Preconditioning Your Way to Better Stem Cells


When stem cells are implanted into injured tissues, they often face a hostile environment that is inimical to their survival. A stroke, for example, can produce brain tissue without ample blood flow, low oxygen levels, and lots of cell debris and inflammation. The same can be said for the heart after a heart attack. If stem cells are going to help anyone we have to find a way for them to survive.

The first hints came in the form of genetically-engineered stem cells that expressed a host of genes that can help cells survive in low oxygen, high stress environments. However, the FDA is unlikely to approve genetically engineered cells for therapeutic purposes. Therefore, a more “user-friendly” way to precondition cells was sought, and found. Instead of loading cells up with extra genes, all you had to do was grow the cells under low oxygen, high stress conditions, and they would adapt and survive when implanted into damaged tissue. This, however, has a drawback: if you want to treat a patient, you do not always have the time it takes for extract and isolate their cells, grow them in culture over a week or two, and then implant them. Is there a better way?

The answer turns out to be yes. Treating cells with particular compounds or growth factors can induce resistance to low-oxygen, high-stress conditions, and two papers show us how it’s done.

The first paper is from the laboratory of Ling Wei at Emory University School of Medicine in Atlanta who has shown in the past that low-oxygen adaptation of mesenchymal stem cells from bone marrow made them better able to treat acute heart attacks in laboratory animals. In this paper, Wei and her colleagues exploited a biochemical pathway known to induce resistance of low-oxygen conditions known as the HIF-1 pathway. The HIF-1 pathway consists of two proteins that work as a pair; HIF1alpha and HIF1beta. HIF1beta is made all the time and HIF1alpha is oxygen sensitive. In the presence of oxygen, enzymes called prolyl hydroxylases modify HIF1alpha, marking it for destruction. In the absence of oxygen, the prolyl hydroxylases do not have enough oxygen to modify HIF1alpha and the HIF1alpha/beta complex activates the expression of a host of genes necessary for increased tolerance to low oxygen levels. Therefore, to make cells more tolerant to low oxygen levels, we need to turn on the HIF1 pathway and to do that we need to inhibit the prolyl hydroxylases.

This turns out to be pretty straight forward. A small molecule called dimethyloxalyglycine or DMOG can effectively inhibit prolyl hydroxylase and induce survival in low-oxygen, high-stress environments. Therefore Wei and her group used DMOG to treat cells and test them out.

In culture, the DMOG-treated cells made proteins known to be important for the establishment of new blood vessels and for survival. When they were compared to cultured stem cells that had not been treated with DMOG, the DMOG-treated cells expressed significantly more of VEGF, Glut-1 and HIF1alpha, all of which are important for surviving in low-oxygen environments. In a Matrigel assay, the DMOG-treated cells also made more blood vessels that were longer than their non-DMOG-treated counterparts.

When used in laboratory animals that had suffered heart attacks, the DMOG-treated cells distinguished themselves once again. They survived better than the control cells and hearts that had received the DMOG-treated cells had much smaller heart scars after heart attacks. Functional assays of heart function illustrated that the DMOG-treated cells helped their heart perform above and beyond what was shown observed in the animals implanted with stem cells that had not bee treated with DMOG.

Thus it is possible to precondition cells without long culture periods or genetic engineering. One compound can accomplish it and the cells only needed to be exposed to DMOG for 24 hours.

In a similar vein, Genshan Ma and others from Zhongda Hospital in Nanjing, China used a small peptide called bradykinin to precondition human umbilical cord endothelial progenitor cells (EPCs); the cells that form blood vessels. In this paper, Ma and colleagues used bradykinin-treated EPCs to treat heart attacks in mice. One nice aspect of this paper is the large number of controls they ran with their experimental runs.

The bradykinin-treated cells outperformed their untreated counterparts when it came to the size of the heart scar, the number of dead cells in the heart, and heart performance parameters. Cell culture experiments established that the bradykinin-treated cells expressed the Akt kinase at high levels, and expressed higher levels of VEGF, the blood vessel-inducing growth factor. Bradykinn-treated cells also were more resistant to being starved for oxygen, and survived better under unusual culture conditions. All of these benefits could be abrogated by inhibiting the activity of the Akt kinase by treating cells with LY294002, a compound that specifically inhibits the activator of Akt.

In this case, cells were treated with bradykinin for 10 minutes to 12 hours.

Two papers, two success stories. Stem cell preconditioning certainly works in laboratory animals. Since stem cell trials have been completed in human patients, it might be time to try preconditioned stem cells in human patients.

Cardiospheres Aid Heart Healing by Secreting Endoglin and Inhibiting TGF-beta signaling


Eduardo Marbán from Cedars-Sinai Heart Institute in Los Angeles and his team have invested a great deal of work into the development of cardiospheres, which are self-assembling heart-derived stem cells that grow as little balls of cells in culture. Several preclinical experiments and a few clinical trials have established the effectiveness of cardiospheres are treatments for the heart after a heart attack. However, Marbán and his co-workers have also worked very hard at determining why cardiospheres heal a damaged heart.

Cardiospheres

Cardiospheres in culture

 

To that end, Marbán and others have returned to their mouse model to do very detailed experiments with their cardiospheres and define exactly why these cells help heal the heart. To date, it is clear that cardiospheres increase the density of blood vessels in the heart tissue, decrease scar deposition, and prevent heart remodeling (the enlargement of the heart after a heart attack to compensate for the increase load placed on smaller amount of heart tissue). Marbán and others wanted to know precisely how cardiospheres managed these feats.

It has been known for some time that scar formation in the heart is largely a consequence of the activation of the TGF-beta signaling pathway (see NG Frangogiannis, Circulation Research 110: 159-173). Inhibition of this pathway can prevent the scar-making cells (fibroblasts) from migrating to the site of damage, dividing, and depositing the protein collagen, which is the main component of heart scars.

An abundant literature on heart scars show that the heart scar plays an important short-term role, but that in the long-run, it prevents the heart from resuming full function because it does not communicate with the rest of the heart muscle cells and does not contract. Therefore, helping the heart get through the first month after the heart attack without a scar is a crucial time.

In a recent paper published by Marbán and his team, cardiospheres were tested in culture and in the heart of mice that had suffered a heart attack. Marbán thought that since the cardiospheres were attenuating scar formation, they must be inhibiting TGF-beta signaling. TGF-beta proteins are secreted by cells and they bind to a receptor complex that then activate intracellular proteins called “SMADs.” These activated SMAD proteins enter the nucleus and activate the transcription of target genes.

TGF-beta signaling

In his co-culture experiments, Marbán and others used normal human fibroblasts from the lower layers of human skin and cultured them with and without cardiospheres. The co-culturing experiments showed that without cardiospheres, the dermal fibroblasts made lots of collagen and activated their internal SMAD proteins. When these human dermal fibroblasts were incubated with cardiospheres, their SMAD proteins were largely inactivated and they made very little collagen.

Such a result is not surprising, but how are the cardiospheres doing this? As it turns out, there is an inhibitor of the TGF-beta receptor complex called “endoglin” that can also be secreted known as sE. When Marbán and others examined their cardiospheres, they were secreting a fair amount of sE.

Thus, the production of sE could definitely prevent dermal fibroblasts from activating their SMADs and making collagen, but what if these sE molecules were inactivated? Marbán and others made antibodies against sE and then used them to inactivate the sE made in culture by the cardiospheres. Under such conditions, the cardiospheres no longer were able to prevent SMAD activation in dermal fibroblasts and the fibroblasts made lots of collagen, even in the presence of cardiospheres.

This is all fine and good, but it is in cell culture, and cell culture experiments must always be confirmed by experiments in a living creature. Therefore, Marbán and his colleagues used cardiospheres to treat mice that had suffered a heart attack. As observed before, the cardiosphere-treated mice showed increases in ejection fraction and fractional shortening, and decreases in end-diastolic and end-systolic volume. The cardiosphere-treated animals also had much less scar tissue after one month and greater blood vessel density. Furthermore, the cardiosphere-treated mice did not show the maladaptive enlargement of the heart muscle cells seen in post-heart attack patients. When the heart tissue was assayed one month after treatment, it was clear that the cardiosphere-treated heart tissue showed increased sE expression and much less TGF-beta signaling. The downstream targets of SMAD activation were much less, and SMADs also showed less activation. Expression of the TGF-beta receptors was also decreased.

This paper shows that endoglin expression plays a key role in preserving and healing the heart after a heart attack. Would it be possible to give soluble endoglin to heart attack patients? This remains to be seen.

One caveat with this paper is that human dermal fibroblasts are similar but different from heart fibroblasts. While it is reasonable to suppose that these two cell types react in a similar way to cardiospheres, such a supposition must be rigorously confirmed experimentally.

Healing Corneal Blindness with Stem Cells from Extracted Teeth


Scientists at the University of Pittsburgh have found a new way to treat corneal blindness, which affects millions of people around the world.

James Funderburgh and his colleagues at the University of Pittsburgh School of Medicine showed that stem cells isolated from the dental pulp of extracted wisdom teeth can be differentiated into specialized cells that can maintain the health and integrity of corneas and rid them of the scars caused by illness or injury that compromise the ability to see clearly.  These cells can be safely injected into the corneas of mice.

According to Funderburgh, who is a professor of ophthalmology, this new approach can replace damaged corneal eye tissue with tissue made from the patient’s own cells rather than cells from a donor. Such a procedure circumvents the problems of immunological rejection that dog the reconstruction of corneal tissue with cells from donors. Furthermore, donor corneas are in short supply in certain parts of the world (e.g., Africa and Asia).

“Our work is promising because using the patient’s own cells for treatment could help avoid these problems,” said Dr. Funderburgh, who is the senior author of a new paper describing the research, in a written statement.

A post-doctoral research fellow in Dr. Funderburgh’s laboratory, Dr. Fatima Syed-Picard, took cells from the pulp of extracted wisdom teeth and chemically processed them to differentiate them into specialized corneal cells. Then Syed-Picard and others injected these “keratocytes” into the corneas of healthy mice. Once in the eyes of laboratory mice, the tooth pulp-derived cells integrated with the existing tissue with no sign of rejection even after several weeks.

Could such a treatment work in human patients? “We are thinking that in the future people may ‘bank’ their extracted wisdom teeth or the cells from those teeth,” Funderburgh told The Huffington Post in an email. “For someone who did not do that it is possible to extract dental pulp with a root canal procedure, but this is still hypothetical. In the worst-case scenario, someone might consider having a tooth extracted to provide cells for this procedure.”

Last year more than 70,000 corneal transplants were performed in the U.S., According to Kevin Corcoran, president and CEO of the Washington, D.C.-based Eye Bank Association of America (EBAA), there were more than 70,000 corneal transplants performed in the US alone.

“There’s a lot of exciting research being done in the area of [corneal] transplant, and EBAA is interested in any outcome that can help restore sight to the blind or visually impaired,” said Corcoran, who was unfamiliar with the Pitt research.

Dr. Syed-Picard stressed that this research is still in the formative stages and the results are preliminary, and added that it would probably take a few years before human testing could begin. The next step, she said, would be to conduct a similar set of experiments in rabbits.

European Knee Meniscus Injury Pilot Trial to Evaluate Cytori Cell Therapy Begins


Cytori Therapeutics is a cell therapy company that is in the process of developing cell therapies from a patient’s own fat tissue that can potentially treat a variety of medical conditions. To date, the preclinical studies and clinical trials suggest that their Cytori Cell Therapy can improve blood flow, modulate the immune system, and facilitating wound repair.

Recently, Cytori has announced that it has enrolled its first patients in an ambitious clinical trial that will test their stem cell product in patients undergoing surgery for traumatic injuries to the meniscus of the knee.  The meniscus is a wedge of cartilage on either side of the knee joint that acts a a shock absorber between the femur and the head of the tibia.

meniscus

Ramon Cugat, who is the Co-Director of the Orthopedic Institute, Hospital Quiron Barcelona, Spain, is the principal investigator for this trial. Dr. Cugat serves as an orthopedic surgeon at Hospital Quiron Barcelona. This trial will test the ability of Cytori Cell Therapy to heal the meniscus and is being conducted in parallel with a similar trial that is testing the Cytori Cell Therapy as a treatment for anterior cruciate ligament (ACL) repairs. The patients treated with Cytori Cell Therapy for ACL repairs are still being evaluated, but to date, no safety related concerns have emerged and the patients seem to have improved. These preliminary results were presented at the Barcelona Knee Symposium in November 2014.

knee_joint

“Dr. Cugat is a leading expert in treating traumatic knee injuries in elite athletes,” said Dr. Marc H. Hedrick, President and CEO of Cytori Therapeutics. “These trials are important to Cytori because, at minimal cost to us, they provide additional clinical evidence that our therapy can be safely used in treating a multitude of knee conditions.”

The meniscus trial is a two-center, phase I study that will assess the safety and efficacy of Cytori’s ECCM-50 adipose-derived regenerative cell therapy in meniscus repair. In this trial, up to 60 patients who have had meniscus surgery to repair the meniscus will receive injection of the cells directly into the meniscus. Each patient will be evaluated by several clinical read outs that assess the recovery of the patient after meniscus surgery. As in the case of the ACL repair study, the goal of this trial is to determine if Cytori Cell Therapy can be safely delivered to the meniscus and whether efficacy can be observed.

“Tears to the meniscus are problematic injuries for active individuals, particularly athletes. Based on the early results from a recent series of 20 patients treated for complete anterior cruciate ligament injuries, we are eager to evaluate whether augmentation surgery with Cytori Cell Therapy will lead to quicker and more complete healing,” said Dr. Cugat.

Injected patients will fill out a patient questionnaire that assesses knee pain, function and activity, This questionnaire is called the Knee Injury and Osteoarthritis Outcome Score (KOOS), but patients will also be physically examined to ascertain the extent of their knee function and the degree of their movement, with or without pain. Patients will be given a visual analogue score to assess knee pain, and knee function will be assessed by the Lysholm Knee Scoring Scale, Tegner Activity Scale, and the Lower Extremity Functional Scale. Each patient will also have their knees examined by Magnetic Resonance Imaging (MRI) in order to examine the structural integrity of their meniscus. These assessments will be taken before and 60, 90, 180 and 365 days after surgery and the MRIs will be done before and at 90, 180 and 365 days after surgery.

The preliminary results of the ACL study showed that the Cytori strategy was feasible and did not result in any significant safety issues above that seen with a standard small volume liposuction. All the injected patients recovered without any complications. The results of the ACL trial were compared to a historical control group of patients who had the same surgical procedure by the same surgical team but without other interventions. Overall, the patient’s recovery from pain and their ability to return to daily activities was accelerated as a result of the therapeutic enriched bone-patellar tendon-bone transplant. Both the patient questionnaires and serial MRI scans of the knees following cell therapy were consistent with accelerated healing of the graft. Presently, Dr. Cugat and his coworkers are obtaining one year follow-up information on the treated patients and they will report their data in a peer-reviewed journal in the future.

ACL and meniscus tears are among the most common sports-related knee injuries and unfortunately, these two injuries often are sustained simultaneously. According to the American Academy of Orthopedic Surgeons, ACL injuries have an annual incidence of more than 200,000 cases with nearly half undergoing surgical reconstruction. Further, an estimated 850,000 patients undergo surgical procedures to address meniscus injuries each year.

Preclinical Study Results Pave the Way for Newly Opened Clinical Trial of Immune Cells Engineered to Attack Protein Found on Tumors in 30 Percent of Patients with Glioblastoma


Scientists from the University of Pennsylvania have engineered immune cells to seek out and attack a type of deadly brain cancer. In an important preclinical study, these souped-up immune cells were shown to be both safe and effective at controlling tumor growth in mice treated with these modified cells. This work is the result of collaboration between a team from the Perelman School of Medicine at the University of Pennsylvania and the Novartis Institutes for BioMedical Research. These results will hopefully be the impetus for a newly opened clinical trial for glioblastoma patients at Penn.

Marcela Maus, assistant professor of Hematology/Oncology at the Penn Abramson Cancer Center, said: “A series of trials that began in 2010 have found that engineered T cells have an effect in treating some blood cancers, but expanding this approach into solid tumors has posed challenges. A challenging aspect of applying engineered T cell technology is finding the best targets that are found on tumors but not normal tissues. This is the key to making this kind of T cell therapy both effective and safe.”

This new preclinical study, which was conducted with Hideho Okada and his colleagues at the University of Pittsburgh, makes use of T cells engineered to express a chimeric antigen receptor (CAR) that specifically binds to a mutant epidermal growth factor receptor protein called EGFRvIII. EGFRvIII is found on the cell surfaces of approximately 30 percent of glioblastoma tumors. Over 22,000 Americans are diagnosed with glioblastoma each year, and those patients whose glioblastomas express the EGFRvIII mutation tend to be more aggressive and are less likely to respond favorably to standard therapies and more likely to recur after treatment.

“Patients with this type of brain cancer have a very poor prognosis. Many survive less than 18 months following their diagnosis,” said M. Sean Grady, who is the Charles Harrison Frazier Professor and chair of the department of Neurosurgery. “We’ve brought together experts in an array of fields to develop an innovative personalized immunotherapy for certain brain cancers.”

This new trial is being led by Donald O’Rourke, associate professor of Neurosurgery, who heads an interdisciplinary collaboration of neurosurgeons, neuro-oncologists, neuropathologists, immunologists, and transfusion medicine experts.

In order to bring this experiment to fruition, Maus and her colleagues had to characterize the EGFRvIII CAR T cell. They had to develop and tested multiple antibodies that bind to cells expressing EGFRvIII on their surface. The single-chain variable fragments or scFvs that recognized the mutant EGFRvIII protein were then extensively tested in order to confirm that they do not also bind to those normal, EGFR proteins that are widely expressed on cells in the human body.

Maus and her group then generated a panel of humanized scFvs and tested their specificity and function in CAR modified T cells. The humanized scFvs have distinct amino acid sequences that more closely resemble human antibodies. From this huge panel of humanized scFvs, they selected one scFv to explore further based on its binding selectivity for EGFRvIII over normal non-mutated EGFR. They also evaluated the EGFRvIII CAR T cells by testing them against normal EGFR-expressing skin cells in mice grafted with human skin. This test showed that the engineered EGFRvIII CAR T cells did not attack cells with normal EGFR, at least under these conditions.

In order to test the selected scFv for its anti-cancer efficacy, Maus and others used human tumor cells that expressed EGFRvIII and showed that the EGFRvIII CAR T cells could multiply and secrete cytokines in response such to tumor cells. When used inside living animals, it was clear that the EGFRvIII CAR T cells ably controlled tumor growth in several mouse models of glioblastoma. The tumors were measured with magnetic resonance imaging (MRI) and the EGFRvIII CAR T cells caused tumor shrinkage, and were also effective with used in combination with the anticancer drug temozolomide, which is normally used to treat patients with glioblastoma.

On the strength of these preclinical successes, this team designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII for both newly diagnosed and recurrent glioblastoma patients who carry the EGFRvIII mutation. “There are unique aspects about the immune system that we’re now able to utilize to study a completely new type of therapy,” said O’Rourke.

For these glioblastoma patients, their T cells were removed by means of apheresis (a process similar to dialysis), and then the T cells were genetically engineered using a viral vector that programs them to find EGFRvIII-expressing cancer cells. The patient’s own engineered cells are infused back into their body, and when the T cells find the EGFRvIII-expressing cells, a signaling domain built into the CAR promotes proliferation of these “hunter” T-cells. This procedure is distinct from other T cell-based therapies that also target some healthy cells, since EGFRvIII seems to only be found on tumor tissue, which the study’s leaders hope will minimize side effects.

This new phase I clinical trial will enroll 12 adult patients whose tumors express EGFRvIII, in two groups: One arm of 6 patients whose cancers have returned after receiving other therapies, and one arm of 6 patients who are newly diagnosed with the disease and still have 1 cm or more of tumor tissue remaining after undergoing surgery to remove it.

The clinical trial is sponsored by the biotech company Novartis. In 2012, the University of Pennsylvania and Novartis announced an exclusive global research and licensing agreement to further study and commercialize novel cellular immunotherapies using CAR technologies. This STM study is the first pre-clinical paper developed within the Penn-Novartis alliance, with Penn and Novartis scientists working collaboratively. Ongoing clinical trials evaluating a different type of Penn-developed CAR therapy known as CTL019 have yielded promising results among some patients with certain blood cancers. In July 2014, the FDA granted CTL019 its Breakthrough Therapy designation for the treatment of relapsed and refractory acute lymphoblastic leukemia in both children and adults.

Long-term Tumorgenicity of Induced Pluripotent Stem Cells


A paper from the Okano laboratory has shown that implantation of neural stem cells made from induced pluripotent stem cells can still form tumors ever after a long period of time.

This paper is an important contribution to the safety issues surrounding induced pluripotent stem cells (iPSCs). As noted in previous posts, iPSCs are made from adult cells by means of genetic engineering and cell culture techniques. In short, by introducing four different genes into adult cells and then culturing them in a special culture medium, a fraction of these cells will de-differentiate into cells that resemble embryonic stem cells in many ways, but are not exactly like them.

The Okano laboratory made iPSCs using viruses that integrate into the genome of the host cell, which is not the safest option. However, because in the four-gene cocktail that is normally used to reprogram these cells (Oct-4, Klf-4, Sox2, and c-Myc), the c-Myc gene is often thought to be the main cause of tumor formation. Okano and his collaborators made their iPSCs without the c-Myc gene, but only used the three-gene cocktail of Oct-4, Klf-4, and Sox2. Such a cocktail is much less efficient that the four-gene cocktail, but it supposed to make iPSCs that are altogether safer.

These iPSCs were differentiated into neural stem cells that grew as tiny spheres of cells, and these “neurospheres” were transplanted into the spinal cords of mice that had suffered a spinal cord injury. The implanted cells differentiated into neurons and glial cells and restored some neural function to these mice. However, the mice were observed for a long period of time after the implantations to assess the long-term safety of these implanted cells.

After 105 days, the implanted mice began to show deterioration of their neural function and their spinal cords showed tumors. It is clear that the Oct-4 gene that was used in the reprogramming procedure was the reason for the tumor transformation.

Graphical Abstract 20141213

This experiment, once again, calls into question the safety of any method for iPSC generation that leaves the transfected genes in the reprogrammed cells. I reported in a previous post that skin cells made from iPSCs that had their transgenes left in them were good at causing tumors and not as good as forming skin cells, but iPSCs without their reprogramming transgenes were safer and more effective tools for regenerative medicine.  This experiment also shows that c-Myc is not the only concern with iPSCs.  Any of the transgenes used for reprogramming can cause problems, and they must be removed if iPSCs are going to produce safe, differentiated cells.  Finally, this experiment pretty much shows that the use of retrovirus tools to introduce genes into cells for the sake of reprogramming is a bad idea if those cells are going to be used for regenerative medicine.  Non-integrating tools are much safer and preferable in these cases.

The Okano paper appeared in Stem Cell Reports.

Human Stem Cells Repair Radiation Damage in Rat Brains


Radiation is a powerful treatment for brain cancer, but this potentially life-saving treatment comes with a heavy cost, which is permanent damage to the brain.

Preclinical work at Memorial Sloan Kettering Cancer Center has shown that human stem cells can be used to make cells that repair radiation-induced damage in the brain.

When rats were treated with radiation and then given cocktails to the human stem cells, they regained the cognitive and motor functions that were lost after brain irradiation.

In the brain, stem cells called OPCs or oligodendrocyte progenitor cells mature into oligodendrocytes that produce the protective myelin coating that surrounds axons in the central nervous system. During radiation treatment, OPCs die off and are depleted. Because OPCs help shield and repair the myelin sheath throughout the life of the organism, depletion of them threatens the integrity of the myelin sheath, which threatens the proper transmission of neural impulses throughout the brain.

A research project led by neurosurgeon Viviane Tabar and her research associate Jinghua Piao wanted to use stem cells to replace these lost OPCs. They used human embryonic stem cells and human induced pluripotent stem cells to make cultured OPCs.

In the next phase of the experiment, Taba, Piao and their coworkers treated rats whose brains had been irradiated with their cultured OPCs. After injection of the stem cell-derived OPCs, brain repair was evident and the rats regained their cognitive and motor function that they had previously lost as a consequence of radiation exposure.

The treatment appeared quite safe since none of the animals developed any tumors or aberrant growths.

The ability to repair radiation damage could mean that the quality of cancer survivors could be greatly improved and it could also expand the therapeutic window of radiation, according to Tabor.

“This will have to be proven further, but if we can repair the brain effectively, we could be bolder with our radiation dosing, within limits,” said Tabor.

Such a treatment scheme could also be very important in children, for whom physicians must use lower doses of radiation to limit brain damage.