Ending the Reliance on Feeder Cells for Stem Cell Growth


A new study, published today in the journal Applied Materials & Interfaces reports the discovery of a new method for growing human embryonic stem cells that does not depend on feeder cells from human or animal cells.

Traditionally, embryonic stem cells are cultivated with the help of feeder cells derived from animals. Feeder cells secrete a host of growth factors and other signaling molecules that prevent the embryonic stem cells from differentiating and maintain their pluripotency. However, the use of animal products in the production of human cells lines rules out their use in the treatment of humans, since they can become contaminated with animal proteins that will cause rejection by the immune system or animal viruses that can infect the patient and cause significant disease.

The team of scientists led by the University of Surrey and in collaboration with Professor Peter Donovan at the University of California have developed a scaffold of carbon nanotubes upon which human stem cells can be grown into a variety of tissues. These nanotube networks mimic the surface of the body’s natural support cells and act as scaffolding for stem cells to grow on. Even cultured cells that have previously relied on feeder cells can now be grown safely in the laboratory, which paves the way for revolutionary steps in replacing tissue after injury or disease.

Dr Alan Dalton, senior lecturer from the Department of Physics at the University of Surrey said: “While carbon nanotubes have been used in the field of biomedicine for some time, their use in human stem cell research has not previously been explored successfully.”

“Synthetic stem cell scaffolding has the potential to change the lives of thousands of people, suffering from diseases such as Parkinson’s, diabetes and heart disease, as well as vision and hearing loss. It could lead to cheaper transplant treatments and could potentially one day allow us to produce whole human organs without the need for donors.”

Controlling Transplanted Stem Cells from the Inside Out


Scientists have worked very hard to understand how to control stem cell differentiation.  However, despite how well you direct stem cell behavior in culture, once those stem cells have been transplanted, they will often do as they wish.  Sometimes, transplanted stem cells surprise people.

Several publications describe stem cells that, once transplanted undergo “heterotropic differentiation.” Heterotropic differentiation refers to tissues that form in the wrong place. For example, one lab found that transplantation of mesenchymal stem cells into mouse hearts after a heart attack produced bone (don’t believe me – see Martin Breitbach and others, “Potential risks of bone marrow cell transplantation into infarcted hearts.” Blood 2007 110:1362-1369).  Bone in the heart – that can’t be good. Therefore, new ways to control the differentiation of cells once they have been transplanted are a desirable goal for stem cell research.

From this motivation comes a weird but wonderful paper from Jeffrey Karp and James Ankrum of Brigham and Women’s Hospital and MIT, respectively, that loads stem cells with microparticles that give the transplanted stem cell continuous cues that tell them how to behave over the course of days or weeks as the particles degrade.

“Regardless of where the cell in the body, it’s going to be receiving its cues from the inside,” said Karp. “This is a completely different strategy than the current method of placing cells onto drug-doped microcarriers or scaffolds, which is limiting because the cells need to remain in close proximity to those materials in order to function. Also these types of materials are too large to be infused into the bloodstream.”

Controlling cells in culture is relatively easy. If cells take up the right molecules, they will change their behavior. This level of control, however, is lost after the cell is transplanted. Sometimes implanted cells readily respond to the environment within the body,. but other times, their behavior is erratic and unpredictable. Karp’s strategy, which her called “particle engineering,” corrects this problem by turning cells into pre-programmable units. The internalized particles stably remain inside the transplanted cell and instruct it precisely how to act. It can direct cells to release anti-inflammatory factors, or regenerate lost tissue and heal lesions or wounds.

“Once those particles are internalized into the cells, which can take on the order of 6-24 hours, we can deliver the transplant immediately or even cryopreserve the cells,” said Karp. “When the cells are thawed at the patient’s bedside, they can be administrated and the agents will start to be released inside the cells to control differentiation, immune modulation or matrix production, for example.”

It could take more than a decade for this type of cell therapy to be a common medical practice, but to speed up the pace of this research, Karp published the study to encourage others in the scientific community to apply the technique to their various fields. Karp’s paper also illustrates the range of different cell types that can be controlled by particle engineering, including stem cells, cells of the immune system, and pancreatic cells.

“With this versatile platform, which leveraged Harvard and MIT experts in drug delivery, cell engineering, and biology, we’ve demonstrated the ability to track cells in the body, control stem cell differentiation, and even change the way cells interact with immune cells, said Ankrum, who is a former graduate student in Karp’s laboratory. “We’re excited to see what applications other researchers will imagine using this platform.”

Stomach Cells Naturally Revert to Stem Cells


George Washington University scientists from St. Louis, Missouri have found that the stomach naturally produces more stem cells than previously realized. These stem cells probably repair stomach damage from infections, the foods we eat, and the constant tissue insults from stomach acid.

The reversion of adult cells to a stem cell fate is one of the goals of stem cell research. Shinya Yamanaka’s research group at the Center for iPS Cell Research and Application and the Institute for Frontier Medical Sciences at Kyoto University won the Nobel Prize in 2012 for his work on reprogramming adult cells into embryonic-like stem cells, otherwise known as induced pluripotent stem cells (iPSCs) that was initially published in 2006.

A collaborative research effort between scientists from Washington University School of Medicine in St. Louis and Utrecht Medical Center in the Netherlands have shown that this reversion from adult cells to stem cells occurs naturally in the stomach on a regular basis.

Jason Mills, associate professor of medicine at Washington University, said, “We already knew that these cells, which are called chief cells, can change back into stem cells to make temporary repairs in significant stomach injuries in significant stomach injuries, such as a cut to damage from infection. The fact that they’re making this transition more often, even in the absence of noticeable injuries, suggests that it may be easier than we realized to make some types of mature, specialized adult cells revert to stem cells.”

Chief cells normally produce a protein called pepsinogen. In the presence of stomach acid, pepsinogen activates itself and once active, the new protein product, pepsin, degrades proteins. Pepsin in an enzyme that is most active in the acidic environment of the stomach. Another enzyme released by chief cells is chymosin, which is also known as rennet. Chymosin curdles the proteins in milk and makes them easier to degrade.

PARIETAL AND CHIEF Cells

Mills and his groups are in the process of studying the transformation of chief cells into stem cells, for injury repair. Mills would also like to investigate the possibility that the potential for growth unleashed by this change may contribute to stomach cancers.

Mills and his collaborator Hans Clevers from the Netherlands have identified stomach stem cell marker proteins that show that chief cells become stem cells even in the absence of serious injury. In the case of serious injury, either in cell culture of in animal models, more chief cells become stem cells, making it possible to repair the damage in the stomach.

Adult Stem Cell Research Has Defeated Embryonic Stem Cells for Funding Priorities


Mallory Quigley from LifeNews has written an article on a report by the Charlotte Lozier Institute, which analyzes funding trends in stem cell research in the state of Maryland. Funding for non-embryonic stem cells greatly outnumbers funding for embryonic stem cells. Read the article here.

FDA Approves the First Stem Cell Clinical Trial for Multiple Sclerosis


The Tirsch Multiple Sclerosis (MS) Research Center of New York has received Investigational New Drug (IND) approval from the Food and Drug Administration to launch a Phase I trial that uses a patient’s own neural stem cells to treat MS.

MS is a chronic disease that results when a patient’s own immune system attacks the myelin insulation that covers many nerves. This damages the myelin sheath and causes degeneration of the nervous system. Some 2.1 million people worldwide are afflicted with MS.

“To my knowledge, this is the first FDA-approved stem cells trial in the United States to investigate direct injection of stem cells into the cerebrospinal fluid of MS patients, and represents an exciting advance in MS research and treatment,” said Saud A. Sadiq, senior research scientist at Tisch and the study’s principal investigator.

The groundbreaking study will evaluate the safety of using stem cells harvested from the patient’s own bone marrow. Once harvested, these stem cells will be injected into the cerebrospinal fluid that surrounds the spinal cord in 20 participants who meet the inclusion criteria for this trial.

Since this is a phase 1 study, it is an open safety and tolerability study. The Tisch MS Research Center and affiliated International Multiple Sclerosis Management Practice (IMSMP) will host all the activities associated with this study.

The clinical application of autologous neural precursors in MS is the culmination of a decade of stem cell research headed by Sadiq and his colleague Violaine Harris, a research scientist at Tisch.

Preclinical testing found that the injection of these cells seems to decrease inflammation in the brain and may also promote myelin repair and neuroprotection.  In a 2012 publication in the Journal of the Neurological Sciences, Harris and others showed that mesenchymal stem cell-derived neural progenitor cells could promote repair and recovery after intrathecal injection into mice with EAE (experimental autoimmune encephalitis), which is a MS-like disease in mice.  They were able to ascertain that intrathecal injection of mesenchymal stem cell-derived neural progenitor cells significantly correlated with reduced immune cell infiltration in the brain, reduced area of demyelination, and increased number of neural progenitor cells in EAE mice.  This successful preclinical study was the impetus for this clinical trial.

Sadiq said, “This study exemplifies the Tisch MS Research Center’s dedication to translational research and provides a hope that established disability may be reversed in MS.” All study participants will undergo a single bone marrow collection procedure, from which mesenchymal stem cell-derived neural progenitor cells (MSC-NPs) will be isolated. expanded, and tested prior to injection.

All patients will receive three rounds of injections at three-month intervals. Safety and efficacy parameters will be evaluated in all trial participants throughout their regular visits with their attending physicians.

Engineered Mesenchymal Stem Cells Make Blood Vessels that Help Heal Ailing Hearts


Another term for a heart attack is a myocardial infarction (MI). A heart attack or an MI occurs when the blood supply to the heart that flows through coronary blood vessels is interrupted. The interruption of blood flow deprives the heart of nourishment and oxygen, and the downstream blood vessels and heart muscle die as a result. The decrease in blood vessel density after a MI can increase cell death, which increases the amount of cell death and the size of the heart scar. Therefore, growing more blood vessels in the heart after a heart attack, which is known as therapeutic angiogenesis, is a potentially strategy in treating an MI (see Ziebart T, et al., (2008) Circ Res 103: 1327–1334)..

To this end, a few clinical trials have attempted to used stem cells that can make blood vessels to reverse heart damage caused by an MI (see Ripa RS, et al. (2007) Circulation 116: I24–I30 and Schachinger V, et al. (2006) N Engl J Med 355: 1210–21).

Among those therapeutic agents for heart attack patients, mesenchymal stem cells (MSCs) are considered excellent candidates. MSCs have the ability to differentiate into smooth muscle, or blood vessels, which means that they can help revascularize the heart after a MI. The problem with MSCs is their tendency to die off rapidly after transplantation into the heart after a heart attack (see Ziegelhoeffer T, et al. (2004) Circ Res 94: 230–38 & O’Neill TT, et al., Circ Res 97: 1027–35; & Perry TE, et al. (2009) Cardiovasc Res 84: 317–25).

To fix this problem, MSCs can be either preconditioned before implantation (see previous posts) or genetically engineered to withstand the hostile conditions inside the heart after a heart attack.

Previously, Muhammad Ashraf and Yigang Wang from the University of Cincinnati genetically engineered MSCs to express a surface protein called CXCR4.  CXC4R is the receptor for a chemokine known as CXCL12/SDF-1.  SDF-1 is a rather potent stem cell recruitment molecule.

When transplanted into the hearts of rodents that had just experienced a heart attack, MSCs that expressed CXCR4 showed increased mobilization and engraftment into the damaged areas of the heart. Also, the pumping abilities of the heart regions into which the MSC-CXCR4s were infused increased, and the MSC-CXCR4 cells cranked up their secretion of blood vessel-inducing growth factors (vascular endothelial growth factor-A or VEGF-A), This led to increased formation of new blood vessels and a decrease in the early signs of left ventricular remodeling (see Zhang D, et al. (2010) Am J Physiol Heart Circ Physiol 299: H1339– H1347; Huang W, et al. (2010) J Mol Cell Cardiol 48: 702–712; &.Zhang D, et al. (2008) J Mol Cell Cardiol 44: 281–292). While these papers show truly stunning results, it was still, even after all this work, unclear if the MSCs were actually differentiating into blood vessel cells and making blood vessels.

To nail this down, Wang and his group used a clever little technique. They engineered MSCs to express CXCR4 and the viral TK gene. TK stands for “thymidine kinase,” which is an enzyme involved in nucleotide synthesis from a virus. The TK enzyme is not found in human cells, and is therefore a target for antiviral drugs. If treated with antiviral drugs that target the TK enzyme, only cells with the TK gene will be killed.

When Wang and his group used their CXCR4-engineered MSCs to treat the heart of mice that had recently suffered a heart attack, they found that their hearts improved and that these same heart were covered with new blood vessels. However, when this experiment was repeated with CXCR4-MSCs that also had the TK gene, Wang his co-workers fed the mice a drug called ganciclovir, which kills only those cells that possess the TK gene. In these mice, their heart failed to improve and also were completely devoid of the new blood vessels.

This paper nicely shows that without viable MSCs, no new blood vessels were made. This strongly suggests that the engineered MSCs are differentiating into blood vessel cells and making new blood vessels, which helps the heart recover from the heart attack and shrinks the size of the dead area of the heart.

What are the implications for human clinical trial\? This is difficult to say. Before clinical trials with genetically engineered cells are approved those cells will need to go through piles of safety tests before they can be used in clinical trials. Once that hurdle is passed, then they can be used in human clinical trials, and they will certainly prove efficacious for human patients.

Making Older Mice Younger with Stem Cell Injections


University of Pittsburgh scientists have used stem cells derived from younger young mice to revitalize older mice. They used mice that were bred to age quickly, but after these stem cell injections, they seemed to have sipped from the fountain of youth. These stem cells were derived from muscles of young, healthy animals, and instead of becoming infirm and dying early as untreated mice did, the injected animals improved their health and lived two to three times longer than expected. These findings were published in the Jan. 3 edition of Nature Communications.

Previous research has revealed stem cell dysfunction, such as poor replication and differentiation, in a variety of tissues in old age. However it is not clear whether that loss of function contributes to the aging process or is a result of it. Senior investigators in this work were Johnny Huard, Ph.D., professor in the Departments of Orthopaedic Surgery and of Microbiology and Molecular Genetics, Pitt School of Medicine, and director of the Stem Cell Research Center at Pitt and Children’s Hospital of PIttsburgh of UPMC, and Laura Niedernhofer, M.D., Ph.D. associate professor in Pitt’s Department of Microbiology and Molecular Genetics and the University of Pittsburgh Cancer Institute (UPCI).

Niedernhofer explained: “Our experiments showed that mice that have progeria, a disorder of premature aging, were healthier and lived longer after an injection of stem cells from young, healthy animals. That tells us that stem cell dysfunction is a cause of the changes we see with aging.”

The research team examined a stem/progenitor cell population derived from the muscle of mice engineered to suffer from a genetic disease called progeria. Progeria is a genetic disease that causes premature aging. Human patients with progeria age extremely quickly and die at a very young age from old age. Muscle-derived stem cells from progeria mice were fewer in number, did not replicate as often, didn’t differentiate as readily into specialized cells and were impaired in their ability to regenerate damaged muscle in comparison to those found in normal rodents. The same defects were discovered in the stem/progenitor cells isolated from very old mice.

Dr. Huard said: “We wanted to see if we could rescue these rapidly aging animals, so we injected stem/progenitor cells from young, healthy mice into the abdomens of 17-day-old progeria mice. Typically the progeria mice die at around 21 to 28 days of age, but the treated animals lived far longer – some even lived beyond 66 days. They also were in better general health.”

As the progeria mice age, they lose muscle mass in their hind limbs, hunch over, tremble, and move slowly and awkwardly. Affected mice received an injection of stem cells just before showing the first signs of aging were more like normal mice, and they grew almost as large. Closer examination showed new blood vessel growth in the brain and muscle, even though the stem/progenitor cells weren’t detected in those tissues. However, the injected cells didn’t migrate to any particular tissue after injection into the abdomen.

Niedernhofer noted: “This leads us to think that healthy cells secrete factors to create an environment that help correct the dysfunction present in the native stem cell population and aged tissue. In a culture dish experiment, we put young stem cells close to, but not touching, progeria stem cells, and the unhealthy cells functionally improved.”

Animals that age normally were not treated with stem/progenitor cells, but these provocative findings urge further research. They hint that it might be possible one day to forestall the biological declines associated with aging by delivering a shot of youthful vigor, particularly if specific rejuvenating proteins or molecules produced by the stem cells could be identified and isolated.