Stem Cell-Based Spinal Cord Repair Enables Robust Corticospinal Regeneration

In the March 28th, 2016 issue of the journal Nature Medicine, Mark Tuszynski and his colleagues from the University of California, San Diego, in collaboration with colleagues from Japan and Wisconsin, report that they were able to successfully coax stem cell-derived neurons to regenerate damaged corticospinal tracts in rats. Furthermore, this regeneration produced observable, functional benefits.

What is the “corticospinal tract” you ask? The corticospinal tracts are part of the “pyramidal tracts” that include the corticospinal and corticobulbar tracts. The pyramidal tracts are the main controllers of voluntary movement and connect their nerve fibers eventually to cells that serve voluntary muscles and allow them to contract. We call such nerves “motor nerves,” and the corticospinal nerve tracts are among the most important of the motor nerve tracts.

These neural tracts are collectively called “pyramidal tracts” because they pass through a small area of the brain stem known as the pyramids, which lie on the ventral side of the medulla oblongata. Both pyramidal tracts originate in the forebrain; specifically from the so-called “motor cortex” of the forebrain. The motor cortex lies just in front of the central sulcus of the forebrain. In the motor cortex, lies thousands of “upper motor neurons” that extend their axons down to the brain stem and spinal cord.

In the brain stem, the majority of these corticospinal tracts crossover (or decussate) to the other side of the brain stem and travel down the opposite side of the spinal cord. The corticospinal axons extend all the way down the spinal cord, until they make a connection (synapse) with a “lower motor neuron” that extends its axon to the skeletal muscles that it will direct to contract. The corticobulbar tract contains nerves that conduct nerve impulses from cranial nerves and these help the muscles of the face and neck contract, and are involved in facial expressions, swallowing, chewing, and so on.

Damage to the upper motor neurons as a result of a stroke can rob a person of the ability to move, since the muscles that are attached to the upper motor neurons cannot receive any signals to contract. Likewise, damage to the axonal tracts (also known as nerve fibers) can paralyze a patient and rob them of their ability to move.

The director of this research project, Mark Tuszynski, MD, PhD, professor in the UC San Diego School of Medicine Department of Neurosciences and director of the UC San Diego Translational Neuroscience Institute, said: “The corticospinal projection is the most important motor system in humans. It has not been successfully regenerated before. Many have tried, many have failed – including us, in previous efforts.”

Dr. Tuszynski continued, “The new thing here was that we used neural stem cells for the first time to determine whether they, unlike any other cell type tested, would support regeneration. And to our surprise, they did.”

In this experiment, Tuszynski, and his colleagues and collaborators used rats that had suffered spinal cord injuries and had trouble moving their forelimbs. Then they implanted grafted multipotent neural progenitor cells (MNPCs) into those sites within the spinal cord that had suffered injury, where corticospinal axonal tracts had been severed or damaged. The MNPCs had been previously treated to differentiate into spinal cord-specific motor neurons. Fortunately, the MNPCs prodigiously formed lower motor neurons that made good, solid, functional synapses with interneurons and upper motor neurons that improved forelimb movements in the rats. This work put the lie to previous beliefs about corticospinal neurons; namely that they lacked any of the internal mechanisms required to regenerate severed or damaged connections.

Even though several previous studies have demonstrated functional recovery in spinal cord-injured rats through the use of stem cell-based treatments, none of these studies has convincingly demonstrated regeneration of corticospinal axons.

“We humans use corticospinal axons for voluntary movement,” said Tuszynski. “In the absence of regeneration of this system in previous studies, I was doubtful that most therapies taken to humans would improve function. Now that we can regenerate the most important motor system for humans, I think that the potential for translation is more promising.”

This is certainly exciting work, but even though it worked in rats, it may not yet work in humans. The road from pre-clinical studies in animals to clinical trials in humans is a long, tedious, frustrating, and uncertain pathway, pockmarked with the failures of past therapies that worked well in animals but failed to translate into successes in human patients.

“There is more work to do prior to moving to humans,” Tuszynski said. We must establish long-term safety and long-term functional benefit in animals. We must devise methods for transferring this technology to humans in larger animal models. And we must identify the best type of human neural stem cell to bring to the clinic.”

Individual Cells of Four Cell-Stage Embryos Show Distinct Genetic Signatures

University of Cambridge and EMBL-EBI researchers have revealed that differences in gene expression begin emerge earlier in human development than originally thought.  According to the Cambridge and EMBL teams, genetic differences arrive as early as the second day after the completion of fertilization.  These four cell-stage embryos consist of four “blastomeres” that appear identical in size and shape.  However, even at these early stages, these four blastomeres are already beginning to display subtle differences in gene expression.

Fertilization of an egg (oocyte) by a sperm is a multistep process that begins with the contact of the sperm with the jelly layer that surrounds the egg (zona pellucida), and the acrosomal reaction of the sperm, contact of the egg and sperm membranes, followed by fusion of the egg and sperm membranes, egg activation, disassembly of the sperm and remodeling of the sperm and egg pronuclei, contact of the sperm and egg pronuclei, and culminating in the initiation of the first mitotic division.  The first cell division or “cleavage” occurs approximately 24 hours after the initiation of fertilization, and forms the two-cell embryo.  The next cleavage occurs about 12 hours later, and the blastomeres initially divide synchromously (at the same time), but eventually divide asynchronously (at different times).  During these early cleavages of the zygote, special embryonic cell cycles and include S phases and M phases that alternate without any intervening G1 or G2 phases.  Therefore individual cell volume decreases.  About day 4, the embryo is a solid ball of 16-20 cells with peripheral cells flattened against the zona pellucida, and compaction occurs forming a cavity that leads to the next blastocyst stage, which is a large free-floating ball of stem cells.

At first, the blastomeres of the early embryo are “totipotent,” which means that each blastomere can potentially divide and grow and produce every single cell of the whole body and the placenta.  After compaction, two cell populations emerge that include, round, slow-dividing cells in the center and fast-growing flatter cells on the outside.  The central cells of the inner cell mass have a “pluripotent” status, which means that they can generate the cells of the whole body, but not the placenta.  However, the point during development at which cells begin to show a preference for becoming a specific cell type is unclear.

At this point, the new study, which was published in the journal Cell, presents rather convincing data that even as early as the four-cell embryo stage, the cells are indeed different.

The EMBL/Cambridge teams utilized the latest sequencing technologies to model embryo development in mice and examined the activity of individual genes at a single cell level.  This analysis showed that some genes in each of the four blastomeres showed distinct genetic signatures.  The expression of one gene in particular, Sox21, differed the most between cells.  Sox21 is part of the so-called “pluripotency network.”  The pluripotency network consists of a cascade of genes that are essential both in culture (in vivo) and in vitro (in the organism) for early development and maintenance of pluripotency.  The EMBL/Cambridge teams discovered that when the activity of Sox21 was reduced, the activity of a master regulator that directs cells to develop into the placenta increased.

“We know that life starts when a sperm fertilizes an egg, but we’re interested in when the important decisions that determine our future development occur,” says Professor Magdalena Zernicka-Goetz from the Department of Physiology, Development and Neuroscience at the University of Cambridge. “We now know that even as early as the four-stage embryo – just two days after fertilization – the embryo is being guided in a particular direction and its cells are no longer identical.”

Dr John Marioni of EMBL-EBI, the Wellcome Trust Sanger Institute and the Cancer Research UK Cambridge Institute, adds: “We can make use of powerful sequencing tools to deepen our understanding of the molecular mechanisms that drive development in individual cells. Because of these high-resolution techniques, we are now able to see the genetic and epigenetic signatures that indicate the direction in which early embryonic cells will tend to travel.”

This research tends to diffuse one of the arguments embryonic stem cell proponents use to justify the destruction of human embryos.  Namely, the early human embryos consist of cells that are all the same and have no interactions with each other.  The embryo is, then, not an individual organism, but a collection of many potential organisms that eventually becomes as unified organism.  This turns out to be incorrect, since the cells of the early blastomere are not all equivalent.  Instead, the blastomeres are interacting with each other and using these interactions to figure what kind of cells their progeny will form.  This is the hallmark of an entity with a unified purpose that has a distinct goal.  Folks, that sounds like a unified organism.  It is simply young.

First Patient Randomized for ACTIsSIMA Trial for Chronic Stroke

SanBio, a regenerative medicine company in Mountain View, California, has announced the randomization of the first enrolled patient in the ACTIsSIMA Phase 2B clinical trial. This trial will examine the efficacy of SanBio’s proprietary SB623 product in patients who suffer from chronic motor deficits as a result of strokes. SB623 consists of modified adult bone-marrow-derived stem cells. A secondary purpose of this trial is to evaluate the safety of SB623 in these patients.

Ischemic strokes account for about 87 percent of all strokes in the United States. Ischemic strokes occur when there is an obstruction in one or more of the blood vessels that provide blood and oxygen to the brain. On the order of 800,000 cases of ischemic stroke occur in the United States every year, and it is the leading cause of acquired disability in the United States. Present drug treatments for stroke either try to prevent strokes or address patients who have recently suffered a stroke. Unfortunately, there are no medical treatments currently available for people who live with the effects of stroke, months or even years after suffering a stroke.

SB623 cells are derived from bone marrow mesenchymal stem cells extracted from healthy donors. These cells are designed to promote recovery from injury by triggering the brain’s natural regenerative ability. SB623 cells have been genetically engineered to express a modified version of the Notch gene (NICD) that conveys upon the cells the ability to promote the formation of new blood vessels and the survival of endothelial cells that form these new blood vessels (see J Transl Med. 2013, 11:81. doi: 10.1186/1479-5876-11-81).

SB623 was tested in a Phase 1/2A clinical trial in which SB623 was implanted into stroke patients and produced some improved motor function.

This follow-up trial, ACTIsSIMA, will treat stroke patients with SB623 cells in order to examine the safety and efficacy of SB623 cells. All patients in this trial have suffered from a stroke anywhere from six months to five years. Also, all patients must exhibit chronic motor impairments.

Damien Bates, M.D., Chief Medical Officer & Head of Research at SanBio, said, “Our previous trial suggested there was potential for SB623 to improve outcomes for patients with lasting motor deficits following an ischemic stroke. Randomization of the first subject marks an exciting step toward further evaluating this treatment as a promising new option for patients.”

For this trial, SanBio is collaborating with Sunovion Pharmaceuticals, Inc. Sunovion is a wholly owned subsidiary of Sumitomo Dainippon Pharma Co., Ltd., and SanBio and Sumitomo Dainippon Pharma have entered into a joint development and license agreement for exclusive marketing rights in North America for SB623 for chronic stroke.

The ACTIsSIMA trial will include approximately 60 clinical trial sites throughout the United States, and total enrollment is expected to reach 156 patients.

Stem Cells Regenerate Human Lens After Cataract Surgery and Restore Vision

Collaboration between scientists from mainland China, the University of California, San Diego School of Medicine and Shiley Eye Institute have developed a new, stem cell-based technique that permits remaining stem cells to regrow functional lenses after the diseased lens was removed. This treatment was initially tested in laboratory animals, but it has now been tested in a small human clinical trial. This procedure produced far fewer surgical complications than the current standard-of-care. The real boost is that this regenerative procedure resulted in regenerated lenses that had superior visual qualities in all 12 of the pediatric cataract patients who served as subjects for this clinical trial.

Kang Zhang, MD, PhD, chief of Ophthalmic Genetics, founding director of the Institute for Genomic Medicine and co-director of Biomaterials and Tissue Engineering at the Institute of Engineering in Medicine, both at UC San Diego School of Medicine, said: “An ultimate goal of stem cell research is to turn on the regenerative potential of one’s own stem cells for tissue and organ repair and disease therapy.” Zhang and his colleagues published their work in the journal Nature.

Cataracts are cloudiness over the lens of the eye that blurs vision. The lens consists mostly of water and protein. When the protein aggregates, it clouds the lens and reduces the light that reaches the retina. This clouding may become severe enough to cause blurred vision. Most age-related cataracts develop from protein clumpings. You do not have to be older to suffer from cataracts. Congenital cataracts occur at birth or shortly after birth. Scarring of the retina or prenatal damage to the eye can cause congenital cataracts. Congenital cataracts are a significant cause of blindness in children. Current treatment for congenital cataracts is limited by the age of the patient. Most pediatric patients require corrective eyewear after cataract surgery.

To address this medical need, Zhang and colleagues examined the regenerative potential of endogenous stem cells on the lens. Unlike other stem cell approaches that involve creating stem cells in the lab and introducing them back into the patient, Zhang decided to use stem cells that are already in place at the site of the injury to do the heavy lifting. In the human eye, lens epithelial stem cells or LECs generate replacement lens cells throughout a person’s life, even though their production declines with age.

Unfortunately, current cataract surgeries essentially remove LECs within the lens. Whatever cells might be left over produce disorganized regrowth in infants and no useful vision. Zhang and his colleagues first confirmed that LECs had regenerative potential. To confirm this, they used laboratory animals. With that knowledge in hand, Zhang and his collaborators devised a novel, minimally invasive surgical procedure that removes the cloudy lens, but manages to maintain the integrity of the membrane that gives the lens its required shape (the lens capsule). With the lens capsule in place, the LECs were activated to replace the missing lens.

Once again, Zhang and his team ensured that their technique worked in animals before they ever tried it on a human patient. Animals with cataracts whose lenses were extirpated, but whose lens capsules were left intact, regenerated new lenses that were devoid of cataracts and provided excellent sight. With their technique honed and ready, Zhang and others tested their procedure on very young human infants in a small human trial. They discovered that their new surgical technique allowed pre-existing LECs to efficiently regenerate functional lenses. In particular, the human trial involved 12 infants under the age of 2 treated with the new method developed by Zhang and others, and 25 similar infants receiving current standard surgical care.

The results were stark: the control group experienced a higher incidence of post-surgery inflammation, early-onset ocular hypertension and increased lens clouding, but those infants who received Zhang’s new procedure showed fewer complications and faster healing. After three months, the 12 infants who underwent the new procedure had a clear, regenerated biconvex lens in all of their eyes.

“The success of this work represents a new approach in how new human tissue or organ can be regenerated and human disease can be treated, and may have a broad impact on regenerative therapies by harnessing the regenerative power of our own body,” said Zhang.

Zhang indicated that he and his colleagues are now looking to apply what they learned in this project to tackling the issue of age-related cataracts. Age-related cataracts are the leading cause of blindness in the world. Over 20 million Americans suffer from cataracts, and more than 4 million surgeries are performed annually to replace the clouded lens with an artificial plastic lens (intraocular lens).

Despite technical advances, a large portion of patients undergoing surgery are left with suboptimal vision post-surgery and are dependent upon corrective eyewear for driving a car and/or reading a book. “We believe that our new approach will result in a paradigm shift in cataract surgery and may offer patients a safer and better treatment option in the future,” said an optimistic Zhang.

Gladstone Institute Scientists Devise New Way to Make Heart Cells from Skin Cells Opening the Door to the Possibility of Personalized Medicine for Heart Attack Patients

Gladstone Institute research scientists have devised a new way to make heart replacement cells. This novel protocol generates cells that lie in between embryonic stem cells and adult heart cells. These induced expandable cardiovascular progenitor cells (ieCPCs) might very well hold the key to treating heart disease. Even though ieCPCs can develop into heart cells, they still have the ability to grow and expand in culture to produce the large numbers of cells required for clinical purposes. When these ieCPCs are injected directly into the hearts of laboratory mice that have recently suffered a heart attack, they formed heart muscle cells and other heart-specific cell types and significantly improved heart function.

Yu Zhang, MD, PhD, lead author on the study and a postdoctoral scholar at the Gladstone Institutes said, “Scientists have tried for decades to treat heart failure by transplanting adult heart cells, but these cells cannot reproduce themselves, and so they do not survive in the damaged heart.” Zhang continued, “Our generated ieCPCs can prolifically replicate and reliably mature into the three types of cells in the heart, which makes them a very promising potential treatment for heart failure.”

CPCs or cardiovascular progenitor cells are the result of embryonic development and help form the embryonic heart. In the embryo, CPCs can differentiate into a wide variety of different heart-specific cells. This Gladstone Institute study, which was published in the journal Cell Stem Cell, Zhang and his colleagues reprogrammed mouse embryonic fibroblasts into CPCs in the laboratory. Once the mouse embryonic fibroblasts had been reprogrammed into CPCs, Zhang and others used a special medium to keep the cells from differentiating into fully-mature, functional heart cells that no longer were able to divide.

CPCs constitute so-called “organ-specific stem cells.” Organ-specific stem cells are special because they can differentiate into adult cells and, under the right conditions, grow, expand and proliferate in culture indefinitely. Zhang and his colleagues were able to expand their ieCPC cultures for over a dozen generations. This generated more than enough cells to treat several patients.

The importance of the ability of these cells to expand in the laboratory cannot be undersold. When a patient suffers a heart attack, over one billion heart cells can die off. Robust cell renewal means ieCPCs can play the role of a sustainable source of cells that can replace the cells that died as a result of the heat attack. Furthermore, ieCPCs can also develop into each of the three different types of heart cells: cardiomyocytes (heart muscle cells), endothelial cells (blood vessel cells), and smooth muscle cells (that surround the blood vessels and regulate their diameter).. When ieCPCs were injected into a mouse hearts, they spontaneously differentiated into each of these heart-specific cell types without requiring any further coaxing or signals.

Previous attempts to treat heart failure by transplanting adult heart cells have produced, for the most part, modest results. Implanted cells tend to survive poorly and do not self-renew, which seriously compromises their ability to repopulate and heal a damaged heart. An additional caveat is that regenerating the heart after a heart attack requires that the heart be supplied with more than just heart muscle cells (cardiomyocytes). Instead the heart needs all three cell types;

Clinical trials that have tested the ability of non-cardiac stem cells to heal the heart after a heart attack have also shown modest, though limited success. In this case, the implanted cells only differentiate into heart-specific cells types rather poorly. Such transdifferentiation events require complex signals that are absent in an adult heart. ieCPCs circumvent these issues since they are already heart-specific progenitor cells that are committed to forming heart-specific cell types.

In this study, 90% of the injected ieCPCs were retained in a mouse heart after a heart attack and successfully differentiated into functioning heart cells. The ieCPCs formed cardiomyocytes that integrated into the myocardium and formed functional connections with existing, surviving cardiomyocytes. The ability to connect with existing heart muscle cells is also crucial to minimize the risk of arrhythmias after a heart attack. The implanted ieCPCs also created new blood vessels that pumped blood and oxygen to newly-forming heart tissues. The ieCPCs significantly improved heart function. The mouse hearts pumped more efficiently, and the benefits lasted for at least three months. Because these cells are generated from skin cells, this procedure also opens the door for personalized medicine in which a heart patient’s own cells are used to treat their heart disease.

ieCPCs Give Rise to CMs, ECs, and SMCs In Vivo and Improve Cardiac Function after MI

(A–E) Immunofluorescence analyses of RFP and CM (A), EC (B and C), and SMC (D and E) markers in tissue sections collected 2 weeks after transplanting RFP-labeled ieCPCs at passage 10 into infarcted hearts of immunodeficient mice. Scale bars represent 100 μm.

(F and G) Ejection fraction and fractional shortening of the left ventricle (LV) quantified by echocardiography. Results from two independent experiments were shown. D, days; W, weeks.

(H–J) Cardiac fibrosis was evaluated at eight levels (L1–L8) by Masson’s trichrome staining 12 weeks after coronary ligation. The ligation site is marked as X. Sections of representative hearts are shown in (I) with quantification in (J). Scar tissue (%) = (the sum of fibrotic area or length at L1–L8/the sum of LV area or circumference at L1–L8) × 100. Scale bars represent 500 μm.

(K) Quantification of LV circumference of mouse hearts 12 weeks after transplantation of 2nd MEFs or ieCPCs. Data were summarized from 48 sections for each group. Data are mean ± SE. ^∗p < 0.05.

“Cardiac progenitor cells could be ideal for heart regeneration,” said senior author Sheng Ding, PhD, a senior investigator at Gladstone. “They are the closest precursor to functional heart cells, and, in a single step, they can rapidly and efficiently become heart cells, both in a dish and in a live heart. With our new technology, we can quickly create billions of these cells in a dish and then transplant them into damaged hearts to treat heart failure.”