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Perturbation of fetal liver haematopoietic stem and progenitor cell development by trisomy 21 Anindita Roy Imperial College London.

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Presentation on theme: "Perturbation of fetal liver haematopoietic stem and progenitor cell development by trisomy 21 Anindita Roy Imperial College London."— Presentation transcript:

1 Perturbation of fetal liver haematopoietic stem and progenitor cell development by trisomy 21 Anindita Roy Imperial College London

2 Leukaemia in children with Down syndrome (DS) ConditionFrequency observed in population Frequency observed in DS Excess risk in DS Acute leukaemia 1 in in x ALL1 in in x AML1 in in x AMKL1 in in x Hasle, 2008

3 TAM +21 GATA1s Gene X DS AMKL Acute leukaemias in Down syndrome +21 GATA1s +21 Birth Fetal haematopoietic cell DS ALL +21 JAK2 R683 CRLF2 m What is the role of trisomy 21 and how does it perturb fetal haematopoiesis in Down syndrome? +21 ? CRLF2 +21 CRLF2 CRLF2 m 20-30%

4 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

5 Fetal Haematopoiesis: Principal Progenitor Populations LMPP CD34+ CD38+CD38-

6 Perturbation of fetal liver HSC/progenitor frequency in DS CBP

7 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

8 Normal Perturbation of fetal liver HSC/progenitor frequency in DS

9 Analysis of fetal liver HSC/progenitors Flow sorted progenitors HSCMPPLMPP 1)Methylcellulose clonogenic assays (100) 2)Lymphoid stromal co cultures (100) 3)Gene expression (Fluidigm-qRT PCR) (50) 4)Xenograft studies ( ,000) Analysis done (no. of cells used/ population)

10 CD14/15/16 Clonogenic assays of FL progenitors Colony readout after 14 days of clonogenic assay of FL HSC/ progenitors

11 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

12 Increased megakaryocyte/erythroid potential of HSC/progenitors in DS NORMAL DOWN SYNDROME COLONIES/ 100 CELLS

13 Impaired B cell differentiation of DS FL progenitors DS FL HSC, LMPP and ELP did not produce CD B cells in MS5 co cultures N T21 CD19

14 NSG mouse engraftment model 200 cGyCD34 + cells ( ,000) Terminate expt at 12 weeks. Analysis of BM, spleen, thymus and liver for human immature and mature haematopoietic cells

15 Qualitative differences in engraftment of normal vs. DS FL CD34 cells in the bone marrow of NSG mice N T21 LYMPHOIDMK/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

16 Altered gene expression in DS FL HSC/ progenitors LYMPHOID GENESMEGA-ERYTHROIDMYELOID GENES

17 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

18 Abnormal fetal liver haematopoiesis in DS ELP LMPP HSC GMPMPP MEP B PROG T PROG Differences in gene priming determine lineage decisions B PROG

19 Acknowledgments Prof Irene Roberts Tassos Karadimitris 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 Ollie Tustall-Pedoe Prof Phillip Bennett Hikoro Matsui Philip Hexley Eugene Ng James Elliott Valeria Melo Oxford: Paresh Vyas Adam Mead Debbie Atkinson SE Jacobsen Manchester: Vaskar Saha Singapore: Jerry Chan Citra Mater

20 Thank you

21 Chr 21 gene expression in DS FL HSC/ progenitors

22 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

23 EBF1 NETWORK EBF1 E2A IL7R PU.1 lo PAX5 CD79a VPREB CEBPa FLT3 GATA1 FOR MYELOID FATE ERYTHROID FATE NOTCH1 T CELL FATE

24 PROPOSED B CELL PATHWAY MPP LMPP ELP BP E2A EBF1 PAX5 IL7R FLT3 GATA1(loss or mega erythroid potential) PU.1 CEBPa (loss or myeloid potential) NOTCH1(loss or T potential) KEY: ELP: early lymphoid progenitor; BP: B cell progenitor


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