Targeting Breast Cancers with Neural Stem Cells

Singapore scientists, in particular researchers at the Institute of Bioengineering and Nanotechnology (IBN) showed that engineered neural stem cells can target and kill breast cancers.

In this study, workers in the laboratory of Shu Wang used mouse induced pluripotent stem cells (iPSCs) and differentiated them into neural stem cells (NSCs). They then engineered the NSCs to express a viral gene called thymidine kinase. Thymidine kinase comes from Herpes viruses, and this is particular gene that is not found in human cells. Therefore it is a target for anti-herpes virus drugs. By using an insect virus called “baculovirus,” Wang and his colleagues introduced thymidine kinase into NSCs. The use of baculovirus makes the NSCs safer for clinical use, since, being an insect virus, it does not grow in human cells, but can introduce genes into them.

By placing the herpes thymidine kinase gene into NSCs, it makes from susceptible to antiherpes drugs. For example, ganciclovir (Cytovene), is phosphorylated by thymidine kinase, and this molecule is quite toxic to cells. Contact between the engineered NSCs and cancer cells, would cause transfer of the toxic molecule to the cancer cells, which would kill cancer cells too. However, this begs the question: Can NSCs home to the tumor and target it?

In order to test the ability of NSCs to target and treat breast cancers, Wu’s group injected NSCs loaded with the suicide gene mice afflicted with breast tumors. Then they treated the mice with ganciclovir. Dual-colored whole body imaging was used to track the distribution and migration of the engineered NSCs.

Imaging showed that the NSCs homed in on the breast tumors in the mice, and accumulated in various organs that were infiltrated by the cancer cells. The survival of the tumor-bearing mice was prolonged from 34 days to 39 days. These data demonstrate that iPS-derived NSCs are able to effectively seek out and inhibit tumor growth and proliferation.

According to Dr Shu Wang, “We have demonstrated that tumor-targeting neural stem cells may be derived from human iPS cells, and that these cells may be used in combination with a therapeutic gene to cripple tumor growth. This is a significant finding for stem cell-based cancer therapy, and we will continue to improve and optimize our neural stem cell system by preventing any unwanted activation of the therapeutic gene in non-tumor regions and minimizing possible side effects.”

Professor Jackie. Y. Ying, IBN Executive Director, said, “IBN’s expertise in generating human stem cells from iPS cells and our novel use of insect virus carriers for gene delivery have paved the way for the development of innovative stem cell-based therapies. With their two-pronged attack on tumors using genetically engineered neural stem cells, our researchers have discovered a promising alternative to conventional cancer treatment.

Palliative Sedation is Not the Same as Euthanasia

Palliative sedation is a medical technique for terminally ill patients who cannot receive adequate pain relief while they are awake. Palliative sedation uses sedative medications to make the patient unaware and unconscious while the disease takes its course. This relieves extreme suffering by placing the patient in a kind of sleep. The sedative medication is gradually increased until the patient is comfortable and able to relax. Palliative sedation is not intended to cause death or shorten life (Erin Brender, MD; Alison Burke, MA; Richard M. Glass, MD. JAMA. 2005;294(14):1850.)

This has not stopped euthanasia advocates from asserting that palliative sedation is euthanasia. The inimitable Wesley Smith has a blog post on this and he refers to an article in the Journal of Pain & Palliative Care Pharmacotherapy that takes this deliberate conflation of these two very different things to the woodshed.  It’s a great read.  Check it out here.

Curing AIDS With Engineereed Stem Cells

Scientists from the UC Davis Health Science HIV team have demonstrated in a proof of principle study the safety and efficacy of transplanting HIV-resistant stem cells into mice.  If this protocol can be replicated in humans, it could signal a way to completely block HIV infection in human patients.

The human immunodeficiency virus (HIV) is a retrovirus.  The retroviruses contain an RNA genome, but once they infect the host cell, the RNA genome serves as a template for the synthesis of a DNA copy of the RNA genome.  The enzyme that performs this task is reverse transcriptase.  The DNA copy of the genome is inserted into the genome of the host cell.  This means that when the host cell divides, the viral DNA is passed to all of its progeny.

HIV infection causes acquired immunodeficiency syndrome (AIDS).  AIDS is characterized by a progressive shutdown of the immune system, which leads to life-threatening infections and cancers.  HIV infection occurs through the transfer of bodily fluids, such as semen, blood, vaginal fluid, saliva, or breast milk.  Sexual transmission, transmission from breast milk, contaminated needles, or from an infected mother to her baby at birth are the four main modes of transmission.  HIV screening of blood products has largely eliminated HIV transmission from blood products.

Since the discovery of AIDS in 1981, more than 25 million people have died from it, and even though antiretroviral treatments have decreased AIDS deaths and new infections, there were still probably at least 2.5 million new cases of AIDS in 2009.

HIV destroys the immune response by infecting helper T cells (CD4+ cells).  HIV can also infect dendritic cell and macrophages.  The mass die off of T helper cells prevents them from mediating cell-mediated immunity, and this makes the patient more susceptible to opportunistic infections.  People with untreated HIV infections usually develop AIDS and die from opportunistic infections or tumors.  Without antiretroviral treatment, someone with AIDS usually dies within a year.

In order to make HIV-resistant blood cell-making stem cells, Joseph Anderson and his co-workers engineered stem cells with three different genes.  First, they introduced into the stem cells, a human/macaque TRIM5 isoform.  In order to understand the significance of this gene, we must understand HIV more deeply.  When a retrovirus enters a host cell, it must “uncoat,” which simply means that the protein coating that surrounds the virus’ genome must be removed so that the reverse transcriptase can convert the RNA genome into a DNA copy.  Macaques are very widespread Old Word nonhuman primates that are immune the infection by HIV.  The reason for the immunity of these animals to HIV infection is that they possess in their cells a form of the TRIM5 protein that binds to bits of the HIV coat proteins and interferes with the uncoating process.  This prevents successful reverse transcription and transport of the viral DNA to the nucleus.  Therefore, the expression of the macaque version of TRIM5 is these blood-making stem cells rendered them resistant to HIV infection.

Secondly, the blood-making stem cells were given a gene that expresses a short hairpin RNA (shRNA).  These shRNAs can bind to the mRNAs are particular genes that prevent their expression.  In this case, the shRNA that was introduced into the blood-making stem cells prevented the production of the CCR5 gene product.  CCR5 is one of the cell surface proteins that HIV uses to gain entry into host cells.  Therefore, these blood-making stem cells will make blood cells that lacked the target for HIV infection.

Third, cells were engineered with a “TAR decoy.”  TAR is a site in the HIV genome that is bound by the HIV-encoded proteins Tat.  Tat binding to TAR activates expression of HIV genes.  However, by introducing TAR sites into the cells, Tat proteins inordinately bind to these non-functional TAR sites and not to the viral TAR site.  This will inactivate any HIV particles that happen to infect these cells.  With all these factors introduced into them, these blood-making stem cells and their progeny are completely resistant to HIV infection.

Introduction of these engineered stem cells into mice allowed these mice to resist infection even after experimental infection with HIV.  In the words of the lead author of this paper, Joseph Anderson, “After we challenged transplanted mice with live HIV, we demonstrated that the cells with HIV-resistant genes were protected from infection and survived in the face of a viral challenge, maintaining normal human CD4 levels.”  Remember the CD4 cells are the class of T cells that are specifically targeted by HIV, although the virus can infect other cell types too.

Anderson continued: “We actually saw an expansion of resistant cells after the viral challenge, because other cells which were not resistant were being killed off, and only the resistant cells remained, which took over the immune system and maintained normal CD4 levels.”  Anderson’s optimism, however, does not end there:  “We envision this as a potential functional cure for patients infected with HIV giving them the ability to maintain a normal immune system through genetic resistance.”  Anderson is an assistant professor of internal medicine and a stem cell researcher at the UC Davis Institute for Regenerative Cures.

This study confirms the safety and efficacy of this protocol, and validates the potential of this treatment for human HIV patients.  A grant application has been submitted by Anderson and his team for human clinical trials, and they are also pursuing regulatory approval for clinical trials.

Richard Pollard, the chief of infectious diseases at UC Davis (and a co-author on the study), said: “This research represents an important step in our fight against HIV/AIDS.  Clinical trials could give us the critical information we need to determine whether our approach truly represents a functional cure for a terrible disease that has affected millions and millions of people.”

Reducing Heart Attack Scars in the Heart – Skip the Stem Cells

Two research groups have independently discovered that the heart scar that forms after a heart attack can be regenerated without stem cell treatments. Li Qian in the laboratory of Deepak Srivastava at the Gladstone Institute, and Victor Dzau’s team at Duke University have shown that the use of various molecules can trigger the conversion of scar tissue into heart muscle.

Dzau’s lab worked in mice and delivered microRNAs into fibroblasts, which are the cells that form the scar tissue in the heart. When these engineered fibroblasts took up the microRNAs, they became heart muscle cells.

MicroRNAs (miRNAs) are found inside cells are usually about 22 nucleotides long. These very small RNA molecules play important regulatory roles in animals and plants by targeting messenger RNAs (mRNAs) for cleavage or translational repression. Thus, miRNAs act as master regulatory molecules for gene expression (See Bartel DP. Cell. 2004;116(2):281-97).

“This is a significant finding with many therapeutic implications,” said Victor J. Dzau, MD, a senior author on the study who is James B. Duke professor of medicine and chancellor of health affairs at Duke University. “If you can do this in the heart, you can do it in the brain, the kidneys, and other tissues. This is a whole new way of regenerating tissue.”

After their experiments in tissue culture, Dzau’s lab showed that this conversion can also occur inside a living animal. Maria Mirotsou, PhD, assistant professor of cardiology at Duke and a senior author of the study commented, “This is one of the exciting things about our study. We were able to achieve this tissue conversion in the heart with these microRNAs, which may be more practical for direct delivery into cells and allow for possible development of therapies without using genetic methods or transplantation of stem cells.”

Since stem cells have proven difficult to manage inside the body, this mode of therapy has distinct advantages over stem cell-based treatments. Notably, the microRNA process eliminates technical problems such as genetic alterations, and also avoids the ethical dilemmas posed by the use of some stem cells.

“It’s an exciting stage for reprogramming science,” said Tilanthi M. Jayawardena, PhD, first author of the study. “It’s a very young field, and we’re all learning what it means to switch a cell’s fate. We believe we’ve uncovered a way for it to be done, and that it has a lot of potential.”

The next step is to test this approach in larger experimental animals. Dzau said therapies could be developed within a decade if additional studies advance in larger animals and humans.

“We have proven the concept,” Dzau said. “This is the very early stage, and we have only shown that is it doable in an animal model. Although that’s a very big step, we’re not there yet for humans.”

Gladstone researchers took a very different approach.  They delivered a cocktail of three genes that are known to direct cells to form heart muscle during embryonic development.  These three genes, Gata4, Mef2c and Tbx5, which are collectively called GMT, were placed into cells at the site of a heart attack.  Srivastava’s group engineered viruses to infect the heart tissue, and after inducing a heart attack, the engineered viruses were injected into the heart, at the site of the heart attack.

The heart contains several resident cell types that are not involved in contraction.  One of these resident populations is the fibroblast, which seems to be able to differentiate into heart muscle cells if properly coaxed.  The GMT-bearing viruses infected the resident fibroblasts and the infected cells differentiated into heart muscle cells that beat, formed connections with existing heart muscle cells, and contracted in synchrony.  The hearts that had suffered heart attacks came roaring back, functionally speaking, and were as good as new.

Dr. Qian, first author on this article, who is also a California Institute for Regenerative Medicine postdoctoral scholar and a Roddenberry Fellow. said, “These findings could have a significant impact on heart-failure patients—whose damaged hearts make it difficult for them to engage in normal activities like walking up a flight of stairs.  This research may result in a much-needed alternative to heart transplants—for which donors are extremely limited. And because we are reprogramming cells directly in the heart, we eliminate the need to surgically implant cells that were created in a petri dish.”

Dr. Srivastava noted, “Our next goal is to replicate these experiments and test their safety in larger mammals, such as pigs, before considering clinical trials in humans.  We hope that our research will lay the foundation for initiating cardiac repair soon after a heart attack—perhaps even when the patient arrives in the emergency room.”  Dr. Srivastava, is also a professor at the University of California, San Francisco (UCSF), with which Gladstone is affiliated.


Stem Cells Inc. Reports Positive Safety Data in Their Spinal Cord Injury Trial With Human Neural Stem Cells

StemCells, Inc., a biotechnology company based in Newark, California, has reported the results of their initial safety review of their human purified neural stem cell line implantations. This report represents the first planned interim safety review of the Company’s Phase I/II spinal cord injury clinical trial. This clinical trial involved a surgical implantation of the stem cells, and suppression of the immune system with anti-rejection drugs. The results of the safety trial show that both parts of the procedures seem to be well tolerated.

This trial was designed to determine the safety and potential, efficacy of the StemCells, Inc. proprietary HuCNS-SC® cells in spinal cord injury patients. HuCNS-SC cells are a purified human neural stem cell line that can form all the cells of the central nervous system (Taupin P. Curr Opin Mol Ther. 2006;8(2):156-63). When these cells are implanted into the retinas for rats that are suffering from retinal degeneration, they form a variety of retinal-specific cell types and seem to aid in retinal regeneration (McGill TJ., et al., Eur J Neurosci. 2012;35(3):468-77).

This clinical trial represents the first time that human neural stem cells have been implanted into the spinal cords of human patients as a potential therapeutic agent for spinal cord injury. The interim data come from the first cohort of patients. All of these first cohort patients suffered a complete spinal cord injury, and show no neurological function below the level of the injury.

All patients in the trial were transplanted with 20 million neural stem cells at the site of injury in the thoracic spinal cord. Observation of the patients revealed that there were no detectable abnormal responses to the cells, and all the patients were neurologically stable through the first four months following transplantation of the cells. Changes in sensitivity to touch were observed in two of the patients. These data merit the continuance of the trial, and further enrollments. Patients with partial spinal cord injuries, who might experience a broader range of improvements are also being sought for enrollment.

Armin Curt, M.D., principal investigator for the clinical trial, said, “We are very encouraged by the interim safety outcomes for the first cohort.”  Dr. Curt is Professor and Chairman of the Spinal Cord Injury Center at the University of Zurich, and Medical Director of the Paraplegic Center at Balgrist University Hospital. Dr. Curt continued, “The patients in the trial are being closely monitored and undergo frequent clinical examinations, radiological assessments by MRI and sophisticated electrophysiology testing of spinal cord function. The comprehensive battery of tests provides important safety data and is very reassuring as we progress to the next stage of the trial.”

Muscle Cells Made from Induced Pluripotent Stem Cells Successfully Treat Mice With Muscular Dystrophy

Work by researchers at the Lillehei Heart Institute at the University of Minnesota have demonstrated the ability of induced pluripotent stem cells (iPSCs) to make muscle-forming cells, and that these cells can be used to treat muscular dystrophy.

Muscular dystrophy refers to a group of inherited diseases that causes muscle fibers to be structurally weak and highly susceptible to damage. The progressive muscle damage causes the muscles to become gradually weaker and weaker until the patient will eventually require a wheelchair.

There are several different types of muscular dystrophy. Most of the varieties of muscular dystrophy causes symptoms appear during childhood, but others cause symptoms to arise during adulthood. The most common form of muscular dystrophy is Duchenne muscular dystrophy (DMD). The symptoms begin early in life (once the child learns to walk), and include frequent falls, difficulty getting up from a lying or sitting position, trouble running and jumping, waddling gait, large calf muscles, and learning disabilities. A less severe and slower progressing form of muscular dystrophy is Becker muscular dystrophy (BMD). Symptoms usually being in the teenage years, but might also not occur until the mid-20s or later. Other types of muscular dystrophy include myotonic (inability to relax muscles at will, most often begins in early adulthood, muscles of the face are usually the first to be affected), Limb-girdle (hip and shoulder muscles are first affected), congenital (apparent at birth or becomes evident before age 2 and varies in severity), fascioscapulohumeral (shoulder blades stick out like wings when the person raises his or her arms, onset occurs in teens or young adults), and oculopharyngeal (drooping of the eyelids and weakness of the muscles of the eye, face and throat, symptoms first appear in a person’s 40s or 50s).

In order to treat muscular dystrophy (MD), many researchers have tried to use gene therapy to place normal versions of the muscular dystrophy gene (which encodes a protein called Dystrophin) into the muscles of MD patients (Romero NB, et al., Hum Gene Ther. 2004;15(11):1065-76 & Mendell JR, et al., Ann Neurol. 2009;66(3):290-7. These types of experiments have met with limited success, since the immune system of muscular dystrophy patients tends to attack the muscles that express dystrophin (Mendell JR, et al., New England Journal of Medicine 2010 7;363(15):1429-37).

In light of the failure of gene therapy trials, researchers have tried stem cell treatments in MD mice. Scientists in the laboratory of Rita Perlingeiro have used muscle precursor cells made from mouse embryonic stem cells to treat MD mice (Radbod Darabi, et al., Exp Neurol. 2009; 220(1): 212–216). Given this early success, Perlingeiro and her co-workers have used mouse iPSCs to make muscle-forming cells that have been used to treat muscular dystrophy in MD mice. In this experiment, suppression of the immune system was not necessary, since the muscle cells were made from cells that came from the patients.

Perlingeiro said of the experiment, “One of the biggest barriers to the development of cell-based therapies for neuromuscular disorders like muscular dystrophy has been obtaining sufficient muscle progenitor cells to produce a therapeutically effective response. Up until now, deriving engraftable skeletal muscle stem cells from human pluripotent stem cells hasn’t been possible. Our results demonstrate that it is indeed possible and sets the stage for the development of a clinically meaningful treatment approach.”

Once transplanted, the muscle-forming cells (myogenic progenitor cells to be exact) moved into the damaged muscles and integrated into them. They formed skeletal muscle and provided extensive and long-term muscle regeneration that resulted in improved muscle function. To make the iPSC cell lines, Perlingeiro and her laboratory workers genetically modified to human iPSC lines with a gene called PAX7. PAX7 encodes a transcription factor that is essential for muscle formation and muscle regeneration. PAX7, with PAX3, designates cells as myogenic progenitor cells. Therefore, inserting the PAX7 gene into iPSCs would drive them to become myogenic progenitor cells.

Once Perlingeiro’s lab perfected the protocol for making myogenic progenitor cells from iPSCs, they found that they could make buckets and buckets of them. The iPSC-derived muscle forming cells were much more efficient at integrating into the muscles and regenerating them than other cell types. Muscle-forming stem cells from human muscle biopsies, for example, failed to persist in the muscle.

Perlingeiro concluded, “Seeing long-term maintenance of these cells without major side effects is exciting. Our research proves that these differentiated stem cells have real staying power in the fight against muscular dystrophy.”

A Patient’s Own Stem Cells Are Beneficial for Peripheral Artery Disease

Nearly 60,000 lower limb amputations occur annually as result of peripheral artery disease (PAD).  There are approximately nine million PAD patients in the United States, and PAD results from hardening of the arteries (atherosclerosis), which is a consequence of diabetes, smoking, high cholesterol, or genetic conditions that cause blood vessel disorders.

In a national study that included 550 patients at 80 different sites, patients had bone marrow stem cells removed from their hip bones, and after processing, these stem cells were re-introduced into the leg muscles to stimulate new blood vessel formation.  Richard J. Powell, M.D., chief of vascular surgery at Dartmouth-Hitchcock, is the lead investigator in this study, which is presently a three-year, third phase clinical study.  The second stage patients showed “remarkable success.”

To delay amputation, patients are usually treated with endovascular therapies like stents or bypass surgery.  Patients with the must severe form of PAD, critical limb ischemia (CLI), have arterial blockage that is so severe that stents or bypass surgery are simply not an option, and amputation is the only treatment possibility.

In the words of Dr. Powell: “All of us have stem cells in our bone marrow, and these stem cells can be utilized to repair other parts of our bodies.  By taking the patient’s own stem cells and injecting them into the ischemic leg, our hope is that we will them improve the blood flow in that part of the leg/”

The stem cells to which Dr. Powell is referring are the ones that bear two protein markers on their cell surfaces: CD133 and CD34.  CD34/CD133 cells are often called “endothelial progenitor cells” or EPCs.  EPCs can form endothelial cells, which are the cells that compose capillaries, the smallest and most delicate of all blood vessels.  EPCs can also form smooth muscle, which is necessary for the production of arteries, which are surrounded by a ring of smooth muscle that regulates the diameter of the artery and therefore its blood flow.  Several studies have shown that EPCs can greatly improve blood flow through the lower limbs in animals with CLI (For example, see Koiwaya H,, et al., J Mol Cell Cardiol.2011;51(1):33-40), but in some studies the stem cells do not survive upon injection and fail to take residence near the vasculature and establish new blood vessels (see  Kawamoto A, Asahara T, Losordo DW.Cardiovasc Radiat Med.2002;3(3-4):221-5 & Murasawa S, Asahara T. Physiology (Bethesda). 2005;20:36-42).  Also several clinic trials have confirmed the efficacy of EPCs for treating patients with vascular disorders (Lara-Hernandez R, et al., Ann Vasc Surg. 2010 Feb;24(2):287-94; Marfella R, et al., Atherosclerosis. 2010 Feb;208(2):473-9; Zhou B, et al., J Thromb Haemost. 2006 May;4(5):993-1002; Kudo FA, et al., Int Angiol. 2003 Dec;22(4):344-8).

In the study directed by Powell, some patients received injections of their own EPCs and other received a placebo.  Six months after treatment, half the patients who had received the placebo died, required an amputation of the affected limb, or had the severity of their leg wounds increase.  However of those patients who received the EPC treatment, only about 25% died, required amputation or saw their leg wound worsen.  The EPC-treated patients also showed improved blood flow in their legs.  Powell summarized these results in this way: “We found that patients who received the stem cell therapy had a significantly lower incidence of amputation at six months than patients who received the placebo.”

Even though, relatively speaking, this study has only a few patients, Powell noticed that he “saw clinically significant improvement in the stem cell-treated patients.”   He noted that these data were “compelling enough that there’s no question that the pivotal trial needs to be done as quickly as possible.”  The phase three trial has begun and once again, half the patients will receive the placebo and half will receive the stem cell treatment.  Powell stated, “we really want to see a therapy that’s effective out to a year.  Nonetheless, the results so far are really promising.”