the buck institute: an inside look sj, cell cycle. 2011...stem cell and somatic cell aging and the...

© 2012 Landes Bioscience.

Do not distribute.

www.landesbioscience.com Cell Cycle 4177

Cell Cycle 10:24, 4177-4188; December 15, 2011; © 2011 Landes BioscienceInsider FEATURE

Dedicated to research on aging, The Buck Institute is advanc-ing a new stem cell initiative. Is there a connection?

The past 20 years have witnessed a considerable interest in the aging phenomenon among researchers in varied fields of biology and medi-cine. The Buck Institute is currently in a phase of growth, and a major focus will be to expand stem cell research. There are two main reasons, and the first is obvious. It is increasingly apparent that stem cells, either embryonic, adult or induced pluripotent, offer exciting possibilities to develop therapeutic approaches to the diseases of aging. The Institute already has programs to explore the potential of stem cells in the treat-ment of a range of neurodegenerative disorders, including Parkinson, Huntington and Alzheimer diseases. With respect to Parkinson disease, Dr. Xianmin Zeng is developing clinical-grade human dopaminergic neurons with the intent to replace those that are lost with disease progression. Furthermore, Dr. Deepak Lamba, who has just joined the Buck Institute faculty, will bring an exciting program to develop stem cell therapeutic approaches to treat macular degeneration, which has a major impact on the quality of life of elders.

The second reason gets at the heart of the Institute’s mission—to understand aging. Adult stem cell populations decline in their ability to repopulate increasingly damaged tissues with age, and defining the molecular events that lead to impaired tissue repair is likely to uncover new insights into the mammalian aging process. This age-specific dys-function of adult stem cells derives from alterations in both the stem cells and the extracellular signals they encounter, but much remains unknown. We at the Buck think this endeavor is an emerging and criti-cal area of research in the aging field. Dr. Victoria Lunyak's recent study demonstrates that molecular mechanisms that drive ex vivo aging (rep-licative senescence) of human adipose-derived stem cells are directly

Brian Kennedy; The Buck Institute for Research on Aging; Novato, CA USA; Email: [email protected]

Figure 1. The Buck Institute in Novato, CA USA.

The Buck Institute: An inside look

Kennedy

linked to the way DNA is pack-aged (chromatin status) within these cells. Cellular or replica-tive senescence is classically seen as the key element of aging and previously thought to be an irreversible event. Quite intriguingly, research in Dr. Lunyak’s lab shows that the aged human adult mesenchymal stem cells can be reprogrammed or rejuvenated by suppression of non-coding RNAs that impede on the correct assembly and function of chromatin. This discov-ery offers additional hope that aging in stem cell populations might be reversible and also provides novel approaches to correct for age-related deficiencies during clinical applications of autologous human adult stem cells.

Ultimately, we at the Buck Institute are well aware that an under-standing of how and why we age must integrate mechanisms of both stem cell and somatic cell aging and the interplay between the two. Another important series of studies at the Buck Institute can be attrib-uted to the work of Judy Campisi and colleagues. Her lab has been at the center of recent studies showing that senescent fibroblasts have an altered secretory program, termed SASP. As fibroblasts become senescent, either by passaging or in response to cell stress signals, this altered secretory program promotes inflammation. Enhanced inflam-mation has both positive and negative effects, stimulating tissue repair and immune clearance of damaged cells, but likely also promoting tumor formation and, given increasing evidence linking inflammatory signals to aging, possibly aging itself. It will be interesting to determine (1) whether adult stem cell populations exhibit altered secretory pat-terns with age and (2) whether adult stem cells are influenced by the signals emanating from other senescent cells.

Studies of aging in invertebrate models have been a driving force in aging research, leading to conserved modulators of longevity, such as the insulin/IGF and TOR pathways. However, there are likely to be aspects of mammalian aging that are not easily accessible in inverte-brates. With due respect to recent studies of intestinal stem cell aging in flies, the contribution of adult stem cells to aging may be one of those poorly accessible pathways. However, the known longevity pathways may be more connected to rejuvenating aging adult stem cells than previously thought. For instance, work by Pan Zheng and colleagues at the University of Michigan has shown that mTOR inhibition with rapamycin serves to rejuvenate aging hematopoietic stem cells and similar effects are being tested in other adult stem cell populations. Several investigators at the Buck Institute have embraced studies of the mTOR pathway, which arguably is the most conserved longevity path-way. It will be interesting to determine how mTOR signaling changes in different populations with aging, and whether reduced mTOR activation mediated by rapamycin confers widespread rejuvenation.

The mechanistic events that drive aging are beginning to be under-stood, and increasing numbers of interventions that mitigate aging


Do not distribute.

4178 Cell Cycle Volume 10 Issue 24

Insier

are being identified. The mission of the Buck Institute is to extend the healthy years of life. Given recent findings with embryonic, induced-pluripotent and adult stem cells, it may not be possible to completely fulfill that mission without significant expertise in these areas.

How does the Buck Institute foster and encourage interdisci-plinary science?

Last year when I was deciding whether to join the Buck Institute as President and CEO, one of the things that went through my mind was whether I could enhance the research in my lab by moving to the Institute. One could see that the quality of the science at the Institute was very strong, but the degree to which different labs support each other and collaborate is less easily discerned. However, very little investigative work was needed to determine the extent to which Buck Institute scientists collaborate and how the interdisciplinary nature of its investigators stimulates these interactions. A large percentage of published manuscripts have contributions from multiple Buck labs, and a number of large, multi-investigator grants have been obtained. The most significant of these for the Institute was a $23 million dollar proposal funded jointly from four National Institutes and the National Center for Research Resources in 2007 to create and stimulate the new field of “Geroscience.” Obviously, gerontology and aging research existed before this proposal, but a new science was emerging at the interface of the basic biology of aging and age-related disease. Geroscience is an attempt to bring together a range of projects and disciplines and explain the relationship between aging to the diseases it enables. The resulting research supported by the Geroscience project has led to new insights and, perhaps more significantly, connected sev-eral previous disparate threads in the aging research field. Furthermore, it has bred a level of collaboration within the Buck Institute as well as with collaborators off site.

More broadly, biological research has trended steadily toward more collaborative, team science in the last two decades for a variety of reasons. First, the challenging problems being addressed by the field (e.g., developing therapies for complex diseases, connecting multiple signaling pathways, understanding transcription on a genome-wide scale) and the varied experimental approaches that are required have pushed the field toward more team-oriented studies that integrate investigators with expertise in a range of disciplines. Second, genetic studies push scientists in unexpected directions. For instance, a genetic screen in yeast for a specific phenotype may identify unexpected genes that push an investigator into an unknown aspect of cell biology, or a mouse gene knockout generated to study one aspect of physiology may give rise to unexpected phenotypes, leading an investigator into another field. Finally, biological research among the “hard sciences” remains the most qualitative, despite recent efforts to make it more

quantitative, and a variety of scientists thinking about a problem in different ways can lead to more creative approaches to solve problems. While many academic departments once tacitly frowned on collab-orative approaches to research, that mode of thinking has largely given way in favor of a mode of thinking in which many groups together working in an interdisciplinary fashion can reach discoveries more quickly and efficiently and bring a deeper understanding.

Aging is perhaps the most complicated “syndrome,” measured by deleterious changes in tissue and organ homeostasis and likely caused by an overlapping set of pathologies. Any one investigator working in one discipline, no matter how insightful, would be hard-pressed to unravel its intricacies. We at the Buck Institute are convinced that a multi-faceted approach designed to tackle aging from many angles is the best approach to understanding the aging process and exploiting that understanding to develop therapeutic strategies to treat the dis-eases of aging, which encompasses most of the chronic disease that are increasing in prevalence.

Figure 2. Research at the Buck Institute is focused on the basic pro-cesses that modulate aging and the relationship between aging and chronic disease. By combining scientists who focus on aging with spe-cialists in chronic diseases, including neurodegenerative syndromes, cancer, diabetes, cardiovascular disease and macular degeneration, the Institute seeks to define how aging promotes the onset and progres-sion of disease. State-of-the-art technical expertise stimulates these studies. The opening of a new facility in 2012 will provide for the build-ing of expertise in stem cell and regenerative medicine research, where focus will be placed not only on developing stem cell-based therapies, but also on how declining function of adult stem cells contributes to the aging process.


Do not distribute.


Insier

Many important scientific advances have been made over the past decade involving the use of stem cells for Parkinson disease (PD) research. These include the generation of new in vitro models that have both greatly aided in our understanding of basic disease processes and been effectively used for drug screening to identify novel PD therapeu-tics. Much progress has also been made towards refining conditions for more effective stem cell-derived replacement therapies for the disorder. However, significant technical obstacles still remain, particularly in terms of clinical use of these cells for transplantation.1 The develop-ment of several innovative methods for the production and analysis of relevant stem cell-derived populations and for their in vivo transplanta-tion, discussed recently at the Buck Institute symposium (“Emerging Innovations to Advance Stem Cell Research and Therapies”), represents an important step toward addressing some of the more confounding technical issues in the PD stem cell field.

The ability to convert both human embryonic stem cells (hESCs) and inducible pluripotent stem cells (iPSCs) to a dopaminergic fate for studying basic disease mechanisms, for drug screening and for cell replacement therapy in PD has undergone rapid technical advances in the last several years (Fig. 1). The laboratory of Xianmin Zeng at the Buck Institute has developed protocols using scalable xeno-free defined conditions to generate dopaminergic neurons from both hESC and iPSC populations and for drug screening to identify compounds that can both enrich for generation of these cells and select against those of a

Prospects and challenges for the use of stem cell technolo-gies to develop novel therapies for Parkinson disease

Shankar J. Chinta and Julie K. Andersen*; The Buck Institute for Research on Aging; Novato, CA USA; *Email: [email protected]

What are the current challenges toward developing stem cell therapies in neurodegenera-tive syndromes like Parkinson disease? Do we still have a basic conceptual barrier to over-come in understanding the molecular events driving pathology in these diseases?

Figure 1.

Andersen

non-dopaminergic fate.2-4 Ongoing efforts by the Zeng laboratory and their collaborators are centered on the development of "good manu-facturing practice" (GMP) compliant protocols for the production and banking of large quantities of these cells for future use. In terms of the use of such cells for transplant therapy, however, many issues still remain (Fig. 2). These include the possibility of allogenic immune rejection and/ or cancer risk associated with transplanted hESC or iPSC-derived dopaminergic neurons. Recently, several protocols have been devel-oped for the direct conversion of somatic cells to dopaminergic neurons that avoid intermediate conversion to iPSCs, overcoming some of these obstacles (Fig. 1).5, 6 However even in these directly converted cells, dif-ferences in gene expression patterns versus those found in endogenous dopaminergic neurons (also likely in most hESC and iPSC-derived lines) may impact on both their usage for drug screening and for transplanta-tion studies. For the latter, producing adequate numbers of cells from aging patients for transplantation may be a particular challenge.

Preclinical studies of PD drug or cell transplantation efficacy and safety often involve evaluation using young animals and in acute neu-rodegenerative models that do not necessarily emulate true disease conditions. Cells transplanted into old diseased brains likely experience the same extrinsic stresses as endogenous cells, and post-mortem autopsies of PD patients who previously underwent transplants suggest that these cells can take on the pathological phenotypes of affected endogenous neurons.7-11 Understanding how intrinsic and extrinsic

aging effects may influence both endogenous cells and those introduced by transplantation (including the identification of specific epigenetic mechanisms involved) is an impor-tant research area discussed in depth at the symposium by Victoria Lunyak from the Buck Institute and Kyung Sun Kang from Seoul National University.12-15 In the context of PD, neuronal stem cells are known to lose proliferative capac-ity with advancing age, and this can impact on their ability to replace damaged, disease-related neurons.16 Developing strategies to enhance neuronal stem cell renewal in the aging brain is, therefore, a critical research area. Additionally, senescent astrocytes may have detrimental non-autonomous effects on both endogenous neurons and on exogenous neuronal transplants via secretion of soluble toxic factors.17 Understanding intrinsic and extrinsic events that affect both endogenous and transplanted cells in the context of aging will be extremely important in terms of designing more affec-tive PD therapies. Excitingly, recent studies in senescent adi-pose-derived adult stem cells have lead to the development


Do not distribute.


Insier

of a novel technology for restoring lost regenerative capacity in these aging cells and their return to a more pluripotent-like state. This involves knockdown of mRNAs produced from genomic non-coding elements known as Alu repeats (Wang et al., Cell Cycle 2011). Excitingly, these "iPSC-like" cells can be differentiated into neuronal lineages. This novel conversion technology could be capable of converting PD patient fibro-blasts (and those from young versus age-matched controls) to dopami-nergic neurons via an iPSC-like route, bypassing standard methods that involve expression of multiple cell lineage-specific growth factors. Use of this simpler method for iPSC conversion may minimize genetic and/or epigenetic differences (including those known to affect senescent programs) in comparison to endogenous dopaminergic neurons. The ability to use newly developed quantitative global mass spectrometry platforms to assess these alterations is an important technical advance for analyzing this possibility (discussed by Mary Lopez, BIRMS, Thermo Fisher Scientific). Depending on how closely genetic and epigenetic profiles from these cells match those of endogenous affected neurons, these cells could conceivably be used in future as a form of “personal-ized medicine” for better optimization of individualized drug treatment programs as well as for exploration of specific disease mechanisms. These cells could also be assessed for whether this novel conversion methodology reduces cancer risks normally associated with iPSC-derived cells during transplantation to determine if they are a better source for cells for this purpose. Efficacy of these and other stem cell-derived populations for cell replacement therapies are likely to be greatly enhanced by the use of newly developed scaffolding matrices or "biosprays" which allow precise cell deposition and maintenance in a secure three dimensional transplantation environment (discussed by Suwan Jayasinghe, University College London).18-22 Continued develop-ment of all of these technologies will undoubtably benefit from innova-tive fund raising programs especially towards very early-stage efforts in these areas (discussed by Ronald Landes, Landes Bioscience).

In conclusion, while the use of stem cells as a tool for the develop-ment of novel PD therapeutics has much potential, there are still many challenges to be addressed. Advances such as those described in this workshop are likely to have a major impact in terms of PD stem cell research being able to reach its full potential.

References

1. Wakeman DR, et al. Mt Sinai J Med 2011; 78:126-58; PMID:21259269; http://dx.doi.org/10.1002/msj.20233.2. Swistowski A, et al. Stem Cells 2010; 28:1893-904; PMID:20715183; http://dx.doi.org/10.1002/stem.499.3. Han Y, et al. PLoS ONE 2009; 4:e7155; PMID:19774075; http://dx.doi.org/10.1371/journal.pone.0007155.4. Swistowski A, et al. PLoS ONE 2009; 4:e6233; PMID:19597550; http://dx.doi.org/10.1371/journal.pone.0006233.5. Pfisterer U, et al. Proc Natl Acad Sci USA 2011; 108:10343-8; PMID:21646515; http://dx.doi.org/10.1073/pnas.1105135108.6. Caiazzo M, et al. Nature 2011; 476:224-7; PMID:21725324; http://dx.doi.org/10.1038/nature10284.7. Freed CR, et al. J Neurol 2003; 250(Suppl 3):III44-6; PMID:14579124; http://dx.doi.org/10.1007/s00415-003-1308-5.8. Olanow CW, et al. Ann Neurol 2009; 66:591-6; PMID:19938101; http://dx.doi.org/10.1002/ana.21778.9. Kordower JH, et al. Exp Neurol 2009; 220:224-5; PMID:19786018; http://dx.doi.org/10.1016/j.expneurol.2009.09.016.10. Kordower JH, et al. Neuropsychopharmacology 2009; 34:254; PMID:19079079; http://dx.doi.org/10.1038/npp.2008.161.11. Gross RE, et al. Lancet Neurol 2011; 10:509-19; PMID:21565557; http://dx.doi.org/10.1016/S1474-4422(11)70097-7.12. Lunyak VV, et al. Hum Mol Genet 2008; 17(R1):R28-36; PMID:18632693; http://dx.doi.org/10.1093/hmg/ddn149.13. Lunyak VV, et al. Cell Metab 2011; 14:147-8; PMID:21803283; http://dx.doi.org/10.1016/j.cmet.2011.07.008.14. Jung JW, et al. Cell Mol Life Sci 2010; 67:1165-76; PMID:20049504; http://dx.doi.org/10.1007/s00018-009-0242-9.15. Bhandari DR, et al. J Cell Mol Med 2011; 15:1603-14; PMID:20716118; http://dx.doi.org/10.1111/j.1582-4934.2010.01144.x.16. Peng J, et al. Aging Cell 2011; 10:255-62; PMID:21108729; http://dx.doi.org/10.1111/j.1474-9726.2010.00656.x.17. Salminen A, et al. Eur J Neurosci 2011; 34:3-11; PMID:21649759; http://dx.doi.org/10.1111/j.1460-9568.2011.07738.x.18. Jayasinghe SN. Macromol Biosci. 2011; PMID:21809448.19. Jayasinghe SN, et al. Macromol Biosci. 2011; In Press.20. Jayasinghe SN. Biomicrofluidics. 2011; 5:13301; PMID:21522490; http://dx.doi.org/10.1063/1.3571478.21. Jayasinghe SN. Analyst (Lond) 2011; 136:878-90; PMID:21271004; http://dx.doi.org/10.1039/c0an00830c.22. Jayasinghe SN. Macromol Biosci 2011; 11:11-2; PMID:20957698; http://dx.doi.org/10.1002/mabi.201000370.

Figure 2.


Do not distribute.


Insier

What role does protein microheterogeneity play in disease and development, and how can we use mass spectrometry to identify, confirm and accurately quantify post-translational modifications on histone proteins?

Over the years, mass spectrometry (MS) has emerged as a powerful analytical technique for the identification of unknown compounds, determination of molecular weights, elucidation of molecular structures and quantification of a wide variety of analytes.1,2 Although mass spec-trometry has been applied to protein biomarker discovery for at least a decade, one of the most difficult problems has been the precise quan-tification and characterization of post-translational modifications and isoforms detected in “shotgun” LC-MS/MS experiments.3,4 Recently, a different type of MS experiment, selected reaction monitoring (SRM), is increasingly being applied to the measurement of protein biomarkers.5 Advantages of SRM-based MS experiments include very high through-put, sensitivity, specificity and absolute quantification using internal isotopically labeled standards. SRM experiments are also a powerful technique for the measurement and quantification of post-translational modifications (PTMs) and disease-specific alterations. MS is the only technique that can deliver the specificity required to detect isoforms associated with protein sequence microheterogeneity and many clini-cally relevant variants.6 Such assays could potentially be used for disease diagnosis and prognosis, and they are typically robust and selective, even in complex matrices.7

In our laboratory at BRIMS and in collaboration with Victoria Lunyak's laboratory at the Buck Institute for Research on Aging, we have devel-oped workflows for label-free, high-resolution LC-MS/MS quantitative global profiling and SRM targeted analysis of histones, histone PTMs and histone modification enzymes.

Histone post-translational modifications (PTMs) are a central theme in the regulation of gene expression. A rapidly growing list of modifica-tions confirms that they play fundamental roles in chromatin modeling processes. These processes are also thought to play a role in stem cell development, cellular senescence and organismal aging.8 To date, most studies in this area have been carried out by genomic analysis, immu-nostaining or top-down LC-MS/MS analysis, where the last two methods are not fully quantitative.Global discovery experiments for PTMs in histones and histone modification enzymes. We applied MS to discover and quantify post-translational modifications in senescent (S) and self-renewing (SR) mes-enchymal stem cells derived from human adipose tissue. LC-MS/MS data were acquired for global discovery experiments using a novel Two-Pass Workflow. This approach was taken, because we found that the chro-matography and instrument methods for optimal full-scan quantitative measurements conflicted with methods for optimal fragmentation

Global profi ling and relative quantifi ction of histones, histone PTMs and histone-modifying enzymes in mesenchy-mal stem cells using LC-MS/MS and a novel PerfectPair mass difference algorithm

Mary F. Lopez,* David Sarracino, Michael Athanas, Bryan Krastins, Amol Prakash and Alejandra Garces; Thermo Scientifi c BRIMS Center; Cambridge, MA USA; *Email: mary.lopez@thermofi sher.com

scans. Therefore, we exploited the mass spectrometer’s mass accuracy and broad dynamic range by taking two distinct passes of data measurement. Pass 1 focused on acquiring uncompromised and optimized full-scan (MS) data for highly reproducible quantification. This first full-scan quantita-tive pass was used to generate an inclusion list of potentially interesting features. The inclusion list was then utilized for targeted fragmentation scan (MS2) acquisition during the second pass of a subset of the data samples. Initially, five technical replicate injections from each condition (either self-renewing or senescent) mesenchymal stem cell lysates were processed in a full-scan optimized configuration. Each replicate set was analyzed using the SIEVE PerfectPair processor, a frame annotation algorithm that determines pairs of frames (features) that are related in mass and retention time differences (Fig. 1). Peak candidate pairs were collected where the mass difference of the pairs was consistent with methylation to within 7 ppm. An inclusion list was generated from these pairs for Pass 2 analysis. This approach was extremely fruitful and allowed us to identify new combinatorial patterns of modifications occurring on histones as well as differential representation of histone-modifying proteins that have not been previously reported in conjunc-tion with ex vivo models of either somatic or stem cell aging (replicative or genotoxic stress-induced senescence).SRM targeted experiments for the quantification of specific post-translational modifications in histones. Chromatin structure plays a key role in DNA damage repair. The response to double-strand breaks (DSB) induced by radiation or DNA-damaging agents typically results in the DNA damage response (DDR) and accumulation of the proteins involved in DNA repair in subnuclear foci. The rapid phosphorylation of Ser139 at the C terminus of the H2AX sequence functions in the recruitment of DDR proteins after DNA damage. This process is also thought to play a role in stem cell development and senescence. We developed a multiplexed SRM assay to precisely measure and quantify the phosphorylated peptide(s) as well as non-phosphorylated pep-tides from elsewhere in the H2AX sequence. The target peptides were differentially expressed in nuclear sample preparations from HCA2 cells exposed to the DNA damaging agent Bleomycin, with the high-est expression occurring shortly after exposure (Fig. 2). This result is consistent with the hypothesis that �H2AX serves as a key regulator of the DDR.Conclusion. Recent advances in mass spectrometry and bioinformat-ics have provided a powerful set of new tools for the analysis of protein isoform heterogeneity and PTMs. Application of these technologies to histones and histone-modification proteins will rapidly advance our understanding of epigenetic mechanisms associated with cellular development and aging.

Lopez


Do not distribute.


Insier

Figure 1. Screen capture illustrating the PerfectPair viewer. Two frames with differences in M/Z and retention time that are consistent with phosphor-ylation are shown.

References

1. Kassel DB. Chem Rev 2001; 101:255-67; PMID:11712247; http://dx.doi.org/10.1021/cr990085q.2. Feng X, et al. Anal Bioanal Chem 2007; 389:1341-63; PMID:17701030; http://dx.doi.org/10.1007/s00216-007-1468-8.3. Aebersold R, et al. Nature 2003; 422:198-207; PMID:12634793; http://dx.doi.org/10.1038/nature01511.4. Kamath KS, et al. J Proteomics 2011; Epub ahead of print; PMID:21983556; http://dx.doi.org/10.1016/j.jprot.2011.09.014.

5. Prakash A, et al. J Proteome Res 2010; 9:6678-88; PMID:20945832; http://dx.doi.org/10.1021/pr100821m.6. Borges CR, et al. Clin Chem 2010; 56:202-11; PMID:19926773; http://dx.doi.org/10.1373/clinchem.2009.134858.7. Lopez MF, et al. Clin Chem 2010; 56:281-90; PMID:20022981; http://dx.doi.org/10.1373/clinchem.2009.137323.8. Lunyak VV, et al. Hum Mol Genet 2008; 17(R1):R28-36; PMID:18632693; http://dx.doi.org/10.1093/hmg/ddn149.


Do not distribute.


Insier

Figure 2. The semi-tryptic (one missed cleavage) C-terminal peptide KATQASQEY and its phosphorylated form were monitored across three nuclear extract sample groups exhibiting a range of DNA DS break damage induced by the DNA damaging agent Bleomycin. Group 1, no DNA damage; Group 2, early after DNA damage; Group 3, DNA DSB . The quantitative amounts of the phosphorylated and non-phosphorylated peptides varied across the groups. Group 2 (early after DNA damage) had the highest expression, with a quantitative ratio of 1.0:2.4:1.3 across groups 1, 2, and 3 This protein expression pattern demonstrates rapid phosphorylation of the Ser139 residue occuring after DNA damage.


Do not distribute.


Insier

How do the physical sciences contribute to regenerative medicine and, in a broader context, to research on aging and age-related disease?

Progress in stem cell research has seen the merger of the physical with the life sciences. Much collaboration at this interface has yielded some remarkable achievements for transforming day to day basic biologi-cal/biomedical research to that with clinical relevance. In the present context, many aspects of stem cell biology, with particular emphasis on aging, were addressed at the basic biological level, from a cellular and whole organism standpoint, revealing many stem cell features we still do not clearly understand. One scenario, in which some stem cell characteristics could be understood, may possibly emerge through the fabrication of synthetic three-dimensional scaffolds that mimic

Biosprays: From the biomedical to the clinical sciences

Suwan N. Jayasinghe; BioPhysics Group; Department of Mechanical Engineering; University College London; London, UK; Email: [email protected]

Figure 1. Characteristic digital images of (A) cell electrospinnning and (B) bio-electrospraying (this particular image also illustrates discharging which takes place at elevated applied voltages and which was observed to have no eff ect on the jetted cells in comparison to controls). Panels (C) and (D) depict the cell electrospun scaff old with multiple cell types and bio-electrosprayed spherical cell-bearing beads, respectively.

Jayasinghe

native microenvironments.1

Therefore, the generation of highly ordered and controlled synthetic living scaffolds require the physical sciences to develop techniques along-side biomaterials (and other exotic molecules) for directly handling living cells with a biopolymer for forming synthetic tissues that could model native tissue.2,3 In addition, these structures demand precision both in the placement of a given cell type in relation to other cells and/or molecules in true three-dimensional space. Hence, to this end, my laboratory has developed the now well-established biotechnology known as cell electrospinning (sister technology of bio-electrosprays) to directly handle living cells in any permutation and combination, with a


Do not distribute.


Insier

wide range of molecules and micro-nanomaterials2,3 for the fabrication of three-dimensional synthetic tissues (Fig. 1). These advances have made it easier for one to appreciate the far-reaching consequences of coupling stem cell biology with advanced cell handling technologies for investigating many aspects most relevant to basic biology and the clinic.4

The physical sciences, in their many manifestations, continue to demonstrate great impact on the health sciences to date. An exempli-fication was summarized above, while another such notable advance-ment in the physical sciences currently undergoing rapid development for the life sciences, are aerodynamically assisted bio-jets.5 The technol-ogy of aerodynamically assisted bio-jets is being investigated for retro-fitting existing flow cells in flow cytometry and for the development of a novel high throughput three-dimensional tissue development and analysis technology. This has implications, from the development of models for understanding age-related diseases (at given stages), to the possible development of drugs (Fig. 2). There are many more implica-tions with such a platform technology.4

Development of novel direct cell handling approaches as those briefly described here have significant applications in basic biology and the clinical sciences. The ability to stack cell-bearing droplets (containing either single or controlled mixtures of cells with micro/nanomaterials)

Figure 2. Panels (A) and (B) schematically represent the development of aerodynamically assisted bio-jets for the creation of synthetic three-dimensional tissues from a single to a multiple needle configuration as a high throughput screening technology, to the development of a sheathless flow cell for retrofitting existing cytometers, respectively. Panels (C) and (D) are, respectively, a representative high-speed digital snap shot and a micrograph of the aerodynamically assisted bio-jet in action and a fabricated three-dimensional multi-cellular tissues.

on one another gives the ability not only to create synthetic fully func-tional tissues in three-dimensions, but also to allow the development of controlled model systems (Fig. 1C and D, Fig. 2D). These have, at present, demonstrated the ability to model some age-related diseases, such as some cardiac, autoimmune and cancers. In parallel, the technol-ogy is undergoing fine-tuning to enhance a whole host of its features. Interestingly, these biotechniques have also been validated for their fea-sibility with whole organisms.6 Fabricated, pre-orientated, model-based studies will not only enable the study of intricacies associated with a wide range of basic biology and disease biology but will also reduce the scarification of larger scale rodent model driven studies, enabling more humane research to be carried out. In addition, the incorpora-tion of the aerodynamically assisted bio-jets into flow cytometry will enhance existing cytometers by reducing its present footprint further while also removing the need for sheath flow. Similar to this technol-ogy, its electric field-driven counterpart, namely bio-electrosprays, has the capacity tof handle highly viscous media with both cells (as singular or heterogeneous cellular populations) and embryos while retaining the ability to form fine cell-bearing droplets. This technology contrib-utes to single cell and embryo analysis as well as diagnostics. From a tissue reconstruction standpoint, the techniques elucidated herein offer the ability to directly rebuild a three-demsnional functional tissue


Do not distribute.


Insier

requiring less bioreactor time, as cells and/or molecules are already within the generated structure. Therefore, combining all these features could see these technologies as cell thereapies, from repairing, replac-ing and rejuvenating damaged or aging tissues/organs to combating diseases through the controlled and targeted delivery of experimental and/or therapeutic genes.7

References

1. Bartolovic K, et al. Analyst 2010; 135:157-64; PMID:20024196; http://dx.doi.org/10.1039/b917813a.2. Townsend-Nicholson A, et al. Biomacromolecules 2006; 7:3364-9; PMID:17154464; http://dx.doi.org/10.1021/bm060649h.

3. Jayasinghe SN, et al. Small 2006; 2:216-9; PMID:17193023; http://dx.doi.org/10.1002/smll.200500291.4. Jayasinghe SN. Analyst 2011; 136:878-90; PMID:21271004; http://dx.doi.org/10.1039/c0an00830c.5. Arumuganathar S, et al. Biomed Mater 2007; 2:158-68; PMID:18458450; http://dx.doi.org/10.1088/1748-6041/2/2/015.6. Joly P, et al. Biomicrofluidics 2009; 3:44107; PMID:20216969; http://dx.doi.org/10.1063/1.3267044.7. Ward E, et al. Analyst 2010; 135:1042-9; PMID:20419255; http://dx.doi.org/10.1039/b923307e.


Do not distribute.


Insier

How will the topic you presented help move this field of research forward?

I presented a series of examples illustrating how the computational approaches we are developing in my lab can be applied to the analysis of genome sequence, chromatin and gene expression dynamics. This was all done in the context of a collaborative study with the group of Dr. Victoria Lunyak on the changes in genome structure, chromatin and gene expression that accompany the aging of adult human stem cells.

How are systems biology approaches being currently applied in the field of regenerative medicine?

The paradigm of systems biology is that biological systems are inte-grated wholes that cannot be adequately understood through a reduc-tionist approach alone. In other words, in addition to understanding the individual parts of a given biological system you must also appreciate how it is that the parts act together to encode the functional whole, whether that whole is a macromolecular protein complex, an entire cell or even an entire tissue or organ system. With respect to regenerative medicine, it is of course critically important to gain an understanding of the biochemical and molecular biology phenomena that underlie the processes of tissue aging and decay. However, the classic experimental approaches that yield such knowledge should also be scaled up to the entire genome and then integrated across levels of biological organiza-tion. Thus, traditional molecular biology must be complemented with a higher-level integrated systems approach in order to truly understand what goes on at the level of tissues and organ systems. Once such an integrated level of understanding has been achieved, then the probabil-ity of successfully intervening in, and possibly even reversing, the pro-cess of tissue degeneration is greatly increased. In short, a systems level understanding of how tissue function declines with aging and disease will help to facilitate medical interventions aimed at tissue regeneration.

The protein coding genes represent only about 2% of the genome. Are we computationally ready for exploring the poorly annotated genomic “dark matter” or “junk DNA,” and if so, can we computationally predict its function?

Analysis and understanding of the functional relevance of the non-coding portion of the genome, particularly transposable element-derived repetitive DNA sequences, has been and continues to be one of the great open challenges in genomics research. Because it

Computational approaches for the transcriptomics, proteomics and epigenetic analysis in adult stem cells

King Jordan; Email: [email protected]

Jordan

was so difficult to understand what these kinds of sequences may be doing in the genome, they were initially dismissed as "junk DNA" with little or no functional relevance. Fortunately, thanks to the work of numerous labs worldwide, it is now abundantly clear that trans-posable element derived repetitive sequences can and do play a variety of critical functional roles for their host genomes, i.e., they are no longer considered as merely "junk DNA" but rather as legitimate players in the regulation of the genome.

Nevertheless, these transposable element-derived sequences still pose a fundamental challenge for computational analysis based largely on the fact that they are repetitive. All of the high-throughput array-based and sequence-based functional genomics assays require specific hybridization between probe and target sequences. Probe sequences can be oligonucleotides laid down on an array or short sequence tags characterized with next-generation sequencing techniques, while target sequences are the messenger RNA or genomic DNA sequences to which these probes map. Repetitive sequences often violate this principle and are thus discarded and simply ignored in subsequent analytical steps. My lab has been developing algorithms that "rescue" multi-mapping sequence probes and, in so doing, allow the transpos-able element-derived repetitive fraction of the genome to be included in genome-level and systems biology analyses.

With respect to the notion of a computational prediction of func-tion for transposable element-derived sequences, I feel the need to be cautious and circumspect in terms of the power of the computational approaches that we use. To whatever extent possible, everything we do in my bioinformatics is based on the analysis of real data rather than ab initio predictions. In other words, even though we use computers in our research, we are empiricists, not theorists, and we always let the data guide any conclusions that we may make. Of course, there is an impor-tant place for prediction in these computational research efforts, but we try to be cautious to ground our predictions in empirical data and, whenever possible, to experimentally validate any predictions that we make. Computational analysis is absolutely essential to high-through-put and systems biology approaches to regenerative medicine, but the experimental approach has been and will remain, the gold standard for generating knowledge in biological research.


Do not distribute.


Insier

What are the roles of DNA methyltransferases (DNMTs) during stem cell aging?

Although DNA demethylation is required to maintain stem cell proper-ties, it has been reported that the role of DNMTs are distinct between embryonic stem cells (ESCs) and adult stem cells. Our recent findings agree that the levels of DNMT1 and DNMT3b decrease, while human umbilical cord blood stem cells (hUCB-SCs) lose their self-renewal and multipotency. However, 5-azacytidine (5-AZA), an inhibitor of DNMT analogous to cystidine, rapidly induced cellular senescence of hUCB-SCs through activation of p16INK4A and p21CIP1/WAF1. The epigenetic characteristics of adult stem cells, as opposed to ESCs, are likely to be more susceptible to DNMTs due to the presence of differential mecha-nisms. Moreover, DNMTs could lead to a delay of the aging process of adult stem cells.

The secret lives of stem cells: Unraveling the molecular basis of stem cell aging

Kyung-San Kang; Adult Stem Cell Research Center; College of Veterinary Medicine; Seoul National University; Seoul, Republic of Korea; Email: [email protected]

Kang

What is the role of stem cell aging in human aging processes?

In terms of stem cells, cellular senescence can be described as a loss of self-renewal, and bypassing replicative senescence is a crucial step for the maintenance of stem cell self-renewal. Loss of self-renewal leads to the breakdown of cellular and tissue homeostasis and the impairment of tissue maintenance and repair. In other words, the regulation of senescence is important not only to extend self-renewal and ex vivo expansion of stem cells, but also to comprehend and fur-ther promote the differentiation and tissue regeneration potential of stem cells. Given that senescence-mediated diminution of regenerative potential of tissues might be caused by the loss of self-renewal in tis-sue-specific adult stem cells, the mechanism of adult stem cell aging in vivo or in vitro is important to understand the regulatory mechanisms of human aging.

How do we get the truly innovative science front and center when it comes to venture capital? You have crafted the term "technology dropped at the firehouse"—is there hope for these "orphans" to be adopted? How do you envision a strat-egy for successful funding?

Attempts to commercialize early-stage bioscience often fail because money is not available. Yet some of the science behind these early-stage companies is good, even excellent. And the social value of the science, which is generally not considered by investors, can be consid-erable. A drug or device that has social value may not be an attractive investment because the cost of development is high or the regulatory obstacles are formidable.

A new drug for amyotrophic lateral sclerosis (ALS), for example, might improve the outcome for the 200,000 patients in the US who suffer from this disease. Very little is available. Riluzole is the only drug approved for the treatment of ALS. It postpones the onset of neuro-muscular symptoms in the SOD mouse model of ALS…for a few days and, even then, only when administered before the onset of symptoms. Clearly, the bar for approval was very low. A new drug for ALS that

Orphan technology: Unfunded early-stage bioscience is dropped off at the fi rehouse

Ronald Landes; Landes Bioscience; Austin, TX USA; Email: [email protected]

Landes

has greater benefit clearly has social value. In fact, an ALS drug developed by a start-up in Israel, Immunity Pharma, is ready for Phase 1 clinical tri-als. When given to SOD mice after the onset of, not before the onset as with Riluzole, it increases survival by 15%. But Immunity Pharma has been unable to raise the $2M–$3M needed to get started.

Investors do not make investment decisions based even in small part on social value. Investors may, however, invest with appropriate incentives. There is a positive role for government here.

Bruce Booth of Atlas Ventures has suggested a couple of programs: (1) a marketplace for tax-deductible Net Operating Losses (NOL), where bioscience industry NOLs could be sold to profitable companies as tax shields and (2) an ultra long-term capital gains rate < 10% for investments of 7–10 years by what he calls "patient capital" (almost an oxymoron).

We must find ways to create safe havens for the commercialization of excellent science with enduring social value.

the buck institute: an inside look sj, cell cycle. 2011...stem cell and somatic cell aging and the...

Documents