Osteoarthritis, a degenerative disease open to future cell ... · Osteoarthritis, a degenerative...

Osteoarthritis,adegenerativediseaseopentofuturecell

therapy?

DrYves-MariePERSMCU-PH

ImmunologiecliniqueetThérapeutiqueostéo-articulaireDépartementdeRhumatologie- CHUMontpellier

[email protected]

• 17%wholepopulation• 10millionspatientsinFrance• Incidence

Ø KneeOA:240/100.000PAØ HandOA:100/100.000PAØ HipOA:88/100.000PA

• Highburdencost+++Ø 3millionsphysicianvisit/yearØ 14millionsdrugprescribed

• Mainriskfactors:Ø AgeØ SexØ Obesity

Bijlsma JWJetal.Lancet2011

Introduction- OA

synoviocytes

monocyteschondrocytes

osteoclasts

OA,pathophysiology:theplayers

Sellam,J.&Berenbaum,F.(2010) Theroleofsynovitisinpathophysiologyandclinic

OApathophysiology:synovialinflammation

UnmetneedinOA

• 3unmetmedicalneeds• Efficientdiseasemodifyingtreatment• Moreeffectivesymptomatictreatment:NSAIDsimprovelessthan50%WOMACscores

• Safertreatment:NSAIDscarrysignificantGIandCVrisk

FINDNEWTHERAPYWITHVARIOUSTARGETS

Cellulartherapy ofrheumatic diseases.EULARText Book2012

Whatisastemcell?

CharacteristicsofMesenchymalStemCells(MSC)

• MSC are defined by :• Adherence to plastic• Fibroblast like morphology• Differentiation potential : fat, bone, cartilage• Phenotype characteristics :

• Negative : CD11b, CD14, CD34, CD45• Positive : CD90, CD105, CD73, ….

• Enhance hematopoietic stem cell engraftment• Tissue reconstitution• Immuno supressive properties• Self-renewal

Selfrenewal

Differentiation

Migration

Releaseofgrowthfactorsandanti-

inflammatorymolecules

Tissueregeneration

CartilageMuscleBoneAdipose

Functionsofstemcells?

Immunecellshomeostasis

Bone marrowhematopoietic niche

preventapoptosis

preventfibrosis

tissuehomeostasis


WherewecanfindMSC– “stromalcells”?

De Girolamo L. Knee Surg Sports Traumatol Arthrosc 2016

McGonagle D. Nat Review Rheum. 2017


Jiang and Tuan., Nat Review Rheum. 2015

• Spontaneousrepairofcartilagedefectsafterrealignmentosteotomyorjointdistraction

• Localjointresidentstromalcells?


Targetinglocalprogenitorscells?

Johnson K. Science 2012

• Kartogenin promoteschondrocytedifferentiationofMSC• Modelofcartilagerepairstrategy

Maumus M, Biochimie. 2013

WhyMSCmakesensesinOA?

§ Synoviocytes orchondrocyteswereco-culturedinmonolayerwithASCfor7days

§ Synoviocytes:ASCssignificantlydown-modulatetheexpressionofIL1β,IL6andIL8

§ Chondrocytes:ASCsignificantlydecreasedIL1β,IL6,MMP13,MCP-1andMIP-1α

Manferdini et al. Arthritis and Rheumatol. 2013

• EffectofASConsynoviocytes andOAchondrocytes

15

ProofofconceptMSCinOA

Collagen I expression Collagen III expression

Col

I ex

pres

sion

(fol

d ch

ange

)

0.0

0.5

1.0

1.5

2.0

****

ChondrocytesASC

aHGF-AbIsotype control

+ + + + +

+- - - -+ + +- -

+-- - -rhHGF (50ng/mL) -- - - +

Col

III e

xpre

ssio

n(f

old

chan

ge)

0.0

0.5

1.0

1.5

2.0

*** *

ChondrocytesASC

aHGF-AbIsotype control

+ + + + +

+- - - -+ + +- -

+-- - -rhHGF (50ng/mL) -- - - +

Maumus M et al. Stem cell Res. 2013

HG

F co

ncen

tratio

n (p

g/m

L)

0

50

100

150

200

ND

Chalone

ASCalone

Co-culture

HG

F ex

pres

sion

(2-D

CT)

0.00

0.01

0.02

0.03

0.04

0.05

Ch Ch-co

ASC

*

ASC-co

HGF induction

ASCmaintainchondrocytephenotypeandpreventchondrocytehypertrophyHGFisakeyfactorforthisparacrineeffect

• HGF-mediatedparacrineeffectofASCsonchondrocytes

16


Luz-Crawford P et al. Stem cells 2015

§ ActivatedMSCexpressIL1RA§MSCdecreasemonocyteactivation

Macrophages MSC WT MSC IL1RA-/-0

500

1000

1500

pg

/ml (

TN

F-α

)

*

Macrophages MSC WT MSC IL1RA-/-0

100

200

300

400

pg/

ml (

IL-1

0 )

*

TNFa IL10

C I A M S C W T M S C I L 1 R A - /-0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

pg

/ml

(IL

-1b)

*

*

IL1b

• MSCswitchmonocytes/macrophagessubsetsthroughIL1RA


MSC

monocytes

IL1RA

IL1b M2polarisation

ê TNFaé IL10ê CD4Tcellsê Bcellsurvival

IFNgTNFaIL1b

CD4

§MSCinduceM2polarisationinvitrothroughIL1binhibition§ SupernatantsfromcocultureofmacrophagesandMSCsinhibitstheproliferationofT-cells

Luz-Crawford P et al. Stem cells 2015

• MSCswitchmonocytes/macrophagessubsetsthroughIL1RA

18


• SingleinjectionofASCsintotheknee-jointatanearlyorlatetimepointafterinductionofOA(Collagenasemodel)

Collagenase injection ASC

injection

Mice sacrificedfor Histology

d0 d14 d42d-7 d-5

• ASCswereisolatedfromfatsurroundingtheinguinallymphnodes(Toulouse:LouisCasteilla)

• ASCswerecharacterizedonMSC-markers§ ASCsstainedpositiveforCD105,CD44andSca-1§ andnegativeforCD11b,CD34andcKit§ ASCs(2.104)wereunilaterallyinjectedintotheOAknee-jointin6µL

Ter Huurne and Van Lent P et al. Arthritis and Rheumatol. 201219


• Collagenase modelinBalb/c• Day-7,ASCtreatmentatday 0

n=10pergroup* p<0.05

0

5

10

15

20

25

Lat Tibia Lat Femur Med Femur

Med Tibia Mean

d42

Ctrl

ASC

* *

0

5

10

15

20

25

Lat Tibia Lat Femur Med Femur

Med Tibia Mean

d14

*

CartilageDestruction

* *

MedFemur

MedTibia

d42Ctrl ASC-treated2independentexperiments

Ter Huurne and Van Lent P et al. Arthritis and Rheumatol. 2012


• Collagenase OAmodelatday -7,ASCtreatmentatday 0

*

n= 10 per group* p< 0.05

d14 d42

Synovial activation

Ctrl ASCCtrl ASC

0.0

0.5

1.0

1.5

2.0

2.5

Syno

vial

act

ivat

ion

(arb

itrar

y sc

ore)

*

Day 42

!"#$%&'(")(*)+,-.)'"&()&/$)0"$$)#('"&)1&)213)4)1*&$5)6+)'"27%&'(").7885$..$.)(.&$(8/3&$)*(591&'(")1&)213):;)1*&$5)&5$1&9$"&)

Osteophyte formation

CtrlASCs

0

1000

2000

3000

4000

Ost

eoph

yt s

urfa

ce (

um2)

Day 14 Day 42

F

T

control F

T

ASC

Osteophyte formation in time

* p< 0.05

*

Collagenase-induced OA

Ter Huurne andVanLentPetal.ArthritisandRheumatol.201221


ASC ASC

ASC

3T3

24 hrs after injection:105 ASC

ligaments

Draining lymph nodes

JS

JS

ASC

Ter Huurne andVanLentPetal.ArthritisandRheumatol.201222


0

4

8

12

16

20

24

CN 2.106ASC 6.106ASC

Follow-up:8 weeks

* ^^L

averty’sscore

*

4%RSA 2x106 ADSC 6x106 ADSC0

4

8

12

16

20

24

4%RSA 2X106ASC 6X106ASC

Follow-up:16weeks

4%RSA 2x106 ADSC 6x106 ADSC

*

*Laverty’sscore

100 μm 100 μm 100 μm 100 μm

2 x10 6 ADSCs4 % RSAOA 6 x10 6 ADSCs

100 μm 100 μm 100 μm 100 μm

F.up= 8 weeks

F.up = 16 weeks

At16weeks:ØIncreasecartilagethickness:

+18.4%group2.106+21%group6.106

ØReductioninLavertyscoreØReductioninostophytevolume:

36± 2group2.10630± 2group6.10659± 2CT

NZWrabbitOAmodelwithASC

Desando Getal.ArthritisRes.Ther.201323


Day11

Day29

Day86

DetectionofhumansequencesbyFISHanalysis

Toupetetal.Arthritis andRheumatol.201324


Invivomechanisms

Protectioncartilage

Osteophyteformation

Inflammation(TNF-α,MMP-1)

Synovitis

Invitromechanisms

Inflammation(IL-1,IL-6,IL-8,MMP-13)

Apoptosis

Fibrosis

CTR ASC

Pers YM et al. HMBCI 2016

ASCareeffectiveinOA

• RegisteredClinicaltrialsofMSCbasedtherapyonClinicalTrials.gov

Phase I31%

Phase II50%

Phase III 15%

Phase IV 4%

Myocardial infraction 23%

GVHD 16%

Diabetes10%Liver cirrhosis

10%

Spinal Cord injury9%

OA 8%

CDMSRA SLE Others

§ Major stem cell used for cell therapy§ Easily isolated and successfully expanded§ large therapeutical applications§ Development of large scale GMP production

From Xin Wei et al. 2013

MSCbasedtherapyinhumans


GVHDImmunedisorders

- Lupus- Arthritis

- Sjögrensyndrome- SystemicSclerosis- Multiplesclerosis

- IBD- Diabetes

TissueRepair- Bone

- Cartilage- Osteoarthritis

Angiogenesis- Ischemic cardiopathy

- Limb ischemia- Stroke

Trophic effect- Skinhealing- Scleroderma


lipoaspiration

ADSCisolation/expansionGMPconditionsCellviabilityReleasecriteria/toxicology

Intra-articularinjectionsSyringe,differentdoses

Evaluation:phase1trialsafety,doseresponseclinicalevaluation(WOMAC…)MRIGemric,

ADIPOAclinicaltrial

Qualitycontrols:§Sterilitytestday8§Endotoxintestday11§Mycoplasmatestday11§Karyotypeperformedon20cultures.Allarenormal.

Releasecriteria:§Cellviability>80%,§CD45+/CD14+cells<5%,§CD90+orCD73+cells>80%§ AbsenceofexpressionofhTERTandOct-4attheendoftheprimaryculturetoassessgenotypestability.

ADIPOAclinicaltrial:cellproduction

ADIPOAclinicaltrial:fatharvesting

AdiposederivedStromalCellsforOsteoArthritis treatment.Aphase1study,bi-centric(Mtp,Wurzburg),doseescalatingstudy

withautologousASCinseverekneeOA(>3K/L)

MRI

Adiposetissue

collection

ASC injection

Arthroscopy + MRI

– 35d - 14d Baseline W1 M1 M3 M6

Clinical assessment

ADIPOAclinicaltrial:design

Pers YM et al. SCTM 2016

48 patients assessed for eligibility

18 consecutively enrolled to receive autologous ASCs

6 received 2 x 106 cells(Low-Dose)

6 received 10 x 106 cells(Mild-Dose)

6 received 50 x 106 cells(High-Dose)

No patients lost to follow-up

6 patients analysed


6 patients analysed


6 patients analysed



• Primarygoal:safety ofasingleinjectionofautologousadiposederivedstemcellsonpatientswithsevereosteoarthritisoftheknee

• Secondaryobjectives:• shorttermefficacyofasingleinjectionofautologousadiposederivedstemcellsonpatientswithsevereosteoarthritisoftheknee

• Besttolerateddose



• n=18• 60%ofpatientswerewomen• Age:64.6years• MeandurationOAwas10.8(± 6)years• MeanstageKL:83.3%stageIVand16.7%stageIII• InitialtotalWOMACscore:48.9(± 18.07)• MeaninitialpainVASscore:61.47(± 12.52)

ADIPOAclinicaltrial:patientscharacteristics


01020304050607080

Baseline 1 Week 3 Months 6 Months

Low Dose

Mild Dose

High Dose

0102030405060708090


Low DoseMild DoseHigh Dose

VAS pain score

KOOS Questionnaire

0

5

10

15

20

25

30

35


Low Dose

Mild Dose

High Dose

SAS score

ADIPOAclinicaltrial

Ø Safeprocedure:4localskinreactioninthefirstmonth

Ø Only2patientsunderwentsurgeryTKAafteroneyearfollow-upand55%after4years

36Pers YM et al. SCTM 2016

Ø dGEMRIC indexincreasein3outof6selectedpatients

Ø Suggestapossiblestructuraleffect

ADIPOAclinicaltrial:structuralassessment


Courtesy F Barry


Tyndal A. Nat Review Rheum. 2013

• MSCcoordinateimmunecellssuchasa“maestroconductor”


§ HLAG5nonclassicHLAclassImolecule,expressedbytrophoblastandhumanMSC

§ IL10andLIFinduceHLAG5expressionbyMSC

§ MSCaddedtoMLRinducedfunctionalCD25+foxp3+Tcells

§ HLAG5inhibitionpreventedTreg induction§ Cell:cell contactiscriticalastranswellpreventedTreg induction

CD4

CD25

MSCinduceTregs throughHLAG5

Selmani et al, Stem cells, 2008

§ BMMSCpreventedlethalsepsis§ PrimedMSCdecreaselunginflammationthroughmonocytesinteractionö IL10ø TNFa,IL6§MSCactivatedthroughTNFa,TLR4§ InteractionsmediatedthroughPGE2

Nemeth, Nature med 2009

MSCinhibitmacrophagesthroughPGE2

TBTreg

M1

PNNM2

Reduce INFLAMMATIONIMMUNEREGULATION

NO, VEGF, PGE2, TSG6, CCL2, IL-10, PDL-1, HO1,

TGFβ1, HGF, HLA-G5

• Which immune cells populations in blood are affected by MSC treatment ?

o Numbero Activation status

• Is there any effect on « target » cells ?

• Are there any surrogate marker for clinical response ?

ADIPOAclinicaltrial:immunemonitoring

Pers YM et al. Theranostics 2018 (in press)

T 4.Treg induction ?• Number• Activation markers

B 3. Alteration of B cells ontogeny?• Maturation markers•Surface Ig (class switch) • Regulatory B cells

1. Alteration of monocyte compartment ?• Subsets

2. Alteration of DC compartment ?• Subsets

1 platform involved8 colors panels with up to 19 various antibodies in the same tubeCANTO II analyser



Phase 1 conducted between March 2012 and December 2014Prospective, single-arm, open-label18 patientsDose escalatingLow dose (n=6): 2x106 ASC IA injection (5 ml)Medium dose (n=6) : 10x106 ASC IA injection (5 ml) High dose (n=6): 50x106 ASC intra-articular injection (5 ml)

Bi centric- Universität Würzburg, GERMANY (7 patients)- CHU Montpellier, France (11 patients)



Harmonization of blood collection Harmonization of blood specimen age prior to staining (max 24hrs)SOP for PBMC preparation and cellular staining

Blood specimen are collected before and after MSC therapy (D0, D7, M1, M3)



CD25+CD127lowFoxP3+in CD4+CD25+FoxP3+inCD4+

D0 D7 M1 M3 D0 D7 M1 M3

Group 1Group 2Group 3



SignificantincreasedinCD25highCD127lowFoxP3+ inCD4

• Treg lymphocytessubsets

• Safety of I.A. injection of ADSCs

• Global switch towards regulatory immune cells

- increased percentage of Treg cells similar results obtained in GVHD and fistulising Crohn disease

- increased percentage of Breg-containing subset

- decreased percentage of classical pro-inflammatory monocytes

• Longer term effect on the immune response?


ParacrinefactorsproducedbyMSCs

RegulationofimmunecellsfunctionsbyMSC-derivedmiRNAs

IdentificationofmiRNAsinvolvedintheregulationofMSCsuppression

• Firststep:TAC(Taqman ArraysCard)• HumanMSC± PBMC(MLRTranswell)• N=3donors• CellsactivatedbybeadsCD3-CD28• CollectRNAatDay3• 384miRNA ineachcard• 8sampleloadingport(48well)• RT-PCR• SoftwareSDSforanalysis• 2pools

• A=miRNA broadlyexpressed,optimization• B=newmiRNA,lessstudied


• TACPoolAanalysisØ Volcano-plotwithupregulatedmiRNAingreen

(n=13)anddownregulatedmiRNAinred(n=3)Ø Belowthebluelinep=NSØ Threshold:FoldChange>2


• Second step: Real Time qPCR validation of the three upregulated miRNAs• Human MSC ± PBMC• N ≥ 4 different donors• Cells activated by BEADS CD3-CD28/PHA• Immunosuppression assay CFSE/ CTV analysis

BM-MSCs

Activated lymphocytes Fresh Blood

Healthy donor

RatiosMSC:PBMC

1:5

Co-culture Day 4Beads

BM-MSC 119Ratio 1:5

BM-MSC 122Ratio 1:5


TAC

up-

regu

late

d

*p<0,05**p<0,01


• Second step: Real Time qPCR validation of the three upregulated miRNAs• Human MSC ± PBMC• N ≥ 4 different donors• Cells activated by BEADS CD3-CD28/PHA• Immunosuppression assay CFSE/ CTV analysis

• TransfectionMSCwithpremiR• Ourhypothesis:

EnhancingMSCimmunosuppression

miRNAupregulated

DecreasePBMCproliferation

ValidationofthefunctionalroleofmiRNAsinMSCimmunomodulation

PHAStimulatedPeripheral blood

mononuclear cell (PBMC)

• TransfectionMSCwithpremiR

DAY0CultureofhBM-MSC

DAY1Transfectionwith premiR

orantagomiR

Oligofectamine andpre/antago-miR mix

DAY2Coculture ofhBM-MSCandPBMC

DAY61)Verification oftransfectionbyRT-qPCR2)Determination ofPBMCproliferation

" CellTrace™Violetlabeling&flowcytometryanalysis

Transfected hBM-MSCover/down-expressing a

candidatemiRNA


• Transfection MSCwithpremiR (gainoffunction)• Focusonup-regulatedmiRNAs• Oligofectamine• TaqMan premiR-Ctrl,premiR-29a,premiR-146a,premiR-155(50nM)

è Efficacy of MSC Transfection with oligofectamine (500 to 10 000 fold)


• TransfectionMSCwithpremiR• CONTACTPHAPBMC/MSC,96-wellplate(Ratio1:10)• ComparisonPBMCproliferationw/womiRNAupregulation

è InhibitionofTcellproliferationissignificantlyhigherafter premiR MSCtransfectionofmiR-29aandmiR-155

*p<0,05


• ParacrinefactorsproducedbyMSCafterpremiR-29atransfection• MSCSupernatant(ELISA):Day4co-culture• RT-PCRwith MSC(ARNm):Day4co-culture

èTGBbetapathwayregulation?


• ParacrinefactorsproducedbyMSCafterpremiR-155transfection• MSCSupernatant(ELISA):Day4co-culture• RT-PCRwith MSC(ARNm):Day4co-culture

èIL-6andPGE2inhibition


• Affymetrix®microarray• TransfectionMSCwithpremiR andantagomiR formicroRNAsupregulated(29a,146aand155)

• MSC=1donor• Aim:identifymRNAsthatarebothupregulatedwithantagomir anddownregulatedwithpremir usingaFCthreshold:

• mRNAdownregulatedwithpremir /pCT (FC<-1.5)• mRNAupregulatedwithantagomir /aCT (FC>1.5) Mergeforanalysis

IdentificationofthemRNAtargetsoftheselectedmiRNAs


• miR-29aandmiR-155targetsPSAT1?

• PSAT1:targetofmiR-29a?– RT-qPCRwithMSC(ARNm)Day4coculture(n=3MSC)

PSAT1maybeatargetofmiR-29aNosignificantdownexpressionaftertransfectionwithpremiR-146aand-155


• PSAT1:targetofmiR-29a?– TransfectionsiRNAPSAT1– ReducePBMCproliferation


PSAT:cellproliferationforSerine/Glycin metabolism

Synthesis

miR-29aandPSAT1arenewfactorsmodulatingMSCimmunosuppressivefunctions

Extracellularvesicles(EVs)derivedfromMSC:afutureoption?




BM-MSC-derived MPs andExosprotected mice from osteoarthritic damagesinthecollagenase-induced OAmodel.

Cosenza S et al. Sci Report 2017

SenescenceandOA

Childs BG et al. Nat Review 2017

SenescenceandOA

McCulloch et al. Aging Cells 2017

ChondrocytesenescenceinOA

Loeser R. et al. OAC 2009

SenescenceandOA

McCulloch et al. Aging Cells 2017

• Chondrocyteskeyplayer• BalanceECMsynthesisanddegradation• Mechanicalstressinducesenescence• Expressionsenescentmarkers• SASP(VEGF,IL1,MMP3,MMP13,IL6)

• Protectiveeffect• Cancer• Skintissuerepair/woundhealing

Senolytics andOA

Hee Jeon O et al. Nat Med 2017

Ganciclovir(selectivekillingofp16-INK4A)reducesOApost-traumaticdevelopment

Senolytics andOA

UBX0101(selectiveclearanceofSNC)attenuatesOApost-traumaticdevelopment


Senolytics andOA

Pellets3DhumanchondrocytesUBX0101reduceslevelsofSNCspromotingachondrocytephenotype


Senolytics andOA

Childs BG et al. Nat Review 2017

• PreclinicaldatasupportefficiencyofMSCinOA• FirststepforASCbasedtherapyinosteoarthritis• LogisticsvalidatedatEUlevel• Proceduresafe• Optimaldosecloseto2.106 ASC• SystemictoleranceinducedafterlocalIAinjection• Next:ADIPOA2,randomizedcontrolledtrialendin2019

Conclusions

10 CLINICAL CENTRES with 15 patients each / 3 PRODUCTION PLATFORM•1. IRELAND, NUIG (F. Shannon) •2. IRELAND SSC (R. Moran),•3. UK CAM (A. McCaskie)•4. NETHERLANDS RUMC (F. van den Hoogen)

•5. ITALY, IOR (R. Meliconi), •6. ITALY, UHP (R. Ramonda) •7. EWK (U. Nöth) GERMANY

•8. MONTPELLIER, CHUM (C. Jorgensen)•9. TOULOUSE, CHUT (A. Cantagrel),•10. PARIS, APHP (F. Berenbaum) FRANCE

NUIG 40 batches

EFS 30 batches

UULM 30 batches

• AphaseIIb,multi-centre,prospective,randomized,double-blindstudy,comparingculture-expandedautologousASCwithplacebo

• 3armstoatotalof153 patientsandfollowedupfor25months(1monthbeforeand24monthsafterkneeinjection)

• Durationofrecruitmentforeachcentre:12monthsTreatment group Dose Frequency Number of patients

Group 1 2.106 ADSC Single injection 51

Group 2 10.106 ADSC Single injection 51

Group 3 Vehicle Single injection 51

!

• PreclinicaldatasupportefficiencyofMSCinOA• FirststepforASCbasedtherapyinosteoarthritis• LogisticsvalidatedatEUlevel• Proceduresafe• Optimaldosecloseto2.106 ASC• SystemictoleranceinducedafterlocalIAinjection• Next:ADIPOA2,randomizedcontrolledtrialendin2019• Next:Allogeneic,EVs,senolytics ?

Conclusions

ADIPOA

MontpellierRosannaFerreira PascalePlenceMarieMaumus FaridaDjouadKarineToupet ClaireBonyDanièleNoël PLuz-CrawfordChristianJorgensen

GalwayFrankBarry

Würzburg– BerlinUlrichNoeth

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