When my guest-bathroom toilet flap valve recently gave out due to old age, I purchased and tried out four different “one size fits all” replacement flap valves, shopping at Home Depot and different hardware stores.  They are very simple devices but none quite fit and all left the toilet leaking.  The process required four different shopping trips and left me with a non-functioning toilet for 10 days.  Then I did the smart thing and found out the brand of my toilet (an Elger) and went to a plumbing supply house and purchased an Elger toilet retrofit kit.  It worked perfectly.  The solution was personalized to the particular design and dimensional specifications of my toilet.

Physicians are trained to diagnose what is going on in an individual and to prescribe a treatment tailored to that individual and his disease condition.  Modern diagnostic tests and instruments such as MRIs have been making this process ever-more precise.  However, until now it has not been possible to make treatment choices based on the molecular, genetic and epigenetic makeup of a given patient.  We are all genomically and epigenomically vastly more different from one another than an Elger toilet is from other toilets.  So, as far as therapies go, medicine has too-often proceeded on the basis of “one size fits all” when the facts have often shown “one size misfits most.”  Finding the right prescription medicine can be like finding the right toilet flap valve, a matter of trial and error and high cost and inconvenience.

According to an article Personalized Medicine Realizing Its Promise by Edward Abrahams, Ph.D. “On average, a drug on the market works for only 50% of the people who take it. The consequences in terms of quality and cost of care are significant, leaving patients to contend with their disease and their medical bills as they switch from one drug to another until they find an effective therapy.”  Trial-and-error of drugs is particularly prevalent in some fields like psychiatry.  A psychiatrist is often unable to make a definitive diagnosis at first between the various shades of depression, bipolar and related mental disorders.  Instead the psychiatrist will keep prescribing different drugs until one or more work to control the symptoms.  Then, based on the drug that works, a diagnosis can be pronounced. 

Now, in the emerging context of personalized medicine, customization of treatment can depend not only on observable diseases conditions but also on the molecular, genomic and epigenomic makeup of the particular patient.  There is increasing evidence, for example, that combinations of genetic and epigenetic markers can be useful in diagnosing mental disease conditions. Genomic, epigenomic and other diagnostic tests are coming on the market which will tell whether a drug treatment or medical procedure is likely to work or not.   For people on an expensive year-long drug therapy, such a test could save their life by indicating that a blockbuster drug is probably not going to work and by preventing exposure to its side effects.  For the government and society, such tests could in aggregate mean hundreds of billions of dollars saved in irrelevant treatments and unnecessary hospital stays.

A special report in Gen provides some examples:

“For example, Genentech/Roche’s Avastin costs $50–$100,000 per year of treatment but works in fewer than 50% of patients. Given that Avastin may generate $12 billion in peak sales, this low rate of efficacy translates into billions of dollars in misdirected healthcare spending. A test for Avastin response, such as that in discovery by BG Medicine, could save the system as much as $6 billion per year if all nonresponders could be removed from the treatment pool. Assuming that a test of this sort is introduced at the beginning of 2013 and is 100% adopted, cumulative savings of $40 billion could be realized by 2019.”  That is not small change. 

“Genomic Health’s Oncotype Dx is a test with compelling cost-saving potential. It is used to predict chemotherapy benefit for patients who have node-negative, estrogen receptor positive (node-, ER+) breast cancer. By averting unnecessary chemotherapy, the test has been shown to save about $2,000 per patient. Extending this cost savings to the roughly 100,000 new cases of node-, ER+ breast cancer in the U.S. each year, this test could save the U.S. healthcare system up to approximately $200 million a year or about $2 billion over the 10-year time horizon under legislative consideration.” Adding up the savings for drug after drug after drug could chop an enormous slice over our annual health care costs.

With the development and adoption of appropriate diagnostic tests, the aggregate effect on health care costs and patient wellbeing could be enormous.  One approach is the development and use of specific tests associated with specific drugs or treatment procedures.  For example.  before prescribing Avastin, the oncologist could order the test being developed by BG Medicine. A better approach that will no doubt be implemented in the longer run involves building databases of patient-specific data, such as their entire genome.  In a recent blog entry, I pointed out that the cost of sequencing the entire genome of a patient is heading down to the $1,000 level probably this year and will be probably at the $100 level within four years or less. If a patient has his or her genome already laid out in such a database, many bad-choice drug treatments could be avoided by a simple computer check against the database, just like transfusions of the wrong types of blood are often avoided now.

Also, the existence of such a database could signal disease susceptibilities and the advisability of preventative actions.  “Over 1,300 genetic tests exist that signal inherited susceptibility to conditions as wide-ranging as hearing loss and sudden cardiac arrest. While not every test has a therapeutic option, a genetic diagnosis often permits targeted prevention or mitigation strategies(ref).”  One example is looking for BRCA1 and BRCA2 genetic mutations indicating a hereditary propensity for breast and ovarian cancer. “Women with BRCA1 or BRCA2 genetic risk factors have a 36% to 85% lifetime chance of developing breast cancer, compared with a 13% chance among the general female population.  For ovarian cancer, women with certain BRCA1 or BRCA2 gene mutations have a 16% to 60% chance of disease, compared with a 1.7% chance among the general population. The BRCA1 and BRCA2 genetic test can guide preventive measures such as increased frequency of mammography, prophylactic surgery, and chemoprevention(ref).

The bottom line for the government is to effectively control health care costs, do everything you can to further implementation of personalized medicine and the building of patient-specific genomic and epigenomic databases..

When I wrote my first blog entries on epigenomics and epigenetics eleven months ago(ref)(ref) , it was clear that these were active areas of extremely interesting academic research.  However, my impression was that it would be years before the knowledge being accumulated could be put to practical work in medicine.  I was wrong.  Not only has the depth and amount of research in these areas increased significantly but epigenetics has entered the commercial medical marketplace in the forms of new therapeutic drugs and diagnostic tools.  This blog entry provides an update.

Background

An introduction to epigenetics and epigenomics and their relevance to aging is in the Feb 2009 blog entry Epigenetics, Epigenomics and Aging.  And, of course, the 13th theory of aging in my treatise is Programmed Epigenomic Changes.  “Epigenetics is the study of changes in phenotype and gene expression arising from mechanisms other than changes in a gene’s DNA sequence.– Over 25 years ago, Surani et al. showed that certain regions of a cell’s genome carry markers over and above the actual gene sequence. This imprint conveys information on differential gene expression, and therefore, shapes the fate of the cell.  Epigenetic information is passed from one cell to another, but the epigenetic code can change through life by interacting with environmental factors. Moreover, unlike gene-sequence mutations, epigenetic changes may be reversible(ref).”

Since cells of all types in an individual have the same DNA, epigenetic information is what gives a cell memory as to the type of cell it is, and many events in the lifetime of a cell are also encoded epigenetically.  And some epigenetic information can be inherited.  The main mechanisms of encoding epigenetic information are DNA methylation and chromatin modifications, such as histone acetylation.  See the blog posts DNA methylation, personalized medicine and longevity and Histone acetylase and deacetylase inhibitors. 

“DNA methylation involves the addition of a methyl group to the 5 position of cytosine (one of the four bases of DNA), which occurs in the context of CpG (cytosine followed by guanine) dinucleotides. This modification can be inherited through cell division. DNA methylation is typically removed during zygote formation and reestablished through successive cell divisions during development. DNA methylation is a crucial part of normal organismal development and cellular differentiation in higher organisms. DNA methylation stably alters the gene expression pattern in cells such that cells can “remember where they have been”; in other words, cells programmed to be pancreatic islets during embryonic development remain pancreatic islets throughout the life of the organism without continuing signals telling them that they need to remain islets. In addition, DNA methylation suppresses the expression of viral genes and other deleterious elements which have been incorporated into the genome of the host over time. DNA methylation also forms the basis of chromatin structure, which enables cells to form the myriad characteristics necessary for multicellular life from a single immutable sequence of DNA. DNA methylation also plays a crucial role in the development of nearly all types of cancer(ref).^[1]”

Of particular interest from the viewpoint of DNA methylation in mammals are the so-called CpG islands, gene promoter sites that are not methylated when a gene is being expressed.  “CpG islands typically occur at or near the transcription start site of genes, particularly housekeeping genes, in vertebrates.^[2]  — “Unlike CpG sites in the coding region of a gene, in most instances, the CpG sites in the CpG islands of promoters are unmethylated if genes are expressed(ref).”

Testing for DNA methylation

A standard approach has been developed for checking out the DNA methylation patterns of humans known as bisulfite sequencing.  “Bisulfite sequencing is the use of bisulfite treatment of DNA to determine its pattern of methylation. — Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. Thus, bisulfite treatment introduces specific changes in the DNA sequence that depend on the methylation status of individual cytosine residues, yielding single- nucleotide resolution information about the methylation status of a segment of DNA.”  Once a DNA sample has been pre-treated with bisulfite, it can be sequenced just like any other DNA.  Software comparison(ref) of a sequenced output against that for a normal human genome will reveal the GpC promoter sites that are methylated.  Thus, orders-of-magnitude increases in power of gene sequencing such as discussed in the blog post My personal longevity – the race between death-stalker and life-prolonger equally benefit testing for DNA methylation. 

Some sequencing platforms have been used extensively for bisulfite sequencing for methylation patterns, for example the Illumina GoldenGate bead array platform. “Through an adaptation of the GoldenGate genotyping assay implemented on a BeadArray platform, the methylation state of 1536 specific CpG sites in 371 genes (one to nine CpG sites per gene) was measured in a single reaction by multiplexed genotyping of 200 ng of bisulfite-treated genomic DNA. The assay was used to obtain a quantitative measure of the methylation level at each CpG site. After validating the assay in cell lines and normal tissues, we analyzed a panel of lung cancer biopsy samples (N = 22) and identified a panel of methylation markers that distinguished lung adenocarcinomas from normal lung tissues with high specificity. These markers were validated in a second sample set (N = 24). These results demonstrate the effectiveness of the method for reliably profiling many CpG sites in parallel for the discovery of informative methylation markers. The technology should prove useful for DNA methylation analyses in large populations, with potential application to the classification and diagnosis of a broad range of cancers and other diseases(ref).”

The June 2009 report CpG Methylation Analysis—Current Status of Clinical Assays and Potential Applications in Molecular Diagnostics provides a snapshot of the technology at that time, a picture that is rapidly changing with the arrival of new ever-more powerful sequencing platforms.

Several methylation studies have been concerned with basic science.  An example is described in a report dated February 3, 2010 International Team Maps Methylation Changes During Cellular Differentiation.  “Using bisulfite sequencing with the Illumina Genome Analyzer, researchers from Singapore and the US mapped and compared DNA methylation patterns in human cells during three progressive stages of differentiation: embryonic stem cells, skin-like cells derived from embryonic stem cells, and primary neonatal skin cells. In the process, the team identified shared and cell type-specific methylation patterns, providing insights into how gene regulation shifts during development.  — With these comprehensive DNA methylome maps, scientists now have a blueprint of key epigenetic signatures associated with differentiation,” co-corresponding author Chia-Lin Wei, a researcher affiliated with the Genome Institute of Singapore and the National University of Singapore, said in a statement.”

DNA methylation and diseases

“DNA methylation is widespread and plays a critical role in the regulation of gene expression in development, differentiation, and diseases such as multiple sclerosis, diabetes, schizophrenia, aging, and cancers (Li et al. 1993; Laird and Jaenisch 1996; Egger et al. 2004). Methylation in particular gene regions, for example in promoters, can inhibit gene expression (Jones and Laird 1999; Baylin and Herman 2000; Jones and Baylin 2002). Recent work has shown that the gene silencing effect of methylated regions is accomplished through the interaction of methylcytosine binding proteins with other structural components of chromatin (Razin 1998), which, in turn, makes the DNA inaccessible to transcription factors through histone deacetylation and chromatin structure changes (Bestor 1998).  Hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes is now firmly established as the most frequent mechanism for gene inactivation in cancers (Esteller 2002; Herman and Baylin 2003 Changes in DNA methylation are recognized as one of the most common forms of molecular alteration in human neoplasia (Baylin and Herman 2000; Balmain et al. 2003; Feinberg and Tycko 2004))(ref).”
 

Many studies have been performed using high-throughput bisulfate sequencing to identify methylation patterns of disease conditions, so many that I can cite only a few examples here.

A December 22 2009 report Twin Epigenetics Study IDs Methylation Differences in Lupus.  “– Epigenetic changes, specifically differences in DNA methylation, may contribute to environmental factors involved in systemic lupus erythematosus risk, according to an online study in Genome Research today. — A Spanish, German, and American research team used bead arrays and targeted bisulfite sequencing to compare DNA methylation patterns in the genomes of more than a dozen sets of identical twins who were discordant for SLE or two other autoimmune diseases. While they did not detect methylation differences for two of the conditions, the team did detect intriguing epigenetic changes when one twin had SLE and the other did not. — “Our study suggests that the effect of the environment or differences in lifestyle may leave a molecular mark in key genes for immune function that contributes to the differential onset of the disease in twins,” Esteban Ballestar, a researcher at the Bellvitge Biomedical Research Institute in Barcelona, said in a statement.”

The September 2009 study Epigenetic silencing of death receptor 4 mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in gliomas had the purpose “To identify and characterize epigenetically regulated genes able to predict sensitivity or resistance to currently tested chemotherapeutic agents in glioma therapy.”  The conclusion is “DR4 promoter methylation is frequent in human astrocytic gliomas, and epigenetic silencing of DR4 mediates resistance to TRAIL/DR4-based glioma therapies.”

Other recent research reports on the epigenetics of disease processes include The ADAMTS12 metalloprotease gene is epigenetically silenced in tumor cells and transcriptionally activated in the stroma during progression of colon cancer, New insights into the biology and origin of mature aggressive B-cell lymphomas by combined epigenomic, genomic, and transcriptional profiling, Epigenetic profiling reveals etiologically distinct patterns of DNA methylation in head and neck squamous cell carcinoma, Aberrant DNA methylation is a dominant mechanism in MDS progression to AML, Large-Scale Profiling of Archival Lymph Nodes Reveals Pervasive Remodeling of the Follicular Lymphoma Methylome, Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation, and Epigenetic Profiles Distinguish Pleural Mesothelioma from Normal Pleura and Predict Lung Asbestos Burden and Clinical Outcome. And this is just a starter list.

Epigenetic drugs

A Feb 1 2010 report in Gen Epigenetics Offers Strategies for New Drugs states “According to a recent report from Business Insights, “Innovations in Epigenetics: Advances in Technologies, Diagnostics & Therapeutics,” epigenetic medicine is already here. — The company puts the epigentic market at over $560 million, derived from the sale of three anticancer products (Dacogen™ from Eisai, Vidaza® from Celgene, and Zolinza® from Merck), which target two epigenetic pathways—DNA methyltransferase (DNMT) and histone deacteylase (HDAC). — Approximately 30 epigenetic drugs are under development by more than a dozen biotechnology companies, according to the Business Insights study. These drugs focus mainly on the treatment of cancer, and neurodegenerative and infectious.”  The $560 million market mentioned above is only for drug sales.  It does not include epigenetics R&D expenditures and the market for epigenetic disease tests.  Whatever the market size is now, I estimate it will double in size every 12 to 18 months being driven by the factors that drive Giuliano’s Law until it gets up into the hundreds of billions of dollars in less than 15years.  Epigenetics and epigenomics are destined to be very big biz.

Epigenetic diagnostic tools

Finally, as DNA methylation patterns are discovered and confirmed for more and more cancers and other diseases, and as the costs of bisulfite epigenomic sequencing continue to drop, it is likely that we will see many methylation diagnostic and predictive tests being integrated into mainline medical practice.  There is evidence, for example, that cancers develop in stages and that methylation tests can provide predictors of premalignant conditions long before the cancers themselves emerge. “One of the advantages of using epigenomic biomarkers is that, in most cases, DNA methylation changes precede clinical symptoms. “If there is a small abnormality that is not yet an invasive cancer, but a precancerous lesion, or a small tumor, many genes will have abnormal methylation, and that is probably true in many tissues,” says Dr. Baylin(ref) .

A number of small biotech companies are moving into this diagnostic area.  As reported in Gen the first of this month “Epigenomics specializes in DNA-methylation technologies and biomarkers and is developing a number of cancer diagnostics based on differences in DNA methylation between healthy and diseased tissue. Constellation Pharmaceuticals is focused on developing therapeutics based on epigenetics. It is currently establishing a preclinical pipeline and developing a technology platform for histone modification. Initial applications will be in oncology. Finally, Epizyme is looking at histone methyltransferases and is developing a pipeline of inhibitors for cancer.”

Like the computer industry was in 1959, the genetic, genomic, epigenetic and epigenomic areas are multifaceted and messy.  They involve far-out ideas, scientific research, hype, university gurus, technology industry developments, entrepreneurial ventures, commercial activities in big companies and sizeable investments. Some approaches and companies will succeed, others will fail.  Some will succeed for a while and then fail.  That is what progress looks like.  Epigenetics, genomics and sequencing are built on the back of the computer and communications revolution.  Without extremely powerful and cheap computers, genetic or epigenetic sequencing would be impossible.  This time around, one net result will be that people will live longer.

Can big multinational corporations buy and own rights to what I do with my nose, my liver, my heart, my little right toe? Possibly, because they can buy or license patent rights for many of my genes.  That is the way it has been for a number of years now.   I have always thought that having someone else hold rights to my natural body parts is completely absurd.   I should be able to do anything with my genes that I want.  But what do I know about such matters?  I am not a lawyer.

Finally, patentability of genes is being challenged in the courts.  According to a Feb 2 news highlight in Gen “Today, the American Civil Liberties Union (ACLU) and the Public Patent Foundation (PUBPAT) will deliver oral arguments asking the court to rule that patents on two human genes associated with breast and ovarian cancer are unconstitutional and invalid. The groups charge that the patents stifle diagnostic testing and research that could lead to cures and that they limit women’s options regarding their medical care. — The lawsuit Association for Molecular Pathology et al. v. U.S. Patent and Trademark Office et al. was originally filed on May 12, 2009, in the U.S. District Court for the Southern District of New York on behalf of breast cancer and women’s health groups, individual women, and scientific associations representing approximately 150,000 researchers, pathologists, and laboratory professionals. — The lawsuit was filed against the PTO as well as Myriad Genetics and the University of Utah Research Foundation, which hold the patents on the genes BRCA1 and BRCA2. The lawsuit charges that patents on human genes violate the First Amendment and patent law because genes are “products of nature” and therefore can’t be patented.”

“The plaintiffs include Breast Cancer Action, The American College of Medical Genetics, the Association for Molecular Pathology, the College of American Pathologists, the American Society for Clinical Pathology, individual researchers, patient advocacy groups, genetic counselors, and individual women(ref).”

So, how did genes get to be patentable in the first case, and what are the main issues involved?  For genes to be patentable, they have to be viewed as inventions.  And they have been so-viewed because attorneys have successfully argued that it has taken inventions to identify them.  Basically, “the US Patent and Trademark Office (USPTO) and the European Patent Office (EPO) have treated isolated and purified nucleotide sequences as if they were the same as man-made chemicals(ref).”  The decision to allow genes to be patented has engendered much controversy and endless opinion papers.  See this list for some of them. 

The economic argument for gene patentability is of course that it provides economic incentive for discovery and invention.  The biotech and pharma companies and universities that hold the gene patents will want to hold onto them.  Increasingly, however, voices are questioning the wisdom of gene patentability and whether it gets in the way of scientific progress, public health and patient care. 

A recent position paper starts out: “Concerns about human gene patents go beyond moral disquiet about creating a commodity from a part of the human body and also beyond legal questions about whether genes are unpatentable products of nature. New concerns are being raised about harm to public health and to research. In response to these concerns, various policy options, such as litigation, legislation, patent pools and compulsory licensing, are being explored to ensure that gene patents do not impede the practice of medicine and scientific progress.  Although gene patents have been granted worldwide for several years, the wisdom of this action is now being questioned.  Lawsuits, proposed legislation, international protests and even patent-office proposals have recently been initiated to eliminate, undermine or otherwise challenge the scope of patents on human genes. The challenges come from various interested parties — people from whom patented genes have been isolated, researchers who wish to undertake genetic epidemiological studies or to develop gene therapies, clinicians and health-care providers who cannot afford expensive licensing fees for genetic tests and policymakers who want to ensure that the patent system actually meets its goal by encouraging invention. Evidence is mounting that gene patents are inhibiting important biomedical research, interfering with patient care and provoking criticisms from international trading partners.” 

My guess is that the case may take 5-10 years to work its way through the courts and may well end up in front of the Supreme Court.  And who knows how they will choose to look at the situation.  In terms of property rights or interstate commerce?  Meanwhile we will have to live with gene patentability. I realize that in working out on the treadmill or taking supplements that either activate or inhibit certain of my genes, I am probably violating somebody or the other’s patent.  So are you.  Bah!

The track record of clinical trials involving human embryonic stem cells (hESCs) is worse than miserable.  A dozen years ago, it was thought that for sure by now in 2010 hESCs would be used in all kinds of regenerative medicinal applications.  Instead, they remain stalled at the gate while other types of stem cells are being used to produce all kinds of intereting results.  In the blog post It’s a long way to stem cell treatment I discussed how the clinical trial of Geron’s proprietary hESC-based product GRNOPC1 was being delayed for the second time in August 2009 because of FDA caution(ref).  That trial, still being delayed, involves the use of hESCs for treating severe spinal cord injuries.

It is very tempting to blame lack of progress in development of hESC therapies on government banning of funding of most embryonic stem cell research during the Bush era.  I personally think the ban was a terrible idea, anti-science and anti-humanistic.  While the ban has been a factor, however, I do not think it had a major impact on worldwide progress in hESC research.  And, simply put, other kinds of stem cells have turned out to be more exciting and more easy to work with.  The stubborn challenges associated with hESC therapies appear to be 1.  possible immune system rejection or reactions because the cells are not derived from the patient (the cells are not autologous), and 2.  assuring that the stem cells differentiate into the desired target cell types and only those types.  More-specialized patient-derived stem cells like haemopoietic and mesenchymal stem cells do not run the risk of immune system rejection and their differentiation can be directed more easily.  And, autologous induced pluripotent stem cells (iPSCs) seem to be able to do everything that hESCs can do without immune system rejection.

There was news last week that GRNOPC1 is emerging again in a new clinical context, this time for treating Alzheimer’s disease.  The report Geron to Study Its hESC Product in Alzheimer Disease with University of California in Gen states “Geron and researchers from the University of California have decided to work together to assess the company’s human embryonic stem cell (hESC) product, GRNOPC1, for Alzheimer disease. The work will be jointly funded by the firm and a university discovery research and training grant. — Geron and the University of California team will now evaluate GRNOPC1 in models of Alzheimer disease. The study is designed to assess whether memory shows recovery after transplantation of GRNOPC1.  — The research will be led by Frank M. LaFerla, Ph.D., director of the Institute for Brain Aging and Dementia at the University of California, Irvine.”

The interest follows in large part from promising mouse studies.  “There are striking parallels between recent data on mouse stem cells in Alzheimer’s disease models and what we know about GRNOPC1(ref).” Dr. LaFerla and his colleagues published a research report in August 2009 entitled Neural stem cells improve cognition via BDNF in a transgenic model of Alzheimer disease.  That report cited data demonstrating “that defects in memory were improved by glial cells derived from mouse neural stem cells transplanted into the hippocampus of rodent models of Alzheimer disease. — GRNOPC1 contains the precursors to human glial cells, which matured and repaired the lesion site in rodent models of spinal cord injury. Additionally, the improvement in memory and the increase in synaptic density observed after injection of neural stem cells were found to be mediated, at least in part, by the neurotrophic factor BDNF, which is secreted from the transplanted cells. GRNOPC1 has been found to secrete BDNF as well as other neurotrophic factors(ref).”  “Taken together, our findings demonstrate that neural stem cells can ameliorate complex behavioral deficits associated with widespread Alzheimer disease pathology via BDNF(ref).”

If and when GRNOPC1 is to be used in a clinical trial for Alzheimer’s disease is yet to be determined as, apparently, whether and when the clinical trial of GRNOPC1 for severe spinal cord injury will be resumed.  The great original glow of hESC-based therapies is now dim and continues to fade while the prospects for other stem cell therapies seem to grow brighter and brighter.

This post is about a race between two exponentially-accelerating complexes of processes which I will refer to here as death-stalker and life-prolonger.   I am very concerned about how this race will go in the coming few years, for my life will depend on that.

Death-stalker

The death-stalker complex includes all those biological changes that exponentially raise the probability of death with advancing age, making sure that every human being is gone by 123.  The death-stalker complex is evolutionarily determined and up to this point we see no clear way around it.  Death-stalker makes no exceptions.  Death-stalker is complicated and thorough, involving age-related changes in the expression of thousands of genes.  Each of the 14 major theories of aging and six candidate theories of aging laid out in my treatise is an aspect of death-stalker.  Death-stalker works through many mechanisms to kill older people.  Cancers, cardiovascular diseases, neurological and muscular degeneration, deadly falls, loss of hearing, eyesight, memory and balance – all are aspects of death-stalker.  Sooner or later he gets everybody and he will surely get me eventually.  At my age of 80, death-stalker is normally kicking in with full force, each year drastically raising the probability that I will contract a deadly cancer, cardiovascular problem, a debilitating fall or something else that will soon kill me.  So, I am very impatient.  Death-stalker is near my door.

My game with death-stalker is to play to live two, three or more times longer than the normal maximum lifespan of 123.  This entails not defeating him but forcing him to bend the rules he has had for humans during our entire history.  If I can win this concession from death-stalker, it will only be possible with the assistance of life-prolonger.

Life-prolonger

On the other side of the scale is the life-prolonger complex, an amorphous collection of contexts, situations, developments and matters which increase the probability of my life and the lives of others becoming ever-longer.  Included in the life-prolonger complex are:

a.     The contexts of the possibility of eliminating many diseases and radical extensions of lifespans, contexts which affect individual behavior and   which give rise to research and activities directed towards making those possibilities real,

b.     Health-supporting changes in the basic behavior patterns in large population segments, an example being great reduction in smoking and, hopefully in the near future, reduction in obesity,

c.       Improvements in the physical, public health, medical and commercial environments, including extensive health education programs, improved nutrition and sanitation, inoculation programs, a massive health establishment, the popularity of health clubs, and the availability and sale of health supplements. 

d.     Massive government, foundation and NGO investments in health care and health care research.

Health care in the US today represents roughly 18% of GDP.

In the May 2009 blog entry Social ethics of longevity I argued that social evolution requires that people live longer – and is in fact leading to longer and longer life spans.  The above kinds of changes along with a possible evolution in the human epigenome have been leading to a rise in life expectancy in advanced societies every year.  See the blog posts Average US life expectancy up 73 days in one year and  Ever-increasing longevity– is epigenomics involved? 

These developments may well help me get to over 100, but not to 235 which has been my age target for a number of years now.  However, there are other life-prolonger factors which exhibit exponential acceleration in growth and are likely to lead to flowering of new anti aging technology, as outlined in my blog post Factors that drive Giuliano’s Law.  These include improvements in life sciences R&D technology and technological infrastructure, increase in scientific knowledge related to longevity drawing from multiple disciplines, evolution in personal behavior and individuals assuming direct responsibility for both life extension and health.

Because I am so aware of the death-stalker complex and because I monitor anti-aging science developments on a daily basis, positive changes in the life-prolonger complex seem to be progressing only at a slow creep while the death-stalker process is proceeding at its usual inexorable rate. Further, it is easy for me to lose sight of who is working on life-prolonger for me, where and how.  And how effectively? The good folk in my HMO are helping a lot in the short term, but they have no notion of how to get me over the 123 year hump.  It seems that unless life-prolonger speeds up a lot, I am not going to make it. 

Viewed over the 20 month period since I generated the first online version of my treatise ANTI-AGING FIREWALLS THE SCIENCE AND TECHNOLOGY OF LONGEVITY, however, a tremendous amount has happened in the life-prolonger complex qualifying as exponentially-accelerating progress.  A single measure that I report on in this blog is genome sequencing, particularly the cost and time for sequencing of entire human or disease genomes.

I have reported before on gene testing, in May 2009, Consumer Genomics and Individual DNA testing. Genetic testing is quite different from genome sequencing.  The former involves scanning a few dozen, hundreds or even thousands of genes for variations.  Genetic testing is typically done to check for the probable efficacy of a drug, to predict susceptibilities to certain cancers, or to provide consumers with personal gene information using a service like 23 and me.   Genome sequencing is far more comprehensive, looking at the entire genome of an individual consisting of about 3 billion base pairs of DNA.  It requires 46 separate chromosome sequences in order to represent the complete genome of a human individual.  Again, the objective is to identify evolutionary and mutational differences from what is expected in a normal human genome like small deletions and insertions, SNPs, multiple copies of genes, interchromosomal translocation events, mutations, gene mislocations, etc.  Here are some benchmark points:

·        For over 30 years there has been continuous improvement in the technology of sequencing, lowering of its cost and improvement of its accuracy.  There have been multiple generations of DNA sequencing technology(ref).

·        Between 1994 and 2004 the cost of sequencing dropped a hundredfold.

·        The cost for sequencing the genome of a single individual in 2004 was about $10 million dollars(ref).  At that time the goal was to reduce the cost to $100,000 in 5 years(ref).  The goal was exceeded, the cost in mid-2009 being around $50,000.

·        Today the cost of sequencing the genome of a single individual has dropped to the $6,000 range and is expected to drop to $1,000 in less than another year.

From the latest issue of Gen: “GEN News Highlights — Life Technologies reports that it is $5,000 away from reaching the hallowed ground of the $1,000 genome. The company has introduced the Applied Biosystems SOLiD™ 4 Sequencing System, which generates up to 100 gigabases of mappable sequence data per run at a cost of $6,000 per genome. Illumina would be another contender in this race, with its HiSeq2000, generating data at $10,000 per genome.”

·        With a continuation of the present trend I expect costs of sequencing an entire human genome to come down to around $100 by 2015, making whole-genome sequencing a routine health-supporting process for everyone with health care.

·        The same economics apply to sequencing the entire genomes of cancer cells and other organisms, making available vast databases of comparative genomic information.

Having everyone’s genome sequenced and on-file will serve the cause of longevity in multiple ways.  Included in these are creation of immense databases that will assist:

·        in clarifying the functions of genes and gene regulation throughout the genome,

·     Understanding variations in the DNA sequences among individuals  and determining what they mean. Understanding small differences may help predict a person’s risk of particular diseases and response to certain medications. For example, see the discussion in the blog post CETP gene longevity variants,

·     Developing treatment protocols related to drug selections, therapy selections for certain cancers and other medical conditions that differentially depend on a patient’s genomic profile,

·     Understanding the 3-dimensional structures of proteins and identifying their functions (see the blog post Protein origami and aging),

·     Exploring how DNA and proteins interact with one another and with the environment to create complex living systems (See the blog post The new omics and longevity research)

·     Understanding the epigenomic factors that are involved in aging and possible anti-aging epigenomic interventions, and

·     Using this knowledge to develop and apply genome-based strategies for lifestyle interventions for longevity and the early detection, diagnosis, and treatment of diseases.  (The blog post Genes discussed or mentioned in this blog provides links to a number of relevant discussions in this blog.)

Researching the above discussion led me to an example of who is out there working on life-prolonger for me, namely the good folk at Life Technologies, the ones out to bring the cost of whole-genome scanning down to $1,000 this year.  From the Life Technologies website “Life Technologies (NASDAQ: LIFE) is a global biotechnology tools company dedicated to improving the human condition. — Our systems, consumables and services enable researchers to accelerate scientific exploration, driving to discoveries and developments that make life even better. — Life Technologies customers do their work across the biological spectrum, working to advance personalized medicine, regenerative science, molecular diagnostics, agricultural and environmental research, and 21st century forensics. The company had sales of more than $3 billion, employs approximately 9,500 people, has a presence in more than 100 countries, and possesses a rapidly growing intellectual property estate of approximately 3,600 patents and exclusive licenses. Life Technologies was created by the combination of Invitrogen Corporation and Applied Biosystems Inc.” 

Wow, they are part of life-prolonger even if I never met any of them, and they are getting breakthrough results.There are hundreds of more biotech companies out there working on various aspects of life-prolonger for me, representing hundreds of billions of dollars in economic activity and utilizing incredible computer and brainpower on my behalf.  Add in the university and hospital researchers, the government labs, the pharma company labs.  The results being produced in many cases represent exponential change, not just linear change.  That’s life-prolonger, getting stronger and meaner every day.  Watch out death-stalker, you are going to have to share the stage when it comes to calling the life-and-death shots.  And, death-stalker, I do recognize that it is more comfortable for most people to call you by your usual name, aging.

A review article published in the February, 9, 2010, issue of the Journal of the American College of Cardiology points out that a number of herbal remedies may become dangerous when their use is combined with taking certain cardiovascular drugs.  How to view this fact depends on your perspective.  One perspective, that of the mainline cardiology profession, places the onus on the herbal supplements.  It says that if your doctor puts you on a life-saving drug, you should avoid herbal supplements that combine dangerously with that drug.  The minority perspective, that of the supplement industry, points out that the supplements of concern are generally a lot safer than the drugs of concern.  For example, if you are taking the drug warfarin (also known as Coumadin), a classical rat poison, you certainly do have to be careful about what you combine it with.  Stay away from garlic, ginkgo biloba, ginger, alfalfa, saw palmetto, green tea, bilberry, fenugreek, ginseng, chondroitin sulfate or vitamin k.  Warfarin kills rats by excessively thinning their blood and those herbal or vitamin substances tend to either potentiate or inhibit warfarin’s blood thinning.  The green tea is only evil when you are already taking the rat poison.

The article Use of Herbal Products and Potential Interactions in Patients With Cardiovascular Diseases starts out “More than 15 million people in the U.S. consume herbal remedies or high-dose vitamins. The number of visits to providers of complementary and alternative medicine exceeds those to primary care physicians, for annual out-of-pocket costs of $30 billion. Use of herbal products forms the bulk of treatments, particularly by elderly people who also consume multiple prescription medications for comorbid conditions, which increases the risk of adverse herb-drug-disease interactions. — In this review, we highlight commonly used herbs and their interactions with cardiovascular drugs. We also discuss health-related issues of herbal products and suggest ways to improve their safety to better protect the public from untoward effects.” 

Drug-herb interactions should be taken seriously for they can be matters of life and death.   According to an accompanying Feb 1 2010 ACC news release entitled As Use of Herbal Remedies Soars, Patients Taking These and Cardiovascular Medications May be at Heightened Risk of Dangerous, Potentially Life-Threatening Interactions “Many people have a false sense of security about these herbal products because they are seen as ‘natural,’” Arshad Jahangir, M.D., Professor of Medicine and Consultant Cardiologist, Mayo Clinic Arizona, — “But ‘natural’ doesn’t always mean they are safe. Every compound we consume has some effect on the body, which is, in essence, why people are taking these products to begin with.” — In addition to their direct effects on body function, these herbs can interact with medications used to treat heart disease, either reducing their effectiveness or increasing their potency, which may lead to bleeding or a greater risk for serious cardiac arrhythmias.  — “We can see the effect of some of these herb-drug interactions—some of which can be life-threatening—on tests for blood clotting, liver enzymes and, with some medications, on electrocardiogram,” Dr. Jahangir said.  — According to the report, a major concern is that patients do not readily disclose their use of herbal remedies, and healthcare providers may not routinely ask about such use. In addition, because these herbs are regarded as food products, they are not subject to the same scrutiny and regulation as traditional medications.”

Other examples of drug-herb interactions exist besides those involving blood thinning.  “For instance, St. John’s wort, which is often taken to treat depression and anxiety, affects how the body absorbs dozens of prescription medications and may diminish the efficacy of statins and beta-blockers, a class of drugs used to treat high blood pressure and heart-rhythm disorders. — Even grapefruit juice, which people often drink for weight loss and heart health, can increase the blood concentration of statins, raising the risk of liver damage and muscle pain, the report notes(ref).”

Any reader of a vampire novel knows that acquiring the blood of a young person is the secret of a vampire’s eternal youth.  In fact, the essence of being a vampire is a constant quest for such acquisition.  According to a news stories that broke today, it seems like scientific knowledge is finally catching up. 

It is also common knowledge among us longevity-science types that somatic stem cells are subject to senescence and that, with aging, these stem cells progressively lose their capability to reproduce and differentiate. (See the discussion in my treatise related to the Stem Cell Supply Chain Breakdown theory of aging.)  “Buildup of levels of Ink4a/P16 associated with aging slows down the rate of differentiation of adult stem cells.” Further, age-related loss of capability to reproduce and differentiate has to do with what is going on in the niches in which stem cells live.  “Our results reveal that aged differentiated niches dominantly inhibit the expression of Oct4 in hESCs and Myf-5 in activated satellite cells, and reduce proliferation and myogenic differentiation of both embryonic and tissue-specific adult stem cells (ASCs). Therefore, despite their general neoorganogenesis potential, the ability of hESCs, and the more differentiated myogenic ASCs to contribute to tissue repair in the old will be greatly restricted due to the conserved inhibitory influence of aged differentiated niches(ref).”  Along with this decline in stem cell renewal capability comes a breakdown in the efficacy of the stem cell supply chain, aging and death.  According to a January 30 2010 news item appearing in Science Daily “A team of Howard Hughes Medical Institute (HHMI) researchers has found that in old mice, a several-week exposure to the blood of young mice causes their bone marrow stem cells to act “young” again.”  Dracula, why are you acting bored? 

The publication related to the new research is Systemic signals regulate ageing and rejuvenation of blood stem cell niches and appeared in the January 28 issue of Nature. “Ageing in multicellular organisms typically involves a progressive decline in cell replacement and repair processes, resulting in several physiological deficiencies, including inefficient muscle repair, reduced bone mass, and dysregulation of blood formation (haematopoiesis). Although defects in tissue-resident stem cells clearly contribute to these phenotypes, it is unclear to what extent they reflect stem cell intrinsic alterations or age-related changes in the stem cell supportive microenvironment, or niche. Here, using complementary in vivo and in vitro heterochronic models, we show that age-associated changes in stem cell supportive niche cells deregulate normal haematopoiesis by causing haematopoietic stem cell dysfunction. Furthermore, we find that age-dependent defects in niche cells are systemically regulated and can be reversed by exposure to a young circulation or by neutralization of the conserved longevity regulator, insulin-like growth factor-1, in the marrow microenvironment. Together, these results show a new and critical role for local and systemic factors in signaling age-related haematopoietic decline, and highlight a new model in which blood-borne factors in aged animals act through local niche cells to induce age-dependent disruption of stem cell function.”

This does not sound much like vampire talk.  Some of the press reports about the work are more lucid if not lurid.  According to the Science Daily writeup “Hematopoietic stem cells give rise to all the cells of the blood system, including immune cells and red blood cells. As animals age, these stem cells become more numerous, but less effective at regenerating the blood system, Wagers says. That translates into a less effective immune system and a greater susceptibility to disease. — To see if younger blood could reverse the sluggishness of aging blood cells, the researchers began by surgically joining the bloodstreams of pairs of mice that were of different ages, but nearly clones of one another.” (Hmmn, joining bloodstreams?  That does sound rather vampire-like.)  “Each mouse carried distinctive genetic markers so that researchers could differentiate between its cells and those of its partner. The technique, called parabiosis, enables researchers to test the long-term effects of one animal’s blood on the tissues and organs of the other. “It’s the only model that really allows us to come close to mimicking an in vivo systemic environment,” Wagers (Amy Wagners, the lead investigator) said. “There is a constant exposure to any cell or soluble factor that circulates, at close to physiologic levels.”  — After several weeks of sharing their blood systems with young mice, the hematopoietic stem cells of the older mice changed markedly. Exposure to a younger animal’s blood somehow pushed the older animal’s hematopoietic stem cells back to a more youthful state, in which they were fewer in number but recovered nearly all of their blood-cell-generating capacity. When transplanted into mice whose own blood-producing cells had been eliminated by radiation, the “rejuvenated” stem cells repopulated the blood with a mixture of cell types similar to that generated by transplanted young stem cells. No such changes occurred in the young mice in these pairings, or among age-matched pairs of animals.”

There is significantly more to the recent research findings, and that is that IGF-1 expression in osteoblasts present in the haematopoietic stem cell bone marrow niches is responsible for the decline in vitality and differentiation capabilities of haematopoietic stem cells in older mice, and neutralizing the IGF-1 in the bone marrow also restores the vitality and differentiation capabilities of these stem cells.

In more detail, ” Wagers and her team haven’t yet discovered the blood-borne factor that triggers this apparent restoration of youthfulness in aged hematopoietic stem cells. But they did find two important clues to how it transmits its effects.  — First, they found evidence that this factor works via bone-forming cells known as osteoblasts, which also are present in bone marrow and help regulate hematopoietic stem cells. When old animals were exposed to young blood, their osteoblasts reverted to more youthful numbers. They also behaved more like younger osteoblasts in their interactions with hematopoietic stem cells. Hematopoietic stem cells grown in cultures with these “rejuvenated” osteoblasts regained the blood-cell-generating capacity characteristic of youthful stem cells. For osteoblasts, the opposite was also true: the bone-forming cells of young animals- from humans as well as mice — showed signs of aging when they were exposed to blood from an older animal. — The team also found that the insulin-like growth factor 1 (IGF-1) hormone appears to be necessary to maintain these stem-cell-regulating osteoblasts in an aged state. When they blocked IGF-1 activity in osteoblast cells in culture or in bone marrow, aged osteoblasts reverted to a “younger” state, and could pass that rejuvenation effect on to hematopoietic stem cells. Blocking IGF-1 activity in the bloodstream of mice didn’t have the same effect, which suggests that IGF-1 acts specifically through osteoblasts. — Oddly enough, IGF-1 is best known for its growth-promoting and potentially anti-aging effects in other tissues, including muscles and bones. “Our findings highlight the fact that IGF-1 signaling is complex and depends in part on the tissue involved,” said Wagers(ref).”

Getting back to vampires, the results of the new study suggests a cure for their centuries-old thirst for blood, since it suggests that blocking the effects of IGF-1 in bone marrow osteoblasts could have the same rejuvenating affect as the blood of a young person.  Of course this would have to be validated by a clinical trial.  Can you imagine a drug company setting up such a trial for vampires where half the participants take a drug that blocks bone marrow IGF-1 and the control group participants go out and hunt human victims and drink their blood in normal vampire fashion? 

Seriously, there just could be some longevity benefit to selective blocking of IGF-1 in osteoblasts.  More must be learned about this possibility.  And the mouse results must be validated in humans.

Do you remember the Monopoly card that says “Go to jail.  Go directly to jail.  Do not pass Go, Do not collect $200?”  Well, imagine that there is a cell reprogramming card that says, say when you land on skin cell, “Go to nerve cell, go directly to nerve cell.  Do not pass iPSC status. Do not collect pluripotent reprogramming factors.”  Very recent research shows that that card exists, and its existence is giving us a broad new perspective on epigenetic regulation of cell fates.  By modifying epigenetic factors, it appears that one type of body cell can be changed, very likely, into any other type of body cell, directly and without a need for reversion into induced pluripotent stem cell (iPSC) status. And the process is efficient.  This post reviews background on cell reprogramming, the new research in context, and speculates on the implications.

Background on cell reprogramming

Research on reprogramming cells from one type to another goes back to the 1980s, long before the first iPSC was produced.  The first work in this area involved fusing two different kinds of cells together to form heterokaryons.  A hetrokaryon is “A cell with two separate nuclei formed by the experimental fusion of two genetically different cells(ref).”  A 1986 publication reports Rapid reprogramming of globin gene expression in transient heterokaryons, where “Interspecific heterokaryons were formed by fusing adult mouse erythroleukemia (MEL) cells and human embryonic/fetal erythroid (K562) cells with each other, or with a variety of mouse and human nonerythroid cell types.”  A series of other publications based on studies of heterokaryons followed.  A 1993 publication Reversibility of the differentiated state in somatic cells reported “Analysis of de novo gene activation in multinucleated heterokaryons has shown that the differentiated state, although stable, is not irreversible, and can be reprogrammed in the presence of appropriate combinations of trans-acting regulatory molecules.”

A 1999 publication Use of somatic cell fusion to reprogram globin genes reports “Experiments with heterokaryons demonstrate that the reprogramming is due to trans-acting factors that are developmental-stage-specific. These results suggest the feasibility of using fusisome-carried sets of nuclear factors to reprogram somatic cells.”  A relatively recent January 2009 study Nuclear reprogramming in heterokaryons is rapid, extensive, and bidirectional reports “Here, we show that hundreds of genes are activated or repressed within hours of fusion of human keratinocytes and mouse muscle cells in heterokaryons, and extensive changes are observed within 4 days.”

Another thread of research related to cell reprogramming was cloning.  Dolly, the world’s most famous sheep, was cloned in 1996.  “The production of Dolly showed that genes in the nucleus of such a mature differentiated somatic cell are still capable of reverting back to an embryonic totipotent state, creating a cell that can then go on to develop into any part of an animal(ref).^[11]”

Another chain of studies in the mid 2000s related to cell reprogramming involved the impact of microenvironment on cell fate.  It was found that when cloned liver stem cells were placed into a cardiac microenvironment, they transformed themselves to acquire a cardiac phenotype and function(ref)(ref)(ref). “Collectively, these results support the conclusion that these adult-derived liver stem cells respond to signals generated in a cardiac microenvironment ex vivo acquiring a cardiomyocyte phenotype and function(ref).”

The hetrokaryon studies, the cloning work and the studies related to the effect of microenvironment indicate that cells of one kind can be directly reprogrammed into cells of another kind and that there is some kind of molecular signaling process involved.  The big more-recent cell reprogramming news of course was the ability to revert any cell to embryonic stem cell-like pluripotency, the creation of induced pluripotent stem cells (iPSCs) starting in 2006.  The first comprehensive discussion of iPSCs in this blog was the March 2009 post Rebooting cells and longevity, and iPSCs have been mentioned or discussed in many subsequent blog posts.  The first studies described the use of four transcription factor proteins to create iPSCs: Oct4, Sox2, Klf4, and c-Myc.  Much progress in creating iPSCs in the last year including use of other transcription factor combinations, safer less-oncogenic vectors for insertion of the transcription factors, induction of stem cell expression without using transgenes, and, most recently, the use of vitamin C to improve the efficiency of reprogramming(ref)(ref). 

The 2008 publication Reprogramming of somatic cell identity summarized the situation as of the time “Nuclear transfer and cell-fusion experiments demonstrate that the epigenetic signature directing a cell identity can be erased and modified into that of another cell type. Furthermore, in the case of cloning, differentiated cells can be reprogrammed back to pluripotency to support the reexpression of all developmental programs. Recent breakthroughs highlight the importance of transcription factors as well as epigenetic modifiers in the establishment, maintenance, and rewiring of cell identity.” 

Nonetheless, the excitement about iPSCs led many researchers to forget or ignore the earlier research on cell reprogramming and assume that if one wants to start with, say, skin cells and end up with nerve (or heart or liver) cells, just about the only practical approach is a two-step one: 1.  Take some skin cells and revert them to being iPSCs, an inefficient process even when using vitamin C, and then  2.  Somehow convince those iPSCs to progressively differentiate to become nerve cells, possibly a quite tricky thing to do in-vivo.  The new research finding suggests that with the right transcription factors it might be possible to start out with any kind of cell and end up with any other kind of cell without going through an intermediate stage. 

Direct cell reprogramming

2008 saw the publication of a breakthrough study In vivo reprogramming of adult pancreatic exocrine cells to beta cells.  “Here, using a strategy of re-expressing key developmental regulators in vivo, we identify a specific combination of three transcription factors (Ngn3 (also known as Neurog3) Pdx1 and Mafa) that reprograms differentiated pancreatic exocrine cells in adult mice into cells that closely resemble -cells. The induced -cells are indistinguishable from endogenous islet -cells in size, shape and ultrastructure. They express genes essential for -cell function and can ameliorate hyperglycaemia by remodeling local vasculature and secreting insulin. This study provides an example of cellular reprogramming using defined factors in an adult organ and suggests a general paradigm for directing cell reprogramming without reversion to a pluripotent stem cell state.”

The new January 2010 research study report Direct conversion of fibroblasts to functional neurons by defined factors reports “Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses.”

The transcription factors used in both studies are different than those used to create iPSCs and the cell-type conversion process is much more efficient than that of reverting cells to iPSC status.  The 2008 study was exciting because it described direct cell reprogramming in-vivo in a way that addresses a disease, albeit in a mouse model.  Regarding the 2010 study, the “neurons could integrate into pre-existing neural networks and form independent synapses with each other.  — This system bypasses production of tumorigenic pluripotent cells, a main barrier to using iPSCs in regenerative medicine, and may provide a platform for more efficient disease modeling and drug discovery(ref).” “They also tested the procedure on skin cells from the tails of adult mice. They found that about 20 percent of the former skin cells transformed into neural cells in less than a week. That may not, at first, sound like a quick change, but it is vast improvement over iPS cells, which can take weeks. What’s more, the iPS process is very inefficient: Usually only about 1 to 2 percent of the original cells become pluripotent(ref).”

Implications include:

·        hESCs, iPSCs and other stem cell types are likely to turn out to be extremely important, but are not the only games-in-town for producing desired cells where and as needed.

·        Cloning taught us that all body cells encompass the same genes and that any one cell encompasses the possibilities in all other cells.  The differences among cells are ones of epigenetic gene expression.  The latest research indicates it may be possible freely to change one cell type to another via introducing highly specific transcription factors.

·        It may turn out to be practical to convert many cell types to other cell types in-vitro, in-vivo or both, allowing the development of many new regenerative medicine applications.  The challenge is discovering the transcription factors and other epigenetic modifiers needed and how to introduce them so as safely get a desired result.

·        The new work probably makes addressing cell senescence even more critical.  I suspect that transforming an old-near-senescent skin cell into a nerve cell will produce an old near-senescent nerve cell unless issues like telomere lengths are also addressed.

·        The new work is likely to contribute to an acceleration in research relating to the discovery and isolation of gene transcription factors(ref), micro-RNAs(ref), HATs and HDACs(ref), DNA demethylases(ref) and other gene regulatory factors.  Unraveling all of those may well take decades. 

I am not worried about running out of work here.

In the blog entry The stem cell supply chain – closing the loop for very long lives, I have suggested that it might be possible to re-introduce fully pluripotent stem cells into the body so as to close the loop in the stem cell supply chain and enable much longer lives. This post reports progress towards that point, again a topic related to Vitamin C suggested by reader jeg3.

Background

Recapitulating the essence of the stem cell supply chain concept: “Stem Cell Supply Chain Breakdown is the newest theory of aging described in my treatise and the one I am currently most excited about.  According to a simplified model of this theory a newly-conceived human embryo consists of pluripotent stem cells (Type A), ones that can potentially divide into any body cells.  With growth, these proliferate and, in a remarkably articulated manner, progressively differentiate into multipotent stem cells (Type B), progenitor cells (Type C), mature body somatic cells (Type D), and many eventually become senescent cells (Type E).”  — ‘According to the best current understanding of stem cells this is an open-loop once-through process.  The above list is in order of increasing cell-type specificity and decreasing cell-type potency to differentiate into other cell types.  Starting at conception and throughout life, all cells on this list except the senescent ones will selectively reproduce and possibly differentiate into cells of types further down in the list.  The state of the body in terms of makeup of cell types continues to change through life and the process goes inexplicably from start (conception) leading to end (death’).  The stem cells themselves are subject to replicative senescence.   Early in life, Type A cells tend to vanish.  With aging, pools of type B and type C cells become exhausted and are less capable of differentiation to renew the supply of Type D cells.  The stem cell supply chain slows down and ceases to function well.  There are fewer healthy Type D cells and more Type E cells, and disease and death soon follow.

The blog entry The stem cell supply chain – closing the loop for very long lives suggests an approach that could conceivably transform the stem cell supply chain from being a once-through process to being a continuous open-loop process.   “There is a possibility of keeping the stem cell supply chain active indefinitely.  The key idea is to use induced Pluripotent Stem cells (iPSCs) which are fully pluripotent and equivalent to embryonic stem cells(ref)(ref)(ref) as feedstock Type A cells in adults to make the stem cell supply chain as a continuous loop process instead of a once-through process.”

I assume the reader is generally familiar with iPSCs and the general approaches to reprogramming cells to iPSC status.  See, for example, the blog posts Rebooting cells and longevity, Update on induced pluripotent stem cells and “Footprint-free” iPSCs – and a crazy wager offer.

This blog post reports research progress on creating iPSCs which may eventually lead to closing the loop.  An old dear friend seems to be involved, Vitamin C.  The study Vitamin C Enhances the Generation of Mouse and Human Induced Pluripotent Stem Cells was timed as a Christmas present and published December 24 2009.  Some of the points related to the new study are:

1.     The approaches to reverting cells to iPSC status have been remarkably inefficient.  “Soon after the exciting discovery of a method to transform human skin cells into stem cells in 2007 came the frustration of actually trying to make a sufficient amount of these induced pluripotent stem (iPSC) cells.  The process is so inefficient that scientists typically only get 0.01 percent of a sample of human skin, or fibroblast, cells to form iPS cell colonies after they infect fibroblasts with the retroviruses used to induce pluripotency(ref).”  The new study report indicates  “However, the low efficiency of iPSC generation is a significant handicap for mechanistic studies and high throughput screening, and also makes bona fide colony isolation time consuming and costly. The efficiency of alkaline phosphatase-positive (AP⁺) colony formation with the four Yamanaka’s factors (Sox2, Klf4, Oct4, c-Myc; SKOM) in mouse fibroblasts is about 1% of the starting population, but only around 1 in 10 of those colonies is sufficiently reprogrammed to be chimera competent – -. ”  This iPSC reprogramming inefficiency has been noted by others as well(ref)(ref).

2.     It is hard to revert old or near-senescent cells to iPSC status given age-related upregulation of tumor suppressor genes. “While our work was in progress, six independent laboratories identified cell senescence as a roadblock for reprogramming (Hong et al. 2009) — “Functional analyses of these genes demonstrate that the p53-p21 pathway serves as a barrier not only in tumorigenicity, but also in iPS cell generation,”  Kawamura et al., 2009, Li et al. 2009 “In murine cells, Arf, rather than Ink4a, is the main barrier to reprogramming by activation of p53 (encoded by Trp53) and p21 (encoded by Cdkn1a); whereas, in human fibroblasts, INK4a is more important than ARF. Furthermore, organismal ageing upregulates the Ink4/Arf locus and, accordingly, reprogramming is less efficient in cells from old organisms,” Marión et al. 2009 “These observations indicate that during reprogramming cells increase their intolerance to different types of DNA damage and that p53 is critical in preventing the generation of human and mouse pluripotent cells from suboptimal parental cells,”  Utikal et al., 2009, Zhao et al., 2008).”  This has led to significant interest in finding compounds that “alleviate cell senescence without increasing the risk of mutations.”  The researchers set out testing antioxidants for this purpose.  The one that worked was vitamin C.

3.    The main finding of the study is that vitamin C can markedly improve the efficiency of the reprogramming process for both mouse and human cells. “We show here that vitamin C, a common nutrient vital to human health, enhances the reprogramming of somatic cells to pluripotent stem cells. By adding Vc to the culture medium, we can now obtain high-quality iPSCs from mouse and human cells routinely.”   Exactly how vitamin C works to achieve this end is not clear.  Other tested antioxidants appeared not to have an effect.  It is highly possible that epigenetic factors are involved.  “Besides reducing p53, Vc accelerates transcriptome changes during reprogramming and allows the conversion of pre-iPSCs to iPSCs. The extent to which these observations relate to cell senescence is unclear, and it is possible that Vc is acting in other ways as well. For example, it could accelerate stochastic events during reprogramming, perhaps by promoting epigenetic modifications that allow further changes to proceed. In this regard, Vc is a cofactor in reactions driven by dioxygenases including collagen prolyl hydroxylases, HIF (hypoxia-inducible factor) prolyl hydroxylases, and histone demethylases (Shi, 2007), and it is interesting to consider that Vc might influence reprogramming by increasing the activity of these enzymes. Histone demethylases are important for development and modulate the expression of the ESC master transcription factor Nanog (Cloos et al., 2008), so it is possible that Vc allows the reprogramming to run more smoothly by facilitating histone demethylation.” 

The new finding can result in increased productivity in creating iPSCs.  However, there is still a way to go before the “closing the stem cell supply chain loop” hypothesis can be tested.  If iPSCs are created outside the body from a person’s tissue, safe ways must be found to introduce them back into the body so they will go about replenishing stocks of Type B and Type C stem cells without creating problems such as tumors or teratomas.

Oh Spirit of Linus Pauling Great Father of Vitamin C, are you listening?  A few days ago I wrote a blog entry Surprise!  Just when we thought we knew everything about vitamin C, pointing to new research indicating that vitamin C could be a cure for Werner’s Syndrome.  It looks like regularly taking vitamin C does a myriad of other things besides serving as a good antioxidant, things like preventing DNA damage induced by renovascular hypertension, and helping to control obesity.  This week’s new finding relates to the usefulness of Vitamin C in creating iPSCs, possibly an important finding for regenerative medicine.

We have new powerful frameworks for looking at old familiar substances like vitamin C, frameworks like epigenetics, proteomics, telomere science and cell cycle molecular biology, and these frameworks are telling us things about vitamin C that Linus may have intuited but could not have put into words.  Because most of the needed words did not exist in his time.

The  responses to my blog posts tell me that many of you readers out there join me in being telomerase life-extension aficionados.  In case you don’t already know about it, you might want to have a look at the Sierra Sciences website.  Sierra Sciences is a small biotech research company (30 scientists), completely devoted to discovering new activators of the telomerase gene to serve the cause of human longevity.  The company was founded by Bill Andrews, one of the discoverers of the telomerase gene back when he was in charge of the molecular biology research group at Geron.

Going to the Sierra Sciences website you are greeted by a video presentation featuring Bill Andrews.  Going on to the home page you are greeted with the slogan “Cure Aging of Die Trying,” which I can personally identify with.  “Sierra Sciences, LLC is a company devoted to finding ways to extend our healthspans and lifespans beyond the theoretical maximum of 125 years.” 

Basically, the company is screening substances, searching for telomerase inducers. The home page reports: “As of January 20, 2010: We have screened 189,264 compounds — We have found 555 telomerase inducers — These represent 34 distinct drug families — Most potent compound = 6% of goal — Check back frequently for updates! — We are screening 4,000 compounds per week”. 

The website features a few fairly current video presentations relating to telomerase as well as older ones.   Finally, the company is seeking the involvement of others:  “Sierra Sciences is seeking individuals passionate about finding the cure for aging to get involved in the company at levels of grant funding, strategy, and management. — We are looking for individuals who will be interested in giving us their input on strategies and helping us to take whatever steps necessary to achieve this cure within our lifetimes.”