Stem Cell-Based Skin Graft for Severe Burns


Severe wounds are typically treated with full thickness skin grafts. Some new work by researchers from Michigan Tech and the First Affiliated Hospital of Sun Yat Sen University in Guangzhou, China might provide a way to use a patient’s own stem cells to make split thickness skin grafts (STSG). If this technique pans out, it would eliminate the needs for donors and could work well for large or complicated injury sites.

This work made new engineered tissues were able to capitalize on the body’s natural healing power. Dr. Feng Zhao at Michigan Tech and her Chinese colleagues used specially engineered skin that was “prevascularized, which is to say that Zhao and other designed it so that it could grow its own veins, capillaries and lymphatic channels.

This innovation is a very important one because on of the main reasons grafted tissues or implanted fabricated tissues fail to integrate into the recipient’s body is that the grafted tissue lacks proper vascular support. This leads to a condition called graft ischemia. Therefore, getting the skin to form its own vasculature is vital for the success of STSG.

STSG is a rather versatile procedure that can be used under unfavorable conditions, as in the case of patients who have a wound that has been infected, or in cases where the graft site possess less vasculature, where the chances of a full thickness skin graft successfully integrating would be rather low. Unfortunately, STSGs are more fragile than full thickness skin grafts and can contract significantly during the healing process.

In order to solve the problem of graft contraction and poor vascularization, Zhao and others grew sheets of human mesenchymal stem cells (MSCs) and mixed in with those MSCs, human umbilical cord vascular endothelial cells or HUVECs. HUVECs readily form blood vessels when induced, and growing mesenchymal stem cells tend to synthesize the right cocktail of factors to induce HUVECs to form blood vessels. Therefore this type of skin is truly poised to form its own vasculature and is rightly designated as “prevascularized” tissue.

Zhao and others tested their MSC/HUVEC sheets on the tails of mice that had lost some of their skin because of burns. The prevascularized MSC/HUVEC sheets significantly outperformed MSC-only sheets when it came to repairing the skin of these laboratory mice.

When implanted, the MSC/HUVEC sheets produced less contracted and puckered skin, lower amounts of inflammation, a thinner outer skin (epidermal) thickness along with more robust blood microcirculation in the skin tissue. And if that wasn’t enough, the MSC/HUVEC sheets also preserved skin-specific features like hair follicles and oil glands.

The success of the mixed MSC/HUVEC cell sheets was almost certainly due to the elevated levels of growth factors and small, signaling proteins called cytokines in the prevascularized stem cell sheets that stimulated significant healing in surrounding tissue. The greatest challenge regarding this method is that both STSG and the stem cell sheets are fragile and difficult to harvest.

An important next step in this research is to improve the mechanical properties of the cell sheets and devise new techniques to harvest these cells more easily.

According to Dr. Zhao: “The engineered stem cell sheet will overcome the limitation of current treatments for extensive and severe wounds, such as for acute burn injuries, and significantly improve the quality of life for patients suffering from burns.”

This paper can be found here: Lei Chen et al., “Pre-vascularization Enhances Therapeutic Effects of Human Mesenchymal Stem Cell Sheets in Full Thickness Skin Wound Re-pair,” Theranostics, October 2016 DOI: 10.7150/ thno.17031.

Activation of the Proteasome Enhances Stem Cell Function and Lifespan


As we age, the capacity of our stem cells to heal and replace damaged cells and tissues decline. This age-associated decrease in adult stem cell function seems to be a major contributor to the physiological decline during aging. A new paper, by Efstathios Gonos and his colleagues at the National Hellenic Research Foundation in Athens, Greece gives one possible technique that might improve the function of stem cells in an aging body.

Cells contain a multiprotein complex called the “proteasome” that degrades unneeded or defective proteins. The proteasome controls protein half-lives, function, and the protein composition of the cell. Functional failure of the proteasome has been linked to various biological phenomena including senescence and aging. The role of the proteasome in stem cells aging, however has received little attention to date.

Proteasome figure

Gonos and his coworkers used mesenchymal stem cells from umbilical cord Wharton’s Jelly and human fat. Because they were able to compare the proteasome activity in very young and aged stem cells, Gonos and others discovered a significant age-related decline in proteasome content and activity between these two types of stem cells. The proteasome from Warton’s Jelly mesenchymal stem cells were consistently more active and displayed more normal function and activity than those from human fat.  In fact, not only were the protease activities of the proteasomes from the aging stem cells decreased, but they also displayed structural alterations.

These differences in proteasomal activity were not only reproducible, but when the proteasome of young stem cells were compromised, the “stemness,” or capacity of the stem cells to act as undifferentiated cells, was negatively affected.

Even more surprisingly, once after mesenchymal stem cells from human donors lost their ability to proliferate and act as stem cells (their stemness, that is) their decline could be counteracted by artificially activating their proteasomes. Activating the proteasome seems to help the cell “clean house,” get rid of junk proteins, and rejuvenate themselves.

proteasomes-and-stem-cells

Gonos and his team found that the stem cell-specific protein, Oct4, binds to the promoter region of the genes that encode the β2 and β5 proteasome subunits. Oct4 might very well regulate the expression of these proteasome-specific genes.

From this paper, it seems that a better understanding the mechanisms regulating protein turnover in stem cells might bring forth a way to stem cell-based interventions that can improve health during old age and lifespan.

This paper was published in Free Radical Biology and Medicine, Volume 103, February 2017, Pages 226–235.

Better Ways to Make Dopamine-Producing Neurons From Stem Cells


Producing dopamine-making neurons from stem cells for transplantation into Parkinson’s disease patients remains challenging. Differentiating stem cells into dopaminergic neurons is not as efficient a process as we would like it to be. While several laboratories have managed to make pretty good batches of dopaminergic neurons, reliably producing large and pure batches of dopamine-making neurons from pluripotent stem cells is still somewhat problematic. Secondly, transplanting dopamine-making neurons into either the midbrain or the striatum of the brain represents another patch of problems because the production of too much dopamine can cause unwanted, uncontrollable movements. Preclinical assessments of stem cell-derived dopamine neurons in laboratory animals have produced positive, but highly varied results, even though the transplanted cells are very similar at the time of transplantation.

“This has been frustrating and puzzling, and has significantly delayed the establishment of clinical cell production protocols,” said Malin Parmar, who led the study at Lund University.

To address this issue, Parmar and his colleagues used modern global gene expression studies to gain a better understand the molecular changes that drive the differentiation of stem cells into dopamine-making neurons. Parmar conducted these experiments in collaboration with a team of scientists at Karolinska Institute. In their paper, which appeared in the journal Cell Stem Cell, Parmar and his colleagues used single-cell RNA seq to construct the neuronal development of dopaminergic neurons.

lmx1a-expressing-cells

These neurons are characterized by the expression of a gene called LMX1a. However, it turns out that LMX1a-expressing neurons includes not only midbrain dopaminergic neurons (see below at the substantia nigra), but also subthalamic nuclear neurons.

midbrain

These findings reveal that markers used to identify midbrain dopaminergic neurons do not specifically isolate midbrain dopaminergic neurons, but isolate a mixture of cells. Is there a way to separate these two populations?

subthalamic-nucleus

Indeed, there is. Parmar and his colleagues in the laboratory of Thomas Perlmann showed that although dopaminergic neurons from the midbrain and subthalamic nuclear neurons are related, they do express a distinct profile of genes that are specific to the two cell types. The authors argue that the application of these distinct marker genes can help optimize those protocols that differentiate dopaminergic neurons from pluripotent stem cells.

See Nigel Kee and others, “Single-Cell Analysis Reveals a Close Relationship between Differentiating Dopamine and Subthalamic Nucleus Neuronal Lineages,” Cell Stem Cell, 2016; DOI: 10.1016/j.stem.2016.10.003.

Hitting Acute Myeloid Leukemia Where It Hurts


Research teams from Massachusetts General Hospital and the Harvard Stem Cell Institute have teamed up to devise a new strategy for treating acute myeloid leukemia (AML). This new strategy is an outgrowth of new findings by these research groups that have identified an enzyme that plays a central role in the onset of AML.

During blood cell development in the bone marrow, hematopoietic stem cells divide to produce daughters cells, one of which remains a stem cells and the other of which becomes a progenitor cell. The progenitor cells can either differentiate toward the lymphoid lineage, in which it will become either a B-lymphocyte, T-lymphocyte, or a Natural Killer cell, or a myeloid precursor that can give rise to neutrophils, megakarocytes (that produce platelets), monocytes, eosinophils, or red blood cells. However, the means by which myeloid cells are formed in the bone marrow of AML patients is abnormal, and the myeloid precursor cells do not differentiate into a specific white blood cells, but, instead, remain immature and proliferate and crowd out and suppress the development of normal blood cells.

David Scadden, MD, director of the MGH Center for Regenerative Medicine (MGH-CRM), co-director of the Harvard Stem Cell Institute (HSCI), and senior author of this Cell paper, had this to say about AML: “AML is a devastating form of cancer; the five-year survival rate is only 30 percent, and it is even worse for the older patients who have a higher risk of developing the disease.” Dr. Scadden continued: “New therapies for AML are extremely limited – we are still using the protocols developed back in the 1970s – so we desperately need to find new treatments.”

What genetic changes cause these developmental abnormalities that lead to AML? As it turns out, a wide range of genetic changes occur in AML (see Medinger M, Lengerke C, Passweg J. Cancer Genomics Proteomics. 2016 09-10;13(5):317-29; and Prada-Arismendy J, Arroyave JC, Röthlisberger S. Blood Rev. 2016 Sep 2. pii: S0268-960X(16)30060-1). In this paper, however, the authors proposed that the effects on differentiation had to transition through a few shared events. Using a method created by lead author David Sykes of the MGH-CRM and HSCI, the team discovered that a single dysfunctional point in the developmental pathway common to most forms of AML that could be a treatment target.

Previous studies had demonstrated that expression of the HoxA9 transcription factor, a transcription factor that must be inactivated during normal myeloid cell differentiation, is actually quite active in the myeloid precursors of 70 percent of patients with AML.  Unfortunately, no inhibitors of HoxA9 have been identified to date.  Therefore, Scadden and others used a different, albeit freaking ingenious, approach to screen small molecules that were potential Hox9A inhibitors based not on their interaction with a particular molecular target but on whether they could overcome the differentiation blockade characteristic of AML cells.

First, they induced HoxA9 overexpression in cultured mouse myeloid cells to design a cellular model of AML.  They also genetically engineered these cultured cells to glow green once they differentiated into the mature white blood cell types.  These groups screened more than 330,000 small molecules to find which would produce the green signal in the cultured cells.  The green glow indicated that the HoxA9-induced differentiation blockade had been effectively overcome. Only these 330,000 compounds, only 12 induced terminal differentiation of these cells, but 11 of then acted by suppressing a metabolic enzyme called DHODH.  DHODH, or dihydroorotate dehydrogenase, is a biosynthetic an enzyme that is a member of the pyrimidine biosynthesis pathway, which catalyzes the oxidation of dihydroorotate to orotate.

dhodh

This is a shocking discovery because the DHODH enzyme is not known to play any significant role in myeloid differentiation.  Corroboratory experiments demonstrated that inhibition of DHODH effectively induced differentiation in both mouse and human AML cells.

The next obvious step would be to use known inhibitors of DHODH in mice with AML.  They were able to identify a dosing schedule that reduced levels of leukemic cells and prolonged survival that caused none of the adverse effects of normal chemotherapy.  Even though six weeks of treatment with DHODH inhibitors did not prevent eventual relapse, treatment for up to 10 weeks seemed to have led to long-term remission of AML.  This remission included reduction of the leukemia stem cells that can lead to relapse.  Similar results were observed in mice into which human leukemia cells had been implanted.

“Drug companies tend to be skeptical of the kind of functional screening we used to identify DHODH as a target, because it can be complicated and imprecise. We think that with modern tools, we may be able to improve target identification, so applying this approach to a broader range of cancers may be justified,” says Scadden, who is chair and professor of Stem Cell and Regenerative Biology and Jordan Professor of Medicine at Harvard University. Additional investigation of the mechanism underlying DHODH inhibition should allow development of protocols for human clinical trials.

Scadden noted that this manuscript describes six years of work and, also, is a true reflection of the collaborative nature of science in pursuit of clinically relevant therapies.

Enrollment Completed in Phase 2 ALLSTAR Cardiac Clinical Trial


Capricor Therapeutics Inc. has announced the completion of patient enrollment in their Phase 2 ALLSTAR clinical trial.  ALLSTAR stands for ALLogeneic Heart STem Cells to Achieve Myocardial Regeneration, and this trial will test Capricor’s CAP-1002 product in patients suffering from cardiac dysfunction following a heart attack.

CAP-1002 cells are cardiosphere derived cells (CDCs) that were isolated from donors.  This investigational therapy is an off-the-shelf “ready to use” cardiac cell therapy that comes from donor heart tissue.  CAP-1002 cells are made to be directly infused into a patient’s coronary artery during a catheterization procedure.

These CDCs were tested in the CADUCEUS clinical trial, in which they were shown to decrease scar size and increase viable heart tissue when implanted into the hearts of heart attack patients.  One-year follow-up examinations of these confirmed the earlier results.

ALLSTAR will study a population similar to the one in the CADUCEUS study (patients who had experienced a heart attack 30-90 days earlier), except that ALLSTAR will treat patients 91-365 days after suffering a heart attack.  The extension of the patient pool was to see if the indication window for CAD-1002 could be extended.

The Capricor CEO Linda Marbàn said, “With the last patient in ALLSTAR having been dosed on September 30, we expect to report top-line 12-month primary efficacy outcome results in the fourth quarter of 2017.”

ALLSTAR is being sponsored by Capricor and is led by Drs. Timothy Henry and Rajendra Makkar of the Cedars-Sinai Heart Institute.  The trial is being conducted at approximately 25-40 sites across the U.S.

The Phase I portion of the trial was funded in part by the National Institutes of Health and completed enrollment in December 2013, and the Phase II portion of the trial is supported in large part by the California Institute for Regenerative Medicine (CIRM).

Positive Results from Mesoblast’s Phase 2 Trial of Cell Therapy in Diabetic Kidney Disease


Mesoblast Limited has announced results from its Phase 2 clinical Trial that evaluated their Mesenchymal Precursor Cell (MPC) product, known as MPC-300-IV, in patients who suffer from diabetic kidney disease. In short, their cell product was shown to be both safe and effective. The results of their trial were published in the peer-reviewed journal EBioMedicine.  Researchers from the University of Melbourne, Epworth Medical Centre and Monash Medical Centre in Australia participated in this study.

The paper describes a randomized, placebo-controlled, and dose-escalation study that administered to patients with type 2 diabetic nephropathy either a single intravenous infusion of MPC-300-IV or a placebo.

All patients suffered from moderate to severe renal impairment (stage 3b-4 chronic kidney disease for those who are interested).  All patients were taking standard pharmacological agents that are typically prescribed to patients with diabetic nephropathy.  Such drugs include angiotensin-converting enzyme inhibitors (e.g., lisinopril, captopril, ramipril, enalapril, fosinopril, ect.) or angiotensin II receptor blockers (e.g., irbesartan, telmisartan, losartan, valsartan, candesartan, etc.).  A total of 30 patients were randomized to receive either a single infusion of 150 million MPCs, or 300 million MPCs, or saline control in addition to maximal therapy.

Since this was a phase 2 clinical trial, the objectives of the study were to evaluate the safety of this treatment and to examine the efficacy of MPC-300-IV treatment on renal function.  For kidney function, a physiological parameter called the “glomerular filtration rate” or GFR is a crucial indicator of kidney health.  The GFR essentially indicates how well the individual functional units within the kidney, known as “nephrons,” are working.  The GFR indicates how well the blood is filtered by the kidneys, which is one way to measure remaining kidney function.  The decline or change in glomerular filtration rate (GFR) is thought to be an adequate indicator of kidney function, according to the 2012 joint workshop held by the United States Food and Drug Administration and the National Kidney Foundation.

nephronanatomy

Diabetic nephropathy is an important disease for global health, since it is the single leading cause of end-stage kidney disease.  Diabetic nephropathy accounts for almost half of all end-stage kidney disease cases in the United States and over 40% of new patients entering dialysis treatment.  For example, there are almost 2 million cases of moderate to severe diabetic nephropathy in 2013.

Diabetic nephropathy can even occur in patients whose diabetes is well controlled – those patients who manage to keep their blood glucose levels at a reasonable level.  In the case of diabetic nephropathy, chronic infiltration of the kidneys by inflammatory monocytes that secrete pro-inflammatory cytokines causes endothelial dysfunction and fibrosis in the kidney.

Staging of chronic kidney disease (CKD) is based on GFR levels.  GFR decline typically defines the progression to kidney failure (for example, stage 5, GFR<15ml/min/1.73m2).  The current standard of care (renin-angiotensin system inhibition with angiotensin converting enzyme inhibitors or angiotensin II receptor blockers) only delays the progression to kidney failure by 16-25%, which leaves a large residual risk for end-stage kidney disease.  For patients with end-stage kidney disease, the only treatment option is renal replacement (dialysis or kidney transplantation), which incurs high medical costs and substantial disruptions to a normal lifestyle.  Due to a severe shortage of kidneys, in 2012 approximately 92,000 persons in the United States died while on the transplant list.  For those on dialysis, the mortality rate is high with an approximately 40% fatality rate within two years.

The main results of this clinical trial were that the safety profile for MPC-300-IV treatment was similar to placebo.  There were no treatment-related adverse events.  Secondly, patients who received a single MPC infusion at either dose had improved renal function compared to placebo, as defined by preservation or improvement in GFR 12 weeks after treatment.  Third, the rate of decline in estimated GFR at 12 weeks was significantly reduced in those patients who received a single dose of 150 million MPCs relative to the placebo group (p=0.05).  Finally, there was a trend toward more pronounced treatment effects relative to placebo in a pre-specified subgroup of patients whose GFRs were lower than 30 ml/min/1.73m2 at baseline (p=0.07).  In other words, the worse the patients were at the start of the trial, the better they responded to the treatment.

The lead author of this publication, Dr David Packham, Associate Professor in the Department of Medicine at the University of Melbourne and Director of the Melbourne Renal Research Group, said: “The efficacy signal observed with respect to preservation or improvement in GFR is exciting, especially given that this trial was not powered to show statistical significance. Patients receiving a single infusion of MPC-300-IV showed no evidence of developing an immune response to the administered cells, suggesting that repeat administration is feasible and may in the longer term be able to halt or even reverse progressive chronic kidney disease. I hope that this very promising investigational therapy will be advanced to rigorous Phase 3 clinical trials to test this hypothesis as soon as possible.”

Patients who received s single IV infusion of MPC-300-IV cells showed no evidence of developing an immune response to the administered cells.  This suggests that repeated administration of MPCs is feasible and might even have the ability to halt, or even reverse progressive chronic kidney disease.

Packham and his colleagues hope that this cell-based therapy can be advanced to a rigorous Phase 3 clinical trial to further test this treatment.

Stem Cell Therapy Might Improve Brain Function of Traumatic Brains Injury Patients


Accidents happen and sometimes really bad accidents happen; especially if they injure your head.  Traumatic brain injuries or TBIs can result from automobile accidents, explosions or other events that result from severe blows to the head.  TBIs  an adversely affect a patient and his/her family for long periods of time.  TBI patients can experience cognitive deficits that prevent them from thinking or speaking straight, and sensory deficits that prevent them from seeing, hearing or smelling properly.  Psychological problems can also result.  Essentially, TBIs represent a major challenge for modern medicine.

According to data from the Centers for Disease Control (CDC), 1.7 million Americans suffer from TBIs each year (of varying severity).  Of these, 275,000 are hospitalized for their injuries and approximately 52,000 of these patients die from their injuries.  In fact, TBIs contribute to one-third of all injury-related deaths in the United States each year.  More than 6.5 million patients are burdened by the deleterious effects of TBIs, and this leads to an economic burden of approximately $60 billion each year.

Currently, treatments for TBI are few and far between.  Neurosurgeons can use surgery to repair damaged blood vessels and tissues, and diminish swelling in the brain.  Beyond these rather invasive techniques, the options for clinicians are poor.

A new study by Charles S. Cox, professor of Pediatric Surgery and co-director of the Memorial Hermann Red Duke Trauma Institute, and his colleagues suggest that stem cell treatments might benefit TBI patients.  The results of this study were published in the journal Stem Cells.

This study enrolled 25 TBI patients.  Five of them received no treatment and served as controls, but the remaining 20 received gradually increasing dosages of their own bone marrow stem cells.  The harvesting, processing and infusion of the bone marrow cells occurred within 48 hours of injury.  Functional and cognitive results were measured with standard tests and brain imaging with magnetic resonance imaging and diffusion tensor imaging.

This work is an extension of extensive preclinical work done by Cox and his coworkers in laboratory animals and a phase I study that established that such stem cell transplantation are safe for human patients.  The implanted stem cells seem to quell brain inflammation and lessen the damage to the brain by the TBI.

Despite the fact that those TBI patients who received the stem cell treatments had greater degrees of brain damage, the treatment group showed better structural preservation of the brain and better functional outcomes than the control group.  Of particular interest was the decrease in indicators of inflammation as a result of the bone marrow cell-based infusions.

Cox said of this trial, “The data derived from this trial moves beyond just testing safety of this approach.”  He continued:  “We now have a hint of a treatment effect that mirrors our pre-clinical work, and were are now pursuing this approach in a phase IIb clinical trial sponsored by the Joint Warfighter Program within the US Army Medical Research Acquisition Activity, as well as our ongoing phase IIb pediatric severe TBI clinical trial; both using the same autonomous cell therapy.”

This an exciting study, but it is a small study.  While the safety of this procedure has been established, the precise dosage and long-term benefits will require further examination.  However it is a fine start to what may become the flowering of new strategies to treat TBI patients.