Stem Cells are cells that have not yet decided what they want to be. Stem cells therefore can be any other cell of the body. The body was developed from embryonic stem cells and is maintained and repaired by adult stem cells. Adult stem cells are located in all tissues of the body and are kept in a resting state until called upon for tissue repair.
When stem cells are transplanted into a new location in the body they can change, adapt and modify their function based on the environmental conditions that they find themselves in. The stem cells are like workers that receive their instructions in the form of chemicals in the local environment. They use the raw materials and structure like a carpenter uses lumber and blueprints to rebuild what is missing, damaged or poorly functional. The stem cells’ DNA is modified by the chemicals surrounding the cells causing each cellular division to more closely approximate the original cell and tissue structure. Therefore, a daughter stem cell may differ in cell type from its parent. For example, a stem cell from fat will form new cartilage cells when placed into a joint that has arthritis.
As well as making new cells stem cells also pump out chemical instructions that regulate and instruct other cells how to participate in the healing regenerative process. Stem cells modulate the inflammation present in a damaged tissue so as to reduce its harmful effects. Stem cells can also resuscitate dying cells by donating missing elements of life from the stem cells’ cytoplasm to an injured cells’ cytoplasm.
If we want to grow replacement organs in the laboratory we need to understand the structure of the tissues. Stem cells live in a body that has three-dimensional structure. Each organ has a structural framework and specific cells attached to it. This 3D structure and the cells and scaffolds that hold it speak a tactile language to the stem cell communicating the needs of the tissue. This communication plus the chemical and cellular communication all happening at a microscopic level determines the ultimate fate of these cells. For example, a lung from a pig can be digested free of cells leaving only the structural collagen “skeleton”. Then stem cells from a human can be applied to this three-dimensional structure and will grow to form a complete functional human lung that can be transplanted into that human with an identical genetic match and without tissue rejection that is commonly seen with donor supplied transplanted lungs.
There are three basic groups of stem cells, embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) and adult stem cells. Embryonic stem cells are found in the placenta and embryos. Induced pluripotent stem cells are genetically modified fibroblasts that have similar properties to embryonic stem cells. Adult stem cells are present in blood, bone marrow and many tissues of the body. In recent years researchers have found that fat tissue has a rich supply of adult stem cells.
While embryonic stem cells have more potential for development into different tissues than adult stem cells they are less predictable and less hardy than the adult stem cells. Embryonic stem cells come from fertilized eggs and must be grown in tissue culture. This is a difficult process requiring strict infection control and strict environmental manipulation. Any stress on these cells causes them to change, to adapt to try to survive by differentiating themselves thereby creating a challenge in the control of the outcome with these cells. Controlled differentiation is possible through the manipulation of the nutrients and chemicals present in their culture so that different cell types can be created. These may be muscle cells, nerve cells or bone cells and so on. Cells that produce insulin have been created in the laboratory from embryonic stem cells. Large numbers of these cells would be necessary for therapy however and growing large numbers is problematic and the longer they grow in culture the less tolerant of normal body conditions they become. Embryonic stem cells have been studied for over three decades with many researchers spending their lives trying to unravel the complexities of the control and characterization of these remarkable cells. Another disadvantage of embryonic cells is that unless our parents had the foresight to have some placental cells stored when we were born we would need to rely on donated cells for treatment.
Induced pluripotent stem cells (iPSCs) can be grown from fibroblasts which are found in skin. Induced pluripotent stem cells are adult cells that have been genetically reprogrammed to an embryonic stem cell–like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. Although these cells meet the defining criteria for pluripotent stem cells, it is not known if iPSCs and embryonic stem cells differ in clinically significant ways. Mouse iPSCs were first reported in 2006, and human iPSCs were first reported in late 2007. I have had the privilege of spending time training in the creation and cultivation of these cells in the laboratory learning techniques for culturing and characterizing human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), as well as reprogramming techniques for the creation of iPSCs. Although there is huge potential for programming these cells there is much research still to be done before we can grow our own replacement body parts from spare skin.
Adult stem cells were first found in the blood and bone marrow. They were found in small numbers and had to be cultured and expanded to larger numbers to be available for research and therapeutics. These cells have been used in the treatment of leukemia since the 1960’s. Radiation is used to remove all cells in the bone marrow including the cancer cells. Then the bone marrow is repopulated with stem cells called hematopoietic stem cells. These stem cells grow all the different constituents of the blood producing cells located in the bone marrow. Adult stem cells have recently been found in other tissues of the body. In fact, most if not all tissues of the body have adult stem cells. These stem cells are for the restoration, repair and regeneration of damaged tissue. In most tissues, such as the brain spinal cord and heart, these cells are few and far between but in some tissues such as fatty (adipose) tissues these cells are present in huge numbers. These adult stem cells are called mesenchymal stem cells and are the hottest topic in regenerative medicine today. Adipose derived stem cells (ADSCs) for the first time offer a therapeutic option yielding enough cells that are robust and ready to heal to make it clinically viable.
Adipose derived stem cells (ADSCs) are collected by harvesting a small amount of fatty tissue from the belly of the pet. This fat is then gently chemically digested and centrifuged to separate the cells from the stroma and vessels. This mixture of stem cells (ADSCs) and highly active chemical mediators of regeneration and healing is called the stromal vascular fraction (SVF). The administration of the stromal vascular fraction into damaged areas such as joints with arthritis, failing kidneys, failing hearts, damaged spinal cords and so on leads to rapid reduction in inflammation, restructuring and rebuilding of tissue and return to function. This is accomplished within a couple of hours of the pet arriving at the hospital for therapy.
Previously adult stem cells were collected by a bone marrow biopsy. Then the cells were cultured and the culture expanded. The process of culturing and growing enough cells to be effective could take three to six weeks. As well as this time delay the bone marrow derived cells had now been grown in culture and are less hardy and may have lost some of their ability to heal. We can harvest enough adipose derived stem cells from our pet’s own fat that there is no need to culture the cells, therapy can be done within a few hours of removing the fatty tissue.
There are over 600 clinics treating humans in the United States today. Because you are essentially transplanting cells from one part of the body to another the process does not need explicit approval from the FDA. People are paying tens of thousands of dollars per treatment and thousands of patients are being treated with their own cells, with very few negative reactions and mostly positive outcomes.
There are several companies that provide services to veterinarians who want to use stem cells for their patients. There are two different systems. One requires the veterinarian to ship the fat to a special laboratory for processing. The other system involves on-site processing of the fat.
Safari stands apart and is well ahead of the curve when it comes to stem cell therapy as compared to other veterinary hospitals or companies providing stem cell therapy to pets. At Safari we use the specialist laboratory equipment and techniques for processing our samples without the disadvantages of shipping and time delays.
Stem Cells are fragile; they do not withstand shipping or handling even if chilled. As a matter of fact, the colder they get the faster they die unless we are talking about liquid nitrogen temperatures of -190°C or -320°F. Even then, controlled cooling in special cryogenic solutions is necessary to maintain their ability to be thawed and still live. This is an issue because many stem cell therapies require multiple treatments. Storing the cells locally in liquid nitrogen is an essential element to proper provision of therapy. At Safari we have a specially designed computer controlled liquid nitrogen freezer that takes the cells down in temperature in a stepwise fashion resulting in minimal fatal crystal formation and a high viability rate. At Safari every pet has several treatment aliquots frozen for future use.
The largest stem cell company in America (Vetstem) requires that the veterinarian collect the fatty tissue, ship it to California for processing. Then they ship the cells back to the practitioner for administration 24 to 48 hours later. The viability of these cells is unknown but studies have shown they start to die very quickly after separated from the supporting fatty tissue that they come from. Other companies (Medivet, Ingeneron, Animal cell therapies, Cellavet, Invitrix) sell a kit and a centrifuge to the practitioner for processing the fatty tissue to extract the stromal vascular fraction in the practice. This process has questionable sterility of the cells because there is no biological safety cabinet which the tissue is processed. Injecting infected stem cells into a damaged joint or spine can have grave consequences. At Safari we use a laminar flow biologic safety cabinet and clean bench to process all the tissues and cells in compliance with cell culture safety techniques established by the FDA. In this we couple the advantages of superior processing with increased viability and same day procedures.
Viability and cell numbers are important. For example, when using stem cells for intervertebral disc disease, the cells are injected directly into the disc. If too many cells are injected they will all die, if too few cells are injected they will not be effective. In addition, it is not just the number of cells but also the number of live cells injected that makes a difference. In the heart or kidney, the number of live cells matter. Unless you can determine at the time of injection, the number of live cells per millimeter of SVF you cannot accurately predict the outcome. At Safari we have live and dead cell counting techniques using special fluorescent dyes and special phase contrast fluorescent microscopes that allow us to see and count the live stem cells. We can also see the morphology of the stem cells giving us subjective information about their health.
Dr. Garner has spent time reading the current research and has been to hands on training in stem cell culture methods. Stem cell processing techniques, administration techniques and cryogenic techniques are evolving very rapidly and we adapt our systems to accommodate current knowledge. For example, some techniques for removing the cells from the fatty tissue may result in live cells but these cells may also be stressed and less active than cells processed differently. Mixing activating chemicals derived from platelets may stimulate and nourish stem cells causing them to multiply faster and more effectively. Real research applies directly benefits our patients and we at Safari adapt these new methods to the betterment of the pet.
Stem Cells are chemotactic. This means they seek out damaged tissue to heal, therefore, administration of stem cells intravenously may be an effective means for therapy. Most stem cells given I.V. are filtered out by the lungs but they may not be killed and may make their way to other areas needing healing. It is also true that the more stem cells placed exactly at the area of disease the better the patient will heal. By placing stem cells directly into damaged tissues, we maximize the healing benefits of the treatment. Injecting the stem cells into the joints without causing damage to the delicate cartilage and surrounding tissues is important but may be beyond the technical capabilities of most veterinarians. C-Arm fluoroscopic guidance of the needle as we can do at Safari is the best way to insure the pet has the best chance of a successful therapy. Administration of stem cells into other organs such as the kidneys or heart requires ultrasound guidance or special catheters placed with the aid of fluoroscopic guidance.
Neurologic disease is very challenging to treat. The location of the damage and the location of the symptoms are often very different places. To understand where the lesion is we use Myelogram and/or MRI to image the spinal cord and locate where there may be compression of the spinal tissue. Many cases of spinal compression are caused by a collapsed intervertebral disc. The disc usually collapses because the structure of the cartilage has prematurely degenerated. If this cartilage and the material within the disc can be regenerated, the disc will re-expand, re-inflate and reduce the pressure on the spinal cord. Injecting a small amount of stem cells into intervertebral discs is almost impossible without special imaging such as the use of a real time C-Arm radiography unit which is not found in many veterinary hospitals. Injecting SVC along the spine and into damaged areas is just as essential for success but cannot be done without the special skills of a neurosurgeon guided by very special imaging equipment.
Rehabilitation is an important component of the success of stem cell therapy. The spinal cord heals by reconnecting long nerve fibers together in the correct order. This is an incredibly complex feat when accomplished by the body. Nerve fibers receive impulses from the brain. These impulses travel down the spinal cord to the legs. When the spinal cord is damaged and the brain wants to move the right leg up it sends impulses down a specific set of nerve fibers which never reach the right leg. Yet when these impulses travel to the point of damage they release chemicals from the ends of the nerves that strive to instruct the stem cells in the area to heal to the proper nerves from that right leg. If at the same time the right leg muscles are moving, then impulses from the leg up the spinal cord are trying to make it to the brain. When stem cells are placed into this damaged area, they are instructed by these chemicals released from these nerves how to reconnect the proper nerves. Without this stimulus, the stem cells will not “Know” what to connect to what. Therefore, I believe it is essential to provide electrostimulation, tactile stimulation, proprioceptive stimulation and mental stimulation together with stem cell therapy to create the environment for proper neurologic healing. Without special training and the equipment for this rehabilitation we would not be as successful as we are with stem cell therapy of the back and spine.
Stem cell therapy has been met with skepticism in the veterinary profession and there are many stories of veterinarians who have sincerely tried this mode of therapy only to be met with less than favorable results. I believe there are rational reasons that some cases of stem cell therapy respond, and some do not. To understand this, we need to look more closely at what is produced when we process fat into stem cells. When the fat tissue is processed it forms three segments: fat, stem cells and everything else or stromal vascular fraction (SVF). So far, over 300 growth factors have been identified in stem cells and the stromal vascular fraction. For example, in cardiovascular disease of humans, it has been shown that the SVF when given alone without any cells at all, has positive effects to the heart muscle. This means that all the stem cells could be dead and there would still be a positive effect. I think many of the cases that were successful with arthritis for example were effective because the growth factors alone but no live cells were placed into the joint. These growth factors caused the resident stem cells to proliferate and heal the joint to a certain point. If these factors were not placed properly they would not be effective. On the other hand, live cells with growth factors such as is found in fresh SVF even if given close to a joint may be effective because the cells will be attracted and will migrate to damaged tissue. Dead cells will not migrate, nor will they produce additional growth factors to carry on the healing process for a longer period of time.
Getting the right number of live cells in the right location is the key to success. Many people do not realize how fragile stem cells are compared to blood cells or other tissue and may handle them improperly. Studies have shown that the size of the needle used to inject stem cells greatly affects if they live through the process of being injected; too small a needle bore and the cells die. Stem cells are 10 times larger than a blood cell and much more fragile. Laboratory techniques must be adhered to – mistakes can be fatal to the cells and how they are handled is important and trying to ship them across the country for administration is ill advised. In addition, the cells that are frozen in laboratories distant to the veterinary practice that processed them are likely freezing dead cells to begin with. Some companies recommend sending cells to them overnight delivery for freezing and for counting. Neither of which would accurately estimate the dose of cells given. The dose matters. In the first studies of cells into an arthritic joint were given 50,000 cells per joint (these were cells from bone marrow that had been expanded) and the results were poor. With SVF cells there are so many cells that it is easy to give between 10 Million cells and 50 million cells per joint. This has proven to be effective. So the number of live cells per joint matters and unless you can determine that at the point of care you cannot know if your stem cell therapy will work.
Doing things right makes a difference. Our stem cell laboratory is state of the art; equipment and training maximize the number of live cells ready for transplanting. Our stem cell administration is state of the art; we use specialized imaging equipment to get stem cells right to the damaged tissues. Our aftercare is also state of the art; we have specially trained staff and all the equipment needed to fully rehabilitate your pet in the post-op period. We look to the future; we have the ability to store the cells, to make multiple administrations because of our cryogenics and our knowledge of how it is properly done.
