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Role of gp130 Activation for HSC Self-renewal

Role of gp130 Activation for HSC Self-renewal. Aurore Degrange and Lisa Treat BE.400 Term Project December 10, 2002. Brief Overview. Background Goals Previous experimental results Computational model Proposed experiments Conclusions. Hematopoietic Stem Cell (HSC) Fate.

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Role of gp130 Activation for HSC Self-renewal

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  1. Role of gp130 Activation for HSC Self-renewal Aurore Degrange and Lisa Treat BE.400 Term Project December 10, 2002

  2. Brief Overview • Background • Goals • Previous experimental results • Computational model • Proposed experiments • Conclusions

  3. Hematopoietic Stem Cell (HSC) Fate www.nih.gov/news/stemcells/chapter5.pdf

  4. Background • Cytokines: soluble proteins • HSC proliferation • HSC differentiation • Some signal through common subunits • LIF: LIFR + gp130 • IL-6: IL-6R + gp130

  5. HIL-6 • Fusion of IL-6 and soluble IL-6 receptor • Fully active at 100- to 1000-fold lower concentrations compared to unlinked Peters et al. (1998)

  6. Goals • Isolate impact of gp130 activation in promoting HSC self-renewal • Adapt ESC computational model by Viswanathan et al. for HSC system • Design experiments • To determine unknown parameters • To validate the model

  7. Important Role of gp130 Activation in HSC Self-renewal • Fixed concentrations of SCF and FL • For HIL-6, narrow range of effective concentrations • Independent of particular ligand used to stimulate gp130 receptor Self-renewing HSC Population Expansion (Fold) Adapted from Audet et al. (2001)

  8. LIF KD1 LIF LIF KC1 LIFR LIFR LIFR gp130 gp130 HIL-6 KC2 KD2 HIL-6 HIL-6 gp130 gp130 gp130 gp130 Model System of gp130 Activation Activation by LIF KD1 = LIF dissociation rate constant KC1 = cross-linking rate constant Activation by HIL-6 KD2 = HIL-6 dissociation rate constant KC2 = cross-linking rate constant Adapted from Viswanathan et al. (2002)

  9. LIF KD1 LIF LIF KC1 LIFR LIFR LIFR gp130 gp130 Activation by LIF At steady-state: where: Adapted from Viswanathan et al. (2002)

  10. HIL-6 KC2 KD2 HIL-6 HIL-6 gp130 gp130 gp130 gp130 Activation by HIL-6 At steady-state: where: Adapted from Viswanathan et al. (2002)

  11. Modeled gp130 Activation by LIF & HIL-6 Signaling Complex Number (M) Ligand Concentration (M)

  12. Effects of Varying Parameters on CLIF Increasing LIF receptor numbers Increasing KD1 Signaling Complex Number (M) Increasing gp130 receptor numbers Increasing KC1 LIF Concentration (M)

  13. Effects of Varying Parameters on CHIL-6 Increasing KD2 Increasing gp130 receptor numbers Increasing KC2 Signaling Complex Number (M) HIL-6 Concentration (M)

  14. Effects of Varying Receptor Numbers Increasing LIF receptor numbers Increasing gp130 receptor numbers Signaling Complex Number (M) LIF Concentration (M) HIL-6 Concentration (M)

  15. Experimental Outline • Getting the parameters for the computational model • Culture of “HSC-enriched” populations under different cytokine concentrations • Assay to quantify HSC self-renewal during culture

  16. Cell Lines • “HSC-enriched” population, using c-kit+Sca-1+lin- cells • Isolation from bone marrow • Immunomagnetically removed lin- BM cells • Fluorescent labelling with antibodies • Fluorescent-activated cell sorter (FACS)

  17. LIF KD1 LIF LIF KC1 LIFR LIFR LIFR gp130 gp130 HIL-6 KC2 KD2 HIL-6 HIL-6 gp130 gp130 gp130 gp130 Getting the Unknown Parameters… • LIFR and gp130 receptor numbers • Dissociation and cross-linking rate constants for LIF and HIL-6 Adapted from Viswanathan et al. (2002)

  18. Parameters: protocol (1) • Radioactive labeling of HIL-6 and LIF • Steady state at 37°C • Free • Cell-bound • Internalized For varying concentrations of ligand Hilton and Nicola (1992)

  19. LIF KD1 KC2 LIF LIF KC1 KD2 LIF KD1 LIF KD2 Parameters: protocol (2) • Isolated membranes • Detergent-solubilized membranes

  20. Culture Conditions • In suspension: serum-free medium • Each is colony derived from a single cell • 100 ng/mL FL + 50 ng/mL SCF • Varying concentrations of LIF or HIL-6 Adapted from Audet et al. (2001)

  21. Measurement of human engraftment by PCR Quantification of human cells by flow cytometry (6 weeks) Secondary transplantation assay Assessment of HSC Culture of human HSC NOD/SCID BM injection Yahata et al. (2002)

  22. Conclusions • gp130 activation plays key role in HSC self-renewal, whether induced by LIF or by HIL-6 • ESC model is adaptable for HSC system • Unknown parameters can be determined from binding experiments • Switch-like response of LIF-induced activation is more convenient for clinical applications • Renewable source of cells in tissue-replacement therapies

  23. Selected References • Audet, J., Miller, C.L., Rose-John, S., Piret, J.M. & Eaves, C.J. (2001) PNAS98, 1757-1762. • Fischer, M., Goldschmitt, J., Peschel, C., Brakenhoff, J.P.G., Kallen, K.J. Wollmer, A., Grotzinger, J. & Rose-John, S. (1997) Nat. Biotechnol.15, 142-145. • Viswanathan, S., Benatar, T., Rose-John, S., Lauffenburger, D.A. & Zandstra, P.W. (2002) Stem Cells20, 119-138. • Hilton, D.J. & Nicola, N.A. (1992) J. Biol. Chem.267, 10238-10247. • de Wynter, E., et al. (2001) J. Biol. Regul. Homeost. Agents 15, 23-27. • Peters, M., Muller, A.M., and Rose-John, S. (1998) Blood. 92, 10:3495-3504. • Yahata et al. (2002) Blood.

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