Non-Randomized Stem Cell Study for Knee Osteoarthritis Yields Positive Results

A peer-reviewed study that was neither placebo-controlled nor randomized, but did examine 840 patients, has shown that the use of a patient’s own bone marrow stem cells are both safe and effective.

Christopher Centeno and his colleagues, who pioneered the Regenexx protocol, use live-imaging to guide the application of stem cells to the site in need of healing. Centeno and others have established several clinics around the United States that utilize the Regenexx system, and the data published in this paper came from these clinics, in addition to Chris Centeno’s own clinic in the Denver, Colorado area.

In this study, patients self-rated their lower extremity functional using a lower extremity functional scale (LEFS), and their knee pain by using a numerical pain scale (NPS). Patients had bone marrow extracted through a bone marrow aspiration. These bone marrow cells were isolated and concentrated, and then prepared for reinvention. In addition, platelet rich plasma (PRP) and platelet lysate (PL) were prepared from the patient’s own blood and these, with the bone marrow cells, were injected into the knee under guided imaging. The frequency and types of adverse events (AE) were also recorded by the physicians.

Some of these patients had fat overlaid on their knee lesions in addition to their bone marrow cells. Of the 840 procedures that were performed, 616 had treatment without additional fat, and 224 had treatment with the fat graft. This was to determine if the use of fat, with its resident stem cell population, augmented healing of the arthritic knee.

When the LEFS scores before and after the Regenexx procedure were compared, an increase of 7.9 and 9.8 in the two groups (out of 80) was observed. The mean NPS score decreased from 4 to 2.6 and from 4.3 to 3 in the two groups. AE rates were 6% and 8.9% in the two groups. An examination of these data showed that pre- and posttreatment improvements were statistically significant. However, the differences between the fat- and fat+ groups were statistically insignificant.

The patients in this study suffered from osteoarthritis. Consequently, they were experiencing significant knee pain and many were candidates for a knee replacement. Many of these patients were able to avoid knee replacement by undergoing the Regenexx procedure.

The study concluded that there was no advantage of adding fat to the joint over the bone marrow cells. Safety in both groups (with and without fat) was excellent compared to knee replacement.

This study used data from patients who were part of the Regenexx registry. Therefore, this study was not a randomized, controlled study, like the kind that are used to test drugs. Randomized controlled trials are being conducted by Centeno and his colleagues at the various Regenexx centers. A knee osteoarthritis study is being studied in Chicago, another study regarding shoulder rotator cuff tears, and a third study examining ACL tears are in progress.

Stem Cell-like Megakaryocyte Progenitors Replenish Platelets After Inflammatory Episodes

A paper that appeared in the journal Cell Stem Cell from the laboratory of Marieke A.G. Esters, from the Heidelberg Institute for Stem Cell Technology and Experimental Medicine in Heidelberg, Germany has answered a long-standing question about how our bodies regenerate platelets using so many of them.

When we suffer damage to our bodies from surgery, accidents, infections, or other physical insults, we tend to use a lot of platelets. Platelets are small cells in the blood that help the blood clot once we cut ourselves. Platelets, however, take some time to form. How then do we rapidly regenerate the platelet pool during such stressful conditions?

Esters and her team have shown that the bone marrow contains stem-like cell called a “single-lineage megakaryocyte-committed progenitor” or SL-MkPs. Platelets bud from a large cell called a “megakaryocyte,” and megakaryocytes form from the hematopoietic stem cells that reside in the bone marrow. Hematopoietic stem cells make all the blood cell that course through our blood vessels and continue to replace those cells throughout our lifetime. Hematopoietic stem cells personify what it means to be a multipotent stem cell.

Haas et al, graphical abstract 5.5x5.5

This newly-identified stem cell population, the SL-MkP actually shares many features with multipotent hematopoietic stem cells and provides a stem cell population that is lineage-restricted (that means they can only form one type of cell) for emergency purposes.

Normally, SL-MkPs are maintained in an inactive, almost sleep-like state. In this state, SL-MkPs do not contribute very much to making platelets in the blood. There is some gene expression in this sleepy state, but protein synthesis is turned way down.

In response to acute inflammation, SL-MkPs wake up and become activated. Upon activation, these cells ramp up protein synthesis and mature into full-blown SL-MkPs that efficiently replenishment of platelets that are lost during high levels of inflammation. Thus, there is an emergency system that accommodates platelet depletion during acute inflammation and replenishes the platelet pool.

Activation of Dormant Viruses May Cause ALS

Inactive viruses that litter the human genome may become reactivated and contribute to the development of motor neuron disease, according to new research published today in the journal Science Translational Medicine.

Human endogenous retroviruses (HERVs) are the flotsam and jetsam of ancient viruses that integrated into our chromosomes long ago as the results of retrovirus infections that occurred over several million years of our history.  These HERV sequences account for about 8% of human DNA and the vast majority of them have acquired multiple genetic mutations that made rendered them innocuous.  Therefore, HERVs are sometimes referred to as “junk” DNA, although some of these sequences have been shown to have function (for example, see Dupressoir A, Lavialle C, Heidmann T. Placenta. 2012 Sep;33(9):663-7).

In 2011, Avindra Nath, the intramural clinical director of the National Institute of Neurological Disorders and Stroke, and his colleagues reported that proteins synthesized by one such HERV known as HERV-K are found in very high concentrations in the brains of patients who died of amyotrophic lateral sclerosis (ALS), which is a progressive and fatal neurodegenerative disease that destroys those motor neurons that control speech, movement, swallowing and breathing, which leads to death between three to five years after the symptoms first appear.

In their new study, Nath’s research group investigated the toxicity of viral proteins to nerve cells. They examined samples of nervous tissue from 11 patients who had died of ALS, 10 Alzheimer’s patients, and 16 people who showed no signs of neurological disease as controls.  They used RNA sequencing to confirm that transcripts of three HERV-K genes are present in tissue samples from the ALS patients but not in those from the Alzheimer’s patients or control patients.  In their next set of experiments, Nath and his coworkers showed that the proteins encoded by these viral genes localized to motor neurons in the brains and front halves of the spinal cords of ALS patients.  This is significant, since the ventral or font portions of the spinal cord contains the cell bodies of motor neurons that send their axonal fibers to the body’s skeletal muscles where they synapse with those muscles.  Thus the presence of the viral proteins strongly correlates with the tendency of these cells to die.

To definitively test the toxicity of these viral proteins to neurons, Nath and others transfected either the entire viral genome, or just the viral env gene, which encodes the virus’s coat protein, into cultured human neurons.  Once integrated into the genomes of the cultured cells, the viral genes were fully activated and used the cell’s molecular machinery to synthesize their respective proteins.  Expression of these viral genes killed off significant numbers of cells and caused them to retract their neural fibers.  Furthermore expression of only the env gene in these cultured neurons was sufficient to kill them.

To test their hypothesis in a living animal, Nath and others generated a strain of genetically engineered mice whose neurons express high levels of the HERV-K env gene.  Behavioral tests showed that these HERV-K env+ animals developed motor function abnormalities; they had difficulty walking and balancing compared to healthy mice.  These symptoms progressed rapidly between 3 and 6 months of age, and half of the animals had died before or shortly after reaching 10 months of age.

Closer examination revealed that neurons in the motor cortex had degenerated.  They also showed a decrease in the length, branching and complexity of dendrites, and a reduction in the number of dendritic spines (small, finger-like extensions that receive chemical signals from other cells).

All of these data strongly suggest that reactivation of dormant HERV-K contributes to neurodegeneration in the brain and spinal cord.  The absence of this virus in the brains of Alzheimer’s patients supports the conclusion that reactivation of it causes degeneration, rather than being a consequence of it, and further suggests that it is specific to ALS.

ALS is associated with genetic mutations in more than 50 different genes.  However, as is the case for Alzheimer’s, these inherited forms of the disease, which account for just 10-15% of cases. But this study only examined patients with sporadic, or non-inherited, ALS, the cause of which have been much harder to pin down.

Further genetic analyses may identify DNA sequence variations, in the HERV-K genes themselves, and others that interact with them, which might make the virus more prone to reactivation.  More work will need to be done to determine exactly how the reactivated virus genes contribute to the disease.

Meanwhile, Nath and his colleagues are collaborating with researchers at Johns Hopkins University to determine if anti-retroviral drugs might alleviate disease symptoms in subsets of ALS patients.

See Li, W., et al. (2015). Human endogenous retrovirus-K contributes to motor neuron disease. Sci. Trans. Med., 7: 307ra153.

Stem-Cell Dental Implants Grow New Teeth in Your Mouth

Dr. Jeremy Mao is the Edward V. Zegarelli Professor of Dental Medicine at Columbia University Medical Center. Mao and his colleagues have published a novel technology that includes a growth factor-infused, three-dimensional scaffold that has the potential to regenerate an anatomically correct tooth in the mouth just nine weeks after implantation. By this procedure, which was developed in the university’s Tissue Engineering and Regenerative Medicine Laboratory, Mao can direct the body’s own stem cells to migrate to the scaffold and infiltrate it. Once these stem cells have colonized the scaffold, they will produce a tooth that can grow in the socket and merge with the surrounding tissue and integrate into it.

Tooth scaffold that is completely composed of natural materials.

Tooth scaffold that is completely composed of natural materials.

Mao’s technique not only eliminates the need to grow teeth in a culture, but it can regenerate anatomically correct teeth by using the body’s own resources. If you factor in the faster recovery time and the comparatively natural process of regrowth (as opposed to implantation), you have a massively appealing dental treatment.

Columbia University has already filed patent applications in regard to this technology. They are also seeking associates to aid in its commercialization. Mao is also considering the best approach for applying his technique to cost-effective clinical therapies.

Repairing Nerves Using Exosomes to Hijack Cell-Cell Communication

Biomedical engineers from Tufts University have discovered a new protocol that can induce mesenchymal stem cells (MSCs) derived from bone marrow to differentiate into neuron-like cells by treating them with exosomes from cultured cells.

PC12 cells are neuron-like progenitor cells derived from rats that can be successfully grown in culture. The Tufts team, led by Qiaobing Xu, found that exosomes extracted from cultured PC12 cells at various stages of differentiation could drive MSCs to differentiate into neuron-like cells.

Exosomes are very small, hollow particles that a wide range of cells types secrete. These tiny vehicles contain proteins, RNA, and other small molecules, and serve as a vehicle for communication between cells. In the nervous system, exosomes guide the direction of nerve growth, and they control nerve connection and direct peripheral nerve regeneration.

Xu and his team showed that these exosomes contain microRNAs (miRNAs), which a small RNA molecules that regulate gene expression and are known to play a role in neuronal differentiation. They hypothesized that these miRNAs activate neuron-specific genes in the MSCs that receive them and this is the reason these cells begin their journey towards differentiating into neurons.

“In combination with synthetic nanoparticles, we may ultimately be able to use these identified miRNAs or proteins to make synthetic exosomes, thereby avoiding the need to use any kind of neural progenitor cell line to induce neuron growth,” said Xu.

This work was published in PLoS ONE 2015; 10(8): e135111 DOI: 10.1371/journal.pone.0135111.

Exosomes Work As Well As Stem Cells to Heal Stroke Damage

A German research team at the University of Duisburg-Essen has published a study in the latest issue of STEM CELLS Translational Medicine that shows tiny membrane-enclosed structures that travel between cells work as well as adult stem cells to help the brain recover from a stroke.

Extracellular vesicles (EVs), which are small, membrane-enclosed structures that pass between cells, which are also referred to as exosomes, were given to one group of stroke-impaired mice and adult stem cells from bone marrow to another. After monitoring these mice for four weeks, both groups experienced the same degree of neurological repair. Besides promoting brain recovery in the mice, the EVs also down-regulated the post-stroke immune responses and provided long-term neurological protection.

This study could lead to a new clinical treatment for ischemic strokes, since exosomes carry far fewer risks than adult stem cell transplants, according to the co-leaders of this research, neurologist Thorsten Doeppner, and Bernd Giebel, a transfusion medicine specialist.

“We predict that with stringent proof-of-concept strategies, it might be possible to translate this therapy from rodents to humans, since EVs are better suited to clinical use than stem cell transplants,” said Doeppner and Giebel.

Scientists think that EVs carry biological signals between cells and direct a wide range of processes. Exosomes are under a good deal of scientific investigation for the role they could play in cancer, infectious diseases, and neurological disorders.

Other studies have shown that exosome administration can be beneficial after a stroke, but the Duisburg-Essen study is the first to supply evidence through a side-by-side analysis that they act as a key agent in repairing the brain.

“The fact that intravenous EV delivery alone was enough to protect the post-stroke brain and help it recover highlights the clinical potential of EVs in future stroke treatment,” Doeppner and Giebel said.

This study included contributions from ten different researchers from Duisburg-Essen’s Department of Neurology and Institute for Transfusion Medicine. The study was supported by the university, Volkswagen Foundation and German Research Council.

“The current research, combined with the previous demonstration that EVs are well tolerated in men, suggests the potential for using this treatment in conjunction with clot-busting therapies for treatment of stroke,” said Anthony Atala, M.D., editor of STEM CELLS Translational Medicine and Director of the Wake Forest Institute for Regenerative Medicine.

Treating a Damaged Liver: Bone Marrow CD45 Cells are Superior to Mesenchymal Stem Cells

Scarring of the liver, otherwise known as “liver fibrosis” usually results when the liver is constantly assaulted by inflammation. Conventional treatments for liver fibrosis are usually not very effective. Therefore, mesenchymal stem cells (MSCs) is an attractive alternative due to their ability to suppress inflammation. Unfortunately, transplanted MSCs tend to show poor survival in the scarred liver, and they have an additional tendency to stimulate the formation of new scar tissue. These characteristics have bred skepticism among many investigators.

New work by Asok Mukhopadhyay and his colleagues from New Delhi, India has compared bone marrow (BM)-derived cells with MSCs as a treatment for liver fibrosis. They used CCl4 to induce liver fibrosis in laboratory mice. Then they treated liver-damaged mice with either BM-CD45 cells or fat-based MSCs.

Liver tests and tissue samples of both sets of mice clearly showed that the BM-CD45 cells did a much job attenuating liver scarring than did the fat derived MSCs. Interestingly, the anti-scarring capacity of the BM-CD45 cells was compromised by the presence of MSCs.

Why did the BM-CD45 cells do a better job? The bone marrow cells expressed rather high level expressions of matrix metalloproteinases. These enzymes chopped through scar tissue and also suppressed the hepatic stellate cells, which are responsible for making the scar tissue in liver. Apparently, the BM-CD45 cells induced the die off of the stellate cells. MSCs, however, released two growth factors (TGFβ and IGF-1) that are known to activate hepatic stellate cells, and promote the formation of scar tissue. As an added bonus, transplantation of CD45 cells led to functional improvement of the damaged liver, and this functional improvement seems to the result of improved liver repair and regeneration.  Thus transplanted MSCs were pro-scarring while transplanted BM-CD45 cells were pro-regeneration, at least in the liver.

To summarize the results of these experiments, BM-derived CD45 cells appear to be a superior candidate for the treatment of liver fibrosis. The structural and functional improvement of CCl4-damaged livers was substantially better in animals that received transplants of BM-CD45 than those who received fat-derived MSCs.