Commentary on Some Recent Theses Televantto Combating Aging: October 2010

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-

completed doctoral theses with particular relevance to thefields covered by Rejuvenation Reseach.1–11 Although it hasbecome common for thesis work to appear in the generalacademic literature, it remains valuable to scan the thesisdatabases for important advances that one might otherwiseoverlook.

Biomedical Imaging of Stem CellsUsing Reporter Genes

Fangjing Wang, Ph.D., Case Western ReserveUniversity, 2010

Mesenchymal stem cells (MSCs), derived from nearly alladult organs, can differentiate into multiple lineages in-cluding osteoblasts, chondrocytes, and adipocytes underdifferent local microenvironments for tissue repair. MSCshave also shown to ameliorate graft-versus-host disease(GVHD) caused by allogeneic hematopoietic stem celltransplantation via suppressing the activation and prolifer-ation of the alloreactive lymphocytes. Therefore, MSCs havegreat potential in bone tissue regeneration and stem cell–based therapies. However, previous techniques to track stemcells in vivo are usually associated with frequent animalsacrifice followed by subsequent histology analysis or re-verse transcriptase polymerase chain reaction (RT-PCR)verification. A continuous, noninvasive, real-time, and lon-gitudinal imaging technique is lacking. In this study, weaimed to establish such a platform technology to image stemcell biodistribution in a disease model (GVHD) in vivo. Atriple-reporter gene containing luc-mrfp-ttk was incorporatedinto human (h) MSCs via lentiviral transduction. The lucgene was imaged with bioluminescence imaging (BLI) andttk was imaged with positron emission tomography (PET).The proliferation rate of the transduced stem cells was re-duced compared to the wild-type cells, although the stemcell differentiation potential was reserved. The transcrip-tional changes between transduced and wild-type stem cellswere examined via gene microarrays to identify the keygenes related to proliferation and mesenchymal differentia-tion to determine the transduction effect. Stem cells that wereloaded into ceramic cubes and implanted into nonobesediabetic/severe combined immunodeficient (NOD/SCID)mice subcutaneously (s.c.) were imaged longitudinally in vivo

by BLI for more than 3 months. PET was also capable ofimaging the implanted stem cells in ceramic cubes. Histologystudies confirmed the formation of bone from the transducedhMSCs in vivo. These results indicate that the imaging tem-plate was developed successfully. In an animal model ofGVHD developed in mice, hMSCs were injected intrave-nously for the treatment of this disease. It was shown thathMSCs enhanced the survival of the GVHD mice comparedto the untreated mice. BLI results showed primary entrap-ment of hMSCs in lung, whereas only a small fraction of cellsmigrated transiently to the intestine. Cells in the mice wereundetectable 7 days after their transplantation. Althougharterial injection led to enhanced cell distribution into dif-ferent organs such as intestines, and prolonged hMSC re-tention in allogeneic mice, no efficacy was observedcompared with the control group, possibly due to the injuryassociated with the surgery procedure that allowed arterialinjection. These data suggest that timely cell injection andrepeated injection may be necessary to effectively prevent oralleviate this disease. In summary, this imaging technologyplays an important role in tracking stem cells, in revealingdisease development mechanisms, as well as in optimizingstem cell treatment strategy.

Comment: Imaging is a critical feature of both the developmentand the delivery of novel therapies, and cell therapies are a pro-minent case in point in regenerative medicine. Notably, as in thisstudy, imaging can provide both positive and negative informationconcerning the efficacy of such therapies.

Small Molecules, Regeneration, and Cell Fate

Simon Hilcove, Ph.D., Scripps ResearchInstitute, 2010

Given appropriate conditions, stem cells can self-renew forlong periods of time while maintaining the ability to differ-entiate into various functional cell types in the body. It isthese characteristics that not only make stem cells a usefulsystem in which to study tissue and organ development, butalso give them great potential for regenerative medicine.Given the success of well-practiced cell-based therapies (e.g.,hematopoietic stem cell transplantation for treating hema-tological diseases and pancreatic islet cell transplantation fortype I diabetes), it is conceivable that this approach could beapplied to many other serious medical conditions where cellsare lost because of disease, injury, or aging. Whether they are

SENS Foundation, Cambridge, United Kingdom.

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cell-based or stimulation of the body’s own repair, it is clearthat a better understanding of stem cell biology is requiredfor these approaches to be realized. Small molecules havelong been associated with biological discoveries. Our un-derstanding of biological processes often develops fromdiscovering or designing ways to perturb a given processand observing the effects of the perturbation. Although ge-netic approaches have been widely used for this purpose, thesmall-molecule approach clearly offers some distinct ad-vantages. Small molecules provide a high degree of temporalcontrol over protein function, which generally acts withinminutes or even seconds, and their effects are often revers-ible, which facilitates both rapid inhibition and activation.Their effect can also be finely tuned by varying concentra-tions of the compound of interest. Moreover, because of theinherent difficulty of genetic manipulation for many types ofstem cells (e.g., low transfection efficiency or poor clonalexpansion), small-molecule tools are especially useful for thestem cell field. Herein, several strategies that aim to uncoverregulators of cardiac development are reported. In particular,the first protocol for monolayer, serum-free differentiation ofembryonic stem cells (ESCs) into cardiomyocytes is de-scribed. Using this protocol, several signaling pathways arefound to be of significance to cardiac development. Our re-sults indicate that bone morphogenetic protein (BMP) andWnt signaling sit high in the order of mesodermal andsubsequent cardiac specification and that inhibition of Wntsignaling is important in later stages of differentiation. Pre-viously, BIO and BMP-4 have been reported to stimulate self-renewal of mouse (m) ESCs. In this study, a novel cardiacmesoderm induction effect of BIO on ESCs was identified,and a strong positive role for BMP-4 in cardiac mesodermformation was observed. Additionally, by conducting asmall-scale screen using our defined cardiac differentiationprotocol, we demonstrate a positive role for calcium/cyclicadenosine monophosphate (cAMP) signaling in the specifi-cation of cardiogenic mesoderm, and a positive role for in-hibition of transforming growth factor-b (TGF-b) signaling inthe specification of true cardiac cells from mesodermal celltypes. Calcium/cAMP signaling is important in variousdifferentiation programs, but its particular importance in thespecification of cardiogenic mesoderm was previously un-known. And while TGF-b has previously been reported topositively impact mesodermal fate specification, its inhibi-tion aiding in specification of cardiac cells from mesodermwas unknown. While monolayer conditions for the differ-entiation of neural 1 and endoderm 2 derivatives have beendescribed, to our knowledge, similar success has yet to beachieved in cardiac differentiation. Use of a similar basaldifferentiation condition should facilitate comparisons ofdifferent ESC-derived populations. A high-throughput screenof primary adult human b-cells for molecules that can induceproliferation is also described. A new serum-free culturecondition that maintains b-cells better than previous condi-tions was formulated and used for screening. Several un-known molecules are described that induce expression ofproliferative markers, as well as several known molecules.In particular, a positive role for inhibition of P38MAPK sig-naling and the importance of MEK/ERK signaling in b-cellcycle regulation are demonstrated. Last, a method for thedirect reprogramming of fibroblasts to a cardiac fate is de-scribed. Using specific combinations of virally overexpressed

transgenes as well as growth factors and small moleculesuncovered in our differentiation studies, efficient cardiacconversion can be achieved. Although the overexpressedgenes are often associated with their role in pluripotency, wedemonstrate that the conversion does not progress through apluripotent intermediate.

Comment: The most remarkable aspect of this study is its sheerbreadth. Four years or so is a remarkably short time in which toderive meaningful and reproducible results, and especially so for abeginning researcher just working toward his or her Ph.D. In thisstudy, small-molecule and transgenic approaches to a remarkablenumber of issues in the manipulation of cell state and cell fate arereported. Particularly interesting is the demonstration of bona fidetransdifferentiation mentioned in the last sentence.

The Study of Stem Cell–Like Fibroblastsin Mouse Digit Regeneration

Yuanyuan Wu, Ph.D., Tulane University, 2010

Adult mouse digit tip regeneration is a level-specific re-sponse. After amputation, the P3 phalangeal element, whichis composed of the most distal phalangeal bone, the nail, softconnective tissue in between, and the ventral skin, can re-generate back into a functional digit tip. The P2 phalangealelement, which is the tissue associated with the phalangealbone immediate behind P3, cannot regenerate after ampu-tation and, instead undergoes a wound healing response andforms scar tissue. Fibroblasts have long been implicated asthe cell population that both bears positional informationand constitutes a potential cell source that can be repro-grammed. To understand the different functions of fibro-blasts in a regenerative event and a nonregenerative event,we designed a series of experiments to compare fibroblastsfrom the regenerative site (P3) and the nonregenerative site(P2) using in vitro culture models and in vivo regenerationmodels. In the first chapter, we isolated P3 and P2 fibroblastsand characterized them as distinct progenitor populationsthat carry positional information and also share some stemcell characteristics. We also established a cell engraftmentmethod to introduce labeled P3 and P2 fibroblasts into themouse digits. Using engraftment, we learned that both P3and P2 fibroblasts can participate in the adult P3 level digitblastema in vivo, and both failed to promote regeneration atthe P2 level. The first chapter introduces the idea that thewound environment, rather than the intrinsic properties ofthe cell, is a major factor in determining whether regenera-tion occurs or not. In the second chapter, we further inves-tigated the participation of P3 and P2 fibroblasts in the P3level regeneration response by evaluating their survival anddifferentiation. P3 cells showed better survival and differ-entiation than P2 cells both in engraftment and during re-generation. Thus, even though the environment plays amajor role in regeneration, the cells maintain intrinsic prop-erties that affect their responsiveness to the local environ-ment during a regeneration event. In the third chapter, wefurther introduced both P3 and P2 fibroblasts into a P2-levelneonatal bone morphogenetic protein-2 (BMP2)-induced re-generation model to test whether they would participate inendochondral ossification. We found that while both P3 andP2 fibroblasts were recruited by stromal cell-derived factor-1(SDF-1) chemotaxis, neither cell type differentiated intochondrocytes. Instead they interfered with the endogenous

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endochondral ossification event by competing for the spacewhere endogenous cells should be recruited. The data sug-gest that recruitment itself is not the answer to promote re-generation, but recruiting the right population is. Theexperimental approach is based on the current knowledge ofstem cell biology and cell therapy. The detailed fundamentalcomparisons based on proliferation, survival, and differen-tiation set a good standard in promoting cell therapy basedregenerative medicine. The results suggest that to fully uti-lize the cell population in regenerative medicine, we mustlearn the balance between intrinsic properties of the cell andinfluences from the environment.

Comment: Most of the studies that I highlight in this spacereport positive results, but in this case it seems highly appropriateto draw attention to a predominantly cautionary tale. The findingsof this study serve to remind us how difficult it is to identify,let alone create, the epigenetic state that is needed for a cell pop-ulation to perform a desired regenerative function. It is essentialthat results of this kind be given more prominence if we are to moveforward optimally to reproducibly effective regenerative medicine.

Engineering Lentiviral Vectors for Gene Therapyand Development of Live Cell Arraysfor Dynamic Gene Expression Profiling

Jun Tian, Ph.D., State University of New Yorkat Buffalo, 2010

With their advantages including wide tropism, high effi-ciency in gene transfer to both dividing and nondividing cells,and stable and long-term expression of transgenes, lentiviralvectors have been used for genes therapies and widely used forbasic biomedical research where gene transfer is required. Be-cause expression of multiple genes from the same target cell isrequired in such applications, this research work focused ondesigning novel multicistronic lentiviral vectors to developgene therapy of diabetes through regulated insulin deliveryfrom skin cells and live cell arrays for analyzing gene expres-sion in a high-throughput and real-time manner. Specifically,first, lentiviral vectors were engineered to produce a fusionprotein between the furin-cleavable proinsulin gene and theself-dimerization mutant of FK506-binding protein to yieldbioactive insulin in keratinocytes that could be released fol-lowing exogenous administration of a small organic molecule,rapamycin. The engineered keratinocytes retained normalmorphology and grew similarly to lentiviral-treated controlcells. Epidermal keratinocytes in culture and in stratifiedbioengineered epidermis released insulin within 30 min afteraddition of rapamycin, and secretion slowed or stopped within2–3 h after removal of the inducer. When the cells were im-planted into athymic mice that were rendered diabetic withstreptozotocin, insulin was detected in the plasma within 1 hafter addition of rapamycin. Concomitantly, glucose decreasedto normal levels, even in diabetic animals suffering severehyperglycemia. Repeated rapamycin administration yieldedsimilar results. These experiments provide proof-of-conceptthat insulin released from the skin in a regulatable manner canbe an effective treatment for diabetic patients. Second, a lenti-viral vector carrying two transcriptional units was designed toreach independent and high level-dual-gene expression. Thetwo transcriptional units were separated by polyadenylation,terminator, and insulator sequences to eliminate promoter in-terference existing between the transcriptional units. With this

design, the expression level of both genes was as high as thatyielded from lentiviral vectors containing only a single tran-scriptional unit. Similar results were observed with severalpromoters and cell types, including epidermal keratinocytes,bone marrow mesenchymal stem cells, and hair follicle stemcells. Notably, this research work also demonstrated quanti-tative dynamic monitoring of gene expression in primary cellswith no need for selection protocols, suggesting that thisoptimized lentivirus may be useful in high-throughput geneexpression profiling studies. Last, using the novel double-promoter lentiviral vector, scalable live-cell microarrays weredeveloped to measure gene expression dynamics in a real-timeand high-throughput manner. To this end, dual-promoterlentiviral vectors were prepared harboring a transcriptionalregulatory element encoding for green fluorescence protein tomonitor cell activation in response to exogenous stimuli and aconstitutive promoter driving red fluorescence protein for in-ternal signal normalization. Lentivirus preparations were im-mobilized in a microarray format, and, after transduction onthe array surface, target cells were treated with cytokines andinterrogated in real-time using automated fluorescence mi-croscopy, providing rich dynamic information over a period ofseveral days. Data normalization by red fluorescence intensityeliminated errors due to spot-to-spot variability in transductionefficiency or changes in cell proliferation upon cytokine treat-ment. These results suggest that the LVA can monitor geneexpression in real-time and high-throughput manner therebyproviding a useful tool for quantitatively measuring gene ex-pression dynamics and deciphering gene regulatory networks.

Comment: The lentivirus is already a mainstay of gene therapyresearch and development by virtue of its versatility, as summarizedat the start of this abstract. However, new applications of this viruswithin gene therapy continue to emerge. The modulation of insulinsensitivity via skin-expressed factors is a supremely imaginative andcounterintuitive approach to one of today’s predominant healthproblems. The fluorescence application also reported here is equallyinnovative and clearly has widespread potential.

Mechanisms of Biomaterial-Mediated FibroticResponses and Strategies to ImproveTissue Reactions to Biomaterial Implants

Paul Thevenot, Ph.D., University of Texasat Arlington, 2010

Despite considerable advancements in biomaterial synthesisand modification techniques, most tissue engineering scaf-folds elicit fibrotic reactions, resulting in implant encapsula-tion, secluding cells and/or therapeutic agents within a thickcollagenous matrix. Therefore, strategies to minimize theseresponses while improving the functionality of recruited/transplanted cells are required. This work set out to improvethe host response to biomaterials and identify what cellularresponses were attributed to biomaterial-mediated fibrosis.Two strategies were investigated involving two different tar-gets. In the first approach, poly-lactic-glycolic acid (PLGA)scaffolds were radiofrequency gas discharge (RFGD) modifiedto bear -NH2 (amine), -COOH (carboxyl), and -OH (hydroxyl)groups altering surface wettability and charge properties.Surface-modified scaffolds altered fibrinogen adsorption andRAW 264.7 (macrophage) cytokine secretion in vitro, subse-quently effecting macrophage chemotaxis to preconditionedmedia from different modified scaffolds. In vivo scaffolds were

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able to slightly alter inflammatory cell influx while increasingcell infiltration density and matrix production. However sur-face modification exerted only minor influence over thethickness of the fibrotic capsule. In the second approach,stromal cell-derived factor 1a (SDF-1a) was incorporated withscaffolds to increase recruitment of host-derived stem cellswhile reducing fibrotic responses. Increased stem cell recruit-ment was able to significantly alter interface collagen deposi-tion, in addition to increasing angiogenic progenitorrecruitment, thus improving angiogenesis and altering the localcytokine environment. By varying cytokine delivery onset andduration, the effects of SDF-1a were linked with a stabilizedmast cell response upstream. This led us to consider whetherthe mast cell responses were primarily regulating the bioma-terial-mediated fibrotic response, and specifically what cellswhere responsible for collagen production at the biomaterialinterface. Fibrocytes and fibrocyte-derived myofibroblastswere identified as primary contributors to collagen deposition.Fibrocytes recovered from the biomaterial implantation sitepredominantly migrated in response to SDF-1a. Targeting thislink with the anti-inflammatory drug dexamethasone did notalter fibrocyte responses; however SDF-1a neutralization sig-nificantly reduced the influx of fibrocytes and generation offibrocyte-derived myofibroblasts. This led to significant re-duction in fibrotic responses. Because mast cells influence theacute inflammatory response to biomaterial implants, we hy-pothesized that creating different degrees of mast cell responseswould result in differential stimulation of SDF-1a–producingcells while reducing inflammatory stimuli. Stabilizing mastcells with Cromolyn was able to significantly reduce macro-phage influx along with fibrocyte and fibrocyte-derived myo-fibroblast influx. This led to significant reduction in fibroticencapsulation and collagen I structure at the biomaterial in-terface, implicating mast cells as the initiator of the biomaterial-mediated fibrotic response. On the basis of these results, wepropose a hypothetical sequence of events leading to the for-mation of fibrotic tissue around biomaterial implants, whichdepends upon the degree of mast cell activation and subse-quent fibrocyte responses. Therefore, this study has identifiedthe critical involvement of the mast cell response in biomaterial-mediated fibrosis, as well as identifying a strategy to reducemast cell activation while improving host-derived stem cellresponses to improve the response to biomaterial scaffolds inthe subcutaneous space.

Comment: Mast cells are a somewhat neglected cell type inregenerative medicine, so this study constitutes an importantcontribution to the body of evidence that they should be morewidely understood and exploited. Fibrosis is a major impediment toa wide variety of procedures in regenerative medicine, and theability of mast cells to control it is worthy of detailed examination.

Timing the Microenvironment: The Effectof Inflammatory Cues on Neural Stem Cell Propertiesand Cell-Mediated Potential for Recoveryand Repair after Spinal Cord Injury

Mitra Hooshmand, Ph.D., University of CaliforniaIrvine, 2010

Cell-based therapies may have promising potential forpromoting recovery following central nervous system (CNS)trauma. However, survival, fate, and migration response of

transplanted populations and their resultant capacity forneurorepair and functional recovery are likely influenced byfactors present in the injured microenvironment. Initialtrauma to the spinal cord results in dynamic recruitment ofdistinct cellular and molecular factors to the injury epicenter.This dissertation investigates the effects of time-specific en-vironmental cues present acutely versus subacutely afterspinal cord injury (SCI) on the properties of transplantedcells and cell-mediated potential for recovery. Fluoresence-activated cell sorted (FACS) multipotent human CNS stemcells propagated as neurospheres (hCNS-SCns) were trans-planted into spinal cord–injured immunodeficient nonobesediabetic/severe immunodeficient (NOD/SCID) mice imme-diately (0 days) post-SCI and at 9 days post-injury (dpi),when the local cues in the injured microenvironment sig-nificantly differ. Our data demonstrate that unlike previouspredictions about the presence of an "optimal window oftransplantation" between 7 and 14 dpi, hCNS-SCns survivalis comparable in both transplantation paradigms. Interest-ingly, we demonstrate that transplantation of hCNS-SCns at0 dpi, at the peak of neutrophil infiltration, results in strikingcell migration toward the injury epicenter, predominant as-trocytic differentiation of hCNS-SCns near the lesion, and nochanges in functional recovery. Although these data areconsistent with the premise of spinal cord being a gliogenicniche, we show that the same cell population transplanted at9 dpi, when macrophage infiltration is high, migrates awayfrom the lesion, predominantly differentiates into oligoden-drocytes and neurons, and promotes locomotor recovery viaremyelination and synapse formation, but not by alternativemechanisms of neurorepair. These findings suggest thatextrinsic cues found at different times postinjury affecthCNS-SCns. We illustrate a previously unidentified effect ofneutrophil-mediated migration and astrogliogenesis, as wellas macrophage-mediated neural fate of hCNS-SCns, andidentify a mechanism for neutrophil-mediated effects viacomplement C1q and C3a synthesis. Identification of specificfactors present at different phases following SCI that limitthe capacity of transplanted cells to restore function in theCNS microenvironment after injury or disease is highly no-vel and may lead to new strategies to expand the therapeuticwindow for successful transplantation paradigms.

Comment: The repair of spinal cord injury is ostensibly of verylimited relevance to aging, but in practice we can learn a great dealabout how to combat aging with regenerative medicine by exam-ining successes in more straightforward contexts. In this case, aconsiderably finer level of detail concerning the evolution of theSCI environment, and thus of its treatment, is elucidated, which ispotentially a major step toward optimization of a therapy that hashistorically reported occasional but very poor reproducible success.

References

1. de Grey ADNJ. A survey of selected recent theses relevant tocombating aging. Rejuvenation Res 2008;11:259–264.

2. de Grey ADNJ. A survey of selected recent theses relevant tocombating aging. Rejuvenation Res 2008;11:689–695.

3. de Grey ADNJ. A survey of selected recent theses relevant tocombating aging. Rejuvenation Res 2008;11:841–846.

4. de Grey ADNJ. A survey of selected recent theses relevant tocombating aging. Rejuvenation Res 2008;11:971–975.

5. de Grey ADNJ. Commentary on some recent theses relevantto combating aging. Rejuvenation Res 2008;11:1073–1078.

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6. de Grey ADNJ. Commentary on some recent theses relevantto combating aging. Rejuvenation Res 2009;12:53–58.

7. de Grey ADNJ. Commentary on some recent theses relevantto combating aging. Rejuvenation Res 2009;12:149–154.

8. de Grey ADNJ. Commentary on some recent theses relevantto combating aging: June 2009. Rejuvenation Res 2009;12:221–225.

9. de Grey ADNJ. Commentary on some recent theses relevantto combating aging: August 2009. Rejuvenation Res 2009;12:289–293.

10. de Grey ADNJ. Commentary on some recent theses relevantto combating aging: October 2009. Rejuvenation Res 2009;12:367–370.

11. de Grey ADNJ. Commentary on some recent theses relevantto combating aging: December 2009. Rejuvenation Res 2009;12:463–467.

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

SENS FoundationCambridge

United Kingdom

E-mail: aubrey@sens.org

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