REJUVENATION RESEARCHVolume 11, Number 3, 2008© Mary Ann Liebert, Inc.DOI: 10.1089/rej.2008.0748

Dissertations

A Survey of Selected Recent Theses Relevant to Combating Aging

Aubrey D.N.J. de Grey

WITH THIS ARTICLE I CONTINUE THE SERIES, begun in Volume 10, of surveys highlighting a small selection of recently com-pleted doctoral theses with particular relevance to the fields covered by Rejuvenation Research.1–4 While it has become

common for thesis work to appear in the general academic literature, it remains valuable to scan the thesis databases forimportant advances that one might otherwise have missed.

Age-related Changes in Neural Stem Cell Properties and Cellular Composition inNeurogenic Niches of the Adult Rat Brain

Tamuna Chadashvili, Ph.D.Rosalind Franklin University of Medicine and Science, 2007

Neurogenesis persists in two distinct regions of the adult mammalian brain, the subventricular zone (SVZ) of the forebrainand the subgranular zone (SGZ) of hippocampal dentate gyrus. In the SVZ, neural stem/precursor cells proliferate, aggre-gate into long chains, and migrate rostrally toward the olfactory bulb via the rostral migratory stream (RMS). The migra-tory neuroblasts proliferate as they traverse the RMS and give rise to granule and periglomerular neurons of the olfactorybulb throughout life. We investigated age-related changes in the neurogenic microenvironment and the neural stem/pro-genitor cell properties in the aging SVZ/RMS. Results revealed a decline in neural stem/progenitor cell proliferation in theSVZ/RMS of mid-aged (12 month old) and aged (22–24 month old) rats compared to young (2 month old) animals. To un-derstand the possible mechanisms for age-related decline in proliferation, we investigated changes in cell cycle kinetics inthe aging RMS and found no changes in cell cycle length. We used Sox-2, a putative neural stem/progenitor cell marker,to evaluate age-related changes in the neural stem cell pool in the aging RMS. Our results showed age-related decline inthe total number of Sox-2-immunolabeled cells, indicating that the reduced pool of neural/stem progenitor cell may con-tribute to the age-related reduction in proliferation. Neurogenic microenvironment undergoes age-related alterations. FGFR-2, a receptor for FGF-2, shows robust expression in neurogenic regions, but is significantly attenuated in the aged brain, in-dicating that decline in FGF-2 signaling may contribute to age-related reduction in neurogenesis. To further explore theenvironmental changes in the neuroenic niches, we evaluated blood vessel density in the aging SVZ/RMS. We found sig-nificant positive correlation between the blood vessel density and neural stem/progenitor cell proliferation in the SVZ ofthe young, mid-aged, and aged brains, suggesting that blood vessels contribute to the neurogenic microenvironment andcontinue to play an important role throughout the aging process. Ablation of the frequently cycling cell population in theRMS with the anti-mitotic drug Ara-C revealed the presence of the endogenous slowly cycling neural stem cell populationin this germinal region of the young and aged brains. This finding suggests that the RMS is a discrete neurogenic regionthat harbors slowly cycling neural stem cells rather than being simply an inert conduit for migratory neuroblasts. Our re-sults also indicate that this region of the aged brain retains the neural stem cell population, which could potentially be stim-ulated for therapeutic brain repair and regeneration.

Comment: There are many reasons why the brain is the most challenging of all our organs to rejuvenate; a major one is the apparentabsence of neurogenesis in most brain regions, without which repair (as opposed to mere retardation5) of damage is limited to such pro-cesses as synaptogenesis6,7 and remylenation.8 Research is ongoing to stimulate neurogenesis in these regions,9 but, as ever, this effortwill be facilitated by the exploitation of improved knowledge concerning what related processes already exist. This elucidation of neu-rogenesis in a region previously presumed to have none is thus a small but significant step forward.

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The Link Between Aging and Tumor Growth in Caenorhabditis elegans

Julie Maddox, Ph.D.University of California, San Francisco, 2007

In nature, there is a strong correlation between physiological aging and tumor susceptibility. Understanding how youthfulanimals resist tumors may provide new insights into tumor biology, particularly if genes that regulate aging influence tu-mor susceptibility. Mutations in the C. elegans gene gld-1 produce a lethal germline tumor. We find that mutations that ex-tend C. elegans’ lifespan, by caloric restriction, mitochondrial dysfunction, or a reduction in daf-2 /insulin/IGF-1 signaling,confer resistance to these tumors. Remarkably, mutations that reduce daf-2 function, which double the lifespan of worms,can completely prevent the shortened lifespan caused by these tumors. daf-2 mutations protect tumorous animals by trig-gering apoptosis through the FOXO transcription factor daf-16 and the tumor suppressor p53, and by activating daf-16 de-pendent and independent processes that inhibit tumor cell division. Interestingly, the inhibition of mitosis is specific to tu-mor cells. Together these findings suggest a fundamental link between mechanisms that promote cell maintenance andanimal longevity and mechanisms that inhibit tumor cell proliferation. To identify downstream components of this pro-cess, we have screened 734 potential daf-16 /FOXO targets and found 16 genes that are necessary for daf-2 mutations tofully suppress tumor growth, and 14 genes that normally promote tumor growth. Approximately one quarter of these genesare known to have roles in human cancer, suggesting that the other genes may be involved in mammalian cancer as well.

Comment: It is often said that the best-studied invertebrates, C. elegans and D. melanogaster, are poor models for many aspects of ag-ing in mammals because the post-mitotic state of the adult soma in these species precludes tumor formation. There is a measure of truthin this logic in that the trade-offs between cancer and other aspects of aging are greatly altered; hence, the differences in anti-cancerpathways between these animals and mammals are probably even greater than those between mice and humans, which are themselvessubstantial, making the development of therapies challenging.10–14 However, the mitotically active germline (and pre-adult) tissues inboth species provide ample opportunity to explore tumorigenesis and its avoidance in these highly tractable organisms. Even simplis-tic models of tumor formation, such as the single-gene model mentioned above, can provide powerful tools to identify more general path-ways, as demonstrated in this study.

Characterization of Human Mesenchymal Stem Cells and Therapeutic Application to aXenotransplant Model of Mucopolysaccharidosis Type VII

Todd Meyerrose, Ph.D.Washington University in St. Louis, 2007

Since their original description as hematopoietic support cells, human mesenchymal stem cells (MSC) have been redefinedas putative stem cells, with various research demonstrating evidence of these cells as the progenitors of not only the myelo-supportive marrow stroma, but also bone, muscle, cartilage, and adipose tissue. This flexibility of lineage commitment pro-vides potential for therapeutic application in cellular or gene therapy. To evaluate the feasibility of using MSC for cell-basedtherapy, we designed a xenotransplant model using the non-obese diabetic severe combined immunodeficient (NOD-SCID)mucopolysaccharidosis type VII (MPSVII) mouse as a recipient. MPSVII is a cumulative, degenerative disease, resulting froma genetic lack of the enzyme beta-glucuronidase (GUSB). GUSB deficiency causes the progressive accumulation of cellularwaste material within lysosomes, leading to multiple severe clinical manifestations in nearly every organ system. Severaltherapies have shown moderate success with delivery of GUSB to affected cells and subsequent cellular uptake of enzymein a process termed “cross-correction.” Efficient cross-correction requires enzyme delivery throughout the body; therefore,the initial study examined the fate of human MSC following transplantation into NOD-SCID mice. Importantly, human MSCwere able to traffic to and persist within multiple tissues, regardless of germline origin. Once distributed, donor cells mustproduce and secrete sufficient enzyme for cross-correction to occur. Extensive in vitro studies were performed to measurethe production and secretion of GUSB from normal MSC and MSC lentivirally engineered to constitutively express GUSB.Early intervention was accomplished by transplantation of MSC into neonatal mice. At 10 and 20 weeks following trans-plantation, animals were assayed for persistence of donor cells, transgene expression, and cross-correction of pathology.Transgene-expressing donor cells were detected in multiple tissue beds, which, combined with lentiviral overexpression ofGUSB, was sufficient to produce moderate cross-correction of MPSVII disease. This was evident through biochemical andhistological examination of multiple tissues, and functional tests of a well-described visual defect associated with MPSVIIdisease. The work of this dissertation demonstrates that the unique characteristics of MSC, coupled with modern molecularengineering techniques, may provide a viable avenue for successful cellular or gene therapy intervention of disease.

Comment: Endocytosis of circulating enzyme is the basis for the standard therapy for lysosomal storage diseases, namely, enzyme re-placement therapy (ERT), in which large quantities of judiciously glycosylated recombinant enzyme are injected intravenously so thataffected cells can traffic a small proportion to the site of accumulation of the offending material.15 Some major diseases of aging are alsoultimately storage diseases,16,17 which could in theory be addressed using similar methods as those used on non-human enzymes.18–21

Cross-correction is potentially a great improvement on ERT since delivery would be continuous and only one-time invasive. Since there

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is now a good understanding of how to target a given protein for secretion, this may soon be a generally applicable approach to thetreatment of such diseases.

Examination of the Engraftment of Exogenous Mesenchymal Stem Cells in OvarianTumors and Their Potential Use as Delivery Vehicles for Therapeutic Genes

Jennifer Dembinski, Ph.D.University of Texas Graduate School of Biomedical Sciences at Houston, 2007

Interactions between neoplastic cells and the host stroma play a role in both tumor cell migration and proliferation. Stro-mal cells provide structural support for malignant cells, modulate the tumor microenvironment, and influence phenotypicbehavior as well as the aggressiveness of the malignancy. In response, the tumor provides growth factors, cytokines, andcellular signals that continually initiate new stromal reactions and recruit new cells into the microenvironment to furthersupport tumor growth. Since growing tumors recruit local cells, as well as supplemental cells from the circulation, such asfibroblasts and endothelial precursors, the question arises if it would be possible to access circulating stromal cells to mod-ify the tumor microenvironment for therapeutic benefits. One such cell type, mesenchymal stem cells (MSC), could theo-retically be engrafted into stroma. MSCs are pluripotent cells that have been shown to form stromal elements such as my-ofibroblasts, perivascular tissues, and connective tissues. Several reports have demonstrated that MSC can incorporate intosites of wound healing and tissue repair, due to active tissue remodeling and local paracrine factors. Given the similaritybetween wound healing and the carcinoma-induced stromal response, one can hypothesize that MSCs have the potentialto be recruited to sites of tumor development. In addition, gene-modified MSC could be used as cellular vehicles to delivergene products into tumors. My results indicate that MSCs home to and participate in tumor stroma formation in ovariantumor xenografts in mice. Additionally, once homed to tumor beds, MSC proliferate rapidly and integrate. My studies aimat understanding the fate of MSCs in the tumor microenvironment, as well as utilizing them for cellular delivery of thera-peutic genes into the stroma of ovarian carcinomas.

Comment: One beauty of biotechnology is its modularity—the frequency with which an advance with some particular intended appli-cation can then be used, often with hardly any change, in a totally unrelated application. The contrast between this study and the pre-vious one is particularly striking in this regard: same cell type,22 same concept (secretion by one cell, uptake by a neighboring one), butnow used to do bad things to the recipient cell rather than good things. It is clearly too soon to say whether this approach will ulti-mately prove to be a powerful anti-cancer therapy, comparable for example to delivery of the same proteins by gene therapy,23 but any-thing that provides a new way to deliver drugs to the deep interior of a solid tumor, especially such a biomedically critical target asovarian cancer,24 is surely worth pursuing.

Analysis of Spontaneous Mutation Frequency and Spectrum in Cloned Mice

Patricia Murphey, Ph.D.University of Texas at San Antonio, 2007

Therapeutic cloning by somatic cell nuclear transfer (SCNT) holds great promise as a vehicle for patient-specific, stem cell-based treatment of a variety of diseases or debilitations. However, this approach will require the use of donor somatic cellsand manipulations in vitro of enucleated oocytes and reconstituted embryos from which embryonic stem (ES) cells can bederived. This is the first work to examine the potential introduction de novo of mutations as a result of the cloning process.To address this, we utilized the Big Blue transgenic mouse system from Stratagene to determine the spontaneous mutationfrequency and spectrum in germ cells, fetuses generated by natural mating or assisted reproductive technologies, three dif-ferent somatic donor cell types typically used for cloning, and cloned fetuses produced from each of these donor cell types.Results from this study show that the spontaneous mutation frequency in female germ cells is similar to that found in malegerm cells at fetal and neonatal stages, and that these frequencies are significantly lower than those in developmentallymatched somatic cells. This result suggests that SCNT embryos produced from somatic cells might carry more mutationsthan those produced by natural reproduction. However, our study found that the frequency of mutations in fetuses gen-erated by assisted reproductive technologies is similar to the frequency found in fetuses conceived naturally, indicating nei-ther maintenance nor manipulation of embryos in vitro introduce additional mutations in surviving embryos. Finally, re-sults of our study of cloned fetuses showed that embryos generated by SCNT maintain a spontaneous mutation frequencycomparable to embryos produced by natural conception, irrespective of the donor cell typed used, due to a “bottleneck ef-fect” that limits propagation of acquired mutations during natural reproduction, assisted reproduction, or cloning. Impor-tantly, our results show that because cloned fetuses maintain a relatively low frequency of point mutations (at least at asingle test locus), comparable to that found in fetuses produced by natural reproduction, epigenetic mechanisms responsi-ble for maintaining genetic integrity appear to be reprogrammed during the cloning process. Collectively, these results sug-gest that the genetic integrity of stem cells derived from therapeutically cloned blastocysts will be similar to that of stemcells derived from blastocysts produced by natural conception or assisted reproductive technologies. This study, conducted

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in a model transgenic system, represents an important step in the examination of the safety of therapeutic cloning beforeapplication to human patients is considered.

Comment: The maintenance of mechanisms that either prevent or eliminate mutations during the creation and manipulation of cells des-tined for therapeutic use is essential if such therapies are to achieve their biomedical potential. There has been evidence in the past, con-firmed here, that the germline is unusually well protected against mutation accumulation (as it must be, according to the disposable somatheory); luckily, there is also evidence that in most tissues the mutation accumulation rate slows during adulthood relative to development,even to undetectable rates in various tissues.25–27 Thus, there is currently good reason for optimism that highly undifferentiated cells gen-erated from adult cells (whether by nuclear transfer28,29 or by direct dedifferentiation30) will indeed be of great therapeutic utility.

Biochemical Characterization of hTRF1 and hTEP1, Two Proteins Involved in Telomere Maintenance

Kambiz Tahmaseb, Ph.D.Wright State University, 2007

Telomeres are the structures that protect the ends of linear chromosomes from fusion and degradation. The telomere con-sists of tandem repeated DNA sequences that can range from hundreds of bases to kilo-bases, depending on the organism.As the cells of an organism replicate their DNA, these repeats are lost due to the end replication problem, where the endsof linear DNA cannot be fully replicated. As the telomeres are shortened through each round of replication, they eventuallyreach a critical point. Once the telomeres are too short and the cell risks losing coding sequences, a signaling pathway is ini-tiated that causes the cell to senesce. However, cells that require continuous replication (i.e., stem cells, germ cells, and can-cer cells) require constant maintenance of their telomeres in order to not enter senescence. The majority of these cells use themultimeric protein telomerase and a host of other proteins to maintain the lengths of their chromosomes. Eukaryotic telo-merase is a nucleoprotein complex consisting of the telomerase RNA (TR), telomere end reverse transcriptase (TERT), andtelomerase associated protein 1 (TEP1). Furthermore, telomeric length is regulated by a host of telomeric binding proteins.This thesis focuses on two proteins important for human telomeric maintenance. The first is human TEP1 (hTEP1), which isa subunit of telomerase. This large protein contains the RNA binding domain that binds hTR. Though the RNA binding sub-unit of hTEP1 has been partially purified before, full-length hTEP1 has been refractory to biochemical analysis due to the in-ability to express and purify this large protein. Here we reveal the very first purification of full-length hTEP1. Furthermore,where the RNA binding domain of hTEP1 alone does not show specific interaction with hTR, we show that full-length hTEP1binds hTR specifically. The second protein of interest in this thesis is the human telomeric repeat binding factor 1 (hTRF1).This protein is one of the telomeric binding proteins that plays a critical role in telomere structure and stability. hTRF1 isalso important as a regulator of telomeric length. hTRF1 has been shown to bind telomeric DNA specifically, and my datareveals details of this surprisingly complex interaction using a sensitive intrinsic fluorescence kinetic technique. Our resultsdemonstrate that hTRF1 binds to both telomeric and non-telomeric DNA. However, hTRF1 exhibits different characteristicsas it binds telomeric DNA and is able to distinguish between telomeric and non-telomeric tracts of DNA. This new infor-mation on these two key players in the maintenance of telomeres will help us further understand how these complex DNAends are preserved in the cell. Through this knowledge, we can devise better tests in understanding how immortal cell lines,such as cancer cells, function and proliferate, making it possible to identify novel therapeutic targets to inhibit this process.

Comment: Telomeric structure is a classic example of an area of science that seemed for some while to be nearing completion and whichthen suddenly burst back into life. With de Lange’s discovery of the “t-loop” began the identification of a number of key players in telo-mere maintenance in addition to the two well-known subunits TR and TERT.31 Detailed structural and functional analysis of the telo-mere and its binding proteins is, as noted above, a highly promising route to the discovery of new ways to impede telomere elongationand thereby control and eliminate tumors while maintaining the functioning of tissues that rely on the periodic division of stem cells.32,33

It will be equally useful in other circumstances when the requirement is to stimulate telomere elongation.34,35

Bone Marrow Stem Cells to Regenerate Injured Murine Renal Tissue

Barbara Imberti, Ph.D.Open University (UK), 2007

Injury to a target organ can be sensed by bone marrow stem cells that migrate to the site of damage, undergo differentia-tion, and promote structural and functional repair. This stem cell capacity prompted us to investigate the potential of mes-enchymal and hematopoietic stem cells to aid regeneration after acute renal failure. We elected to use the model of renalinjury induced in mice by the anticancer agent cisplatin. Injection of mesenchymal stem cells of male bone marrow originremarkably protected cisplatin-treated syngeneic female mice from renal function impairment and severe tubular injury.Y-chromosome containing cells localized to the tubular epithelial lining and displayed binding sites for Lens culinaris lectin,indicating that mesenchymal stem cells had engrafted the damaged kidney and differentiated into tubular epithelial cells,

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thereby helping to restore renal structure and function. Moreover, mesenchymal stem cells markedly accelerated tubularproliferation in response to cisplatin-induced damage, as revealed by higher numbers of Ki-67 positive cells within thetubules in comparison to cisplatin-treated mice given saline. Hematopoietic stem cells failed to exert these beneficial effects.The mechanism by which mesenchymal stem cells prevent cisplatin-induced tubular cell damage was studied in vitro. Wefound that mesenchymal stem cells protected proximal tubular cells from cisplatin-induced damage. Indeed, mesenchymalstem cells, when in co-culture with cisplatin-treated tubular cells, induced tubular cell proliferation. This phenomenon wasmediated by the soluble factor insulin-like growth factor 1, expressed by mesenchymal stem cells, known for its mitogenicproperties and regenerative-promoting capacity. Therefore, mesenchymal stem cells exerted their beneficial effect on cis-platin-damaged tubular cells through a paracrine mechanism. These results offer a strong case for exploring the possibil-ity that mesenchymal stem cells, by virtue of their renotropic properties and tubular regenerative potential, may providethe basis for a new therapeutic modality for the treatment of acute renal failure in humans.

Comment: The kidney is a complex tissue in which different cell types exhibit a range of frequencies of cell division and failure cur-rently relies on transplant.36 Like all mitotically active tissues, it is vulnerable to collateral damage when a cancer patient receives treat-ment designed to target rapidly dividing cells, such as chemotherapy. The mesenchymal stem cell has shot to prominence in recent yearsas evidence of its remarkable versatility (multipotency) has continued to pile up.37–39 Thus, in a sense it is no surprise that the kidneyis another potential beneficiary of that effect—but it is certainly a relief.

Enrichment of Therapeutic Hematopoietic Stem Cell Populations from Embryonic Stem Cells

Jennifer Gilner, Ph.D.University of North Carolina at Chapel Hill, 2007

Embryonic stem cells (ESC) serve as a versatile and infinite source of primitive stem cells of any developmental lineage,made readily available for study or therapeutic application. The hematopoietic lineage in particular may benefit from sucha level of accessibility since therapeutic applications from conventional sources of hematopoietic stem cells (HSC) are of-ten limited by the inability to isolate or expand sufficient numbers of cells. However, the potential of alternative ESC-de-rived generation of HSCs has not yet been fulfilled. Our fundamental goal in the work presented in this dissertation wasto define and characterize a method for the isolation of transplantable HSC differentiated from ESC. Our initial study wasdesigned to isolate early hematopoietic progenitors based on flow cytometric sorting of cells expressing a lineage-restrictedfluorescent transgene. However, incomplete genetic regulation of the transgene at the necessary stage of differentiation pre-cluded its use as a molecular marker of HSCs. We then conducted studies in an adult source of HSC (i.e., bone marrow)to further characterize a stem cell enrichment method that separates a population of cells based on efflux of the DNA-bind-ing fluorescent dye Hoechst 33342 (side population cells [SP]). Our findings demonstrate that within the SPs, the exposureof cells to commonly used cell surface marker antibodies can reduce the engraftment of HSCs. Returning to our originalgoal of isolating ESC-derived HSCs, we applied the SP sorting method to early-stage embryoid bodies (EB) differentiatedfrom ESCs. Our results show that the SPs from early EBs contain long-term repopulating hematopoietic stem or progeni-tor cells, as evidenced by sustained (�16 weeks) lympho-myeloid engraftment of transplanted recipients. Furthermore, wehave found that inclusion of a truncated erythropoietin receptor transgene in ESCs used to derive the graft can improvethe efficiency of transplantation in a ligand-dependent fashion. In summary, we have employed genetic and cell sortingtechniques to successfully isolate and expand a population of HSCs differentiated from ESCs, and these studies may beuseful in the design of future therapeutic applications from embryonic stem cells.

Comment: A major reason why the ethical arguments against the use of embryonic stem cells have gained such traction is that themedical utility of such cells is acknowledged by researchers to be still quite far off.40 Arguably the primary obstacle to early therapiesis our poor state of knowledge of how to differentiate such cells reliably to just the right degree—specifically, into the mono- or oli-gopotent states that allow them to support a specific tissue of interest without also developing into multiple undesired and uncontrolledlineages (teratoma formation).41,42 The HSC has always been one of the most important targets for such work, given the wide range ofdiseases of the hematopoietic system and the limited options (including in aging, especially immunosenescence43,44) for treating them.This report of significant progress in that direction is highly welcome.

Temperature-responsive Hydroxybutyl Chitosan as a Dynamic Scaffold for Tissue Engineering and Regenerative Medicine

Jiyoung Dang, Ph.D.Johns Hopkins University, 2007

In the field of tissue engineering, the desire to recreate the complexity of the interplay between the cell and its surroundingmatrix has long been a driving force in biomaterials development. The natural matrix environment provides multiple cues

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that play a critical role in regulating cellular behavior. Reciprocally, cells are capable of modulating their environment throughsynthesis and degradation of matrix proteins. Hydroxybutyl chitosan (HBC) is a temperature-sensitive polymer with no pre-viously reported use in tissue culture systems. With a polysaccharide backbone, HBC should be an attractive candidate forartificial tissue matrices. The versatility of this polymer allows for exploration of different, dynamic scaffold platforms thattake advantage of its thermally responsive property. The ability of this polymer to transition from liquid to solid states withchanges in temperature can be exploited as a delivery vehicle for injectable therapies. Upon injection in vivo, the polymer so-lidifies in situ and entraps at the site of injection any biologically active moieties, relevant to regenerative tissue repair, in-corporated into the polymer solution. In vitro studies evaluate the efficacy of a stem cell-based therapeutic with a sustainedrelease of growth factors to support the growth of intervertebral disc cells. In vivo application of the HBC gel delivery sys-tem in a rabbit model confirms surgical feasibility. Combined, the results of these studies set a foundation for building anoptimal therapeutic for promotion of tissue regeneration and replacement. The HBC polymer can also serve as a culture plat-form that can be dissolved away, via cooling, leaving behind a polymer-free engineered cell sheet or tissue. The electrospunHBC aligned nanofibers provide a surface that is both biologically and topographically active. These aligned nanofibers areable to provide topographical cues for induction of cellular alignment as well as genotypic changes in mesenchymal stemcells. The resultant differentiated stem cells can be recovered as a cell sheet by dissolution of the culture substrate. The abil-ity to introduce topographical cues to create an aligned stem cell sheet, activated to differentiate to a specific lineage, mayhave a tremendous impact on engineering of tissue constructs for regenerative medicine applications.

Comment: Tissue engineering relies, arguably more than any other area of biotechnology, on the in-built ability of our cells to rectifyproblems in their environment.45–47 Our ability to fabricate structures in which cells can operate properly is still primitive, and onlywhen we approximate real tissue with reasonable fidelity can those cells finish the job. Accordingly, even though the mainstays of tis-sue engineering scaffolds have been with us for decades, there is still ample need for further fundamental discovery of new materialsthat our cells may find more acceptable.

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DISSERTATIONS 695

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