Research related to induced pluripotent stem cells (iPSCs) is proceeding at warp speed.  I have posted multiple blog entries related to iPSCs, ten so far in 2010.  These are all listed in the November 2010 blog post Past blog postings on stem cells and epigenomics.  In a recent series of comments however(ref), my reader B has made it clear to me that there were still major gaps in my coverage of important iPSC developments over the last year.  I fill-in on several of these developments here, occasionally also mentioning research already covered. 

Key Developments 

Taken together, a series of publications seem to establish: 

·        Initial passages (test-tube generations) of iPSCs tend to have certain epigenomic differences from corresponding hESCs (human embryonic stem cells) depending on their donor tissue of origin and exhibit a tendency to differentiate back into those donor tissues.   

·        Genome-wide transcriptional profiles of ESCs and iPSCs can reveal the disparities between them.

·        Assuring full pluripotency and equivalence of iPSCs to hESCs can be accomplished by multiple passages or by use of hDAC inhibitors.  Thus, there may be a genomic and functional difference between freshly created iPSCs and ones subject to subsequent treatment to assure full pluripotency.

·        While there are molecular differences between various strains of iPSCs and ESCs and possible functional differences, some iPSCs are sufficiently pluripotent to create fully functional mice.

·        At least some lines of iPSCs exhibit signs of epigenetic youth and significantly delayed senescence.  Tissues created from them are correspondingly young.  However, several reports of what iPSC reversion does to telomere lengths appear to be contradictory.

·        There is at least one report that iPS cells display mitochondrial rejuvenation compared to their fibroblast source cells.  This mitochondrial rejuvenation was passed on to fibroblast cells derived from the iPSCs.

·        Researchers have succeeded reverted a large number of cell types to iPSC status.

·        More reliable and efficient means for cell reprogramming that yield more consistent “higher-fidelity” iPSC are being discovered.   

·        iPSC research might well lead to therapies for HIV.

·        The iPSC research field is still young, vibrant and full of surprises.  Although new research results keep pouring in, there is much still to be learned.

·        The promise for the future is bright. 

Here are some of the relevant publications supporting the above points.  Paying attention to the dates of some of these publications and what they say illustrates how thought processes about iPSCs have been evolving just in the course of one year. 

Epigenomic characteristics of iPSCs compared to fully pluripotent hESCs 

The February 2010 publication Persistent donor cell gene expression among human induced pluripotent stem cells contributes to differences with human embryonic stem cells.  “– although hiPSCs have been described as “embryonic stem cell-like”, these cells have a distinct gene expression pattern compared to hESCs, making incomplete reprogramming a potential pitfall. It is unclear to what degree the difference in tissue of origin may contribute to these gene expression differences. To answer these important questions, a careful transcriptional profiling analysis is necessary to investigate the exact reprogramming state of hiPSCs, as well as analysis of the impression, if any, of the tissue of origin on the resulting hiPSCs. In this study, we compare the gene profiles of hiPSCs derived from fetal fibroblasts, neonatal fibroblasts, adipose stem cells, and keratinocytes to their corresponding donor cells and hESCs. Our analysis elucidates the overall degree of reprogramming within each hiPSC line, as well as the “distance” between each hiPSC line and its donor cell. We further identify genes that have a similar mode of regulation in hiPSCs and their corresponding donor cells compared to hESCs, allowing us to specify core sets of donor genes that continue to be expressed in each hiPSC line. We report that residual gene expression of the donor cell type contributes significantly to the differences among hiPSCs and hESCs, and adds to the incompleteness in reprogramming. Specifically, our analysis reveals that fetal fibroblast-derived hiPSCs are closer to hESCs, followed by adipose, neonatal fibroblast, and keratinocyte-derived hiPSCs. — Conclusions and outlook:  After analyzing, in detail, genome-wide transcriptional profiles of starting cell populations, partially reprogrammed cells and iPSCs and comparing these with ESCs, we conclude that iPSCs and ESCs share a well-defined core pluripotency network. However, some core genes often seem expressed at lower levels in iPSCs. In addition, this network comprises not only the usual pluripotency transcription factors, but also genes not yet described as, but likely to be, involved in pluripotency and/or self-renewal and genes responsible for many other biological processes, such as cell-cell communication and metabolism.” 

he May 2010 publication Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells.  “By comparing genetically identical mouse ES cells and iPSCs, we show here that their overall messenger RNA and microRNA expression patterns are indistinguishable with the exception of a few transcripts encoded within the imprinted Dlk1-Dio3 gene cluster on chromosome 12qF1, which were aberrantly silenced in most of the iPSC clones. Consistent with a developmental role of the Dlk1-Dio3 gene cluster, these iPSC clones contributed poorly to chimaeras and failed to support the development of entirely iPSC-derived animals (‘all-iPSC mice’). In contrast, iPSC clones with normal expression of the Dlk1-Dio3 cluster contributed to high-grade chimaeras and generated viable all-iPSC mice. Notably, treatment of an iPSC clone that had silenced Dlk1-Dio3 with a histone deacetylase inhibitor reactivated the locus and rescued its ability to support full-term development of all-iPSC mice. Thus, the expression state of a single imprinted gene cluster seems to distinguish most murine iPSCs from ES cells and allows for the prospective identification of iPSC clones that have the full development potential of ES cells.” 

The July 2010 publication Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. “Here we show that iPSCs obtained from mouse fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Notably, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and altered differentiation capacity.”–– “iPSCs derived from different somatic cell types retain a transient epigenetic and transcriptional memory of their cell types of origin  at early passage, despite acquiring pluripotent gene expression, transgene-independent growth and the ability to contribute to tissues in chimeras.  Continuous passaging resolves these differences giving rise to iPSCs that are molecularly and functionally indistinguishable.” 

The August 2010 publication Chromatin structure and gene expression programs of human embryonic and induced pluripotent stem cells.  “Recent studies have suggested that ESCs and iPSCs represent different pluripotent states with substantially different gene expression profiles. We describe here a comparison of global chromatin structure and gene expression data for a panel of human ESCs and iPSCs. Genome-wide maps of nucleosomes with histone H3K4me3 and H3K27me3 modifications indicate that there is little difference between ESCs and iPSCs with respect to these marks. Gene expression profiles confirm that the transcriptional programs of ESCs and iPSCs show very few consistent differences. Although some variation in chromatin structure and gene expression was observed in these cell lines, these variations did not serve to distinguish ESCs from iPSCs.” 

The September 2010 publication Analysis of human and mouse reprogramming of somatic cells to induced pluripotent stem cells. What is in the plate?  For one thing, the introduction to this paper provides a concise summary of progress in generating iPSCs up to the date of the publication.  “Briefly, cornerstone publications in the reprogramming field have described the following attributes of iPSCs: they can be transmitted to the germ line [2], generated without the oncogenic factor c-Myc [3], [4], obtained from human cells using the same set of factors [5], [6] as well as other factors [7], obtained without permanent genomic manipulation [8], [9], [10], [11], [12], [13], produced from patient cells [14], [15], [16] even with the correction of a genetic disease [17], and more recently, a study demonstrated that iPSCs can give rise to viable mice by tetraploid complementation assays [18], [19], [20]. Similar to ESCs, iPSC lines have been shown to differentiate into derivatives of the three embryonic germ layers. More specifically, studies have demonstrated iPSC’s ability to generate cells of the cardiovascular and hematopoietic lineages [21], [22], insulin-secreting islet like structures [23], functional cardiomyocytes [24], cells of the neural lineages [25], cells of the adipose lineage [26] and retinal cells [27]. Moreover, a number of papers have began to decipher the mechanisms involved in reprogramming [28], [29], [30], [31], [32], [33], [34], [35], [36], a phenomenon that will likely require significant effort in order to be fully understood.” 

“There are a large number of reprogramming experiments published so far encompassing genome-wide transcriptional profiling of the cells of origin, the iPSCs and ESCs, which are used as standards of pluripotent cells and allow us to provide here an in-depth analysis of transcriptional profiles of human and mouse cells before and after reprogramming. When compared to ESCs, iPSCs, as expected, share a common pluripotency/self-renewal network. Perhaps more importantly, they also show differences in the expression of some genes(ref).”

Among things stated under Conclusions and Outlook are: “After analyzing, in detail, genome-wide transcriptional profiles of starting cell populations, partially reprogrammed cells and iPSCs and comparing these with ESCs, we conclude that iPSCs and ESCs share a well-defined core pluripotency network. However, some core genes often seem expressed at lower levels in iPSCs. In addition, this network comprises not only the usual pluripotency transcription factors, but also genes not yet described as, but likely to be, involved in pluripotency and/or self-renewal and genes responsible for many other biological processes, such as cell-cell communication and metabolism. — Although we cannot answer the question of whether iPSCs are truly functionally equivalent to ESCs, it seems increasingly obvious that there exists more than one state of pluripotency. This would explain why we can distinguish between ESCs and iPSCs, but also between iPSCs generated with different protocols. As we believe it is important to select the best iPSCs in terms of their differentiation potential, we propose that checking the newly generated iPSC lines for the silencing of a number of genes marked with bivalent domains would assist in preselecting the most promising iPSCs for further studies. Importantly, even though the field of somatic cell reprogramming moves incredible fast and brings us closer every day to getting the “perfect” protocol for iPSC generation in terms of efficiency, a crucial question remains: will we be able to get cells which are safe to use for therapeutical applications? To answer this question, not only will different cell types, ages and origins have to be tested, but also the protocol used for the generation of the iPSCs. Moreover, understanding the path through which somatic cells arrive to a pluripotent state should allow us to evaluate, more accurately, the potential risks inherent in the use of iPSCs in therapy. The propensity of iPSCs to differentiate and not to go wayward after transplantation, judged by the integrity of their genome and epigenome, will need to be evaluated in great detail(ref).” 

The September 2010 publication Epigenetic memory in induced pluripotent stem cells relates “Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an ‘epigenetic memory’ of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modeling or treatment.” 

The December 2010 publication  Induced pluripotent stem cells: epigenetic memories and practical implications.  “It was generally assumed that iPSCs are functionally equivalent to their embryonic stem cell (ESC) counterparts. Recently, a number of research groups have demonstrated that this is not the case, showing that iPSCs retain ‘epigenetic memory’ of the donor tissue from which they were derived and display skewed differentiation potential. This raises the question whether such cells are fit for experimental, diagnostic or therapeutic purpose. A brief survey of the literature illustrates that differences at both epigenetic and transcriptome level are observed between various pluripotent stem cell populations. Interestingly, iPSC populations with perceived ‘anomalies’ can be coaxed to a more ESC-like cellular state either by continuous passaging-which attenuates these epigenetic differences-or treatment with small molecules that target the machinery responsible for remodelling the genome.  This suggests that the establishment of an epigenetic status approximating an ESC counterpart is largely a passive process. The mechanisms responsible remain to be established. Meanwhile, other areas of reprogramming are rapidly evolving such as, trans-differentiation of one somatic cell type to another by the forced expression of key transcription factors.” 

At least some forms of iPSCs exhibit significantly delayed senescence though reports of what iPSC reversion does to telomere lengths appear to be contradictory.

The October 2010 publication The LARGE principle of cellular reprogramming: lost, acquired and retained gene expression in foreskin and amniotic fluid-derived human iPS cells has a lot to say, including about this topic.  “Cellular reprogramming is a means of assigning greater value to primary AFCs (human amniotic fluid cells) by inducing self-renewal and pluripotency and, thus, bypassing senescence. Here, we report the generation and characterization of human amniotic fluid-derived induced pluripotent stem cells (AFiPSCs) and demonstrate their ability to differentiate into the trophoblast lineage after stimulation with BMP2/BMP4. We further carried out comparative transcriptome analyses of primary human AFCs, AFiPSCs, fibroblast-derived iPSCs (FiPSCs) and embryonic stem cells (ESCs). This revealed that the expression of key senescence-associated genes are down-regulated upon the induction of pluripotency in primary AFCs (AFiPSCs). — — Our study aimed at a more detailed molecular characterization of AFiPSCs. To this end, we generated AFiPSCs and demonstrated their ability to differentiate into the extraembryonic trophoblast lineage. This study also highlights the potential of cellular reprogramming to avert replicative senescence observed in bulk primary AFCs.”

Going on:  “— Results: Senescence is bypassed by the derivation of AFiPSCs from human AFCs  — To investigate the effect of reprogramming on bypassing senescence observed in primary AFC cultures (Figure 1A-II, -III), we analyzed the expression of senescence and telomere-associated genes in young primary AFCs (P6) and senescent AFC (P17) compared to AFiPSC lines (approximately P20). From a list of 116 senescence-associated genes (Table S4) derived from the Gene Ontology database [35], including those described by Vaziri et al. [45], we identified 64 genes as significantly differentially expressed in AFCs at passage 17 compared to the union of all AFiPSC lines (Figure 5). Of these, telomere-associated genes and genes involved in regulating the cell cycle, e.g. MAD2L2, PARP1, RPA3, DKC1, MSH6, CHEK1, PLK1, POU2F1, CDC2, BLM, WRN, DNMT1, DNMT3B, LMNB1, and CDT1, were down-regulated in primary AFCs compared to AFiPSCs and ESCs. In contrast, PIN1, LMNA, GADD45A, CBX6, NOX4, ENG, HIST2H2BE, CDKN2A, CDKN1A, GDF15 and SERPINE1, among others, were up-regulated in primary AFCs compared to AFiPSCs and ESCs. — Cellular reprogramming bypasses senescence of bulk primary AFCsOne of the great advantages of AFiPSCs over their bulk primary counterparts for any desirable application is their acquisition of the ability to propagate indefinitely. The data presented herein suggest, that this phenotypically rejuvenated appearance of AFiPSCs is based on a gene regulatory network, which averts or at least markedly delays the onset of senescence. This is based on the fact that primary AFCs and AFiPSCs and ESCs exhibit opposing expression patterns related to a large number of senescence-associated genes. In particular, we could detect high expression levels of various cell cycle and telomere elongation-associated genes, such as MAD2L2, PARP1, RPA3, DKC1, MSH6, CHEK1, PLK1, POU2F1, CDC2, LMNB1 and CDT1, as well as TERT itself in AFiPSCs in contrast to primary AFCs(ref).”

This same publication concerned itself with a core pluripotency network shared by ESCs and iPSCs.  “AFiPSCs, FiPSCs and ESCs share a core self-renewal gene regulatory network driven by OCT4, SOX2 and NANOG. Nevertheless, these cell types are marked by distinct gene expression signatures. For example, expression of the transcription factors, SIX6, EGR2, PKNOX2, HOXD4, HOXD10, DLX5 and RAXL1, known to regulate developmental processes, are retained in AFiPSCs and FiPSCs. Surprisingly, expression of the self-renewal-associated gene PRDM14 or the developmental processes-regulating genes WNT3A and GSC are restricted to ESCs. — This supports the idea that the main function of KLF4 and c-MYC in the process of reprogramming is to accelerate or enhance the efficiency by increasing a balanced cellular proliferation, while in pluripotent cells they seem to be dispensable [61]–[63]. — Among the expressed genes, which are universally acquired during reprogramming processes, independent of the cell source, are key pluripotency-regulating factors, such as POU5F1, SOX2 and NANOG. These establish a core gene regulatory network essential for maintaining self-renewal and pluripotency [46] (ref).” 

A December 2009 publication Telomere dynamics in human cells reprogrammed to pluripotency reported : “IPSCs, like ESCs, have been shown in several reports to display increased activity of at least one important enzymatic component of telomere homeostasis – the reverse transcriptase telomerase (TERT) – compared to the activity seen in somatic cell types (i.e. [14], [15]). More recently it was shown that mouse fibroblasts reprogrammed to pluripotency have both TERT activity and elongated telomeres [9]. This group further demonstrated that although one component of the reprogramming cocktail, the oncogene c-myc, had been shown to directly activate telomerase expression in human cells [17], [18], it was not required for telomere elongation in mouse IPSCs. Marion and colleagues further demonstrated that fibroblasts from both young (6 month) and old donor mice (2.3 yr) elongate telomeres to a similar degree following IPSC conversion.” — Together, these results led the authors to justifiably conclude that “most telomere elongation occurs postreprogramming”[9]. — We examined telomere length in human skin fibroblasts from young and old donor subjects, IPSCs derived from these cells, and IPSCs returned to a differentiated phenotype. We found that like animal cells reprogrammed by either somatic cell nuclear transfer or direct reprogramming, human fibroblasts converted to the IPSC phenotype generally displayed significantly elongated telomeres, and after re-differentiation, displayed a loss of telomere length. Like the mouse, this process was observed irrespective of the inclusion of c-myc in the reprogramming cocktail, and occured to approximately the same degree in cells derived from either young and old subjects. Unlike the mouse however, we observed greater heterogeneity between cell lines, both in the magnitude of telomere elongation during IPSC conversion and telomere shortening following redifferentiation. Also unlike the slow and progressive telomere elongation reported in mouse IPSCs, based on the seven human IPSC lines we analyzed, hIPSC telomeres achieved the 14–15 Kb length characteristic of human ESCs as early as P5.”

The March 2010 paper Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients reported consistent results.  “Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state, that several telomerase components are targeted by pluripotency-associated transcription factors, and that in autosomal dominant DC, transcriptional silencing accompanies a 3′ deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase, and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients.”

The May 2010 publication Spontaneous reversal of the developmental aging of normal human cells following transcriptional reprogramming expresses a quite different view of telomere length restoration in iPSCs.  “An hES-derived mortal clonal cell strain EN13 was reprogrammed by SOX2, OCT4 and KLF4. The six resulting induced pluripotent stem (iPS) cell lines were surveyed for telomere length, telomerase activity and telomere-related gene expression. In addition, we measured all these parameters in widely-used hES and iPS cell lines and compared the results to those obtained in the six new isogenic iPS cell lines. —  We observed variable but relatively long TRF lengths in three widely studied hES cell lines (16.09-21.1 kb) but markedly shorter TRF lengths (6.4-12.6 kb) in five similarly widely studied iPS cell lines. Transcriptome analysis comparing these hES and iPS cell lines showed modest variation in a small subset of genes implicated in telomere length regulation. However, iPS cell lines consistently showed reduced levels of telomerase activity compared with hES cell lines. — CONCLUSION: Prematurely aged (shortened) telomeres appears to be a common feature of iPS cells created by current pluripotency protocols. However, the spontaneous appearance of lines that express sufficient telomerase activity to extend telomere length may allow the reversal of developmental aging in human cells for use in regenerative medicine.”

Previous blog posts have also cited contradictory reports as to whether telomere lengths in iPSCs are disappointingly short(ref) or amply long (ref).  My own conjecture is that all these reports are accurate and the differences are due to differences in the iPSC cell lines studied.  A take-away message here is that iPSCs are far from all being the same thing.

More reliable and efficient means for cell reprogramming that yield more consistent “higher-fidelity” iPSC are being discovered.   

See the discussions of several additional publications in the July 2010 blog entry   Induced pluripotent stem cells – developments on the road to big-time utilization  and in the October 2010 entry  A breakthrough in producing high-fidelity induced pluripotent stem cells.  

iPSC cells display mitochondrial rejuvenation

The November 2010 publication Mitochondrial rejuvenation after induced pluripotency reports “We have examined the properties of mitochondria in two fibroblast lines, corresponding IPSCs, and fibroblasts re-derived from IPSCs using biochemical methods and electron microscopy, and found a dramatic improvement in the quality and function of the mitochondrial complement of the re-derived fibroblasts compared to input fibroblasts. This observation likely stems from two aspects of our experimental design: 1) that the input cell lines used were of advanced cellular age and contained an inefficient mitochondrial complement, and 2) the re-derived fibroblasts were produced using an extensive differentiation regimen that may more closely mimic the degree of growth and maturation found in a developing mammal.  CONCLUSIONS/SIGNIFICANCE: These results – coupled with earlier data from our laboratory – suggest that IPSC conversion not only resets the “biological clock”, but can also rejuvenate the energetic capacity of derived cells.” 

More is constantly being learned about efficiently reprogramming cells to iPSCs

The October 2010 publication Generation of iPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules  reports “Here, we identify a specific chemical combination, which is sufficient to permit reprogramming from mouse embryonic and adult fibroblasts in the presence of a single transcription factor, Oct4, within 20 days, replacing Sox2, Klf4 and c-Myc. The iPSCs generated using this treatment resembled mouse embryonic stem cells in terms of global gene expression profile, epigenetic status and pluripotency both in vitro and in vivo. We also found that 8 days of Oct4 induction was sufficient to enable Oct4-induced reprogramming in the presence of the small molecules, which suggests that reprogramming was initiated within the first 8 days and was independent of continuous exogenous Oct4 expression.”

Researchers have succeeded in reverting a large number of cell types to iPSC status

I cite only a few 2010 and late 2009 publications as examples: Generation of induced pluripotent stem cells by efficient reprogramming of adult bone marrow cells, Effective generation of iPS cells from CD34+ cord blood cells by inhibition of p53, Reprogramming adult hematopoietic cells, and Conversion of ancestral fibroblasts to induced pluripotent stem cells.

More is being learned relative to directing the differentiation of ESCs and iPSCs into target cell lines.

I cite only an example publication, the January 2010 report SIP1 mediates cell-fate decisions between neuroectoderm and mesendoderm in human pluripotent stem cells.  Highlights are “* Smad-interacting protein 1 (SIP1) regulates hESC differentiation.  * SIP1 upregulation promotes neuroectodermal differentiation.  * SIP1 inhibits mesendodermal and endodermal differentiation.  * SMAD2/3 and NANOG/OCT4/SOX2 cooperatively regulate SIP1 expression.”  Also, “In turn, Activin-Nodal signaling cooperates with NANOG, OCT4, and SOX2 to control the expression of SIP1 in hESCs, thereby limiting the neuroectoderm-promoting effects of SIP1.” The mesendoderm is embryonic tissue which differentiates into mesoderm and endoderm.  The mesoderm is “The middle embryonic germ layer, lying between the ectoderm and the endoderm, from which connective tissue, muscle, bone, and the urogenital and circulatory systems develop(ref).”  The endoderm is “The innermost of the three primary germ layers of an animal embryo, developing into the gastrointestinal tract, the lungs, and associated structures. Also called hypoblast(ref).” And the ectoderm is “The outermost of the three primary germ layers of an embryo, from which the epidermis, nervous tissue, and, in vertebrates, sense organs develop(ref).”

iPSC research might well lead to therapies for HIV 

The November 2010 publication Generation of HIV-1 Resistant and Functional Macrophages From Hematopoietic Stem Cell–derived Induced Pluripotent Stem Cells relates “By developing iPSCs to treat HIV, there is the potential for generating a continuous supply of therapeutic cells for transplantation into HIV-infected patients. In this study, we have used human hematopoietic stem cells (HSCs) to generate anti-HIV gene expressing iPSCs for HIV gene therapy. HSCs were dedifferentiated into continuously growing iPSC lines with four reprogramming factors and a combination anti-HIV lentiviral vector containing a CCR5 short hairpin RNA (shRNA) and a human/rhesus chimeric TRIM5α gene. Upon directed differentiation of the anti-HIV iPSCs toward the hematopoietic lineage, a robust quantity of colony-forming CD133⁺ HSCs were obtained. These cells were further differentiated into functional end-stage macrophages which displayed a normal phenotypic profile. Upon viral challenge, the anti-HIV iPSC-derived macrophages exhibited strong protection from HIV-1 infection. Here, we demonstrate the ability of iPSCs to develop into HIV-1 resistant immune cells and highlight the potential use of iPSCs for HIV gene and cellular therapies.”  Note that since the original donor cells and the redirected iPSC cells were both in the hematopoietic lineage, some of the possible problems of epigenetic traces were obviated. 

The scheduled January 2011 publication Human Pluripotent Stem Cells Produce Natural Killer Cells That Mediate Anti-HIV-1 Activity by Utilizing Diverse Cellular Mechanisms offers an additional hope for the battle against HIV.  “Natural killer (NK) cells are a key component of the innate immune system with the ability to kill diverse tumor cells and virus-infected cells. While NK cells have been shown to play an important role in the control of HIV-1 replication, their functional activities are often compromised in HIV-1-infected individuals. We have previously demonstrated the derivation of NK cells from human embryonic stem cells (hESCs) with the ability to potently kill multiple types of tumor cells both in vitro and in vivo. We now demonstrate the derivation of functional NK cells from human induced pluripotent stem cells (iPSCs). More importantly, both hESC- and iPSC-derived NK cells are able to inhibit HIV-1 NL4-3 infection of CEM-GFP cells. Additional studies using HIV-1-infected human primary CD4+ T cells illustrated that hESC- and iPSC-derived NK cells suppress HIV-1 infection by at least three distinct cellular mechanisms: killing of infected targets through direct lysis, antibody-dependent cellular cytotoxicity, and production of chemokines and cytokines. Our results establish the potential to utilize hESC- and iPSC-derived NK cells to better understand anti-HIV-1 immunity and provide a novel cellular immunotherapeutic approach to treat HIV/AIDS.” 

Promise for the future is bright 

The July 2010 publication Evolution of induced pluripotent stem cell technology summarizes progress and the challenges remaining.  “iPSC technology provides unprecedented opportunities in biomedical research and regenerative medicine. However, there remain a great deal to learn about iPSC safety, the reprogramming mechanisms, and better ways to direct a specific reprogramming process. The iPSC field will flourish on its mechanistic studies, iPSC-based disease modeling, and identification of new small molecules that modulate reprogramming.” 

iPSCs have not been ready yet for use in human regenerative medicine experiments although I believe the start of that time is rapidly approaching.  I conjecture that If the furious 2010 pace of iPSC research continues we might see animal experiments in the major longevity intervention Closing the loop in the stem cell supply chain in as little as 5-7 years.  And the pace appears to be accelerating.

In several earlier blog entries I have advocated the concept that social evolution is leading now to rapid biological evolution in humans, the entries including Social evolution and biological evolution – another dialog with Marios Kyriazis, and Social ethics of longevity.  How could this happen given that evolution has taken millions of years?  I have suggested that the evolution involved is epigenetic evolution which moves far faster than Darwinian genetic evolution.  This blog entry is about a new theory that explains how this evolution is taking place: stochastic epigenetic evolution.

Stochastic epigenetic evolution is a new and different theory offering explanations for both aging and the current rapid pace of human evolution.  It is based on the notion that components of the epigenome are not stable but are in constant flux due to random events.  This flux may be responsible for variable disease susceptibilities, ability of the organism to evolve very rapidly to accommodate to new conditions, and perhaps even aging itself. 

It is not that there is already a scarcity of theories of aging.  I have laid out 14 major theories of aging and 6 additional candidate theories in my treatise ANTI-AGING FIREWALLS – THE SCIENCE AND TECHNOLOGY OF LONGEVITY.  The new theory of stochastic epigenetic evolution is interesting because it is different than the others, and because it explains some things otherwise not well explained.  I present an overview of the theory here and discuss some of its ramifications.

Background on evolution

Classic “evolution (also known as biological, genetic or organic evolution) is the change in the inherited traits of a population of organisms through successive generations.^[1] This change results from interactions between processes that introduce variation into a population, and other processes that remove it. As a result, variants with particular traits become more, or less, common. A trait is a particular characteristic—anatomical, biochemical or behavioural—that is the result of gene–environment interaction(ref).” 

According to the classical view of evolution “The main source of variation is mutation, which introduces genetic changes. These changes are heritable (can be passed on through reproduction), and may give rise to alternative traits in organisms. Another source of variation is genetic recombination, which shuffles the genes into new combinations which can result in organisms exhibiting different traits. Under certain circumstances, variation can also be increased by the transfer of genes between species,^[2][3] and by the extremely rare, but significant, wholesale incorporation of genomes through endosymbiosis^[4][5] (ref).” 

So, in the classical view evolution is based on mutations in the genome and the process is likely to be extremely slow taking many generations to take hold.  “Two main processes cause variants to become more common or rarer in a population. One is natural selection, through which traits that aid survival and reproduction become more common, while traits that hinder survival and reproduction become rarer. Natural selection occurs because only a small proportion of individuals in each generation will survive and reproduce, since resources are limited and organisms produce many more offspring than their environment can support(ref).” I comment that this classical description obviously does not match what is happening in many modern countries like Japan or Italy where too-few children are being born to maintain the population size.

Going on, “Over many generations, heritable variation in traits is filtered by natural selection and the beneficial changes are successively retained through differential survival and reproduction. This iterative process adjusts traits so they become better suited to an organism’s environment: these adjustments are called adaptations.^[6]  — However, not all change is adaptive. Another cause of evolution is genetic drift, which leads to random changes in how common traits are in a population(ref).”

This classical view of evolution is far too slow to explain many examples of observed evolution.  Why for example are Americans now significantly taller and longer-lived than they were a couple of generations back?  And consider for examples the case of lizards transported from one Caribbean island to another.  “An experiment with lizards in the Caribbean has demonstrated that evolution moves in predictable ways and can occur so rapidly that changes emerge in as little as a decade. — The experiment involved the introduction of one species of lizard to fourteen small, lizard-free Caribbean island near the Exumas in the Bahamas. The lizards were left for fourteen years. The original intent of the experiment was to study extinction. The experiment, started by Thomas Schoener of the University of California at Davis, would have provided scientists with important information as they observed the extinction of the introduced lizards. Unfortunately, the lizards adapted to their new environments, and the focus of the experiment changed to study this rapid evolution.”  The lizards evolved different lengths of legs to be optimal for the vegetation of the islands concerned.   And they did this fast enough to survive in their new island homes.  “The rate of evolutionary change is measured in units called darwins. Darwins provide a measure of the proportional change in a given organ over time. Changes typically seen over millions of years in the fossil record usually amount to 1 darwin or less. The transplanted lizards evolved at rates of up to 2000 darwins(ref).”

Another example of observed rapid evolution is in finches “Over a ten year period, three natural selection events occurred, suggesting that evolutionary change might be more rapid than ever before suspected(ref).”  There are multiple other examples of rapid evolution.  “Acting as super-predators, humans are forcing changes to body size and reproductive abilities in some species 300 percent faster than would occur naturally, a new study finds. — In a review of 34 studies that tracked 29 species across 40 different geographic systems, harvested and hunted populations are on average 20 percent smaller in body size than previous generations, and the age at which they first reproduce is on average 25 percent earlier(ref).”  The ability of many diseases to evolve rapidly also challenges the classical evolution model.  “ — the classic model also has significant limitations in explaining common human disease; common variants can explain only a small fraction of a given disease phenotype, even the most well understood, such as adult-onset diabetes and height(ref).”

These evolutionary changes and many others are happening far too rapidly to be explained by changes in genes which remain stable for large numbers of generations.  We have largely the same genes our ancestors did millions of years ago.   Instead, the rapid evolutionary changes must represent inheritable changes in the respective epigenomes, not in the underlying DNA sequences.  

Stochastic epigenetic evolution

Back in 2007, the publication Combinatorial epigenetics, “junk DNA”, and the evolution of complex organisms suggested a possible strong role for epigenetic shifts in determining the evolution of complex organisms. The authors suggest that epigenetic shifts facilitate classical mutations in the evolutionary process.  “It is proposed that, in eukaryotes, changes in epigenetic trends and epigenetically transforming encounters between alternative chromatin structures could arise frequently enough so as to render probable particular conjunctions of changed transcription rates.– The chances for two or more particular epigenetically determined regulatory trends to occur together in a cell are increased thanks to the proposed low specificity requirements for most of the pertinent sequence changes in intergenic and intronic DNA or in the distribution of middle repetitive sequences that have teleregulatory impact. Inheritable epigenetic changes (“epimutations”) with effects at a distance would then perdure over the number of generations required for “assimilation” of the several regulatory novelties through the occurrence and selection, gene by gene, of specific classical mutations. These mutations would have effects similar to the epigenetic effects, yet would provide stability and penetrance. The described epigenetic/genetic partnership may well at times have opened the way toward certain complex new functions. Thus, the presence of “junk DNA”, through co-determining the (higher or lower) order and the variants of chromatin structure with regulatory effects at a distance, might make an important contribution to the evolution of complex organisms.”  Several of the later papers listed below see evolution as also taking place purely at the epigenetic level. 

The case for the theory of stochastic epigenetic evolution is laid out in the 2009 publication Epigenetic gambling and epigenetic drift as an antagonistic pleiotropic mechanism of aging.  “I suggest that random changes in cellular gene expression (cellular epigenetic gambling or bet hedging) evolved as an adaptive mechanism to ensure survival of members of a group in the face of unpredictable environmental challenges. Once activated, it could lead to progressive epigenetic variegation (epigenetic drift) amongst all members of the group. Thus, while particular patterns of gene expression would be adaptive for a subset of reproductive individuals within a population early in life, once initiated, I predict that continued epigenetic drift will result in variable onsets and patterns of pathophysiology–perhaps yet another example of antagonistic pleiotropic gene action in the genesis of senescent phenotypes. The weakness of this hypothesis is that we do not currently have a plausible molecular mechanism for the putative genetic ‘randomizer’ of epigenetic expression, particularly one whose ‘setting’ may be responsive to the ecology in which a given species evolves. I offer experimental approaches, however, to search for the elusive epigenetic gambler(s).”

Proposed mechanisms for stochastic epigenetic evolution

The 2009 publication Stochastic epigenetic variation as a driving force of development, evolutionary adaptation, and disease discusses the stochastic epigenetic evolution theory in detail and proposes molecular mechanisms for the “genetic ‘randomizer’ of epigenetic expression” discussed in the previously-cited paper.  The discussions in this paper are technical and rather tough for a layman to follow but I quote selectively. “It has occurred to us that increased variability with a given genotype might itself increase fitness. This could arise by genetic variants that do not change the mean phenotype but do change the variability of phenotype. A natural mechanism to use to consider such a model is epigenetic plasticity during development, for example, varying DNA methylation patterns. This idea differs from Lamarckian inheritance, in that in our model the genetic change is inherited, and this change leads to increased epigenetic variation. It also differs from the likely role of epigenetics in modifying mutation rate, –.  As a proof of principle, we revisited previously generated data sets (14) of genome-scale analysis of DNA methylation in human and mouse tissues and explored them in two new ways. First, we investigated whether there were regions of variable methylation across individuals for a given tissue type. Then we explored whether tissue-specific differentially methylated regions (T-DMRs) differed across species and whether the underlying DNA sequence could account for these differences. — To assess the degree of intrinsic variability in DNA methylation of a given tissue, we set out to identify the location of the most highly variable regions of DNA methylation in mouse liver from four individuals. We chose this specific tissue because it is relatively homogeneous. We examined newborns in whom polyploidy is minimal, although copy number would not be expected to affect DNA methylation, because our method controls for copy number (15). Environmental effects were minimized by examining inbred mice (indeed, littermates from the same cage). Surprisingly, many loci throughout the genome showed striking variations in DNA methylation, which we term variably methylated regions (VMRs). Surprisingly, these VMRs were significantly enriched in the vicinity of genes with Gene Ontogeny (GO) functional categories for development and morphogenesis (Table 1) when using either all genes for comparison or all regions present on the CHARM array, indicating that enrichment is not explained solely by high CpG content, because the array itself is designed to assay high-CpG regions. Examples of developmental genes with VMRs—Bmp7, involved in early embryogenic programming and bone induction, Pou3f2, involved in neurogenesis and stem cell reprogramming, and Ntrk3, involved in body position sensing—are shown in Fig. 1. — Next, we were interested in whether changes in differential methylation across species (mouse and human) could be traced back to an underlying genetic basis. To address this question, we focused on T-DMRs, given the wealth of data gathered in previous studies and their relevance to human diseases, such as cancer. Previously we reported that DMRs that distinguish colorectal cancer from normal colonic mucosa (C-DMRs) are enriched for T-DMRs, and this finding was validated in a large independent set of samples. In many cases, the loss of differential methylation in one species was related to an underlying loss of CpGs at the corresponding CpG island or nearby CpG island shore (14). A typical example of an evolutionary change in differential methylation involved LHX1, a transcriptional regulator essential for vertebrate head organization and mesoderm organization, (shown in Fig. 5). Note the T-DMR in human that is not in mouse on the left of the TSS. The human has gained CpGs at a CpG island shore (with the island shown in orange tick marks in the bottom panel). In contrast, both species have a moderate CpG count to the right of the TSS, and both have DMRs in this region. This is an example of how a genetic variation (i.e., gain of CpGs) allows for development-relevant tissue-specific differences in a highly conserved gene. Thus, differential methylation that itself differs across species may be due to underlying sequence variation at the site of these DMRs. Additional examples of this are available at rafalab.jhsph.edu/evometh.pdf. – Discussion Here we have proposed a model in which increased variability with a given genotype might increase fitness not by changing mean phenotype, but rather by changing the variability of phenotype with a given genotype. We also have provided a possible mechanism by which such enhanced variability could be genetically inherited and lead to increased stochastic epigenetic variation during development. Note that the genomic loci for such variation would be well defined in our model; we have provided examples of these loci. Although these loci do not represent the primary engine of development, they do provide plasticity in the developmental program by virtue of the stochastic variation that they impart through the genes in their proximity. — Our model differs from that of a transgenerational epigenetic effect on phenotypic variation and disease risk (16), in that in our model, the genetic variant is inherited and contributes to enhanced phenotypic variation, which can be mediated epigenetically in each generation. It also differs from a hypermutable genetic-switching model, in which the genotype itself changes from generation to generation, increasing phenotypic plasticity (17). — Our model provides a mechanism for developmental plasticity and evolutionary adaptation to a fluctuating environment. Although the model is general and does not necessitate epigenetic variation, we have demonstrated the existence of VMRs that affect phenotype (i.e., gene expression) in isogenic mice raised in an identical environment, and have shown that similar VMRs exist in humans as well. We also have reported a potential genetic mechanism for differences in tissue-specific methylation across species—namely, the gain or loss of a CpG island or the associated shore. The localization near a specific gene would provide specificity of the effect of variation, but the mechanism for variation could entail the relationship to tissue-specific promoters, transcription factor binding sites, population variation in CpG density in these regions, or a combination of such factors. Distinguishing among these possibilities will require further experimentation.”

Another paper that suggests a mechanism for stochastic epigenetic evolution is the 2010 publication Epigenetics in the Extreme: Prions and the Inheritance of Environmentally Acquired Traits.  “Prions are an unusual form of epigenetics: Their stable inheritance and complex phenotypes come about through protein folding rather than nucleic acid-associated changes. With intimate ties to protein homeostasis and a remarkable sensitivity to stress, prions are a robust mechanism that links environmental extremes with the acquisition and inheritance of new traits.”  A 2009 paper Protein folding sculpting evolutionary change forwards the same theme. “Because changes in protein homeostasis occur with environmental stress, prions can be cured or induced by stress, creating heritable new phenotypes that depend on the genetic variation present in the organism. Both prions and Hsp90 provide plausible mechanisms for allowing genetic diversity and fluctuating environments to fuel the pace of evolutionary change. The multiple mechanisms by which protein folding can influence the evolution of new traits provide both a new paradigm for understanding rapid, stepwise evolution and a framework for targeted therapeutic interventions.”

Implications of stochastic epigenetic evolution 

Rapidity of evolution 

As discussed above, epigenomic evolution can happen much faster than could happen due to changes only in the genome.  This is consistent with observations in both humans and other species.

Epigenetic regulation and variability in aging

There are a number of studies relating epigenetic changes to aging.  The honeybee is a well-studied example of an organism where epigenetic mechanisms appear to be the main determinants of aging.  Environmental conditions and specialization of functions can have major impacts on lifespans. 

Examples are given in the 2004 publication Epigenetic Regulation of Aging in Honeybee Workers.  “Aging and longevity are complex life history traits that are influenced by both genes and environment and exhibit significant phenotypic plasticity in a broad range of organisms. A striking example of this plasticity is seen in social insects, such as ants and bees, where different castes can have very different life spans. In particular, the honeybee worker offers an intriguing example of environmental control on aging rate, because workers are conditionally sterile and display very different aging patterns depending on which temporal caste they belong to (hive bee, forager, or a long-lived caste capable of surviving for several months on honey alone). The ubiquitous yolk protein vitellogenin appears to play a key role in the regulatory circuitry that controls this variation.” 

As stated in the publication Handbook of models for human aging for the honeybee ,  “Epigenetic regulation is responsible for the differentiation of females into workers and queens – two cases with strongly diverging lifespan potential – and a plastic pattern of worker longevity that appears to be determined by the social colony setting rather than chronological age.”

Epigenetics and senescence

The 2010 publication The curious case of aging plasticity in honey bees reports “Curiously, aging progresses slowly in workers that engage in nursing and even slower when bees postpone nursing during unfavorable periods. We, therefore, seek to understand how senescence can emerge as a function of social task performance.”

As a matter fact, in the honeybee worker the regulation of aging appears to be mainly epigenetic and have or little to do with functional senescence.  The 2007 paper Aging without functional senescence in honey bee workers reports “The limited existing data support a direct connection between old age, increased mortality rate and decreased behavioral or physiological performance in organisms ranging from flies [2] to humans [3]. A recent study [4], however, suggests that the linkage may be less universal than previously postulated. To investigate this linkage directly in the non-traditional aging model Apis mellifera [5], old honey bee workers were studied with respect to survival and performance. A test battery of behavioral assays showed a significant increase in experimental mortality rate with chronological age, but no evidence for an age-dependent performance decline in locomotion, learning or responsiveness to light or sucrose. The explanation for this decoupling of intrinsic mortality and functional decline may lie in the social evolution of honey bees [6].”

It appears that in some cases epigenetic regulation can reverse cellular senescence.  The 2005 paper Social reversal of immunosenescence in honey bee workers relates “A striking example of immunosenescence is seen in the honey bee (Apis mellifera) worker caste. The bees’ age-associated transition from hive duties to more risky foraging activities is linked to a dramatic decline in immunity. Explicitly, it has been shown that an increase in the juvenile hormone (JH) level, which accompanies onset of foraging behavior, induces extensive hemocyte death through nuclear pycnosis. Here, we demonstrate that foragers that are forced to revert to hive-tasks show reversal of immunosenescence, i.e. a recovery of immunity with age. This recovery, which is triggered by a social manipulation, is accompanied by a drop in the endogenous JH titer and an increase in the hemolymph vitellogenin level. Vitellogenin is a zinc binding glycolipoprotein that has been implicated in the regulation of honey bee immune integrity. We also establish that worker immunosenescence is mediated by apoptosis, corroborating that reversal of immunosenescence emerges through proliferation of new cells. The results presented here, consequently, reveal a unique flexibility in honey bee immunity–a regulatory plasticity that may be of general biological interest.”  Loss or gain of longevity or immunity with change of circumstances requires a fast-acting epigenetic mechanism, and stochastic epigenetic evolution is a candidate for that mechanism.

There is a body of interesting literature relevant to DNA methylation changes in humans as a function of age, changes that are quite possibly due to stochastic epigenetic evolution.  I cannot review these here but mention specifically the 2010 publication Widespread and tissue specific age-related DNA methylation changes in mice.  “Our findings demonstrate a surprisingly high rate of hyper- and hypomethylation as a function of age in normal mouse small intestine tissues and a strong tissue-specificity to the process. We conclude that epigenetic deregulation is a common feature of aging in mammals.”  Of course this is the premise of the 13^th theory of aging outlined in my treatise Programmed Epigenomic Changes.  If the stochastic epigenomic evolution theory is correct, then the “programming” would consist of epigenetic drift due to accumulation of multiple random changes in the epigenome.

Rapid changes in disease susceptibility

It has also been suggested that stochastic epigenomic evolution may be responsible for the development of disease susceptibilities.  The concern is that it can drive rapid “epigenomic drift” as mentioned above. The 2008 paper Age-Specific Epigenetic Drift in Late-Onset Alzheimer’s Disease relates “Despite an enormous research effort, most cases of late-onset Alzheimer’s disease (LOAD) still remain unexplained and the current biomedical science is still a long way from the ultimate goal of revealing clear risk factors that can help in the diagnosis, prevention and treatment of the disease. Current theories about the development of LOAD hinge on the premise that Alzheimer’s arises mainly from heritable causes. Yet, the complex, non-Mendelian disease etiology suggests that an epigenetic component could be involved. Using MALDI-TOF mass spectrometry in post-mortem brain samples and lymphocytes, we have performed an analysis of DNA methylation across 12 potential Alzheimer’s susceptibility loci. In the LOAD brain samples we identified a notably age-specific epigenetic drift, supporting a potential role of epigenetic effects in the development of the disease. Additionally, we found that some genes that participate in amyloid-β processing (PSEN1, APOE) and methylation homeostasis (MTHFR, DNMT1) show a significant interindividual epigenetic variability, which may contribute to LOAD predisposition. The APOE gene was found to be of bimodal structure, with a hypomethylated CpG-poor promoter and a fully methylated 3′-CpG-island, that contains the sequences for the ε4-haplotype, which is the only undisputed genetic risk factor for LOAD. Aberrant epigenetic control in this CpG-island may contribute to LOAD pathology. We propose that epigenetic drift is likely to be a substantial mechanism predisposing individuals to LOAD and contributing to the course of disease.”

The 2010 papers Epigenetic Epidemiology of Common Complex Disease: Prospects for Prediction, Prevention, and Treatment, Epigenetics in molecular epidemiology of cancer a new scope, Emerging role of epigenetics in stroke: part 1: DNA methylation and chromatin modifications, Epigenetics in neurodegeneration: A new layer of complexity and the 2009 paper Prospects for epigenetic epidemiology are among many other recent publications relating epigenetic changes to disease processes.

As is the case for many of my blog entries, I have been able here to cite only a few of the very many relevant literature citations.  I believe they have been sufficient, however, to illustrate the importance of epigenomic changes in aging and disease processes and to render plausible the new stochastic epigenomic theory of evolution.  I expect that I will be writing more blog entries related to epigenomics as the amount of research in this area continues to explode.

A longevity news story breaking today was widely published in the world press.  Some of the 123 headlines listed on Google are Ageing Process “reversed” in mice, Die hard: Scientists reverse aging in mice,  Scientists unlock secret of eternal youth,  Secret to eternal youth ‘found’, Harvard scientists reverse aging in mice, Virility, Mental Ability Restored in Aged Mice in Gene Study,Nature Says, Science Makes Old Mice Young Again, Scientists Find Fountain of Youth … in Mice, Harvard University Doctor Turns Back Time on Aging: Will an Anti-Aging Pill Be .., Enzyme Reverses Aging in Mice and The Curious Case of the Backwardly Aging Mouse.

I think the research reported is quite important though these headlines sensationalize and misrepresent that research.  I comment here both on the research and on the press coverage it received.

The publication that initiated this press flurry is a 28 November online publication that appeared in Nature Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice.  “An ageing world population has fuelled interest in regenerative remedies that may stem declining organ function and maintain fitness. Unanswered is whether elimination of intrinsic instigators driving age-associated degeneration can reverse, as opposed to simply arrest, various afflictions of the aged. Such instigators include progressively damaged genomes. Telomerase-deficient mice have served as a model system to study the adverse cellular and organismal consequences of wide-spread endogenous DNA damage signalling activation in vivo. Telomere loss and uncapping provokes progressive tissue atrophy, stem cell depletion, organ system failure and impaired tissue injury responses1.  Here, we sought to determine whether entrenched multi-system degeneration in adult mice with severe telomere dysfunction can be halted or possibly reversed by reactivation of endogenous telomerase activity. To this end, we engineered a knock-in allele encoding a 4-hydroxytamoxifen (4-OHT)-inducible telomerase reverse transcriptase-oestrogen receptor (TERT-ER) under transcriptional control of the endogenous TERT promoter. Homozygous TERT-ER mice have short dysfunctional telomeres and sustain increased DNA damage signalling and classical degenerative phenotypes upon successive generational matings and advancing age. Telomerase reactivation in such late generation TERT-ER mice extends telomeres, reduces DNA damage signalling and associated cellular checkpoint responses, allows resumption of proliferation in quiescent cultures, and eliminates degenerative phenotypes across multiple organs including testes, spleens and intestines. Notably, somatic telomerase reactivation reversed neurodegeneration with restoration of proliferating Sox2+ neural progenitors, Dcx+ newborn neurons, and Olig2+ oligodendrocyte populations. Consistent with the integral role of subventricular zone neural progenitors in generation and maintenance of olfactory bulb interneurons2, this wave of telomerase-dependent neurogenesis resulted in alleviation of hyposmia and recovery of innate olfactory avoidance responses. Accumulating evidence implicating telomere damage as a driver of age-associated organ decline and disease risk and the marked reversal of systemic degenerative phenotypes in adult mice observed here support the development of regenerative strategies designed to restore telomere integrity.”

So, mice were genetically engineered to be born with knocked-out telomerase genes and these mice aged in an accelerated fashion  They eventually exhibited most or all the degenerative conditions known to be associated with aging.  Such effects have been observed for many years.  The new discovery is that reactivation of telomerase in these mice reverses many of the phenomena of aging and, in effect, makes these mice younger again.  This is an extremely important discovery because it strongly suggests that a single intervention can reverse the multiple deleterious manifestations of aging which have always been thought to be permanent and, once occurred, irreversible.  At least this seems to be true for aging induced by telomerase deficiency.  To me it comes close to being a mind-boggling proof-of-concept that aging can be reversed, establishing that aging is a two-way street.  It is also additional evidence that aging is not just due to accumulation of random damage due to wear-and tear.  It would be impossible to repair so many and so radically different forms of random damage with a single intervention. 

The writeup in Nature News is more plain-spoken. “ — When mice are engineered to lack telomerase completely, their telomeres progressively shorten over several generations. These animals age much faster than normal mice — they are barely fertile and suffer from age-related conditions such as osteoporosis, diabetes and neurodegeneration. They also die young. “If you look at all those data together, you walk away with the idea that the loss of telomerase could be a very important instigator of the ageing process,” says DePinho (Ronald DePinho, an author of the research publication and a cancer geneticist at the Dana-Farber Cancer Institute and Harvard Medical School). — To find out if these dramatic effects are reversible, DePinho’s team engineered mice such that the inactivated telomerase could be switched back on by feeding the mice a chemical called 4-OHT. The researchers allowed the mice to grow to adulthood without the enzyme, then reactivated it for a month. They assessed the health of the mice another month later. — “What really caught us by surprise was the dramatic reversal of the effects we saw in these animals,” says DePinho. He describes the outcome as “a near ‘Ponce de Leon’ effect” — a reference to the Spanish explorer Juan Ponce de Leon, who went in search of the mythical Fountain of Youth. Shrivelled testes grew back to normal and the animals regained their fertility. Other organs, such as the spleen, liver and intestines, recuperated from their degenerated state. — The one-month pulse of telomerase also reversed effects of ageing in the brain. Mice with restored telomerase activity had noticeably larger brains than animals still lacking the enzyme, and neural progenitor cells, which produce new neurons and supporting brain cells, started working again. — “It gives us a sense that there’s a point of return for age-associated disorders,” says DePinho. Drugs that ramp up telomerase activity are worth pursuing as a potential treatment for rare disorders characterized by premature ageing, he says, and perhaps even for more common age-related conditions. – “

Let me turn to the sensationalism in the press reporting for a moment.  If you have read my recent blog post When reading press releases and newspaper articles about research discoveries, beware!  then you know I am sensitive to misrepresentation of research results in headlines.   I hate to be nitpicking but most of the press reports for this research do not make clear:

1.     The study shows only that accelerated aging from one cause can be reversed, that cause being lack of telomerase expression.  We know that many causes can accelerate apparent aging (like exposure to radiation and some chemicals) but we do not know yet whether aging from such causes can be reversed.  And we do not know if natural aging, however that comes about, can be reversed.  I hope and suspect that aging-is-aging and aging from all causes can be reversed, but as a scientist I don’t know yet that this is true.  

2.     The apparent age reversal observed was not in normal mice who aged normally.  It was in mice genetically engineered not to have a natural feature, an active telomerase gene, who age (or do something very much like aging) prematurely.  The age-reversal was initiated by restoring what had been taken away, natural telomerase production.  We do not know if the age reversal would work in normally aged mice let alone in normally-aged humans.   

3.     Despite the fact that telomerase-deficient mice age more rapidly, neither this work nor any other research I know about establishes that telomere-lengths are critical for limiting normal lifespans in either mice or humans.  It was not reported that the mutant mice who underwent age-reversal lived longer than normal mice.  There is no indication that extended lifespan let alone eternal youth was involved despite the headlines claiming it. 

4.     The genetic-engineering approach to turning the telomerase genes back on in the mutant mice can’t be used in humans since any genetic engineering of humans is taboo.

“Harrison (David Harrison, who researches ageing at the Jackson Laboratory in Bar Harbor, Maine) also questions whether mice lacking telomerase are a good model for human ageing. “They are not studying normal ageing, but ageing in mice made grossly abnormal,” he says. Tom Kirkwood, who directs the Institute for Ageing and Health at Newcastle University, UK, agrees, pointing out that telomere erosion “is surely not the only, or even dominant, cause” of ageing in humans(ref).”

So, while this research is important and very encouraging from the viewpoint of life extension, it is not at all clear that it will lead to life-extending interventions in humans.  I agree with DePinho who “says he recognizes that there is more to ageing than shortened telomeres, particularly late in life, but argues that telomerase therapy could one day be combined with other therapies that target the biochemical pathways of ageing. “This may be one of several things you need to do in order to extend lifespan and extend healthy living,” he says(ref).”

For background you can read about the Telomere Shortening and Damage theory of aging in my treatise.  For updates on recent research on telomerase, you can see my recent blog entries:

* Telomere lengths, Part 3: Selected current research on telomere-related signaling,

* Telomere lengths, Part 2: Lifestyle, dietary, and other factors associated with telomere shortening and lengthening, and

* Telomere lengths, Part 1: Telomere lengths, cancers and disease processes.

Having reliable sets of predictive biomarkers for diseases is at the heart of a new emerging paradigm for medicine, a paradigm I have called Personalized Predictive Preventative Participatory Medicine (PPPPM).  See the blog entries  Harnessing the engines of finance and commerce for life-extension,  Personalized medicine – reducing the cost and improving the effectiveness of health care, and Transformed State of Medicine – 2025.  This blog entry reports on progress for identifying reliable biomarkers for cardiovascular diseases.

A disease biomarker is in general a condition or substance used as an indicator of a biological state that describes a disease susceptibility or that is predictive of a disease . “It is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention(ref).”  In the general sense I am using here, a biomarker can be age, gender, childhood adiposity, a clinical test score like a measurement of cholesterol or blood pressure, body-mass index, a genetic condition such as mutation of a specified gene, an epigenetic condition, a condition related to family or ethnic history, X-ray and MRI results, existing or previous disease conditions,  or personal habits such as cigarette smoking.   

Of the hundreds of relevant publications, I have selected only a limited number for review here to illustrate what I see as the major developmental trends for cardiovascular biomarkers. 

Framingham study risk scores for cardiovascular heart disease

A biomarker system for scoring risks of various cardiovascular heart disease (CHD) was developed years ago as part of the Framingham Heart Study.  “Determining 10-year (short term) risk for developing CHD is carried out using Framingham risk scoring. The risk factors included in the Framingham calculation are age, total cholesterol, HDL cholesterol, systolic blood pressure, treatment for hypertension, and cigarette smoking. Because of a larger database, Framingham estimates are more robust for total cholesterol than for LDL cholesterol. Note, however, that LDL cholesterol remains the primary target of therapy. The Framingham risk score gives estimates for “hard CHD” which includes myocardial infarction and coronary death(ref).” 

As listed on the Framingham Heart Study website “Risk prediction estimates for the risk of various cardiovascular disease outcomes in different time horizons are available as score sheets and direct risk functions. The choice of the appropriate risk prediction algorithm should take into account the following components: cardiovascular outcome, population of interest, time horizon and risk factors. Outcome-specific algorithms preceded by the descriptions of the above four components are available for the following:

Atrial Fibrillation (AF) (10-year risk) and calculator
Congestive Heart Failure
Coronary Heart Disease (10-year risk)
Coronary Heart Disease (2-year risk)
General Cardiovascular Disease
Hard Coronary Heart Disease and calculator (10-year risk)
Intermittent Claudication
Recurring Coronary Heart Disease
Stroke
Stroke after Atrial Fibrillation and calculator
Stroke or Death after Atrial Fibrillation and calculator”

C-reactive protein, a CHD biomarker but not a cause

Significant research attention has been paid to finding additional biomarkers that can improve  the sometimes-weak predictive capabilities of the traditional biomarker combinations used in Framingham risk scoring.  One such candidate extensively studied has been C-reactive protein (CRP), known since the 1960s as a measure of inflammation and long suspected to be a predictor of CHD.  A 1999 study C-Reactive Protein, a Sensitive Marker of Inflammation, Predicts Future Risk of Coronary Heart Disease in Initially Healthy Middle-Aged Men reports “We used a sensitive immunoradiometric assay to examine the association of serum C-reactive protein (CRP) with the incidence of first major coronary heart disease (CHD) event in 936 men 45 to 64 years of age. The subjects, who were sampled at random from the general population, participated in the first MONICA Augsburg survey (1984 to 1985) and were followed for 8 years. — Conclusions—These results confirm the prognostic relevance of CRP, a sensitive systemic marker of inflammation, to the risk of CHD in a large, randomly selected cohort of initially healthy middle-aged men.”

“In the Harvard Woman’s Health Study, results of the CRP test were more accurate than cholesterol levels in predicting heart problems. Twelve different markers of inflammation were studied in healthy, postmenopausal women. After three years, CRP was the strongest predictor of risk. Women in the group with the highest CRP levels were more than four times as likely to have died from coronary disease, or to have suffered a nonfatal heart attack or stroke compared to those with the lowest levels. This group was also more likely to have required a cardiac procedure such as angioplasty (a procedure that opens clogged arteries with the use of a flexible tube) or bypass surgery than women in the group with the lowest levels(ref).”

There is a significant literature relating CRP to CHD.  The 2005 study C-Reactive Protein and the 10-Year Incidence of Coronary Heart Disease in Older Men and Women reports “Background— High C-reactive protein (CRP) is associated with increased coronary heart disease risk. Few long-term data in the elderly are available. — Methods and Results— Baseline CRP was measured in 3971 men and women 65 years of age without prior vascular diseases; 26% had elevated concentrations (>3 mg/L). With 10 years of follow-up, 547 participants developed coronary heart disease (CHD; defined as myocardial infarction or coronary death). With elevated CRP, the 10-year cumulative CHD incidences were 33% in men and 17% in women. — Conclusions— In older men and women, elevated CRP was associated with increased 10-year risk of CHD, regardless of the presence or absence of cardiac risk factors. A single CRP measurement provided information beyond conventional risk assessment, especially in intermediate-Framingham-risk men and high-Framingham-risk women.”

 The 2006 publication The relative strength of C-reactive protein and lipid levels as determinants of ischemic stroke compared with coronary heart disease in women reported “OBJECTIVES: We sought to determine the relative strength of high-sensitivity C-reactive protein (hs-CRP) and lipid levels as markers for future ischemic stroke compared with coronary heart disease (CHD) in women. — BACKGROUND: Although hs-CRP and lipid levels are established risk determinants for vascular disease, the relative strength of these biomarkers for ischemic stroke compared with CHD is uncertain. — METHODS: Among 15,632 initially healthy women who were followed for a 10-year period, we compared hs-CRP, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), non-high-density lipoprotein cholesterol (non-HDL-C), high-density lipoprotein cholesterol (HDL-C), apolipoproteins A-I and B100, and lipid ratios as determinants of ischemic stroke compared with CHD. — CONCLUSIONS: In this large prospective cohort of initially healthy women, lipid levels are significant risk determinants for ischemic stroke, but with a magnitude of effect smaller than that observed for CHD. High-sensitivity CRP associates more closely with ischemic stroke than with CHD. Concomitant evaluation of lipid levels and hs-CRP may improve risk assessment for stroke as well as CHD.” 

The clear association of CRP with CVD led researchers to speculate that perhaps high CRP is a cause of CVD.  A series of studies shot down this speculation.  The 2008 publications The association of C-reactive protein and CRP genotype with coronary heart disease: findings from five studies with 4,610 cases amongst 18,637 participants.   “CONCLUSIONS: We found no association of a genetic variant, which is known to be related to CRP levels, (rs1130864) and having CHD. These findings do not support a causal association between circulating CRP and CHD risk, but very large, extended, genetic association studies would be required to rule this out.

Another 2008 study C-reactive protein (CRP) gene polymorphisms, CRP levels, and risk of incident coronary heart disease in two nested case-control studies. reported “CONCLUSIONS: Common variation in the CRP gene was significantly associated with plasma CRP levels; however, the association between common SNPs and CRP levels did not correspond to a predicted change in CHD risk. The underlying inflammatory processes which predict coronary events cannot be captured solely by variation in the CRP gene.”

Finally, a  2009 JAMA publication Genetic Loci Associated With C-Reactive Protein Levels and Risk of Coronary Heart Disease concluded “The lack of concordance between the effect on coronary heart disease risk of CRP genotypes and CRP levels argues against a causal association of CRP with coronary heart disease.”So, CRP is a biomarker of inflammation and of CHD.  Inflammation may well be a cause for CHD.  “Inflammation plays a key role in the pathogenesis of CHD at every stage from initiation to progression and rupture of the atherosclerotic plaque. but its marker, CRP, it not such a cause(ref).”  The 2005 publication C-reactive protein comes of age concludes “It is our contention that the future will see much wider use of CRP and CRP-driven therapies in clinical medicine, improving our ability to identify and manage cardiovascular disease.”  The authors were right about CRP as a biomarker but, in the light of the new knowledge, wrong about CRP as a target of therapies.  This illustrates the incremental nature of biomedical knowledge. 

The search for improved biomarkers

The search for better biomarkers predictive of cardiovascular diseases has continued for over 30 years now.  Many publications have appeared on the topic such as the 2009 review publication Biomarkers and Cardiovascular Disease.   By 2009 some researchers were starting to think that further search was unproductive.  The 2009 paper Novel and Conventional Biomarkers for Prediction of Incident Cardiovascular Events in the Community reported on a  “Cohort study of 5067 participants (mean age, 58 years; 60% women) without cardiovascular disease from Malmö, Sweden, who attended a baseline examination between 1991 and 1994. Participants underwent measurement of C-reactive protein (CRP), cystatin C, lipoprotein-associated phospholipase 2, midregional proadrenomedullin (MR-proADM), midregional proatrial natriuretic peptide, and N-terminal pro-B-type natriuretic peptide (N-BNP) and underwent follow-up until 2006.”  — Results  During median follow-up of 12.8 years, there were 418 cardiovascular and 230 coronary events. Models with conventional risk factors had C statistics of 0.758 (95% confidence interval [CI], 0.734 to 0.781) and 0.760 (0.730 to 0.789) for cardiovascular and coronary events, respectively. Biomarkers retained in backward-elimination models were CRP and N-BNP for cardiovascular events and MR-proADM and N-BNP for coronary events, which increased the C statistic by 0.007 (P = .04) and 0.009 (P = .08), respectively. The proportion of participants reclassified was modest (8% for cardiovascular risk, 5% for coronary risk). Net reclassification improvement was nonsignificant for cardiovascular events (0.0%; 95% CI, –4.3% to 4.3%) and coronary events (4.7%; 95% CI, –0.76% to 10.1%). Greater improvements were observed in analyses restricted to intermediate-risk individuals (cardiovascular events: 7.4%; 95% CI, 0.7% to 14.1%; P = .03; coronary events: 14.6%; 95% CI, 5.0% to 24.2%; P = .003). However, correct reclassification was almost entirely confined to down-classification of individuals without events rather than up-classification of those with events. The conclusions were “Selected biomarkers may be used to predict future cardiovascular events, but the gains over conventional risk factors are minimal. Risk classification improved in intermediate-risk individuals, mainly through the identification of those unlikely to develop events.”

Progress reported during 2010

Natriuretic peptides 

“Natriuretic peptides are peptide hormones that are synthesized by the heart, brain and other organs. The release of these peptides by the heart is stimulated by atrial and ventricular distension, as well as by neurohumoral stimuli, usually in response to heart failure(ref).”  I cite a few 2010 publications relating them to cardiovascular risk.

A 2010 publication Assessment of Conventional Cardiovascular Risk Factors and Multiple Biomarkers for the Prediction of Incident Heart Failure and Atrial Fibrillation reports “Objectives: The purpose of this study was to assess the predictive accuracy of conventional cardiovascular risk factors for incident heart failure and atrial fibrillation, and the added benefit of multiple biomarkers reflecting diverse pathophysiological pathways. — Background: Heart failure and atrial fibrillation are interrelated cardiac diseases associated with substantial morbidity and mortality and increasing incidence. Data on prediction and prevention of these diseases in healthy individuals are limited. — Methods: In 5,187 individuals from the community-based MDCS (Malmö Diet and Cancer Study), we studied the performance of conventional risk factors and 6 biomarkers including midregional pro-atrial natriuretic peptide (MR-proANP), N-terminal pro–B-type natriuretic peptide (NT-proBNP), midregional pro-adrenomedullin, cystatin C, C-reactive protein (CRP), and copeptin. — Results: During a mean follow-up of 14 years, 112 individuals were diagnosed with heart failure and 284 individuals with atrial fibrillation. — Conclusions: Conventional cardiovascular risk factors predict incident heart failure and atrial fibrillation with reasonable accuracy in middle-age individuals free from disease. Natriuretic peptides, but not other biomarkers, improve discrimination modestly for both diseases above and beyond conventional risk factors and substantially improve risk classification for heart failure.” 

Another 2010 study of natriutic peptides is Amino-Terminal Pro–B-Type Natriuretic Peptide Improves Cardiovascular and Cerebrovascular Risk Prediction in the Population.  “Increased circulating amino-terminal pro–B-type natriuretic (NT-proBNP) levels are a marker of cardiac dysfunction but also associate with coronary heart disease and stroke. We aimed to investigate whether increased circulating NT-proBNP levels have additive prognostic value for first cardiovascular and cerebrovascular events beyond classic risk factors. In a community-based cohort of 5063 participants free of cardiovascular disease, aged 55 years, circulating NT-proBNP levels and cardiovascular risk factors were measured. Participants were followed for the occurrence of first major fatal or nonfatal cardiovascular event. A total of 420 participants developed a first cardiovascular event (108 fatal). — We conclude that, in an asymptomatic older population, NT-proBNP improves risk prediction not only of heart failure but also of cardiovascular disease in general beyond classic risk factors, resulting in a substantial reclassification of participants to a lower or higher risk category.”

Calcium scores

The 2010 publication Coronary Artery Calcium Score and Risk Classification for Coronary Heart Disease Prediction reports: “Context  The coronary artery calcium score (CACS) has been shown to predict future coronary heart disease (CHD) events. However, the extent to which adding CACS to traditional CHD risk factors improves classification of risk is unclear. — Objective  To determine whether adding CACS to a prediction model based on traditional risk factors improves classification of risk. — Design, Setting, and Participants  CACS was measured by computed tomography in 6814 participants from the Multi-Ethnic Study of Atherosclerosis (MESA), a population-based cohort without known cardiovascular disease. Recruitment spanned July 2000 to September 2002; follow-up extended through May 2008. — Conclusion  In this multi-ethnic cohort, addition of CACS to a prediction model based on traditional risk factors significantly improved the classification of risk and placed more individuals in the most extreme risk categories. “

Imaging biomarkers

CT scanning for calcification indicating subclinical coronary atherosclerosis may be a useful biomarker for patients already known to have other CVD risk factors.  The 2010 publication  Coronary Risk Stratification, Discrimination, and Reclassification Improvement Based on Quantification of Subclinical Coronary Atherosclerosis reports “Objectives: The purpose of this study was to determine net reclassification improvement (NRI) and improved risk prediction based on coronary artery calcification (CAC) scoring in comparison with traditional risk factors.  — Background: CAC as a sign of subclinical coronary atherosclerosis can noninvasively be detected by CT and has been suggested to predict coronary events. — Methods: In 4,129 subjects from the HNR (Heinz Nixdorf Recall) study (age 45 to 75 years, 53% female) without overt coronary artery disease at baseline, traditional risk factors and CAC scores were measured. Their risk was categorized into low, intermediate, and high according to the Framingham Risk Score (FRS) and National Cholesterol Education Panel Adult Treatment Panel (ATP) III guidelines, and the reclassification rate based on CAC results was calculated. — Results: After 5 years of follow-up, 93 coronary deaths and nonfatal myocardial infarctions occurred — Conclusions: CAC scoring results in a high reclassification rate in the intermediate-risk cohort, demonstrating the benefit of imaging of subclinical coronary atherosclerosis. Our study supports its application, especially in carefully selected individuals with intermediate risk.”

So far the use of advanced imaging biomarkers for predicting cardiovascular disease has been relatively disappointing.  The 2010 paper Cardiac computed tomography and myocardial perfusion scintigraphy for risk stratification in asymptomatic individuals without known cardiovascular disease: a position statement of the Working Group on Nuclear Cardiology and Cardiac CT of the European Society of Cardiology states “ — From available data, the use of MPS (myocardial perfusion scintigraphy) as first line testing modality for risk stratification is not recommended in any category of primary prevention subjects with the possible exception of first-degree relatives of patients with premature CAD in whom MPS may be considered. However, the Working Group recognizes that neither the use of computed tomography for calcium imaging nor of MPS have been proven to significantly improve clinical outcomes of primary prevention subjects in prospective controlled studies.”

Combinations of multiple new and old biomarkers

For the many new as well as traditional biomarkers known to be weakly predictive of CHD, 2010 saw several publications relating to different ways of combining them to create more robust predictive tests.

The 2010 publication Multimarker Prediction of Coronary Heart Disease Risk reports “Objectives: The aim of this study was to investigate whether multiple biomarkers contribute to improved coronary heart disease (CHD) risk prediction in post-menopausal women compared with assessment using traditional risk factors (TRFs) only. — Background: The utility of newer biomarkers remains uncertain when added to predictive models using only TRFs for CHD risk assessment. — Methods: The Women’s Health Initiative Hormone Trials enrolled 27,347 post-menopausal women ages 50 to 79 years. Associations of TRFs and 18 biomarkers were assessed in a nested case-control study including 321 patients with CHD and 743 controls. Four prediction equations for 5-year CHD risk were compared: 2 Framingham risk score covariate models; a TRF model including statin treatment, hormone treatment, and cardiovascular disease history as well as the Framingham risk score covariates; and an additional biomarker model that additionally included the 5 significantly associated markers of the 18 tested (interleukin-6, D-dimer, coagulation factor VIII, von Willebrand factor, and homocysteine). —   Results: The TRF model showed an improved C-statistic (0.729 vs. 0.699, p = 0.001) and net reclassification improvement (6.42%) compared with the Framingham risk score model.  The additional biomarker model showed additional improvement in the C-statistic (0.751 vs. 0.729, p = 0.001) and net reclassification improvement (6.45%) compared with the TRF model. Predicted CHD risks on a continuous scale showed high agreement between the TRF and additional biomarker models (Spearman’s coefficient = 0.918).”

The 2010 publication Multiple marker approach to risk stratification in patients with stable coronary artery disease found an interesting result for the population studied. “Aims: Multimarker approaches for risk prediction in coronary artery disease have remained inconsistent. We assessed multiple biomarkers representing distinct pathophysiological pathways in relation to cardiovascular events in stable angina. — Methods and results We investigated 12 biomarkers reflecting inflammation [C-reactive protein, growth-differentiation factor (GDF)-15, neopterin], lipid metabolism (apolipoproteins AI, B100), renal function (cystatin C, serum creatinine), and cardiovascular function and remodelling [copeptin, C-terminal-pro-endothelin-1, mid-regional-pro-adrenomedullin (MR-proADM), mid-regional-pro-atrial natriuretic peptide (MR-proANP), N-terminal-pro-B-type natriuretic peptide (Nt-proBNP)] in 1781 stable angina patients in relation to non-fatal myocardial infarction and cardiovascular death (n = 137) over 3.6 years. — Conclusion Comparative analysis of 12 biomarkers revealed Nt-proBNP, GDF-15, MR-proANP, cystatin C, and MR-proADM as the strongest predictors of cardiovascular outcome in stable angina. All five biomarkers taken separately offered incremental predictive ability over established risk factors. Combination of the single markers slightly improved model fit but did not enhance risk prediction from a clinical perspective.”

The 2010 publication Contribution of 30 Biomarkers to 10-Year Cardiovascular Risk Estimation in 2 Population Cohorts reports “Background— Cardiovascular risk estimation by novel biomarkers needs assessment in disease-free population cohorts, followed up for incident cardiovascular events, assaying the serum and plasma archived at baseline. We report results from 2 cohorts in such a continuing study. ^—  Methods and Results— Thirty novel biomarkers from different pathophysiological pathways were evaluated in 7915 men and women of the FINRISK97 population cohort with 538 incident cardiovascular events at 10 years (fatal or nonfatal coronary or stroke events), from which a biomarker score was developed and then validated in the 2551 men of the Belfast Prospective Epidemiological Study of Myocardial Infarction (PRIME) cohort (260 events). No single biomarker consistently improved risk estimation in FINRISK97 men and FINRISK97 women and the Belfast PRIME Men cohort after allowing for confounding factors; however, the strongest associations (with hazard ratio per SD in FINRISK97 men) were found for N-terminal pro-brain natriuretic peptide (1.23), C-reactive protein (1.23), B-type natriuretic peptide (1.19), and sensitive troponin I (1.18).  A biomarker score was developed from the FINRISK97 cohort with the use of regression coefficients and lasso methods, with selection of troponin I, C-reactive protein, and N-terminal pro-brain natriuretic peptide. Adding this score to a conventional risk factor model in the Belfast PRIME Men cohort validated it by improved c-statistics (P=0.004) and integrated discrimination (P<0.0001) and led to significant reclassification of individuals into risk categories (P=0.0008). — Conclusions— The addition of a biomarker score including N-terminal pro-brain natriuretic peptide, C-reactive protein, and sensitive troponin I to a conventional risk model improved 10-year risk estimation for cardiovascular events in 2 middle-aged European populations. Further validation is needed in other populations and age groups.”

The challenge of combining multiple biomarkers into reliable standardized tests is a significant one.  An editorial from the American Heart Association’s magazine Circulation addresses this issue, Separating the Contenders From the Pretenders – Competitive High-Throughput Biomarker Screening in Large Population-Based Studies.  “Despite great enthusiasm for biomarkers as tools to enhance risk prediction and to lead the way in a transformation towards personalized cardiovascular medicine, progress in the biomarker field has been painstakingly slow, particularly in the area of population screening. Some individual biomarkers such as C-reactive protein (CRP) have demonstrated consistent associations with incident cardiovascular events across multiple studies, but the magnitude of these associations is modest,¹ and only small improvements in discrimination and reclassification are seen.^2,3 One attractive solution to the limitations of individual biomarkers is to combine nonredundant biomarkers into panels to enhance risk assessment. However, results of studies testing multiple biomarkers for risk prediction in primary prevention populations have not provided a clear picture, with some studies showing qualified promise^4–6 and others suggesting limited value.^2,7,8”  — Although the study represents a qualified victory for multiple biomarker panels that include CRP, NT-proBNP, and cTnI, does it represent the end of the road for the other biomarkers that were tested and failed? More important, does the failure here and in prior studies of the more novel biomarkers suggest that biomarker discovery in this area is likely to be futile? We believe such a conclusion would be premature. — Progress forward requires movement in several directions. For the established biomarkers, further clinical validation of panels containing CRP, NT-proBNP (or BNP), and a sensitive troponin assay is required in different age, race, and sex groups, given the known influences of these factors on levels of these biomarkers. We also need carefully designed observational and interventional studies to help us understand the full implications of reclassification based on these biomarkers. In particular, it is critical to establish the safety of deferral of preventive therapies for individuals who are reclassified to a lower risk category by biomarkers. With regard to the more novel biomarkers, careful thought is needed with regard to the appropriate target populations for discovery and validation, as well as the criteria used to sort out the contenders from the pretenders. ^“

Genomic biomarkers – SNPs

A presentation at the American Heart Association meeting a couple of weeks ago reports on progress in using single nucleotide polymorphisms (SNPs) in gene sequences to strengthen the discriminatory power of existing biomarker panels for predicting CHD.  “A single-nucleotide polymorphism (SNP, pronounced snip) is a DNA sequence variation occurring when a single nucleotide — A, T, C, or G — in the genome (or other shared sequence) differs between members of a species or paired chromosomes in an individual(ref).”

As reported in Genomeweb News on November 17 2010 article Study Suggests Genetic Data May Improve Heart Attack Risk Prediction, “Incorporating genetic information into heart attack risk prediction models based on traditional risk factors can help to more accurately classify a subset of individuals, according to a team of Mayo Clinic researchers.  — In a study done through the National Human Genome Research Institute-funded Electronic Medical Records & Genomics, or eMERGE, Network, the investigators brought together information on traditional heart attack factors from medical records with data on 11 heart attack risk SNPs for nearly 1,300 individuals. — Their findings, presented at the American Heart Association Scientific Sessions meeting last night, indicate that this genetic information refined heart attack risk classifications for almost a third of those evaluated. — “This study tells us that genetic information may be helpful in screening people for their risk for having a heart attack,” Mayo Clinic cardiologist Iftikhar Kullo, who is leading the study, said in a statement. — Heart attack risk is typically determined from a set of risk factors such as age, cholesterol levels, blood pressure, smoking behavior, and more. But such factors, which are brought together in a Framingham Risk Score for predicting heart risk over a decade, don’t always classify individuals accurately.”

Going on, — “The method we have been using for decades to predict heart attack risk is not ideal,” Kullo said. “[M]any people thought to be at low risk experience a heart attack.” — In an effort to find ways to refine heart attack risk profiles, Kullo and his colleagues evaluated Framingham Risk Scores for 1,262 individuals with no history of heart disease based on their medical record data. — They also genotyped the individuals at 11 SNPs thought to be associated with heart disease using DNA isolated from the individuals’ blood samples and compared the predictive value of genetic data alone with Framingham Risk Score predictions and models that included both Framingham Risk Score and SNP information. — By incorporating the SNP information, the researchers reported, they were able to reclassify 50 of the 197 individuals from the low-risk group into an intermediate-risk group and move 86 of 397 individuals in the intermediate risk group up to a higher risk (“intermediate-high”) group. — Similarly, the team found that 54 of the 430 individuals considered intermediate-high risk belonged in the high risk category. — On the other hand, 77 intermediate risk, 79 intermediate-high risk, and 39 of 238 high risk individuals were bumped down to a lower risk category when their SNP data was added to their heart attack risk profiles. — If the findings pan out in future clinical studies, the researchers said, it may be possible to provide more accurate heart attack risk information to patients — particularly those who fall into intermediate risk categories based on traditional risk factor data. — Previous research evaluating half a dozen protein biomarkers for cardiovascular disease found only modest improvements in risk prediction when these markers were combined with traditional risk factor information.”

Summary

·        Biomarker combinations for identification of cardiovascular disease risk have been used for many years, particularly the Framingham heart study risk scores.   

·        Identification of additional predictive CVD biomarkers for has been an ongoing process for many years and several powerful newer ones have been identified starting with C-reactive protein, going on to natriuretic peptides and, more recently SNP gene variations. There appear to be well over 30 potential CVD biomarkers most of which show fairly weak associations and then in many cases only to specific cardiovascular diseases.

·        Emphasis is turning to finding specific combinations of biomarkers which offer the greatest predictive power.  The process is very slow because there are so many possible combinations, because there are several different cardiovascular diseases and because large cohorts of people must be followed for a number of years to get results.

·        While progress is slow it appears to be steady and already various studies have already suggested predictive biomarker panels that are significantly improved over the traditional ones.  Reported progress in 2010 alone appears to be significant.

Press releases and resulting newspaper articles about biomedical research can be misleading, even when they are from the most respectable institutions.  The problem is most often with what they leave out.  By ignoring a whole stream of prior research by others or parallel current research, they often give the impression that an incremental research result is a fundamental new breakthrough.  Such releases are often picked up and republished by hundreds or thousands of newspapers and other publications worldwide.  I examine one example of this kind of happening here.  And I suggest a few things to keep in mind when reading press releases or newspaper articles about research discoveries.  

The press release I will use as an example was dated yesterday and from the Harvard Medical School, not exactly a shabby organization.  The press release appeared in whole or part in a great many news publications worldwide.  Here it is as seen in Eurekaalert:  

“Rare disease reveals new path for creating stem cells  — BOSTON, Mass. (November 21, 2010)—As debilitating as disease can be, sometimes it acts as a teacher. — Researchers at Harvard Medical School and the Harvard School of Dental Medicine have found that by mimicking a rare genetic disorder in a dish, they can rewind the internal clock of a mature cell and drive it back into an adult stem-cell stage. This new “stem cell” can then branch out into a variety of differentiated cell types, both in culture and in animal models. — “This certainly has implications for personalized medicine, especially in the area of tissue engineering,” says Bjorn Olsen, the Hersey Professor of Cell Biology at Harvard Medical School and Dean of Research at the Harvard School of Dental Medicine. — These findings appear November 21, online in Nature Medicine. — Fibrodysplasia Ossificans Progressiva (FOP), which affect fewer than 1,000 people worldwide, is a horrific genetic disease in which acute inflammation causes soft tissue to morph into cartilage and bone. Over the course of a few decades, patients gradually become thoroughly ossified, as though parts of their body have turned to stone. There is no cure or treatment. — Damian Medici, an instructor of medicine at Harvard Medical School and Beth Israel Deaconess Medical Center, found that, unlike normal skeletal tissue, the pathological cartilage and bone cells from these patients contained biomarkers specific for endothelial cells—cells that line the interior of blood vessels. This led him to question whether or not the cartilage and bone growing in soft tissues of FOP patients had an endothelial origin. — Medici and his colleagues transferred the mutated gene that causes FOP into normal endothelial cells. Unexpectedly, the endothelial cells converted into a cell type nearly identical to what are called mesenchymal stem cells, or adult stem cells that can differentiate into bone, cartilage, muscle, fat, and even nerve cells. (Embryonic stem cells have the potential to become any type of cell, whereas adult stem cells are limited.) — What’s more, through further experiments the researchers found that instead of using the mutated gene to induce the transformation, they could incubate endothelial cells with either one of two specific proteins (growth factors TGF-beta2 and BMP4) whose cellular interactions mimicked the effects of the mutated gene, providing a more efficient way to reprogram the cells. — Afterwards, Medici was able to take these reprogrammed cells and, in both culture dishes and animal models, coax them into developing into a group of related tissue types. — “It’s important to clarify that these new cells are not exactly the same as mesenchymal stem cells from bone marrow,” says Medici. “There are some important differences. However, they appear to have all the potential and plasticity of mesenchymal stem cells.”  — “The power of this system is that we are simply repeating and honing a process that occurs in nature,” says Olsen. “In that sense, it’s less artificial than other current methods for reprogramming cells.” — According to study collaborator Frederick Kaplan, Isaac & Rose Nassau Professor of Orthopaedic Molecular Medicine at the University of Pennsylvania School of Medicine and a world expert on FOP, “While we want to use this knowledge to stop the renegade bone formation of FOP, these new findings provide the first glimpse of how to recruit and harness the process to build extra bone for those who desperately need it.” — Medici and Olsen echo this, stating that the most direct application for these findings is the field of tissue engineering and personalized medicine. It is conceivable that transplant patients may one day have some of their own endothelial cells extracted, reprogrammed, and then grown into the desired tissue type for implantation. Host rejection would not be an issue.”

This press release is well written and largely though not completely accurate.  However, read even carefully by a person without the right technical background, that person is likely to get certain impressions, including:

1.     The researchers discovered that endothelial cells can revert or be reverted into mesenchymal stem cells (MSCs) or adult stem cells or ones very much like them.  The researchers discovered that the clock of cell differentiation can be run backwards.

2.     The researchers discovered that reversion of endothelial cells into MSC-like cells can be accomplished by using a mutant version of the FOP gene.

3.     The researchers discovered that reversion of endothelial cells into MSC-like cells can also be accomplished using either of two proteins (growth factors TGF-beta2 and BMP4), and this was a more efficient process than using the FOP gene variant.

Of these statements, only the second one is correct.  Let’s look at some of the prior research.

It has been known for a long time that endothelial cells can be converted into MSC-like cells

The phenomenon of epithelial-mesenchymal transition has been known to exist for some time; it was not discovered as part of the new research.  It is part of developmental biology and is also observed in some cancers.  “Epithelial-mesenchymal transition or transformation (EMT) is a program of development of biological cells characterized by loss of cell adhesion, repression of E-cadherin expression, and increased cell mobility. EMT is essential for numerous developmental processes including mesoderm formation and neural tube formation(ref).”  A few of the many prior papers I have found on the topic are:

(2008) The epithelial-mesenchymal transition generates cells with properties of stem cells.  “The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. Induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits, but in addition the expression of stem-cell markers– These findings illustrate a direct link between the EMT and the gain of epithelial stem-cell properties.” 

(2008) Molecular signature and therapeutic perspective of the epithelial-to-mesenchymal transitions in epithelial cancers

(2004) NF-kappaB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression

(1996) Epithelial-mesenchymal transitions in cancer progression 

(1995) Epithelial-mesenchymal transitions in development and tumor progression

EMT has been around and studied as seen in these and many other publications since before the mid 90s and statement #1 above is plain false.  Also of course, the whole field of induced pluripotent stem cells (iPSCs) I have written about so often is directly concerned with running the clock of cell differentiation backward.  In that field, the reported research is a teaspoon full that is poured into a 50 gallon drum of existing research(ref). 

The capabilities of the growth factors TGF-beta2 and BMP4 to induce epithelial-mesenchymal transition have been known for some time

The 2005 paper Transforming Growth Factor-β Signaling during Epithelial-Mesenchymal Transformation: Implications for Embryogenesis and Tumor Metastasis  states “–However, more recent studies have implicated a significant role of the transforming growth factor-β (TGF-β) in causing EMT in both development and pathology.”

The 2004 paper Endogenous TGF-beta signaling suppresses maturation of osteoblastic mesenchymal cells also deals with the relationship of the two growth factors to mesenchymal cells.  “Thus, signaling cross-talk between BMP and TGF- pathways plays a crucial role in the regulation of osteoblastic differentiation – “

TheNovember 2010 paper Low Doses of Bone Morphogenetic Protein 4 Increase the Survival of Human Adipose-Derived Stem Cells Maintaining Their Stemness and Multipotency is another current study relating BMP4 to stem cell maintenance instead of differentiation.  “Our results therefore support BMP4 as a promising factor for expanding human adipose tissue-derived MSCs maintaining their properties of stemness and multipotency.”

The 2008 paper BMP4 induces an epithelial-mesenchymal transition-like response in adult airway epithelial cells reports “We conclude that the activity of BMP4 in EMT during development is recapitulated in adult airway epithelial cells and suggest that this activity may contribute to inflammation and fibrosis in vivo.”

So, statement #3 above is also untrue “The researchers discovered that reversion of endothelial cells into MSC-like cells can also be accomplished using either of two proteins (growth factors TGF-beta2 and BMP4).”  This was well known beforehand. 

In all fairness, the paper described in the press release did make significant incremental contributions to existing knowledge:

a.     The EMT process, well established in biology, can be practically harnessed to create mesenchymal-type adult stem cells.

b.     This can be accomplished by using a mutant version of the FOP gene.

c.     The authors demonstrated in the laboratory that a more practical approach is to use either TGF-beta2 or BMP4.

My problem is with the press release.  I have no dispute with the content of the actual publication in question Conversion of vascular endothelial cells into multipotent stem-like cells.  “Mesenchymal stem cells can give rise to several cell types, but varying results depending on isolation methods and tissue source have led to controversies about their usefulness in clinical medicine. Here we show that vascular endothelial cells can transform into multipotent stem-like cells by an activin-like kinase-2 (ALK2) receptor–dependent mechanism. In lesions from individuals with fibrodysplasia ossificans progressiva (FOP), a disease in which heterotopic ossification occurs as a result of activating ALK2 mutations, or from transgenic mice expressing constitutively active ALK2, chondrocytes and osteoblasts expressed endothelial markers. Lineage tracing of heterotopic ossification in mice using a Tie2-Cre construct also suggested an endothelial origin of these cell types. Expression of constitutively active ALK2 in endothelial cells caused endothelial-to-mesenchymal transition and acquisition of a stem cell–like phenotype. Similar results were obtained by treatment of untransfected endothelial cells with the ligands transforming growth factor-β2 (TGF-β2) or bone morphogenetic protein-4 (BMP4) in an ALK2-dependent manner. These stem-like cells could be triggered to differentiate into osteoblasts, chondrocytes or adipocytes. We suggest that conversion of endothelial cells to stem-like cells may provide a new approach to tissue engineering.”

This research, though possibly important, is far from the bottom line when it comes to practical approaches for reverting ordinary body cells into stem cells.  See Past blog postings on stem cells and epigenomics for blog entries on many other research efforts aimed at reverting normal cells into stem cells.  New research findings related to this subject are now being reported in the literature practically every week.

About reading research press releases or newspaper articles

·        Press releases and newspaper articles are prone to building up incremental research findings so they sound like basic breakthroughs.  When you see a headline like “Fountain of youth – University of Gokomursk scientists discover how to turn the clock back on skin cells to make stem cells,” be aware that 20 other research groups may have already found ways to do the same thing.  The writers don’t like all the nitpicking caveats that the scientists themselves would give.  University PR people want to make their institutions look good and newspaper writers like to make their articles simple.

·        When reading press releases, particularly ones trumpeting research breakthroughs, be aware of what is left out, perhaps a rich history of past research, perhaps that the discovery being reported is not unique.  Perhaps the research being reported is just another large stone in a large edifice still being built.  Perhaps it is only a tiny stone.  Almost all key discoveries are based on highly-related past discoveries. 

·        Don’t assume that everything said is accurate.  Most press releases and newspaper articles are written by professional writers, people who might not have a good grasp of a technical subject.

·        To find the strait scoop on the research, when the press release or newspaper article is triggered by a research publication, go directly to the research publication.

Having said these things press releases and newspaper articles are often very useful because when responsibly written:

·        they might convey interesting otherwise-unpublished information based on interviews with the scientists who did the work; they may include fascinating quotes and personal opinions;

·        they can explain highly technical developments in plain language understandable to all.

For these reasons, I will continue occasionally to quote from press releases or newspaper articles.  However, I will continue to rely for information primarily on the published scientific research literature, on direct interactions with researchers, and on presentations I have seen at respectable research conferences.

Many interesting studies have been conducted in the field of sleep medicine. You can check out the journal Sleep Medicine and the website of the American Academy of Sleep Medicine. However, the exact roles of sleep and the relationship of sleep to longevity are not well understood. Most studies in the literature appear to have to do with sleep deprivation, sleep disorders like insomnia or restless legs syndrome or the relationship of sleep to certain diseases like diabetes and immune system disorders. This blog entry offers a brief general introduction to the roles of sleep and then focuses on a few studies that relate amount of sleep to general health and longevity.

Roles of sleep

Sleep is a very multifaceted subject. A number of interesting talks from a 2007 conference on sleep at the Salk Institute, Waking up to Sleep can be found here. Particularly, Jerry Siegel’s talk provides a general introduction to the topic. In many species of animals, including ourselves, dogs, cats and primates, evolution has given a high priority to sleeping, so it must fulfill important survival needs. Whatever sleep does, it is known that prolonged sleep deprivation or abnormal sleep can have serious consequences. In rats, prolonged sleep deprivation can lead to a physical damage including skin lesions, subcortical damage, weight loss, blood plasma changes and slowed wound healing(ref). In humans, sleep deprivation appears to be associated with decreased cognitive function(ref), psychotic-like states(ref) and other negative situations reported below.

About 20 species are known to sleep like we do, a small percentage of those out there. And whether or how animals sleep in thousands of other species has not been systematically studied.

Sleep is complicated, typically consisting of several phases. “REM (rapid eye movement) sleep in adult humans typically occupies 20–25% of total sleep, about 90–120 minutes of a night’s sleep. During a normal night of sleep, humans usually experience about four or five periods of REM sleep; they are quite short at the beginning of the night and longer toward the end. Many animals and some people tend to wake, or experience a period of very light sleep, for a short time immediately after a bout of REM. The relative amount of REM sleep varies considerably with age. A newborn baby spends more than 80% of total sleep time in REM.^[2] During REM, the activity of the brain’s neurons is quite similar to that during waking hours, but the body is paralyzed due to atonia; for this reason, the REM-sleep stage may be called paradoxical sleep.^[3] This means there are no dominating brain waves during REM sleep. — REM sleep is physiologically different from the other phases of sleep, which are collectively referred to as non-REM sleep (NREM). Vividly recalled dreams mostly occur during REM sleep(ref).” REM sleep is a state in which the brain stem is highly activated, brain metabolism is high and in which twitching and male erections sometimes occur. The brain is doing some kind of work during REM sleep. REM sleep does not occur in fish, reptiles and insects but does appear to occur in some mammal species in addition to humans.

Needs for sleep and optimal sleep patterns may vary from individual to individual. “Sleep is a complex phenotype and as such it is possible that there are numerous genes which may each have a number of effects that control an individual’s sleep pattern(ref).” Truthfully, there is very much we don’t know about it.

The various roles of sleep in normal and pathological conditions is the subject of much study and discussion but is relatively ill-understood. One role of sleep that is fairly well agreed-on is memory consolidation(ref). “– converging evidence, from the molecular to the phenomenological, leaves little doubt that offline memory reprocessing during sleep is an important component of how our memories are formed and ultimately shaped(ref).” However details of this process and exactly what happens during sleep remain murky. “–the term ‘‘memory consolidation’’ refers to a poorly defined set of processes which take an initial, unstable memory representation and convert it into a form that is both more stable and more effective. At this time, it is unclear how memories are altered after initial encoding, and no consensus as to which of the processes contributing to this alteration should be included under the umbrella of memory consolidation(ref).

Sleep – how many hours are best?

Many studies have sought to correlate the average number of hours spent daily in sleeping with mortality for individuals in various populations and in different age and gender categories. The studies point to somewhat inconsistent but interesting results.

Recent population studies

To begin, the 2010 publication Sleep Duration and All-Cause Mortality: A Systematic Review and Meta-Analysis of Prospective Studies analyzes the results of 16 studies which included “– 27 independent cohort samples. They included 1,382,999 male and female participants (follow-up range 4 to 25 years), and 112,566 deaths. Sleep duration was assessed by questionnaire and outcome through death certification. In the pooled analysis, short duration of sleep was associated with a greater risk of death (RR: 1.12; 95% CI 1.06 to 1.18; P < 0. 01) with no evidence of publication bias (P = 0.74) but heterogeneity between studies (P = 0.02). Long duration of sleep was also associated with a greater risk of death (1.30; [1.22 to 1.38]; P < 0.0001) with no evidence of publication bias (P = 0.18) but significant heterogeneity between studies (P < 0.0001).”

— “Short duration of sleep was defined differently in different studies. It: “was ≤5 h per night,^6,7,20,22 ≤6 h,^10,19,23,25 <7 h,^{21,24,26–29} ≤4 h.^9” The studies likewise defined long duration of sleep differently > 8 h per night,^10,19,24,30 ≥9 h,^{6,7,20,22,23,25–28} ≥10 h,^9,21 and ≥ 12h.²⁹  And “the reference category, being 7 h per night in the majority of studies,^{6,7,9,19–22} 7 to 8 h,^23–26 7 to 9 h,^27,28 6 to 8 h,¹⁰ and 9 h.²⁹” Therefore, since “short” and “long” are variably-defined, the exact meaning of the conclusion of this meta study is not clear “Both short and long duration of sleep are significant predictors of death in prospective population studies.” I would take it to mean something like “Less than 5.5 hours of sleep per night or more than 9 hours are significant predictors of death in prospective population studies”

A new study reported in an October issue of Science Daily, Women’s Study Finds Longevity Means Getting Just Enough Sleep contributes another piece to the puzzle that may not exactly fit with the rest. “A new study, derived from novel sleep research conducted by University of California, San Diego researchers 14 years earlier, suggests that the secret to a long life may come with just enough sleep. Less than five hours a night is probably not enough; eight hours is probably too much. — A team of scientists, headed by Daniel F. Kripke, MD, professor emeritus of psychiatry at UC San Diego School of Medicine, revisited original research conducted between 1995 and 1999. In that earlier study, part of the Women’s Health Initiative, Kripke and colleagues had monitored 459 women living in San Diego (ranging in age from 50 to 81) to determine if sleep duration could be associated with mortality.”

“Fourteen years later, they returned to see who was still alive and well. — Of the original participants, 444 were located and evaluated. Eighty-six women had died. Previous studies, based upon questionnaires of people’s sleep habits, had posited that sleeping 6.5 to 7.5 hours per night was associated with best survival. Kripke and colleagues, whose 1990s research had used wrist activity monitors to record sleep durations, essentially confirmed those findings, but with a twist. — “The surprise was that when sleep was measured objectively, the best survival was observed among women who slept 5 to 6.5 hours,” Kripke said. “Women who slept less than five hours a night or more than 6.5 hours were less likely to be alive at the 14-year follow-up. — Kripke said the study should allay some people’s fears that they’re not getting enough sleep. “This means that women who sleep as little as five to six-and-a-half hours have nothing to worry about since that amount of sleep is evidently consistent with excellent survival. That is actually about the average measured sleep duration for San Diego women.” 

This UC study has a consistent result with the above-mentioned 2010 metastudy Sleep Duration and All-Cause Mortality: A Systematic Review and Meta-Analysis of Prospective Studies in that too much or too little sleep increases mortality. However, the amount of nightly sleep corresponding to the lowest mortality – 5 to 6.5 hours – seems to be surprisingly low in this UC study when compared to the least mortality ranges in the meta-study or when compared to other studies or conventional wisdom. 

A large study in China is reported in the 2010 publication Sociodemographic and health correlates of sleep quality and duration among very old Chinese. “STUDY OBJECTIVES: To examine factors associated with self-reported sleep quality and duration among very old adults in China. — DESIGN: Cross-sectional analysis of the 2005 wave of the Chinese Longitudinal Healthy Longevity Survey (CLHLS). — SETTING: In-home interview with older adults in 22 provinces in mainland China. — PARTICIPANTS: A total of 15,638 individuals aged 65 and older (5,047 aged 65-79, 3870 aged 80-89, 3927 aged 90-99, and 2794 aged 100 and older, including 6688 men and 8950 women). — Sixty-five per cent of Chinese elders reported good quality of sleep. The average number of self-reported hours of sleep was 7.5 (SD 1.9), with 13.1%, 16.2%, 18.0%, 28.0%, 9.2%, and 15.5% reporting < or = 5, 6, 7, 8, 9, and > or =10 hours, respectively (weighted). Multivariate analyses showed that male gender, rural residence, Han ethnicity, higher socioeconomic status, and good health conditions were positively associated with good quality of sleep. All other factors being equal, octogenarians, nonagenarians, and centenarians were more likely to have good sleep quality than the young elders aged 65-79. Elders with poorer health status or older age were more likely to have either relatively shorter (< or = 6 h) or longer (> or = 10 h) sleep duration. Married elders were more likely to have an average duration between these two values. Except for some geographic variations, associations between all other factors and sleep duration were weak compared to the effects of health. — CONCLUSIONS: Age and health conditions are the two most important factors associated with self-reported sleep quality and duration. Good quality of sleep among long-lived old adults may have some implications for achieving healthy longevity.”

A series of recent news reports are appearing sharing some of the new findings, like The perils of too much sleep. “10 Hours Or More May Increase Stroke Risk, Researchers Say. — Women who get more than 10 hours of sleep a night may increase their risk of incident stroke, researchers said here at the American Heart Association meeting. Additionally, women who had six or less hours of sleep did not have an associated increased stroke risk, Dr. Alan Flint of the Harvard School of Public Health reported. — Flint and colleagues performed a prospective cohort study of 69,794 female nurses ages 40 to 65, measuring self-reported sleep data from 1986 to 2006 to an endpoint of fatal or nonfatal stroke. — Patients were asked to report total hours of actual sleep — ranging from less than five to 11 or more — as well as any confounding factors, such as alcohol intake, fruit and vegetable consumption, physical activity, and smoking status. Body mass index and the presence of diabetes or hypertension were recorded as potential intermediary factors. — At the 20-year follow-up, a total of 2,303 strokes were reported. After adjusting for confounders, Flint and colleagues found those who had slept 10 or more hours a night had a 63 percent increased risk of stroke compared with a baseline average risk with seven hours a night of sleep.

Comparatively, patients who slept six or fewer, or from eight to nine hours a night, had insignificant increases in stroke risk after adjusting for confounders, when compared with baseline.   Researchers were unable to determine any of the underlying biological mechanisms that may cause the increased risk in patients with higher sleep duration. “We do find that identifying long sleep duration is useful in marking risk, although it doesn’t immediately lead to any clinical recommendation,” Flint told MedPage Today. Future research should investigate the possible causes of the increased risk of stroke in women, he said. “We’d like to update [the study] and get an idea of whether a pattern of sleep over a lifetime that accounts for the risk, or whether there are other factors that account for that, like clinical depression, jobs, family, or other interaction with that risk.”

Correlations of sleep lengths and quality with pathological conditions

A November 15 news release Poor Sleep Quality Increases Inflammation, Community Study Finds reports “People who sleep poorly or do not get enough sleep have higher levels of inflammation, a risk factor for heart disease and stroke, researchers have found. — Data from a recent study were presented Sunday, Nov. 14 at the American Heart Association Scientific Sessions in Chicago by Alanna Morris, MD, a cardiology fellow at Emory University School of Medicine. –The results come from surveying 525 middle-aged people participating in the Morehouse-Emory Partnership to Eliminate Cardiovascular Health Disparities (META-Health) study on their sleep quality and sleep duration. — Acute sleep deprivation leads to an increased production of inflammatory hormones and changes in blood vessel function, but more research is needed on the physiological effects of chronic lack of sleep, Morris says.– “Most of the studies looking at the body’s response to lack of sleep have looked at subjects who have been acutely sleep deprived for more than 24 hours in experimental sleep laboratories,” she says. “Nothing of this sort has been investigated in epidemiologic studies.” — In the META-Health study, the researchers assessed sleep quality using the Pittsburgh Sleep Quality Index survey, where a score over six (based on the median sleep score of the study population) is considered poor. They also analyzed their data based on hours of sleep. –Individuals who reported six or fewer hours of sleep had higher levels of three inflammatory markers: fibrinogen, IL-6 and C-reactive protein. In particular, average C-reactive protein levels were about 25 percent higher (2 milligrams per liter compared to 1.6) in people who reported fewer than six hours of sleep, compared to those reporting between six and nine hours.– That difference was still significant even when the data is corrected for known risk factors such as smoking, blood pressure, diabetes and obesity, Morris says. –“

“For people who got little sleep, the C-reactive protein levels were increased, but still in the range of what health authorities would consider low to intermediate risk,” she says. “However, our study population represents a community-based population [as opposed to patients in the hospital or with known cardiovascular disease], so they have overall lower risk and lower C-reactive protein levels than many of the high risk populations in other studies.” — Inflammation may be one way poor sleep quality increases the risks for heart disease and stroke, Morris says.– “It remains uncertain whether short sleep duration contributes directly to cardiovascular mortality, or whether it is a mediating or moderating factor,” she says.–Previous research has shown that people who sleep between seven and eight hours per night live longest, and that especially short or especially long sleep durations bring higher mortality. Researchers find that short and long sleep durations are often seen together with high blood pressure, obesity, diabetes and psychological stress – all risk factors for heart disease and stroke. — Long sleep duration may reflect a compensation for sleep apnea, which the sleep quality survey does not directly address. However, in the META-Health study, people who slept for more than nine hours didn’t show significantly higher levels of inflammation markers. — In a separate poster, Morris is also presenting research on a difference between men and women in the interaction between sleep quality and arterial stiffness. Her results show that both men and women with poor sleep quality had higher blood pressures, but only men with poor sleep quality had a higher arterial stiffness, a lack of blood vessel flexibility which drives hypertension and puts more burden on the heart.”

I point out that in this study and others, correlations between sleep quality and sleep lengths with health factors like inflammation and presence of C-reactive protein do not establish causation in either direction.

The relationship between abnormal sleep durations and potential cardiovascular and other problems has been noted before, for example in the 2007 publication Review: Inflammation, sleep, obesity and cardiovascular disease. “Sleep loss can also have consequences on safety related behaviours both for the individuals and for the society, for example the increased risk of accidents when driving while drowsy. In this case there might appear to be a causal chain. Accidents don’t create prior sleep loss and drowsiness. However, other factors besides sleep loss could lead to both lack of sleep and accident proneness, such as intense emotional stress.

While conventional wisdom holds that amount of sleep is correlated with cognitive functioning, this may not necessarily be the case. For example the 2006 publication Poor sleep is associated with impaired cognitive function in older women: the study of osteoporotic fractures concludes “Objectively measured disturbed sleep was consistently related to poorer cognition, whereas total sleep time was not. This finding may suggest that it is disturbance of sleep rather than quantity that affects cognition.” “Results are from 2932 women (mean age 83.5 years) in the Study of Osteoporotic Fractures between 2002 and 2004. Cognitive function was measured by Mini-Mental State Examination (MMSE) and Trail Making B Test (Trails B). Cognitive impairment was defined as MMSE < 26 or Trails B > 278 seconds. Sleep parameters measured objectively using actigraphy included total sleep time, sleep efficiency, sleep latency, wake after sleep onset (WASO), and total nap time.”

Inadequate sleep has also been thought to affect glucose metabolism and raise the risk for diabetes. Example publications addressing these issues are the 2009 publication Do sleep disorders and associated treatments impact glucose metabolism?, the 2008 report The reciprocal interaction between sleep and type 2 diabetes mellitus: facts and perspectives and the 2005 study Sleep loss and the development of diabetes: a review of current evidence. “Emerging evidence suggests that short duration of sleep and sleep disturbances increase the risk of developing diabetes.”

The association of too-short or abnormal sleep with overproduction of inflammatory cytokines is a repeated theme in the research literature. For example, the 2005 publication Experimental studies on the interaction between sleep and the immune system in humans reports on the causative effect of inflammatory cytokines on sleep. “Sleep-wake behavior is very sensitive to experimental host defense activation, for example, by bacterial endotoxin. When the injection of endotoxin is accompanied by fever and a prominent neuroendocrine activation, sleep continuity will be disturbed. When the production of inflammatory cytokines is stimulated by smaller amounts of endotoxin, but no fever and no neuroendocrine activation are apparent, the nonREM-sleep amount will increase. This is possibly due to changes in the biological activity of the tumor necrosis factor-alpha (TNF-alpha) system. Besides their important function in sleep regulation during acute immune response, cytokines also seem to be involved in physiological sleep regulation, although there still is not very much data on this issue.”

In fact, the communication between the brain and the immune system with respect to sleep seems to flow both ways as pointed out in the 2006 report Bidirectional communication between the brain and the immune system: implications for physiological sleep and disorders with disrupted sleep.

The 2008 publication Sleep, insomnia and falls in elderly patients Points out the difficulty of interpreting sleep data. Do old patients who have insomnia fall and experience fractures because of lack of sleep or because of their medications? “Insomnia is common in older people and can be associated with significant daytime dysfunction. Sleep problems, and the medications used to treat them, may contribute to the risk of falls and fractures in this population; however, the independent effects of disturbed sleep or the risk of hypnotic use are not well understood.”

The 2008 study report Actigraphy-measured sleep characteristics and risk of falls in older women comes up with a somewhat clear result. “METHODS: Study subjects were participants in the Study of Osteoporotic Fractures. In 2978 primarily community-dwelling women 70 years and older (mean age, 84 years), sleep and daytime inactivity were estimated using wrist actigraphy data collected for a minimum of 3 consecutive 24-hour periods (mean duration, 86.3 hours). Fall frequency during the subsequent year was ascertained by a triannual questionnaire. Use of medications was obtained by examiner interview. — RESULTS: In multivariate-adjusted models, relative to those with “normal” nighttime sleep duration (>7 to 8 hours per night), the odds of having 2 or more falls in the subsequent year was elevated for women who slept 5 hours or less per night (odds ratio, 1.52; 95% confidence interval, 1.03-2.24). This association was not explained by the use of benzodiazepines. Indexes of sleep fragmentation were also associated with an increased risk of falls. For example, women with poor sleep efficiency (<70% of time in bed spent sleeping) had 1.36-fold increased odds of falling compared with others (odds ratio, 1.36; 95% confidence interval, 1.07-1.74).” In this study 5 hours or less of sleep per night correlated with increased risk for falls, while the new UC study described above suggests that the lowest mortality for women is associated with 5 – 6.5 hours of sleep.

These are but a sample of a large collection of publications implicating disease and pathology associations with abnormal sleep conditions. For purposes here, the main take-away message is that a number of pathological conditions are associated with insufficient or disturbed sleep. Many researchers are content to see this as an association. Other researchers see a causal connection. They see   insufficient or disturbed sleep as causing a pathological condition.

Diet and sleep

Diet too seems to be a factor with respect to sleep. The 2009 publication Relationships among dietary nutrients and subjective sleep, objective sleep, and napping in women particularly implicates the intake of dietary fats with shortening of sleep time. “Participants were 459 post-menopausal women enrolled in the Women’s Health Initiative. Objective sleep was estimated using one week of actigraphy. Subjective sleep was prospectively estimated with a daily sleep diary. — CONCLUSIONS: Actigraphic total sleep time was negatively associated with intake of fats. Subjective napping, which may be a proxy for subjective sleepiness, was significantly related to fat intake as well as intake of meat.”

There is probably much more that can be said about diet and sleep and I will possibly take that topic up in another blog post.

No causative effects determined

Note that in all of these studies there are correlations of lengths of sleep time or quality of sleep with mortality or pathological conditions, but not causative links. For example, yes “those who had slept 10 or more hours a night had a 63 percent increased risk of stroke compared with a baseline average risk with seven hours a night of sleep.” But the long sleeping times may not have caused the increased risk of stroke. Some underlying illnesses may have both induced the longer sleeping times and the increased risk of strokes. Yet, some researchers have inferred the presence of causation which seems sensible to them.

Bottom lines

What I get from all of the above is:

·       Much is yet to be learned about the relationships between sleep, health and longevity.

·       Multiple population studies indicate longevity is correlated with not too-much and not too-little sleep. However the studies are inconsistent in defining “too much” and “too little” sleep. Somewhere between 5.5 and 7 hours of sleep a night is the average sweet-spot depending on the study.

·       Multiple pathological states appear to be correlated with too-little or too-much sleep. Again, the definitions of too-little or too-much sleep vary according to the study.

·       The population studies establish only correlations, not that amount of sleep increases or decreases mortality or any health risk. Certain sicknesses could lead to health hazards and at the same time either interfere with sleep or lead to prolonged sleeping.

·       However some researchers infer that there is a causal connection where sleep duration and quality is a determinant of longevity or health conditions.

·       The population studies provide averages but do not establish that “one size fits all” with respect to sleeping durations. Optimum length of nightly sleeping time is likely to vary among individuals.

·       Personally, I typically enjoy between 7.5 and 9 hours of sleep a night, relatively long times for my age group. According to some studies this is fine. According to other studies so much sleep puts me in a higher-mortality risk category.

Whether a variant of the longevity proposal in the recent blog post Closing the loop in the stem cell supply chain – presented graphically will come to fruition will depend critically on research progress related to pluripotent stem cells (iPSCs) and adult stem cells.  This post provides a listing of earlier blog posts covering topics in stem cell research and epigenetics broadly relevant to that longevity proposal.

The posts are listed in reverse order of date.

·        Interesting recent stem cell research (November 2010)

·        A breakthrough in producing high-fidelity induced pluripotent stem cells (October 2010)

·        Induced pluripotent stem cells – developments on the road to big-time utilization (July 2010)

·        A near-term application for iPSCs – making cell lines for drug testing (June 2010)

·        Induced pluripotent stem cells – second-rate stem cells so far (April 2010)

·        DNA Methyltransferases, stem cell proliferation and differentiation (April 2010)

·        Epigenetics going mainstream (February 2010)

·        IPSCs, telomerase, and closing the loop in the stem cell supply chain (February 2010)

·        Direct cell reprogramming (January 2010)

·        Important new mesenchymal stem cell therapies (January 2010)

·        Progress in closing the stem cell supply chain loop (January 2010)

·        DNA demethylation (November 2009)

·        It’s a long  way to stem cell treatment (Novenber 2009)

·        “Footprint-free” iPSCs – and a crazy wager offer (October 2009)

·        Homicide by DNA methylation (October 2009)

·        Who is doing gene reprogramming? (October 2009)

·        The stem cell supply chain – closing the loop for very long lives (September 2009)

·        An emerging new view of aging – the stem cell supply chain (August 2009) 

·        Treating genetic diseases with corrected induced pluripotent stem cells (August 2009)

·        Research evidence for the Decline In Adult Stem Cell Differentiation theory of aging (July 2009)

·        Hair stem cells (July 2009)

·        On cancer stem cells (July 2009)

·        Embryonic Stem cell research news (July 2009)

·        Dental Pulp Stem Cells – the big needle vs the tooth fairy (June 2009)

·        Update on induced pluripotent stem cells (June 2009)

·        Inflammation, cancer and stem cells in autoimmune diseases (June 2009)

·        Simple but powerful non-invasive adult stem cell cures (June 2009)

·        Epigenomic complexity (June 2009)

·        Histone acetylase and deacetylase inhibitors (May 2009)

·        State of autologous stem cell therapies (May 2009)

·        Trojan-horse stem cells might offer an important new cancer therapy (May 2009)

·        Gene therapy for fruit flies with Parkinson’s Disease (May 2009)

·        The new omics and longevity research (April 2009)

·        Rebooting cells and longevity (March 2009)

·        DNA methylation, personalized medicine and longevity (March 2009)

·        Epigenetics, epigenomics and aging (February 2009)

·        Protein origami and aging (February 2009)

·        Stem cell differentiation and nanotubes (February 2009)

Also relevant are discussions of theories of aging in my treatise:

·        Stem Cell Supply Chain Breakdown

·        Programmed Epigenomic Changes

If you want to learn more you can review stem cell basics here.

Of the hundreds of publications in the last year relating to stem cells not already reviewed in earlier blog entries, I have selected a few that are particularly interesting for inclusion here.  I start out with three publications that appeared only a few days ago.

Prevention of muscle aging by adult stem cell transplantation

A brand-new publication (November 10 2010) reports an interesting and exciting result Prevention of Muscle Aging by Myofiber-Associated Satellite Cell Transplantation. “We demonstrate that engraftment of myofiber-associated satellite cells, coupled with an induced muscle injury, markedly alters the environment of young adult host muscle, eliciting a near-lifelong enhancement in muscle mass, stem cell number, and force generation. The abrogation of age-related atrophy appears to arise from an increased regenerative capacity of the donor stem cells, which expand to occupy both myonuclei in myofibers and the satellite cell niche. Further, these cells have extensive self-renewal capabilities, as demonstrated by serial transplantation. These near-lifelong, physiological changes suggest an approach for the amelioration of muscle atrophy and diminished function that arise with aging through myofiber-associated satellite cell transplantation.”

Science news reports on the same study in more detail: “The experiments showed that when young host mice with limb muscle injuries were injected with muscle stem cells from young donor mice, the cells not only repaired the injury within days, they caused the treated muscle to double in mass and sustain itself through the lifetime of the transplanted mice. “This was a very exciting and unexpected result,” said Professor Bradley Olwin of CU-Boulder’s molecular, cellular and developmental biology department, the study’s corresponding author. — Muscle stem cells are found within populations of “satellite” cells located between muscle fibers and surrounding connective tissue and are responsible for the repair and maintenance of skeletal muscles, said Olwin. The researchers transplanted between 10 and 50 stem cells along with attached myofibers — which are individual skeletal muscle cells — from the donor mice into the host mice. — “We found that the transplanted stem cells are permanently altered and reduce the aging of the transplanted muscle, maintaining strength and mass,” said Olwin.”

Continuing “ Olwin said the new findings, while intriguing, are only the first in discovering how such research might someday be applicable to human health. —  In healthy skeletal muscle tissue, the population of satellite stem cells is constantly maintained, said Olwin. — “In this study, the hallmarks we see with the aging of muscles just weren’t occurring,” said Olwin. “The transplanted material seemed to kick the stem cells to a high gear for self-renewal, essentially taking over the production of muscle cells. But the team found that when transplanted stem cells and associated myofibers were injected to healthy mouse limb muscles, there was no discernable evidence for muscle mass growth. — “The environment that the stem cells are injected into is very important, because when it tells the cells there is an injury, they respond in a unique way,” he said. “We don’t yet know why the cells we transplanted are not responding to the environment around them in the way that the cells that are already there respond. It’s fascinating, and something we need to understand.” — At the onset of the experiments the research team thought the increase in muscle mass of the transplanted mice with injured legs would dissipate within a few months. Instead, the cells underwent a 50 percent increase in mass and a 170 percent increase in size and remained elevated through the lifetime of the mice — roughly two years, said Olwin.”

I find this result fascinating, a) because the positive changes in muscle mass and strength  induced by the stem transplantation seemed to be permanent and lifelong and, it appeared, free from aging, b) the cell-donor mice had to be young, c) the process would not work unless the recipient mice had muscle injuries.  Because of the similarity of human and mouse muscle biology and stem cell biology, I think the process will quite possibly work the same in humans.  Olwin is quoted as saying “With further research we may one day be able to greatly resist the loss of muscle mass, size and strength in humans that accompanies aging, as well as chronic degenerative diseases like muscular dystrophy.”  I agree with him.

Adult stem cells populations depend on nutrient availability

There is another new (November 2010) study that offers a new perspective on the behavior of adult stem cells in their niches.  The study is based on work with fruit flies.  While the applicability of its results to humans requires further research, it is possible that the pathways involved are evolutionarily conserved.  The relevant publication is Stem Cell Dynamics in Response to Nutrient Availability.  “When nutrient availability becomes limited, animals must actively adjust their metabolism to allocate limited resources and maintain tissue homeostasis [1-3]. However, it is poorly understood how tissues maintained by adult stem cells respond to chronic changes in metabolism. To begin to address this question, we fed flies a diet lacking protein (protein starvation) and assayed both germline and intestinal stem cells. Our results revealed a decrease in stem cell proliferation and a reduction in stem cell number; however, a small pool of active stem cells remained. Upon refeeding, stem cell number increased dramatically, indicating that the remaining stem cells are competent to respond quickly to changes in nutritional status. Stem cell maintenance is critically dependent upon intrinsic and extrinsic factors that act to regulate stem cell behavior [4]. Activation of the insulin/IGF signaling pathway in stem cells and adjacent support cells in the germline was sufficient to suppress stem cell loss during starvation. Therefore, our data indicate that stem cells can directly sense changes in the systemic environment to coordinate their behavior with the nutritional status of the animal, providing a paradigm for maintaining tissue homeostasis under metabolic stress.”

According to a Salk Institute press release as reported in Science News: “When the researchers fed their flies a “poor,” proteinless diet, the levels of circulating insulin-like peptides plummeted, the testes of starved flies became progressively thinner, and stem cell numbers started to decline. Upon re-feeding, insulin-like peptide expression and stem cell numbers recovered quickly. “We found that in starved flies there are fewer stem cells and they divide slower,” says postdoctoral researcher and co-first author Lei Wang, Ph.D. “However, a small pool of active stem cells remained even after prolonged starvation.” — Since germline stem cells are the only cells capable of passing genetic information on to the next generation, the researchers suspected that unique strategies might have been adapted during evolution to protect these stem cells from temporary environmental changes. However, as they discovered, a similar response to protein starvation and re-feeding was demonstrated by another stem cell population—intestinal stem cells present in the midgut. This suggests that the coordination of stem cell maintenance in response to environmental changes represents a conserved strategy utilized across multiple tissues.”

Getting iPSCs to differentiate into desired somatic cell types

The November 2010 publication Differentiation of Functional Cells from iPS Cells by Efficient Gene Transfer discusses how an andovirus vector can be used to transduce transgenes into mouse iPS cells to get those cells to differentiate efficiently into desired somatic cell types, particularly demonstrating differentiation into adipocytes or osteoblasts.   The publication reports “Although establishment of an efficient gene transfer system for iPS cells is considered to be essential for differentiating them into functional cells, the detailed transduction characteristics of iPS cells have not been examined. By using an adenovirus (Ad) vector containing the cytomegalovirus enhancer/beta-actin (CA) promoters, we have developed an efficient transduction system for mouse mesenchymal stem cells and embryonic stem (ES) cells. Also, we applied our transduction system to mouse iPS cells and investigated whether efficient differentiation could be achieved by Ad vector-mediated transduction of a functional gene. As in the case of ES cells, the Ad vector could efficiently transduce transgenes into mouse iPS cells. We found that the CA promoter had potent transduction ability in iPS cells. Moreover, exogenous expression of a PPARγ gene or a Runx2 gene into mouse iPS cells by an optimized Ad vector enhanced adipocyte or osteoblast differentiation, respectively. These results suggest that Ad vector-mediated transient transduction is sufficient to promote cellular differentiation and that our transduction methods would be useful for therapeutic applications based on iPS cells.” 

At the nanoscale level, embryonic stem cells and induced pluripotent stem cells appear to be the same

A May 2010 study Induced pluripotent stem cells at nanoscale reports “Reprogramming of mouse and human somatic cells into induced pluripotent stem (iPS) cells has been made possible with the expression of the transcription factor quartet Oct4, Sox2, c-Myc, and Klf4. Here, we compared iPS cells derived from mouse embryonic fibroblasts with the 4 factors to embryonic stem cells by electron microscopy. Both cell types are almost indistinguishable at the ultrastructural level, providing further evidence for the similarity of these 2 pluripotent stem cell populations.”

iPSCs can themselves reprogram other body cells into iPSC status

Induced pluripotent stem cells (iPSCs) are normally made by exposing normal somatic (body) cells to various combinations of transcription factors in a lengthily laboratory procedure.  But does an iPSC have the power within itself to reprogram normal body cells into iPSC status simply by contact?  The answer appears to be yes.  The February 2010 publication  Reprogramming of somatic cells after fusion with induced pluripotent stem cells and nuclear transfer embryonic stem cells reports “In this study we examine whether a somatic cell, once returned to a pluripotent state, gains the ability to reprogram other somatic cells. We reprogrammed mouse embryonic fibroblasts by viral induction of oct4, sox2, c-myc, and klf-4 genes. Upon fusion of the resulting iPS cells with somatic cells harboring an Oct4-GFP transgene we observed, GFP expression along with activation of Oct4 from the somatic genome, expression of key pluripotency genes, and positive immunostaining for Oct4, SSEA-1, and alkaline phosphatase. The iPS-somatic hybrids had the ability to differentiate into cell types indicative of the three germ layers and were able to localize to the inner cell mass of aggregated embryos. Furthermore, ntES cells were used as fusion partners to generate hybrids, which were also confirmed to be reprogrammed to a pluripotent state. These results demonstrate that once a somatic cell nucleus is reprogrammed, it acquires the capacity and potency to reprogram other somatic cells by cell fusion and shares this functional property with normal embryonic stem (ES) cells.”

Using neural stem/progenitor cells derived from iPSCs to repair spinal cord injuries

The March 2010 oublication Are induced pluripotent stem cells the future of cell-based regenerative therapies for spinal cord injury? Reports “Despite advances in medical and surgical care, current clinical therapies for spinal cord injury (SCI) are limited. During the last two decades, the search for new therapies has been revolutionized by the discovery of stem cells, inspiring scientists and clinicians to search for stem cell-based reparative approaches for many disorders, including neurotrauma. Cell-based therapies using embryonic and adult stem cells in animal models of these disorders have provided positive outcome results. However, the availability of clinically suitable cell sources for human application has been hindered by both technical and ethical issues. The recent discovery of induced pluripotent stem (iPS) cells holds the potential to revolutionize the field of regenerative medicine by offering the option of autologous transplantation, thus eliminating the issue of host rejection. Herein, we will provide the rationale for the use of iPS cells in SCI therapies.”

The June 2010 publication Neural stem cells in regenerative medicine: bridging the gap reports “Repair of the chronically injured spinal presents with multiple challenges, including neuronal/axonal loss and demyelination as a result of primary injury (usually a physical insult), as well as secondary damage, which includes ischemia, inflammation, oxidative injury and glutamatergic toxicity. These processes cause neuronal loss, axonal disruption and lead to a cystic degeneration and an inhibitory astroglial scar. A promising therapeutic intervention for SCI is the use of neural stem cells. Cell replacement strategies using neural precursor cells (NPCs) and oligodendroglial precursor cells (OPCs) have been shown to replace lost/damaged cells, secrete trophic factors, regulate gliosis and scar formation, reduce cystic cavity size and axonal dieback, as well as to enhance plasticity, axonal elongation and neuroprotection. These progenitor cells can be obtained through a variety of sources, including adult neural tissue, embryonic blastocysts and adult somatic cells via induced pluripotent stem cell (iPSC) technology. The use of stem cell technology – especially autologous cell transplantation strategies – in regenerative therapy for SCI holds much promise; these therapies show high potential for clinical translation and for future disease treatment.”

These all appear to be interesting contributions to our knowledge base related to iPSCs and adult stem cells, and strengthen the possibility of realizing the proposed longevity intervention of closing the loop in the stem cell supply chain.

This dialog is focused on how rapid social evolution is  driving biological evolution and how the result is increasing longevity in advanced countries.  I sent Marios* an e-mail with the paragraphs marked VG which appear in this blue font.  And Marios  responded with the paragraphs in black font marked MK.

VG.     I propose an explanatory framework that can lend clarity to discussions relating evolution to longevity.  That framework views social evolution as a separate and important matter in addition to biological evolution.  Social evolution takes place in cultural niches, that is, societies.  By social evolution I would mean the evolution of all key aspects of the environment and behavior of people in a society: how people live, work and communicate, their social, government, economic production and family systems, their institutions of all kinds, the technologies they have and how they have adapted to use of those technologies, what they eat and drink, their belief systems, their expectations and how they think.  For my purpose here I focus on common elements of advanced industrial and post-industrial societies, including those in most European countries, the US and Canada, Japan, Australian  and large sections of Chinese and Indian societies.Some key points are: 

VG.   1.  While biological evolution of humans appears to be very slow, social evolution is happening very rapidly, the use of cell phones and Internet and the resulting changes in communication capabilities and availability of knowledge being recent examples.

MK.   This is a very good point. Rapidly-evolving social developments will augment and complement biological evolution, particularly post-Darwinian evolution. 

VG.   2.   The principles of human social evolution cannot be derived from the principles of human biology, just as the laws of biology cannot be derived from those of chemistry and chemistry cannot be derived from physics.  However, chemistry must be compatible with physics, biology must be compatible with chemistry and the rules of social evolution must be compatible with the laws of biology.

MK.   The two concepts are interdependent. I believe that one drives the other and vice-versa

VG.  3.    Most dramatic increases in longevity have come about through social evolution such as the adoption of sanitation systems, recognizing the existence of diseases, inoculations, cleaner are and cleaner water, laws, dentistry, medicines, seat belt laws, stopping smoking, improved nutrition,  safer cars and products of all kinds.  The list goes on and on and there is still very far to go.

MK.   Yes, however we are likely to witness a much more far-reaching social and cultural influence on human longevity.  Technological developments will inextricably modify our society, I hope for the best. The internet is changing everything.  New technology makes it easy for people to exercise at home, aids their diet, and promotes psychological well-being via remote interactions with friends.  I believe that increased external inputs (emanated from the interaction between technology and society) are essential in stimulating human brain evolution, which in turn, must result in increased lifespans, for reasons I explain elsewhere.

VG.  4.   It is far too narrow to see biological evolution of humans only in terms of genetics.  It is mainly happening through changes in the epigenome.  And evolution due to changes in the epigenome can happen very rapidly compared to evolution due to changes in genes.  This can be seen in certain animals which rapidly adapt to changed environments in a few generations, including dramatic changes in their body characteristics, without any changes in their genetics.  And, with better nutrition and a healthier environment, children now are growing up to be taller adults than their parents were in several developing countries.   

MK.   There is increasing evidence supporting the role of epigenetic changes in shaping our evolution. Here, I am not referring to the slow, cumbersome process of evolution by natural selection, but to the new type of human evolution based on self-organising complexity and intellectual development. The way we stimulate our brain, our thoughts, actions and lifestyle all have an epigenetic effect on our genes, which helps in initiating events that result in progressively longer lifespans.

VG.   5.   Social evolution is driving biological evolution.  The changes in the epigenome of humans result from social evolution.  And by evolutionary standards the change is very fast as can be seen in terms of human longevity. 

MK.   I agree, as mentioned above. Social evolution is a much more efficient and quick way to evolve, compared to the Darwinian model. By social evolution I also mean cultural and technological changes that influence the entire process. 

VG.  6.   Complex societies demand greater longevity because they demand preservation and extension of knowledge.  That is why in advanced and advancing societies, for years now average lifespan has been increasing about 4 hours every day.  Life expectancy at birth approximately doubled between 1850 and 2004 and in primitive times it was perhaps 22 years for those who survived childhood with 30 years being very old.  Our genes have not changed during this time.  The evolution has been social and in our epigenomes.  

MK.  We will witness a gradual increase of our lifespans, with a few individuals breaking through the existing maximum lifespan (around 120 years), and an increasing number of super-centenarians (those aged 110 years plus), over and above what is currently predicted. Subsequently, it may become the norm for people to live to 130 plus. However, this seems a relatively slow process for achieving truly indefinite lifespans of centuries or even millennia. Perhaps there will be a quicker mechanism, which will suddenly augment (inflate) the process. I don’t know. 

VG.   7.    As longevity has been increasing, so have the main events of life been spread out: getting married, having the first children, finishing education and starting work, and end of the time when productive work is possible.  Back in the bronze age, most women started having babies when they were biologically able to do so – at 13 to 15; now the average age of marriage is over 30. 

MK.  This also will have a profound effect on societal evolution and thus biological evolution. Issues of fertility, decline in male sperm and hormonal changes are influenced by society and are relevant to longevity. For example, technological developments have caused pollution, which has now (according to some) caused a decline of function of human sperm, and thus lower fertility. Lower fertility is (according to some views) positively correlated with longer lifespans. This is an intricate example of how society changes affect biological evolution.  

VG.   8.     I therefore agree with Marios that longer and longer lives will result from our social and epigenetic evolution – assuming no cataclysmic events that are serious enough to destroy or seriously set back our societies.    I also believe we can accelerate the trend to greater longevity and believe it would be a very good thing to do so from a social and economic viewpoint. 

MK.  Personally I am only interested in the biology of the process, but will consider any influences derived from social, cultural and technological domains. I will leave the economical, ethical and psychological implications of super-longevity for others to debate. 

VG.  9. Finally, I have to acknowledge that this discussion itself is a manifestation and instrument of social evolution.  “If you find yourself riding on a horse, the best thing is to ride the horse in the direction in which it is going.”  

MK.   It is indeed. But my advice is also “try not to fall off”! 

VG.  10. So let’s get on with discovering how we can support people living healthy lives longer.  I see one possible approach which conceivably could crack the human aging barrier of 123 years and keep people alive and healthy for several hundred years.  It is described in the blog entry Closing the loop in the stem cell supply chain – presented graphically.

MK. I read all your blogs relating to stem cells, and I believe that this is a way forward. I am suggesting some ways for possible help, such as making use of transposons that may eventually be used in order to influence stem cell DNA along the lines you suggest. Also, newer developments in Synthetic Biology may provide the tools for accelerating any interventions upon stem cell production and function. These are just two areas of possible interest but there are many others. We need to encourage scientific dialogue in this respect and get interested scientists to take part. There is a lot of research currently going on but I find the approach rather fragmented. Many, if not all, researchers are unaware of the relevance their results have upon achieving indefinite lifespans. Their vision is restricted by funding constraints. We need to break through this barrier, but fortunately there are some platforms where we can expect dialogue (see www.imminst.org for example).

* Dr. Kyriazis is a well-known physician and researcher in the field of anti-aging medicine with a long history of research, scientific and popular publications in this field.   You can find the Wikipedia entry on him here and get a sense of some of his accomplishments from this google search.  This dialog appears here and on Dr. Kyriazis’ web site www.elpistheory.info.

Robert Pappas is an independent filmmaker currently in the final stage of updating his film To Age or Not to Age. This is a film on longevity research featuring interviews with prominent researchers. Two days ago, Robert asked me to generate a short presentation on my own pet theory for extraordinary longevity for inclusion in the film. This is the Closing the loop in the stem cell supply chain theory described in the blog entries The stem cell supply chain – closing the loop for very long lives , Progress in closing the stem cell supply chain loop, and mentioned in several others. The presentation had to be simple and suitable for non-technical viewers and was needed immediately. It will be shown on Dish TV and will likely be exposed to a couple of million viewers. I prepared the presentation yesterday and for that purpose I created two explanatory graphics. For the benefit of my less-technical readers I present the graphics and more or less the same explanation here, though not quite as simplified for the film and with some hyperlinked references.

NORMAL CELL TYPE PROGRESSION (SIMPLIFIED)

Normal Progression of Cell Types

The first diagram is a simplified explanation of how cells relate to one another in the course of a normal lifetime. At CONCEPTION of fertilization (upper left corner) the GERMLINE CELLS, sperm from father and egg from mother come together to form an embryo containing at a very early stage EMBRYONIC STEM CELLS. These are pluripotent cells, which means they are capable of eventually differentiating into all of the body’s some 200 different types of cells (blood cells, nerve cells, heart cells, skin cells, etc.). During EMBRYONIC DEVELOPMENT this cell differentiation occurs. In this simplified description three categories of cells are produced: ADULT STEM CELLS, PROGENITOR CELLS and SOMATIC BODY CELLS. These categories of cells will be around the rest of our lives although the specific cells in them will continue to change due to cell division, cell death and, except for adult stem cells, cell replacement.

SOMATIC BODY CELLS for the purpose of this presentation are the working cells of the body: neurons, glia, red blood cells, immune system cells, muscle cells, skin cells, liver cells, thyroid epithelial cells, heart cells, etc. etc. – excluding stem and progenitor cells.

ADULT STEM CELLS can differentiate on an as-needed basis to make replacement SOMATIC BODY CELLS or PROGENITOR CELLS, and they do this throughout a lifetime. ADULT STEM CELLS come in different varieties, such as hematopoietic stem cells which are found in the bone marrow and give rise to all the blood cell types and neural stem cells which give birth to new neurons in the brain and other parts of the body and other related nerve-associated cells like astrocytes and oligodendrocytes. Typically, ADULT STEM CELLS in any category can differentiate into a limited number of target BODY SOMATIC CELL types. They divide like all other cell types and can also morph into PROGENITOR CELLS and SOMATIC BODY CELLS in response to signaling. ADULT STEM CELLS tend to live in stem cell niches which care for them and function like storehouses. Adult stem cells may remain quiescent (non-differentiating although dividing) for substantial periods of time until they are activated by a normal need for more cells to maintain tissues, or by an emergency need induced by disease or tissue injury(ref). The storehouses are always ready to replenish SOMATIC BODY CELLS – that is, as long as a supply of quality ADULT STEM CELLS still exists.

PROGENITOR CELLS are ones intermediate in their range of target differentiation capability between ADULT STEM CELLS and SOMATIC BODY CELLS. They tend to be more specific in what they can differentiate into. For some cell types there is a hierarchy of progenitor cell types and some PROGENITOR CELLS are short-lived intermediates created in the process of differentiation of ADULT STEM CELLS into SOMATIC BODY CELLS. For simplicity of presentation I have shown them as a single box in the diagram although their properties vary widely.

A few factors are important for this discussion:

1.    In us humans and other mammals, renewal of our SOMATIC BODY CELLS through differentiation of ADULT STEM CELLS and PROGENITOR CELLS is required throughout life. In the course of a year, virtually all of our blood cells turn over. If ADULT STEM CELL and PROGENITOR CELL differentiation were to stop, we would soon die. No problem during most of normal life, as long as the stocks of ADULT STEM CELLS and PROGENITOR CELLS are plentiful and the cells are young enough that they will readily differentiate. With advanced aging, those conditions may no longer exist. Disease and death may follow.

2.    Cells in all of these three categories (ADULT STEM CELLS, PROGENITOR CELLS and SOMATIC BODY CELLS) age with progressive cell divisions. That is: a) there are major shifts in their global gene expression patterns ; hundreds if not thousands of genes get upregulated or downregulated, b) their telomeres (caps at the ends of chromosomes) generally get shorter, c) there are multiple changes in the epigenome, i.e. methylation of promoter regions of genes and changes in histone (spools around which DNA is wrapped) acetylation and histone morphology, and d) once a critical number of replications occur the cells become senescent or dysfunctional or destroy themselves or turn cancerous.

3.    With aging ADULT STEM CELLS and PROGENITOR CELLS become less ready to differentiate resulting in the stream of renewal for SOMATIC BODY CELLS slowing down and also the resulting replacement cells being epigenetically older. As cell renewal slows down and the number of senescent cells increases, so does organ renewal slow down or become impaired and disease susceptibility increases. The processes shows up as what we normally call aging and accelerates as the years roll on. Eventually life cannot be sustained and the organism dies.

4.    The process is once-through in nature, as is life as we know it. All our cells age and there is nothing in place to keep them young. Up to this point aging has been absolutely insurmountable. But we may be able to change that.

CLOSING THE LOOP IN THE STEM CELL SUPPLY CHAIN

The longevity intervention – closing the loop

The second diagram is the same as the first with additional boxes overlaid and shaded in orange showing the possible longevity intervention. I have described the intervention in previous blog entries(ref)(ref)(ref) but will repeat it here with reference to the diagram.

The first step is to COLLECT A SMALL SAMPLE of SOMATIC BODY CELLS from an individual of any age, perhaps in a drop of blood perhaps in a tiny scraping of skin. All of us have experienced collection of such samples.

The next step is to CORRECT THE COLLECTED CELLS FOR GENETIC DEFECTS. This is now a fairly well-understood procedure(ref)(ref). Any mutated genes that confer disease susceptibilities is snipped out of the DNA and a healthy versions of the same gene is re-inserted in its place. See the blog entry Treating genetic diseases with corrected induced pluripotent stem cells.An option is to perform the last two steps in reverse order. Also, at either stage the number of cells could be multiplied by standard laboratory processes.

The next step is REVERSION TO IPSC STATUS, that is, reversion of the collected corrected cells to being induced pluripotent stem cells. Using one of a number of known approaches, the cells are reverted to being undifferentiated cells that are, apart from correction for damaging genetic mutations, functionally, genetically and immunologically equivalent to the original EMBRYONIC STEM CELLS for the donor of the tissue sample. Reversion to iPSCs is an exciting stream of technology development that has been underway 3-4 years now. See the recent blog entry A breakthrough in producing high-fidelity induced pluripotent stem cells. A key point is that the iPSC cells at this point are young cells like the original EMBRYONIC STEM CELLS. It the reversion job is done right they no longer have any of the markers of aging that were associated with the cells they were made from. This is “the fountain of youth” on the cellular level.

The next step is to INDUCE DIFFERENTIATION INTO ADULT STEM CELLS. Much research has been going on oriented to direction of differentiation of pluripotent cells (hESCs or iPSCs) into specific cell types. Research has shown that such pluripotent cells can be directed to become motor neurons(ref), dopaminergic neuronal subtypes(ref), functional dendritic cells(ref), functional hepatic cells(ref), thyroid follicular cells(ref), chondrocytes(ref), glial, and pancreatic endocrine cells(ref), and many other cell types. While most research has been concerned with direct differentiation into SOMATIC BODY CELLS there should be no barrier in principle to inducing differentiation into ADULT STEM CELL and PROGENITOR CELL types. “The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced(ref).” Note that the thus-derived ADULT STEM CELLS, being derived from young iPSC cells will also be young cells.

The final step of the process is INTRODUCTION INTO ADULT STEM CELL NICHES of these young adult stem cells into the same individual that provided the sample. The idea is to create introduction of each type of ADULT STEM CELL into its respective niches.   I do not know how easy this will be. We do know the places where several types of adult stem cells reside like in bone marrow and in vascular niches. It may be that adult stem cells will more or less know on their own where to go if introduced into the general vicinity of a niche. We do know, for example, that nerve stem cells introduced in the vicinity of damaged nerve tissue will go to that tissue and forthwith start to repair it. An important thing we also know is that the young ADULT STEM CELLS will not generate an immune rejection because they are rejuvenated versions of the original donor’s own cells, immunologically identical. Further research will be required on how best to achieve this step. My intuition says it is achievable.

As can be seen in the diagram, what was a once-through process of aging now is supplemented by a closed-loop process of rejuvenation. The ADULT STEM CELLS introduced in the steps highlighted in orange are young and when they differentiate into SOMATIC BODY CELLS, those cells will by young too. Periodic rejuvenation created by use of this process could be repeated to keep a person young, perhaps if is repeated every ten years. Could the process enable breaking through the historical limit of 123years for human lifespans? Could it enable lifespans of hundreds of years? I don’t know but of all the proposed longevity interventions I am aware of, I think this one provides the best shot.

Every scientist in history has been hampered by not knowing what he does not know, that is, being completely ignorant of and oblivious to important knowledge that emerges later. And, the same is true for me with regard to the longevity intervention I am proposing and what I write. At present I see no reason in principle for why closing the loop in the stem cell supply chain could not work. But the proof will be in the doing. I am optimistic because of the rapid progress I am seeing in the science and practice of each step of the process that I have proposed. Most of the current research on application of iPSCs is in the field of regenerative medicine is aimed at correcting genetic disease-causing defects or at treatment of diseases. While the research is not specifically aimed at longevity, much of the detailed technical knowledge acquired should be applicable to making real the longevity intervention described here.

 

For research background for closing the loop in the stem cell supply chain as a proposed longevity intervention, please see the November 13, 2010 listing of past blog postings on stem cells and epigenetics.