Inhibition of AKT Kinase Increases Umbilical Cord Blood Growth in Culture and Engraftment in Mice

Dr. Yan Liu from the Department of Pediatrics and the Herman B Wells Center for Pediatric Research at the Indiana University School of Medicine in Indianapolis, Indiana and his colleagues have increased the engraftment efficiency of umbilical cord hematopoietic (blood cell-making) stem cells in immunodeficient mice. The technique developed by Lui and his colleagues is simple and increases the proliferation of umbilical cord blood hematopoietic stem cells (UCB-HSCs) in culture, which potentially solves several long-standing problems with umbilical cord blood transplantation.

Umbilical cord blood has been used in the clinic for more than 40 years in hematopoietic stem cell transplantation therapies to treat patients with bone marrow diseases or to reconstitute the bone of those cancer patients who had to have theirs wiped out to cure their leukemia or lymphoma.

One of the problems with umbilical cord blood transplantations, however, is the small amount of material in a typical cord blood collection and, therefore, the small number of hematopoietic stem cells (HSCs) available for transplantation. To ameliorate these shortcomings, hematologists will transplant more than one lot of cord blood (a so-called “double umbilical cord blood transplantation”), which, unfortunately, also increases the risk of immunological rejection (so-called Graft Versus Host response).

A second strategy to get around the low numbers of UCB-HSCs is to expand them in culture, which has proven difficult. However, some experiments have given us more than enough hope to suspect this this is a feasible option (see Flores-Guzmán P, et al., Stem Cells Transl Med. 2013 Nov;2(11):830-8; Bari S., et al., Biol Blood Marrow Transplant. 2015 Jun;21(6):1008-1; Pineault N, Abu-Khader A. Exp Hematol. 2015 Jul;43(7):498-513).

Dr. Lui and his coworkers wanted to examine the role of the signaling protein AKT (also known and protein kinase B) in UCB-HSC expansion in culture. To this end, they used silencing RNAs to knock-down AKT gene expression in cultured UCB-HSCs. AKT knock-down enhanced UCB-HSC quiescence and growth in culture. In a separate experiment, Lui and others treated human UCB-HSCs (so-called CD34+ cells) with a chemical that specifically inhibits AKT activity. Then they subjected these cells to a battery of tests in culture and in laboratory mice.

The results were astounding.  Treatment of human UCB-HSCs did not affect the identity of the HSCs and enhanced their ability to form isolated colonies in cell culture growth tests known as “replating assays.”  Additionally, the short-term inhibition of AKT with drugs also enhanced the ability of UBC-HSCs to repopulate the bone marrow of immunodeficient mice.

In summary, inhibition of AKT increases human UCB-HSC quiescence, growth potential, and engraftment in laboratory mice.

These interesting pre-clinical results suggest that AKT inhibitor can increase the expansion of UCB-HSCs in culture and potential increase their tendency of these cells to engraft in patients.

Fat-Based Mesenchymal Stem Cell-Seeded Matrix Heals Bronchopleural Fistula in Female Cancer Patient

Bronchopleural fistulae, mercifully abbreviated as BPF, refers to an opening or hole in the respiratory tree that causes continuity between the pleural space that surrounds the lungs and the bronchial tree. BPH is a highly feared complication of surgery on the respiratory system.

BPH can complicate surgical resection of the pulmonary system. Patients with lung cancers may require lung resection in order to remove tumorous lung tissue. The rate of BPH incidence after lung surgery varies widely, with reported incidences ranging from 1.5 to 28%. Necrosis or death of lung tissue as a result of infection can also cause BPH, as can tuberculosis. Chemotherapy or radiation therapy for lung cancers can also result in BPF. Finally, BPF may caused by persistent spontaneous pneumothorax, which refers to an abnormal build up of air or other gases in the pleural space, which causes an uncoupling of the lung from the chest wall.

To date, treatment for BPF is only partially effectively. The main treatment includes surgery, but the rate of recurrence of the fistulae remains rather high as do the rate of mortality. Can stem cells show us a better way?

Perhaps they can. Dennis A. Wigle, a surgeon at Mayo Clinic, and his collaborators used a synthetic bioabsorbable matrix seeded with the patients one fat-based mesenchymal stem cells to heal a BPF in a 63-yr old woman. Mind you, this is a case study (the lowest quality clinical evidence) and not a controlled study,. However, the success of this case study is at least suggestive that such an approach might prove useful for patients who suffer from BPFs.

Microscopic assessment of matrix cell seeding. (A): Ethidium bromide (red) and Syto-13 (green) costain demonstrating live and dead cells on mesenchymal stem cell seeding on matrix. (B): Confocal microscopy with CD90 (Thy-1) fluorescein isothiocyanate (green) and Hoechst 33342 (trihydrochloride trihydrate) (blue) fluorescent nuclear staining. These images were captured using a ×20 objective and a ×10 eyepiece, for a combined magnification of ×200. Scale bar = 150 µm.

A 63-yr old woman who had surgical resection of the lung in order to treat her lung cancer had, as a consequence of her surgery, a BPF. Some 30 different surgical attempts were made to repair the BPF, but all of them failed. The woman’s health declined and her medical team started to think of alternative treatments.

Fortunately, Mayo Clinic has been participating in an ongoing clinical trial to use fat-based mesenchymal stem cells to treat anal fistulae in Crohn’s disease patients. Therefore Dr. Wigle and his team considered using the protocol utilized with Crohn’s patients to repair this woman’s BPF.

Fat biopsies were taken from the patient and the fat was washed, minced, digested with enzymes, and then grown in special culture media. The adipose tissue-derived mesenchymal stem cells (AD-MSCs) grew and were isolated, characterized and shown to be MSCs.

These cells were then seeded on a matrix of synthetic bioabsorbable poly(glycolide-trimethylene carbonate) copolymer and then placed in a bioreactor to grow. After about 4 days, the matrix was flush with AD-MSCs, and this cell-seeded patch was then used in a subsequent surgery to seal the opening in the respiratory tree. This time the surgery worked. The patient was discharged 25 days after the surgery and sent home.

MRIs of the respiratory system showed that the BPF had indeed closed and properly resolved.

Preoperative imaging showing size and location of fistula, and postoperative imaging demonstrating disease resolution. (A): Preoperative bronchoscopy demonstrating large bronchopleural fistula (BPF) cavity and lateral extension of fistula tracts. (B): Postoperative bronchoscopy (3 months) demonstrating progressive healing of BPF site. (C): Preoperative computed tomography scan demonstrating large BPF with connection to atmosphere (additional axial slices inferiorly). (D): Postoperative computed tomography scan (16 months) demonstrating resolution of BPF.

This case study might confirm what was previously observed in large animal studies by Petrella and others, namely that AD-MSCs can be used to heal BPF. Petrella and others theorized that implanted MSCs induce the proliferation of fibroblasts that then deposit collagen, which seals the BPF (see Ann Thorac Surg 97:480–483.  Alternatively, AD-MSCs might differentiate into cell types  required for regeneration of the airways (Dominici M, and others, Cytotherapy 8:315–317).  Either way, this paper seems to suggest that AD-MSCs can be isolated from a patient’s fat (even a very sick patient like this one) without incident and used for tissue engineering applications that can repair very serious wound like BPF. 

This paper was published in: Johnathon M., Aho, et Al., “Closure of a Recurrent Bronchopleural Fistula Using a Matrix Seeded With Patient-Derived Mesenchymal Stem Cells.” Stem Cells Trans Med October 2016 vol. 5 no. 10 1375-1379. 

Induced Pluripotent Stem Cell-Based Model System of Hypertrophic Cardiomyopathy Provides Unique Insights into Disease Pathology

A research team at the Icahn School of Medicine at Mount Sinai led by Bruce Gelb created a model of hypertrophic cardiomyopathy (HCM) by using human induced pluripotent stem cells.

Patients who suffer from an extreme thickening of the walls of the heart exhibit HCM. This excessive heart thickening is associated with a several rare and common illnesses. There is a strong genetic component to the risk for developing HCM. Can stem cell-based model system be used to study the genetics of HCM?

The answer to this question seems to be yes, since laboratory-generated induced pluripotent stem cells lines that have been differentiated into heart cells that, in many cases, closely resemble human heart tissue. Studies with such stem cell-based model systems have reaped useful insights into disease mechanisms (see F Kamdar, et al., J Card Fail. 2015 Sep;21(9):761-70; Lee YK, Ng KM, Tse HF. J Biomed Nanotechnol. 2014 Oct;10(10):2562-85).

In this paper, Bruce Gelb and his colleagues examined a genetic disorder called cardiofaciocutaneous syndrome (CFC). CFC is caused by mutations in a gene called BRAF. It is a rare condition that affects fewer than 300 people worldwide, and causes head, face, skin, and muscular abnormalities, including abnormalities of the heart.

Gelb and his coworkers isolated skin cells from three CFC patients and reprogrammed them into induced pluripotent stem cells, which were then differentiated into heart cells. In this disease model system, the heart muscle cells enlarged, but this seemed to be due to the interaction of the heart muscle cells with heart-specific fibroblasts. Fibroblasts constitute a significant portion of total heart tissue, even though the heart muscle cells are responsible for the actual pumping activity of the heart. In their model system, Gelb and others observed that these fibroblast-like cells produce an excess of a protein growth factor called TGF-beta, which causes the cardiomyocytes to undergo hypertrophy or abnormal enlargement.

This model system has relevance for research on several related and more common genetic disorders, including Noonan syndrome, which is characterized by unusual facial features, short stature, heart defects, and skeletal malformations.

There is no cure for HCM in patients with these related genetic conditions, but if these findings are correct, then scientists might be able to treat HCM by blocking specific cell signals. This is something that scientists already know how to do. Approximately 40 percent of patients with CFC suffer from HCM (two of the three participants in this study had HCM). This suggests a pathogenic connection, though the link has never been adequately researched.

“We believe this is the first time the phenomenon has been observed using a human induced pluripotent stem cell model of the disease,” said Bruce Gelb.

Please see Rebecca Josowitz et al., “Autonomous and Non-Autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC -Derived Cardiomyocytes,” Stem Cell Reports, 2016; DOI: 10.1016/j.stemcr.2016.07.018.

USC Researchers Isolate Human Nephon Progenitor Cells – Future Possibilities for Kidney Regeneration

Researchers at the Saban Research Institute of Children’s Hospital of Los Angeles and the University of Southern California (USC) have reported the isolation of human nephron progenitor (NP) cells. These results, which were published in the journal Stem Cell Translational Medicine, might very well elucidate how progenitor cells differentiate into become renal cells and then develop into kidneys. Such insights could, possibly provide new strategies to promote renal regeneration after chronic kidney failure or acute kidney injury.

Kidneys are composed of about a million tiny filtration units known as “nephrons.” These diminutive structures filter waste and concentrate those wastes into urine, which is leaked into the bladder. In humans, approximately 500,000 to 1,000,000 nephrons are generated before week 34 – 36 of fetal gestation. However, at this point in development, the NP cells are exhausted and kidney development (known as “nephrogenesis”) effectively ceases. If the kidney loses a large enough quantity of nephrons after this time period, such losses may lead to irreversible kidney failure, since no further cell repair or regeneration is possible.

In past studies, NPs were made from induced pluripotent stem cells, or by utilizing animal models. Scientists at USC and Children’s Hospital of Los Angeles (CHLA), chose a different tactic; they designed an efficient protocol by which they could directly isolate human NPs. To accomplish this, Dr. Laura Perin and her colleagues used RNA-labeling probes to obtain cells that expressed the SIX2 and CITED1 genes. Cells expressing both of these genes are almost certainly NPs, since SIX2 and CITED1 are master regulatory genes that promote renal development.

Dr. Perin, co-director of CHLA’s GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics in Urology, added, “In addition to defining the genetic profile of human NP, this system will facilitate studies of human kidney development, providing a novel tool for renal regeneration and bioengineering purposes.”

On a rather sanguine note, Perin noted that these experiments, which constitute proof-of-concept work, may create new applications to researchers who might be able to use her laboratory’s techniques to isolated progenitor cells for other organs, the pancreas, heart, or lung. “This technique provides a ‘how to’ of human tissue during development,” said Perin.

“It is an important tool that will allow scientists to study cell renewal and differentiation in human cells, perhaps offering clues to how to regulate such development,” added first author of this paper, Stefano Da Sacco.

Functional, Though Not Completely Structurally Normal Tissue-Engineered Livers Made from Adult Liver Cells

Tracy C. Grikscheit and her research team from the Saban Research Institute at the Children’s Hospital Los Angeles have produced functional, tissue-engineered human and mouse liver from adult stem and progenitor cells.

The largest organ in our bodies, the liver executes many vital functions. It is located in the upper right portion of the abdomen protected by the rib cage. The liver has two main lobes that are divided into many tiny lobules.

Liver cells are supplied by two different sources of blood. The hepatic artery provides oxygen-rich blood from the heart and the portal vein supplies nutrients from the intestine and the spleen. Normally, veins return blood from the body to the heart, but the portal vein allows chemicals from the digestive tract to enter the liver for “detoxification” and filtering prior to entering the general circulation. The portal vein also delivers the precursors liver cells need to produce the proteins, cholesterol, and glycogen required for normal body activities.

The liver also makes bile. Bile is a mixture of water, bile acids (made from stored cholesterol in the liver), and other sundry chemicals. Bile made by the liver is then stored in the gallbladder. When food enters the duodenum (the uppermost part of the small intestine), the gallbladder contracts and secretes bile is secreted into the duodenum, to aid in the digestion of fats in food.

The liver also stores extra sugar in the form of glycogen, which is converted back into glucose when the body needs it for energy. It also produces blood clotting factors, processes and stores iron for red blood cell production, converts toxic nitrogenous wastes (usually in the form of ammonium) into urea, which is excreted in urine. Finally, the liver also metabolizes foreign substances, like drugs into substances that can effectively excreted by the kidneys.

Both adults and children are affected by various types of liver disease. Liver can be caused by infectious hepatitis, which is caused by a variety of viruses, chronic alcoholism, inherited liver abnormalities (e.g., Wilson’s disease, hemochromatosis, Gilbert’s disease) or various types of liver cancer. One in ten people in the United States suffer from liver cancer and need a liver transplant. Liver transplantation is the only effective treatment for end-stage liver disease, but the scarcity of liver donors and the necessity of life-long immunosuppressive therapy limit treatment options. In some cases (such as inborn errors of metabolism or acute bouts of liver insufficiency), patients may be effectively treated by transplanting small quantities of functional liver tissue.

Alternate approaches that have been investigated, but these protocols have significant limitations. For example, “hepatocyte transplantation” involves the infusion of liver cells from a donated liver. This protocol, however, wastes many cells that do not integrate into the existing liver and such a treatment is usually little more than a stop-gap solution, since most patients require a liver transplant within a year of this treatment.

Human-induced pluripotent stem (iPS) cells are another possibility but, so far, iPS cells differentiate into immature rather than mature, functional, proliferative hepatocytes.

A need remains for a robust treatment that can eliminate the need for immunosuppressive theory. “We hypothesized that by modifying the protocol used to generate intestine, we would be able to develop liver organoid units that could generate functional tissue-engineered liver when transplanted,” said Dr. Grikscheit.

Grikscheit and her co-workers extracted hardy, multicellular clusters of liver cells known as liver organoid units (LOUs) from resected human and mouse livers. These LOUs were loaded onto scaffolds made from nonwoven polyglycolic acid fibers. These scaffolds are completely biodegradable and they provide a structure upon which the LOUs can grow, fuse, and form a structure that resembles a liver.

After transplantation of the LOU/scaffold combinations, they generated tissue-engineered livers or TELis. Tissue-engineered livers developed from the human and mouse LOUs and possessed a variety of key liver-specific cell types that are required for normal hepatic function. However, the cellular organization of these TELis did differ from native liver tissue.

The tissue-engineered livers (TELis) made by Grikscheit’s laboratory contained normal liver components such as hepatocytes that properly expressed the liver-specific protein albumin, CK19-expressing bile ducts, vascular structures surrounded by smooth muscles that expressed smooth muscle-specific actin, desmin-expressing stellate cells, and CD31-expressing endothelial cells. The production of albumin by the TELi hepatocytes indicated that these cells were executing their normal secretory function. In a mouse model of liver failure, their tissue-engineered liver provided some hepatic function. In addition, the hepatocytes proliferated in the tissue-engineered liver.

A cellular therapy for liver disease that utilizes technologies like this would completely change the treatment options for many patients. In particular, children with metabolic disorders and require a new liver to survive might see particular benefits if such a treatment can come to the clinic. By generating functional hepatocytes comparable to those in native liver, establishing that these cells are functional and proliferative, Grikscheit and her colleagues have moved one step closer to that goal.

To access this paper, please see: Nirmala Mavila et al., “Functional Human and Murine Tissue-Engineered Liver Is Generated From Adult Stem/Progenitor Cells,” Stem Cells Translational Medicine, August 2016 DOI: 10.5966/sctm.2016-0205.

Factor From Umbilical Cord Blood Could Treat Harmful Inflammation

Umbilical cord blood turns out to have a factor that can potentially fight inflammation, according to scientists at the University of Utah School of Medicine. This study was published online Sept. 6, 2016, in The Journal of Clinical Investigation.

“We found something we weren’t expecting, and it has taken us to new strategies for therapy that didn’t exist before,” says Guy Zimmerman, M.D., a professor of internal medicine at the University of Utah School of Medicine, who was also the senior author of this work. Dr. Zimmerman collaborated with associate professor of pediatrics, Christian Con Yost, M.D., and their colleagues for this work.

Inflammation is well-known to anyone who has whacked their leg, been stung by a bee or a wasp, or anyone who over-stressed their muscles. The redness, heat pain, and swelling are signs that the body is cleaning up damaged cells and their debris, fighting invading microorganisms, and beginning the healing process. However, under certain circumstances, inflammation can go overboard and turn against us and seriously and chronically damage healthy tissues. Out-of-control inflammation is probably the culprit behind several different conditions ranging from rheumatoid arthritis to sepsis. In fact, the inflammatory overreaction to infections is one of the most common causes of hospital deaths.

Dr. Yost and his coworkers successfully isolated a cord blood factor, called “neonatal NET inhibitory factor” or nNIF. This name comes from the ability of this factor to inhibit “NETs” or neutrophil extracellular traps. NETs or neutrophil extracellular traps are composed of processed chromatin bound to granular and selected cytoplasmic proteins that are released by white blood cells called neutrophils. NETs seem to be a kind of last resort that neutrophils turn to in order to control microbial infections. Even though NETs usually help our bodies ward off infectious bacteria and viruses, they can also damage blood vessels and organs during sepsis.

As physicians who have treated critically ill patients suffering from out-of-control inflammation, Drs. Zimmerman and Yost recognized the therapeutic potential of nNIF. “We knew we were onto something that could be very meaningful,” recalls Yost.

To test if this cord blood-based factor could control sepsis, Zimmerman and Yost and others treated groups of mice that suffered from laboratory-induced inflammatory disease. In the absence of treatment, only 20 percent of the mice survived longer than two to four days. However, 60% of those mice treated with nNIF survived after the same amount of time.

“Sepsis is a case where the body’s reaction to infection is lethal,” says Yost. “nNIF is offering insights into how to keep the inflammatory response within prescribed limits.” He adds that they will carry out additional studies to test the therapeutic properties of nNIF.

Anti-Inflammatory Agent Isolated From Umbilical Cord Blood
Infection fighting cells from umbilical cord blood (left) and circulating blood three days after birth (right) from the same prematurely born baby. Umbilical cord blood has high levels of a factor, called neonatal NET inhibitory factor (nNIF), which inhibits a specific inflammatory response called NETs. Within two weeks after birth, nNIF levels drop and NETs can form. True to their name, they consist of a net-like substance that traps infectious agents like bacteria, as seen on the right. nNIF is showing promise as a potential therapy against harmful inflammation and sepsis.

nNIF seems to be present for just a brief window of time at the beginning of life. It circulates in cord blood and persists in the baby’s own bloodstream for up to two weeks after birth. However, after two weeks, nNIF disappears and is not found in older babies and is completely absent from the blood of adults. Scientists in Yost’s laboratory also discovered that the placenta also contains a similar, albeit less potent, anti-inflammatory agent. The evanescent nature of these factors possibly indicates that inflammation is under tight control during this time, since the fragility of young babies might make extensive amounts of inflammation deleterious to their health.

“The beginning of life is a delicate balance,” says Yost. “Our work is showing that it is important to have the right defenses, but they have to be controlled.”

AUF1 Gene Important Inducer of Muscle Repair

A new study in the laboratory of Robert J. Schneider at NYU Langone and his collaborators has uncovered a gene that plays integral roles in the repair of injured muscle throughout life. This investigation shows that this previously “overlooked” gene might play a pivotal role in “sarcopenia,” which refers to the loss of muscle tissues with age.

This collaboration between scientists at NYU Langone Medical Center and the University of Colorado at Boulder showed that the levels of a protein called AUF1 determine if stem cell populations retain the ability to regenerate muscle after injury and as mice age.

Changes in the activity of AUF1 have also been linked by past studies to human muscle diseases. More than 30 genetic diseases, known collectively as myopathies, show defective muscle regeneration and these anomalies cause muscles to weaken or waste away.

For example, muscular dystrophy is a disease in which abnormal muscles fail to function properly and undergo normal repair. Although the signs and symptoms of Duchenne Muscular Dystrophy vary, in some cases wildly, this disease develops in infants and affects and weakens the torso and limb muscles beginning in young adulthood. Sarcopenia, in healthy individuals occurs in older patients.

Skeletal muscles have a stem cell population set aside for muscle repair known as satellite cells. These cells divide and differentiate into skeletal muscle when skeletal muscle is damaged, and as we age, the capacity of muscle satellite cells to repair muscle decreases.

AUF1 is a protein that regulates muscle stem cell function by inducing the degradation of specific, targeted messenger RNAs (mRNAs). According to Robert Schneider, “This work places the origin of certain muscle diseases squarely within muscle stem cells, and shows that AUF1 is a vital controller of adult muscle stem cell fate.” He continued: “The stem cell supply is remarkably depleted when the AUF1 signal is defective, leaving muscles to deteriorate a little more each time repair fails after injury.”

The experiments in this study demonstrated that mice that lack AUF1 display accelerated skeletal muscle wasting as they age. These AUF1-depleted mice also showed impaired skeletal muscle repair following injury. When the molecular characteristics of these AUF1-depleted muscle satellite cells were examined, Schneider and his collaborators showed that auf1−/− satellite cells had increased stability and overexpression of so-called “ARE-mRNAs.” ARE mRNAs contain AU-rich elements at their tail-ends. AUF1 proteins bind to these ARE mRNAs and induce their degradation. In the absence of AUF1, muscle satellite cells accumulate ARE mRNAs. One of these ARE mRNAs includes that which encodes matrix metalloprotease, MMP9. Overexpression of MMP9 by aging muscle satellite cells causes degradation of the skeletal muscle matrix, which prevents satellite-cell-mediated regeneration of muscles. Consequently, the muscle satellite cells return to their quiescent state and fail to divide and repair skeletal muscle.

When Schneider and his coworkers and collaborators blocked MMP9 activity in auf1−/− mice, they found that they had restored skeletal muscle repair and maintenance of the satellite cell population.

These experiments suggest that repurposing drugs originally developed for cancer treatment that blocks MMP9 activity might be a way to dial down age-related sarcopenia.

“This provides a potential path to clinical treatments that accelerate muscle regeneration following traumatic injury, or in patients with certain types of adult onset muscular dystrophy,” said Schneider.

This work was published here: Devon M. Chenette et al., “Targeted mRNA Decay by RNA Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate, Promoting Skeletal Muscle Integrity,” Cell Reports, 2016; DOI: 10.1016/j.celrep.2016.06.095.

http://newdrugapprovals.org/2016/09/05/novel-isoform-selective-inhibitor-of-histone-deacetylase-8-hdac8/

Intravenous Administration of CardioCell’s Ischemic-Tolerant Mesenchymal Stem Cells Decreases Inflammation in Heart After Heart Attack and Prevents Remodeling

The San Diego, California-based biotechnology company, CardioCell, has sponsored a preclinical study in laboratory mice that explored giving preconditioned mesenchymal stem cells intravenously to animals that had suffered large heart attacks. This study “Mesenchymal stem cells grown under chronic hypoxia traffic to regions of myocardial infarction, suppress splenic natural killer cells, and attenuate adverse remodeling in mice with large acute MI” at the European Society of Cardiology (ESC) Congress by one of the authors, Dr. Michael Lipinski, ‎Interventional Cardiologist at MedStar Washington Hospital Center. Lipinski collaborated with Drs. Dror Luger, Research Scientist at Washington Hospital Center and Stephen Epstein, Director, Translational and Vascular Biology Research at MedStar Heart and Vascular Institute, Chair of CardioCell’s Scientific Advisory Board and Member of CardioCell’s Heart Failure Advisory Board.

The vast majority of stem cell-based studies to treat heart attacks have examined stem cell injections directly into the heart muscle or using techniques associated with stent placement to administer stem cells through the blood vessels that surround the heart. Stem cell injection directly into the heart muscle requires special equipment and personnel who have special training. This procedure also carries the risk of rupture of the wall of the heart, even though skilled practitioners can reduce such risks. Intracoronary administration of stem cells does not require specialized training or equipment, since any cardiologist can perform such a procedure. However, several studies have shown that the vast majority of the stem cells administered in this fashion end up in the lung. Intravenous administration would potentially be the safest and easiest way to administer such stem cells. The problem with intravenous (IV) administration of stem cells is that no one has been able to show that IV administration of stem cells works for heart attack patients.

The problem is that IV stem cells do not know where to go. However, some IV-administered stem cells do find their way to the damaged heart. How can we make the majority of such stem cells go to where they are so badly needed?

Several experiments have examined ways to do this. In particular, mesenchymal stem cells (MSCs) move towards increasing concentrations of a small protein called “stromal cell-derived factor-1” (SDF-1). The receptor for SDF-1, CXCR4 (a member of the Src family of protein kinases, for those who are interested) binds SDF-1, and when it does so, it kicks the cell in the rump, wakes it up, and drives toward higher and higher concentrations of SDF-1. Several laboratories have forced the expression of higher levels of CXCR4 in stem cells in order to improve their ability to home to damaged tissues, because damaged tissues express SDF-1. Such a strategy works (one example, see Cheng, M., et al., J Mol Cell Cardiol. 2015;81:49-53), and even increases tissue healing, but increasing CXCR4 levels in stem cells without resorting to genetic engineering techniques is preferable, since such a procedure would not only be safer and less expensive, it would require fewer regulatory hurdles to pass FDA muster (see Park JS, et al., Methods. 2015;84:3-16).

CardioCell’s new preclinical study has examined the ability of ischemia-tolerant mesenchymal stem cells (itMSCs), when administered intravenously, to heal the heart after a heart attack. They examined the ability of IV administration of itMSCs to prevent the deterioration of the left ventricle and the onset of “remodeling” that occurs after a heart attack (i.e. “remodeling” refers to the enlargement of the heart that leads to heart failure). Secondly, this study examined the ability of itMSCs to reduce the inflammation that develops after a heart attack and responsible for the continued death of heart muscle and heart blood vessel cells.

It has been well established that itMSCs secrete factors that have marked abilities to staunch inflammation. Therefore this study was specifically designed to determine if intravenously administered itMSCs can improve cardiac function following a heart attack and if such improvements were mediated by systemic anti-inflammatory activities. According to Stephen Epstein, “The study impressively demonstrates the validity of these concepts. IV itMSC administration indeed improves cardiac function, and the itMSCs achieve this – at least, in part, – by their anti-inflammatory effects and abilities to decrease NK cells. These findings can profoundly impact future strategies for treating patients with AMI.”

For this study, CD1 male mice were given heart attacks by surgically occluding the left anterior descending artery (LAD). After surgery, these mice were given infusions of itMSCs into their tail veins. These itMSCs were continuously grown at 5% O2. Stem cell infusions were given 24 hours following the experimentally-induced heart attacks. The infused stem cells were labeled with a radioisotope (indium-111 oxine), which allowed them to be easily visualized in the bodies of the mice. Each animal was infused with one million cells. So-called “ex vivo phosphor” imaging of the heart was performed 24 hours following itMSC injection.

In a separate study, mice were subjected to baseline echocardiography followed by surgery in which they were given heart attacks. Then 24 hours later, the mice were randomized to two groups, one of which received an infusion of two million itMSCs and the other of which received infusions of physiological saline solution. There were 16 mice in both groups.

Echocardiography was repeated at 3, 7 and 21 days after the induction of the heart attack. Blood, spleen and hearts were then harvested and the heart were stained with TTC staining of the hearts — TTC staining identifies dead versus live tissue. Live tissue stains a deep red color and dead tissue appears white.

Radiolabeled itMSCs preferentially homed to regions of myocardial injury. However, it must be admitted that the total number of the injected cells that engrafted in the myocardium was small. There was minimal homing in control mice that had been given stem cell infusions but were not given heart attacks. The size of the infarction was no different in the two groups as ascertained by TTC staining (28±3% for itMSC group vs. 25±3% for control group). As expected, control saline-treated mice with large infarcts that covered more than 25% of the left ventricle showed an increase in adverse compared to mice with smaller infarcts. However, mice treated with itMSCs did not demonstrate an increase in adverse remodeling, regardless of the size of their infarcts. This seems to demonstrate that itMSCs prevented the adverse LV remodeling occurring in mice with large infarcts.

Additionally, the heart wall thickness were greater in the itMSC group both during contraction and relaxation compared with the control group. Importantly, itMSC injection caused a significant decrease in splenic Natural Killer cells compared with control injection (2.6±0.13 vs. 3.4±0.36, p<0.04). This is important, because Natural Killer (NK) cells play a major role in post-heart attack inflammation and cause to good deal of the damage to the heart after a heart attack. Other experiments in cell culture showed that itMSCs significantly suppress NK cell proliferation as a consequence of the cocktail of molecules they secrete.

This study was predicated upon and entirely different strategy than the paradigms that have driven previous stem cell experiments. The vast majority of previous stem cell experiments assumed that implanted stem cells improve cardiac outcomes by regenerating heart tissue or by stimulating endogenous stem cell populations to engraft into the myocardium and regenerate the damaged heart. Thus, the greater the number of stem cells that engraft into the heart, the better.

In this study, a different paradigm was being tested. In this case, IV delivery results in very low numbers of cells engrafting into the damaged myocardium. However, the stem cells are not expected to contribute to myocardial regeneration; instead they are expected to stem the excessive immune or inflammatory responses that cause the progressive deterioration of the heart that dogs heart attack patients. In this study, the authors wished to examine if IV administered itMSCs grown under chronic hypoxic conditions could improve myocardial function and adverse remodeling, and if a functional benefit occurs, does the anti-inflammatory effects of itMSCs play an important mechanistic role in this improvement. In this study, both hypotheses seem to be valid.

CardioCell’s itMSCs secrete higher levels of growth factors and other important proteins associated with neoangiogenesis and healing and also seem to home to damaged tissues better than MSCs grown under normal conditions. This preclinical study might provide fodder for further experiments and discussions in the months and years to come.

 

Behavior Of Brain Stem Cells Controlled By Cerebrospinal Fluid Signals

The choroid plexus is a network of blood vessels in each ventricle of the brain. It is derived from the pia mater and produces the cerebrospinal fluid.  The choroid plexus, unfortunately, has been ignored to some degree when it comes to brain research.  However, CSF turns to be an important regulator of adult neural stem cells, research indicates.

A new study led by Prof. Fiona Doetsch at the Biozentrum of the University of Basel, Switzerland has shown that signals secreted by the choroid plexus dynamically change during aging, and these different signals affect the behavior of aged stem cells.

In the adult brain, neural stem cell populations in various places throughout the central nervous system divide to give rise to neurons and glial cells throughout our lives. These stem cells reside in unique micro-environments (known as niches) that provide key signals that regulate stem cell self-renewal and differentiation. Stem cells in the adult brain contact the ventricles, which are cavities in the brain filled with CSF. CSF bathes and protects the brain and is produced by the cells of the choroid plexus.

Doetsch and her coworkers have shown that the choroid plexus is a key component of the stem cell niche, and that the properties of this stem cell niche change throughout life and affect stem cell behavior.

Doetsch’s group discovered that the choroid plexus secretes a cocktail of important signaling factors into the CSF. These CSF-secreted growth factors are important in stem cell regulation throughout life. As we age, the levels of stem cell division and formation of new neurons decrease. They also showed that although stem cells are still present in the aged brain, and have the capacity to divide, their ability to do so have significantly decreased.

“One reason is that signals in the old choroid plexus are different. As a consequence, stem cells receive different messages and are less capable to form new neurons during aging. In other words, compromising the fitness of stem cells in this brain region,” said Violeta Silva Vargas, first author of the paper that appeared in the journal Cell Stem Cell. “But what is really amazing is that when you cultivate old stem cells with signals from young fluid, they can still be stimulated to divide, behaving like the young stem cells.”

In the future, Doetsch and her group plans to tease out the composition of the signaling factors secreted by the choroid plexus.  They would also like to know how the composition of this growth factor cocktail changes as a result of changes in brain states and how these changes affect neural stem cells. This could provide new ways to understand brain function in health and disease.

“We can imagine the choroid plexus as a watering can that provides signals to the stem cells. Our investigations also open a new route for understanding how different physiological states of the body influence stem cells in the brain during health and disease, and opens new ways for thinking about therapy,” said Doetsch.

This work was published here: Violeta Silva-Vargas et al., “Age-Dependent Niche Signals from the Choroid Plexus Regulate Adult Neural Stem Cells,” Cell Stem Cell, 2016; DOI: 10.1016/j.stem.2016.06.013.

New Antibody Drug Clears Brain of Amyloid Plaques and Delays Onset of Alzheimer’s Disease Symptoms in Small Clinical Trial

An experimental drug called aducanumab seem to be able to remove the toxic proteins that build up and cause the onset of Alzheimer’s disease in the brain, according to findings from a small clinical trial. Because of the small size of this trial, I must stress that these results, though potentially exciting, should also elicit some caution.

The results of this small clinical trial were reported in the journal Nature on August, 31, 2016. In this trial, aducanumab dissolved amyloid-β proteins in patients suffering from early-stage Alzheimer’s disease. This was a Phase I clinical trial, and therefore, was designed mainly to test the safety of aducanumab in human patients. Thus, the final word on whether aducanumab works to mitigate the memory losses and cognitive decline associated with Alzheimer’s disease must be subjected to clinical trials specifically designed to test such things. Two larger phase III trials are presently in progress, and are planned to be completed approximately in 2020 (note: this is an estimate).

The latest study enrolled 165 subjects who were split into different groups; subjects in one group received aducanumab and subjects in the other group were administered a placebo. In the group that received aducanumab infusions, 103 patients were given the drug once a month for up to 54 weeks. These patients experienced a reduction in the amount of tangled amyloid-β in their brains. These clinical recapitulated the results of pre-clinical experiments in laboratory mice that were actually reported in the same paper. Aducanumab seems to clear amyloid-β plaques from the brains of laboratory mice and human patients.

“This drug had a more profound effect in reversing amyloid-plaque burden than we have seen to date,” says psychiatrist Eric Reiman, who serves as executive director of the Banner Alzheimer’s Institute in Phoenix, Arizona. Reiman and his colleagues are in the process of testing other approaches for Alzheimer’s prevention and treatment. “That is a very striking and encouraging finding and a major advance.” Reiman wrote a commentary accompanying the article.

“This is the best news we’ve had in my 25 years of doing Alzheimer’s research, and it brings hope to patients and families affected by the disease,” says neurologist Stephen Salloway of Butler Hospital in Providence, Rhode Island, who is a member of the clinical team that ran the trial.

Patients in those groups that received aducanumab were divided into different subgroups that were given one of four different doses. Those patients who received the highest doses also had the highest reductions in plaques, and a group of 91 patients who had been treated for 54 weeks saw slower cognitive declines than did those who received placebo infusions.

Neuroscientists have had a long-standing and often spirited debate over the significance of the accumulation of amyloid-β in the pathology of Alzheimer’s disease. The memory loss and other symptoms of Alzheimer’s disease almost certainly result from the die-off of neurons in the brain, but do the amyloid-β plaques form as a consequence of this massive neuronal die-off or are they the cause of it? This clinical trial seems to provide good evidence for the “amyloid hypothesis,” since the elimination of amyloid-β protein seems to ameliorate the symptoms of Alzheimer’s disease.

Reiman however, cautions, wisely I think, that this trial is too small to definitively demonstrate that aducanumab actually works. Several other drugs for Alzheimer’s disease have shown promising results in the early-stage of clinical trials only to end in failure, and even in the deaths of patients.

Aducanumab led to abnormalities on brain-imaging scans in less than one-third of the patients. Researchers must closely monitor these anomalies in Alzheimer’s trials, because some participants in previous Alzheimer’s antibody trials have died as a result of brain inflammation. Fortunately, all of the reported imaging abnormalities eventually disappeared in about 4 to 12 weeks, and none of the patients who showed such abnormalities were hospitalized. Curiously, some of the patients who showed imaging anomalies continued to take the drug despite these side effects. Patients who received higher doses of the drug, or who had genetic risk factors for Alzheimer’s, were more likely to develop the brain anomalies.

Biogen, the company that makes aducanumab, has adjusted the drug’s dosage and the monitoring schedule for patients who have an increased genetic risk for Alzheimer’s in its phase 3 trials. According to Reiman, drug makers, like Biogen, must determine if a particular dosage that hits a “sweet spot” that is strong enough to work without causing potentially lethal brain inflammation.

Aducanumab is a bright spot in the field of Alzheimer’s therapeutics after years of failed antibodies and other types of drug trials. The antibody drug solanezumab failed to slow cognitive decline in two large 2013 clinical trials.  However solanezumab may have a second life and is being tested in multiple other trials, one of which includes individuals with mild Alzheimer’s disease. Results from this trial might be reported as early as the end of 2016.

Other therapeutic strategies undergoing clinical trials include strategies that target enzymes called β-secretase 1 that processes amyloid proteins, antibodies that attack the so-called the microtubule-binding tau protein, which is found in high concentrations in the neurofibrillary tangles found in the brains of many Alzheimer’s disease patients.

“The fact that we now have an antibody that gets into the brain sufficiently enough to engage its target and remove plaques is an important development, and we look forward to seeing results from this and other phase 3 trials,” Reiman says.