Place: Keystone, CO
Date: 15^th to 20^th February 2010

Keystone Symposia has served the bioscience community for 38 years by providing a forum to publicise high quality scientific data in a relaxing environment conducive to the establishment of links between researchers. One of the best features of Keystone meetings is that they are not too large and one gets a chance to talk to some of the best researchers in the field and more often than not they are willing to share as yet unpublished results. The current meeting which took place in the Keystone conference centre was no exception to this.

The focus of the meeting was to examine recent developments in stem cell differentiation and dedifferentiation and one has to say that these aims were largely achieved. The scientific organisers (Shinya Yamanaka, Kyoto, Japan and Fiona Watt, Cambridge, UK) had invited several high quality presenters. Vast amounts of data were presented so naturally this has led us to produce quite a lengthy report. Our objective in producing this report is to give an overview of the conference proceedings rather than an exhaustive description of every presentation, so we apologise if anyone feels they have been missed out or if there are any omissions of data that were described.

Day One

The first day of the meeting saw the keynote addresses by Shinya Yamanaka (Kyoto University) and James Thomson (University of Wisconsin) which effectively set the tone for the entire meeting since the bulk of presentations were concentrated on pluripotent cells.

Day Two

On the second day we got down to the real business of the meeting. The first talk saw Shinya Yamanaka once again take the podium to describe some of his lab´s results in the derivation of induced pluripotent stem cells (iPSCs). However, in a manner that has become characteristic of Yamanaka, he was keen to stress the possible problems associated with iPSC rather than trying to underline their potential usefulness. The key theme of his talk was that human iPSC (hiPSC) have a degree of transcriptional heterogeneity, like their mouse counterparts, but that microarray studies have not shown any common misregulated genes. In short his argument was that we really know very little about the characteristics of hiPSC and the only measure we have of their pluripotency is that they are able to form teratomas. In view of this we need to define which qualities the best hiPSC lines should have to produce ´safe´ iPSC. This means we will need to specify the best donor somatic cells and reprogramming methods.

Next up was Rudolf Jaenisch who described some results from his reprogrammable iPSC mouse model. Many of these data have been published prior to the meeting so we can restrict our reporting of Prof Jaenisch´s talk to his treatment of X-inactivation. The most interesting observation was that although human embryonic stem cells (hESC) should normally have one inactive and one active X-chromosome (as opposed to mouse ESC (mESC) which often have both x-chromosomes active), culture under 5% oxygen conditions appears to repress the XIST gene which is involved in x-inactivation. However, if the cells are exposed to normal 20% oxygen first, they undergo x-inactivation that is not reversible by transferring the cells to 5% oxygen.

Helen Blau was next to present. She had gone back to an older method for studying the reprogramming of a somatic genome which involves fusion of the somatic partner with a pluripotent cell, in this case an mESC, to examine what happens to the epigenetic status of the somatic chromosomes. The ESC phenotype seems to be dominant in such heterokaryons. Fusions of human fibroblasts and mESCs are a good model because the RNA products of the human and mouse genomes can be distinguished by PCR. By this method, they showed that OCT4 and NANOG are rapidly upregulated from the human genome following fusion which was, perhaps not unexpectedly, accompanied by extensive DNA demethylation at the promoters of these genes. Such demethylation turned out to the principal theme of this talk as she went on to describe the AID (or AICDA) gene which may be important for DNA demethylation, since it can deaminate 5-methylcytosine to produce thymidine which is then detected as a mismatch by the DNA repair machinery of the cell. This results in replacement of thymine by cytosine which amounts to demethylation of the 5-methylcytosine at this position. The data presented included siRNA knockdown of AID in the heterokaryons which seemed to block the demethylation activity quite effectively. Helen Blau pointed out that their model still requires absolute proof particularly with regard to the exact mechanism of AID mediated demethylation and what targets this enzyme to one set of genes and not others. However, she did also point out that Wolf Reik in Cambridge has shown AID activity in primordial germ cells (PGCs) that are also capable of extensive DNA demethylation during their development. This point was to be addressed in a later presentation by Azim Surani.

After a brief description of methods for making pig iPSC by Jenny Liao there was an interesting talk from Christoph Bock who is trying to develop functional genomics assays to quantify the utility and safety of human hiPSC. Basically his method involved differentiating both iPSC and ESC as embryoid bodies for 16 days followed by genome wide DNA methylation mapping to see how differentiation affects this important epigenetic modification. Dr Bock argued that while there are other methods to measure epigenetic changes such as histone modifications patterns; this would require the use of techniques such as ChIP-on-chip or ChIP sequencing which although powerful, requires large numbers of cells. In contrast DNA methylation mapping can be done with a few as 5000 cells which lends itself nicely to the quantitative differentiation assay proposed in Dr Bock´s talk.

So what were his conclusions? Essentially his data suggest that every ESC and iPSC line has some differentially methylated promoters but no single locus subject to this type of epigenetic modification can be used to distinguish and iPSC from an ESC. He also suggested that most of the genes that should not be expressed in ESC are more or less the same in iPSC lines. Such inactive genes tend to have hypermethylated promoters, however, although there was some variability in the numbers of genes showing hypermethylation between ESC and iPSC, there was a very good correlation between genes showing hypo-methylation (and therefore gene activation) in ESC and iPSC. This point was put forward as evidence that iPSC retain very little epigenetic memory of their somatic origins however, this doesn't explain why there should be variability of DNA hypermethylation at some genes. Dr Bock argued that there is possibly some degree of random hyper- or hypo-methylation at genes that are not immediately involved in the pluripotency maintenance network of ESC or iPSC.

After lunch (or more appropriately the skiing break!) Austin Smith (Cambridge University) presented some of his findings on the differences between mESC and the epiblast stem cells (epiSCs) and how these data relate to the ground state of pluripotency in mESC. Essentially the bulk of Austin´s presentation related to his recent publications on the subject but it was particularly well delivered and explained the subject matter clearly.

The next presentation from Ihor Lemischka (Mount Sinai School of Medicine) also described some of his recently published data on systems level analyses of fate changes in mESC. Ultimately he described an enormous amount of work performed on pluripotent and differentiating mESC to characterise their transcriptomes (mRNA), epigenomes (histone acetylation and RNA polymerase II binding maps) and their proteomes. This was all valuable data since it could be a useful tool to compare hESC and hiPSC, not only to each other but also to confirm that they differentiate in predictable ways to give cell types that do what they are supposed to do. However, the presentation covered a vast amount of published data and that we will not report here.

Amanda Fisher (Imperial College, London) was next to present some data that again put forward the idea that heterokaryons are a good model in which to study epigenetic reprogramming. Fusion of human B-cells with mESCs produces heterokaryons in which all the expected pluripotency genes are upregulated from the formerly somatic human genome. This is accompanied by downregulation of genes specific to the B-cell phenotype. The great advantage of this experimental system for studying reprogramming is that mESC with knockouts for various genes that might function as epigenetic modifier proteins can be used to observe what effect this has on the ability of ESC to reprogram a somatic genome in the heterokaryon. They showed that ESC lacking activity of Polycomb Repressive Complexes 1 and 2 (PRC1 and 2) were substantially less able to reprogram than wild type ESC and led them to hypothesise that the reprogramming effect might be due to PRC1 and 2 repressing genes that would otherwise interfere with reprogramming. They tested this by using RNAi to knockdown genes normally repressed by PRC1 and 2 but this failed to rescue a reprogramming phenotype in the PRC1 and 2 knockout cells. They did show that the gene JARID2 seems to be involved in reprogramming and demonstrated its enrichment at the promoters of genes known to have bivalent chromatin domains in pluripotent cells. Quite how this is able to mediate reprogramming they were unable to say. The presentation raised more questions than it provided answers, as is often the case in science, but it does provide a useful pointer for future research and the Fisher group are currently interrogating the factors required for successful reprogramming of human blood cells.

The first day ended with a couple of short talks from promising newcomers to the field. The last of these by Li Fang Chu (Baylor College) put forward an interesting hypothesis that primordial germ cells (PGCs) were actually the source of ESCs cultured from the inner cell mass (ICM). This was unusual, but certainly interesting, since there is documented evidence that PGCs are able to convert into embryonic germ cell lines, although these are a slightly different form of pluripotent cell. The hypothesis put forward here implies that PGCs, or at least a form of precursor cell type, exists in the ICM which is counter to the accepted wisdom that PGCs arise from the proximal epiblast at a later stage of development. However, Chu provides evidence for his idea by showing that clusters of cells expressing the PGC gene BLIMP1 (or PRDM1) appear in outgrowths of ICMs plated onto feeder cells. The cells were able to generate colonies of alkaline phosphatase positive cells whereas BLIMP negative areas of the ICM apparently could not do this. Moreover, he showed that these BLIMP1 positive cells were able to colonise the genital ridges of murine E8.5 embryos after in utero injection which lends credence to their PGC identity. This unusual idea was formulated into a hypothesis that ESC do not arise directly from pre-implantation epiblast cells but transition through an intermediate PGC stage. This is quite difficult to test from his current data as no other markers that are characteristic of the PGC phenotype was shown, but the idea seemed to stick with several other members of the meeting since it came up in subsequent presentations.

Day Three

The first slot of day three fell to Azim Surani (Cambridge University), which conveniently followed on from the germ cell topic of the last talk on day two. Surani is of course well known for his work on PGCs from mouse embryos and he began by describing new results from his investigations of the regulation of the Blimp1 gene by Lin28 and Let7. He also alluded to the idea that a form of PGC might be an ESC precursor by noting that Blimp1 negative epiblast cells are unable to produce PGCs in vivo. However he quickly moved on to more defined and published work on the changes in histone modification patterns and nuclear morphology during PGC specification in vivo. His novel results were concerned with the possibility that deamination of 5-methycytosine by Aid/Apobec genes, followed by base excision repair to replace the resulting thymidine with cytosine, might be the mechanism of the rapid DNA demethylation that occurs either soon before or immediately after arrival of PGCs in the genital ridge. This followed on from Helen Blau´s talk on day one, but Surani provided some nice data showing the coincident expression of Aid and Apobec genes with the known timing of DNA demethylation in ex vivo PGCs and the appearance of chromatin bound Xrcc1 which is known to be involved in the base excision repair process.

Surnani´s presentation resulted in a number of questions, but probably the most interesting one was that since base excision repair is a fundamentally error prone mechanism, how would this be acceptable in cells of the germline that are supposed to maintain genome integrity to a very high level? Also Helen Blau asked if loss of function studies should be performed but Surani countered both of these questions by saying that neither of these points could be easily examined with the small numbers of PGCs available from the mouse embryo.

The next slot was Hans Scholer (Max Planck Institute for Molecular Biomedicine, Germany) who began by asking how hESC relate to mESC and EpiSCs but in a short while the topic changed to Prof Scholer´s principal research focus of adult germline stem cells (GSCs). The argument of this section of the talk was that precise selection of culture conditions is able to convert GSCs from mouse testis into pluripotent cells that have a very similar transcriptome to mESC with the exception of their imprinted genes which show an androgenetic pattern as one might expect given the origin of the cells. In effect this amounts to cellular reprogramming by alterations in culture conditions without addition of extrinsic reprogramming factors such as viruses. Interesting enough in its own right but Prof Scholer then presented a critique of the one paper that claimed to have achieved similar results from human testis biopsies and showed that at a transcriptomic level the cells obtained in this latter paper were more similar to fibroblasts than to hESC. Clearly there is still much to be achieved in the field of pluripotent GSCs. Staying with the idea of non viral reprogramming, the next talk by Kevin Eggan (Harvard University) described his group’s efforts to replace viral reprogramming vectors with small molecules that alter the epigenome. His principal finding was the molecule he has named RepSOX due to its ability to replace SOX2 in fibroblast reprogramming experiments which seems to work as a TGFb inhibitor. These experiments still require the other vectors of course so the search is on for small molecules that can replace all of the Yamanaka factors and Eggan appears to have some candidates that can replace OCT4 and KLF4. Since RepSOX can replace c-MYC in the reprogramming process and a combination of the OCT4 and KLF4 vectors with this molecule is sufficient to generate iPSC, there are high hopes that we might be able to dispense with viral reprogramming in the near future.

Chad Cowan (Harvard University) is into fat! More precisely he is interested in obesity and how we can understand the pre-disposition of some individuals to accumulate excessive adipose tissue by examining the characteristics of adipocytes differentiated from iPSC of obsese individuals. This is an interesting model system and Dr Cowan´s presentation represents the first mention of the use of iPSC technology for disease modelling in this Keystone meeting. At present he has been able to establish methods to differentiate adipocytes from hESC and some limited data on hiPSC and these data formed the body of presentation.

The morning session continued with a couple of short talks from Azadeh Golipur (Samuel Lundefeld Research Institute, Canada) who was looking at RNAi screens to identify genes regulating the initial stages of reprogramming in iPSC generation and Colin Melton (USCF) who has examined the changes in miRNA expression during differentiation of mESC. To end we had a presentation by Larry Stanton (The Genome Institute of Singapore) describing the structure-function relationships of SOX2 and SOX17 and how these could be interconverted by removal of certain structural motifs.

The afternoon session on day three began with Takashi Shinohara (Kyoto University, Japan) and his description of the positive and negative regulators of mouse GSCs (mGSCs) and how they used this information to develop defined culture media incorporating glial cell line derived neurotrophic factor and bFGF to maintain GSCs indefinitely in vitro.

Anthony Atala (Wake Forest University) was up next with an impressive review of his work over the last decade concerning the usefulness of multipotent cells derived from the amniotic fluid and placenta. These stem cells express OCT4 and SSEA4 but their profile of other surface markers suggests that they may represent a stage somewhere between ESC and lineage restricted adult stem cells. Despite this, they seem to have many characteristics of pluripotent cells including the ability to differentiate into cell types of all three germ layers but crucially they do not form teratomas in vivo and so have been suggested as a possible alternative to hESC/hiPSC.

We ended day three with a talk by Thomas Zwaka (Baylor College, Texas). This was a departure from the published programme but was interesting nevertheless because of the hypothesis that pluripotent cells rely more upon a tightly controlled, but dynamic, network of gene expression to maintain their phenotypes, whereas somatic cells may be more reliant upon the activities of their housekeeping genes. One of the principal genes in this network seems to be Ronin which has been shown to bind 866 sites throughout the mESC genome and seems to be an activator of the mTOR pathway. Approximately one third of Ronin target genes are also occupied by Oct4, so there is a possible link into the pluripotency gene network, although its principal functions seems to be regulating the key metabolic processes (such as ribosome biogenesis) in mESC. This may be one of the mechanisms by which they prevent their differentiation.

Day Four

The focus of the meeting changed today. Days one, two and three were concerned largely with pluripotent cells but today we switched our attention to adult stem cell types. The first talk of the day was given by Fiona Watt (Cambridge University, UK) describing her admirable studies of the epidermal stem cell niche. Much of her data on the identification of epidermal stem cells on the basis of Lreg1 expression has been aired before, but her description of collagen islands printed onto gold coated glass slides as a means of controlling epidermal stem cell differentiation was excellent and highlighted many interesting observations on the control of stem cell differentiation by shape and stiffness of the cells 3D environment. Data was also presented showing how the actin cytoskeleton can mediate these effects.

Haematopoietic stem cells (HSCs) were the focus of the next talk given by Toshio Suda (Keio University, Japan), wherein data were presented to show how hypoxic microenvironments are necessary to maintain long term haematopoietic progenitor cells in a quiescent and multipotent state. It has been suggested that increasing levels of reactive oxygen species (ROS) are one of the triggers causing differentiation of such HSCs and Suda´s data show that HIF1a (hypoxia inducible factor 1 alpha) may be involved in mediating this trigger, since HIF1a knockout mice show loss of quiescence in their HSC population and also decreases in HSC numbers as they age. This is the converse of normal ageing where HSC numbers actually increase (although they may not function so well as will see from the next talk) and suggests that the HSC pool size may be controlled at least in part by HIF1a.

In an interesting parallel with pluripotent cells, they showed that quiescent HSC are more dependent upon glycolysis than oxidative phosphorylation as their ATP source which fits well with the need to restrict the production of ROS. However a switch to oxidative phosphorylation occurs in cycling or expanding HSC. On a similar note Dr Suda also observed that the mitochondrial mass of quiescent HSC is quite low which draws another parallel with pluripotent cells.

The haematopoietic focus was maintained by the next speaker in what was perhaps one of the most though provoking lectures of the whole meeting. Amy Wagers (Harvard University) has recently published her findings on a possible contribution of serum borne factors from younger mice to a process that could be loosely termed “rejuvenation” of the stem cell population of older mice. These data arose from a set of heterochronic parabiosis experiments, which involves connecting the circulatory systems of one mouse to another to see what happens to the individuals concerned. However, when old mice were exposed to the circulatory environment of young mice, their skeletal muscle progenitor cells and HSCs began to behave in a similar fashion to those in the younger parabiont.

Decline in tissue function is a normal facet of ageing, with muscle atrophy and haematopoietic dysfunction being among the principal characteristics of this decline, and it is normally thought to be irreversible. Interestingly the work presented by Amy Wagers draws upon earlier developments by Thomas Rando and Irina Conboy (Berkelely CA) that showed some improvement of skeletal muscle satellite cell function in this heterochronic parabiosis model but this is the first demonstration that the effect may apply to other stem cell compartments. Wager´s data seem to imply that the circulating factors may affect the cells that define and create the HSC niche rather than the HSC themselves, since exposure of ageing osteoblasts to factors within young serum was needed to ensure that the HSC pool size reduced to youthful levels and that the skewing of myeloid versus lymphoid differentiation was reversed. Regarding the nature of such “rejuvenating” factors, rather than undertake a laborious analysis of the differences between old and young sera, the Wagers group undertook a small molecule screen to see if any promising candidates might be able to replicate this effect in vitro and in vivo. One ALK4 receptor kinase inhibitor did seem to be able to partially recapitulate the effect!

Like many developments this presentation raises more questions than it answers. A reasonably well tested theory of ageing is that many of its problems arise through the accumulation of harmful mutations in the DNA. If this is true how does parabiosis reverse this accumulation (if indeed it does so at all)? Similarly can it restore the telomeres of aged HSC? Even in the light of such questions the data is still a fascinating new addition to the investigation of ageing that might help us to elucidate the “genetic or epigenetic” basis of the ageing phenomenon.

Fred Gage (Salk Institute) had some interesting data on the expression of Sox2 in the self renewing cells of the dentate gyrus. Progeny of these cells differentiate into neurons within one month of the cells “birth” and this process appears to continue throughout adult life. The Gage group is studying the cellular, molecular and environmental influences that regulated neurogenesis in the adult brain as well as looking at methods to modulate the levels of adult neurogenesis. But perhaps the most interesting part of their work is using iPSC from humans and non human primates to see if there are differences in their neuron forming capabilities that could explain differences between the human and primate brains.

The afternoon session saw Deepak Srivastava (UCSF) discuss his work on miRNA regulation of cardiac stem cell fate. His group has identified individual miRNAs that govern differentiation of ESC and iPSC into mutlipotent cardiac progenitors, and some of these also direct subsequent differentiation into cardiomyocytes, endothelial and smooth muscle cells. These miRNAs appear to regulate transcriptional networks and signal transduction pathways central to the adoption of a cardiomyocyte cell fate and are amenable to manipulation which opens up more possibilities for directing the differentiation of pluripotent cells.

We had a brief reversal back to iPSC related topics for the talk by Hideyuki Okano (Keio UIniversity, Japan) who described the possible uses of iPSC for regenerating the damaged central nervous system. They found that miPSC could be induced to form neural stem cells using the same methods for mESC (Naka et al, Nature Neuroscience, 2008) and that these were able to contribute to the repair of damaged spinal cord in immunodeficient mice. Crucially these animals showed significant improvements in their ability to control their hindlegs after transplant and the only difference to transplant of cells derived from hESC was that fewer glial cells were produced from the iPSC. Hans Kierstead (UC Irvine) continued the spinal cord injury theme with similar data concerning the recent clinical trial by Geron using hESC to treat this condition.

The fourth day was finished off by Catherin Niemann (University of Cologne, Germany) who changed the focus completely with her description of sebaceous gland homeostasis by hair follicle stem cells.

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Day Five

The last day of the meeting saw talks on a mixture of subjects ranging from differentiation of ESCs into cell types of potential clinical use to more immediate possibilities for applying ESCs to the discovery of new drug leads. The first talk of the morning was the one of the more industrial presentations of the meeting given by Emmanuel Baetge (Novocell, San Diego) who described the impressive progress made by Novocell in developing and validating protocols to produce pancreatic islets for treatment of type I and II diabetes.

Through a stepwise differentiation protocol modelled after pancreatic development, Novocell have been able to generate pancreatic endoderm progenitor cells which could produce glucose responsive endocrine cells upon transplantation into the epididymal fat pad, kidney capsule or subcutaneous or omental sites of SCID mice. These glucose responsive islet cells were capable of maintaining stable blood glucose levels in streptozotocin treated mice (which destroys their exisiting beta cells and induced hyperglycemia) for more than one year. Dr Baetge continued by describing Novocell´s efforts to scale up production of islets to deliver a safe and affordable treatment for diabetes.

Ron Mckay (NIH, Bethesda) was up next and although starting with some of his older data on neural stem cell differentiation from ESCs, he really wanted to make the point that hESC lines seem to differentiate in a predictable and uniform fashion and that hiPSC are basically the same except for one or two notable genes (such as glutathione-S-transferase (GST)). He debated the point at some length whether or not this might be due to individual genetic differences. but went on to form an interesting idea that iPSC might be a valuable way to answer questions about evolutionary biology since we have the opportunity to study an enormous number of iPSC lines derived from many individuals in a way that is somewhat impractical using hESC. After introducing this point he went back to the slightly more mundane, but undoubtedly valuable, differentiation of hESC to hepatocytes and indicated that some of the iPSC lines he was using were rather better for making hepatocytes than hESC.

Probably the real focus of the day was the possibility of translating hESC research into clinical realities. Consistent with this the next presentation came from Rita Perlingeiro (University of Minnesota) who discussed the differentiation of ESCs into muscle progenitors. Basic methods for differentiating mESC as embryoid bodies seem to be very inefficient for making myogenic cell types. To improve on this situation her group produced Doxycycline inducible Pax3- and Pax7-GFP constructs and stably transfected these into mESC. They differentiated these to give GFP-expressing PDGFRa–FLK1 cell populations that were able to engraft in the MDX mouse model for Duchenne muscular dystrophy, although in the case of Pax3-GFP, this nascent mesodermal population may have been transplanted too early after induction of differentiation, since teratomas were produced. The results with the Pax7-GFP line were rather more encouraging since not only were they able to contribute to repair and regeneration of dystrophic MDX muscle, but they also took up anatomical locations typical of the skeletal muscle satellite cells. Furthermore, these cells showed transcriptomic profiles similar to those of satellite cells isolated ex vivo. Interestingly miPSC showed almost identical Pax7 expression to mESC upon differentiation and preliminary evidence suggests that the Pax7 expressing cells may be able to engraft and repair MDX muscles albeit with a lower efficiency as the same cells derived from mESC.

An industrial focus continued with the talk given by Nobuko Uchida (StemCells Inc, Palo Alto) although the data was more involved with neuroprotection, that is using derivatives of ESC to either replace enzyme deficiencies or re-myleination strategies. The first example was of the neuronal ceroid lipofuscinoses, a rare group of fatal neurodegenerative diseases in which a lysosomal enzyme crucial to the removal of the products of lipid damage such as lipofuscin. Failure to remove this material effectively can lead to neuron death, but the therapeutic options are limited since the enzyme cannot cross the blood brain barrier if administered into the peripheral vasculature. To get around this problem, Stem Cells Inc are developing methods to transplant human central nervous system stem cells (Hu-CNS-SC) into mouse models of these diseases. The company has generated cell banks from CD133-positive cells enriched from the human CNS and suggests that unlike ESC or iPSC these cells do not require prior differentiation before transplant and they do not form teratomas. Another potential area of application for these cells is re-myelination of damaged axons. When injected into the shiverer mouse model (which has a myelination defect) these cells were able to contribute to axon repair. The company has recently received FDA approval to start a phase I study for the use of Hu-CNS-SC in Pelizaeus-Merzbacher disease, a fatal myelin disorder. A third potential application of this adult stem cell population is protection of the photoreceptors in an animal model of retinal degeneration. The company has hailed this as a possible treatment for age related macular degeneration.

The morning session ended with a return to more fundamental ESC studies with a presentation from Michael Drukker (Stanford medical school) on early ESC differentiation.

The first three talks of the afternoon were devoted to drug and biomarker discovery using ESC. Amy Sinor (Harvard Stem Cell Institute) was investigating spinal muscular atrophy using ESCs isolated from a mouse model of this disease and uncovered various pathways regulating the survival of motor neuron gene (SMN) in ESC derived motor neurons. In addition, her group has screened a large number of small molecules to find candidates capable of increasing SMN levels and decrease motor neuron death. Zhong Zhong (Glaxo SmithKline R & D, China) was doing broadly similar research aimed at drug discovery in neuroscience but Gabriela Cezar (University of Wisconsin) took a different approach based upon the identification and quantitative analysis of biochemical processes in neurodevelopmental disorders (a process she refers to as metabolomics) to identify new biomarkers for disease processes.

Conclusions

The meeting was very engaging since many of the presentations provided us with data that has not yet been published. Keystone meetings often provide the participants with the opportunity to reflect upon their current research projects in the light of findings from other workers in the same or similar fields and this meeting was no exception. We look forward to seeing more data from the labs of those who presented over the last few days and to some of the exciting developments, protocols and technologies that may well arise from their work.

The secret to eternal youth has been under pursuit for centuries. But now, results reported in Nature by a group at Harvard University shed new light on just how the ageing process might be co-ordinated across tissues and, astonishingly, that we may be able to reverse it (Mayack et al., 2010).

The extent to which ageing affects the stem cell compartment and tissue function reflects how much the tissue relies on resident stem cells for normal tissue homeostasis. Tissues with high turnover (for example blood, skin and gut) contain a prominent stem cell compartment and demonstrate a high regenerative capacity, whilst those with low cell turnover but high regenerative capacity (E.g. pancreas, skeletal muscle and liver) contain fewer stem cells and use different mechanisms for tissue homeostasis and repair. Thus, each tissue contains its own specialized environment, or niche, to preserve stem cell potential. There are many hypotheses as to why various changes occur in the stem cell compartment with ageing. Discrete cell-intrinsic or population changes are implicated, with concomitant alterations in differentiation potential and hence tissue restoration capability. The stem cell niche provides a tight regulatory and supportive microenvironment for resident stem cells and it has been demonstrated that alterations in extrinsic signals can modulate the niche and may even coordinate the ageing process across various host tissues. The aged hematopoietic system exhibits reduced immune function and increased malignancy, particularly that of the myeloid subtype. There is an increase in the hematopoietic stem cell (HSC) population in aged bone marrow (Sudo et al., 2000), however these usually demonstrate impaired hematopoietic engraftment, reduced reconstitution of peripheral blood leukocytes and differentiation capacity (favouring myeloid over other phenotypes) when compared with young HSCs. Profiling studies demonstrate altered stem cell regulatory gene expression in aged ‘dysfunctional’ HSCs alongside the suppression of lymphoid and increased expression of myeloid specification genes (Rossi et al, 2005).

Using an in vivo parabiotic system coupled with the in vitro study of regulatory HSC niche cells, Mayack et al. studied the role of local microenvironmental and systemic factors on hematopoietic stem and progenitor cell (HSPC) ageing. The authors generated heterochronic pairs (where ‘young’ mice were surgically joined to ‘aged’ mice) and compared these with isochronic parabionts (age matched surgically joined pairs). Parabiosis allows a common blood circulation between the pair and thus assessment of the effects of age-regulated circulating cells or factors on tissue function (Harrison et al., 1977; Hotta et al., 1980; Carlson & Faulkner, 1989; Conboy et al., 2005). In isochronic parabionts and in young-heterochronic partners exposed to an aged circulatory system, the frequency and number of primitive and long-term reconstituting HSCs (LT-HSCs) was unaltered, as shown by long-term multi-lineage reconstruction of irradiated recipients. However in aged-heterochronic partners, the authors reveal that exposure of aged bone marrow to young systemic factors restored the engraftment and lineage potential of donor LT-HSCs to that of youthful levels.

The authors have demonstrated previously that bone forming osteoblasts, a component of the HSC niche, convey physiologically appropriate signals to modulate HSC activity (Mayack & Wagers, 2008). Like HSCs, the number and frequency of osteoblastic niche cells are increased in aged mice. Their current results show that short exposure of young bone marrow cells to isolated osteoblastic niche cells from aged mice is sufficient to significantly increase the HSC population, akin to levels observed in aged mice. To test whether the ‘age-reversal’ effects observed in heterochronic pairs result from a reversion of age-related changes in the osteoblastic niche by a young systemic environment, the authors determined osteoblast frequency and number in aged-heterochronic parabionts and found not only that the osteoblast niche was restored to youthful levels, but that there was a significant reduction in HSPC accumulation as a result of the ‘age-reversed’ niche cells. Although the number and frequency of niche cells was not affected in young mice that were heterochronically joined to aged partners, isolated niche cells from these animals induced increased expansion of HSPCs when compared with young isochronic pairs, which the authors attributed either to circulating ‘ageing’ factors from the elder, or a dilution of ‘youthful’ factors in the young-heterochronic parabiont.

Furthermore, young HSCs exposed in vitro to aged osteoblastic niche cells subsequently acted like aged HSCs, showing reduced capacity for hematopoietic reconstruction and myeloid-skewed differentiation potential, indicating that local niche cells regulate HSC function and specify their physiological ‘age’. However, the reconstituting activity of young HSCs exposed to osteoblastic niche cells from aged-heterochronically paired animals showed a youthful profile of hematopoietic engraftment, demonstrating that age-associated alterations in the HSC niche can be reversed by a young circulation. Similar results were demonstrated in vitro using short term co-culture assays with young HSPCs; increased accumulation of HSPCs was observed following co-culture with niche cells from young mice exposed ex vivo to serum from old mice or aged human donors, whilst aged niche cells exposed to young serum displayed reduced capacity to induce LT-HSC accumulation.

In order to determine the mechanism by which this occurs, Mayack et al. studied the survival and transcription profiles of HSCs exposed to young or aged osteoblasts. Whilst no change in LT-HSC apoptosis was observed, real-time PCR analysis revealed differential expression of stem cell regulatory genes (Sox4, Notch1 and Notch2) and significant upregulation of age-regulated myeloid markers in young LT-HSCs exposed to aged-isochronic niche cells, alongside decreased expression of lymphoid markers – a result which was not observed when young LT-HSCs were exposed to niche cells from aged-heterochronic parabionts.

The authors then sought to determine the role of insulin-like growth factor-1 (IGF-1), implicated in the regulation of ageing and longevity across multiple tissue types. Neutralisation of IGF-1 in aged osteoblastic niche cells or young HSCs with an anti-IGF-1 antibody ameliorated the ‘ageing’ effects of an old niche on young HSCs and the response of young niche cells to aged serum, however had no effect on isochronically-matched HSCs and niche cells, suggesting that IGF-1 signalling in niche cells impairs HSC regulation. Further, they went on to demonstrate in vivo that this effect occurs locally via changes in IGF-1 signalling within the niche. Whilst direct delivery of anti-IGF-1 antibody into the bone marrow of aged mice markedly decreased the subsequent HSC accumulation capacity of treated niche cells in vitro, systemic IGF-1 neutralisation had no effect on the osteoblastic niche or HSC population dynamics.

This study demonstrates that age-specific changes within the regulatory niche initiate age-related stem cell dysfunction and that this can be reversed by youthful systemic factors. Their results implicate a novel and important role for IGF-1 signalling in the regulation of HSPCs within the ageing hematopoietic niche. Although the systemic mechanism by which IGF-1 is regulated during ageing remains unclear, the involvement of Wnt signalling in other age-associated pathology highlights this pathway as a probable candidate, as both Wnt and IGF-1 signalling are implicated in age-related changes across various tissues in a tissue-specific manner. However this assumption cannot be transferred across all tissue types. Skeletal muscle for example, relies solely on resident satellite cells for tissue repair (Morgan & Partridge, 2003), which remain quiescent unless responding to injury or disease. In line with the current study, heterochronic parabiosis also elicits age-reversal effects on aged muscle (Conboy et al., 2005), however in this system the age-reversal effects of a youthful circulation are mediated by enhanced Notch signalling (Conboy et al., 2003). Notably, in contrast to its effects on the hematopoietic stem cell compartment, local expression of IGF-1 maintains regenerative capacity in aged muscle (Musarò et al., 2001).

Although the role of stem cells in overall longevity remains controversial, these impressive results indicate that the age-related decline in stem cell function is modulated by local and systemic cues and that therapeutic strategies utilising the systemic milieu to rejuvenate the niche, at least in the case of the hematopoietic system, might significantly extend the period of normal stem cell function and promote hematopoietic longevity. The observations of Mayack et al. could also support the idea that cellular ageing may have an epigenetic component. The relative contributions of genetic and epigenetic factors (either inherited or acquired) to the ageing process is a fascinating subject and if changes in epigenetic gene regulation are significant this offers the possibility of reversing at least some of the age related changes. Elucidation of the stem cell-niche interaction dynamics in other contexts will allow us to determine whether similar results can be achieved to promote the prolonged health of other tissue types. Given that aged human serum elicited similar effects to an aged mouse circulation on the HSC niche, it will be interesting to determine whether this effect also works in humans.

References

Mayack S.R. et al. Systemic signals regulate ageing and rejuvenation of blood stem cell niches. 2010(463):495-500.

Sudo, K. et al. Age-associated characteristics of murine hematopoietic stem cells. J. Exp. Med. 2000(192):1273–1280.

Rossi D.J. et al. Cell intrinsic alterations underlie hematopoietic stem cell ageing. PNAS 2005(102):9194-9199

Harrison D.E. et al. Cell lines from old immunodeficient donors give normal responses in young recipients. J. Immunol. 1977(118):1223–1227.

Hotta T. et al. Age-related changes in the function of hemopoietic stroma in mice. Exp. Hematol. 1980(8):933–936.

Carlson B.M. & Faulkner, J.A. Muscle transplantation between young and old rats: age of host determines recovery. Am. J. Physiol. 1989(256):C1262–C1266.

Conboy, I.M. et al. Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature 2005(433):760–764.

Mayack S.R. & Wagers A.J. Osteolineage niche cells initiate hematopoietic stem cell mobilisation. Blood 2008(112):519-531.

Morgan J.E. & Partridge T.A. Muscle satellite cells. Int. J. Biochem. Cell Biol. 2003(35):1151–1156.

Conboy I.M. et al., Notch-mediated restoration of regenerative potential to aged muscle. Science 2003(302):1575–1577.

Musarò A. et al., Localised Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nature Genetics. 2001(27):195-200.

DNA methylation is an epigenetic mechanism which allows the regulation of gene expression, genome structure and genome stability. Current dogma dictates that DNA methylation mainly occurs on cytosines at CG di-nucleotides, which are found clustered together forming dense patches within the genome. Such regions are known as CpG islands and usually coincide with regulatory regions in promoter sequences, with methylation of cytosines within these regions positively correlated to gene repression. Previous studies of DNA methylation status of various cell types have provided snapshots of this epigenetic modification having mainly concerned themselves with analysing the DNA methylation status of these CpG islands.

However, in a great leap forward in epigenomics, an article in Nature by Lister et al has put DNA methylation back under the spotlight, as it is the first to report complete DNA methylation maps of the entire genome at single base resolution. This colossal undertaking was established in two cell types; human embryonic stem cells (hESCs) and human foetal lung fibroblasts, using the MethylC-Seq technique combined with the Illumina Genome Analyzer II platform. This system allowed for a huge number of reads and the scale of the work presented is breathtaking. Around 178 giga-bases of sequence was generated, equal to 57 times the base content of the entire genome and so covering 86% of all bases, allowing 94% of all cytosines to be identified. In total, 45 million methylated cytosines were analysed in foetal fibroblasts (IMR90) and 62 million in hESCs (line H1) (See Figure 1). This huge number of reads allows for confidence in the methylation status of each cytosine residue and affords the study of strand-specific methylation and transcription. The detailed analysis of DNA methylation alone is impressive enough, but the authors go on to link this with messenger RNA/small RNA expression, as well as the location of various histone modifications (tri-methylation of lysine’s 4, 27 and 36 on histone H3, mono-methylation of Lysine 4 Histone H3 and acetylation of Lysine 27 histone H3) and DNA-binding factors (TAF1, SOX2, NANOG, p300 and OCT4). Such detailed, integrated maps allow for the generation of substantial amounts of data and detailed correlations between the various factors studied.

The striking initial finding was the prevalence of non-CpG cytosine methylation (being in the form methyl-CHG or methyl-CHH, where H is Adenosine, Cytosine or Thymine) in hESCs, where around 25% of total methylation was outwith the CpG context whereas, in the foetal fibroblasts, 99.98% of methylation was in the CpG form (See Figure 1). A comparison of methylation states between two different hESC lines (H1 and H9) showed a remarkable similarity, and suggests that this mode of methylation may be a conserved feature in hESC linked to pluripotency. Further analysis uncovered that non-CpG methylation was more common in gene bodies, rather than promoters, and was correlated to the expression status of the gene. Enrichment was also observed on the anti-sense strand, and further correlated to increased intronic transcription. Interestingly, it was further noted that non-CpG methylation was significantly enriched at genes involved in RNA processing, splicing and metabolism and depleted at sites of DNA-binding factors and enhancer elements.

Figure 1. Cytosine Methylation in IMR90 Foetal Lung Fibroblasts and H1 hESCs (Adapted from Lister et al)

To further establish a connection between the pluripotent state and non-CpG methylation, iPSC were generated from the fibroblasts which previously showed very little non-CpG methylation; and accordingly, non-CpG methylation was re-established to the levels observed in hESCs. The next obvious task was to understand the mechanisms behind this modification pattern in hESCs. Thorough analysis of DNA methylation patterns found a consensus sequence for the DNMT3 DNA methyltransferases, while the periodicity of this sequence observed in the genome is consistent with spacing between the active sites in the DNMT3A and DNMT3L heterotetramer complex, which mediates de novo DNA methylation. Gene expression analysis supported these findings, showing the over-representation of DNMT3A in hESCs when compared to fibroblasts.

This paper describes a monumental step forward in epigenetics with respect to the scale, analytical techniques, findings and, importantly, a paradigm shift in relation to the way we think about DNA methylation.

In another recent paper in Nature Genetics, Doi et al (2) also report that DNA methylation outwith CpG islands is potentially an important regulatory mechanism. In this article, DNA methylation was studied by comparing regions of methylation which differ between fibroblasts, hESCs and iPSCs. Differentially methylated regions (DMRs) were identified between iPSCs and fibroblasts of origin which exhibited comparatively low densities of CpG di-nucleotides but lay close to CpG islands (referred to as CpG “shores”). These regions were described as being Reprogramming-Differentially Methylated Regions (R-DMRs) and were often located near developmental and regulatory genes. A significant proportion of R-DMRs exhibited hypomethylation in iPSCs and overlapped with bivalent domains (regions of tri-methylation of lysine 4 and 27 of histone H3) and SOX2, NANOG, and OCT4 binding sites. This suggests that sites of demethylation during reprogramming of fibroblasts to iPSC are tightly linked to genes involved in pluripotency.

A further part of this study compared the presence of DMRs when comparing iPSC and hESC to determine the similarity of the epigenome in these two apparently “similar” cell types. DMRs were found (both under and over-methylation) between iPSC and hESC with 50% of DMRs lying close to genes of interest, suggesting that iPSC could occupy a distinct and possibly aberrant epigenetic state. However, these regions were relatively small in number (71 DMRs), and perhaps a comparison across different hESCs lines and across iPSCs lines will be required in order to uncover a more representative number of DMRs that “normally” exist, or indeed may uncover regions which “normally” differ. In the future, extended studies similar to this may allow certain regions to be used as epigenetic “Quality Controls” for validating the likeness of iPSCs to hESCs.

1. Nature - Direct conversion of fibroblasts to functional neurons by defined factors (2009)
2. Nature Genetics - Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts (2009)

Mesenchymal stem cells (MSCs) are well known to suppress immune function in vitro. However,
there have been variable results regarding their immune modulatory function after transplantation
in vivo.  It is not known exactly how MSCs suppress immune function.  Presumably, MSCs come
into contact with lymphocytes in the lymph nodes and immature dendritic cells in target organs.
However, when implanted intravenously, MSCs have shown a variable ability to migrate to these
tissues, which could explain the variable results regarding their immune modulatory function in vivo.  
Our hypothesis was that MSCs can modulate ocular inflammation efficiently if they can be delivered
in an appropriate way. Therefore, we chose direct application of MSCs onto the corneal surface to
investigate the immune suppressive effect in the eye.  Our results indicate that MSCs in situ can
effectively modulate ocular inflammation in a chemical burn model.  

Give some background rationale, explaining why this hypothesis was important in stem cell research.

I believe the fundamental goal of stem cell research is to establish clinically applicable therapeutics
to treat conditions such as such as immune‐associated disease and cancer.  Our hypothesis
supports the evidence that MSCs can be used to treat inflammatory disease in vivo.  

Briefly outline your experimental approach to test your hypothesis.

To investigate the anti‐inflammatory and anti‐angiogenic effects of MSCs, we used a chemical burn
model of the cornea. We applied MSCs directly to the burned corneas and evaluated
neovascularization, corneal opacity, inflammatory cell infiltrates and cytokine changes.

Was there a specific methodological technique that was very important in these studies?

We developed this animal model to represent acute chemical burns which result in corneal stem cell
deficiencies in their later stages. We also developed the method of topical application of the MSCs
on to the corneal surface.  These two methods were critical in evaluating the in vivo effect of MSCs
on acute inflammation, and  investigating whether MSCs can restore the micro‐niche that is
necessary to support corneal epithelial stem cells in the inflamed eye.

How do you interpret these results? What does this mean for stem cell biology?

I believe these findings provide evidence that stem cells can be used therapeutically to modulate
immune function.  It extends the possible therapeutic role of stem cells; they can be used not only
as a source for cellular differentiation and replacement, but for modulation of the microenvironment.    

What hypotheses should the field test now?

We should continue to examine the mechanisms of the immune modulatory function of MSCs is, by
studying factors such as soluble mediators (e.g., IL‐10 or indoleamine‐2,3 dioxygenase) which may
be critical in the development of tolerance. Meanwhile, clinical trials using MSCs in various stem cell
deficient or autoimmune diseases should continue to verify the clinical relevance of the application
of MSCs.  

Why did you select the journal Stem Cells for your paper?

We selected your journal because of its reputation as one of the leading journals in the field of stem
cell biology, and because of its high impact factor.

Finally, on a more personal note, tell us a little about you, your education, and training. What is your
position right now? What would you like to do in the near future? What impact do you expect this
award to have on your career aspirations?

I graduated from the Medical College at Seoul National University in 1994, and then earned my Ph.D.
in Ophthalmology at Seoul National University. I joined Dr. Jin Hak Lee in the Seoul Artificial Eye
Center at Seoul National University Hospital Clinical Research Institute, and have been there since
1999. I have been working to develop Seoul‐type keratoprosthesis for patients with corneal
blindness, and to investigate the feasibility of using porcine cornea xenografts as a substitute for
human corneas.  I am also studying immune‐modulatory therapeutics using MSCs and TLR‐
associated signals for epithelial stem cell deficient patients, immune‐related corneal disease patients,
and cornea transplant patients. Recently, I joined Charles Surh’s laboratory at the Scripps Institute to
investigate mucosal tolerance.  I believe the Stem Cells Young Investigator Award will greatly help
to advance my scientific career.  It also encourages my devotion to stem cell research, and I am
very grateful to the Editorial Board of Stem Cells for this award.

Induced pluripotent Stem Cells (iPSC) are widely believed to share many of the characteristics of Embryonic Stem Cells (ESC) and as such have been credited with the potential to revolutionise regenerative medicine. The potential benefit of iPSC exists because of their genetic similarity to the individual from whom they were derived, implying that if differentiated and clinically useful cell types produced from iPSC were transplanted back into the individual, the likelihood of immune rejection should be greatly reduced.

The way in which iPSC were created in early experiments cast doubts upon the clinical usefulness of these cells because integrating retroviruses or lentiviruses were used to reprogram somatic cells, but the development of non-integrating reprogramming methods had generated some degree of optimism that iPSC could be created that might be truly safe for clinical application. Now a new study has cast doubts upon the characteristics of iPSC themselves, since it seems that somatic cells produced from iPSC may have a tendency to undergo apoptosis or become senescent much earlier than similar cells derived from ESC.

This newly published research from the group of Robert Lanza in Stem Cells compared 25 human ESC (hESC) and 8 human iPSC (hiPSC) lines which had similar abilities to differentiate into blood precursor cells, blood vessels, and cells of the eye. Comparing the characteristics of cells derived from hiPSC and hESC, researchers found that blood and vascular derivatives from hiPSC display abnormal molecular and/or cellular processes compared to their corresponding hESC counterparts. The hiPSC showed notably decreased growth and differentiation efficiency, sometimes 1000 fold less than hESC when prompted to differentiate into haematopoietic stem cells (HSC). The differentiation method in this case made use of a well established technique that causes any HSC arising from the hiPSC to expand and differentiate further into colonies of more mature blood cell types such as granulocytes and macrophages, which appear as highly visible colonies in a soft agar medium. This method usually works well with most hESC lines, but the hiPSC lines examined typically gave much fewer and much smaller colonies.

Much more disturbing is the propensity for apoptosis in the cells that differentiate from hiPSC. Blast cells, an early type of haematopoietic progenitor sometimes called haemangioblasts, showed fragmented morphology and presence of the cleaved form of Caspase 3 that clearly indicate an apoptotic phenotype. Worse still, endothelial cells and cells of the retinal pigmented epithelium (RPE, which supports the photoreceptors of the retina) became senescent very soon after differentiating from iPSC. Over 50% of endothelial cells derived using the group´s well established protocol expressed senescence associated β-galactosidase and many, if not all, displayed flattened morphology typical of senescent cells. RPE develops quite slowly from hESC but can be routinely expanded for approximately five passages. This was not the case for RPE derived from hiPSC since they could only survive as far as the first passage before adopting a senescent morphology and β-galactosidase expression.

There have been numerous reports, both in the scientific literature and media, suggesting hiPSC may be identical to hESCs but these current data from Lanza´s group are among the first to suggest that this may not be absolutely true. Global gene expression profiling shows that many transcripts are common to both hESC and hiPSC but also that there are significant numbers of genes (approximately 4%) whose expression levels differ. The consequences of this are currently unclear, as are the mechanisms that might lead to the early senescence of hiPSC derived cells, but there have been other reports describing differences between hESC and hiPSC, such as aberrant maintenance of gene imprinting and expression of oncofetal antigens, so we would be well advised to investigate such differences in exhaustive detail before contemplating any clinical applications of hiPSCs.

It must be noted that the hiPSC used in Lanza´s study were all derived using integrating retroviral or lentiviral vectors, so it is possible that their observations reflect the genomic changes caused by viral insertions, which means that a similar investigation of hiPSCs derived using non integrative methods is both desirable and timely. If these future studies suggest that the problem lies with inefficient or incomplete reprogramming of the somatic genome then we must consider the possible applications of hiPSC with great care. To quote Robert Lanza, the senior author of this study, “Although there is excitement that iPSC can serve as an embryo-free source of stem cells, it would premature to abandon research using hESC until we fully understand what’s causing these problems.” We look forward to the insight gained from future developments in this field with great interest.