Navrmc 2010 Abstracts

8/9/2019 Navrmc 2010 Abstracts

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In collaboration with

1st North American Veterinary Regenerative Medicine Conference March 56, 2010, Santa Ynez Valley, California

Abstracts of Presentation


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A BSTRACTS OF P RESENTATIONS

Characterization of Tendon Progenitor Cells .............. ............. ............. ............. ............. .............. ......... 3

Immunomodulatory Activity of Allogeneic Bone Marrow, Adiposeand Placentally Derived Equine Mesenchymal Stem Cells ............. ............. ............. ............. ............ 4

MSCs are the New Medicine .................................................................................................................. 5

Stem Cell-Derived Ocular Cells for the Treatment of Human Eye Disease.................... ............. ............ 6

Clinical Follow-up of Horses Treated Intra-articularly with Bone Marrow-DerivedMesenchymal Stem Cells for Orthopaedic Lesions ............ ............. ............. ............. ............. ............ 7

Role of Growth Factors in Mesenchymal Stem Cell-Mediated BloodFlow Restoration and Bone Repair ............ .............. ............. ............. ............. ............. .............. ......... 8

Regenerative Medicine Techniques for Treatment of Deep Digital Flexor Tendinopathy.............. ......... 9

Possible Clinical Applications for Stem Cell Therapy from ResearchBased in Equine Veterinary Practice ................................................................................................. 10

Clinical Experience with Autologous Bone Marrow for Treatment of Orthopedic Injuries ......................................................................................................................... 11

Equine Cord Blood MSCs in Bone and Cartilage Engineering ........................................................... 12

Osteoprogenitor Cell Therapy in an Equine Fracture Model....... ............. .............. ............. ............. .. 13

Stem Cell Gene Programming for Musculoskeletal Repair.................. ............. ............. ............. ......... 14

Evaluation of the MarrowXpress System for Red Cell Depletion,

Volume Reduction and Mononuclear Cell Recovery of EquineUmbilical Cord Blood and Bone Marrow ............. .............. ............. ............. ............. ............. ......... 15

Immune Effect of Equine Bone Marrow-Derived Mesenchymal Stem Cells ............ ............. ............. .. 16

Combining Processed Stem Cells and PRP for the Treatment of Acute SevereTendon and Ligament Injuries in Thoroughbred Racehorses ........................................................ 17

Platelet-Rich Plasma as Regenerative Therapy: Methods of Useand Growth Factor Dynamics ............. ............. ............. ............. .............. ............. ............. ............. 18

Evaluation of Senescence in Mesenchumal Stromal Cells Isolated from EquineBone Marrow, Adipose and Umbilical Cord Tissue ........................................................................ 19

Hypoxia and Stem Cell Biology ............. ............. ............. ............. ............. .............. ............. ............. .. 20

Self-Complementary Adeno-Associated Viral Vectors Exhibi t HighEfficiency in Joint Tissues Depending on Serotype Selection ............. ............. .............. ............. .... 21

The Effect of Tendon-derived Progenitor Cells on a Collagenase-Induced Model of Tendinitis in Horses .......................................................................................... 22


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Characterization of Tendon Progenitor Cells

J. G. Barrett and J. A. BrownMarion duPont Scott Equine Medical Center

Virginia-Maryland Regional College of Veterinary Medicine, VA

The objective of our study was to compare cell growth kinetics and biosynthetic capabilities of bonemarrow mesenchymal stem cells (BM-MSCs) and tendon-derived progenitor cells (TPCs) cultured onstandard culture plates versus bovine, highly purified bovine, porcine, and rat tail collagen-coatedsurfaces.

Cells from six young-adult horses were isolated, expanded, and cultured on collagen-coated tissue culture plates for 7 days. Samples were analyzed for cell viability and number on days 4 and 7, and for mRNAexpression of collagen type I, collagen type III, cartilage oligomeric matrix protein (COMP), and decorinon day 7.

We found that tendon-derived progenitor cells cultured on all collagen types yielded more cells thansimilarly cultured BMMSCs on day 4. A statistically significant (P=0.05) increase in cell number was

observed for TPCs grown on rat tail collagen versus standard culture on day 4. BM-MSCs expressedmore collagen type I mRNA when cultured on control, porcine and highly-purified collagen, and morecollagen type III when cultured on control, porcine, highly-purified collagen, and rat-tail collagen, thandid TPCs. Tendon-progenitor cells expressed significantly more COMP when cultured on control and allcollagen types, and decorin when cultured on porcine, highly purified bovine and bovine collagen whencompared with BM-MSCs. No difference in expression of collagen type I, collagen type III, COMP or decorin mRNA was observed between collagen groups and standard culture plate controls for both celltypes on day 7.

Based on this study, we learned that there is an advantage to culturing TPCs on randomly organized rat-tail collagen during the early growth phase. Tendon progenitor cells showed superior growth kinetics andexpression of the matrix organizational components, COMP and decorin than did similarly cultured BM-

MSCs, which preferentially expressed more collagen types III and I. Further in vitro studiescharacterizing factors that influence BM-MSC and TPC gene expression are warranted.

Addendum - To validate the potential use of TPCs to t reat tendinitis, a pilot study was performed in tworace horses (2-3 yo) with severe superficial digital flexor tendinitis. Lateral digital extensor tendon

biopsies were obtained; TPCs were cultured and injected into each SDFT. No adverse effects were seenwith biopsies or with injection site reactions. One horse is 4 months post-injury at this time, and the other horse is 9 months post-injury. Both lesions are filling in with near-normal echogenicity, but linear fiber

pattern is abnormal though improving for both 4- and 9-month follow-up of each horse. However, thecross-sectional area of each tendon remains enlarged (greatest CSA is 2.82 cm 2 for the horse at 4 months;and 1.77 cm 2 for the horse at 9 months post injury). The horse at 9 months post-injury is sound at the trot.

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Immunomodulatory Activity of Allogeneic Bone Marrow, Adiposeand Placentally-Derived Equine Mesenchymal Stem Cells

D.D. Carrade, N.J. Walker, D.L. Borjesson

Department of Pathology, Microbiology and Immunology

School of Veterinary Medicine, University of California, Davis

Tissue sources of equine multipotent mesenchymal stem cells (MSCs) include fat, bone marrow,umbilical cord tissue and umbilical cord blood. Currently, patient tissues are harvested and MSCs areexpanded ex vivo for autologous therapeutic use. However limiting cellular therapies to autologous use

precludes treatment of acute lesions (MSC expansion takes 2-3 weeks) and complicates quality controland standardization due to variations in MSC recovery and growth kinetics. Our long-term objective is todevelop a safe and efficacious allogeneic (third party) MSC product for equine regenerative therapy.

The use of allogeneic, unmatched MSCs may be possible because studies with human and rodent MSCshave demonstrated that MSCs (1) are of inherently low immunogenicity, (2) do not elicit lymphocyte

proliferation, and (3) suppress the immune response. As a first step to assess preclinical safety for allotransplantation of MSCs, we determined lymphocyte proliferative responses to allogeneic MSCs andcompared surface expression of major histocompatibility complex (MHC)-I and MHC II on equine MSCsderived from bone marrow, fat, umbilical cord blood and cord tissue. Four cell lines from each tissuetype were assessed. Similar to rodent and human MSCs, equine MSCs from bone marrow, cord blood,cord tissue and fat all express high levels of MHC-I (>90%) with minimal to no expression of MHC-II(one adipose line and one cord blood line showed low 16-20% MHC-II expression). All equine MSClines were negative for the T cell costimulatory molecule CD86. Bone marrow- and adipose-derivedallogeneic MSCs significantly decreased lymphocyte proliferation in a one-way mixed lymphocytereaction (30-40% of positive control). Allogeneic MSCs derived from cord tissue and cord blood neither elicited nor suppressed lymphocyte proliferation. These findings suggest that MSCs derived fromdifferent tissues vary in their capacity to modulate lymphocyte proliferation and that alterations inlymphocyte proliferation in vitro are independent of MSC MHC expression.

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MSCs are the New Medicine!

Arnold I. Caplan, Ph.D.

Professor of Biology and Director, Skeletal Research Center Case Western Reserve University, Cleveland, Ohio

Marrow-derived adult mesenchymal stem cells (MSCs) can be isolated and culture expanded. Althoughthese cells are capable of differentiating into lineages that result in the fabrication of bone, cartilage,muscle, marrow stroma, tendon/ligament, fat and other connective tissues, MSCs have recently beenshown to be intrinsically therapeutic. Such culture-expanded adult MSCs are immunomodulatory,especially in muting T-cells and, thus, allogeneic MSCs have been used to mute or cure graft-versus-host-disease and Crohns disease and are now being tested in certain autoimmune diseases. Furthermore, theseallo-MSCs set up a regenerative microenvironment that is anti-apoptotic, anti-scarring, mitotic for tissueintrinsic progenitors and angiogenic. These immuno and trophic activities result from the secretion of

powerful bioactive molecules which, in combination, support localized regenerative event. The MSCsreside in every tissue of the body and function as perivascular cells (pericytes) until a focal injury occurs.

At sites of injury, the pericyte is released and functions as a MSC that provides molecular assistance inactivities leading to tissue regeneration. Such assistance involves many tasks involving the immuno-

protection and trophic activities provided by the MSCs.

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Stem Cell-Derived Ocular Cells for the Treatment of Human Eye Disease

Dennis Clegg, PhD Neuroscience Research Institute

University of California, Santa Barbara

Human induced pluripotent stem cells (iPSCs) have great promise for cellular therapy, and ocular cellssuch as the retinal pigmented epithelium (RPE) are of particular interest because of their potential for treating degenerative eye diseases, including age-related macular degeneration. We show here that iPSCsgenerated using Oct4, Sox2, Nanog, and Lin28 can spontaneously differentiate into RPE cells, which canthen be isolated and cultured to form highly differentiated RPE monolayers. RPE derived from iPSCs(iPS-RPE) were analyzed with respect to gene expression, protein expression, and rod outer segment

phagocytosis, and compared with cultured fetal human RPE (fRPE) and RPE derived from hESCs (hESC-RPE). iPS-RPE expression of marker mRNAs was quantitatively similar to that of fRPE and hESC-RPE,and marker proteins were appropriately expressed and localized in polarized monolayers. Levels of rodouter segment phagocytosis by iPS-RPE, fRPE, and hESC-RPE were likewise similar and dependent onintegrin alpha v beta 5. After transplantation into the Royal College of Surgeons (RCS) dystrophic rat,these cells facilitated short-term maintenance of photoreceptors and rescued visual function. Thus, iPS-RPE represent a potential source for cellular therapy for macular degeneration and provide a new avenuefor the study of RPE diseases.

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Role of Growth Factors in Mesenchymal Stem Cell-MediatedBlood Flow Restoration and Bone Repair

Fernando FierroUC Davis Medical Center

Recent gene expression profiling highlighted basic fibroblast growth factor (bFGF), platelet-derivedgrowth factor (PDGFB) and transforming growth factor beta (TGF-b 1) signaling as critical pathwaysduring proliferation and differentiation of mesenchymal stem cells (MSC). In addition, MSCs releasehigh levels of vascular endothelial growth factor (VEGF), a critical pro-angiogenic signal. Usinglentiviral-mediated overexpression of these growth factors, we observe that bFGF or PDGFB reduce bymore than two-fold the doubling times of MSCs (p


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Clinical Follow-up of Horses Treated Intra-articularly with Bone Marrow-Derived MesenchymalStem Cells for Orthopaedic Lesions

Dora J. Ferris*, David D. Frisbee*, John D. Kisiday*, C. Wayne McIlwraith*,Brent A. Hague**, Michael D. Major**, Robert K. Schneider***, Chad J. Zubrod**,

Christopher E. Kawcak* and Laurie R. Goodrich*

*Gail Holmes Equine Orthopaedic Research Center, College of Veterinary Medicine & BiomedicalSciences, Colorado State University, Fort Collins, CO

**Oakridge Equine Hospital PC, Edmond, OK ***Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State

University, Pullman, WA

In 40 orthopaedic (joint/collateral ligament injury) cases treated intra-articularly with bone marrow-derived mesenchymal stem cells (BMSCs), 73% resulted in return to function when followed up anaverage of 21 months post-treatment. In horses with Grade 3 meniscal injuries, two of five horses wereable to return to or exceed their previous level of soundness.

BMSCs have gathered increasing attention as a viable therapy for musculoskeletal lesions in horses,humans, and other animals. Clinical follow-up has been limited mainly to case reports with low numbers.The goal of this study was to follow-up a modest number of horses suffering orthopaedic lesions andtreated intra-articularly with BMSCs.

Horses (N=60) receiving BMSCs expanded at Colorado State Universities, Orthopaedic ResearchLaboratory or Advanced Regenerative Therapies were retrospectively followed based on medical recordanalysis and follow-up with the attending veterinarian/owner. Five separate centers participated.

Follow-up information (mean=21, range 7-39 months post treatment) was obtained on 40 horses. Jointflares were reported in 3 horses that were not pretreated with nonsteroidal anti-inflammatory medications

prior to cell injection. All horses improved and, similar to other cases, returned to work. Of 40 horseswith orthopaedic injuries, 73% returned to work. Age, sex, breed, or discipline was not significantlyassociated with outcome. The most impressive outcome was seen in horses treated for Grade 3 meniscaltears. In the current study, 2/5 horses with Grade 3 lesions were able to return to or exceed their previouslevel of soundness following arthroscopic debridement of the lesion and intra-articular injection of BMSCs.

This study confirms anecdotal reports of good clinical outcomes post BMSC treatment for orthopaediclesions, especially in cases suffering from severe meniscal damage. Results of this study support futurecontrolled trials to be undertaken for the use of BMSCs in horses.

Acknowledgements to Gary Baxter, Kurt Harris, and Bob Racich for contribution of cases ( 5) and to

Sangeeta Rao and Francisco Oleo-Popelka for statistical analysis. Dr.s Frisbie, Kisiday, McIlwraith,Kawcak, Goodrich, Watkins, Hague, Major and Schneider have stock in Advanced RegenerativeTherapies.

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Regenerative Medicine Techniques for Treatment of Deep Digital Flexor Tendinopathy

Robin Bell, Sabine Buechler, Kaitlin Clark, Alexandra Clifton, Jack Snyder,Melinda MacDonald, Sarah Puchalski, Mary Beth Whitcomb, Larry Galuppo

Department of Surgical and Radiological SciencesSchool of Veterinary Medicine, University of California, Davis

With the advent of MRI and contrast-enhanced CT in equine practice, in conjunction with more traditionaltechniques such as diagnostic ultrasonography, diagnosis of deep digital flexor tendinopathy (DDFT) in the regionof the foot is common in performance horses. The most commonly identified lesion types are core lesions,abrasions (dorsal or ventral) and longitudinal tears. Traditional therapy includes rest and rehabilitation, correctiveshoeing, nonsteroidal anti-inflammatory drugs, and administration of intra-articular medications into the distalinterphalangeal joint or intrasynovial medications into the navicular bursa or tendon sheath. In some casesendoscopic debridement of the damaged tissue is also indicated.

Regardless of the type of traditional therapy used, the prognosis for horses with primary DDFT to return to full performance is reported to be only 29%. If there is combined pathology (other lesions identified in the surroundingligaments and bones) the prognosis is worse, with the majority of those horses exhibiting persistent lameness.Regenerative medicine technology may offer the potential for improved tendon and ligament healing. Currentregenerative medicine techniques include cellular therapy with bone marrow-derived mesenchymal stem cells andadipose-derived stromal vascular fraction, bone marrow aspirate concentrate and platelet-rich plasma.

The objective of our study was to determine whether regenerative medicine techniques improved the outcomes for horses diagnosed with primary DDFT or DDFT combined with other related lesions. To accomplish this, weconducted a retrospective analysis of horses seen between 2004 and 2009 at the UC Davis Veterinary MedicalTeaching Hospital that had been treated with one of three regenerative medicine techniques: bone marrow-derivedmesenchymal stem cells (MSCs), adipose-derived stromal vascular fraction (SVF), or bone marrow aspirateconcentration (BMAC).

Follow-up information was available for 40 horses, with a minimum follow-up time of six months. In addition tothe primary diagnosis of DDFT and DDFT in combination with other pathology, lesion grade was also considered

in relation to treatment outcome. In general, all three regenerative medicine techniques had good success for mildand moderate lesions, with decreased efficacy for severe lesions in all cases. However, horses with severe lesionsthat were treated with SVF had a good outcome. Horses with bilateral disease had a poor prognosis for return to

performance regardless of treatment type.

The overall treatment outcome was comparable for horses with primary DDFT and those with combined pathology,regardless of lesion grade. In fact, horses with DDFT combined with other pathology tended to have a better outcome for each lesion grade. This finding was unexpected and further study is needed to determine whether thereis any relationship between DDFT combined with other pathology and better treatment outcomes. As expected,horses with more severe lesions had a worse prognosis for return to full performance.

All three treatment techniques had reasonable success with mild and moderate lesions. With more severe lesions,SVF had a better outcome compared with horses treated with BMAC or MSCs. However, this result may have

been biased by the fact that fewer horses were treated with SVF and a majority of the horses treated with MSCswere bilaterally affected.

Overall, all horses had a better success for return to performance compared with published data for horses treatedwith traditional therapy techniques. This study supports the use of regenerative medicine techniques for treatmentof DDFT, although further investigation is needed to evaluate the effects of multiple treatments and combined

products to optimize healing. Regardless of the regenerative medicine technique used, early diagnosis and therapyshould improve the overall success of horses afflicted with DDFT of the distal extremity.

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Possible Clinical Applications for Stem Cell Therapyfrom Research Based in Equine Veterinary Practice

Dr. Doug HerthelAlamo Pintado Equine Hospital

Los Olivos, CA

Our practice group historically was one of the first to become involved with stem cell-based therapies for equine orthopedic injuries. At the 2001 AAEP Convention we reported positive results for horses treated

between 1995 and 1998 with autologous bone marrow aspirates for suspensory desmitis. The report of these cases has stimulated multiple investigators to further analyze and improve upon our originaltechniques.

Since our original casework, we have continued to treat and document stem cell treatments for orthopedicinjuries using autologous bone marrow concentrate and adipose vascular fraction product produced byVet Stem. More recently, we have begun to use autologous MSCs derived and expanded from cultured

bone marrow concentrate.

We have based our clinical decisions for treatment on two hypotheses. The first is that stem cells are the paramedics of the body and that injected MSCs can replace, rejuvenate or repair injured or dying tissueswithin the body. The second hypothesis is that adipose vascular fractions, bone marrow concentrates andcultured MSCs contain and/or stimulate tissue-healing trophic components. Based on these hypotheses,we have assumed that high numbers of MSCs would accelerate healing in damaged tissue and that theseincreased numbers and concentration of cells improves, exponentially, their ability to communicate andsecrete signal molecules and trophic factors.

Based on these scientific assumptions, we have used various stem cell and regenerative medicinetechniques to treat tendon and ligament injuries of all types, osteoarthritis and laminitis. The results have

proven to be universally rewarding. We expect that with increasing research to improve our

understanding of proper dosage, intervals for treatment and preferred modes of administration, theseinitial positive results could be improved upon. Therefore, we are encouraging a strong and continuingcollaboration between basic research scientists and equine clinicians to improve the health care of horses.

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Clinical Experience with Autologous Bone Marrow for Treatment of Orthopedic Injuries

CR Johnson, DVM, MS, DACVSWoodford Equine Hospital

Versailles, KY

Over the past nine years we have been treating career threatening and historically frustrating orthopedicinjuries of the equine athlete with locally administered, autologous bone marrow. Cases treated includedsoft tissue and bony tissue lesions. The soft tissue cases treated included severe superficial flexor tendonitis, refractory proximal suspensory desmitis, and suspensory branch desmitis. The bony tissuecases were subchondral cysts of the stifle and fetlock, and surgically irreparable sesamoid fractures.

The majority of the soft tissue cases were severe superficial digital flexor tendonitis in racingThoroughbreds. We defined severe lesions as 30-50% core lesions that were at least 10 cm long. Caseswere treated by collecting and directly injecting autologous bone marrow into the core lesions usingultrasound guidance for needle placement. My experience has been that tendons treated with autologous

bone marrow decrease in size (grossly and ultrasonographically) faster than traditionally treated tendons,and the ultrasonographic appearance of the fiber pattern improves much faster than traditionally treated

tendons. Despite the relatively fast return to normalcy of ultrasonographic appearance of these large corelesions, the overall time of healing is not reduced (nine to twelve months). In my experience, largelesions of the suspensory branches respond similarly to superficial flexor tendon lesions. The cases of

proximal suspensory desmitis treated with autuologous bone marrow injection were either severe or refractory to more conservative therapies (rest, shockwave, etc.)

Bony tissue lesions treated with direct injection of autologous bone marrow were subchondral cysts andsurgically irreparable sesamoid fractures (comminuted, abaxial). The treated cases with subchondralcysts (stifles and fetlocks) were all lame at presentation ranging from Grade 2 to Grade 4 of 5 of theaffected limb and most were Thoroughbred racing stock (weanlings, yearlings, and 2-year-old horses).My experience with treating these cases is that approximately 75% become sound and stay sound longer than do cases treated with surgical debridement of the cysts or direct injection of the cysts with Vetalog.

The fractured sesamoid cases treated with autologous bone marrow injection were weanling and yearlingswith fibrous unions (at least four months after occurrence of the fracture), and, more recently, race horseswith large basilar or comminuted sesamoid fractures. My experience has been that bony union can beinduced in the cases of fibrous union and the bony union of older horses is occurring faster thantraditionally treated cases. No data on effect of performance is available to date, but, in the fibrous unioncases of the horses intended for sale at auction, the radiographic appearance of the sesamoid wasimproved.

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Equine Cord Blood MSCs in Bone and Cartilage Engineering

Thomas G. Koch, DVM, PhD, ACVIM CandidateSeniorforsker, Orthopedic Research Laboratorium, Aarhus University, Denmark

Adjunct Professor, Department of Biomedical Sciences, University of Guelph, Canada

Orthopedic injuries are the most common causes of lost training days or premature retirement in theequine athlete, and bone and joint cartilage problems constitute a significant proportion of these problems.Cell-based therapies are emerging treatment options for equine orthopedic injuries, but at this time a morewidespread use of cell-based therapies in the horse is restricted to traumatic ligament and tendon injuries.A number of studies have shown so-called trilineage potential of multipotent mesenchymal stem cells(MSCs) from neonatal and adult tissues to differentiate into cartilage, bone and tendon in vitro suggestiveof their in vivo potential for cartilage and bone repair.

Cord blood MSC-based therapies may offer advantages when compared with adult MSCs, including bonemarrow MSCs (BM-MSCs). Studies on human cord blood-derived MSCs suggest that these cells havehigher replicative potential and broader potency, as illustrated by their longer telomeres, compared with

bone marrow-derived cells, and the ability to become tissues of endoderm and ectoderm origin in addition

to the common mesoderm cell lineages.

We recently reported the isolation of MSCs from fresh equine cord blood (eCB-MSCs). These cells retaintrilineage potency after cryopreservation and therefore can likely be banked in cryostorage for later use.In addition to their potential superior cellular properties, eCB-MSCs would be available at the time of injury allowing treatment time to be dictated by the clinician, as opposed to BM-MSCs where time for cellular expansion must be allowed for. Safety concerns to horse and clinician associated with the harvestof tissues from adult horses would also be eliminated with the use of cord blood-derived cells. Reducedisolation success of MSCs from umbilical cord blood compared with that of bone marrow aspirates have

been a concern in the past, but we recently demonstrated isolation of MSCs from all processed eCB units.

Comparative chondrogenic studies of eCB- and eBM-MSCs showed that both cell sources were capable

of producing hyaline-like cartilage in vitro . The results of this study suggest that eCB-MSCs have greater potential for chondrogenesis than eBM-MSCs. Significant differences were noted in pellet size, geneexpression and protein secretion assays and the histological morphology was more hyaline-like andstained more strongly for extracellular matrix GAGs in the CB-MSC cultures.

The capacity of CB-MSCs to proliferate and differentiate osteogenically within resorbable Pro Osteoncoralline hydroxyapatite scaffolds was tested. The proliferation of undifferentiatied CB-MSCs seeded inscaffolds and dynamically cultured in non-induction medium was shown to recover after an initialdecrease after seeding. Scaffolds seeded with eCB-MSCs and statically cultured in osteogenic inductionmedium demonstrated improved differentiation as indicated by cell morphology and matrix depositionusing SEM; high alkaline phosphatase (ALP) activity and osteocalcin concentration; and increasedexpression of osteogenic genes compared to non-induced scaffolds. The results from this study show that

eCB-MSCs are capable of in vitro

proliferation and osteogenic differentiation within corallinehydroxyapatite scaffolds.

The results of these in vitro chondrogenic and osteogenic studies supports continued research of thereparative potential of equine CB-MSCs for cartilage and bone defects. Research in the use of equine CB-MSCs in engineering osteochondral constructs in vitro for mosaic arthroplasty of focal cartilage defects isongoing and scheduled for in vivo assessment in the horse.

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Osteoprogenitor Cell Therapy in an Equine Fracture Model

Laurie McDuffieUniversity of Prince Edward Island

Research conducted in our laboratory has shown that fibroblast-like cells can be isolated readily fromequine periosteal tissue. Based on osteogenic, adipogenic, and chondrogenic differentiation as well asCD marker evaluation, we consider these cells to include populations of mesenchymal stem cells andosteoprogenitors. The ability of these periosteal-derived cells to become bone-producing cells wasconfirmed based on the presence of multiple bone markers and bone nodule production. In an attempt todetermine the capacity of these cells to produce bone in the live animal and promote bone healing, a livehorse study including simulated fractures was conducted.

Initially, a preliminary study was conducted using five adult horses. Horses had fractures created in thelateral splint bones (MC4) of both front limbs. While horses were under general anaesthesia, a half

centimetre of bone was removed from the upper portion of the splint bone using a bone saw. At the timeof fracture creation, periosteal tissue was collected from the medial aspect of the tibia and used for cellisolation and expansion. Preparation of periosteal-derived cells prior to implantations included labellingwith DiI and induction with osteogenic media.

Simulated fractures were treated with periosteal-derived cells in fibrin glue (treatment limb) or fibrin gluealone (control). Implantation of cells was conducted with the horses standing and sedate using ultrasoundguidance every 2 weeks up to 6 weeks post-operatively. Radiographic data were collected weekly.Histological data were collected at various times for each horse (8, 9, 10, 11 and 12 weeks).

The results of the pilot study were promising. Radiographic results showed an increase (p


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Stem Cell Gene-Programming for Musculoskeletal Repair

Dr. Alan J. NixonSchool of Veterinary Medicine

Cornell University

Our research investigations have focused on the use of autologous, bone marrow-derived MSCs for cartilage repair in an equine model. Cartilage defects were created in the lateral trochlear ridge of bothstifles. One defect was treated with 20 million MSCs suspended in an autologous cryoprecipitateactivated with thrombin and CaCl. The opposite joint was treated with the same fibrin substrate withoutMSCs.

At four weeks, the MSC-treated joint showed improved arthroscopic appearance over the fibrin-onlytreated control group. Biopsies taken at 4 weeks also demonstrated an enhanced response compared withthe control group. However, at 8 months the histologic and immunohistologic appearance of the cartilagerepair of both the MSC-treated and control joint were not significantly different.

The results this investigation demonstrate that MSCs alone have only moderate effects on improvinglong-term quality of cartilage healing. However, other studies conducted by this researcher and othershave given indications that growth factor gene-transcripted MSCs may demonstrate enhancedchondrogenesis in cartilage resurfacing. Consequently, further investigative work to determine whichgenes best drive chondrogenic transformation would seem to be in order.

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Evaluation of the MarrowXpress System for Red Cell Depletion,Volume Reduction and Mononuclear Cell Recovery of

Equine Umbilical Cord Blood and Bone Marrow

Sean D. Owens, DVM, DACVP, Danielle D. Carrade, BS, Jennifer L, Johns, DVM, DACVP, Julie

Burges, BS, MS, Larry D, Galuppo, DVM, DACVS, Fred Librach, BS, Dori L. Borjesson, DVM, PhD,DACVPDepartment of Pathology, Microbiology and Immunology (Owens, Johns, Carrade, Borjesson)

Department of Surgical and Radiological Sciences (Galuppo)UC Davis Veterinary Blood Bank (Burges, Librach)

School of Veterinary Medicine, University of California, Davis, CA

After collection, cord blood and bone marrow must be volume reduced and RBC depleted in order todevelop therapeutic cellular products and prepare cells for cryopreservation and storage. TheMarrowXpress System (MXP) is an automated, closed, sterile system that uses flow control optical

sensors to achieve separation of a concentrated buffy coat. The purpose of this study was to evaluate theability of the MXP to reliably volume reduce, RBC deplete and recover mononuclear cells (MNCs) fromequine cord blood and bone marrow. Cord blood was obtained from 60 thoroughbred foals within 10minutes of parturition, and bone marrow was obtained from 48 horses of both sexes representing variousages and breeds. Cord blood was collected into either a 250-ml blood bag or three 60-ml syringescontaining CPDA-1. Bone marrow was collected into one or two 60-ml syringes containing heparin. Afull CBC was performed on the cord blood and bone marrow pre- and post-MXP processing using theADVIA 120 Hematology System. The results obtained were as follows: Cord blood - Percent volumereduction: 84.5 ( 4.2). Percent HCT depletion: 62.4 ( 6.6). Percent Mononuclear Cell recovery: 87.6 ( 10.5). Bone marrow - Percent volume reduction: 75.8 ( 8.0). Percent HCT depletion: 72.3 ( 10.0).Percent Mononuclear Cell recovery: 88.3 ( 31.5). In conclusion, the MXP System efficiently andreproducibly recovered bone marrow and cord blood MNCs, with RBC reduction, to a consistent and

uniform volume.

Corresponding author: Dr. Sean Owens, Department of Pathology, Microbiology and Immunology,School of Veterinary Medicine, University of California, Davis, California 95616 (e-mail:[email protected]).

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Immune-Effect of Equine Bone Marrow-Derived Mesenchymal Stem Cells

John Peroni, DVM, MSCollege of Veterinary Medicine

University of Georgia

Adult mesenchymal stem cells (MSCs) have attracted attention in past years in the areas of tissueengineering, as vehicles for gene therapy, as support cells for hematopoietic stem cell engraftment and asanti-proliferative and immunomodulating cells. The use of allogenic cells is an attractive proposition

because these cells are readily available as an off the shelf treatment and can be grown to behomogeneous populations of cells, thereby providing a more predictable cell-based treatment approachand increased quality control. Prior to accomplishing an allogenic approach, research needs to becompleted to assess the immunoreactivity of the cell transplant.

In this brief presentation, we delineate some of our results in investigating the complex relationship thatexists between the immune system and bone marrow MSCs (BM-MSCs). There is growing evidence thatone of the mechanisms through which adult stem cells contribute to the healing of injured tissues is by

modulating inflammation at the site of injury. For example, these cells reduce the damaging effects of inflammatory proteins released in tissues after injury or trauma. In addition, there is compelling evidencefrom work in laboratory animals that these beneficial effects can be achieved in inflammatory diseasessuch as colitis and renal disease by the administration of stem cells derived from the injured animal itself (autologous cells) or another animal of the same species (allogenic cells).

In our laboratory, we identify adult equine stem cells isolated from bone marrow aspirates by theexpression of the following genes: CD105, CD73, CD44 and CD90. Based on evidence in the literatureand our own experience, approximately 85% of adult equine stem cells are immunoreactive for antibodiesdirected against these gene products. To ensure that the isolated cells are, in fact, stem cells and nothematopoietic in origin, we ensure that they do not express markers associated with hematopoietic cells(e.g., CD45, CD 34 and CD31). Finally, we differentiate these cells into bone, fat and cartilage, using

techniques standardized for stem cells derived from other species.

In recent in vitro experiments, we tested the hypothesis that co-culture of equine BM-MSCs withallogenic lymphocytes would modulate the proliferative effects of phytohemagglutinin (PHA) on thelymphocytes. The results of these experiments indicate that equine BM-MSCs in co-culture reduce therate of lymphocyte proliferation in a manner that is dependent upon the concentration of BM-MSCs

present. Furthermore, BM-MSCs in co-culture with lymphocytes markedly reduce production of TNFalpha by lymphocytes stimulated with PHA. These results provide the basis for ongoing experimentsin which we determine whether these anti-inflammatory effects are dependent on the presence of the BM-MSCs themselves or are due to a soluble factor released by those cells.

Our studies indicate that co-culturing of adult stem cells with lymphocytes reduces proliferative responses

of allogenic lymphocytes and their synthesis of inflammatory mediators.

In those studies, a profoundanti-inflammatory effect could be elicited by exposing cells to supernatants obtained from co-cultures of adult stem cells and stimulated lymphocytes. For example, adult stem cells by themselves or co-culturedwith lymphocytes secrete IL-10, a cytokine that regulates T-cell activity and antagonizes the pro-inflammatory effects of IL-12.

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Combining Processed Stem Cells and PRP for the Treatment of Acute Severe Tendon and Ligament Injuries in Thoroughbred Racehorses

Wesley Sutter Ocala Equine Hospital

Combining PRP and processed stem cells is an attempt to provide the three components of tissueregeneration; a provisional matrix and growth factors via PRP and cells via cBMA or processed adiposetissue. These treatments can be administered patient side when cBMA and PRP are combined or within 2days of lipectomy with PRP and processed adipose tissue. We hypothesized that in acute, severe tendonand ligament lesions in thoroughbreds a combination of PRP and processed stem cells would result in ahigher percentage of success than PRP injected alone. Success was defined as 5 or more starts followingtreatment, partial success was 1-4 starts, and failures were horses that failed to have a start within 18months of treatment. The records of horses treated from 2007-2009 with PRP were examined. We foundthat 166 horses met the criteria having acute core lesions greater than 15% in the SDF tendon or suspensory body or branch. Of those, 112 horses were treated with PRP alone, and 54 were treated withPRP and processed stem cells. The superficial digital flexor tendonitis group showed 33% success, 21%

partial success, and 46% failure in horses treated with PRP alone (n=59). In horses where PRP and processed stem cells were combined to treat SDF tendonitis (n=37), the results were 48% success, 12% partial success, and 40% failure. Severe lesions of the suspensory body and branch treated with PRP(n=53) showed 69% success, 7% partial success and 23% failure. When processed stem cells werecombined with PRP for suspensory injuries (n=17), the success was 44%, partial success 22%, and failure33%. There appears to be a trend toward a beneficial effect of PRP and processed stem cells in the SDFtendonitis. This trend does not appear to present in the severe suspensory branch and body injuries. Atthis writing, 44% (n= 76) of the treated horses are in rehab without re-injury. More time is necessary for the data to mature before complete analysis can be undertaken.

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20/22

Platelet-Rich Plasma as Regenerative Therapy:Methods of Use and Growth Factor Dynamics

Jamie Textor, DVM, DACVSSchool of Veterinary MedicineUniversity of California, Davis

Platelet-rich plasma (PRP) is widely used to augment healing of musculoskeletal tissues in horses and in people. The intent of intralesional PRP use is to administer a physiologic cocktail of concentratedgrowth factors, which are released from the alpha granules of activated platelets. PRP has manycomponents of an ideal regenerative therapy: it is autogenous, collection is minimally invasive(venipuncture), preparation is automated and rapid, and it is effective. Results indicate improved tissuehealing in notoriously unforgiving sites, such as equine or human tendon. Since preparation is not labor-intensive, PRP is available for same-day stall-side or intra-operative use. However, commonly usedadministration strategies for horses may not provide the maximum available benefit from PRP; data will

be presented to explain growth factor release profiles. This talk will also provide a review of theliterature and discuss the effect of preparation and administration methods, with the aim of improving our use of PRP in equine regenerative medicine applications.

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21/22

Evaluation of Senescence in Mesenchymal Stromal Cells Isolated fromEquine Bone Marrow, Adipose and Umbilical Cord Tissue

M. A. Vidal, N. Walker, E. Napoli, D. C. Genetos, D. L. Borjesson

Department of Pathology, Microbiology and Immunology (Borjesson, Walker)

Department of Surgical and Radiological Science (Genetos, Napoli, Vidal)School of Veterinary Medicine, University of California, Davis

Mesenchymal stem cells (MSCs) from adult and neonatal tissues are intensively investigated for their usein regenerative medicine. The purpose of this study was to compare the onset of senescence in stem cellsisolated from equine bone marrow (BMSCs), adipose tissue (ASCs) and umbilical cord tissue (UCMSCs).The MSCs harvested from tissues of four donors were used to assess cell proliferation, senescenceassociated -galactidose staining, and telomere length as well as stemness and lineage-specific marker expression. The results showed that before senescence ensued, all cell types proliferated at approximately1 day/cell doubling. BMSCs significantly increased cell doubling rate after passage 8 and ceased

proliferation after 32 total cell doublings, while UCMSCs and ASCs achieved 66 and 80 total celldoublings, respectively. UCMSCs and ASCs from each donor showed marked -galactidose staining at

passage 20 to 21, while BMSCs stained positive by passage 10. These passages were also associated withsignificantly reduced telomere length for all three cell types, which ranged from 10.2 to 4.5 kbp in

passage 3 and senescent cultures, respectively. Expression levels of Oct 4 appeared to decrease inBMSCs toward senescence, while they increased in ASCs. Oct 4 expression was significantly higher inall MSC types compared with Sox2 and Nanog. MSCs from each tissue appeared to stain intensively for osteonectin at the stage of senescence compared with earlier passages, while vimentin and low levels of smooth muscle actin were consistently expressed. In conclusion, MSCs from bone marrow appear tosenesce much earlier than those from adipose and umbilical cord tissue. These results demonstrate thelimited passage numbers of subcultured stem cells from bone marrow available for use in research andtissue engineering.

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22/22

Hypoxia and Stem Cell Biology

Clare Yellowley, PhDSchool of Veterinary MedicineUniversity of California, Davis

Oxygen is a critical regulator of bone development, maintenance and repair. In most cases cellsdetect changes in regional oxygen using the hypoxia-inducible transcription factor pathway (HIF)and alter their gene expression profile and protein production for a variety of reasons, includingconservation of energy (1). We are interested in the effects of oxygen tension on the biology of the osteoblast cell lineage and ultimately on bone physiology. We have demonstrated that under hypoxic conditions, osteoblasts change their prostaglandin expression profile, in particular upregulating prostaglandin E 2 levels and the expression of its receptor EP1 (2,3). Hypoxicosteocytes upregulate the expression of osteopontin, which is chemotactic for mesenchymal stemcells (MSCs) (4). Interestingly, we have also shown that cellular differentiation in human (5) andcanine MSCs is impaired as the oxygen level drops and that hMSC ability to migrate is impaired.Clearly these cells are exquisitely sensitive to the oxygen environment and effects on cell

physiology are diverse.

Oxygen plays an important role in bone development (6), but its influence may extend into adultlife where it appears to regulate bone repair (7). Fracture sites are regions of reduced oxygentension, primarily as a result of vasculature disruption (8,9). Using a well-characterized method of inducing a transverse femoral fracture in mice (10) we have demonstrated regional hypoxia in thefracture callus. We have also investigated the role of the SDF-1/CXCR4 signaling axis onfracture healing. SDF-1 is a chemokine that data suggest is released at sites of ischemic injury(11). SDF-1 interaction with its receptor CXCR4 on MSCs is thought to initiate and potentiatetheir migration to the site of injury to participate in repair. Using our femoral fracture model, wedemonstrate that an inhibitor of SDF-1/CXCR4 signaling, AMD3100, inhibits fracture repair byreducing callus specific gene expression and callus size. We postulate that disruption of SDF-1

signaling to its receptor inhibits MSC homing to the site of injury and slows repair.

In summary, oxygen levels have a significant impact on cell function. Regulation of oxygentension is often overlooked in tissue regeneration protocols but may have a significant impact on

proliferation, differentiation and ultimately survival of stem and progenitor cells. The regionaloxygen levels should be carefully considered when propagating cells and generating tissues invitro , and during implantation in vivo .

References1. Semenza, G.L. (2008) Curr. Opin. Genet. Dev. Oct;8(5):588-94.2. Lee, C.M. et.al. (2010) J Bone Miner Metab. 28(1):8-16.3. Genetos, D.C. et. al. (2009) J Cell Biochem. May 15;107(2):233-9.4. Raheja, L.M. et. al. (2008) Biochem Biophys Res Commun. Feb 22;366(4):1061-6.5. Raheja, L.M. et. al. (2010) Cells Tissues Organs. 191(3):175-84.6. Schipani, E. (2006) Ann N Y Acad Sci. Apr;1068:66-73.7. Shen, X. (2009) J Orthop Res. Oct;27(10):1298-305.8. Brighton, C.T, and Krebs, A.G. (1972) J Bone Joint Surg Am. 1972 Mar;54(2):323-32.9. Heppenstall, R.B. et. al. (1975) Clin Orthop Relat Res. Jan-Feb;(106):357-65.10. Marturano, J.E. et. al. (2008) J Biomech. 41(6):1222-8.11. Askari, A.T. et. al. (2003) Lancet. 2003 Aug 30;362(9385):697-703.

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Navrmc 2010 Abstracts