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Cellules stromales mésenchymateuses
Intérêt thérapeutique Production et contrôles
DU Médecine Régénérative 19 novembre 2015
Unité U917 MIcroenvironnement et CAncer
MICA
Karin Tarte [email protected]
MSC Identified by Fridenstein in early 70’s
Adherent fibroblastic cells able to form colonies (CFU-F) and to differentiate into bone
MSC Rare cells expressing no specific marker Found in the bone marrow, adipose tissue, and
Wharton’s jelly (umbilical cord) + many tissues Strong in vitro expansion (self-renewal) Multipotency Very few data on native MSC
Mesenchymal stem cells Frenette Ann Rev Immunol 2013; 31:285
Ectopic organized
hematopoietic bone
Native mesenchymal stem cells CD146pos
Crisan Cell Stem Cell 2008, 3:301
Sacchetti Cell 2007, 131:324
Corselli Blood 2013, 121:2891
Native mesenchymal stem cells
CD271posCD44neg
Qian JBC 2012, 287:25795
Cultured MSC
Self-renewal?
Heterogeneity Mesenchymal stromal cells
Capacity of differentiation?
Paracrine activity, immunosuppression/anti-inflammatory effect
Ez dissociation
Centrifugation
Production of MSC
Adherence
- Selected FCS
+/- FGFb
- Platelet Lysate Adipocytes
Chondrocytes
Osteoblasts
Numerous enrichment approaches
(Stro-1, CD73, CD49a…) without
current clinical application Vascular cells
Neurons...
SVF
Qualification of initial product
CFU-F
Collagenase
DNAse
CD45/CD235a/CD14 C
D73
CD271
Nb of CFU/ml in the BM
% CD14negCD45negCD34negCD235anegCD73posCD271pos
within viable cells
Qualification of initial product
%MSC (FCM)
Correlation between CFU-F number and % of MSC
Nb o
f C
FU
-F/m
L
Correlation quantification par CMF ou par comptage des CFUF
0.001 0.01 0.1 1 1010
100
1000
10000
% CSM CMF
Nb
CF
UF
/mL
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)
MSC for autoimmune disorders
BM-MSC FCS 1-3 x 106/kg IV 10 patients
MSC for autoimmune disorders
BM-MSC PL 2-5 intrafistular injections 106 MSC/cm2
10 patients
MSC for alloimmune disorders
Tan et al. JAMA 2012;307:1169
BM-MSC FCS 1-2 x 106/kg IV D0 et W2
MSC for alloimmune disorders
Le Blanc et al. Lancet 2004;363:1439 Le Blanc et al. Lancet 2008;371:1579
Allogeneic BM-MSC FCS 0,4-9 x 106/kg IV 55 patients
MSC for critical limb ischemia
Powell et al. Mol Ther 2012;6:1280
Randomized clincial trial Ixmyelocel-T (BM-derived MSC + macro, 12-d culture) IM inj (20 points)
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
IDO (iNOS, HO-1)
sHLA-G,
PGE2, IL-10, TGFb
Galectins
Jagged-1
TGFb,
sHLA-G
Monocyte
iDC
mDC
PGE2
IL-6, IL-10
IL-6, IL-10
Treg
Treg
Treg
Tab Tab
Tab
Differenciation
Maturation
IL-12 T-cell activation
CCL2
B
B
B
Survival Proliferation
Differenciation
Survival
Proliferation
IFN-g
Adaptive
Immunity
IFN-g
TNF-a
General mechanisms of immunoregulation
Cytokines: IL-10, TGFb, HGF, VEGF...
Membrane molecules: PD1/PD-L1,PD-L2, CD200R/CD200...
Enzymes IDO-1, IDO-2 (Trp) iNOS & ARG-1 (Arg) HO (heme)
PGE2 HLA-G (soluble and membrane forms)
Activated T cells
Activated T cells + CSM
0
2
4
6
Ind
ex
de
pro
lif
T + antiCD3/28 CSM CSM + L-1MT CSM + D-1MT
+ - -
- - - -
- +
+
+
+ + + +
+
T-cell proliferation
Activated T cells + CSM
+ L-1MT
Activated T cells + CSM
+ D-1MT
MSC and B cells
Support resting and activated B-cell survival Maintain the proliferative potential of resting B cells Support activated B-cell survival & proliferation
B cells B + MSC B cells
28%
Active caspase-3
19%
B + MSC
CFSE
Abso
lute
num
ber
of
viable B
cells
(x10
5)
0
2
4
6
8
10 ***
B cells B + MSC
B
ACTIVATION
STEP
DIFFERENTIATION
STEP
MSC and B cells Alter normal B-cell differentiation
Naive B cells
ACTIVATION
STEP
DIFFERENTIATION
STEP
CD
38
0
2.5
5
7.5
10
MSC _
+
% o
f CD138+
cells
n.s.
50000
25000
0
100000
50000
0
5000
15000
0
10000
25000
20000 * * **
IgA
ASC p
er
10
6 B
cells
IgG IgM
BCL-6
PRDM1 IRF4 XBP1
B
CD20
MSC and B cells
B
TNFa
IFNg
94% 31% 51%
B cells B + MSC B +
TNF-primed MSC
Active caspase-3
B + MSC B +
IFN-primed MSC
CFSE
Functional heterogeneity of MSC depending on the cytokine context
IDO
ADSC-P
L
BM
MSC-P
L
BM
MSC-F
CS
pADSC-P
L
pBM
MSC-P
L
pBM
MSC-F
CS
-40
-20
0
20
40
60
80
100
***
Inh
ibit
ion
of
B-c
ell
pro
life
rati
on
(%
)
In-vitro expanded MSC and immunology
IL-6
Neutrophil
Survie Migration
Burst oxydatif
TSG-6 IDO
PGE2
sHLA-G
Tgd
Tgd
Tgd
IL-12
TNF-a
IL-10
Macrophage
IL1Ra PGE2
PGE2 Survie
Prolifération
Cytotoxicité
IFN-g
Prolifération
Cytotoxicité
IFN-g
IFN-g
TNF-a
NK
NK
NK
IFN-g
TNF-a
Immunité
Innée
NK activés
KILLING
NK NK
Activated T cells
Activated T cells + CSM
0
2
4
6
Ind
ex
de
pro
lif
T + antiCD3/28 CSM CSM + L-1MT CSM + D-1MT
+ - -
- - - -
- +
+
+
+ + + +
+
T-cell proliferation
Activated T cells + CSM
+ L-1MT
Activated T cells + CSM
+ D-1MT
NK + IL-2 CSM CSM + L-1MT CSM + D-1MT
+ - -
- - - -
- +
+
+
+ + + +
+ 0
2
4
6
8
Ind
ex
de
pro
lif
NK-cell proliferation
MSC and immunoregulation
Critical parameters Species (mouse versus human)
Tissue origin
Culture conditions (PL versus FCS, FGF-2, whole BM versus BMMC…)
Number of PD
Validation of immunological assays
STANDARDIZATION
MSC-related parameters
Immune cell-related parameters
NK
B Th CTL
DC
MO
PMN
Tissue origin
Species
Culture conditions
Culture expansion (PD, senescence)
Donor-related variability
MSC to immune cell ratios
Cell purification
Viability Proliferative potential
Read-out
Stimulation cocktail
Specific inhibitors
MSC
Priming
Mech
anisms?
Sensebé Hum Gen Ther 2011 Ménard Stem Cell Dev 2013 Ménard Stem Cell Res Ther 2013 Krampera Cytotherapy 2013
Tab Tab
Tab
B B
B
NK
NK
Mo
TNF/IFN-g
- IDO - NO - HO - PGE2 -TSG-6
PMN
Clinical-grade MSC & Immunoregulation
Various processes
Phenotype
Various
sources
What is the best clinical-grade MSC?
MSC heterogeneity: production process
T cells
90% 29%
T + MSC
ADSC-P
L
BM
MSC-P
L
BM
MSC-F
CS
pADSC-P
L
pBM
MSC-P
L
pBM
MSC-F
CS
0
20
40
60
80
100
% i
nh
ibit
ion
% i
nh
ibit
ion
ADSC-P
L
BM
MSC-P
L
BM
MSC-F
CS
pADSC-P
L
pBM
MSC-P
L
pBM
MSC-F
CS
0
20
40
60
80
100
% i
nh
ibit
ion
NK + MSC NK cells
92% 47%
T
NK
Clinical-grade MSC display various immune properties depending on the production process
ADSC-P
L
pADSC-P
L
BM
MSC-P
L
pBM
MSC-P
L
BM
MSC-F
CS
pBM
MSC-F
CS
100
1000
10000
100000
MSC heterogeneity: production process T
NFA
IP6
** ** **
** **
*
AD
SC
-PL
1 1.4 8.4 1.4
BM
MS
C-P
L
1 1 8.6 5.5
BM
MS
C-F
CS
4.3 10.6 2.7 5.3
HLA DR CD200 CD54 CD106
MSC heterogeneity: tissue of origin
Tab Tab
Tab
B B
B
NK
NK
Mo
TNF/IFN-g
- IDO - PGE2 - TSG-6 - HO - NO
PMN
Affymetrix microarrays
Role of tissue origin?
474 differentially expressed genes 276 upregulated in ADSC 198 upregulated in BM-MSC
MSC heterogeneity: tissue origin
0
5
10
15
20
25
kynu
renin
(µM
)
IFNg
0
20
40
60
80
BM-MSCADSC
rMFI
HLA-DR IDO
ICAM-1
MO n
s
AT n
s
0
2
4
6
8
10
rMFI2.91 0.076
VCAM-1
MO n
s
AT n
s
0
5
10
15
rMFI
BM-MSC ADSC
MHC class II
MO n
s
AT n
s
0
2
4
6
rMFI
0.303
BM-MSC ADSC BM-MSC ADSC
VCAM-1 ICAM-1 MHC class II
rMF
I
rMF
I
rMF
I
Fold change ADSC/
BM-MSC
IFNg
ADSC-P
L
pADSC-P
L
BM
MSC-P
L
pBM
MSC-P
L
BM
MSC-F
CS
pBM
MSC-F
CS
100
1000
10000
100000
MSC plasticity: Licensing by inflammatory context
TN
FA
IP6
ADSC-P
L
pADSC-P
L
BM
MSC-P
L
pBM
MSC-P
L
BM
MSC-F
CS
pBM
MSC-F
CS
0.1
1
10
100
PT
GS
2
Prevention of aGVHD in mice
Untreated MSC MSC treated with 5U IFN-g
MSC treated with 50U IFN-g
MSC treated with 500U IFN-g
Polchert et al. Eur J Immunol 2008; 38:1745
MSC plasticity: Licensing by inflammatory context
Influence of TLR ligands Effect on MSC-mediated reprogramming of
macrophages in sepsis
LPS TNF-a
PGE2
TNF-a
NO
IL-10
TNF-a
Németh et al. Nat Med 2009;15:42
Safety of MSC uses
Short Term Safety
donor / starting
material
Processes
controls for release
Long term Safety
transformation ?
Senescence ?
Favoring tumor growth
?
Unwanted homing &
differentiation ?
Rubio et al, Cancer Res 2005, 65:3035-9
hTERT
TMC
Transformation of ADSC ?
Rosland GV et al Cancer Res 2009
Transformation of BM MSC ?
MSC production & control: French experience
Prevention of aGVHD post allo-HSCT using MSC (double blinded versus placebo)
74 patients, 22 centers
Strong efforts in: Standardization of MSC production and QC
Centralization of specialized MSC qualification Immunogenicity, Immunosuppression
Centralization of monitoring MSC engraftment (CFU-F chimerism by Q-PCR)
Immunological properties of MSC (in vitro, in vivo)
Immune reconstitution (Phenotype, PBMC proliferation)
SFGM-TC
MSC p
roduc
tion
& c
ontr
ol:
Fre
nch e
xpe
rienc
e
Culture
protocol
Donor
number
(Age)
PD
(Proliferation
rate)
Karyotype
P1 Karyotype P2 hTERT P1
FCS/FGF-2 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]/
46,XX[28]/
ND
13A2 (56 y) 17 (9.44) 46, XY [30] ND ND
Platelet
Lysate
12B (47 y) 18 (6.2) 46,XX [28]/
47,XX,+5 [2] 47,XX +5[2]/
46,XX [28]
Neg
13B (56 y) 16 (1.5) 46, XY [30] 46,XY [30] Neg
14B* (27 y) 17 (16.2) 46,XY [30] 46,XY [30] Neg
15B* (33 y) 15 (1.3) 46,XY [30] 46,XY [30] Neg
16B* (23 y) 19 (16.5) 46,XY [30] ND ND
Recurrent 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
b-Gal Staining
No anchorage independent growth, no tumor in SCID mice
12A2
11A
Early Late
0%
0%
94%
97%
-
Karyotype is not relevant Further study on MSC stability Definition of guidelines at French and European levels
BM MSC = ADSC
Impact of senescence
- Senescence affects
immunoregulatory properties of MSC
NK cells
T cells
Impact of senescence
- Senescence affects
immunoregulatory properties of MSC
No stim IFN 20 IFN 100 IFN 100
+ TNF
IDO
Actine
Stromal cells and cancer
Direct & Indirect roles of CAF
- Cell growth - Metastasis - Angiogenesis - Drug resistance
- Immune escape
Joyce et al. Nat Rev Cancer 2009; 9:239
MSC and FL
1. Stromal cells recruit and support directly malignant B cells 2. Malignant B cells confer a supportive phenotype to stromal cells 3. Stromal cells organize malignant B-cell niche (interactions with TAM, T cells…)
FL B cell
CXCL12 BAFF Hh TNF/LT
IL-4
TFH
P- STAT6
PD-1
TNF, LT IFNg
TFH
CD40L
CD40
P- STAT5
IL-15/ IL-15Ra
TAM
IL-15Rb/ gc
TNF, IL4 IFNg
CCL2
Ame-Thomas Blood 2007; 109:693 Guilloton Blood 2012 ; 119:2556
MSC and multiple myeloma
Specific GEP (GDF-15, IL-6…)
Increased expression of XBP1s
Increased capacity to sustain MM cell growth
Increased capacity to induce osteoclast formation
MSC and AML transformation
“Niche-induced oncogenesis”
Origin Mesoderm vs neuroepithelium Stem Cells Self-renewal Plasticity/Heterogeneity
Immunosuppression and MSC Immunosuppression vs immunoprivilege Inflammatory context Specificity (target, MSC subset)
Native MSC Localization (between tissues, within tissues) Roles (immunoregulation?) Progeny
MSC and tumor stroma Homing to tumor site Direct supportive functions Angiogenesis Immunosuppression Malignant niche
MSC subsets with different functions?
MSC plasticity?
Clinical-grade MSC production: Processes? Controls?