Keeping Stem Cells in Place for Better Bone Healing

Stem cell-based treatments for bone injuries have made some remarkable strides in the past few years. Unfortunately, a common pitfall of bone-making stem cells is the tendency of these cells to wander away from the site of injury. This “wander lust” among stem cells can inhibit healing and reduce stem cell efficacy. How to keep the cells home? The answer seems to be encasing them in a water-retaining gel that keeps them in place, but degrades once the cells have done their job.

Cartilage production has benefitted from the use of these so-called “hydrogels” that encase cells and keep them at the site of injury. However, hydrogels have yet to be tried with bone regeneration.

Assistant Professor of Biomedical Engineering, Danielle Benoit, said, “For example, we should not be able to pinpoint repairs within the periosteum, or outer membrane of bone material.”

The hydrogels used by Benoit and her colleagues mimic the body’s natural tissues, but they also are biodegradable and disappear before the immune system recognizes them as foreign substances.

Benoit believes that the special properties of hydrogels could direct bone-making mesenchymal stem cells to make bone mad repair bone fractures at the site of injury, and then leave the site once the cells have completed their mission.

In previous work (M.D. Hoffman, and others, Biomaterials, 34 (35) (2013), pp. 8887–8898), ) Benoit and her co-workers transplanted hydrogel-encased stem cells onto the surface of mouse bone grafts. However, Benoit’s group not only closely observed the behavior of these implanted cells in the animal, but also in culture dishes outside the animal.

In these experiments, Michael Hoffman and others grafted decellularized bone into the long bones of mice. Because these grafts had all their living material removed, all the bone healing that occurred would be solely due to the implanted stem cells.

Then stem cells that had been genetically engineered to glow a fluorescent green color. The bone material was also coated with hydrogels to keep the stem cells at the site of the bone graft. Then Benoit’s group monitored the bone regeneration process to determine the loss or retention of stem cells at the site of the bone graft in the presence or absence of hydrogels. They used the amount of fluorescence to ascertain the number of cells present at the site of repair. Strangely, Benoit and her colleagues were unable to demonstrate the ability of the PEG hydrogels to control spatiotemporal MSC localization. Therefore, it seemed to be due to the hydrogels and their properties.

As it turns out, depending on how the hydrogels are made, they have different rates of degradation. Benoit, therefore, decided to synthesize gel fibers that underwent biodegradation at different rates. Once the hydrogel began to experience degradation, the spaces between the hydrogels fibers increased and this allowed cells to exit the hydrogel.

In a series of experiments Hoffman, Van Hover and Benoit showed that the faster the rates of hydrogels degradation, the poorer the retention of the cells within the hydrogels. Retention rates were directly proportional to the hydrogels rate of degradation, since longer-lived hydrogels showed higher levels of cell retention and shorter-lived gels showed shorter retention times. In the words of Benoit and her colleagues: “cell localization at allograft surfaces decays in close agreement with network degradation kinetics both in vitro and in vivo.

Such hydrogels with variable degradation rates show promise in not only in bone regeneration, but also in heart attacks in which the initiation of healing might be instigated without invasive surgical procedures that can greatly weaken an already incredibly sick patient.

Mesoblast Clinical Trial Shows Stem Cell Treatments Improve Glycemic Control in Type 2 Diabetics

Mesoblast Ltd has announced the results of their clinical trial in type 2 diabetics at the annual meeting of the American Diabetes Association.

Mesoblast has developed a proprietary adult stem cell they call a mesenchymal precursor cell or MPC, which they are attempting show can be used as an “off the shelf” medical product. MPCs seem to act like immature mesenchymal stem cells that can modulate the immune response and have greater flexibility.

In this trial, Mesoblast was banking of the ability of administered MPCs to suppress inflammation. Type 2 diabetes results from an insensitivity of tissues to secreted insulin. Consequently, cells do not receive enough of the insulin signal to take up sugar and make protein, glycogen, and fat. Another prominent feature of type 2 diabetes is chronic, low-level inflammation, which is largely due to the chronically high blood glucose concentrations that damages cells, blood vessels, nerves, and connective tissue. By treating type 2 diabetics with MPCs, Mesoblast was hoping to ascertain the ability of MPCs to quell chronic inflammation.

The trial was conducted across 18 sites in the US. 61 patients with type 2 diabetes received either one intravenous infusion of 0.3, 1.0 or 2.0 millions MPCs per kilogram body weight over 12 weeks. One group of patients were given a placebo. Patients had suffered from diabetes an average of 10 years and had poor control with the drug metformin (Glucophage), which is one of the most widely-used drugs for type 2 diabetes.

The results were largely positive:
When it comes to safety, there were no safety issues observed during the 12-week study period. The MPC cell infusions were well tolerated (with a maximal dose of 246 million cells). With regard to efficacy, there were dose-dependent improvement in glycemic control as evidenced by a decrease at all time points after week 1 in hemoglobin A1c (HbA1c) in MPC- treated patients compared with an increase in HbA1c in placebo treated subjects. HbA1c is a blood test that determines how much damage the high sugar levels are doing to the body. The test uses the blood protein hemoglobin to assess the damage that high glucose levels are doing to the rest of the body. In this clinical trial, significant reductions in HbA1c were observed after 8 weeks in the 2 M/kg MPC group compared to placebo (p<0.05) which was sustained through 12 weeks. The reduction in HbA1c was most pronounced in subjects with baseline HbA1c ≥ 8% (i.e. those patients with relatively poorer glucose control).

Fasting insulin levels were reduced in the 1 million and 2 million/kg groups compared to placebo (P<0.05), and reduced levels of inflammatory cytokines TNF-alpha and IL-6 (which are made at high levels during inflammation) were observed at 12 weeks in MPC groups compared to placebo.

The scientists and physicians involved in this clinical trial concluded there was sufficient evidence to support further evaluation into the use of MPCs in the treatment of type 2 diabetes and its complications. They also thought that there were grounds for exploring other therapeutic venues in which MPCs might prove useful.

Mesoblast Chief Executive Silviu Itescu said: “We are very pleased with these results which are consistent with an immunomodulatory mechanism by which our MPCs may have glucose-lowering effects in patients with type 2 diabetes. We are evaluating whether similar effects may be seen with the use of MPCs in the treatment of kidney disease and other complications of type 2 diabetes.”

While it is improbable in the extreme that this one-time treatment will improve the long-term clinical outcomes of diabetics, it is possible that repeated treatments will provide better Glycemic control for poorly controlled diabetics, and that these repeated treatments will produce long-term improvements in the health of these patients.

Engineered Stem Cells from Human Umbilical Cord Blood Eradicates Pancreatic Tumor

Tissue-specific stem cells called mesenchymal stem cells (MSCs) are a very efficient way to delivery new drugs to cancer sites. One of the reasons these cells do such a good job with cancers in that MSCs have a liking for tumors, and once MSCs are injected into a patient or laboratory animal with tumors, the MSCs make a “B-line” for the tumor and get cozy with it.

Interleukin-15 (IL-15) is a small protein synthesized by white blood cells in our bodies, and IL-15 has a demonstrated ability to stop tumors in their tracks. Unfortunately, IL-15 is broken down quickly once it is injected into the body and consequently, has to be given in very high quantities for it to work. At such high concentrations, IL-15 causes severe side effects, and therefore, it has not been pursued as an anti-tumor agent to the degree that it deserves.

To get around this problem, a Chinese group led by Kexing Fan from the International Joint Cancer Institute in Shanghai, China, genetically engineered MSCs isolated from human umbilical cord blood so that they expressed IL-15. When these engineered MSCs that expressed a mouse version of IL-15 were subjected to experimental verification, the expressed IL-15 activated white blood cells to divide just like native IL-15.

Next, Fan’s group used these souped-up cells to treat In mice afflicted with pancreatic tumors. Pancreatic cancer is an indiscriminate killer, since by the time it causes any symptoms, it is usually so advanced, that there is little to be done in order to treat it. Thus new strategies to treat this yep of cancer are eagerly being sought. Systemic administration of IL-15-expressing MSCs significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice. The tumors of these mice showed extensive cell death, and other types of immune cells known to fight tumor cells (NK and T cells) had also accumulated around the tumor. Other experiments confirmed that the injected MSCs did indeed migrate toward the tumors and secrete IL-15 at the site of the tumors.

Interestingly, those mice that were cured from the pancreatic tumors, appeared to have a kind of resistance of these tumors. Namely, when Fan and his colleagues tried to reintroduce the same tumor cells back into the cured mice, the tumor cells would not grow. Thus the engineered MSCs not only tuned the immune system against the tumor, but they effectively vaccinated the mice against it as well.

Overall, these data seem to support the use of IL-15-producing MSCs as an innovative strategy for the treatment of pancreatic tumors.

Enzyme Helps Stem Cells Improve Recovery From Limb Injury

Ischemia refers to the absence of oxygen in a tissue or organ. Ischemia can cause cells to die and organs to fail and protecting cells, tissues and organs from ischemia-based damaged is an important research topic.

Perfusion refers to the restoration of the blood flow to an organ or tissue that had experienced a cessation of blood flow for a period of time. Even though the restoration of circulation is far preferable to ischemia, perfusion has its own share of side effects. For example, perfusion heightens cells death and inflammation and this can greatly exacerbate the physical condition of the patient after a heart attack, traumatic limb injury, or organ donation.

“Think about trying to hold onto a nuclear power plant after you unplug the electricity and cannot pump water to cool it down,” said Jack Yu, Chief of Medical College of Georgia’s Section of Plastic and Reconstructive Surgery. “All kinds of bad things start happening,”

Earlier studies in the laboratory of Babak Baban have shown that stem cells can improve new blood vessel growth and turn down the severe inflammation after perfusion (see Baban, et al., Am J Physiol Regul Integr Comp Physiol. 2012 Dec;303(11):R1136-46 and Mozaffari MS, Am J Cardiovasc Dis. 2013 Nov 1;3(4):180-96). Baban is an immunologist in the Medical College of Georgia and College of Dental Medicine at Georgia Regents University.

The new study from the Baban laboratory shows that an enzyme called indolamine 2,3,-dioxygenase or IDO can regulate inflammation during perfusion. IDO is widely known to generate immune tolerance and dampen the immune response in the developing embryo and fetus, but it turns out that stem cells also make this enzyme.

In their study, Including IDO with bone marrow-derived stem cells increased the healing efficiency of injected stem cells.

 Treatment Effect on Toe Spread Ratio Averages (48–72 hours after treatment). The outcome of stem cell (SC) therapy indicates that IDO may improve recovery. IDO-KO mice treated with SC demonstrated an accelerated recovery compared with IDO-KO treated with PBS (p-value <0.05). However, the WT mice treated with SC showed the greatest recovery of intrinsic paw function when expressed as a ratio comparing it to the non-injured paw (p-value = 0.027). Functional recovery from ischemia-reperfusion (IR) injury in the different treatment groups was measured, using a modified version of walking track analysis. For each subject, toe spread was measured in the IR limb (Ti) and control contralateral limb (Tc). The ratio of the toe spread in the injured limb (Ti) to the control limb (Tc) was then calculated by Ti/Tc. A ratio of 1 indicates 100% recovery or equal width and thus normal intrinsic function. When comparing the WT group treated with stem cells to those treated with PBS, a 45% increase in recovery was seen demonstrating the efficacy of stem cell therapy alone in the presence of an environment where IDO expression is present. doi:10.1371/journal.pone.0095720.g001
Treatment Effect on Toe Spread Ratio Averages (48–72 hours after treatment).
The outcome of stem cell (SC) therapy indicates that IDO may improve recovery. IDO-KO mice treated with SC demonstrated an accelerated recovery compared with IDO-KO treated with PBS (p-value <0.05). However, the WT mice treated with SC showed the greatest recovery of intrinsic paw function when expressed as a ratio comparing it to the non-injured paw (p-value = 0.027). Functional recovery from ischemia-reperfusion (IR) injury in the different treatment groups was measured, using a modified version of walking track analysis. For each subject, toe spread was measured in the IR limb (Ti) and control contralateral limb (Tc). The ratio of the toe spread in the injured limb (Ti) to the control limb (Tc) was then calculated by Ti/Tc. A ratio of 1 indicates 100% recovery or equal width and thus normal intrinsic function. When comparing the WT group treated with stem cells to those treated with PBS, a 45% increase in recovery was seen demonstrating the efficacy of stem cell therapy alone in the presence of an environment where IDO expression is present.

Also indicators of inflammation, swelling, and cell death decreased in animals that received bone marrow-derived stem cell injections and had IDO.  Baban’s group also showed that the injected stem cells increased endogenous expression of IDO in the perfused tissues.

BMDScs can enhance IDO and regulatory T cells while reducing inflammatory cytokines in the hind limb IR injury. Immunohistochemical analysis of paraffin embedded tissues from murine model with IRI of hind limb showed that treating the animals with BMDSCs in an IDO sufficient microenvironment first: increased IDO and FOXP3 expression (panels A and B, red arrows), while decreased the inflammatory cytokines, IL-17 and IL-23 (panels C and D). Anti inflammatory cytokine, IL-10, was increased as demonstrated in panel E. All together, these analysis suggest a potential therapeutic role for BMDSCs, re-enforced by possible IDO dependent mechanisms. All pictures are 400X magnification
BMDScs can enhance IDO and regulatory T cells while reducing inflammatory cytokines in the hind limb IR injury.
Immunohistochemical analysis of paraffin embedded tissues from murine model with IRI of hind limb showed that treating the animals with BMDSCs in an IDO sufficient microenvironment first: increased IDO and FOXP3 expression (panels A and B, red arrows), while decreased the inflammatory cytokines, IL-17 and IL-23 (panels C and D). Anti inflammatory cytokine, IL-10, was increased as demonstrated in panel E. All together, these analysis suggest a potential therapeutic role for BMDSCs, re-enforced by possible IDO dependent mechanisms. All pictures are 400X magnification

Baban thinks that even though these experiments were performed in mice, because mice tend to be a rather good model system for limb perfusion/ischemia, these results might be applicable in the clinic.  “We don’t want to turn off the immune system, we want to turn it back to normal,” said Baban

According to Baban’s collaborator, Jack Yu, even a short period of inadequate blood supply and nutrients results in the rapid accumulation of destructive acidic metabolites, reactive oxygen species (also known as free radicals), and cellular damage.  The power plant of the cell, small structures called the mitochondria, tend to be one of the earliest casualties of ischemia/perfusion.  Since mitochondria require oxygen to make a chemical called ATP, which is the energy currency in cells, a lack of oxygen makes the mitochondria leaky, swollen and dysfunctional.

“The mitochondria are very sick,” said Yu. ” When blood flow is restored, it can put huge additional stress on sick powerhouses.  “They start to leak things that should not be outside the mitochondria.”

Without adequate energy production and a cellular power plant that leaks, the cells fill with toxic byproducts that cause the cells to commit a kind of cellular hari-kari.  Inflammation is a response to dying cells, since the role of inflammation is to remove dead or dying cells, but inflammation can give the coup de grace to cells that are already on the edge.  Therefore, inflammation can worsen the problem of cell death.

Even though these results were applied to limb ischemia perfusion, Baban and his colleagues think that their results are applicable to other types of ischemia perfusion events, such as heart attacks and deep burns.  Yu, for example, has noticed that in the case of burn patients, the transplantation of new tissue into areas that have undergone ischemia perfusion can die off even while the patient is still in the operating room.

“It cuts across many individual disease conditions,”  said Yu.  Transplant centers already are experimenting with pulsing donor organs to prevent reperfusion trauma.

The next experiments will include determining if more is better.  That is, if giving more stem cells will improve the condition of the injured animal.  In these experiments, which were published in the journal PLoS One, only one stem cell dose was given.  Also, IDO-enhancing drugs will be examined for their ability to prevent reperfusion damage.

Even though stem cells are not given to patients in hospitals after reperfusion, stem cell-based treatments are being tested for their ability to augment healing after reperfusion.  Presently, physicians reestablish blood flow and then give broad-spectrum antibiotics.  The results are inconsistent.  Hopefully, this work by Baban and others will pave the road for future work that leads to human clinical trials.

Induced Pluripotent Stem Cells Used to Make New Bone In Monkeys

Cynthia Dunbar, MD and her colleagues at the National Heart, Lung, and Blood Institute, which is a division of the National Institutes of Health (NIH) in Bethesda, Maryland have shown for the first time that it is possible to make new bone from induced pluripotent stem cells that are derived from a patient’s own skin cells.

This study, which was done in monkeys, shows that there is some risk that induced pluripotent stem cells (iPSCs) can form tumors, but that the risk of tumor formation is less than what was shown in immuno-compromised mice.

iPSCs are made from adult cells by means of a process called “reprogramming.” To reprogram adult cells, genetic engineering techniques are used to introduce specific genes into adult cells. These introduced genes drive the adult cells to de-differentiate into a less mature state, until they eventually become pluripotent, much like embryonic stem cells.

Originally, discovered by Nobel-prize winner Shinya Yamanaka, reprogramming was initially done with genetically engineered viruses that insert genes into the genome of cells. Even though these viruses do a passable job of reprogramming cells, they also introduce insertion mutations. Yamanaka and others originally used four transcription factors (Oct4, Sox2, Klf4, c-Myc) to reprogram adult cells. Several of these genes are overexpressed in a variety of tumors, and therefore, the use of these genes does create a risk of forming cells that overgrown and become tumorous. Secondly, The reprogramming process does put cells under the types of stresses that increase the mutation rate, and these mutations can also increase the risk of forming tumor cells. However, it is clear that not all reprogramming protocols cause the same rate of mutations, and that the mutation rate of iPSCs was originally overestimated. What is required is a good way to screen iPSC lines for mutations and for safety, especially since not all iPSC lines are equal when it comes to their safety.

The advantage of using iPSCs over embryonic stem cells is that the immune system of the patient should not reject tissues and cells made from iPSCs. This would eliminate the need for immune suppression drugs, which can be rather toxic.

Cynthis Dunbar from the National Heart, Lung, and Blood Institute said of her experiments, “We have been able to design an animal model for testing of pluripotent stem cell therapies using the rhesus macaque, a small monkey that is readily available and has been validated as being closely related physiologically to humans.

Dr. Dunbar continued: “We have used this model to demonstrate that tumor formation of a type called a ‘teratoma’ from undifferentiated autologous iPSCs does occur; however, tumor formation is very slow and requires large numbers of iPSCs given under very hospitable conditions. We have also shown that new bone can be produced from autologous iPSCs as a model for their possible clinical application.”

Dunbar and her team used a excisable polycistronic lentiviral vector called STEMCCA (Sommer et al., 2010) that expressed four genes: human OCT4, SOX2, MYC, and KLF4 to make iPSCs from skin cells. After they had derived culturable iPSCs from rhesus monkeys (made under feeder-free conditions), Dunbar and her group seeded them on ceramic scaffolds that are used by reconstructive surgeons to fill in or rebuild bone. Interestingly, these cells regrew bone in the monkeys.

The differentiated iPSCs formed no teratomas, but monkeys that had received implantations of undifferentiated iPSCs formed teratomas in a dose-specific manner.

Dunbar and her colleagues note that this approach might be beneficial for people with large congenital bone defects or other types of traumatic injuries. Having said that, it is doubtful that bone replacement therapies will be the first human iPSC-based treatment, since bone defects are not life-threatening, even though they can seriously compromise the quality of a patient’s life.

“A large animal preclinical model for the development of pluripotent or other high-risk/high-reward generative cell therapies is absolutely issues of tissue integration of homing, risk of tumor formation, and immunogenicity,” said Dunbar. “The testing of human-derived cells in vitro or in profoundly immunodeficient mice simply cannot model these crucial preclinical safety and efficiency issues.”

This NIH team is now collaborating with other labs to differentiate macaque iPSCs into liver, heart, and white blood cells for to test them for eventual pre-clinical trials in hepatitis C, heart failure, and chronic granulomatous disease, respectively.

Effective Stem Cell-Based Treatment for Lupus

Chinese physicians and stem cell researchers from Shenzhen, China have reported on their clinical trial that treated 40 patients with severe and refractory lupus systemic erythematosus with mesenchymal stem cells isolated from umbilical cord.  This 40-patient, multicenter study targeted patients with active and difficult-to-treat lupus.

Systemic lupus erythematosus (SLE), which is also simply known as lupus, is an autoimmune disease in which the body’s immune system mistakenly attacks healthy tissue.  Lupus can affect the skin, joints, kidneys, brain, or even other organs.

What causes lupus is uncertain, but tissue damage seems to predispose some people to the onset of lupus.  Lupus commonly affects women than men, and it can occur at any age.  It most commonly appears most often in people between the ages of 10 and 50, and African-Americans and Asians are affected more often than people from other ethnic groups.  Particular drugs have also been linked to the onset of lupus or lupus-like conditions (e.g., isoniazid, hydralazine, procainamide, and less commonly anti-seizure medicines, capoten, chlorpromazine, etanercept, infliximab, methyldopa, minocycline, penacillamine, quinidine, and sulfasalazine).

The symptoms of lupus vary tremendously and they usually come and go.  Almost all lupus patients have some joint pain and swelling, and some develop arthritis.  Typically the joints of the fingers, hands, wrists, and knees are most often affected.  Other symptoms include chest pain when taking a deep breath, fatigue, fever, general discomfort, uneasiness, or ill feeling (malaise), hair loss, mouth sores, swollen lymph nodes, and sensitivity to sunlight.  Also, a specific type of skin rash known as a “butterfly” rash occurs in about half of lupus patients.  The butterfly rash is most often seen over the cheeks and bridge of the nose, but can be widespread and gets worse in sunlight.  Some people have only skin symptoms and have what is known as discoid lupus. 

Chronic lupus usually becomes concentrated in specific organs, which can cause secondary symptoms.  These symptoms can include:

1.  Brain and nervous system: headaches, numbness, tingling, seizures, vision problems, personality changes.

2.  Digestive tract: abdominal pain, nausea, and vomiting, and the symptoms of liver failure.

3.  Heart: abnormal heart rhythms (arrhythmias).

4.  Lung: coughing up blood and difficulty breathing.

5.  Skin: patchy skin color, fingers that change color when cold (Raynaud’s phenomenon)

To treat lupus, powerful anti-inflammatory drugs are usually used.  These include systemic steroids such as prednisone (Deltasone and others), hydrocortisone, methylprednisolone (Medrol and others), or dexamethasone (Decadron and others).  Other drugs include nonsteroidal anti-inflammatory drugs (NSAIDS), such as ibuprofen (Advil, Motrin and other brand names) or naproxen (Aleve, Naprosyn and others).  However, other drugs include antimalarial drugs such as hydroxychloroquine (Plaquenil), chloroquine (Aralen), or quinacrine. Recent studies suggest that lupus patients treated with antimalarial medications have less active disease and less organ damage over time. Therefore, many experts now recommend antimalarial treatment for all patients with systemic lupus unless they cannot tolerate the medication. If these do not work, then the “big guns” include immunosuppressives, such as azathioprine (Imuran), cyclophosphamide (Cytoxan, Neosar), mycophenolate mofetil (CellCept), or belimumab (Benlysta) and Methotrexate (Rheumatrex, Folex, Methotrexate LPF).  These drugs have long lists of side effects and drug interactions.  Even then, some patients are not helped by these drugs.

Thus more efficacious and safe ways to treat recalcitrant cases of lupus have included stem cell treatments.  In particular, mesenchymal stem cells and their ability to suppress inflammation.  To that end, several pre-clinical and clinical trials have tested mesenchymal stem cells from bone marrow, fat and umbilical cord to reduce the chronic inflammation associated with particular autoimmune diseases (see P Connick, et al., Lancet Neurol. 2012 Feb;11(2):150-6; MM Bonab, et al., Curr Stem Cell Res Ther. 2012 Nov;7(6):407-14; J Voswinkel, et al., Immunol Res. 2013 Jul;56(2-3):241-8; P Connick P, et al., Trials. 2011 Mar 2;12:62; D Karussis, et al., Arch Neurol. 2010 Oct;67(10):1187-94; B Yamout, et al., J Neuroimmunol. 2010 Oct 8;227(1-2):185-9).

This new Chinese trial provides some very interesting and welcome data on the use of mesenchymal stem cells from umbilical cord to treat lupus.

Dr. Xiang Hu, who founded the biotechnology company Beike, said, “We are pleased with the results we have seen in our clinical trial.  While some severe cases experienced relapse after 6 months, the results show a markedly improved quality of life expectation.  This is a big step forward in combating autoimmune disease.  We will now be looking to further our SLE research efforts to find even better results.”

The forty patients who participated in the study were recruited from four centers in China.  The umbilical cord-derived mesenchymal stem cells used in the study were processed by Beike Biotech’s scientists at the company’s new state-of-the-art Jiangsu Stem Cell Regenerative Medicine Facility in Taizhou, China.  Stem cells from sources other than the patient’s own body are known as allogeneic stem cells, and these forty patients, all of whom have refractory lupus were infused with umbilical cord mesenchymal stem cells intravenously at the beginning of the study and one week later.  To score each patient’s disease progression, a clinical test called a SLEDAI or Systemic Lupus Erythematosus Disease Activity score.  The SLEDAI score results from a compilation of multiple clinical and laboratory tests.

After 6 months, all patients showed significant improvement in their SLEDAI scores, but after six months, several patients experienced relapse, which required a repeat treatment with mesenchymal stem cells. Also, the safety profile of these cells was superior to many of the drugs used to treat lupus.

This study is suggests that the mesenchymal stem cells suppress active lupus without causing severe adverse effects.  However, in order to show that definitively, a double-blinded, placebo-controlled study must be conducted.  Also, trying to provide longer periods of relief rather than just six months of relief is another factor that further work will hopefully address.

A Three-Dimensional-Printed, Stem Cell Implant Repairs a Hip

Physicians and stem cell scientists at Southampton, UK have completed a hip surgery in which a 3D printed implant and stem cell graft were used to replace a diseased hip.

The 3D printed hip was made from titanium but it was designed using the patient’s CT scan and CAD CAM (computer aided design and computer aided manufacturing) technology. By printing the hip bone by means of CAD CAM technology the manufactured hip was designed to the patient’s exact specifications and measurements.

This implant will provide a new socket into which the ball of the femur bone is inserted. Between the titanium implant and the pelvis bone, the surgeons inserted a graft containing bone-making stem cells.

The stem cell graft should act as a filler for the loss of bone. The patient’s own bone marrow stem cells were added to the graft in order to provide a source of bone-making stem cells to encourage bone regeneration behind and around the metal implant.

Douglas Dunlop, a consultant orthopedic surgeon, who conducted this operation at the Southampton General Hospital, thinks that this type of procedure could be a genuine game changer. “The benefits to the patient through this pioneering procedure are numerous. The titanium used to make the hip is more durable and has been printed to match the patient’s exact measurements – this should improve the fit and could rescue the risk of having to have another surgery. The bone graft material that has been used has excellent biocompatibility and strength and will fill the defect behind the bone well, fusing it all together.”

Over the past decade Dr. Dunlop and University of Southampton scientist Professor Richard Oreffo have developed a translational research program that aims to use a patient’s own skeletal stem cells to replace damaged or lost bone during orthopedic procedures.  For example, see A Aarvold, et al., J Tissue Eng Regen Med. 2012 Oct 5. doi: 10.1002/term.1577; JO Smith, et al., J Tissue Eng Regen Med. 2014 Apr;8(4):304-13; ER Tayton, et al., J Bone Joint Surg Br. 2012 Jun;94(6):848-55; E Tayton, et al., Acta Biomater. 2012 May;8(5):1918-27; A Aarvold, et al., Regen Med. 2011 Jul;6(4):461-7. doi: 10.2217/rme.11.33; and JO Smith, et al., Tissue Eng Part B Rev. 2011 Oct;17(5):307-20.

In this particular operation, the graft is made up of a bone scaffold that allows blood to flow through it. Stem cells from the bone marrow attach to this material and grow new bone. This implant will support the 3D printed hip implant.

Professor Oreffo comments: “The 3D printing of the implant in titanium, from CT scans of the patient and stem cell graft is cutting edge and offers the possibility of improved outcomes for patients.

“Fractures and bone loss due to trauma or disease are a significant clinical and socioeconomic problem. Growing bone at the point of injury alongside a hip implant that has been designed to the exact fit of the patient is exciting and offers real opportunities for improved recovery and quality of life.”

For the patient, Meryl Richards, from Hampshire, the procedure means an end to her hip troubles. In 1977 she was involved in a traffic accident and since then has had to have six operations to repair her injured hip.

She says: “The way medicine has evolved is fantastic. I hope that this will be the last time that I have to have a hip operation. I feel excited to have this pioneering surgery and I can see what a benefit it will have to me.”

Making Functional Neurons from Skin Cells

A new method for deriving fully functional neurons from skin cells could provide essential model systems for studying neurodegenerative diseases, and testing new drugs and stem cell-based treatments.

Genetic engineering experiments with skin cells can produce cells with distinct neuron-like characteristics. By using viruses to introduce three neuron-specific genes (achaete-scute complex-like 1 (Ascl1), brain-2 (Brn2a), and myelin transcription factor-like 1 (Myt1l)) skin cells can be effectively and directly converted into induced neurons or iN cells. Unfortunately, iN cells are not fully functional neurons and are also produced in very low numbers (see O. Torpor, et al., Proc Natl Acad Sci U S A. 2013 Apr 23;110(17):7038-43).

This new technique, however, can produce neurons that are fully functional, which makes them better models for the study of age-related diseases such as Parkinson’s disease and Alzheimer’s disease.

Cambridge University researchers devised this new technique by carefully observing the way neurons developed on the nervous systems of tadpoles. In particular, the Cambridge group was interested in the maturation of neurons after their formation.

According to Anna Philpott, who is a member of the Department of Oncology, “When you reprogram cells, you’re essentially converting them from one form to another but often the cells you end up with look life they come from embryos rather than looking and acting like more mature cells. In order to increase our understanding of diseases like Alzheimer’s, we need to be able to work with cells that look and behave like those you would see in older individuals who have developed the disease, so producing more ‘adult’ cells after reprogramming is really important.”

Instead of simply adding transcription factors to skin cells, Philpott and her colleagues paid close attention to the activities of those transcription factors once they are expressed inside the cell. Dividing cells, for example, modify their transcription factors with attacking phosphate groups to them. This process of phosphate attachment helps with cell growth but prevents cells from maturing to fully adult neurons.

However, Philpott and her group engineered transcription factors that could were unable to receive phosphate group attachments. Such modified transcription factors produced neurons that were more mature and more useful as model systems for neurodegenerative diseases.

Such controls are also at work in other tissues such as the pancreatic islet cells, and Philpott and her co-workers are already attempting to use her strategy to make mature pancreatic beta cells that secrete insulin in response to increased glucose concentrations.

“We’ve found that not only do you have to think about how you start the process of cell differentiation in stem fells, but you also have to think about what you need to do to make differentiation complete – we can learn a lot from how cells in developing embryos manage this,” said Philpott.

Cartilage Production From Fat-Based Stem Cells Without Exogenous Growth Factors

Making cartilage from fat-based stem cells would be so much more attractive if we didn’t have to use exogenous sources of growth factors. Nevertheless, fat-based stem cells remain quite attractive as a source of cartilage since these cells can be grown in culture to large numbers and can also be readily differentiated into chondrocytes if they are stimulated with the growth factor transforming growth factor-β1 (TGF-β1). Using exogenous TGF-β1, however, has side undesirable effects. Is there another way?

Maybe. A new study by Loran Solorio and Eben Alsberg at Case Western Reserve University has used a culture medium containing TGF-β1-loaded microspheres to make cartilage from fat-based stem cells in culture. This technique can make cartilage without any exogenous growth factors, since all growth factors required for cartilage production are found within the culture system.

In this study, Solorio and Alsberg used exogenous TGF-β1 to induce cartilage formation in fat-based stem cells that were grown in sheets. These sheets of cells made cartilage after 3 weeks. Once it was clear that their experimental system worked well, they used TGF-β1-loaded gelatin microspheres to deliver the growth factor. By tweaking the quantity of microspheres and the concentration of TGF-β1 required for this to work, Solorio and Alsberg showed that the use of TGF-β1-loaded microspheres could induce cartilage formation as well as exogenous TGF-β1. Staining for cartilage-specific molecules and detailed microscopic observation of the cartilage showed that it was indeed, good, solid cartilage.

This publication is the first demonstration of the self-assembly of fat-derived stem cells into high-density cell sheets capable of forming cartilage in the presence of TGF-β1-releasing microspheres. The incorporation of these microspheres might bypass the need for extended culture of the stem cells, potentially allowing stem cells sheets to be implanted more rapidly into defects to regenerate cartilage in a living organism.

Differentiation of Induced Pluripotent Stem Cells Decreases Immune Response Against Them

The goal of regenerative medicine is to replace dead or damaged cells, tissues and even organs with living, properly functioning cells tissues and organs. However, this goal has a few genuine barriers that include tumor formation in the case of pluripotent stem cells, poor cell survival, or even immunological rejection of the transplanted cells before they can render any long-term benefits. Induced pluripotent stem cells (iPSCs), which are made from adult cells by a combination of genetic engineering and cell culture techniques, can be made from a patient’s own mature cells and the differentiated into almost any tissue in the adult body. However, research with mouse iPSCs has shown that even stem cells produced from the subject’s own tissues can be rejected by the subject’s own immune system.

Immune rejection of iPSCs is a legitimate concern, but research from the Stanford University School of Medicine has shown that differentiation of iPSCs into more mature cells before transplantation into mice allows them to be tolerated by the immune system.

Joseph Wu, MD, PhD, director of the Stanford Cardiovascular Institute, said, “Induced pluripotent stem cells have tremendous potential as a source for personalized cellular therapeutics for organ repair. This study shows that undifferentiated iPS cells are rejected by the immune system upon transplantation in the same recipient, but that fully differentiating these cells allows for acceptance and tolerance by the immune system without the need for immunosuppression.”

Wu is the senior author of this publication, which appeared online on May 30th in Nature Communications. Lead authorship of this paper is shared by Patricia Almeida, PhD, and Nigel Kooreman, MD, and assistant professor of medicine Everett Meyer, MD, PhD.

Several other studies have suggested that differentiation of iPSCs can reduce their tendency to activate the immune system after transplantation. However, this study of Wu and others is the first to closely examine, at the molecular and cellular level, how this works.

“We’ve demonstrated definitively that, once the cells are differentiated, the immune response to iPS-derived cells is indistinguishable from its response to unmodified tissue derived from elsewhere in the body,” said lead author Nigel Kooreman.

Pluripotent stem cells have the capacity to differentiate into any cell in the adult body. Of the two types of pluripotent stem cells, embryonic stem cells are made from embryos and iPSCs are made in the laboratory from existing adult cells (e.g., skin or blood). Induced pluripotent stem cells are easier to come by than embryonic stem cells, they match the genetic background of the person from whom they were obtained, and they are not as ethically dubious as embryonic stem cells. Thus, in theory, iPSCs are a good option for any physician who wants to make patient-specific stem cells for potential therapies.

Previous studies in mice have shown, however, that even genetically identicaliPSCs can trigger an immune response after transplantation. Thus, Wu and his colleagues have, for the past six years, been investigating how to use immunosuppressive medications to dampen the body’s response to both embryonic andiPSCs and render them more amenable for clinical use (see AS Lee, et al., J Biol Chem 2011 286(37):32697-704; Durruthy-Durruthy L, et al.,PLoS One, 2014 9(4):e94231 and others).

In this recent study, Kooreman and his co-lead authors decided to examine the immune response against transplanted stem cells. They first transplanted undifferentiated iPS cells into the leg muscles of genetically identical recipient mice. These grafts were rejected and no iPSCs were detected six weeks after transplantation.

Next, Wu and his co-workers differentiated the iPSCs into blood vessel-making endothelial cells that line the interior of the heart and blood vessels and then transplanted them into genetically-identical mice. Kooreman, Almeida, and Meyer then compared the acceptance by the immune system of these iPSC-derived endothelial cells with that of naturally occurring endothelial cells derived from the aortic lining of genetically-identical donor mice. To emphasize once again, all the transplanted cells were genetically identical to the mice in which they were injected. Unlike the undifferentiated iPS cells, both the iPS-derived endothelial cells and the aortic endothelial cells survived for at least nine weeks after transplantation.

Next, Wu and his group repeated the experiment, but they removed the grafts 15 days after transplantation. They observed immune cells called lymphocytes in all grafts, but these immune cells were much more prevalent in the grafts of undifferentiated iPS cells. When the lymphocytes that infiltrated the grafts of undifferentiated iPSCs were compared with those in the differentiated iPSC-derived grafts and the endothelial grafts, their gene expression profiles differed significantly. Those lymphocytes in the undifferentiated iPSC grafts expressed high levels of genes known to be involved in robust immune responses, but lymphocytes in both types of endothelial cell grafts expressed higher levels of genes known to be involved in dampening the immune response and inducing self-tolerance.

Finally, Wu and others directly examined a specific type of lymphocyte called a T cell. Grafts of undifferentiated iPS cells harbored large numbers of T cells that were largely homogeneous, which is characteristic of a robust immune response. Conversely, T cell from grafts of the two types of endothelial cells were more diverse, which suggests a more limited immune response which is typically associated with a phenomenon known as self-tolerance.

“The immune response to the iPS-derived endothelial cells and the aortic endothelial cells, and the longevity of the grafts, was very similar,” said Kooreman. “If we specifically look at the T cells, we see they’re also very similar and that they look much different from grafts that are rejected.”

Wu, who is also a professor of cardiovascular medicine and of radiology, said, “This study certainly makes us optimistic that differentiation — into any nonpluripotent cell type — will render iPS cells less recognizable to the immune system. We have more confidence that we can move toward clinical use of these cells in humans with less concern than we’ve previously had.”

Laser-Activation of Dental Stem Cells Spurs Dentine Regeneration

A variety of experiments, clinical trials, and strategies have attempted to exploit stem cells as therapeutic agents in regenerative medicine. However, once stem cells are removed from their niches within the body and grown in artificial culture systems their properties can change. Such culture-acquired changes can often compromise the therapeutic potential of some stem cells. For this reason, the development of relatively simple but effective stem cell isolation and manipulation techniques represents someone of the prominent technical hurdles to the clinical use of stem cells.

Several laboratories have used exogenous factors to direct the differentiation of tissue-resident stem cells, but these exogenous factors can often cause unwanted side effects. For this reason, simpler manipulation techniques are always a welcome addition to the armamentarium of stem cell scientists.

To that end, Ashok B. Kulkarni from the National Institute of Dental and Craniofacial Research in Bethesda, MD and David J. Mooney from the Harvard School of Engineering and their colleagues and co-workers have used non-ionizing, low-power laser (LPL) treatments to activate host stem cells and promote tissue regeneration. This is a minimally invasive treatment that directs stem cells already present in tissues to heal damaged tissues.

LPL treatment was used to activate human dental stem cells in a laboratory culture system. Upon LPL treatment, the dental stem cells began to synthesize a powerful growth factor called transforming growth factor–β1 (TGF-β1). The endogenous synthesis of TGF-β1 and its receptor drove the dental stem cells to form dentin tubes.

When Kulkami and Mooney used an assay in animals called a “pulp capping model,” they discovered that LPL-activated dental stem cells were able to regenerate dentin after laser activation. To further demonstrate that these regenerative effects were the result of TGF-β1, Kalkami and Mooney and others made cells that did not have a functional TGF-β receptor II. This mutation completely abrogated the effects of LPL treatments. Also, if the dental stem cells were incubated with a TGF-βRI inhibitor, the effects of LPL on the dental stem cells was attenuated.

Thus, there is a simple and non-invasive way to activate a resident stem cell population in our bodies. Furthermore, the mechanisms by which LPL activates these stem cells has been defined as TGF-β mediated. These experiments also outlines the mechanism by which resident stem cells might be harnessed by means of light-activated endogenous cues for clinical regenerative applications. Exciting, huh?

STAP Author Agrees to Retract Both Nature Papers

STAP cells or Stimulus-Triggered Acquisition of Pluripotency cells were allegedly derived from adult mouse cells by subjecting those adult cells to a variety of environmental stresses. Even though the derivation of STAP cells was not terribly efficient, the ability to make pluripotent stem cells without viruses or the introduction of new genes seemed to be a godsend for stem cell scientists. Unfortunately, further testing and inquiries into STAP cells has revealed multiple problems and several labs have been completely unable to recapitulate the results of the researchers who reported the derivation of STAP cells. These problems have led many scientists to question the factuality of STAP cell derivation.

STAP cells took another hit this week when genetic tests of STAP cells indicated that those cells do not match the mice from which they were allegedly derived, according to a report from Nature News Blog.

The derivation of STAP cells were initially reported by Haruko Obokata from the RIKEN center and her colleagues. Given the remarkable nature of the claims in those papers, many scientists were skeptical and moved to test the protocols utilized by Obokata and others in those paper to make STAP cells from adult mouse cells. Unfortunately, these independent tests universally flopped, and an internal investigation by the Riken Center came to the conclusion that Dr. Obokata was guilty of research misconduct, which she has denied.

Teruhiko Wakayama, a scientist from Yamanashi University and one of the co-authors on the STAP papers, subjected some of the cell lines that he derived using the STAP approach back in March to a battery of genetic tests. He was dismayed to discover that some of these cell lines did not match the adult mice from which they were supposed to have been generated. This raises the possibility that the STAP cells are the result of contamination, which is a perennial problem in cell culture laboratories. Wakayama did not observe any anomalies with the lines reported in the Nature papers, but, just to be safe, he sent those and other lines to an independent, and unnamed, lab for further examination and corroboration.

These independent tests, according to reports from Japanese media sources, have found that none of the STAP cell lines match the mouse strains they were supposed to be from. This calls “into question whether the STAP phenomenon has ever been demonstrated.”

Last week, the Nature News Blog reported that Dr. Obokata had agreed to retract one of the two STAP papers, even though the retraction has yet to appear in print. Now, according to the ScienceInsider, Obokata has consented to retracting both Nature papers. The ScienceInsider added this will not end the STAP story, since Riken is doggedly trying to determine whether the STAP phenomenon exists and as some critics are asking how these flawed papers were published in the first place.

“The science of the two papers was rigorously, robustly peer-reviewed as part of our usual editorial procedures. Any inaccuracies in the presentation of data that may have come to light since the peer review are being investigated,” a spokesperson from Nature told ScienceInsider.