Perturbation of fetal liver haematopoietic stem and progenitor cell development by trisomy 21...
Transcript of Perturbation of fetal liver haematopoietic stem and progenitor cell development by trisomy 21...
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Perturbation of fetal liver haematopoietic stem and progenitor cell development by
trisomy 21
Anindita RoyImperial College London
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Leukaemia in children with Down syndrome (DS)
Condition Frequency observed in population
Frequency observed in DS
Excess risk in DS
Acuteleukaemia
1 in 2800 1 in 100-200 10-20 x
ALL 1 in 3500 1 in 300 12 x
AML 1 in 14 000 1 in 300 46 x
AMKL 1 in 233 000 1 in 500 466 x
Hasle, 2008
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TAM+21
+21GATA1sGene X
DS AMKL
Acute leukaemias in Down syndrome
+21GATA1s
+21
Birth
Fetalhaematopoietic
cell
DS ALL
+21JAK2R683
CRLF2 m
What is the role of trisomy 21 and how does it perturb fetal haematopoiesis in Down syndrome?
+21? CRLF2
+21CRLF2CRLF2 m
20-30%
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Role of trisomy 21 in fetal haematopoiesis
Aim 1:Characterise 2nd trimester normal and T21 human fetal liver (FL) haematopoiesis
Aim 2:Study in vitro behaviour of normal and T21 FL HSC and progenitors.
Aim 3:Define gene expression signature of normal vs T21 FL HSC and progenitors
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Fetal Haematopoiesis: Principal Progenitor Populations
LMPP
CD34+
CD38+CD38-
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Perturbation of fetal liver HSC/progenitor frequency in DS
CBP
CBP
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Perturbation of fetal liver B progenitor frequency in DS
N FL
T21 FL
CD34+CD19+ B progenitors were significantly reduced in DS FL, especially CD34+CD19+CD10- Pre pro B cells
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Normal
Perturbation of fetal liver HSC/progenitor frequency in DS
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Analysis of fetal liver HSC/progenitors
Flow sorted progenitors
HSC MPP LMPP
1) Methylcellulose clonogenic assays (100)
2) Lymphoid stromal co cultures (100)
3) Gene expression (Fluidigm-qRT PCR) (50)
4) Xenograft studies (1000- 30,000)
Analysis done (no. of cells used/ population)
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CD
14/1
5/16
Clonogenic assays of FL progenitors
Colony readout after 14 days of clonogenic assay of FL HSC/ progenitors
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Increased clonogenicity of progenitors in DS FL
In vitro clonogenic assays showed significantly increased clonogenicity of T21 HSC,CMP and MEP compared to normal FL
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Increased megakaryocyte/erythroid potential of HSC/progenitors in DS
0
10
20
30
40
50
60
70
80
HSC MPP LMPP CMP MEP GMP
No
. o
f co
lon
ies/
100 c
ell
s p
late
d
CFU-GEMMERYTHROIDCFU-MkECFU-MkCFU-G/M/GMMYELOID BLAST
0
10
20
30
40
50
60
70
80
HSC MPP LMPP CMP MEP GMP
No
. o
f co
lon
ies/
100 c
ell
s p
late
d
NORMAL
DOWN SYNDROME
CO
LO
NIE
S/
10
0 C
EL
LS
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Impaired B cell differentiation of DS FL progenitors
DS FL HSC, LMPP and ELP did not produce CD34-19+ B cells in MS5 co cultures
N
T21
CD
19
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NSG mouse engraftment model
200 cGy CD34 + cells (1000- 30,000)
Terminate expt at 12 weeks. Analysis of BM, spleen, thymus and liver for human immature and mature haematopoietic cells
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Qualitative differences in engraftment of normal vs. DS FL CD34 cells in the bone marrow of NSG mice
N
T21
LYMPHOID MK/Ery
Further characterisation of engrafted hCD45 cells
DS FL CD34 cells demonstrate reduced (lymphoid) engraftment in NSG mice suggesting cell intrinsic abnormalities caused by T21
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Altered gene expression in DS FL HSC/ progenitors
LYMPHOID GENES MEGA-ERYTHROID MYELOID GENES
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1. Demonstrated HSC, MPP and LMPP for the first time in human FL
2. Demonstrated lymphoid progenitors and mature B cells in human FL for the first time (including novel CD34+CD19+CD10- progenitor which may be key to understanding pathogenesis of childhood ALL) and showed that mature B cells can be generated in vitro
3. Comprehensive gene expression analysis of normal FL HSC and progenitors.
Summary: defining normal FL haematopoiesis
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Abnormal fetal liver haematopoiesis in DS
ELPLMPP
HSC GMPMPP
MEP
B PROG
T PROG
Differences in gene priming determinelineage decisions
B PROG
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AcknowledgmentsProf Irene RobertsTassos Karadimitris
Gillian CowanSarah FilippiGeorg BohnKaterina GoudevenouAris ChaidosMing HuLuciana GarguiloSubarna ChakravortyKate XuValentina CaputoMauritius KleijnenKelly MakaronaDavid O’ConnorJoanna CostaSuhail ChaudhuryRebecca Babb
Ollie Tustall-Pedoe
Prof Phillip BennettHikoro Matsui
Philip HexleyEugene NgJames ElliottValeria Melo
Oxford:Paresh Vyas Adam Mead Debbie AtkinsonSE Jacobsen
Manchester:Vaskar Saha
Singapore:Jerry ChanCitra Mater
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Thank you
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Chr 21 gene expression in DS FL HSC/ progenitors
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•
1. Xenograft data for HSC compartment (may need better mouse model than NSG for mega-erythroid engraftment)
2. Explore significance of microenvironment in more detail (FL vs FBM)
3. Lymphoid defect: -RAG1 (overexpression: ? B lymphoid block/ DNA damage)-functional studies with mature B cells-Fetal BM lymphoid development in more detail
4. Explore cytokine receptor pathways such as IGF1R and IGF2R
Future research directions
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EBF1 NETWORK
EBF1
E2A
IL7R
PU.1 lo
PAX5
CD79aVPREB
CEBPa
FLT3
GATA1
FOR MYELOID FATE
ERYTHROID FATE
NOTCH1
T CELL FATE
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PROPOSED B CELL PATHWAY
MPP
LMPP
ELP
BP
E2A
EBF1
EBF1PAX5
IL7R
FLT3
GATA1 (loss or mega erythroid potential)
PU.1CEBPa
(loss or myeloid potential)
NOTCH1 (loss or T potential)
KEY: ELP: early lymphoid progenitor; BP: B cell progenitor