Cellules stromales mésenchymateuses

Intérêt thérapeutique Production et contrôles

DU Médecine Régénérative 19 novembre 2015

Unité U917 MIcroenvironnement et CAncer

MICA

Karin Tarte karin.tarte@univ-rennes1.fr

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?