Bone marrow stroma: biology and therapeutic exploitation

Bone marrow stroma: biology and
therapeutic exploitation
!
Francesco Dazzi
!
[email protected]
Outline
1. Bone marrow stroma and the physiology of the
haemopoietic niche
!
2. The malignant niche: potentials for therapeutic
intervention?
!
3. Mesenchymal stromal cells for HSCT complications
Bone marrow stroma
MSC induce the differentiation of GMP and CMP
into macrophages
103
100
104
2.69
100
104
41.3
101
102
103
17
3
10
3
10
2
10
2
100
4
Gr-1 high
29
103
Gr-1 int-low
30.6
102
101
100
101
102
103
57.2
101
102
103
104
100
104
4
14.3
10
102
102
100
25.3
101
102
103
104
101
102
103
0.5
0.0
104
39.3
32.9
100
6.86
100
20.9
101
102
103
0
Gr-1 HIGH
C&
Gr-1 INT-LOW
Gr-1 NEG
egr1
100
104
*
4
101
102
103
0
104
CD11b&
F4/80&
CD115&
Relative fold expression
100
20
0
F4/80
CD115
r
Gr-1 neg
3.84
100
20
te
ND#
BM+MSC
af
10
ND#
Gr-1 int-low
0.732
1
40
P
102
HSC+MSC&
103
40
M
104
CMP+MSC
G
103
Gr-1 high
4.7
re
102
CD11b
9.35
fo
101
4
be
100
10
60
P
Gr-1 neg
0.419
0
M
101
*
60
G
ND#
af
te
r
102
80
C
M
P
ND#
Gr-1 int-low
0.254
be
fo
re
Gr-1 high
0.524
MEP+MSC&
CD11b
1.2
103
10
20
1.0
54
101
3.17
40
* 1.5
4
103
10
28.9
11.8
100
103
0
5.26
104
C
M
P
10
52.1
101
Gr-1 neg
27.4
0
103
Relative fold expression
10
10
102
GMP+MSC&
CD11b
84.4
36.6
101
101
13
100
10
104
10
101
7.36
100
CMP+MSC&
18
100
104
Relative fold expression
102
r
101
te
100
60
af
33.9
100
101
P
101
*
2.0
M
2
G
10
re
2
2.5
fo
10
80
be
3
P
10
B&
M
3
20
G
101
10
36.1
af
te
r
11.7
104
C
M
P
2
7.51
be
fo
re
10
48.5
C
M
P
3
104
% in CD11b+
10
31.2
76.2
% in CD11b+
Gr,1&
104
BM+MSC&
A&
Relative fold expression
Sfp1 (PU.1)
6
4
2
MSC-induced macrophages exhibit a ‘tissue healing’ profile
M1#markers#
Ido
Cox2
Marco
Il12b
Nos2
0.0
****
1.0
0.5
0.0
****
1.0
0.5
0.0
1.5
****
****
1.0
0.5
0.0
Relative fold expression
0.5
1.5
***
1.5
Relative fold expression
***
1.0
2.0
Relative fold expression
Relative fold expression
Relative fold expression
*
1.5
50
**
40
30
20
10
0
M2#markers#
20
10
0
**
1.5
1.0
0.5
0.0
10
8
6
4
2
0
*
3
2
*
1
0
Relative fold expression
30
2.0
Fizz1
Irf4
Relative fold expression
40
Relative fold expression
****
50
Ccl12
Chia
Relative fold expression
Relative fold expression
Arg-1
1.5
1.0
0.5
0.0
CD11b+ - BM
CD11b+ - BM+MSC
CD11b+ - BM+GM-CSF+IL-6
Imatinib produces complete remissions in most CML patients
MSC protect CML cells from imatinib via CXCL-12/CXCR4
Vianello et al. Haematologica 2010
AMD3100 sensitises APL to Ara-C.
Leukaemia boosts MSC induced myeloid generation
Macrophages (CD68+ CD14+ CD206+ CD163+ HLA-DR-)
Frequency of live cells
8
6
4
2
0
-
+
-
+
-
+
+
-
+
+
-
+
+
-
+
+
hMSC
CML sample
K562
MDSC (Lin- CD33+ CD11b+ HLA-DR-)
Frequency of live cells
2.0
1.5
1.0
0.5
0.0
-
+
-
+
-
+
+
-
+
+
-
+
+
-
+
+
hMSC
CML sample
K562
Conclusions
• Bone marrow stroma regulates self-renewal
and differentiation of HSC
• Niches are different and consist of various
components
• The bone marrow niches are hijacked by the
leukaemia process
Mesenchymal stromal cells
Bone
Cartilage
Fat tissue
MSC
Therapeutic applications
Functions
Tissue support
Tissue homeostasis
3D structure and
mechano-transduction
Modulation of
inflammation
Regulation of
stem cell renewal
Ex-vivo expansion
of HSC
Treatment of
inflammatory conditions
Improve tissue
repair
Manipulate the malignant niche
MSC can promote haemopoietic reconstitution
Jaganathan, Dazzi, Bonnet Leukemia 2010
The therapeutic effect is not associated with donor cell
engraftment
The MSC ‘facilitating effect’ does not seem to be
related to haemopoietic engraftment
‘MSC’
Stemness
Niche
activity
Immunomodulation
Mesenchymal stromal cells exhibit
immunosuppressive activities
T
Cognate antigen
Mitogen
CD3/CD28
Allogeneic MHC
Krampera et al, Blood 2003
MSC have ‘tissue
‘anti-inflammatory’
repair’ activity
activity
• Renal ischaemia
!
• Lung fibrosis
!
• Toxic liver injury
!
• Stroke
!
• Myocardial infarction
• Arthritis
!
• Multiple sclerosis
!
• IBD
!
• Sepsis
!
• Asthma
Aminoacid starvation is the main
effector mechanism
MSC
ARG NOS
IDO HDC
T cells (HSC?)
mTOR?
GCN2?
Arginine
Tryptophan
His3dine
Cell cycle
arrest
?
Proliferative response to HY (cpm)
The inhibitory effect is not dependent
on MHC class I expression
15,000
11,250
7,500
3,750
0
Control
MSC
3T3-F442A
Krampera et al, Blood 2003
MSC increase survival of aGVHD patients
Le Blanc et al, Lancet 2008
Incidence and severity of GVHD after prophylactic
administration of MSC
BM
PBSC
MSC + BM/
PBSC
aGVHD > II
25%
29%
28%
cGVHD lim
42%
22%
40%
cGVHD ext
21%
21%
19%
Lazarus et al, BBMT 2005
MSC for acute GvHD: preliminary data
Adults''
Data'
'
!!
Total'(n)'
Age'(y)'
Median'(Range)'
Sex'
21'
'
49,2'(25;70)'
'
Children'
Data'
!!
Total'(n)'
Age'(y)'
16'
'
Median'(Range)'
7,3'(1;15)'
Sex'
'
Male'
Female'
17'
4'
Male'
Female'
7'
9'
Disease'
'
Disease'
'
AML'
ALL'
MDS'
NHL/HL/MM'
6'
3'
2'
7'
AML'
ALL'
MDS'
AplasRc'Anemia/Other'Anemias'
7'
1'
1'
3'
Others'
3'
Others'
4'
Number'of'Doses'
1'
2'
3'
4'
Dose'(x10^6/Kg)'
Mean'(Range)'
'
6'
10'
4'
1'
'
3,6'(1,6;8,1)'
Number'of'Doses'
1'
2'
3'
4'
Dose'(x10^6/Kg)'
Mean'(Range)'
'
2'
3'
7'
4'
'
3,7'(1,7;7,4)'
Dazzi and Apperley
Overall Outcome
Adults
Children
Adults
Children
MSC for GVHD: the need for ‘licensing’
Tissue affected by GVHD
DC
PC
2
T
Chemokines
3
4
IFN-γ
1
5
T
MSC
POSTCAPILLARY
VENULE
Dazzi & Marelli-Berg, EJI 2008
The efficacy of MSC on GVHD depends on time of infusion
Day 0
Day 2
Day 20
Day 30
Polchert et al, EJI 2008
In vivo macrophage depletion impairs MSC
therapeutic effect on GvHD
Control
Clodronate
MSC+clodronate
MSC
1.0
0.5
time (weeks)
11
10
9.
5
9
8
7.
5
6.
5
6
0.0
5.
5
GvHD score (average)
1.5
MSC
Chronic inflammation
Acute inflammation
Fibrosis
iMSC
aMSC
‘Licensing’
Alternative
‘licensing’
Th1
M1
Th2
M2
ClinicalImmunosuppression
responses to
MSC
Persistent antigen stimulation
Dazzi, Best Practice Haem 2011
Graft-versus-host disease
Acute GvHD
Chronic GvHD
Acute inflammation Alloimmunity Necrotic changes
Confined to specific tissues
Th1
Chronic inflammation
Autoimmunity
Fibrosis
Multisystemic
Th2
Pilot study
!
Total of 40 patients over 2 years
Grade II (visceral) GvHD
• 7 Clinical Centres (KCL-Marsden-ICLSouthampton-RFH-Bristol)
• KCL-Imperial for cell manufacturing
!
Primary aim of the study:
!
Identify a biomarker predictive of response to MSC
Responders
Non-Responders
Transcriptomic profile in peripheral blood monocytes
Conclusions
• MSC exhibit a potent anti-inflammatory effect
!
• Such an effect requires a conducive inflammatory
environment
!
• Treatment of acute GvHD with MSC results into a significant
clinical benefit
!
• A stratified approach is likely to produce better results

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