REJUVENATION RESEARCHVolume 12, Number 4, 2009© Mary Ann Liebert, Inc.DOI: 10.1089/rej.2009.0915

Commentary on Some Recent Theses Relevant to Combating Aging: August 2009

Aubrey D.N.J. de Grey

289

SENS Foundation, Cambridge, United Kingdom.

Dissertations

IN THIS ARTICLE I CONTINUE the series, begun in issue 10(1), of surveys highlighting a small selection of recently completeddoctoral theses with particular relevance to the fields covered by Rejuvenation Research.1–9 Although it has become com-

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

Biomimetic Composite Scaffolds for the Functional Tissue Engineering of Articular Cartilage

F. Moutos, Ph.D., Duke University, 2009

Articular cartilage is the connective tissue that lines the ends of long bones in diarthrodial joints, providing a low-friction,load-bearing surface that can withstand a lifetime of loading cycles under normal conditions. Despite these unique and ad-vantageous properties, the tissue possesses a limited capacity for self-repair due to its lack of vasculature and innervation.Total joint replacement is a well-established treatment for degenerative joint disease; however, the materials used in theseprocedures have a limited lifespan in vivo and will likely fail over time, requiring additional—and increasingly compli-cated—revision surgeries. For younger or more active patients, this risk is unacceptable. Unfortunately, alternative surgi-cal options are not currently available, leaving pain management as the only viable treatment. In seeking to discover a newtherapeutic strategy, the goal of this dissertation was to develop a functional tissue-engineered cartilage construct that maybe used to resurface an entire diseased or damaged joint. A three-dimensional (3-D) woven textile structure, produced ona custom-built miniature weaving loom, was used as the basis for producing novel composite scaffolds and cartilage tis-sue constructs that exhibited initial properties similar to those of native articular cartilage. Using polyglycolic acid (PGA)fibers combined with chondrocyte-loaded agarose or fibrin hydrogels, scaffolds were engineered with anisotropic, inho-mogeneous, viscoelastic, and nonlinear characteristics prior to cultivation. However, PGA-based constructs showed a rapidloss of mechanical functionality over a 28-day culture period, suggesting that the inclusion of other, less degradable bio-material fibers could provide more stable properties. Retaining the original 3-D architecture and fiber/hydrogel compositeconstruction, poly ([varepsilon]-caprolactone) (PCL)-based scaffolds demonstrated initial biomechanical properties similarto those of PGA-based scaffolds. Long-term culture of 3-D PCL/fibrin scaffolds seeded with human adipose-derived stemcells (ASCs) showed that scaffolds maintained their baseline properties as new, collagen-rich tissue accumulated within theconstructs. In an attempt to improve the bioactivity of the PCL scaffold and further induce chondrogenic differentiation ofseeded ASCs, we produced a hybrid scaffold system by embedding the 3-D woven structure within a porous matrix de-rived from native cartilage. We then demonstrated how this multifunctional scaffold could be molded, seeded, and cul-tured to produce an anatomically accurate tissue construct with potential for resurfacing the femoral head of a hip. In sum-mary, these findings provide valuable insight into a new approach for the functional tissue engineering of articular cartilage.The results of this work will hopefully lead to the discovery of new strategies for the long-term treatment of cartilage pathol-ogy.

Comment: Historically, treatments for damaged articular cartilage have tended to fall into two types—biological and nonbiological.In other words, either one to cajole the body’s existing machinery to do a better job than it naturally does to repair the material, or onesimply replaces the material with an artificial sunstitute. The loss of mechanical functionality referred to in the above abstract is, un-fortunately, still a feature of all nonbiological approaches. Conversely, the “cajoling” approach has thus far failed to meet the challengesposed by the fact that cartilage contains a remarkably low density of cells, and these cells exhibit particularly severe signs of senescence(similar in phenotype to replicative senescence). In this study, the author has adopted a hybrid approach, in which cells are seeded in-tyo an initially artificial scaffold. Such approaches are becoming increasingly attractive throughout tissue engineering.

Human Endothelial Progenitor Cells: A Novel and Promising Cellular Therapy for Regenerative Medicine

J. Wood, Ph.D., University of Nevada, 2009

Endothelial progenitor cells (EPCs) represent a novel and promising therapy for myriad tissues and conditions, includingdiseases and disorders of the liver and small intestine. Cirrhosis and other diseases have created a need for a readily avail-able supply of hepatocytes and supporting cells in diseased and scarred liver. Following chemo/radiation therapy and in-flammatory bowel disease, the cell populations of the small intestine are diminished and a cell therapy for the replenish-ment of these populations is needed. Additionally, the cellular makers to identify both EPCs and mesenchymal stem cells(MSCs) have been defined in the literature, but a debate remains as to the heterogenic versus homogenic nature of the cellpopulations. This dissertation investigates the engraftment potential of EPCs in the liver when transplanted (Tx) in uterointo the preimmune sheep model via two routes of injection, intrahepatic (IH) and intraperitoneal (IP). Upon finding en-graftment, the contribution of these cells to vasculature and parenchymal tissue as well as their differentiative potential incontribution to the developing liver was investigated. Tx EPCs engraft, albeit at low levels, but associate preferentially withvasculature. In addition to their association with vasculature, the EPCs maintain the expression of endothelial markers inaddition to expressing markers ranging from fully differentiated hepatic cells to liver stem cells. In addition to their con-tribution to the liver, EPCs not only engraft into the small intestine but do so in a preferential manner in the area contain-ing the crypts of Lieberkühn (above the muscularis mucosa and below the crypt–villus junction). Upon transplantation,these cells actively engraft and differentiate into both intestinal stem cells (ISCs) and into the supporting cell types of theISC niche, as well as mature cells of the intestinal parenchyma. Finally, linear amplification-mediated polymerase chain re-action (LAM-PCR) and ligation-mediated (LM)-PCR were employed to identify vector integration sites in both MSCs andEPCs transfected with a variety of retroviruses. These experiments are designed to address the existence of a heterogeneousor homogeneous population in both the EPC and MSC populations. Further testing on an experimental sample reveals thepresence of chimeric DNA in the sample, and successful amplification of integration sites in this sample is pending furtherinvestigation.

Comment: The conflict between the goals of maintaining proliferation of stem cells and restricting proliferation of cancer cells con-stitutes the most daunting challenge to the biomedical gerontologist. Approaches that have been proposed to resolve it have often fo-cused on management of telomere shortening, either by stimulating telomere elongation and combating cancer by other means, or bysuppressing telomere elongation and combating stem cell dysfunction by other means. The extreme form of the latter approach involvesgenetically eliminating all telomere elongation potential and compensating with periodic stem cell repopulation. Accordingly, the dis-covery described here that EPCs readily repopulate crypts is of great significance. The gut has historically seemed to be the most chal-lenging of all the continuously renewing tissues to replenish, ostensibly requiring surgery or at least highly invasive endoscopic ma-nipulation. The fact that this study was performed in sheep, a species whose telomere dynamics and telomerase profile resembles humans,further enhances its significance.

Synthetic and Endogenous Small Molecules That Regulate Stem Cell Fate

J. Clark, Ph.D., Scripps Research Institute, 2009

Given appropriate conditions, stem cells can self-renew for long periods of time while maintaining the ability to differen-tiate into various functional cell types in the body (Kim et al. 2007). These characteristics not only make stem cells a usefulsystem in which to study tissue and organ development but also give them great potential for regenerative medicine. Giventhe success of well practiced cell-based therapies (e.g., hematopoietic stem cell transplantation for hematological diseases(Adamkiewicz et al. 2006; Oringanje et al. 2009) and pancreatic islet cell transplantation for type I diabetes (Vaithilingamet al. 2008), it is conceivable that this approach could be applied to many other serious medical conditions where cells arelost as a result of disease, injury, or aging. Realization of the therapeutic potential of stem cells will require a better un-derstanding of the signaling pathways that control stem cell fate as well as an improved ability to manipulate stem cellproliferation, differentiation, and reprogramming. Work presented in this dissertation describes our efforts toward ex-panding our understanding of stem cell biology. Presented is a review of various discovery-based approaches (Zhao et al.2005), including high-throughput chemical approaches and mass spectrometry, in the stem cell field. In chapter two we re-port the implementation of an unbiased or untargeted high-throughput screen to identify a synthetic small molecule (JC12)that induces differentiation of murine embryonic stem cells to Lmx1a-positive dopamine neuron progenitor cells. The pro-genitor cells generated in the presence of JC12 express the combination of transcription factors (En1, Otx2, Lmx1a, Lmx1b,Msx1, Mash1, and Ngn2) associated with dopamine neurons with midbrain identity. Initial mechanism-of-action studiessuggest that JC12 does not function via the known hedgehog pathway but does involve the inhibition of Wnt signaling. Inchapter three we provide evidence for a hypothesis of stem cell regulation by metabolites herein termed the “plasticmetabolome.” Metabolites are endogenous small molecules that are the products of active biological pathways. Metabolomicanalysis complements transcriptome and proteome analysis. The metabolome is an important unexplored dimension to un-

DISSERTATIONS290

derstanding the pluripotent or “stemness” phenotype. We conclude with a review of small molecules applied to stem cellbiology with an emphasis on self-renewal, differentiation, and reprogramming. Within this review, we place the discoveryof JC12 and the “plastic metabolome” in the context of other small molecules.

Comment: While efforts to create extremely primitive stem cells (whether through nuclear transfer or by dedifferentiation) have oc-cupied the scientific headlines for some years, many stem cell researchers have been occupied with the opposite process—coaxing prim-itive cells along desired developmental pathways, so that they can repopulate specific tissues after transplantation. This is a huge chal-lenge, not least in view of the large number of different target states that may be of therapeutic value. Systematic approaches to thistask, such as discussed in this study, are thus urgently required.

Technologies Enabling Autologous Neural Stem Cell-Based Therapies for Neurodegenerative Disease and Injury

S. Bakhru, Ph.D., Carnegie Mellon University, 2009

The intrinsic abilities of mammalian neural stem cells (NSCs) to self-renew, migrate over large distances, and give rise toall primary neural cell types of the brain offer unprecedented opportunity for cell-based treatment of neurodegenerativediseases and injuries. This thesis discusses development of technologies in support of autologous NSC-based therapies, en-compassing harvest of brain tissue biopsies from living human patients; isolation of NSCs from harvested tissue; efficientculture and expansion of NSCs in three-dimensional (3-D) polymeric microcapsule culture systems; optimization of mi-crocapsules as carriers for efficient in vivo delivery of NSCs; genetic engineering of NSCs for drug-induced, enzymatic re-lease of transplanted NSCs from microcapsules; genetic engineering for drug-induced differentiation of NSCs into specifictherapeutic cell types; and synthesis of chitosan/iron oxide nanoparticles for labeling of NSCs and in vivo tracking by cel-lular magnetic resonance imaging (MRI). Submillimeter scale tissue samples were harvested endoscopically from subven-tricular zone regions of living patient brains, secondary to neurosurgical procedures including endoscopic third ventricu-lostomy and ventriculoperitoneal shunt placement. On average, 12,000 � 3,000 NSCs were isolated per mm 3 ofsubventricular zone tissue, successfully demonstrated in 26 of 28 patients, ranging in age from 1 month to 68 years. Toachieve efficient expansion of isolated NSCs to clinically relevant numbers (e.g., hundreds of thousands of cells in Parkin-son disease and tens of millions of cells in multiple sclerosis), an extracellular matrix-inspired, microcapsule-based cultureplatform was developed. Initial culture experiments with murine NSCs yielded unprecedented expansion folds of 30� in5 days, from initially minute NSC populations (154 � 15 NSCs per 450-mm-diameter capsule). Within 7 days, NSCs ex-panded as almost perfectly homogenous populations, with 94.9% � 4.1% of cultured cells staining positive for Nestin, amarker for NSCs, 81.4 � 3.7% of cells staining positive for KI67, a proliferation marker, and 0% of cultured cells stainingpositive for glial fibrillary acidic protein (GFAP), a marker indicative of undesired astrocytes. The same microcapsules usedfor expansion were designed to contain NSCs beyond delivery to the brain, maintaining NSC phenotype and suppressingundesired astroglial differentiation during the acute phase of inflammation beyond surgical implantation. In vitro, �80%of encapsulated cells challenged with 0.1% fetal calf serum over 5 days in culture showed persistent Nestin expression,compared to �20% under the same conditions outside of microcapsules, indicating that the microcapsule interior can pre-serve phenotype in the presence of serum concentrations at least an order of magnitude greater than those estimated to bepresent in cerebrospinal fluid (CSF) after surgical implantation. To release transplanted NSCs on cue from microcapsulesafter the acute inflammatory response, NSCs were genetically engineered using the Tet-on® drug-inducible gene expres-sion system to produce and secrete the enzyme alginase in response to the inducer drug doxycycline. Engineered NSCs,exposed to 1 mg/mL doxycycline, produced sufficient alginase to digest alginate, a structural component of the microcap-sule wall, within 8 h, effectively dissolving microcapsules and releasing encapsulated NSCs. To direct differentiation oftransplanted NSCs toward therapeutically valuable cell types (e.g., dopaminergic neurons in case of Parkinson disease andoligodendrocytes in case of multiple sclerosis), NSCs were genetically engineered to inducibly express the proneural tran-scription factors NGN1 and Olig1 on demand. Induced expression of NGN1 yielded �90% neurons, induced expression ofOlig1 yielded �80% oligodendrocytes, compared to neuron/oligodendrocyte yields �10% for GFP-expressing controls.NSCs with the capacity to inducibly express these transcription factors showed preservation of therapeutically valuable mi-gratory capacity (average rostral migratory stream [RMS] migration rate of approximately 40 mm/h before induction). Dif-ferentiating NSCs, however, showed largely arrested migration within 12 h of induction for Olig1 cells and 36 h of induc-tion for NGN1 cells. Finally, tracking of NSCs at the single cell level via high-resolution (11.7 T) cellular MRI, was madepossible through development of contrast-enhancing, chitosan-functionalized ultrasmall superparamagnetic iron oxide (US-PIO) nanoparticles that are rapidly uptaken by NSCs. Chitosan, a positively charged derivative of chitin, promotes elec-trostatically driven attachment of chitosan-USPIO nanoparticles to negatively charged domains on the outer leaflet of thecellular membrane, enhancing uptake by clathrin-mediated endocytosis (�10� increase in uptake efficiency relative to un-modified USPIO). Uptaken USPIOs remained in cells for at least 8 days due to charge-induced endosomal escape of nanopar-ticles into the cytosol. In combination, all developed technologies offer a basis for clinical evaluation of autologous NSC re-placement therapies, the future of which promises to shift the present paradigm for treatment of neurodegenerative diseasesand injuries.

DISSERTATIONS 291

Comment: It seems that many, perhaps all, tissues of elderly individuals retain small numbers of stem cells with the same function-ality as those present in early adulthood or even earlier. Thus, the motivation for developing sophisticated methods to differentiate em-bryonic stem cells along defined developmental pathways in vitro, so as to create replacement adult stem cells for transplantation, isbased on the insufficient numbers of such cells and their inability to function in the aged environment, rather than on their completeabsence. However, if such cells can be extracted and expanded in vitro without losing their “stemness,” the daunting complexi-ties associated with dedifferentiation and redifferentiation can be side stepped. This study is remarkable not only for theefficiency with which NSCs were isolated from tissue and expanded, but also for the fact that they could be extracted inthe first place from so delicate a tissue as the brain.

Multipotent Human Hair Follicle Stem Cells for Cardiovascular Tissue Engineering

S. Gopinath, Ph.D., State University of New York at Buffalo, 2009

Adult stem cells that were obtained from human hair follicles (hHF-SC) exhibited high proliferation potential and expressedsurface markers characteristic of mesenchymal stem cells (MSCs), such as CD44, CD73, and CD90. Notably, hHF-SC coulddifferentiate toward the adipogenic, chondrogenic, and osteogenic lineages, suggesting that these cells are similar to MSCsfrom bone marrow. Immunohistochemistry of the hair follicle tissue confirms the isolation of MSC-like cells, showing sim-ilar expression profile for the surface markers and smooth muscle �-actin (SMa) expression in the outer root sheath (ORS).Addition of basic fibroblast growth factor (bFGF) significantly increased proliferation potential of the hHF-SC. We testedthe ability of the hHF-SC to compact fibrin gels. Our results show that bFGF completely blocks compaction of fibrin gels.Transforming growth factor-� (TGF-�) is able to restore compaction partially in cells treated with bFGF, but does not showany difference in untreated cells. Immunocytochemistry confirms lack of SMa fibers in bFGF-treated cells compared to un-treated cells, with few cells showing fibers in cells treated with bFGF and TGF-�. Differentiation toward SMCs was furtherexplored using tissue-specific promoters, namely SMa and myosin heavy chain (MHC) promoters driving expression of agreen fluorescence protein variant (ZsGreen) or red fluorescence protein (RFP), respectively. Compaction assay on the hHF-SMC showed that bFGF treatment showed no difference in compaction ability, suggesting the isolation of mature SMCs.In conclusion, hHF-SC exhibit high proliferation ability and multilineage differentiation potential and, therefore, they pro-vide an easily accessible source of stem cells for tissue engineering and regenerative medicine.

Comment: The recent discovery that some stem cells of the epidermis are as primitive as the mesenchymal stem cells discovered inthe bone marrow is of obvious operational significance, in view of the much greater ease of access to the skin. However, much work re-mains to be done to characterize these cells and establish whether the same protocols being developed for the manipulation of bone mar-row-derived MSCs achieve the same results with follicle-derived ones. This study provides considerable encouragement in that regard.

Multipotent Mesenchymal Stromal Cells and Apolipoprotein E Secretion: A Potential Treatment for Atherosclerosis and Alzheimer Disease

S. Zeitouni, Ph.D., Tulane University, 2009

Apolipoprotein E (ApoE) is a molecular scavenger in the blood and brain with three human isoforms of ApoE ([varep-silon]2, [varepsilon]3, [varepsilon]4) each differentially affecting the function of the protein. ApoE [varepsilon]2 and [varep-silon]3, tend to be protective in the case of atherosclerosis and Alzheimer disease but ApoE [varepsilon]3 has other diseaseimplications. Therefore, introduction of ApoE [varepsilon]3 may be beneficial. Multipotent mesenchymal stromal cells(MSCs) are adult progenitor cells, which are easily expanded in culture and engraft into host tissues. We tested whethermurine (m) MSCs and human (h) MSCs from the bone marrow could be used as an expression system to provide ApoE inanimal models of atherosclerosis and Alzheimer disease. In initial experiments, mMSCs were injected into the lateral ven-tricles of postnatal ApoE null mice, which constitutively secreted small amounts of ApoE (Peister et al. 2006). The presenceof mMSCs resulted in improved cognitive behavioral testing compared to control groups. Polymerase chain reaction (PCR)assays demonstrated that the MSC levels required to elicit the change were extremely low, at the detection limit of the as-say. Unmanipulated hMSCs did not synthesize ApoE mRNA in vitro. Therefore, we examined the potential for inductionof expression. A molecular screen revealed that dexamethasone induced and sustained expression of ApoE. The ApoE couldbind to lipid, low-density lipoproteins (LDLs), and the LDL receptor, demonstrating functionality (Zeitouni et al. 2008). Wenext tested the ability of hMSCs to secrete ApoE in the blood, as a strategy for the treatment of severe atherosclerosis. Uponimplantation of a fibrin construct containing hMSCs, the cells persisted for 1 week, causing clear vascularization of the siteand high levels of ApoE were secreted into the blood of the mice. Unfortunately, the hMSCs did not survive in immune-competent hosts, suggesting that an immune-compromised ApoE null mouse is necessary. The data here demonstrate thatadministration of MSCs homozygous for ApoE [varepsilon]3 and dexamethasone may represent a novel therapy for ApoE-related diseases. Furthermore, MSC conditioned medium may be a future preparation for the administration of humanApoE. These results are therefore a significant step forward for the study and treatment of ApoE-related diseases.

DISSERTATIONS292

Comment: When a given protein is congenitally absent, there are two obvious ways to introduce it: Gene therapy, in which cellsare genetically altered in situ so that they now produce it, or enzyme therapy (which can also, of course, be used for proteins that arenot enzymes), in which the protein is injected into the circulation (or the cerebrospinal fluid) and taken up by the cells that need it viaendocytosis. The gene therapy approach can be achieved more easily in continuously renewing tissues, particularly the blood, by exvivo genetic alteration of stem cells, which then repopulate the tissue. A relatively unexplored variation on this theme is to modify andinject not stem cells but terminally differentiated cells that secrete the protein of interest, which is then taken up by nearby cells. Thisin many ways gives the best of both worlds of the other approaches. In this study, that concept is used to provide a protein that is al-ready being expressed in the recipient, but in a different allelic variant. It is unclear whether expressing ApoE3 late in life will benefitindividuals who have suffered accelerated cardiovascular and/or neurodegenerative aging due to the presence of apoE4, but this is justthe sort of therapy that would be required if so.

References

1. de Grey ADNJ. Dissertations. Rejuvenation Res 2007;10:117–122.2. de Grey ADNJ. A survey of selected recent theses relevant to combating aging. Rejuvenation Res 2007;10:245–251.3. de Grey ADNJ. A survey of selected recent theses relevant to combating aging. Rejuvenation Res 2007;10:339–343.4. de Grey ADNJ. A survey of selected recent theses relevant to combating aging. Rejuvenation Res 2007;10:641–646.5. de Grey ADNJ. A survey of selected recent theses relevant to combating aging. Rejuvenation Res 2008;11:259–264.6. de Grey ADNJ. A survey of selected recent theses relevant to combating aging. Rejuvenation Res 2008;11:689–695.7. de Grey ADNJ. A survey of selected recent theses relevant to combating aging. Rejuvenation Res 2008;11:841–846.8. de Grey ADNJ. A survey of selected recent theses relevant to combating aging. Rejuvenation Res 2008;11:971–975.9. de Grey ADNJ. Commentary on some recent theses relevant to combating aging. Rejuvenation Res 2008;11:1073–1078.

Address correspondence to:Dr. Aubrey D.N.J. de Grey

SENS FoundationUnited Kingdom

E-mail: aubrey@sens.org

DISSERTATIONS 293

This article has been cited by:

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