/ . Embryol exp. Morph. Vol. 56, pp. 191-200, 1980
Printed in Great Britain © Company of Biologists Limited 1977
Fibroblast progenitor cells of the embryonic
chick limb
By STUART A. NEWMAN 1
From the Department of Biological Sciences, State University of
New York at Albany
SUMMARY
A population of mesenchymal cells derived from the stage-25 chick wing tip gives rise to
progeny of a similar morphology and to authentic fibroblasts when grown in low density
culture. Mixed clones containing both cell types are often observed. As the more rapidly
proliferating fibroblasts begin to predominate in these cultures, collagen biosynthesis rises
from the basal mesenchymal level to a level characteristic of mature fibroblasts. The fibroblast progenitor is discussed relative to the other cell types of the mesodermal lineage of the
developing limb.
INTRODUCTION
During early development of the chick limb bud, diversification of the mesenchymal cells results in the production of myoblasts, the chondroblasts of the
skeletal anlagen, and the fibroblasts of the tendons, dermis, and musculature.
Though studies have been conducted that bear on the lineage relationships of
these cells to one another and to their progenitor cells (Searls, 1967; Caplan,
1970; Dienstman, Biehl, Holtzer & Holtzer, 1974; Abbott, Schiltz, Dienstman &
Holtzer, 1974; Newman, 1977; Ahrens, Solursh, Reiter & Singley, 1979)
unequivocal patterns of descent have not been determined, partly because of the
difficulty of isolating stem cells that exhibit their multipotential character
in clonal culture (Dienstman et al. 191 A; Newman, 1977). Though cells can be
derived from leg muscles of 8-day embryos which give rise to both myogenic
and fibrogenic cells in clonal culture, these cells are not thought to represent
primitive members of the mesodermal lineage, but rather are considered to be
committed members of the myogenic 'compartment' (Abbott et al. 1974).
Taking account of the results of the experimental morphology of the embryonic
limb bud allows the identification of primordia that are developmentally immature at various stages of development (Saunders, 1977; Newman, 1977). Cell
populations containing progenitor cells of interest can thus be isolated and
studied in culture. Here I report the isolation of a cell type from the distal
tip of the stage-25 wing bud with a characteristic 'early mesenchymal' morphology, which demonstrably gives rise by division in low-density culture both
1
Author's address; Department of Anatomy, New York Medical College, Valhalla,
N.Y. 10595, U.S.A.
13-2
192
S. A. NEWMAN
to cells of a similar morphology, and to fibroblasts. The latter cell type is
identified by its distinctive morphology and its increased level of collagen
biosynthesis relative to the progenitor cells. Since a majority of cells from the
same source can differentiate into chondrocytes when grown as explants or
as reaggregates (Newman, 1977), it is possible that the same stem cell can have
alternative fates depending on its interactions with other cells.
MATERIALS AND METHODS
Preparation of tissue and cells. Wing buds of stage-25 (Hamburger & Hamilton,
1951) chick embryos were excised and placed in Simm's balanced saline solution
(BSS) (Simms & Saunders, 1942). The apical tips of these wing buds, obtained
by cutting the limb with a chisel-bladed scapel (Clay-Adams Instrument Co.)
parallel to and 0-2-0-3 mm proximal to a line tangent to the apical ectodermal
ridge at its thickest point, were pooled in BSS. This tissue was dissociated into
cell suspensions by incubation for 20 min in a solution of 0-25 % trypsin in
calcium- and magnesium-free BSS, transfer of the intact tissue pieces to Ham's
nutrient medium F-10 as modified by Coon (1966), containing 10% fetal calf
serum (FCS) (both obtained from Grand Island Biological Company) and 1 %
bovine serum albumin (BSA) followed by 12 gentle passes through a syringe
with an 18-gauge needle. The resulting suspension was filtered through sterile
lens paper and counted in a hemocytometer. When prepared by this method the
filtered suspensions routinely contained 5 x 104 cells per wing tip. In some cases
the ectoderm was removed from the explanted limb tips with watchmaker's
forceps after brief trypsinization, prior to cell dissociation. This had no effect
on the observations reported below.
Cultures. Suspensions were diluted in the medium described above and plated
on to 35 mm tissue-culture dishes (Falcon No. 3001) at either 5 x 103 or 5 x 104
cells per dish, in 1 ml of medium. Plating efficiencies were typically 1 % at the
former and 10-20% at the latter density. At the lower density single attached
cells were circled and observed for up to 2 weeks with the replacement of 0-5 ml
medium every 3-4 days. At the higher density the medium was completely
replaced every 3-4 days. Short-term organ cultures of nondissociated limb tips
were also prepared. Each of 20 to 40 wing tips were cut in half and the pooled
fragments were distributed between two organ culture dishes (Falcon No.
3010) containing supplemented F-10 medium (including FCS and BSA) made
up with 1-2% Bacto agar (Difco Corp.) (Gordon & Lash, 1974). Radioactive
isotope was added to these cultures in 0-25 ml of liquid medium.
Photomicrography. Living cultures were photographed under phase-contrast
optics. In those cases where the progeny of a single cell were to be examined, a
cell that was well isolated from others was selected after 1-2 days of culture
on a dish on which 5 x 103 cells were initially seeded. Such cells were circled and
examined daily.
Fibroblast progenitor cells
193
Fig. 1. Characteristic cell type in low- and medium-density (5 x 103 or 5 x 104 cells
per 35 mm dish) culture prepared from stage-25 chick wing-tip mesenchyme
after 1-2 days in culture. Arrow indicates cell whose nucleoli are separated by a
septum. Scale bar represents 50/*m.
Collagen analysis. Explant or monolayer cultures were incubated for 6 h
in supplemented F-10 containing sodium ascorbate (50/ig/ml), /?-amino
propionitrile (100/Ag/ml) and [3H]proline (New England Nuclear Corp.,
NET-323, 50 /iCi/ml). The values [(hydroxyproline)/(hydroxyproline + proline)]
x 100 were obtained for the cells and medium by hydrolysis of trichloroacetic
acid precipitated material in 6 N-HC1, separation of amino acids on Dowex
50 resin and determination of radioactivity by scintillation counting, as described by Schiltz, Mayne & Holtzer (1973).
RESULTS
Morphology of the progenitor and progeny cells in culture
When plated at densities of 5 x 103 or 5 x 104 cells per 35 mm dish, all the
cells of the stage-25 subridge mesoderm that adhered and spread after 1-2
days in culture had the broad, flattened morphology pictured in Fig. 1. These
194
S. A. NEWMAN
2a
Fig. 2. 0 ) + 0 ) Two clones of mesenchymal cells in which the morphology of the
progeny resembles that of the founder cell. Cells were seeded at 5 x 103 per 35 mm
dish. Photographed after 9 days in culture. Scale bar represents 50 fim.
Fibroblast progenitor cells
195
Fig. 3. Three 'mixed' clones in which some progeny have mesenchymal morphology
and some are fibroblastic. Cells were seeded at 5 x 103 per 35 mm dish. Arrows in
(a) indicate a dividing pair, both of which exhibit mesenchymal morphology. Arrows
in (b) indicate a dividing pair, one of which is mesenchymal and one of which is
fibroblastic. Arrows in (c) indicate a mesenchymal cell which is connected by
cytoplasmic bridges both to a cell with a similar morphology and to a bipolar
fibroblastic cell. Cellular vacuolation is characteristic of these low-density cultures.
Scale bar represents 50 /tm.
cells were notable in the regularity of their shape, their slow division rate,
particularly at the lower density, and the prominance of their nucleoli, which,
when more than one were apparent, were often separated by septa within the
nucleus (Fig. 1).
As reported (Newman, 1977), if these cells are reaggregated as little as one
day after sparse growth, they give rise to uniformly fibroblastic colonies under
196
S. A. NEWMAN
Fig. 4. Region of fibroblastic monolayer formed after 2 weeks in culture by cells
such as those in Figs. 2 and 3, but seeded at 5 x 104 cells per 35 mm dish. Scale bar
represents 50/*m.
culture conditions permissive for chondrogenesis. This contrast with the chondrogenie fate of wing-tip cells which have been reaggregated within 6 h suggests that
the non-interacting mesenchymal cells rapidly lose their chondrogenic potential.
After 2-3 days in culture, cells in the higher density (5xlO 4 cell) plates
exhibited a moderate increase in cell number from day 1, and a mixture of
morphologies, though even at this stage the proximity of the original cells
prevented the assignment of specific progeny to specific founders. At 2-3 days
the cells in the lower density (5 x 103 cell) plates exhibited little change from day
one. After 9-14 days in culture many of the cells in the lower density plates
had still failed to divide or had divided only once or twice. Among those that
had divided in these plates, cells of the original morphology were still in evidence.
The photographs in Fig. 2 show the maintenance of the original mesenchymal
morphology in all the members of two clones. Frequently, however, clones
were observed in which cells with the mesenchymal morphology had divided
to give rise to a mixed clone, some of whose members exhibited the founder
cell's appearance, others taking on the appearance of mature chick fibroblasts,
such as those observed after serial subculture of embryonic chicken muscle.
From the photographs in Fig. 3 it is clear that cells of either morphology can be
derived from the same founder cell: mesenchyme cells photographed during
cytokinesis are attached to cells of both predominant progeny types. In one of
the cases shown (Fig. 3 c) a single mesenchyme cell is attached to a cell of each
progeny type.
Fibroblast progenitor cells
197
Table 1. Synthesis of collagen by mesenchymal cells and
their in vitro progeny
Stage-25 wing-tip
mesenchyme
3-day mono-disperse
cultures of mesenchyme
Total
incorporation
(cpm)
(Hypro/
Hypro + Pro)
xlOO
170980
1-72
Cell layer
26897
2-89
Medium
Cells + medium
8796
35693
16-26
610
Cel Is + medium
Mesenchymal explants, or dissociated cells after 3 days in culture (initially seeded at 5 x
104 cells per dish) were pulsed for 6 h with [3H]proline. Incorporation of radioactivity into proline and hydroxyproline was determined for TCA-precipitable material after hydrolysis and
separation on Dowex 50 resin (Schiltz et al. 1973).
When cells of the fibroblastic morphology have appeared in a clone, they proliferate at a more rapid rate, eventually giving rise to large colonies, and, when
the original inoculum is at least 5 x 104 cells per 35 mm dish, a confluent
monolayer of fibroblastic cells (Fig. 4).
Collagen biosynthesis in the progenitor and progeny cells
Authentic fibroblasts synthesize type-I collagen in characteristically large
amounts (Abbott et al. 1974). In contrast, chick limb-bud precartilage mesenchyme synthesizes low levels of a collagen which has also been identified as type
I by fluorescent antibody staining (von der Mark, von der Mark & Gay, 1976)
and by cyanogen bromide peptide analysis (R. Mayne & S. Newman, unpublished). As chondrogenesis proceeds in situ and in vitro, high levels of type-II
collagen begin to be produced (Linsenmayer, Toole & Trelstad, 1973; von der
Mark et al. 1976; von der Mark & von der Mark, 1977). The transition to a new
collagen type does not occur when the cells are initially seeded at subconfluent
densities (R. Mayne & S. Newman, unpublished). That is, the monolayer
cultures continue to synthesize type-I collagen. For the purposes of the present
study I was interested in the quantitative question of whether the level of collagen
biosynthesis as a proportion of the total protein produced rises to the high levels
characteristic of mature fibroblasts, concurrent with the predominance of that cell
type in the differentiating cultures.
As shown in Table 1, the amount of collagen as a percentage of total protien
synthesized by the wing-tip mesenchyme cells and their progeny after 3 days
in monolayer culture (seeded at 5 x 104 cells per 35 mm dish) was significantly
enhanced over that synthesized by the fresh explants. The three-day figure is
in line with that reported for authentic fibroblasts (Abbott et al. 1974).
198
S. A. NEWMAN
DISCUSSION
The capability of preparing a pure population of precursor cells to a given
terminal cell type would be a benefit to studies of development at both the
molecular and the histological levels. At the molecular level the opportunity
would be afforded of correlating changes in chromatin structure and composition with programmatic changes in a cell's differentiated state, as reflected in
altered macromolecular syntheses (Newman, Birnbaum & Yeoh, 1976; Perle
& Newman, in preparation). At the histological level it is important to determine what options a given precursor cell type has at particular points during
organogenesis so that one has a way of deciding among competing models for
the control of this process (Zwilling, 1968; Caplan & Koutroupas, 1973;
Summerbell, Lewis & Wolpert, 1973; Newman, 1977, Newman & Frisch, 1979).
A uniform population of progenitor cells which express alternative fates under
different culture conditions would help provide needed information in this
area.
In the case of the cell population described here it is uncertain whether the
small percentage of cells which ' take' in culture are representative of the population which is seeded, the vast majority of which will form cartilage cells if
initially grown under aggregated conditions (Newman et al. 1976; Newman,
1977). Furthermore, it is not known whether the cells which do thrive are a
homogeneous population of fibroblast progenitor cells, since a variable proportion of the clones observed at the end of two weeks or more contained only
cells of the original mesenchymal morphology. (Data not shown.) These questions can only be addressed if culture conditions can be found that assure a
large initial attachment and survival rate of the seeded population (Millis,
Hoyle & Field, 1977) and/or the expression of a uniform cell morphology
among the progeny. Nevertheless, the finding reported here, of a transition
from a mesenchymal to a fibroblastic cell type, is not dependent on statistical
evidence, but relies on the appearance of mixed clones (including mixed pairs
undergoing cytokinesis) in the low-density cultures, as shown in Fig. 3.
The data presented on relative collagen synthesis in Table I indirectly support
the clonal evidence. Since whole stage-25 wing tips synthesize collagen as 1-2 %
of their total protein, while their surviving progeny after 3 days in culture
synthesize three times as much collagen, one can provisionally conclude that a
change to a more fibroblastic cell type has occurred. The only alternative to this
conclusion would be that a minor subpopulation of the wing tip is responsible
for all the collagen produced in that tissue, and that these are the only cells that
survive in culture. Then the transition observed from fibroblast progenitor to
authentic fibroblast could be taking place with no quantitative enhancement of
type-I collagen synthesis in cells competent to produce this protein. That this
is probably not the case can be determined by examining the early limb bud
sections stained with anti-type-I collagen antibody by von der Mark et al.
Fibroblast progenitor cells
199
(1976). It is apparent from that study that mesenchymal cells prior to overt
differentiation into muscle, cartilage or connective tissue produce type-I
collagen at a low but uniform level. Thus with regard to the typs and quantitative level of collagen biosynthesis it is likely that the cells in our cultures that
we identify as fibroblast progenitors are representative of the mesenchymal cells
of the stage-25 wing tip. It is of course possible that several covertly differentiated
cell types would be similar in these properties and in their morphology.
The question of whether the surviving cells are representative of the original
mesenchymal population is of interest because any reasonable model of skeletal
pattern formation requires a bi- or multi-potential population of cells whose
fates can be assigned in a spatially dependent fashion (Newman & Frisch, 1979).
I have suggested that the choice of chondrocyte v. fibroblast results from an
interaction-dependent modulation of the fate of a single progenitor cell type
(Newman, 1977) but the present data do not allow us to decide between that
possibility and differential selection to explain the presence of fibroblasts and
absence of chondrocytes in plates seeded at subconfluent densities.
Previous work has shown that the stage-25 wing tip contains no cells with
myogenic potential, under conditions where one muscle cell among several
thousand non-muscle cells would have been observed (Newman & Mayne, 1974;
Newman, 1977). This is confirmed by anatomical evidence that the myogenic
and chondrogenic primordia of the chick limb bud constitute disjoint lineages
which start out spatially separated (Christ, Jacob & Jacob, 1977; Chevallier,
Kieny, Mauger & Sengel, 1977). Thus, the choice of material has provided an
important simplification of the lineage problem. Whether the fibroblast progenitors of the stage-25 wing tip are identical to the cartilage progenitors that
can be derived from the same tissue, and if so, by what mechanism they diversify
to form the skeletal pattern of the adult hand, are questions that warrant further
study.
REFERENCES
J., SCHILTZ, J., DIENSTMAN, S. & HOLTZER, H. (1974). The phenotypic complexity of
myogenic clones. Proc. natn. Acad. Sci., U.S.A. 71, 1506-1510.
AHRENS, R. B., SOLURSH, M., REITER, R. S. & SINGLEY, C. T. (1979). Position-related capacity
for differentiation of limb mesenchyme in cell culture. Devi Biol. 69, 436-444.
CAPLAN, A. (1970). Effects of the nicotinamide-sensitive teratogen 3-acetylpyridine on chick
limb cells in culture. Expl Cell Res. 62, 341-355.
CAPLAN, A. & KOUTROUPAS, S. (1973). The control of muscle and cartilage development in
the chick limb; the role of differential vascularization. J. Embryol. exp. Morph. 29, 571583.
CHEVALLIER, A., KIENY, M., MAUGER, A., SENGEL, P. (1977). Developmental fate of the
somitic mesoderm in the chick embryo. In Vertebrate Limb and Somite Morphogenesis
(ed. D. A. Ede, J. R. Hinchliffe & M. Balls), pp. 421-432. Cambridge University Press.
CHRIST, B., JACOB, H. J., & JACOB, M. (1977). Experimental analysis of the origin of the wing
musculature in avian embryos. Anat. Embryol. 150, 171-180.
COON, H. (1966). Clonal stability and phenotypic expression of chick cartilage cells in vitro.
Proc. natn. Acad. Sci., U.S.A. 55, 66-73.
DIENSTMAN, S. R., BIEHL, J., HOLTZER, S., & HOLTZER, H. (1974). Myogenic and chondrogenic lineages in developing limb buds grown in vitro. Devi Biol. 39, 83-95.
ABBOTT,
200
S. A. NEWMAN
J. S. & LASH, J. W. (1974). In vitro chondrogenesis and cell viability. Devi Biol.
36, 88-104.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. /. Morph. 88, 49-92.
LINSENMAYER, T. F., TOOLE, B. P. & TRELSTAD, R. L. (1973). Temporal and spatial transitions
in collagen types during embryonic chick limb development. Devi Biol. 35, 232-239.
MiLLis, A. J. T., HOYLE, M. & FIELD, B. (1977). Human fibroblast conditioned media
contains growth promoting activities for low density cells. /. cell. Physiol. 93, 17-24.
NEWMAN, S. A. (1977). Lineage and pattern in the developing wing bud. In Vertebrate Limb
and Somite Morphogenesis (ed. D. A. Ede, J. R. Hinchliffe & M. Balls), pp. 181-197.
Cambridge University Press.
NEWMAN, S. A., BIRNBAUM, J. & YEOH, G. C. T. (1976). Loss of a non-histone chromatin
protein parallels in vitro differentiation of cartilage. Nature, Lond. 259, 417-418.
NEWMAN, S. A. & MAYNE, R. (1974). Axial differences in the progress of chondrogenesis
in the embryonic limb bud. /. Cell Biol. 63, 245a.
NEWMAN, S. A. & FRISCH, H. L. (1979). Dynamics of skeletal pattern formation in developing chick limb. Science, N.Y. 205, 662-668.
SAUNDERS, J. W., Jr. (1977). The experimental analysis of chick limb bud development. In
Vertebrate Limb and Somite Morphogenesis (ed. D. A. Ede, J. R. Hinchliffe & M. Balls),
pp. 1-24. Cambridge University Press.
SCHILTZ, J. R., MAYNE, R. & HOLTZER, H. (1973). The synthesis of collagen and glycosaminoglycans by dedifferentiated chondroblasts in culture. Differentiation 1, 97-108.
SEARLS, R. (1967). The role of cell migration in the development of the embryonic chick
limb bud. /. exp. Zool. 166, 39-50.
SIMMS, H. S. & SAUNDERS, M. (1942). The use of serum ultrafiltrate in tissue culture for studying fat deposition and for virus propagation. Archs Pathol. 33, 619-635.
SUMMBERBELL, D., LEWIS, J. H. & WOLPERT, L. (1973). Positional information in chick
limb morphogenesis. Nature, Lond. 244, 492-496.
VON DER MARK, H., VON DER MARK, K. & GAY, S. (1976). Study of differential collagen
synthesis during development of the chick embryo by immunofluorescence. I. Preparation
of collagen type I and type II specific antibodies and their application to early stages of the
chick embryo. Devi Biol. 48, 237-247.
VON DER MARK, K. & VON DER MARK, H. (1977). Immunological and biochemical studies
of collagen type transition during in vitro chondrogenesis of chick limb mesodermal cells.
J. Cell Biol. 73, 736-747.
ZWILLING, E. (1968). Morphogenetic phases in development. Devi Biol. (Suppl.) 2, 184—
207.
GORDON,
{Received 25 May 1979, revised 8 October 1979)