Researchers Grow Retinal Ganglion Cells in the Laboratory

Researchers from laboratory of Donald Zack at The Johns Hopkins University in Baltimore, Maryland have used genome editing methods to efficiently differentiate human pluripotent stem cells into retinal ganglion cells. Retinal ganglion cells are found in the retina that and helps transmit visual signals from the eye to the brain. Abnormalities or death of ganglion cells can cause vision loss, and conditions such as glaucoma and multiple sclerosis can wreak havoc on ganglion cells.

“Our work could lead not only to a better understanding of the biology of the optic nerve, but also to a cell-based human model that could be used to discover drugs that stop or treat blinding conditions,” said Zack, who is the Guerrieri Family Professor of Ophthalmology at the Johns Hopkins University School of Medicine. “And, eventually it could lead to the development of cell transplant therapies that restore vision in patients with glaucoma and MS.”

Published in the journal Scientific Reports, Zack and his team genetically modified a line of human embryonic stem cells so that they would fluoresce once they differentiated into retinal ganglion cells. Then they used these cells to develop new differentiation methods and characterize the resulting cells.

To genetically modify their cells, Zack and others used the CRISPR-Cas9 system. CRISPR stands for “clustered regularly interspaced short palindromic repeats” and these are short segments DNA, which are found in bacteria, contain short repeated sequences. Following each repeated sequence is a short spacer that usually comes from previous exposures to a bacterial virus or plasmid. Bacteria use the CRISPR/Cas system as a kind of immune system that prevents cells from being invaded by foreign DNA. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaeal genomes.

When bacteria are invaded by a virus, the particular Cas nucleases capture the viral DNA, cut it and insert it into the CRISPR array. When the bacterial cell is infected by a virus, an RNA is transcribed from the CRISPR array called the crRNA. This crRNA then hybridizes with the invading DNA or RNA and the double-stranded RNA or DNA/RNA hybrid is degraded by Cas proteins.

The CRISPR/Cas system is a useful laboratory tool for gene editing or adding, disrupting or changing the sequences of particular genes. If Cas9 and the appropriate crRNA are delivered into cells, you can cut a genome almost anywhere. CRISPR has a huge number of potential applications.

Zack and his group used the CRISPR/Cas system to insert a fluorescent protein gene into the DNA of their stem cells line. This red fluorescent protein would be expressed if a gene called BRN3B (POU4F2) was also expressed. BRN3B is expressed by mature retinal ganglion cells. Therefore, once these cells differentiated into retinal ganglion cells, they would glow red when viewed with a fluorescence microscope.

After differentiating their cells, Zack and his coworkers used a technique called fluorescence-activated cell sorting to isolate fully differentiated cells from other cells. The pure cell culture contained cells that displayed the biological and physical properties observed in retinal ganglion cells produced naturally, according to Zack.

As an added bonus, Valentin Sluch, a former graduate student in Zack’s laboratory, and her colleagues discovered that soaking the pluripotent stem cells in a chemical called “forskolin” at the commencement of the differentiation protocol significantly improved the efficiency of differentiation. Forskolin is a labdane diterpene found in the roots of the Indian Coleus plant (Coleus forskohlii), which belongs to the mint family.  It is used by some people as a weight loss supplement by some people.

“By the 30th day of culture, there were obvious clumps of fluorescent cells visible under the microscope,” said Sluch, who is now a postdoctoral scholar working at Novartis. Sluch continued, “I was very excited when it first worked. I just jumped up from the microscope and ran [to get a colleague]. It seems we can now isolate the cells and study them in a pure culture, which is something that wasn’t possible before.”

“We really see this as just the beginning,” adds Zack. In follow-up studies using CRISPR, his lab is looking to find other genes that are important for ganglion cell survival and function. “We hope that these cells can eventually lead to new treatments for glaucoma and other forms of optic nerve disease.”

To use these cells to develop new treatments for Multiple Sclerosis, Zack is collaborating with Dr. Peter Calabresi, professor of neurology and director of the Johns Hopkins Multiple Sclerosis Center.

Next-Generation Cell Therapy for Graft-Versus-Host Disease

Endonovo Therapeutics, Inc has announced its development of a cell-based treatment for Graft-versus-Host Disease (GvHD). This treatment utilizes umbilical cord blood stem cells that have been grown and enhanced by specific treatments.

GVHD occurs when newly transplanted donor cells attack the recipient’s body. It can occur after a bone marrow or stem cell transplant if the cells have not been properly matched or even if the donor and recipient are relatively well matched. The chances of suffering GVHD are around 30 – 40% if the donor and recipient are genetically related and close to 60 – 80% when the donor and recipient are not related.

GVHD can be either acute or chronic and the symptoms of GvHD can be either mild or severe. Typically, acute GVHD comes on within the first 6 months after a transplant. Common acute symptoms include: Abdominal pain or cramps, nausea, vomiting, and diarrhea, Jaundice (yellow coloring of the skin or eyes) or other liver problems, skin rash, itching, redness on areas of the skin. Chronic GVHD usually starts more than 3 months after a transplant, and can last for the lifetime or the patient. The symptoms of chronic GvHD include: dry eyes or vision changes, dry mouth, white patches inside the mouth, and sensitivity to spicy foods, fatigue, muscle weakness, and chronic pain, joint pain or stiffness, skin rash with raised, discolored areas, as well as skin tightening or thickening, shortness of breath, weight loss.

Endonovo uses a novel method to enhance stem cells. Their so-called “Cytotronics platform” utilizes Time-Varying Electromagnetic Field (TVEMF) technology to expand and enhance the therapeutic properties of stem cells and other types of cells for regenerative treatments and tissue engineering. This platform can potentially optimize cell-based therapies so that they have greater therapeutic potential than they had prior to their treatment.

The Cytotronics™ platform dates back to experiments conducted at NASA to expand stem cells in culture. NASA’s goal was to create stem cell therapies that could be used to treat astronauts during long-term space exploration. NASA scientists showed that Time-Varying Electromagnetic Fields (TVEMF) could stimulate the expansion of stem cells in the lab. Additionally, TVEMF increased the expression of dozens of genes related to cell growth, tumor suppression, cell adhesion and extracellular matrix production.

By testing and tweaking this technology over a period of 15 years, Endonovo scientists created a novel protocol for augmenting the therapeutic properties of cells in culture through physics rather than genetic engineering. The Cytotronics™ platform seems to be able to make stem cells that express higher levels of key genes necessary for tissue healing and regeneration.

As an example of the efficacy of this technology. Endonovo scientists have shown that Cytotronic™ expansion of peripheral blood stem cells resulted in an over 80-fold expansion of CD34+ cells in as little as 6 days.

Endonovo is using the Cytotronic platform to enhance the regenerative properties of mesenchymal stem cells (MSCs), which have the capacity to staunch inflammation in patients with GvHD and other inflammatory diseases.

However, despite their promise, MSC-based therapies suffer from poor engraftment and short-term survival when transplanted into sick patients. These remain major limitations to the effective therapeutic use of MSCs. If there was a safe and effective way to beef up the survival and regenerative properties of MSCs, such a technique would be indispensable.  This makes MSCs prime candidates for the Cytotronic Platform.

Dr. Donnie Rudd, Chief Scientist & Director of Intellectual Property at Endonovo, said: “Our Cytotronics platform is particularly suited to address many of the issues that have plagued stem cell therapies that have recently failed, such as their loss of potency and self-renewal when expanded ex vivo, their poor engraftment and their limited ability to survive when transplanted.”

Earlier this year, Endonovo announced a protocol for the creation of a cell mixture from a portion of the human umbilical cord co-cultured with adipose-derived stem cells. This resulting cell mixture contains a rich source of highly-proliferative, immunosuppressive cells that are not recognized by the patients immune system, since they contain neither of the major histocompatibility markers (HLA double negative). These cells are “immune privileged,” which means that are not recognized as foreign cells by the patient’s immune system, and therefore are a significant source of cells for MSC-based therapies.

Endonovo Therapeutics has used this new technology to create a biologically potent, off-the-shelf, allogeneic treatment for Graft-Versus-Host disease and a wide-array of other conditions. They would like to test these products in clinical trials eventually.

Endonovo hopes that stem cells enhanced by the Cytotronics™ platform will become a major innovation in the regenerative medicine market.

“We are very excited to be a leader in the development of next-generation, ex vivo enhanced cells for regenerative medicine,” stated Endonovo CEO, Alan Collier. “We have seen several stem cell therapies fail in clinical trials over the last couple of years, which points to a critical need for the development of methods to increase the biological and therapeutic properties of stem cells.”

“We believe that enhancing the biological and therapeutic properties of stem cells using bioelectronics is the future of cell-based therapies,” concluded Mr. Collier.

Capricor Reports Encouraging Results in its DYNAMIC Trial

Capricor Therapeutics, Inc., located in Beverly Hills, CA, has announced their six-month safety and adverse event data from a Phase I clinical trial of their CAP-1002 product for patients with advanced heart failure. This clinical trial is part of the DYNAMIC or which is short for Dilated cardiomYopathy iNtervention with Allogeneic MyocardIally-regenerative Cells trial whose goal is to evaluate CAP-1002 in patients with advanced heart failure.

CAP-1002 is Capricor’s lead investigational allogeneic, cardiosphere-derived cell (CDC) therapy. Allogeneic means that the cells come from someone other than the patient. The advantage of allogeneic cells is that they come from healthy donors whose cells have not been ravaged by old age or other conditions. These cells do not need to be matched to the patient’s immune system in this case because they help the heart through indirect means (see Tseliou E, et al., J Am Coll Cardiol. 2013 Mar 12;61(10):1108-19).  Cardiospheres are cells taken from the hearts of healthy patients that grow in culture as small balls of cells. Because these cells are derived from the heart and grow as spheres, they are called cardiospheres (see Cheng K, et al., JACC Heart Fail. 2014 Feb;2(1):49-61.).

Cardiospheres have been shown in small clinical trials (the CADUCEUS trial) to replace the heart scar with heart muscle (see Malliaras K, et al., Am Coll Cardiol. 2014 Jan 21;63(2):110-22).  Animal studies in rats showed similar results (see above).

CAP-1002 is an off-the-shelf “ready to use” cardiac cell therapy that consists of cells that come from donor heart tissue and is infused directly into a patient’s coronary artery during a catheterization procedure. This Phase I study is meant to determine if CAP-1002 is safe and effective in treating heart function and structure. In particular, Capricor scientists are interested in determining if CAP-1002 cells can decrease heart scar tissue and promote the growth of heart muscle. In doing so, this regenerative treatment might delay or even prevent the onset of heart failure. The US Food and Drug Administration has granted CAP-1002 an orphan drug designation for the treatment of cardiomyopathy associated with Duchenne Muscular Dystrophy.

Capricor’s Cardiosphere-Derived Cells are a unique therapeutic product that were created in the laboratory of company Co-Founder and Scientific Advisory Board Chairman, Dr. Eduardo Marbán, who is the Director of the Heart Institute at Cedars-Sinai Medical Center.

All patients in this trial have advanced heart failure and have progressed to a more advanced stage of the disease. Patients received CAP-1002 in up to three coronary arteries, which delivers the cells to the more of the diseased parts of the heart. Since these patients have significant fibrosis in all areas of the heart, this delivery system is optimal for these patients. Cell delivery will also utilize methods that do not stop blood flow, which will decrease patient discomfort during cell delivery.

The data from this trial, so far, comes from 14 patients who were diagnosed with either dilated cardiomyopathy or non-ischemic dilated cardiomyopathy. These patients have ejections fractions of 35% or less and are classified as New York Heart Association class III or Ambulatory Class IV heart failure.

The data collected to date show that CAP-1002 cells are safe and well tolerated and produced no adverse cardiac events at one month or six months after they were infused into the patient’s hearts. Although DYNAMIC was designed as a Phase I clinical trial that does not assess the efficacy of CAP-1002 cells, patients have also been tested for their subject wellbeing, exercise capacity (six-minute walk test), ejection fraction, and ventricular volumes.

According to the principal investigator Dr. Raj Makkar of Cedar-Sinai Medical Center, the data so far are rather encouraging, even beyond the positive safety data, since they are seeing “concordance between the clinical improvement and the physiological measurements of trends for improved ejection fraction and reverse re-modeling.” Dr. Makkar, however, emphasized that this clinical trial only tested a small cohort of patients, and these data must be confirmed in larger clinical trials.

Mesoderm Progenitor Cells With Reduced Tumor-Causing Potential Derived from Human Pluripotent Stem Cells

Karl Willert, PhD, associate professor in the Department of Cellular and Molecular Medicine at the University of California, San Diego and his colleagues have generated a new cell line in his laboratory that can potentially all the tissues in our bodies that are generated from mesoderm.

During embryonic development, 14 days after fertilization, the embryo is transformed from a single-cell thick sheet to a three-layered structure by a process called gastrulation. Gastrulation forms an outer layer of cells known as the ectoderm, which forms the skin and the nervous system, a middle layer of cells known as the mesoderm, which forms the muscles, heart, blood vessels, kidneys, gonads, dermis, adrenal glands, bones, and several other important tissues, and an innermost layer of cells called the endoderm, which forms the gastrointestinal tract as its associated structures. These three layers, the ectoderm, mesoderm, and the endoderm, are collectively known as the “primary germ layers” and they are formed at gastrulation.

Willert, in collaboration with co-corresponding author David Brafman from Arizona State University, used a high-throughput screening platform that had been previously developed in Brafman’s laboratory to define the exact cellular microenvironment that would drive pluripotent stem cells efficiently differentiate into mesodermal progenitor cells. Such cells could theoretically differentiate into any of the derivatives of the mesodermal germ layer, and these cells would also show a greatly reduced capacity to form tumors, since they are no longer pluripotent, but only multipotent.

After using their screening platform to differentiate human embryonic stem cells into cells that expressed mesodermal-specific genes, Willert and his team settled upon a microenvironment that differentiated these stem cells into intermediate mesodermal progenitor (IMP) cells that could be propagated in culture. Interestingly, these IMP cells had the ability to differentiate into mature kidney cells, without the risk of forming tumors. Oddly, these cells were not able to differentiate into other types of mesodermal derivatives.

“This work nicely complements recent advances in tissue engineering and the goal of rebuilding or recreating functional organs, such as what we’ve seen with the creation of ‘mini-kidneys’,” said Willert. “It represents a novel source of cells.” This study was published November 10, 2015 in the online journal eLIFE.

Extensive analyses showed that their IMP cells lacked tumor-forming potential. However, they retained the ability to differentiate into cells that compose the adult kidney. The ability to generate expandable populations of IMPs cells with limited differentiation have several advantages over pluripotent human stem cell cultures. First, pluripotent stem cell cultures can be differentiate into specific cell types but even under the best of conditions, such cell preparations can harbor undifferentiated cells that retain the potential to seed tumor growth. Secondly, it is much easier to manipulate and differentiate IMP cells than pluripotent stem cells. That simplifies the protocols for handling these cells, which also decreases the time and expense required to make anything from these cells.  Third, since IMP cells have limited differentiation capabilities, they are less likely than pluripotent stem cells to differentiate into unwanted cell types.

“Our cells can serve as building blocks to generate kidneys that may one day be suitable for cell replacement and transplantation,” said Willert. “I think such a therapeutic application is still a few years in the future, but engineered kidney tissue can serve as a powerful model system to study how the human kidney interacts with and filters drugs. Such an application would be of tremendous value to the pharmaceutical industry.”

Even though Willert’s IMP cells differentiated into kidney cells, Willert is optimistic that they are capable of differentiating into other mesodermal-derived cell types, like gonads. “We have only characterized their potential to differentiate into cells that contribute to the kidney. We are now investigating to what extent these cells can generate other tissues and organs that derive from intermediate mesoderm, including reproductive organs.”

Willert and his colleagues are using the same protocol to generate other expandable progenitor cell lines from pluripotent stem cells derived from other germ layers, such as ectoderm and endoderm.