Mesenchymal Precursor Cells Reduce Cardiac Scar in Heart Failure Patients


Heart failure is a life-limiting condition that affects over 40 million patients worldwide. Fortunately, people who suffer from heart disease now may have new hope. A new study suggests that damaged tissue can be regenerated by means of a stem cell treatment that was injected into the heart during surgery.

This small-scale study was published in the Journal of Cardiovascular Translational Research. It treated and then followed 11 patients who, during coronary artery bypass graft surgery, had stem cells directly injected into their heart muscle near the site of the tissue scars that had resulted from previous heart attacks.

The most common cause of heart failure is “Ischemic cardiomyopathy” or ICM. ICM occurs when the heart has enlarged to such a degree that the vasculature can no longer supply the heart with adequate blood. ICM can also result from multiple sites of blockage in the coronary arteries of the heart that prevent adequate circulation in the heart.

In this study, researchers delivered a novel stem cell mesenchymal precursor cell type (iMP) during coronary artery bypass surgery (CABG) in patients with ICM whose ejection fractions were below 40%. The iMP cells are derived from what seem to be very young mesenchymal stem cells that lack the typical cell-surface proteins of mesenchymal stem cells. The cells have the ability to form a variety of mesodermal-derived tissues. Also, these cells suppress immunological rejection by the patient’s body, and therefore, they can be implanted into a patient’s body, even though their tissue types do not match. Therefore, these cells can not only be expanded in culture, but can also potentially differentiate into heart-based cell types, including heart muscle and blood vessels.

This study was a phase IIa safety study that was NOT placebo-controlled, double-blinded. It enrolled 11 patients, all of whom underwent scintigraphy imaging (SPECT) before their surgery. SPECT is an effect means to detect “hibernating myocardium” that does not properly contract. Hibernating myocardium is not suitable for iMP implantation.

During the CABG surgery, iMP cells were implanted in the heart muscle (intramyocardially). Stem cells were injected into predefined areas that were viable and close to infarct areas that usually showed poor vascularization. Such areas, because of their poor vascularization could not be treated with grafting because of their poor target vessel quality.

After surgery, SPECT imaging was used to identify changes in scar area. Fortunately, Intramyocardial implantation of iMP cells with CABG was safe. The huge surprise came with the reduction of the heart scar. Subjects showed a 40% reduction in the size of scarred tissue. Remember that heart scars form after a heart attack, and can increase the chances of further heart failure. This scarring, however, was previously thought to be permanent and irreversible. The patients also showed improved myocardial contractility and perfusion of nonrevascularized areas of the heart in addition to significant reduction in left ventricular scar area at 12 months after treatment.

“Quite frankly it was a big surprise to find the area of scar in the damaged heart got smaller,” said Prof Stephen Westaby from John Radcliffe hospital in Oxford, who undertook the research at AHEPA university hospital in Thessaloniki, Greece, with Kryiakos Anastasiadis and Polychronis Antonitsis.

Clinical improvement was correlated with significant improvements in quality of life at 6 months after the treatment all patients.

Jeremy Pearson, the associate medical director at the British Heart Foundation (BHF), said: “This very small study suggests that targeted injection into the heart of carefully prepared cells from a healthy donor during bypass surgery, is safe. It is difficult to be sure that the cells had a beneficial effect because all patients were undergoing bypass surgery at the same time, which would usually improve heart function.

“A controlled trial with substantially more patients is needed to determine whether injection of these types of cells proves any more effective than previous attempts to improve heart function in this way, which have so far largely failed.”

Dr. Westaby noted that improvements in the health of their patients were partly a result of the heart bypass surgery. However, he added that the next study would include a control group who will undergo CABG but not receive stem cell treatment, in order to measure exactly what impact the treatment has.

“These patients came out of heart failure partly due to the bypass grafts of course, but we think it was partly due to the fact that they had a smaller area of scar [as a result of the stem cell treatment]. Certainly this finding of scar being reduced is quite fascinating,” he said.

These results suggest that the delivery of iMP cells can induce regeneration of heart muscle and other heart tissues in patients with ischemic heart failure.

This paper was published: Anastasiadis, K., Antonitsis, P., Westaby, S. et al. J. of Cardiovasc. Trans. Res. (2016) 9: 202. doi:10.1007/s12265-016-9686-0.

Gamida Cell Announces First Patient with Sickle Cell Disease Transplanted in Phase 1/2 Study of CordIn™ as the Sole Graft Source


An Israeli regenerative therapy company called Gamida Cell specializes in cellular and immune therapies to treat cancer and rare (“orphan”) genetic diseases. Gamida Cell’s main product is called NiCord, which provides patients who need new blood-making stem cells in their bone marrow an alternative to a bone marrow transplant. NiCord is umbilical cord blood that has been expanded in culture. In clinical trials to date, NiCord has rapidly engrafted into patients and the clinical outcomes of NiCord transplantation seem to be comparable to transplantation of peripheral blood.

Gamida Cell’s two products, NiCord and CordIn, as well as some other products under development utilize the company’s proprietary NAM platform technology to expand umbilical cord cells. The NAM platform technology has the remarkable capacity to preserve and enhance the functionality of hematopoietic stem cells from umbilical cord blood. CordIn is an experimental therapy for those rare non-malignant diseases in which bone marrow transplantation is the only currently available cure.

Gamida Cell has recently announced that the first patient with sickle cell disease (SCD) has been transplanted with their CordIn product.  Mark Walters, MD, Director of the Blood and Marrow Transplantation (BMT) Program is the Principal Investigator of this clinical trial. The patient received their transplant at UCSF Benioff Children’s Hospital Oakland.

CordIn, as previously mentioned, is an experimental therapy for rare non-malignant diseases, including hemoglobinopathies such as Sickel Cell Disease and thalassemia, bone marrow failure syndromes such as aplastic anemia, genetic metabolic diseases and refractory autoimmune diseases. CordIn potentially addresses a presently unmet medical need.

“The successful enrollment and transplantation of our first SCD patient with CordIn as a single graft marks an important milestone in our clinical development program. We are eager to demonstrate the potential of CordIn as a transplantation solution to cure SCD and to broaden accessibility to patients with rare genetic diseases in need of bone marrow transplantation,” said Gamida Cell CEO Dr. Yael Margolin. “In the first Phase 1/2 study with SCD, the expanded graft was transplanted along with a non-manipulated umbilical cord blood unit in a double graft configuration. In the second phase 1/2 study we updated the protocol from our first Phase 1/2 study so that patients would be transplanted with CordIn as a standalone graft, which is expanded from one full umbilical cord blood unit and enriched with stem cells using the company’s proprietary NAM technology.”

Somewhere in the vicinity of 100,000 patients in the U.S suffer from SCD; and around 200,000 patients suffer from thalassemia, globally. The financial burden of treating these patients over their lifetime is estimated at $8-9M. Bone marrow transplantation is the only clinically established cure for SCD, but only a few hundred SCD patients have actually received a bone marrow transplant in the last ten years, since most patients were not successful in finding a suitable match. Unrelated cord blood could be available for most of the patients eligible for transplantation, but, unfortunately, the rate of successful engraftment of un-expanded cord blood in these patients is low. Therefore, cord blood is usually not considered for SCD patients. Without a transplant, these patients suffer from very high morbidity and low quality of life.

Eight patients with SCD were transplanted in the first Phase 1/2 study performed in a double graft configuration. This study is still ongoing. Preliminary data from the first study will be summarized and published later this year. A Phase 1/2 of CordIn for the treatment of patients with aplastic anemia will commence later this year.

NurOwn, Modified Mesenchymal Stem Cells, Show Clinical Benefit in Phase 2 Trial in ALS Patients


BrainStorm Cell Therapeutics Inc. (BCLI) has developed a cell-based product they call “NurOwn.” NurOwn consists of mesenchymal stem cells that have been cultured to secrete a variety of neurotrophic factors (NTFs). These NTFs are a collection of different growth factors that promote the survival of neurons. NurOwn cells were originally developed in the laboratories of Professor Dani Offen and the late Professor Eldad Melamed, at Tel Aviv University. NurOwn cells have been studied extensively and they clearly have the capacity to migrate to damaged areas in the central nervous system (Sadan O, et al., Stem Cells. 2008 Oct;26(10):2542-51), decrease dopamine depletion in a Parkinson’s disease model system (Barhum Y, et al., J Mol Neurosci. 2010 May;41(1):129-37), can promote the survival of photoreceptors in the retina of animals who optic nerves were damaged (Levkovitch-Verbin H, et al., Invest Ophthalmol Vis Sci. 2010 Dec;51(12):6394-400), decrease quinolinic acid toxicity in an animal model of Huntington’s disease (Sadan O, et al., Exp Neurol. 2012 Apr;234(2):417-27), and improve motor function and survival in a genetic model of Huntington’s disease.

On the strength of these experiments, NurOwn cells have also been tested in clinical trials. Because NTF-secreting MSCs (or, MSC-NTF cells) are designed specifically to treat neurodegenerative diseases, most of the clinical trials, to date, have examined of safety and efficacy of MSC-NTFs in patients with neurological disorders. The safety of NurOwn cells was established in a small phase I/II trial with amyotrophic lateral sclerosis (ALS) patients. This was a small study (12 patients), but showed that, at least in this patients population, intrathecal (injected into the central nervous system) and intramuscular administration of MSC-NTF cells in ALS patients with is safe and patients even showed some indications of clinical benefits, but the study was too small to be definitive about the efficacy of these cells.

Now a recently completed randomized, double-blind, placebo-controlled phase 2 study of NurOwn in ALS patients has found that NurOwn is safe and well tolerated and may also confer clinical benefits upon ALS patients.

According to BrainStorm, this phase 2 study achieved its primary objective (safety and tolerability). No deaths were reported in the study and no patients discontinued participation because of an adverse event. All patients in both active treatment and placebo groups experienced at least one treatment-emergent adverse event that tended to be mild-to-moderate in intensity in both groups. Treatment-related adverse events, as determined by a blinded investigator, occurred slightly more frequently in active-treated patients than in placebo-treated patients (97.2 percent vs. 75.0 percent). The largest differences in frequencies were for the localized reactions of injection site pain and back pain, and fever, headache, and joint pain.

However, NurOwn also achieved multiple secondary efficacy endpoints in this trial. NurOwn showed clear evidence of a clinically significant benefit. Most significantly, the response rates were higher for NurOwn-treated subjects compared to placebo at all time points in the 24 weeks during which when the study was conducted.

This clinical trial conducted at three sites in the U.S: Massachusetts General Hospital, UMass Medical School and the Mayo Clinic. 48 patients were randomized to receive NurOwn cells administered via combined intramuscular and intrathecal injection (n= 36), or placebo (n=12). They were followed monthly for approximately three months before treatment and six months following treatment, and were assessed at 2, 4, 8, 12, 16 and 24 weeks.

The primary investigator in this trial, Robert H. Brown of the University of Massachusetts Medical Center and Medical School said, “These exciting findings clearly indicate that it is appropriate to conduct a longer study with repetitive dosing.”

Subjects treated with NurOwn in this trial showed slowing of progression of ALS and no safety concerns. NurOwn-treated patients also displayed increased levels of growth factors in the cerebrospinal fluid and decreased signs of inflammation after two weeks. These are good indicators that the MSC-NTF cells are orchestrating some kind of beneficial biological effect.

Based on these results, new trials are warranted that will examine repeat dosing at 8 to 12 weeks and employ a larger number of subjects.

LIF Increases Muscle Satellite Expansion in Culture and Transplantation Efficiency


Transplantation of satellite stem cells, which are found in skeletal muscles, might potentially treat degenerative muscle diseases such as Duchenne muscular dystrophy. However, muscle satellite cells have an unfortunate tendency to lose their ability to be transplanted then they are grown in culture.

In order to generate enough cells for transplantation, the cells are isolated from the body and then they must be grown in culture. However, in order to properly grow in culture, the cells must be prevented from differentiating because fully differentiated cells stop growing and die soon after transplantation. Several growth factors, cytokines, and chemicals have been used in muscle satellite cell culture systems. Unfortunately, the optimal culture conditions required to maintain the undifferentiated state, inhibit differentiation, and enhance eventual transplantation efficiency have not yet been established satisfactorily.

Because it is impossible to extract enough satellite cells for therapeutic purposed from biopsies, these cells must be expanded in culture. However this very act of culturing satellite cells renders them inefficient for clinical purposes. How can we break away from this clinical catch-22?

Shin’ichi Takeda from the National Center of Neurology and Psychiatry and his colleagues have used growth factors to maintain muscle satellite cell efficiency during cell culture. In particular, Takeda and others used a growth factor called leukemia inhibitory factor (LIF). LIF effectively maintains the undifferentiated state of the satellite cells and enhances their expansion and transplantation efficiency. LIF is also thought to be involved in muscle regeneration.

This is the first study on the effect of LIF on the transplantation efficiency of primary satellite cells,” said Shin’ichi Takeda of the National Center of Neurology and Psychiatry. “This research enables us to get one step closer to the optimal culture conditions for muscle stem cells.”

The precise mechanisms by which LIF enhances transplantation efficiency remain unknown. Present work is trying to determine the downstream targets of LIF. Identifying the precise mechanisms by which LIF enhances satellite cell transplantation efficiency would help to clarify the functional importance of LIF in muscle regeneration, and, even more importantly, further its potential application in cell transplantation therapy.

The reference for this paper is: N. Ito et al., “Enhancement of Satellite Cell Transplantation Efficiency by Leukemia Inhibitory Factor,” Journal of Neuromuscular Diseases, 2016; 3 (2): 201. DOI: 10.3233/JND-160156.

Umbilical Cord Blood Mesenchymal Stem Cells do Not Cause Tumors in Rigorous Tests


Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) have the ability to self-renew and also can differentiate into a wide range of cell types. However, many clinicians and scientists fear that even these very useful cells might cause tumors.

To that end, Moon and colleagues from the Korean Institute of Toxicology have rigorously tested the tendency for hUBC-MSCs to cause tumors. They used a large battery of tests in living organisms and in culture. hUCB-MSCs were compared to MRC-5 and HeLa cells. MRC-5 cells are known to have no ability to cause tumors and HeLa cells have a robust ability to form tumors, and therefore, constitute negative and positive controls,

To evaluate the ability of cells to cause tumors, Moon and others examined the tendency of these cells to grow without being attached to a substratum. This is a hallmark of tumor cells and is called “anchorage-independent growth” (AIG). To assess AIG, the cells were grown in soft agar, which is a standard assay for AIG. hUCB-MSCs and MRC-5 cells formed few colonies in soft agar, but HeLa cells formed a greater number of larger colonies. This indicated that hUCB-MSCs and MRC-5 cells do not show AIG, a common trait of tumorigenic cells.

The next assay implanted these cells into live laboratory animals. hUCB-MSCs were implanted as a underneath the skin of BALB/c-nu mice (nasty creatures – they bite). All the mice implanted with hUCB-MSCs and NRC-5 cells showed any sign of tumors. Both gross and microscopic examination failed reveal any tumors. However, all mice transplanted with HeLa cells developed tumors that were clearly derived from the implanted cells.

These experiments, though somewhat mundane, rigorously demonstrate that hUCB-MSCs do not exhibit tumorigenic potential. This provides further evidence of these cells clinical applications.

The paper appeared in Toxicol Res. 2016 Jul;32(3):251-8. doi: 10.5487/TR.2016.32.3.251.

German Group Uses Induced Pluripotent Stem Cells to Model Nonalcoholic Fatty Liver Disease


A German research group has used pluripotent stem cells to design a new in vitro model system for investigating nonalcoholic fatty liver disease (NAFLD).  NAFLD, or steatosis, is a liver disease whose prevalence is probably much higher than estimated, and the new cases of it are increasing every year throughout the world.  NAFLD is typically associated with obesity and type-2 diabetes.  An estimated one-third of the general population of Western countries is thought to be affected with NAFLD, with or without symptoms.  It usually results from a high caloric diet in combination with a lack of exercise.  The liver begins to accumulate fat as lipid droplets.  Initially, this is a benign state, but it can develop into nonalcoholic steatohepatitis (also known as NASH), an inflammatory disease of the liver.  Then many patients develop fibrosis, cirrhosis or even liver cancer.  However, in many cases patients die of heart failure before they develop severe liver damage.

A major obstacle that dogged NAFLD research was that biopsies of patients and healthy individuals were required.  Researchers from the Institute for Stem Cell Research and Regenerative Medicine at the University Clinic of Düsseldorf, Germany solved this problem by reprogramming skin cells into induced pluripotent stem cells (iPSCs) that they differentiated into hepatocyte-like cells.

“Although our hepatocyte-like cells are not fully mature, they are already an excellent model system for the analysis of such a complex disease”, said Nina Graffmann, first author of the paper that appeared in the journal Stem Cells and Development.

The researchers recapitulated important steps of the disease in cultured cells.  They demonstrated up-regulation of PLIN2, a protein called perilipin that surrounds lipid droplets. Mice without PLIN2 do not become obese, even when overfed with a high fat diet.  Also the key role of PPARα, a transcription factor involved in controlling glucose and lipid metabolism, was reproduced in the tissue culture system.  “In our system, we can efficiently induce lipid storage in hepatocyte-like cells and manipulate associated proteins or microRNAs by adding various factors into the culture.  Thus, our in vitro model offers the opportunity to analyse drugs which might reduce the stored fat in hepatocytes,” Graffmann said.

Senior author James Adjaye and his colleagues hope to expand their model by deriving iPSCs from NAFLD patients.  They hope to discover differences that might explain the course of NAFLD.

“Using as reference the data and biomarkers obtained from our initial analyses on patient liver biopsies and matching serum samples, we hope to better understand the etiology of NAFLD and the development of NASH at the level of the individual, with the ultimate aim of developing targeted therapy options,” said Adjayer.

This paper can be found at Nina Graffmann et al., “Modeling NAFLD with human pluripotent stem cell derived immature hepatocyte like cells reveals activation of PLIN2 and confirms regulatory functions of PPARα,”Stem Cells and Development, 2016; DOI: 10.1089/scd.2015.0383.

A New Tool for Gene Editing In Stem Cells Can Drive Changes in Cell Fate Without Causing Mutations


Recently, a new tool is now available to control gene expression in order to understand gene function and manipulate cell fate. This new tool is called CRIPSR/Cas9, which is a gene-editing tool that employs a genetic system that naturally occurs in bacteria, who use it as a protection against viruses. CRISPR/Cas9 allows scientists to precisely add, remove or replace specific sequences of DNA. It is the most efficient, inexpensive and easiest gene editing tool available to date.

Several laboratories have tried to use CRISPR/Cas9 to activate genes in cells, but such an effort has not always succeeded. However a research team at Hokkaido University’s Institute of Genetic Medicine has developed a powerful new method that uses CRISPR/Cas9 to do exactly that.

In cells, genes have an expression switch called “promoters.” Genes are switched off, or silenced, when their promoters are methylated, which means that islands of C-G bases have a methyl group (a –CH3 group) attached to the cytosine base. The Hokkaido University team wanted to turn an inactivated gene on. The ingeniously combined a DNA repair mechanism, called MMEJ (microhomology-mediated end-joining), with CRISPR/Cas9 to do this. They excised a methylated promoter using CRISPR/Cas9 and then used MMEJ to insert an unmethylated promoter. Thus, they replaced the off-switch signal with an on-switch signal.

DNA Methylation

The gene that was activated was the neural cell gene OLIG2 and the embryonic stem cell gene NANOG in order to test the efficiency of this technology in cultured cells. Within five days, they found evidence that the genes were robustly expressed. When they activated the OLIG2 gene in cultured human stem cells, the cells differentiated to neurons in seven days with high-efficiency.

Toru Kondo and his colleagues also discovered that their editing tool could be used to activate other silenced promoters. They also found that their system didn’t cause unwanted mutations in other non-target genes in the cells. According to Kondo, this gene editing tool has wide potential to manipulate gene expression, create genetic circuits, or to engineer cell fates.

See Shota Katayama et al., “A Powerful CRISPR/Cas9-Based Method for Targeted Transcriptional Activation,” Angewandte Chemie International Edition, 2016; 55(22): 6452 DOI: 10.1002/anie.201601708.