Prospects in Stem Cell Research – Reflections on Clones, Chimera, and Minotaur

Prospects in Stem Cell Research – Reflections on Clones, Chimera, and Minotaur

Lecture Presenter
Roger Orth, MD -428×607

Roger Orth, M.D.

Dr. Orth was born in Chicago, Illinois, and received his Bachelor of Science Degree at Illinois Institute of Technology. His Medical Degree is from Tulane University School of Medicine and he completed his internship and residency at the Univ…Full Profile

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The future ain’t what it used to be. Signed, Yogi Berra

If you don’t know where you are going, you may end up someplace else. Signed, Yogi Berra

More than any other time in history, mankind faces a crossroads. One path leads to despair and utter hopelessness. The other, to total extinction. Let us pray we have the wisdom to choose correctly. Signed, Woody Allen

I am giving this lecture as an offshoot of several personal experiences. A previous lecture in this series was in regard to the human genome project and its future aspect as related to evolving medicines. During that lecture series I did review a number of aspects of the genome project but also noted the remarkable ability of current scientists to make what they want in regard to new organisms. One example I described at that time was the combination of hepatitis C and dengue fever into a new organism. The latter was developed in London in the hopes of developing a usable cellular model to study hepatitis C. The obvious ramifications of a highly infectious disease capable of transmission independent of blood product mixing with a particularly indolent but difficult to treat hepatitis C should be obvious. An additional lecture I gave on bioterrorism described the remarkable work done by Soviet scientists through the 1980’s in their Siberian bioterrorism genetic work. A host of diseases, new in character, were defined and developed. The combination of pathogens was attempted, the results of which have never been fully revealed by either the Russian or United States government. Of note is that when the Russian government changed, the scientists in Siberia were actively working on such combination diseases as Ebola plus small pox. Again, the idea of new organisms, particularly chimeric organisms, represents my ongoing interest in some of the work that has been done with stem cells as well as various cloning work that I will discuss further in this lecture. Actually, I developed interest in biochemistry very early in my medical school training. I was in a combination MD/PhD program with an emphasis on cellular enzymes based in the liver. I have a strong recollection of some coworkers who in the early 1970’s were also interested in obtaining human organs for research. These coworkers were busy at the Charity Hospital morgue not just for obtaining various organs but also for specific organs from human fetus. I well reflect when this type of work was outlawed in 1972 and 1973 in regards to obtaining human fetus tissue. In any event, many of the aspects of stem cell research have been very much played in the lay press, and some of it was consolidated in a lecture I gave to a local church group.

I am reminded of an article that appeared this year in the Wall Street Journal in regards to Fidel Castro and his interest in cloning. Apparently, Mr. Castro had hopes early on of bring back a particular cow, ubre blanc. This remarkable cow was able to produce the world record for milk in a single day–240 pounds. The latter occurred in 1982 at a time in which Castro was quite desperate to find some distinctive nature to the Cuban situation as Russian support of his regime was dwindling. When the cow died in 1986 he had it stuffed and preserved in the Center for Genetic Engineering and Technology in Havana. Interestingly, in 1987 Castro decided to play up his political system’s victories and proposed future victories claiming that his communist system would allow the state to develop a new breed of cow. He proposed that his genetic researchers could proceed with a group of pint-sized cows that might live in apartments. This novel idea included that the cows would graze on grass that was grown in drawers fixed with artificial light. This would allow the children of Cuba to have milk in their own apartment rather than being dependent on centralized sources. Interestingly, during the time of the Cuban government interest, the milk consumption of children in Cuba had been well documented to be far behind the preceding opportunity available in the country. I bring up this story because his Havana Genetics Institute announced planned attempts for cloning of the highly productive cow when the “Dolly” sheep cloning events occurred in 1997. Reportedly, this cow is still in a freezer somewhere in Havana awaiting the cloning by the socialist state. In any event, if this process interests such diverse groups as a Unitarian church as well Fidel Castro it certainly deserves further comment.

Microchimerism and autoimmune diseases

I think it is particularly apt to start with a discussion of a new form of pathophysiology recently described in the rheumatology literature as well as cell researcher literature in regard to a problem of microchimerism and its possible relation to autoimmune diseases. Stem cells have long been known to be existent, particularly in bone marrow, since clinical use of this process has been ongoing for at least 40 years in regard to the now well established role of bone marrow transplants in various white blood cell and bone marrow-related states. The current controversy of stem cell, embryonic cell, and embryonic germ cells leads to an interest in basic stem cell processes. The potential that microchimerism, which includes stem cells from a non-self source, could be involved in diseases is particularly interesting. One can think of pregnancy as a remarkable process that includes a “fetal allograft.” The meaning of allograft includes the suffix allo which reflects tissue from a same species but different than self source. Of course, a pregnancy involves the growth and development in utero of a new individual with genetic markers coming from both the father and mother source and therefore a requirement that the body develop a certain tolerance to the fetus while it is growing in utero. The immune mechanisms of fetus growth and development certainly reflect a whole field of study in themselves. Many of these immune mechanisms that allow a fetus to grow involve the HLA (human leukocyte antigen) system. The latter is a series of immunologic markers that allow the body to recognize self from non-self. During growth and development of a fetus there is a remarkable mixing of cells which is truly bidirectional. The bidirectional cells can include measurement in the mother’s blood of fetal cells within the first 5-6 weeks from implantation. The concentration of fetal cells goes up dramatically in the last weeks of pregnancy. Through a process of observation it has become apparent that these fetal cells can have persistence in the mother, even years after the delivery of the child. In 1992 Dr. Bianchi in Boston was working as a medical geneticist. She was looking for CD-34 markers which are known to be present on stem cells. She had hopes of developing a prenatal test of the fetus by looking at the mother’s blood. Subsequent tests have been shown effective since various disorders can now be described in regard to the fetus–by just simply measuring the fetal blood cells in the mother’s blood. However, as Dr. Bianchi was making her observations she noted that some of the fetal cells, which included a Y chromosome (unique to male embryos), were present in mothers who did not have a current male fetus. This led her to do further work in identifying whether fetal cells can persist in the mother beyond the actual postpartum period. On further review, she was able to demonstrate fetal cells present up to 27 years after the last delivery. Of course, she used an interesting marker which is the Y chromosome which would be unique in the presence of a female as her test mechanism. This is not to invoke any further thought that the Y chromosome itself is active as a pathogen or disease-causing problem in the mother but rather a simple marker that can easily be measured using PCR type techniques. As is often the case in science, there was convergence of research from a completely different source. A Dr. Lee Nelson is a practicing academic rheumatologist out in Seattle. She is at a facility that is frequently well known for bone marrow transplants. Bone marrow transplants have been complicated over the years by a disorder known as graft versus host disease. In this disorder, the recipient of the transplant develops a reaction against the transplant. Interestingly, the graft versus host disease looks remarkably similar to scleroderma. Scleroderma, of course, is a systemic disorder long thought to be part of the autoimmune family of disorders. Additionally, other observations had been made in these graft versus host patients which would include other potential autoimmune disorders. As well, some patients receiving a bone marrow transplant who had previous immunologic- based diseases such as myasthenia gravis, scleroderma, and even Crohn’s disease have been shown to have remarkable improvement when their bone marrow was ablated and a new bone marrow instilled in the transplant. I suspect this keen observation caused many local conferences in Seattle to speculate why these disorders look similar to known disorders of autoimmune texture as above. In any event, in 1995 Dr. Nelson began looking at issues of fetal cells and their possible relationship to autoimmune disease. In pursuing that information she did speak with a scientist at a company in California called Cell Pro. That company had reported fetal cells in tissue documented well after a delivery had occurred. Dr. Hall at that company pointed Dr. Nelson towards Dr. Bianchi since the latter was very much involved in her work as a medical geneticist with stem cells as a marker for underlying disease. This has led to a fruitful merging of these two seeming disparate lines of research. These two scientists by themselves, as well as now with confirmation from a number of groups, have defined that fetal cells do have a strong tendency to be associated with prolonged presence in patients who have documented scleroderma. A number of groups now have confirmed that the incidents of cells from the fetus in scleroderma patients may well be present 20 and 30 years after the last delivery. This is interesting since there seems to be a bimodal frequency in age distribution of scleroderma. We have a group of children who developed scleroderma, usually in the 9©12 range, and we have a group of adults who developed scleroderma often in the 45-55 range. Stem cell transfer can occur as a bidirectional component, and the children may have exposure to stem cells interrelated to cells from the mother. The latent stem cells showing up after delivery may have a relation to the second peak of scleroderma in the 45-50 age group. It has long been noted that various autoimmune diseases have specific genotypes that have an increased risk for autoimmune disease. Further studies in several centers have now shown that scleroderma has a strong tendency for a secondary similarity in HLA types with the mother and fetus. Specifically, HLA DRB1 and some alternate DRB genotypes apparently are associated with a significant increased frequency of scleroderma. It is possible that the immune process that results in scleroderma is really a process of lack of immunologic recognition of the stem cells by the patient. Having very similar HLA subtypes may result in a tolerance that leaves the stem cell to be active in the patient long after the mixing occurred. In February this year, a rheumatology journal reported for the first time that the stem cells could be harvested and shown to be toxic against patient tissue when reacted in a cell culture system. This does suggest that the stem cells have become a form of lymphocyte, or T-Cell, that can be associated with cellular damage and is very actively involved in known mechanisms of cellular immunity. Other disorders of an autoimmune type are actively being investigated including Hashimoto’s disease, and primary biliary cirrhosis in regards to possible role of microchimerism and etiology of those disorders. There have been other disorders that have well been known to occur during pregnancy that might also be interrelated to stem cell deranged activities. One disorder is polymorphic eruption of pregnancy. The latter is an eruption that occurs during the pregnancy and looks very much like severe hives. In 1998 research work did demonstrate the presence of fetal stem cells in the skin, and the inference has been that of potential relationship to this eruption. A far more common problem may be that of preeclampsia. It has been shown now that women with preeclampsia have up to a 1000 fold frequency of fetal DNA being measured in their blood system. The cause of preeclampsia has long been sought to try to identify what process is activating the endothelial damage that is so characteristic of this disorder. It is possible that in appropriate mixing of stem cells and subsequent release of either DNA or other cell-based mechanisms may account for some of the pathology related to preeclampsia. An additional examination has been fetal cells that have been found in myositis, particularly in children. These cases of dermatomyositis have an increased frequency of HLA-DQ in the child and the mother. Again, a form of tolerance may be existent which allows this specific form of autoimmune problem. Of note is that T-Cells, as in many cells, tend to home to various tissues, and it is not surprising that various forms of tolerance are related to specific overlap in the HLA haplotypes. One additional example of long-term cells present is that of massive blood transfusions. Donor cells may exist for many years after an episode with 20-30 blood transfusions. The cells present can be measured and found to represent up to 3% of the recipient’s blood cells rather than the very low cell count that is seen in the latter stages of normal pregnancy. The fetal maternal cell mixing may not always be pathologic. It has been shown in isolated case reports that thyroid tissue, when checked, can show a karyotype that shows that actual thyroid tissue appears to have come from an allo source. Similarly, in liver tissue it has again been shown that actual liver cells are sometimes of an origin other than the patient from whom the liver is obtained.

All of this information at this point just leads to further speculation that various pathophysiologic mechanisms related to maternal fetal stem cell interchange may play a significant role in explaining some of the very difficult disorders, particularly those in the autoimmune family. It is also very interesting to take a moment to speculate about various treatments that might be geared towards this emerging obstruction. Apparently, the group in Seattle has actively performed stem cell transplants on a selective number of scleroderma as well as autoimmune disorders with some anecdotal responses that are quite impressive. Certainly, bone marrow transplant can never go mainstream as a treatment for scleroderma, even if it is a devastating manifestation. However, the active possibility of monoclonal therapy or other highly specific immunotherapy cannot be ignored in the future as one looks to solutions for what has previously been a series of difficult, if not impossible, to treat disorders. Much is likely to occur in the next decade regarding this subject. Graft versus host disease may include a number of disorders including the more common bone marrow transplant, but certainly autoimmune disorders such as scleroderma, Hashimoto’s disease, myasthenia gravis, primary biliary cirrhosis, Sjogren’s syndrome, as well as polymyositis, pruritic eruption of pregnancy, and problems related to blood transfusion and organ transplants. It will be interesting to note in the future if the similarities and dissimilarities in graft versus host disease and scleroderma can be explained on the basis of various genotypes. Interestingly, though the two disorders are quite similar for some clinical features of scleroderma, there is significant variance in regard to manifestation of problems in the renal system and in Raynaud’s disease.

Plasticity: Time for a reappraisal?

I would next like to venture into some discussion of stem cells in relation to plasticity which is actually a process in which a stem cell becomes differentiated into any of a host of end organ cells. Increasingly it is apparent that stem cells from several sources can be differentiated into end organs with various growth factors as the stimulus. Certainly, bone marrow cells have been shown to evolve into a number of potential end organ cells including blood vessels, muscle, heart, and even liver. Other end organs have also been suggested. We have an opportunity potentially to move into a paradigm in regards to future treatment in regenerative medicine. The combination of stem cells and a further understanding of genome derived signals may well lead to a remarkable opportunity and an emerging field to treat diseases in a completely different fashion. Certainly, in crude therapy such as chemotherapy and radiation therapy, a healing stem cell can be utilized to replace damaged tissue in an end organ. This would lend the opportunity to mend “like with like.” The cell, after all, is nothing but a form of self-assembling system, and by using stem cells one can use nature itself with the healing process rather than the blunt tools of drug therapy. The human body has over one trillion cells. There are 260 cell types that have been identified, and an estimated 2000 cell types are present. Interestingly, there are somewhere between 1500 and 11,000 signaling and receptor genes. It is the latter genes that probably hold the greatest promise for future therapeutic choices. Once stem cells from a source can be identified and harvested, the use of these signaling genes can be applied to allow differentiation of the stem cells into the needed and appropriate tissue. Much excitement is in evolution right now since stem cells can not only be harvested from bone source but other sources as well. Various companies are aggressively looking for ways of identifying the growth factors for the obvious reason of patenting new therapeutic choices in regards to stem cell treatments. A company Human Genome Sciences is using its now famous shotgun technique to identify cells that are activating and releasing signaling molecules in the process of differentiation. By checking which parts of the genome are turning on and off and amplifying these parts they hope to use this technique to identify and subsequently patent these signaling proteins. A more established gene company–Genentech has a freezer someplace in California, reportedly with 4200 types of signaling proteins that they have available but don’t known where they belong. In conjunction with various boutique stem cell research facilities such as University of Florida they hope to selectively test these agents in hopes of finding angioblastic factors, liver-promoting factors, nerve growth factors, etc, etc, etc. Actually, the latter looks quite attractive as a method of treatment and evaluation since it merges specific research groups with the uncharted resources already existent. These stem cells are also being sought in other organs, especially those organs that are known to regenerate. The gastrointestinal tract regenerates many cells a day as does skin. Of note is that one nerve stem cell that has recently been shown to be accessible is that of the olfactory nerve. The latter is very superficial, and specimens can be obtained with a minimal nasal biopsy. Researchers in Australia already have a program to use that stem cell with appropriate growth factors in spinal cord victims. I think a particularly interesting stem cell is that that is associated with the hippocampus. The latter part of the brain is well known to be the basic “hard drive” in the brain. When one develops new memories they are basically being formed in the hippocampus and stored there. Most interestingly the hippocampus is associated with identifiable stem cells where many parts of the brain do not have readily identifiable stem cells. It would be most interesting to see if this stem cell availability is related to the need for developing new memories, and therefore the application of stem cells in developing these memories. The actual mechanism of memory development has never been fully evaluated, but stem cells may prove an answer for not just therapeutic neurological intervention but also supplemental intervention to improve memory function. Stem cells are unique in the form of reproduction. They have what is described as asymmetric reproduction in which the division of the cell is accomplished with a residual new cell that can go on to be differentiated into an end organ and a residual stem cell. In private discussion I have been told that the process of asymmetric division may involve some form of asymmetric passage of DNA strands which allow for the original template cell, similar to the government keeping the plates while they may new money for general circulation. If that is true, the molecular observation of an asymmetric distribution of DNA may prove remarkably beneficial if the underlying biochemistry of that process can be further defined.

I recently had the opportunity to visit with Dr. Ed Scott at University of Florida who is establishing a boutique stem cell research group. His group includes those that can make stem cells for subsequent use in various end organs. He is actively working on an angioblastic stem cell which has a great deal of potential use in cardiac cases as well as retinal disease. He is also working with a group that can apparently make a stem cell that is able to differentiate into hepatic organ, and subsequent organ treatment will be a great boom. In July of this year a group in Germany used a somewhat crude technique in obtaining patient stem cells and subsequently injecting them back into coronary arteries using angiographic techniques. This was just recently reported to be effective in diminishing the amount of heart damage as well as increasing blood perfusion in heart attack victims. Similar work apparently is being done at University of Texas in Houston as reported in the Wall Street Journal…I can certainly envision a stem cell with hepatic function being injected into patients with cirrhosis and allowing us to better treat our cirrhosis victims.

I think the immune process that rejects foreign material will make adult stem cell research using specific growth factors a far more likely approach than the subsequent work that is also being performed involving embryonic cells.

Embryonic stem cells

The whole process of embryonic stem cells has been remarkably controversial since it involves not just science but also a potential political conflict. The lay press has been fraught with information regarding stem cells in general, and politicians as well as lay readers have a tendency to confuse the issues of stem cells in regard to the potential relationship to embryonic cells. I will discuss a few issues regarding embryonic cells which are cells that derive from an embryo. In 1998 Compton, at the University of Wisconsin, showed for the first time that they could grow embryonic stem cells in the lab. They were able to grow those using specific inhibitor factors that kept the cells from ongoing differentiation and could arrest their development and leave them for redirection into other cell lines. A current study by a Dr. Gearhart at Johns Hopkins used a separate cell line of human embryonic germ cells. Germ cells could be isolated with his technique again coming from a fetus, but in this case from the egg or sperm source. Differentiation could be arrested.

Interestingly, germ cells, if allowed to grow further, do have a tendency to develop into embryoid body cells and subsequently a form of tumor cell known as teratoma. During the process of differentiation, these embryonic cell lines have been recently described as showing increased levels of immune system proteins. The more immune system protein manifested the more likely concurrent immunotherapy will be requisite if cell lines are planned for therapeutic treatments. There are those that suggest that future therapeutic treatments will include embryonic stem cell banks similar to the current blood bank in which a requesting physician can ask for an embryonic stem cell line and subsequently arrange for specific cell differentiation and cellular replacement therapy. Certainly, the manifestation of immune system proteins would need to be controlled, possibly by specific genetic cloning techniques, to remove these proteins prior to use in a foreign patient program–allo grafts. In the near term I think this approach is likely to be relatively nonfruitful because of this protein immune manifestation that is part of the intrinsic cellular machinery. The embryonic cell lines have been legislated as to a restricted number of lines by President Bush a year ago in his address to the nation regarding embryonic cell tumor lines and the supposed 65 lines already made that are “grandfathered in.” Though those lines exist and likely will be useful for further therapeutic research treatment option, I suspect that the adult stem cell lines which have no such limitations since they come from the patient, will be more fruitful in the future. The original funding for University of Wisconsin studies on embryonic cells was actually paid for by a private company, Geron. This company is a world leader in trying to identify the cellular factors that are responsible for differentiation. The company has recently absorbed the Roslin Group that was responsible for the now famous sheep Dolly. They are burning their research investment money at a remarkable rate and have recently redirected all of their research towards more limited goals which will include identification of differentiation factors rather than some of the other research of a more broad range option that they have previously discussed. Their web site is quite interesting and worth spending time

reading. One must remember that Dolly was a cloning experiment that included a need for remarkable cellular plasticity. In that now famous mammalian cloning process cells from a mammary gland cell line from a 6 year old ewe were merged with denucleated egg cell from a sheep. This did allow the reprogramming of the adult cell and subsequently development of a true embryo. There are really three approaches to cell reprogramming. In therapeutic cloning the patient adult nucleus will be harvesting from some tissue such as skin, etc. It will then be mixed with a human egg cell and allowed to grow to a blastocyst stage. At the blastocyst stage an anterior embryoid cellular extract can be obtained which will allow cultivation of the embryonic cells. This does involve human eggs which are hard to get, and also this is a fairly controversial approach. There is a second approach that involves a human nucleus from an adult differentiated cell. This approach includes mixing the nucleus with a cow egg with the distinct advantage that many cow eggs can be obtained. Additionally, this approach does evade laws that are made or will be made related to human cloning since it involves a non-human component of DNA and therefore not subject to laws as written. Developing this Minotaur has actually been performed at a company, Advanced Cell Technology, which is known to be interested in agricultural uses of cloning as well. This approach does have distinct psychological disadvantages but also includes the problem of cow mitochondria. The cow mitochondria are present in the cow egg as all eggs have to have an energy source which will subsequently drive the process of cellular division. The mitochondria in an egg are what not only drives the egg in regards to future energy needs but are the same mitochondria that can be involved in cellular function in the course of division, etc. Mitochondrial DNA is fairly unique since the mitochondria itself is felt to be a possible organelle that has ancient roots in a merger that gave great strength to the eukaryotic cell lines that merged with bacteria eons ago to allow the cell line to have available energy sources in the mitochondria and allow much further development of this primitive cell form.

Certainly, mitochondria have been documented in essentially all eukaryotic cell lines since this merger occurred, reportedly two billion years ago. The company that made the human/cow chimera also had a solution for the mitochondria coming from DNA of the cow. They proposed making a cow clone line that would actually have human mitochondria. Therefore, if one were to use the cells from that cow line the DNA from the nucleus transplant (all human) would be merged with the eggs from this cow line which has mitochondria of human origin in its mitochondrial DNA. Though they have not proceeded in making that cell line partly because of the remarkable uproar in lay press and political circles regarding this approach they have not ruled out future applications. The third approach to cell reprogramming is that which is alluded to previously, and that is to isolate reprogramming factors. These reprogramming factors would not require a cloning operation and could be used with the Pluri-potential cells that are well known now to exist including in adult stem cell research previously mentioned. It takes only minutes to think about dozens of diseases that could be utilized if cell lines could be identified and placed, whether in example such as diabetes missing insulin producing cells, or problems such as Parkinson’s disease or as alluded, the angiopblasts needed to repair coronary artery damage in acute heart attacks. Certainly, spinal cord injury stories, osteoporosis stories, and even genetic diseases such as cystic fibrosis could be addressed using these techniques. There are many new potential spinal cord therapies that have been discussed, some of which I have alluded to in regard to the olfactory stem cell. As well there have been bone marrow stromal cells that are felt to potentially be utilized in the future to bridge the wound of a spinal cord injury and allow a frame work to occur which can have axon regrowth to occur. There are also mixtures of stem cells being proposed. An Australian group has recommended that thymus precursor cells be grown in vitro and subsequently placed into the kidney capsule and grow a new thymus. The thymus, or course, is critical in T-Cell function. These artificially grown thymus in the patient can be turned off to reaction to various cell lines if they are grown concurrently with stem cells of other sources–this would allow “off the shelf” stem cell use. I suspect that will be more of a novelty, though the desire continues in the business world to have an off the shelf cell line that can be utilized in cell banking for therapeutics.

Genetic engineering companies I would like to touch on just a few companies that have caught my attention as I prepared for this lecture. These companies are different than the way research as evolved in the past post World War II era. In the Post World War II era much research of a basic science nature often occurred in university or isolated research institutes without the goal of fiduciary return as a necessary step for the research itself. The advent of bioengineering has actually led to a completely new form of funding in regards to research which also defines the continuing problem the federal government is going to have in controlling types of research since they will not be subject to withholding funds if someone doesn’t like what they are doing. One interesting company is Geron Corporation. They have the remarkable claim to immortality. With Dr. Jerry Shay and team they have managed to work on telomerase as an enzyme that can regenerate the cellular telomere. This very critical cap of DNA is a 6-nucleotide sequence that appears in almost all life forms at the tips of DNA. The presence of this repeating cap of DNA is necessitated because of the unique way that DNA is regenerated and the recurrent need for losing just a little bit of DNA at every copying cycle. When the cell runs out of the telomere cap–the cell will have apoptosis process occur–cellular programmed death. Geron Corporation made great news when they were able to splice the telomerase enzyme into a cell line and “immortalize” the cell line. Cell lines historically have had what is noted as the Hayflick limit which basically is an observation from the early 1960’s by a Dr. Hayflick that cells can only reproduce a limited number of times before cell death occurs. Telomerase is able to regenerate the cap of DNA so that multiple cell divisions can occur. One would speculate that this will improve aging, but also of note is that telomerase deranged activity in differentiated cells is also noted to be correlated with cancer, another process that has uncontrolled growth of cells. It remains to be seen if this will be highly useful for making us age with less restriction. Geron Corporation has moved its activities more towards cytoplasmic growth factors which are likely to yield therapeutic choices and future treatment modalities that can generate financial benefit in a nearer term horizon. Another company of note is P.P.L. Therapeutics which actually I believe has recently been absorbed into the Geron Corporation. In any event, they are linked to the Roslin Group which is famous for the Dolly cloning story. They also, however, are noted to have interesting products such as cloned cows that can make various human proteins and milk. They have been successful in albumin as well as alka-1 antitrypsin, both of which have potential therapeutic uses when purified and given back to humans. They are also the ones who recently identified that they could genetically remove a alpha11 3 galactosyl moiety which is part of the major histocompatibility complex and associated with marked reactions if xeno transplants are attempted.

Specifically, organs for animals have been tried in the past as sources of human organs, but are often rejected forthwith because of these major histocompatibility complex antigens. By removing this galactose moiety through deletion of the galactocele transferase enzymes they were able to develop a piglet line that purportedly could be used for human organs. However, as I referenced in my human genome project lecture, much of the DNA that is present in any animal line includes DNA that is of retrovirus origin. This retroviral origin cell line may include 10%-13% of the actual DNA and has been more than a nuisance–it is the cause of human HIV infections. I think substituting a porcine HIV infection for a simian HIV infection will potentially be of great difficulty in controlling. Certainly, the company has already speculated that they would have to chop out that part of the DNA related to the retroviral portion, but this is a very large part of the genome and is not likely to occur in a near term process.

Another company is Advanced Cell Technology. I have already alluded to their work on the human clone story with regard to cows. One particularly slick idea seems to be Genzyme Transgenics. They are specializing in milk proteins of human origin, but apparently are also the group that have now cloned spider silk into goat milk. Spider silk is a particularly interesting compound itself since it is stronger than Kevlar and may represent a remarkable source of this very interesting protein. I am particularly interested in a company named Chromos Molecular Systems. I would challenge anyone to look at their web site since they are the ones who have patented the application of artificial mammalian chromosomes. This is a particularly slick use of the genome since it involves the empty segments of DNA known as satellite DNA. Using these repeating segments often that have high adenosine–thymine components they can harvest a “empty” chromosome of mammalian origin. This chromosome can be reprogrammed to hold genes from other mammalian source and can be re-injected into the animal cell using vector technology that is an offshoot of basic bacterial vector lambda research that is so very important in the history of DNA molecular biology. In any event, they have actually made chromosomes that can be passed in other animals from generation to generation. Much of the speculative, if not science fiction type, writing that I will allude to further utilized this remarkable technology and our ability to use the designer genes to generate future designer populations and children. Another company that actually looks quite interesting is Osiris Therapeutics whose line of stem cells can be utilized to facilitate bone marrow transplant and joint surgery. They have also been working on heart muscle and angiopblasts. They are often involved with matrix work on which stem cells would subsequently grow. I would suspect that they will be again related to the boutique stem cell labs who develop stem cells that need a place to grow in various therapeutic options. A completely different way of DNA transfer has been developed by Copernicus Therapeutics. They have packaged DNA in small 25 nanometer positively charged clusters, and in conjunction with larger liposomes can transfer DNA into a cell that is already functioning. They are using this in cystic fibrosis and hoping to generate DNA transfer and use DNA washes which will improve cellular function in active patients without having to go through other steps of gene therapy. There is a company, Metamorphix which is a division of Cargill, a large well known food source company.

They are involved with genetic tests and cloning for beef tenderness and have actually produced a cell line of clone cattle that apparently will have tenderness as its prime directive and allow for a higher yield of more expensive beef. A small company named Aegen Biosciences has developed a silicon chip that allows mass denucleation of eggs and would be utilized for using robots to make batch lots of clones. I have already alluded to the role that Genetech and Human Genome Sciences may be playing in a number of issues including the definition of genome-based cellular differentiation factors that are likely to be remarkably important in stem cell research. I will also mention a specific group of researchers in Australia and Denmark who recently pooled their efforts to make a marked improvement on the actual process of cloning. With their approach, no need for micromanipulation is present. They simply divide the egg and that part which does not have a nucleus can be fused with electric current to an appropriate donor nucleus. They fuse this with electric current and take second egg tissue to make up for their smaller egg-derived cytoplasm. This fusion can occur both in the field, and is very easily adapted to the Aegen Biosciences system for future roboticising processes that may be requisite for batch lot cloning processes.

Road to utopia/road to dystopia

In my final section, I would just like to make a brief review of some of the aspects of genetic engineering and comments that have been made in regard to this process. Much has occurred since Watson and Quick described the DNA structure in their now famous paper in 1953. Certainly, the process of cloning was begun in 1960’s concurrent with the publication of Rachel Carson’s “Silent Spring.” I mention the later because some of the unseen consequences may not yet be clear in regard to genetic engineering. The debate regarding genetic engineering really began full force in 1972 as a response to a popular book on human cloning at the time. The sides wind up with Joshua Lederberg in an affirmative position publishing some articles in popular press as well as Leon Cass answering these publications as an ethicist against such manipulations. Interestingly, Dr. Cass remains very active in the process since he heads up President Bush’s bioethics panel. Much came of a series of articles that culminated with William Gaylin in an article in New York Times Magazine in 1972. This was in response to processes of cloning which again was stimulated by a popular book on cloning. Mr. Gaylin stated in his article, “What will we do with the discarded messes along the line? What will we do with those pieces and parts, near-successes, and almost-persons? What will we call the debris? At what arbitrary point will the damaged “goods” become damaged “children” requiring nurture rather than disposal? The more successful one became at this kind of experimentation, the more horrifyingly close to human would be the failures, the whole thing seems beyond contemplation for ethical and aesthetic, as well as scientific reasons.” It was also in 1972 that the house subcommittee on science, research, and development asked the science policy research division of the Library of Congress to study genetic engineering. They did study cloning and human parthenogenesis. The result was the statement that, “It is not now possible.” However, they recommended forethought and evaluation rather than acceptance of a haphazard evolution of genetic engineering. I mention this committee because it turns out that government does not have a standard means of monitoring genetic engineering. Multiple subsequent committees and congressional panels have been charged with information gathering and recommendations. One recurring theme, however, has been that this technology has gone forward at such a speed that there is no natural forethought available so that recommendations can be made in advance of any achievements. Additional sets of hearings, this time by a subcommittee on health and the environment, of the house committee on interstate and foreign commerce, studies cloning in 1978. This was in response to a book, David Rorvicks, “Cloning of a Man.” Their results in 1978 suggested there was not any need for regulation since it wasn’t going to be possible. Later in 1978, the world was shaken, however, when baby Louise Brown was born as the first in vitro fertilization baby. Extensive articles in regards to the combination of five parents related to a single baby, both the genetic and rearing father as well as the genetic, gestational, and rearing mother, as well as the subsequent technology related to IVF became very important. This technique would play quite a role if anyone was doing human cloning, and of note is that 200,000 worldwide births related to IVF have been accomplished. Process of IVF has now been incorporated into every day vernacular of medicine, but again the world was shaken in February 1997 with “Hello Dolly.” Certainly, the announcement by Dr. Ian Wilmut that a Scottish group had developed the first mammalian clone, in this case a sheep, caused quite a bit of stir. This became evident as the process itself was studied in which 277 reconstructed embryos were generated, 29 were implanted, resulting in one live birth. The embryo that grew was Dolly–#137. Dr. Wilmut was quick to define that this was not a process for humans but was for agricultural purposes. However, speculation in the process as to stages in which gene targeting and gene engineering would occur were certainly discussed. An interesting paper was reported in early January 1998. At that time the first knock out technology was utilized to delete a gene from an organism in regards to the control for head production. Two different groups reported headless mice and tadpoles could be raised–though at the time of birth they would of course not remain alive. The deleted embryologic control gene for head development, however, would allow the technologic line for an organ procurement program. Lee Silvert was one of the many commentators who stated that if this were applied to people, “These human bodies without any semblance of consciousness would not be considered persons, and thus it would be perfectly legal to keep them “alive” as a future source of organs.” His further comment included, “Cloning is the technology of narcissism and nothing satisfies narcissism like immortality. Headlessness will be the cloning’s crowning achievement. If we flinch in the face of this high-tech barbarity, we’ll deserve to live in the hell it heralds.” Further furor occurred with the publication of Nicholas Wade in his book, “Life Script.” In 2001 this popular science journalist went on to suggest that, “Though today’s genomic revolution is being conducted in the name of medicine, it will not necessarily stop at its implied goal, the attainment of perfect health. The power to reshape the human clay has no clear limits. How far should we go in enhancing qualities other than health such as physique or intelligence?”

By October 2000 the occasion of mixing genomes including human had included the actual making of a human/cow and human/pig mix. I previously described how that occurred elsewhere in this paper. I thought the commentary by a J. Bottum in October 2000 in an article “The pig-man cometh” was of note. He said, “You and I will live for many years in youthful health; our cancers, our senilities, our coughs, and our infirmities all swept away on the triumphant cresting wave of science. But our sons and our daughters will mate with the pig-men, if the pig-men will have them, and our swine-snouted grandchildren, the fruit not of our loins, but of our arrogance and our bright test tubes will use the story of our generation to teach a moral to their frightened litters.”

In answer to fears from watchful consumers, the Geron Corporation CEO, Dr. O’Karma, reassured that “Our objective is to increase the health span, not the life span.” Well known biologist at Harvard, Edward O’Wilson stated that any attempt to change the apparent imperfections in human nature “would lead to the domestication of the human species we would turn ourselves into lap dogs.” No less a dignitary in science, a James Watson has long advocated germ line engineering, “We can talk principles forever, but what the public actually wants is not to be sick. And if we help them not be sick, they will be on our side.” In fact, the ethics of supplying the ability for gene engineering has been more than that in regards to the process itself. Many writers have made the statement that the ability to perform genetic engineering will at some point potentially become mandated for an opportunity for equalization of misses chances in our society. Speculation that government could invoke a requirement that genetic engineering be available to all economic strata rather than just the rich who could afford such treatment has been a recurrent theme in the readings that I have noted. Nicholas Wade again comments, “The sequencing of the human genetic makes it possible to envision for the first time the creation of a genetically more just society, one in which the most fundamental kind of wealth–the genes that confer health and fitness–would be for the first time accessible to all.” This approach also, however, has polarized numerous members of congress to make various statements. Tom Harkin, Democrat of Iowa stated in 1998, “To those who suggest that cloning research is an attempt to play God, I invite you to take your rank alongside Pope Paul V who in 1616 persecuted the great astronomer Galileo for heresy–for saying that the earth indeed revolved around the sun and did not otherwise.” He went on to state, “I do not think that any attempt to limit the pursuit of human knowledge is demeaning to human nature.” Christopher Bond who has actually authored legislation in regard to cloning has stated, “For reasons just as obvious to anyone with any moral sense, such practices must be outlawed, for otherwise, our society would produce a generation of human beings purely for the purpose of producing spare parts for others and those to be destroyed. Some may call this a “slippery slope” I believe sheer cliff would be more accurate.”

So how does this process evolve? The most telling collection of combination science and science fiction is a book I have read by a Dr. Stock from UCLA titled “Redesigning Humans.” He has done a fairly cogent job of describing genetic therapy–germinal choice technologies, by his description. Genetic engineering can involve several approaches. Certainly we already use viral vectors. Viral vectors have the advantage of being designed to move DNA into a cellular genome, and just simply using the already existing viruses has been actively used in certain gene treatment programs including on people. These are often designed for replacing missing genes in cancer cells but also for replacing missing genes in single protein malfunction such as muscular dystrophy or glycogen storage disease.

Active therapeutic trials in that regard are already underway. The disadvantage of immune reaction against a virus after several treatments as well as the random injection site on the genome in regards to DNA have been drawbacks. Having the genes in a non natural site may interfere with underlying control mechanisms for the gene expression. Another approach for genetic therapy has been DNA vectors. I have already mentioned the unusual approach being tested on cystic fibrosis patients with DNA in a special coating that is allowed to be injected into cells through liposomes, the subsequent incorporation into the cell cystoplasm of this positive charge protein coat, 25 nanometers in size, containing a specific missing gene, has allowed the theoretical incorporation of the DNA into the nucleus. Once in the nucleus it can be merged into the genome and allow manifestation of the missing protein. These studies are currently underway but not likely to prove broadly beneficial, though they have the advantage of being active on somatic cells alone and not being germinal in nature cannot be carried through to any subsequent children. Gene therapy using germinal choice technology has already been alluded to in regards to the stem cell. One can immortalize the specific stem cell with telomerase and end the limitation on rebuilding that cell. In a recent publication, stem cell repair processes involving DNA repair processes have been noted to potentially be the actual life limiting component in whole organism, in this case a mouse. If one can have additional life span for a stem cell it is postulated that the underlying organism requiring replacement from stem cells can have a far longer life span. This has been shown to be true in mice in which a specific DNA repair enzymes is required for a prolonged stem cell life, and will give the benefit of a longer life span than expected for the mice who carry that gene.

I think the most telling process from review of the literature, however, is that regarding artificial chromosomes. The artificial chromosome for mammalian system as previously discussed does offer a remarkable opportunity to do genetic manipulation and use the sub cellular structures that are already in place to recognize such manipulation attempts. One can anticipate that artificial chromosomes can be utilized to incorporate a longevity factor such as telomerase but also more importantly to incorporate signaling proteins which can assimilate host regeneration. Host regeneration, of course, has been a major issue in spinal cord injury as well as a number of other end organ damage episodes. Those who have speculated on its use have also suggested that it may be requisite to have an inducible toxin in the artificial chromosome. By putting in an inducible toxin such as diphtheria into the chromosome, a fail safe mechanism can be generated in which the affected cells can be killed off when no longer needed. Certainly, replacement therapy for protein problems such as insulin deficit using artificial chromosomes would be of great utility, and there is even a remarkable discussion of docking techniques that already are available with the artificial chromosome that would allow updated new versions of the software of DNA when new options become available. It would only be right if this technology were utilized that those who buy it would want to have the opportunity for an updated version, somewhat similar to the updated versions seemingly endlessly available on America Online.

As the opportunity exists, I do note that the lessons in complexity alluded to in Rachel Carson’s “Silent Spring” can also be seen in other proven examples. Two quick examples would include a wonderful book entitled “The Call of the Distant Mammoth” by Peter Ward. In this book he describes the transmigration of Clovis people as they crossed from Siberia into the Americas during the last ice age. Only the passage of a thousand years resulted in more than 50 large mammal species in North and South America being extinct. He also discussed the push- pull that may have been interrelated to the actual elimination of the Neanderthal and homo erectus subspecies that occurred as homo sapiens became dominant many years later. The unseen consequences were certainly not seen. Probably the biology student’s best known extinct species is raphus cucullatus–the dodo. For whatever mechanism, in this case natural selection presumably, the organisms selected flightless status. Unanticipated, unknown predator known as man forced the rapid extinction of the species. The conflicts of Charles Darwin’s natural selection with his concurrent economist Adam Smith who wrote on the laissez-faire economics is markedly noted in the end result, free market maximization of the short term gain with the higher efficiency of flightlessness but ignorance of the subsequent consequence loss of the species. Genetic engineering is now possible, it is just simply not clear what will happen.

Roger Orth, M.D.

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