Mesenchymal Stem Cells from lab to
bench side:
the example of Multiple Sclerosis
Luc Sensebé
UMR5273 ² U1031 STROMALab
[email protected]
EBMT Autoimmune Diseases & Immunobiology Working Parties
November 16, 2012 Paris
Numerous clinical applications
‡ Immune-mediated diseases
‡ Autoimmune diseases
‡ aGVHD, organ transplant
‡ Sepsis (mouse models)
‡ Regenerative medicine
‡ Cardiovascular disease (cardiac muscle, vessels)
‡ Epithelium (skin, cornea)
‡ Skeletal tissue (long bone, cartilage, mandibula)
Common mechanisms of action
‡ Few evidence for cell replacement
‡ Poor long-term persistance
‡ Poor transdifferentiation capacities
‡ Touch-and-go paracrine effects
‡
‡
‡
‡
Trophic factors
Cell recruitment
Immunomodulatory & antiinflammatory
Vasculature improvment
In-vitro expanded MSC and immunology
Macrophage
Proliferation
Survival
Differentiation
Neutrophil
B
B
B
Survival
IL-12
TNFIL-10
Migration
Oxydative burst
Monocyte
Differenciation
Treg
Treg
Treg
iDC
IFNTNF-
Maturation
NK
Survival
Proliferation
IFNCytotoxicity
T
T
T
T
T
T
IDO (iNOS, HO-1)
sHLA-G,
PGE2,
IL-10, IL-6, TGF
Galectins,
Jagged-1,
TSG-6
mDC
NK
NK
IL-12
T-cell activation
MSC for immune disorders
Allogeneic BM-MSC FCS
0,4-9 x 106/kg IV
55 patients
Le Blanc et al. Lancet 2004;363:1439
Le Blanc et al. Lancet 2008;371:1579
Efficacy
in vitro
MSC plasticity: Licensing by
inflammatory context
Prevention of aGVHD in mice
Untreated MSC
MSC treated with 5U IFNMSC treated with 50U IFNMSC treated with 500U IFNPolchert et al. Eur J Immunol 2008; 38:1745
Inhibition of B-cell
proliferation (%)
***
100
80
60
40
20
0
-20
C
S
L
-F
-P
SC
SC
M
M
pB
pB
M
M
D
SC
-P
L
S
C
pA
-P
L
-F
SC
B
M
M
M
M
B
A
D
El Hajjami et al. Cancer Res 2009; 69: 3228
SC
SC
-P
L
-40
MSC heterogeneity: Production process
100
Inhibition of T-cell
proliferation (%)
80
60
40
20
-F
SC
M
M
20
0
**
M
pB
M
40
0
**
*
**
5
10
15
**
**
1000
K Tarte & M Krampera
C
-F
-F
-P
SC
pB
M
M
M
M
B
S
C
S
L
L
SC
M
M
pB
B
M
M
D
SC
SC
-P
-P
L
-P
L
100
pA
20
Kynurenine/Tryptophan ratio
10000
A
D
SC
TNFAIP6
100000
r = 0.729
60
C
S
L
L
-P
-P
SC
SC
D
pA
pB
L
C
S
-F
-P
M
M
SC
SC
M
B
M
B
A
D
SC
-P
L
0
80
SC
Inhibition of T-cell
proliferation (%)
100
MSC heterogeneity: tissue origin
VCAM-1
VCAM-1
MHC
MHC class
classII
II
ICAM-1
ICAM-1
15
6
10
5
rMFI
rMFI
10
rMFI
rMFI
rMFI
rMFI
8
6
4
4
2
2
A
M
A
2.91
BM-MSC
ADSC
HLA-DR
kynurenin (µM)
80
60
40
20
0
ns
ADSC
T
ns
BM-MSC
A
T
ADSC
O
T
BM-MSC
O
ns
ns
ns
ns
M
O
Fold change BM-MSC ADSC
ADSC/
0.076
BM-MSC
rMFI
0
0
M
0
0.303
IDO
25
20
15
10
5
0
IFN
Efficacy in MS
in vivo models
IFN
« Mesenchymal stem cells ameliorate experimental auto-immune encephalomyelitis
inducing T-cell anergy » E Zappia et al. Blood 2005, 106:1755-1761
Antonio Uccelli
A. Uccelli & M. Introna
MSCs for multiple sclerosis
BM-MSC FCS
1-3 x 106/kg IV
10 patients
GMP - Clinical scale
‡ Producing cells in conditions approaching
pharmaceutical standards to obtain
efficient Advanced Therapy Medicinal
Products (European Commission [EC] No. 1394/2007)
‡ Large scale for clinical use
Safety
‡ quality assurance (QA) and quality control (QC)
Æ reproducibility and traceability of processes
Æ appropriate standards for QC
‡ long term follow up of patients
Safety of MSC uses
‡ Short Term Safety
² donor / starting
material
² Processes
² controls for release
‡ Long term Safety
² transformation ?
² Senescence ?
² Favoring tumor growth
?
² Unwanted homing &
differentiation ?
Defining the risks
Genetic instability is linked with two main processes
² Transformation
‡ p53 is the main player
‡ Sarcomas (Ewing, osteosarcomas) originate from MSC
² Senescence
‡ Senescent cells could not perform ´the jobµ
‡ Senescent cells can transform
‡ Risk linked to senescence-associated secretory phenotype
(SASP) Î sustaining in situ cancer cells and promoting tumor
progression
Transformation of ADSC ?
TMC
hTERT
Rubio et al, Cancer Res 2005, 65:3035-9
Transformation of BM MSC ?
Rosland GV et al Cancer Res 2009
MSC production & control:
French experience
Culture
protocol
FCS/FGF-2
Platelet
Lysate
Donor
number
(Age)
PD
(Proliferation
rate)
Karyotype
P1
Karyotype P2
hTERT P1
1A (55 y)
23 (29.5)
46, XX [15]
ND
Neg
2A (38 y)
23 (209.2)
46, XX [17]
46,XX [21]
Neg
3A (32 y)
22 (170.8)
46, XY [17]
46,XY [20]
Neg
4A (29 y)
20 (145.8)
46, XX [14]
ND
Neg
5A (41 y)
24 (20.9)
46, XY [16]
ND
Neg
6A (33 y)
21 (78.2)
49, XY, +5, +8, +20 [3]/
46, XY [19]
46,XY [31]
Neg
7A (50 y)
19 (4.6)
46, XY [21]
ND
Neg
8A (30 y)
21 (47.9)
46, XY [6]
46,XY [21]
Neg
9A (61 y)
20 (18.1)
49, XY, +5, +8, +20 [3]/
46, XY [18]
46,XY [29]
Neg
10A (28 y)
19 (39.8)
46, XY [27]
ND
Neg
11A (48 y)
24 (34)
46, XX [30]
46,XX [30]
Neg
12A (47 y)
22 (44.6)
47, XX, +5 [15]/
46, XX [5]
ND
Neg
13A (56 y)
20 (54.2)
46, XY [20]
ND
Neg
12A2 (47 y)
20 (57.5)
47, XX, +5 [3]/
46, XX [17]
47,XX,+5[2]/
ND
13A2 (56 y)
17 (9.44)
46, XY [30]
46,XX[28]/
ND
ND
12B (47 y)
18 (6.2)
46,XX [28]/
47,XX,+5 [2]
47,XX +5[2]/
Neg
46,XX [28]
46,XY [30]
46,XY [30]
46,XY [30]
ND
Neg
Neg
Neg
ND
13B (56 y)
14B* (27 y)
15B* (33 y)
16B* (23 y)
16 (1.5)
17 (16.2)
15 (1.3)
19 (16.5)
46, XY [30]
46,XY [30]
46,XY [30]
46,XY [30]
¾Recurrent aneuploidy, not dependent on the culture
process, donor effect?, growth disavantage
MSC production & control:
French experience
‡ In vitro senescence
‡ Growth arrest at PD 35-52
‡ No hTERT induction, No c-myc modulation
‡ Induction of p16 between P4 et P7
0%
12A2
฀ -Gal Staining
Late
Early
0%
94%
97%
11A
‡ No anchorage independent growth, no tumor in SCID
mice
Karyotype is not relevant
Further study on MSC stability
Definition of guidelines at French and European levels
Research team
biology and culture of MSC
(efficacy in vitro & in vivo)
defining controls
R & D
adapting culture processes
to clinical grade production and GMP
(safe & efficient processes)
learning from trials
Clinical trials
phase 1 & 2
Clinical trials
phase 3
« final GMP process »
closed systems / bioreactors
major needs
xenofree medium/ serum free medium
relevant controls
´the path to safety controlsµ
search for stability and senescence markers
nb of population doublings
‡ easy, more biologically
relevant than nb of passages
karyotype & CGH array ?
‡ low or very low sensitivity
‡ relevance ?
gene expression
SASP
‡ panel of genes (Q-PCR or
dedicated microarray):
SSS««VKRXOGEH
defined
‡ Proteomic studies
Î Controls should be reproducible and fast
EFS & UMR5273/INSERM U1031
(Toulouse)
‡ Frédéric Deschaseaux
‡ Audrey Varin
‡ Philippe Bourin
‡ Sandrine Fleury
‡ Julien Gaillard
‡ Alain Langonné
Genova (Italy)
‡ Antonio Uccelli (Coordinator)
Bergamo (Italy)
‡ Martino Introna
Department of Neurology
Toulouse University Hospital
‡ Michel Clanet
INSERM U1043 & CNRS U5282
‡ Roland Liblau
‡ Raphaelle Romieu-Mourez
INSERM U917 & EFS
(Rennes)
‡ Karin Tarte
‡ Joëlle Dulong
‡ Cédric Menard
DRK & University of Ulm (Germany)
‡ Hubert Schrezenmeier
‡ Markus Rojewski
‡ Anita Fekete
University of Verona (Italy)
‡ Mauro Krampera