Perturbation of fetal liver haematopoietic stem and progenitor cell development by

trisomy 21

Anindita RoyImperial College London

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

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%

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

LMPP

CD34+

CD38+CD38-

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

Normal

Perturbation of fetal liver HSC/progenitor frequency in DS

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)

CD

14/1

5/16

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

0

10

20

30

40

50

60

70

80

HSC MPP LMPP CMP MEP GMP

No.

of c

olon

ies/

100

cel

ls 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.

of c

olon

ies/

100

cel

ls p

late

d

NORMAL

DOWN SYNDROME

CO

LON

IES/

100

CEL

LS

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

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

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

Altered gene expression in DS FL HSC/ progenitorsLYMPHOID GENES MEGA-ERYTHROID MYELOID GENES

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

Abnormal fetal liver haematopoiesis in DS

ELPLMPP

HSC GMPMPP

MEP

B PROG

T PROG

Differences in gene priming determinelineage decisions

B PROG

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

Thank you

Chr 21 gene expression in DS FL HSC/ progenitors

•

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

EBF1 NETWORK

EBF1

E2A

IL7R

PU.1 lo

PAX5

CD79aVPREB

CEBPa

FLT3

GATA1

FOR MYELOID FATE

ERYTHROID FATE

NOTCH1

T CELL FATE

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