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Flow cytometric analysis of growth factor receptor expression on hemopoietic progenitors

Flowcytometrische analyse van groeifaktor receptor expressie op hemopoietische voorlopercellen.

PROEFSCHRIFT . ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prot. Dr P.W.C. Akkermans M.A. en volgens het besluit van het college voor promoties. De open bare verdediging zal plaatvinden op woensdag 24 september 1997 om 11 :45 uur

door

Margrethe Olga de Jong geboren te Rotterdam

Promotlecommissie Promotor:

Prof. Dr B. Lowenberg

Overige leden:

Prof. Dr W. van Ewijk Prof. Dr H.J. Tanke Dr G. Wagemaker

Co-promotor:

Dr AW. Wognum .

The work described in this thesis was performed at the Radiobiological Institute TNO, the Institute of Radiobiology, Erasmus University Rotterdam (Rijswijk, The Netherlands) and at the Department of Hematology, Erasmus University Rotterdam (Rotterdam, The Netherlands) and was supported by grants of the Netherlands Organization for Scientific Research NWO, the Dutch Cancer Society Koningin Wilhelmina Fonds, the Royal Netherlands Academy of Arts and Sciences, and contracts of the Commission of the European Communities.

Theories come and go, the frog stays Frallfois Jacob,

Instiflll Pasteur, Paris, France

am. Robbert

ISBN 90-9010860-2

This thesis was· wrilten, edited, and produced on a desktop publishing system using ti Apple Macintosh computers. Texl types are Times® and Helvelica®, Printed by FEBODRUK B.V., Enschede, the Netherlands, on recycled (90g) paper.

Cover "kernboodschapR by Karola van Roayen and Robbert Paulussen.

© 1997 M.O. de Jong All parts of this publication may be reproduced, stored in a retrieval system, or transmitted In

any form or by any means, mechanical, photocopying recording, or othelWise, provided that the source is mentioned.

Marg de Jong

Table of contents

TABLE OF CONTENTS

Abbreviations. "'"'''''''''''''''''''''' ............................................................................ 7 1.

General introduction .................................................................................. 9

2.

A sensitive method to detect cell surface receptors using biotinylated growth factors ....................................................................... 57

Progress in Histochel1listl)' alld Cytoc/iemistl)1 26: 119-123 (1992), and Jaquemin-Sablon A (cd.) Flow Cytometry - New Developments. Springer-Verlag Berlin Heidelberg New York Tokyo (1992) 3.

Biotinylation of interleukin-2 (IL-2) for flow cytometric analysis of IL-2 receptor expression: comparison of different methods ................ 57 loul'llal of ImmmlOlogical Methods

4.

184: 10 1-112 (1995)

Purification of repopulating hemopoietic cells based on binding of biotinylate(l Kil ligand........................................................................... 85 Leukemia 10: 1813-1822 (1996)

5.

FDC-Pl cells as a model to examine differential binding of stem cell factor (SCF) and anti-Kit antibodies to hemopoietic cells ............... 109

6.

Separation of myeloid and erythroid progenitors based on expression of CD34 and Kit. .................................................................. 131 Blood 86 (11): 4076-4085 (1995)

7.

Coexpression of Kit and the receptors for erythropoietin, interleukin·5 and granulocyte/ macrophage· colony stimulating factor on hemopoietic cells .................................................................... 151 Stem Cells (in press t 997)

8.

Differential expression of receptors for hemopoietic growth factors on subsets of CD34 + hemopoietic cells ..................................... 173 Leukemia and Lymphoma 24: 11-25 (1996)

9.

Summary and discussion ...................................,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 195 Samenvatting .(Summary in Dutch) ... :.................................................... 209 Curriculum vitae ..................................................................................... 218 Publications ........................................................................................... 219 Dankwoord ...............................................................................:............ 220

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Abbreviations

Marg de Jong

ABBREVIATIONS

AA

amino acids

B:P

biotin:protein (ratio)

BFU-E

erythroid burst~forming unit

BM BSA

bone marrow bovine serum albumin

eDNA

complementary DNA

CFU-C

colony-forming unit in culture

CFU-E

erythroid colony-forming unit

CFU-GM

granulocyte! macrophage colony~forming unit

CFU-S

spleen colony-forming unit

CML

chronic myeloid leukemia

D:P

DIG:protein (ratio)

DIG

digoxigenin

DMSa

dimethyl sulfoxide

EPa

erythropoietin

FACS

fluorescence activated cell sorter

FCS

fetal calf serum

FDC-PI

factor~dependent

FISH

fillorescent in situ hybridization

FIrC

FLS

fluorescein isothiocyanate forward light scatter

GAM

goat atiti-mouse antibodies

GARa

goat anti-rat antibodies

GF

G-CSF

growth factor gro~vth factor receptor granulocyte! macrophage colony~stiml1lating factor granulocyte colony-stimulating factor

H58

Hoechst 33258

HH

Hanks' Hepes buffered salt solution HID serum! azide hemopoietic stem cell

GF-R

GM-CSF

HSA

HSC

cell line - clone PI

7

Flow cytometric analysis of growth factor receptor expression on hemopoleUc progenitors

lL-

interleukin-

IP

immunoprecipitation

LTRA

long-term repopulating ability

M-CSF

macrophage colony-stimulating- factor

mAb

monoclonal antibody

NBS

N- hydroxy succinimide

PB

peripheral blood

PBS

phosphate-buffered saline solution·

PCR

polymerase chain reaction

PE

PhycoErythrin

PerCP·

peridinin chlorophyll protein

PHA

phytohemagglutinin

PI

PO

propirlium iodide perpendicular light scatter pregnant mouse uterus extract peroxidase

PSA

PBS; semml azide

R

RELACS

receptor Rijswijk experimental light activated cell sorter

Rhl23

Rhodamine 123

RT-PCR

reverse transcriptase polymerase chain reaction

PLS PMUE

SCF

stem cell factor

(~KL,

Kit ligand; MGF, mast cell growth factor; SF,

Steel factor)

scm STRA

severe combined immunodeficiency short-term repopulating ability

TPO

Tris-buffered saline solution thrombopoietin

WGA

wheat germ agglutinin

TBS

8

CHAPTER 1

General introduction

1.1

Hemopoiesis

1.2 Hemopoietic growth factors (GFs) 1.3 Hemopoietic GF receptors 1.4 Biological activities of hemopoietic GFs 1.5 Examination of GF receptor expression on hemopoietic celis 1.6 Objectives and outline of this thesis 1.7 Literature cited

9

Flow cytometrlc analysis of growth factor receptor expression on hemopoietic progenitors

1.1 Hemopoiesis Blood cells fulfill a number of important functions, including 02 and C02 transport (erythrocytes), blood clotting (platelets), specific immunity and antibody production (lymphocytes), and nonspecific defense against pathogens (monocyte,/ macrophages and granulocytes). Most blood cells have a limited life span, varying from several hours to several months. Replacement of these cells requires a daily production of approximately 2 x 1011 red and 1.5 x lOti white blood cells in an average human adult. Under changed

env!ronmental conditions (e.g., low atmospheric oxygen tension), or pathologic cond,itions, such as blood,loss, tissue damage, or infections, this production capacity may be increased at least IO-fold. The process of blood cell formation, hemopoiesis, takes

place mainly in the bone marrow (EM) in adult mammals. In'fetal hemopoiesis, liver and spleen also play an important role. There is residual hemopoiesis in the spleen of adult mice. All blood cells are derived from a small pool of pluripotent hemopoietic stem cells (HSC) IMcCulloch, 1983) IMetcalf, 1989). Most of these are kept in a quiescent state, only a small subset of HSC is actively proliferating at any moment [Becker et a!., 1965] [Moore, 19911. This population is responsible for the formation of 'daughter cells, which after

progressive cell divisions gradually lose their multipotent differentiation potential and acquire the characteristic phenotypi y and functional properties of the individual blood cell

lineages (figure 1.1). These committed progenitors undergo terminal differentiation into mature blood cells, which are released into the circulation. The mechanism by which the HSC population is maintained and develops into mature blood cells is still incompletely understood. It is possible that blood cell formation is initiated by donal expansion df activated HSC with asymmetrical cell divisions, resulting in simultaneous formation of new HSC that maintain the stem cell pool as well as committed descendants that eventually differentiate into mature blood cells [Holtzer ct ai., 19721. Alternatively, analogous to oocyte development, there might be a limited pool of

HSC, whi~h are acti~ated successively and undergo terminal differentiation into mature blood cells IRosendaal et al .. 19791. According to this theory, the daughter cells are not identical to the parent cell, indicating that 'self renewal' of HSC does not take place. In agreement with such a hypothesis, a decrease in the mean telomere length during proliferation of immature hemopoietic cells into mote differentiated precursors has bee'n observed {Vaziri ct aI., 1994] [Lansdorp, 1995]. Supported by experimental data that demonstrated limits to the self renewal capacity of the stem cell compartment after stress to the hemopoietic system [Mauch et ai., 1988], this would make depletion of the HSC compartment conceivable. However, since the daughter cells that result from the first divisions of a HSC may still be pluripotent, the HSC compartment could be effectively replenished when part of these cells return to a quiescent state.

10

Marg de Jong

Chapter 1

General introduction

figure 1.1 . Schematic representation of hemopoietic stem ceH differentiation.

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11

Flow cytometric analysis of growth factor receptor expression on hemopoietic progenitors

Several models for the mechanism that regulates differentiation of HSC into the different blood cell lineages have been proposed. Commitment to differentiation may be' the result of a stochastic process [Till ct ai., 19641 [Nakahata et aI., 1982] [Tsuji and Nakahata, 1989]. According to this hypothesis, commitment to a specific lineage is determined by intrinsic properties of the cell itself and cannot be jnfluenced by external factors. Alternatively, cells may be directed to enter a particular lineage by specific microenvironmental stimuli and! Or lineage-specific hemopoietic growth factors {Curry and . Trentin, 1967] [Van Zant and Goldwasser, 1977]. According to this model, the direction HSC

differentiation wiII take is determined by the relative concentration of stimulatory and inhibitory signals. However, although in the stochastic model HSC commitment is an intrinsic property of the HSC itself, whether or not these cells will proliferate or differentiate is still dependent on the availability in the microenvironment of the appropriate OFs and other stimuli to which those cells can respond. Therefore, these models are not necessarily mutually exclusive.

1.2 Hemopoietic. growth factors (GFs) Growth factors (GFs) play an imp