Perturbation of fetal liver haematopoietic
stem and progenitor cell development by
trisomy 21
Anindita Roy
Imperial College London
Leukaemia in children with Down syndrome (DS)
Condition
Frequency observed in
population
Frequency observed in
DS
Excess risk in DS
Acute
leukaemia
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
Acute leukaemias in Down syndrome
Birth
TAM
+21
+21
GATA1s
Fetal
haematopoietic
cell
+21
? CRLF2
DS AMKL
+21
20-30%
+21
GATA1s
Gene X
+21
CRLF2
CRLF2 m
+21
JAK2R683
CRLF2 m
DS ALL
What is the role of trisomy 21 and how does it perturb fetal
haematopoiesis in Down syndrome?
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
Fetal Haematopoiesis: Principal Progenitor Populations
CD34+
CD38-
CD38+
LMPP
Perturbation of fetal liver HSC/progenitor frequency in DS
CBP
CBP
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
Perturbation of fetal liver HSC/progenitor frequency in DS
Normal
Analysis of fetal liver HSC/progenitors
Flow sorted progenitors
Analysis done
(no. of cells used/ population)
1) Methylcellulose clonogenic assays (100)
2) Lymphoid stromal co cultures (100)
3) Gene expression (Fluidigm-qRT PCR) (50)
4) Xenograft studies (1000- 30,000)
HSC
MPP
LMPP
CD14/15/16
Clonogenic assays of FL progenitors
Colony readout after 14 days of clonogenic assay of FL HSC/ progenitors
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
Increased megakaryocyte/erythroid potential of
HSC/progenitors in DS
100 CELLS
No. of colonies/ 100 cells plated
No. of colonies/ 100 cellsCOLONIES/
plated
80
70
CFU-GEMM
ERYTHROID
CFU-MkE
CFU-Mk
CFU-G/M/GM
MYELOID BLAST
NORMAL
60
50
40
30
20
10
0
HSC
MPP
LMPP
CMP
MEP
GMP
LMPP
CMP
MEP
GMP
80
DOWN SYNDROME
70
60
50
40
30
20
10
0
HSC
MPP
Impaired B cell differentiation of DS FL progenitors
N
CD19
T21
DS FL HSC, LMPP and ELP did not produce CD34-19+ B cells in MS5 co cultures
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
Qualitative differences in engraftment of normal vs. DS FL
CD34 cells in the bone marrow of NSG mice
Further characterisation of engrafted hCD45 cells
LYMPHOID
MK/Ery
N
T21
DS FL CD34 cells demonstrate reduced (lymphoid) engraftment in NSG mice
suggesting cell intrinsic abnormalities caused by T21
Altered gene expression in DS FL HSC/ progenitors
LYMPHOID GENES
MEGA-ERYTHROID
MYELOID GENES
Summary: defining normal FL haematopoiesis
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.
Abnormal fetal liver haematopoiesis in DS
MEP
Differences in gene
priming determine
lineage decisions
HSC
MPP
GMP
B PROG
LMPP
ELP
T PROG
Acknowledgments
Prof Irene Roberts
Tassos Karadimitris
Prof Phillip Bennett
Hikoro Matsui
Gillian Cowan
Sarah Filippi
Georg Bohn
Katerina Goudevenou
Aris Chaidos
Ming Hu
Luciana Garguilo
Subarna Chakravorty
Kate Xu
Valentina Caputo
Mauritius Kleijnen
Kelly Makarona
David O’Connor
Joanna Costa
Suhail Chaudhury
Rebecca Babb
Philip Hexley
Eugene Ng
James Elliott
Valeria Melo
Ollie Tustall-Pedoe
Oxford:
Paresh Vyas
Adam Mead
Debbie Atkinson
SE Jacobsen
Manchester:
Vaskar Saha
Singapore:
Jerry Chan
Citra Mater
Thank you
Chr 21 gene expression in DS FL HSC/ progenitors
Future research directions
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
EBF1 NETWORK
ERYTHROID FATE
GATA1
E2A
FLT3
IL7R
VPREB
CD79a
PAX5
EBF1
PU.1 lo
CEBPa
FOR MYELOID FATE
NOTCH1
T CELL FATE
PROPOSED B CELL PATHWAY
MPP
FLT3
GATA1 (loss or mega erythroid potential)
E2A
IL7R
LMPP
PU.1
(loss or myeloid potential)
CEBPa
EBF1
ELP
NOTCH1 (loss or T potential)
EBF1
PAX5
BP
KEY: ELP: early lymphoid progenitor; BP: B cell progenitor