tracking algorithm, we calculated the fractional anisotropy (FA) and apparent diffusion coefficient (ADC) values. We used FiberTrack Software (Philips, Best, The Netherlands) and, after a test/re-test for intra-individual variability evaluation, we measured FA and ADC at the implant site (T11-T12) and in normal tissue in the upper cervical and lower lumbar spinal cord segments, by placing single point ROIs. To ensure that the chosen ROIs were effectively inside the spinal cord and part of its architectural structure we detected and reconstructed a spinal fiber bundle at each ROI. If not evoked, we discarded that ROI changing its position (Figure1 upper panel). To ensure that at every follow-up the same axial level would be selected, we chose the vascular cleft of the vertebral body and the midline inside the intervertebral disk between the somatic endplates as reference sites (Figure1 lower panel).

Figure 1. Spinal cord MRI. Upper Panel: Diffusion Tensor Imaging (DTI) overlaid with the STIR pulse sequence, in a MPR algorithm. For each of single point (colored box) ROIs, a correspondent fiber is evoked and reconstructed to ensure that the level examined and the ROI adopted is inside the spinal cord. Lower Panel: DTI post-processing by mean of a MPR algorithm, with overlaying of the STIR pulse sequence, to select the exact levels to be studied at this time of examination and in further MR follow-up scans. Disk and somatic vascular cleft are adopted to select the proper planes.

We overlaid, inside a Multi-Planar Reformat (MPR) program, a high resolution axial T2 weighted image on the volumetric DTI data. Measurements were carried out placing two ROIs, right and left, localized in the supposed site of implantation (the grey matter of the anterior horns of the spinal cord). The mean of the two measures was considered in the analysis for the anterior part of spinal cord. Another two ROIs were drawn in correspondence to the posterior spinal bundles, making for a total of four ROIs for each level.

Blood routine laboratory tests (renal function, liver function, glucose) and test for Hepatitis B or C, HIV and TBC were performed at the study entry and 7days before surgery. Routine and haematological (full blood count and CD3+, CD3+/CD8+, CD4+/8+ counts) laboratory tests and tacrolimus dosage were performed weekly during the first post-operative month and then monthly during the 6months of the immunosuppression period.

Bladder ultrasound, measuring the post void residual volume, was performed at study entry and on the 2nd and 21st days after surgery and then every three months, for one year.

The hNSCs used in this clinical trial were produced according to good manufacturing practice protocols (GMP), as dictated by the European Medical Agency (EMA) guidelines. The tissue collection procedure, the cell factory, the production procedure and the cell validation criteria received formal approval and certification by the appropriate regulatory body, namely the Agenzia Italiana del Farmaco, protocol number aM 101/2010 (updated in 2014 after AIFA inspection to number aM 2/2014). Tissues and cells were procured and handled as described below [24].

Human fetal brain tissue specimens, all derived from the forebrain, were routinely collected from fetuses at gestational ages greater than the 8th post-conceptional week. They were immediately dissected and used to gene
rate hNSC lines under sterile conditions. Tissue procurement was approved by the ethical committee of the Institute Casa Sollievo della Sofferenza di San Pio da Pietrelcina and was possible exclusively upon the mother giving informed, written consent. Of note, no tissue from therapeutic or procured abortion was used in this study and all of the specimens were collected from fetuses that underwent natural, spontaneous in utero death, (miscarriage). Also, specimen collection and medical procedures were in full accord with the Helsinki declaration (WMA Declaration of Helsinki – Ethical Principles for Medical Research Involving Human Subjects). Dissociation of brain tissue, primary culturing and cell propagation were carried out according to the procedure described previously by Vescovi and colleagues [8]. As a whole, the results reported in this study refer to cells from a maximum of two donors, whom died at the 15th and 16th gestational week. The two different cell lines were used randomly to treat patients.

Tissue was washed in a PBS solution (Dulbeccos PBS 1X, PAA plus 50ng/ml of gentamicin) and mechanically dissociated. Cells were seeded at a density of 104 cells/cm2 as described previously [8]. Cultures were maintained in a humidified incubator at 37C, 5% O2 and 5% CO2 and allowed to proliferate as free-floating clusters (neurospheres).

710 days after the primary cell seeding the neurospheres that had formed were collected and mechanically dissociated and replated at the same initial density. This step (passaging) was routinely repeated up to 17 times before the cells were supplied to the surgery site on the day of transplantation. Throughout these passages aliquots of cells were frozen as neurospheres, which were cryopreserved in 10% DMSO (Dimethyl sulfoxide, LiStarFish) culture medium as a pharmaceutical intermediate product, with an assigned batch number. This freezing step was conducted in order to coordinate the timing of cell production with the surgery schedule. It also allowed for the establishment of an Intermediate Product to warrant the series of quality controls required to certify the safety, identity, potency and the pharmaceutical grade of the donor hNSCs, to satisfy the AIFA, GMP regulatory process criteria. Once the date of the surgery had been determined, the intermediate product was thawed and cells underwent additional passages and expansion (a maximum of 17 times overall, as stated above), until they were delivered to the surgery room as the final product (cell drug) to be transplanted.

In agreement with the AIFA certification criteria, we subjected cells to serial quality control testing throughout the whole production process (IPCs-In Process Controls). Additional control tests were performed on the Intermediate Product and Finished Product before their final release.

The in process controls were meant to pursue a microbial monitoring of the product (Total Microbial Count) and to evaluate cellular growth parameters (cell counting and trypan blue exclusion method) throughout all cell passages. Standard haematoxylin and eosin staining and histological evaluations were also carried out on the fetal donor tissue to confirm the neural nature of the starting material. This was in addition to the precise anatomical dissection criteria used to isolate the initial tissue. The release tests, performed at the very last passage, were meant to verify the sterility of the product, the absence of mycoplasma and a low level of endotoxin contamination (LAL test;

In the culture system at hand, this could be extrapolated by determining the cells growth kinetic. In fact, in the neurosphere system, the slope of the growth curve provides an indirect index of the hNSCs inherent ability for self-renewal [25],[26]. We determined such kinetic curve by calculating the estimated overall cell number increase at each passage, as described earlier [8] and did confirm the long-term stability of these cultures with respect to this parameter (Figure2A).

Figure 2. Cell quality control. (A) Growth kinetics of a set of hNSCs lines showing the increasing, estimated overall cell number at each passage. (B) Clonal efficiency assay showing the percentage of cells that retain the ability to form clonal neurospheres over the total cell number plated is reported (Replicates n=3), bars describe standard error. (C) hNSCs differentiate into astrocytes (left, green, GFAP), neurons (left, red, III-tubulin) and oligodendrocytes (right, GalC, red); nuclei are counterstained in blue (DAPI). (Bar=50m). (D) All of the hNSCs lines tested undergo extinction in vitro upon growth factor removal, as shown by the negative growth kinetic in which the total cell number approaches zero in a few passages.

The analysis of the clonogenic capacity throughout passaging was used to directly confirm the stability of the self-renewal capacity of our hNSCs. This was determined by the hNSCs clonal efficiency assay, which shows the percentage of plated cells that retains the ability to form clonal neurospheres under stringent culture conditions. Briefly, neurospheres were mechanically dissociated and cells were counted, also assessing cell vitality by trypan blue exclusion. Cells were seeded at a density of 250 viable cells/cm2 on a Poly-L-lysine layer (Sigma-Aldrich) in a flat bottom 24-well dish and incubated at 5%O2 5%CO2 and 37C in DMEM/F12, in the presence of both mitogens, human recombinant EGF and basic FGF. After 7days of incubation, neurospheres that measured between 50m and 100m in diameter were counted under an inverted microscope. Clonal efficiency was determined as the ratio between the total cell seeded and the number of neurospheres obtained. An example of this analysis and of stable clonogenic ability in serially subcultured hNSCs is shown in Figure2B.

A critical feature of hNSC is their multipotency, i.e. their ability to give rise to the three major neural lineages [8]. To show that hNSCs retained stable differentiation capacity throughout culturing, their multipotency was determined by in vitro differentiation tests, in which the simultaneous presence of neurons, astrocytes and oligodendrocytes was detected by immunocytochemical labeling (-tubulin III, neuron marker; GFAP, astroglial marker; GalC, oligodendroglial marker). Neurospheres were mechanically dissociated and the cells were resuspended in the same culture medium used during their routine culturing but in the absence of EGF and in the presence of the sole bFGF. Cells were seeded on a cultrex layer (Cultrex Basement Membrane Extract, Trevigen) and incubated at 5% O2, 5% CO2 and 37C for 3days. Then culture media was replaced with DMEM/F12 supplemented with 2% FBS, w/o growth factors [27]. Cells were incubated for an additional 7days, after which they were fixed with 4% paraformaldheyde and immunostained using a standardized method [8]. An example of such a differentiation test is shown in Figure2C.

Neural stem cells appear
to be quite resilient to transformation. Both mouse and human cells depend on the presence of mitogens to undergo proliferation and promptly differentiate upon their removal from the culture [28]. Thus, ensuing growth factor independence is considered as a sign of potential hNSCs transformation. Hence, we routinely monitored growth factor dependence in our GMP-grade hNSCs, in order to provide indirect evidence to their lack of transformation and tumorigenicity. This was evaluated by shifting expanding cells to a culture media without EGF and bFGF and analyzing their growth curve upon further sub-culturing. The latter ought to display a negative slope, confirming the cells inability to sustain self-renewal in the absence of mitogenic stimulation, as shown in Figure2D.

We also monitored the stability of the hNSCs karyotype all throughout passaging in culture. Chromosome G-banding confirmed that the two cell lines used in this study retained their normal, 46 XX or 46 XY karyotype profile all throughout culturing (Figure3). Forty eight to 120hours from seeding, cell cultures (higher than 1,000,000 cells) were treated for three hours with colcemid solution (KaryoMAX Colcemid Solution, 10g/ml) at a final concentration of 0.05g/ml to arrest cells in metaphase. Cells were then treated with hypotonic solution (0.075M potassium chloride solution, SIGMA) and with a fixative solution, prior to proceeding with the karyotype analysis.

Figure 3. The cell lines used in this study were confirmed to retain a normal karyotype all throughout passaging. The figure shows the example of a karyogram performed on the hNSC line from a female donor (46, XX) after seventeen passages. Chromosome G-banding was routinely performed on both the Intermediate Product and the Finished Product. In addition we also tested for karyotype stability the cells that were left in the needle, post-transplantation.

The lack of tumorigenicity in hNSCs was also evaluated directly by in vivo safety testing. Immunodeficient, athymic nude mice, were injected into the lateral striatum (Figure4A) with 3 105 hNSCs. They were sacrificed six months later. Immunohistochemistry analysis showed that, as seen previously [8], hNSCs engraft efficiently, with only a few sporadic cells remaining positive for the human proliferation antigen Ki67. Engrafted cells differentiated into neuronal cells expressing the TubulinIII antigen and into astrocytes expressing the glial fibrillary acidic protein (GFAP) (Figure4D, E, F). We observed neither signs of hyperproliferation (only an average of 4.831.12% of Ki67 positive cells per transplant were found in the 20 animals analyzed per each cell line (n=15)) nor tumor formation (Figure4B). We also ran positive controls to verify that the system used here was truly capable to detect the presence of engrafted cells with tumorigenic ability (positive controls). To this end, we set up a parallel group of transplanted nude mice that received between 50 and 50.000 transformed human neural cancer stem cells [29]. In these animals, immunohistochemistry analysis promptly revealed formation of hypercellular, invasive tumor cell masses as early as 3months after transplant at the highest concentration and approximately 5months for the lowest (Figure4C).

Figure 4. hNSCs transplant into Nude Mice CNS. The lateral striatum of nude mice was the target area (A, arrow) for the transplantation of normal hNSCs (B) or glioblastoma cancer stem cells (GBM; positive graft controls; C). Mice were sacrificed six (B) and two months (C) after transplantation, respectively. The hematoxilyn/eosyn stain showed that structural organization of the transplanted regions was well preserved in mice tranplanted with hNSCs (B), whereas hypercellularity and anomalous growth and necrosis ensued in regions receiving GBM cells (C). Confocal microscopy of anti-human nuclei staining (huN, green, D) showed that hNSCs engrafted efficiently, with only a few human cells retaining residual proliferation activity as shown by co-labeling with the proliferation marker ki67 (red). hNSCs labeled with huN (E, green) differentiate into TubIII+ neurons (E, red) and GFAP+ astrocytes (F, red). Nuclei are shown by DAPI staining. Scale bars: D-E = 15 m; all insets: 10 m, bar in inset D.

The details of the surgical apparatus and transplantation protocol have been published previously [18]. Briefly, a platform was fixed to the spine through percutaneous posts. After laminectomy (T8-T11) and dural opening a floating retracting cannula design was used to place a needle precisely into the ventral horn, using a rigid conformation. After placement, the cannula was retracted and converted to its flexible form to allow it to float jointly liable with the spinal cord. Unlike the previous trial we decided to use a cannula with a broader, 25 gauge diameter (instead of 30) to facilitate cells loading and to minimize their reflux along the needle track during infusion, also in consideration of the high cell concentration of our cell suspension, which was 5 folds higher than those previously used [30]. On average, the time required for the entire surgery procedure was approximately 4hours. Before implantation cells were counted and suspended in HBSS buffer (Hanks BSS 1X, PAA) at a concentration of 50,000 cells/l. After batch release, cells were maintained at 2-8C for not more than 1hour prior to implantation. Patients received either unilateral (Group A1) or bilateral (Group A2) NSCs microinjections (3 microinjections on each side) into the lumbar spinal cord. Each microinjection consisted of 15l of the above 50,000cells/l suspension, yielding a total of 750,000 cells per injection site. Standard Monitoring techniques were applied during surgery and included lower extremity somatosensory and motor evoked potentials, when readings proved reliable. All surgical procedures were performed under general anaesthesia using previously described procedures [31]. Cefazolin 1g IV was administered at the time of dural opening and immediately after surgery.

Methyprednisolone 125mg IV was administered preoperatively at 2hours before incision. Patients subsequently received oral prednisone, with a 28-day taper and a dose change each week, declining from 60mg to 40mg, 20mg, and 10mg orally, every day.

Tacrolimus was administered orally at 0.1mg/kg twice a day, beginning on post-operative day 1. The drug was titrated so as to maintain blood levels comprised between 510ng/ml for 6months, after which the treatment was terminated.

After surgery patients were admitted to the Intensive Care Unit and mobilized. On postoperative day 3 patients were admitted in the neurosurgical unit and on day 5 or 7 (depending on the patient conditions) were transitioned to physical rehabilitation department for a 20-day period of intensive physical therapy. Neurological status, including assessment of muscle strength by MRC and spasticity by Ashworth scale and general examination (including blood pressure), biochemistry (renal function, liver function, glucose, tacrolimus levels) and haematological assessments (full blood and CD4 count) were performed daily during the first week post-surgery. This procedure was carr
ied out weekly for the first three weeks and monthly, thereafter. FVC was performed monthly.

The study could be halted at any time either by the Safety Monitoring Board, by the ISS, by the investigator(s) or by the ethics committees, based on any medical event experienced by the patient, which was deemed to be of clinical significance and that might occur after the treatment. Severity was graded according to the modified WHO criteria.

We received a total of 227 applications to participate in this study, 185 of which turned out to be ineligible since they did not meet the inclusion criteria. A total of 18 eligible patients were summoned for the initial medical screening at the recruitment centres. After the visit and detailed discussion of the informed consent 9 patients withdrew their request to participate. Three of the patients that were enrolled developed respiratory failure during the three months of observation of the disease natural history prior to transplantation. This made it impossible for them to safely undergo surgery. The final cohort of patients included 6 sporadic ALS patients (2 females and 4 males). The principal characteristics of the recruited patients are described in Table2.

Table 2. Clinical characteristics of patients

Their median age was 46years (Range: 3067). The median duration of the disease from the onset of symptoms to recruitment was 48months (Range: 1672). The median ALS-FRS-R score at the study entry was 28 (Range: 2435), while the median FVC was 82% (Range: 6483).

The treatment caused no severe adverse events. All patients were extubated without problems in the operating room and showed no immediate, post-operative respiratory difficulties. The most common adverse event was post-surgical pain, as reported immediately after surgery. This was confined to the injection sites and to the corresponding dermatomes, with only one patient (number 753) experiencing transient, painful spasms in the lower limbs. Pain was mild (WHO II-III) and well compensated with narcotic and non-narcotic analgesics, disappearing an average 4days after surgery (Range: 16). There was no correlation with the number of the injection sites. One patient (number 779) developed deep vein thrombosis in the leg 3months after surgery, which required anticoagulant treatment. Subsequently a hematoma at the site of surgical scar was observed. MRI confirmed that the blood and oedema at this site were superficial and confined to the epidermal layer. The hematoma showed no clinical neurological correlate and readily resolved upon drainage.

No patients suffered side effects from immunosuppressive treatment. Tacrolimus was well tolerated and did not have to be suspended prematurely. All patients showed tacrolimus blood levels that were within the therapeutic target range, confirming compliance. Renal and liver function, blood and CD3+; CD3+/CD8+ CD4+/8+ counts remained within normal range.

SEPs and MEPs showed no changes of the sensory and motor conduction time during and following surgery. Bladder ultrasound showed no abnormal post-void residual volumes.

There were no post-procedural complications. In all patients post-surgical MR scans revealed an expected extradural fluid collection at the site of surgery, which resolved spontaneously after 3 to 6months (Figure5A-G). Serial MRI showed no structural changes (including tumor or syrinx formation) within the brain and the spinal cord after transplantation relative to the baseline (Figure5). A 12months follow-up analysis in 2 out of 6 patients portrayed no significant, long lasting changes in the Apparent Diffusion Coefficient (ADC) and Fractional Anisotropy (FA). At post-surgery day 21 a slight reduction of the FA values was observed, followed by a subsequent recovery at later follow-up scans. ADC, which probes the interstitial space restriction in the case of ischemia or cytotoxic oedema or, conversely, facilitation of water diffusion in the case of increased extracellular interstitial water, showed no evidence of post-transplant damage. Neither brain morphological and signal alterations nor pathological enhancements were detected 12months after surgery.

Figure 5. MRI Follow-up. T2 weighted sequences acquired on sagittal plane before surgery (images A-B) and respectively 21days (image C), 3 (image D), 6 (image E), 9 (image F) and 12months (image G) after transplantation. Post-surgical MR scans revealed an expected extradural fluid collection at the site of surgery, which resolved spontaneously. No structural changes were detected after hNSCs transplantation relative to the baseline.

The few microliters of residual hNSCs suspension that were left inside the injection cannula were harvested at the end of the whole transplantation procedure and put back into culture. By applying the same set of analysis adopted for their initial GMP certification, we were able to determine that the implanted hNSCs had retained the full complement of stem cell properties, including self-renewal and multipotency and lack of tumorigenicity upon in vivo implantation into immunodeficient mice (Figure6).

Figure 6. Post Transplant hNSCs Test. (A) an example of human neural stem cells that were leftover from the transplant and put back in culture were they re-established typical neurosphere, expanding lines, with a growth profile that mirrored that of the very same cultures prior to the transplant, as shown in B. These cells differentiated into neurons (III-Tubulin, green, C) and astrocytes (GFAP, Red, C) and oligodendrocytes (GalC, Red, D). E: an example of whole brain reconstruction from one out of 10 nude mice that were transplanted into the right lateral striatum with the cells recovered from the transplant and recultured, showing no hyperplastic areas or tumor formation. Bars: A, 100m C,D 50m, Bar in D.

An aliquot of the very same cells that were injected into the patientss spinal cord were fixed the day of the intervention and tested for karyotype stability, as described into methods section. All of the cells used in this study showed a normal chromosomal asset (see example in Figure3).

Clinical assessments ranging from 6 to 18months after transplantation showed no acceleration in the course of progression of the disease due to the treatment. No significant modifications in the decline of all clinical and instrumental measures were observed between the pre- and post-transplantation phases (Figure7).

Figure 7. Clinical follow-up. Changes of the Forced Vital Capacity (upper panel) and of the ALS-FRS score (lower panel) in the 3-month period of natural history observation and after transplantation. The arrow indicates the time of NSCs transplantation.

Patients 740 and 779 showed a transitory improvement in the ambulation sub-score on the ALS-FRS-R scale (from 1 to 2), which persisted 3 and 1month after surgery, respectively. Patient 753 showed an improvement of the MRC score for tibialis anterior (from 1 to 2) that persisted for up to 7months after surgery.

Eight months after surgery, patient 753, together with two additional patients (746 and 7
67), died due to the progression of respiratory failure related to the natural course of the disease. All the three of them had refused PEG and invasive ventilation. The autopsies of patients 746 and 753 documented no other causes of death. The autopsy of patient 767 was not carried out because the relatives declined the necessary consent. Histopathological, post-mortem analysis from the one patient from whom the spinal cord tissue could be harvested showed neither tissue abnormalities nor the presence of hyper-cellular regions (Figure8).

Figure 8. Representative cross section of the spinal cord in the region of transplantation stained with luxol fast blue and periodic acid Schiff. There is no apparent disruption of tissue due to injection. Note the degeneration of the cortico-spinal tracts (lateral sclerosis). The inset demonstrates a phosphorylated TDP43 inclusion in a remaining motor neuron. Scale bars are 1mm for the low power and 20 microns for the inset.

No negative reactions on the patients mood and quality of life were observed. Only patient 753 experienced alterations in his mood, accompanied by depressive symptoms in the post-surgery period. This emerged as a reaction to the paucity of assistance that she/he received, from her/his family and the progressive deterioration of the relationship with her/his spouse. The mood improved after the activation of the domiciliary assistance from the public health services. All patients were interviewed at the last visit and confirmed that they were satisfied by their participation in this study.

This study on six patients with ALS shows that transplantation of hNSCs into the anterior horns of the lumbar spinal cord appears to be a safe procedure that causes no major, short- or medium-term, deleterious effects. Our data are consistent with those reported previously [18],[19],[32]. Of importance, our findings emerge from a similar experimental and clinical protocol that is comparable to these studies but we, in turn, employed a much higher cell dosage and a milder immunosuppression regimen.

The first result is to be found in the fact that the surgery underpinning intraspinal cell injection in humans appears to be a safe, reproducible and reliable procedure. Also, perspective improvements were made here, particularly concerning the number of implanted cells, that in our study was four and a half times higher than in previous approaches a maximun of 4.5106 cells/patient used here versus the previous 1106 cells [32]. In addition, we adopted a floating cannula with a larger diameter. This further reduced the risk of the cells clogging the needle, and minimized their flowing back along the walls of the needle track through the cell infusion procedure, irrespective of their higher concentration in our cell suspension.

Second, MRI scans revealed no structural changes or tumor formation in the spinal cord parenchyma all throughout the follow-up period. The analysis of Apparent Diffusion Coefficient (ADC) and Fractional Anisotropy (FA) did not show any damage to the spinal tissue. Of note, FA is a parameter that probes the axonal density. Thus a lesion of the spinal cord should induce a definite and long lasting reduction of FA. In our 18month-long follow up we observed no significant, long-lasting changes in the FA values. This was further confirmed by the histopathological, post-mortem analysis.

At present, any interpretation of the slight clinical improvement observed in three patients remains highly speculative. Notwithstanding, by taking the evidence available from the literature into account, some cautious hypothesis can still be put forth, if only tentatively. Such effects could be related to the well known ability of NSCs to ameliorate the pathophysiological and neurological traits in many animal models of neurological disorders, including ALS [13]. This may occur through the release of neurotrophic and growth factors, cytokines and immunomodulatory molecules [33] that diffuse into the pathological tissue, thereby eliciting multifaceted, anti-inflammatory and anti-gliosis effects. In agreement with this view, we have recently found that the same cell used in this trial can effectively reduce reactive microglia in the spinal cord of SOD1G93A rats (Vescovi et al., manuscript in preparation). Intriguingly, compelling evidences now exist that implanted NSCs, which integrate and survive predominantly as astroglial cells in the brain parenchyma, exert their immunomodulation ability through secretion of extracellular membrane vesicles (EMVs), influencing the microenvironment through the traffic of bioactive molecules [34],[35]. If the above were the case, it would also help to explain as to why these mild clinical improvements may occur irrespective of the total number of injections, as observed here.

An additional, significant finding from this study is the lack of acute or delayed toxicity that might have ensued from the transplantation of our hNSCs lines. The latter are routinely generated anew and banked as tissue becomes continuously available over time. This approach has now allowed us to establish a GMP-certified, clinical grade bank of hNSCs, which is constantly expanding both in size and donors repertoire. Each single hNSCs line is steadily and plentifully expandable ex vivo, with multipotentiality and functional characteristics remaining stable and certified throughout time. Thus, not only does this approach provide a broad array of different hNSCs lines for future studies allowing for determining if cells from different regions or developmental ages may be more amenable for cell therapy in different pathological contexts but also enables us to carry out this whole phase I trial using cells from a maximum of two donors.

Previous pre-clinical data from our lab using these same hNSCs pointed to a low expression profile of HLA molecules. Furthermore, negligible immunological reaction was observed following intracerebral transplantation, upon transient (peri-transplantation) immunosuppression [10]. Hence, in considering that ALS patients at advanced stages of the disease might be weaker and more vulnerable to pharmacological toxicity, in the study reported here we adopted a reduced immunosuppression regimen as compared to that employed by Glass and colleagues [19]. As expected, we observed a smaller cohort of side effects arising from the immunosuppressive treatment.

While transplantation of cells from human foetuses is an accepted technique, it is undeniable that such an approach is often met with ethical and moral concern, particularly outside the scientific community. At times, heated public debates have escalated to worrisome levels due to the fact that, to date, this tissue has always been derived from therapeutic or procured abortions. This also explains as to why approval from ethical committees may encounter significant hurdles when the use of foetal material is involved. To the best of our knowledge, this is the first time that a procedure for the isolation of hNSCs is successfully carried out from foetuses that were certifiably deceased in utero due to natural causes. Also, tissue procurement in this study took place according to the same international guidelines that are in place for the donation of organs for therapeutic transplantation. Hence, this provi
des an approach to hNSCs transplantation that is free from the ethical concerns that may arise from the use of foetal human tissues.

In all experimental and clinical transplantation studies on hNSCs, the lack of univocal hNSC markers makes it difficult to precisely define the actual composition of the transplanted cell suspension. This matter is further compounded by the fact that implanted cells undergo significant stress due to their preparation for transplantation, their transportation and loading into the needle and throughout the injection process. Up until now, it could not be ruled out that the cells death that inevitably occurs throughout these procedures might significantly or even prevalently affect the hNSCs pool in the injected cell suspension. As paradoxical as it may seem, the chances still exist that the stem cell content in the cell suspension that eventually reaches the patients own tissue in hNSCs transplantation procedures may drop significantly, even to the point that the final graft might actually contain negligible amounts of bona fide brain stem cells. Here we were able to recover the few cells that were left in the injection needle following transplantation and to examine them thoroughly. By this, we were able to show that the cells that were injected into the ventral horns of the spinal cord of ALS patients were, in fact, the same hNSCs suspension that were released from the cell factory that, as such, did retain key features of stemness and lack of tumorigenicity (Figure6).

We are now routinely carrying this procedure out. This introduces an additional level of control on the cell composition and quality that should help improve standardization of hNSC-based cell therapies in future trials.

We have been able to reproducibly and quite extensively expand hNSCs ex-vivo. Thus, this whole trial is actually based on the use of only two donors. The use of such a limited number of donors inherently reduces the inter-treatment variability that may arise from the implantation of different hNSC lines into different patients.

Very few studies in the scientific literature report the results of clinical trials with stem cell transplantation. These results are not definitive and no trial has been replicated in multiple centres. Experimental neural transplantation inevitably involves only relatively small groups of patients, with a corresponding loss of statistical prowess. Hence, only international collaboration may accomplish the most effective progress in stem cell clinical trial. This is the first report of an international coordinated effort about the cell therapy and transplantation approach in ALS patients. By utilizing a methodology similar to that adopted by our collaborators [18], we have reproduced the safety of the approach and provided an improved ability to compare the relative efficacy of the different cell types, also factoring out variance in the approach to delivery. A large multidisciplinary team of cell biologists, neurologists and neurosurgeons with significant experience in experimental cell therapy and in the treatment of ALS are involved in our studies. These researchers worked closely with regulatory agencies (AIFA, ISS), patient advocacy groups and ethical regulatory bodies. Moreover this is the first clinical trial with hNSCs that is run entirely by a not-for-profit organization, strictly on a charitable fund raising basis.

We are now broadening the import of this trial by testing intraspinal injections into the cervical spinal cord (C3-C4 level) of 12 ambulatory patients.

We can conclude that, while these results are not definitive, it appears that transplantation of human fetal neural cells in the lumbar spinal cord is a safe procedure. This technique has now been freed of any ethical concerns arising from the use of human fetal tissue. In addition, up to five fold more cells can now be safely implanted into the human spinal cord and a milder immunosuppression regimen adopted in ALS patients in the absence of related adverse effects.

We will next compare our results with the findings from the recently concluded trial at Emory University, which will help both groups to extend and deepen their conclusion on the use of cell therapy in ALS.

The results from this investigation also describe a cell therapy platform that will allow broadening the number and reproducibility of cell therapy clinical trials for ALS and, other neurological disorders. These can now be carried out under more standardized conditions and will be based on a more homogenous repertoire of clinical grade, donor hNSCs, which also avoid ethical concerns.

ALS: Amyotrophic lateral sclerosis

hNSC: Human neural stem cell

GMP: Good manufacturing practice

ALS-FRS-R: Amyotrophic lateral sclerosis-functional rating scale-revised

MRC: Medical Research Council

PEG: Percutaneous Endoscopic Gastrostomy

MN: Motoneuron

MSC: Mesenchymal stem cell

NPC: Neural progenitor cell

FDA: Food and Drug Administration

ISS: Istituto Superiore di Sanit

AIFA: Agenzia Italiana del Farmaco

EudraCT: European Clinical Trials Database

FVC: Forced vital capacity

POMS: Profile of mood state

SEIQoL-DW: Schedule evaluation of individual quality of life direct weighting

SC: Spinal cord

MRI: Magnetic resonace imaging

DTI: Diffusion tensor imaging

FA: Fractional anisotropy

ADC: Apparet diffusion coefficient

ROI: Region of interest

MPR: Multi planar reformat

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From stem cells to billions of human insulin-producing …

Posted: August 19, 2015 at 2:45 pm

Harvard stem cell researchers today announced that they have made a giant leap forward in the quest to find a truly effective treatment for type 1 diabetes, a condition that affects an estimated 3 million Americans at a cost of about $15 billion annually:

With human embryonic stem cells as a starting point, the scientists are for the first time able to produce, in the kind of massive quantities needed for cell transplantation and pharmaceutical purposes, human insulin-producing beta cells equivalent in most every way to normally functioning beta cells.

Doug Melton, who led the work and who 23 years ago, when his then infant son Sam was diagnosed with type 1 diabetes, dedicated his career to finding a cure for the disease, said he hopes to have human transplantation trials using the cells to be underway within a few years.

We are now just one pre-clinical step away from the finish line, said Melton, whose daughter Emma also has type 1 diabetes.

A report on the new work has today been published by the journal Cell.

Felicia W. Pagliuca, Jeff Millman, and Mads Gurtler of Meltons lab are co-first authors on the Cell paper. The research group and paper authors include a Harvard undergraduate.

You never know for sure that something like this is going to work until youve tested it numerous ways, said Melton, Harvards Xander University Professor and a Howard Hughes Medical Institute Investigator. Weve given these cells three separate challenges with glucose in mice and theyve responded appropriately; that was really exciting.

It was gratifying to know that we could do something that we always thought was possible, he continued, but many people felt it wouldnt work. If we had shown this was not possible, then I would have had to give up on this whole approach. Now Im really energized.

The stem cell-derived beta cells are presently undergoing trials in animal models, including non-human primates, Melton said.

Elaine Fuchs, the Rebecca C. Lancefield Professor at Rockefeller University, and a Howard Hughes Medical Institute Investigator who is not involved in the work, hailed it as one of the most important advances to date in the stem cell field, and I join the many people throughout the world in applauding my colleague for this remarkable achievement.

For decades, researchers have tried to generate human pancreatic beta cells that could be cultured and passaged long term under conditions where they produce insulin. Melton and his colleagues have now overcome this hurdle and opened the door for drug discovery and transplantation therapy in diabetes, Fuchs said.

And Jose Oberholzer, MD, Associate Professor of Surgery, Endocrinology and Diabetes, and Bioengineering at the University of Illinois at Chicago, and its Director of the Islet and Pancreas Transplant Program and the Chief of the Division of Transplantation, said work described in todays Cell will leave a dent in the history of diabetes. Doug Melton has put in a life-time of hard work in finding a way of generating human islet cells in vitro. He made it. This is a phenomenal accomplishment.

Melton, co-scientific director of the Harvard Stem Cell Institute, and the Universitys Department of Stem Cell and Regenerative Biology both of which were created more than a decade after he began his quest said that when he told his son and daughter they were surprisingly calm. I think like all kids, they always assumed that if I said Id do this, Id do it, he said with a self-deprecating grin.

Type 1 diabetes is an autoimmune metabolic condition in which the body kills off all the pancreatic beta cells that produce the insulin needed for glucose regulation in the body. Thus the final pre-clinical step in the development of a treatment involves protecting from immune system attack the approximately 150 million cells that would have to be transplanted into each patient being treated. Melton is collaborating on the development of an implantation device to protect the cells with Daniel G. Anderson, the Samuel A. Goldblith Professor of Applied Biology, Associate Professor in theDepartment of Chemical Engineering, the Institute of Medical Engineering and Science, and the Koch Institute at MIT.

Melton said that the device Anderson and his colleagues at MIT are currently testing has thus far protected beta cells implanted in mice from immune attack for many months. They are still producing insulin, Melton said.

Cell transplantation as a treatment for diabetes is still essentially experimental, uses cells from cadavers, requires the use of powerful immunosuppressive drugs, and has been available to only a very small number of patients.

MITs Anderson said the new work by Meltons lab is anincrediblyimportant advance for diabetes. There is no question that ability to generate glucose-responsive, human beta cells through controlled differentiation of stem cells will accelerate the development of new therapeutics. In particular, this advance opens to doors toan essentially limitless supply oftissue for diabetic patients awaiting cell therapy.”

RichardA.Insel, MD, chief scientific officer of the JDRF, a funder of Meltons work, said the JDRF is thrilled with thisadvancementtoward large scale production of mature, functional human beta cells by Dr. Melton and his team. This significant accomplishmenthas the potentialto serve as a cell source for islet replacement in people with type 1 diabetes and mayprovide a resource for discovery of beta cell therapies that promote survival or regeneration of beta cells and development of screening biomarkers to monitor beta cell health and survival to guidetherapeutic strategies for all stages of the disease.

Melton expressed gratitude to both the Juvenile Diabetes Research Foundation and the Helmsley Charitable Trust, saying their support has been, and continues to be essential. I also need to thank Howard and Stella Heffron, whose faith in our vision got this work underway, and helped get us where we are today.

While diabetics can keep their glucose metabolism under general control by injecting insulin multiple times a day, that does not provide the kind of exquisite fine tuning necessary to properly control metabolism, and that lack of control leads to devastating complications from blindness to loss of limbs.

About 10 percent of the more than 26 million Americans living with type 2 diabetes are also dependent upon insulin injections, and would presumably be candidates for beta cell transplants, Melton said.

There have been previous reports of other labs deriving beta cell types from stem cells, no other group has produced mature beta cells as suitable for use in patients, he said. The biggest hurdle has been to get to glucose sensing, insulin-secreting beta cells, and thats what our group has done.

In addition to the institutions and individual cited above, the work was funded by the Harvard Stem Cell Institute, the National Institutes of Health, and the JPB Foundation.

Cited: Pagliuca, F., Millman, J. and Grtler, M, et. al. Generation of functional human pancreatic beta cells in vitro. Cell. October 9, 2014.

Dr. Melton has made an author’s proof available. Click here to download the PDF.

The beginning shows a spinner flask containing red culture media and cells, the cells being too small to see. Inside the flask you can see a magnetic stir bar and the flask is being placed on top of a magnetic stirrer.

This is followed by a time cours
e series of images, magnified, showing how cells tart of as single cells and then grow very quickly into clusters over the next few days. The size of the clusters is the same as the size of human islets at the end.

The final image shows 6 flasks, enough for 6 patients, spinning away. If you look closely, you can see particles spinning around, the white dust or dots are clusters of cells, each containing about 1000 cells.

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Why I'm sure human stem cell trial will be safe – New …

Posted: August 9, 2015 at 6:45 pm

(Image: Natasha Little)

The new kind of stem cell announced yesterday may be the future of regenerative medicine, but Masayo Takahashis pilot safety study using a type of stem cell to treat age-related blindness is at the cutting edge

Later this year, you will make history when you begin the first ever human trial of induced pluripotent stem cells. Why is this such a big deal? Stem cells have enormous medical potential because they can become any other type of cell. If we can use them to replace old or damaged cells, this could have huge implications for treating degenerative diseases.

Stem cells can be harvested from embryos, but this is ethically controversial. Despite this, there are several trials of these embryonic stem cells under way. Their use often requires drugs to stop the immune system from rejecting them, which can cause complications for elderly patients. Induced pluripotent stem (iPS) cells offer an alternative. These are made from a patients own cells, removing the need for the immunosuppressant drugs. Plus there are no ethical issues.

How would treatment with iPS cells work? iPS cells are made by injecting several reprogramming genes into adult cells that have been removed from the body. This makes them rewind to an embryonic state. Then, we can make iPS cells differentiate into the cell type we need by injecting proteins that instruct embryonic stem cells to become liver, retina or any other type of cell. The idea is that these reprogrammed cells can then be inserted in the body to replace damaged cells. We are at least 20 years from any clinical treatments, but the potential is exciting.

Are there any potential pitfalls with iPS cell treatments? Yes, we have to be very careful because iPS cells multiply endlessly. This means that if any undifferentiated iPS cells were accidentally put into someone, they could cause tumours. Thats why this study is so important. It is not a clinical trial, but a six-subject pilot study to confirm the safety of putting cells derived from iPS cells into humans.

Who are the participants in the study? The six people all have age-related macular degeneration in their eyes. This weakens the vision in the central field, eventually leaving people with only peripheral vision. In the type of degeneration we are working with, this is caused by the deterioration of the retinal pigment epithelium (RPE) the layer of cells that clears away extra-cellular debris that lands on the retina.

We aim to replace the damaged section of the RPE with cells created from skin taken from the patients arm. The skin cells will be reprogrammed into iPS cells and then differentiated into RPE cells. It will take a year to grow enough RPE cells to introduce them to a damaged eye. Although I am excited to see if there is any improvement in sight, this study aims only to demonstrate the safety of RPE cells derived from IPS cells.

How confident are you that the pilot will be a success? Very confident. We have trialled this intervention on mice, rats and monkeys, and observed no tumours. I chose to work with RPE cells because of their characteristic brown pigment. This means we can avoid injecting tumour-causing iPS cells by selecting only the clumps of pure brown RPE cells. Of course, we do have to pick out around 50,000 RPE cells, so it can be a bit tough.

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Stem Cell Trial ALS Clinic

Posted: August 1, 2015 at 10:40 am

Stem cell transplantation study for the treatment of ALS Phase 2

The phase 2 trial focused on the safety and maximum tolerated dose of Human Spinal Cord Derived Neural Stem Cell Transplantation. This study expanded on the work of the phase 1 study directed by Eva L. Feldman, M.D., Ph.D., who is the principal investigator and director of the first-ever FDA-approved human clinical trial of stem cells injected directly into the spinal cords of ALS patients.

Phase 1 of the trial, designed to study the safety of the procedure, was completed in 2013 with no significant adverse side effects to patients. And follow-up patient evaluations have produced some extraordinary data: Several participants in the trial, who were treated early in their disease, were determined to have had little or no significant progression of ALS for more than 700 days post-surgery.

Updated July 2015: Fifteen patients were studied in the phase 2 study, the last 3 of which received 8 million stem cells injected into the lumbar spinal cord followed by 8 million stem cells injected into the cervical spinal cord.

All procedures have been completed and the trial is still ongoing. No data has been released.

Future trials

Updated July 2015: No details are avakilable for a possible next pahse of the study at this time and we are not enrolling patients. We encourage any interested persons to continue to monitor the Neuralstem website for additional details as they are released.

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Stem Cell Trial ALS Clinic

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