There's a magic in the distance, where the sea-line meets the sky. Alfred Noyes (1880-1958)
If you lose blood new blood cells of all types are formed from stem cells in the bone marrow, and after a time the composition of blood cells in the body will be entirely normal. This regeneration of adult cell types from simpler adult stem cells in bone marrow happens with blood and with other cells types too, but can it be for all cells?
If you break a bone and the bone is set and immobilized, after a month there will be new bone cells which bridge the gap. Where did these new specialized (differentiated) cells come from? The repair is expected, and even taken for granted, but exactly how does this happen?
There are cells in many areas of the body, which are primitive (undifferentiated) because they have not developed into specialized cells like neurons or muscle fibers, but they can, under special circumstances, differentiate and repair damaged tissue. Is it possible that we can collect and manipulate these cells in the lab, and grow these undifferentiated stem cells to make other specialized (differentiated) cell types? Cell biologists have been doing this for a while. Figure 1 gives a broad outline of the process.
Pluripotent Stem Cells are cells that can change into (differentiate into)
several different cell types. 2,4,5
This article will explain how these regenerative cell processes occur and will present much of what we have learned about controlling and directing new tissue regeneration in the lab (in vitro), and in test animals (in vivo). We will also discuss the prospects for the future with regard to Stem Cell Technology. Finally, we will examine the outlook for the application of Stem Cell therapy, and its derivative discoveries, toward mitigation and support of cells damaged by old polio. Finally, we will examine the conceivability of stopping, reversing, or improving Post-Polio Syndrome with Stem Cell Technology.
EMBRYONIC STEM CELLS
There are two broad categories of stem cells: adult stem cells and embryonic stem cells. When the sperm enters the egg the sperm and egg nuclei fuse and the genes from both sperm and egg mix. This fertilized egg, the zygote, is totipotent2. The totipotent cell has the capability to divide to form new cells and/or transform (differentiate) such cells into all the different cells and tissues in an adult. It is a developing embryonic cells that, when acted upon by cellular hormones and the environment2, can switch blocked human genes on, and some active embryonic genes off and thereby transform (differentiate). Toti, the prefix, indicates the potential for transformation into all the cell types of the body to produce a new individual. If the human being is to develop normally this is imperative, as all the tissues must derive from the zygote. As this fertilized egg goes through the stages of embryo formation, growth, and differentiation of cells the process is called embryogenesis3
Before a sperm fertilizes a human egg it contains a single copy of all the genes a human being possesses. When the sperm penetrates the outer covering of the egg the sperm's genes mix with the egg's component of all the genes needed to be human. Now there are two sets of all the genes, which is the normal situation for every cell in the adult human body. The information each gene possesses, say for blood type, may be different in sperm and egg, but after fertilization there are two genes for blood type and for virtually every other human genetic characteristic. In the adult, however, there are different cell types, which make up different tissues. There are bone cells, skin cells, nerve cells, blood cells and the cells that compose organs. And, each general cell type, like a blood cell, has several different subtypes. Each differentiated adult cell expresses different genes and suppresses others1. In the adult body there are several trillion cells.
Embryonic stem cells, in the center of the spherical five day embryo, given the proper nutrients and growth factors, seem to be able to divide and grow in a laboratory dish for a year or more without differentiating. Stem cells from an adult cannot.4 The reasons for this have not been clarified but early embryonic stem cells signal to one another, and while many of these signal chemicals have been identified, and are used to direct cells to develop into particular tissue cells; bone, cartilage, muscle, or neurons, the distinctive chemical differences between adult and embryonic cells are unknown.5
Figure 2 below illustrates early embryo formation from which embryonic stem cells are derived. The source of these embryos has been in vitro fertility clinics where the growth of the embryo has been stopped at the 5-day stage. Embryonic stem cells, from the center of the ball of cells of the blastocyst, are pluripotent, which means they can differentiate into very many different cell types of the body6. Many adult stem cells have failed in this regard but some adult stem cells, which are rare, but present in bone marrow, blood, brain, skin, and other tissues, have proven to be multipotent, and even pluripotent meaning they can differentiate many or a few tissues.6
The five-day blastocyst, used to harvest embryonic stem cells, abundant inside it's ball of cells, is equivalent in size to a fraction of the size of the period at the end of this sentence. There are no specialized tissues, organs, nor self-awareness. However, the use of embryonic stem cells presents ethical concerns to many because of beliefs that this structure is a human individual and has potential to develop into a human being. The center of the ball contains about 30 stem cells.
EMBRYONIC STEM CELLS, ETHICS, AND LAW
Many in our country contend that the fertilized egg is an individual and that life begins with fusion of sperm and egg. If after cell division the two resulting cells are separated, naturally or mechanically, identical twins can result. So the fertilized egg is certainly not an individual. Also, both the sperm and the egg are living human cells prior to fertilization. The strongest ethical argument limiting the use of embryos seems to be the fact that, if left alone, embryos implanted into a mother would develop into unique human beings. However, limiting the scope of ES research to a few embryos already having produced stem cells, while discarding thousands of unused embryos produced by couples within in vitro fertility clinics seems by many to be misguided.
On July 31, 2001, the House of Representatives of the United States voted for a broad ban on human cloning which included the ban on cloning for research purposes, including cloning embryos that could be used for stem cell therapies. The ban includes penalties of 10 years in prison and fines of 1 million dollars for anyone who generates cloned human embryos7.. As this was passed the Department of Health and Human Services stated that there were about 64 cell lines that could be used. Later this estimate was decreased to 24 or 25. However, many stem cell researchers doubt that any of these stem cell lines will be useful for therapy.8
These political and legal issues complicate the existing technical hurdles to developing stem cell therapies. For example, there is a knotty problem using embryonic stem cells in an adult recipient. These stem cells are foreign tissue with foreign markers on the cell surface. These markers alert the immune system to muster an attack against the foreign cells17. This must be solved for effective embryonic stem cell therapies to become reality.
One remedy would be to use a cell nucleus from the recipient of therapy and switch it with the nucleus in a zygote. If this could be done all the cells produced from that cell would be genetically identical to the therapy recipient. That should mitigate some rejection problems. However, you may realize at this point that the procedure described here is cloning (therapeutic cloning). Such research is not eligible for federal dollars in the United States. However, very recently Zwanka et al successfully altered the genetic composition of a human stem cell by removing a disease-producing gene.16 This very recent breakthrough could lead to the genetic manipulation of stem cells instead of cloning zygotes. It is a workaround. This had been done with mouse embryos earlier but the technical obstacles to genetic alteration of human stem cells prohibited the same kind of success in humans. Zwanka's group used an electroporetic approach to allow for gene insertion by recombination within a healthy cell genome.16
The British, who have no laws against Embryonic Stem Cell Research, draw the line of life at implantation in the uterus9, which takes a middle ground approach between life beginning at fertilization and life beginning at birth. In any event these issues will be of significant importance as we learn more about how embryonic stem cells differentiate, with laboratory manipulation, to viable adult cell types. It is likely that some successful procedures will be more readily approached with Embryonic Stem Cells and some with Adult Stem Cells. In addition to the ethical and political barriers, as you can see, there are significant technical and scientific problems, which must be solved.
THE EMBRYONIC STEM CELL IN THE TREATMENT OF NEURODEGENERATIVE DISEASES
Animal models, particularly rodents, have served valuably in much of the preliminary work on correcting neurodegeneration using stem cells. In 2002 American Scientists reported in Nature success in using stem cells from mouse embryos to cure Parkinson's disease in rats.10 Parkinson's Disease affects about 5 million people worldwide and results from the degeneration of specialized brain neurons that produce the chemical dopamine. The most evident symptoms are movement and walking difficulties. Ron McKay of the National Institutes of Health transplanted a gene into a rodent embryonic stem cell that continuously reproduces, by cell division, into a large number of the correct type of dopamine secreting nerve cells.10 McKay's group transplanted these cells into a rat with Parkinson's symptoms. The animals resumed normal movements and stayed healthy for the equivalent to a lifetime for a human being.10 Despite the fact that McKay was successful in rats we have a long way to go before such success occurs with humans. Rats and mice can be genetically engineered so that rejection of the tissue is not a problem. The adult human has an immune system that has to be sidestepped by therapeutic cloning or stem cell genetic recombination, and immune suppression17. And rat cells behave differently than human cells. McKay subsequently tried to do the same experiment by transplanting human embryonic stem cells into a monkey with Parkinsons but researchers were unable to get these stem cell derived dopamine neurons to secrete a large enough amount of dopamine. So some difficulties remain along with other problems discussed above. 11 The neurons within the brain, which die in Parkinsons disease, are all clumped together. So if the proper replacement neurons are cultured in large enough number, and if inflammation during transfer and immune rejection can be prevented, the cells can colonize the area in the proper number and allow for normal secretion of dopamine.
This problem becomes much more complex for polio-damaged neurons and their contingent muscles. Although we will talk about some strategies and therapies for polio we should understand that motor neurons from the anterior horn have their cell bodies in the spinal cord but long axons, sometimes over a meter long, must wend their way down, horizontally, or vertically to innervate microscopic skeletal muscle fibers, each of which is like a strand of hay in a haystack a mile away. To date, state of the art researchers in neuronal stem cell biology, like John Gage at the Salk Institute, cannot conceive of a way to guide these neurons to the muscle fibers to positively affect function, even if all the other problems can be solved. 12 Dr. Gage said to me, in response to a question I posed to him: I agree that it is unlikely that in the damaged cord of any kind, that the transplanted cells will differentiate into functional neurons and send axons peripherally to the appropriate muscles.12 There is also the problem of regenerating skeletal muscle, which has died or become dysfunctional due to years of atrophy. We will consider these topics, but we must understand the additional difficulties that present for Polio, when compared to neurodegenerative Parkinsons and other brain cell function problems, where the neurons are all in one place.
USE OF ADULT STEM CELLSEmbryonic stem cells have some disadvantages. As mentioned, they are readily rejected by the immune system of the recipient. Also, they convert more often to tumors than do stem cells derived from adult tissues.11 Recently stem cells have been isolated from a number of adult tissues (Figure 2). Some of these cells are monopotent, able to produce only one type of adult cell, but some adult stem cells are pluripotent13. Some cells, reported by Catherine Verifaillie, are pluripotent cells from bone marrow, which resemble stem cells but have other characteristics. She calls them Multipotent Adult Progenitor Cells or MAPC's22. However, the work has not been published in the United States and has been criticized because it has not been repeated in other labs.23 Nevertheless, there is an improving outlook for the use of adult cells in the arsenal of therapeutic applications of adult tissue remediation, particularly for neuron cells in the brain and elsewhere. It is more difficult to isolate and characterize adult cells because they are a very small component of the adult tissue, and there seem to be several lines of cells mixed together. Adult stem cells have actually been used for years when bone marrow is transplanted for malignancies or bone marrow disease. Within the bone marrow are hematopoietic stem cells as well, which replace all of the types of blood cells, red and white. In addition it has been demonstrated that these stem cells also have multi and pluripotency and have even produced several other kinds of tissue cells including neurons.14 Stem cells from an adult tissue type that can produce another different tissue type are said to be plastic, or exhibit plasticity. For example, nascent umbilical cord has stem cells that can produce human neurons15.
Adult Stem Cells only recently were discovered to be suitable for use in therapy. Two recent discoveries buttressed this:
- The cells were found and cultured in brain and other organs and can be grown and maintained in the laboratory.
- Differentiation of these cells has been demonstrated in the lab (in vitro). So the adult stem cell is more plastic* than previously realized. 14
In addition to adult stem cells, stem cells derived from the Wharton's Jelly of the umbilical cord of humans shows great promise in cultivability and plasticity.15*Plasticity is the ability of an adult stem cell of one tissue to generate a specialized cell type of another tissue.
THE PROSPECTS FOR OLD POLIO REPAIR WITH STEM CELL THERAPY
Polio, as far as I can see, has never been mentioned in the Stem Cell literature as a disease that can be helped with Adult or Embryonic Stem Cell Therapy. There is spinal cord injury, diabetes, Parkinson's disease, blood diseases and cancers (which have been treated successfully for 40 years with hematopoietic stem cells with marrow transplants), and even psychiatric illnesses, which result from poorly functioning brain cells, or damaged cells. Other diseases have also been mentioned as possible targets for repair including skeletal muscle in muscular dystrophy, and organ replacement. There are
several aspects of old polio damage that may be amenable to improvement with stem cell therapy. In talking to researchers, working in neuronal stem cell therapy, some seem to feel the prospects for polio repair are very promising given enough time and research18, while others express doubt, particularly about guiding large numbers of the long motor neuron axons, feet, to its target muscle fiber.12 Muscle fibers themselves would also need to be replenished because of damage by atrophic processes over a long period of time.
Figure 5. Motor Neurons produced in the lab from embryonic stem cells. Note the stringy axons and small bushy end fibers. Printed with permission of Dr. Musharov.18, 20
Skeletal muscle has been replaced in animal models and this should be possible, in time, for human polio.1 Work on Stem Cell Therapy for muscles destroyed by dystrophin in Muscular Dystrophy have to be cloned or genetically altered to remove the faulty genes. Work is progressing in this field. For polio there is no need to genetically alter stem cells because polio is not a genetic problem. Also, stem cells are present in the adult muscle tissue, which can produce viable muscle. It may be possible to guide stem cell derived neurons (Figure 5) using biological materials such as chondroitin or other biologically based scaffolding 18. Of course, the shorter the distance from the cord to the muscle, the better the results should be. Post-Polio muscle damage can be much more disabling at the hip or above rather than lower, for example at the calf.
Figures 4a and 4b
Weakened hip, buttock, or paraspinal muscles (Figure 4a,b) can be very disabling, and are closer to the spinal cord. They provide support for muscle movement below. These critical muscles would be easier to tackle with neuron engraftment and could, if successful, provide significant support and improvement in function. So the easiest muscles to enervate could provide the most substantial improvement. But all this is still theoretical and many hurdles remain. Nevertheless, there are several ways that stem cell technology can be used to improve the outlook if the research and therapies bring clinical trials in the next 10 years. We are an aging population so time is controlling with regard to potential polio therapies.
Weakness of the hip if more disabling, generally, than weakness lower in the leg. Muscles are also closer to the cord where new anterior horn cells could be coaxed, theoretically, to new striated muscle fibers.
Deep muscles of the torso support the spine and are in close proximity to the cord. These would be easiest to strengthen and may provide significant improvement.
Many Polio survivors have weak paraspinal and deep muscles that support the spine. This can be very disabling and destabilize the spine, resulting in impingement on adjacent nerves which compromise function. These muscles are very close to the cord and may be enervated by newly grafted motor neurons, which, because of the close proximity, would be more easily connected to these critical muscles. These new ideas in remediation of Post-Polio Syndrome should be considered in the context of stem cell therapy.
Several signaling factors act between stem cells allowing them to differentiate and grow in vitro and in vivo. As stem cell research progresses we should uncover more of these growth and differentiation factors needed for cell differentiation, adaptation, connection to other cells within a tissue, and proper function. Imagine, if you will, a concoction of factors (some of which are already known) that can signal motor neurons to form synapses (connections) with new muscle fibers. Muscle signaling Cell Adhesion Molecules (CAM) attract the placement of synapses on muscle. Other factors may be used to guide cells to the proper muscle fibers. Without using stem cells some of these new derivative cellular hormones could be perfused into a trouble area. There are many possibilities; the only question is how long it will be until effective therapies are derived from Stem Cell Research.
Much of the advancement in stem cell therapies, and much of the realization of future promise will come as a result of lab work using model organisms like mice. A model of Spinal Cord damage, resulting in complete paralysis, has been mitigated in a mouse with neurons derived from stem cell engraftment so that after treatment the mouse uses its hind legs in walking motions where prior to treatment it could not. Rodents can be more easily engineered genetically and cloned so that rejection of implanted cell grafts does not occur. With a new model organism for polio, a mouse, reported in Polio Network News by Dr. Jubelt, there might be new opportunity to study post-polio rehabilitation with stem cell grafts. The possibility of using this polio mouse model did not escape my attention because of the success we have had using rodents to further our understanding of cell differentiation and the possibilities stem cell therapy.24
As this research progresses scientists are finding the cells that produce new signaling chemicals and cellular growth hormones as they examine how to prompt cells to differentiate in the lab.19 It is conceivable that support cells can be implanted in the spinal cord alongside marginally functional motor neurons to provide nutrients and growth factors. Strangely researchers have found stem cell muscle engrafts produce a lot of glial and astrocyte cells instead of muscle. Those are the very cells that support neurons. So these cells could be used for support of motor neurons either by fusion, or by secretion of helpful nutrient chemicals. Or, if the axons cannot reach distal muscle we could fuse new neurons, or other nerve support cells to existing giant motor units to support them, make them more healthy or even help them to produce and maintain more sprouts to muscle. This could result in a second recovery similar to the sprouting events that occurred during the first recovery from acute polio. All this is within the realm of existing stem cell research and its future possibilities. Dr. Murashov, of Eastern Carolina Medical College, an active stem cell researcher, was happy to know this article for polio survivors was being written and he felt this should be discussed because of the possibilities he saw25. Dr. Murashov is working on spinal cord injury and is trying to produce sensory cells in the dorsal horn. With polio we need anterior horn cells where the effector muscles are further away and atrophied, or cell support for overburdened neurons living, but compromised or moribund. This is a new and an exciting prospect, but as with most polio research the need is grant money, access by researchers to stem cell lines, and increased interest in solving therapeutic problems.
- 1. HHMI News: Researchers Produce Motor Neurons From Embryonic Stem Cells,
- 2. Stem Cells: Scientific Progress and Future Research Directions.
Department of Health and Human Services. June 2001. http://www.nih.gov/news/stemcell/scireport.htm
- 3. M. Schuldiner et al.; "Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells"; Proc. Natl. Acad. Sci. USA 97, 11307-11312; Oct. 10, 2000. ABSTRACT
- 4. Stem Cells, A Primer, National Institutes of Health, September 2002
- 5. Stem Cells: Dream or Reality? Mariani, Sara, M., Highlights from the American Society of Hematology 44th Annual Meeting, December 6-10, 2002, Philadelphia, Pennsylvania.
- 6. Stem Cells: Scientific Progress and Future Research Directions, National Institutes of Health, Department of Human Services, 2001.
- 7. Ezzell, Carol, Cloning and The Law: Will therapeutic cloning end up being against the law? Scientific American, November 24, 2001
- 8. Ezzell, Carol, Stem Cell Showstopper: Without Cloning They Aren't Likely To Work. Scientific American, December 16, 2001
- 9. Mariani, Sara. Stem Cells: A Plenary Policy Forum: Highlights from the American Society for Hematology 44th Annual Meeting; December 6-10, 2003, Philadelphia, Pennsylvania. Medscape General Medicine 5(1), 2003
- 10. McKay, R. et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's Disease. Nature, 417, published on-line 20 June; doi: 10.1038/nature00900,(2002). ABSTRACT
- 11. Pearson, Helen. Stem Cell Hopes Double, Nature Science Update, June 21, 2002
- 12. Fred H. Gage, Director of the Laboratory of Genetics of the Salk Institute at La Jolla, California, Personal Communication, February 8, 2003.
- 13. Kaji, Eugene, H. and Leiden, Jeffrey, M. Gene and Stem Cell Therapies JAMA, Vol. 285, No 5, February 7, 2001
- 14. Bjornson CR, Rietze RL, Reynolds BA, Magli MC, Vescovi AL, Turning Brain into Blood: a hematopoietic fate adopted by adult neural stem cells in vivo. Science. 1999;283;:534-537. ABSTRACT
- 15. Mitchell Kathy E. et al, Matrix Cells from Wharton's Jelly Form Neurons and Glia Stem Cells, 2003; 21:50-60
- 16. Thomas P. Zwaka & James A. Thomson, Homologous recombination in human embryonic stem cells. Nature Biotechnology, Published online: 10 February 2003, doi:10.1038/nbt788,
- 17. Drukker, Micha et al. Characterization of the expression of MHC proteins in human embryonic stem cells -PFD FILE Proc. Natl. Acad. Sci. USA, Vol. 99, Issue 15, 9864-9869, July 23, 2002
- 18. Murashov, Alexander K. Development of a Method for Generation of Spinal Cord Neurons from Embryonic Stem Cells for Treatment of Spinal Cord Injury, Murashov's Lab Page,February 13, 2003, http://www.ecu.edu/physio/labakm/Stem Cells.htm
- 19. Villa, A. et.al. Genetic perpetuation of in vitro expanded human stem cells: Properties and Therapeutic Potential., Brain Res. Bulletin 2002 Apr. 57: 789-94 ABSTRACT
- 20. Murashov, Alexander, M.D., Ph.D., Assistant Professor in Physiology, East Carolina School of Medicine, Personal Communication
- 21.Cossu, Giulio, Mavilo, Fulvio, Myogenic Stem Cells for the therapy of primary myopathies: wishful thinking or therapeutic perspective?
- 22.Verfaillie, Catherine, M. et al., Pluripotency of mesenchymal stem cells derived from adult marrow, Nature 418, 41 - 49 (2002).
- 23. Wade, Nicholas, Scientists Make 2 Stem Cell Advances New York Times, June 21, 2002. Synopsis
- 24. Mouse Model Developed for Post-Polio Research, Polio Network News, Fall, 2002, Vol. 18., No. 4
- 25.Personal Communication Dr.. Murashov follows:
Thank you for writing to me about this important matter. I am very pleased
that you are going to bring up the issue of stem cell research and its
possible practical implications for therapy of neurological disorders
including old polio. While I am not working in animal model of polio, I am
optimistic about future of stem cell research in general. By changing
properties of stem cells (either, embryonic or adult) one may be able to
generate sufficient quantities of specialized cells to replace
malfunctioning or missing cells in the tissue. The fascinating plasticity
of stem cells together with directed differentiation may help to establish
functional integration between transplanted cells and the host tissue. To
me the possibilities are almost unlimited. I think that stem cells may be
used for treatment of old polio as well. Theoretically, support cells may
be developed to support compromised motor neurons. Surprisingly, in some
cases stem cells "sense" the problem in the organism and differentiate
accordingly to repair the damage. Of course more studies must be done
before proposing cell therapy in patients. Unfortunately, scarce funding in
stem cell research significantly slows the progress in developing cell
You are more then welcome to use images from my website.
Alexander K. Murashov, M.D., Ph.D.
Assistant Professor in Physiology
East Carolina University School of Medicine
Brody Bldg. #6N-98
600 Moye Blvd.
Greenville, NC 27858
Contact Professor Edward P. Bollenbach
Academic Web Pages: Home: http://www.angelfire.com/mi/nccc/
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