cell therapy sciences · “Caregiver placebo effect for dogs with lameness from...
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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, Michael G., and Richard B. Evans. “Caregiver placebo effect for dogs with lameness from osteoarthritis.” Journal of the American Veterinary Medical Association241.10 (2012): 1314-1319. 2. Guercio, Annalisa, et al. “Production of canine mesenchymal stem cells from adipose tissue and their application in dogs with chronic osteoarthritis of the humeroradial joints.” Cell biology international 36.2 (2012): 189-194. 3. Lee, Jienny, et al. “Effect of donor age on the proliferation and multipotency of canine adipose-derived mesenchymal stem cells.” Journal of veterinary science 18.2 (2017): 141-148. 4. Perry, K. www.vettimes.co.uk/article/using-cellular-therapies-for-canine-joint-treatment-part-1/ 5. Harman et al. “A Prospective, Randomized, Masked, and Placebo-Controlled Efficacy Study of Intraarticular Allogeneic Adipose Stem Cells for the Treatment of Osteoarthritis in Dogs.” Front Vet Sci. 3 (2016) : 81 6. Kuah et al. “ Safety, tolerability and efficacy of intra-articular Progenza in knee osteoarthritis:a randomized double-blind placebo-controlled single ascending dose study. “ J Transl Med 16 (2018) :49 AFFILIATIONS AND CONFLICT OF INTEREST: Authors work in Research and Development at CTSL ACKNOWLEDGEMENTS: To all of the small animal vet clinics in the UK who have kindly sent us samples and cases for this study. 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 sciences Developing Regenerative Medicine for Vets CLINICAL DATA: RESULTS: INTRODUCTION: MATERIALS AND METHODS: Osteoarthritis is a leading cause of pain and lameness in dogs. Regenerative medicine and in particular MSC treatments are increasingly being used to treat OA because not only do they alleviate pain, they also decrease inflammation, improve structural damage and enhance tissue repair. MSC therapy provides an important option when conventional therapies do not provide adequate control of arthritic pain or where the dog cannot tolerate high levels of pain relieving medication and in cases where there is no suitable, proven surgical option (for example in elbow disease) or to delay the need for salvage surgeries (Perry 2016). MSC culture-expansion selects for high numbers of viable, adhesive MSCs and removes residual connective tissue debris, thus providing a quality- controlled, standardised, viability-tested stem cell injection to ensure the highest level of safety and efficacy in the clinic. There are some reports particularly in human medicine that MSCs extracted from older people are less able to proliferate and are less efficacious than those from younger people. Cell Therapy Sciences is in a unique position to be able to study the culture characteristics of MSCs from a wide range of dogs of different ages and to follow this up with a case series of dogs treated with these MSCs. Dogs of various ages, each with a documented history of moderate to severe OA inadequately controlled on conventional therapy, underwent adipose tissue harvest for MSC extraction and culture-expansion. MSCs were separated from the adipose tissue using an optimised, standard protocol (Guercio 2012) and the adherent cells from the SVF were culture-expanded until 90% confluent. The cultured cells were harvested to prepare quality controlled, autologous stem cell injections for each dog, which were checked for stem cell numbers, morphology, point-of care viability ( > 90%) and confirmed as pathogen-free. Cell-cultures were photographed daily using the same optical field, cells were counted and population doubling-time calculated. Cell Therapy Sciences use only early passage stem cells (P0-P2) to prepare injections for clinical use and therefore P0-P2 cells were tested in this study. Dogs were monitored for up to 6 months following intra-articular MSC injections into their affected joints. A global score, based on clinical examinations and owners’ feedback on pain and mobility, was used by the treating veterinarian to evaluate treatment response. Results were analysed using a two tailed student t-test for the population doubling time comparison and a Mann-Whitney U-test for the clinical outcome data. Authors: Joanna Miller 1 , Christine Standen 1 , Lucy Frost 2 1 Cell Therapy Sciences Ltd, Coventry, United Kingdom. 2 University of Warwick, Warwick, United Kingdom. • This study provides compelling evidence from 52 dogs that canine adipose-derived MSCs can be successfully and rapidly expanded in culture at early passages regardless of the age of the donor dog. • There is accumulating published evidence , including recent double blind, randomised placebo- controlled studies in OA ( Harman et al, 2016; Kuah et al 2018) that intra-articular injections containing high numbers of MSCs can bring about significant improvements in clinical signs and symptoms such as pain and overall mobility. • All treated dogs in the cell culture study were followed up as a case series to determine response to therapy in the opinion of the treating vet, based on clinical assessments and owner feedback. This data indicates that 98% of dogs with moderate to severe OA showed clinical improvement, with 60% demonstrating an excellent improvement. • This was a case series with no blinded control group and will be subject to “care-giver bias” ; whilst studies have reported that care-giver placebo effects can be as high as 40% in canine OA studies ( Conzemius 2012) this alone cannot explain the 98% clinical improvement reported here. • Efficacy results in the older dogs suggested that whilst the overall response rate was high ( 94%), fewer of the older dogs achieved an excellent improvement. This is possibly due to the overall disability level of the older dogs and the need for complimentary rehabilitation therapy. Additionally, treatment of only one or two of the worst affected joints may highlight problems in other joints or soft tissues. • At Cell Therapy Sciences we only prepare stem cell therapies cultured at early passage (P0-P2) and therefore these were tested in this study. It has been reported in a previous small study ( 5 dogs per group) that donor age could have an affect on cumulative population doublings at later passages ( Lee et al 2017).” • Age of the donor does not adversely affect the ability of MSCs to grow normally in culture. • Following intra-articular injections of MSCs, 98% of dogs with moderate to severe OA showed a clinical improvement. • Even in older dogs (10-15 years) 94% showed clinical improvements, with dogs <10 years most likely to demonstrate an excellent response. ABBREVIATIONS • OA: Osteoarthritis • MSC: Mesenchymal Stem Cell • SVF: Stromal Vascular Fraction • FCS: Foetal Calf Serum • PDT: Population Doubling Time ABSTRACT: OBJECTIVES: To evaluate whether a dog’s age influences either the growth rate of autologous adipose-derived Mesenchymal Stem Cells (adMSCs) during culture-expansion, or clinical efficacy outcomes following treatment with intra-articular (IA) adMSC injections. METHODS: Dogs of various ages, each with a documented history of moderate- severe OA inadequately controlled on conventional therapy, underwent adipose tissue harvest for stem cell extraction and culture-expansion. Cell-cultures were photographed daily using the same optical field, cells counted and population doubling-time calculated. Dogs were monitored for up to 6 months following IA adMSC injections into all affected joints. A Global Score, based on clinical examinations and owners’ feedback on pain and mobility, was used to evaluate treatment response. RESULTS: Fifty-two dogs were included in this analysis. No significant affect of dog’s age was seen on population doubling time of low passage (P0 & P1) cultures of canine adMSCs. The majority of dogs (98%) demonstrated a clinical improvement (51/52 cases) following adMSC injections, with 60% evaluated as demonstrating an excellent response (31/52 cases). In the oldest dogs (10-15 years) 94% showed a clinical improvement, with 6/18(33%) demonstrating an excellent response. In younger dogs (< 10 years) a greater number showed an excellent response (25/34;74%). STATEMENT: Age of the donor does not adversely affect the ability of adMSCs to grow normally in culture. Following intra-articular injections of adMSCs, 98% of dogs with moderate to severe OA showed a clinical improvement. Even in older dogs (10-15 years) 94% showed clinical improvements, with dogs <10 years most likely to demonstrate an excellent response. 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). Clinical Response: % of dogs treated 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 Population doubling times (hours) for adMSCs cultured from adipose samples taken from 52 dogs of different ages (range : 1 year to 15 years). The cultures were early passage (P0-P2). No significant difference was found between the PDTs for young dogs compared with dogs of 10 years and older ( Mean 18.74 +/- 6.20 and 17.93 +/- 6.89 respectively). < 10 years (n= 34) p = 0.665 5 0 0 10 15 20 25 30 35 40 ≥10 years (n= 18) Population Doubling Time ( Hours) 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 Table 1. This describes the relationship between the number of cells counted per field and the estimated total number of cells on the culture flask. The blue figure is the final harvest cell count using a haemocytometer. Population doubling time is calculated according to the formula: PDT =T(Log2/Log(Cf/Cs)) where T is the time in hours between cell counts, Cf is the final cell count, Cs is the initial cell count. 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 0.69 5 34 3.93 6 48 5.55 7 99 11.44 8 216 24.96 9 389 44.950 A Typical Canine MSC Growth Curve No of days in culture Estimated no. of MSCs present each day 5 0 0 10 2 4 6 8 10 15 20 25 30 35 40 45 50 Clinical Response: % of dogs treated
Transcript of 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).
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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).
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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
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