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

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

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)

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

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 Adapted from Audet et al. (2001) Self-renewing HSC Population Expansion (Fold)

Model System of gp130 Activation LIF LIFR gp130 K C1 K D1 gp130 K C2 K D2 gp130 HIL-6 Activation by LIF K D1 = LIF dissociation rate constant K C1 = cross-linking rate constant Activation by HIL-6 K D2 = HIL-6 dissociation rate constant K C2 = cross-linking rate constant Adapted from Viswanathan et al. (2002)

Activation by LIF LIF LIFR gp130 K C1 K D1 Adapted from Viswanathan et al. (2002) At steady-state: where:

Activation by HIL-6 Adapted from Viswanathan et al. (2002) At steady-state: where: gp130 K C2 K D2 gp130 HIL-6

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

Effects of Varying Parameters on C LIF Increasing LIF receptor numbers Increasing gp130 receptor numbers LIF Concentration (M) Signaling Complex Number (M) Increasing K D1 Increasing K C1

Effects of Varying Parameters on C HIL-6 Increasing K C2 Increasing gp130 receptor numbers HIL-6 Concentration (M) Signaling Complex Number (M) Increasing K D2

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

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

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)

Getting the Unknown Parameters… LIFR and gp130 receptor numbers Dissociation and cross-linking rate constants for LIF and HIL-6 gp130 K C2 K D2 gp130 HIL-6 LIF LIFR gp130 K C1 K D1 Adapted from Viswanathan et al. (2002)

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)

Parameters: protocol (2) Isolated membranes Detergent-solubilized membranes K C2 K D2 LIF K C1 K D1 LIF K D1 K D2 LIF

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)

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

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

Selected References 1. Audet, J., Miller, C.L., Rose-John, S., Piret, J.M. & Eaves, C.J. (2001) PNAS 98, Fischer, M., Goldschmitt, J., Peschel, C., Brakenhoff, J.P.G., Kallen, K.J. Wollmer, A., Grotzinger, J. & Rose-John, S. (1997) Nat. Biotechnol. 15, Viswanathan, S., Benatar, T., Rose-John, S., Lauffenburger, D.A. & Zandstra, P.W. (2002) Stem Cells 20, Hilton, D.J. & Nicola, N.A. (1992) J. Biol. Chem. 267, de Wynter, E., et al. (2001) J. Biol. Regul. Homeost. Agents 15, Peters, M., Muller, A.M., and Rose-John, S. (1998) Blood. 92, 10: Yahata et al. (2002) Blood.