GRANDS PRINCIPES THERAPEUTIQUES DE LA

MEDECINE REGENERATRICE
= THERAPIE CELLULAIRE
Dr Yves-Marie
Marie PERS
MCU-PH,
PH, MD, PhD
Immunologie clinique et Thérapeutique ostéo-articulaire
ostéo
Département de Rhumatologie - CHU Montpellier
IRMB-INSERM
INSERM U1183
ympers2000@yahoo.fr
 Regenerative medicine

Pancreas
Autoimmune
disease
Hematologia
Liver
Cardiovascular
diseases Retina - Cornea
Stem cells
Regenerative
Medicine Cancers
Neurodegenerative
diseases Repair
Renew Muscular
Skin Restore diseases
Regenerate

Osteo-articular
articular diseases

This medicine is based on cell therapy

→ stem cell interest
 Regenerative medicine
• La thérapie cellulaire consiste en l'injection de cellules humaines dont
le but est de prévenir, traiter ou atténuer une maladie.
• Le terme thérapie cellulaire autologue est employé lorsque les
cellules proviennent du patient lui--même.
• Allogénique: autre individu
 Regenerative medicine
• Impasses de la médecine actuelle :
• Lésions médullaires
• Accident vasculaire cérébral constitué
• Insuffisance cardiaque et ischémie myocardique
• Diabète insulino-dépendant
• Démence : Alzheimer, Parkinson
• Arthrose,
• Maladies auto-immunes rares
• Insuffisance hépatique
• Brulures étendues
• …
 Regenerative medicine

• Remplacer des cellules âgées, non

fonctionnelles ou détruites par des
cellules en bonne santé pour restaurer
la fonction de l’organe

Fontaine de jouvence
Myth and dreams
Myth and dreams
 Main principles
• Collecte simple
• Disponible en grande quantité
• amplification in vitro
• Intégration au sein du tissu cible et y être fonctionnel

Cellules souches
What is a stem cell ?
 What is a stem cell ?
• Cellules souches multipotentes
• Cellules souches tissu-spécifique
• Hématopoïétiques
• Peau
• Neurales
• Hépatocytaires Cellules souches Hématopoïétiques
CD34 + (cytométrie de flux)
• Pancréatiques, etc.
• Cellules souches mésenchymateuses : MSC

• Cellules souches pluripotentes

• Embryonnaires : cellules « ES »
• Adultes : « iPSc » reprogrammation
 Advantages
• Mieux comprendre le développement normal et pathologique
humain (=étude des étapes de développement)
• Modèles cellulaires pour les maladies génétiques rares + test
médicament
• Étude différenciation et fonctions des cellules
• Thérapie cellulaire
 Limits
• Tératogène
• Intérêt des modèles animaux : le long terme
• Source de cellules embryonnaires
• Allogénique (banque de lignées)
• Problème: incompatibilité HLA: immunosuppression
• Autologue
• Clonage thérapeutique
• Equivalent chez l’adulte (?) : iPS
• Ethique
• Manipulation d’embryon (ES)
• Les CS du fœtus : prélevés sur des fœtus avortés
• Les CS « bébés » : prélevées dans le cordon ombilical
What is a stem cell ?

MSC
Characteristics of Mesenchymal Stem Cells (MSC
- Adherent to plastic

- Immunophenotype → No specific marker

CD31 CD34 CD106

Counts

Counts

Counts
CD11b CD14 CD45 HLA-DR
(High expansion in vitro)

Counts

Counts
Counts
Counts

CD13 CD73 CD90 CD105

Counts

Counts
Counts
Counts

- CD73+, CD90+, CD105+, (CD13+)

b-, CD14-, CD19-, CD34-, CD45-, HLA-DR-, (CD31-, CD106-)
- CD11b
Characteristics of Mesenchymal Stem Cells (MSC
- Multipotency: ability to differentiate into adipocytes (adipose tissue), osteoblasts (bone)
and chondrocytes (cartilage)

Adipocyte Ostéoblaste Chondrocyte

 Functions of MSC

Self renewal Migration

Release of growth
factors and anti-
Differentiation
inflammatory molecules

Cartilage Muscle Bone Adipose

Tissue regeneration
 Functions of MSC
• Fonctions des CSM

Support progéniteur

Anti-fibrotique

Pro-chimiotactisme

Anti-apoptotique

Pro-angiogénique

Immunomodulation

Courtesy Barry F et al. The Biology and Therapeutic Applications of Mesenchymal cells. Volume 2. Chapter 29.
Where we can find MSC – “stromal cells” ?
Bone marrow sampling Adipose tissue sampling
(liposuccion)

Human
Adipose tissue

Collagenase digestion

Stroma Vascular Fraction Mature adipocytes

SVF

Filtration, centrifugation, counting and seeding

De nombreuses approches d’enrichissement existent
(Sto1, CD73, CD271…) mais pas d’utilisation clinique

Adhesion and proliferation of MSC/ASC

Fœtal calf serum selected +/- bFGF
Platelet lysat
 ASC isolation/preparation (example)

eh N. Stem Cell Investigation 2019

 ASC isolation/preparation (example)

eh N. Stem Cell Investigation 2019

 Where we can find MSC – “stromal cells” ?

mo L. Knee Surg Sports Traumatol Arthrosc 2016

 Therapeutic applications of MSC
• Registered Clinical trials of MSC based therapy on ClinicalTrials.gov / PubMed

SLE Others Myocardial infraction

 Major stem cell used for cell therapy RA 23%
MS
 Easily isolated and successfully CD
expanded OA GVHD
 large therapeutical applications 8% 16%
 Development of large scale GMP
Spinal Cord injury
production
9%
Diabetes
Liver cirrhosis 10%
10%

Phase III Phase IV 4%

15% Phase I
31%

Phase II
Wei et al. 2013 50%
 Therapeutic applications of MSC

T et al. Cell Transplantation 2016

 Therapeutic applications of MSC

Trophic effect Angiogenesis

- Skin healing - Ischemic cardiopathy
- Scleroderma - Limb ischemia
- Stroke

Immune disorders
Tissue Repair GVHD - Lupus
- Bone - Arthritis
- Cartilage - Sjögren syndrome
- Osteoarthritis - Systemic Sclerosis
- Multiple sclerosis
- IBD
- Diabetes
 Osteoarthritis = ARTHROSE
• 17% whole population
• Incidence

Knee OA: 240/100.000 PA

Hand OA: 100/100.000 PA

Hip OA: 88/100.000 PA
• Overweight +++

RR 1.9 (hand OA + weight-bearing
joints)

High risk joint replacement (X 5)

t al. Lancet 2011

5 collaboration. N Engl J Med 2017;377:13-27
 OA epidemiology
• High burden cost +++

13 millions physician visit/year

14 millions drug prescribed

1.7% expenses French health system

High cost for joint replacement (22.6
billions in USA)

Knee OA: 83% of total OA burden
• Disability 11th cause over the world
• 14.2 total Years Lived with Disability
• 64.8% increase / 1990
• Underestimates
• Lack of data…

2012
n Phys Rehabil Med 2016
Rrheum Dis 2014
 Joint PHYSIOLOGY

AC 1999
tier J. Nat Rev Disease Primers 2016
 OA Pathophysiology
All the components of the joint are
involved in the process:

Cartilage ≈ chondrocytes + ECM

Subchondral bone ≈ OC/OB

Synovial ≈ inflammation

Muscles, ligaments
 Unmet need in OA
• 3 unmet medical needs
• Efficient disease modifying treatment
• More effective symptomatic treatment:
NSAIDs improve less than 50% WOMAC
scores
• Safer treatment: NSAIDs carry significant GI
and CV risk

FIND NEW THERAPY

WITH VARIOUS TARGETS
Clinical applications of MSC in rheumatology

MSC Immunoregulatory
Differentiation functions
capacities Trophic functions

Synovial
Formation TGFβ Synoviocytes
d’osteophytes BMP Fibroblaste

Dégradation IL1β, IL8,

du cartilage TNF PGE2, NO
TNFα,
MSC Chondrocyte

MMP
ADAMTS
Chondrocyte

Differentiation in To block inflammation

chondrocytes using Chondroprotection and Anti-inflammatory cell
biomaterials stimulation of endogenous therapies
repair
Tissue engineering:
Repair of focal Application to Application to
defects osteoarthritis osteoarthritis
 Different types of cartilage lesions
Focal defect Wide zones

Mainly post-traumatic Associated with rheumatic diseases

- 20% of arthroscopic procedure - Rheumatoid arthitis:
(knee: 5-10% young patients and >60% 0,3% of total population
old patients) - Osteoarthritis: 35% of women and
20% of men > 65 years

Few or no tissue repair by chondrocytes and / or endogenous stem cells →

interest in using mesenchymal stem cells in regenerative medicine for cartilage !
Treatments for focal defects
Microfracture
Mosaicoplasty ou
osteo-chondral graft

ACI: autologous
chondrocyte
implantation

MACI: matrix-induced
chondrocyte
implantation
Treatments for focal defects
Results: ACI = microfracture > mosaicoplasty
But some limitations

Limitated to small
defects

Osteophyte Low integration

formation and
fibrocartilage Mortality at the
formation in lesion donnor site

Mortality at the
donnor site - New approaches
for tissue
Perioste engineering
hypertrophy
- New cell sources
 Treatments for focal defects
BM-MSCs efficacy compared to autologous
chondrocyte implantation ?
Col I Col II
MSCs are as efficient as chondrocytes for
cartilage repair (n=36)

- improvement of patient quality of life

and activities in everyday life and sports Col X PG

- hyalin cartilage formation (1 year)

Nejadnik et al, Am J. Sports Med, 2010

MSCs can be used as an alternative to chondrocytes for cartilage repair

- reduced costs,
- only one surgery,
- minimize mobidity at the donor site
Cell type
MSC chondrogenesis

MSC Chondroprogenitors Chondroblasts Chondrocytes Hypertrophic

chondrocytes

Condensation Proliferation Differentiation Terminal

& differentiation & maturation differentiation
Factors

TGFβ IGF-1 BMPs VEGF

BMPs BMPs FGFs BMP-2
FGF-2 FGF-2 etc FGF-2
ECM proteins

Col 1, Col 2a Col 2b, Col 9, Agg Col 10, MMP13

 MSC for cartilage engineering
Optimal combination of 3 essential components :

Mesenchymal
Stem Cell

Biomaterial Inductive
(synthetic or signals
natural) (growth
H2 O factors)

H2 O

 Biocompatibility & degradability

 Permeability
 Cell adhesion and differentiation
 Porosity for cell migration and mechanical properties
erspectives: bio-printing
printing for cartilage engineerin

Costantini, 2016, Biofabrication

Multi head tissue/organ building

system for regeneration of
osteochondral defects

16, Biofabrication
erspectives: bio-printing
printing for cartilage engineerin
erspectives: bio-printing
printing for cartilage engineerin
“Biopen”
 Why MSC make senses in OA ?

M, Biochimie. 2013
 ASC are effective in OA

CTR ASC

In vivo mechanisms In vitro mechanisms

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

Osteophyte formation Apoptosis

Inflammation (TNF-α, MMP-1) Fibrosis

Synovitis

et al. HMBCI 2016

 ADIPOA clinical trial

lipoaspiration ADI

ADSC
isolation/expansion
GMP conditions
Cell viability
Release
criteria/toxicology

Intra-articular injections
Syringe, different doses

Evaluation: phase 1 trial

safety, dose response
clinical evaluation (WOMAC…)
MRI Gemric,
ADIPOA clinical trial: cell production

Quality controls:
Sterility test day 8 ADI
Endotoxin test day 11
Mycoplasma test day 11
Karyotype performed on 20
cultures. All are normal.

Release criteria:
Cell viability >80%,
CD45+ / CD14+ cells < 5%,
CD90+ or CD73+cells > 80%
 Absence of expression of hTERT
and Oct-4 at the end of the
primary culture to assess
genotype stability.
ADIPOA clinical trial: fat harvesting

ADI
 ADIPOA clinical trial: design
Adipose derived Stromal Cells for OsteoArthritis treatment.
A phase 1 study, bi-centric (Mtp,, Wurzburg), dose escalating study ADI
with autologous ASC in severe knee OA (>3 K/L)
ASC injection

Adipose
tissue
MRI collection Arthroscopy + MRI

– 35d - 14d Baseline W1 M1 M3 M6

Clinical assessment
et al. SCTM 2016
 ADIPOA clinical trial: design
48 patients assessed for eligibility

ADI

18 consecutively enrolled to receive autologous ASCs

6 received 2 x 106 cells 6 received 10 x 106 cells 6 received 50 x 106 cells

(Low-Dose) (Mild-Dose)
Dose) (High-Dose)

No patients lost to follow-up No patients lost to follow-up

follow No patients lost to follow-up

6 patients analysed 6 patients analysed 6 patients analysed

et al. SCTM 2016

 ADIPOA clinical trial
90
80 VAS pain score Low Dose

Safe procedure: 4 local skin 70 Mild Dose
reaction in the first month 60
High Dose ADI
50

Only 2 patients underwent 40
surgery TKA after one year 30
20
follow-up and 55% after 4 years 10
0
Baseline 1 Week 3 Months 6 Months

80 KOOS Questionnaire
35 SAS score Low Dose
70
60 30 Mild Dose
50 25 High Dose
40 20
Low Dose
30 15
Mild Dose
20
10
10 High Dose
5
0
Baseline 1 Week 3 Months 6 Months 0
Baseline 1 Week 3 Months 6 Months
47
et al. SCTM 2016
 ADIPOA clinical trial: structural assessment

ADI

dGEMRIC index
increase in 3 out of 6
selected patients

Suggest a possible
structural effect

et al. SCTM 2016

 ADIPOA clinical trial: long-term
long success

• Long-term success?
ADI
• Follow-up
up of the 11 patients in Montpellier
• Depends on the initial stage: TKR incidence
increases with the importance of joint
space narrowing (=x-ray score)

Total knee replacement (TKR) 1 YEAR 2 YEARS 4 YEARS

ADIPOA 1 18% (n=2) 36% (n=4) 55% (n=6)

Nielsen FK et al. 2017 25% 45% 68%

K et al. BMC Musculoskeletal Disorders. 2017

 ADIPOA 2 clinical study

CT Management group Overall management of

the study (study doc, VHP
CHUM : Sponsor initiation, monitoring
activities, …)
Pharmacovigilance ,

ECRIN coordination Management /

Regulatory Support,
SAE Reporting

Coordination of ECRIN
partners

ECRIN national partners

Italy : National reg. and
Germany : Ireland : Netherland :
UK : Fondazione ethical submissions,
KKS
HRB Clinical The Clinical Trial Local monitoring and
CTCM Dusseldorf GIMEMA
Research Facility Unit of Nijmegen, vigilance
ONLUS,
CRCN Data management
Rome

CHUM CHUT APHP CAM EWK NUIG SSC UNIPD IOR RUMC Design, Execution of CT

Clinical Centres
 ADIPOA 2 clinical study
centre, prospective, randomized, double-blind
• A phase IIb, multi-centre,
study, comparing culture-expanded
expanded autologous ASC with placebo
• 3 arms to a total of 153 patients and followed up for 25 months (1
month before and 24 months after knee injection)

Treatment group Dose Frequency Number of patients

Group 1 2.106 ADSC Si
Single injection 51

Group 2 10.106 ADSC Si

Single injection 51
Group 3 Vehicle Si
Single injection 51

 Perspectives for MSC therapy

M et al. Medecine Sciences, 2018

 Perspectives for MSC therapy

M et al. Medecine Sciences, 2018

tracellular vesicles (EVs) derived from MSC: a future option

BM-MSC-derived MPs and Exos protected mice from osteoarthritic damages in t

collagenase-induced OA model.
S et al. Sci Report 2017
tracellular vesicles (EVs) derived from MSC: a future option
 Perspectives for MSC therapy

M et al. Medecine Sciences, 2018

Perspectives for MSC therapy
 Perspectives for MSC therapy

M et al. Medecine Sciences, 2018

Perspectives for cell therapy
Darvadstrocel (Alofisel®)
Perspectives for cell therapy
 Conclusions
• Preclinical data support efficiency of MSC in OA
• First step for ASC based therapy in osteoarthritis
• Logistics validated at EU level
• Procedure safe
• Optimal dose close to 2.106 ASC
• Systemic tolerance induced after local IA injection
• Next: ADIPOA2, randomized controlled trial end in 2022
• Next: ADIPOA3…
• Next: Allogeneic, EVs, senolytics ?
 IRMB - Inserm U1183 – Equipe 1
Biologie de la cellule souche mésenchymateuse et
thérapies du cartilage

Danièle Noël
Christian Jorgensen
Claire Bony
Karine Toupet
Marie Maumus
Claire Lousouarn
Jérémy Boulestreau
Pauline Rozier
Jérémy Salvador
Yves-Marie Pers
Noémie Petitjean
Alexandre Théron
Alexandre Maria
Philippe Guilpain ADIPOA