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ISOLATION AND CHARACTERIZATION OF VERY EARLY OSTEOPROGENITOR CELLS
Susan Allison Steckle
A thesis submitted in conformity with the requirements for the degree of Master of Science
Graduate Departnient of Anatomy and Cell Biology University of Toronto
8 Copyright by Susan Allison Steckle (1997)
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ISOLATION AND CHARACTERIZATION OF VERY EARLY OSTEOPROGENITOR CELLS
Master of Science, 1997.
Susan Allison Steckle Graduate Department of Anatomy and Ce11 Biology University of Toronto
The purpose of ihis work was to use a previously established technique. poly(A)-
PCR. on freshly plated primary cells to aid in the characterization of very early
osteoprogenitor cells. Establishing a molecular profile representative of the
osteoprogenitor phenotype will help in the identification of morphologically indistinct
osteoprogenitor cells prior to their overt morphological differentiation. In the rat calvarial
(RC) cell mode1 of osteoblast differentiation. RC cells proliferate and differentiate in
vitro over a time span of 21 - 25 days, culminating in the formation of mineralized bone
nodules. Very early (day 4 to 6 aller plating) in the culture period, individual colonies
arising from single cells were identified microscopically, and a cellular sample (1 -3 cells)
was micrornanipulated fiom each colony. This cellular sample was used to produce a
poly(A) cDNA library, while the remaining cells in the colonies were left undisturbed and
allowed to differentiate. After bone-forming colonies had been retrospectively identified,
their respective cDNA libraries were used for Southem blotting for the presence of
messages for bone matrix proteins and other putative osteoprogenitor markers.
Osteoprogenitors were compared with fibroblastic cells h m similarly identified colonies
that did not differentiate to bone nodules. There was intercellular heierogeneity in the
expression profiles of the very early osteoprogenitor cells, which expressed al1 or some
combination of osteopontin, osteonectin, type 1 collagen and type III collagen; none
expressed bone sialoprotein. alkaline phosphatase or osteocalcin. Fibroblastic cells were
found to express some combination of type 1 collagen, osteopontin, and osteonectin; only
one sarnple expressed alkaline phosphatase. Results suggest that the fibroblast and
osteoprogenitor phenotypes express several cornmon markers. This technique will aid in
the fùrther molecular characterization of early cells in the osteoblast lineage.
iii
ABSTRACT ................................................................................................................................................. ................................................................................................................................ TABLE OF CONTENTS iv
LIST 01: FIGUES .......................................................................................................................................... v ........................................................................................................................... LISI- OF ABBREVIATIONS vi
OBJECTIVE ................................................................................................................................................... I ...................................................................................................................................... ~NTRODUCTION 1
I . Histology of Bone Cclls ..................................................................................................................................... I 7 .............................................................................................................................................................. Figure I -
1.. Ostroblast Diffrrentiation .............................................................................................................................. 4 .. kigure 2 ............................................................................................................................................................. 5 III . DiffcrentiotionMarkers .................................................................................................................................. 7
.................................................................................................................................................. Osteopontin 7 ................................................................................................................................................. Osreoncctin 9
........................................................................................................................................... Collagen type 1 I O Coltagen type I I 1 ...................................................................................................................................... I I Osteocalcin ............................. .. ......................................................................................................... 13
.................................................................................................................................... Bonc Sialoprotein 1 4 ................................................................................................................................. Alkaline Phosphatase 15
L32 ........................................................................................................................................................... 16 IV . Poly(A) PCR and Low Density Cultures .................................................................................................... 16
..................................................................................................................................................... V . Objectives 1 8 ............................................................................................................................................... MET~IODS 19
Ccll Culture ............................................................................................................................................... 19 Single Csll Isolaiion .................................................................................................................................. 19
............................................................................................................................................. Poly(A)-PCR 2 0 7 7 Southem Analysis ................................................................................................ ........................................ -
............................................................ ............................... cDNA Probes .. 2 3 .............................................................................................................................................. Data Analy sis 24
......................................................................................................................................... R ~ s u ~ r s -26 Figure 3 ............................................................................................................................................................ 27 Table I ............................................................................................................................................................. 29 Figurc 4 .......................................................................................................................................................... 30 Figure 5 ............................................................................................................................................................ 32 Figure 6 ............................................................................................................................................................ 33
............................................................................................................................................... Discussio~ 36 I . The Poly(A)-PCR Approach ............................................................................................................................ 37
Tcchnical Limitations ................................................................................................................................. 37 Rcproducibility and Deiection Limits ......................................................................................................... 39
..................................................................................................... . II Phenotypc of Early Osteoprogcnitor Cclls 40 .................................................................................................................. Phenoiypic Heterogeneiîy 42
..................................... III . Rctrospective Analysis of Single RC Cells .. ............................. 44 SUMMARY AND CONCLUSIONS .................................................................................................................. 47
............................................................................................................................................. REFERENCES 48
Proposed lineage diagram with differentiation steps for cells of the osteoblast lineage.
Ostcoblast lineage diagram showing known expression profiles for each stage in the lineage.
Representative RC culture dishes stained by the Von Kossa technique showing mineralized nodules and other designated colonies.
Composite of Phosphorimager data for molecular markets tcsted on each sample.
3-Dimensional graph of al1 markers (as a ratio to total cDNA), showing relative expression levels.
Graphs of individual molecular markers for each sarnple tested.
a-MEM ALP BSA BSP cDNA COLL-1 COLL- I II dATP, dA dCTP dGTP dNTP DTT dTTP, dT ECM FGF-R kd M-MLV m mRNA OB OCN OD ON OP OPN PCR PDGF-R PTH-R RC RGD RT SDS SPARC SSC TBE
a-minimum essential medium alkaline phosphatase bovine serum albumin bone sialoprotein - complementary deoxyribonuclcic acid collagen type 1 collagen typc III 2'-deoxyrib adenosine triphosphate 2'-deox yribo c ytidine triphosphate 2'-deoxyribo guanosine triphosphate 2'-deoxyribonuc leotides dithiothrtitol Z'deoxyribo thymidine triphosphate extracellular matri x fibroblast growth factor nceptor kilodalton Moloncy Murine Leukemia V i w mi llimcûe messenger ribonucleic acid ostcoblast o s t d c i n optical density units osttonectin osteoptogenitor osteopontin polymcrase chah reaction plateletderived growth factor nceptor parathymid hormone rwcptor rat calvarial argininc-gl ycine-aspartate reverse mnscription sodium docecyl sulfate secretcd pmtein acidic and rich in cystcine sodium chloridt/sodium cime b& tris-boratc/EDTA electrophorcsis bufkr
The purpose of this work was to identiQ retrospectively and characterize very
emly osteoprogenitor cells in the rat calvarial (RC) ce11 mode1 of osteoblast
di fferentiation.
During osteoblast differentiation, morphologically indistinct osteoprogenitor cells
proliferate and differentiate into morphologically identifiable preosteoblasts, then
osteoblasts and osteocytes. An increasing number of molecular markers are known to be
up- and dom-regulated during this differentiation sequence, and describing their
expression profiles aids in characterization of a phenotype for these cells as they progress
fiom less mature to more mature cells. In this Introduction, 1 will describe various
aspects, including histological and molecular features, of the osteoblast lincage.
1. Hbtology of Bone Celh
The cumnt mode1 of bone ce11 development is a linear dif'ferentiation sequence
h m the mesenchymal stem ce11 to cornmitteci osteoprogenitor to pmsteoblast to newly
diffcrentiated osteoblast to mature osteoblast to osteocyte as depicted in Figure 1 (Owen
et al, 1985; Aubin et al, 1993, 1995).
Mesenchymal E a r l ~ Late PreOB/ OB Osteocyte Stem Cell OP OP Early OB
Figure 1: A proposed lineage diagram with differentiation steps for cells of the osteoblast lineage. Adapted h m Aubin et al, 1993. (OP: osteoprogmitor; OB: osteoblast)
Although its location in vivo is uncertain and its molecular profile not yet
established, evidence for the existence of a mesenchymal stem ce11 includes the isolation
of clonal rnultipotential ce11 lines with capacity to differentiate into various mesenchymal
tissues including muscle, fat, cartilage and bone (reviewed in Aubin et al, 1993). By
definition, the osteoprogenitor is considered commitîed to the osteoblast lineage. At least
some osteoprogenitors are thought to reside in the periosteum of bone and the stroma1
compartrnent of bone marrow. The spindle-shaped cells resembling fibroblastic cells and
with plump oval nuclei and abundant cytoplasm in growing bone are thought to be
osteoprogenitors (Scott, 1967). The osteoprogenitor cells in the fibrous periosteum are
thought to have undergone little differentiation; they are morphologically
indistinguishable fiom fibroblastic cells and it is not possible to identiQ them on the basis
of their microscopic appearance alone.
Osteoblasts, on the other hand, have a distinct cuboidal or polygonal morphology
whm actively foming bone. They have a basophilie cytoplasm that is rich in rough
endoplasmic reticulum, and they line the bone surface while laying dom bone matrix.
Preosteoblasts resemble osteoblasts morphologically and are Iocated behind the layer of
osteoblasts on the bone surface (farther away h m the newly developing osteoid).
Osteocytes are the terminally diffetcntiated ce11 of the osteogenic lineage. They are
flattened, stellate-shaped cells that lie in lacunae in bone. As such, they arc completely
surroundcd by the bone maûix, and have long cytoplasmic processes that extend through
canaliculi in bont to aid in communication with other cells and ensure nourishment.
Bone lining cells are thought to be inactive osteoblasts that have flattened and
elongated dong bone surfaces where neither formation nor rcsorption is occurring. Bone
lining cells communicate amongst themselves and with neighbouring osteocytes through
gap junctions.
II. Osteoblast Differentiation
Osteoblasts are bone-fonning cells which are readily identified by their distinct
cuboidal morphology and their location on the bone surface during bone formation.
During osteogenesis, they actively synthesize a variety of bone matrix proteins such as
collagen, osteopontin, SPARCIosteonectin, osteocalcin, bone sialoprotein, as well as the
membrane-bound enzyme alkaline phosphatase (reviewed in Aubin et al, 1993, 1995).
Expression of these proteins has helped in the molecular identification of osteoblasts.
Early osteoblasts and preosteoblasts have also been extensively characterizcd recently.
For exarnple, they express many of the same proteins that mahue osteoblasts express, but
there is marked heterogeneity in the expression profiles at the single ce11 level at al1 these
nlatively mature stages (Liu et al, 1994). Individual cells stageci as early osteoblast,
pmsteoblast or osteoprogenitor have varied expression patterns of the repertoire of bone
matrix proteins as well as cytokine receptors, such as PDGF-R, FGF-R and PTH-R (Liu
et al, 1994). The known expression profiles for the osteoblast lineage are depicted in
Figure 2, and will be individually discussed below.
Mesenchymal Earl~ Late Pre-OB/ OB Osteocyte Stem Ce11 OP OP Earl y OB
MARKERS
Osteopontin ?
Collagen type 1 ?
Ostcocalcin ?
Alkaline Phosphatase ?
Bone Sialoprotein ?
Figure 2: A proposed lineage diagram with differentiation steps for cells of the osteoblast lineage. Known expression profiles for each stage in the osteoblast lincage are shown. Adapted fmm Aubin et al, 1993,1995
LEGEND: 3 Not known - - - - - - - - Not preseni ----------------- Present at low or heterogenous levels Present Present at high levels
Whereas the mature osteoblast phenotype is considered reasonably well-
characterized, recently. more attention has been directed towards the delineation of the
osteoblast differentiation sequence and questions related to osteoblast development. It is
unknown how many discrete steps or stages exist as a putative mesenchymal stem ce11
diflerentiates into a cornmitted osteoprogenitor, and on to a mature osteoblast. Reasons
why this differentiation sequence has been difficult to characterize include the inability to
distinguish morphologically earlier cells in the lineage and the lack of molecular markers
for the early cells and transition points. Nevertheless, limiting dilution analysis has shown
that the osteoprogenitor is present at a very low frequency, approximately 1 in 300 cells,
in the RC ce11 population (Bellows and Aubin, 1989). one mode1 cornmonly used to study
osteoblast differentiation in vitro. The RC cell population is obtained by enzymatically
digesting rat bone tissue to yield a mass population including cells at different stages of
differentiation, such as more mature osteoblasts and fibroblasts, as well as
osteoprogenitors and other ce11 types with high proliferative potential (Bellows et al,
1990). Although their fiequency in the population is very low, the presence of the
osteoprogenitor is defined by its proliferative capacity and the fact thnt its progeny have
the ability to differentiate into bone nodule forming osteoblests in viîro. Their low
fiequency has also contributed to the difficulty in ncognizing osteoprogcnitor cells.
III. Differentiation Markers
Osteoblasts express many different molecules, but some of the better studied ones
are extracellular matrix molecules that the osteoblast lays down in the bone matrix, such
as types 1 and III collagen, osteopontin, osteonectin, bone sialoprotein, osteocalch, and
the membrane-bound enzyme alkaline phosphatase. As osteoblasts develop, more of these
molecules are upregulated, and are thus used as markers of osteoblastic differentiation.
The following sections will describe the current state of characteriuition of these markers,
their tissue distribution, putative fùnctions and their expression profiles throughout the
osteoblast lineage. The final section will address L32, not as a marker of osteoblastic
differentiation. but as a general marker of the presence of amplified cDNA fiom each of
the cells analyzed.
Osteopontin
Osteopontin (OPN) is a sialic-acid rich, 44 kd phosphorylated, ca2+-binding
glycoprotein that contains an ArgGly-Asp (RGD) adhesion motif (Oldkrg et al, 1986).
It was originally purified fiom bone as sialoprotein 1 (Franzen and Heinegard, 1985;
Fisher et al, 1987) or 44 kd bone phosphoprotein (Prince et al, 1987), and h m other
cells as 2ar (Smith and Denhardt, 1987). Although OPN was originally purified fiom
bone, it has sincc been identified in non-mineralizing tissues in such diverse otgans and
tissues as kidney (Yoon et ai, 1987; Lopn et ai, 1993; Giachelli et al, 1994; Shiraga et
al, 1992; Worcester et al, 1992). imer ear (Lopez et al, 1995; Swanson et ai, 1989),
utew, decidu, placenta and metrial glands (Young et ai, 1990; Nomura et al, 1988;
Waterhouse et al, 1992), artenal smooth muscle (Giachelli et al, 1991, 1993; Liaw et al,
1995), and epithelium of several organ systems (Brown et al, 1992). It is also expressed
in numerous tumour ce11 types and cells stimulated to proliferate by treatment with
tumour promoters (Craig et al, 1988; Khokha et al, 1991).
Since it is secreted by osteoblasts? and expressed in osteoclasts and osteocytes.
OPN is considered to be important in both mineralization and resorption of the bone
matrix (Reinholt et al, 1990; Weinreb et al, 1990; Flores et al, 1992; Tezuka et al, 1992;
Chen et al, 1993; Sodek et al, 1992) though a definitive huiction has not yet been
discovered. One possibility, suggested by the fact that OPN is found
imrnunohistochemically at the mineralization front in developing bone, is that OPN may
influence the rate of rnineralization rather than nucleation of hydroxyapatite crystal
growth (McKee el al, 1990; Sodek et al, 1992; nviewed in Denhardt and Guo, 1993). A
role in resorption has also been proposed (Reinholt et al, 1990; Ross et al, 1993). OPN is
reporteci to associate with fibronectin (Nemir et al, 1989; Singh et al, 1990), type 1
collagen (Chen et al, 1992) and osteocalcin (Ritter et al, 1992). It is also regarded as a
ceIl attachrnent factor, as it promotes the adhesion and spreading of osteoblasts,
osteoclasts, mesenchymai cells, fibroblasts, nontransformecl calvarial ce11 lines,
osteoblast-like osteosarcorna cells and many transfomiad fibroblastic ce11 lines
(Somerman et al, 1987, 1989; Oldberg et al, 1986), and is a ligand of the CD44 receptor
(Weber et al, 1 996).
OPN immunoreactivity has k e n found in osteoblasts and osteocytes of both
endochondral and membranous bones, as well as in fibroblast-shaped cells, refemd to as
preosteoblasts, which were not in contact with the bone itself but in osteogenic sites
(Mark et (11, 1987a). During the osteoblast differentiation sequence, OPN typicaily
appears prior to other bone matrix proteins, including bone sialoprotein (BSP) and
osteocalcin (Mark et al, 1 987% 1 987b, 1 988; Stein et al, 1 989; Owen et al, 1 990; Chen et
al, 199 1 a; Moore et al, 199 1 ; Pockwinse et al, 1 992; Liu et al, 1994).
Osteonectin
Osteonectin (ON) is a 32 kd phosphorylated glycoprotein that can simultaneously
bind to hydroxyapatite and type 1 collagen (Termine et al, 1981). ON is identical to
SPARC (Secreted Protein Acidic and Rich in Cysteine; Mason et <II, 1986a), to a 43 kd
protein secreted by epitheliai cells in culture (Sage et al, 1984) and to BM-40, a protein
produced by the murine Englebrah-Holm-Swarm basement membrane tumor (Dziadek et
al, 1986). While ON is expressed in osteoblasts (Holland et al, 1987) and odontoblasts
(Fujisawa et al, 1989, Reichert et al, 1992) and is abundant in bone of the axial skeleton
and skull (Termine et al, 198 1, Nomura et al, 1988, Holland et d, 1987), it is also present
in a variety of non-mineralizing tissues. SPARC was described as a product of the
parietai endoâerm cells during mouse development (Bolander et al, 1988, Holland et al,
1987, Mason et al. 1986b). ON has a widespread dishibution in otha tissues, particularly
those undergohg rapid proliferation and matrix production, as well as tissues involvcd in
the processes of rnindization and steroid production (Mundlos et al, 1992, Holland et
al, 1987).
While some investigators have proposed a role for ON in the process of
mineralization (Jundt et al, 1987, Tennine et al, 1981), its distribution in non-
mineralizing tissues (Holland et al, 1987, Nomura et al, 1988, Wasi et al, 1984, Bolander
et al, 1988, Dziadek et al, 1986, Sage et al, 1989% Mundlos et al, 1 W2), and its
inhibition of hydroxyapatite-seeded crystal formation in vitro (Doi et al, 1989, Romberg
et al, 1985) argue against a specific role for ON which is limited to the initiation of
mineralization (Sage et d. 1989a, Holland et al, 1987). Although ON is clearly linked to
proliferation in fetal tissues (Mundlos et al, 1 W2), it has also been shown to inhibit ce11
spreading (Sage et al, 1989b, Lane and Sage, 1990). Of particular interest to the present
study, ON is expressed in human marrow-derived osteoprogenitor cells (Long et al, 1995)
as well as in spindle-shaped cells, thought to be newly-recruited osteoblastic precursors,
probably at an intermediate stage between the so called "detemined osteoprogenitor
cells" (Friedenstein 1973) and already completely developed active osteoblasts (Jundt et
al, 1987).
Collagm @pe I Type 1 collagen (COLL-I) is the most abundant of the bone matrix proteins, and it
is prcvalent in other tissues such as sicin, tendon, ligament, cornea and most other
connective tissues as well. Fibrillar or fibril-forming collagens, which include both type 1
and type III, are similar in size, containhg large triple-belid domains of about 1000
amino acids per chah Type 1 collagen is a trimeric molecule composed of two a l(1)
chains and one a2(1) chain.
COLL-1 is the major structural component of the bone matrix. It can fom cross
links with other collagen types, particularly type II1 in bone (Cheung et al, 1983), and it
associates with OPN (Chen et al, 1992) and BSP (Fujisawa et al, 1995). Given its wide
distribution throughout the body, COLL-1 is not cxpected to play a rolc in the nucleation
of hydroxyapatite formation during mineralization of the bone matrix, although
hyàroxyapatite crystals do sit in the hole zone regions of collagen fibrils once the
mineralization process has begun (reviewed in Glimcher, 1984).
In some studies, expression of COLL-1 has k e n shown to be initially high, then
gradually falling to lower levels as osteoblasts mature (Stein et al, 1989; Gentenfeld el
al, 1988; Owen et al, 1990, 1991), while in others it is found to increase as osteoblasts
mature (Liu et al, 1994), more consistent with results of in situ hybridization which show
high COLL-1 expression in mature osteoblasts including both younger and older
osteoblasts (Heersche et al. 1 992).
Colfagen @pe 111 Type III collagen (COLL-III) is a homotrimer of three identical collagen al(II1)
chains which can becorne rapidly cross-linked through the formation of inttrmolecular
disulphide bonds (Cheung et al, 1983). There is some controversy regarding COLL-III
expression in bone and bone cells. While there is relatively littîe data in the literatute
describing COLL-III expression in osteoblasts and other cells in theu lincage, many have
used low or lack of COLL-III expression as indicative of osteoblast populations free of
fibroblast contamination (Robey and Termine, 1985). Indeed, some have stated that
"osteoblasts produce exclusively type 1 collagen" (Rodan and Noda, 199 1). However,
others report the pnsence of small amounts of COLL-III in the organic matrix of bone
(Niyibizi and Eyre. 1989; Keene et al, 1988), particularly in discrete fiber bundles
throughout the bone cortex, mainly concentrated at the Haversian canal surface and the
bone-periosteal interface (Keene et al, 1991), although whether of osteoblast or other ce11
origin is not clear. Mouse odontoblasts express COLL-III (Lukinmaa et al, 1993). COLL-
III is the major collagen of the fibrous matrix that foms dong the periosteal surface
(reviewed in Ashhurst, 1990) and is expressed in the upper demis, as well as in the
fibroblasts of the periosteurn and fibrous mesenchyme between bone spiculas (Sandberg
et al, 1988). Various subpopulations of osteoblast-like cells isolated fiorn chick calvaria
produced only COLL-1 in vitro, while early digestion cells with a more "tibroblast-like"
appearance produced COLL-I as well as COLL-III (Wiesîner et al, 1981). Bone nodules
formed Ni vitro fiom fetal rat calvarial cells immunolabel for COLL-III (Bellows et al,
1986). Also, COLL-III expression has been demonstrated in mouse osteoblast-like cells
(Scott et al, 1980). clonal osteoblastic lines fiom fetal rat calvaria (Aubin et al, 1982),
chick embryo osteoblests (Majmudar et al, 1991), and in cultures of 7-day old rat tibia1
ceils (Stringa et al, 1995). The alkaline phosphatase-rich rat osteosarcorna ce11 line ROS
3 712.8 was shown to synthesi~ approh te ly 98% COLL-1, while a more 'bfibroblastic"
ce11 line h m the seme tumot, ROS 291, produced 7096 COLLI and 304b COLL-III (P.
Bernstein, unpublishcd observation; discussed in Rodan and Rodan, 1983).
Osteocalcin
Osteocalcin (OCN), also known as bone gla protein, is a 6 kd vitamin-K
dependent matrix protein that contains yîarbxyglutarnic acid and binds to ca2+ and
hydroxyapatite (Gundberg et d, 1984; Hauschka et al, 1975). OCN is synthesized by
osteoblasts and odontoblasts in bone and dentin, mspectively, and, while largely
restricted to mineralizing tissues (Bronckers et al, 1 985. 1 987; 1 keda et al, 1992; Ohta et
rrl, 1989), recently Thiede et al (1994) reported osteocalcin expression in
megakaryoc ytes.
While generally thought to be first expressed with the onset of mineralization
(Broncken et al, 1985, 1987; Groot et al, 1986; Gerstenfeld et al, 1987; Mark et al,
1 W b , 1988; Stein et al, 1989; Boivin et al, 1990; Owen et al, 1990, 1991 ; Pockwinse et
al, 1992), single ce11 poly(A)-polymerase chain reaction (PCR) (Liu et al, 1994) and
immunohistochemistry studies (Bronckers et al, 1987) have demonstrateci that young
osteoblasts are positive for OCN prior to detectable mineralization. A definitive fùnction
has not been discovered for OCN, however in vitro evidence shows thac OCN inhibits the
transition from brushite to hydroxyapatite (Romberg et al, 1986) which suggests that
OCN may act as a negative regulator of bone mineralization. Since OCN associates with
OPN in vitro (Ritter et al, 1992), and OPN may act as a ce11 attachrnent factor for
ostaoclasts, OCN may play a role in bone resorption (reviewd in G e h n Robey, 1989).
However, recent studics on mice lacking OCN expression support a role for OCN in
growth plate development, bone formation and cortical thickness (Desbois et al, 1995).
Botte Sioloprofein
Bone sialoprotein (BSP) is a 33-34 kd protein that undergoes extensive ps t -
translational modifications to make it approximately 75 kd (Oldberg et al, 1988; Ecarot-
Chamer et al, 1989; Fisher et al, 1990; Zhang et al, 1990). BSP contains an RGD-ce11
attachent motif, as well as several stretches of polyglutamic acid that may be involved
in hydroxyapatite binding (Oldberg et al, 1988) since BSP is able to nucleate
hydroxyapatite crystal formation (Hunter and Goldberg, 1993). BSP also binds to
collagen (Fujisawa et al, 1995) and cnhances attachent and spreading of gingival
fibroblasts in vitro (Somennan et al, 1988).
BSP expression was first thought to be restncted to bone tissue (Oldberg et al,
l988), but it has bem found in trophoblasts in placenta (Bianco et al, 1991) and recently
in tumour cells metastasizing to bone (Bellahcéne et uf, 1996). Its expression is
associated with new bone formation by differentiated osteoblasts (Chen et ai, 1199 b). It
has been proposeci that the appearance of the ability to produce BSP marks the boundary
between the preosteoblast and osteoblast stages of osteoblast development (Bianco et al,
1993) and its expression ciearly p d e s that of osteocalcin (Liu et al, 1994). BSP
expression increascs with the development of nascent bone nodulcs in rat bone marrow
stroma1 ce11 cultures, and declines in older mineralizing nodules (Malaval et al, 1994).
Alkaline Phosplratose
Alkaline phosphatase (ALP) is a characteristic product of osteoblasts (Rodan and
Rodan, 1983). There are three alkaline phosphatase isozymes (placenta1 (Kam et al,
1985); intestinal (Henthom et al, 1987; Berger et al, 1987); and tissue non-specific
isozyme which is highly expressed in bone, liver and kidney (Weiss et al, 1986)) that
mise From three separate genes. ALP catalyzes the hydrolysis of monoester phosphates. at
a pH optimum of 8 to 10. The enzyme is linked to the extracellular ce11 surface by a
phosphatidyl inositol linkage (Noda et al, 1987) and is removed by cleavage with
phosphatidyl inositol-specific phospholipase C (Fedde et al, 1988; Turksen and Aubin,
199 1 ). Though its physiological function in bone remains uncertain, it is thought that
ALP is cleaved from osteoblasts and circulates in the bloodstream during penods of
active osteogenesis, allowing senim ALP activity to be used as an indicator of bone
formation and turnover (McComb et al, 1979). ALP activity in bone cells has been
reported to change with the ce11 cycle, dropping during G2 and M phases, rising during G ,
and peaking during S phase (Fedarko et al, 1990). By histochemistry and biochemical
analysis of some cells lines, ALP activity is found to be high in osteoblasts and young
osteocytes and low in more mature osteocytes (Doty and Schofield, 1976; Rodan and
Rodan, 1983).
ALP expression is thought to appear quite early and clearly pnor to the onset of
mineralization in differentiating osteoblastic cells, tending to increase as osteoblasts
mature then decrease when mineralization is well progressed (Bronckers et al, 1987;
Aronow et al, 1990; Owen et al, 1990; Turksen and Aubin, 1991 ; Mark et al, 1987a).
Studies on single cells and isolated colonies confimed early onset of ALP expression;
ALP increased as colonies became osteogenic and acquired mineral, but rnRNA levels
did not decrease relative to other markers of osteoblast differentiation during the time
period investigated (Liu et al, 1994).
We sought a specific molecule to serve as a general control for the amplification
process. An appropriate control would be a molecule that is known to be present at
relatively stable levels, regardless of ce11 cycle stage or cellular maturity. L32 is a
ubiquitous protein that is an integral component of the ribosome in both prokaryotic and
eukaryotic cells, and has a poly(A) tail. making it suitable for amplification by Poly(A)-
PCR. Thus, L32 appeared to fit the criteria for a control of the amplification process.
although its signal intensity varied more markedly than anticipated in cornparison to total
cDNA signal intensity. It was thetefore not used to nomalize the data for cornparisons
(total cDNA signal intensity was used for this purpose - see Methods/Data Analysis).
IV. Poly(A) PCR and Low Density Cultures
Work already done in ow lab has characterized several early stages in the
osteoblast lineage with a novel technique. When fetal RC cells are plated at very low
densities, single isolated colonies arise from single cells, whose differentiation fate can be
followed, Le. to colonies of osteoblastic, fibroblastic, or other mesenchymal ce11 types.
Since one osteoprogenitor ce11 gives rise to one bone nodule with the characteristics of
woven bone under standard RC culture conditions (Bhargava et al, 1988), and since
colonies are derived from clonal, normal (i.e.: non-established, non-transformed) cells, it
can be concluded that each ceIl in the colony had the same osteoprogenitor ceIl as an
ancestor. By combining a replica plating technique to these low density cultures and by
using poly(A)-PCR, this lab has been able to molecularly characterize some
osteoprogenitors, preosteoblasts and early osteoblasts; osteoprogenitor stages investigated
included ones well before the cells had acquired the morphological characteristics of
osteoblastic cells and prior to upregulation of ALP and the matrix molecules discussed
above (Liu and Aubin, 1994). The technique involved placing a polyester cloth over the
forming individual colonies. Some cells fiom each colony attached to the cloth after
several days and division cycles; the cloth was then removed, placed in its own culture
dish, and incubated at 37OC. Meanwhile, the original, or master dish, was placed in an
incubator at a lower temperature (generally 30°C) to stall the growth of the cells until the
colonies growing on the replica cloth had matureci. Once bone colonies had formed
mineralized bone nodules on the cloth, their sister colonies were identified by matching
their locations on the dish with that on the cloth. Either single cells or whole colonies
were collected by trypsinizing the colonies, and the cells were subjected to molecular
analysis by poly(A)-PCR. This approach allowed characterization of stages wlier in the
osteoblast lineage than was ever pnviously possible (Liu and Aubin, 1994; Aubin et al,
1995; Liu et al, 19%). however the cells were removed h m their in vivo ages by 8-1 l
days and several divisions in vitm. It was my objective to adapt this technique to
characterize still earlier cells in the osteoblast lineage, removed fiom their in vivo ages by
only 3 to 5 divisions in vitro.
V. Objectives
The purpose of this work was to retrospectively identify and characterize very
early osteoprogenitor cells in the RC ce11 mode1 of osteoblast differentiation. In this
approach, RC cells were plated at low density and a cellular sarnple was taken fiom each
colony after 4 to 5 days of growth. This cellular sample was used to produce a poly(A)-
PCR-based cDNA library, while the remaining cells in the colonies were allowed to
proliferate and differentiate in vitro for 21 - 25 days, during which time mineraiized bone
nodules formed in some colonies. Once bone-fonning colonies were retrospectively
identified, the cDNA libraries were used for Southem blotting to establish a molecular
profile based on the presence of messages for bone matnx proteins and other putative
osteoprogenitor marken.
Cell Culture Fetal RC cells were obtained by sequential enzymatic digestion of 21-day fetal
Wistar rat calvariae as previously described (Bellows et al, 1986, 1987). Briefly,
calvariae were cleaned of skin and connective tissue, minced and sequentially digested in
collagenase (Sigma Chemical Company. Si. Louis, MO). Cells obtained from the last
four of the five digestion steps (populations II - V) were pooled and plated in T-75 flasks
in a-MEM containing 1 5% heat-inactivated fetal bovine serum (Imrnunocorp, Montreal,
QC), as well as penicillin G (100 pg/ml; Sigma), gentamycin (50 pg/ml; Life
Technologies, Grand Island, NY) and huigizone (0.3 pg/rnl; Life Technologies). After 24
hours, attached cells were washeâ with phosphate-buffered saline to remove unattached
cells and other debris, then collected by trypsinization and an aliquot counted on an
electronic Coulter Counter. Cells were then plated at very low densities (500 to 1000 cells
per 100 mm dish) and grown in a-MEM medium (as above) supplemented with ascorbic
acid (50 pglml; Fisher Scientific, Fair Lam, NI), sodium P-glycerophosphate (10 mM;
Sigma Chemical Co.) and dexarnethasone (10 nM; Sigma Chemical Co.) as described
(Liu et al, 1994). Medium was changed every 2-3 days. Dishes were incubated at 37'C in
a humidifid atrnosphere of 95% air, 5% CQ.
Single Celf Isolation Four to five days aftcr plating, incipient colonies comprising 8 to 50 cells were
located in phase conûast micmscopy and thtù locations markecî. Each colony was
assigned an arbitrary number for later identification. A Gilson P2 micropipette was used
to manually scrape a small number of cells (1-3) fiom each of the colonies; the cell
sample was drawn up into the pipette in 1 )il of medium. The remaining cells in each
colony were left intact. The micromanipulated cells were released directly into lysis
buffer in a 0.5 ml Eppendorf tube and kept on ice. Lysis buffer consisted of 50 mM Tris
HCI, pH 8.3, 75 mM KCI, 3.0 m M MgCl,, 0.50% Nonidet P-40 (Sigma), 2 FM each in
dATP, dCTP, dGTP, and d'ITP (Boehringer Mannheim), 2000 u/ml RNAguard
(Pharmacia), 1 00 dm1 Inhibitlice (5'-3' Incorporated), and 100 ng/ml [dTIz4 cDN A
primer. When al1 cells from growing colonies had been sampled, the mRNA was reverse
transcribed and amplified by poly(A)-PCR (see below), and stored at -70°C.
Cells remaining in the individual colonies were grown to maturity, generally 21 -
25 days, in medium as above, with medium changes each 2-3 days. The cells were then
fixed and stained by the Von Kossa technique to distinguish mineralized bone colonies
fkom colonies comprising fibroblastic cells.
Po&(A)-PCR First-strand cDNA synthesis and poly(A)-PCR were performed essentially as
described ( B d y et al, 1990, Liu et al, 1994). First-strand cDNA synthesis was
perfonned within one hour of ce11 micromanipulation. Samples were heated to 6S°C for 1
minute, cooled to roorn temperature for 3 minutes, then put on ice untilO.5 FI of M-MLV
reverse transcriptase (200 uni&/@; Life Technologies Ltd.) was dded. Samples were
incubatcd at 37OC for 15 minutes, thcn the enzyme was heat-inactivated a 6S°C for 10
minutes. Following first strand synthesis, a poly(dA) tail was added to the 3' end of the
cDNA with terminal transferase. 4 pl of 2x tailing buffer (consisting of 200mM
potassium cacodylate (pH 7.2), 4 m M CoCI,, 0.4 mM DTT, and 200 pM dATP) and 1 pi
(25 units) of teminal transferase enzyme (Boehnnger Mannheim) were added; samples
were incubated for 15 minutes at 37*C, then heat-inactivated for 10 minutes at 65°C. The
entire mRNA population was then amplified by PCR using primen with a poly(T)
stretch. The sequence of the PCR primer used was 5' ATG TCG TCC AGG CC0 CTC
TGG ACA AAA TAT GAA 'l'TC 3' followed by a stretch of 24 dT residues. A l x Taq
buffer mixture was preparcd, consisting of 10 mM Tris HCI (pH 8.3). 50 mM KCI, 1.5
rnM MgCl,, 100 pg/ml bovine serum albumin (BSA), 1 mM each dATP, dCTP, dGTP,
and dTTP, 0.05% Triton X-100, and a 0.1 ODZm units per sample of PCR primer. 50 pl
of this l x Taq buffer mixture was aliquoted to each sample, plus 1 pI of Taq DNA
Polymerase (5 units/pl; Sangon Limited Canada, Scarborough. Canada). Samples were
overlaid with mineral oil (Peikin-Elmet Cetus) and arnplified for 25 cycles of 1 minute at
94T, 2 minutes at 42OC, and 6 minutes at 72OC, followod by an additional 25 cycles of 1
minute at 94°C' 1 minute at 42OC. and 2 minutes at 72OC, followed by a 10 minute
extension at 72'C. Samples were then stored at -70°C. Samples useâ for Southem
blotting underwent a secondary PCR amplification. 0.5 pl of the original PCR-amplifid
sample was diluted to 100 pl with lx Taq buffer, as above but 0.2 rnM in dNTPs, and
0.06 units of PCR primer and 0.5 pl Taq DNA polymerase per sample. No mineral
oil was used for secondary amplifications. Samples werc amplified foi 25 cycles of 1
minute at 94"C, 1 minute at 42OC, and 2 minutes at 72OC, followed by a 10 minute
extension at 72°C. Samples were stored at -70°C.
Southern Ana&sis Following the secondary poly(A)-PCR amplification, selected samples were used
for Southern blots. Before preparing Southem blots, jpi of each of the poiy(Aj-PCR
arnplified cDNA samples were stained with ethidium bromide, electrophoresed through a
1.5% agaroseI0.5X TBE gel and viewed under ultraviolet light. Those samples that were
subjectively judged to have very little to no cDNA present relative to other samples were
excluded From further analysis (results not show). Next, Southem blots were prepared
from the remaining selecied samples. 5 pl of secondary PCR products were
electrophoresed approximately 3cm in a 1.5% agarose gel in 0 . 5 ~ TBE. The gels were
denatured in a solution of 1.5 M NaCVO.5 N NaOH, neutralized in a solution of 1 M
Tris/1 .5 M NaCI, and DNA transferred to a nylon membrane (0.2 Fm; ICN Biotrans) by
capillary transfer. Membranes were baked at 80°C for 2 hours and blots were probed with
specific cDNA probes produced from the 3' end of cDNA clones for various mRNA
messages (sec below), as previously described (Liu et al, 1994). Briefly, cDNA probes
were labeled with [ 3 2 ~ ] d ~ ~ ~ using an oligolabelling kit (Phannacia, Uppsala, Sweden).
All prehybridizations and hybridizations were perfomed at 42OC. AAer hybridization, the
blots were washed once for 30 minutes each at 42°C and 50°C in 2x SSC/O.i% SDS, and
once for 15 minutes each at 55°C and 60°C in lx SSC/O.l% SDS. Results of Southern
blotting were visualized by Phosphorimager or x-ray film (Kodak X-OMAT).
Phosphorimager data were quantitated and analyzed by IPLab Gel Software.
cDNA Probes The cDNAs comprising the 3' sequences were prepared and provided by F. Liu in
the lab. Rat al COLL-1 (palR.2) (Genovese et d.. 1984; kindly provided by Dr. D.
Rowe, Farmington, CT) was a 900-bp cDNA Pst1 fragment containing the entire 3'
noncoding region and one-half of the C-terminal of the propeptide of the a 1 chain of type
1. Rat bone/liver/kidney ALP (Noda et al, 1987; gift of Dr. G. A. Rodan, Merck Sharpe,
and Dohme Research Laboratories, West Point. PA) was a 600-bp cDNA EcoRi fragment
obtained by digesting pRAP54 with BssHII-XhoI to remove 1.8 kb of the 5' region and
religating the blunt ends. Rat OPN (kindly provided by Dr. R. Mukhejee, Montréai,
Qutbec) was a 700-bp cDNA BomHI-Ecoiü hgrnent obtained by digesting full length
cDNA with PvuI to remove 800 bp of the 5' region and ligating the blunt ended fragment
into Sniol cut pGEM-7Zf(+) vector (Promega, Madison, W?). Mouse SPARCION (Mason
et al, 1986a; kindiy pmvided by Dr. B. Hogan, London, üK) was a 600-bp cDNA
EcoRVHindII fragment obtained by digesting pG43 with AvaI and SmuI to remove 1400
bp of the 5' ngion and nligating the blunt ended fragment of the cut pGEM-1 vector
(nomega). Rat COLL-III (Glurnoff et al, 1994; kindly provided by Dr. E. Vuorio, Turku,
Finland) was a 500-bp cDNA fragment obtained by digesting pRGRS with BamHI to
mnove 1700 bp of îhe 5' region. Rat BSP was a partial cDNA containing 500 bp of 3'
region isolated with BSP-specific pnmcts h m a Agt 1 1 library pmpared h m RC ails
forming bone nodules (prepared by A. Gupta and J.E. Aubin). Rat OCN was a partial
cDNA containing 350 bp of 3' region isolated with OCN-specific primers from a hgtl 1
library prepared from ROS 17/2.8 cells (prepared by A. Gupta and J.E. Aubin). Rat L32
was a partial cDNA containing 400 bp of 3' region isolated with a mouse L32 probe
(Meyuhas and Peny. 1980; kindly provided by Dr. N.N. Iscove, Ontario Cancer Institute,
Toronto, Canada) from a Agt 1 1 library prepared from RC ceils forminy bune nodules.
Total cDNA probe was prepared as described by Sambrook et al (1989) from poly(~) '
mRNA isolated (Auffray and Rougeon, 1980) from mass populations of fetal RC cells
grown in the presence of dexamethasone in which bone nodules were beginning to
mineralize.
Data Analysis Phosphorimager data were recorded and images analyzed by IPLabGei Software
for Macintosh. A rectangle (37x30 pixels) was drawn on the most intensely labeled
portion of each lane of the blots and the intensity of the image inside the rectangles was
digitally recorded. A background value was taken from the negative (cell-free) control
lane. This background value was subtracted from the values obtained for sample lanes.
Data were expressed graphically as a ratio of the southem blot labeling intensity to the
total cDNA labeling intensity for each marker examined. Ratios were plotted in arbitrary
Expression data were subjectively judged for each marker examined such that
visi bl y discernible signals were designated as king expressed for that particular sample.
In order to have a consistent lower limit of detection, the ratio of the signal intensity of a
marker to the total cDNA value for a sample had to be greater than or equal to 10% of the
corresponding mature OB sarnple ratio (which was the highest ratio for al1 markers
except OPN and by far the most intense signal for each marker). Those markers whose
ratios were less than 10% of the mature OB ratio were designated as not expressed, or not
significantiy difkrent fiom background. Cornparison of du subjective visual inspection
and the values deteminrd in this manner showed that they agreed well.
When RC cells were plated at low density, nascent colonies were easily identified
at day 4 or 5 of the culture period, and their locations recorded. Samples comprising I to
3 cells were micromanipulated fiom the colonies and subjected to poly(A)-PCR. When
the remaining cells in each colony were allowed to continue growth for 21 to 25 days, a
few bone colonies were identified aller staining by the Von Kossa technique and non-
bone colonies aller staining with Bromophenol Blue. Two representative culture dishes
stained by the Von Kossa technique, each with one bone colony, are shown in Figure 3;
circles on the bottom of the dish mark the locations of other colonies fiom which cells
were also sampled. Samples 2 and 4 through 9 were recovered h m colonies that
subsequently formed mineralized bone nodules; samples 3 and 10 through 14 were
recovered frorn colonies that also formed bone which had not mineralized by the time of
fixation. Samples 15, 17. and 18 were recovered h m larger (greater than 500 cells)
colonies comprising cells of fibroblastic morphology, while samples 16, 19, and 20 were
fiom smaller (under 100 cells) colonies of non-osteogenic cells. In three cases, two
samples were taken h m the same colony (samples 10 and 1 1; 12 and 13; 19 and 20).
Southem blots were prepared with poly(A)-PCR amplified cDNA h m both the
samples repmenting early osteoprogenitor cells and other early precursor cells (here
designated as fibmprogenitors based on morphology of the resuliing cells). Controls that
were also included on the blots were a sarnple that was processed without any ceils
prcsent, and a sample amplifieà h m mRNA h m a mature, mineralid osteoblast
Figure 3: Two represmtative culture dishes stained by the Von Kossa technique, each with one bone colony (arrow). Circles on the bottom of the dish mark the locations of other colonies fiom which cells were sarnpled.
colony (kindly provided by F. Liu). The lane with no ce11 present for the poly(A)-PCR
amplification was used as a negative control lane, and was subtracted as the background
reading for quantitation by the IPLabGel program. The mature mineralized osteoblast
colony lane served as a positive control for al1 of the markers tested by Southem blotting.
Relative levels of cDNA loading on Southem blots were compared by ethidium bromide
staining, L32 expression, and a total cDNA 3' probe prepared as in Mcthods. The
expression level of specific markers was qwtified by detennining the ratio of the
intensity of the scanned gel for that marker over the intensity of total cDNA labeling
(data show in Table 1).
The Southem blot (Figure 4) and comsponding nomalized data from the
Phosphorimager (Figures 5 and 6) indicate that OCN, ALP, and BSP were either
undetectable or expressed at very low levels in ail samples except for the positive control,
the sample arnplified fiom a mineralized, mature osteoblast colony. Only one sample
(sample 16) showed expression of any of these markers (ALP) at a level above
background. Levels of expression of other marken were variable. OPN and ON were
expressed at moderate levels in most samples tested, with high expression in a few
sarnples. For example, OPN was detectable in al1 osteoprogenitor samples tested, and at
quitc high levels in some samples (samples 6,7,9, 1 1, 13 and 14). Expression was lower,
but detectable, in most fibroblastic sampks as well. Expression of OPN and ON were, on
average, higher in osteoprogenitor cells than in fibmprogenitor cells. COLL-1 was
expressed at moderate to high levels in a few osteoprogenitors (3 ,S to 7,9 and 1 1). and at
low levels in several other samples (samples 8, 13 and 14). COLL-III was e x p t e d at
high levels in 2 osteoprogenitor samples (samples 7 and 4). and at low levels in two other
Table I : Ratio of marker signal intensity to total cDNA signal intensity for each sample. Numbers in bold print indicate sarnples with expression levels above background (designated as greater than or equal to 10% of the ratio for sample 1 ). These data were used to plot Figures 5 and 6.
Fi yre 4: Composite of al1 Phosphorimager data. Each column represents the same sample; data in each row have the same exposure time to the Phosphorimager cassette. Sample 1 : mature osteoblast colony; samples 2- 14: osteoprogenitor samples; sarnples 1 5- 20: non-osteogenic samples.
COLL- III
SPARC ON ~~~ . .. ..; , , ,*A.
umpie v i l w / total CONA
Figure S: 3-dimensional representation of nomaiized Southern bloning data showing I
expression levels of dl markers. Sample 1: mature osteoblast colony; samples osteoprogenitor samples; samples 1 5-20: non-osteogenic samples.
dative 2- 14:
Figure 6: Individual graphs of normalized Southem blotting data for each marker. Data is expressed as a ratio of marker expression to total cDNA for the same sample.
Figure 6 conthued ...
osteoprogenitor samples (samples 5 and 8). No fibroprogenitor samples had COLL-III
expression at a level above baseline.
These data indicate a great deal of intercellular heterogeneity in both
osteoprogenitor and fibroprogenitor cells. However, five general osteoprogenitor
phenotypes emerged: cells with (1) OPN expression only (samples 10 and 12); (2) OPN
and ON expression (sample 2); (3) OPN, ON and COLL-1 expression (samples 3, 6, 9,
11. 13, 14); (4) OPN, ON, and COLL-III expression (sample 4); and (5) OPN, ON,
COLL-1 and COLL-III expression (samples 5, 7 and 8). Four general fibroprogenitor
phenotypes also emerged: cells with (1) ON expression only (samples 19 and 20); (2) ON
and OPN expression (sarnple 18); (3) OPN, ON and COLL-I expression (samples 15 and
1 7); and (4) ON. OPN, COLL-I and ALP expression (sample 16).
This is the first report of the isolation and partial characterization of determined,
normal (i.e., non-established, non-transfomieci), early (recovered at day 4-5, division
number 4 to 6 hom in vivo lifetime) osteoprogenitor cells.
The population of cells isolated fiom 21-day fetal rat calvaria is a mixed
rnesenchymal population which consists of cells at many varying stages of
differentiation. There are osteoprogenitor cells, cells more advanced or differentiated in
the osteoblast lineage, as well as cells associated with colonies designated here as
fibroblastic based on their spindle-shaped to pleiomorphic morphologies and the fact that
they did not fom bone. By enzymatically digesting fetal rat calvaria, the extracellular
matrix is effectively removed, and, once the cells are plated, any remaining debris and
non-adherent cells are removed as well. This makes it much easier to select a cellular
sample fiom the population for molecular analysis than if the cells were housed in vivo,
although, when isolated and plated in this manmr, al1 spatial information about the origin
of the cells is lost. In this regard, the procedure intmduced hem is a way to obtain cells
removed h m their in vivo age by only 4 to 6 ce11 divisions in vitro, and to study low
kquency osteoprogenitor cells whose pnçisc in viw location is unlnown. Since discnte
colonies arise from single cells, the sibling cclls to the ones that are selected for analysis
will be of clonal origin, and their fate can be followed to detexmine the lineage of the
early cells.
1. The Poly(A)-PCR Approach
Both the duration of the reverse transcription reaction and the concentration of the
deoxyribonucleoddes used for the poly(A)-PCR approach used were limited to restrict the
length of the resulting cDNA to approximately 300 to 800 base pairs. This allows the
relative sequence abundances to be preserved in the subsequent PCR amplification by
preventing selection against long sequences or those with extensive secondary structures.
Although it is now possible to amplify sequences of up to 35 kb in length (Bames. 1994),
the current poly(A)-PCR protocol was employed for the following reasons: it has been
established to work on rat calvarial cells in this laboratory (Liu et al, 1994); it is more
economical considering the large numbets of samples that are processed at each time; and
it is sufficient in that it produces a 3' cDNA library which can be used for Southem
blotting to assess expression patterns in carly osteoprogenitor cells. The technique has
been used to obtain representative cDNA libraries for analysis of hematopoetic cells
(Brady et al, 1990, 1995), pre- and post-implantation stage embryonic tissues (Rambhatla
et al, 1995, Varmuza and Tate, 1992), and recently, in our lab, for the analysis of both
early and mature osteoblasts and preosteoblasts (Liu et al. 1994) and osteoprogeniton
identified by replica plating (Liu and Aubin, 1994).
Tech nicul Limitations It has been documented that the poly(A)-PCR technique is very sensitive to slight
variations in amplification conditions (Brady and Iscove, 1993.). This sensitivity is due in
part to the minute quantities of RNA serving as a template for the amplification reaction,
and the non-specificity of the technique. Several factors may have contributed to the
observed variations in ampli fication efficient y. The material being examined was
amplified from proliferating single cells, which results in varying message levels
depending on what stage of the ce11 cycle the cells are in at the time of isolation. This is
one variable that is beyond our control as we are unable to determine before selecting an
individual ce11 what stage it is at. Another variable that is dificuit to control is c d
adhesion. When cells were picked from sparsely populated young colonies, generally
only ceIl was isolated at a time. However, in more confluent colonies cells had often
made very strong contacts amongst themselves, and it is very likcly that more than one
ce11 was selected for analysis. This may also affect the amplification reaction efiiciency
since there is more RNA to serve as a template for the reaction. Several samples were
excluded from the final analysis because they did not ampli@ well. as assessed by
ethidium bromide staining, total cDNA and L32 signal intensities. If the total cDNA and
L32 signal intensities were very low to not discemible when subjectively comparing them
relative to other samples on the same Southern blot, the sample was excluded from
further analysis. Since some cellular samples were more efficient1 y amplified than others
by the poly(A)-PCR technique, uneven arnounts of cDNA, for each sample relative to
others, were loaded on Southem blots. Also, the material king examined was amplified
from proliferative single cells and we had found earlier that no known message including
several genes considered housekeeping was present at a steady-state in al1 sarnples (Liu
and Aubin, unpublished data) to allow direct cornparison of cDNA quality between
samples. Therefore, we used total cDNA as a standard against which al1 samples were
normalized as a ratio to compare relative expression levels between samples.
Reproducibiliîy and Detection Limits
The results presented in the current study are based on digital Southem blotting
data obtained with a phosphorimager system. Ideally, one would prepare one Southem
blot to be repeatedly probed with the individual marken in succession, in order to ensw
that amounts of cDNA on the blot were precisely consistent for al1 markers tested.
However, one limitation of the technique is that despite stringent stripping methods.
small amounts of radioactive probe remain on the blots after attempts to refiesh the blots
for subsequent probing; these minute amounts of radioactivity are detectable by the
extremely sensitive phosphorimager system. As a result, a separate Southem blot was
used for each individual marker that was examined. To assess the reproducibility of
sample loading in these individual blots, four separate blots were prepared and probed for
COLL-III. A consistent pattern of COLL-III expression was observed when these
multiple blots were compared. Similarly, OPN was repeatedly tested on four separate
blots, also yielding a consistent pattern of expression among the samples examined.
The data obtained when testing markers multiple times were not averaged for
statistical analysis since the raw values varied greatly fiorn blot to blot, while relative
differences between samples remained consistent. Factors that affected this variability
include the cassette exposure time. the "fieshness" of the radioisotope at the time of
labeling the blots, and the background labeling of the blots.
As introduced in Methods, marker signals were subjectively judged by comparing
signals with the overall background of the blots and with other samples. This subjective
determination of expression was followed with a numerical analysis of one blot for each
market. The lower limit of signal intensity that designated a signal as representing
expression of a marker was set arbitrarily to 10% of the positive control (mature OB)
sample, afier first normalking the signal to standardizc: difkrences in the aiiiount of
cDNA loaded per sample. Due to the subjective nature of the analysis, there are some
samples whose expression pattern may be different had the baseline expression level been
set to 5%, for exarnple, rather than 10% of the control. However, the 10% limit chosen
gave agreement between visuai inspection and the Phosphorimager values.
The data generated from the blots already have the background (the signal
intensity of the negative control lane, the cell-fiee reaction products) subtracted From each
sarnple value. It is of note that al1 sample lanes resulted in positive values, indicating that
every lane. even those with no expression of the marker of interest, had some background
level of signal detectable by the highly sensitive phosphorimager system.
II. Pbenotype of Early Osteoprogenitor Cells
My data indicate that early osteoprogenitor cells express combinations of COLL-1
and COLL-III, OPN and ON. That these cells did not express BSP, OCN or ALP,
markers characteristic of more mature osteoblasts, is consistent with and confirms their
immature stage. Notably, however, there is considerable heterogeneity in the expression
profiles of the markea that are expressed by the early osteoprogenitor cells, from cells
expressing al1 of COLL-I, COLL-III, OPN and ON (Le.: sarnple 7). to ones expressing
different combinations of one, two or three markers at diverse levels. This heterogeneity
will be discussed further below.
Of particular note is the expression of COLL-III. Among non-osteogenic or
fibroprogenitor cells examined, COLL- I II was not expressed. However, severai
osteoprogenitor sarnples showed moderate to high levels of COLL-III expression
(samples 4, 5, 7 and 8). Reports that COLL-III mRNA is detected by in situ hybridization
and imrnunohistochemistry in fibroblastic cells of the periosteum (Sandberg et al, 1988).
and that COLL-III is the major collagen of the fibrous matrix that forms dong the
periosteal surface (Keene, 1991; reviewed in Ashurst 1990) lend credence to the
designation of COLL-III as a marker of early osteoprogenitor cells. The high level of
COLL-III expression found in the mature osteoblast sarnple is consistent with a few
reports (see Introduction) of COLL-III in bone nodules formed by fetal RC cells (Bellows
et al, 1986), and in certain clonal osteoblastic lines h m fetal RC (Aubin et al, 1982) and
adult rat tibia1 osteoblastic populations (Strhga et al, 1995). It also suggests that
detection of COLL-III expression in sorne isolated osteoblastic populations is as
consistent with the presence of immature osteoprogenitoa as with fibroblastic
contamination, to which it has oAen been atûibuted (Robey and Termine, 1985).
The various phenotypes that have emergcd here for both osteoprogenitor and
fibroprogenitor cells are compriscd of different combinations of the 4 markers used
(COLL-1, COLL-III, OPN, ON). However, it is of note that only two phenotypes are
common to cells of both colony types; coexpression of OPN and ON and of OPN, ON
and COLL-1 was seen in both some osteoprogenitors and some fibroprogeniton. These
cornmon phenotypes may be representative of cells earlier or more primitive (less
differentiated) than tlie other cells exarnined here. This possibility is consistent with the
fact that the colonies resulting from these particular fibroprogeniton (samples 15, 17 and
18) became very large, indicative of extensive proliferative capacity and that few of the
bone colonies from these particular osteoprogenitors had yet mineralized by the time of
fixation (only samples 2, 6 and 9 had mineralized; samples 3, 11, 13, 14 had not),
suggesting that they may have arisen from more immature osteoprogenitors.
Phenorypic Heterogenee A gteat deai of intercellular heterogeneity is evident in both osteoprogenitor and
fibroprogenitor colonies. Duplicate samples pickeâ fiom the same colony, such as
samples 10 and 1 1, and 12 and 13 (these pairs h m two separate bone colonies), and
samples 19 and 20 h m a colony that did not fom a bone nodule, are informative. One
explanation for the phenotypic heterogeneity observeâ here is non-synchronous
differentiation progression for sibling cells within colonies, in other words, cells wen
picked at different stages in their progression through the diffeientiation sequence. A
second possibility is that even cells at the same stage of differentiation may have some
flexibility in both the repertoin of genes expressed and their levels of expression. Liu et
al, (1994; Liu and Aubin, 1994) found support for this by both the poly(A)-PCR
technique and by imrnunocytochemistry with sntibodies against bone markers, including
ALP, BSP and OCN. The technique 1 have used for ce11 isolation is biased to examine
cells at an early osteoprogenitor stage, i.e. afkr only 4 to 6 divisions in vitro. Within the
specific window on which these techniques focus, there are discrete developmental stages
as well. Some colonies, when picked, had as few as 5 to 15 cells while others had as
many as 50 to 80 cells, despite having been in vitro for the same length of time and
despite having the same end resuit. bone nodule formation. These differences, whiçh may
reflect progenitor cells of different developmental or proliferative lifetimes, could also
account for some of the heterogeneity in the expression pattern observed.
One further point that should be made with regard to intercellular heterogeneity is
that of sample size. When, for exarnple, 10 cells are selected fiom a colony for molecular
analysis by poly(A)-PCR. al1 of the messages expressed in those 10 cells will be shown in
the phenotype, regardless of whether al1 10 cells or just one ce11 is expressing a particular
message. On the other hand, during single ce11 analysis, it is not an average phenotype
that is s h o w but a specific phenotype of a cell at a particular stage in its differentiation
sequence, which could account for intercellular differences. Some of the observed
heterogeneity may be a result of this averaging effect, as more than one ceIl was obtained
by micromanipulation in some cases.
My data are limited by the relatively small number of ce11 samples available for
analysis. It would be of interest to repeat the selection of cells in multiple independent
experiments and determine whether the apparently separate phenotypes are reproducible
in every ce11 isolate examined.
III. Retrospcctive Analysis of Single RC Cells
Osteoprogenitor cells proliferate in vitro for several rounds of cell division
resulting in bone nodules whose sizes Vary. Cells fiom such developing bone colonies
(sarnples 2-14), the focus of this study, express COLL-1 and -III, OPN and ON to varying
degrees. Arnongst possible cxplanations for thc intcrccllular variations observed (see
above) some rnay result from osteoprogenitor cells being more or less mature in the
osteogenic lineage relative to othen at the time of sampling. While al1 osteoprogenitor
samples examined here ultimately give rise to bone nodules in vitro, some bone colonies
at the time of fixation were very heavily mineralized (samples 2, 5, 6, 8 and 9), others
were sparsely mineralized (samples 3, 4, 7, 10, 1 l), and othen had no detectable
mineralization (samples 1 2- 14). These devcloprnental differences could reflect that these
osteoprogenitor cells were at varying stages of differentiation when isolated. However,
not al1 bone nodules are identical in size. Whether size reflects relative developmental
maturity (Le. large colonies result fiom very immature progenitors) is not currently
known. Data h m Liu et al (in pnparation) and Aubh (in preparation) suggest this may
not be so and that colony sizc and differentiation progression rnay instead reflect a
stochastic process.
Thm are also more mature cells of the osteoblast lineage in the RC population.
These cells, while they have a roughly fibroblastic or pleiomorphic appearance when
initially isolated i~ vitro, would be expected to prolifemte to only a limited extent in viîro
but to express markers consistent with a relatively ma- phenotype. An example of this
type of cell may be sample 16. The colony from which this sample came formed a colony
of only approximately 80 cells by day 21 of culture. Yet, the cell isolated at 4 days in
vitro already expressed the more mature osteoblast marker ALP to a small degree, more
than any other immature cellular sample examined during these experiments. In fact, it
was the only sample, apart from the mature osteoblast colony which served as a positive
control, to have any of the more mature matkers (ALP, BSP and OCN) detectably
expressed. The expression of this more mature marker, along with the very small size of
the resulting colony, suggest that sarnple 16 may have been an early preosteoblast at the
time of sampling. This is in accordance with other work currently king performed in this
lab, in which some cells isolated by replica plating express ALP and other more mature
markers, but do not yet have the morphologid characteristics of osteoblasts (Liu et al.
manuscript in preparation).
A third ce11 type that cm be described in the RC population is one in which cells
are undifferentiated or uncomrnitted to the osteoblast limage or immature enough that
they will be very proliferative in vitro, i.e. they give rise to very large so-called
fibroblastic colonies. Examples of thesc are samples 15, 17 and 18. None of these cells
expressed detectable levels of ALP, BSP, OCN or COLL-III, but they did express ON,
OPN and COLL-1 to varying degrces.
These studies have provided somc initial steps to allow molecular chanicterization
of early osteoptogenitor cells and preparation of corresponding cDNA libraries chat can
be used to investigate novel genes and markers of this stage in the osteoblast
differentiation scqucnce. The techniques can be extended to investigate expression of any
markers whose 3' sequences are known by repmbing the Southem blots or n-ampliQing
the cDNA to prepare new blots. ln faci, the potential application of the technique in this
regard is limited only by the number of probes that can be obtained with a full 3'
sequence, since cDNA material can be reamplified many times.
SUMMARY AND CONCLUSIONS
1. I t is possible to isolate early osteoprogenitor cells by plating RC cells at low density
and manually micromanipulating cells fiom the culture dish.
2. Within the limitations set by the small sample size available, early osteoprogenitor
celis isolated in this marner are morphologically fibroblastic or pleiornorphic and
exhibit one of five molecular phenotypes:
0 expression of OPN only
a expression of ON and OPN
expression of ON, OPN and COLL-1
a expression of ON, OPN and COLL-III
a expression of ON, OPN, COLL-1 and COLL-III
3. Within the limitations set by the small sample size available, early cells associated
with colonies that did not fom bone nodules in vitro that are morphologically
fibroblastic or pleiomorphic also exhibit one of four molecular phenotypes:
expression of ON only
a expression of ON and OPN
0 expression of ON, OPN and COLL-1
a expression of ON, OPN, COLL-1 and ALP
4. It is possible to examine cells in the osteoblast lineage at earlier cimes in vitro than
previously possible. This work will make a signifiaint contribution to our cumntly
limited knowledge of very eatly osteoprogenitor cells.
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