Safety and tracking of intrathecal allogeneic mesenchymal stem cell transplantation in healthy and diseased horses
Barberini et al. Stem Cell Research & Therapy
Safety and tracking of intrathecal allogeneic mesenchymal stem cell transplantation in healthy and diseased horses
Danielle Jaqueta Barberini 0
Monica Aleman 2
Fabio Aristizabal 1
Mathieu Spriet 1
Kaitlin C. Clark 0
Naomi J. Walker 0
Larry D. Galuppo 1
Rogério Martins Amorim 3
Kevin D. Woolard 0
Dori L. Borjesson 0
0 Veterinary Institute for Regenerative Cures and the Department of Pathology, Microbiology & Immunology, University of California , Davis , USA
1 Department of Surgical & Radiological Sciences, University of California , Davis , USA
2 Department of Medicine & Epidemiology, University of California , Davis , USA
3 Department of Veterinary Clinics, São Paulo State University “Julio de Mesquita Filho” - UNESP , Botucatu, SP , Brazil
Background: It is currently unknown if the intrathecal administration of a high dose of allogeneic mesenchymal stem cells (MSCs) is safe, how MSCs migrate throughout the vertebral canal after intrathecal administration, and whether MSCs are able to home to a site of injury. The aims of the study were: 1) to evaluate the safety of intrathecal injection of 100 million allogeneic adipose-derived MSCs (ASCs); 2) to assess the distribution of ASCs after atlanto-occipital (AO) and lumbosacral (LS) injection in healthy horses; and 3) to determine if ASCs homed to the site of injury in neurologically diseased horses. Methods: Six healthy horses received 100 × 106 allogeneic ASCs via AO (n = 3) or LS injection (n = 3). For two of these horses, ASCs were radiolabeled with technetium and injected AO (n = 1) or LS (n = 1). Neurological examinations were performed daily, and blood and cerebrospinal fluid (CSF) were evaluated prior to and at 30 days after injection. Scintigraphic images were obtained immediately postinjection and at 30 mins, 1 h, 5 h, and 24 h after injection. Three horses with cervical vertebral compressive myelopathy (CVCM) received 100 × 106 allogeneic ASCs labeled with green fluorescent protein (GFP) via AO injection and were euthanized 1-2 weeks after injection for a full nervous system necropsy. CSF parameters were compared using a paired student's t test. Results: There were no significant alterations in blood, CSF, or neurological examinations at any point after either AO or LS ASC injections into healthy horses. The radioactive signal could be identified all the way to the lumbar area after AO ASC injection. After LS injection, the signal extended caudally but only a minimal radioactive signal extended further cranially. GFP-labeled ASCs were not present at the site of disease at either 1 or 2 weeks following intrathecal administration. Conclusions: The intrathecal injection of allogeneic ASCs was safe and easy to perform in horses. The AO administration of ASCs resulted in better distribution within the entire subarachnoid space in healthy horses. ASCs could not be found after 7 or 15 days of injection at the site of injury in horses with CVCM.
Mesenchymal stem cells; Intrathecal; Neurology; Scintigraphy; Adipose tissue; Cerebrospinal fluid
Mesenchymal stem cells (MSCs) have been evaluated as
a potential treatment for a variety of diseases, including
], musculoskeletal [
], and inflammatory  disorders, in many species.
The central nervous system (CNS) has a limited
capacity for regeneration making stem-cell based therapy a
promising alternative due to its immunomodulatory,
anti-inflammatory, neuroprotective, antiapoptotic, and
proneurogenic characteristics [
Several neurological disorders in humans have raised
particular interest for stem cell-based therapy, including
multiple sclerosis, spinal cord injury, Parkinson’s disease,
stroke, Huntington’s disease, amyotrophic lateral
sclerosis, and Alzheimer’s disease, which have been studied
in experimental models and clinical trials [
equine medicine, neurological disorders affecting the
brain and spinal cord can present a therapeutic
] and many horses with neurological
diseases such as equine protozoal myeloencephalitis (EPM)
and cervical vertebral compressive myelopathy (CVCM
or ‘Wobbler’s disease’) can have neurological sequelae
even after the recommended treatment.
An important first step when considering a cell type
for use in regenerative medicine is to investigate the
ability of that cell to migrate, engraft, and survive at sites
of injury without causing significant adverse side effects.
Preliminary studies have demonstrated the safety of
intrathecal MSC administration in rats [
], humans [
], rabbits [
], and horses [
]. MSCs could
potentially be administered intrathecally to horses at three
different sites: the atlanto-occipital (AO) cisterna [
intervertebral space between the first and second cervical
vertebrae (C1–C2) [
], and the lumbosacral (LS) space
]. Site selection could depend on neuroanatomical
localization of lesions (the administration site preferred
closest to the lesion), the clinician’s expertise, patient
cooperation, and pharmacological protocol (e.g., sedation
versus general anesthesia) [
]. It is currently unknown
whether MSCs administered intrathecally would be able to
migrate throughout the subarachnoid space and home to a
diseased site. Developing protocols to administer allogeneic
MSCs would permit immediate cell therapy in acute and
subacute neurological diseases and would eliminate
variation in ex vivo expansion that can hinder autologous cell
use, especially in older animals and humans [
The objectives of this study were: 1) to determine the
safety of a relatively high dose of intrathecal
adiposederived MSCs (ASCs) in healthy horses; 2) to track
ASCs after AO and LS administration in healthy horses;
and 3) to determine if ASCs would migrate to the
diseased spinal cord site in horses with severe neurologic
disease and survive in the central nervous system for
2 weeks following inoculation.
We hypothesized that the intrathecal administration of
allogeneic ASCs into healthy horses would be safe, as
measured by clinical neurological evaluation, blood work,
and cerebrospinal fluid (CSF) analysis, that the ASCs
would distribute throughout the subarachnoid space after
both LS and AO injection, and that ASCs administered in
horses with severe neurological disease would migrate to
the neurological injury site and would survive for 2 weeks.
For healthy horses, six clinically healthy adult mares of
Thoroughbred (n = 2), Quarter Horse (n = 2), and
Warmblood (n = 2) breeds from the Center of Equine
Health (CEH), UC Davis, USA, were selected for the
study. The ages of the mares ranged from 6 to 21 years
(mean 12 years). All mares were determined to be
healthy based on a normal physical and neurological
examination, and by a complete cell blood count and
serum biochemical profile.
For diseased horses, three client-owned horses who
were going to be euthanized (per the owner’s request)
due to moderate to severe neurological signs were
donated to the CEH for this study. These horses were
donated with presumed CVCM. These horses had a
normal physical examination. The neurological
examination revealed signs consistent with a symmetrical
cervical (C1 to C6) myelopathy. Their neurological deficits
were graded as 3 according to a published grading scale
]. The protocol was approved by UC Davis
Institutional Animal Care and Use Committee (IACUC) on 24
June 2015 (protocol no. 18801).
This was a non-blinded, randomized study.
For healthy horses, three horses were randomly assigned
to receive ASCs via AO injection and three horses to
receive ASCs via LS injection. One horse from each group
received ASCs radiolabeled with
99mtechnetium-hexamethylpropylene-amine-oxyme (99mTc-HMPAO; AnazaoHealth,
Tampa, FL, USA) for cell tracking studies. A control AO
injection of 99mTc-HMPAO without ASCs was performed
in one horse. Blood (jugular venipuncture) and CSF were
collected prior to and after ASC injection (day 0 and day
30, end of study). Complete neurological clinical
examinations were performed prior to ASC injection and
once a day for 30 days (the end of the study).
For neurologically diseased horses, three horses with
CVCM received ASCs labeled with noninfectious viral
vectors transduced with green fluorescent protein (GFP)
via AO injection. Blood and CSF were collected prior to
and 7 (n = 1) or 15 (n = 2) days after ASC injection.
Additionally, neurological examinations were performed
until the end of the study (7 or 15 days). Horses were
euthanized at 7 or 15 days postinjection and submitted
for a full nervous system necropsy by a board-certified
CSF collection and analysis
CSF was collected per routine protocol utilizing published
anatomical landmarks from the AO or LS space prior to
ASC injection and at 30 days postinjection. The CSF was
submitted for complete cytological analysis at the William
R. Pritchard Veterinary Medical Teaching Hospital
(VMTH), UC Davis. Due to the observed and reported
seroprevalence in our area [
], an immunofluorescent
antibody test (IFAT) for the detection of antibodies against
Sarcocystis neurona and Neospora hughesi, the causative
agents of EPM, was preformed concurrently on serum
and CSF. If antibodies were present in serum and/or CSF,
serum to CSF IFAT antibody titer ratios were calculated to
further determine the likelihood of EPM infection in
horses with neurologic disease [
Mesenchymal stem cells (MSCs)
ASCs (passage (P)2–P4) were used for injections into
healthy and neurologically diseased horses. These cells
were obtained from the UC Davis VMTH Regenerative
Medicine Laboratory (RML). These samples were
originally submitted for MSC expansion for autologous patient
treatment. Excess cells not used for treatment were
donated for research purposes with written consent of the
owner. Cryopreserved MSCs were thawed, washed, and
expanded in culture exactly as previously described [
Equine MSCs were tested for purity and identity using
CD44 (clone CVS18; AbD Serotec, Raleigh, NC, USA),
CD29 (clone 4B4LDC9LDH8; Beckman Coulter, Brea,
CA, USA), F6B (white blood cell label; gift of Dr. Jeffrey
Stott, UC Davis), CD90 (clone DH24A; VMRD, Pullman,
WA, USA), MHC I (clone CVS22; AbD Serotec), and
MHC II (clone CVS20; Bio-Rad Laboratories, Hercules,
CA, USA) (Additional file 1). Prior to administration
they were enumerated and confirmed to be viable
(trypan blue), sterile (bacterial culture), and negative for
endotoxin (PYROGENT Plus LAL Gel Clot Assay;
Lonza, Walkersville, MD, USA) and mycoplasma
(MycoScope PCR Kit; Genlantis, San Diego, CA, USA).
The eGFP/luciferase lentivirus
] was a gift from Dr. Jan Nolta.
Equine MSCs were transduced as previously described
for human MSCs [
]. Briefly, equine MSCs were
thawed for at least 3 days prior to transduction. Cells
were trypsinized, pelleted, resuspended in transduction
media (Dulbecco’s modified Eagle’s medium (DMEM),
10% fetal bovine serum (FBS), and 10 μg/mL protamine
sulfate) and plated in a T75 flask. Lentivirus was added
to the flask (multiplicity of infection (MOI) = ~ 10) and
the cells were incubated overnight. After 24 h, 2 volumes
of standard media were added to the flask. Cells were
passed and counted 3 days after transduction and again
5 days later. eGFP efficiency was determined by
fluorescent microscopy and flow cytometry at both time points.
Cells were then frozen and/or expanded for injection.
Radiolabeling with 99mTc-HMPAO
ASCs were labeled with 20 mCi of 99mTc-HMPAO as
previously described [
] with minor modifications as
follows. ASCs were resuspended in a small volume of
media with 10% FBS (∼ 0.5 mL). HMPAO (reconstituted in
saline) was added to the 99mTc and incubated for 5 min.
Cells were added to make a final concentration of 125 μg/
mL HMPAO, 20 mCi99m Tc for cell loading (22 mins).
After cell loading, ASCs were washed twice with saline
(first wash contained 5% autologous serum), and
resuspended in sterile saline. Labeled cells were injected at
a concentration of 100 million cells in 5 mL. An aliquot of
cells was used to determine viability and label persistence.
Labeling efficiency and label persistence at 6 h were
assessed as previously described [
]. Cell viability at
6 h was assessed on a sample of ASCs using the trypan
blue exclusion test. For the control injection (no ASCs),
HMPAO was combined with 99mTc (125μg HMPAO +
10 mCi Tc for 5 min) and then 5 mCi 99mTc-HMPAO was
diluted to 5 mL with saline for injection.
Intrathecal ASC injection
For healthy horses, 100 × 106 ASCs were injected in
5 mL of sterile saline either AO or LS (depending on the
group, n = 3 each) immediately following CSF collection.
One horse in each group received Tc-labeled ASCs and
one horse was injected AO with free label (Tc-HMPAO
without ASCs; control) after the study was completed.
For neurologically diseased horses (n = 3), 100 × 106
ASCs transfected with a retrovirus GFP-bioluminescence
construct were injected in 5 mL of sterile saline via AO
tap immediately following CSF collection.
Tracking of ASCs
Short-term tracking in healthy horses
Scintigraphic imaging Images were acquired using a
gamma camera (IS2 medical Systems, Ottawa, Canada)
with a low-energy all-purpose collimator set at 140 keV
photoelectric peak and 20% symmetrical window. Lateral
images of the spine from the head to the sacrum were
obtained using a 1-min static acquisition immediately
postinjection and at 30 min, 1 h, 5 h, and 24 h after
injection. Small radioactive markers were placed as
landmarks for scintigraphic acquisition. Radiographs (Eklin
EDR-6, Sound-Eklin, Carlsbad, CA, USA) of the
vertebral column from the head to the sacrum were
obtained the day after scintigraphy with radiopaque
markers in a similar location to the radioactive markers.
Scintigraphic interpretation The presence and
distribution of the radioactive signal was assessed subjectively by
a board-certified radiologist using the radioactive markers
for anatomical localization based on correlation with the
radiographs. The persistence at the injection site was
quantified over time. A region of interest (ROI) was drawn
using the free-hand ROI tool of the DICOM viewer
software (Osirix Foundation, Geneva, Switzerland).
Persistence was defined as the ratio of detected counts corrected
for decay in the ROI at each time point divided by the
number of counts initially present in the ROI.
ASCs were transfected with a retrovirus
GFPbioluminescence construct prior to injection exactly as
described previously [
]. Equine MSCs are readily transfected
with this viral vector [
]. Intrathecal injection was
performed in horses under general anesthesia and 100 × 106
labeled ASCs suspended in saline solution was injected into
the AO space.
Tracking in neurologically diseased horses
Necropsy and sample collection A full nervous system
necropsy was performed 15 days (n = 2) and 7 days (n = 1)
after ASC injection. Tissue sections were collected and
fixed in 4% paraformaldehyde (PFA) for 3 days (PFA
changed daily). Following fixation, samples were cryoprotected
in 30% sucrose and then sectioned for routine
hematoxylin and eosin (H&E) tissue processing and frozen
sections. In brief, each spinal cord segment was sampled
for two H&E cross-sections and three frozen
crosssections in an alternating pattern (frozen–H&E–frozen–
H&E–frozen), followed by an approximate 1-cm
longitudinal section for frozen. Approximately 150 frozen
sections were evaluated for each horse. Routine H&E
sections were processed for regular histology and stained
on an autostainer (Leica Biosystems Inc., IL, USA). Frozen
sections were cut on a cryotome (Life technologies,
Carlsbad, CA, USA) at 10 μm thickness and stained with
4,6diamidino-2-phenylindole (DAPI) at 2 μg/mL for 5 min at
room temperature prior to mounting. Sections of the
nervous system (brain and spinal cord) were imaged on an
Evos FL inverted epifluorescence scope (Life technologies,
Carlsbad, CA, USA) to detect bioluminescent ASCs.
To assess for the presence of occult GFP-labeled cells,
we performed additional immunofluorescence assays
using a rabbit-anti-GFP primary antibody (clone D5.1; Cell
Signaling, Danvers, MA, USA), visualized using an
Alexa594-conjugated goat-anti-rabbit IgG secondary
antibody (Leica Biosystems Inc.). Sections from three horses
were imaged on both the GFP and 594 (red) channels.
Anti-ASC alloantibody flow cytometric crossmatch assay
To determine if healthy horses developed any anti-MSC
alloantibodies after intrathecal administration of allogenic
ASCs, we performed a crossmatch assay exactly as
previously described [
]. In brief, cryopreserved ASCs were
thawed, washed, incubated in blocking solution, washed,
and incubated with horse sera (ASC recipient serum). After
incubation, ASCs were washed and incubated with the
secondary antibody (rabbit polyclonal antibody to equine
IgGFITC; Abcam, Cambridge, MA, USA). Cells were washed,
resuspended in flow buffer, and analyzed on a flow
cytometer (Cytomics FC 500; Beckman Coulter). Flow
cytometry data were analyzed using FlowJo flow cytometry
software (Tree Star Inc., Ashland, OR, USA). Each recipient
horse sera was incubated with the ASCs that they received
as well as ASCs that they did not receive (irrelevant ASCs)
to determine binding specificity. Pre- and post-sera were
available for four of the recipient horses.
Detection of anti-BSA antibodies
An enzyme-linked immunosorbent assay (ELISA) was
adapted to detect antibodies directed against the primary
bovine protein in FBS, bovine serum albumin (BSA)
]. This ELISA was performed as previously described
to determine if the intrathecal administration of ASCs
cultured in FBS resulted in increased anti-BSA titers in
healthy horses [
]. Serum samples collected prior to
ASC administration and at 30 days and 14 months
following ASC administration were available from four of
the horses. The fold-increase in color relative to the
negative control was determined for each sample.
CSF parameters prior to and after ASC injection were
compared using a paired student’s t test. A P value < 0.
05 was considered significant for all analyses.
ASC injection into healthy horses
High-dose intrathecal ASC injection is safe and well
ASC administration was readily performed in both
standing (LS) and laterally recumbent anesthetized horses
(AO). No adverse events were noted during or after ASC
administration. The complete blood count and
biochemical profile showed no alterations prior to or after ASC
injection (with or without 99mTc-HMPAO) for either AO
or LS groups. All mares had normal physical and
neurological examinations prior and after the
administration of ASCs, including the 30 day recheck
High-dose intrathecal administration of ASCs does not alter
There were no statistical differences between AO
and LS CSF parameters prior to or after ASC
administration; as such, the groups were combined for
further analysis. In addition, there were no significant
differences in CSF total protein, nucleated cell count,
or cell differential prior to or after ASC
administration (P > 0.05). The majority of horses had normal
cell counts, total protein, and cell differential both
prior to and after ASC administration. Prior to ASC
administration, 2/6 horses (33%) had increased
nucleated cell count (9 and 13 cells/μL, reference
range ≤ 5 cells/μL) and all horses had a normal total
protein concentration (reference range < 100 mg/dL).
These changes were not considered clinically
relevant in light of normal neurological and physical
examination. Thirty days after ASC administration,
1/6 horses (17%) had an increased CSF protein
(121 mg/dL) and 5/6 horses (83%) had an increased
nucleated cell count (6, 6, 7, 13, and 26 cells/μl).
There were no large or atypical cells (potential MSCs)
noted on highly concentrated CSF fluid submitted for
cytological review. Antibody titers on IFAT for both blood
and CSF were negative for S. neurona and N. hughesi.
Therefore, antibody titer ratios were not calculated.
Data from CSF analyses prior to and after ASC
administration are detailed in Table 1.
ASCs administered AO distributed caudally throughout the
vertebral canal whereas ASCs administered LS failed to
Immediately after AO injection of radiolabeled ASCs, a
radioactive signal was identified with the maximal intensity
at the site of injection and extending cranially into the
caudal aspect of the cranial vault and caudally to the level of
the second cervical vertebra (Fig. 1a, c). One hour after
injection, a radioactive signal was present at the level of the
first thoracic vertebra and, at 5 h, the radioactive signal
could be identified reaching the lumbar spine (Fig. 2). At
24 h postinjection, the radioactive signal was still present
and strong in the cranial cervical vertebral canal, with a
weaker signal seen within the lumbar spine. The radioactive
signal remained the most intense at the injection site at all
After LS injection, a radioactive signal was
immediately visible at the level of the last lumbar vertebra and
cranial sacrum (Fig. 1b, d). Over time, the signal
extended caudally to the posterior dural sac, while a
minimal radioactive signal extended cranially (Fig. 2). The
signal was still identified at 24 h postinjection.
The distribution and progression of the free
99mTcHMPAO radioactive signal after AO administration was
similar to the injection of labeled ASCs, with the signal
reaching the cranial thoracic area after 1 h and the sacral
area after 5 h (Fig. 3). Mild uptake was apparent in the
thyroid gland after the control injection (Fig. 3a, b). This
was not seen with the labeled ASC injection.
Radiolabel persistence at the injection site was
quantified (Table 2). Persistence decreased over time with all
three injections. The persistence after AO injection at
the later time points was lower than after LS injection.
The persistence after the control injection was lower at
all time points except for that at 24 h.
Horses did not develop anti-ASC antibodies after intrathecal
No horses (0/4) developed detectable anti-ASC
alloantibodies. Percent specific IgG binding ranged from
0 to 2.8% at all time points with no change from
baseline (time 0), 30 days, or 14 months following
Ad-MSC administration in any of the horses.
Horses did not develop anti-BSA antibodies after intrathecal
Three of the four horses had high titers to BSA at
day 0 and those titers remained high at day 30 and
at 14 months following Ad-MSC administration. One
horse had a low positive titer to BSA at day 0 and
that titer remained low at day 30 following Ad-MSC
administration. This horse converted to a high BSA
titer at the 14 month recheck, likely due to recent
ASC injection into neurologically diseased horses
ASCs labeled with GFP (Fig. 4) were administered via
AO injection with no adverse effects. The complete
blood count and biochemical profile showed no
alterations prior to or after ASC injection. CSF analysis
showed a mild increase in protein concentration before
ASC injection in one horse.
The neurological status of the diseased horses
remained unchanged throughout the different time
points of examination following the administration of
ASCs. The C1–C6 myelopathy was characterized by
ataxia, tetraparesis, hypermetric gait, and proprioceptive
deficits of all limbs. The neurological deficits were
symmetrical and graded 3 of 5.
Post-mortem examination findings
All diseased horses were subject to a complete
postmortem examination that showed lesions consistent with
equine CVCM. Chiefly, spinal cords from affected
animals exhibited swollen myelin sheaths, axonal swelling
and spheroid formation, digestion chamber formation,
and multifocal glial nodules. Affected spinal cord
segments ranged in severity but were typically most severely
affected in C4 to C6 (Additional file 2). Spinal cord
segments showed no GFP-labeled ASCs at the site of the
lesions and no immunolabeling towards GFP was detected
in any patients (data not shown).
AO, atlanto-occipital, AO-C, atlanto-occipital control, LS, lumbosacral
Neurological diseases of horses are important causes of
ataxia, weakness, and decreased performance. The
pathophysiology of various neurologic diseases can be complex,
multifactorial, and/or poorly understood. Depending on
etiology, certain neurologic conditions can represent a
therapeutic challenge, especially those that result in
profound brain and spinal cord injury [
20–22, 33, 43
overcome the limited capacity of CNS regeneration, a
possible alternative would be to use MSCs to assist in
modulating neuroinflammation and neuroregeneration.
Data from this exploratory study suggest that the
intrathecal administration of relatively high doses of
allogeneic, culture-expanded ASCs is well tolerated in
healthy horses regardless of whether the cells are
administered at the AO or LS space. In addition, ASCs after
AO injection appear to distribute more efficiently
through the subarachnoid space. Another study in
horses also showed the safety of intrathecal
transplantation through the AO space of bone marrow-derived
MSCs (BM-MSCs) with a much lower cell dose (1 × 106
]. Other human studies have
demonstrated the safety of intrathecal injection of autologous
6, 27, 28, 44
] with a few studies using allogeneic
A single intrathecal administration of allogenic ASCs
to healthy horses did not elicit an anti-MSC alloantibody
response. These findings may suggest that, in the
absence of disease and breakdown of the blood–brain
barrier, a systemic response to allogenic cells will not be
mounted. In our previous study, low-dose cell
administrations or a single dose also did not elicit an anti-MSC
antibody response whereas multiple high doses into a
tendon lesion did elicit a response [
]. The percent
of background binding of equine IgG to equine
AdMSCs was similar in this study (< 2.8%) to what we
noted previously (background IgG binding varied from
2.4–7.5%). These findings suggest that the assay is fairly
robust and repeatable.
In agreement with our previous study, the majority of
horses (89%) were positive for anti-BSA antibodies prior
to and after MSC injection. Anti-BSA antibodies develop
in horses due to frequent vaccination with products
created in FBS-containing media [
]. Similar to our
previous study, anti-BSA titers did not increase in the horses
in this study.
There is no consensus on the best route to deliver MSCs
to the neurological system [
]. Studies have variably used
], intrathecal [
], intranasal [
lesional, and intracerebral [
] injections. Theoretically,
intrathecal injection could be more effective since
relatively large MSCs would not have to pass the blood–brain
barrier. In support of this, several studies have reported
beneficial effects in mice and humans after intrathecal
MSC administration for CNS diseases [
10, 18, 51–54
However, at least one serious adverse event (acute
disseminated encephalomyelitis) has been reported after
intrathecal BM-MSC administration in a human .
In this study, both AO and LS transplantation of ASCs
was technically easy and safe to perform, with no
demonstrated adverse effects in horses. Furthermore, the
neurologic status was not altered in any of the horses
(healthy or diseased) in this study. The caudal
distribution of the labeled ASCs after AO injection and the lack
of cranial migration after LS injection suggest that the
cells are progressing in the direction of CSF flow in
the absence of any identified spinal cord lesions. The
lower persistence at the AO injection site when
compared to LS is consistent with the subjective
assessment of migration of ASCs. The caudal migration of
ASCs injected at the AO space is likely responsible
for this lower persistence.
Based on our study, atlanto-occipital centesis is the
preferred route of administration to facilitate getting the
highest concentration of MSCs to the cervical area.
Given that both routes are safe, determining the best site
for ASC transplantation should take into account the
safety of the horse (for example, general anesthesia) and
lesion location [
]. Lumbosacral puncture is a relative
easy procedure that does not require general anesthesia
]. This approach may be preferable in animals
showing neurological signs localized caudal to T3 spinal cord
segments. Although AO puncture is routinely performed
under general anesthesia [
], it has been reported that
it could be performed in the standing horse with
appropriate sedation [
]. However, puncture of the spinal
cord or brainstem is a potential risk. Another technique
that has been described in the standing horse is the
collection of CSF between the atlas and axis [
The relatively low persistence at 24 h also observed
with the LS injection despite the lack of demonstrated
local migration is similar to the persistence that has been
reported with local intralesional injection of MSCs in
the equine superficial digital flexor tendon (24% at 24 h)
]. This might be due to systemic distribution of the
MSCs after injection but this might also reflect a
limitation of the tracking technique. Suboptimal label
persistence has been demonstrated after injection of
99mTcHMPAO-labeled MSCs in dogs [
]. Although it is
harder to demonstrate in horses due to physical
limitations in imaging the whole body (size and signal
attenuation), it is likely that the measured persistence is
an underestimation of the actual cell persistence due to
loss of label from some of the cells.
The concern about the presence of the free label was
the justification for performing a control injection. The
similar distribution of the labeled ASCs and the free
label suggest that both are distributing following the
CSF flow which is mainly unidirectional from cranial to
]. The lower initial persistence of the free label
can be explained by a faster distribution or higher
systemic absorption that can be explained by the smaller
size of the label compared with the ASCs. The higher
persistence at 24 h with free label might be explained by
fixation of some of the label in local cells. The uptake in
the thyroid region observed with the control injection
confirms systemic distribution of the free label and
absorption of free technetium by the thyroid gland.
We were unable to find GFP-labeled ASCs
transplanted into diseased horses 7 or 15 days after ASC
injection. This finding could be due to the fact that: 1) the
neurologic lesions were chronic and did not attract
ASCs to the site; 2) ASCs were not restricted to the
spinal cord area and they could have migrated outside
the CSF or distributed throughout the subarachnoid
space; or 3) ASCs could have undergone cell death.
A study with ASCs labeled with Qtracker 655
quantom dots showed that ASCs were still found at an
equine tendon lesion site 1-week postinjection [
Another study compared administration of GFP-labeled
ASC trough intravenous, intraperitoneal, and
subcutaneous routes in mice, and after 75 days eGFP-bright cells
were found in the brain, heart, liver, lung, kidney, and
omental fat by polymerase chain reaction (PCR) and
cytospin analyses [
]. It has also been demonstrated in
mice that MSCs injected intra-arterially selectively
engraft in the bone marrow after a localized radiation up
to 33 weeks. In this study, the authors used
bioluminescent imaging to measure cell distribution of monomeric
red fluorescent protein/luciferase MSCs [
Some studies have reported MSC apoptosis after in
vivo injection [
], but despite this it was shown to
be crucial for the MSC immunosuppression function
 and to improve cardiac function through secretion
of the anti-inflammatory factor tumor necrosis factor
(TNF)-α-induced protein 6 (TNAIP6 or TSG-6) [
Limitations of the study include the small number of
horses, making interpretation of quantitative data
difficult (for example, the increase in CSF protein in one
horse). There were small changes in the CSF nucleated
cell count both prior to and after ASC injection in four
out of five horses. These findings might represent
normal variation or mild subclinical disease, although all
animals were neurologically normal on examination.
Our results demonstrate that AO and LS intrathecal
injection of allogeneic ASCs is safe and easy to perform in
horses. Additionally, due to the flow of CSF from cranial
to caudal, AO administration of ASCs had a better
distribution within the subarachnoid space and presumably
to the spinal cord from the cervical to the lumbosacral
region, suggesting that this approach might be more
suitable for cranial lesions in the spinal cord. ASCs
could not be found at 15 days after injection at the site
of injury in horses with CVCM, suggesting that ASCs
did not have time to reach the lesion site or that ASCs
did not stay/survive in the spinal cord for this period of
time in a high enough number to be detected.
Additional work needs to be performed to determine if
multiple intrathecal allogeneic MSC injections are well
tolerated as well as the efficacy of MSCs to treat horses
with inflammatory or degenerative lesions of the nervous
Additional file 1: Representative image of equine ASC phenotype.
Equine ASC phenotype panel. Positive markers: CD44, CD29, CD90, and
MHC I. Negative markers: F6B and MHC II. (JPEG 46 kb)
Additional file 2: Representative images of neurologic patients, spinal
cord cross-sections, H&E stain. A) Several dilated myelin sheathes are
evident that B) contain macrophages consistent with digestion chambers
(arrows). C) Swollen axons are present with spheroid formation (*). D)
Multifocal glial nodules (arrowhead) are present within the white matter.
Scale bar = 100 μm. (JPEG 92 kb)
AO: Atlanto-occipital; ASC: Adipose-derived mesenchymal stem cell;
BMMSC: Bone marrow-derived mesenchymal stem cell; BSA: Bovine serum
albumin; CEH: Center of Equine Health; CNS: Central nervous system;
CSF: Cerebrospinal fluid; CVCM: Cervical vertebral compressive myelopathy;
DAPI: 4,6-Diamidino-2-phenylindole; ELISA: Enzyme-linked immunosorbent
assay; EPM: Equine protozoal myeloencephalitis; FBS: Fetal bovine serum;
GFP: Green fluorescent protein; H&E: Hematoxylin and eosin;
IACUC: Institutional Animal Care and Use Committee;
IFAT: Immunofluorescent antibody test; LS: Lumbosacral; MSC: Mesenchymal
stem cell; PFA: Paraformaldehyde; RML: Regenerative Medicine Laboratory;
ROI: Region of interest; VMTH: Veterinary Medical Teaching Hospital
This project was supported by a generous gift from the Haas Family and by
the Center for Equine Health with funds provided by the State of California
satellite wagering fund and contributions by private donors. Danielle J.
Barberini was supported by grant #2014/20550-8, São Paulo Research
Foundation (FAPESP/CAPES), Brazil. The funding agencies played no role in
the development of the study, collection, analysis and interpretation of the
data, in the writing of the manuscript, or in the decision to submit the
manuscript for publication.
Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated
or analyzed during the current study.
The study was designed by all authors. DJB cultured and prepared ASCs for
injection in healthy horses and wrote the manuscript. KCC cultured and
prepared ASCs for injection in diseased horses. FA performed sedation and
anesthesia of the horses. MA performed neurologic examinations. FA, MA,
and DJB performed collections of cerebrospinal fluid and cell injection,
clinical and neurological examinations, and the majority of the data
acquisition. NJW performed cell labeling with technetium and GFP. MS
supervised scintigraphic and radiographic imaging acquisition and
performed interpretation. KDW performed nervous system necropsies. All
authors contributed to data interpretation and manuscript preparation. LDG,
MS, KDW, and RMA revised the manuscript. DLB provided funding and
mentorship and revised the manuscript. All authors read and approved the
The protocol (#18785, #18801) was reviewed and approved by the UC Davis
IACUC on 2 June 2015.
Consent for publication
The authors declare that they have no competing interests.
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