Abstracts of Presentations

1st North American Veterinary

Regenerative Medicine Conference
March 5–6, 2010, Santa Ynez Valley, California

In collaboration with
 ABSTRACTS OF PRESENTATIONS

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

Immunomodulatory Activity of Allogeneic Bone Marrow, Adipose

and 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-Derived

Mesenchymal Stem Cells for Orthopaedic Lesions ............................................................................ 7

Role of Growth Factors in Mesenchymal Stem Cell-Mediated Blood

Flow Restoration and Bone Repair ..................................................................................................... 8

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

Possible Clinical Applications for Stem Cell Therapy from Research

Based 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 Equine
Umbilical 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 Severe
Tendon and Ligament Injuries in Thoroughbred Racehorses ........................................................ 17

Platelet-Rich Plasma as Regenerative Therapy: Methods of Use

and Growth Factor Dynamics ......................................................................................................... 18

Evaluation of Senescence in Mesenchumal Stromal Cells Isolated from Equine

Bone Marrow, Adipose and Umbilical Cord Tissue ........................................................................ 19

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

Self-Complementary Adeno-Associated Viral Vectors Exhibit High

Efficiency 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
 Characterization of Tendon Progenitor Cells

J. G. Barrett and J. A. Brown

Marion 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 bone
marrow mesenchymal stem cells (BM-MSCs) and tendon-derived progenitor cells (TPCs) cultured on
standard culture plates versus bovine, highly purified bovine, porcine, and rat tail collagen-coated
surfaces.

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 mRNA
expression of collagen type I, collagen type III, cartilage oligomeric matrix protein (COMP), and decorin
on day 7.

We found that tendon-derived progenitor cells cultured on all collagen types yielded more cells than
similarly 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 expressed
more collagen type I mRNA when cultured on control, porcine and highly-purified collagen, and more
collagen type III when cultured on control, porcine, highly-purified collagen, and rat-tail collagen, than
did TPCs. Tendon-progenitor cells expressed significantly more COMP when cultured on control and all
collagen types, and decorin when cultured on porcine, highly purified bovine and bovine collagen when
compared 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 cell
types 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 and
expression 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 studies
characterizing factors that influence BM-MSC and TPC gene expression are warranted.

Addendum - To validate the potential use of TPCs to treat tendinitis, a pilot study was performed in two
race 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 seen
with 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, the
cross-sectional area of each tendon remains enlarged (greatest CSA is 2.82 cm2 for the horse at 4 months;
and 1.77 cm2 for the horse at 9 months post injury). The horse at 9 months post-injury is sound at the trot.

3
 Immunomodulatory Activity of Allogeneic Bone Marrow, Adipose
and 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 are
expanded 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 control
and standardization due to variations in MSC recovery and growth kinetics. Our long-term objective is to
develop 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 MSCs
have 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 and
compared surface expression of major histocompatibility complex (MHC)-I and MHC II on equine MSCs
derived from bone marrow, fat, umbilical cord blood and cord tissue. Four cell lines from each tissue
type 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 MSC
lines were negative for the T cell costimulatory molecule CD86. Bone marrow- and adipose-derived
allogeneic MSCs significantly decreased lymphocyte proliferation in a one-way mixed lymphocyte
reaction (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 from
different tissues vary in their capacity to modulate lymphocyte proliferation and that alterations in
lymphocyte proliferation in vitro are independent of MSC MHC expression.

4
 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. Although
these 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 been
shown 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 Crohn’s disease and are now being tested in certain autoimmune diseases. Furthermore, these
allo-MSCs set up a regenerative microenvironment that is anti-apoptotic, anti-scarring, mitotic for tissue
intrinsic progenitors and angiogenic. These immuno and trophic activities result from the secretion of
powerful bioactive molecules which, in combination, support localized regenerative event. The MSCs
reside 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 in
activities leading to tissue regeneration. Such assistance involves many tasks involving the immuno-
protection and trophic activities provided by the MSCs.

5
 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 cells
such 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 iPSCs
generated using Oct4, Sox2, Nanog, and Lin28 can spontaneously differentiate into RPE cells, which can
then 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 rod
outer segment phagocytosis by iPS-RPE, fRPE, and hESC-RPE were likewise similar and dependent on
integrin 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 avenue
for the study of RPE diseases.

6
 Role of Growth Factors in Mesenchymal Stem Cell-Mediated
Blood Flow Restoration and Bone Repair

Fernando Fierro
UC Davis Medical Center

Recent gene expression profiling highlighted basic fibroblast growth factor (bFGF), platelet-derived
growth factor (PDGFB) and transforming growth factor beta (TGF-b1) signaling as critical pathways
during proliferation and differentiation of mesenchymal stem cells (MSC). In addition, MSCs release
high levels of vascular endothelial growth factor (VEGF), a critical pro-angiogenic signal. Using
lentiviral-mediated overexpression of these growth factors, we observe that bFGF or PDGFB reduce by
more than two-fold the doubling times of MSCs (p<0.05), while TGF-b1 and VEGF did not significantly
affect cell growth. This effect on proliferation correlated with morphological changes in MSCs:
Overexpression of bFGF or PDGFB reduces the cell size in a 15-20% as compared with controls
(p<0.05). TGF-b1 did not significantly affect the size of MSCs, but increased actin-stress fibers in MSCs.
VEGF did not exert evident effects on MSC morphology. bFGF does not affect significantly the
adipogenic differentiation of MSCs but strongly inhibits calcium precipitation during osteogenesis.
PDGFB plays opposite effects during MSC differentiation: it strongly enhances osteogenic differentiation
and blocks adipogenesis in all measured parameters. In contrast, TGF-b1 inhibits both adipogenic and
osteogenic differentiation in all parameters measured (p<0.005), while VEGF did not affect MSC
differentiation.

Our results demonstrate the critical role of bFGF, PDGFB and TGF-b1 in MSC growth and differentiation
and confirm the existence of common signaling pathways between bFGF and PDGFB. Indeed, using
western blot methodology, we observe that exclusively PDGFB activates the AKT pathway, while both
bFGF and PDGF activate ERK1/2. Finally, using a hind limb ischemia model, we show that enforced
expression of VEGF enhances blood flow restoration, mediated by MSCs on early time points (2 weeks
and less) as compared with controls, while MSCs over-expressing PDGF showed a great positive effect
on blood flow restoration 8 weeks after injury, presumably favoring MSC survival at the site of injury.
Overall, our data support the rationale of genetically modifying MSCs in order to direct their
differentiation fates, affect their proliferation and survival and enhance their trophic, therapeutically
relevant, signals.

7
 Clinical Follow-up of Horses Treated Intra-articularly with Bone Marrow-Derived Mesenchymal
Stem 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 & Biomedical
Sciences, 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 an
average of 21 months post-treatment. In horses with Grade 3 meniscal injuries, two of five horses were
able 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 and
treated intra-articularly with BMSCs.

Horses (N=60) receiving BMSCs expanded at Colorado State Universities, Orthopaedic Research
Laboratory or Advanced Regenerative Therapies were retrospectively followed based on medical record
analysis 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. Joint
flares 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 horses
with orthopaedic injuries, 73% returned to work. Age, sex, breed, or discipline was not significantly
associated with outcome. The most impressive outcome was seen in horses treated for Grade 3 meniscal
tears. In the current study, 2/5 horses with Grade 3 lesions were able to return to or exceed their previous
level 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 orthopaedic
lesions, especially in cases suffering from severe meniscal damage. Results of this study support future
controlled 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 Regenerative
Therapies.

8
 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 Sciences

School of Veterinary Medicine, University of California, Davis

With the advent of MRI and contrast-enhanced CT in equine practice, in conjunction with more traditional
techniques such as diagnostic ultrasonography, diagnosis of deep digital flexor tendinopathy (DDFT) in the region
of 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, corrective
shoeing, nonsteroidal anti-inflammatory drugs, and administration of intra-articular medications into the distal
interphalangeal joint or intrasynovial medications into the navicular bursa or tendon sheath. In some cases
endoscopic 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 surrounding
ligaments 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. Current
regenerative medicine techniques include cellular therapy with bone marrow-derived mesenchymal stem cells and
adipose-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, we
conducted a retrospective analysis of horses seen between 2004 and 2009 at the UC Davis Veterinary Medical
Teaching Hospital that had been treated with one of three regenerative medicine techniques: bone marrow-derived
mesenchymal stem cells (MSCs), adipose-derived stromal vascular fraction (SVF), or bone marrow aspirate
concentration (BMAC).

Follow-up information was available for 40 horses, with a minimum follow-up time of six months. In addition to
the 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 mild
and moderate lesions, with decreased efficacy for severe lesions in all cases. However, horses with severe lesions
that 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 there
is 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 MSCs
were bilaterally affected.

Overall, all horses had a better success for return to performance compared with published data for horses treated
with traditional therapy techniques. This study supports the use of regenerative medicine techniques for treatment
of 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 therapy
should improve the overall success of horses afflicted with DDFT of the distal extremity.

9
10
 Possible Clinical Applications for Stem Cell Therapy
from Research Based in Equine Veterinary Practice

Dr. Doug Herthel

Alamo 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 original
techniques.

Since our original casework, we have continued to treat and document stem cell treatments for orthopedic
injuries using autologous bone marrow concentrate and adipose vascular fraction product produced by
Vet 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 tissues
within the body. The second hypothesis is that adipose vascular fractions, bone marrow concentrates and
cultured 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 these
increased numbers and concentration of cells improves, exponentially, their ability to communicate and
secrete signal molecules and trophic factors.

Based on these scientific assumptions, we have used various stem cell and regenerative medicine
techniques 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, these
initial positive results could be improved upon. Therefore, we are encouraging a strong and continuing
collaboration between basic research scientists and equine clinicians to improve the health care of horses.

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

CR Johnson, DVM, MS, DACVS

Woodford Equine Hospital
Versailles, KY

Over the past nine years we have been treating career threatening and historically frustrating orthopedic
injuries of the equine athlete with locally administered, autologous bone marrow. Cases treated included
soft 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 tissue
cases 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 racing
Thoroughbreds. We defined severe lesions as 30-50% core lesions that were at least 10 cm long. Cases
were treated by collecting and directly injecting autologous bone marrow into the core lesions using
ultrasound 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 core
lesions, the overall time of healing is not reduced (nine to twelve months). In my experience, large
lesions 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 and
surgically irreparable sesamoid fractures (comminuted, abaxial). The treated cases with subchondral
cysts (stifles and fetlocks) were all lame at presentation ranging from Grade 2 to Grade 4 of 5 of the
affected 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 yearlings
with fibrous unions (at least four months after occurrence of the fracture), and, more recently, race horses
with large basilar or comminuted sesamoid fractures. My experience has been that bony union can be
induced in the cases of fibrous union and the bony union of older horses is occurring faster than
traditionally treated cases. No data on effect of performance is available to date, but, in the fibrous union
cases of the horses intended for sale at auction, the radiographic appearance of the sesamoid was
improved.

12
 Equine Cord Blood MSCs in Bone and Cartilage Engineering

Thomas G. Koch, DVM, PhD, ACVIM Candidate

Seniorforsker, 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 the
equine 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 more
widespread 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 suggestive
of their in vivo potential for cartilage and bone repair.

Cord blood MSC-based therapies may offer advantages when compared with adult MSCs, including bone
marrow MSCs (BM-MSCs). Studies on human cord blood-derived MSCs suggest that these cells have
higher 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 retain
trilineage 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 harvest
of tissues from adult horses would also be eliminated with the use of cord blood-derived cells. Reduced
isolation 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, gene
expression and protein secretion assays and the histological morphology was more hyaline-like and
stained more strongly for extracellular matrix GAGs in the CB-MSC cultures.

The capacity of CB-MSCs to proliferate and differentiate osteogenically within resorbable Pro Osteon®
coralline hydroxyapatite scaffolds was tested. The proliferation of undifferentiatied CB-MSCs seeded in
scaffolds and dynamically cultured in non-induction medium was shown to recover after an initial
decrease after seeding. Scaffolds seeded with eCB-MSCs and statically cultured in osteogenic induction
medium demonstrated improved differentiation as indicated by cell morphology and matrix deposition
using SEM; high alkaline phosphatase (ALP) activity and osteocalcin concentration; and increased
expression 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 coralline
hydroxyapatite scaffolds.

The results of these in vitro chondrogenic and osteogenic studies supports continued research of the
reparative 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 is
ongoing and scheduled for in vivo assessment in the horse.

13
 Osteoprogenitor Cell Therapy in an Equine Fracture Model

Laurie McDuffie
University of Prince Edward Island

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

Initially, a preliminary study was conducted using five adult horses. Horses had fractures created in the
lateral 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 time
of fracture creation, periosteal tissue was collected from the medial aspect of the tibia and used for cell
isolation and expansion. Preparation of periosteal-derived cells prior to implantations included labelling
with DiI and induction with osteogenic media.

Simulated fractures were treated with periosteal-derived cells in fibrin glue (treatment limb) or fibrin glue
alone (control). Implantation of cells was conducted with the horses standing and sedate using ultrasound
guidance 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<0.05) in the
bone density of the fracture site in treated limbs compared with control limbs, indicating an increase in
bone formation. Although histological data were not analyzed statistically, objective measurements
showed that the percent area of bone in sections from fracture sites treated with cell-based therapy was
greater than that of control limbs. We concluded that a full study using a higher number of horses should
be conducted to confirm these initial results.

A full study using 10 adult horses was conducted following the pilot study. Methods were similar except
for a few changes. The fracture was increased to 1 cm in length in attempt to increase the severity of the
fracture model. All fracture sites were examined and analyzed at an end point of 12 weeks.

At this time, all horses have had fractures created and cell injections performed. Radiographic data have
been scored by two authors and some histological sections have been subjectively evaluated. Objective
grey-scale fracture data have not been analyzed, and all histological sections have not been anlayzed
objectively at this time.

Results of radiographic scoring show that the treated fractures do not have increased bone production
compared with controls. Subjective evaluation of histology sections support the radiographic results. We
conclude that further basic science research needs to be conducted prior to use in clinical cases.

14
 Stem Cell Gene-Programming for Musculoskeletal Repair

Dr. Alan J. Nixon

School 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 both
stifles. One defect was treated with 20 million MSCs suspended in an autologous cryoprecipitate
activated with thrombin and CaCl. The opposite joint was treated with the same fibrin substrate without
MSCs.

At four weeks, the MSC-treated joint showed improved arthroscopic appearance over the fibrin-only
treated control group. Biopsies taken at 4 weeks also demonstrated an enhanced response compared with
the control group. However, at 8 months the histologic and immunohistologic appearance of the cartilage
repair 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 improving
long-term quality of cartilage healing. However, other studies conducted by this researcher and others
have given indications that growth factor gene-transcripted MSCs may demonstrate enhanced
chondrogenesis in cartilage resurfacing. Consequently, further investigative work to determine which
genes best drive chondrogenic transformation would seem to be in order.

15
 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,
DACVP
Department 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 to
develop therapeutic cellular products and prepare cells for cryopreservation and storage. The
MarrowXpress 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 the
ability of the MXP to reliably volume reduce, RBC deplete and recover mononuclear cells (MNCs) from
equine cord blood and bone marrow. Cord blood was obtained from 60 thoroughbred foals within 10
minutes of parturition, and bone marrow was obtained from 48 horses of both sexes representing various
ages and breeds. Cord blood was collected into either a 250-ml blood bag or three 60-ml syringes
containing CPDA-1. Bone marrow was collected into one or two 60-ml syringes containing heparin. A
full CBC was performed on the cord blood and bone marrow pre- and post-MXP processing using the
ADVIA 120 Hematology System. The results obtained were as follows: Cord blood - Percent volume
reduction: 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 and
reproducibly 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:
sdowens@ucdavis.edu).

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

John Peroni, DVM, MS

College of Veterinary Medicine
University of Georgia

Adult mesenchymal stem cells (MSCs) have attracted attention in past years in the areas of tissue
engineering, as vehicles for gene therapy, as support cells for hematopoietic stem cell engraftment and as
anti-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 be
homogeneous populations of cells, thereby providing a more predictable cell-based treatment approach
and increased quality control. Prior to accomplishing an allogenic approach, research needs to be
completed to assess the immunoreactivity of the cell transplant.

In this brief presentation, we delineate some of our results in investigating the complex relationship that
exists between the immune system and bone marrow MSCs (BM-MSCs). There is growing evidence that
one 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 evidence
from work in laboratory animals that these beneficial effects can be achieved in inflammatory diseases
such 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 the
expression of the following genes: CD105, CD73, CD44 and CD90. Based on evidence in the literature
and our own experience, approximately 85% of adult equine stem cells are immunoreactive for antibodies
directed against these gene products. To ensure that the isolated cells are, in fact, stem cells and not
hematopoietic 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 with
allogenic lymphocytes would modulate the proliferative effects of phytohemagglutinin (PHA) on the
lymphocytes. The results of these experiments indicate that equine BM-MSCs in co-culture reduce the
rate 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 experiments
in 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 profound
anti-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-cultured
with lymphocytes secrete IL-10, a cytokine that regulates T-cell activity and antagonizes the pro-
inflammatory effects of IL-12.

17
18
 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 tissue
regeneration; a provisional matrix and growth factors via PRP and cells via cBMA or processed adipose
tissue. These treatments can be administered patient side when cBMA and PRP are combined or within 2
days of lipectomy with PRP and processed adipose tissue. We hypothesized that in acute, severe tendon
and ligament lesions in thoroughbreds a combination of PRP and processed stem cells would result in a
higher percentage of success than PRP injected alone. Success was defined as 5 or more starts following
treatment, partial success was 1-4 starts, and failures were horses that failed to have a start within 18
months of treatment. The records of horses treated from 2007-2009 with PRP were examined. We found
that 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 with
PRP 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 were
combined with PRP for suspensory injuries (n=17), the success was 44%, partial success 22%, and failure
33%. There appears to be a trend toward a beneficial effect of PRP and processed stem cells in the SDF
tendonitis. This trend does not appear to present in the severe suspensory branch and body injuries. At
this 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.

19
 Platelet-Rich Plasma as Regenerative Therapy:
Methods of Use and Growth Factor Dynamics

Jamie Textor, DVM, DACVS

School of Veterinary Medicine
University 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 concentrated
growth factors, which are released from the alpha granules of activated platelets. PRP has many
components 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 tissue
healing 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 used
administration 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 the
literature and discuss the effect of preparation and administration methods, with the aim of improving our
use of PRP in equine regenerative medicine applications.

20
 Evaluation of Senescence in Mesenchymal Stromal Cells Isolated from
Equine 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 use
in regenerative medicine. The purpose of this study was to compare the onset of senescence in stem cells
isolated 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, senescence
associated β-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 approximately
1 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 cell
doublings, respectively. UCMSCs and ASCs from each donor showed marked β-galactidose staining at
passage 20 to 21, while BMSCs stained positive by passage 10. These passages were also associated with
significantly 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 in
BMSCs toward senescence, while they increased in ASCs. Oct 4 expression was significantly higher in
all 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 to
senesce much earlier than those from adipose and umbilical cord tissue. These results demonstrate the
limited passage numbers of subcultured stem cells from bone marrow available for use in research and
tissue engineering.

21
 Hypoxia and Stem Cell Biology

Clare Yellowley, PhD

School of Veterinary Medicine
University of California, Davis

Oxygen is a critical regulator of bone development, maintenance and repair. In most cases cells
detect 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, including
conservation 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 E2 levels and the expression of its receptor EP1 (2,3). Hypoxic
osteocytes upregulate the expression of osteopontin, which is chemotactic for mesenchymal stem
cells (MSCs) (4). Interestingly, we have also shown that cellular differentiation in human (5) and
canine 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 adult
life where it appears to regulate bone repair (7). Fracture sites are regions of reduced oxygen
tension, 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 the
fracture callus. We have also investigated the role of the SDF-1/CXCR4 signaling axis on
fracture 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 potentiate
their migration to the site of injury to participate in repair. Using our femoral fracture model, we
demonstrate that an inhibitor of SDF-1/CXCR4 signaling, AMD3100, inhibits fracture repair by
reducing 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 oxygen
tension 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 regional
oxygen levels should be carefully considered when propagating cells and generating tissues in
vitro, and during implantation in vivo.

References
1. 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.

22