Stem Cell Gene Therapy For An Inherited Neurological Disease


Scientists at the University of Manchester in the United Kingdom have used stem cell gene therapy to treat a fatal genetic brain disease in mice. Sanfilippo is a fatal, inherited condition that causes progressive dementia in children. This particular treatment strategy could also be used to treat other types of neurological, inherited diseases. The Manchester team hopes to bring this strategy to a clinical trial within two years.

Sanfilippo afects one in 89,000 children in the United Kingdom and is an untreatable “mucopolysaccharide disease ” or MPS disease. MPS diseases involve an abnormal storage of mucopolysaccharides. This abnormal storage results from the absence of a specific enzyme. Without the enzyme, the breakdown process of mucopolysaccharides is incomplete. Partially broken down mucopolysaccharides accumulate in the body’s cells causing progressive damage. The storage process can affect appearance, development, and the function of various organs of the body. Each MPS disease is caused by the deficiency of a specific enzyme.

Patients with Sanfilippo are unable to degrade heparan sulfate. There are four different types of Sanfilippo, which is also called MPS type III. MPS type IIIA results from a deficiency in the enzyme N-sulfoglucosamine sulfohydrolase, MPS type IIIB lacks N-Acetylglucosaminidase, MPS type IIIC has an absence in Acetyl-CoA:alpha-glucosaminide-acetyltransferase, and MPS type IIID lacks N-acetylglucosamine 6-sulphatase. In all four forms of MPS III, excessive heparan sulphate storage occurs in the brain, leading to its progressive deterioration; the amount of heparan sulphate storage in other tissues influences the extent of physical symptoms. Children eventually lose the ability to walk and swallow.

Brian Bigger from the University of Manchester’s Institute of Human Development led this research into therapies for MPS type IIIA. According to Bigger, bone marrow transplants have been used to treat similar diseases (e.g., Hurler syndrome). In this case, gene therapy was used to introduce the missing enzyme into the transplanted cells. Unfortunately, this did not work terribly well because the white blood cells from the bone marrow did not make enough of the enzyme to treat the disease.

A fraction of the white blood cells made bone marrow are called monocytes, and some of the monocytes traffic to the brain to become microglia. Since microglia are made by hematopoietic stem cells (HSCs) in the bone marrow, genetic engineering of cultured HSCs should increase expression of the missing enzyme in microglia. In previous experiments, HSCs were engineered with viruses to express the missing enzyme, but this expression was poor in microglia.

To fix this problem, Bigger and his team increased enzyme expression in the engineered HSCs in bone marrow. They used a gene control region from the “pyruvate kinase” gene, which is a very highly expressed gene. This increased expression of the missing enzyme to five times the normal levels and to 11% of normal levels in the microglia cells in the brain. The enzyme

This type of treatment corrects the inflammation in the brain and completely corrects the hyperactivity behavior in mice with Sanfilippo. Bigger adds, “We now hope to work to a clinical trial in Manchester in 2015.”

Bigger and his colleagues are manufacturing a viral vector to deliver genetic material into cells for use in humans and they hope to use this in a clinical trial with patients at Central Manchester University Hospital NHS Foundation Trust by 2015.

This stem cell gene therapy approach was recently shown by Italian scientists to improve conditions in patients with a similar disease that affects the brain called metachromatic leukodystrophy. Bigger refined the vector used bythe Italian group.

According to Bigger, this approach might have the potential to treat several neurological genetic diseases.

Nanometer Scaffolds Regulate Neural Stem Cells


In the laboratory, stem cells can grow in liquid culture quite well in many cases, but this type of culture system, though convenient and rather inexpensive, does not recapitulate the milieu in which stem cells normally grow inside our bodies. Inside our bodies, stem cells stick to all kinds of surfaces and interact with and move over a host of complex molecules. Many of the molecules that stem cells contact have profound influences over their behaviors. Therefore, reconstituting or approximating these environments in the laboratory is important even though it is very difficult.

Fortunately nanotechnology is providing ways to build surfaces that approximate the kinds of surfaces stem cells encounter in our bodies. While this field is still in its infancy, stem cell-based nanotechnology may provide strategies to synthesize biologically relevant surfaces for stem cell growth, differentiation, and culture.

One recent contribution to this approach comes from Jihui Zhou and his team from the Fifth Hospital Affiliated to Qiqihar Medical University. Zhou and his co-workers prepared randomly oriented collagen nanofiber scaffolds by spinning them with an electronic device. Collagen is a long, fibrous protein that is found in tendons, ligaments, skin, basement membranes (the substratum upon which sheets of cells sit), bones, and is also abundant in cornea, blood vessels, cartilage, intervertebral disc, muscles, and the digestive tract. Collagen is extremely abundant in the human body; some 30% of all the proteins in our bodies are collagen. It is the main component in connective tissues.

There are many different types of collagen. Some types of collagen form fibers, while others for sheets. There are twenty-eight different types of collagen. Mutations in the genes that encode collagens cause several well-known genetic diseases. For example, mutations in collagen I cause osteogenesis imperfecta, the disease made famous by the Bruce Willis/Samuel T. Jackson movie, “Unbreakable.” Mutations in Collagen IV cause Alport syndrome, and mutations in either collagen III or V cause Ehlers-Danlos Syndrome.

Wen cells make fibrous collagen, they weave three collagen polypeptides together to form a triple helix protein that is also heavily crosslinked. This gives collagen its tremendous tensile strength.

Collagen fibers
Collagen fibers

In this experiment, electronic spinning technology made the collagen fibers and these fibers had a high swelling ratio when placed in water, high pore size, and very good mechanical properties.

Zhou grew neural stem cells from spinal cord on these nanofiber scaffolds and the proliferation of the neural stem cells was enhanced as was cell survival. Those genes that increase cell proliferation (cyclin D1 and cyclin-dependent kinase 2) were increased, as was those genes that prevent cells from dying (Bcl-2). Likewise, the expression of genes that cause cells to die (caspase-3 and Bax) decreased.

Thus novel nanofiber scaffolds could promote the proliferation of spinal cord-derived neural stem cells and inhibit programmed cell death without inducing differentiation of the stem cells. These scaffolds do this by inducing the expression of proliferation- and survival-promoting genes.

Neural Stem Cell Proliferation Increased By Herbal Extract


When it comes to herbal medicine, count me a skeptic. Some people swear by many herbs, but when these same herbs are objectively tested under controlled conditions, they fail spectacularly or they only show modest effects.

For example, a lady in my church is absolutely certain that Echinacea will cure your cold. However, a paper by Barrett in Phytomedicine, 2003 Jan;10(1):66-86 reviews several Echinacea trials and concludes that: “Although suggestive of modest benefit, these trials are limited both in size and in methodological quality. Hence, while there is a great deal of moderately good-quality scientific data regarding E. purpurea, effectiveness in treating illness or in enhancing human health has not yet been proven beyond a reasonable doubt.” Also the prestigious Cochrane database has examined many human trials that tested Echinacea and concluded that “Echinacea preparations tested in clinical trials differ greatly. There is some evidence that preparations based on the aerial parts of Echinacea purpurea might be effective for the early treatment of colds in adults but results are not fully consistent. Beneficial effects of other Echinacea preparations, and for preventative purposes might exist but have not been shown in independently replicated, rigorous randomized trials.” For this study, see Linde K, Barrett B, Wölkart K, Bauer R, Melchart D. Echinacea for preventing and treating the common cold. Cochrane Database Syst Rev. 2006 Jan 25;(1):CD000530,

When it comes to Ginkgo biloba extracts, the use of Ginkgo for age-related dementia has a veritable history, but the Cochrane reviews concluded: “The evidence that Ginkgo biloba has predictable and clinically significant benefit for people with dementia or cognitive impairment is inconsistent and unreliable.” See Birks J, Grimley Evans J. Ginkgo biloba for cognitive impairment and dementia. Cochrane Database Syst Rev. 2009 Jan 21;(1):CD003120. doi: 10.1002/14651858.CD003120.pub3.

Therefore, it is with some skepticism that I relate the following report to you.

Neural stem cells in the subventricular zone of the hippocampal dentate gyrus on adult mammals are responsible for learning and memory. These cells stop dividing during severe depression and dementia and expand during learning.

Hippocamus anatomy

The natural growth of these stem cells is insufficient to replenish cells after a severe stroke or in the event of serious brain disease. Therefore finding a way to stimulate these is important from a clinical standpoint.

Professor Yuliang Wang from Weifang Medical University has used an extract of Ginkgo biloba called EGb761 to treat rats with dementia. In their hands, this materials seems to safely treat memory loss and cognitive impairment (see Zhang Z, Peng D, Zhu H, Wang X. Experimental evidence of Ginkgo biloba extract EGB as a neuroprotective agent in ischemia stroke rats. Brain Res Bull. 2012 Feb 10;87(2-3):193-8).

Wang and his co-workers took this work one step further and examined the effects of EGb761 on the proliferation of neural stem cells in the subventricular zone and dentate gyrus of rats with vascular dementia.

According to Wang and others, the extract promoted and prolonged the proliferation of neural stem cells in the subventricular zone and dentate gyrus of rats with vascular dementia. The cells continued to proliferate for four months and improved learning and memory in rats with vascular dementia.

If you do not believe it, see Wang JW and others, Neural Regeneration Research 2013; 8 (18): 1655-1662.

Artificial Bones From Umbilical Cord Stem Cells


I am back from vacation. We visited some colleges in Indiana for my daughter who will be a senior this year. She really liked Taylor University and Anderson University. We’ll see if the tuition exchange works out.

Now to blogging.

Scientists from Granada, Spain have patented a hew biomaterial that consists of activated carbon cloth that just happens to be able to support the growth of cells that have the ability to regenerate bone. These results came from experiments that were conducted outside any living animals, but they hope to confirm these results in a living animal in the near future.

This new biomaterial facilitates the growth of bone-making cells derived from umbilical cord stem cells. This activated carbon cloth acts as a scaffold for cells that differentiate into “osteoblasts,” which are bone-building cells. This activated carbon cloth gives the osteoblasts a proper surface upon which to promote the growth of new bone.

Bone loss as a result of cancer, trauma, or degenerative bone diseases requires replacement bone to heal to damaged bone. Making new bone in the laboratory that can be transplanted is an optimal strategy for treating these patients.

Even though this laboratory-made bone was not used in living laboratory animals to date, the laboratory results look quite impressive. In the future, such techniques could help manufacture medicines or other sources of material to repair bone or lost cartilage. Once such artificial bone has been made in the laboratory, the Spanish team hopes to transplant it into rats or rabbits to determine if it can regenerate bone in such creatures.

Presently, no materials exist to replace lost bone. The method used to make bone by the research team from Granada uses a three-dimensional support that facilitates the production of those cell types that regenerate bone without the need for additional growth factors.

The growth of these umbilical cord stem cells on activated carbon cloth produced a product that could produce organic bone, but also mineralize the organic bone matrix. This patent could have numerous clinical applications in regenerative medicine and the Granada group hopes to obtain funding to continue this work and achieve their ultimate objective: to regenerate bones by implanting biomaterial in patients with bone diseases.

Using Human Induced Pluripotent Stem Cells to Study Diamond Blackfan Anemia


Diamond-Blackfan Anemia or DBA results from mutations in a gene on chromosome 19 (in most cases). Mutations in the ribosomal protein S19 affects the ability of blood cells to make protein and causes low numbers of red blood cells. DBA patients are dependent on blood transfusions, but some are cured, to some extent at least, by bone marrow transplants. Unfortunately, some DBA patients have severe side effects from bone marrow transplants, which means that bone marrow transplants are not a panacea for all DBA patients.

Fortunately, Michell J. Weiss and his colleagues at the Children’s Hospital of the Philadelphia (CHOP) have used human induced pluripotent stem cells (iPSCs) to study DBA at the molecular level and even develop the beginnings of a cure for DBA patients. Weiss collaborated with Monica Bessler, Philip Mason, and Deborah French, all of whom work at CHOP.

Remember that red blood cells are made inside the bone marrow of the patient by hematopoietic stem cells (HSCs). HSCs divide to renew themselves, and to produce a daughter cell that will differentiate into one of several different types of blood cells. As a kind of gee-wiz number, a healthy adult person will produce approximately 10[11]–10[12] (100 billion to 1 trillion) new blood cells are produced daily in order to maintain steady state levels in the peripheral circulation.

In DBA patients, the bone marrow is empty of red blood cells. In order to get a better idea why, Weiss and his team isolated fibroblasts from the skin of DBA patients, and used genetic engineering techniques to convert them into iPSCs. When Weiss and his group tried to differentiate these iPSCs derived from DBA patients into red blood cells, they were not able to make normal red blood cells. However, Weiss and his colleagues used different genetic engineering techniques to fix the mutation in these iPSCs. After fixing the mutation, these cells could be differentiated into red blood cells. This experiment showed that it is possible to repair a patient’s defective cells.

This is a proof-of-principle experiment and there are many hurdles to overcome before this type of experiment can be done in the clinic to DBA patients. However, these iPSCs can play a vital role in deciphering some of the mysteries surrounding this disease. For example, two family members may have exactly the same mutation, but only one of them shows the disease whereas the other does not. Since iPSCs are specific to the patient from whom they were made, Weiss and his group hope to compare the molecular differences between them and understand the difference in expression of this disease.

Also, these cells offer a long-lasting model system for testing new drugs or gene modifications that may offer new treatments that are personalized to individual patients.

Weiss and his research group used this same technology to test drugs for the often aggressive childhood leukemia, JMML or Juvenile Myelomonocytic Leukemia. Once again, iPSCs were made from JMML patients and differentiated into myeloid cells, which divided uncontrollably just as the original myeloid cells from JMML patients.

Weiss and his colleagues used these cells to test two drugs, both of which are active against JMML. One of them is an inhibitor of the MEK kinase that was quite active against these cells. This illustrates how iPSCs can be used to test personalized treatment regimes for patients.

The stem cell core facility at CHOP is also in the process of making iPCS lines for several inherited diseases: dyskeratosis congenita, congenital dyserythropoietic anemia, thrombocytopenia absent radii, Glanzmann’s thrombasthenia, and Hermansku-Pudlak syndrome.

The even longer term goal is the use these lines to specifically study the behavior of such cells in culture and under certain conditions, test various drugs on them, and to develop treatment strategies on them as well.

A More Efficient Way to Make Human Induced Pluripotent Stem Cells


Stem cell researchers at the University of California, San Diego have designed a simple, reproducible, RNA-based method of generating human induced pluripotent stem cells (iPSCs). This new technique broad applications for the successful production of iPSCs for use in therapies and human stem cell studies.

Human iPSCs are made from adult cells by genetically engineering adult cells to overexpress four different genes (Oct4, Klf4, Sox2, and c-Myc). This overexpression drives the cells to de-differentiate into pluripotent stem cells that have many of the same characteristics as embryonic stem cells, which are made from embryos. However, because iPSCs are made from the patient’s own cells, the chances that the immune system of the patient will reject the implanted cells is low.

The problem comes with the overexpression of these four genes. Initially, retroviruses have been used to reprogram the adult cells. Unfortunately, retroviruses plop their DNA right into the genome of the host cell, and this change is permanent. If these genes get stuck in the middle of another gene, then that cell has suffered a mutation. Secondly, if these genes are stuck near another highly-expressed gene, then they too might be highly expressed, thus driving the cells to divide uncontrollably.

Several studies have shown that in order to reprogram these cells, these four genes only need to be overexpressed transiently. Therefore, laboratories have developed ways of reprogramming adult cells that do not use retroviruses. Plasmid-based systems have been used, adenovirus and Sendai virus-based systems, which do not integrate into the genome of the host cell, have also been used, and even RNA has been used (see Federico González, Stéphanie Boué & Juan Carlos Izpisúa Belmonte, Nature Reviews Genetics 12, 231-242).

The UC San Diego team led by Steven Dowdy has used Venezuelan equine virus (VEE) that they engineered to express the reprogramming genes required to make iPSCs from adult cells. Because this virus does not integrate into the host genome, and expresses RNA in the host cell only transiently, it seems to be a safe and effective way to make buckets of messenger RNA over a short period of time.

The results were impressive. The use of this souped-up VEE produced good-quality iPSCs very efficiently. Furthermore, it worked on old and young human cells, which is important, since those patients who will need regenerative medicine are more likely to be young patients than old patients. Also, changing the reprogramming factors is rather easy to do as well.

Japanese first Ever Induced Pluripotent Stem Cell Clinical Trial Given the Green Light


The first clinical trial that utilizes induced pluripotent stem cells has been given a green light. For this clinical trial six patients who suffer from age-related macular degeneration will donate skin biopsies and the cells from these skin biopsies will be used to generate induced pluripotent stem (iPS) cells in the laboratory. After those iPS cell lines are screened for safety (normal numbers of chromosomes, no mutations in critical genes, etc.), they will be differentiated into retinal cells. The retinal cells will be transplanted into the retinas of these six patients.

This clinical trial was approved by Japan Health Minister Norihisa Tamura and it will be next summer by Masayo Takahashi. Dr. Takahashi is a retinal regeneration expert and a colleague of the man who first developed iPS cells, Shinya Yamanaka. Yamanaka won the Nobel Prize for his discovery of iPSCs last year. In fact, this clinical trial epitomizes, in the eyes of many, the determination of Japanese scientists and politicians to dominate the iPS cell field. This national ambition kicked into high gear after Yamanaka shared the Nobel Prize for Physiology or Medicine last October for his iPS cell work.

Norhisa Tamura, Japanese Minister of Health
Norihisa Tamura, Japanese Minister of Health
Masayo Takahashi, MD, PhD, Riken Center for Developmental Biology.
Masayo Takahashi, MD, PhD, Riken Center for Developmental Biology.

“If things continue this way, this will be the first in-clinic study in iPS cell technology,” says Doug Sipp of the Riken Center for Developmental Biology (CDB). The CDB, Takahashi’s institute, will co-run the trial with Kobe’s Institute for Biomedical Research and Innovation. “It’s exciting.”

Sipp, however, also noted that this move has not surprised anyone in Japan, since the Japanese stem cell community has heavily invested in iPS cells. Nevertheless, since Takahashi yet to formally publish the details of her trial, some have questioned whether she is actually ready to move forward. IPS cells are viewed as the perfect compromise for regenerative medicine. They are adult, and therefore do not require the destruction of human embryos for their establishment, and they are also pluripotent like an embryonic cell, which makes them relatively powerful sources for regenerative medicine.

Critics, however, warn that iPS cells were only discovered in 2007. To date, they remain difficult to create and culture and they can become tumorous in many hands. However, many labs have a great deal of expertise and skill when it comes to handling and deriving iPS cells. These labs derive and culture iPS cells routinely. In fact, Sipp notes that Riken’s CDB alone has produced world-class work with all kinds of stem cells, including embryonic stem (ES) cells, which are the models for iPS cells.

Additionally, Sipp and others point out that a scientist who has collaborated with Takahashi in the past, Riken’s Yoshiki Sasai, is doing groundbreaking work with ES cells and the eye. The British journal Nature has called Sasai “The Brainmaker,” and has said that his research is “wowing” the world.

The Japanese government has also soundly funded Takahashi’s trail. The health ministry’s recent stimulus plan set aside more money for stem cells (in particular iPS cells) than anything else. According to the journal Nature, the Japanese government sequestered 21.4 billion yen ($215 million) for stem cell research. Of this pot of money, the health ministry provided 700 million yen ($7 million) for a cell-processing center to support Takahashi before her trial was even approved. Two centers devoted to iPS cells are slated to be built with 2.2 billion yen ($22 million). The AFP reports the prime minister has set aside a breathtaking $1.18 billion, for iPS-cell work. Yamanaka has told Nature that the Japanese government seems to be “telling us to rush iPS cell-related technologies to patients as quickly as possible.”

Robert Lanza, CSO of Advanced Cell Technology, might once have been the logical bet to be first to the clinic with iPS cells. Unlike Takahashi, he has three ES cell trials under his belt, and has started talks with the FDA about transplanting iPS cell-derived platelets, but his iPS proposal is taking longer. Lanza bitterly noted, not without justification, “We don’t have the prime minister and emperor to speed things along for us.”

Since 2007, the year that Yamanaka reported the derivation of iPS cells from adult cells, Japan has focused on iPS cells. Yamanaka showed that increasing the expression of four genes could change limited adult human cells into potent, embryonic-like cells. “At Yamanaka’s institute alone, there are at least 20 teams focusing on iPS cells now,” Sipp says. There are teams at Riken, the Universities of Tokyo and Keio, and others. “A lot is happening here.” In fact, the Center for IPS Cell Research and Application was created expressly for Yamanaka.

Takahashi has reported part of the design of her clinical trial at scientific meetings. She told the International Society for Stem Cell Research in June 2012 she had created iPS-cell derived retinal pigment epithelial (RPE) cells for transplantation. RPE cells lie behind the photoreceptors in the retina, and the photoreceptors have their ends embedded into the RPE. The RPE cells replenish and nourish the photoreceptors, and without the RPE cells, the photoreceptors die from the damage incurred by exposure to light.

Retinal Pigmented Epithelium

Death of the RPE cells cause eventual death of photoreceptors and that results in blindness. At the International Society for Stem Cell Research conference, Takahashi reported her that her iPS cell-derived RPEs possess proper structure and gene expression. They also do not produce tumors when transplanted into mice, and survive at least six months when transplanted into the retinas of monkeys. The vision of these animals, however, was not tested. She did note that some AMD patients’ sight improves when RPE cells are moved from the eye’s periphery to its center.

Retinal pigment epithelial cells derived from iPS cells.
Retinal pigment epithelial cells derived from iPS cells.

Takahashi has published many iPS and ES cell papers. These papers include two papers with Yamanaka: one on creating retinal cells from iPS cells, and one on creating safe iPS cells. However she has not published trial details, which is not required, but such a landmark trial should be transparent, as argued by many stem cell experts.

Still, according to Sipp, Takahashi has submitted a relevant paper to a top journal for review, which shows that this clinical trial is purely a determination of the safety of the procedure. Lanza has reported his trials in the journal The Lancet, and similar, but small, trials are doing well. His three ES cell trials treated Stargardt’s macular dystrophy and Age-related Macular Degeneration. Lanza’s trial, however, treated “dry” macular degeneration, while Takahashi’s trial will treat “wet” Age-related Macular Degeneration, which is good news for Takahashi.

Paul Knoepfler, a UC Davis stem cell scientist who runs a widely read blog site, has written that the ministry overseeing Takahashi’s trial will reportedly monitor some key factors: gene sequencing and tumorigenicity. But Knoepfler, like others, would like to see more details.

The Japanese Health Ministry and the US FDA recently agreed to devise a joint regulatory framework for retinal iPS cell clinical trials, which will come on line 2015. Takahashi’s trial is set for 2014.