Safety and efficacy of the mesenchymal stem cell in feline eosinophilic keratitis treatment

13 Pages·2017·9.1 MB·English

by  Antonio J. Villatoro

Villatoroetal.BMCVeterinaryResearch (2018) 14:116
https://doi.org/10.1186/s12917-018-1413-4
RESEARCH ARTICLE Open Access
Safety and efficacy of the mesenchymal
stem cell in feline eosinophilic keratitis
treatment
Antonio J. Villatoro1,2†, Silvia Claros2,3†, Viviana Fernández1,2, Cristina Alcoholado3,2, Fernando Fariñas1,2,
Antonio Moreno4, José Becerra2,3,5 and José A. Andrades2,3*
Abstract
Background: Felineeosinophilickeratitis(FEK)isachronickeratopathycausedbyasuspectedimmunemediated
responsetoanunknownantigenicstimulus.Thepurposeofthisstudywastoinvestigatethesafetyandtherapeutic
effectsofallogeneicfelineadipose-derivedmesenchymalstromalcells(fAd-MSCs)implantedsubconjunctivalaround
theocularsurfacelesioninfivecatswithFEKrefractorytocurrentavailabletreatments.
Results:FEKwasdiagnosedbyclinicalappearanceandevidenceofeosinophiland/ormastcellsincornealcytology.
Eachanimalwastreatedwithtwoapplicationsof2×106millionoffAd-MSCs2monthsapart.Ocularsurfaceintegritywas
assessedbeforetreatmentandat1,3,6and11monthsaftertreatment.Clinicalsignsshowedasignificantchangeduring
thefollow-upwithresolutionofthecornealandconjunctivalesionsandtherewerenosignsofregressionorworsening.
Conclusions:Implanted cells were well-tolerated and effective reducing clinical signs of FEK with a sustained
effect during the study period. None of the animals showed systemic or local complications during the study.
To our knowledge, this is the first time in literature that local implantation of allogeneic fAd-MSCs has been
found as an effective therapeutic alternative to treat cats with FEK.
Keywords: Cat, Adipose mesenchymal stem cell, Lacrimal gland, Feline eosinophilic keratitis, Feline herpes
virus, Allogeneic cell therapy
Background The diagnosis is based on clinical appearance and cor-
Feline eosinophilic keratitis (FEK) or feline proliferative neal cytology. Clinical signs start with a superficial
keratitis (FPK) is a unique, chronic and progressive, infil- vascularization of the perilimbal corneal, and as the dis-
trative keratopathy in cats [1, 2], but is also described in easeprogresses,thelesionsappearasanedematousarea,
horses [3], rabbits [4], and dogs [5]. This disease involves an irregular and vascularized mass with pink to white
the ocular surface in cats of any age, but is most com- infiltrates that form gritty yellow-white corneal plaques
monly diagnosed in young to middle-aged cats, and extending axially from thelimbus.
appears unilaterally 66% of the time, but eventually The occurrence of eosinophil and/or mast cells in a cor-
becomea bilateral condition [1, 2, 6].Ingeneral, the con- nealcytologicspecimenisdiagnosticforproliferativefeline
dition only affects the cornea, though it can also involve eosinophilickeratitis[1,2,7].Theetiologyofthiscondition
thethirdeyelidandtheconjunctiva[1]. is not clearly understood. FEK is believed to be an
immune-mediated disorder in which there is an exagger-
*Correspondence:[email protected] atedimmuneresponsetoanantigenicstimulus,withatype
†Equalcontributors IortypeIV(subtypeIVb)hypersensitivityreaction[8].
2LaboratoryofBioengineeringandTissueRegeneration(LABRET),
There may be an underlying viral infection, feline
DepartmentofCellBiology,GeneticsandPhysiology,FacultyofSciences,
UniversityofMálaga,BiomedicineResearchInstituteofMalaga(IBIMA), herpes virus-1 (FHV-1) that plays an initial role in
CampusUniversitariodeTeatinos,29071Málaga,Spain the pathogenesis of the disease [2, 6, 9, 10], having
3NetworkingBiomedicalResearchCenterinBioengineering,Biomaterialsand
Nanomedicine(CIBER-BBN),28029Madrid,Spain
Fulllistofauthorinformationisavailableattheendofthearticle
©TheAuthor(s).2018OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0
InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinkto
theCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver
(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Villatoroetal.BMCVeterinaryResearch (2018) 14:116 Page2of13
been described that at least 76.3% of cats with FEK cats with an 11-month follow-up. fAd-MSCs were previ-
are positive to FHV-1 [6]. ously characterized through in vitro tests, according to
The current treatment of FEK typically consists of top- theInternationalSocietyofCellTherapy.
icalapplicationsofcorticosteroidsorsimilarandimmuno-
suppressive drugs as cyclosporine A, several times a day Methods
for long periods of time with variability in efficacy and This was an uncontrolled open-label study in the treat-
safety[1,2,6,11].Inaddition,recurrencesofclinicalsigns mentofFEK.
arecommonaftercessationoftreatment[6,10,11]. All animal procedures were conducted by licensed vet-
Adipose-derived mesenchymal stromal cells (Ad- erinarysurgeonsandcomplywithbothnationalandEuro-
MSCs) are multipotent stem cellswith capacity to differ- pean legislation (Spanish Royal Decree RD1201/2005 and
entiate into osteogenic, adipogenic, chondrogenic, myo- EU Directive 86/609/CEE as modified by 2003/65/CE, re-
genic, among other cell lineages, and with important spectively) for the protection of animals used for research
secretory abilities of different bioactive molecules with experimentation and other scientific purposes. Likewise,
trophic, paracrine and immunomodulatory functions the protocols were approved by the Institutional Animal
[12–16]. Although the mechanisms of immunomodula- CareandUseCommitteeofBIONAND(AndalusianCen-
tion remain partially elusive, recent studies have demon- ter for Nanomedicine and Biotechnology) Málaga, Spain,
strated the capacity of MSCs to modulate both the innate andwritingconsentwasobtainedfromallowners.
andadaptiveimmunesystems[13,17].MSCsinhibitT-cell
proliferation, alter B-cell function, downregulate major Animals
histocompatibilitycomplex(MHC)II,andinhibitdendritic Five client-owned cats (5 eyes) of European breeds, 3
cellmaturationanddifferentiation[13,18,19]. males and 2 females, aged between 3 and 6 years, and
Their low immunogenicity and their immunoregula- weighting from3.2to4kgwere selected(Table 1).
tory potential allow their allogeneic use, which makes All individuals were affected by FEK, at least during
them an alternative to be a promising new treatment for 6 months and refractory to the current treatment, with
severe refractory autoimmune diseases [16, 20]. They duration of the clinical signs from 6 to 12 months (mean
have been extensively studied as a cellular therapy for 9 months). Only one eye was affected in all cases. The
different pathological conditions, with the cat and dog righteyewasaffectedin3cats(60%),thelefteyein2cats
asanimalmodels[21–24]. (40%). They received previous treatment (corticosteroids,
The purpose of our study was to evaluate the safety cyclosporine, and others) with partial improvement and
and the therapeutic effects of local implantation of allo- recurrenceswithsomelevelofintolerancetocyclosporine
geneic feline Ad-MSCs (fAd-MSCs) subconjunctivally in administration without any viable therapeutic alternatives
Table1Summaryofsignalment,clinicaldataandevolutionforveterinarypatients
Parameter Cat-1 Cat-2 Cat-3 Cat-4 Cat-5
Age(years) 4 4 5 6 3
Sex MC MC MC FS FS
Breed DSH DSH DSH DSH DSH
Weight(kg) 3.2 3.8 3.8 4 4.1
Duration(months) 12 9 8 10 6
Eye R L L R R
Lesionlocalization ST ST IN IN ST
FHV-1 – + + + +
Clinicalsigns PL,V,H PL,V,ED,H PL,V,ED,H PL,V,H PL,V,H
STT 14 18 16 21 19
Cytology E,M E,M E,M,N E,M E,M
Previoustreatment C,Ca,Ao Ca,Ly,Ao Ca,C,Ly,Ao Ca,C,Ly,IΩ,Ao Ca,Ly,IΩ,Mg,
Ao
Clinicalremission 2months 2months 6months 4months 5months
Hematologicchanges No No No No No
MC,malecastrated;FS,femalespayed;DSH,domesticshorthair;R,righteye;L,lefteye;ST,superotemporalquadrant;IN,inferonasalquadrant;FHV-1,felineher-
pesvirus1;PL,cornealinfiltrativeplaque;V,vascularization;H,hyperemia;ED,cornealedema;STT,Schirmerteartest;E,eosinophils;M,mastcells;N,neutrophils;
C,topicalcorticosteroids;Ca,topicalcyclosporineA;Ao,antibiotics;Ly,L-lysine;IΩ,topicalinterferonomega;Mg,oralmegestrolacetate
Villatoroetal.BMCVeterinaryResearch (2018) 14:116 Page3of13
(Table1).Untreatedorplaceboanimalswerenotincluded
in the present study, since only cats with naturally occur-
ring FEK were admitted, and spontaneous recovery has
neverbeenreportedincatswithrefractoryFEK[6].
Cats did not receive any kind of anti-inflammatory or
immunomodulatorymedicationsforatleast2weeksbefore
cell therapy treatment, and for the entire duration of the
study. All cat owners signed a written consentbefore initi-
ation of this experimental procedure and were fully in-
formed that long-term outcome, safety, complications, and
efficacyofthecellimplantationinFEKwerenotknown.
Clinicalevaluation
All animals underwent a full veterinarian clinical and
Fig.1Cornealcytology.Abundanteosinophilsandmastcellswere
ophthalmicexamination.
detectedinallanimals
Initial complete ophthalmic examination consisted of
Schirmer tear test (STT-1) at one minute, fluorescein
staining,applanationtonometry,slit-lampbiomicroscopy Follow up was at 1, 2, 3, 6 and 11 months after fAd-
and indirect ophthalmoscopy. The corneal and conjunc- MSCs implantation (Fig. 2). During this time, recur-
tival lesions were described in each animal, affected eye rences of the corneal lesion, additional ocular diseases,
andposition oftheocularsurfacelesion (Table1). and the health of the contralateral cornea were moni-
Diagnosis of FEK was based upon the clinical appear- tored. Corneal cytology, complete blood cell count and
ance of infiltrates in one of the eyes and the corneal and serum biochemistry profile were performed during all
conjunctivalcytology. follow upappointments.
Clinicalfindings:Cornealvascularizationandinfiltration
was present in all cases. Proliferative, white-pink, edema- IsolationandinvitrocultureoffAd-MSCs
tous, irregular and vascularized ingrowth of tissue and Adipose tissue was aseptically collected from abdominal
gritty, white-yellow cornealplaques wereprominent inall subcutaneous fat under general anesthesia with isoflur-
cases, spreading fromthe limbustothe centerofthe cor- ane of a single specific pathogen-free cat during a rou-
nea. Corneal lesions were found in the superotemporal tine ovariectomy procedure, and maintained at 4 °C in a
quadrantin3oftheeyes(60%),andintotheinferiornasal tube with culture medium. Under a laminar flow hood,
quadrant in two eyes (40%). None of the cases showed after weighing the harvested adipose tissue, 5 g of fat
corneal ulcer and the fluorescein staining was negative. was minced and mixed with 20 mL of Hanks solution
The adjacent conjunctiva was hyperemic near to the cor- (Sigma-Aldrich, Madrid, Spain) containing 0.1% collage-
neal lesion in all cases. The ocular alteration was limited nase type II (Sigma-Aldrich) by incubating at 37 °C for
tothe corneaandconjunctiva,andalltheothersophthal- 90 min in orbital agitation. After digestion, the cell sus-
mologicparametersevaluatedwerenormal. pension was filtered through a 100 μm cell strainer. The
Cytology was obtained in all cases after application cell suspension was centrifuged at 400 g for 5 min to
of a topical anesthetic with the blunt end of a scal- discard the lipid layer and the obtained cell pellet was
pel blade. Abundant eosinophils and mast cells were washed withculturemedium. Primary cultures were car-
detected in all animals (Fig. 1). Besides, one of the ried out in T175 flasks with Dulbecco’s modified Eagle’s
cats showed the existence of a remarkable number medium (DMEM) containing 10% (v/v) fetal bovine
of neutrophils. The Wright-Giemsa stains were nega- serum (FBS), 2.5 mM L-glutamine, 100 U/mL penicillin,
tive to microorganisms. 100 μg/mL streptomycin, and 1.25 μg/mL fungizone (all
A polymerase chain reaction (PCR) for FHV-1 was from Sigma-Aldrich). The culture medium was changed
performed by Laboratory Idexx (Barcelona, Spain), ap- twice per week and cells were selected by their capacity
plying a hydrophilic polyethersulfone membrane on the toattachtotheflask surface,discardingthefloating cells
cornea. FHV-1wasdetected infourcatsofthis study. in the first medium change at 72 h. When culture flasks
A complete blood cell count and serum biochemistry became 80% semiconfluence, cells were detached with
profile priortotreatmentwereperformedonallanimals, 0.25% trypsin containing 1 mmol/L EDTA and subse-
withoutshowinganyalterationoftheparametersevaluated. quently replated at a concentration of 104 cells/cm2 for
Acutephasemarkerswerenotincludedinthestudy,asno continued passaging. The remaining cells were cryopre-
specificparameterisdescribedforthisdisease. served in cryopreservation media (10% dimethyl sulfoxide
Villatoroetal.BMCVeterinaryResearch (2018) 14:116 Page4of13
Fig.2Summaryofthecelltherapyandclinicalfollow-up
and90%FBS),frozenat−80°Cinanisopropanoljacketed blue (TB), alcian blue (AB), and immunohistochemically
closed container (Nalgene Cryo freezing container), and for type II collagen, using standard techniques as previ-
storedinliquidnitrogenuntilthenextday.Allexperiments ouslydescribed[24].
andinvivoimplantationwereconductedatpassage2.
Karyotype
Cellproliferation fAd-MSCs at passage 2 were karyotyped as previously
Cell proliferation was measured using MTS (CellTiter 96 described [28]. fAd-MSCs were seeded and cultured in a
AqueousOneSolutionCell Proliferation Assay,Promega) T175 flask until semi-confluence was achieved. Then,
assayaccordingtomanufacturerprotocol.Briefly,ina96- cells were harvested and treated with 0.05 μg/ml of col-
well plate, 3000 fAd-MSCs per well were seeded using 8 cemid (Thermo Fisher Scientific, Inc.) for 20 min to ar-
wells as replicates of each sample. Cells were allowed to rest mitotic cells in metaphase. Subsequently, pelleted
proliferateperformingthemediumchangetwiceperweek cells were resuspended in a hypotonic solution (0.075 M
and making readings on days 0, 3, 5, 7 11, 14, 18, 21, 25 potassium chloride solution) for 5 min to swell cells.
and 28. Supernatants were collected and absorbance was fAd-MSCs were then fixed in cold methanol: glacial
measured at 490 nm using a microplate reader (ELx800, acetic acid (3:1) and washed three times to ensure
Bio-Tekinstruments,Winooski,VT,USA). complete removal of cytoplasmic debris. Afterward, they
were stained in 2% Giemsa and analyzed with ordinary
Flowcytometryanalysis bright-field microscopy. Analysis included scanning all
Fluorescence-activated cell sorting (FACS) was used to slides, counting a minimum of twenty metaphases, ana-
characterize fAd-MSCs from passage 2, and was per- lyzing a minimum of seven metaphases, and karyotyping
formed as previously described in detail [25]. The anti- aminimumoftwo metaphases.
bodies usedarelistedinTable 2.
Table2Antibodiesusedforflowcytometry
Invitromultilineagecelldifferentiation Antibody Supplier Clone Isotype Fluorochrome
To assess the multipotentiality, fAd-MSCs at passage 2 CD29 MiltenyiBiotech TS2/16 IgG1κ PE
were differentiated long adipogenic, osteogenic, and
CD34 MiltenyiBiotech AC136 IgG2a FITC
chondrogenic lineages according to standard protocols,
CD44 MiltenyiBiotech DB105 IgG1 APC
as previously reported [26, 27]. Cells were cultured in
CD45 MiltenyiBiotech 5B1 IgG2a APC
specific induction medium for 21 days. Afterward, adi-
pogenic differentiation was evaluated by Oil red O stain- CD73 BDPharmingen AD2 IgG1κ PE
ing on day 21 after induction. Alkaline phosphatase CD90 MiltenyiBiotech DG3 IgG1 APC
(ALP) activity and calcium deposition were analyzed on MHC-I BDPharmingen G46–2.6 IgG1κ FITC
days 7, 14 and 21 to evaluate osteogenic differentiation. MHC-II BDPharmingen G46–6 IgG2aκ PE
For chondrogenic differentiation, a 3D pellet culture
STRO-1 R&DSystems STRO-1 IgMλ Pure
modelwasused.Onday21,pelletsweresubjectedtorou-
Anti-mouseIgM AbDSerotec 3A6 Polyclonal PE
tine histological processing and then stained by toluidine
Villatoroetal.BMCVeterinaryResearch (2018) 14:116 Page5of13
Immunomodulatorypotential monolayer and maintained their proliferation capacity
Inhibition of lymphocyte proliferation assays were withoutindicationofsenescence (Fig.3b).
performed using peripheral blood mononuclear cells
(PBMCs)harvestedfromahealthydonorcat.PBMCswere Cellproliferation
separatedusingFicoll-Hypaquedensitygradientcentrifuga- Cell proliferation wasstudied by MTS assay. Thegrowth
tion and stained with 4 μM 5-chloromethylfluorescein dia- curve showed that the cellsstarted proliferating immedi-
cetate (CMFDA, Cell Tracker Green Kit C2925, Thermo ately after being plated (there was no lag phase), initiat-
Fisher Scientific, Inc.). PBMCs were plated in a 96-well ing the logarithmic growth phase and reaching its
plate at a concentration of 5×104 cells/well. fAd-MSCs at plateauphase inapproximately 21days(Fig. 3c).
passage2wereharvestedandseededinthe96-wellplateat
1×104 cells/well, previously inactivated with mitomycin C Flowcytometryanalysis
for3h.ConcanavalinA(ConA;Sigma-Aldrich)wasadded Once the secondary cultures at passage 2 reached 70%
to experimental wells at a final concentration of 5 μg/ml. of confluence, cells were subjected to FACS analysis.
Thefollowingexperimentalgroupswereused(intriplicate): The profiles of fAd-MSCs were uniformly and strongly
PBMCs control; PBMCs and fAd-MSCs; PBMCs and positive for mesenchymal markers CD29, CD44, CD73,
ConA;PBMCs,fAd-MSCsandConA.Cellswereincubated CD90andMHC-I(Fig.3d),andnegativeforhematopoietic
for 72 h at 37 °C and 5% CO2, and then, the amount of markers CD34, CD45 and MHC-II. Additionally, a minor
lymphocyte proliferation was analyzed by flow cytometer populationofSTRO-1-expressingcellswasobserved.
(Beckman Coulter). For the purpose of comparison, lym-
phocytesstimulatedwithConAweresetto100%prolifera- Invitromultilineagecelldifferentiation
tion. Flow cytometry data were analized using FlowJo Adipogenic differentiation was confirmed by Oil Red O
cytometrysoftware. staining. After culturing cells with adipogenic-inducing
media for 21 days, red-stained lipid droplets were
present inthe cytoplasm(Fig. 4a-f).
Celltransplantation
The osteogenic differentiation potential was confirmed
The procedure was performed under sedation with meto-
by histochemical localization of ALP and Alizarin Red S.
midine0.005mg/Kg(Sedator®Lab.DVF,Barcelona,Spain).
In these cultures, morphological changes were observed;
Alleyeswereimplantedaseptically,withtwoinjections
in a time interval of 2 months, of 2×106 allogeneic fAd- non-supplemented cells showed the spindle-shaped
morphology, while osteoinduced cells formed numerous
MSCs in 0.4 mL DMEM, using a 21G needle, subcon-
nodules, which were stronger stained for ALP activity
junctivally near the lesion, with preliminary vitality test
(Fig. 4g-h) and Alizarin Red S (Fig. 4i-j). Consistent with
with trypan blue staining. All cats were hospitalized for
these results, quantitative measurement of ALP activity
24 h after local transplantation to monitor any potential
showed that osteoinduced cells exhibited significantly
adverse effect.
higher levels (p <0.001) compared with the controls
The animals did not receive any kind of systemic and
(Fig. 4k). Moreover, a significantly calcium deposition
topical medicationduringthefollow upperiod.
wasonlydetected intreatedcells (p <0.001) (Table 3).
For chondrogenic differentiation, a 3D pellet culture
Statisticalanalysis
model was used. Histological study showed that fAd-
Means and standard deviations were performed using
MSCs were able to differentiate into chondrocytes. After
SigmaStatsoftware(SPSSInc.,Chicago,IL)withStudent’s 21 days of culture, pellets incubated with rhTGF-β1
ttestsorone-wayanalysisof variance(ANOVA)afterthe
showed characteristic chondrogenic phenotype with
data passed normality and equal variance tests. Results
lacunae formation. Besides, their extracellular matrix
wereconsideredsignificantlydifferentatp<0.05.
exhibitedmetachromasiawhenstainedwithTB andhigh
affinity for AB, indicating accumulation of glycosaminogly-
Results cans and proteoglycans (Fig. 5). Immunohistochemical
IsolationandcultureoffAd-MSCs staining revealed that pellets stimulated with rhTGF-β1
The abdominal subcutaneous fat was processed and producedadepositionofcartilage-specifictypeIIcollagen.
MSCs were successfully isolated from the donor sample
Table3Quantificationofcalciumcontentasindicationof
using their ability to adhere to tissue culture plastic. In
mineralization
primarycultures,the cells grew rapidlyandalarge num-
Time(days) Treatment Calcium(mg/dl)
ber of colony-forming units was observed 2 days after
initial seeding and 80% of semiconfluence was achieved 21 Control 9.468±0.275
on day 14 (Fig. 3a). On secondary cultures, fAd-MSCs Osteoinduced 13.496±0.892**
appeared as spindle-shaped cells that were grown in a Valuesareexpressedasthemean±SD(n=3).**p<0.01
Villatoroetal.BMCVeterinaryResearch (2018) 14:116 Page6of13
Fig.3Cellmorphology,proliferationandrepresentativeFACSanalysisoffAd-MSCsforseveralmesenchymalandhematopoieticmarkers.aInprimary
cultures,alargenumberofadherentcellswithfibroblasticmorphologyandcolony-formingunitswereobservedtwodaysafterinitialseeding.bOn
secondarycultures,fAd-MSCsappearedasspindle-shapedcellsthatgrowninamonolayer.cRepresentativecurveobtainedwithMTScellproliferation
assayatpassage2.Cellsstartedproliferatingimmediatelyafterbeingplated,initiatingthelogarithmicgrowthphaseandreachingitsplateauphase
around21days.dTheimmunophenotypeprofilesrevealedahomogeneouscellpopulation,characterizedbythestrongpositiveexpressionofCD29,
CD44,CD73,CD90andmajorhistocompatibilityclassI(MHC-I),andlackexpressionofCD34,CD45andMHC-II.Aswell,aminorpopulationof
STRO-1-expressingcellswasobserved.Bars,200μm
Villatoroetal.BMCVeterinaryResearch (2018) 14:116 Page7of13
Fig.4Assessmentofadipogenic(a-f)andosteogenic(g-k)differentiation.Control(a-c)andadipo-induced(d-f)culturesat7,14and21days.
PositiveOilRedOstainingconfirmedthepresenceoflipiddropletsonlyinadipogenic-inducedcells.Bars,200μm.Insertsrepresenthigher
magnificationofthespecificstain.HistochemicallocalizationofALP(g-h)andAlizarinRedS(i-j)stainingatday21.Osteoinducedcellsformed
numerousnoduleshighlypositiveforALPstaining.ControlcellsremainedAlizarinRedSnegativebyday21whereasredcalciumnodules
clearlyappearedontheosteoinducedcultures.kQuantitativemeasurementofALPactivityat7,14and21days.Osteoinducedcellsexhibited
significantlyhigherlevelsofALPactivity(p<0.001)comparedwiththecontrols.Valuesrepresentthemeans±SD,n=3.Asterisk(*)indicatesa
statisticallysignificantdifference(p<0.001)forthesameconditionatdifferenttimepoints,whereas(●)representsp<0.001betweentwogroups
atthesametimeperiod.Bars,200μm
Villatoroetal.BMCVeterinaryResearch (2018) 14:116 Page8of13
Fig.5Assessmentofchondrogenicdifferentiation.Histologicalsectionsofpelletsafter21daysintheabsence(a-c)orpresence(d-f)ofrhTGF-β1.
ThedegreeofmaturationafterchondrogenicdifferentiationwasassessedbyTB,ABstainingandtypeIIcollagenimmunohistochemistry.Pellets
incubatedwithrhTGF-β1clearlydisplayedimprovedchondrogenesis.Bars,200μm
Karyotype allogeneic fAd-MSCs with 11-months follow-up. FEK
fAd-MSCshadanormalmetaphasespreadandkaryotype. is a chronic, progressive, inflammatory disease of the
cornea and conjunctiva. Its manifestation is through
Immunomodulatorypotential superficial vascularization with formation of gritty
The PBMCs proliferation was assessed by flow cytom- yellow-white plaques and stromal infiltration with
etry of CMFDA stained cells and their growth was edema of the cornea [1, 10, 29]. The disease seems
compared to the presence or absence of fAd-MSCs at tohavenoage,breedorsexpredilection,andappearsuni-
72 h post co-culture. As depicted in Fig. 6, fAd-MSCs laterally 80% of the time [6, 29]. If left untreated, it may
significantly reduced PBMCs proliferation (p <0.05) eventually become a bilateral condition, as being a pro-
in direct contact. gression of the disease [1, 10]. The characteristics and
clinical signs described in our cases are consistent with
Celltransplantation publishedresultsaboutthisdisease[1,6].
After the first implantation, 2 of the 5 cases (Fig. 7a-b) Theetiologyofthediseaseisstillundetermined,butap-
presented decrease of ocular signs before the second im- pearstobeanimmuneresponsetoanunknownantigenic
plantation. Their clinical evaluation showed a significant stimulus[30],thattriggersinflammationwithaprominent
improvement during the 4 first weeks after cell trans- roleinthedevelopmentofthesymptomsandsigns.
plantation with progressive disappearance of ocular In this study, the cytology results of the affected areas
signs, beginning with a decrease of corneal plaque and a showed eosinophils and mast cells in all cases (100%),
subsequent decrease in the corneal vascularization and similar to other studies [2, 6]. These cells are not found
hyperemia (Fig. 7a). In these animals, the corneal on healthy feline corneas, and their presence has been
cytology was negative for eosinophils and mast cells assumed to be pathognomonic of FEK. It is described a
from the 2-month follow-up. Rest of animals showed a correlation between FHV-1 and FEK, where the FHV-1
complete remission of clinical signs and cytology at is the most frequent cause of conjunctivitis and keratitis
6 months. This recovery remained stable until the last indomestic cats[31].
follow-up where the cornea was totally transparent with The current treatment for this disease consists of top-
complete regression of corneal plaque, blood vessels and ical anti-inflammatory agent (corticosteroid) or drugs
hyperemia, and did not show signs of worsening without with glucocorticoid-like activity (megestrol acetate) and/
topic treatment (Fig. 7). or immunosuppressant (cyclosporine A) [2, 6, 11]. These
There were no local or systemic complications dur- agents are associated with deleterious and undesirable
ing all follow-ups, without any alteration of the blood side effects that limit the long-term use of these drugs
cell count and serum biochemistry profile, with re- [26]. Therefore, corticosteroids are a poor choice in cor-
spect to initial values. neal diseases with FHV-1 infection, because to induce
virus reactivation, potentiate corneal penetration of
Discussion virus, and increase susceptibility of keratocytes to viral
This is the first clinical study in FEK that evaluates infection [7, 32].On the other hand, topical cyclosporine
results after subconjunctival implantation of in cats has been related with certainly grade of
Villatoroetal.BMCVeterinaryResearch (2018) 14:116 Page9of13
Fig.6Suppressionofperipheralbloodmononuclearcells(PBMCs)proliferation.PBMCswerestimulatedwithconcanavalinA(ConA)andthen
incubatedwithfAd-MSCs.Valuesrepresentsthemeans±SD,n=3.Asterisk(*)indicatesastatisticallysignificantdifference(p<0.05)
intolerance and adverse effects like irritation, chemosis, osteogenic, and chondrogenic differentiation similar to
conjunctivalhyperemia andblepharitis [1,6]. thosedescribedforthisspecie[40–43].
The refractory cases of eosinophilic keratitis have been Our FACS data are consistent with other studies, al-
treated with megestrol acetate, however the condition thoughthe immunophenotype ofthe feline cells involves
can only be control but not cured, since after the cessa- difficulty because of the limited availability of antibodies,
tion of treatment, recurrence of clinical signs is com- which cross-react with this species. The expression pro-
mon. [6, 10] Also different side effects such as neoplasia, file corresponding with those is widely described in the
diabetes mellitus, adrenocortical suppression, behavioral literaturefor MSCs,asshown byourresults:positiveex-
changes and mammary hyperplasia have been associated pression of CD29, CD44, CD73, CD90, STRO-1 and
with its use. [33] All these contraindications entail the MHC-I,andlackofexpressionof hematopoieticmarkers
need tofinda safe, simple andeffective treatment. CD34, CD45 and MHC-II [21, 23]. There is controversy
Due to the chronic disease character, the condition in the literature about the use of STRO-1 as a good
can only be controlled but not cured. In addition, recur- MSCs marker; however, some authors recommend this
rences of clinical signs are common after treatment ces- profile, among others, as the most useful markers for the
sation [6, 10]. All this leads to the need to find a safe, optimal identification of MSCs [44]. The positive popula-
simple andeffectivetreatment. tion has shown ability to differentiate into multiple mes-
Feline Ad-MSCs are multipotent stem cells with cap- enchymal lineages, such as chondroblasts, adipoblasts,
acity to differentiate, with important secretory faculty of osteoblasts in addition to hematopoiesis supportive cells
different bioactive molecules with trophic, paracrine, withavascularsmoothmuscle-likephenotype[45].
anti-inflammatory and immunomodulatory functions Furthermore, fAd-MSCs have shown in vitro both
[13, 34, 35]. Ad-MSCs have been isolated and character- their capacity to inhibit mitogen-stimulated PMBC pro-
ized from several domestic species and are currently be- liferation as well as the genomic stability of their karyo-
ing used as therapeutics for a number of clinical type. These results were consistent with others recently
applicationsinveterinary medicine [23,24,26,36–39]. published[46,47].
In our study, we used adipose tissue because it is an The effective use of allogeneic MSCs in patients is
easilyaffordable andplentiful sourcefor obtaining MSCs a new reality possible due to their low immunogen-
withahighproliferationcapacity[26,40].Veryfewstud- icity by lack of MHC-II [13]. This allows a rapid ini-
ies have investigated the isolation and characterization of tiation of therapy without the need for harvesting
felineMSCs[21,23,24,40,41].FelineAd-MSCsobtained MSCs from each patient, screening the best donors,
fromourdonorhaveshownconsistencyintheirisolation, preventing transmission of infectious diseases, and evalu-
expansion, high ratio proliferation, plastic adherent, and ating in vitro their MSC profile and immunosuppressive
behavior in vitro, exhibiting its ability of adipogenic, features[26,46].
Villatoroetal.BMCVeterinaryResearch (2018) 14:116 Page10of13
Fig.7Follow-upeyeimagesinfivecats(a-e)withchroniceosinophilickeratitis.Animalsshowedacompleteremissionofclinicalsignsat
6monthsandthisrecoveryremainedstableuntilthelastfollow-upat11months