cell therapy sciences · “caregiver placebo effect for dogs with lameness from...

Effect of a Dog’s Age on Stem Cell Culture Times and Efficacy Outcomes in Dogs with Osteoarthritis Treated with Intra-articular Injections of Autologous Mesenchymal Stem Cells.

REFERENCES:1. Conzemius,MichaelG.,andRichardB.Evans.“Caregiverplaceboeffectfordogswithlamenessfromosteoarthritis.”JournaloftheAmericanVeterinaryMedicalAssociation241.10(2012):1314-1319.2. Guercio,Annalisa,etal.“Productionofcaninemesenchymalstemcellsfromadiposetissueandtheirapplicationindogswithchronicosteoarthritisofthehumeroradialjoints.”Cellbiologyinternational36.2(2012):189-194.3. Lee,Jienny,etal.“Effectofdonorageontheproliferationandmultipotencyofcanineadipose-derivedmesenchymalstemcells.”Journalofveterinaryscience18.2(2017):141-148.4. Perry,K.www.vettimes.co.uk/article/using-cellular-therapies-for-canine-joint-treatment-part-1/5. Harmanetal.“AProspective,Randomized,Masked,andPlacebo-ControlledEfficacyStudyofIntraarticularAllogeneicAdiposeStemCellsfortheTreatmentofOsteoarthritisinDogs.”FrontVetSci.3(2016):816. Kuahetal.“Safety,tolerabilityandefficacyofintra-articularProgenzainkneeosteoarthritis:arandomizeddouble-blindplacebo-controlledsingleascendingdosestudy.“JTranslMed16(2018):49 AFFILIATIONS AND CONFLICT OF INTEREST: AuthorsworkinResearchandDevelopmentatCTSLACKNOWLEDGEMENTS: ToallofthesmallanimalvetclinicsintheUKwhohavekindlysentussamplesandcasesforthisstudy.

OBJECTIVES:

To evaluate whether a dog’s age influences either the growth rate of adipose-derived Mesenchymal Stem Cells (MSCs) during culture-expansion, or clinical efficacy outcomes following treatment of the dog’s osteoarthritis using intra-articular injections of autologous MSCs.

cell therapy sciencesDeveloping Regenerative Medicine for Vets

CLINICAL DATA:

RESULTS:

INTRODUCTION: MATERIALS AND METHODS:

Osteoarthritis isa leadingcauseofpainand lameness indogs.Regenerativemedicineand inparticularMSCtreatmentsareincreasinglybeingusedtotreatOAbecausenotonlydotheyalleviatepain,theyalsodecreaseinflammation,improvestructuraldamageandenhancetissuerepair.MSCtherapyprovidesanimportantoptionwhenconventionaltherapiesdonotprovideadequatecontrolofarthriticpainorwherethedogcannottoleratehighlevelsofpainrelievingmedicationandincaseswherethereisnosuitable,provensurgicaloption(forexampleinelbowdisease)or todelay theneed forsalvagesurgeries (Perry2016).MSCculture-expansionselects forhighnumbersofviable,adhesiveMSCsandremovesresidualconnectivetissuedebris,thusprovidingaquality-controlled,standardised,viability-testedstemcellinjectiontoensurethehighestlevelofsafetyandefficacyintheclinic.TherearesomereportsparticularlyinhumanmedicinethatMSCsextractedfromolderpeoplearelessabletoproliferateandarelessefficaciousthanthosefromyoungerpeople.CellTherapySciencesisinauniquepositiontobeabletostudytheculturecharacteristicsofMSCsfromawiderangeofdogsofdifferentagesandtofollowthisupwithacaseseriesofdogstreatedwiththeseMSCs.

Dogs of various ages, each with a documented history of moderate to severe OA inadequately controlled onconventionaltherapy,underwentadiposetissueharvestforMSCextractionandculture-expansion.MSCswereseparatedfromtheadiposetissueusinganoptimised,standardprotocol(Guercio2012)andtheadherentcellsfromtheSVFwereculture-expandeduntil90%confluent.Theculturedcellswereharvestedtopreparequalitycontrolled,autologousstemcellinjectionsforeachdog,whichwerecheckedforstemcellnumbers,morphology,point-ofcareviability(>90%)andconfirmedaspathogen-free.Cell-cultureswerephotographeddailyusingthesameopticalfield,cellswerecountedandpopulationdoubling-timecalculated.CellTherapySciencesuseonlyearlypassagestemcells(P0-P2)toprepareinjectionsforclinicaluseandthereforeP0-P2cellsweretestedinthisstudy.Dogsweremonitoredforupto6monthsfollowingintra-articularMSCinjectionsintotheiraffectedjoints.Aglobalscore,basedonclinicalexaminationsandowners’feedbackonpainandmobility,wasusedbythetreatingveterinariantoevaluatetreatmentresponse.Resultswereanalysedusingatwotailedstudentt-testforthepopulationdoublingtimecomparisonandaMann-WhitneyU-testfortheclinicaloutcomedata.

Authors: Joanna Miller1, Christine Standen1, Lucy Frost2 1CellTherapySciencesLtd,Coventry,UnitedKingdom.2UniversityofWarwick,Warwick,UnitedKingdom.

•Thisstudyprovidescompellingevidence from52dogsthatcanineadipose-derivedMSCscanbesuccessfullyandrapidlyexpandedincultureatearlypassagesregardlessoftheageofthedonordog.

•Thereisaccumulatingpublishedevidence,includingrecentdoubleblind,randomisedplacebo-controlled studies in OA ( Harman et al, 2016; Kuah et al 2018) that intra-articular injectionscontaininghighnumbersofMSCscanbringaboutsignificantimprovementsinclinicalsignsandsymptomssuchaspainandoverallmobility.

•Alltreateddogsinthecellculturestudywerefollowedupasacaseseriestodetermineresponsetotherapyintheopinionofthetreatingvet,basedonclinicalassessmentsandownerfeedback.Thisdataindicatesthat98%ofdogswithmoderatetosevereOAshowedclinicalimprovement,with60%demonstratinganexcellentimprovement.

•Thiswasacaseserieswithnoblindedcontrolgroupandwillbesubjectto“care-giverbias”;whilststudieshavereportedthatcare-giverplaceboeffectscanbeashighas40%incanineOAstudies(Conzemius2012)thisalonecannotexplainthe98%clinicalimprovementreportedhere.

•Efficacyresultsintheolderdogssuggestedthatwhilsttheoverallresponseratewashigh(94%),feweroftheolderdogsachievedanexcellentimprovement.Thisispossiblyduetotheoveralldisabilityleveloftheolderdogsandtheneedforcomplimentaryrehabilitationtherapy.Additionally,treatmentofonlyoneortwooftheworstaffectedjointsmayhighlightproblemsinotherjointsorsofttissues.

•AtCellTherapySciencesweonlypreparestemcelltherapiesculturedatearlypassage(P0-P2)andthereforetheseweretestedinthisstudy.Ithasbeenreportedinaprevioussmallstudy(5dogspergroup)thatdonoragecouldhaveanaffectoncumulativepopulationdoublingsatlaterpassages(Leeetal2017).”

• AgeofthedonordoesnotadverselyaffecttheabilityofMSCstogrownormallyinculture.

• Followingintra-articularinjectionsofMSCs,98%ofdogswithmoderatetosevereOAshowedaclinicalimprovement.

• Eveninolderdogs(10-15years)94%showedclinicalimprovements,withdogs<10yearsmostlikelytodemonstrateanexcellentresponse.

ABBREVIATIONS • OA: Osteoarthritis• MSC: MesenchymalStemCell• SVF: StromalVascularFraction

• FCS: FoetalCalfSerum• PDT: PopulationDoublingTime

ABSTRACT: OBJECTIVES:Toevaluatewhetheradog’sageinfluenceseitherthegrowthrateofautologousadipose-derivedMesenchymalStemCells(adMSCs)duringculture-expansion,orclinicalefficacyoutcomesfollowingtreatmentwithintra-articular(IA)adMSCinjections. METHODS:Dogsofvariousages,eachwithadocumentedhistoryofmoderate-severeOAinadequatelycontrolledonconventionaltherapy,underwentadiposetissueharvestforstemcellextractionandculture-expansion.Cell-cultureswerephotographeddailyusingthesameopticalfield,cellscountedandpopulationdoubling-timecalculated.Dogsweremonitoredforupto6monthsfollowingIAadMSCinjectionsintoallaffectedjoints.AGlobalScore,basedonclinicalexaminationsandowners’feedbackonpainandmobility,wasusedtoevaluatetreatmentresponse.RESULTS:Fifty-twodogswereincludedinthisanalysis.Nosignificantaffectofdog’sagewasseenonpopulationdoublingtimeoflowpassage(P0&P1)culturesofcanineadMSCs.Themajorityofdogs(98%)demonstratedaclinicalimprovement(51/52cases)followingadMSCinjections,with60%evaluatedasdemonstratinganexcellentresponse(31/52cases).Intheoldestdogs(10-15years)94%showedaclinicalimprovement,with6/18(33%)demonstratinganexcellentresponse.Inyoungerdogs(<10years)agreaternumbershowedanexcellentresponse(25/34;74%). STATEMENT:AgeofthedonordoesnotadverselyaffecttheabilityofadMSCstogrownormallyinculture.Followingintra-articularinjectionsofadMSCs,98%ofdogswithmoderatetosevereOAshowedaclinicalimprovement.Eveninolderdogs(10-15years)94%showedclinicalimprovements,withdogs<10yearsmostlikelytodemonstrateanexcellentresponse.

DISCUSSION: CONCLUSION:

FIG 3 : CLINICAL EFFICACY OF MSC TREATMENT IN DOGS WITH OA FIG 4 : DOES AGE INFLUENCE THE CLINICAL EFFICACY OF MSC TREATMENT IN DOGS WITH OA ?

Veterinary Global Efficacy Score for dogs with OA treated with intra-articular culture expanded MSC injections (n=52). 60% achieved an excellent clinical improvement (31/52 dogs), 21% achieved a moderate clinical improvement (11/52 dogs), 17% achieved a mild clinical improvement (9/52 dogs) and only 2% had no clinical improvement (1/52 dogs).

Veterinary Global Efficacy Scores for dogs with OA treated with culture-expanded MSC therapy; responses presented according to the age of the dog. In the younger age group (< 10 years) , 100% of dogs were assessed as achieving a clinical improvement; 74% of these achieved an excellent clinical improvement (25/34 dogs), 21% achieved a moderate clinical improvement (7/34 dogs), 6% achieved a mild improvement (2/34 dogs). In the older age group (≥ 10 years) 94% were assessed as achieving a clinical improvement; 33% of these achieved an excellent clinical improvement (6/18 dogs), 22% achieved a moderate clinical improvement (2/34 dogs), 39% achieved a mild clinical improvement (7/18 dog) and 6% did not improve (1/18 dogs).

Clin

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Res

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e: %

of d

ogs

trea

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2%

17%

21%

60%

6%0% 6%

39%

22%21%

33%

74%<10 years (n=34) ≥10 years (n=18)

FIG 2 : MSC PDTs FOR YOUNG DOGS COMPARED WITH OLDER DOGS

Populationdoublingtimes(hours)foradMSCsculturedfromadiposesamplestakenfrom52dogsofdifferentages(range:1yearto15years).Thecultureswereearlypassage (P0-P2). No significant difference was found between the PDTs for youngdogscomparedwithdogsof10yearsandolder(Mean18.74+/-6.20and17.93+/-6.89respectively).

< 10 years (n= 34)

p = 0.665

5

00

10

15

20

25

30

35

40

≥10 years (n= 18)

Popu

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n D

oubl

ing

Tim

e ( H

ours

)

Photomicrographs of canine MSCs in culture taken using the same field of view on sequential days. Bar = 0.1mm.

FIG 1 : ESTIMATION OF MSC POPULATION DOUBLING TIMES (PDTS)

Day 2 Day 5 Day 6 Day 7 Day 8 Day 9

Table1.Thisdescribestherelationshipbetweenthenumberofcellscountedperfieldand theestimated totalnumberofcellson thecultureflask.Thebluefigureisthefinalharvestcellcountusingahaemocytometer.Populationdoublingtimeiscalculatedaccordingtotheformula:PDT=T(Log2/Log(Cf/Cs))whereTisthetimeinhoursbetweencellcounts,Cfisthefinalcellcount,Csistheinitialcellcount.

Growth rate of a typical case of canine MSCs

Population Doubling Time (hrs): 27.91

Days in culture

No cells in photo field

No MSCs (million)

2 6 0.695 34 3.936 48 5.557 99 11.448 216 24.969 389 44.950

A Typical Canine MSC Growth Curve

No of days in culture

Estim

ated

no.

of M

SCs

pres

ent e

ach

day

50

0 102 4 6 8

1015

2025

30354045

50

Clin

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