the rat bone marrow stromal cell osteogenic … · 111 dose- and time-dependent biphasic effects on...
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THE RAT BONE MARROW STROMAL CELL OSTEOGENIC SYSTEM: CHARACTERIZATION OF SUBPOPULATIONS, FRACTIONATION,
AND EFFECTS OF PDGF
Alexandra knore Herbertson
A thesis submitted in conformity with the requiremen ts for the degree of Doctor of Philosophy
Graduate Department of Dentistry
University of Toronto
Q Copyright by Alexandra Herbertson 1998
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THE RAT BONE MARROW STROMAL CELL OSTEOGENIC SYSTEM: CHARACTERIZATION OF SUBPOPULATIONS, FRACTIONATION,
AND EFFECTS OF PDGF
by Alexandra Lenore Herbertson
Doctor of Philosophy, 1998
Graduate Department of Dentistry, University of Toronto
ABSTRACT
The objective of this thesis was to better define and characterize the heterogeneous cellular
environment of the rat bone marrow (RBM) stromal ce11 osteogenic system. 1 began by
identifying and quantifying the major subpopulations present when RBM stromal cells are
grown under conditions stimulating bone formation. ie., supplementation with
dexamethasone (dex). As expected, dex significantly increased the number of alkaline
phosphatase (AP) positive colonies and von Kossa positive bone nodules, but concomitantly
stimulated the number of macrophage colonies, while reducing the number of hemopoietic
cells expressing leukocyte common antigen. Since the presence of these multiple ce11 types
rnay cornplicate determination of mechanisms of regulation of the osteoprogenitors, we next
investigated methods for purification of the osteoblast lineage cells. Flow cytometric
sorting perfotmed on the basis of AP expression resulted in A P ~ B ~ and A P ~ O W fractions of
cells: within the ~ 9 h i g h fraction. the number of AP-positive colonies and bone nodules were
signi ficantly enriched. Adipocyte and macrophage colonies were signi ficantly depieted in
the M h i g h fraction and consistently enriched in the A P ~ W fraction of cells.
Finally, 1 investigated the effect of PDGF. a Factor produced in abundance by macrophages.
i n fractionated and unfractionated RBM stromal populations. While stimulating
proliferation and differentiation of the macrophage and adipocytic lineages, PDGF had
..- 111
dose- and time-dependent biphasic effects on bone nodule formation. stimulating at low
doses and at early stages of osteoblast differentiation but inhibiting at high doses and later;
suggesting that PDGF has pleiotropic effects on bone metabolism, and that its effects on
bone might be direct on osteoblast lineage cells.
Together, my data validate methods for enrichment of osteoprogenitors in RBM stroma1
cultures and provide a stronger b a i s for investigating the nature of their regulaiion by
hormones, cytokines. and growth factors.
ACKNOWLEDGEMENTS
First and foremost, 1 wish to thank my supervisor. Dr. Jane E. Aubin, for her
unending support, guidance, patience, and understanding during my time in her laboratory.
1 consider myself privileged to have been able to work under her.
1 also wish to thank fellow laboratory mernbers Usha Bhargava, Harry Moe, and Sui-
Wah Fung for their patience in teaching and their excellent technical assistance. As well, 1
wou1d like to thank fellow students Fina Liu and Shiva Shadmand for their friendship and
support during this time.
1 also wish to acknowledge the financial support of the Medicai Research Council of
Canada through their Dental Fellowship program, and the suppon of the Harron Trust Fund,
without which this work would not have been possible.
Finally. 1 would like to extend my love and thanks to my husband, Dale Herbertson.
for al1 the years of support and understanding.
TABLE OF CONTENTS
Page
ABSTRACT
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
LIST OF FIGURES
LIST OF TABLES
ABBREVIATIONS
C)IAPTER 1 Introduction
1.1 General Introduction
1.2 Non-Osteogenic Stromal Ce11 Types
1.3 Osteogenic Stromd Cells .
1 -4 S tromal CeIl Interactions
1.5 Purification of osteoblasts from stroma
1.6 Cytokines and Growth Factors in the Stromal Environment
1 -7 Platelet-Derived Growth Factor (PDGF)
1.8 Thesis Objectives
C HA PTER 2 Dexarnetliasone Alters the Subpopulution Make- Up of
Rat Bone Marro w Stromal Cell Cultures
2.1 Introduction
2.2 MateriaIs and Methods
2.3 Results
2.4 Discussion
V
vii
viii
CHAPTER 3 CeU Sorting Enriches Osteogenic Popularions in Rat Bone
Marrow Struma1 Cell Cultures 49
3.1 Introduction 50
3.2 Materiais and Methods 53
3.3 Results 55
3 -4 Discussion 69
CHAPTER 4 PDGF has Biphosic Effecfs on the Differenfiation of
Osteoprogenilon in RBM Sbomal Ce11 Populations
4.1 Introduction
4.2 Materiais and Methods
4.3 ResuIts
4.4 Discussion
CHAPTER 5 Geneml Surnrnmy and Conclusions
5.1 Conclusions and Future Experiments
vii
LIST OF FZGURES
Figure 2.1
Figure 2.2
Figure 2.3
Figure 2.4
Figure 2.5
Figure 2.6
Figure 2.7
Figure 3.1
Figure 3.2
Figure 3.3
Figure 3.4
Figure 4.1
Figure 4.2
Figure 4.3
Figure 4.4
Figure 4.5
Figure 4.6
Overviews of 35 mm RB M strornai ce11 culture dishes
Time course of bone noduie formation in RBM stromal ce11 cultures
AP positive colonies in RBM stroma1 ce11 cultures
aNE3E colonies in RBM strornai ce11 cultures
Micrographs of the different cell types present in RBM stroma
Micrographs of immunohistochemical staining of RBM stromal cells
Flow cytometry frequency vs. fluorescence intensity histograms
Flow cytometric set up for sorting
Flow cytometry analysis
Adipocyte colony numbers for sorted RBM stromal populations
Bone noduleslaNBE colonies vs. plating density (soned populations)
Ce11 number vs. time cultured, with and without PDGF
Bone nodules vs. PDGF concentration
Number and size of aNBE colonies vs. PDGF concentration
Adipocyte colonies vs. PDGF concentration
Bone nodules vs. PDGF pulses
PDGF's effects on sorted RBM stromal populations
Page
37
38
39
40
41
42
43
63
64
66
67
84
85
86
87
88
89
LIST OF TABLES
Page
Table 1.1 PDGFs effects on bone formation in osteoblastic ce11 cultures 20
Table 2.1 Quantitation of colony types in RBM stroma1 ce11 cultures 36
Table 3.1 Percentage of AP-positive colonies in sorted RBM stromai populations 60
Table 3.2 Net changes in various colony types in ~ ~ h i g h and ~ ~ 1 0 ~ sorted cells 61
Table 3.3 Net changes in bone nodules/aNBE colonies for sorted cells 62
Table 4.1 PDGFs ffects on bone nodule, adipocyte and aNBE colonies 82
Table 4.2 Effect of continuous exposure to PDGF on RBM stroma1 populations 83
aNBE AP BM CDR CFU-F
CFU-O
CFU-ST
d,
dex
ED2
EGF GAM GM-CSF
HPR hr.
IL- 1
IGFBP
LCA
LNGFR
M
mAB
M-CSF
MRC OX- 1
MRC OX-22
PDGF
PGE2
m RBM 21 1.13
RBM
RECA- 1
TNF
a-naphthyl butynte esterase
aikaline phosphatase
bone marrow
colour development reagent
fibroblastic colony-forming unit
osteoprogenitor colony-forming unit
stroma1 colony-forming unit
day(s) dexamethasone
monoclonal antibody specific against rat macrophage cells
epidermal growth factor
goat anti-mouse
granuIocyte/rnacrophage colony-stimulating factor
horseradish peroxidase hour(s ) interleukin 1
insulin-like growth factor binding protein
leukocyte comrnon antigen
low-affinity nerve growth factor receptor
molar
monoclonal an tibody
macrophage colony stirnulating factor
monoclonal an tibody against leukocyte cornmon ant igen
monoclonal antibody against nt lymphocytes
platelet-det-ived growth factor
prostaglandin E2
parathyroid hormone
monoclonal antibody specific against rat alkaline phosphatase
rat bone marrow
monoclonal antibody specific against rat endothelial cells
tumor necrosis factor
CHAPTER 1
Introduction
1.1 Generd Introduction
Bone manow (BM) is a complex tissue consisting of many different ce11 types with distinct
functions existing together in a coordinated, regulated and inter-dependent fashion. BM can
be divided into two broad cornpartments: the hemopoietic cells. a primarily nonadherent
population in ce11 cultures, and the strornal cells, primarily adherent in ce11 cultures.
Regulation of mammalian hemopoiesis is dependent on a microenvironment composed of a
variety of hemopoietic cells and strornal cells and their extracellular rnatrix (Dexter, 1982;
Ross et al., 199 1 ; Long, 1992). it also appears that stromal ce11 growth and development.
including those of osteogenic cells. is regulated by the local microenvironment. as
hemopoietic cells c m produce cytokines known to affect the growth and differentiation of
stromal cells, and BM stromal cells can release factors affecting other strornal cells (Ogiso et
al., 1991: Yanai et al.. 1991; Zhang et al., 1991; Sasaki and Hong, 1993).
1.2 Non-Osteogenic Stroma1 Ce11 Types
It has been recognized that BM stroma contains a mixed population of ceIl types (Dexter,
1982). but many published reports remain focused on only one or a few of the ce11 types in
the culture. In mouse hemopoieticaily active long-term BM stromal cultures. the cell
populations have been identified as: 2 1 + 2% fïbroblastoid. 3 I 0.3% endothelial, 26 + 3%
adipocytic. and 49 + 1% macrophage (Hauser et al., 1993, accounting for essentially al1
(99%) of the stroma. In rat bone marrow (RBM) stromal ce11 cultures, Iittle is known about
the subpopulation make up. except that in 10 - 12 d. primary cultures the colony distribution
was found to be 85% fibroblast-like, 10.3% rnacrophage/dendritic/granulocyte, and 2.5%
endothelial (Sirnmons et al., 1991). Maniatopoulos et al. (1988) also noted in primary RBM
stromal cultures the presence of small, mononuclear (hemopoietic) cells (Maniatopoulos et
3 al., 1988); the repeated medium changes in the 7 d. pnmary culture period in Maniatopoulos'
method is used to reduce hemopoietic contamination and cellular debris while expanding the
numbers of CFU-F. Even in the culture conditions selected to maxirnize osteogenesis, only
a proportion of the cells in RBM stromal ce11 cultures are osteogenic, and the presence of
other morphologically and histochemically/irnmunohistochemically distinguishable ceIl
types has been reported (Malaval et al., 1994). The multiple ceIl types present reflect, at
least in part, the multiple l ineages in marrow stroma, including also muitipotential
mesenchymal stem cells with the ability to differentiate dong various pathways. As the
presence of multiple subpopulations complicates the elucidation of cell-ce11 interactions
within osteogenic stroma, documentation of which cells are present and in what quantities is
needed to unravel the interactions. A brief summary follows of the ce11 types that have been
reported in BM stromal ce11 cultures (of a variety of species).
This group includes definitive fibroblasts involved in forming the supporting connective
tissue of marrow. However, the designation of "fibroblast" has also been used as a general
category of BM stromal cells which are undefined or undifferentiated. The term colony
forming unit - fibrobIast(oid) or CFU-F has been used in reference to colonies which c m
include fibroblasts, adipocytes, endothelial cells, chondro- and osteoprogenitor cells, smooth
muscle cells, reticular and reticular-fibroblastoid cells, and mesenchymal stem cells often in
combination wi th macrophages. mast cells, myeloid cells, and hemopoietic stem cells
(Wang et al., 1990; Benayahu et al., 1991; Simmons et al., 1991). Another term used is
colony forming unit strornal or CFU-ST for strornal or adherent cells (McIntyre and
Bjornson, 1986). Assessrnents of results from experiments designating colonies as CN-F
can be difficult to interpret i n light of this potential variety in ce11 composition. For
example, PDGF is known to increase growth of CFU-ST (McIntyre and Bjornson, 1986),
but whether this is a response from multiple populations of cells or just one ce11 type is not
4 known. A monoclonal antibody (mAb), STRO-1, was developed which (in combination
with a glycophorin A negative screen) sorts CFü-Fs from other human BM cells (Simmons
and Torok-Storb, 199 1 ). Cells sorted on this bais (glycophorin-negative, STRO- 1 -positive)
give rise to fibroblasts, smooth muscle cells, and adipocytes which results in an adherent
layer able to support hemopoiesis (Simmons and Torok-Storb, 199 1 ). Osteoprogenitors
capable of differentiating into functional osteoblasts are also present in adult human BM-
derived STRO- 1 -positive C N - F (Gronthos et al., 1994). Taken together, the data suggest
that STRO- 1 may be associated - although not exclusively - with stroma1 mesenchymal stem
cells. More recently, Joyner et al. (1997) have also isolated a mAB (HOP-26) reactive
against human CFU-F, including osteoprogeniton (Joyner et al., 1997). Amongst a series of
mABs developed against RBM stroma, ST3 and ST4 are reactive against fibroblasts
(Sullivan et al., 1989) but ST3 labels primarily marrow fibroblasts and ST4 labels pnmarily
fibroblasts in other tissues. suggesting that the fibroblastic cells of the marrow are different
from those of non-hemopoietic tissues (Sullivan et al., 1989). Treatment with ST3 and
complement pnor to culture decreases CFU-F (and hemopoietic colony formation) (Sullivan
et al.. 1989). While the development of mAbs has aided in identification of fibroblastic cells
in BM, many cells are labelled fibroblastic for want of other identifying characteristics, and
the term undoubtedly encompasses a variety of ce11 types.
Endothelial cells
Endothelial cells have been frequently. although not consistently, identified in BM ce11
cultures. lsolated endathelial ce11 colonies can be obtained from mouse BM with low
plating numbers of less than 20 BM stroma1 cells/35mrn dish in medium supplemented with
GM-CSF (10 UII) (Wang and Wolf, 1990). Mouse endothelial cells have dendritic
cytoplasmic processes, round nuclei, and overlapping cytoplasms in confluent cultures
(Yanai et al., 199 1). Histo- and irnmunohistochemical identification of endothelial cells has
historically posed a problem. Endothelial cells, like macrophages, will take up low density
5 lipoprotein. In mice, endothelial cells bind Ulex europaeus 1 lectin (Yanai et al.. 1991).
Factor VI11 antigen may or may not be detected in mouse endotheliai cells (Wang et al.,
1990; Yanai et ai., 199 1). Polyclonal antibodies against von Willebrand factor are not
endothelial cell-specific and do not identify al1 endothelial cells (Duijvestijn et al.. 1992).
While alkaline phosphatase (AP) activity has been reported in endothelial cells, high activity
appears to be associated with isolated brain capillary endothelial cells in vivo. while
endothelial cells lining blood vessels elsewhere do not contain significant levels (Meyer et
al., 1990). The recent development of mAbs specifically recognizing endothelial cells in
mouse marrow (Hasthorpe et al., 1990) and in rats (RECA- 1 = rat endothelial ce11 antigen)
(Duijvestijn et al., 1992) has made definitive identification of these cells easier.
Reticular ceils
Reticular cell is a histomorphornetric term for the adventitial ce11 which forms the outer
layer surrounding the sinuses in the BM; the reticular ce11 is thought to send out sheet-like
extensions containing reticular fibres to fom a three dimensional network (reticulum) of
cytoplasmic extensions throughout the marrow (Westen and Bainton, 1979). The reticular
ce11 is fibroblastic in appeannce, is characterized by its membrane-bound AP. and associates
mainly with granulocytic precursors (Westen and Bainton. 1979). Antibodies against low-
affinity nerve growth factor receptor (LNGFR) label stroma1 reticular cells with dendritic
features in fresh smears and in formalin-fixed. paraffin embedded hurnan BM (Cattoretti et
al., 1991). These LNGFR positive cells are also positive for AP, reticulin. collagen type iII,
and are negative for neural, endothelial, and leukocyte markers (Cattoretti et al.. 1991).
LNGFR positive cells have an oval nucleus, a scanty cytoplasm with long dendrites that
interrningle with hemopoietic ceils, line the abluminal side of sinus endothelial cells. and
provide the scaffold for the hemopoietic marrow (Cattoretti et al.. 1991). The reticular
stroma1 cells appear in the fetal BM before the hemopoietic activity begins, originate from
the vesse1 adventitia, and radiate in the BM cavity (Cattoretti et al., 1991). Bamer ceIl is
another term for a reticular cell described by Weiss and Geduldig (199 1 ) in a microscopie
histological study of mouse BM. Bamer cells run from the bone-lining layer to subosteal
blood vessels and appear associated with al1 stages of hemopoiesis (Weiss and Geduldig,
1991). Recent works have described ce11 correlates in vitro to the reticular ce11 in vivo.
Anti-LNGFR antibodies label 7 - 11% of the fibroblastoid population in 4 week-old long-
term human BM cultures, suggesting that these cells are reticular cells (Cattoretti et al.,
199 1). In another study. LNGFR positive cells with multiple, long dendritic processes were
similarly found in long-tenn human BM cultures; while few were seen in 2 week old
cultures, their numbers increased up to 6 weeks (Wilkins and Jones, 1995). Since AP is a
comrnon cytochemical marker of both 'reticular' cells and preadipocytes, it has been
suggested that reticular cells may convert to adi pocytes when marrow cellularity abruptly
decreases (Bianco et al., 1988). In murine BM cultured in methylcelluIose/plasma dot,
white blood ceIl conditioned medium, dexamethasone (dex) and serum, extensive branching
colonies developed which were called reticulofibroblastoid and were thought to become
adipocytic by d. 14 of culture (Ross et al., 1991). It is unclear whether these cells represent
a differentiation stage in a preadipocytic lineage, or a multipotential stem ce11 lineage, or
mixed colonies derived from multiple lineages as whole marrow ceIl cultures were used.
However, the authors felt that reticulofibroblastoid ceils are a distinct lineage on the basis of
morphology, adipocyte formation, and production of collagen type IV, and with their long
branching arms rnay be important in setting up compartments for hemopoiesis in mouse BM
(Ross et al., 1991).
Adipocytes
Adipocytes are a well-known component of BM. In long-term BM cultures, the presence of
fat ceIIs is taken as a marker of a hemopoietically productive culture (Ross et al., 1991).
Adipocytes have been descnbed in combinations with many other populations of marrow
cells, e-g., reticulofibroblastoid cells as previously mentioned (Ross et al., 199 1). Bennett
7 and others ( 199 1) described stroma1 colonies from rabbit BM with fibroblastic morphology
that had differentiated in an adipocytic direction, but that subsequently reverted to a more
proliferative stage and differentiated dong the osteogenic pathway (Bennett et al., 1991).
These, and other data, have given rise to the notion of a bipotential adipocyte-osteoblast
progenitor or adipocyte-osteoblast switch. For example, long-term munne marrow culture
stromal clone (BMS2) has been produced which can differentiate into adipocytes; while
BMS2 has been used to examine the regulation of preadipocytic cells (Gimble et al., 1991),
it also demonstrates osteogenic characterstics (Dorheirn et al., 1993). The data are
interesting, but further work will be needed to fully characterize the nature of the adipocyte-
osteoblast phenotypic transformation (see, for e-g.. (Gimble et al., 1996; Aubin and
Heersche, 1997), for reviews).
Smooth muscle cells
In long-term human BM cultures. fibroblastoid cells expressing a-smooth muscle actin have
also been described (Wilkins and Jones, 1995). These cells appear at approximately 2
weeks, and form a majority of the stromal population within 4 - 6 weeks, under at least some
culture conditions (Wilkins and Jones, 1995). In long-term adult human BM cultures,
stromal myoid componenü were also present when the stromal cells were cultured withour
hydrocortisone and horse serurn: the presence of the latter two additives actually inhibited
myoid ce11 growth and development (Bonanno et al., 1994).
Hemopoietic Ceils
Hemopoietic cells exist in intimate association with stroma1 cells in BM in vivo and in vitro.
To remove the majority of well-differentiated hemopoietic cells when culturing stromal
cells, centrifugation or adherence techniques are commonly used. Hemopoietic stem cells in
rnouse BM cm be enriched by adherence to tissue culture plastic, but it is not known if the
cells are adhering to the plastic or to a stromal ce11 (Kiefer et al., 199 1). In mouse BM,
hemopoietic stem cells are present at a frequency of 1 in 25,000 in the adherent ce11 layer
(Kiefer et al., 199 1). and their subsequent differentiation is influenced by the existing culture
conditions. Hernopoietic cell types described in BM stromal cultures include macrophages
(Dexter, 1982: McIntyre and Bjomson, 1986: Wang and Wolf. 1990; Keller et al., 1993;
Wilkins and Jones, 1995)- mast cells (Mclntyre and Bjornson, 1986), dendritic cells (Giesler
et al., 199 1 ; Miyagi and Kimun. 1993, osteoclasts (Udagawa et al., 1990), granulocytes
(Simmons et al., L99 1), and megakaryocytes (Briddell et al., 1992).
Macrophages have long been known as a component of long-term BM stroma1 cultures
(Dexter, 1982). In rnice, macrophages are frequently seen in BM stromal cell cultures and
isolated macrophage colonies can be obtained at the low plating density of 20 or less BM
stroma1 cells/35mrn dish using media supplernented with GM-CSF at 10 UA (Wang and
Wolf, 1990). Mouse macrophages cm be identified with mAbs or histochemical stains such
as berberine sulphate or a-naphthyl butyrate esterase ( aNBE) (Kanemoto et al., 199 1 ). In
human BM cultures. macrophages have been identified in the adherent layer (Keller et al.,
1993; Wilkins and Jones. 1995). While one study found a rather homogeneous macrophage
population (Keller et al., 1993)- another identified two types of macrophages (Wilkins and
Jones, 1995). One type had a rounded morphology and abundant phagocytosed debris,
while the other type had cells that were elongated and flattened, dispalyed several short
cytoplasmic processes, and lacked phagocytosed debris (Wilkins and Jones, 1995). The
second type of macrophage was seen only in more mature cultures (Wilkins and Jones.
1995). The number of macrophages present varied significantly between long-term BM
culture isolates. and did not correlate with the number of hemopoietic foci present (Wilkins
and Jones, 1995).
1.3 Osteogenic Stromal Cells
Friedenstein and colleagues first demonstrated that BM stroma contains ceils with the
capacity to f o m bone when they are transplanted in vivo in diffusion chambers
(Friedenstein et al., 1968). This has been confirmed repeatedly in vivo with BM cells in
diffusion chambers (Ashton et al., 1984; Owen, 1988) and, more recently, conditions have
been established in which bone formation is observed in vitro in cultures of BM stromal
cells isolated from several species. Tibone first reported bone production in vitro by BM
strornal cells in aspirated femoral marrow from adult rabbits (Tibone and Bernard, 1982).
Since then, other nonclonal animal models of BM osteogenesis, with bone nodules as the
terminal product, have also been characterized and optimized for more reproducible and
more copious osteoblastic differentiation. Under culture conditions previously established
to support and rnaximize osteogenesis in cultures of folded chick periostea (Tenenbaum and
Heersche, 1982) and osteobtastic cells derived from calvaria (Bellows et al., 1986), bone
nodule formation in cultures of stromal cells has been seen in young-adult rat femoral BM
(Maniatopoulos et al.. 1988), chick embryo tibial and femoral BM stroma (Kamalia et aI.,
1992), neonatal pig tibial and femoral BM stromal cells (Thomson et al.. 1993). young adult
mouse femoral BM stroma (Van Vlasselaer et al.. 1993), and adult human BM (Vilamitjana-
Amedee et al., 1993). However, optimal conditions for the formation of discrete bone
nodules in these different BM strornal cultures differ, likely refiective of species or donor
age differences. For example, culture conditions similar to those used in rat stroma
(Maniatopoulos et al., 1988) result in rnineralization without nodule formation in adult
human stroma (Cheng et al., 1994). This may in part be a reflection of the age of the BM
donor. as age has been reported to affect the yield of nodules and/or minenlized matrix (see
discussion in (Vilamitjana-Amedee et al.. 1993)), and discrete bone nodules have been
reported in young adult human BM stroma (Vilamitjana-Amedee et al., 1993). The
importance of species differences is obvious when looking at dex as an additive: 10-8 M dex
10 increases the apparent number of osteoprogenitor cells which give rise to bone nodules by 5 -
50-fold in RBM stromd cells (Aubin et ai., 1990) while 10-7 M dex has been detennined to
be optimal for minerdization in a human system (Cheng et ai.. 1994), and concentrations up
to 10-10 M significantiy reduce bone nodule formation in mouse BM stroma (Falla et al..
1993). As well, mouse BM stroma needs to be plated at higher densities to obtain CFU-F
numbers comparable to those in the rat system (unpublished observations), resulting in at
least some attempts to develop strategies for enrichment of osteoprogenitors after general
isolation protocols (Falla et al., 1993).
The mineralized bone nodules formed in these mixed stromai population systems have
morphological characteristics similar to authentic woven bone with its associated cells.
Specifically. in M M stromal cultures, a bone nodule has rows of cuboidal cells with oval
nuclei and prominent nucleoli, well-developed rough endoplasmic reticulum, and prominent
Golgi (Satomura and Nagayarna, 199 1 ) on the surface, separated from the mineralized
material by an osteoid-like matrix; cells resernbling osteocytes are embedded in the
rnineralized matrix (Maniatopoulos et al.. 1988) and sorne cells protmde ce11 processes
toward neighbouring cells through the extracellular matrix (Satomura and Nagayama, 199 1 ).
X-ray microanalysis and X-ray diffraction respectively have been used to show the presence
of Ca and P in the matrix. in the fom of hydroxyapatite (Maniatopoulos et al.. 1988). RBM
stromal cells express PTH receptors (Zhang et al.. 1995), and the cells associated with
nodules exhibit high AP activity (Maniatopoulos et al.. 1988) and produce type 1 collagen
and SPARC/osteonectin, dong with the non-collagenous bone proteins osteocalcin and bone
sialoprotein (Maniatopoulos et al., 1988; Kasugai et al.. 199 1 ; Malaval et al.. 1994).
1.4 Stroma1 Ce11 Interactions
General cell-cell interactions
While different BM stromal cells are known to interact, in most cases the mechanism of the
interaction is not clear, although multiple cytokines and growth factors produced by
different ce11 types present are likely to play roles (see Section 1.6 below). For example,
adipocytes can stimulate the growth of endothelial cells. while endothelial cells can inhibit
the growth and developrnent of preadipocytes (Yanai et al.. 1 99 1 ). Rat marrow stromal
fibroblast-like cells produce a number of osteoiropic factors that at low concentrations
stimulate, or at high concentrations inhibit, calvarial osteoblast growth and there is evidence
for the involvement of insulin-like growth factors (Zhang et al., 199 1). Rat skin fibroblasts
and periodontal ligament fibroblasts release soluble factors into their conditioned medium,
including prostaglandins, that inhibit osteoblast differentiation in BM stroma (Ogiso et al..
1991). On the other hand, bone formation is increased in stroma1 cell cultures when
conditioned medium from rat calvaria cells is added, suggesting that mature osteoblasts
produce a paracrine growth factor that can stimulate the differentiation of osteoblasts from
precursor cells (Hughes and McCulloch, 1991). It has also been suggested that factors
produced by BM cells may affect other BM stromal cells that are distant from the factor
source; healing RBM produces a growth factor activity with a preferential effect on
osteogenic cells (Bab et al., 1988). As well. marrow ablation in rat long bones induces an
increase in osteogenesis in distant skeletal sites and the systemic osteogenic response is
thought to be rnediated by circulating factors produced by the healing marrow (Bab et al.,
1988; Gazit et al., 1990; Bab et al., 1992).
Limiting dilution studies
In rat calvaria and RBM stromal bone mode1 systems, the nurnber of nodules or colonies
capable of forming bone can be counted for an assessment of osteoprogenitor numbers
12 recoverable from the tissue and able to undergo differentiation in vitro under certain test
conditions (e-g.. rat calvaria (Bellows and Aubin, 1989); RBM stroma (McCulloch et al.,
1991)). Limiting dilution analysis was used to investigate the differentiation process of
these restricted monopotential osteoprogenitor cells in these primary cultures. In rat calvaria
ce11 populations, limiting dilution indicated a "single-hit" phenornenon, i.e. that only one
ce11 type was limiting for nodule formation; the data are consistent with the lirniting cell type
being the osteoprogenitor itself, and with such cells being present at a measurable but low
frequency ( 4 % ) of the total population under standard isolation and culture conditions
(Bellows and Aubin, 1989). In contrast, in RBM stromai secondary populations expression
of neither total CFU-F nor the osteoprogenitor subfraction (Cm-O) follows a linear
relationship in limiting dilution analysis until very high ce11 densities are reached, suggesting
cooperativity of ce11 types within the population and a multi-iarget phenornenon (Aubin et
al., 1990). In multi-target events. the response (production of a bone nodule) is observed
only when at least one ce11 of each of rn categories is present in the sample (Lefkovits and
Waldmann. 1979). i.e., that at least two ce11 types are limiting for bone nodule formation in
RBM stroma. When a constant number of non-adherent (hemopoietic) BM cells was added
back to the limiting dilution analyses of the stroma1 cells, about 11100 CFU-O were
measured and their expression now followed a Iinear, single-hit relationship (Aubin et al.,
1990). Endothelial cells or endothelial ce11 conditioned medium had similar effects on CFU-
O expression (Aubin et al.. 1990). Similarly. formation of bone nodules in vitro from
murine marrow CN-F plated nt low density requires the presence of irradiated feeder celis,
and PDGF. L-3. and EGF appeared not to be able to substitute for feeder cells. leading to
the suggestion that another as yet unidentified factor produced by the imdiated cells was
required (Friedensrein et al.. 1992). These data suggest that stroma1 osteogenesis in RBM
may be under the regulation of other cells in the BM which can exert either stimulatory or
inhibitory activities.
1.5 Purification of osteoblasts from stroma
Given the multiple subpopulations reported to be present in stroma, methods for enriching or
separating the osteogenic subpopulations is desirable. Several strategies have been used to
produce enriched osteoblast populations from mixed stromal populations, but these can be
grouped into adherence techniques or antibody-based techniques.
Adherence characteristics
While nonadherence to plastic has been reported to be characteristic of the 'majonty' of
osteoprogenitor cells in mouse (Falla et al., 1993) and human (Long et al., 1990; Long et al.,
1995) BM, most BM stromal nodule systems rely on (slow) adherence to plastic for isolation
of osteoblastic cells. There is comparable osteoprogenitor plating efficiency in control and
nonadherent (1 hr. at 37" C in a tissue culture flask) rnouse marrow cultures (Falla et al.,
1993) and in control and nonadherent (24 hr. at 37' C in a tissue culture flask) primary RBM
cultures (Scutt and Bertram, 1995). In the original descriptions of osteogenic rat
(Maniatopoulos et al.. 1988) and chick (Kamalia et al., 1992) BM, stromal cells were
allowed to attach for 24 hr. prior to the first medium change (and medium is changed
cornpletely 3 times per week thereafter). The frequent medium changes are thought to
reduce hemopoietic contamination (Maniatopoulos et al., 1988; Kamalia et al.. 1992) while
expanding the CFU-F (Maniatopoulos et al., 1988). of which osteoprogenitors are a
subgroup; however, significant numbers of hemopoietic cells continue to be present into the
secondary culture even with this protocol (Herbertson and Aubin. 1995). In protocols
described for adult human BM (Cheng et al., 1994). mouse BM (Van Vlasselaer et al.,
1993). and porcine BM (Thomson et al.. 1993). longer adherence times are used. and the
medium is changed only after 5 - 7 d. of primary cultures. Scutt and colleagues have
reported recently that PGE2 can induce cells in the nonadherent population in RE3M to fom
adherent CFU-F, a proportion of which are osteoprogenitors which give rise to osteoblasts
14 which synthesize bone-like tissue (Scutt and Bertram. 1995). In these studies, the action of
PGE2 was shown to be dependent on the glucoconicoid (exogenously added dex) levels
present in the cultures. suggesting that both time and cytokine conditioning are factors to be
considered in the establishment of protocols for enrichment of osteoblastic populations.
An tibodies
The finding that cells at different stages of the osteoblast lineage express antigenic
determinants unique or distinctive to these stages is beginning to be useful for identification
and isolation of osteoblastic cells at various stages of differentiation (Aubin and Turksen,
1995). Thus, antibodies may be useful not only for identification of diverse ce11 lineages
within stroma1 populations, but also to charactenze differentiation stages within the
osteoblast lineage and to fractionate populations based on epitope expression. Of antibodies
raised against osteoblastic cells and reported to date, those recognizing the more mature cells
in the lineage (osteoblasts and osteocytes) have been more commonly found than those
recognizing less mature cells. Enrichment of relatively mature osteoblastic cells has been
reported. Van Vlasselaer and colleagues used irnmunopanning with mAbs to first deplete
populations of hernatopoietic cells, then used flow sorting with Sca-1 and wheat germ
agglutinin binding to purify osteoblastic cells from the 5-fluorouracil treated femoral BM of
young adult mouse (Van Vlasselaer et al., 1994). Two mAbs (OB 7.3 and SB-5) have been
nised that label the surface of chick osteocytes specifically and OB 7.3-coupled magnetic
beads have been used to isolate relatively pure osteocytes from chick mixed bone ceIl
populations (Van der Plas and Nijweide, 1992). Several an tibodies are available that
recognize early as well as late stage osteoblastic cells, e.g. RCC455.4. which we recently
isolated (Turksen et al., 1992) and found by expression cloning to recognize galectin 3
(Aubin et al., 1996). lmmunopanning of human BM cells with osteocalcin and osteonectin
enriches for osteoblastic cells (Long et al., 1995). Antibodies against AP have been raised
by several labs (in Our labontory, RBM 2 1 1.13 was raised against RBM stroma (Turksen et
15 al., 1992)); these label preosteoblastic cells as well as osteoblasts. In the rat calvarial
system, AP expression has been used successfully to fractionate osteoblastic populations
into AP-positive and -negative populations (Turksen and Aubin, 1991). Most of the
osteoprogenitors giving rise to osteoblasts making bone in vitro in the absence of added
glucocorticoids (dex) reside in the AP-positive pool. while osteoprogenitors present in the
AP-negative pool require dex to differentiatiate. AP expression has also been used for flow
cytometric sorting in female Rl3M stromal ce11 cultures, but production of a mineralized
matrix after sorting was only descibed in cells which were AP negative at the time of sorting
(Rickard et al., 1994). There are few reports that describe antibodies recognizing ceils
earlier than the preosteoblast. Haynesworth et al. (1992) irnmunized with human marrow
stromal cells to isolate antibodies that react on a subset of marrow stromal cells and a variety
of tissues in vivo, but do not label osteobiasts or osteocytes (Haynesworth et al., 1992). The
authors concluded that the antibodies may recognize molecules present on early progenitors
or stem cells and that the epitope(s) is lost during osteogenic differentiation, but conclusive
support for this awaits further characterization (Haynesworth et al., 1992). STRO-1
recognizes mesenchymal progeniton in human marrow stromal cells with the ability to form
cells with the phenotype of fibroblasts, adipocytes. smooth muscle cells. and osteoblasts
(Simmcns and Torok-Storb. 199 1 ; Gronthos et al.. 1994). Recently. HOP-26 was described
as an antibody strongly reactive with cells in marrow stromal colonies at early stages of
differentiation, before the induction of AP, and HOP-26 does not bind to mature osteoblasts;
immunopanning with HOP-26 selected the marrow stroma1 fibroblastic colony-forming
units (CN-F) (Joyner et al., 1997). Bruder and colleagues (Bmder et al., 1997) have also
recently reported an antibody which they believe recognizes human mesenchymal stem
cells. As yet, similar antibodies to early progenitors have not been reported for the RBM
stromal system.
1.6 Cytokines and Growth Factors in the Stroma1 Environment
The rich cytokine milieu resulting from production by both hernatopoietic and stromd celIs
creates a complex environment in which both immature osteoprogeni tors and mature
osteoblasts reside. For example, G-CSF (Felix et al., 1988; Greenfield et al., 1993;
Taichman and Emerson. 1994), GM-CSF (Felix et al., 1988; Horowitz et al., 1989; Horowitz
et al., 1989; Weir et al., 1989: Felix et al., 1991; Benayahu et al.. 1992; Greenfield et al.,
1993), M-CSF (Sato et al., 1 986; Shiina-Ishimi et al.. 1986; Elford et al., 1987; Sato et al.,
1987; Felix et al.. 1989; Greenfield et al., 1993; Weir et al.. 1993; Lacey et al.. 1994), IL- la
(Greenfield et al., 1993). ILIP (Birch et al.. 1993; Dodds et al., 1994; Bilbe et al., 1996;
Shadmand and Aubin, 1997), IL-3 (Felix et al., 1988; Birch et al., 1993), IL-5 (Greenfield et
al., 1993), IL-6 (Ishimi et al., 1990; Birch et al.. 1993; Greenfield et al., 1993; Dodds et al.,
1994; Lacey et al., 1994; Bilbe et al., 1996; Malaval et al., 1997; Xiong et al., 1997). IL-7
(Greenfield et al.. 1993). lL-8 (Birch et al., 1993; Chaudhary and Avioli, 1994; Bilbe et al..
1996), IL- 1 1 (Bilbe et al., 1996), LIF (Greenfieid et al., 1993; Bilbe et al., 1996; Malaval et
al.. 1997), ONC-M (Bilbe et al., 1996). PDGF-u (Graves et al., 1989: Zhang et al.. 1991:
Bilbe et al., 1996), PDGF-P (Bilbe et al., 1996: Canalis and Rydziel, 1996). SCF (Bilbe et
al., 1996), TGF-P (Birch et al., 1993; Greenfield et al., 1993; Dodds et al., 1994; Bilbe et al.,
1996). TNF-a (Birch et al., 1993; Greenfield et al., 1993; Bilbe et al.. 1996), and TNF-8
(8 ilbe et al.. 1 996) are produced by osteoblastic cells. Glucocorticoids are known important
regulators of cytokine and growth factor production in many cell types. and this laboratory
has been particularly interested in glucocorticoid regulation of osteoprogenitor
differentiation in both calvarial ce11 cultures and in the RBM stroma1 populations. In RBM
stromal cultures, there is little bone formation in the absence of dex. Work reported in this
thesis will address whether the effect of dex is direct on osteoprogenitor cells or indirect
through its ability to alter the numbers and kinds of other lineages present which would
concomitantly alter the cytokine and growth factor milieu of the stromal cultures. In
17 particular. the ability of dex to stimulate the maturation of monocyte lineage cells into
macrophages (see Chapter 2 and (Herbertson and Aubin. 1995)) would be expected to alter
the levels and kinds of cytokines and factors produced by cells of this lineage, including
PDGF, whose effects on osteoprogenitor differentiation 1 have investigated in detail
(Chapter 4).
1.7 Platelet-Derived Growth Factor (PDGF')
PDGF - General Notes
PDGF is well-known as a mitogen for mesenchymal cells and appears to have broad roles in
tissue development. PDGF exists as 30 kDa disulphide bonded homo- or hetero-dimen of A
and B chains which interact with the two PDGF receptors, a and P. PDGF-B, the normal
counterpart of the oncogene v-sis of the simian sarcoma virus (Westermark and Heldin,
199 l) , has been shown to have stimulatory effects on primitive pluripotential hemopoietic
cells in mouse BM (Yan et al., 1993) and overexpression of PDGF-B in murine hemopoietic
cells induces a lethal myeloproliferative syndrome in vivo with anemia, neutrophilia and
monocytosis (Yan et al.. 1994). PDGF and its receptors show appositional expression in
epitheliallstromal tissues. and appear to be involved in stromal-mesenchymal interactions
during normal embryonic development and in basal ce11 carcinomas (Ponten et al.. 1994).
Measurements of transcript levels during mouse development have shown that PDGF A
chain and PDGF a-receptor are expressed early, while PDGF B chain and PDGF fi-receptor
appear to becorne important later in development (Bowen-Pope et al., 1991). In normal
embryos, PDGF a-receptor is expressed in both mesodermal and neural crest derived
mesenchyme (Schatteman et al.. 1992). The PDGF a-receptor gene is deleted in the Patch
mutant mouse (Bowen-Pope et al., 199 1 ): in hornozygous mutants many mesodermal
derivatives are poorly developed and the embryos do not survive until birth (Bowen-Pope et
18 al., 1991). Specifically in terms of skeletai effects, homozygous embryos demonstrate
severe vertebral malformations. spina bifida and cleft face. and distoriions of other bones
formed by endochondral ossification (Schatteman et al., 1992). The Patch heterozygotes
express half the wild-type level of PDGF a-receptor transcripts and protein and suivive with
minimal defects: an increase in the width of the prefrontal bone and absence of functional
melanocytes (Bowen-Pope et ai., 1991). The Patch mutant mice suggest that PDGF is very
important in mesodemal and skeletal development.
PDGF in Non-Os feogenic Stroma
Fibroblasts and macrophages are sources of PDGF (Bowen-Pope et al.. 1991) and we have
previously reported that both ce11 types are present in RBM stroma1 cells under conditions
which prornote bone formation (Herbertson and Aubin. 1995). PDGF appears to be stored
in the bone matrix. and the trapping or sequestering of PDGF could affect its biological
action through complex modes of release and presentation to responding cells (Hauschka et
al., 1986).
PDGF in Osteoblasts
Normal human osteoblastic cells produce PDGF-A (Graves et al., 1989; Zhang et al.. 1991),
PDGF a- and p-receptors (Zhang et al., 199 1). PDGF B is reportedly not made by human
osteoblastic cells (Graves et al., 1989; Zhang et al.. 199 1: Bilbe et al.. 1996). although there
is a recent report of its production by rat osteoblasts (Canalis and Rydziel, 1996) and mRNA
for PDGF B have been detected in several human osteoblastic ceil lines (MG-63 ce11 line,
SaOs-2. TE-85) (Bilbe et al., 1996). The effect of PDGF on osteogenic cells in vitro has
been investigated by several groups. PDGF increases proliferation (fetal rat calvaria organ
cultures (Canalis and Lian, 1988; Pfeilschifter et al., 1990). fetal rat calvaria ce11 cultures
(Centrella et al., 1989; Centrella et al., 199 1 ; Pfeilschifter et al., 1992). human osteoblastic
celis (Graves et al.. 1989; Gilardetti et al.. 199 1 ; Zhang et al., 199 1 ; Abdennagy et al., 1992).
19 mouse trabecular explant cells (Abdennagy et al., L 992) and the murine osteoblastic ce11 line
MC3T3-E 1 (Davidai et al., 1992)), consistent with PDGFs well known activity as a rnitogen
for mesenchymal cells including fibroblasts. glial cells, and srnooth muscle cells. However,
the evidence in terms of osteoblast proliferation is not conclusive, as most experiments with
nontransformed cells involve possibly mixed cultures, with fibroblasts and other stromai
cells present which are dso known to respond mitogenically to PDGF (Hirata et al., 1985)-
and it has been shown that in fetal rat calvarial tissue culture, PDGF selectively stirnulated
Fibroblast replication and function (Hock and Canalis, 1994).
Effects of PDGF on Osteoblacric Cells In Vitro
The reported effects of PDGF on osteoblast differentiation have been controversial (no effect
- (Canalis and Lian, 1988; Cassiede et al., 1996); positive effect - (Centrella et al., 1989;
Pfeilschifter et al., 1990); negative effect - (Centrella et al., 1991 ; Pfeilschifter et al.. 1992;
Hock and Candis, 1994)). With respect to the effect of PDGF on differentiation, there are
several possible sources of variation, including the PDGF isoform used. Although the two
PDGF receptors are structurally and functionally related, there are important differences
between them in ligand binding: the a-receptor binds al1 three PDGF isoforms (PDGF-AA,
-AB, -BB) with similarly high affinities, but the p-receptor binds only PDGF-BB with high
affinity and binds PDGF-AB with a 10-fold lower affinity (Hart, 1990; Grotendorst et al.,
199 1). The proportions of PDGF receptor types can Vary between different ce11 types (Hart,
1990). In fetal rat calvaria ce11 populations, osteoblasts express primarily @ receptor
cornpiexes, with 30% pp receptor and 10% aa receptor complexes (Pfeilschifter et al., 1992).
There also appear to be important differences between PDGF receptor types in responses
elicited (Pfeilschifter et al., 1992) and the two receptors initiate traverse of the ce11 cycle by
distinct mechanisms (Coats et al.. 1992). In human fibroblasts the PDGF p-receptor, and not
Table 1.1 The Effects of PDGF on Proliferation and Differentiation in
Osteobiastic Ce11 Cui tures
S tudy Culture Prolifn Dserentiation (#)
Candis and Lian (1988) FRC* organ culture + no effect (collagen/osteocalcin)
Graves et al. ( t 989) adult human ceIl cultures + not determined
Centrella et al. (1989) FRC ce11 cultures + + (collagen/totai protein)
Pfeilschifter et al. (1 990) FRC organ cultures + + (collagen)
Zhang et al. (1991) explant human bone cells + not deterrnined
Centralla et al. ( 199 1) FRC ce11 culture + - (ml
Gilardetti et al. ( 199 1) adult human ce11 culture + not determined
Pfeilschifter et al. (1992) FRC ce11 culture + + (collagen)
- Hock and Candis ( 1994) FRC organ cultures + - (collagen)
- (bone rnatrix formation)
Cassiede et al. ( 1996) RBM stroma1 ce11 cultures + - (Ap)
no effect (osteogenesis)
*FRC = fetal rat calvaria
# = markers used as an indicator of bone differentiation are included in parentheses
21 the a-receptor, mediated induction of actin reorganization and membrane mffling
(Westermark and Heldin, 199 1). U-2 OS cells migrated in the presence of PDGF AB and BB
dimers but not in the presence of PDGF AA dimers (Allam et al., 1992). suggesting the
chernotactic effect is rnediated through the $-receptor. Thus the use of a particular isoform,
or a mixture of isoforms, may yield different results depending on the levels and kinds of
PDGF receptors a particular ceIl type expresses.
The fact that different culture systems (e-g., fetnl versus adult, tissue versus cell, clonal
versus mixed population) and different culture conditions (e-g., semm supplementation or
not) are being used to monitor osteoblast responsiveness to PDGF exacerbates the dificulty.
The maturational status or differentiation of progenitor populations can also lead to changes
in receptor levels: although PDGF a-receptor appears to be present throughout osteoblast
development, the levels appear to increase with osteoblast differentiation (Liu and Aubin,
1996). The duration of exposure to PDGF may also be important; most investigations have
used short exposure times (24 hr.) (Candis and Lian, 1988; Centrella et al., 1989; Zhang et
al., 199 1 ) or 48 hr. (Pfeilschifter et al., 1990) to 72 hr. (Hock and Canalis, 1994). Given
earlier data with other factors such as EGF (Antosz et al., 1987). which show biphasic
effects depending upon the time and duration of exposure. longer exposures rnay have
significantly different effects from shorter exposures. The timing of both the pulse and the
phenotypic assessments are also important, since recentIy it was reported chat assessrnents of
proliferation and AP activity of marrow stroma1 cells immediately after exposure to PDGF
were different from assessments done later (Cassiede et al., 1996). Another possible reason
for reported differences is that different markets of osteoblast development have been used
in different studies. For example, total protein synthesis, collagen synthesis, AP and
osteocalcin production have al1 been used as differentiation markers. While PDGF has been
shown to increase collagen synthesis (Centrella et al., 1989; Pfeilschifter et al.. 1990), the
collagen may be incorporated into a fibrous tissue matrix instead of bone matrix
22 (Pfeilschifter et al., 1990). Experiments which use the more specific bone markers such as
AP (although not unique to the osteoblast lineage) or the highly specific osteocalcin suggest
that PDGF inhibits bone formation in vitro (Centrella et al., 1992; Pfeilschifter et al., 1992),
consistent with reports that in calvaria tissue culture, PDGF inhibits bone matrix formation
(Hock and Canalis, 1994). This may be in keeping with the effect of PDGF on smooth
muscle cells: PDGF-BB increases proliferation while inhibiting expression of the
differentiated smooth muscle ce11 phenotype (Ho1 ycross et al., 1992).
Effects of PDGF on Osteobiastic Crlk In Vivo
Similarly to the in vitro studies, there are discrepancies in terms of the effect of PDGF on
bone in vivo. PDGF (A and 8) is expressed at sites of normal human fracture repair,
suggesting a role in regulation of this process (Andrew et al.. 1995). Local administration of
PDGF-BB at the time of osteotomy in rabbits had a stimulatory effect on bone fracture
healing in terms of increased callus density. increased mechanical bone strength. and
osteogenic differentiation (Nash et al.. 1994). In contrast, local administration of PDGF-BB
in rat craniotorny defects was found to inhibit osteogenin induced bone regeneration and
stimulated a soft tissue repair wound phenotype (Marden et al., 1993). Systemic
administration of PDGF-BB to adult fernale rats significantly increased osteoblast number.
bone density and strength, although premature closure of the growth plate, decreased body
fat. and extraskeletal collagen deposition were also noted (Mitlak et al., 1996). The differing
results in the in vivo studies may also be due to the different systems and methods of
administration of PDGF. PDGF is not normally found in plasma and plasma appears to
contain PDGF binding protein(s) that would serve to inactivate PDGF released into plasma
(Bowen-Pope et al.. 1984). Intravenously injected PDGF is rapidly cleared frorn the plasma
in baboons (t 112 = 2 min. (Bowen-Pope et al.. 1984)) and in rodents (90% removed after I
hr. (Cohen et al.. 1990). These results suggest that release of PDGF at sites of vascular
injury would greatly increase the local concentration of PDGF but PDGF not localized to the
23 site of injury would be rapidly cleared frorn the circulation (Bowen-Pope et al., 1984).
Thus, systemic administration of PDGF likely results in only short pulse exposures. In
summary, while PDGF is thought to have critical roles in proliferation during developrnent
and during fracture healing, its role in osteoprogenitor differentiation and bone formation is
stiI1 poorl y understood.
1.8 Thesis Objectives
The objective of this thesis w u to better define and characterize the heterogeneous cellular
environment of the rat bone marrow (RBM) stromal cell osteogenic system. To this end, I
f i n t used histochemical and immunohistochemical methods to identify and quantify the
main subpopulations present in RBM stroma and determine their tirne course of
development under conditions supporting osteogenesis (Chapter 2). I then applied methods
to separate and enrich the osteogenic population from the other stromal populations (Chapter
3). Finally, 1 investigated the effects of PDGF on RBM stromal osteogenesis using both the
mixed populations and the enriched populations (Chapter 4).
CHAPTER 2
Y
Dexamethasone Allers the Subpopulation Make-Up of Rat Bone
Marro w Stroma1 Ce11 Cultures
This chapter has been published in Journal o f Bone and Mineral Research ( 1995) 10.285- 94 as an original paper with the above title (Alexandra Herbertson and Iane E. Aubin)
25
2.1 Introduction
The stromal system of BM refers to the connective tissue elernents which provide structural
and functional suppon for hemopoiesis. Osteogenic cells are known to be present in the BM
stroma of various animal models (Maniatopoulos et al.. 1988; Benayahu et al., 1989;
Friedenstein. 1990; Leboy et al.. 199 1) including human marrow (Haynesworth et al.. 1992).
and production of a bone-like mineralized tissue from marrow cells has been demonstrated
both in vivo, ie.. in diffusion charnkrs loaded with BM cells (Friedenstein. 1968), and in
vitro, where bone-like tissue is synthesized by marrow stromal cells cultured in medium
containing ascorbic acid, 8-glycerophosphate and the glucocorticoid dex (Maniatopoulos et
a 1988; Kasugai et al., 199 1; Satomura and Nagayama. 199 1 ; Malaval et al.. 1992).
However, in addition to osteogenic cells, BM stroma contains other ce11 types: those widely
acknowledged include fibroblasts. adipocytes, and endothelial cells (Dexter. 1 982; McIntyre
and Bjornson, 1986: Owen, 1988; Hasthorpe et al., 1990: Wang and Wolf, 1990; Benayahu
et al., 1991; Bennett et al.. 1991). Other ce11 types identified as components of BM stroma
include srnooth muscle cells (Peled et al., 1 99 1 ; S immons and Torok-S torb, 1 99 1 ; Galmiche
et al., 1993), reticular (Dexter. 1982; Benayahu et al.. 199 1 ) and reticulo-fibroblastoid cells
(Ross et al., 1991).
Hernopoietic cells exist in intimate association with stromal cells in BiM in vivo and in vitro.
Long tenn BM cultures have been used to unravel the differentiation process of many white
blood cell lineages, as well as the role of stromal lineages in this process (Dexter, 1982).
When stromal cells are cultured, the majority of well-differentiated hemopoietic cells are
thought to be removed by centrifugation or. as in our system. by adherence techniques.
However, hemopoietic stem cells are present at a frequency of 1 in 25,000 adherent rnarrow
cells (Kiefer et al.. 199 1 ). and culturing the adherent BM fraction results in the culturing of
early hemopoietic cells whose development is guided by the existing culture conditions. In
27 mice, macrophages are frequently seen in cultures of BM stromal cells (Dexter, 1982) and
isolated macrophage colonies c m be obtained at low plating density (Wang and Wolf,
1990). Dendritic cells, which belong to the myeloid lineage, have also been described in
stromal cultures (Giesler et al., 199 1 : Inaba et al,, 1992).
Although there are some data describing the different ce11 types in RBM stroma, there is
little quantitative information on the subpopulation make-up. One study of pnmary RBM
cultures after 10 d. growth in vitro reponed largely fibroblast-tike (85%) colonies, and by
immunoperoxidase staining 10.3% macrophages-dendritic and granulocytic cells, and 2.5%
endothelial cells (Simmons et al., 199 1 ). Since it is known that the culture conditions have a
significant effect on what lineages proliferate and differentiate, the subpopulations present
need to be determined for each system. There are scant data on the subpopulation make-up
when stromal cells are grown under conditions stimulating bone formation. When RBM
stromal cells were cultured under such conditions (Maniatopoulos et al.. 1988), fibroblast
colony forming units (CFU-F) were found to comprise about 11100th of the adherent ce11
fraction, while osteoprogenitor cells giving rise to bone nodules comprise about 1/300th of
the same plated population i n the presence of dex (Aubin et al., 1990).
Immunocytochemistry, in combination with Northern analysis and morphological
description, confirmed that only a portion of the cells contnbute to osteogenesis (Malaval et
al.. 1994). Neither CN-F nor osteoprogenitor expression followed a linear relationship in
limiting dilution analysis suggesting cooperativity of ce11 types within the population and a
multi-target phenornenon (Aubin et al., 1990). In support of interacting ce11 types, when a
large constant number of nonadherent (hemopoietic) BM cells was added back to adherent
stromal cells, osteoprogenitor cells were measured at a frequency of lllOOth adherent cells
and their expression followed a linear. single-hit relationship. Endothelial cells or
endothelial cell-conditioned medium aiso increased expression of osteoprogenitors giving
rise to bone nodules (Aubin et al.. 1990). These data and others (Hughes and McCulloch.
28 199 1 ; Ogiso et ai., 199 1 ; Zhang et al., 199 1) indicate that the expression of osteogenesis by
BM osieoprogenitor ceils may be under the replation of oîher cells in the BM, which cm
exert either stimulatory or inhibitory activities. As a step toward understanding further the
cell-ce11 interactions affecting bone formation in the RBM stromal systern, we have analyzed
quantitatively the subpopulations over a temporal sequence during which osteoprogenitors
differentiate and bone nodules forrn.
2.2 Materials and Methods
Cell culture and pluring
BM stromal cells from the femora of young 40 - 43-day-old, 1 10 - 120 gram, male Wistar
rats were cultured essentially as described (Maniatopoulos et al., 1988). The rats were
sacrificed by cervical dislocation, and the femora were dissected under aseptic conditions
and placed in medium (a-modified essential medium, a-MEM) containing antibiotics ( 1000
uglml penicillin G (Sigma Chernical Co.. St. Louis, MO), 500 ug/ml gentamycin sulfate
(Sigma). and 3.0 @ml Fungizone (Flow Laboratories, McLean, VA)) (lOxAB). The
adherent connective tissue and muscle were removed from the exterior of the femora, the
femora were pIaced in fresh antibiotic medium as above, and their ends were cut off with a
scalpel. Each fernur was flushed with a-MEM until the bone appeared blanched (about six
or eight times). This suspension was passed through a syringe several times to produce a
largely single ce11 suspension; cells recovered from two femora were added to a T-75 tissue
culture flask (Falcon. Becton Dickinson, Oxnard, CA) and incubated for one week in a 37'C
hurnidified 95% air/5% CO2 incubator. Growth medium. consisting of a-MEM containing
10% fetal calf serum. antibiotics (100 ug/ml pen G, 50 ug/ml gentamycin, and 0.3 uglml
Fungizone), 50 ug/ml ascorbic acid, was changed every 2 - 3 d. After 7 d., each T-75 ce11
culture flask was washed with 15 ml warrn PBS and adherent cells were recovered with a
29 mixture of 10 ml of 0.25% trypsin (w/v in citrate saline) and 5 ml of collagenase (Rao et al.,
1977). Recovered ceils were passed through a syringe with a 22 gauge needle to ensure a
singletell suspension. Cells were counted electronicaily on a Coulter Counter then plated
at densities between 8 x 103 and 5 x 104 cells per 35mm dish (Falcon), or at a range of these
densities to obtain a balance between colony isolation and adequate colony numben for
quantitation (see Results section). For flow cytometry. cells were plated in 100 mm dishes
(Falcon). Cells were cultured in a-MEM supplemented as above, and where indicated, IO-^
M dex (Sigma) for various penods of time from 7 - 35 d. To promote mineralization. 10
m M Na-$-glycerophosphate (Sigma) was added either continuously or for the 2 d. of culnire
prier to fixation. Cultures were used to determine total ce11 number, stained, or prepared for
flow cytometry as below.
Ce11 Counrs
Cells were recovered from a minimum of triplicate dishes by trypsinizing as above and
counted on a Coulter Counter.
Stn in ing
AP/von Kossa/toluidine blue
The histochemical stain for AP is a modification of Pearse's (1960) method (Drury and
Wallington. 1967). Cells were rinsed once with cold PBS and fixed in 10% coid neutral
buffered forrnalin for 15 min., rinsed with distilled water, and left in distilled water for 15
min. Fresh substrate (10 mg Naphthol AS MX-PO4 (Sigma) dissolved in 400 ul N.N-
dirnethylformarnide, added to 50 ml distilled water and 50 ml Tris-HCl (0.2 M. pH 8.3) and
60 mg Red Violet LB salt (Sigma)), filtered through Whatman's No. 1 filter immediately
before use, was added and incubated for 45 min at 20°C. The dishes were then rinsed in
distilled water, drained and stained with 2.5% silver nitrate for 30 min. at 20°C (von Kossa
stain). The dishes were again rinsed in distilled water and drained, toluidine blue was
30 applied for 2 seconds and the dishes were then rinsed 3 times with tap water. The dishes
were finally air dried.
ccNBE
The procedure from Sigma Diagnostics Kit 181 (St. Louis. USA) for aNBE was
followed, except that the counterstaining was omitted. The kit describes the staining
patterns for monocytes and T lymphocytes, but macrophage staining with this stain has been
described in several studies (Giesier et al., 199 1 ; houe et al., 199 1 ; Kanemoto et aI., 199 1 ).
Sudan IV
Sudan IV staining method is modified frorn Clark (Clark, 198 1). Medium was suctioned
from the culture dishes and celIs were fixed in 10% neutral buffered formalin for 1 hr. After
rinsing with distilled water, followed by rinsing twice rapidly with 70% alcohol, Sudan IV
solution (0.1 gm Sudan IV, 50 ml acetone, 50 ml alcohol) was added for 5 - 10 minutes.
Differentiation of the stain was done with 70% alcohol for 2 - 5 seconds, then the dishes
were rinsed with water and allowed to air dry.
Horseradish Peroxidase Coniueated Antibodv S taininq
Medium was suctioned from the culture dishes, ceils were fixed in 3.7% formalin in TBS
(10 mM TRIS pH 7.5, 150 mM NaCI) for 5 minutes, followed by -20' methanol for 5
minutes if permeabilization was needed. Endogenous peroxidase activity was blocked with
3% Hz02 in TBS. The first antibodies, either RBM 2 1 1.13, anti-rat AP (Turksen et al.,
1992) (x 1000 dilution) or ED2 anti-rat macrophage (Dijkstra et al., 1985) (Serotec Ltd..
Toronto, Canada, diluted x500) were diluted in TBS containing 3 9 BSA prior to addition,
and ce11 cultures incubated at 37' for 45 - 60 minutes. After washing, horseradish
peroxidase-conjugated (HPR) CAM (diluted x300) was added and incubation con tinued at
37'C for 30 - 45 minutes. HPR Colour Development Reagent (0.3 g. BioRad CDR in 100
3 1 ml of -20' methanol plus 60 pl Hz02 (30%) in 100 ml TBS, mixed in equal proportions
immediately before using) was added at 20' for one hr.. cells were washed in Hz0 and
ailowed to air dry.
Quan ritarion
Quantitation of colony types involved identification of AP positive colonies by staining as
above, bone nodules by morphology and von Kossa staining, macrophages by aNBE and
ED2 staining, and adipocytes by rnorphology and Sudan IV staining. For quantitation, only
foci containing >10 cells (approximately three population doublings) were classified as
colonies; both colony counts and grid point counts (percentage of grid cross points covered
by colonies) for proportion of the culture dish covered were done. In some experiments.
colonies were graded according to their size (10 - 25 ce11 foci, 25 - 100 cells, or > LOO cells).
For statistical analysis. the Student t test (unpaired, double-sided) was used to determine
levels of significance.
Flow cytornetry
Cells were recovered from dishes by a mixture of trypsin-collagenase as above. passed
through a syringe with a 22 gauge needle, and centrifuged. The cells were resuspended in
Fresh growth medium as above and incubated at 37'C for 30 - 60 minutes to allow the cells
to recover from trypsinization. Cells were centrifuged then resuspended in sterile primary
mouse anti-rat antibodies (mouse myeloma Ig's (Organon Teknika Co.. West Chester, PA)
as a control, RBiM 2 L 1.13 for AP(Turksen and Aubin, 199 1). ED2 (Serotec) for
macrophages (Dijkstra et al.. 1985). MRC OX- 1 (Cedarlane Laboratories Ltd.. Canada) for
leukocyte common antigen (Sunderland et ai.. L979) or MRC OX-22 (Serotec) for leukocyte
common antigen restricted to T and B lymphocytes (Woollett et al.. 1985) diluted in 3%
BSA in PBS, and incubated at 37°C for 30 minutes. After incubation, cells were washed.
incubated with FITC-conjugated sheep anii-mouse antibodies (Serotec) for 30 minutes at
3 2 37'C. and then washed with PBS. Immediately before analysis. cells were filtered through a
fine mesh to remove any ceIl clumps. Propidium iodide was added to determine ceIl vitality.
Sarnples were mn on a Coulier EPICS Profile Analyzer equipped with an Argon laser
operated at 488 nm for fluorescence excitation. Fluorescence was detected with a 525 nm
band pass filier (+/- 10 nrn) and samples gated for size and low propidium iodide staining.
RBM stroma1 cells cultured for 21 d. in the presence of 10% FCS, ascorbic acid, P-
glycerophosphate. and dex, produced mineralized bone nodules similar to those previously
characterized as bone-like (Maniatopoulos et al., 1988; McCulloch et al., 199 1). When
marrow stroma1 cells were cultured as above but without dex, bone nodules were also seen,
but at a much lower frequency. The difference in bone nodule formation in cultures with
and without dex is apparent macroscopically (see Figure 2.1. A and B). Microscopic
assessrnent of bone nodule nurnbers showed significantly greater numbers in the cultures
with dex. compared to those without, in six out of six separate experiments. three of which
are shown in Table 2.1. An increase in the numbers of bone nodules in cultures with dex
was accompanied by a significant decrease in total cell number in these cultures (data not
shown). consistent with previous studies (Maniatopoulos et al.. 1988). Cells associated with
early bone nodules exhibited typical polygonal osteoblast rnorphology and stained positively
for AP (see Figure 2.5A). Bone nodules formed in these cultures over several days: the
number of bone nodules formed plateaued by d. 2 1 (Figure 1.2) but mineralization detected
by von Kossa staining continued to increase up to at least 35 d. when P -glycerophosphate
was present continuously. Because of the variability of the frequencies of different ce11
types within each BM isolation. each separate experiment used a minimum of three different
plating densities in order ro obtain bone nodules in numben satisfactory for both counting
33 and statistics (ie.. 10-150 nodules per 35 mm dish). In Table 2.1. the plating density for
each separate experiment is noted. Both tirne at which new nodule formation ceased and
especially the onset of mineralization were plating ce11 density-dependent: both occurred
earlier when higher densities of bone nodules were present (data not shown).
In cultures in which bone nodules formed, many other ce11 morphologies and colony types
were also evident. Using the same ce11 cultures as those in which bone nodules were
analyzed and the same temporal sequence, we identified and quantitated these other marrow
stroma1 populations with a variety of histochernical stains. In six out of six experiments, a
large percentage of the colonies present were positive for AP, with significantly higher
nurnbers of AP positive colonies in cultures grown with dex (three of which are shown in
Table 2.1). Similarly to the time course of bone nodule formation, the percentage of a
culture dish covered by AP positive colonies (as determined by grid point count) plateaued
after 21 d. However. oniy a proportion of the AP positive colonies were coïncident with and
corresponded to bone nodules (Figure 2.3A. 2.5A). A micrograph of an AP positive colony
with a nonbone appearance is shown in Figure 2-58 . Although the total number of AP
positive colonies appeared to decrease after d. 14 (Figure 2.3A), this is likely due to an
increase in numbers of large AP positive colonies which merge with and become
indistinguishable from the srnaller colonies (see Figure 2-38).
Pronounced differences in aNBE positive (monocyte/macrophage) colonies (Figure 2.5D) in
cultures with and without dex was also apparent. The number of aNBE positive colonies
was significantly higher in dex-containing cultures in six out of six experiments, three of
which are shown in Table 2.1. In contrast to the time course of formation of AP positive
colonies and bone nodules, al1 sizes and total number of aNBE positive
(monocyte/macroph~ge) colonies continued to increase up to ai least d. 28 of culture, the last
34 time point analyzed (see Figure 2.4). There was no apparent consistently close physical
association of bone nodules and aNBE positive colonies (Figure 2.5D).
Fat colonies (Figure 2 . X ) were present at very low frequency under Our culture conditions,
and dex significantly increased the numbers of fat colonies in only one out of six of these
experiments, and only slightly in that particular experiment (see Table 2.1).
Immunolabelling was used to identify more specifically the hemopoietic colonies. ED2
(macrophage) imrnunohistochemically stained colonies (Figure 2.6B) were of similar ce11
size and morphology as those strongly positive with aNBE. However, ED2 positive colonies
were present at similar or higher frequencies to strongly positive aNBE colonies, probably
reflecting the increased sensitivity of the immunohistochemical stain over the histochemical
stain. Quantitation of ED2 positive colonies indicated that macrophage number increased in
cultures grown with dex compared with those without dex in four out of four separate
experiments, three of which are shown in Table 2.1.
We also used flow cytometry to further quantify the stromal ce11 make-up under these
growth conditions. The stromal population exhibited a broad distribution of ce11 sizes
consistent with the many subpopulations present. The number of cells positively labeled
were compared with control cells incubated with nonspecific mouse myeloma Ig's. Similar
to the results from the colony counts. there were more AP positive cells and more
macrophages in the cultures with dex compared with the cultures without dex. For example,
in the experiment shown in Figure 2.7. in the cultures with dex compared to those without,
29.7% venus 12.5%. respectively, of total cells were labeled positively for AP, and 8.3%
versus 2.5%. respectively, of total cells were positive with ED2 antibody. Flow cytometry
was more sensitive than immunohistochemical staining in that it identified relatively more
macrophages in the cultures without dex than were apparent by colony counting (the results
35 from Figure 2.7 are from the same experiment shown as experiment 3 in Table 2.1).
consistent with the cells seen in culture dishes (compare Figure 2.6C to 2.6A), flow
cytometry identified more hemopoietic cells (MRC OX-1 positive) in the cultures without
dex compared to those with dex (Figure 2.7D versus 2.7H). While the majority of the
hemopoeitic cells in the cultures with dex are macrophages (8.3% macrophages out of
1 1.5% of cells identified as hemopoietic cells), a much smaller proportion of the
hemopoietic cells in the cultures without dex are macrophages (2.5% macrophages out of
36.9% hernopoietic cells). The bulk of the hemopoeitic cells in the cultures without dex are
as yet unidentifieci, but do not appear to be lymphocytes as determined by lack of labeling
with MRC OX-22 (data not shown). They do not appear to be of the granulocytic lineage as
they lack the typical morphology and previous histochemical staining trials with naphthol
AS-D chloroacetate esterase (Sigma) and myeloperoxidase (Sigma) for neutrophils and their
precursors were also negative (data not shown).
Table 2.1 Quantitation of colony types identified in RBM stroma1 ce11 cultures grown for 21
d. in vitro with ascorbic acid. P-glycerophosphate, and with or without dex (10-8 M)
Experiment Plating density (+) or # bone % covered with # fat # aNBE+ # ED2+ (35mm dishl (4 dex nodules AP+ colonies colonies colonies colonies
1 5 x 104 + 3444 89.1 +2-1 4 1 2 73k10 91kIO
5 x 104 - O k O 82.9 k 1.1 lkl 3+,1 O+O
2 5 x 1 0 4 + 112k7 98.4i0.2 O k O 119+5 259+1I
5 x 104 * 1 + 1 95.8 f 0.2 1+1 7 + 2 O I O
3 2 x 104 + 1 1 + 2 92.4 k0.7 O+,O 52k5 81+4
2 x 104 4 O f O 87.4 + 1.2 O f O 1 1 + 4 0.4st0.2
Experirnents with + dex > - dex 3/3*** 3/3** 1/3* 3/3*** 3/3***
T-Test of (+) dex venus (-) dex: * p 5 .OS; ** p < -01; *** p 1 .O01
Figure 2.1 Overviews of 35 mm culture dishes grown for 21 d. in medium containing
ascorbic acid and 8-glycerophosphate. Dishes were stained with AP/von Kossa/toluidine
blue; cells cultured with dex (A) or culnired without dex (B). Black von Kossa positive
nodules are apparent in A, while AP positive colonies appear gray in both (magnification
I -6x).
7 14 2 1 28 35
Days in culture
Figure 2.2 Time course of formation of bone nodules in RBM stroma1 cells cultured with
10-8 M dex. Bone nodules first appeared during the second week of u.dture and their
number appeared to plateau during the third week of culture; the proportion of nodules
mineralized appeared to increase with time in culture. In this experirnent, p -
glycerophosphate was present continuously. W mineralized nodules;. total bone nodules
O 10 20 30 40
Days in culture
Days in culture
Figure 2.3 (A) Cornparison of the nurnber of AP positive colonies (u ), the area of
the culture dish they cover (i), and the number of bone nodules (+ ) in RBM
stroma1 cells cultured for various periods of time with dex. Only a proportion of the AP
positive colonies are coincident with bone nodules. (B) Size distribution of AP positive
colonies seen in (A) with time. While the number of small colonies and foci decrease with
time, the number of large colonies increase with time. Colony size distributions: 61 10 - 25
cells; 25 - 100 cells; . > 100 cells
7 14 21 28
Days in cuiture
Figure 2.4 Al1 size classes of strongly positive aNBE colonies in RBM stroma1 cells,
cultured with dex, continued to increase at least up to d. 28, the latest point analyzed.
Colony size: u 10 - 25 cells; h-- 25 - I û û cells; > 100 cells. Total
colonies: &
Figure 2.5 Photomicrographs of the different cell types present in RBM stromal cells
cultured for 21 d. with dex. (A) AP/von Kossaltoluidine blue staining of a bone nodule
where * marks mineralized von Kossa positive nodule and AP positive (appear gray in this
black and white photomicrograph) cells with typical polygonal morphology can be seen at
the periphery of the mineralized nodule (magmfkation ~100) . (B) AP positive cells (appear
gray) (mawcation x100). (C) Sudan N positive (fat) cells showing a colony with two
foci; stained lipid droplets appear black in this black and white photomicrograph
(magnification x30). Inset shows an adipocyte with typical morphology (magmfication
x300). @) aNBE positive (monocytelmacrophage) cells (appear black) not associated with
a bone nodule (magnification x 170)..
1 S . . - .
f -. \ - m. . : -- - a . * - - \ .* -- -- O * . &? * - .'i ,* . . - t - . O -
N .. \
Y;. . 4 8 - - - - * . -. . 0 I - \ -. \ - .- , . - = . i * b
r . * \ . : . ' b . \ . r - - . ' u- 4 - * . - t 0 -. L\- - . *
Figure 2.6 Photomicrographs of irnmunohistochemical staining of RBM stromal cells
grown for 21 d. (A) Control cells of cultures with dex, stained with non-specific myelorna
IgGs. The black in the upper left corner is a portion of a bone nodule. (B) The same
cultures as in (A), but stained with ED2 mAb; a positively stained macrophage colony can
be seen. (C) Control cells for the same experiment as above, but grown without dex. stained
with non-specific myeloma IgGs. In addition to the stromal ceiis, many small unstained
hemopoietic ceiis can be seen - these appear dark when stained with control antibodies (non-
specific IgGs) andor second antibody alone or without any antibodies. (D) The same
cultures as in (C), but stained with ED2 mAb; no positive colonies are seen. Al1
magnifications x 120.
Aw - Dex, conirol 'i 1
B. - Dex. RBM 211.13 D. - Dex. MRC OX-1
E. + Dex. control F. + Dex. RBM 211.13 G. + Dex. ED2 Hw + Dex. MRC OX-1
Figure 2.7 Flow cytometry: frequency histogrm vs fluorescent intensity. (A) and (E) Controls stained with non-specific
myeloma IgGs, for cells grown without dex and with dex, respectively, in the sarne experiment. (B) and (F) Staining with
RBM 2 1 1.13 for AP in crlls grown without dex and with dex, respectively. (C) and (G) Staining with ED2 (macrophages) in
cells grown withoui dex and with dex, respectively. (D) and (H) Staining with MRC OX- 1 (leukocytes) in cells grown
without dex and with dex, respectively. Al1 cells were collected for analysis at d. 21 of culture.
2.4 Discussion
In this paper, we provide quantitative evidence that RBM stromai populations grown under
conditions prornoting osteogenesis and bone formation comprise a heterogeneous mixture of
ce11 types. We confirmed that the addition of dex, similar to its effects in other osteogenic
models (Candis, 1983; Bellows et al., 1986) stimulates osteogenesis and the formation of
bone-like tissue or bone nodules in vitro from adult RBM stroma1 cells (Maniatopoulos et
al., 1988; Kasugai et al., 199 1 ; Leboy et al., 199 1). The presence of other morphologically
distinguishable ceIl types in stromal cultures making bone has been noted on occasion but
not carefully investigated. For example, previously we reportecl that the predominant cells
under similar culture conditions with dex were of spindle-shaped rnorphology (fibroblastic)
or were pleomorphic and the majority did not srain for AP activity (McCulloch et al., 199 1).
More recently, in a molecular and imrnunohistochemical study of osteoprogenitor
differentiation, we confirmed that only a proportion of the cells in RBM stromal cultures are
osteogenic (Malaval et al., 1994). In this paper. we characterized and quantified the
presence of cells of the monocyte-macrophage lineage and adipocytes, both lineages known
to be present in stromal cultures of mouse and human (Dexter. 1982; Wang and Wolf.
1990), but not previously identified in adult RBM stromal cultures undergoing osteogenesis.
Dex significantly increased the numbers of AP positive colonies and cells in RBM stromal
cultures, a finding consistent with reports that dex increased AP enzyme levels in the mouse
B M stromal ce11 line, LMBA- 15 (Benayahu et al., 1989), and in other marrow stromal-derived
ce11 lines (Benayahu et al., 199 1). Interestingly, we found that both the percentage area of
the culture dish covered by AP positive colonies and bone nodule production plateaued in
the RBM stromal cultures at similar times, but only a small proportion (about 1/4) of AP
positive colonies were coincident with bone nodules. Likewise in chick BM stromal
cultures, only a proportion, albeit higher than in rat (55% venus 25%) of the AP positive
45 ceIl population contnbutes to osteogenesis (Kamalia et al.. 1992). This low proportion of
bone AP to total AP colonies could be due to AP positive osteoblastic cells being present,
but either lacking the ability to proliferate sufficiently to form a bone nodule andor being at
stages of differentiation which they cannot form bone in vitro (see aiso discussion in
Malaval et al. (Malaval et al., 1994)). Alternatively, while AP is a well-known marker of
bone cells, it is not exclusive to them. and the presence of other, non-bone, AP positive
lineages is possible. For example. several preadipose ce11 lines are AP positive (Udagawa et
al., 1989; Benayahu et al., 199 1; Dorheim et al., 1993).
Only low numbers of adipocyte colonies were seen under Our culture conditions. This is in
contrat to other systems where production of adipocytes is stimulated by the addition of
glucocorticoids (Dorheim et al., 1993; Bellows et al., 1994). Bennett et al. (Bennett et al.,
199 1) have suggested that there is a bipotential progenitor in rabbit BM stroma which can
differentiate in an adipocytic direction or along the osteogenic pathway. When Beresford et
al. (Beresford et al., 1992) grew adult rat marrow stroma1 cells in the presence of FCS and
dex, both adipocytic and osteogenic cells differentiated, but an inverse relationship between
adipocytic and osteogenic cells was seen. The latter authors showed that with continuous
exposure to dex in both the primary and secondary cultures. the differentiation of osteogenic
cells predominated, whereas the differentiation of adipocytes predominated when dex was
present in secondary culture only. Although dex was present only in the secondary cultures
in our experiments, few adipocyte colonies were seen, suggesting that other factors in our
culture conditions favor differentiation of osteogenic cells over adipocytes, whether there is
a bipotential adipo-osteogenic ce11 present or not (for funher discussion, see Bellows et al.
(Bellows et al., 1994). For example. Beresford et al. (Beresford et al., 1992) used female
rats (we used male), an initial period in the primary where the medium was not changed for
5 - 6 d. (we changed medium 3 tirnes/week), and a shorter secondary culturing time of 8 - 10
d. (versus our tirne of 21 d.), al1 conditions that could markedly affect the subpopuiation
46 make-up of the cultures and influence the ability of multiple celi populations to interact
andfor differentiate.
As previously mentioned, Simmons et al. (Sirnrnons et ai.. 199 1) reported that ptimary RBM
stromal cultures contained 10% "residual marrow components" of macrophagidgranulocytic
leukocytes. Maniatopoulos et al. (Maniatopoulos et al.. 1988) also noted the presence of
small, mononuclear round cells in their primary cultures, and attributed them to the
hemopoietic component of BM. but did not comment on them in the secondary cultures, in
which they analyzed osteogenesis. However, the repeated medium changes in the 7 d.
primary culture penod adapted from Maniatopouios et al. (Maniatopoulos et al.. 1988) and
used here is thought to reduce hemopoietic contamination while expanding the numbers of
CFU-F and decreasing cellular debtis. Clearly, the hemopoietic population recognized by
MRC 0x4 (Cedadane). which detects leukocyte common antigen, is present in the stromal
layer of cells in cultures grown bath in the absence and presence of dex. However, dex
reduced the fraction of hemopoietic cells that express this antigen in these cultures. Of
interest, the majority of the hernopoietic cells present in dex-containing cultures are
macrophages, while a much srnaller proportion are macrophages in the absence of dex.
These other MRC OX- 1 positive cells appear not to label with MRC OX-22, suggesting that
they are not lymphocytes, and by histochemical stains appear not to be neutrophils. They
may be monocytes or more primitive hemopoietic cells, however, the paucity of specific
mAbs to rat hemopoietic cells, especially when compared with those available for mouse
and human, currently hinders their identification.
The finding that macrophage production is stimulated under conditions also stimulating
bone formation is interesting. While macrophages have been reported in murine BM
secondary cultures (Wang and Wolf, 1990), we are unaware of any definitive identification
of hemopoietic cells in secondary cultures of RBM stroma grown under conditions favoring
47 bone formation. Notably, clearly concomitant with stimulating the differentiation of
osteoprogenitors, dex stimulated the maturation of the monocytic lineage into macrophages
in our cultures, possibly while inhibiting proliferation of more primitive precunors or
altering the proportion of other lineages in the hemopoieitic fraction. An aitemate
explanation could be that dex stimulates osteogenic cells or other strornal cells to produce
factors which indirectly stimulate macrophage production. Murine osteoblastic cells are
known to produce M-CSF and GM-CSF (Felix et al., 1988; Horowitz and Jilka, 1992), and
recent reports descnbe the detection of rnRNA transcripts for IL- 1 P , IL-6, IL-8, and TGF-
p 1,2,3 in normal human bone (Birch et al., 1993) and for IL-5, IL-6, IL-7, M-CSF, GM-CSF.
and TGF- P 1 in the MC3T3-E 1 ce11 line and rat primary osteoblastic cultures (Greenfield et
al., 1993). As well, rnouse BM stromal cells produce stimulators such as M-CSF and GM-
CSF (Benayahu et al., l992), and constitutively produce mRNAs for CSF-I . GM-CSF. kit
ligand, and IL-6 (Kittler et al., 1992). Thus it appears that osteoblasts or other stromal cells
secrete many factors that could affect the hemopoietic lineages, including macrophage
production. Of course, the inverse is also possible: with dex altenng hernopoietic lineages,
stimulating production of mature macrophages and altering cytokine Ievels in such a way
that stimulation of bone nodule production results. As well, the differentiation of monocytes
into macrophages could result in the loss of production of inhibitory factors. For example,
IL-IP, which has biphasic effects on bone formation but is inhibitory when present
continuously during the differentiation sequence (Ellies and Aubin, 1990), is secreted by
monocytes, but its secretion decreases with differentiation into macrophages (Kreutz et al.,
1993). In any case. it appears crucial to identify the ce11 populations present in this system,
as the effecrs of any factor on bone formation in this system may be direct or indirect and
mediated through the other ce11 lineages present.
In RBM stromal populations, the culture conditions used to promote growth and
differentiation of AP positive colonies, a subpopuIation of which includes osteoprogenitor
48 cells that differentiate to form bone nodules (colonies of bone), concornitantly reduce the
proportion of hemopoietic cells expressing leukocyte common antigen, while promoting the
growth and differentiation of macrophages. These data show conclusively for the first time
the presence of macrophages in RBM stroma1 cultures grown under conditions favounng
bone formation, extend the information on the subpopulation rnake-up and heterogeneity
under these culture conditions, and contribute to an understanding of the multitarget
phenornenon observed in osteogenic differentiation in RBM stroma.
CHAPTER 3
Ce11 Sorting Enriches Osteogenic P o p lations in Rat Bone Marro w Stroma1
Cell Cultures
This chapter has ken accepted for publication in Bone (December, 1997) as an onginal paper with the above tide (Alexandra Herbertson and Jane E. Aubin)
49
3.1 Introduction
While a number of clonai, established and irnrnortdized bone-derived ce11 lines have been
isolated. many of these appear to show anomalies in aspects of their phenotype and
regulation by hormones and growth factors (for review, see (Aubin et al.. 1993)). It is
therefore of considerable importance to identify the normal osteoprogenitors in populations
freshly isolated from tissue. Cultures of whole marrow stromal cells were first shown to
contain cells with the capacity to fonn bone when they were transplanted in vivo in diffusion
chambers by Friedenstein and colleagues (Friedenstein et al.. 1968). This has been
confirmed repeatedly in vivo with BM cells in diffusion chambers (for example, see (Ashton
et al., 1984; Owen, 1988)) and, more recently. conditions have been established in which
bone formation is observed in vitro in cultures of BM strornal cells from diverse species
including aduk rabbit (Tibone and Bernard, 1982), adult male (Maniatopoulos et al.. 1988)
and female (Leboy et al.. 1991) rat, chick embryo (Karnalia et al.. 1992). neonatal pig
(Thomson et al., 1993). adult mouse (Van Vlasselaer et al., 1993). and adult human
(Vilamitjana-Amedee et al.. 1993). In stroma (McCulloch et al., 199 I), as in calvariai
populations (Bellows and Aubin, 1989; Bellows et al., 1990). the bone nodule assay can be
used as an indirect functional assay of the osteoprogenitor. the end product being a nodule of
osteoblasts/osteocytes embedded in a mineraiized matrix characterized as bone on the buis
of light and phase contrast microscopy, histochemistry including von Kossa staining.
scanning electron microscopy. and transmission electron rnicroscopy (Tibone and Bernard,
1982; Maniatopoulos et al., 1988). The RBM stroma1 ce11 system has been well
characterized in terms of morphological characteristics of differentiating osteoblasts and
their matrix (Maniatopoulos et al., 1988; Satomura and Nagayama. 199 1), in terms of
molecular and irnmunohistochemical details of the expression of rnarkers associated with
bone formation (Kasugai et al., 199 1; Malaval et al.. 1994). and with respect to the effect of
exogenous agents on bone formation (Ogiso et al.. 1991; Zhang et al., 199 1; Notoya et al..
1994; Tenenbaum et al., 1995). However, in RBM stromal ce11 cultures. only a small
5 1 proportion of the cells are osteogenic, and the presence of other morphologicaily
distinguishable ce11 types has been reported (Malaval et al., 1994; Herbertson and Aubin,
1995). For example, in these culture conditions, while some osteoprogenitors can be
assayed in the absence of dex. the assayable number increases markedly in dex,
concomitantly with the growth and differentiation of macrophage colonies (Herbertson and
Aubin, 1995). The presence of multiple ce11 types in RBM stroma1 populations and their
varying proportions from one isolation to another despite ngorous attention to isolation
protocol may account for the considerable variation in the absolute number of bone nodules
formed in different experiments, and may complicate interpretation of cytokine and hormone
effects as direct or indirect on the osteoprogenitors present. The presence of these multiple
subpopulations suggests a need for a method which can punfy the osteogenic component,
including osteoprogenitors, from other lineages in the RBM stroma. To remove the majority
of well-differentiated hematopoietic cells when culturing, centrifugation or adherence
techniques are commonly used. Maniatopoulos et al. (1988) noted in their primary cultures
the presence of small hematopoietic cells and the repeated medium changes in the 7 d.
primary culture period were used to reduce their presence (Maniatopoulos et al.. 1988;
Kamalia et al.. 1992) and cellular debris while expanding the C N - F (Maniatopoulos et al.,
1988), of which osteogenic colonies are a subgroup. However. it is also known that
hematopoietic stem cells are present at low frequency in the adherent marrow ceIl fraction
(Kiefer et al.. 1991) and their subsequent development is guided by the existing cuiture
conditions. Agents for hernolysis and density gradients are other commonly used strategies
in other systems for decreasing hematopoietic contamination (Falla et al., 1993). Nij weide
and collragues reported selec tive cyanide killing of monocyte-macrophage lineage ce1 1s in
mouse marrow stroma1 ce11 cultures (Modderman et al.. 1994); in Our hands, no specific
killing effects were achievable with this method in RBM stroma1 cultures (unpublished
observations). Macrophage and endothelid ce11 cornponents c m also be separated from the
fibroblast-like population by their phagocytic properties (Stein and Stein, 1980; Pitas et al.,
52 198 1 ; Yanai et ai., 199 1 ) and magnetic bead separation techniques based on phagocytosis
(Zhang et al., 1995) have been reported. Row cytometric analysis based on phagocytosis of
fluorescently labeled acetylated low density lipoprotein was found to be Iess effective in
identifying macrophages ccjmpared to mAB labelling (unpublished observations).
In addition to the strategies used to decrease the hematopoietic population, there has also
been investigation into methods to enrich selectively for osteoblast populations from stromal
populations. Selective adherence has been one approach, as sumrnarized in Chapter 1,
Section 1 .S. Increasingly, the availability of mAbs recognizing subpopulations of
osteoblasts (Aubin and Turksen. 1995) is providing means for antibody-based rnethods for
purification. AP expression has been reported previously to be useful in fractionation of
osteoblastic populations into an AP-positive population and an AP-negative population in
the rat calvaria system; interestingly amongst the former were dex-independent progenitors
and in the latter were progeniton which required dex to differentiate (Turksen and Aubin,
1991). AP expression has also been used for flow cytometric sorting in female RBM
stromal ce11 cultures, but production of a mineralized matrix after sorting was only descibed
in cells which were AP-negative at the time of sorting (Rickard et al., 1994). In this paper
we describe flow cytometnc sorting of RBM stroma based on AP expression, with resultant
enrichment for osteoprogenitors capable of dividing and differentiating to f o m osteoblasts
which synthesize bone.
3.2 Materiais and Methods
Ceil culture und plating
BM stroma1 cells from the femora of young 40 - 43-day-old, 1 10 - 120 gram. male Wistar
rats were cultured essentially as described previously ((Maniatopoulos et al., 1988), see
Chapter 2). After 7 d., adherent cells were recovered in a singIe ceIl suspension, then either
counted electronically on a Coulter Counter (Coulter Electronics. Hiaieah, FL) and plated at
densities between 2 x 104 and 4 x lo4 cells per 35 mm dish (Falcon) (unsorted controls), or
labelled and processed through the sorter (see below) prior to plating. After plating. cells
were cultured for an average of 21 d., then stained (APIvon Kossa/toluidine blue, N E , and
Sudan IV) as previously described in Chapter 2.
Flow cytometry
Cells were recovered from flasks or dishes were passed through a syringe with a 22 gauge
needle, and centrifuged. The cells were resuspended in fresh growth medium and incubated
at 37°C for 30 - 60 minutes to allow the cells to recover from the trauma of the procedure.
Cells were centrifuged then resuspended in stenle primary mouse anti-rat antibodies diluted
in 1.5% BSA in PBS: mouse myeloma Ig's (Organon Teknika Co., West Chester. USA) for
controfs, RBM 2 1 1.13 for AP (Turksen et al., 1992), MRC OX- 1 (Cedarlane Laboratories
Ltd., Hornby, Canada) for leukocyte common antigen (Sunderland et al., 1979). ED2
(Serotec Canada, Toronto, Canada) for macrophages (Dijkstra et al.. 1985), or RECA
(Medac GmgH, Hamburg, Germany) for rat endothelial cells (Duijvestijn, 1992), and
incubated at 37°C for 30 minutes. After incubation, celIs were washed twice then incubated
with FITC-conjugated sheep anti-mouse antibodies (Serotec) diluted in 1.5% BSA in PBS
for 30 minutes at 37'C and then washed with PBS twice. Ce11 suspensions were then placed
on ice and flow cytometric sorting was begun immediately; the sorting process took on
average 2 - 4 hr. Immediately before analysis, cells were passed through 22 gauge needles
54 to remove any ce11 clumps. For flow cytometric counting. propidium iodide was added and
populations were dou ble-gated for low propidium iodide staining and size. For ceIl sorting,
samples were also gated for size but propidium iodide was added only to a small control
sample to determine cell vitality and plating numbers were varied accordingly (one sorting
trial with small amounts of propidium iodide - 5 Wrnl cell suspension - left in demonstrated
its toxicity in these populations). Samples were run on a EPICS Ceil Sorter (Coulter)
equipped with an Argon laser operated at 488 nm for fluorescence excitation. Fluorescence
was detected with a 525 nm band pass filter (f 10 nm). Sorting regions were set a minimum
of 3% away from the border of the 1% negative control region to exclude overlapping
populations. Percentage of positive cells (FITC versus Count) was monitored throughout
the sort as a check for antigen capping, although this was not observed to be a problem
dunng the sorting periods. Sorted cells were collected in tubes with a-MEM containing
30% fetal calf serum and antibiotics as above; they were then diluted in medium as above
and plated accordingly. For son controls, cells were stained as above. one gate expanded to
include 100% of the population, and cells were passed through the machine prior to
collection.
Quantitation
Quantitation of colony types involved identification of AP-positive colonies by
staining (red) as above, identification of bone nodules by black von Kossa stained mineral
deposits surrounded by cuboidal AP-positive stained cells. monocytes/rnacrophages by
aNBE staining (dark brown). and adipocytes by (red) Sudan IV staining and morphology.
For quantitation, only foci containing >10 positively stained cells (approximately three
population doublings) were classified as colonies; both colony counts and gnd point counts
for proportion of the culture dish covered were done. For statistical analysis, the Student t
test (unpaired. double-sided) was used to determine levels of significance. unless the
standard deviations differed significantly. in which case a Welch t test was used.
When RBM strornal cells were analyzed by flow cytometry after either 7 d. primary culture
or after 7 d. primary and 18 d. secondary culture. they exhibited a broad distribution of ce11
sizes consistent with the multiple hematopoietic and mesenchymal subpopulations present
(Herbertson and Aubin, 1995). necessitating the use of both log forward scatter (related to
ce11 size) and log side scatter (related to ce11 granulaiity) in analysis and sorting parameters.
In both counting and sorting experiments, the populations were first gated for size: using log
forward scatter, the gated area was set to remove smdl fragments and debris or discard any
ohvious ce11 clumps, with a lower limit consistent with platetelet size (approximately 2 pm)
and an upper limit consistent with large cultured cells (approximately 30 pin). On the basis
of fluorescence, control cells incubated with mouse myeloma Ig's as the ptimary antibody
were gated to 1 .O% (see Figure 3.2A) to eliminate 99% of the background autofluorescence
(a normal characteristic of a11 viable mammalian cells (Aubin, 1979) and marked in sorne
BM stromal cells (Keller et al., 1993)) and any non-specific staining (see Figures 3.1 A and
3. IB. and control in Figure 3.2). With these conditions, RBM populations labelled with the
anti-rat AP antibody (RBM 21 1.13) displayed a broad range of labelling intensity (see
Figure 3.1 C).
In RBM stromal cells grown for 7 d. primary and then 18 d. secondary culture. multiple cell
lineages are represented, with subpopulations varying according to the presence or absence
of dex, as we previously reported (Herbertson and Aubin, 1995). Typically. more cells
labeled with RBM 2 11.13 (for AP) when RBM strornal cells were grown in the presence
(74.7%) than in the absence of dex (13.9%) (see Figure 3.2A and 3.2B). In contrast, cells
staining for MRC OX I (for leukocyte common antigen) were present at lower frequency in
cultures with dex (2.8%) than without dex (21.2%). A small percentage of cells stained
positively with ED2 (anti-macrophages) only in the cultures with dex (2.0%). RECA, an
56 antibody specific for rat endothelial cells (Duijvestijn et al.. 1992), stained few cells in either
cultures with (1.1 %) or without dex (0.9%). representing < 1% of cells over backgound. [n
multiple experiments, by either flow cytometry or immunohistochemical staining (data not
shown). negligible numbers of ceils stained with RECA, suggesting that endothelial cells are
present in Iow numbers in RBM stroma1 ce11 cultures under these growth conditions.
For ce11 fractionation after 7 d. primary culture, sorting gates were set to capture cells either
~ ~ h i s h (cells intensely labeled with RBM 2 11.13) or A P ~ O W (clearly negative for RBM
2 1 1.13 labeling) as indicated in Figure 3.1 ; in this experirnent 3 1.2% of cells were in the
former category and 32.7 % in the latter (see Figures 3.1B and 3. ID). A P ~ ~ ~ , A P ~ O ~ , or
control cells were plated to determine osteoprogenitor distribution and frequency. Similar to
results we reported previously with flow cytometrk sorting of fetal rat calvarid populations
(Turksen and Aubin. 1991), and consistent with reports that murine osteoblastic cells (Van
Vlasselaer et al., 1994) and stroma initiating cells are depleted by passage through a flow
cytometer (Deryugina and Muller-S ieburg, 1 993; Deryugina et al., 1995)- approximately
50% (on a per plated ce11 basis) of nodule forming cells are lost during processing/sorting
when compared to unsorted controls. In some experiments, we therefore added an
additional control, measuring ceIl viability with propidium iodide at the end of labelling
manipulations and then adjusting plating densities to compensate for reduced ceIl viability.
The nurnber of AP-positive colonies was enriched in the ~ ~ h k h fraction of RBM cells
cornpared to either the unfractionated cells or the AP'OW fraction. whether or not the sorted
cells were cultured in the presence or absence of dex (see Table 3.1). However, since
numbers of bone nodules and macrophage colonies are low i n cultures without dex
(Herbertson and Aubin, 1995) and even in the A P ~ @ fraction bone nodule numbers were
significantly higher (p 1 -05) when cultured with dex versus without dex, and since trends in
bone nodule or macrophage colony numbers were not signifiant in minus-der cultures,
subsequent results are reported only for dex-containing cultures. Table 3.2 summarizes 5
57 separate expenments. It is clear that within the A P ~ ~ ~ ~ fraction the number of AP-positive
colonies and osteoprogenitors (giving rise to bone nodules) were signifkantly enriched
compared to control, but as we reported earlier. bone colonies comprise only a srnall
proportion of the total AP-positive colonies (Herbertson and Aubin, 1995). The increase in
osteoprogenitor frequency ranged from approximately 2 to 100 fold (see Table 3.3). The
fold increase in osteoprogenitor frequency did not appear to correlate with either the initial
percentage of cells positive for AP in the culture at the time of sorting, nor did it appear to
correlate with any fold change in macrophage colonies (as determined by N E staining)
(see Table 3.3). In addition, the fold increase in osteoprogenitor frequency did not appear to
correlate with the post-sorting purity check; both experiments B and C in Table 3.3 had fold
increases of 1 . 7 ~ but different post-sorting purïties (B post-sorting purity was 95.8%. C
post-sorting purity was 96.5%). while in experiment D in Table 3.3 the post-sorting purity
was lower, 93.5%. but the fold increase was higher at 3.5~.
Concomitantly with the significant enrichment of osteoprogenitors, the ~ P h i g h fraction of
cells was usually significantly depleted for macrophages (in 415 experiments listed in Tables
2 and 3). In the anomalous experiment. experiment C in Table 3.3. macrophage numbers
were very high in the unfractionated control; while the total number of macrophage colonies
increased in the A P @ ~ fraction, in this experiment the number of large (A00 cells)
macrophage colonies in the AP hieh fraction (3.3 k1.5 colonies) significantly decreased (p 5
.O\) cornpared to the unfractionated control (13.0 t 2.0 colonies). The apparent increase in
macrophage colonies is likely due to the decrease in number of large macrophage colonies.
which tend to overgrow and obscure srnaller colonies that may also be present. Thus.
although total colony number suggests othenvise. in experiment C the actual number of
individual macrophages probably decreased. In contrast. macrophages appear to be
consistently ennched in the APIOW fraction of cells, a fraction with no consistent changes
from control in either AP-positive colonies or osteoprogenitors (see Table 3.2).
58 Very low numbers of adipocyte colonies fonn in RBM stromal ce11 cultures under these
growth conditions (Herbertson and Aubin. 1995). Of the 3 experirnents in which adipocyte
colonies were quantitated, 2 had fat colonies present in large enough numbers (> 4 colonies
per dish in the unfractionated cells) to be analyzed statistically. Within the fraction
of cells. adipocyte colonies were significantly depleted, while they were significantly
enriched in the AP~OW fraction (see Table 3.2 and Figure 3.3).
While enrichment of bone nodules and AP-positive colonies is seen within the ~ ~ h i g h
fraction, it is also apparent thar this fraction is still a mixed population. For example. culture
dishes are not completely covered with AP-positive colonies (see Table 3.1). reflecting the
presence of AP-negative colonies. and while the macrophage colony number is generally
decreased, some are still present (see Table 3.3). As mentioned earlier, post-sort purity
checks (re-analyzing the sorted population on the flow cytometer) of the A P ~ @ cells
showed that this fraction was minimally 93 - 97% pure; thus some of the non-AP-positive
colonies may reflect the 3 - 7 9% "contaminating" cells. Post-sort purity checks of the AP~OW
cells showed that this population was minimally 99% pure, reflecting the relative ease in
exciuding a highly stained cell.
MRC OX-1 is a mAB reactive against leukocyte common antigen, which is known to be
present on > 95% of thymocytes, BM cells, and thoracic duct lymphocytes (Sunderland et
al., 1979). Two sorting trials with this antibody were perfomed to see if they yielded
"cleaner" RBM stromal populations, ie.. populations better depleted of hematopoietic cells.
While the removal of the majority of the hematopoietic ce11 fraction increased AP-positive
colony and bone nodule numbers, macrophages were also present in numbers equal to or
greater than those in unfractionated control populations (results not shown).
59 Previous work in our labontory has shown that in RBM stroma1 populations osteoprogenitor
expression, as determined by bone nodule formation. does not follow a linear relationship in
limiting dilution analysis until very high ce11 densities are reached, suggesting cooperativity
of ce11 types within the population and a multi-target phenomenon (Aubin et al., 1990)
(Aubin, 1997. submitted). When we analyzed osteoprogenitor frequencyhodule number
versus plating density in control versus A P ~ @ and A P ~ O W populations, osteoprogenitor
expression still followed a non-linear relationship (Figure 3.4A). Similarly, but not to the
same degree, the aNBE positive macrophage colonies appear to follow a non-linear
relationship with decreasing plating densities (Figure 3.4B).
Table 3.2
Net clianges (enrichnient/depletion) in various coloiiy types in AP high and AP low fractions, compured io unfractionated controls.
Al1 cultures contüined dex üfter plating. Changes indicated by the ürrows were significant at minimally p 5 .O5 as determined by t
tests, or the trend seen i s indicated.
I
Al kaline Phosphatase
Bone Nodules
aNBE i- Colonies
Fat Colonies
A P High
1' 515
AP L o i v
$315 (trend & 1/5) ?1/5
'r 5/5
4 315 (trend 4 115) 1' 115
5 212
1 3/5 215
1' 415 (trend 7 115)
7' 2/2
Figure 3.1
Flow cytometric sorting. (A) and (C) are frequency (count) histograms versus ce11
fluorescence (FITC) intensity for control (incubated with mouse myeloma I, 0's as the
primary antibody) and RBM 2 1 1.13 (AP) stained RBM stroma1 populations respectively. In
(A), the control cells have the lower cut-off of cursor 'a' set to I .O%. 1 n (C) , the number of
cells in the area 'a' represents those positively stained for RBM 2 1 1.13 (38.8%). (B) and
(D) are forward scatter log (FS log; representing size) venus FITC intensity contour maps
for control and RBM 2 1 1.13 stained populations respectively. The sorting gates were set to
capture cells with either high (gate 'c') or low (gate 'b') expression of RBM 21 1.13. In (B)
gate 'b' contains 66.4% and gate 'c' contains 1 % of the total population, whereas in (D) gate
'b' contains 32.7% and gate'c', which has been shifted right relative to the control, contains
3 1.28 of the total population.
65 Figure 3.2 Legend
Flow cytometnc analysis. (A) and (B) are frequency (count) histograms versus ce11
fluorescence (FITC) intensity in RBM stromal cell cultures grown in secondary cultures for
18 d. without or with dex, respectively. From top to bottorn: RBM stromal cells are stained
with control mAb (incubated with rnouse myeloma Ig's as the primary antibody) and RBM
21 1.13 (anti-AP), MRC 0x4 (anti-LCA), ED2 (anti-macrophage), and RECA (anti-rat
endothelid cell antigen).
ControI High Low
RBM 211.13 Expression
Figure 3.3
Adipocyte colony numbers for RBM stroma1 populations sorted according to RBM 2 1 1.13
(AP) expression. Data is frorn the experiment designated "D" in Table 3. T tests show that
adipocyte colonies are significantly lower (p 1 -01) in AP high (high RBM 21 1.13 binding),
and are significantly higher (p 5 -01) in AP low (low RBM 2 1 1.13 binding) populations.
Control Low High
RBM 211.13 Expression
# cells platedn5 mm dish
O 2 x lW4)
0 IxlO(4)
5 x l q 3 )
2 5 x lO(3)
R 1 x W 3 )
Y cdls p l a t e d / 3 ~ m dish 2 x lO(4)
1 x lO(4)
5 x lO(3)
2.5 x 10(3)
1 x W 3 )
Control Low High
RBM 21 1.13 Expression
68 Figure 3.4 Legend
Bone nodules (A) or aNBE colonies (B) versus plating density for RBM stroma1 populations
sorted according to RBM 2 1 1.13 (AP) expression. Data are from the experiment designated
"C" in Table 3.3. In (A), t tests show that osteoprogenitor (capable of giving rise to a bone
nodule) numbers are significantly lower (p S .O 1) in the AP low (low RBM 2 1 1.13 binding),
and are significantly higher (p 5 -01) in the AP high (high RBM 21 1-13 binding) at al1
densities from 2 x 104 to 2.5 x 103. In (B), t tests show that aNBE positive colony numbers
are significantly lower (p 5 .O 1) in AP low (low RBM 2 1 1.13 expression) populations only
at the 2 x 104 density, and are significantly higher (p < .05) in AP high (high RBM 21 1.13
expression) populations at densities from 2 x 104 to 2.5 x 103.
3.4 Discussion
Previous work from Our lab has shown the presence of multiple ce11 types in RBM stroma1
popul;itions, including cells of the monocyte-macrophage lineage, fibroblasts, and
adipocytes (Herbertson and Aubin. 1995). Notably the addition of dex, concomitant with
stimulating the differentiation of osteoprogenitors to bone nodule forming cells, stimulated
the maturation of the monocytic lineage into macrophages in our cultures, possibly while
inhibiting proliferation of more primitive precurson (Herbertson and Aubin. 1995). In
cultures with dex, macrophages appear to make up the majority of the hematopoietic cells
present (Herbertson and Aubin, 1995). Despite rigorous attention to ce11 isolation
techniques, the proportions of hematopoietic versus mesenchymal subpopulations present in
RBM stroma appear to Vary from one isolation to another and rnay account for the
considerable variation in the absolute number of bone nodules seen in different experiments.
Consistent with the hypothesis that heterotypic cell-cell interactions influence osteogenesis
in the RBM system (Aubin et al., 1990; Aubin, 1997, submitted), the presence of these
multiple subpopulations also complicate the interpretation of direct versus indirect cytokine
effects in BM systems (Thomson et al., 1993; Herbertson and Aubin, 1995). Notably, in Our
fractionated populations, more than one cell type remains limiting in the populations,
suggesting that cell-ce11 interactions still appear to influence osteoprogenitor differentiation
and nodule formation; in addition, i t is possible that homotypic ce11 interactions, i.e.
"community effects" may also play a role as hypothesized recently based on limiting
dilution and other frequency analyses (Aubin, 1997, submitted).
Clearly, in the ~ ~ h i g h fraction of cells we ennched for osteoprogenitors which divide and
differentiate to give rise to bone nodules, although the resultant cultures still comprised a
mixture of lineages. This mixture of lineages may reflect the 3 - 7 8 "contaminating" cells
in the ~ ~ h k h fraction, and the 1% "contaminating" cells in the A P ~ O W fraction. Although
70 flow cytometric sorting can yield populations of high purity, 100% purity is only ever
approximated unless single ce11 cloning methods are added to the isolation step. Given the
large range of ce11 sizes seen in the RBM stromal populations, Our sort gates were
accordingly set wider, which would also tend to reduce the stringency of the sort.
Nevertheless, the 93 - 978 purity obtained in our sorting experiments are sirnilar to those
achieved by an improved immunopanning technique applied to isolate CD 34+ cells from
human BM (93.54 i 3.4 % purity). and better than the 57 - 80% purities obtained in some
other immunopanning procedures (Cardoso et al., 1995). Multiple antibody sorting steps
and an imposition of single ce11 collection methods may help to reduce the presence of the
contarninating cells further.
The mixed nature of the ~ ~ h ~ g ~ population could result not only from limitations in the
efficacy of sorting, but also from cells that have reverted from AP-positive osteoblastic cells
to a more immature, AP-negative phenotype, or the presence of other, non-bone, AP-
positive lineages. The presence of AP-positive colonies and osteoprogenitors (giving rise to
bone nodules) in the AP**W fraction is likely representative of the presence of immature
progenitors which, at the time of sorting, are not yet expressing AP (see also (Turksen and
Aubin. 1991)). While AP is a well known marker of the mature osteoblast lineage. AP is
also a cytochemical marker of 'reticular' cells in marrow which make up a large proportion
of the marrow stroma (Westen and Bainton, 1979; Cattoretti et al., 1991): these cells have
often been postulated to be preadipocytes (Bianco et al., 1988; Ross et al., 1991) which are
known to be AP-positive (Udagawa et al.. 1989; Benayahu et al., 1991; Dorheim et al.,
1993). However, in our stromal ce11 cultures with the highest numbers of fat colonies
present in the controls, fat colony number was decreased in the ~ ~ h g h and increased in the
AP'OW fraction of cells. Although isolated brain capillary endothelial cells express high AP
activity which decreases with ce11 culturing (Meyer et al., 1990). bone endothelial cells have
been reported to express oniy low levels of AP (Rickard et al., 1995). We would thus expect
71 these not to be found in the ~ ~ h i g h fraction of cells. especially since direct quantitation of
endothelial cells by endotheliai specific antibodies suggested that they do not form a
significant proportion of the RBM stromal ce11 cultures under our culture conditions.
While the finding that cells at different stages of the osteoblast lineage express antigenic
determinants unique or distinctive to these stages is beginning to be useful for identification
and isolation of osteoblastic cells at various stages of differentiation (Aubin and Turksen,
1995), we are still limited by a lack of specific antibodies definitely recognizing cells earlier
than the preosteoblast. Haynesworth et al. (1992) reported isolation of antibodies that react
on a subset of human rnarrow stromal cells and a variety of tissues in vivo, but do not label
osteoblasts or osteocytes (Haynesworth et al.. 1992). The authon concluded that the
antibodies may recognize molecules present on early progeniton or stem cells and that the
epitope(s) is lost during osteogenic differentiation (Haynesworth et al., 1992). STRO- 1
recognizes human marrow stroma1 mesenchymal progenitors with the ability to form cells
with the phenotype of fibroblasts. adipocytes. srnooth muscle cells. and osteoblasts
(Simmons and Torok-Storb. 199 1 ; Gronthos et al.. 1994). Very recently. HOP-26. a mAE3
against hurnan marrow stromal osteoprogeniton, has been described (Joyner et al.. 1997).
HOP-26 labels an epitope present on osteoprogenitors before the induction of APT but dues
not bind to osteoblasts, and its presence on the ce11 surface allowed enrichment of human
marrow CN-F including osteoprogenitors (Joyner et al .. 1997). As ye t, similar antibodies
to early progenitors do not exist for the rat nor have the macromolecules recognized by these
antibodies been identified to allow preparation of rat-specific reagents. Enrichment of
relatively mature osteoblastic cells has also been reported. Immunopanning of human BM
cells with osteocalcin (normally considered a marker of mature osteoblasts) and osteonectin
has yielded sorne enrichment for cells with osteoprogenitor properties (Long et al., 1995).
Van Vlasselaer and colleagues used immunopanning with mAbs to first deplete populations
of hematopoietic cells, then used flow soning with Sca- 1 and wheat germ agglutinin binding
72 to enrich for osteoblastic cells from the 5-fluorouracil treated femoral BM of young adult
mouse (Van Vlasselaer et al.. 1994). Two mAbs (OB 7.3 and SB-5) that label the surface of
chick osteocytes specifically have been used (with magnetic beads) to isolate relatively pure
osteocytes from chick mixed bone ceIl populations (Van der Plas and Nijweide, 1992).
MAbs against AP have been raised by several labs (in Our lab, RBM 2 1 1.13 was raised in an
injection of RBM stroma (Turksen et al., 1992)); these label preosteoblastic celis as well as
osteoblasts. In the rat calvarial system, AP expression has been used successfully to
fractionate osteoblastic populations into AP-positive (highest 10% of cells) and AP-negative
populations (Turksen and Aubin, 1991) and this report extends that work into the RBM
stromal system. In the rat calvarial system, the fractionation resulted in osteoprogenitors
being separated into two "classes": one capable of expressing bone formation without
exogenous dex stimulation (high AP-positive). and one expressing bone formation only in
its presence (AP-negative) (Turksen and Aubin. 199 1). These data are consistent with the
hypothesis that these two populations of progenitor cells represent different stages of
differentiation. with the latter representing a less mature ceIl than the former and requiring
different regulatory signals for its expression than the latter (Turksen and Aubin, 199 1).
Given that few bone nodules f o m in RBM stromal ce11 cultures in the absence of dex, and
the marked stimulation of bone nodule formation noted with dex. it is tempting to speculate
that the majority of osteoprogenitors in stromal cultures. especially as assayed in the
secondas, cultures. may be of the relatively immature phenotype. In our experiments, we
Found few assayable osteoprogenitors (little bone nodule production) in the APL& fraction
in the absence of dex. suggesting that RBM stroma contains largeiy dex-dependent
osteoprogenitor populations and the possibility that dex is required later than when AP is
first expressed.
73 Flow cytometric sorting of RBM stroma1 populations according to high or low AP
expression is an effective technique for ennchment of AP colonies and osteoprogenitors
which c m divide and differentiate to give rise to bone nodules, and should prove useful for
further studies of the nature of the osteoprogeniton and their regdation in this tissue.
CHAPTER 4
PDGF has Biphasic Effects on the Differentiation of Osteoprogenitors
in Rat Bone Marrow Stroma1 Ce11 Populations
This chapter has been submitted for publication with the above title (Aiexandra Herbertson and Iane E. Aubin)
7 4
4.1 Introduction
PDGF, 30 kDa disulphide bonded homo- or hetero-dimers of A and B chains which interact
with the two PDGF receptors, a and P, appears to have broad roles in tissue development
and repair (Bowen-Pope et ai., 199 1; Schatteman et al.. 1992; Yan et al., 1993; Ponten et al.,
1994; Yan et ai., 1994). With respect to bone, normal human osteoblastic cells produce
PDGF-A (Graves et al., 1989; Zhang et ai., 199 1) and PDGF a- and fi-receptors (Zhang et
al ., 1 99 1 ). PDGF B is reportedly not made by human osteoblastic cells (Graves et al ., 1 989;
Zhang et al., 1991; Bilbe et al.. 1996), although there is a recent report of its production by
rat osteoblasts (Candis and Rydziel. 1996) and mRNA for PDGF B has been detected in
several human osteoblastic celi lines (MG-63 ce11 line, SaOs-2, TE-85) (Bilbe et al., 1996).
PDGF has been reported to affect both proliferation and aspects of the differentiated
phenotype of osteoblastic populations in vitro. It increases proliferation in many
osteoblastic mode1 systems (fetal rat calvaria organ cultures (Canalis and Lian, 1988;
Pfeilschifter et al., 1 WO), fetal rat calvaria ceIl cultures (Centrella et al., 1989; Centrella et
aI., 199 1; Pfeilschifter et al., 1992), human osteoblastic cells (Graves et al., 1989; Gilardetti
et al.. 1991; Zhang et al., 1991; Abdennagy et al., 1992), mouse trabecular explant cells
(Abdennagy et al., 1992) and the murine osteoblastic cell line MC3T3-El (Davidai et al.,
1992)), consistent with its well known activity as a mitogen for other mesenchymal cells
inctuding fibroblasts, glial cells, and smooth muscle cells. However. whether it is
osteobiastic cells alone responding in most of these models is not clear, as most experiments
with primary ce11 cultures involve mixed cet1 populations. with fibroblasts and other stroma1
cells present which are also known to respond mitogenically to PDGF (Hirata et al., 1985).
In this regard. PDGF was found to stimulate seiectively fibroblast replication and function in
fetal rat calvarial organ culture (Hock and Candis, 1994).
76 The reported effects of PDGF on parameters of osteoblast differentiation have k e n diverse
(no effect - (Canalis and Lian, 1988; Cassiede et al., 1996); positive effect - (Centrella et al..
1989; Pfeilschifter et al., 1990); negative effect - (Centrella et al.. 199 1 ; Pfeilschifter et al.,
1992; Hock and Candis. 1994)). These conflicting results may mise from several different
sources, including the PDGF isoform used (there are isoform differences in ligand binding
(Hart. 1990; Grotendorst et al., 199 l)), and differing proportions of ceIl subpopulations with
different PDGF receptor types (Hart, 1990). which mediate different responses (Westermark
and Heldin. 1991; Allam et al., 1992; Coats et al., 1992; Pfeilschifter et al., 1992). The fact
that different culture systems (e-g.. fetal versus adult. orgadtissue versus ceil. clonal venus
mixed populations) and different culture conditions (e-g., serum supplementation or not) are
being used to monitor osteoblast responsiveness to PDGF exacerbates the difficulty. The
maturational status or differentiation of progenitor populations can also lead to changes in
receptor levels: although PDGF a -receptor appears to be present throughou t osteoblast
development. the levels appear to increase with osteoblast differentiation (Liu and Aubin.
1996). The duration of exposure to PDGF may also be important; most investigations have
used short exposure times (24 hr. (Canalis and Lian, 1988; Centrella et al., 1989; Zhang et
al., 199 1 ), 48 hr. (Pfeilschifter et al., 1990) or 72 hr. (Hock and Canalis. 1994)). Given
earlier data with other factors such as EGF (Antosz et al., 1987; Aubin et al., 1992). which
elicit biphasic effects depending upon the time and duration of exposure. and when during
the proliferation/maturation sequence they were given, the effects of PDGF might be
expected to Vary depending on the treatment protocol. This was found to be the case. for
example. when the effects of PDGF on proliferation and AP activity of marrow stroma1 cells
were compared immediateiy after exposure to PDGF versus later (Cassiede et al.. 1996).
Finally. different parameters to measure osteoblast development have been used in different
studies. For example. total protein synthesis. collagen synthesis. AP andor os teocalcin
production have variously been used as differentiation markers. While PDGF has been
shown to increase collagen synthesis (Centreth et al., 1989; Pfeilschifter et al.. 1990), the
77 collagen may be incorporateci into a fibrous tissue matrix rather than a bone matnx
(Pfeilschifter et al., 1990). Consistent w ith this. experiments which use more specific bone
markers. such as AP (although not unique to the osteoblast lineage) and osteocalcin, suggest
that PDGF inhibits bone formation in vitro (Centrella et al., 1992; Pfeilschifter et ai., 1992;
Hock and Candis, 1994).
Osteoprogenitor cells present in the stromal population of young-adult RBM, cultured in
medium containing ascorbic acid. P-glycerophosphate and dex, divide and differentiate to
give nse to osteoblasts which synthesize bone-like tissue (Maniatopoulos et al., 1988): the
number of bone nodules is quantifiable and provides a very specific, terminal differentiation
product by which to measure the effects of PDGF on osteoblast differentiation. Given the
established proliferation-differentiation sequence charactenstic of the RBM mode1 (see, e.g.,
(Malaval et al., 1994)), this system provides an opportunity to target specific events by
varying the exposure times to PDGF. Further, BM stromal cells may mimic the in vivo
environment, with its mu1 tiple subpopulations and different lineages present. For example,
fibroblasts and macrophages, which we have previously reported to be present in RBM
stromal cells grown under conditions which promote bone formation (Herbertson and
Aubin, 1995). are also sources of and responders to PDGF (Bowen-Pope et al.. 199 1). Thus,
while BM stromal ce11 cultures rnay recapitulate the complex cellular interactions that occur
in vivo. establishing underlying mechanisrns as direct or indirect on particular
subpopulations can be difficult. Flow cytometnc soning of RBM stroma1 cells on the basis
of AP expression, however. allows separation of the stroma into A P ~ ~ and A P ~ O W
populations. Within the A P ~ ~ ~ ~ fraction, the number of AP-positive colonies and
osteoprogenitors (giving rise to bone nodules) are significantly enriched and adipocyte and
macrophage colonies depleted compared to the unfractionated control (Herbertson and
Aubin, 1997). In contras, within the M l o w fraction of cells. adipocyte and macrophage
colonies are consistently enriched (Herbertson and Aubin, 1997). We have used the RBM
78 stromal system, in combination with flow cytometric sorting, as a mode1 for investigating
the role that PDGF plays in osteoprogenitor differentiation.
4.2 Materials and Methods
S e m
Levels of PDGF in semm Vary depending on how semm is prepared and the species
used (e-g.. PDGF levels in whole blood semm from normal humans (17.5 t 3.1 ng/ml),
baboons (2.7 ng/rnl) (Bowen-Pope et al., 1984) and fetal bovine serum (0.929 f 0.23 ng/mI)
(Hyclone Defined Fetal Bovine Serum)). In these experiments. we used fetal bovine serum
at a final concentration of 10%; even with the higher concentrations of PDGF reported (17.5
ng/ml for human whole blood serum), this would yield basal PDGF levels of approximately
1 - 2 ng/ml. In experiments in which Hyclone "defined fetal bovine serum" was used, the
serurn would contribute only ,093 ng/ml PDGF, negligible compared to the additional
concentrations (0.25 - 10 ng/ml) added. PDGF-BB is the major isoform seen in semm from
most non-primate species (Bowen-Pope et al.. 1989); accordingly. the porcine PDGF (R &
D S ystems. Inc.) used in these experiments contains only PDGF-BB homodimers.
Cell culttlrr and plnting
BM stromal cells from the femora of young 40 - 43-day-old. 1 10 - 120 gram, male
Wistar rats were cdtured essentially as previously described ((Maniatopoulos et al.. 1988).
see Chapter 2). After 7 d.. adherent cells were recovered in a single ce11 suspension, and
either counted electronically o n a Coulter Counter (Coulter Electronics, Hialeah, FL) and
plated at densities between 2 x 104 and 4 x 104 cells per 35 mm dish (Falcon) or stained and
processed through the flow cytornetric ce11 sorter (see Chapter 3) prior to plating. After
plating. cells were cultured in growth medium plus porcine PDGF (1 - 10 @ml) in pulses as
79 indicated in the Figures or continuously as indicated for an average of 21 d., then stained
(APIvon Kossa/toluidine blue, nNBE, and Sudan IV) as previously described in Chapter 2.
Quantitation
Quantitation of colony types was as prevously described in Chapter 3. In addition,
for analysis of multiple sets of data, the Wilcoxon signed rank test was used.
As we reported previously (Herbertson and Aubin. 1995), dex markedly affects both the
subpopulation make-up and osteogenic differentiation in RBM stromal ce11 cultures. While
a small number of bone nodules form in cultures without dex, dex increases significantly the
number of bone nodules and the number of macrophage colonies present, and decreases the
proportion of cells expressing leukocyte common antigen. In al1 experiments reported here,
PDGF was added to cultures also supplemented with dex.
Cunfin~ious expasure to PDGF
The continuous presence of PDGF (4 ng/ml) was mitogenic in RBM stromal ce11 cultures,
significantly increasing late log phase growth rate and saturation density (see Figure 4.1).
PDGF at the same concentration decreased the number of bone nodules, but concomitantly
increased macrophage and adipocyte colony number (see Table 4.1). suggesting that the
decrease in bone nodules is not due to generalized ce11 toxicity. PDGF dose response
analysis was then undertaken for bone nodule (Figure 4.2), macrophage (Figure 4.3), and
adipocyte (Figure 4.4) colony types. Multiple experiments were done over a three year
period in regular and defined serum and data from al1 experiments are summarized in Table
4.2. PDGF had biphasic effects on bone nodule formation: the number of bone nodules was
80 stimulated by very low doses of PDGF (I L ng/ml in regular or defined semm experiments)
and inhibited by higher doses (2 4 ng/ml); in multiple repeat experiments the intermediate
concentrations either had no affect over control (Figure 4.2) or showed a sharper transition
between stimulatory and inhibitory doses (e.g., Table 4.1). In three experiments in which
complete dose response curves were available. the ID50 for bone nodule formation was
approximately 3 ng/ml PDGF (range in al1 experiments was approximately 2 - 10 nghl) . In
contrast, the continuous presence of PDGF consistently stimulated aNBE positive
(previously confimed to be macrophage colonies by specific antibody (ED2) labelling
(Herbertson and Aubin. 1995)) colonies and adipocyte colonies at al1 concentrations tested
in al1 experiments (see Tables 4.1. 4.2). Total macrophage colony number appeared to be
maximally stimulated a PDGF concentrations of 2 - 2.5 ng/ml. however. the number of
large macrophage colonies continued to increase dose dependently (see Table 4.1 and Figure
4.3) and may have overgrown and obscured smaller colonies. Likewise, adipocyte colonies
appear to increase in a dose-dependent manner in al1 experiments (see Tables 4.1. 4.2 and
Figure 4.3): in three experiments in which a plateau of adipocyte stimulation was seen
(corresponding to the same experiments as for bone nodules above). the EDso was
approximately 1.5 ng/rnl.
Pulse exposrrre to PDGF
The development of bone nodules is characterized by a proliferative period followed by a
period of morphologically and molecularly defined osteoblast differentiation and matrix
deposition and mineraiization (Liu et al., 1994; Lian and Stein, 1995; Aubin and Liu, 1996).
To discriminate the developmental stages at which the biphasic effects of PDGF occurred.
we pulse-treated cultures at different stages of the nodule formation process. 2 d. pulses
with PDGF (4 nglml or 6 nglml) early during log phase growth (d. 1 - 3. d. 3 - 5)
significantly stimulated bone nodule numbers. PDGF pulses during late log phase growth
(d. 5 - 7 or d. 5 - 8) either increased (2/4 experiments) or did not alter (2/4 experiments)
8 1 bone colony formation. PDGF pulses later in the culture penod (d. 7 - 9 onwards), when
saturation density was reached and nodules were forming, tended to decrease or significantly
decreased (d. 13 - 15) nodule formation (see Figure 4.5).
The effects of PDGF on RBM cells fractionaied on the busis of AP expression
Flow cytometric soning of 7 d. primary RBM strornal ceIl cultures was performed on the
basis of AP expression with a mAb against AP (RBM 2 1 1.13). The resultant ~ ~ h i g ~ . APiow,
or control cells were plated to determine osteoprogenitor (giving rise to bone nodules),
macrophage. and adipocyte distribution and frequency . As descri bed in detai 1 elsew here
(Herbertson and Aubin, 1997). wi thin the ~ p h i g h fraction, the number of AP-posit ive
colonies and osteoprogenitoa were significantly enriched while adipocyte and macrophage
colonies were depleted compared to the unfractionated control. In contrast. within the
APiow fraction of cells, adipocyte and macrophage colonies were consistently enriched
(Figure 4.6). When these sorted populations were cultured with PDGF (4 ng/ml), results
similar to those with unfractionated control populations were obtained. Specifically, in two
separate experiments. the same treatment with PDGF (4 ng/ml. continuous exposure)
significantly decreased bone nodules while significantly increasing macrophage and
adipocyte colonies in the fractions (see Figure 4.6). In the A P ~ O W fraction of cells, 4
ng/ml PDGF significantly decreased bone nodules in one experiment (see Figure 4.6) (no
statistically significant difference was seen in the other). significantly increased adipocyte
colonies, and significantly increased large macrophage colonies (often to an extent that
colonies merged making counting of total colonies difficult) while slightly decreasing or not
significantly affecting total macrophage colony nurnbers (see Figure 4.6).
4 Table 4.1 RBM stroma1 cells plated at 2x 10 B5 mm dish were grown for 2 Id., with dex and
the PDGF concentrations indicated present continously from d. 1 - 21. PDGF had dose-
dependent biphasic effects on bone nodule formation. while over the same concentration
range both adipocyte and aNBE positive colonies increased. Results are means f one
standard deviation.
** t tests venus PDGF O ng/ml: 'p I .OS, *p 5 .O 1, p I -00 1.
1 Condition 1 Bone nodules 1 Fat colonies Total aNBE
PDGF O ng/ml
PDGF 1 ndml
PDGF 2.5 ng/rnl
PDGF 5 ng/mi
PDGF 10 nghl
25.7 (+ 10.2)
57.3 (k 16.4)#
42.5 (f 9.1)#
4.0 (k 7.4)#
0.3 (1- 0.5) *
1 .O (I 0.0)
9.0 (f 0.0)
59.7 (+ 13.1)"
63.7 (f 1 1.5) **
74.3 (ir4.0) **
Table 4 2 Effect of continuous exposure to PDGF on RBM stromd populations
Net changes (stimulation = up arrow. inhibition = down arrow) with continuous exposure to
PDGF at the doses indicated, compared to vehide controls in RBM stroma1 ce11 cultures
with dex. Changes indicated by the arrows were signifiant at minimally p 5 .O5 as
determined by t tests, or the trend seen is indicated.
Adi~ocvte colonies 3DGF n g / d
0.25 (not deterrnined)
Bone nodules
7. 111
* Wilcoxon signed rank test of al1 experiments at 4 ng/ml PDGF show that overail patterns
are significant, ie., bone nodule numbers decrease (p = .0020), aNBE positive (macrophage)
colony numbers increase (p = .0020), fat colony numbers increase (p = .O 156).
&BE + colonies
T 111
0.50
1
2 - 2.5
4*
T 1/3 (trend 7 2/3)
't 1/6 (trend T 416) (trend 1/6)
k 5/8 ? 1 /8 (trend 1' 2/8)
J6110 (trend & 4/10)
2/3 (trend f in)
?' 5/6 (trend f 1/6)
? 6/'7 (trend T In)
? 8/10 (trend f 2/10)
7' 111
f 414
7- 414
T 7n
Days cultured
Figure 4.1 Ce11 number (log scale) versus time cuitured. The continuous presence of 4
ng/rnI of PDGF (PDGF 4) significantly increased ceIl numbers present in RBM stroma1
cultures at days 4, 6, and 8 (late log phase growth) and the final saturation density when
cornpared to the vehicle control (PDGF O). t test: #p 5 -05, *p I .O 1.
O 0.25 O 5 1 2 4 6 10
PDGF (ng/mI)
p p p p p p p p p p p p p p - - - - - - - - - - - - - - - -
- - - - -
Figure 4.2 The number of bone nodules present in RBM stroma1 ce11 cultures grown for
2 Id. in the continuous presence of dex and PDGF concentratiocs as indicated. PDGF 0.25
nglml significantly increased the number of bone nodules. while PDGF concentrations > 2
ng/rnI dose-dependently decreased the number of bone nodules setn in dex treated cultures.
t test versus vehicle control (PDGF O): #p 5 -05, *p < .O 1 , **p S .O0 1.
....... Coloay size:
msma11( 10-25 cells) medium (25- 100 cells)
' ~large(>100celIs) """'
1 . Totai number .......
PDGF concentration (ng/ml)
Figure 4.3 The number and size of aNBE positive (macrophage) colonies present in RBM
stroma1 ce11 cultures grown for 21d. in the continuous presence of dex and PDGF
concentrations as indicated. The total colony number increases start with PDGF 0.25 ng/ml
and appear to plateau at PDGF concentrations 2 2 ng/rnl. however. large colony numbers
continue to increase. t test versus vehicle control (PDGF 0) for the corresponding size or for
total number: #p 1 -05, *p I .O 1. **p S -00 1 .
O 0.5 1 2 4
PDGF concentration (ng/ml)
Figure 4.4 The number of adipocyte colonies present in RBM stroma1 cell cultures grown
for 21d. in the continuous presence of dex and PDGF concentrations as indicated. PDGF
concentrations > 1 ng/ml dose-dependently increased the number of adipocyte colonies.
Since vehicle control (PDGF 0) had a rnean and standard deviation of 0, one column tests
versus a theoretical mean of O were used: #p 5.05, *p I .O 1. **p 2 .ûû 1.
O 1 - 3 3-5 5 - 7 7 - 9 9- 11 11-13 13-15
PDGF pulse (days exposed)
Figure 4.5 The number of bone nodules present in RBM stroma1 ce11 cultures grown for
21d. in the continuous presence of dex and PDGF 4 n g h l pulses as indicated. 2 d. PDGF
pulses at d. 1 - 3, d. 3 - 5, and d. 5 - 7 significantly increased the number of bone nodules,
while PDGF pulse at d. 13 - 15 showed a (srnaIl) decrease in the number of bone nodules. T
test versus control (O days exposed): #p 5.05, *p S .O 1, **p S -001.
controi controi + PDGF A$.'* A@& + PDGF
90 Figure 4.6 Legend
The number of bone nodules present in RBM stroma1 cell cultures grown for 7d. primary
culture, then sorted according to alkaline phosphatase (AP) expression (high expression =
~ ~ k g h , low or negative expression = AP~OW, unfractionated cells = control) and grown for
21d. in the continuous presence of dex and PDGF 4 @ml. Bone nodules are decreased
significantly by PDGF in both ~ p h i g h and AP~OW fractions; adipocyte colonies are
significantly increased in al1 populations; total aNBE (macrophage) colonies increased in
control and A P ~ ~ ~ ~ populations. While the total uNBE colony number decreased in the
AP~OW fraction in ihis experiment, the number of large colonies increased significantly,
likely obscuring srnaller colonies. t test versus same condition without PDGF: #p < -05, *p I
.Ol, **p 1.001.
Our data suggest that PDGF affects both proliferation and differentiation in RBM stromal
ce11 cultures. m i l e the main purpose of our study was to investigate an effect of PDGF on
osteoprogenitors and bone fomation, we also monitored other lineages which are present in
these cultures, regulated by some of the same factors and hormones (e-g., dex (Herbertson
and Aubin, 1995)), and whose presence may modulate osteoblast lineage ce11 expression
(Aubin, 1997, submitted). The mitogenic effects of PDGF on late log phase growth rate and
saturation density of RBM stromal cultures. together with its ability to stimulate the
numben of macrophage and adipocytic lineage colonies, suggest that PDGF stimulates
proliferation and differentiation in these lineages. In contrat. continuous exposure to PDGF
decreased AP-positive colony and bone nodule number. suggesting that PDGF inhibits
osteoprogenitor differentiation to mature bone-forming osteoblasts. The ability of pulse
treatment with PDGF in early to late log phase growth to increase bone nodule numbers
while pulse treatment late in the differentiation sequence inhibits bone nodule numbers
suggests, however, that PDGF is stimulatory for progenitors at an early stage of osteoblast
differentiation and is inhibitory at later stages. This is consistent with data on calvaria organ
cultures in which the cells proliferating in response to PDGF appeared to be in the periosteal
fibroblast and osteoprogenitor ce11 zones (Hock and Canalis, 1994), and with the suggestion
that PDGF may stimulate a fibrous tissue, rather than bone tissue, phenotype (Marden et al.,
1993). The biphasic nature of the effects of PDGF was also evident over different
concentration ranges. with low doses stimulating and high doses inhibiting bone nodule
fomation. These in vitro studies may shed light on the conflicting data reported from
PDGF treatments in vivo. Continuous exposure to PDGF may be considered analogous to
studies with local administration of PDGF-BB (e.g.. in rat craniotomy defects), which was
found to inhibit bone regeneration (Marden et al.. 1993). Since PDGF is rapidly cleared in
rats (90% within 1 hr. (Cohen et al.. 1990)). pulse exposures rnay mimic systemic
92 administration which has been shown to significantly increase osteoblast number (Mitlak et
al., 1996)). as with early PDGF pulses in our culture system.
Previous studies reported that 24 - 48 hr. pulses with PDGF-BI3 (approximately 10 - 100
ng/ml) decreased AP activity in fetal rat calvaria ce11 cultures (Centrella e t al., 1991;
Pfeilschifter et al.. 1992). Cassiede and colleagues found that a 48 hr. pulse of 5 ng/ml
PDGF-BB on preconfluent rat marrow stromal cells had no significant effect on
osteochondrogenic potential (as measured by bone nodule formation) and observed that a
decrease in AP activity directly after pulse treatment is not predictive of subsequent
osteochondrogenic potential on removal of the growth factor (Cassiede et al.. 1996). We
cannot account for why our data are different from Cassiede and colleagues, but since their
pulse window (d. 4 - 6) falls between our d. 3 - 5 pulses (which were consistently
stimulatory) and Our late log phase growth (d. 5 - 7 or 5 - 8) pulses (which showed mixed
increaselno difference result). it is possible that they treated at a particular developmental
tirne/differentiation stage which is insensitive to a PDGF effect. Taken together with Our
biphasic PDGF concentration effects and the exquisitely sensitive timing required to target
particular differentiation stages, the data argue for cornplex. multiphasic effects of PDGF on
this bone ceIl lineage.
While dex. concomitantly stimulated bone nodule formation and macrophage development in
RBM stromal ce11 cultures (Herbertson and Aubin. 1995), continuous exposure to PDGF
stimulated production of macrophage colonies but inhibited bone nodule formation. Both
macrophages (Yan et al.. 1993) and osteoblasts (Zhang et al.. 1991) are known to express
PDGF p-receptors. and thus both lineages should respond to porcine PDGF which is
primarily PDGF-BB. Macrophages produce cytokines and growth regulating factors (eg.,
PGEz, TNF, IL-6 (Horowitz and Gonzaieç, 1996); IGFBP-4 (Li et al.. 1996)) which may
affect osteoblastic cells. and inhibit stromal development (Goliaei et al., 1995). Since PDGF
93 strongly up-regulates IL- 1 mRNA levels in munne macrophages (Yan et al., 1993), and the
continuous presence of both IL-if3 and IL-la are known to inhibit bone formation (Eliies
and Aubin, 1990), it seemed possible to us that the inhibitory effect of PDGF on bone
nodules might be indirect and mediated through macrophages and IL- 1 production. IL- 1 has
been shown to have biphasic effects on bone formation in RBM stroma1 ce11 cultures, and
the dose response curve varies according to the endogenous IL4 levels (Shadmand and
Aubin, 1995), similar to the biphasic response we measured here with PDGF and the small
shifts in dose responsiveness seen between independent RBM isolations. On the other hand,
the fact that osteoblasts express PDGF-P receptors suggests that PDGF could have direct
effecü on osteoblast iineage cells. To test this hypothesis, we sorted RBM stroma1 cells on
the bais of expression of AP. We showed previously that this resulted in populations 93 -
97% pure in the M h i g h fraction, a fraction significantly enriched for osteoprogenitors and
depleted in adipocytes and macrophages, while the AP[OW fraction was significantly
enriched in macrophages and fat cells (Figure 4.6, and Herbertson and Aubin, 1997). In two
separate experiments, the effects of PDGF on osteoprogenitors (giving rise to bone nodules)
in the M h i g h fractions appeared quantitatively sirnilar to what we observed in the
unfractionated control. suggesting a direct effect of PDGF on the osteoblast lineage. Of
course, we cannot nile out that the fewer remaining monocyte-macrophage lineage cells in
the ~ ~ h i g h fraction are sufficient to effect a cytokine cascade rnediating the PDGF response,
but this seems unlikely given the similar fold-changes in bone in response to PDGF in
populations with markedly different initial numbers and fold-changes in monocyte-
macrophage cells. The reciprocal question of whether the effects of PDGF on macrophages
are a11 direct is also of interest since osteoblastic cells are known to produce some of the
factors thought to be involved in hematopoiesis including M-CSF (Shiina-Ishimi et al.,
1986; Elford et al., 1987: Benayahu et al., 1992; Greenfield et al., 1993)). Again, there
appears to be no direct relationship between the number of bone nodules and the number of
macrophage colonies presen t in unfractionated or fractionated populations, suggesting that
94 the stimulatory effects of PDGF on macrophages are direct. In this regard, other authors
have reported that PDGF-BB has a stimulatory effect on primitive pluripotential
hematopoietic cells (preCFCmulti), mediated by up-regulation of U-1 in marrow
macrophages. and showed conclusively the presence of PDGF $-receptors on normal
marrow macrophages (Yan et al., 1993).
Exposure to PDGF-BB has been reported to inhibit expression of differentiated phenotypes
in certain other mesenchymal ceil types, e.g., smooth muscle (Holycross et al., 1992). While
chronic exposure to high concentrations inhibits osteogenesis, it is clear that low doses and
puisatile exposure of early progenitors leads to stimulation of differentiation to mature
matrix-synthesizing osteoblasts. In addition, we consistently found a stimulation of
adipocyte colony numbers between 1 - 10 ng/ml PDGF. which appears to be a novel
observation. Indeed, PDGF has been reported to inhibit adipocyte differentiation in human
female adipose tissue cultures, although the concentrations required to see inhibition were 2
10 ng/ml (Hauner et al., 1995). concentrations higher than we used here. It is not clear why
Hauner et al. did not see a stimulatory effect although differences in the cultures and culture
conditions used (including species differences - human versus rat: serum-free venus serum
containing medium, and differences in anatomic sites (rneduflary versus non-medullary
adipocytes)) are al1 possibilities. It is also interesting that PDGF is stimulatory to both
adipocyte precursors and osteoblast precursors under at least some conditions, e.g., very low
dose treatment, but that the responsiveness of the lineages diverges at higher concentrations
such that PDGF stimulates adipogenesis white concomitantly inhibiting osteogenesis in the
same cultures. Since we found that PDGF is mitogenic at early stages of culture during log
phase growth, it is possible that progenitors for both lineages, including, e.g.. a putative
bipotential adipocyte-osteoblast progenitor (reviewed in (Gimble et al., 1996)), are
comparably stimulated to undergo division, but that subsequent differentiation steps are
modulated quite differently by this growth factor. In this regard, the differences in
95 sensitivity to PDGF (EDso for adipocyte colony stimulation, 1.5 n g / d PDGF; ID50 for bone
nodule inhibition, 3 ng/ml PDGF, in the sarne experiments) of these lineages are notable and
together with the marked differences in absolute nurnber of adipocyte and osteoblast
colonies, suggest that the stimulation in adipocyte formation is not at the expense of
osteoblast formation. as suggested with some hormone treatment protocols, including
1,25(OH)2D:, treatment (reviewed in (Gimble et al., 1996; Aubin and Heersche, 1997)).
Our data suggest that the effects of PDGF on the differentiation of osteoprogenitors to
mature osteoblasts in RBM stromal populations are biphasic, with both stimulation and
inhibition of differentiation and bone nodule formation being observed, probably through
the growth factor targeting different events early and late in the differentiation sequence.
These complex effects, which occur concomitantly with modulation of other Iineages
present in the stroma1 environment, suggest that PDGF has pleiotropic effects on bone
metabolism.
CHAPTER 5
Conclusions and Future Experirnents
97 5.1 Conclusions and Future Experiments
The studies presented in this diesis were aimed at furthering knowledge on whether cell-ce11
interactions affect osteogenesis in the heterogeneous cellular environment of RBM ce11
cultures. To this end, RBM stromal ceils were grown under conditions stimulating bone
nodule formation, then histochemical and immunohistochemical staining, and flow
cytometric analysis and sorting were used for description and analyses. Finally, the effect of
PDGF was examined in this system using these techniques.
In Chapter 2, 1 began to elucidate and quantify some of the subpopulations present when
RBM stromal cells are grown with or without dex under conditions favoring bone formation.
Culture dishes were analyzed for ce11 counts, or stained with either histochemical or
immunohistochemical stains, and colony types were quantitated. or cells were processed for
flow cytometry. Dex significantly increased the number of AP positive colonies and von
Kossa positive bone nodules, aNBE positive colonies, and ED2 positive (macrophage)
colonies. The number of adipocyte foci was largely unaffected in these experiments. Flow
cytornetry confirmed colony counts and showed stimulation by dex of AP positive cells and
macrophages, and in addition, the reduction of hemopoietic cells expressing leukocyte
common antigen. These data show that when RBM stromal populations are grown under
conditions stimulating osteoprogenitor differentiation and bone formation, the stroma1
subpopulation rnake-up, including expression of hemopoietic lineages, is markedly altered.
Specifically, dex stimulated osteoprogenitor differentiation concomitant with promoting the
growth and differentiation of macrophages. These data also showed for the first tiine the
presence of macrophages in RBM stroma1 cultures grown under conditions favouring bone
formation, and extended the information on the subpopulation make-up and heterogeneity
under these culture conditions.
98 Since the presence of multiple ceIl types in RBM stromai populations rnay modulate or even
mask, and complicate interpretation of, cytokine and hormone effects on the
osteoprogenitors present. 1 looked for a method for purification of the osteoprogenitor
population. In Chapter 3, 1 described experiments in which flow cytometric sorting of 7 d.
primary RBM stromal ce11 cultures was performed on the basis of AP expression with an
antibody against AP (RBM 21 1.13 (Turksen and Aubin, 1991)). The resuitant ~ ~ h i p h .
AP'OW, or control cells were plated to determine osteoprogenitor. macrophage, and adipocyte
distribution and frequency. Approximately 50% of osteoprogenitor cells (capable of giving
rise to bone nodules) were lost during processing/sorting when compared to unsorted
controls. Nevertheless. within the kiPhigh fraction. the number of AP positive colonies and
bone nodules were signi ficantly enriched compared to the unfractionated control; the
increase in osteoprogenitor frequency ranged from approximately 2 to 100 fold. There were
few assayable osteoprogenitors in either the ~ ~ h i g h or APlow fractions in the absence of dex,
suggesting that RBM stroma contains largely dex-dependent osteoprogenitor populations,
and that dex rnay regulate osteoprogenitors subsequent to the upregulation of AP. Bone
nodule numbers in either the or A P I ~ W fraction did not follow a linear relationship
w i th decreasing plating densi ty . Adipocyte and macrophage colonies were depleted in the
M b g h fraction of cells, but were enriched in the AP~OW fraction. I thus showed that flow
cytometric sorting of RBM stromal populations according to high or low Ai? expression is
an effective technique for enrichment of AP positive colonies and osteoprogenitors capable
of dividing and differentiating into bone nodule-forming cells, and should prove useful for
further studies of the nature of the osteoprogenitors and their regulation in this tissue.
In Chapter 4. 1 combined the techniques of the previous two chapters to investigate the
effects of PDGF in RBM stroma1 ceil populations, again grown under conditions promoting
growth and differentiation of osteoprogenitors to mature bone-forming osteoblasts. PDGF
was mitogenic during late log phase growth and increased the saturation density of RBM
99 stromal cultures, as well as dose-dependently stimulating the numbers of macrophage and
adipocytic lineage colonies, suggesting that PDGF stimulated proliferation and
differentiation in these lineages. The continuous presence of PDGF over the same
concentration range had biphasic effects on bone nodule formation, increasing bone nodules
at low doses (6 1 ng/ml) and dose-dependently decreasing formation at 2 4 ngfml. When
present as a short-duration pulse in early to Iate log phase only, PDGF increased bone
nodule numbers at al1 doses tested, but inhibited bone nodule formation late in the
differentiation sequence, suggesting that PDGF is mitogenic during an early stage of
osteoblast differentiation but inhibitory later. Comparison of controVmixed RBM stroma1
cultures with populations fractionated on the bais of AP expression suggested that the
inhibitory effects of PDGF may be elicited by a direct action on osteoblast lineage cells.
These complex effects on bone. which occur concomitantly with modulation of other
lineages (macrophages and adipocytes) present in the stromal environment, suggest that
PDGF has pleiotropic effects on bone metabolism.
The histochemical and immunohistochemical techniques described. dong with the ability to
fractionate RBM stroma1 populations on the basis of AP expression, have facilitated the
characterization of the RBM stromal system, and will be of use when the effects of other
cytokines or growth factors are analyzed in this system. We are currently limited in the rat
system by a lack of other specific antibodies to early osteoprogenitors as recently described
for human osteoblastic cells (Sirnmons and Torok-Storb, 199 1 ; Haynesworth et al., 1992;
Gronthos et al.. 1994; Joyner et al., 1997), especially given that the specific macromolecules
recognized by these antibodies are only beginning to be identified (see, e.g.. (Bruder et al.,
1997)) to allow preparation of rat-specific reagents. As other specific reagents are identified
by antibody or molecular approaches, future sorting experiments can be designed to achieve
even better enrichment for cells earlier in the osteoblast lineage. While the 93 - 97% purity
obtained in Our sorting experiments is comparable to other sorting and irnmunopanning
1 0 0 techniques, it has left open some arnbiguity and necessitated some caution in interpretations
of the data as discussed in Chapter 3 and Chapter 4. Nevertheless, the approaches have been
established and they lead to reasonable ce11 yields, so that future experiments can al1 be
airned at improving the purity of the osteoblast lineage rather than development of
techniques. Methods for improving purity could include double sorting with one specific
antibody. use of both positive and negative immunoselection in sorting, andlor a
combination of sorting with irnmunopanning techniques. Likewise, as antibodies become
available recognizing other rat early hemopoietic cells. it will become possible to identify
more precisely and manipulate the leukocyte antigen positive cells, whose numben are also
markedly altered by dex in osteogenic RBM cultures, to determine more clearly their
roIe/interactions with osteoprogenitors in this system. The ability to identify and then
fractionate the subpopulations present in RBM stroma1 ce11 populations represents an
important step towards understanding the cell-ce11 interactions which affect bone formation
in BM.
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