Rejuvenation (Aging)

The treatment of the aging process and of the diseases associated with it could lead to the rejuvenation of the body, life-extension, and immortality. Such an endeavor, however, would be extremely difficult, and the mere suggestion of it is highly controversial. Many scientists believe there is no such thing as a treatment that will reverse the aging process. Indeed, a coalition of 51 gerontologists and biologists, led by S. Jay Olshansky, took an unprecedented step of publishing a paper titled "No Truth to the Fountain of Youth," which was sharply critical of antiaging medicines and the companies that market them. The following is an excerpt from their position statement:

There has been a resurgence and proliferation of health care providers and entrepreneurs who are promoting antiaging products and lifestyle changes that they claim will slow, stop or reverse the processes of aging. Even though in most cases there is little or no scientific basis for these claims, the public is spending vast sums of money on these products and lifestyle changes, some of which may be harmful. Scientists are unwittingly contributing to the proliferation of these pseudoscientific antiaging products by failing to participate in the public dialogue about the genuine science of aging research. The purpose of this document is to warn the public against the use of ineffective and potentially harmful antiaging interventions.

Although there is some truth in this statement, the tone is such that it could discourage other scientists and the general public from seeking an authentic rejuvenation therapy. This debate continues to the present day and is currently focused not only on "entrepreneurs who are promoting antiaging products" but also on a critique of the British gerontologist Dr. Aubrey de Grey, who has chastised the biogerontology community for being too conservative in their approach to rejuvenation. De Grey has proposed what he calls "strategies for engineered negligible senescence" or SENS, which involves an aggressive battery of preventative and therapeutic treatments. Robert Butler, S. Jay Olshansky, George Martin, and other prominent members of the biogerontology community have dismissed the SENS protocol as mere science fantasy, and in a published statement they have declared that they "wish to dissociate ourselves from the cadre of those impressed by de Grey’s ideas in their present state."


Many scientists who have entered the field of gerontology have done so with the stated intention of finding cures for age-related diseases, but not therapies to reverse the aging process. These scientists often shy away from any talk of rejuvenation for fear of being ridiculed by their colleagues. When Orville and Wilbur Wright were working secretly on their motorized kite at Kitty Hawk, North Carolina, most scientists thought the attempt to build a flying machine was pure folly and a complete waste of time. In the 1980s many scientists were equally contemptuous of any attempt to clone a mammal. Indeed, in 1984 James McGrath and Davor Solter, two leaders in the field of animal cloning, published an article in the journal Science, which claimed that cloning a mammal was biologically impossible. Just 12 years later, Ian Wilmut and his team proved them wrong.

This topic will explore the controversial topic of rejuvenation and the science that could make it a practical reality. There is, of course, no way to reverse the aging process at the present time, but there are therapies available that treat age-related diseases. Some of these therapies, particularly those involving hormone supplements, can have dangerous side effects and should never be used without the supervision of a physician. But the goal of rejuvenation therapy is to treat and reverse the aging process itself, so that age-related diseases never occur in the first place. Accomplishing this feat will make the flight at Kitty Hawk seem like child’s play and will dwarf all other scientific endeavors, including the exploration of Mars and the Human Genome Project. Nevertheless, the following discussion will show that the necessary theories, tools, and techniques are now at hand to produce a viable rejuvenation therapy within the next 20 years.

Turning back the clock

The basic goal of any rejuvenation therapy is to reset the clock in aged cells or tissues in order to move them back to a preexisting, youthful state. Anti-aging medicines, such as estrogen or testosterone supplements, do not reverse the aging process, nor do they alleviate all of the symptoms associated with a loss of those hormones. This is due to the age-related changes that occur in all of the cells in the body. Old cells do not respond to hormones the same way they did when they were young. Hormone receptors in the membranes of every cell change with time, as does the translation machinery that uses mRNA to synthesize proteins.

The large number of aging theories suggests that rejuvenation would have to consist of many therapies designed to reverse the aging process simultaneously at the cell, tissue, and physiological levels—a task that would be almost impossible to accomplish. A better approach is to focus the therapy at the level of the cell nucleus, which is, after all, at the heart of the age-related changes that occur in humans and other animals. Nuclear rejuvenation therapy (NRT) would require a gene expression profile for every kind of cell in the body, as well as the identification of all gene regulatory molecules. With this information at hand, gene expression could be manipulated in order to rejuvenate the cells, which would lead automatically to the rejuvenation of the whole body.

Identification of all human genes, of which there are an estimated 30,000, is already under way. This information is being obtained by research laboratories in several countries around the world as part of the Human Genome Project.Once the genes are identified, several procedures can be applied in order to determine expression profiles, the identity of gene-regulatory molecules, and the manipulation of specific genes. These procedures include DNA microarray analysis, nuclear transfer technology, cell fusion technology, stem cell analysis, and gene therapy.

DNA MICROARRAY ANALYSIS

Based on information provided by the genome project, a short piece of every available gene can be spotted onto a solid support (usually, a specially treated glass microscope slide) to produce a microarray of gene fragments. The microarray can then be hybridized with labeled mRNA isolated from chosen cells. If a gene is active in the cell, its mRNA will bind to the piece of that gene attached to the microarray, effectively labeling that particular point, or pixel, on the array.

Microarray analysis of gene expression. Fragments of genes are spotted onto a glass microscope slide to produce a two-dimensional array. Labeled mRNA is hybridized to the array to determine which genes are active (yellow spots) and which are not (blue spots). This simulated array shows the expression of 100 genes.

Microarray analysis of gene expression. Fragments of genes are spotted onto a glass microscope slide to produce a two-dimensional array. Labeled mRNA is hybridized to the array to determine which genes are active (yellow spots) and which are not (blue spots). This simulated array shows the expression of 100 genes.

Computers are used to compare the young and old cells, spot by spot, to gain a final estimate of expression for every gene represented on the array. Microarrays were used recently by University of Wisconsin scientists, who evaluated the activity of 20,000 genes in cells from the prostate gland, before and after the cells attained replicative senescence.

Microarray analysis provides an extremely powerful method for analyzing the aging process in an unbiased manner. That is, until the genome project was completed, gerontologists using available theories as a guide had to make an educated guess as to which genes might be involved in cellular senescence. Studies were then designed around these genes in a few of the animal’s tissues or organs. It is clear now that such a limited approach is doomed to failure. Aging is a highly integrated phenomenon, involving all of the organs and tissues of the body. Some tissues or organs may age at their own rates, but they are all part of the same process.

Nuclear transfer technology

This technology involves the transfer of a nucleus from one cell into another cell that has had its own nucleus removed. This procedure has been used to clone amphibians and mammals and was originated by the great 19th-century German embryologist Hans Spemann to test two theories of cell differentiation. All animals originate from a single cell, which grows and divides in a process called embryogen-esis to produce an animal consisting of billions of cells.

Embryogenesis is also associated with cellular differentiation. That is, as the embryo grows, some of the cells become neurons while others give rise to the heart, skin, bones, and all other cells and tissues of the body. If all the cells originate from the same cell, the egg, they must have the same genome (i.e., the same genes). But if that is the case, how can they differentiate? One theory suggests that cells differentiate by losing genes. A second theory states that all cells in an adult have the same genes, but some are repressed and thus nonfunctional. Spemann reasoned that if the second theory was true, a nucleus from an advanced embryo (containing 16 cells) should be able to support development when transferred into a 4-cell embryo. Spemann’s experiment, using salamanders, produced two healthy embryos, thus supporting the second theory.

Full proof for the second theory, however, did not come until 1996, when Ian Wilmut and his team cloned Dolly the sheep. Wilmut’s experiment differed from Spemann’s in that the donor nuclei came from adult cells. If a nucleus from an adult cell can support embryonic development, leading to the birth of a normal lamb, then all cells in the body must have the full complement of genes, which are retained throughout the life span of the individual.

Cloning sheep. The Poll Dorset provides the nucleus, which is obtained from cultured ovine mammary gland epithelial (OME) cells. The blackface provides the egg, which is subsequently enucleated. If the cloning process is successful, the clone will look like a Poll Dorset.

Cloning sheep. The Poll Dorset provides the nucleus, which is obtained from cultured ovine mammary gland epithelial (OME) cells. The blackface provides the egg, which is subsequently enucleated. If the cloning process is successful, the clone will look like a Poll Dorset.

Cloning technique. Light micrograph of a sheep egg being injected with an embryonic cell during sheep cloning. The egg (at center) has had its DNA genetic material removed. At left a pipette holds the egg; at right a microneedle injects an embryonic sheep cell into it. The implanted egg is then stimulated to grow into a lamb by a spark of electricity, nourished in the womb of a surrogate sheep. In 1996 this research at the Roslin Institute in Edinburgh, Scotland, produced the world's first cloned (genetically identical) sheep.

Cloning technique. Light micrograph of a sheep egg being injected with an embryonic cell during sheep cloning. The egg (at center) has had its DNA genetic material removed. At left a pipette holds the egg; at right a microneedle injects an embryonic sheep cell into it. The implanted egg is then stimulated to grow into a lamb by a spark of electricity, nourished in the womb of a surrogate sheep. In 1996 this research at the Roslin Institute in Edinburgh, Scotland, produced the world’s first cloned (genetically identical) sheep.

These cloning experiments not only proved the validity of the second theory of differentiation, but they also proved that it is possible to rejuvenate a nucleus, and that the molecules necessary to effect this dramatic transformation are located in the cytoplasm of the oocyte. In other words, animal cloning is a form of nuclear rejuvenation. When a nucleus from an adult cell is placed inside an enucleated egg, the environment of the egg reprograms, and rejuvenates, the older nucleus. Thus nuclear transfer, combined with mi-croarray analysis, provides a powerful tool for developing a nuclear rejuvenation therapy.

Cell fusion technology

This technology is closely related to nuclear transfer in that the nucleus of one kind of cell is brought under the influence of the cytoplasm of a second type of cell. The two cells are exposed to a Sendai virus that stimulates fusion of the cells’ membranes. This procedure was first used by cell biologists in the 1960s to study the reactivation of avian erythrocyte nuclei exposed to a HeLa cell (a type of cancer cell). The HeLa cell is a highly active, immortalized cell, with a large round nucleus composed of decondensed chroma-tin. By contrast, the avian erythrocyte is highly differentiated with a small, inactive nucleus, consisting mostly of condensed chromatin. Fusion of these two cells results in a dramatic transformation of the erythrocyte nuclei, characterized by a reduction in the amount of condensed chromatin and an increase in nuclear volume.

Scientists at Harvard University have reprogrammed somatic cell nuclei by fusing them with embryonic stem cells. Analysis of genome-wide transcriptional activity, along with additional tests, showed that the somatic genome was reprogrammed to an embryonic state. The use of stem cells in this way provides a very powerful method for identifying the factors responsible for nuclear rejuvenation.

Stem cell analysis

Stem cells are capable of differentiating into many different kinds of cells and are currently being used to regenerate normal bone marrow in patients suffering from leukemia. Future applications involve therapies to treat damaged spinal cords, Parkinson’s disease, Alzheimer’s disease, and cardiovascular disease.

Stem cells may also be used to identify nuclear rejuvenation factors. This can be done in two ways: cell fusion experiments (described above) and directed differentiation, a process whereby cultured stem cells are induced to differentiate by exposing them to a variety of molecules. Experiments such as these will make it possible for scientists to identify cell-specific expression profiles. Studies have shown that stem cell differentiation to a neuron passes through several stages or levels, each of which is characterized by activation of a unique set of nuclear transcription factors (molecules that control gene expression).

Cell Fusion. A HeLa cell was fused with an avian erythrocyte and allowed to incubate for 48 hours. Molecules in the HeLa cell cytoplasm induced a dramatic change in the erythrocyte nucleus, which included an increase in size, a decrease in the amount of condensed chromatin, and a reactivation of transcriptional activity.

Cell Fusion. A HeLa cell was fused with an avian erythrocyte and allowed to incubate for 48 hours. Molecules in the HeLa cell cytoplasm induced a dramatic change in the erythrocyte nucleus, which included an increase in size, a decrease in the amount of condensed chromatin, and a reactivation of transcriptional activity.

Differentiation of embryonic stem cells. Embryonic stem cells are obtained from the inner cell mass of a blastocyst. When cultured, these cells can differentiate into many different kinds of cells, representing the three germ layers.

Differentiation of embryonic stem cells. Embryonic stem cells are obtained from the inner cell mass of a blastocyst. When cultured, these cells can differentiate into many different kinds of cells, representing the three germ layers.

Aging is no doubt associated with a similar stage-specific set of nuclear factors. In this case a stage is likely to be a period of about 10 years, so that each decade will be accompanied by a unique set of nuclear factors. Stem cell analysis, combined with cell fusion and cloning technologies, would provide a way to identify these factors, after which they could be used to reset the apparent age of a nucleus to any decade desirable.

Micrograph of a human embryo soon after fertilization. The cells, or blastomeres, result from divisions of the fertilized egg and are surrounded by the protective zona pellucida layer. The cells of embryos like these are sometimes harvested as stem cells.

Micrograph of a human embryo soon after fertilization. The cells, or blastomeres, result from divisions of the fertilized egg and are surrounded by the protective zona pellucida layer. The cells of embryos like these are sometimes harvested as stem cells.

Gene therapy

This therapy is used to modify or replace specific genes within the nucleus. Gene therapy would be used to correct random mutations that are likely to appear with age throughout the genome. Rejuvenating the nucleus with an age-specific set of transcription factors would not undo this damage. Indeed, genetic mutations could accelerate aging or trigger cancer development. Since the location of these mutations is expected to vary among individuals, it would be necessary to sequence the genome of every person who is interested in receiving rejuvenation therapy. Ultrafast sequencing machines are being developed now. The ultimate goal is a machine that can sequence the entire human genome in one day at a cost of about $1,000. These machines are expected to be available within the next 10 years.

THE FINAL PACKAGE

Delivery of rejuvenation therapy could be accomplished using li-posomes. Each liposome would contain the factors necessary to rejuvenate one type of cell. As a consequence, it would be necessary to produce a separate liposome package for each of the 200 known cell types in the human body. Liposomes can be targeted to specific cells by embedding special recognition proteins in their membrane. Thus the liposomes would only bind to and enter specific cells. For example, a liposome package intended for cardiac muscle would have a cardiac muscle recognition protein embedded in its membrane. These recognition proteins can be designed to preclude cross-reactivity.

The exact contents of each liposome package would vary depending on the outcome of the above analysis for each individual. All individuals would receive the basic package needed to rejuvenate cells that have suffered age-related effects. But some patients may have a genetic predisposition for Alzheimer’s disease, which would require the inclusion of a gene therapy vector for the affected neurons. Others may suffer from random genetic mutations, cardiovascular disease, osteoporosis, or diabetes, and thus would require special packages.

The liposome packages could rejuvenate an individual, but they would not stop the clock. Immediately after the therapy, the cells would begin to age again. Consequently, rejuvenation therapy would have to be repeated from time to time.

There would, of course, be many dangers associated with such a radical therapy. As the liposome packages are produced, they would have to be tested in a series of clinical trials before they could be used as a medical therapy. If the mosaic theory of the aging process holds true, only a fraction of the known cell types would have to be treated. In this case the 20-year estimate quoted above, to obtain a safe and effective therapy, would likely hold. On the other hand, if 200 packages had to be tested, then rejuvenation therapy of the kind described here would not be available for at least 50 years. At this rate 40 packages would have to be developed and tested every 10 years. This estimate assumes an accelerated pace in testing and validation procedures and an absence of serious complications in the trials themselves.

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