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Adipose-Derived Stem Cells:

Physiology, Application, & Speculation

A Report By James A. Mentz

November 20, 2012

Four years ago, my Uncle Creighton was diagnosed with a rare blood disease called multiple myeloma, which is a cancer affecting plasma cells. With no available treatment for his unfamiliar disease in Chicago, Creighton moved to Houston to undergo an experimental stem-cell procedure that saved his life. The cancerous Plasma cells, which are a kind of white blood cells that produce antibodies, accumulate in the bone marrow and disturb production of red blood cells, casing a number of problems.[i] Like most cancer treatments, his procedure was painfully slow. Since the cancerous plasma cells accumulate in the bone marrow, the doctor suctioned out as much infected bone marrow as he could and isolated the mesenchymal stem cells from Creighton’s marrow to save for later implantation. The bone marrow that Creighton still had in his body was eventually killed by radiation and chemotherapy.  Injecting the isolated mesenchymal stem cells back into Creighton’s hollow bones was the quickest way to restore the tissue needed to produce healthy white blood cells. Creighton’s own stem cells were able to restore the lost bone marrow and for that reason he is alive today. Post-natal (adult) stem cells possess a unique ability that makes them one of the most important healing factors in humans and animals. Three aspects contribute to stem cells’ significance: the capacity to self-renew, long-term viability, and multilineage potential.[ii]^,[iii] Multilineage potential is the ability of a cell to differentiate (change into) a range of cell types. The multilineage potential of mesenchymal stem cells from bone marrow has been studied abundantly, but the full understanding of adipose-derived stem cells is still unfolding.  Adipose tissue (or fat) is among the most accessible tissues in the human body and is seeded with tiny stem cells. This discovery has opened the doors to a whole new realm of research involving the use of adipose-derived stem cells for treatment in animals. Adipose-derived stem cells’ ability to differentiate along multiple lineage pathways, combined with their accessibility and self-renewing capabilities, make ASCs a platform for the future of medicine.

 

Ever since their discovery, stem cells have been a focal point in regenerative medicine. In 1963, Canadian scientists Ernest McCulloch and James Till discovered a self-renewing cell found in the bone marrow of mice. The late 60’s brought about the discovery of mesenchymal stem cells in bone marrow by Friedenstein.It was not until 1978 that similar cells were found in human umbilical cord blood and named hematopoietic stem cells.[iv] Until 2001, stem cells were believed to be limited to hematopoietic stem cells and bone marrow mesenchymal stem cells.However, phenomena like progressive osseous heteroplasia evidenced otherwise. The condition, usually in children, causes the formation of ectopic bone within subcutaneous adipose tissue as seen in this picture. Analyzing the chunks of hard bone strewn among fat tissue, the presence of osteoblasts (bone producing cells) can be detected.[v]  Since normal osteoblasts are permanently embedded into naturally occurring bones, the osteoblasts found in the adipose tissue must have formed there on their own. Future research would reveal that the genetic disorder causes stem cells in the subcutaneous fat to differentiate into osteoblasts. In 2001, a team of researchers from the University of California Los Angeles Regenerative Bioengineering Laboratory proved that adipose tissue is a source for stem cells.⁴ The list of uses of adipose-derived stem cells has grown exponentially in the years since. Applications in the veterinary field have in many aspects passed human application. One reason for this unusual fact is that the Food and Drug Administration has many restrictions on the use of stem cell treatments for humans. In addition, racehorses, many of which are extremely expensive and prone to tendon injuries, are popular candidates for treatment.[vi] In Texas, the rigid policies on stem cell treatments for humans were changed in April of 2012, making Texas a leader in the development of stem cell treatments. Interestingly, Governor Rick Perry posed the proposal after he underwent stem cell treatment himself for his back pain. The bill allows stem cell treatment with consent from the patient and approval from an institutional review board.[vii] Today, people travel abroad to escape stiff FDA policies and receive treatment. In the future we can hope for more trusted treatments available to everyone.

 

The process of deriving stem cells in adipose tissue makes ASCs an extremely valuable asset to regenerative medicine. Liposuction is the standard technique used to obtain human fat. The liposuctioned fat is washed thoroughly with a sterile phosphate-buffered saline to remove debris. The clean adipose tissue is treated with an enzyme called collagenase to loosen the stem cells from their embedding in the fat. The enzyme is inactivated and the tissue sample is spun in a centrifuge, filtered, and spun again until the plasma and red blood cells are separated from the stem cells, which form a solid pellet at the bottom of the test tube.^2,5 The following picture illustrates the cycle of adipose-derived stem cell isolation and differentiation.³ The isolated stem cells go on to be injected into a patient where damage has been done, and a self-sufficient supply of stem cells help repair tissue quickly. For every 300 mL of fat harvested, somewhere between 1 x 10⁷ and 6 x 10⁸ stem cells can be isolated (90% of which are viable).³With roughly 400, 000 liposuction surgeries in the United States each year, adipose tissue to derive stem cells from is abundant.⁵ Cosmetic surgery clinics, with the consent of their patients, have started to sell and donate liposuctioned fat to privately owned research clinics.

 

Multipotent cells like adipose-derived stem cells have not yet differentiated, giving them many diverse uses for matured adults. As zygote develops into a fetus with over 200 unique cell types, certain cells differentiate, or become more specialized.[viii] Generally, there is no going back from a more specialized cell to a less specialized one. Stem cells are non-differentiated cells and luckily have the ability to self-renew. Differentiation affects a cell’s size, shape, membrane potential, and responses to specific signals. Tracing back to the foundation, a human egg is fertilized with a sperm cell and

the zygote begins to multiply into identical cells. When about 100 cells have been produced, they take the shape of a hollow sphere called a blastocyst. The blastocyst consists of an inner cell mass comprised of cells, which are totipotent, meaning they can differentiate into virtually anything. Cells within the cell mass take shape of morula, consisting of the three distinguished germ layers, ectoderm, endoderm, and mesoderm. From these layers, a variety of cell types form as seen in this diagram. Stem cells remain undifferentiated, so that, there will be a cache of undifferentiated cells to repair damaged tissue. These cells, when isolated, can be placed back into a sample of damaged tissue and the stem cells will differentiate and heal the tissue. In an experiment headed by Patricia Zuk of the University of California Los Angeles, stem cells were placed into cultures containing an osteogenic medium, adipogenic medium, and chondrogenic medium.² At the end of a six-week period, the osteogenic cell culture was washed and centrifuged. The cultures with added stem cells produced a higher level of calcium bone matrix than the control, which did not have any stem cells. The stem cells in the experimental culture differentiated into osteoblasts and produced more calcium matrix. In another test, calcified bone matrix, which can only be produced by osteoblasts, was found (and dyed red for identification) in an osteogenic medium with added stem cells. Interestingly, this experiment was attempted using bone marrow derived stem cells and adipose-derived stem cells. Both sets of stem cells yielded equal amounts of extracellular matrix.³ The results of these tests can be seen in this picture. In another sample, stem cells were added to an adipogenic medium and left to culture for five weeks. Increased activity of glycerol-3-phosphate dehydrogenase, an enzyme involved in the production of lipid biosynthesis and higher concentration of glycerol-3-phosphate were found in the stem cell affected sample. Like before, the stem cells differentiated into adipocytes producing adipose tissue matrix.  In the figure below, the results of the experiment are charted, clearly showing more active glycerol-3-phosphate dehydrogenase (GPDH) along with a picture of the denser adipose matrix. Finally, after only 3 weeks of the stem cells living in the chondrogenic sample, the sample was assayed for sulfated proteoglycans, the structural proteins in cartilage. Not surprisingly, the stem cell affected sample had notably higher levels of sulfated proteoglycans because stem cells had differentiated into chondrocytes to produce cartilaginous matrix.² In another experiment, samples of a chondrogenic medium were cultured, the control without stem cells, and the experimental, with stem cells. The cultures were studied under a microscope, and after fourteen days, ASCs differentiated and changed their structure, forming dense chondrogenic accumulations of cells as seen in the figure above.³

 

Adipose-derived stem cells can be applied for clinical treatment to cure a number of damages tissues. As seen in the experiments listed previously, adipose derived stem cells can very effectively differentiated into bone, adipose tissue, cartilage, and muscle, and be used for numerous orthopedic tissue repair treatments.[ix] P. A. Zuk, in a speculative journal article, states, “With its mesodermal origin, the application of ASCs to bone and cartilage defects is obvious along with their use in tendon and intervertebral disk repair. However, the use of ASCs is expanding to both the ectodermal and endodermal lineages.” The following describes a series of trials done to test stem cells effect on nervous tissue. In a group of mice, the common peroneal nerve, a peripheral nerve stimulating muscle contraction in the hind leg, was isolated from all surrounding tissue and damaged by “crushing.” In one sample, human stem cells in a nutrient rich solution were applied over the damaged nerve (the mice were immunosuppressed to prevent antibodies from attacking the human cells), and the other sample was supplemented with the same solution without the stem cells. After a number of days, the hind paws of both sets of mice were dyed, and the mice were allowed to run down a tunnel. The footprints left by the dyed hind paws of the mice evidenced that the stem cell treated mice had gained more normal neuro-muscular function in their legs. These results appear graphed in this figure. Further, the nervous tissue was taken from the two samples, and the conductivity velocity and amplitude were tested by stimulating the tissue between electrodes. Results were also graphed and can be seen in the figure. The resulting information proves that adipose-derived stem cells stimulate the repair of injured neurons. Increased signal conduction is a result of more healthy nerve fibers. Although the adipose-derived stem cells could not differentiate into nerve cells, the stem cells took on another important role, secreting neurotrophins and matrix components to allow the nerve to heal itself much faster.[x] Future developments in neurological stem cell research could offer treatment and help to countless neurodegenerative diseases like Huntington’s disease and Alzheimer’s as well as damage-induced blindness and deafness. Additionally, the National Cancer Research Institute of Japan concluded after a series of trials that, “Adipose tissue is a source of multipotent stem cells that can be easily isolated, selected, and induced into mature, transplantable hepatocytes.”[xi] The potential differentiation of adipose-derived stem cells into hepatocytes, a functional cell in the liver, could lead to a permanent treatment for hepatitis, which can lead to liver failure (the inability to remove toxins from the blood) and death. Similar effects have been observed as the ASCs take on a pancreatic endocrine phenotype, perhaps becoming a treatment for diabetes.³ In addition, the potential for ASCs to repair cardiac muscle after a heart attack should not be overlooked. An Australian research institute found that adipose-derived stem cells drove the recovery of dead cardiac tissue by vascularizing the tissue and differentiating into cardiac muscle cells.[xii] The possibilities of adipose-derived stem cell application are boundless, but the research must be put in before most treatments can be publically administered. Much of the problem with injecting stem cells into an animal in vivo is due to stem cells’ imperfect requirements to differentiate. An ASC’s differentiation is triggered by extracellular factors. This could lead to trouble if a colony of stem cells, injected into a damaged intervertibral cartilage disk, began differentiating into osteoblasts because of their juxtaposition with vertebral bone.

 

The ethics of stem cell research and use have been heartily questioned. When the field of study took off with the developments in embryonic stem cell research, many people feared that the need for human fetuses would become an issue. Luckily, as embryonic stem cell research was being restricted, the uses of a new kind of stem cell were discovered. Adult stem cell research was ethically much more easy to accept, but before adipose-derived stem cells were pioneered, there was still a problem. Adult stem cells were only known derive from bone marrow, so acquisition made research slow. Adipose-derived adult stem cells provided a practical, safe, and abundant source for stem cells. Today, nearly no one has ethical dilemmas concerning adipose-derived stem cells in research (except for animal rights groups opposed to experimenting with animals). The ethical trouble lies in this question: When is it okay to administer ASC treatment to the public? As outlined earlier, Texas Governor Rick Perry believes that with approval from a review board and patient consent, treatment should be allowed. Dr. William Smythe, a member of the Texas Medical Board, comments, “You want to add a layer of protection? Put a moratorium on the use of these agents until they’re proven.”⁷ Hopefully the review board put in place will be an effective measure to provide safe treatment for those in need and prevent more experimental treatments. Stem cell banks provide the service of holding your umbilical blood stem cells and are generally supported by the scientific community. However, these banks are not actually benefitting anyone at the time. They will simply hold on to your stem cells so if an actual, clinically proven way to use them is developed, you will have access to the new treatment.

 

The realm of stem cell research is surrounded by excitement, speculation, and hope for the future (and rightfully so). Promising research provides new insight every day and the list of potential uses only grows longer. The adipose-derived stem cell, in its availability, safe derivation, self-renewal, and multilineage potential, make it the future of medical healing. The illustrious history encompassing adipose-derived stem cells brings the biological phenomenon into an even brighter light. A chain of unlikely events has led to developments in stem cell research and set the foundation for future discovery. ASCs easy derivation process is yet another reason it is being studied so universally. This essay alone sources articles from California, New York, Russia, New Zealand, and Japan. The physiology behind stem cell’s unique multilineage potential gives hope to immeasurable applications. Experiments like those headed by the University of California Los Angeles and the Department of Biochemistry and Molecular Medicine in Moscow provide the scientific community proof of adipose-derived stem cell’s possibilities in regenerative medicine. Finally, though the field has seen its share of criticism, adipose-derived stem cells provide an ethically reasonable means of stem cell research.

 

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