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Perturbation of fetal liver haematopoietic stem and progenitor cell development by trisomy 21

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). Hasle, 2008. Acute leukaemias in Down syndrome. Birth. TAM. DS AMKL. +21. 20-30%. +21 GATA1s Gene X. +21

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Perturbation of fetal liver haematopoietic stem and progenitor cell development by trisomy 21

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  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) Hasle, 2008

  3. Acute leukaemias in Down syndrome Birth TAM DS AMKL +21 20-30% +21 GATA1s Gene X +21 GATA1s +21 Fetal haematopoietic cell +21 ? CRLF2 +21 JAK2R683 CRLF2 m +21 CRLF2 CRLF2 m DS ALL What is the role of trisomy 21 and how does it perturb fetal haematopoiesis in Down syndrome?

  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 CD34+ CD38- CD38+ LMPP

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

  9. Analysis of fetal liver HSC/progenitors Flow sorted progenitors Analysis done (no. of cells used/ population) • Methylcellulose clonogenic assays (100) • Lymphoid stromal co cultures (100) • Gene expression (Fluidigm-qRT PCR) (50) • Xenograft studies (1000- 30,000) HSC MPP LMPP

  10. Clonogenic assays of FL progenitors CD14/15/16 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 COLONIES/ 100 CELLS DOWN SYNDROME

  13. Impaired B cell differentiation of DS FL progenitors N T21 CD19 DS FL HSC, LMPP and ELP did not produce CD34-19+ B cells in MS5 co cultures

  14. 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

  15. 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

  16. Altered gene expression in DS FL HSC/ progenitors LYMPHOID GENES MEGA-ERYTHROID MYELOID GENES

  17. Summary: defining normal FL haematopoiesis • Demonstrated HSC, MPP and LMPP for the first time in human FL • 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 • Comprehensive gene expression analysis of normal FL HSC and progenitors.

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

  19. 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 Acknowledgments 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. Future research directions • Xenograft data for HSC compartment (may need better mouse model than NSG for mega-erythroid engraftment) • Explore significance of microenvironment in more detail (FL vs FBM) • 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

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

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

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