Phase I Clinical Trial of Fat-Based Mesenchymal Stem Cells for Severe Osteoarthritis

In the July 2016 edition of the journal Stem Cells Translational Medicine, a report has been published that lays out the results of a phase I clinical trial that used mesenchymal stem cells from a patient’s own fat tissues to treat osteoarthritis of the knee.  This study was not placebo controlled, but did examine the effects of escalated doses on the patient.  The main  investigator for this trial was Dr. Christian Jorgensen from Lapeyronie University Hospital in Montpellier, France.

Osteoarthritis (OA) is the most common musculoskeletal condition in adults and it can cause a good deal of pain and disability.

Joints like the knee consist of a junction between two or more bones.  The ends of these bones are capped by layer of cartilage called “hyaline cartilage” that serves as a shock absorber.  Larger joints like the knee, shoulder, and hip are encased in a sac called the “bursa” that is filled with lubricating synovial fluid.


OA involves damage and/or destruction of the cartilage caps at the ends of long bones, and erosion and ultimately permanent changes in the structure of bone that underlies the cartilage at the end of the bone. The knee loses its shock absorbers and lubricators and becomes a grinding, inflamed, painful caricature of its former self.

To treat OA, most orthopedic surgeons will replace the damaged knee with an artificial knee that is attached the upper (femur) and lower (tibia and fibula) bones of the leg.  This procedure, arthroplasty, reconstructs the knee with artificial materials that form synthetic joints.  Alternatively, some enterprising physicians have tried to use stem cells from bone marrow to repair eroded cartilage in the knees of OA patients.  Christopher Centeno and his colleagues at his clinic near Denver, CO and affiliated sites have pioneered procedures for OA patients.  However, Dr. Centeno remains skeptical of the ability of stem cells from fat to treat patients with OA.

In animal studies, OA of the knee can be induced by injected tissue-destroying enzymes.  If laboratory mice that received injectionof these enzymes into their knees are then treated with fat-based mesenchymal stem cells, the effects and symptoms of OA do not appear (ter Huurne M, et al. Arthritis Rheum 2012; 64:3604-3613).  In another study in rabbits, injections of 2-6 million fat-derived mesenchymal stem cells into the knee-joint of rabbits suffering from OA improved cartilage health and inhibited cartilage degradation.  These administered cells also reduced inflammation in the knee (Desando G., et al., Arthritis Res Ther 2013; 15:R22).  Therefore, fat-based mesenchymal stem seem to have some ability to ameliorate the effects and consequences of OA, at least in preclinical studies.  This trial is the beginnings of what will hopefully be a series of experiments that will assess the ability (or inability) to treat OA patients.

18 patients were enrolled from an initial pool of 48 candidates who all suffered from severe, symptomatic OA of the knee.  Six patients received 2 million mesenchymal stem cells isolated from their own fat, 6 others received ten million mesenchymal stem cells isolated from their own fat, and the final group of 6 OA patients received 50 million mesenchymal stem cells isolated from their own fat tissues.  These mesenchymal stem cells were isolated from the patient’s fat that was collected by means of liposuction.  The fat was then processed by means of a standard protocol that is used to isolated mesenchymal stem cells from human fat (see Bura A, et al., Cytotherapy 2014; 16:245-257).  All patients received their stem cells by means of injection into the knee-joint (inter-articular injections).

Because this is a Phase I clinical trial, assessing the safety of the procedure is one of the main goals of this study.  No adverse effects were associated with either the liposuction or the interarticular injections.  The article even states: “Laboratory tests, vital signs and electrocardiograms indicated no local or systemic safety concerns.”. Four patients experienced slight knee pain and joint effusion that either resolved by itself or with treatment with a nonsteroidal antinflammatory drug (think ibuprofen).  Therefore it seems fair to conclude that this procedure seems safe, but a larger, placebo-controlled study is still required to confirm this.

As to the patient’s clinical outcomes, 17 of the 18 patients elected to forego total knee replacement.  All patients showed improvement in pain and knee functionality at 1 week, 3 months and 6 months after the procedure.  However, only the low-dose group showed improvements that were statistically significant.

WOMAC pain and function improvement during the study (WOMAC = Western Ontario and McMaster Universities Arthritis Index)

WOMAC pain and function improvement during the study. Abbreviation: WOMAC, Western Ontario and McMaster Universities Arthritis Index.

Seven of the patients treated in Germany (11 patients were treated in France and 7 were treated in Germany) were also examined with Magnetic Resonance Imaging (MRI) before and 4 months after the procedure.  Six of the seven patients showed what could be interpreted as improvements in cartilage.

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dGEMRIC and T1rho magnetic resonance imaging (MRI) of selected patients. The graphs on the left show the dGEMRIC (n = 6) and T1rho (n = 5) values before and 4 months after cell therapy. Increasing dGEmRIC and decreasing T1rho values are each known to correspond to increasing glycosaminoglycan/proteoglycan content and thus improved cartilage condition. On the right, the corresponding dGEMRIC and T1rho maps are shown as a color-coded overlay on an anatomical MRI for a patient receiving a low cell dose. The observed values in the cartilage change in the time course can be easily seen and correspond to an increase in cartilage condition. Abbreviation: dGEMRIC, delayed gadolinium-enhanced magnetic resonance imaging of cartilage.

Tissue biopsies of 11 of the 18 patients revealed an absence of significant inflammation, but some patients (4-5) showed signs of weak or moderate inflammation.  One patient showed what seemed to be a sheet of MSC cells on the surface of the cartilage.

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Histologic findings. (A): Vascular congestion and weak lymphocytic infiltrate of the synovial (case 8) (magnification, ×50). (B): Osteoarthritic cartilage OARSI grade >3 (case 4) (×25). (C): Toluidine blue staining (case 2) (magnification, ×100). (D): Stem cell stroma shows an Alcian blue depleted matrix compared with the strong staining of osteoarthritic cartilage (case 2) (magnification, ×100). (E): Weak PS100 staining of possible stem cells on the cartilage surface and strong PS100 staining of chondrocytes (case 2) (magnification, ×100). Abbreviations: OARSI, Osteoarthritis Research Society International.

The primary outcome of this study – the safety of interarticular injections of fat0-based mesenchymal stem cells – seems to have been satisfied.  This is similar to the safety profiles of such cells in clinical trials that have used fat-based mesenchymal stem cells to treat fistulae in inflammatory bowel disease (Bura A, et al., Cytotherapy 2014; 16: 245-257) or critical limb ischemia (Lee WY and others, Stem Cells 2013; 31:2575-2581).  Also, patients showed improvements in pain and functionality.  Even though there was no placebo in this study, a double-blinded, placebo-controlled study that examined the use of efficacy of interarticular hyaluronic acid injections showed a smaller decreased in pain score that what was observed in this case (22.9 ± 1.4 vs 30.7 ± 10.7).  It is doubtful that the injected mesenchymal stem cells made much cartilage but instead quelled inflammation and stimulated resident stem cell populations to repair damage in the knee.

This study is small and is not placebo controlled, however, the hopeful results do warrant a larger, phase 1/2 placebo-controlled study that is apparently already underway.

An even more intriguing project might be to prime the isolated mesenchymal stem cells to make cartilage and then use live fluoroscopy to overlay the cells on the actual cartilage lesions.  While this is a more exacting procedure, it is the way Centeno and his group are using stem cells to treat their patients, and a true head-to-head study of the efficacy of fat-based mesenchymal stem cells versus bone marrow-based mesenchymal stem cells would be immensely useful.

Pancreatic Cancers Treated Better By Breaking Up Scar Tissue

Despite advances in cancer treatment, tumors of the pancreas remain among the most difficult to treat. To date, pancreatic cancers remain largely resistant to immune-based therapies, despite the successes of immunotherapies in treating lung cancers and melanomas.

A new study from Washington University School of Medicine in St. Louis that was published in the journal Nature Medicine, has shown that immunotherapy against pancreatic cancer can shrink these tumors if they are given in combination with drugs that break up the fibrous scar tissue in these tumors.

Physicians at Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital are using the strength of these data to conduct a phase 1 clinical trial in patients with advanced pancreatic cancer. This clinical trial will test the safety of this drug combination when given alongside standard chemotherapy.

“Pancreatic tumors are notoriously unresponsive to both conventional chemotherapy and newer forms of immunotherapeutics,” said senior author David G. DeNardo, PhD, an assistant professor of medicine. “We suspect that the fibrous environment of the tumor that is typical of pancreatic cancer may be responsible for the poor response to immune therapies that have been effective in other types of cancer.”

Pancreatic cancers are unusual among cancers since they characteristically consist of large swaths of scar tissue. These balls of fibrous tissue that surround the tumor create a protective environment for cancer cells. These scar tissue-based capsules prevent the immune system accessing the tumor cells and also limit the exposure of these tumors to chemotherapies that have been administered through the bloodstream. DeNardo and his colleagues used a mouse model of pancreatic cancer to determine if disrupting these fibrous capsules could sensitize pancreatic tumors to chemotherapy regimens.

Pancreatic tumors are surrounded by a protective "nest" made of fibrotic scar tissue and the cells that manufacture it (red). A new study demonstrates that disrupting this fibrous tissue makes immune therapy and chemotherapy more effective in attacking tumors of the pancreas. (Image: DeNardo Lab)
Pancreatic tumors are surrounded by a protective “nest” made of fibrotic scar tissue and the cells that manufacture it (red). A new study demonstrates that disrupting this fibrous tissue makes immune therapy and chemotherapy more effective in attacking tumors of the pancreas. (Image: DeNardo Lab)

“Proteins called focal adhesion kinases are known to be involved in the formation of fibrous tissue in many diseases, not just cancer,” DeNardo said. “So we hypothesized that blocking this pathway might diminish fibrosis and immunosuppression in pancreatic cancer.”

Focal adhesion kinase (FAK) is a protein (encoded by the PTKs gene) that controls cell adhesion and cell motility. Inhibiting FAK activity in breast cancer cells makes them less likely to spread to other organs (see Chan, K.T., et al. 2009. J. Cell Biol. doi:10.1083/jcb.200809110). Small molecules have been designed that can readily inhibit FAK, and DeNardo and his colleagues used FAK inhibitors against pancreatic cancer in combination with immunotherapy.

Focal Adhesion Kinase

In their mouse study, an investigational FAK inhibitor was administered to mice in combination with a clinically approved immune therapy that activates the patient’s own T-cells so that they can effectively attack tumor cells.

Mice that had pancreatic cancer survived no longer than two months when given either a FAK inhibitor or immune therapy alone. If the FAK inhibitors were added to standard chemotherapy, the tumor response improved over chemotherapy alone. However, the three-drug combination that consisted of FAK inhibitors, immune therapy and chemotherapy, displayed the best outcomes in laboratory studies and more than tripled survival times in some mice. Some were still alive without evidence of progressing disease at six months after treatment and beyond.

The success of this mouse study provided a strong rationale for testing this drug combination in patients with advanced pancreatic cancer, according to oncologist Andrea Wang-Gillam, MD, PhD, an associate professor of medicine, who was involved with this research.

“This trial is one of about a dozen we are conducting specifically for pancreatic cancer at Washington University,” she said. “We hope to improve outcomes for these patients, especially since survival with metastatic pancreatic cancer is typically only six months to a year. The advantage of our three-pronged approach is that we are attacking the cancer in multiple ways, breaking up the fibers of the tumor microenvironment so that more immune cells and more of the chemotherapy drug can attack the tumor.”

Mesenchymal Stem Cells from Bone Marrow Improve Liver Function and Reduce Liver Scarring in Patients with Alcoholic Cirrhosis

Dr Soon Koo Baik from the Yonsei University Wonju College of Medicine, and Dr. Si Hyun Bae from The Catholic University of Korea and their colleagues have conducted an important phase 2 clinical trial that tests the ability of mesenchymal stem cells from bone marrow to treat cirrhosis of the liver. In this trial, seventy-two patients who had established cirrhosis of the liver participated in a multicenter, randomized, open-label, phase 2 trial (published in the journal Hepatology, DOI:10.1002/hep.28693).

The liver is a hugely important organ. Not only is it the largest internal organ in our bodies, but it serves as the main metabolic factory of the body because of the outsized role it plays in metabolism. The liver takes up and stores and processes nutrients from food. Once it processes fats, sugars, and amino acids, the liver delivers them to the rest of the body. The liver also makes new proteins, such as clotting and immune factors, produces bile, which helps the body absorb fats, cholesterol, and fat-soluble vitamins, and removes waste products the kidneys cannot remove, such as fats, cholesterol, toxins, and medications.

The condition known as cirrhosis is a condition in which the liver gradually deteriorates and becomes unable to function normally due to chronic, or long-lasting, injury. The accumulation of scar tissue in the liver is typically slow and gradual and as scar tissue replaces more healthy liver tissue, the liver begins to fail. Scar tissue also partially blocks the flow of blood through the liver. Chronic liver failure (also known as end-stage liver disease) culminates in the inability of the liver to perform important functions. Since the liver is an organ that have a good deal of regenerative ability, end-stage liver disease essentially becomes so damaged that it cannot effectively replace damaged cells.

Cirrhosis is most commonly called by chronic alcoholism, but so can chronic viral infections by viruses like hepatitis B virus and hepatitis C virus.  Additionally, particular genetic diseases can also cause cirrhosis in children or young adults.

Mesenchymal stem cells have the ability to secrete cocktails of pro-healing molecules that might be able to support the growth and survival of liver cells. A variety of experiments in animals have established that the administration of mesenchymal stem cells (MSCs) from bone marrow (Truong, NH, et al., Stem Cells Int. 2016;2016:5720413. doi: 10.1155/2016/5720413; Almeida-Porada G, et al., Exp Hematol. 2010;38:574–580; Berardis S, et al., World J Gastroenterol. 2015;21:742–758), and other sources (De Ugarte DA, et al., Cells Tissues Organs. 2003;174:101–109; in ‘t Anker PS, etr al., Haematologica. 2003;88:845–852; Lee OK, et al., Blood. 2004;103:1669–1675) can decrease inflammation within the liver, inhibit the death of liver cells and promote their survival, and promote the regeneration of residential liver cells.

In clinical trials, administration of MSCs to cirrhosis patients has established the safety of MSC-based treatments (Amin MA, et al., Clin Transplant. 2013;27:607–612; El-Ansary M, et al., Stem Cell Rev. 2012;8:972–981; Jang YO, et al., Liver Int. 2014;34:33–41; Kharaziha P, et al., Eur J Gastroenterol Hepatol. 2009;21:1199–1205; Mohamadnejad M, et al., Arch Iran Med. 2007;10:459–466). Unfortunately, the design of these trials involved the mixing of patients with alcohol-based cirrhosis, viral-based cirrhosis, and other types of cirrhosis. Therefore, it is impossible to draw any conclusions about the efficacy of MSC transplantations on the basis of these trials. However, one trial, by Jang, et al, examined the effect of MSCs from bone marrow in patients with alcoholic cirrhosis. After 11 patients received MSC implantations, improvements in liver tissue architecture were observed in 6/11 patients, and 10 patients showed recovery of liver function. These 10 patients had decreased expression of molecules that induce scarring in the liver (i.e. TGF-β1, collagen type I, and α-smooth muscle actin). Significantly, Jang and others observed these improvements in the absence of significant complications or side effects during the study period. On the strength of these results, a larger phase 2 study is certainly warranted (see F. Ezquer, et al., World J Gastroenterol. 2016 Jan 7; 22(1): 24–36).

In this Bak and Bae clinical trials, 72 patients were randomly assigned to three groups that consisted of a control group and two autologous bone marrow-based MSC groups that underwent either one-time or two-time hepatic arterial injections of 5 × 10[7] MSCs, 30 days after bone marrow aspiration. All patients also underwent a follow-up biopsy 6 months after enrollment and adverse events were monitored for 12 months.

The primary endpoint in this study was the improvement in the amount of scar tissue in biopsies (as assayed by Picrosirius-red staining). The secondary endpoints included liver function tests, a measure of the severity of cirrhosis called the Child-Pugh score, and another score called the Model for End-stage Liver Disease (MELD) score. The outcomes were analyzed by per-protocol analysis.

When it comes to the amount of scar tissue in the patient’s livers, patients that received one-time and two-time bone marrow-based MSC administrations, showed 25% (19.5±9.5% vs. 14.5±7.1%) and 37% (21.1±8.9% vs. 13.2±6.7%) reductions in the amount of liver scar after MSC administration, respectively (P0.05). The Child-Pugh scores of both BM-MSC groups (one-time: 7.6±1.0 vs. 6.3±1.3 and two-time: 7.8±1.2 vs. 6.8±1.6) were also significantly improved following BM-MSC transplantation (P<0.05) compared to the control group that did not receive MSCs. Most significantly, perhaps, is that the proportion of patients with adverse events did not differ among the three groups.

From this larger phase 2 study, it seems that transplantation of a patient’s own bone marrow-based MSCs can safely improve the degree of scarring in the liver of cirrhosis patients and also improve liver function in patients with alcoholic cirrhosis. This study seems to confirm what was observed in preclinical studies in laboratory animals and extends what was observed in the phase 1 studies. While more work is certainly required, these results are certainly hopeful.

New Autoimmune Treatment Removes Rogue Immune Cells Without Suppressing the Immune System

New preclinical experiments by scientists at the University of Pennsylvania have established that genetically engineered T-cells can drive severe autoimmune diseases into remission without suppressing the patient’s immune system. If the principles applied in this study also prove to be true in human patients, they can potentially revolutionize the treatment of autoimmune diseases.

Autoimmune diseases result when your immune system recognizes your own cells and tissues as foreign and mounts and immune response against them. Autoimmune diseases like systemic lupus erythematosus (also known as “lupus”), rheumatoid arthritis, scleroderma, multiple sclerosis, celiac disease, Sjögren’s syndrome, polymyalgia rheumatic, or ankylosing spondylitis can deeply affect the health of an individual and can also cause large amounts of tissue damage.

Treatment of autoimmune diseases usually requires high doses of drugs that suppress the immune system, such as corticosteroids, or various types of biological agents that also cause a host of undesirable side effects.

This new study, however, by scientists from the Perelman School of Medicine at the University of Pennsylvania have adapted an already-existing technology to remove the subset of antibody-making cells that cause the autoimmune disease. This strategy removes the rogue immune cells without harming the rest of the immune system.

In these experiments, the University of Pennsylvania team examined an autoimmune disease called pemphigus vulgaris or PV. PV results when the immune system recognizes a protein called desmoglein-3 (Dsg3) as foreign and attacks it. Dsg3 helps form attachment sites called “desmosomes” that normally adhere skin cells together to form tight, tough sheets. Desmosomes are also found between epithelial cells, myocardial cells, and other cell types.


Current therapies for autoimmune diseases like PV use drugs like prednisone and rituximab, which suppress large parts of the immune system. Consequently, prednisone and rituximab can leave patients vulnerable to potentially fatal opportunistic infections and cancers.

To treat PV, University of Pennsylvania researcher Aimee Payne and her colleagues used a mouse version of PV that is fatal in mice. Their experimental treatment, however, successfully treated this otherwise fatal autoimmune disease without causing any unintended side effects, which might harm healthy tissue. The results from these experiments were published in the journal Science.

“This is a powerful strategy for targeting just autoimmune cells and sparing the good immune cells that protect us from infection,” said Dr Payne, who serves as the Albert M. Kligman Associate Professor of Dermatology at the Perelman School of Medicine.

In collaboration with Dr. Michael Milone, assistant professor of Pathology and Laboratory Medicine, Payne and her colleagues adapted the Chimeric Antigen Receptor T-Cell (CART-Cell) technology that is being successfully used to experimentally treat malignant cells in certain leukemias and lymphomas. “Our study effectively opens up the application of this anti-cancer technology to the treatment of a much wider range of diseases, including autoimmunity and transplant rejection,” Milone said.

Aimee Payne, Michael Milone, Christoph Ellebrecht, left to right
Aimee Payne, Michael Milone, Christoph Ellebrecht, left to right

CART-Cells are T-lymphocytes that have been extracted from the peripheral blood of cancer patients and then genetically engineered to express a receptor that specifically recognizes a protein on the surface of tumor cells. These chimeric antigen receptor (CAR)-expressing cytotoxic T-lymphocytes have the ability to recognize and destroy tumor cells, which shrinks the tumor and potentially cures the patient.

CAAR technology

The core concepts behind CAR T-cells were first described in the late 1980s. Unfortunately, technical challenges prevented the development of this technology until later. However, since 2011, experimental CAR T cell treatments for B cell leukemias and lymphomas have been successful in some patients for whom all standard therapies had failed.

Antibody-producing B-lymphocytes or B-cells can also cause autoimmunity. A few years ago, a postdoctoral researcher in Payne’s laboratory named Dr. Christoph T. Ellebrecht came upon CAR T cell technology as a potential strategy for deleting rogue B-cells that make antibodies against a patient’s own tissues. Soon Payne and her team had teamed up with Milone’s, which studies CAR T cell technology. Their goal was to find a new way to treat autoimmune diseases.

“We thought we could adapt this technology that’s really good at killing all B cells in the body to target specifically the B cells that make antibodies that cause autoimmune disease,” said Milone.

“Targeting just the cells that cause autoimmunity has been the ultimate goal for therapy in this field,” noted Payne.

Because an excellent mouse model existed for PV, Payne and Milone decided to examine pemphigus vulgaris. Since PV consists of a patient’s antibodies attacking those molecules that normally keep skin cells together, it can cause extensive skin blistering and is almost always fatal. PV is treatable with broadly immunosuppressive drugs such as prednisone, mycophenolate mofetil, and rituximab.

However, to treat PV without causing broad immunosuppression, the Penn team designed an artificial CAR-type receptor that would home the patient’s own genetically engineered T-cells exclusively to those B-cells that produce harmful anti-Dsg3 antibodies.

Payne and Milone and their colleagues developed a “chimeric autoantibody receptor,” or CAAR, that displays fragments of the Dsg3 on their cell surfaces. Since the Dsg3 protein is the target of the PV-causing B-cells, the CAAR acts as a lure for the rogue B cells that target Dsg3. The CAAR effectively brings the cells into fatal contact with the therapeutic T cells.

After testing a battery of different cultured, genetically engineered T-cells, these teams eventually found a CAAR that worked well in cell culture and enabled host T cells to efficiently destroy anti-desmoglein-producing B-cells. These cultured cells worked so well that they even killed B-cells isolated from PV patients. The engineered CAAR T cells also performed successfully in a mouse model of PV. The CAAR T-cell effectively killed desmoglein-specific B cells, prevented blistering, and other manifestations of autoimmunity in the animals. “We were able to show that the treatment killed all the Dsg3-specific B cells, a proof of concept that this approach works,” Payne said.

Not only were these treatments devoid of undesirable side effects in the laboratory mice they studied, but they maintained their potency despite the presence of high levels of anti-Dsg3 antibodies that might have swamped out their CAARs.

Next, Payne plans to test her treatment in dogs, which can also develop PV and often die from it. “If we can use this technology to cure PV safely in dogs, it would be a breakthrough for veterinary medicine, and would hopefully pave the way for trials of this therapy in human pemphigus patients,” Payne said.

Penn scientists would also like to develop applications of CAAR T cell technology for other types of autoimmunity. Organ transplant rejection, which is also related to autoimmunity, complicates organ transplants, and normally requires long-term immunosuppressive drug therapy, may also be treatable with CAAR T cell technology.

“If you can identify a specific marker of a B cell that you want to target, then in principle this strategy can work,” Payne said.