Culture Medium from Human Amniotic Membrane Mesenchymal Stem Cells Promotes Cell Survival and Blood Vessel Production in Damaged Rat Hearts

The laboratory of Massimiliono Gnecchi at the Fondazione IRCCS Policlinico San Matteo in Pavia, Italy has used the products of amniotic mesenchymal stem cells to treat heart attacks in laboratory rodents. The results are rather interesting.

In a paper published in the May 2015 edition of the journal Stem Cells Translational Medicine, Gnecchi and his colleagues grew human amniotic mesenchymal stem cells derived from amniotic membrane (hAMCs) in cell culture.

These cells were isolated from amniotic membrane donated by mothers who were undergoing Caesarian sections. The membranes were removed, and grown in standard culture media under standard conditions. Once the cells grew out, they were collected and grow in a medium known as DMEM (Dulbecco’s modified Eagle Medium). After the cells had grown for 36 hours, they culture medium was filtered, concentrated, and readied for use.

The first experiments included the use of this conditioned culture medium to treat H9c2 embryonic heart muscle cells with in culture and then expose the heart muscle cells to low oxygen conditions. Normally, low oxygen conditions kill heart muscle cells. However, the cells pre-treated with conditioned medium from hAMCs showed much more robust survival in low-oxygen conditions. This shows that molecules secreted by hAMCs had promote the survival of heart muscle cells.

Next, Gnecchi and his team used their conditioned medium to treat laboratory rats that had suffered heart attacks. Some of the rats were treated with conditioned culture medium from cultured skin cells and others with sterile saline. The culture medium was injected directly into the heart muscle.  The rats treated with conditioned medium from hAMCs showed far less cell death than the other rats. The rats treated with the hAMC-treated culture medium also had vastly denser concentrations of new blood vessels.

It is well-known that mesenchymal stem cells from many sources are filled with small vesicles known as exosomes that are loaded with healing molecules. Mesenchymal stem cells release these exosomes when they home to damaged tissues. The culture medium from the hAMCs were almost certainly filled with exosomes. The molecules released by these cells helped promote heart muscle cell survival in the oxygen-depleted heart, and induced the recruited large numbers of EPCs (endothelial progenitor cells), which established large numbers of new blood vessels. These new blood vessels gave oxygen to formerly depleted heart tissue and promoted heart healing. The size of the heart scar was smaller in the rats treated with hAMC-conditioned medium.

Unfortunately there were no measurement of cardiac function so we are not told if this treatment affected ejection fraction, or other physiological parameters. Nevertheless, this paper does show that exosomes from hAMCs do promote the production of blood vessels and cell survival.

Gene Therapy Increases Stem Cell Recruitment to Heart and Improves Heart Function

Data from a Phase 2 clinical trial is creating quite a stir in cardiology circles. According to the findings of this study, the single administration of a gene on a non-viral-derived plasmid improves cardiac structure, function, serum biomarkers and clinical status in patients with severe ischemic heart failure one year after treatment.

The results from the final 12-months of the Phase 2 STOP-HF clinical trial for the JVS-100 treatment were presented at the European Society of Cardiology – Heart Failure 2015 meeting by the developer of this technology: Juventas Therapeutics Inc. The founder of Juventas, Marc Penn, M.D., Ph.D., FACC, is also the medical officer and director of Cardiovascular Research and Cardiovascular Medicine Fellowship at Summa Health in Akron, Ohio. Dr. Penn presented the results of this randomized, double-blind, placebo-controlled STOP-HF trial, which included treatments on 93 patients at 16 different clinical centers in the United States.

“The results from STOP-HF demonstrate that a single administration of 30 mg of JVS-100 has the potential to improve cardiac function, structure, serum biomarkers and clinical status in a population with advanced chronic heart failure who are symptomatic and present with poor cardiac function,” stated Dr. Penn. “These findings combined with our deep understanding of SDF-1 biology will guide future clinical trials in which we plan to prospectively study the patient population that demonstrated the most pronounced response to JVS-100. In addition, we will further our understanding of JVS-100 by determining if a second administration of drug may enhance benefits beyond those we observed with a single administration.”

These study. some patients received a 30 mg dose of JVS-100 while others received a placebo.  Patients who received JVS-100 showed definite improvements 12 months after treatment.  The cardiac function and heart structure of the patients who received JVS-100 were far better than those who had received the placebo.  JVS-100-treated patients showed a changed in left ventricle ejection fraction of 3.5% relative to placebo, and left ventricular end-systolic volume of 8.5 ml over placebo.  When patients were asked to walk for six minutes, the JVS-100-treated patients were better than patients who had received the placebo.  Likewise, when patients were given the Minnesota Living with Heart Failure Questionnaire, the JVS-100-treat patients had a better score than those who had received the placebo.  Also, there were no unanticipated serious adverse events related to the drug reported for the study.

JVS-100 is a non-viral DNA plasmid gene therapy. Plasmids are small circles of DNA that are relatively easy to manipulate, grow and propagate in bacterial cells. In the case of the JV-100 treatment, the plasmid encodes a protein called stromal cell-derived factor 1 (SDF-1). SDF-1 is a naturally occurring signaling protein that recruits stem cells from bone marrow to the site of SDF-1 expression. SDF-1, therefore, acts as a stem cell recruitment factor that summons stem cells to the places where they are needed.

When JV-100 is delivered directly to a site of tissue injury, it induces the expression of SDF-1 protein into the local environment for a period of approximately three weeks. SDF-1 secretion creates a homing signal that recruits the body’s own stem cells to the site of injury to induce tissue repair and regeneration.

Juventas is developing JVS-100 into a treatment of advanced chronic cardiovascular disease, including heart failure and late stage peripheral artery disease.

These improvements in heart function are relatively modest.  Therefore, it is difficult to get too excited about these results.  Also, Alexey Bersenev, a umbilical cord stem cell researcher, noted that the primary end points (or goalposts) for this trial were not met, and that makes this an unsuccessful trial.  Despite this bad news, JV-100 does seem to be safe, and the theory seems sound, even if the results are more than a little underwhelming.

Stem Cell-Tweaking Drug Might Treat Osteoporosis

A research group from the Florida campus of The Scripps Research Institute (TSRI) has identified a new therapeutic approach that could promote the development of new bone-forming cells in patients suffering from bone loss.

The study was published in the journal Nature Communications, and it focused on a protein called PPARγ, which is a master regulator of fat, and the impact of this molecule on the fate of mesenchymal stem cells derived from bone marrow. Since these mesenchymal stem cells can differentiate into several different cell types, including fat, connective tissues, bone and cartilage. Consequently mesenchymal stem cells have a number of potentially important therapeutic applications.

A partial loss of PPARγ in a genetically modified mouse model led to increased bone formation. Could the use of drugs to inhibit PPARγ and potentially mimic that effect? This group combined a variety of structural biology approaches and then tried to design drugs that could fit PPARγ. This type of strategy is called “rational design,” and this yielded a new compound that could repress the biological activity of PPARγ.

The new drug, SR2595 (SR=Scripps Research), when applied to mesenchymal stem cells, significantly increased bone cell or osteoblast formation, a cell type known to form bone.

“These findings demonstrate for the first time a new therapeutic application for drugs targeting PPARy, which has been the focus of efforts to develop insulin sensitizers to treat type 2 diabetes,” said Patrick Griffin, chair of the Department of Molecular Therapeutics and director of the Translational Research Institute at Scripps Florida. “We have already demonstrated SR2595 has suitable properties for testing in mice; the next step is to perform an in-depth analysis of the drug’s efficacy in animal models of bone loss, aging, obesity and diabetes.”

In addition to identifying a new, potential therapeutic use for bone loss, this study may have even broader implications.

“Because PPARG is so closely related to several proteins with known roles in disease, we can potentially apply these structural insights to design new compounds for a variety of therapeutic applications,” said David P. Marciano, first author of the study, a recent graduate of TSRI’s PhD program and former member of the Griffin lab. “In addition, we now better understand how natural molecules in our bodies regulate metabolic and bone homeostasis, and how unwanted changes can underlie the pathogenesis of a disease.” Marciano will focus on this subject in his postdoctoral work in the Department of Genetics at Stanford University.

Experimental Drug Can Stimulate the Regrowth of Damaged Tissues

Research at Case Western Reserve, in collaboration with scientists from UT Southwestern Medical Center has identified yet another stem cell-activating drug. In animal models, this drug has helped mice regrow damaged liver, colon, and bone marrow tissue. The experimental drug examined in these experiments might open new possibilities for regenerative medicine. If clinical trials show that this drug therapy works in humans, it might save the lives of critically ill people with liver or colon disease or even some cancers.

This study was published in the journal Science. Even this work is exciting, this research is in the early stages and more work is necessary for the drug can be tested in people.

“We are very excited,” said co-author Sanford Markowitz, professor of cancer genetics at Case Western Reserve’s School of Medicine. “We have developed a drug that acts like a vitamin for tissue stem cells, stimulating their ability to repair tissues more quickly,” he added. “The drug heals damage in multiple tissues, which suggests to us that it may have applications in treating many diseases.”

This new drug is called SW033291. SW033291 works by inhibiting an enzyme with the formidable name of 15-hydroxyprostaglandin dehydrogenase, which is mercifully shortened to 15-PGDH. This enzyme degrades regulatory molecules called “prostaglandins.” One of these prostaglandins, known as prostaglandin E2, stimulates stem cell growth and differentiation. Inhibition of 15-PGDH increases the concentrations of prostaglandin E2 and stimulates the growth of tissue stem cells, which promotes healing.


Markowitz and his colleagues first showed that SW033291 inactivated 15-PGDH in a test tube. When they fed the drug to cells, it also inhibited 15-PGDH. Finally, they gave the drug to lab animals and showed that even in a living body, SW033291 inhibited 15-PGDH.

Does the drug augment healing? To determine this, Markowitz and others subjected mice to lethal doses of radiation, followed by a partial bone marrow transplant. Some of the mice were given SW033291 plus the bone marrow transplant while others received only the transplant. The mice that received SW033291 survived, while the others died.

In other studies, mice that had lost large amounts of blood were given SW033291, and mice given SW033291 recovered normal blood counts six days faster than mice that did not get the treatment.

Mice with an inflammatory disease called ulcerative colitis were given SW033291 and the drug “healed virtually all the ulcers in the animals’ colons and prevented colitis symptoms,” said the study’s authors.

“In mice where two-thirds of their livers had been removed surgically, SW033291 accelerated regrowth of new liver nearly twice as fast as normally happens without medication.” Additionally, SW033291 produced no adverse side effects.

Researchers who were not involved with the work said the study showed promise, but urged a heavy dose of caution. For example, Dusko Illic, a stem cell expert at Kings College London, said: “The drug seems to be too good to be true. We would have to be sure that nothing else was wrong with any organ in the body,” because if there were cancer cells present, the treatment would likely cause tumor cells to grow along with other tissue.

However, Ilaria Bellantuono, an expert in stem cell science and skeletal ageing at the University of Sheffield, said a key part of the drug’s promise could be in helping cancer patients, if it is proven safe. The “treatment has the potential of boosting patents’ resilience and improving their response to cancer treatment,” said Bellantuono. “This study is a proof of concept in mice and more experimental work is needed to verify the long-term safety of such an approach but it surely shows promise.”

The author of this study said that the first people to receive the experimental treatment in clinical trials would likely be patients who are receiving bone marrow transplants, have ulcerative colitis, or are undergoing liver surgery.

A Small RNA that Increases Bone Formation in Osteoporotic Bone-Making Cells

We normally think of bone as a very static tissue that does not change very much. However bone is actually a very dynamic tissue is constantly being remodeled in response to the needs of the organism. Bone remodeling is mediated by two different types of cells: osteoblasts that build bone and osteoclasts that resorb bone. Osteoblasts are derived from mesenchymal stem cells in the stroma of the bone marrow. The differentiation of mesenchymal stem cells into osteoblasts is mediated by molecules made by bone cells when bone is damaged. Osteoclasts come from pre-osteoclast cells that are monocyte-derived cells that fuse into multinucleate osteoclasts in response to the death of osteocytes (bone cells).

In healthy bone, osteocytes secrete a molecule called sclerostin, which prevents any new bone deposition. A break in bone causes the death of osteocytes near the site of the break, and the nearby osteocytes stop secreting sclerostin and start producing growth factors, nitric oxide and prostaglandins.

Bone deposition

The lining cells of the bone marrow cavity detach and fuse with blood vessels. The mesenchymal stromal cells, under influence from IL-1, become pre-osteoblasts, and they start to secrete M-CSF, which prepares the pre-osteoclasts to fuse and become multinucleate osteoclasts. Pre-osteoclasts then express a molecule called RANKL, which binds to the RANK receptor on the surface of pre-osteoclasts and this induces them to fuse, and become mature osteoclasts. The osteoclasts secrete acid and cathepsin K to dissolve the damaged bone. The osteoclasts stop eating bone when the pre-osteoblasts mature into full-fledged osteoblasts that stop making RANKL and start making OPG, which binds to RANK, but does not activate it. Without this stimulation, the osteoclasts die. Then the osteoblasts divide, fill the cavity made by the now-deceased osteoclasts, and remake the bone. Some of the osteoblasts become entrapped in the bone matrix and become osteocytes. The bone takes several months to remineralize and 3-4 years to completely remineralize.  See here for a video of this.

Bone resorption-deposition

If there is a relative increase in bone resportion relative to bone deposition, the result is fragile, poorly mineralized bones, and this condition is known as osteoporosis. Decreased bone mass and bone strength causes an increased incidence of bone fractures, which often leads to further disability and early mortality. Bone healing is also impaired.

To treat osteoporosis, clinicians usually prescribe anti-resorptive agents that exert their effect by decreasing the rate of bone resorption. This strategy, however, has drawbacks, since as noted above, bone deposition relies on bone resorption. Inhibition of bone resorption also inhibits bone deposition, and bone tends to remain static and heal poorly.

A new paper has examined osteoporosis from the perspective of osteoblasts. It has been well established that in osteoblasts function is diminished in osteoporotic patients. Therefore increase osteoblast function is of chief interest. Work from the laboratories of Jihua Chen and Yan Jin from the Fourth Medical University has shown that a miniature RNA molecule called miR-26a plays a critical role in modulating bone formation during osteoporosis. Chen and Jin and others discovered that miR-26a treatment of mesenchymal stem cells effectively improved the osteogenic differentiation capability of these mesenchymal stem cells. In these experiments, they isolated mesenchymal stem cells from female mice that had their ovaries removed. Such mice are prone to undergo osteoporosis because they lack the hormone estrogen that stimulates osteoblast function. When these stem cells were treated with MiR-26a, they increased their bone-making capacities by in culture and when injected into live mice.

Further work showed that MiR-26a directly targets a gene called Tob1. Tob1 negatively regulates the BMP/Smad signaling pathway, and MiR-26a binds to the rear mRNA (3′-untranslated region) of Tob1, and prevents Tob1 translation.

These findings indicate that miR-26a is a potentially promising therapeutic candidate to enhance bone formation in order to treat osteoporosis and to promote bone regeneration in osteoporotic fracture healing.

For the article, go here.

Cartilage-Making Stem Cells from Joints

Chiharo Akazawa from the Tokyo Medical and Dental University and his colleagues have tested two types of mesenchymal stem cells from human patients for their ability to make bone, cartilage, or fat. Their tests illustrated what has been shown several time before; mesenchymal stem cells tend to differentiate into the tissues that most closely resemble their tissue of origin.

Akazawa and his colleagues previously discovered a way to effectively isolated mesenchymal stem cells from bone marrow, which is no small feat because mesenchymal stem cells (MSCs) are a minority of the cells in bone marrow (Mabuchi and others (2013), Stem Cell Reports 1: 152-165). In a recent paper in the journal PLoS ONE, Akazawa and others used this technique to isolate MSCs from bone marrow and from synovial membrane – the fluid-filled sac that encases joints. In large joints, this synovium is large and called a “bursa.”.

In culture, the bone marrow-derived MSCs from several different human donors showed a marked tendency to form bone, but they did not make good cartilage or fat. The synovial MSCs, on the other hand, did not do so well at making bone, but made very good fat and cartilage. These differentiation trends were observed in MSCs culture for several different human donors. All cells were collected during arthroscopic surgery.

Since the synovial membrane of patients suffering from osteoarthritis undergoes, increased cell division, it is possible that the number of stem cells also increases. Alternatively, using MSCs from healthy donors who do not have arthritis may be even more preferable. Nevertheless, MSCs from synovial membrane show excellent cartilage-making potential and they may be a suitable source of cell for cartilage regeneration.

Stem Cell Treatment Improves the Skin Quality of Children With inherited Skin Blistering Disease

A new stem cell-based therapy has shown some very promising results. This therapy was designed to treat a rare and debilitating skin condition that affects children, for which no cure currently exists. This cell-based therapy provided pain relief and reduced the severity of the skin condition for patients who participated in the clinical trial.

The clinical trial was led by scientists at King’s College London, who collaborated with researchers from the Great Ormond Street Hospital (GOSH). They recruited 10 children afflicted with a disease called recessive dystrophic epidermolysis bullosa (RDEB).

RDEB is a painful skin disease in which very minor skin injury leads to blisters and wounds that tend to heal very slowly or not at all. The skin of RDEB patients is quite fragile and it tends to scar, develops contractures, and is also prone to life-threatening skin cancers.

Dystrophic epidermolysis bullosa
Dystrophic epidermolysis bullosa

This clinical trial, known as the EBSTEM trial, is a The Phase I/II trial whose results were published early online in the Journal of Investigative Dermatology. This study was designed to test the safety of infusions of stem cells and to determine if this treatment could help diminish the severity of the disease and improve quality of life for these patients.

During the first six months of the trial, participants were given three infusions of bone marrow- derived mesenchymal stromal cells from unrelated donors. Mesenchymal stem cells (MSCs) have been shown to home to wounded tissue and mediate wound healing in several previous studies. Although these infused stem cells do not survive permanently, they may still deliver therapeutic benefits.

The treated children were then monitored for a year after these cell infusions. Several different clinical tests failed to reveal any serious adverse effects in patients as a result of the stem cell treatment. When the pain levels of patients were measured, patients consistently reported lower pain levels after the treatment than before the treatment. Also the severity of their disease was also reported to have lessened following the stem cell infusions. Parents of these children reported better wound healing in their children and they also showed less skin redness and fewer blisters.

Overall, the outcomes of the trial are promising. However, this is an unblinded study of participants and may, therefore, contain positive biases in the way the information is reported. In interviews with families, participants reported a range of benefits from sleeping better, to the parents being able to return to work part-time because their children required less intense care. In fact, one family was actually able to plan their first vacation together.

Thus, further work is required to better understand the mechanisms that helped patients improve. Did the stem cells trigger the production of a growth factors and immune system regulators? Did these secreted compounds stimulate wound healing and reduce inflammation in the skin? Or did the presence of the cells somehow improve skin quality? Further studies are also required to confirm the efficacy of the treatment and establish the optimal dose of cells to give RDEB patients.