role of gp130 activation for hsc self-renewal aurore degrange and lisa treat be.400 term project...
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Role of gp130 Activation for HSC Self-renewal
Aurore Degrange and Lisa Treat
BE.400 Term ProjectDecember 10, 2002
Brief Overview
BackgroundGoalsPrevious experimental resultsComputational modelProposed experimentsConclusions
Hematopoietic Stem Cell (HSC) Fate
www.nih.gov/news/stemcells/chapter5.pdf
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 receptorFully 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-renewalAdapt ESC computational model by Viswanathan et al. for HSC systemDesign experiments To determine unknown parameters To validate the model
Important Role of gp130 Activation in HSC Self-renewal
Fixed concentrations of SCF and FLFor HIL-6, narrow range of effective concentrations Independent of particular ligand used to stimulate gp130 receptor
Adapted from Audet et al. (2001)
Self-r
en
ew
ing H
SC
Popula
tion
Expansi
on (
Fold
)
Model System of gp130 Activation
LIF
LIF LIF
LIFR
LIFR
LIFR
gp1
30
gp1
30
KC1
KD1
gp1
30
gp1
30
KC2KD2
gp1
30
gp1
30
HIL-6
HIL-6
HIL-6
Activation by LIFKD1 = LIF dissociation rate
constant
KC1 = cross-linking rate constant
Activation by HIL-6KD2 = HIL-6 dissociation rate
constant
KC2 = cross-linking rate constant
Adapted from Viswanathan et al. (2002)
Activation by LIFLIF
LIF LIF
LIFR
LIFR
LIFR
gp1
30
gp1
30
KC1
KD1
Adapted from Viswanathan et al. (2002)
111 CRL DK
LIFK CRC C 1
21
1
11 C
LRKD
At steady-state:
LIFC C
RCK 21
1
2
12
2
1111
θ4χαα1χαα1
2
1T
CD
LIFLIF
CD
LIFLIFLIF RL
KKKKLC
where:
LIFT
LIFT
CRR
CCRR
22
111
211
1
χ
α
TTC
DLIF
RRK
LK
2
1θT
T
R
R
Activation by HIL-6
Adapted from Viswanathan et al. (2002)
122 CRL DK
6212
HILK CRC C
1
22 C
LRKD
At steady-state:
6
212
HIL
C C
RCK
2
1γα8α1α1
α8
α16
266
26
66
HILHILHIL
CHIL
HILHIL KC
where:
6122 2 HILT CCRR LKDHIL 26α 22γ TC RKgp1
30
gp1
30
KC2KD2
gp1
30
gp1
30
HIL-6
HIL-6
HIL-6
10-13
10-12
10-11
10-10
10-9
10-8
10-7
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1x 10
-10
LIFHIL-6
Modeled gp130 Activation by LIF & HIL-6
Sig
nalin
g C
om
ple
x N
um
ber
(M)
Ligand Concentration (M)
10
-1310
-1210
-1110
-1010
-910
-810
-70
0.2
0.4
0.6
0.8
1
1.2x 10
-10
10-13
10-12
10-11
10-10
10-9
10-8
10-7
0
0.2
0.4
0.6
0.8
1
1.2x 10
-1010
-1310
-1210
-1110
-1010
-910
-810
-70
0.5
1
1.5
2
2.5x 10
-10
Effects of Varying Parameters on CLIF
Increasing LIF receptor numbers
Increasing gp130 receptor numbers
LIF Concentration (M)
Sig
nalin
g C
om
ple
x N
um
ber
(M)
10-13
10-12
10-11
10-10
10-9
10-8
10-7
0
0.2
0.4
0.6
0.8
1x 10
-10
Increasing KD1
Increasing KC1
10-13
10-12
10-11
10-10
10-9
10-8
10-7
0
1
2
3
4
5
6x 10
-11
10-13
10-12
10-11
10-10
10-9
10-8
10-7
0
0.2
0.4
0.6
0.8
1
1.2x 10
-10
10-13
10-12
10-11
10-10
10-9
10-8
10-7
0
0.2
0.4
0.6
0.8
1x 10
-9
Effects of Varying Parameters on CHIL-6
Increasing KC2
Increasing gp130 receptor numbers
HIL-6 Concentration (M)
Sig
nalin
g C
om
ple
x N
um
ber
(M)
Increasing KD2
10-13
10-12
10-11
10-10
10-9
10-8
10-7
0
0.5
1
1.5
2
2.5x 10
-10
10-13
10-12
10-11
10-10
10-9
10-8
10-7
0
0.2
0.4
0.6
0.8
1x 10
-9
Effects of Varying Receptor Numbers
Increasing LIF receptor numbers
Increasing gp130 receptor numbers
LIF Concentration (M)
HIL-6 Concentration (M)
Sig
nalin
g C
om
ple
x N
um
ber
(M)
Experimental Outline
Getting the parameters for the computational modelCulture of “HSC-enriched” populations under different cytokine concentrationsAssay to quantify HSC self-renewal during culture
Cell Lines
“HSC-enriched” population, using c-kit+Sca-1+lin- cellsIsolation 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 numbersDissociation and cross-linking rate constants for LIF and HIL-6
gp1
30
gp1
30
KC2KD2
gp1
30
gp1
30
HIL-6
HIL-6
HIL-6
LIF
LIF LIF
LIFR
LIFR
LIFR
gp1
30
gp1
30
KC1
KD1
Adapted from Viswanathan et al. (2002)
Parameters: protocol (1)
Radioactive labeling of HIL-6 and LIFSteady 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
KC2KD2
LIFLIF LIF
KC1KD1
LIFKD1 KD2
LIF
Culture Conditions
In suspension: serum-free mediumEach is colony derived from a single cell100 ng/mL FL + 50 ng/mL SCFVarying concentrations of LIF or HIL-6
Adapted from Audet et al. (2001)
Assessment of HSC
Measurement of human engraftment by PCRQuantification 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-6ESC model is adaptable for HSC systemUnknown parameters can be determined from binding experimentsSwitch-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, 1757-1762.
2. 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.
3. Viswanathan, S., Benatar, T., Rose-John, S., Lauffenburger, D.A. & Zandstra, P.W. (2002) Stem Cells 20, 119-138.
4. Hilton, D.J. & Nicola, N.A. (1992) J. Biol. Chem. 267, 10238-10247.
5. de Wynter, E., et al. (2001) J. Biol. Regul. Homeost. Agents 15, 23-27.
6. Peters, M., Muller, A.M., and Rose-John, S. (1998) Blood. 92, 10:3495-3504.
7. Yahata et al. (2002) Blood.