Induced Pluripotent Stem Cells Repair Spinal Cord Injury in Mice

Induced pluripotent stem cells (iPSCs), are made from adult cells by means of genetic engineering and share many, though not all, of the characteristics of embryonic stem cells. The regenerative capacity of iPSCs is truly remarkable, but there are definitely safety concerns with them. The procedure that makes iPSCs from adult cells drives them to divide quickly and often. These cell divisions produce high rates of genetic mutations, some of which are of little consequence, and others that are. Also prolonged culture of iPSCs can select for cells that acquire cancer-causing mutations. Laboratory tests have established that iPSCs have a capability to cause tumors in laboratory animals that at least equals that of embryonic stem cells.

Nevertheless, some labs have designed protocols to screen iPSC lines for tumor-causing or non-tumor-causing lines. Also, iPSCs have been successfully used in therapeutic experiments in laboratory animals without generating tumors. Therefore, iPSCs might be closer to therapeutic use than we think.

With this comes a fascinating publication from the Laboratory of Molecular Neuroscience in the Graduate School of Biological Sciences at the Nara Institute of Science and Technology in Ikoma, Japan; specifically from the laboratory of Kinichi Nakashima. In this experiment, workers in Nakashima’s laboratory used iPSCs that were made from mouse adult cells to make neural stem cells (NSCs).

NSCs are found in the central nervous system and they replace cells in the central nervous system or augment the central nervous system in response to learning and memory or things like that. NSCs are not a monolithic cell population, since some NSCs have the ability to make specific populations of neurons (the cells responsible for neural impulses), while others form glial cells (the cells that support and maintain the neurons).

Nakashima’s laboratory has designed a highly efficient protocol for converting iPSCs into NSCs. They predicted that these NSCs would represent a much less mixed population. Nakashima surmised that such NSCs would almost certainly do a better job of repairing a spinal cord injury. Therefore, led by Yusuke Fujimoto, his colleagues produced several iPSC lines and converted them into NSCs. They called these cell lines “neuroepithelial-like stem cells from human iPS cells” or hiPS-lt-NES cells.

Characterization of these cells in culture showed that they were a homogeneous population that differentiated into many different types of spinal-specific neurons and glial cells. Next, as predicted by Nakashima, Fujimoto and his colleagues transplanted these hiPS-lt-NES cells into the spinal cords of mice that had suffered spinal cord injuries.

The results were remarkable. The transplanted hiPS-lt-NES cells differentiated into neural cells in the spinal cord and promoted functional recovery of hind limb motor function. This is a remarkable finding, but perhaps the transplanted cells only secreted growth factors that helped heal the spine and played no real role in regenerating the spinal cord. Nakashima was not satisfied with this result.

To determine if the transplanted cells were actually regenerating the spinal cord, Fujimoto and the rest of his laboratory workers used two different tracers and also killed off the transplanted cells. The nerve cell tracers showed that the transplanted cells and nerve cells that were already in the spinal cord formed the new neural networks and connections to restore normal hind limb function. Neurons native to the spinal cord and the newly introduced neurons hat were formed from transplanted hiPS-lt-NES cells reconstructed the corticospinal tract by forming proper connections with other neurons and integrating neuronal circuits. Then, when they deliberately killed off the transplanted cells, no neural regeneration occurred. Thus the transplanted hiPS-lt-NES cells not only contributed to the regeneration of the spinal cord and its neural circuits, but they initiated and drove the process.

These fascinating findings suggest a new way to treat spinal cord injury and it does not require the killing of embryos.

The FOCUS-CCTRN Trial – Transendocardial Delivery of Bone Marrow Stem Cells Improves Heart Function in Heart Attack Patients

Mayo Clinic researchers have completes a Phase II clinical study that demonstrates that bone marrow stem cells can fix a sick heart. They discovered that stem cells derived the bone marrow of heart patients, when isolated and injected into their hearts, improved heart function. These researchers also found that particular types of the stem cells seemed to be responsible for the largest patient improvement, and, therefore, warrant further study.

This clinical study is an extension of earlier work in Brazil that treated a small number of patients with fewer stem cells (Perin EC, Dohmann HF, Borojevic R, et al. Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation. 2003;107(18):2294-2302.). The earlier study treated 21 patients, seven of whom received placebo treatments and fourteen others who received injections of bone marrow stem cells into the walls of their hearts.  In this study, a total of 92 (82 men; average age: 63 years) were randomly assigned to the placebo or experimental groups (n=61 in Bone Marrow Cell transplant group and n=31 in the placebo group).  This patient group suffered from coronary artery disease or LV dysfunction, and limiting heart failure or angina.  These patients had weakened hearts as a result of previous heart attacks.

These 92 patients received either a placebo (sterile saline bereft of any cells) or 100 million bone marrow-derived stem cells that were extracted from the patient’s hips. In all cases the treatment consisted of a one-time injection into the wall of the heart.  This injection procedure actually consisted of 15 small injections in stem cells into regions of the ventricle wall that were known to consist of live cells as demonstrated by previous “electromechanical mapping” studies of the heart (see Willerson JT, Perin EC, Ellis SG, et al. Intramyocardial injection of autologous bone marrow mononuclear
cells for patients with chronic ischemic heart disease and left ventricular dysfunction (First Mononuclear Cells injected in the US [FOCUS]). Am Heart J. 2010;160(2):215-223
for a description of this mapping).  The injections were made performed with a NOGA catheter.  This clinical trial is the first clinical to use such a large a dose of stem cells.

NOGA Catheter

The significance of using these patient’s own bone marrow stem cells is not lost on cardiologists, since previous reports have shown that bone marrow from patients with chronic heart conditions or who have suffered heart attacks show diminished stem cell populations and activities (see Heeschen C, Lehmann R, Honold J, et al. Profoundly reduced neovascularization capacity of bone marrow mononuclear cells derived from patients with chronic ischemic heart disease. Circulation. 2004;109(13):1615-1622 & Kissel CK, Lehmann R, Assmus B, et al. Selective functional exhaustion of hematopoietic progenitor cells in the bone marrow of patients with postinfarction heart failure. J Am Coll Cardiol. 2007;49(24):2341-2349).  If higher doses of stem cells can still help improve the function of such heart patients, then perhaps such a protocol would be helpful for them.

Mayo Clinic cardiologist Robert Simari, who was part of this study, said “We found that the bone marrow cells did not have a significant impact on the original end points that we chose, which involved reversibility of a lack of blood supply to the heart, the volume of the left ventricle of the heart at the end of a contraction, and maximal oxygen consumption derived through a treadmill test.” Simari is chairman of the Cardiovascular Cell Therapy Research Network (CCTRN), which is a network of five academic centers and associated satellite sites that conducted the study. The CCTRN is supported by the National Heart, Lung, and Blood Institute, which also funded the study.

Simari described the results of this study: “But interestingly, we did find that the very simple measure of ejection fraction was improved in the group that received the cells compared to the placebo group by 2.7 percent.” Ejection fraction refers to the average percentage of blood pumped from the left ventricle each time the heart pumps.  You can listen to Simari discuss this clinical trial here.

Emerson Perin, and James Willerson of the Texas Heart Institute, who were the principal investigators in this study, noted that although 2.7 percent does not seem like a large number, it does represent a statistically significant increase and this means an improvement in heart function for chronic heart failure patients who have no other options.

Dr. Perin noted, “This was a pretty sick population. They had already had heart attacks, undergone bypass surgery, and had stents placed. However, they weren’t at the level of needing a heart transplant yet. In some patients, particularly those who were younger or whose bone marrows were enriched in certain stem cell populations had even greater improvements in their ejection fractions.”

The study participants had an average age of 63 years old, but this study showed that those patients who are younger than the average participant age improved more than the average. In these patients, the ejection fraction improved by 4.7 percent. The variable that seemed to predict whether or not the patient would benefit from this procedure was the quality of their bone marrow stem cells.  Detailed examinations of bone marrow stem cell populations from each patient showed that younger patients who showed greater improvements have large quantities of CD34+- and CD133+-type stem cells in their bone marrow isolates.  Stem cells with these particular markers tend to produce blood vessels and making more blood vessels, increases the flow of oxygen and nutrients to the heart muscle.  This spares the damaged heart muscle from experiencing more damage and shores the existing heart muscle to improve its function.

Dr. Simari concluded, “This tells us that the approach we used to deliver the stems cells was safe. It also suggests new directions for the next series of clinical trials, including the type of patients, endpoints to study and types of cells to deliver.”

Dick Cheney’s Heart Transplant

Because people have asked me to comment on the Dick Cheney heart transplant, I thought I would make one entry about it. Readers of this blog will recognize that I have very conservative leanings when it comes to subjects such as politics and health care. Also, the organ transplant waiting lists are local and federal. The decision to put someone on the organ recipient list is a decision that is between the patient and their physicians. I do not think the government has any right to intercede in the decision because it is a private decision. The shortage of organs can be addressed in other ways, but it seems to me that rationing by the government is simply wrong and contrary to the founding principles of our constitutional republic.

Having said all that, Cheney waited 20 months to receive his heart, and he was given no special treatment. You can argue that a younger person should have received this heart, but why? Cheney waited his turn. His age was, in his doctor’s opinion, not an important factor. Therefore, we should go with his doctor and not some bureaucrat.

Nevertheless, the best story on this comes from the inimitable Wesley Smith.  Read his view here.  It says it all.

GABA-Making Neurons Made from Stem Cells Reverse Motor Defects in Mice with a Form of Huntington’s Disease

Huntington disease is a horrible, slow, relentless and progressive death sentence. This disease is inherited, and if one of your parents has Huntington’s disease (HD), you have a 50% chance of inheriting the disease. HD is cause by mutations in a gene found on human chromosome 4. This mutation resides in a gene that encodes the Huntingtin protein. However, these mutations are unusual in that they are due to excessive number of repeats of the triplet sequence, CAG. CAG codes for the amino acid glutamine, and normally, there is a stretch of 10-28 glutamines in normal versions of the Huntingtin protein. However, CAG repeats tend to cause the enzymes that make DNA to slip and resynthesize the repeat, thus causing the number of consecutive CAG triplets in this gene to expand. In persons with Huntington’s disease, the CAG triplet is repeated anyways from 36 to 120 times. This expands the stretch of glutamine residues and creates and toxic protein that is cut into smaller fragments that kill nerve cells.

The symptoms of Huntington’s disease usually begin with behavioral disturbances that show up before the onset of movement disorders. These behavioral symptoms can include hallucinations, irritability, moodiness, restlessness or fidgeting, paranoia, and even psychosis. Abnormal movements begin and these include facial movements, including grimaces, the turning of the head to shift eye position rather than moving the eyes, quick, sudden, sometimes wild jerking movements of the arms, legs, face, and other body parts, slow, uncontrolled movements, and an unsteady gait. The dementia slowly gets worse and other symptoms eventually emerge that include disorientation or confusion, loss of judgment, loss of memory, personality changes, and speech changes.

This disease has no treatments and no cure, but researchers have published a paper in the journal Cell Stem Cell that is a starting block of further research that might lead to a treatment. In this paper, a special type of brain cell generated from stem cells seems to help ameliorate the muscle coordination deficits that eventually lead to uncontrollable spasms (choreas) that are so characteristic of the disease.

Su-Chun Zhang, a neuroscientist at the University of Wisconsin-Madison and senior author of the new study said: “This is really something unexpected.” This work suggests that locomotion could be restored in mice with a Huntington’s-like condition.

Zhang’s laboratory has a great deal of experience and expertise at making different types of brain cells from human embryonic stem cells or induced pluripotent stem cells. In the newly published article, Zhang and his colleagues reported the production of neurons that use a neurotransmitter called “gamma-amino butyric acid,” which thankfully goes by the acronym “GABA.” GABA is one of the most heavily used neurotransmitters in the central nervous system, and GABA receptors come in many shapes and sizes, but virtually all of them are chloride channels. While this may not mean anything to you, to a neuron that is trying to generate a nerve impulse, chloride ions are inhibitory and they cut the neuron off at the knees. GABA, therefore, is an extremely important inhibitory neurotransmitter that shuts neurons down when they need to be shut down.

This significance of making GABA-using neurons in the laboratory cannot be lost on Huntington’s patients, because GABA-making neurons are the ones that take the biggest beating during the onset of Huntington’s disease. Without these GABA-using neurons, it is impossible for various portions of the brain to properly coordinate movement. According to Zhang, GABA-producing neurons produce one the key neurotransmitters for coordinating movement.

At the UW-Madison Waisman Center, Zhang and his colleagues discovered how to make large quantities of GABA neurons from human embryonic stem cells. They then tested these neurons in mice that had an induced condition that resembled Huntington’s disease. They implanted these cells in the brains of mice, and they were very surprised to see that the implanted cells not only integrated into the brain, but also projected axons to the correct targets and effectively reestablished the broken communication network. This largely restored motor function.

Zhang noted that these results surprised so because GABA-making neurons are found in a part of the brain called the basal ganglia. The basal ganglia play a central role in voluntary motor coordination. However, GABA-making neurons, however, exert their influence at a distance on cells in the midbrain through neural circuits that are fueled by the GABA-making neurons.

Zhang explained it this way: “This circuitry is essential for motor coordination, and it is what is broken in Huntington patients. The GABA neurons exert their influence at a distance through this circuit. Their cell targets are far away.”

Zhang, however, did not stop there. Many neuroscientists do not think that the results Zhang and his co-workers observed are even possible. He explained further: “Many in the field feel that successful cell transplants would be impossible because it would require rebuilding the circuitry. But what we’ve shown is that the GABA neurons can remake the circuitry and produce the right neurotransmitter.”

This new study has profound implications for regenerative therapy of neurodegenerative disease. One day, it might be possible to treat Huntington’s disease with cell transplants that capitalize on the plasticity of the adult brain. Zhang noted that the adult brain is considered by some neuroscientists to be stable and not easily susceptible to therapies that try to correct things like broken neural circuits. For a therapy to work, it has to be engineered so that it targets only specific cells. Zhang added, “The brain is wired in such a precise way that if a neuron projects the wrong way, it could be chaotic.”

This new research is indeed promising, but it must be worked up and correlated from the mouse model to the condition found in human patients, and this type of very hard, tedious work will take a great deal of time, people hours, and a whole lot of trial and error. However, for a disease that now has no effective treatment, this work could become the next best hope for Huntington’s disease patients.

A caveat to this research is that the mice with Huntington’s disease-like symptoms were given the disease by means of the chemical called quinolinic acid. Administration of this chemical by means of “bilateral intrastriatal microinjections,” which is a fancy way of saying injecting really small amounts of this stuff into a specific part of the basal ganglia, generates mice that display the movement disorders similar to those seen in humans with this disease (see Sanberg PR, et al., Experimental Neurology 1989 Jul;105(1):45-53). Also, the pathology of the brains of these mice shows some similarity to that observed postmortem in the brains of Huntington’s disease patients.

The problem is this: implanting cells into the brains of mice that have been subjected to quinolinic acid results in those cells living and taking up residence in the brain of the mouse and somewhat reconstructing the striatum of the mouse brain (see Dunnett SB. Novartis Found Symp. 2000;231:21-41; discussion 41-52). This is due to the fact that quinolinic acid lesions in the brain specifically kill off particular parts of the brain, but the environment of the brain is still relatively normal. When similar experiments are attempted in human patients, the implanted tissue takes a beating and dies because the brains of Huntington’s disease patients are not chemically altered, but genetically altered. These brains are a toxic waste dump, so to speak, and implanted tissue or cells die (see Francesca Cicchetti, Denis Soulet, and Thomas B. Freeman. “Neuronal degeneration in striatal transplants and Huntington’s disease: potential mechanisms and clinical implications,” Brain (2011) 134 (3): 641-652. doi: 10.1093/brain/awq328).

It seems to me that the environment of the brain must be improved before cell therapy is going to work, and that is a much more difficult problem to address. Dying neurons spill their neurotransmitters into the intracellular space. Huge neurotransmitter overdose can kill nearby neurons and this contributes to the toxic environment in the brain of Huntington’s disease patients. Finding a way to quell the poisonous products released by dead neurons is the next great unanswered quest for these patients.

SanBio Tests Its Mesenchymal Stem Cell Line SB623 as a Treatment for Strokes

California-based biotechnology company, SanBio Inc., has announced the successful enrollment of its first patients in a Phase 1/2a clinical trial that will test the safety and efficacy of a novel stem cells product in the treatment of chronic deficits that are the product of strokes. This stem cell product is called SB623, and so far, 6 of 18 patients have been given this product. This clinical trial is being conducted at the University of Pittsburgh and Stanford University. The trial is being conducted at Stanford University and the University of Pittsburgh. Thus far, no safety concerns have been reported.

SB623 is a stem cell line that was originally derived from adult bone marrow stem cells. Specifically, mesenchymal stem cells were isolated from bone marrow and cultured. They were then genetically engineered to express a modified version of the “Notch” gene. The Notch gene encodes a protein that is embedded into the membrane and has regions that extend to the cell exterior and another portion that extends to the cell interior. The scientists who made SB623 forced the mesenchymal stem cells to express the internal portion of the Notch protein. The significance of this is simple; the internal portion of the Notch protein does all the work and the external portion of it regulates the internal portion. By expressing only the internal portion of the Notch protein, the scientists made a version of the Notch protein that is always active. When active, the Notch protein turns mesenchymal stem cells into cells that support neurons, which are the main functional cells of the nervous system that transmit neural impulses.  Therefore, SB623 cells are derivatives of mesenchymal stem cells that have the capacity to form neurons or cells that greatly resemble neurons.

SanBio scientists and others have used SB623 cells in laboratory rodents that have sufered strokes. In all animal studies, SB623 cells appear to be safe and efficacious. Tate and colleagues published a paper in the journal Cell Transplantation in 2010 entitled, “Human mesenchymal stromal cells and their derivative, SB623 cells, rescue neural cells via trophic support following in vitro ischemia.” This paper (Cell Transplant. 2010;19(8):973-84), SB623 cells were co-cultured with brain slices after those slices had been deprived of oxygen, which is exactly what happens to the brain during a stroke. SB623 cells or the medium that was used to grow SB623 cells rescued cells in the brain slices from dying. This effect was also dosage dependent.

In an earlier paper, SanBio scientists showed that SB623 cells made scaffolds of molecules that supported the growth of neurons (Aizman et al., J Neurosci Res. 2009 Nov 1;87(14):3198-206). In other work, scientists at Northwestern University implanted SB623 cells into the brains of rats with Parkinson’s disease and showed that they prevented the death of dopaminergic neurons; the cells that usually die during Parkinson’s disease (Glavaski-Joksimovic A,, et al., Cell Transplant. 2009;18(7):801-14). Therefore, the use of SB623 cells in rodents points to a potential for these cells as therapeutic agents in human disease.

The Chief Executive Officer for SanBio, Keita Mori, said of this trial, “This represents a major milestone in the human clinical testing of this important new approach for regenerative medicine. We are pleased to learn that the initial dose level was well tolerated.” In this clinical trial, SB623 is implanted into the damaged region of the brains of stroke patients. Product safety is the primary focus of the study; however, particular tests and measurements of efficacy are also being tested.

SanBio’s Vice President of Development, Ernest Yankee said, “The successful completion of the initial dose cohort is a major step in any first-in-human study. We are looking forward to initiating the next two dose cohorts and wrapping up the study. The safety findings thus far are very encouraging”

Neuronal Stem Cells Made from Mature Skin Cells

Stem cell researcher Hans Schöler and his colleagues at the Max Planck Institute for Molecular Biomedicine in Münster, Germany, have successfully isolated neural stem cells from completely differentiated skin cells. Workers and Schöler’s lab procured skin cells from mice and exposed them to a cocktail of special proteins called “growth factors,” and concurrently subjected them to specific culture conditions. This induced the skin cells to differentiate into neuronal somatic stem cells. Schöler noted that their research “shows that reprogramming somatic cells does not require passing through a pluripotent stage.” These new approaches to regenerative medicine can produce stem cells in a shorter time period and are also safer for human clinical use.

Pluripotent stem cells have definitely been the darling of stem cell science since their discovery. When exposed to the right environment, pluripotent stem cells differentiate into every type of cell in the body. However, the pluripotency of these cells, while being their grace is also their curse. According to Schöler, “pluripotent stem cells exhibit such a high degree of plasticity that under the wrong circumstances they may form tumors instead of regenerating a tissue or an organ.” However reprogrammed stem cells can provide a way around these dangers, since they are not pluripotent, but Multipotent (they can only give rise to select subset of cell types rather than any cell type). This can give them an edge in terms of safety and therapeutic potential.

To convert skin cells into stem cells, the Max Planck researchers invented an ingenious protocol that combined several different growth factors (proteins that direct cellular growth) in a culture system that grows the cells and encourages their differentiation into stem cells. One of these growth factors is called Brn4, and Brn4 had never been used in reprogramming experiments before. However, Schöler’s group discovered that Brn4 is one of the most powerful inducers of the stem cell fate in skin cells. The reprogramming of mature skin cells into neuronal stem cells is even more effective if the growth factor-treated skin cells are grown in specific culture conditions. Such culture conditions drive the cells to divide faster and, according to Schöler, the cells gradually “lose their molecular memory that they were once skin cells.” Only after a few cell divisions, the newly produced neuronal somatic stem cells are, for the most part, indistinguishable from neuronal stem cells extracted from neural tissue.

There are other reasons that this work from Schöler’s laboratory might be readily applicable to clinical settings. According the Schöler, “The fact that these cells are multipotent dramatically reduces the risk of neoplasm formation, which means that in the not-too-distant future they could be used to regenerate tissues damaged or destroyed by disease or old age; until we get to that point, substantial research efforts will have to be made.” However, these experiments were done with mouse skin cells. In order to show that this protocol could work for human regenerative medicine, Schöler and his colleagues must demonstrate that human skins cells can also undergo a similar transformation. Additionally, it is crucial to show that these skin cell-derived neuronal stem cells are stable over long periods of time in culture and when implanted into laboratory animals.

Schöler concluded with these remarks: “Our discoveries are a testament to the unparalleled degree of rigor of research conducted here at the Münster Institute. We should realize that this is our chance to be instrumental in helping shape the future of medicine.” At this point, the project is still in its initial, basic science stage although “through systematic, continued development in close collaboration with the pharmaceutical industry, the transition from the basic to the applied sciences could be hugely successful, for this as well as for other, related, future projects. The blueprints for this framework are all prepped and ready to go – all we need now are for the right political measures to be ratified to pave the way towards medical applicability.”

Fat-Derived Mesenchymal Stem Cells Aid Wound Healing

Breaches in the skin produce wounds that have the ability to heal, but take time to do so. During the time prior to healing, the wound is subject to irritation, pain, and infection. Speeding up wound healing is a necessary to prevent wound infections and other wound-related morbidities.

Wound healing requires a somewhat complicated chain of events that includes interactions with nearby cells and tissues. Wound healing is slower in patients with conditions such as type 1 diabetes mellitis. New therapeutic methods are available for chronic wounds, but there are no satisfactory methods for treating chronic wounds that stubbornly refuse to heal.

Stem cell treatments have been tested as potential treatments for chronic wounds. Mesenchymal stem cells (MSCs) from bone marrow can accelerate wound healing in a rodent model system (see Wu Y, et al., “Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells 2007;25:2648-2659, & Chen L, et al., “Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing.” PLoS One 2008;3:e1886). However, the acquisition of a patient’s bone marrow MSCs requires bone marrow aspirations, and if a patient already has a chronic wound that refuses to heal, introducing anther lesion is probably not a good idea.

Therefore, a South Korean research group as tried to use fat-based MSCs, or adipose tissue-derived MSCs (ADSCs) to accelerate wound healing in a rodent model system. The paper reference is Seung Ho Lee, et al., “Effects of Human Adipose-derived Stem Cells on Cutaneous Wound Healing in Nude Mice,” Ann Dermatol Vol. 23, No. 2, 2011 DOI: 10.5021/ad.2011.23.2.150, and it can be found at this link.

In this work, Lee and his team counted on earlier work that showed that ADSCs improved wound healing in mice that suffered from an inherited form of type 2 diabetes mellitis (Nambu M, et al., “Accelerated wound healing in healing-impaired db/db mice by autologous adipose tissue-derived stromal cells combined with atelocollagen matrix,” Ann Plast Surg 2009;62:317-321.). In other experiments, human ADSCs accelerated the closure of wounds in nude mice (Kim WS, et al., “Wound healing effect of adipose-derived stem cells: a critical role of secretory factors on human dermal fibroblasts,” J Dermatol Sci 2007;48:15-24). Nude mice have a mutation in the FOXN1 gene, which causes them to be born without a thymus gland or body fur. The lack of a thymus gland means that they do not have T cells and this makes them unable to reject tissues that are transplanted into them.

In this study Lee and his colleagues determined the benefits of human ADSCs in wound healing on a nude mice. They used a contraction-preventing splint method, and covered each wound with either ADSC-populated collagen gels (CG), human dermal fibroblast (DFs)-populated CG, or CG alone. They measured the size and thickness of the wounds after healing, and examined the histology of the wounds once they had healed.

The results were rather clear. Wound sizes after ASC treatment was significantly smaller than those wounds that were treated with CG alone (28.63±5.05 mm2, 54.63±5.69 mm2, p<0.05). Wounds treated with DFs healed significantly faster than wounds treated with either ASCs and CG alone (11.09±2.71 mm2, p<0.05). However, when the healed tissue was excised and examined under the microscope, the dermal portion of ASCs-treated wounds was thicker than the others, but the DF-treated wounds was thicker than those treated with CG alone (84.50±4.39μm, 75.78±4.52μm, 51.61±2.31μm, p<0.05).

Dermal fibroblasts (DFs) accelerated wounds faster than ADSCs.   Several reports in the literature have shown that DFs can accelerate cutaneous wound healing.  When seeded in a collagen sponge matrix, DFs facilitated dermal and epidermal wound healing better than wounds treated with the collagen sponge only.  Skin substitutes with dermal components that contain DFs induce the proliferation and differentiation of skin cells (keratinocytes) and increase formation of basement membrane.  Both of these accelerate wound re-epithelialization (Okamoto E, Kitano Y. Expression of basement membrane components in skin equivalents–influence of dermal fibroblasts. J Dermatol Sci 1993;5:81-88; Maruguchi T, Maruguchi Y, Suzuki S, Matsuda K, Toda K, Isshiki N. A new skin equivalent: keratinocytes proliferated and differentiated on collagen sponge containing fibroblasts. Plast Reconstr Surg 1994;93:537-546; Medalie DA, Eming SA, Collins ME, Tompkins RG, Yarmush ML, Morgan JR. Differences in dermal analogs influence subsequent pigmentation, epidermal differentiation, basement membrane, and rete ridge formation of transplanted composite skin grafts. Transplantation 1997;64:454-465).

Thus, the Lee paper supports this previous work, but other work suggests that DFs may not help patients with diabetes mellitis and have chronic wounds (Wu Y, Chen L, Scott PG, Tredget EE. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells 2007;25:2648-2659).  Therefore, even though DFs seem to accelerate wound healing in non-diabetic mice, ADSCs are able to accelerate wound healing in diabetic mice, and therefore might be even more useful for patients.

Furthermore, DFs have other clinical limitations in that they must be isolated from a patient’s own skin.  Also, the ability of DFs to be detected by the immune system could also limit their ability to heal wounds.  ADSCs, on the other hand, are easily isolated by liposuction, are poorly recognized by the immune system, and might accelerate wound healing.  In conclusion, ADSCs might provide a treatment regime for chronic wounds and help those who suffer from such things experience wound closure.

Treating Hypoplastic Left Heart Syndrome with Tissue Engineered Blood Vessels

Angela Irizarry is four-years old and was born with a congenital heart condition called Hypoplastic Left Heart Syndrome (HLHS). HLHS causes the main pumping chamber of the heart, the left ventricle to be abnormally small and stunted. Therefore, the heart only has one pumping chamber, and such a condition is potentially fatal.

HLHS affects approximately 3,000 babies in the US alone each year. Since babies with HLHS have an underdeveloped left side of the heart, the right side of the heart must pump blood to both the lungs and the rest of the body. Before the baby is born, the lungs are not being used because the placenta provides the oxygen for the baby and the baby is surrounded by amniotic fluid. Therefore, the lungs are bypassed by a connection between the vessels that extend from the right side of the heart to the lungs and the vessel that extends from the left side of the heart. This bypass is called the “ductus arteriosus.” The ductus arteriosus and a hole is the septum that separates the left and right side of the heart close very soon after birth (1-2 days after birth). In some children, the ductus arteriosus does not close, which is called patent ductus arteriosus (PDA). Once the ductus arteriosus closes in children who have HLHS, the right side of the heart can’t pump blood out to the rest of the body. The undeveloped heart cannot pump efficiently enough to support the life of the child, and the baby becomes very sick and may die within the first days of life.

Without two heart chambers pumping blood throughout the entire body, HLHS babies can’t deliver sufficient levels of oxygen to their organs and extremities. This severely affects their development and also causes them to turn blue and suffer from a lack of energy. According to Dr. Breuer, without a surgical repair, 70% of them die before their first birthday.

Surgical treatment of HLHS occurs in three stages. The first stage is the Norwood procedure, which is done during the first week of life. The Norwood procedure reconstructs the aortic arch, which is the main blood vessel that supplies blood to the body. Surgeons also insert a tube to connect the aorta to the blood vessel that supplies the lungs (the pulmonary artery). This shunt allows the right side of the heart to pump blood into the aorta.

The second stage is performed when the baby is 4-6 months old and is called the bidirectional Glenn procedure or hemi-Fontan. In this surgery, some of the veins that carry blood from the body are connected to blood vessels that carry blood to the lungs. This allows most of the blood to flow directly from the body into the lungs, and reduces the workload of the right side of the heart. Because blood with higher levels of oxygen is pumped into the aorta, it supplies the rest of the body with oxygen-rich blood.

The third stage is carried out when the child is 18-48 months old, and is known as the Fontan procedure. The Fontan procedure takes the remaining blood vessels that carry blood from the body and connects them to the blood vessels that carry blood to the lungs. This ensures that ALL the blood returning from the body receives oxygen in the lungs and also ends the mixing of oxygen-rich blood with oxygen-poor blood. This operation improves the general health of the child and also prevents from having the blue look.

These surgeries are traumatic, and expensive. Not all children survive them. Is there a better way? In Angela’s case, physicians have used stem cells to help Angela grow a new blood vessel in her body. This experimental treatment could rapidly advance the burgeoning field of regenerative medicine.

In August of 2011, Doctors at Yale University implanted a bioabsorbable tube into Angela’s chest. This tube is designed to dissolve over time, but before the implantation procedure, the tube was seeded with stem cells and other cell types that had been harvested from Angela’s bone marrow. Doctors are quite confident that the tube has disappeared, but in its place, a new blood vessel was built from the bones of the bioabsorbable tube. Apparently, this tube functions like a normal blood vessel.

Christopher Breuer, the Yale pediatric surgeon who led the 12-hour procedure to implant the device, commented, “We’re making a blood vessel where there wasn’t one. We’re inducing regeneration.” Before the procedure, Angela had little energy or endurance. Now, even though she is on several medications, she has the spunk of a regular child her age. Dr. Breuer and her parents are confident that she will be able to start school in the fall.

Recent advances in stem-cell science, regenerative medicine, and tissue engineering suggest that regenerative forces in our bodies that are lost soon after birth might be reawakened with strategically implanted stem cells and other tissue. This hope is fueling research at many academic laboratories and dozens of start-up companies. At these laboratories, scientists are racing to find effective ways to treat previously intractable maladies including paralysis due to spinal cord injuries, poor-functioning kidneys and bladders, and heart muscle damaged from heart attacks.

Also, regenerative medicine seeks to improve presently available treatments. For example, in the case of the Fontan procedure, pediatric surgeons implanting a synthetic blood vessel made of Gore-Tex in order to reroute blood from the lower extremities directly to the lungs instead of through the heart. While this works, this device prone to causing blood clots, infection and in some cases, the child needs additional surgeries later in life to increase the size of the blood vessels to accommodate the growth of the child. Dr. Breuer wants to create a natural conduit for blood that reduces the complications associated with a synthetic tube and grows with the child.

Though not involved in this study, Robert Langer, a researcher at Massachusetts Institute of Technology and a regenerative-medicine pioneer, called Angela’s case a “real milestone and broadly important for the field of tissue engineering.”  Langer also added, “It gives you hope that when you combine cells with a scaffold and [put] them in the body, they will do the right thing.”

According to Claudia, Angela’s mother, the heart defect was diagnosed when she (Claudia) was five months pregnant. Angela had her first operation when she was 5 days old, and the second when she was 8-months old. However, she heart defect still sapped her energy and stunted her growth. Angela was shy, small for her age and lacked the stamina of a normal 3-year-old. According the Claudia, “If she ran from [the living room] to the kitchen, she got tired and she had purple lips.”

Dr. Breuer and other Yale staff met with Angela and her family four times. They discussed the advantages and risks associated with conventional synthetic tubes versus this new, bioengineered approach. Dr. Breuer said that a tissue-engineered blood vessels can still narrow or become blocked and other complications might also arise (e.g., cancer, immune system troubles etc.) that are difficult to foresee. According to Claudia Irizarry, who works as a church secretary, the family’s faith in God and their doctors influenced them to choose the bioengineered version over the synthetic version.

To say the least they are glad they did. According to Angela’s father, Angel Irizarry, who works as a carpenter, his daughter seems more like a regular kid, according to her. “It’s a huge difference,” he says. “It’s like going from a four-cylinder to an eight-cylinder car in one operation.” Before the surgery, he added, “her eyes weren’t as happy as [they are] now.”

It took Dr. Breuer four years of tedious work after he joined Yale in 2003 to develop his bioengineered blood vessel. After those four years, he sought approval from the U.S. Food and Drug Administration in 2007 to test his approach on patients. It took another four years and 3,000 pages of data before the agency allowed him to conduct his first human trials. Breuer’s clinical trial builds on the cases of 25 children and young adults who were successfully treated in Japan a decade ago with a similar approach. Dr. Breuer hopes to implant his tissue-engineered blood vessel into a second patient soon as part of a six-patient Phase I/II clinical trial that examines the safety of the procedure and determine if the blood vessels actually grow as the child gets grows. Breuer hopes that treatment in these patients is non-problematic. If so, then it might qualify for special FDA humanitarian device exemption.

Stem Cells Allow Kidney Transplant Recipients to Live Without Anti-Rejection Drugs

Researchers from Northwestern Medicine And University of Louisville are in the midst of a clinical trial to examine the use of stem cell infusions to re-educate the immune system of recipients of transplanted organs. Such re-education of the immune system might completely eliminate the need for anti-rejection medicines.

Organ transplant recipient must take several pills each day for the remainder of their lives. These medicines are drugs that suppress the immune system, and these drugs have many undesirable side effects. Prolonged use of these drugs can cause high blood pressure, diabetes, infections, heart disease, and cancer. Therefore a stem cell-based approach that obviates the need for drugs that inhibit the immune system would offer transplant recipients better quality of life and few health risks for transplant patients.

Joseph Leventhal, a transplant surgeon at Northwestern Memorial Hospital said, “The preliminary results are exciting and may have a major impact on organ transplantation in the future. With refinement, this approach may prove to be applicable to the majority of patients receiving the full spectrum of solid organ transplants.” Leventhal is the main author of this study in collaboration with Suzanne Ildstad, who is the director of the Institute of Cellular Therapeutics at the University of Louisville. The study is, in fact, one of the first of its kind, since it does not require that the organ donor and recipient do not have to be tissue matched.

For standard kidney transplants, the organ donor, who has agreed to donate a kidney, provides their kidney for transplantation to the recipient. In this study, the organ donor not only provides a kidney, but also a small quantity of blood cells. Approximately one month before the transplant, the organ donor gives some bone marrow by means of a procedure called “apheresis.”

Apheresis removes whole blood from a patient, and then uses a centrifuge-like instrument to separate blood components. These separated portions are removed and the remaining components used for retransfusion. The blood components are separated into fluids, otherwise known as plasma, platelets, and white blood cells. From the white cell fraction, a group of cells that the study cells “facilitating cells” are isolated. The organ recipient’s bone marrow is partially ablated with radiation.

The kidney is then transplanted into the recipient’s body, and one day later, the facilitating cells are given to the recipient. Because the organ recipient’s bone marrow has been semi-ablated, the facilitating cells have space to grow without competition from the recipient’s bone marrow. The goal of this is to make within the recipient two bone marrow stem cell systems that are completely functional in one person. The patient is given anti-immune system drugs, but he or she is slowly weaned off them, with the goal of all anti-rejection drugs being ended within one year of the transplant. To qualify for this study, patients must have compatible blood types

Ildstad provided this insight, “This is something I have worked for my entire life.”  Ildstad pioneered the discovery of the “facilitating cell.”  This trial is ongoing, but the initial results are immensely encouraging, since some transplant patients seem to not need their anti-rejection medicines anymore even though they now have a kidney inside them that was not tissue matched.  Specifically, five of eight people who underwent this treatment protocol were able to stop all immunosuppressive therapy within a year after their kidney and stem-cell transplants,. Note that four of these five patients received kidneys that came from unrelated donors. Notably, all of these patients maintained entirely donor-derived immune systems with no signs of Graft-versus-Host disease.  Ildstad and her team have since treated seven more people. “We continue to see good results,” she says. This could easily revolutionize solid organ transplantation.

Amniotic Stem Cells Are Used With Biomaterials to Fabricate Functional New Heart Valves

When children are born with abnormally formed heart valves, their prognosis is poor and surgery is the only option. What if we could fix the heart valves before the baby is ever born? “Science fiction,” you say. Fortunately fetal surgery, the use of surgical treatment on an unborn baby afflicted with certain life-threatening congenital abnormalities, is a procedure that has been used for decades, and the technology to do these procedures is always improving. Fetal surgery attempts to correct problems that are too severe to correct after the baby is born.

There are two main techniques used in fetal surgery. Open fetal surgery used a Cesarean section (hysterotomy) to expose the portion of the baby that requires surgery. After completion of the surgery, the baby is returned to the uterus and the uterus is closed. Sometimes the surgery is scheduled to coincide with the delivery date, and surgery is done before the cord is cut. This way, the baby is sustained by the mother’s placenta and doesn’t need to breathe on his own.

If the baby’s airway it blocked, a procedure called EXIT (ex utero intrapartum treatment) is used. During EXIT procedures, an opening is made in the middle of the anesthetized mother’s belly. The baby is partially delivered through the opening but remains attached by the umbilical cord. Now the surgeon clears the airway so the fetus can breathe. After the procedure, the umbilical cord is cut and clamped, and the infant is fully delivered. EXIT is used to give the surgeon time to perform multiple procedures to clear the baby’s airway, so that once the umbilical cord is cut, the baby can breathe with an unblocked airway.

Fetoscopic surgery makes use of fiber-optic telescopes and specially designed instruments to enter the uterus through small surgical openings to correct congenital malformations without major incisions or removing the fetus from the womb. Fetoscopic surgery is less traumatic and reduces the chances of preterm labor.

Now that we have some clue about fetal surgery, how do we use this to fix heart valves? To fix heart valves, we must replace them with something else. The best alternative would be to grow new heart valves, but these do not grow on trees. What then should we do? The answer is, construct new ones from stem cells.

Tissue engineering uses organic polymers that can be molded into the shape of particular organs and seeded with cells. These polymers are nontoxic and biodegradable. Therefore, once they are seeded with cells, the cells will degrade the polymers and replace them, and grow into the shape originally established by the mold. A special class of fetal stem cells called amniotic fluid stem cells have proven to be especially good at making heart valves and a recent publication shows the feasibility of using laboratory-fashioned heart valves as replacements in fetal sheep.

Weber and colleagues from the Swiss Center for Regenerative Medicine and Clinic for Cardiovascular Surgery, University Hospital Zurich, used stem cells from amniotic fluid to fashion new heart valves. Amniotic fluid comes from a sac that surrounds the embryo and the fetus and is filled with fluid. The embryo and then fetus is suspended in this fluid and the membrane is called the amnion and the fluid is called amniotic fluid.

The Swiss group isolated amniotic fluid cells (AFCs) from pregnant sheep between 122 and 128 days of gestation by means of a technique called “transuterine sonographic sampling.” This technique is rather precise and does not represent a severe risk to the fetus. They then made stented, three-leafed heart valves from a scaffold made from a biodegradable polymer called PGA-P4HB, which stands for poly-glycolic acid dipped in about 1% poly-4-hydroxybutyrate. This material formed a composite matrix that was used to form a heart valve-shaped mold that was then seeded with AFCs. The AFCs grew into the mold, degraded the polymer matrix and assumed the shape of the mold (Weber B., et al., Biomaterials. 2012 Mar 13).

These fabricated heart valves with then implanted into their natural position by means of an in-utero closed-heart hybrid approach. Other sheep fetuses had heart valves implanted that were not seeded with AFCs as a control. 77.8% of the animals implanted with AFC-seeded heart valves survived. Heart functionality tests were measured with echocardiography and angiography, and 1 week after implantation, the fabricated heart valves were completely functional and showed structural integrity (they weren’t falling apart), and also showed no signs of blood clots forming on them (which occurs when heart valves have structural imperfections that allow clotting proteins to stick to them and form clots).

While this experiment represents an interesting approach for fixing fetal hearts, it is still in the experimental stages. Nevertheless, this provides the experimental basis for future human fetal prenatal heart treatments that use completely biodegradable materials seeded with a baby’s own stem cells to make a replacement tissue.

Induced Pluripotent Stem Cells Form Layered Retina-Like Structure in Culture

Embryonic stem cells can form several different types of eye-specific cells. In the early years of the 21st century, reproducible and efficient methods for differentiating embryonic stem cells into lens cells, retinal neurons, and retinal pigment epithelial (RPE) cells were developed (Haruta M., Embryonic stem cells: potential source for ocular repair. Semin Ophthalmol. 2005 Jan-Mar;20(1):17-23).

Other experiments showed that embryonic stem cells could be differentiated into neural progenitor cells (NPCs). These NPCs differentiated in culture and some of them even expressed genes characteristic of developing retinal cells. Although it must be noted that this was uncommon and cells expressing markers of mature photoreceptors were not observed. Implantation of these differentiated NPCs into the retinas of laboratory animals allowed them to survive for at least 16 weeks, migrate over large distances, and form photoreceptor-like cells that made blue-absorbing pigments. These cells also integrated into the host retina (Banin E, Retinal incorporation and differentiation of neural precursors derived from human embryonic stem cells. Steem Cells. 2006 Feb;24(2):246-57).

These early experiments were followed by several others that showed equally remarkable promise. Workers in Takahashi’s laboratory in Kobe, Japan found that embryonic stem cells could form retinal precursors, but that they rarely formed photoreceptors unless they were treated with extracts from embryonic retinas. However in a follow-up paper in 2008, Takahashi, research group found that specific cocktails of small molecules and/or growth factors could push retinal precursors to form photoreceptors (Osakada, et al., Nat Biotechnol. 2008 Feb;26(2):215-24). Kunisada’s lab in Gifu, Japan used various techniques to differentiate embryonic stem cells in culture so that they would form an elaborate retinal-like structure. When this structure was transplanted into the eyes of rodents with inherited eye diseases, these transplanted cells regenerated the ganglion cells in the retina (Aoki H, et al., Graefes Arch Clin Exp Ophthalmol. 2008 Feb;246(2):255-65). Yu’s lab from Seoul National University, Seoul, South Korea made pure RPE cell cultures from embryonic stem cells and then transplanted them into the eyes of rodents with RPE-based retinal degeneration diseases (Park UC, et al., Clin Exp Reprod Med. 2011 Dec;38(4):216-21). The transplanted cells formed RPEs and integrated into the retinas of the laboratory animals. Sophisticated functional assays definitively showed that the RPEs made from embryonic stem cells gobbled up the old segments from photoreceptors and recycled the components back to the photoreceptors (Carr AJ, et al., Mol Vis. 2009;15:283-95).

Using embryonic stem cells to make retina-like structures in culture can provide a model for testing new drugs and procedures to treat degenerative eye diseased such a macular degeneration. Also, such structures might be used to transplant sections of retina into the eyes of individuals where the retina has died off.

With this goal in mind, researchers at the University of Wisconsin-Madison have succeeded in making made early retina structures that contain growing neuroretinal progenitor cells. The novelty in this experiment is that they did it using induced pluripotent stem (iPS) cells that were derived from human blood cells.

In 2011, the laboratory, of David Gamm lab, pediatric ophthalmologist and senior author of the study whose lab is at the Waisman Center, created structures from the most primitive stage of retinal development using embryonic stem cells and iPS cells derived from human skin. These structures generated the major types of retinal cells, including photoreceptors, they did not possess the layered structure found in more mature retina. Clearly something was missing t form a retinal-like structure.

The iPS cells used in this study were made by scientists at a biotechnology company called Cellular Dynamics International (CDI) of Madison, Wisconsin. CDI pioneered the technique to convert blood cells into iPS cells, and they extracted a type of blood cell called a T-lymphocyte from donor samples. These T-lymphocytes were reprogrammed into iPS cells (full disclosure: CDI was founded by UW-Madison stem cell pioneer James Thomson).

With these iPS cells, Gamm and postdoctoral researcher and lead author Joseph Phillips, used their previously-established protocol to grow retina-like tissue from iPS cells. However, this time, about 16% of the initial retinal structures developed distinct layers, which is the structure observed in a mature retina. The outermost layer primarily contained photoreceptors, whereas the middle and inner layers harbored intermediary retinal neurons and ganglion cells, respectively. This particular arrangement of cells is reminiscent of what is found in the back of the eye.

At 72 days, stem cells derived from human blood formed an early retina structure, with specialized cells resembling photoreceptors (red) and ganglion cells (green) located within the outer and inner layers, respectively. Nuclei of cells within the middle layer are shown in blue. These layers are similar to those present during normal human eye development.

These retinal structures also showed proper connections that could allow the cells to communicate information. In the retina, light-sensitive photoreceptor cells along the back wall of the eye produce impulses that are ultimately transmitted through the optic nerve and then to the brain, and this allows. Because these layered retinal structures not only had the proper cell types, but also the proper connections, these findings suggest that it is possible to assemble human retinal cells into the rather complex retinal tissues found in an adult retina. This is extremely stupefying when one considers that these structures all started from a single blood sample.

There are several applications to which these structures might be subjected. They could be used to test drugs and study degenerative diseases of the retina such as retinitis pigmentosa (a major cause of blindness in children and young adults). Also, it might be possible one day to replace multiple layers of the retina in order to help patients with more widespread retinal damage.

Gamm said, “We don’t know how far this technology will take us, but the fact that we are able to grow a rudimentary retina structure from a patient’s blood cells is encouraging, not only because it confirms our earlier work using human skin cells, but also because blood as a starting source is convenient to obtain. This is a solid step forward.” He also added, “We were fortunate that CDI shared an interest in our work. Combining our lab’s expertise with that of CDI was critical to the success of this study.”

This work was published in the March 12, 2012 online issue of Investigative Ophthalmology & Visual Science. The research is supported by the Foundation Fighting Blindness, the National Institutes of Health, the Retina Research Foundation, the UW Institute for Clinical and Translational Research, the UW Eye Research Institute and the E. Matilda Ziegler Foundation for the Blind, Inc.

Fat-Based Mesenchymal Stem Cells Reduce Ischemic Damage to Organs

Ischemia is a term used in medicine to refer to conditions under which organs are deprived of oxygen. Oxygen deprivation causes cells to die and if enough cells die, then the organ is unable to perform its designed function; a condition known as organ failure. Mesenchymal stem cells (MSCs) have been shown in several animal studies to provide significant therapeutic benefit in ischemic organ injuries. Three recent papers have examined the ability of fat-derived MSCs to mitigate ischemic organ damage in lungs, kidneys, and livers. While these studies are in animals, they might provide the foundation for future clinical studies in human patients.

In the first paper (Sun CK, et al., Crit Care Med. 2012 Feb 14), three groups of male rats were either 1) operated on without inducing liver damage; 2) operated on so that the main blood supply to the liver was interrupted for 60 minutes, followed by re-opening the blood supply and treating the rats with fresh culture media that was used to grow the fat-based MSCs; and 3) operated on to cut off the blood supply to the liver for 60 minutes, followed by releasing the blood flow and treatment with fat-derived MSCs at 6 hours and 24 hours after surgery. Three days later, all animals had their livers assayed for damaged, stress and cell death.

In the first group, no sign of liver damage or stress or cell death was observed. In the second group, all the markers for cell death, liver damage and stress were significantly elevated. However in the third group, the markers for cell death, liver damage and stress were significantly lower than those in group two and other markers of liver cell health were increased in the third group relative to the second group.

These results show that fat-derived MSCs preserve liver health and decrease inflammation after ischemic damage to the liver.

The second paper (Furuichi K, et al. Clin Exp Nephrol. 2012 Mar 8), used a similar strategy to examine the ability of fat-derived MSCs to ameliorate kidney function and health after suffering ischemic conditions. Here again, the renal artery to the kidney was clamped for 45 minutes and then injected with either MSCs or buffer at 0, 1, and 2 days after surgery.

The results were a little strange in that the administered MSCs mainly went to the lung. However, those animals that were injected with buffer showed inflammation in the kidney and lots of cell death in the kidney. However those injected with MSCs showed significantly reduced signs of inflammation and greatly reduced amounts of inflammation.

Thus, despite homing to the lung, adipose-derived mesenchymal cells seem to present a reasonable cell-based therapy option for ischemic kidney injury.

Finally, a third paper (Sun CK, et al., J Transl Med. 2011 Jul 22; 9:118), examined the use of fat-derived MSCs to reduce damage during ischemic injury to the lungs. This paper used rats that were divided into three groups. The first group underwent surgery, but no damage was done to the blood supply to the lung. In the second group, the left bronchus of the lung was clamped for 30 minutes, after which the lung was unclamped and the blood allowed to flow for 3 days (known as reperfusion) followed by treatment with fat-derived MSC culture medium. Animals in the third group underwent the same procedure, but were treated with one million and a half fat-derived MSCs at 1, 6, and 24 hours after lung injury. Three days later, animals from all three groups were examined for markers of lung damage and inflammation.

In the first group, the lungs were normal in their function, cell structure, and biochemical markers. No signs of inflammation were observed. The second group, however, had left lung (the one that had been clamped) that worked much more poorly than the right lung. Also, the blood pressure required to push blood through that damaged lobe was much higher in the second group than the other two groups. The more damaged a lung has suffered, the harder it is for the heart to pump blood through it, and the right ventricle much work harder to pump blood through it, which raised the blood pressure in the lung.

The third group showed lungs that worked better and had lower blood pressure than those in the second group. Tissue sections of lungs from group 2 and three animals showed much more damaged in lungs from group two animals than those in group three. Measurement of gene expression in the tissues also showed that lungs from group two animals had much higher levels of genes expressed during inflammation and cell death than those from group three.

This paper presents evidence that fat-derived MSCs might decrease lung damage after ischemic injury.

Trauma to the body from car accidents or work-related injuries can cause organ ischemia. If this damage is significant, acute organ damage can result. Fortunately, fat-derived MSCs are relatively easy to isolate with little additional trauma to the patient. These papers might provide the impetus for future preclinical experimental and, eventually, clinical trials in human patients to alleviate ischemic damage to organs in accident victims.

The Cells=Drugs Argument Has Suffered A Significant Blow

The Regenexx blog site has a fascinating article on tow approaches to reducing transplantation rejection. Osiris Corporation has tried to market a stem product that is a kind of one-size-fits-all stem cell approach for regenerative medicine. This takes mesenchymal stem cells from the bone marrow of young patients and concentrated them in a vial for use. Unfortunately, once these stem cells differentiate into other cell types, they are rejected by the patient’s immune system. While using mesenchymal stem cells from a different person can provide regeneration under particular circumstances, the transplants that use a patient’s own stem cells are always the best from the perspective of the immune system.

A study from Northwestern showed that kidney transplant patients who were also given transplants of bone marrow from the kidney donor did not require any immunosuppressive drugs to prevent the immune system from rejecting their new kidney. This shows that instead of stem cells in a vial (a one-size-fits-all approach to regenerative medicine), an individualized approach seems to be far superior. However, the stem cells = drugs dictum of the FDA argues for the stem cells in a vial approach. Unfortunately, in a Phase III clinical trial, Osiris’ Prochymal product spectacularly failed to provide relief to patients suffering from “Graft versus Host Disease (GVHD). Therefore the stem cells in a vial approach failed, but the individualized worked. This shows that the stem cells = drugs ideology is not one that is tied to reality.

To read Regenexx’s fascinating blog post, go here.

Mesenchymal Stem Cells Can Potentially Treat Non-Union Fractures

Sometimes bone fractures have trouble healing. Such fractures are called “stable non-union fractures,” and they represent major clinical challenges. There are few treatment options for stable non-union fractures, and such conditions represent a major health issue. Fracture treatment options include bone grafting and/or remodeling of the fracture through open reduction and internal fixation (ORIF). In general, ORIF involves the use of plates, screws or even an intramedullary rod to stabilize the bone. Other, less-invasive care options such as treatment with bone morphogenic proteins (BMPs) and other types of bone stimulators are also available.

Can mesenchymal stem cells help such fractures heal better? Centeno and his colleagues at Regenexx conducted their own original research study that shows that some patients probably can be helped by the same sorts of procedures that they use to treat knees. This procedure includes bone marrow aspiration from the crest of the top of the pelvis (the ilium). The mesenchymal stem cells are isolated from the bone marrow and cultured for a few days. Then the expanded and prepared mesenchymal stem cells are applied precisely to the area that needs healing by means of c-Arm fluoroscopy. Sounds good? Yes it does, but to show that it works requires a tried and true clinical study. Centeno’s group has done exactly that, but the number of patients in this study is small. Still this paper represents one of the first examinations of stem cells treatments for stubborn fractures they resist healing.

In this paper, six patients were evaluated. All six had chronic fractures that had not healed (chronic fracture non-unions). There were four women and two men in this experimental group, and they had suffered from these fractures for an average of 8.75 months. The range of the times the patients had lived with these fractures ranged from 4- 18 months, but one patient had lived with their fracture for over 100 months.

All six patient were treated with their own stem cells that were extracted by means of bone marrow aspirations, cultured in the laboratory for 3- 7 passages, and then suspended in phosphate-buffered saline and lysate from peripheral blood platelets. All mesenchymal stem cells were assessed by microscopic examination and flow cytometry to ensure that they expressed the proper surface proteins. Mesenchymal stem cells were then injected percutaneously by means of a sterile trocar, guided by fluoroscopic imaging into the site of the stubborn fracture. To determine if the fractures healed, patients were scanned with X-rays, and computerized tomographic (CT) imaging.

Only five of the patients could be contacted for follow up, but the results are somewhat encouraging. The first patient was a 37-year old smoker (1/4 pack a day) who had suffered with a non-healing fracture for 9 months, but only 2 months after the treatment, was back to “full activities.” An X-ray at 14 months after healing showed excellent healing of the fracture.

The second patient was an 82-year old woman who had suffered from several fractures because of osteoporosis. She had stem cells implanted into her fractured back, and by eight months after the treatment regime, she showed advanced healing of her back fracture. Within four to six weeks after the transplant, the patient walked normally for her age and enjoyed new activities, albeit with age restrictions.

The third patient was a 68-year old woman with a long-time history of multiple sclerosis. She had an 18-month fracture that had not healed in her foot and had to walk with a walking boot immobilizer. Follow-up X-rays showed that after 2 and 6 months she had moderate healing of her fracture and returned to normal activities by 4-6 weeks after the transplant. Unfortunately, she dropped an object on the same foot at 7 months after the procedure and no further follow-up seemed practical.

The fourth patient is a 59 year old woman who had a 40-year history of a traumatic hip fracture and hamstring tear. Unfortunately, her follow up x-rays failed to show any signs of healing.

The fifth patient is a 67-year old man with a 4-month lower leg fracture. He also had type II diabetes mellitus, and coronary artery disease. This patient returned to full walking 4-6 weeks after the procedure. 5 months after the transplant, his x-rays showed signs of healing. No further follow up was possible.

Four of the six patients treated with their own mesenchymal stem cells showed good healing of the fractures that resisted healing through conventional means. The only fracture that showed no signs of healing was a 40-year old fracture that was difficult to immobilize. It is possible that the lack of immobilization caused the bone, which reacts to stress forces, caused this fracture that had adapted to being broken, and could no longer produce signals necessary for repair.

While this study is preliminary, the results support the hypothesis that a patient’s own mesenchymal stem cells are a potential alternative treatment for the treatment of stubborn, fractures that refuse to heal.

Wrongful Birth Lawsuit

According to the publication New Scientist, estimates by the Israeli medical profession postulate that there have been at least 600 ‘wrongful life’ lawsuits since the first case in 1987. A ‘wrongful life’ lawsuit occurs when the parents of a child with some kind of developmental abnormality or genetic disease sue the doctors who helped birth the child in the name of the child. The lawsuits allege that had the parents known about fetus’ severe genetic problem, they would have chosen to terminate their pregnancy.

“Wrongful life” claims are generally brought by the children, or much more typically, parents acting on behalf of the children.  Essentially, the lawsuit specifies that the children are suing for the right to have never been born.  They are suing doctors for NOT putting them to death. According to an article in BioNews, the psychological implications of such lawsuits on the children named in them have been noted by several medical ethicists.  Professor Rabbi Avraham Steinberg of University Hadassah Medical School, Jerusalem, commented: “I find it very difficult to understand how parents can go on the witness stand and tell their children ‘it would have better for you not to have been born. What are the psychological effects on the children?”

Now in the state of Oregon, a “wrongful life” lawsuit in Portland was put forward involving a Down syndrome child. According to the newspaper, the Oregonian, in June 2007, Ariel and Deborah Levy were excited by the birth of their daughter, when then experienced profound shock and anger when hospital staff told them their daughter had Down syndrome.  When asked if she had had a prenatal test in the form of a chorionic sampling test, Mrs. Levy answered in the affirmative.  Unfortunately, the results showed that they were going to have a normal, healthy child.  Several days after being born, a blood test confirmed that the Levy’s little girl, Kalanit Levy, had Down syndrome.  Therefore, the Levys filed suit against legacy Health, claiming that they would have aborted the pregnancy if they had known that their daughter had Down syndrome.  The Levys say that they “dearly love their daughter, who is now 4 years old, but they want Legacy to pay for the extra life-time costs of caring for her, which are estimated to be about $3 million.

With all respect to the Levys, but this, “We dearly love her but would have killed her before she was born” schtick does not wash.  What if she learns that her parents brought this case.  Doctors cannot guarantee outcomes.  We do not have a right to a particular child and no one should have to be legally declared wrongfully born.  If the jury has any sense in this matter, they will throw this case out.  It is a clear-cut case of chasing deep pockets with a detestable premise.

Different Kinds of Stem Cells in the Heart

For almost a century, the sciences of human physiology, cardiology, and medicine have believed that the heart is a terminally differentiated organ whose cells do not undergo further cell division. Essentially however many heart cells you were born with persisted throughout your own personal lifespan. Any increases in the size of the heart were thought to result from expansion of the size of the heart muscle cells .

Work from several labs over the last 15 years have shown that this dogma does not stand further scrutiny. In 1995, Peiro Anversa and his colleagues at New York Medical College in Valhalla, NY examined the differences in heart size between men and women at various ages and found that heart mass was stable in women, but in men, loss of heart mass was due to cell loss and not a decrease in cell size. Also, cell size was stable in women, but tended to increase in men. This increase in cell volume compensated for the loss of heart muscle cells and kept the thickness of the heart walls the same in older and younger men. However, the mass of the heart still decreased in men as they age. This finding does not support the assumption that heart muscle cells are born during development and stay with you throughout your life (for this study see Giorgio Olivetti, et al., Gender Differences and Aging: Effects on the Human Heart. JACC Vol. 26, No. 4 (1995): 1068-79).

Other work that contradicted the commonly accepted dogma examined hearts of people who had experienced “acromegalic cardiomyopathy.”  In a nutshell, individuals with acromegalic cardiomyopathy had a problem with too much growth hormone.  This growth hormone imbalance caused the patient to be really tall, and suffer from bone abnormalities.  This also causes enlargement of the heart.  This patient died at the age of 65, and had a heart that weighed 800 grams.  This is six times the weight of a normal heart.  However, when the size of the heart muscle cells from the man who had died of acromegaly were compared with that of a 99-year old women who had died of pneumonia, the volume of their cells was similar (Leri, Kajstura and Anversa, Role of Cardiac Stem Cells in Cardiac Pathophysiology: A Paradigm SHift in Human Myocardial Biology. Circulation Research 109 (2011): 941-61).  This strongly calls into question the notion that heart enlargement is due to an increase in cell size.

Why was the acromegalic heart larger?  The answer seems to be that it contained far more cells, and recent work has demonstrated that hearts have a stem cell population that can divide and generate new heart cells.  At all ages, the heart contains heart muscle cells that are dividing and expressing a host of genes found only in dividing cells (CDC6, Ki67, MCM5, Phospho-H3, aurora B kinase).  When the heart enlarges for pathological reasons, the proportion of heart muscle cells that expresses these genes increases (see Levi P, Kajstura J and Anversa P, Cardiac Stem Cells and Mechanisms of Myocardial Regeneration. Physio Rev 85 (2005): 1373-1416).

There is not one population of heart stem cells, but four of them, and they all possess different characteristics, and, possibly, different embryological origins.  The first group is “side population cells.”  Side population cells (SPCs) are identified by their ability to expel toxic compounds and dyes (Hierlihy AM, Seale P, Lobe CG, Rudnicki MA, Megeney LA. The post-natal heart contains a myocardial stem cell population. FEBS Lett.2002 Oct 23;530(1-3):239-43).  SPCs have a membrane protein that pumps such molecules from the cell, and when cultured in a semisolid medium, they will differentiate into heart muscle cells.  The exact genes expressed by SPCs is uncertain, since there seem to be, at least in rodents, a few subclasses of SPCs.  In mice, 2% of all heart cells are SPCs, and they have an expression pattern that looks like this: Sca1[high], c-kit[low], CD34[low], and CD45[low].  Cells that express Sca1 normally form blood vessels, but SPCs do not seem to form blood vessels.  This is the conclusion of cell tracing experiments that marks cells and then places them into damaged hearts.  Once the stem cells have divided and integrated into the heart, the animals are sacrificed and their hearts are stained for the marker that characteristic of the implanted stem cells.  Such experiments show that SPCs do not make blood vessels in mice (Tara L. Rasmussen, et al., Getting to the Heart of Myocardial Stem Cells and Cell Therapy. Circulation. 2011; 123: 1771-1779).

In rats, the data is less clearly interpretable, because rat SPCs express a gene called Bcpr1, but the Bcpr1-positive cells either express CD31 and can form blood vessels or do not express CD31 and cannot form blood vessels.  It appears that only the Sca-1[positive] CD31[negative] cells have a pronounced ability to form heart muscle cells.  SCPs might come from neural crest cells, and this hypothesis comes from their behavior in culture (Oyama et al., Cardiac side population cells have a potential to migrate and differentiate into cardiomyocytes in vitro and in vivo.  J Cell Biol 176 (2007): 329-41).

The second population of cardiac stem cells is the Sca-1 cells.  Sca1 cells do not possess the functional properties of stem cells, but they represent 2% of all heart cells.  While they can be grown in culture, only a very small percentage of the cells express any heart muscle-specific proteins (3%-4%), and when delivered to a damaged heart, Sca1 cells will fuse with existing cells to modestly improve heart function (Matsuura K., et al., JBC 279 (2004): 11384-91).  When injected into damaged hearts, Sca1 cells form blood vessels but their ability to survive in the heart is very poor (Li Z., et al., JACC 53 (2009): 1229-40).  Also, Sca1 cells seem to secrete molecules that help the heart and its other stem cell populations to work better.  Most of the heart muscle turnover seems to result from c-kit[positive] cells and the role of SPCs and Sca1 cells is, to date, uncertain.

On the cardiac surface, a third heart stem cells exists, the epicardial progenitors.  There are several different subtypes of epicardial progenitors; Flk1-expressing cells form blood vessels, WT1- and Tbx18-expressing cells make heart muscle (Zhou B., et al., Nature 454 (2008): 109-13 and Cai CL., et al., Nature 454 (2008): 104-108).  There is also a pool of c-kit[positive] cells in the human heart that can differentiate into heart muscle cells and blood vessels (Castaldo C, et al., Stem Cells 26(7), 2008: 1723-31).

The final heart stem cell population is the cardiosphere derived cell (CDC) population.  “Cardiospheres” are balls of cells formed by CDCs in culture.  While in these spheres, a variety of cells form around a core of primitive, c-kit[positive] cells.  Cardiospheres do not consist of a uniform mass of cells, but a pastiche of cells.  Some of these cells have gap junction proteins that are found in mature heart muscle cells that allow them to connect with each other and pass ions from one cell to another.  Others are highly uncommitted and have tremendous growth potential.  These cells express c-kit at high levels.  CDCs are the stem cells that have been used in the recent CADUCEUS and SCIPIO clinical trials.  They are capable of forming heart muscle cells and blood vessels.  They are also easily extracted from hearts by biopsies that are out-patient procedures.

Thus even though there are several different types of heart stem cells, they play a role in repair, and pathology.  They can also be exploited to heal hearts, shrink heart scars, and make a denser collection of blood vessels in the heart.  Further work on them will increase the ability of cardiologists to heal the hearts of patients with failing hearts.

Defunding of the “Snowflake” Frozen Embryo Program

The consummate bio-ethicist Wesley Smith has a column on the defunding of the Snowflake program by the Obama administration. According the a story in the Washington Times, the “White House has sought to defund the Embryo Adoption Awareness Campaign in its fiscal 2013 budget. The Department of Health and Human Services “is not requesting funds for this program” because “the Embryo Adoption program will be discontinued in FY2013,” HHS officials said in a February funding report to Congress.”

In vitro fertilization generates thousands of frozen embryos every year. There are probably something like a half a million frozen embryos in the United States alone. The complete lack of regulation of this industry is a very poor model for other countries and, additionally, is a national disgrace. The Snowflake program brought couples who wanted to adopt embryos together with available embryos. The Embryo Adoption Awareness Campaign provided funds to agencies to create videos about embryo adoption awareness, maintain a blog, about embryo adoption and the embryo adoption agency, generate embryo adoption materials, and to help pay the salaries of staff members that were employed by the agencies.  Essentially, these funds were used to advertise for the embryo adoption agency.  The embryos available for adoption were designated by the genetic parents of the embryos as being available for adoption.  Embryo adoption agencies include; Snowflakes Embryo Adoption Program, Embryos Alive, National Embryo Donation Center: A Centralized Clinic/Adoption Service Provider (NEDC), Adoption and Fertility Resources, Embryo Adoption Services of Cedar Park, Crystal Angels, Embryo Adoption Services, and Blessed with Infertility.

Apparently, genetic parents who wish to give up their embryos for adoption must contact the adoption agencies themselves.  The Embryo Adoption Awareness Campaign helped fund advertising for those agencies to make potential adoptive couples aware of the availability and option of embryo adoption.  If the embryos were not designated by the genetic parents for adoption, they were designated for research or flushed down the sink.

The program was terminated because it there was a lack of interest in it.  Those agencies that used the funds also apparently did not do very much with those funds.  Therefore, termination of a government program is a potentially good thing as money-saving is certainly a good thing.  However, there are hundreds of federally funded projects whose performances are worse than poor, but still these programs exist.  Given this administrations’ spendthrift ways, it is doubtful in the extreme that termination of this program was to save money. This is almost certainly a poke in the eye at pro-life conservatives and it is a rather childish one at that. Read Smith’s column here.

PS – A hearty thanks to the commenter Sheila who corrected much of the erroneous information in the original post.

Planarian Stem Cell Genes Provide Insight into Human Stem Cells

When I was an undergraduate, I cut up planarians in the laboratory and watched them regenerate. Planarians are a type of free-living flatworm that has an uncanny ability to regenerate. Cut them in half, and the tail half will grow a head and the head half will grow a tail. Cut them in half, and the left half will grow a new right side and the left half will grow a new left side. They are truly remarkable critters. How do these worms do this? It appears that the cells of this worm can de-differentiate and become like embryonic cells that originally made the damaged structures. Essentially, the bit of the worm that needs to regrow, recapitulates the developmental process that made it in the first place.

In this way, the bodies of planarians act like stem cells. Stem cells have the potential to regenerate tissues that have been irreparably damaged. One of the problems with stem cells is how to control them. Yet planarians have a genome full of genes that have human homologs. Therefore, planarians seem a logical choice as a model system to study stem cell behavior. Yet, until now, scientists have been unable to efficiently identify the genes that regulate the planarian stem cell system.

At the Whitehead Institute, Peter Reddien‘s lab has revealed some unique insights into planarian biology. These discoveries might help stem cell scientists deliver on a promising role in regenerative medicine. Published in the journal Cell Stem Cell, Reddien and his co-workers used a novel approach to identify and study those genes that control stem cell behavior in planarians. Perhaps unsurprisingly, at least one class of these genes has a counterpart in human embryonic stem cells.

Once injured, planarians (Schmidtea mediterranea) use stem cells, called cNeoblasts, to regrow missing tissues and organs. Within about a week after being injured, the worms have formed two complete planarians. These cNeoblasts are similar to embryonic stem cells in that they are “pluripotent,” which simply means that they have the capacity to form almost cell type in the body. In order to regrow damaged tissues, researchers want to be able to turn on pluripotency and then turn it off after cells have replaced the damaged or missing adult cells.

Reddien, associate professor of biology at MIT and a Howard Hughes Medical Institute (HHMI) Early Career Scientist, said this about his paper: “This is a huge step forward in establishing planarians as an in vivo system for which the roles of stem cell regulators can be dissected. In the grand scheme of things for understanding stem cell biology, I think this is a beginning foray into seeking general principles that all animals utilize. I’d say we’re at the beginning of that process.”

Dan Wagner, a postdoctoral research fellow in Reddien’s lab, and Reddien constructed a protocol to identify genes that regulate the differentiation and renewal of the stem cell population. After identifying genes active in cNeoblasts, Wagner exposed the planarians to ionizing radiation. This left only one surviving cNeoblast in each planarian. After this treatment, each cNeoblast can divide and form colonies of new cells that will differentiate into distinct cell types and divide at specific rates.

Now Wagner and his colleagues eliminated each of the active genes, one per planarian, and observed to determine the behavior of the surviving cNeoblasts without that missing gene. Because the cNeoblasts divide and differentiate at a reproducible rate, the research group could easily determine the role of each gene in cNeoblast behavior. If a colony cNeoblasts was missing a particular gene and had fewer stem cells than the controls, that gene plays in stem cell renewal. Conversely, if the colony had fewer differentiated cells than normal, then the missing gene played a role in differentiation.

Wagner explained, “Because it’s a quantitative method, we can now precisely measure the role of each gene in different aspects of stem cell function. Being able to measure stem cell activity with a colony is a great improvement over methods that existed before, which were much more indirect.”

This screen identified 10 genes that affect cNeoblast renewal, and two of these genes also play roles in cNeoblast renewal and differentiation. Three of the stem cell renewal genes are rather interesting because they code for proteins that are similar to components of Polycomb Repressive Complex 2 (PRC2). PRC2 is known to regulate stem cell biology in mammalian embryonic stem cells and other types of stem cells as well.

These data suggests that the mechanisms that control stem cells in planarians and mammals certainly share some similarities. This might even extend to the mechanisms by which cNeoblasts and embryonic stem cells maintain their naive developmental state. Such work might lead to more insights into stem cell biology that will allows better control and manipulation of stem cells, which will make their use in regenerative medicine much safer.

University of Georgia Lab Generates Blueprint for Stem Cell Responses to Signaling Molecules

What makes a stem cell a stem cell? This is not a trivial question, but an answer to this question is essential in order to understand how to make adult cells stem cells and how to find, and manipulate other stem cells in the body to amplify their healing properties.

Fortunately a great deal of work has been done in this area – genes expressed by stem cells under particular conditions. However, data from different labs tends to conflict with each other. What is a stem cell scientist to do?

From this morass of cacophony comes a very satisfying study from the University of Georgia at Athens, GA. This study, which comes from the laboratory of Stephen Dalton, professor of cellular biology, has generated a wiring diagram of sorts that describes how stem cells respond to external signaling molecules. In one paper, Dalton and his band of intrepid scientists have managed to reconcile several conflicting observations from many different labs.

This paper, which appeared in the March 2 edition of the journal Cell Stem Cell, can potentially provide stem cell scientists with the ability to control precisely the differentiation of particular stem cells into specific cell types. Dalton offered this assessment of his publication: ‘We can use the information from this study as an instruction book to control the behavior of stem cells. “We’ll be able to allow them to differentiate into therapeutic cell types much more efficiently and in a far more controlled manner.”

Many researchers have tended to view signaling in stem cells in an atomistic way. In other words, a single type of signaling molecule sets in motion a specific signal transduction pathway that culminates in maintaining or changing the fate of the stem cell. This, however, appears to be far too simplistic. In the Dalton paper, evidence is presented that several signaling molecules work together in complex ways to control a variety of molecular switches that specified is a stem cell continues to divide and renew itself, or becomes a specific cell type, such as a neuron, heart muscle or skin cell.

To paint of picture of our understanding of stem cell signaling before the publication of the Dalton paper, let us take the “Wnt” signaling molecule as an example. Approximately half the published studies presented evidence that Wnt signaling molecules drove stem cells to renew themselves and not differentiate, but remain in the naïve development state. For example:
1. Cai C, Zhu X. The Wnt/β-catenin pathway regulates self-renewal of cancer stem-like cells in human gastric cancer. Mol Med Report. 2012 Feb 21. doi: 10.3892/mmr.2012.802.
2. Miki T, Yasuda SY, Kahn M. Wnt/β-catenin signaling in embryonic stem cell self-renewal and somatic cell reprogramming. Stem Cell Rev. 2011 Nov;7(4):836-46.
3. Bisson I, Prowse DM. WNT signaling regulates self-renewal and differentiation of prostate cancer cells with stem cell characteristics. Cell Res. 2009 Jun;19(6):683-97.
4. Shimizu T, Kagawa T, Inoue T, Nonaka A, Takada S, Aburatani H, Taga T. Stabilized beta-catenin functions through TCF/LEF proteins and the Notch/RBP-Jkappa complex to promote proliferation and suppress differentiation of neural precursor cells. Mol Cell Biol. 2008 Dec;28(24):7427-41.

However, several other papers argued just the opposite. Instead Wnt drove stem cells to differentiate and not stay in the developmentally naïve state:
1. Davidson KC, et al., Wnt/β-catenin signaling promotes differentiation, not self-renewal, of human embryonic stem cells and is repressed by Oct4. Proc Natl Acad Sci U S A. 2012 Mar 5.
2. Li HX, Luo X, Liu RX, Yang YJ, Yang GS. Roles of Wnt/beta-catenin signaling in adipogenic differentiation potential of adipose-derived mesenchymal stem cells. Mol Cell Endocrinol. 2008 Sep 10;291(1-2):116-24.
3. Munji RN, Choe Y, Li G, Siegenthaler JA, Pleasure SJ. Wnt signaling regulates neuronal differentiation of cortical intermediate progenitors. J Neurosci. 2011 Feb 2;31(5):1676-87.
4. Kirton JP, Crofts NJ, George SJ, Brennan K, Canfield AE. Wnt/beta-catenin signaling stimulates chondrogenic and inhibits adipogenic differentiation of pericytes: potential relevance to vascular disease? Circ Res. 2007 Sep 14;101(6):581-9

Could Wnt molecules drive cells to do both differentiate and remain in the naive state? According to Dalton the answer is yes and there is a simple reason why. Dalton’s research team showed that at low concentrations, Wnt signaling keeps the stem cell in its naive, developmental, pluripotent state. However, at higher concentrations, Wnt signaling does just the opposite and drives the stem cell to stop dividing and differentiate.

However, we must avoid viewing Wnt signaling in a linear fashion because Wnt does not work alone. Other signaling molecules, such as fibroblast growth factor (FGF2), Activin A, and insulin-like growth factor (IGF), work with Wnt to modify stem cell behavior. If that doesn’t make things complicated enough, these signaling pathways can amplify or inhibit each other to cause what would be a two-fold increase under one set of conditions to become a 10-fold increase under another distinct set of conditions. The timing of cell signaling (when the cells are given the signaling molecule) also plays a crucial role with respect to the outcome.

Dalton remarked on his findings: “One of the things that surprised us was how all of the pathways ‘talk’ to each other. You can’t do anything to the IGF pathway without affecting the FGF2 pathway, and you can’t do anything to FGF2 without affecting Wnt. It’s like a house of cards; everything is totally interconnected.”

In another example, when activated, the PI3K/Akt signaling pathway maintains stem cell self-renewal, and it does so by inhibiting Raf/Mek/Erk and Wnt signal transduction pathways. The PI3K/Akt pathway also drives another signal transduction pathways called the “Activin A/Smad2,3” pathway to promote self-renewal, and this is mediated by stimulating the expression of a gene long known to be essential for stem cell self-renewal called Nanog. However, at low levels of PI3K/Akt signaling, the Wnt pathway is activated an, in combination with the Smad2,3 pathway, promotes differentiation.

Why is it that the Smad2,3 signaling proteins promote stem cell self-renewal and differentiation? When PI3K/Akt signaling decreases, the Wnt signal transduction pathway teams up with the Raf/Mek/Erk signal transduction pathway, which was suppressed by PI3K/Akt. Together, these two pathways target the protein kinase Gsk3β, which drives cells to differentiate. Thus, the signal to self-renew or differentiate revolves around Smad2,3 and the state of this signaling pathway determines if the stem cell differentiates of continues in its naïve developmental state, self-renewing with abandon.

This paper is the result of five years of generating hypotheses, testing them, and then revising the hypotheses in light of new data. This painstaking process was continued until the discrepancies were properly resolved. Fortunately, these data can provide scientists with a better grasp of that first step that stem cells might take as they differentiate. Furthermore, Dalton is quite confident that the same approach can be used to dissect and elucidate the molecular events that underlie other developmental steps that occur as the cells in an embryo divide and differentiate into more specific cell types.

Dalton sounded a hopeful note: “Hopefully this type of approach will give us a greater understanding of cells and how they can be manipulated so that we can progress much more rapidly toward the routine use of stem cells in therapeutic settings.” Dalton said.

Marion Zatz, who is chief of the Developmental and Cellular Processes Branch in the Division of Genetics and Developmental Biology at the National Institutes of Health (NIH), oversees stem cell biology grants awarded by the NIH (which partially supported Dalton’s work). Zatz made this comment about Dalton’s paper: “This work addresses one of the biggest challenges in stem cell research—figuring out how to direct a stem cell toward becoming a specific cell type. In this paper, Dr. Dalton puts together several pieces of the puzzle and offers a model for understanding how multiple signaling pathways coordinate to steer a stem cell toward differentiating into a particular type of cell. This framework ultimately should not only advance a fundamental understanding of embryonic development, but facilitate the use of stem cells in regenerative medicine.”

Dalton’s paper is truly a remarkable achievement that will allow a deeper and more accurate understanding of stem cell biology and development.

Induced Pluripotent Stem Cell Treatments for Heart Attacks

Several recent papers have used induced pluripotent stem cells (iPSCs) to treat heart attacks in laboratory animals. These papers followed similar strategies that included culturing iPSCs, differentiating those iPSCs into heart muscle cells, surgically inducing a heart attack in laboratory rodents, and then transplanting the iPSC-induced heart muscle cells into the hearts of the animals that suffered a heart attack. The results are beyond encouraging; they are remarkable.

The first paper is from James Thomson’s laboratory at the University of Wisconsin, Madison (Zhang J, et al., Circ Res. 2009 Feb 27;104(4):e30-41). In this paper, human iPSCs were differentiated into heart muscle cells. These heart muscle cells expressed many heart muscle-specific genes and proteins, and also had mixed characteristics. Some of the cells resembled heart muscle from the upper part of the heat (atrial), some looked like heart muscle from the lower part of the heart (ventricular), and still others had similarities to heart pacemaker cells. These iPSC-derived heart muscle cells also showed the same response to heart medicines that normal, native heart muscle would show. Thus human iPSCs can form functional heart muscle cells.

While experiment paralleled experiments with mouse iPSCs (see Mauritz C, et al., Circulation. 2008 Jul 29;118(5):507-17; & So KH, et al., Int J Cardiol. 2011 Dec 15;153(3):277-85), it begged the question: “Could these iPSC-derived heart muscle cells integrate into a working heart and act like normal heart muscle cells?” These answer to this question in a certifiable “Yes!”

The first paper – Mauritz C., et al., Eur Heart J. 2011 Nov;32(21):2634-41; examined mouse iPSCs and their ability to differentiate into heart muscle cells that could be used to treat laboratory animals with heart attacks.  These workers from Kutschka’s lab found that iPSC-derived heart muscle came in two forms; those that expressed an enzyme called “fetal liver kinase-1” and those that did not.  Fetal liver kinase-1 or Flk-1 is a receptor for a growth hormone called vascular endothelial growth factor (VEGF).  VEGF is a major stimulator of blood vessel formation, and Flk-1 confers upon cells the ability to respond to VEGF and form blood vessels.

Kutschka’s lab workers isolated the Flk-1-positive cells from the Flk-1-negative cells by means of a cell sorter, but they did not other extremely important experiment.  They used cells that expressed a fluorescent protein if and only if they had not completely differentiated.  This way, all incompletely differentiated cells were removed by the cell sorter.  Since incompletely differentiated iPSCs can cause tumors, this is an important safety consideration if this technology is ever to see the light of clinical trials.  The two heart muscle cell populations were then implanted into the hearts of laboratory mice that had suffered heart attacks.  Control mice were injected with saline.

The results showed that both populations of iPSC-derived heart muscle cells integrated into the injured hearts and increased heart function and structure.  However the Flk-1-positive cells conferred even more benefits onto the hearts.  Furthermore, because these iPSC-derived heart muscle cells were produced from adult cells that came from the animals that had suffered heart attacks, there was no need to use mice that had defective immune systems, or were given immunosuppressive drugs.  This definitely shows that iPSC-based treatments are ready for human clinical trials at some time in the near future.

The second paper – Singla DK, et al., Mol Pharm. 2011 Oct 3;8(5):1573-81; takes a slightly different approach.  This research group from the University of Central Florida made iPSCs from a cultured heart muscle cell line called H9c2.  Singla and colleagues transfected these cells with four genes (Oct3/4, Sox2, Klf4, and c-Myc) and this successfully transformed the cultured heart muscle cells into iPSCs.  Then they differentiated those iPSCs into heart muscle cells that beat in culture and also expressed essential heart muscle proteins.  When transplanted into the hearts of laboratory animals that had recently suffered a heart attack, these iPSC-derived muscle cells made proper contacts with other heart muscle cells, properly communicated with them, and improved heart function much better than transplantation of H9c2 cells, or those injected with no cells.  Once again, we have the same strain of mouse from which H9c2 was made.  These mice did not require any suppression of the immune system to receive this treatment because they received cells made from their own genetic stock, and the immune system recognized them as part of themselves.

Finally, a paper by Nelson TJ, et al., Circulation. 2009 Aug 4;120(5):408-16 – from the Mayo Clinic in Rochester, Minnesota also shows the feasibility of iPSCs for regenerative therapy in laboratory animals.  In this paper, undifferentiated iPSCs were transplanted into the hearts of laboratory rodents that had recently suffered heart attacks.  The iPSCs were placed into differentiation media and transplanted into the hearts of mice with poorly operating immune systems and those with normal immune systems.  In the mice with poorly functional immune systems, the implanted iPSCs formed aggressive tumors that overtook the heart and eventually killed the animal.  However, in those animals with normally-operating immune systems, no tumors formed, and the iPSCs form heart-specific cell types and properly engrafted into the heart without disrupting te structure of the heart.  iPSC treatment also regenerated cardiac, smooth muscle, and endothelial tissue, and restored post-heart attack function when it came to contractile performance, the thickness of the ventricular wall, and the electrophysiology of the heart.

These experiments show that iPSCs can fix injured hearts.  There are even protocols to safely differentiate them into heart muscle cells.  Clearly the safety of these must be better investigated and established before they can transition to clinical trials.  However, these papers are definitely a good start to what will hopefully become, some day, personalized stem cells to treat an ailing heart.