Groundbreaking Microscopic and Macroscopic Discoveries


February 9, 2017
by Janice Fisher

The road to discovery of the Myo/Nog lineage began almost 30 years ago with the identification of 10 skeletal muscle stem cells that were hidden among a hundred thousand other cells—the veritable needle in the haystack.

Mindy George-Weinstein, PhD, chief research and science officer, has spent three decades discovering, targeting and closely observing Myo/Nog cells.“We were asking a relatively simple question, using the chick embryo as the model,” says Mindy George-Weinstein, PhD, chief research and science officer, Philadelphia College of Osteopathic Medicine. “The question was: When does skeletal muscle begin to develop in the embryo? The dogma at the time was that embryonic cells were naive until they received signals from the developing spinal cord to become programmed to form muscle.” But two reports in the literature caught the eye of Dr. George-Weinstein. Researchers Howard Holtzer, PhD, and Michael Solursh, PhD, had found that muscle could develop from early embryos before the formation of the spinal cord if cells were grown in the presence of a rich, undefined mixture of molecules. “We decided to take a closer look.”

The first step involved culturing embryonic cells in the absence of molecules seen within the embryo itself. Lo and behold, muscle appeared. Next, the George-Weinstein lab found 10 to 20 cells that contained MyoD, a molecule that drives skeletal muscle development. Visualizing MyoD was possible using three-dimensional DNA nanoparticles (3DNA) developed by PolyProbe, a company whose laboratory was housed at PCOM. The group then discovered that the cells with MyoD released a molecule called Noggin, an inhibitor of bone morphogenetic proteins (BMPs) that are present in the embryo and adults. (The scientists who had discovered Noggin applied this name because a mutated form produces malformations of the head.)

The cells found in the George-Weinstein lab were named Myo/Nog for their dual roles as precursors to muscle and factories for Noggin production. Myo/Nog cells were also identified with an antibody called G8. “The beauty of this antibody,” says Dr. George-Weinstein, “is that it binds to the cell surface. We could apply the antibody to living embryos to track Myo/Nog cells, isolate the cells to study their properties in culture, and kill them.”

For almost 20 years, the George-Weinstein lab studied the roles of Myo/Nog cells during embryonic development. “As organs are forming,” explains Dr. George-Weinstein, “Myo/Nog cells become integrated into many different tissues, some that have muscle and some that don’t.” The tiny population of Myo/Nog cells present in the early embryo is critical for muscle formation and development of multiple organs, including the eyes, heart and brain.

“One unique feature of Myo/Nog cells,” says Dr. George-Weinstein, “is their stubbornness to remain as both a muscle precursor cell and a cell that produces Noggin regardless of their environment.” As it turns out, Myo/Nog cells are also found in normal and diseased tissue of adult mice, rats, rabbits and humans. They not only produce Noggin; they migrate very quickly to sites of injury. That’s where the story gets even more interesting.


Overturning Dogma in New Discovery

Based on the lab’s discovery that killing Myo/Nog cells disrupts normal eye development in the chick embryo, they began to study their roles in the more mature lens and retina. In collaboration with a group of researchers at Thomas Jefferson University, they found that Myo/Nog cells developed into a type of muscle cell called a myofibroblast, in response to wounding of the chick lens. The George-Weinstein lab then made the leap to adult human eyes, where Myo/Nog cells were found in low numbers.

Again, the group found themselves overturning a dogma in the field. Myofibroblasts in the lens were presumed to develop from epithelial cells that along with their mature derivatives, the lens fiber cells, were thought to be the only cells in the lens. Using human lens tissue removed during cataract surgery, they discovered that Myo/Nog cells are the source of contractile myofibroblasts that resemble skeletal muscle cells. “The problem with myofibroblasts is that their contractions produce wrinkles in the capsule that surrounds the lens. These wrinkles can affect the pathway of light as it passes through the lens on its way to the retina,” says Dr. George-Weinstein. This phenomenon occurs in a disease called posterior capsule opacification (PCO), or secondary cataract. Between 15 and 30 percent of adults, and almost all children, get PCO after cataract surgery. This vision-impairing disease can be treated with laser, but side effects, some serious, can occur, and laser surgery isn’t available worldwide.

Since it can’t be predicted which adults will get PCO, ideally a drug that kills Myo/Nog cells could be given to everyone having cataract surgery. The George-Weinstein lab developed such a drug in collaboration with Robert Getts, PhD, vice-president of research and development and chief scientific officer at Genisphere LLC, a company that evolved from PolyProbe. The G8 antibody used for targeting the drug to Myo/Nog cells was attached to 3DNA nanoparticles containing a toxic molecule called doxorubicin that’s commonly used for cancer chemotherapy. In a recently published paper, the team reported that this novel drug specifically kills Myo/Nog cells and prevents the emergence of myofibroblasts in cultures of human lens tissue. In collaboration with veterinarians at the University of Utah, the drug is currently being tested for its ability to reduce PCO in rabbits undergoing cataract surgery.

Myo/Nog cells are also present in the retina. Arturo Bravo-Nuevo, PhD, at Jefferson University, and Alice Brandli, PhD, and Jonathan Stone, PhD, both at the University of Sydney, have explored the roles of Myo/Nog cells in two models of retinal disease—retinopathy of prematurity, which afflicted Stevie Wonder and Ray Charles, and light damage. Their publications describe the effects of either eliminating or adding Myo/Nog cells to the diseased retina. Myo/Nog cells were shown to be important for preserving the viability and function of photoreceptor cells that respond to light. The next step is to determine how Myo/Nog cells protect the retina. “If their protective effects are mediated by releasing a molecule, then hypothetically the molecule itself could be injected into the eye instead of the cells,” says Dr. George-Weinstein. “This approach would circumvent the formation of muscle from injected Myo/Nog cells whose contractions could have devastating effects on vision.”


Myo/Nog Cells: Good, Bad or Both?

“The take-home message from our work is that Myo/Nog cells might be good guys or bad guys—or even both, in the same organ. That’s what makes these cells so interesting to study,” says Dr. George-Weinstein. In the embryo and the retina, Myo/Nog cells orchestrate normal development and protect cells from damage. While Myo/Nog cells are critical for lens development, they contribute to PCO with their contractions. “In some wounds, such as diabetic ulcers, the addition of Myo/Nog cells may help with healing,” suggests Dr. George-Weinstein. On the other hand, sometimes injury leads to scar tissue formation, for example after a myocardial infarction. “We know that Myo/Nog cells are present in the heart, kidney and lungs. Do they mediate normal wound healing, deposit scar tissue, or both—and if the answer is both, what flips the switch?”

Myo/Nog cells are also present in tumors. The group has proposed that Myo/Nog cells may indirectly affect the behavior of cancer cells. In animal models, an increase in Noggin promotes skin cancer, and in human skin tumors, the number of Myo/Nog cells correlates with the extent of invasion. While Myo/Nog cells may act indirectly in some tumors, a second hypothesis, and one that Dr. George-Weinstein has been eager to test for years, is that the Myo/Nog cell itself might be the stem cell for cancers with muscle-like properties. In both scenarios, eliminating Myo/Nog cells may be therapeutic.

The road to discovery of the Myo/Nog lineage began almost 30 years ago with the identification of 10 skeletal muscle stem cells that were hidden among a hundred thousand other cells—the veritable needle in the haystack.

Mindy George-Weinstein, PhD, chief research and science officer, has spent three decades discovering, targeting and closely observing Myo/Nog cells. Her pioneering work is presently the focus of a multi-institutional consortium consisting of four separate yet interrelated research projects.


 

The Myo/Nog Cell Consortium

These questions and hypotheses will be addressed in a multi-institutional consortium of Myo/Nog cell researchers. Dr. George-Weinstein and Ms. Gerhart will continue their studies of targeting Myo/Nog cells in the lens to prevent PCO. Mark Byrne, PhD, of Rowan University is working with the scientists at Genisphere and Dr. George-Weinstein and Ms. Gerhart to develop a more effective method of drug delivery. Dr. Bravo-Nuevo and Nancy Philp, PhD, of Thomas Jefferson University will be studying the neuroprotective properties of Myo/Nog cells. The role of Myo/Nog cells in fibrosis and sarcomas will be investigated by Kathryn Behling, MD, PhD, of Cooper Medical School of Rowan University.

Dr. George-Weinstein describes the multi-institutional consortium as “an integrated and collaborative fact-finding mission that promotes the flow of ideas and data between investigators focused on a single cell type in multiple diseases.” Dr. George-Weinstein notes that the entire body of work was made possible by her 30-year collaboration with Ms. Gerhart.

“Jackie invented the techniques required for the experiments, generated and interpreted data, and contributed to the overall vision of the projects that were supported by PCOM, the NIH and multiple foundations over the years. Jackie’s work was described by reviewers of our manuscripts as a technical tour-de-force.” Many talented MS and DO students were also instrumental in uncovering the mysteries of Myo/Nog cells.

“It’s incredible,” reflects Dr. George-Weinstein, “that what appeared to be a simple question which we answered in the chick embryo has led to the development of potential therapies for human diseases. This extremely fulfilling journey, like most research, has had its struggles that were overcome with persistence and hard work. The winding path began at PCOM. We are thrilled to have returned to our roots for the next stage of the journey.”


Decades of Collaboration

When Jacquelyn Gerhart became PCOM’s laboratory coordinator of the scientific support staff and bio-imaging core facility in 2015, it was a definitive homecoming. Her campus home, the bio-imaging core facility in Evans Hall, is precisely where she began her PCOM career in 1987, right out of college. It was then the electron microscopy suite; she recalls two electron microscopes, one of them room-sized. Today, a range of more sophisticated devices have joined those microscopes—now much smaller and more powerful.

Ms. Gerhart is responsible for helping faculty and research staff from the Philadelphia and Georgia campuses with microscopy, cytology and related techniques. She also coordinates the efforts of the research support staff, participates in interviewing applicants for research positions, and orients new employees. Ms. Gerhart spends approximately 20 percent of her time conducting her own research.

In 1990, Ms. Gerhart began working with Dr. George-Weinstein when “the chick system was up and running. We were looking for an antibody that we could use to study skeletal muscle, and that’s where it all began.” The productivity and longevity of their collaboration (which continued even when they were not at PCOM) is perhaps no longer so unusual among contemporary women scientists. But Ms. Gerhart remembers chatting at a professional meeting with the late Elizabeth Hay, MD, a pioneering cell and developmental biologist. As they stood in a line for the women’s bathroom, Dr. Hay commented that she hadn’t always had to wait in such lines because there were so few women in the field.

The collaborative culture of PCOM was a powerful lure for Ms. Gerhart. She knows that if she’s not able to check on the well-being of her cultures containing human lens tissue, for example, “there’s always someone who is willing to help out, from the people we’ve hired right out of college to staff that have been here for 20 or more years.” Students volunteer to work at night; they tell her, “I live right here, and I can come in and take care of this for you.” Students who worked in the lab with Ms. Gerhart and Dr. George-Weinstein stay in touch, seeking updates on their research as well as opportunities to network among the web of connections formed in their nurturing environment. Ms. Gerhart has mentored biomedical studies students on the research track, as well as research-loving DO students. She also welcomed undergraduates from Cabrini College and high school students from Lower Merion High School, her alma maters.

The continuous opportunity to innovate sweetened her return to PCOM. “Some of the things we do haven’t been done before,” she notes, “or we had to modify someone else’s procedure to fit our project. Sometimes you have to sit there and think: ‘This is what we need to do. But how are we going to do it?’” In the early work with chick embryos, she recalls, “we had to figure out a way to grow the embryos so we could watch them develop in the absence of Myo/Nog cells. Right after the egg was laid we would cut a window in the shell and cover it with plastic wrap to watch them grow.” Ms. Gerhart, an artist, relied regularly on an art supply catalogue, following a recommendation by a fellow artist, the late Camille DiLullo, PhD, professor of anatomy, department of bio-medical sciences. “Tools for fine work in sculpture helped me manipulate embryos,” says Ms. Gerhart. “Crocus cloth, used to sand art projects, was perfect for sharpening our knives.”

Ms. Gerhart points to a desk drawer. “I have my box of tools right here, and no one is allowed to touch them; they are mine,” she says mildly. “I made them 20 years ago, and they still work and I still pull them out if I need them.” She remembers working with tissue sections “that are so thin you can barely see them; you have to float them on water and get the water to reflect just right to find them. To move them, you had to pull out an eyelash and glue it to a stick.”

Years ago, learning how to perform a dissection under the microscope, Ms. Gerhart worried, “I can’t even see what we’re cutting!” Finally, “the light was just right, and I saw it. So I tell students, don’t worry; eventually it’s just going to light up for you.” What’s necessary, she stresses, is patience. “You have to be willing to sit and try things, or it’s not going to work.

“Just the other day I called Mindy to say, ‘You’ve got to come over and see this!’ Mindy was as excited as I was. Leadership comes from the top down, so a leader who is excited will get everyone else excited. I think research at PCOM will do really well under her leadership as the chief research and science officer.”

Ms. Gerhart and Dr. George-Weinstein continue to work together to test potential therapeutics that target Myo/Nog cells. Ms. Gerhart collaborates with Dr. Getts and his staff at Genisphere; Dr. Byrne, at Rowan University; and Liliana Werner, MD, PhD, at the University of Utah, to develop a drug that kills Myo/Nog cells to prevent secondary cataract formation.


The George-Weinstein Lab: "A Brilliant Place to Learn"

Ms. Gerhart recalls “a fantastic PCOM student” who was recommended to the lab by his former undergraduate research mentor. The student was Mitchell B. Crawford, DO ’15. Dr. Crawford quickly learned how to dissect the delicate tissues of the embryo, a variety of histological procedures and novel approaches to culturing human tissue. “We relied upon Mitch’s imagination and ability to integrate information during brainstorming sessions in which we designed and interpreted experiments,” says Dr. George-Weinstein. Like other lab members, he coauthored several papers with Ms. Gerhart and Dr. George-Weinstein.

Dr. Crawford describes Ms. Gerhart and Dr. George-Weinstein as great teachers. “The lab was such a welcoming environment; Jackie and Mindy were so kind and so eager to teach and have me be a part of anything I was willing to help out with. It was just a brilliant place to learn.

“The lens project was pretty exciting,” he says. “We were doing things that we didn’t know how to do—and no one else did, either. How do you take human tissue straight from the OR without damaging it? What kinds of solutions should be used? What’s the right temperature and humidity for the incubators? We were learning as we went along.”

While Dr. Crawford misses working in the lab, he puts to use the skills he developed at the bench in multiple clinical research projects that engage him now, as a second-year psychiatry resident at the Harvard South Shore Psychiatry Training Program, Harvard Medical School/VA Boston Healthcare, Brockton, Massachusetts.

Dr. George-Weinstein’s concern with children who suffered from ocular diseases made a strong impression on Dr. Crawford. “We talked about an eye drop that might be given on mission trips to kids in Third World countries. Some people say that psychiatry isn’t the most prestigious or financially rewarding specialty. My experience with Mindy and the lab helped orient me and remember that it’s important to do what you think is important in life.”


Research Experience as a Springboard to a Career in Medicine

When Jordanna Perlman Quinn, DO, MS/Biomed ’02, joined the George-Weinstein lab, she began at the very beginning: by learning what a stem cell is. “It wasn’t yet known that stem cells programmed to produce skeletal muscle are present in the very early embryo,” she says. Dr. George-Weinstein and Ms. Gerhart “took me under their wing, and taught me how to work in a lab.”

Today, Dr. Quinn’s practice of regenerative medicine in Golden, Colorado, includes stem cell injections into her patients to help them grow cartilage.

Dr. Quinn, who received her DO from Western University of Health Sciences in Pomona, California, is board certified in physical medicine and rehabilitation, and maintains a musculoskeletal/sports-based practice with a special interest in a natural medical approach. She describes herself as “having one foot in Western medicine and another in the holistic approach.”

“Jordanna had a broad view of medicine and healing, and she embraced the osteopathic philosophy,” says Dr. George-Weinstein.

Dr. Quinn recalls “living in the lab, day in and day out, with Jackie or by myself, extracting stem cells from quail eggs and chick eggs. It was a lot of work—late nights, and intense.” She developed the protocol for isolating Myo/Nog cells, a procedure that is currently utilized for analyses of their properties in normal and diseased adult tissues. “Jordanna’s contributions were enormous. She was exceptionally smart, dedicated and an absolute pleasure to work with,” recalls Dr. George-Weinstein.

Dr. Quinn won the Sigma Xi Award for outstanding research, and was the coauthor (as Jordanna Perlman) of several papers with Ms. Gerhart and Dr. George-Weinstein. But her experiences in the lab influenced her beyond the realm of science. Throughout her career, Dr. Quinn has pondered how to inspire the people who work for her.

“I see now that Mindy was a great work model,” she says. “The atmosphere in the lab was wonderful, and that makes you want to work harder. We had so much fun, but not at expense of hard work.” Dr. Quinn adds, “Jackie and Mindy were both so passionate about their work. That’s infective; you can’t help but be interested.”


Expanding the Work of the George-Weinstein Lab

Besides Dr. George-Weinstein and Ms. Gerhart, the following PCOM-affiliated students, faculty and technicians performed experiments that led to publications, presentations and grants. Most are coauthors on one or more of 26 peer-reviewed published papers arising from the work of the George-Weinstein lab. The first of these papers was published in Developmental Biology in 1991. Their most recent manuscripts will appear in PLOS ONE and the Journal of Pharmacology and Experimental Therapeutics early in 2017.

Brian A. Bast, DO ’02
Michael C. Baytion, MS/Biomed ’98
Joanna N. Capparella, DO ’97
Eric T. Cochran, MS/Biomed ’97, DO ’01
Mitchell B. Crawford, DO ’15
Rocco J. Crescenzo, DO ’92, clinical assistant professor, internal medicine, PCOM
Jeffrey T. Dare, MS/Biomed ’97, DO ’02
Steven M. DeLuca, MS/Biomed ’98, DO ’02
Camille DiLullo, PhD, professor of anatomy, bio-medical sciences [deceased]
Kevin M. DuPrey, DO ’10
Justin L. Elder, DO ’07
Joseph M. Flynn, DO ’96
Gerard J. Foti, DO ’94
Carolyn E. Miehle Ianieri, DO ’95
Stephanie K. Iem, MS/Biomed ’03
Tage Nielsen Kvist, PhD, professor emeritus, bio-medical sciences, PCOM
Christian S. Lopez, MS/Biomed ’99
Luis Alberto Narciso, DO ’08
Christine Neely, technician
Robert Niewenhuis, PhD, professor emeritus, bio-medical sciences, PCOM
Michele E. Paessler, DO ’97
Jessica Pfautz, technician
Jordanna Perlman Quinn, MS/Biomed ’02
Rebecca A. Reed, DO ’97
Beth A. Ricci, DO ’95
Jared S. Schure, MS/Biomed ’05
Eileen P. Simak, DO ’00
Jennifer Leigh Sobel, DO ’99
Larry Wayne Spector, DO ’94
Adam C. Steinberg, DO ’98
Benjamin Leslie Stewart II, DO ’05
Robert J. Strony, DO ’03
Dolores R. Tornambe, MS/Biomed ’06, DO ’10