Stem Cell Research

Virus-Free Technique Enables Researchers To Easily Make Fat Stem Cells Pluriotent

Stem cells from human fat are multipotent to begin with, meaning they are basically designed to turn into fat, bone or muscle tissue. While skin cells have been extensively used to create induced pluripotent (iPS) cells in the two plus years since iPS cells were first demonstrated, the ready multipotency and available supply of fat stem cells were seen by Stanford researchers as perhaps an easier approach.

Tiny circles of DNA turned out to ve the key to a new and easier way to transform stem cells from human fat into induced pluripotent stem (iPS) cells.  Unlike other commonly used techniques, the method, which is based on standard molecular biology practices, does not use viruses to introduce genes into the cells or permanently alter a cell's genome.

The new technique is the first example of reprogramming adult cells to pluripotency in this manner, and is hailed by the researchers as a major step toward the use of such cells in humans. They hope that the ease of the technique and its relative safety will smooth its way through the necessary FDA approval process.

"This technique is not only safer, it's relatively simple," said Stanford surgery professor Michael Longaker, MD, and co-author of the paper. "It will be a relatively straightforward process for labs around the world to begin using this technique. We are moving toward clinically applicable regenerative medicine."

The Stanford researchers used the so-called minicircles - rings of DNA about one-half the size of those usually used to reprogram cells - to induce pluripotency in stem cells from human fat. Pluripotent cells can then be induced to become many different specialized cell types. Although the researchers plan to first use these cells to better understand - and perhaps one day treat - human heart disease, induced pluripotent stem cells, or iPS cells, are a starting point for research on many human diseases.

"Imagine doing a fat or skin biopsy from a member of a family with heart problems, reprogramming the cells to pluripotency and then making cardiac cells to study in a laboratory dish," said cardiologist Joseph Wu, MD, PhD. "This would be much easier and less invasive than taking cell samples from a patient's heart." Wu is the senior author of the research.  Research assistant Fangjun Jia, PhD is the lead author of the work.
      
The minicircle reprogramming vector works so well because it is made of only the four genes needed to reprogram the cells (plus a gene for a green fluorescent protein to track minicircle-containing cells). Unlike the larger, more commonly used DNA circles called plasmids, the minicircles contain no bacterial DNA, meaning that the cells containing the minicircles are less likely than plasmids to be perceived as foreign by the body. The expression of minicircle genes is also more robust, and the smaller size of the minicircles allows them to enter the cells more easily than the larger plasmids. Finally, because they don't replicate they are naturally lost as the cells divide, rather than hanging around to potentially muck up any subsequent therapeutic applications.
      
The researchers chose to test the reprogramming efficiency of the minicircles in stem cells from human fat because previous work in Wu and Longaker's lab has shown that the cells are numerous, easy to isolate and amenable to the iPS transformation, probably because of the naturally higher levels of expression of some reprogramming genes. They found that about 10.8 percent of the stem cells took up the minicircles and expressed the green fluorescent protein, or GFP, versus about 2.7 percent of cells treated with a more traditional DNA plasmid.
      
When the researchers isolated the GFP-expressing cells and grew them in a laboratory dish, they found that the minicircles were gradually lost over a period of four weeks. To be sure the cells got a good dose of the genes, they reapplied the minicircles at days four and six. After 14 to 16 days, they began to observe clusters of cells resembling embryonic stem cell colonies - some of which no longer expressed GFP.
      
They isolated these GFP-free clusters and found that they exhibited all of the hallmarks of induced pluripotent cells: they expressed embryonic stem cell genes, they had similar patterns of DNA methylation, they could become multiple types of cells and they could form tumors called teratomas when injected under the skin of laboratory mice. They also confirmed that the minicircles had truly been lost and had not integrated into the stem cells' DNA.
      
Altogether, the researchers were able to make 22 new iPS cell lines from adult human adipose stem cells and adult human fibroblasts. Although the overall reprogramming efficiency of the minicircle method is lower than that of methods using viral vectors to introduce the genes (about 0.005 percent vs. about 0.01-0.05 percent, respectively), it still surpasses that of using conventional bacterial-based plasmids. Furthermore, stem cells from fat, and, for that matter, fat itself, are so prevalent that a slight reduction in efficiency should be easily overcome.

Adapted from the Stanford University School of Medicine announcement.

New Form of Stem Cell Communication Rescues Diseased Neurons

Researchers using mouse models have determined that transplanted stems cells, when in direct contact with diseased neurons, send signals through specialized channels that rescue the neurons from death. These direct cell-to-cell connections may also play a role in normal development by laying down the blueprint for more mature electrical connections between neurons and other cells.

While it was already known that stem cells will seek out diseased cells in the brain, this international research group of scientists showed -- both in tissue culture and in mice -- that the stem cells actively bring diseased neurons back from the brink via cross-talk through gap junctions, the connections between cells that allow molecular signals to pass back and forth. The international team included researchers from Sanford-Burnham Medical Research Institute (Sanford-Burnham, formerly Burnham Institute for Medical Research), the Karolinska Institutet, Beth Israel Deaconess Medical Center (BIDMC), Harvard Medical School and Université Libre de Bruxelles.

Significantly, the stem cells do not need to differentiate into the specific type of neuron to provide this therapeutic effect.

Possibility Of Embryonic Stem Cell Transplant Rejection Narrowed

Researchers have discovered a new strategy for making embryonic stem cell transplants less likely to be rejected by a recipient's immune system. The new strategy involves fusing bone marrow cells to embryonic stem cells. Once fused, the hybrid cells have DNA from both the donor and recipient, raising hopes that immune rejection of embryonic stem cell therapies can be avoided without drugs.

"Our study shows that transplanted bone marrow cells fuse not only with bone marrow cells of the recipient, but with non-hematopoietic cells, suggesting that if we can understand the process of cell fusion better, we may be able to target certain organ injuries with the patient's own bone marrow cells and repair the tissues," said Nicholas Zavazava, M.D., Ph.D., a University of Iowa researcher involved in the work.

Although the study holds promise for future embryonic stem cell therapies, the results may be even more far reaching. Zavazava and colleagues used two different mouse strains, one as the donor and the other as the recipient. When bone marrow cells were engrafted into the recipient, they tested for the presence of both donor and recipient cells and found three different types of cells: donor cells, recipient cells, and fused cells that had DNA from the donor and recipient. They then discovered that these cells could fuse with many different types of cells in addition to embryonic stem cells, including those from the liver, kidney, heart, and gut. Although more work is necessary to determine the exact clinical outcomes, the discovery raises the possibility that bone marrow cells could be fused to transplant organs to reduce the likelihood of rejection. They could also be fused to failing organs to support regeneration.

Adapted from the Federation of American Societies for Experimental Biology announcement.

Researchers Transform Mouse Skin Cells Into Neurons, Skipping Induced Pluripotent Stem Cell Stage

The future of regenerative medicine continues to depend on the type of stem cell that is proven most effective, most scalable, and most time and cost efficient to produce.  Approaches include embryonic stem cells, adult stem cells, Induced pluripotent stem cells, from both autologous and allogeneic sources.  One of our Sector Companies, International Stem Cell Corp., is attempting to create a stem cell bank based on parthenogenic stem cells derived from unfertilized eggs (oocytes).  Such a bank would have the following advantages:

1. A scalable bank would contain a manageable number of stem cell lines that will be immunological matches for large patient populations of different ethnic origin.


2. Unlike induced pluripotent stem (iPS) cells these would not involve extensive gene manipulation, which to this point in time have largely unknown biological impact.

We point this out in order to ask ourselves what the potential impact on such a stem cell bank might be from new study results obtained by researchers at Stanford University.  Since human iPS cells were first created in late 2007, the emphasis has been on developing these cells in a faster, cheaper way that also provides safe and predictable biological results when used in regenerative therapies.

But what if the iPS phase can be skipped entirely?  Will adult cells converted, for example, directly  into nerve cells be an eventual solution to Parkinson's Disease?  Or will parthenogenic, or some other more basic, more embryonic-like, cell provide the answer.  Perhaps both will be a solution and success in the market, as well as in therapy, will be a function of efficiency from origin to therapeutic use, and resultant lower cost. Perhaps neither will be universally useful in general therapy, but will be more effective for therapies in particular parts of the body.  At this stage no one knows for sure.  What we do know is that knowledge is expanding rapidly as shown in this current Stanford study.

Stanford researchers have succeeded in transforming mouse skin cells in a laboratory dish directly into functional nerve cells with the application of just three genes. The cells make this transition without first becoming a pluripotent type of stem cell.

Mesenchymal Stem Cells: A New Understanding Of How We Age

Throughout adulthood stem cells work to replace mature cells lost to turnover, injury or disease. Some of the genes responsible for keeping stem cells active and productive modify the chromatin, the complex combination of DNA and fibers that make up chromosomes.

Now, researchers from the Sbarro Institute for Cancer Research and Molecular Medicine at Temple University and the Human Health Foundation in Spoleto, Italy have studied the impact of MECP2 on mesenchymal stem cells, or MSCs, multipotent stem cells that can differentiate into a variety of cell types.  MECP2 is one of the chromatin modifier genes whose mutations underlie RETT syndrome, a severe X-linked neurodevelopmental disorder.

The study's conclusion is that inhibition of MECP2 induces senescence, or aging, in MSCs. In general, MSCs are of particular interest to researchers, because of the multiple roles they perform (They are the stem cell utilized in stem cell therapies being developed by Aastrom Biosciences, Athersys Inc., Opexa Therapeutics and Osiris Therapeutics, among others) . MSCs support hematopoiesis, or blood production, and contribute to the maintenance of many organs and tissues. Their aging has profound consequences on body physiology.

“Our studies suggest that MECP2 is a factor whose expression must be tightly regulated to avoid alteration in the cell’s function,” said Dr. Umberto Galderisi, Department of Experimental Medicine, Biotechnology and Molecular Biology Section, Second University of Naples, Italy and lead author of the study.

“Studies on in vitro stem cell senescence (aging) can be of interest in order to dissect molecular events leading to a decline of stem cell functions with advancing of age,” said Dr. Antonio Giordano, director of the Sbarro Institute and the Center for Biotechnology at Temple University.

Adapted from the S.H.R.O. announcement.

Researchers Generate Abundant Functional Blood Vessel Cells From Embryonic And Induced Pluripotent Stem Cells

Researchers have developed a new technique -- and described a novel mechanism -- for turning human embryonic and pluripotent stem cells into plentiful, functional endothelial cells, which are critical to the formation of blood vessels. Endothelial cells form the interior "lining" of all blood vessels and are the main component of capillaries, the smallest and most abundant vessels.

The new approach allows scientists to generate virtually unlimited quantities of durable endothelial cells -- more than 40-fold the quantity possible with previous approaches.

"This technique is the first of its kind with serious potential as a treatment for a diverse array of diseases, especially cardiovascular disease, stroke and vascular complications of diabetes," said Dr. Shahin Rafii, the study's senior author and Arthur B. Belfer Professor in Genetic Medicine and co-director of the Ansary Stem Cell Institute at Weill Cornell Medical College. Dr. Rafii is also an investigator of the Howard Hughes Medical Institute.

Researchers first screened for molecular factors that come into play when stem cells turn into endothelial cells. Their findings led them to a strategy that significantly boosts the efficiency of producing these cells.

They then tracked the differentiation process in real-time using a green fluorescent protein marker developed by Dr. Daylon James, the study's first author and assistant research professor in the Department of Reproductive of Medicine at Weill Cornell Medical College. They found that when they exposed stem cells to a compound that blocks TGF-beta (a growth factor involved in cell specialization) at just the right time during cell culturing, the propagation of endothelial cells dramatically increased.

Molecule Identified That Inhibits Stem Cell Differentiation

It turns out that stem cells have need for long-term memory to help them know who they are and what they should become. Researchers at the Stanford University School of Medicine have now identified a molecule involved in keeping skin stem cells on the straight and narrow path.

“We’re starting to understand the molecular mechanism of cellular memory,” said Paul Khavari, MD, PhD, professor of dermatology. “How a stem cell remembers what it is, and why it might go astray.”

The molecule, called DNMT1, helps the stem cells know whether to self-renew to create more stem cells, or to differentiate into specialized, non-dividing adult skin cells. It’s important because too much self-renewal can lead to cancer, and too little can inhibit wound healing.

Much ado is made over pluripotent cells — such as embryonic stem cells and their laboratory doppelgangers, induced pluripotent stem (iPS) cells — which have the potential to differentiate into any of the body’s different cell types. But adult stem cells, while more limited in their ability to create new types of cells, still have important roles in the body. They’re particularly vital in skin, blood and other tissues that must constantly regenerate new cells.

Biologists Develop Efficient Genetic Modification Of Human Embryonic Stem Cells

One of the non-ethical problems with embryonic stem cell research has been the lack of efficient ways to study genetics using these cells.  Now, using bacterial artificial chromosomes  (BACs) to swap in defective copies of genes, researchers have successfully transferred a defective copy of the gene p53, which suppresses cancer, into a human embryonic stem cell line.

"This will help open up the whole human embryonic stem cell field. Otherwise, there are really few efficient ways you can study genetics through them," said Yang Xu, professor of biology at the University of California, San Diego who directed the research.

BACs are synthesized circles of human DNA, which bacteria will replicate just like their own native chromosomes. Commercially available BACs can be modified within bacterial cells to insert altered copies of specific genes. Once the modified BACs are introduced into human cells, they will sometimes pair up with a matching segment of a human chromosome and swap segments of DNA, a process called homologous recombination.

Ethnic Origin Of Stem Cells Found Important To Safety And Effectiveness Of Specific Future Drugs

Analysis of a variety of human embryonic stem cell lines determined that the cells originated largely from Caucasian and East Asian populations, with little representation from populations originating in Africa.  The lines analyzed are currently in use in research laboratories around the world.  In response to these results, scientists from Scripps Research Institute used skin cells from an individual of West African Yoruba heritage to create a new stem cell line, the first to carry the genetic profile of this ethnic group.

Why is this important? Research has shown that discordance between the ethnic origin of organ donors and recipients can influence medical outcomes for tissue transplantation, and that the safety and effectiveness of specific drugs can vary widely depending on ethnic background.

"Ethnic origin is a critical piece of information that should come with every cell line," said Scripps Research Professor Jeanne Loring, who is senior author of the paper resulting from this research. "Everyone who works with stem cells should be doing this kind of analysis."

One of our Sector Companies, International Stem Cell Corp. (ISCO) announced a collaboration with Dr. Jeanne Loring and Scripps Research in November of last year.  ISCO has developed a parthenogenetic homozygous stem cell line and is hopeful that this research will provide immune-matched stem cells for human transplant which will allow the building of a "stem cell bank" that could offer on-demand delivery of stem cells matched to a patient’s own immune system.

"Knowing that a big push in the future is using stem cell lines in the clinic and in drug development, there's a need to have an ethnically diverse population of cells," added Louise Laurent, assistant professor at the University of California, San Diego (UCSD) and research associate at Scripps Research, who is first author of the paper with Caroline Nievergelt, also an assistant professor at UCSD.

China's Star Rising Despite World Skepticism Of Its Past Stem Cell Research Approaches

Despite world skepticism of China's research approach to stem cell therapies, Medical Tourism continues to grow as China's government pours generous dollars into research on regenerative medicine and aggressively recruits high-caliber scientists trained abroad in pursuit of its ambition to become a world leader in the field.  This According to a study by the Canadian-based McLaughlin-Rotman Centre for Global Health (MRC).  As a result of its expanded investment, Chinese contributions to scientific journals on RM topics leapt from 37 in year 2000 to 1,116 in 2008, exceeded only by the contributions of experts in the USA, Germany, Japan and the UK.

The accomplishment is all the more interesting given that China's international credibility has been and still is severely hindered by global concerns surrounding Chinese clinics, where unproven therapies continue to be administered to thousands of patients, including an increasing number of desperate Westerners fueling Medical Tourism which has become a growth industry.

"When you look at the issue of stem cells in China, you see the Yin-Yang of a scientific powerhouse mixed with controversial clinical application of stem cell therapies," said Dr. Peter Singer, MRC's Director. "The overall picture at the moment is ambiguous but in the future, given the measures that have been put in place, the science can be expected to rise and the controversy to fall."

Until May 2009, clinical trials to determine the effectiveness of stem cell therapies were not required in China. Now proof of safety and efficacy through clinical trials is required by China's Ministry of Health for all stem cell and gene therapies.

The change was made after international experts, joined by top Chinese researchers, protested that treatment centers were acting "against commonly accepted principles of modern scientific research" and successfully called on China to regulate new treatments and ensure patient safety.

Despite the new rules, however, stem cell treatments are still available at over 200 hospitals across China to patients of diseases such as ataxia, Lou Gehrig's disease, traumatic brain and spinal cord injury, diabetes, Parkinson's, multiple sclerosis, autism, cerebral palsy, stroke, optic nerve hypoplasia and many others.

"To our knowledge, Chinese policy makers and ethicists are working out the regulation details,” says Ms. McMahon, the study's lead author. "Once that is accomplished, we still expect a delay, during which the therapies currently administered by clinics and hospitals will be evaluated individually to determine whether they meet the criteria of China's Ministry of Health."

Stem Cell Therapy 2010: Where Are We Headed?

2009 was marked by five main trends (not necessarily in order of importance):

1. The credit crisis and economic downturn which prompted Sector Companies to reduce costs or provide greater investment incentives to raise money from new investors.  The good news is that most Stem Cell Sector Companies that needed cash were able to raise it in a generally hostile financing environment.


2. Research moved into the clinical stage with confusing results.  For the most part this is because U.S. clinical disease related trials have addressed only patients with diseases where there is no hope and stem cells are a last resort.  Alternatively, trials have been run in heart and osteopathic arenas where results have generally been positive but not total therapeutic cures.  Where the FDA has been involved, trials have been small in number of participants as proving safety is the FDA's first priority with efficacy a distant second.


3. Big pharma made its move to be part of future stem cell therapy by investing in three of our Sector Companies with mesenchymal stem cell products.


4. The new administration loosened the guidelines for stem cell lines that could be used for research funded by federal grants. 


5. Several Stem Cell Sector Companies expanded their product offerings in the sale of stem cells and stem cell lines to other researchers.

What will 2010 bring?

2009, The Year Big Pharma Bought The Idea Of Mesenchymal Stem Cell Therapy

In November of 2008,  Genzyme Corp. and Osiris Therapeutics (one of our Sector Companies) announced a "strategic alliance for the development and commercialization of Prochymal and Chondrogen." Prochymal and Chondrogen are both biologic drug candidates. Prochymal is a preparation of mesenchymal stem cells (MSCs) formulated for intravenous infusion. Chondrogen is a preparation of MSCs formulated for direct injection into the knee. The mesenchymal stem cells n these products are taken from the bone marrow of healthy young adult donors. Both product candidates are allogeneic and scalable, two important qualities from Big Pharma's perspective.

Osiris's deal with Genzyme made it the Sector Company with the highest market capitalization at December 31, 2008, a distinction that returned to Geron Corp. after Osiris suffered clinical trial setbacks in 2009.  Following Genzyme and Osiris came a deal for an autologous biologic drug product between Opexa Therapeutics (a Sector Company) and Novartis.

The latest deal is Phizer's purchase of Athersys, Inc.'s stem cell research. Athersys, one of our Sector companies, has 18.9 million shares outstanding with a float of 14.5 million shares.  Athersys' deal with Phizer was announced on the 21st of December.  During that week, roughly 110 million shares changed hands, and that includes a short trading day on the 24th and no trading on the 25th.  So basically 110 million shares traded hands in three and one half trading days.  It seems a substantial amount of money is on the sidelines waiting for anything positive on the stem cell front.