By Tom Imerito
If Pittsburgh's renowned leadership in the fields of engineering, manufacturing and health care appear to be unrelated, they are not so for Dr. Alan Russell, Director of the McGowan Institute for Regenerative Medicine. "While we were living and breathing engineering and manufacturing, Pittsburgh pioneered organ transplantation," he said. "If you put the three together, you get regenerative medicine," he said.
Broadly speaking, regenerative medicine uses the body's innate healing mechanisms to promote faster, better healing of wounds caused by injury and disease. At a deeper scientific level, the discipline seeks to discover the most basic molecular factors and forces that make our cells form, grow, function, reproduce and die.
The task is daunting because our ever-changing cells live inside our ever-changing bodies, which in turn, live in an ever-changing world. To make things even more complex, each of our bodies is unique.
Bodies, Tissues, Cells, Molecules
For purposes of reducing complexity, refreshing memories and visualizing our bodies’ systems, from the top-down: We are made of muscle, bone, skin, fat and organ tissues composed of cells that are supported within a matrix of non-cellular material, known as the extracellular matrix. Cells are made of a membrane, cytoplasm and a nucleus. The membrane holds the cell together and protects its interior from the very different environment on the outside. Cytoplasm contains numerous sub-compartments called organelles that behave like a neighborhood of everyday folks going about their humdrum lives, doing things like nutrient uptake (eating), gas exchange (breathing), energy creation (working) and excretion (powdering their noses). The nucleus, acts like a commander-in-chief, making decisions about the big picture stuff, like when to eat, grow, fight and surrender. To make all these decisions and then do something about them, the nucleus relies on DNA: two strands of sugar connected by paired molecules called bases, that connect the two strands, giving the DNA molecule its famous double helix configuration. DNA is divided into sections, called chromosomes that act like the individual cars of a very long supply train that can be decoupled and dropped at a siding or rushed to the front lines when necessary. Inside the cars are genes, specialists communicating by walky-talky, firing big artillery, driving tanks, flying helicopters, administering medical supplies and doling out extra rations, all directed by drill sergeants, called factors, barking orders at platoons of specialist protein-infantrymen to initiate, promote and inhibit responses to changes in local conditions on the ground.
In order to function properly the four bases must match each other in one of two ways: adenine (A) must pair with thymine (T) for (A-T or T-A) and cytosine (C) must pair with guanine (G) for (C-G or G-C). Other configurations are called single nucleotide polymorphisms (SNPs), and they frequently spell trouble.
With only 23 chromosomes, two strands of sugar and four bases to connect them, the system looks simple until the fact arises that the human genome contains about 3 billion base pairs, comprising an estimated 20,000-25,000 genes. Although there is still a lot more to learn about the human genome, two things are apparent: 1) virtually no two sequences are alike from one individual to the next and; 2) some mutated genes are indicators of disease.
Driven by the correlation between gene mutation and disease, North Side startup Redpath Integrated Pathology has centered its business on molecular diagnosis of recalcitrant types of cancer. “We get the cases when the pathologist can't make the call,” Redpath CEO, Mary Del Brady said. “And it's becoming more difficult, because of the advances in imaging that enable earlier diagnoses." Redpath has developed a patented method of diagnosing small cancer biopsy specimens by isolating highly specific areas of a tumor from routinely collected hospital samples; reverting the fixed sample to a duplicable state; isolating its DNA; repeatedly duplicating the sequences by means of a laboratory process called polymer chain reaction (PCR); and assaying the aggregated mass to determine the relative quantity of mutated base pairs at between eight and 15 DNA locations known to correlate positively for cancer, thereby rendering a definitive diagnosis.
Redpath Founder and Chief Scientific Officer, Dr. Sidney Finkelstein, elaborated on Brady's comments: "Most specimens are examined under a light microscope, and in most cases, that is enough to make a reliable diagnosis. But in a portion of cases the microscope does not provide adequate information to make a diagnosis, so the physician is left in the position of not knowing what to do next. Detecting molecular mutations that are at the base of cancer can provide very important, very discriminating information that allows a patient to be treated better.
“Up until the present time, the biologist looked indirectly at cancer,” Finkelstein continued. “Today, we're looking at biological markers attached to mutations, the damage in DNA that is at the very root of cancer.” The biomarkers to which Finkelstein refers are chemical substances that bond with specific disease molecules and alter their colors to facilitate more differentiated and accurate observation.
Across the Allegheny River at Carnegie Mellon University’s Molecular Biosensors and Imaging Center, Professor Marcel Bruchez develops both dye and fluorescent molecules for use as biomarkers. “We're developing quantum dots and dye molecules,” Bruchez said. “Dye molecules are organic molecules made of carbon, hydrogen, nitrogen and sulfur that absorb light very intensely and transmit a narrow band of color. We use quantum dots as inorganic fluorescent dyes that absorb light in one color and very efficiently emit light in a number of different colors. By designing molecules with narrower emission bands, we are able to get as much information as possible from a single specimen.”
A Molecular View of Cancer
Not far away, at the Hillman Cancer Center in Shadyside, Dr. Michael Lotze uses biomarkers to investigate the signaling characteristics of a new-found protein called micro-RNA, a very small molecule that regulates immune response; and a recently identified class of immune-response factors, called DAMPs (Damage Associated Molecular Pattern) molecules, which, it is hypothesized, respond to danger, damage or injury, rather than intrusion by external entities, collectively known as none-self, which was until about five years ago, the widely accepted model.
Lotze and his team, work under the hypothesis that the underlying cause of cancer is not a simple matter of uncontrolled cell growth, as it is conventionally defined, but rather a manifestation of a failure of the body's normal mechanism of scheduled cell death, called apoptosis (a-POP-toh-sis), which results in a vicious cycle of increased inflammation and subsequent unscheduled cell death, called necrosis.
While Lotze is working on the problem of how to get cancer cells to stop growing, other of his colleagues at the McGowan Institute are trying to figure out how to make them grow for purposes of healing wounds.
Cells, Scaffolds and Factors
Dr. Stephen Badylak, McGowan's Director of the Center for Preclinical Studies, explains tissue regeneration thus: “There are three pillars that investigators take when approaching their particular organ of interest. They either start with the cells that form the organ; or with the scaffold that serves as the organ’s structural support; or with some type of a biologically active molecule such as a growth factor. So you've either got cells, scaffolds or growth factors.”
Growth factors are proteins produced by cells that tell them what to do, such as morph into specific functional types, like kidneys, skin, muscles and eyes; continue to divide just long enough to make their intended tissue or organ; stop doing so, when the job is done; dispose of unnecessary cells, like the vestigial webs we all had between our fingers and toes before we came into the world; carry away dead invaders during immunity skirmishes; and repair the wounds when the battle is over.
Badylak is conducting tissue reconstruction trials using Urinary Bladder Matrix (UBM), a scaffold material produced by removing all the cells from layers of pig bladder tissue, thereby leaving behind an extracellular matrix, empty of cells, but retaining essential growth factors. “We're getting the scaffold plus all the inherent growth factors that are part and parcel of the normal extracellular matrix.” Badylak said. The matrix normally has growth factors and other instructive molecules that have been secreted by the cells that made the matrix in the first place. So we get not only the structure, but we've got the right mixture of different growth factors. The body responds to an acellular matrix in a very friendly way because the molecules of which it is composed are so common that the body doesn't even recognize that the matrix doesn’t have cells.
Somewhat surprisingly, in addition to providing support and growth factors with minimal rejection response, Badylak’s matrices also serve to promote tissue growth as a direct consequence of degrading. “We have learned recently that in addition to the growth factors that are released, as the scaffolds proteins degrade, the ECM forms new molecules that are degradation products of the parent molecules that cause a second wave of biologic activity. So Mother Nature has been very clever in the way she has set all of this up.”
Badylak and his team suspect that natural tissue repair may be dependent on the functions of our bodies’ killer and cleanup cells, known as macrophages. “Macrophages go to the area where they're needed, engulf and digest offenders and then send out the appropriate signals to replace the damaged tissue,” Badylak said. “Basically they kill what's in front of them and repair the damage in their wake. But we have learned recently that there are other macrophages that look identical to the killers under the microscope but they facilitate constructive remodeling. The two macrophage types are classified as M1, killer macrophages, or M2, friendly constructive macrophages.
“Recently we have found that when we decellularize an extracellular matrix, it almost always induces an M2 friendly response. If we don't get it totally decellularized or if we try to manipulate it too much, then it goes back to the M1 killer response. We think that most of the inflammation signals reside in the cells and the friendly signals reside in the matrix.
Synthetic Scaffolds and a New Kind of Stem Cell
Taking a somewhat different tack, University of Pittsburgh adjunct faculty member, Dr. Anthony Atala is a pediatric urologic surgeon and Director of the Wake Forest Institute for Regenerative Medicine in North Carolina. Atala's research spans the spectrum of tissue generation and regenerative medicine. His researchers are working on tissue and organ replacement therapies for virtually every part of the human body.
Atala summarized his approach to tissue generation: “If you have a patient who has a defect or disease in a tissue or organ, you would go to that specific organ and take a very small piece of tissue from that organ and from that piece of tissue you would harvest a normal cell and expand that cell outside the body and then you would place those cells with biomaterials in a mold and seed the biomaterials with the cells. Very much like a layer cake, one layer at a time. And then you would put the cells in an incubator in a very specific atmosphere and temperature and then you would start cooking the mold, if you will, until it's ready for implantation.”
In generating tissue that is missing from a human body or organ, Atala’s multidisciplinary team uses synthetic porous scaffolds made of a polymer material called polylactic-co-glycolic acid (PLGA) to form the required body part. Because the material doesn’t have any cells, the body tends to accept it. “The materials we use are manufactured because we can reproduce the same materials time and time again,” Atala said. “We have used materials from animals, but there's a lot of variability because every animal is genetically different. These raw materials don't have any cells, so they are not rejected.”
Although Atala’s synthetic scaffolds adequately address the problem of tissue support, in large wounds, they require the addition of cells. “The maximum diffusion distance for cells to cross without scars is about one cubic centimeter – about the size of a jelly bean. For a small defect you can use a scaffold alone. But for a larger defect you need cells,” he said.
In response to the need for implantable cells that are able to differentiate into numerous functional types (a characteristic called pluripotency), Atala's group has developed a new class of stem cell derived from amniotic fluid that appears to have the most desirable characteristics of both adult and embryonic stem cells. They do not destroy life as some ethicists argue embryonic stem cell therapies do.
They are relatively easy to expand in culture, as organ-specific adult stem cells are disinclined to do; and they do not cause tumors after implantation, as embryonic stem cells tend to do. The new amniotic fluid-derived stem (AFS) cells are isolated from the excess fluid drawn from an expectant mother’s womb during pregnancy to detect birth defects. Because a large fraction of the fluid is reserved for extra procedures in the event additional testing is needed, in most cases there's a lot left over after the test is done. The harvested fluid can either be frozen for use later on in the child’s life or, as Atlala hopes, an AFS bank will be established to harvest about 100,000 specimens, at least one of which will be compatible with more than 99 percent of the United States population.
Recombinant DNA Techniques
Employing yet another approach, at Pitt’s Molecular Medicine Institute, Dr. Paul Robbins and colleagues are using recombinant DNA to alter damaged and dysfunctional cells outside the body and re-injecting them into the joints of arthritis patients. “We do things in the context of orthopedics, so we're thinking about cartilage, bone or tissue that you might want to repair either using stem cells, genetics or a combination of the two, Robbins said. “We have worked out a method of locally inserting a gene that blocks one of the mediators of inflammation. We do so by taking cells out of the joint, modifying them and putting them back in. You can use the same methods to improve cartilage. We have been working with people in veterinary medicine, to treat osteoarthritis in horses with the goal being the expansion or regeneration of cartilage by delivering growth factors locally to that site.”
Improving the Quality of Life
Although most research in the field of molecular and regenerative medicine is still either in the laboratory or in clinical trials, Dr. Johnny Huard, Director of the Stem Cell Research Center at UPMC’s Children’s Hospital, has actually improved the quality of life for patients suffering from urinary incontinence. Huard works closely with Pittsburgh startup Cook MyoSite, a company he co-founded, that licenses the technology from the University of Pittsburgh. Huard’s patented tissue regeneration process calls for extracting muscle tissue from a patient’s thigh, isolating a population of stem cells, growing the cells in a flask over a period of two to three weeks and injecting them into the patient’s urethral neck, thereby causing the body to repopulate the urethral sphincter with newly functional muscle, and restoring control. Although the procedure has been limited to women thus far, Huard says that the success of the trials combined with demonstrations of safety will accelerate the FDA approval process, allowing the procedure to be applied to men, as well as to be adapted to other organs, including cartilage, bone and cardiac tissue.
Although McGowan’s Director, Dr. Russell, makes reference to putting casts on broken bones and doing bone marrow transplants as the more recent antecedents of molecular and regenerative medicine, from a longer historical perspective, he views them as mythological in origin. “The idea of re-growing and culturing organs to harness biological power in order to induce a beneficial effect in the body goes back to Greek mythology,” he said. “As punishment for giving man fire from the gods, Prometheus was sentenced to have his liver eaten by an eagle every day and every day it regenerated. Even then they identified that the liver could regenerate. So the principle is very old, but the tools that we have are becoming more and more refined as time goes by.”
This October, hundreds of researchers and clinicians in the fields of molecular and regenerative from around the world will gather at the Seventh Annual Stem Cell Conference at the David L. Lawrence Convention Center in Pittsburgh. Whether the origins of molecular and regenerative medicine are distant or recent, it is evident that a good part of their future will be here.