Commentary on Some Recent Theses Relevantto Combating Aging: December 2009

Aubrey D.N.J. de Grey

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 While it has become common for

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

Conductive Polymers for Controlled Release and Treatment of Central NervousSystem Injury

Rajiv Saigal, Ph.D., Harvard University, 2009

As one of the most devastating forms of neurotrauma, spinal cord injury remains a challenging clinical problem. Thedifficulties in treatment could potentially be resolved by better technologies for therapeutic delivery. To develop newapproaches to treating central nervous system injury, this dissertation focused on using electrically conductive polymers,controlled drug release, and stem cell transplantation. We first sought to enhance the therapeutic potential of neural stem cellsby electrically increasing their production of neurotrophic factors (NTFs), important molecules for neuronal cell survival,differentiation, synaptic development, plasticity, and growth. We fabricated a new cell culture device for growing neuralstem cells on a biocompatible, conductive polymer. Electrical stimulation via the polymer led to upregulation of NTFproduction by neural stem cells. This approach has the potential to enhance stem cell function while avoiding the pitfalls ofgenetic manipulation, possibly making stem cells more viable as a clinical therapy. Seeing the therapeutic potential ofconductive polymers, we extended our studies to an in vivo model of spinal cord injury (SCI). Using a novel fabrication andextraction technique, a conductive polymer was fabricated to fit to the characteristic pathology that follows contusive SCI.Assessed via quantitative analysis of magnetic resonance (MR) images, the conductive polymer significantly reducedcompression of the injured spinal cord. Further characterizing astroglial and neuronal response of injured host tissue, wefound significant neuronal sparing as a result of this treatment. The in vivo studies also demonstrated improved locomotorrecovery mediated by a conductive polymer scaffold over a nonconductive control. We next sought to take advantage ofconductive polymers for local, electronically controlled release of drugs. Seeking to overcome reported limitations in drugdelivery via polypyrrole, we first embedded drugs in poly(d,l-lactide-co-glycolide]-co-polyethylene glycol) (PLGA-PEG)nanoparticles and then demonstrated scalable incorporation and controlled release. In a functional application, electronicallycontrolled release of minocycline nanoparticles was used to rescue primary spinal cord neurons from an excitotoxic envi-ronment in vitro. This approach offers a wide range of therapeutic possibilities, especially for treating traumatic lesions of thecentral nervous system. Finally, we explored use of conductive polymers for directed differentiation of progenitor cells.Retinal progenitors were seeded on custom polypyrrole cell culture devices and subjected to a biomimetic pattern of electricalstimulation. Stimulated cells showed phenotypic changes, increased neurite outgrowth, increased immunocytochemicalexpression of cone rod homeobox (CRX) and protein kinase C (PKC), and decreased expression of glial fibrillary acidicprotein (GFAP). Biomimetic stimulation thus led cells toward early photoreceptor and bipolar cell fates and away from anastrocytic cell fate. Electrical stimulation via a conductive polymer offers a novel approach for directing differentiation ofprogenitor cells.

Comment: It is well known that the interaction between the nervous system and other cell types is critical to the latter’s function andeven survival: The classic example is the degeneration of muscle fibers following extended failure of excitation via a neuromuscularjunction. Thus, there is a clear motivation to explore electrical stimulation in the fabrication and culture of materials intended fortransplantation in tissue engineering, both prior to transplantation and following it. However, to my knowledge such an approach has notbeen applied hitherto. This study examines the potential of that concept at numerous levels and offers many reasons for optimism that itcan become a key tool in the tissue engineer’s arsenal.

SENS Foundation, Cambridge, United Kingdom.

REJUVENATION RESEARCHVolume 12, Number 6, 2009ª Mary Ann Liebert, Inc.DOI: 10.1089=rej.2009.1008

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Innovative Approaches to Regenerative Medicine: Tissue Repairand Generation of Stratified Squamous Epithelia

Cathy Rasmussen, Ph.D., University of Wisconsin–Madison, 2009

Cell-based therapeutics generated from somatic or pluripotent stem cell sources have the potential to revolutionize thetreatment of debilitating diseases and medical conditions previously believed to be untreatable. This dissertation describes amultifaceted investigation of regenerative medicine approaches ranging from basic to translational research in the field ofcutaneous wound repair employing both somatic and pluripotent stem cell sources. Allogeneic=autologous chimericbioengineered skin substitutes offer an innovative approach for the treatment of severe cutaneous trauma. Mixed populationsof green fluorescent protein-labeled NIKS and unlabeled primary keratinocytes were used to model the allogeneic andautologous components of chimeric cultures. These proof-of-principle studies demonstrated that organotypic co-culture ofNIKS and primary keratinocytes generated a fully stratified epidermal component of composite skin substitute tissue,providing wound coverage and restoring cutaneous barrier function while delivering ‘‘autologous’’ keratinocytes to thewound site. Initial studies examining the therapeutic potential of pluripotent stem cells for skin regeneration and repairhighlighted the technical challenges associated with cultivating pluripotent cell types. A continuous, uniform, adventitiousagent-free feeder layer cell source (3T3 CG) was evaluated for the ability to support human embryonic stem cell serialcultivation. 3T3 CG feeder layers sustained long-term serial cultivation of karyotypically normal, undifferentiated humanembryonic stem cells that retained the ability to differentiate into ectodermal, mesodermal, and endodermal cell types. Use ofthis cell source would facilitate basic stem cell research and support early-stage development of stem cell-derived thera-peutics. Although several studies describe the generation of human embryonic stem cell-derived keratinocyte-like cells, nolaboratory has reported the successful generation of three-dimensional tissue structure from human embryonic stem-derivedcell sources. This dissertation describes the morphogenesis of a stratified squamous epithelium from human embryonic stemcell-derived keratinocytes. This tissue exhibited keratinocyte terminal differentiation up to the final formation of a definitivecornified layer with epidermal-specific gene and protein expression patterns, although a functional epidermal permeabilitybarrier was not established and tissue did not stably engraft in an in vivo model of skin transplantation. These findingsrepresent a significant advance in the generation of human tissues and organs from human embryonic stem cell-derivedprogenitor cells.

Comment: The classical demonstration that a cell population consists of (or at least contains) pluripotent cells is its ability todifferentiate into examples of all three germ layers, either in an organized manner after appropriate stimulation or as a teratoma. However,for practical use, these tests are only preliminary, since the semidifferentiated cell (the oligopotent stem cell) to be transplanted into thepatient must have a sufficiently precisely defined epigenetic state to ensure that it gives rise to all the cell types in the relevant tissue in amanner that allows them to function as normal. As this study notes, in the case of the epidermis (and in many other cases) this has notbeen fully achieved. This study constitutes a large step toward that goal, even though the failure to achieve stable engraftment means thatit cannot be counted as a complete success.

Elucidating the Role of Mesenchymal Stem Cell Participation in the Tumor Microenvironment

Shannon Kidd, Ph.D., University of Texas Graduate School of Biomedical Sciences at Houston, 2009

To meet the requirements for rapid tumor growth, a complex array of nonneoplastic vascular, fibroblastic, and immunecells are recruited to the tumor microenvironment. Understanding the origin, composition, and mechanism(s) for recruitmentof these stromal components will help identify areas for therapeutic intervention. Previous findings have suggested thatex vivo–expanded bone marrow-derived mesenchymal stem cells (MSCs) home to the sites of tumor development, re-sponding to inflammatory signals, and can serve as effective drug delivery vehicles. Therefore, we first sought to fully assessconditions under which MSCs migrate to and incorporate into inflammatory microenvironments and the consequences ofmodulated inflammation. MSCs delivered to animals bearing inflammatory insults were monitored by bioluminescenceimaging and displayed specific tropism and selective incorporation into all tumor and wound sites. These findings wereconsistent across routes of tumor establishment, MSC administration, and immunocompetence. MSCs were then used asdrug delivery vehicles, transporting interferon-b to sites of pancreatic tumors. This therapy was effective at inhibitingpancreatic tumor growth under homeostatic conditions, but inhibition was lost when inflammation was decreased with2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid (CDDO-Me) combination treatment. Next, to examine the endogenous tumormicroenvironment, a series of tissue transplant experiments were carried out in which tissues were genetically labeled andengrafted in recipients prior to tumor establishment. Tumors were then analyzed for markers of tumor-associated fibroblasts(TAFs): a-smooth muscle actin (a-SMA), nerve glia antigen 2 (NG2), fibroblast activation protein (FAP), and fibroblast-specificprotein (FSP), as well as endothelial marker CD31 and macrophage marker F4=80. We determined the majority of a-SMAþ,NG2þ, and CD31þ cells were non-bone marrow derived, whereas most FAPþ, FSPþ, and F4=80þ cells were recruited from thebone marrow. In accord, transplants of prospectively isolated bone marrow MSCs prior to tumor development indicated thatthese cells were recruited to the tumor microenvironment and co-expressed FAP and FSP. In contrast, fat transplant ex-periments revealed recruited fat derived cells co-expressed a-SMA, NG2, and CD31. These results indicate TAFs are a

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heterogeneous population composed of subpopulations with distinct tissues of origin. These models have provided a plat-form upon which further investigation into tumor microenvironment composition and tests for candidate drugs can beperformed.

Comment: The simplistic model of a tumor as a purely clonal population of cells, infiltrated only by blood vessels, has for some timebeen known to be incorrect. In particular, immune cells are present in large numbers in solid tumors, and there is much interest in themechanisms by which their antitumor potential is suppressed and the tumor thus allowed to survive and grow. In this study, the cell typebeing investigated is one whose recruitment to the tumor appears, by contrast, to favor tumor growth. The exploitation of suchrecruitment as a ‘‘trojan horse’’ for delivery of anticancer drugs is an ingenious method for treating particularly challenging cancers suchas pancreatic cancer, as explored here; additionally, this study provides important new data on the types of bone marrow–derived cell thatinfiltrate the tumor, which may allow us to manipulate such cells so that recruitment does not occur and the tumor is thereby weakened.

Mechanistic Studies and Practical Applications of Direct Reprogramming

Christopher Rodolfa, Ph.D., Harvard University, 2009

Recent studies have demonstrated that primary human fibroblasts can be directly reprogrammed to a pluripotent stateupon the retrovirus-mediated addition of four transgenes. The three studies presented in this thesis have sought to extendthis finding to provide therapeutically relevant resources for amyotrophic lateral sclerosis (ALS) research and expand ourunderstanding of the underlying mechanism of this direct reprogramming. In the first study, we demonstrate that theseinduced pluripotent stem (iPS) cells can be produced directly from elderly patients with chronic disease by generating themfrom two sisters, ages 82 and 89, afflicted with familial ALS. We further show that these patient-specific iPS cells can bedirected to differentiate into motor neurons, the cell type destroyed in ALS. These results provide for the large-scale pro-duction of disease-relevant cells directly from affected patients, a valuable substrate for disease modeling, drug discovery,and eventually autologous cell-replacement therapies. However, these initial lines may be of limited utility to the study andtreatment of ALS because the vast majority of ALS cases are sporadic with no known genetic correlation. In the second study,we report the generation of a bank of 51 iPS cell lines from 15 individuals, including sporadic and familial ALS cases as wellas healthy controls. In addition to providing a powerful resource for the study and potential treatment of this debilitatingdisease, the collection of cell lines reported here will also provide for a more comprehensive study of the quality andconsistency of iPS cells. Finally, in a third study, we turn our attention to the mechanisms of nuclear reprogramming itself,taking two approaches to this investigation. First, we demonstrate that human fibroblasts can be partially reprogrammed byintroduction of two of the iPS-generating factors. Characterization of this partially reprogrammed state suggests a decouplingof the two hallmark stem cell properties—self-renewal and pluripotency. Second, a time series during iPS induction revealsmolecular milestones involved in this process and illustrates that changing the number of factors used to produce iPS cellshas a profound effect on the kinetics of the process while maintaining the order of these milestone events. Taken together, thedata presented in this thesis provide an initial perspective on the processes underlying direct reprogramming and achievetwo key goals of regenerative medicine—the establishment of pluripotent stem cells directly from patients and their directeddifferentiation into cell types relevant to the patient’s disease. These accomplishments will play an essential role in laying thefoundation for the burgeoning field of personalized regenerative medicine.

Comment: Though ALS is not a feature of aging, its generally sporadic genetic etiology and its progressive degenerative course make ita highly relevant condition for the development and testing of therapies for various aspects of age-related degeneration. The successfulcreation of new motor neurons (via iPS cells) from cells of elderly patients allays a number of concerns with regard to the feasibility ofautologous stem cell therapy in aging (though the daunting remaining task of transplanting such cells or their progenitors must not beunderestimated). This thesis additionally reports some novel data characterizing the dedifferentiation process whereby iPS cells arecreated: The more we discover about that mysterious process, the more broadly we will be able to manipulate it for therapeutic purposes.

Strategies for Prevention of Antibody-Mediated Allograft Rejection

Hao Wang, Ph.D., University of Western Ontario, 2009

Antibody-mediated rejection (AMR) remains the primary obstacle in presensitized patients following organ transplanta-tion, as it is refractory to anti-T cell therapy and can lead to early graft loss. In the first part of this thesis, we report on a novelapproach aimed at preventing antibody-mediated acute vascular rejection (AVR), one of the major unresolved hurdles ofclinical transplantation. We demonstrate in a murine model that free bone transplantation combined with cyclosporinesuppresses antidonor antibody responses, induces indefinite cardiac allograft survival (>100 days), and preserves graftarchitecture. In contrast, untreated, or cyclosporine alone treated recipients rejected their cardiac grafts in 7–16 days withgraft histology indicative of AVR. To determine whether graft survival was associated with hematopoietic or stromalelements of the transplanted free bone, we administered isolated bone marrow (BM) mononuclear cells or free bone that wasirradiated to deplete hematopoietic elements. Although BM mononuclear cells had no effect on cardiac graft survival,irradiated free bone transplantation containing mesenchymal stem cells was capable of prolonging graft survival. Mostinterestingly, the prolongation effect was genotype-independent because third-party irradiated bone graft was also effective.

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Due to the fact that current immunosuppressive approaches are clinically ineffective at preventing AVR, this study providespromise for further investigations on BM components as a means of addressing a currently unmet medical need. In thesecond part of this thesis, we investigated complement blockade in prevention of antibody-mediated accelerated humoralrejection (ACHR) in presensitized recipients. BALB=c recipients were presensitized with C3H donor skin grafts 1 week priorto kidney transplantation from the same donor strain, a model designed to mimic AMR in presensitized patients. This modelwas used to evaluate the effect of blocking the fifth complement component (C5) with an anti-C5 monoclonal antibody onprevention of graft rejection. Anti-C5 administration completely blocked terminal complement activity and local C5 depo-sition, and in combination with cyclosporine (CsA) and short-term cyclophosphamide (CyP) treatment, it effectively pre-vented heart graft rejection. These recipients achieved permanent graft survival for more than 100 days with normal histologydespite the presence of systemic and intragraft antidonor antibodies and complement, suggesting ongoing accommodation.Furthermore, double-transplant experiments demonstrated that immunological alterations in both the graft and the recipientwere required for successful graft accommodation to occur. These data suggest that terminal complement blockade with afunctionally blocking antibody represents a promising therapeutic approach to prevent AMR in presensitized recipients. Thestudies in this thesis suggest that free bone grafting and anti-C5 therapy are both promising therapeutic approaches to treatand=or prevent acute and chronic AMR of solid organs, capable of inducing long-term graft survival and thus indicating theirpotential future utility in clinical transplantation.

Comment: Despite the massive progress in autologous transplantation that has resulted from the discovery of iPS technology, thereremains a clear need to improve our ability to manipulate the immune system so that allogeneic transplantation is also pratical withoutunacceptable immunosuppression. Such ‘‘tolerization’’ of a recipient to a donor’s cells or organs will allow the much greater use oftraditional organ transplant procedures (especially as we become more proficient at cryopreserving whole organs), and perhaps even moreimportantly it will allow us to develop xenotransplantation of organs from pigs or other suitable animals, as well as gene and enzymetherapy using nonhuman genes with therapeutically useful properties. The success reported here in controlling two major mechanisms ofrejection is a fine demonstration that comprehensive tolerization is a foreseeable goal.

Zinc Finger Nuclease Knockout of CCR5 in Hematopoetic Stem Cells As an Anti-HumanImmunodeficiency Virus Gene Therapy

Nathalia Holt, Ph.D., Tulane University, 2009

CCR5 is the major co-receptor for human immunodficiency virus type 1 (HIV-1) entry and an important target for drugdevelopment. The recent finding that hematopoietic stem cell (HSC) transplantation from a CCR5-negative donor to an HIV-infected patient produces long-term virus control in the absence of antiretroviral drugs suggests the potential of stem cell andgene therapies targeting CCR5. To this end, we are developing a protocol to knock-out CCR5 in a patient’s own HSC usingCCR5-targeted zinc finger nucleases (ZFNs). ZFNs are sequence-specific proteins that generate a double-stranded break inDNA, which is converted into a gene-disrupting lesion by host repair processes. We have optimized the delivery and functionof ZFNs targeting CCR5 in human CD34þ HSCs, achieving up to 27% disruption of CCR5 alleles. We confirmed that ZFN-treated HSCs remain fully functional by transplanting a mouse model of human hematopoiesis, the NOD=SCID=IL2gcnull(NSG) mouse, where the modified HSCs retained the ability to differentiate into multiple hematopoietic lineages. HumanizedNSG mice are additionally capable of supporting HIV-1 infection. Following challenge with an R5-tropic virus, controlanimals demonstrated altered CD4:CD8 ratios, profound loss of human cells in the thymus and gut-associated lymphoidtissues (GALT), and high viral loads in multiple tissues sampled. In contrast, ZFN-treated animals had significantly loweracute viral loads and very low levels of virus in tissues by 10–12 weeks postinfection. At this stage, the numbers of humancells in tissues that are targets for HIV-1 infection had normalized, including the GALT and thymus. Fluorescence-activatedcell sorting (FACS) and polymerase chain reaction (PCR) analysis revealed a rapid and dramatic selection for CCR5� cells inthese populations. These findings demonstrate that ZFN-treated HSCs can generate HIV-resistant cells in vivo that rapidlyreplace cells depleted by HIV-1 infection, and importantly, preserve GALT populations. Transient ZFN treatment resulting inpermanent disruption of CCR5 in autologous HSCs could therefore represent a viable clinical approach for HIV-infectedpatients.

Comment: Although most gene therapy work has historically been focused on the insertion of new genes into a congenitally deficientgenome, there are also many applications in which the requirement is to remove or modify an existing gene. This concept has become evenmore relevant with the ppreciation of the dangers of random integration of inserted DNA, because it can also be used to insert new genesin defined locations. The currently most promising method for this ‘‘gene targeting’’ procedure uses a three-part system: ReplacementDNA in an episome, a designed zinc finger protein to target the desired genomic location, and an endonuclease to induce homologousrecombination using the episome as substrate. The term ‘‘zinc finger nuclease’’ arises because the zinc finger protein (ZFP) and theendonuclease are encoded as a fusion protein. This thesis reports the use of ZFNs in a context not related to aging, but its unequivocalsuccess paves the way for similar work targeting (for example) telomerase as part of a comprehensive anticancer therapy.

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.

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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:Aubrey D.N.J. de Grey

SENS FoundationP.O. Box 304

El Granada, CA 94018

E-mail: aubrey@sens.org

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