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Ultrastructure and growth of human limb mesenchyme (HLM15) in vitro.

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Ultrastructure and Growth of Human Limb
Mesenchyme (HLM15) In Vitro
ROBERT 0. KELLEY, THOMAS I. BAKER, HARRY A. CRISSMAN
AND CAROLYN A. HENDERSON
Departments of Anatomy and Microbiology, T h e University of N e w Mexico
School of Medicine, Albuquerque, N e w Mexico 871 06 and Los Alamos Scientific Laboratory, University of California, Los Alamos, New Mexico 87544
ABSTRACT
Human embryonic hand and foot plates (horizon XVI) exhibit
localized differences in mitotic activity between developing digital and interdigital zones of mesenchyme. To investigate development of regulation of cell
reproduction, mesenchymal cells from lower limb buds dissected from a human
embryo (stage XV) were cultured. Electron microscopy reveals that HLM15 cells
in situ are fibroblastic, exhibit euchromatic nuclei, one to two peripheral nucleoli, extensive granular ER, free ribosomes, and microfilaments and microtubules oriented in the long axis of cellular processes. Cells released from
substratum with trypsin-EDTA become rounded and microfilaments and microtubules become disoriented, perinuclear bundles. HLMl5 has a mean generation
time of 24 hours during logarithmic growth and karyotype analysis indicates
that cells retain diploid chromosomal number after 17 passages. DNA-fluorochrome analysis on the Los Alamos Flow Microfluorometer (FMF) demonstrates
that HLM15 cells possess no detectable polyploidy. Computer fit of FMF distributions reveal that random populations of HLM15 during exponential growth contain 42.6% cells in GI, 45.9% in S and 11.5% in G2-l-M. As confluency is reached
these data become 57.6%, 37.5% and 4.9% respectively. Disaggregated limb
mesenchyme (in vivo) analyzed without culturing exhibit FMF distributions of
56.1% in GI, 36.7% in S and 7.2% in GI+M. Similarities in cell cycles between
near-confluent HLMl5 cultures and limb mesenchyme in vivo suggest that regulation can be achieved by increasing or decreasing the traverse time through GI
(both in vitro and in vivo). The proportion of non-cycling cells (GO)in the FMF
GI distribution is presently not known.
In man, limb rudiments appear as localized swellings of dorsal somatic mesoderm (mesenchyme) which push the adjacent trunk ectoderm (epithelium) into
hillocks. These early limb buds soon project beyond the embryonic body wall and
begin their lengthwise elongation (ORahilly, Gardner and Gray, '56). Growth of
early vertebrate limb buds is thought to
be apical (Saunders, '48), although many
investigators (Hornbruch and Wolpert, '70;
Janners and Searls, '70; Searls and Janners, '71) discount the idea that differential proliferation provides a mechanism for
early distal growth of the bud. However,
later limb development (viz., individuation
of digits) proceeds by at least two different
morphogenetic processes : (a) maintenance
of five growth centers at the distal tip
ANAT. REC., 175: 657-672.
of mesenchymal digital blastemata (Milaire, '65) and (b) regional necrosis of
mesenchyme in presumptive interdigital
zones (Kelley, '70a). In human embryonic
hand and foot plates (horizon XVI, Streeter,
'48) localized differences in mitotic activity
exist between developing digital and interdigital zones of mesenchyme, mitotic indices being higher beneath the apical epidermal rim (a.e.r.) than in more proximal
regions (Kelley, '70b ).
It is well known that embryonic cells
develop characteristic rates of reproduction
during differentiation. Mechanisms which
control development of mitotic regulation
are unclear, however. To investigate the
problem of differential growth during inReceived Sept. 13, '72. Accepted Nov. 22, '72.
657
658
R. 0. KELLEY, T. I. BAKER, H. A. CRISSMAN A N D C. A. HENDERSON
dividuation of digits we have begun a study
of cycle kinetics of mesenchymal cells both
in vitro and during limb morphogenesis
in man. In this paper we present techniques
for culturing human limb mesenchyme
(termed HLM15, cultured from an embryo
staged at horizon XV, Streeter, '48), their
ultrastructure in vitro and their life cycle
analysis as determined by flow microfluorometry (FMF, Kraemer, Deaven, Crissman
and Van Dilla, '70). These in vitro techniques may provide a system by which the
development of regulation of cell reproduction essential to human limb morphogenesis can be studied.
Trypsin was removed by centrifugation at
200 x g for ten minutes and the pellet was
resuspended in culture medium. The suspension, containing approximately 1 x lo5
disaggregated cells and several small fragments, was transferred to a culture flask
and incubated until monolayered (to confluence ).
Maintenance (subcultivation of
monolayer cultures)
Cells were transferred every four days
with complete medium exchange on the
second day. Cultures were propagated on
flat surfaces of 8 oz glass bottles. At each
transfer, cell monolayers were washed with
GKN and removed from substrate with a
MATERIALS A N D METHODS
solution of trypsin-EDTA in GKN (0.45
Growth conditions
mg/ml trypsin, 1.0 mM EDTA). After cells
Culture medium used in all experiments had detached (usually within 5 minutes)
was Eagle's MEM supplemented with 10% aliquots were counted in a hemocytometer.
tryptose phosphate broth (Difco), 10% Approximately 1 X lo6 cells were transfetal calf serum (NABI), and low concen- ferred to culture bottles containing fresh
trations of antibiotics (25 units/ml Nysta- medium.
tin, 50 pg/ml Neomycin Sulfate, U.S.P.,
Preservation by freezing
and 10 pg/ml Aureomycin). Cells were inAt
passage
five, ampules of cells were
cubated in a humidified incubator (37°C)
frozen
in
liquid
nitrogen. Cells were susin an atmosphere of 7.5% CO,. Manipulations of cells were conducted aseptically in pended in dimethyl sulphoxide (DMSO)
reagent (10% DMSO, 20% fetal calf
a UV sterilized chamber (Lab Con).
serum, 70% Earle's Balanced Salt Solution) to a cell concentration of 2 x lo6 per
Procurement of material
milliliter. One milliliter of this suspension
Lower limb buds were dissected from was transferred to an ampule which was
human embryos (horizon XV) following then sealed, frozen (-50°C for 30 minutes,
therapeutic interruption of pregnancy and -80°C for 2-4 hours), and stored in liquid
placed in culture medium at 37°C. Develop- nitrogen. Cells were recovered by rapid
mental stage was determined by matching thawing and sterile transfer to tissue culstructures with corresponding descriptions ture tubes. Fresh medium was added slowly
of Streeter ('45, '48) and ORahilly et al. to a volume of 10 ml of fresh medium,
('56). Careful examination revealed that transferred to culture bottles, and incudigital blastemata were present in upper bated to confluence.
limb buds but no structural differentiation
of mesenchyme was apparent in lower
Karyotype analysis
limbs. Epithelium was removed with Puck's
Cells were cultured on glass cover slips
Saline GM containing 0.1 mg/ml trypsin in dishes containing 5 ml of medium.
and 0.5 mM EDTA.
When cells were nearly confluent, 0.2 ml
of 0.01% colchicine was added to the meEstablishment of primary cultures
dium. After four to six hours the inhibitor
Small pieces of mesenchymal tissue were was removed and coverslips were incubated
rinsed several times with a glucose-potas- in hypotonic solution ( 5 ml of Earle's Balsium-sodium solution (GKN, McLaren, Hol- anced Salt Solution diluted l :9with H,O)
land and Sylverton, '59) and incubated in for 20 minutes. Cells were fixed with 3 : l
GKN containing 0.25% trypsin. Fragments methanol-acetic acid, stained with Geimsa,
were then minced and incubated at 37°C and karyotypes prepared from the mitotic
with intermittent shaking for one hour. figures.
HUMAN LIMB MESENCHYME I N VITRO
Life cycle analysis
HLM15 cells, in exponential and stationary phases of growth, were rinsed with
Pucks saline GM and removed from walls
of culture flasks by incubation for 30 minutes (37°C) in 10 ml of dispersing solution
(Puck's saline GM containing 0.5 mM
EDTA and 0.1 mg/ml trypsin). Neutralization medium (Puck's saline G containing
0.01 mg/ml DNase, 0.2 mg/ml soybean
trypsin inhibitor and 0.1 mg/ml bovine
serum albumin) was added to the flask
and the cell suspension transferred to conical centrifuge tubes for pelleting (700 X g).
Neutralizing solution was then aspirated,
cells were rinsed in saline G, and fixed
overnight at 4°C in formalin (20 ml of
40% formalin in 80 ml of saline G adjusted to pH 7.0). In addition, apical epithelium was mechanically removed from
paired lower limb buds (horizon XV), mesenchyme was disaggregated in dispersing
solution and cells (designated in vivo) were
prepared for FMF analysis as controls.
After hation, preparations were rinsed
in water and hydrolyzed in 4N HC1 for
20 minutes at room temperature. Following a rapid rinse in distilled water, cells
were stained for 20 minutes at room temperature with acraavine, rapidly rinsed
in acid alcohol and resuspended in HzO
immediately preceding flow microfluorometry (Trujillo and V a n Dilla, '72). The
data-processing program written at the
Los Alamos Scientific Laboratory makes a
least-squares best-fit of a normal distribution function to the GI peak, a second degree polynomial to the S distribution and
another normal distribution function to the
G2+M peak. Areas under GI, S and Gz+M
portions of FMF distributions, determined
by computer, are in agreement with results
obtained by autoradiographic cell cycle
analysis (Kraemer, Deaven, Crissman and
Van DiUa, '70). Cultures in exponential
growth were pulse-labeled (10 minutes)
with H3-thymidine to determine the percentage of cells in S. Mitotic figures/lOO
cells provided the mitotic index. These
data, when applied to a graphic plot by
the technique of Okada ('67), permit calculation of times for the GI, S, Gz and M
periods.
659
Mean generation time
The mean generation time was obtained
by following population doubling times
during exponential growth. Several bottles
were each inoculated with 4 X 10' cells.
Cells from four identical culture flasks
were then removed with trypsin-EDTA at
16, 24, 40 and 72 hours of incubation and
the number of cells in each bottle was determined by hemocytometer count.
Electron microscopy
Growth medium was removed from confluent cultures grown in plastic dishes and
replaced with 3.0% glutaraldehyde in
0.1 M phosphate buffer (pH 7.4) at room
temperature €or two hours. Preparations
were postfixed in 2.0% osmium tetroxide
in phosphate buffer at room temperature
for one hour, rapidly dehydrated through
an ethanol series, gently scraped from the
substrate with a wooden policeman, and
flat embedded in Epon 812. Other cell
samples were removed from glass culture
bottles with 0.45 mg/ml trypsin and 1.0
mM EDTA, centrifuged at 700 X g, and
pellets were preserved and prepared for
electron microscopy as described. Thin sections (mounted on uncoated grids) were
stained for one hour in saturated aqueous
uranyl acetate at 35"C, for ten minutes in
alkaline lead citrate at room temperature
and examined in an Hitachi HU-1lC electron microscope.
RESULTS AND DISCUSSION
Techniques herein described illustrate
the capability and simplicity of culturing
human embryonic mesenchyme with maintenance of diploid karyotype. The fine
structure of limb mesenchyme in vivo has
been reported previously (Kelley, '70).
HLM15 cells in vitro are elongated fibroblasts which average 20-30
in length
and 1-2 in width (fig. 1 ) . Figure 1 illustrates profiles of cells fixed in situ. Nuclei
are euchromatic, spherical to ovoid in
shape and contain one to two nucleoli. Peripheral heterochromatin can be observed
in figure 2 in addition to dense bodies (autophagic vacuoles?) which are present in
both perinuclear cytoplasm and cell processes. Perinuclear cytoplasm exhibits mitochondria, elongated Golgi centers and
flattened granular endoplasmic reticulum
660
R. 0. KELLEY, T. I. BAKER, H. A. CRISSMAN AND C. A. HENDERSON
(GER) which extend into cytoplasmic
processes (fig. 3 ) . Figure 3 illustrates a
cell process sectioned to demonstrate surface and substratum orientation. Microfilaments (50-80 A in diameter) extend along
the long axis of the process adjacent to
the cell surface abutting the culture medium. Mitochondria, dense bodies, and cisternae of GER (filled with flocculent electron dense material) are present in the
body of the process. Surfaces abutting the
substratum exhibit an extracellular material with features of protein polysaccharide. In addition to microfilaments, microtubules are also oriented in the long axis
of the cell. Microfilaments course beneath
nuclei forming dense fibrillar bundles
(fig. 4 ) .
When cell-substrate relationships are
altered with trypsin-EDTA, HLM15 cells
lose fibroblastic configuration and become
rounded (fig. 5 ) . Nuclei retain peripheral
heterochromatin but are folded and indented. Nucleoli remain adjacent to nuclear envelopes (fig. 6). Cytoplasmic reorganization is exhibited by presence of
disoriented microfilaments in perinuclear
zones (figs. 5, 7), convoluted cisternae of
GER and disruption of large Golgi complexes into numerous small Golgi centers
(fig. 8). Microtubules in association with
Golgi zones are apparent. Centrioles retain perinuclear orientation, although nuclei have undergone alteration in shape
(fig. 9 ) . These changes in organization
suggest that HLM15 cells are under tension when attached to the substrate and
when surface materials are removed (weakened), cells return to a relaxed, spherical
configuration. Oriented microfilaments and
microtubules may play a role in the assumption of cell shape relative to the extracellular environment (Comings and
Okada, '70; Goldman and Follett, '69).
Under the described culture conditions,
HLM15 has a population doubling time of
24 hours during the logarithmic growth
phase (fig. 10). This mean generation time
is constant through 17 passages. Passages
18 and 19 show a marked decline in growth
rate and confluent growth has not been obtained beyond the twentieth passage. These
growth properties have been consistent for
ten ampuIes recovered from liquid nitrogen
at passage five. In two instances the final
passage has been retained for several
months with complete medium exchange
every three days but a continuous or transformed line has not yet been established.
Figure 11 illustrates fluorochrome-DNA
distribution for HLM15 in exponential and
stationary growth phases and in vivo (without a.e.r. ) respectively. Graphs consist of
two peaks: the first represents cells with
diploid DNA content (G1), and the second
represents cells with tetraploid DNA content (G2+M phases). The region between
peaks illustrates cells synthesizing DNA
(S phase). Computer fit of FMF distributions reveals that random populations of
HLM15 during exponential growth contain
42.6% cells in GI, 45.9% in S and 11.5%
in G2+M (autoradiographic analysis yields
44.4% cells in S after pulse label with H3thymidine). As confluency is reached these
data become 57.6%, 37.5% and 4.9%
respectively. Disaggregated limb mesenchyme (in vivo) analyzed without culturing exhibit FMF distributions of 56.1%
in GI,37.6% in S and 7.2% in G2+M.
Application of FMF distribution data
from random populations of HLM15 in
exponential growth to a graphic Okada
plot is illustrated in figure 12. Traverse
times through G1,S and G2+M are 8.4 h,
12.2 h and 3.4 h respectively. Using the
mitotic index as the percentage of cells
in M, M
t is 0.8 h and G
t 2 is calculated to be
2.6 h.
Similarities in FMF distributions between near-confluent HLM15 cultures and
limb mesenchyme in vivo suggest that
regulation can be achieved by increasing
or decreasing the traverse time through GI
(both in v i m and in vivo). Since Rubin
('71) has demonstrated that cell growth
in vitro is inhibited through the combined
effects of both lowered pH and high cell
density, it is not unreasonable to suggest
that mesenchymal cell cycles in developing
limbs and HLM15 in vitro respond to population density by increasing GI. Unfortunately, the proportion of non-cycling cells
(GO)in the FMF GI distribution is presently
not known.
To determine if heteroploid or aneuploid
classes had emerged with subcultures of
HLM15, karyotype analyses of metaphase
configurations were prepared. Figure 13 illustrates normal diploidy visualized in 97%
HUMAN LIMB MESENCHYME IN VITRO
of 63 metaphase determinations. There is
no evidence of alteration in number of morphology of the diploid chromosomal complement. Furthermore, additional peaks at
high channel numbers (fig. 1 1 ) were absent, also suggesting absence of heteroploidy.
In contrast to these observations on an
embryonic cell during early histogenesis,
Gamow and Prescott ('70) have demonstrated that cell cycles in 4, 8 and 16 celled
mouse embryos lack a measurable GI phase.
In view of other investigations (Dalcq and
Pasteels, '55; Wimber and Lamerton, '65),
they generalize that cell reproduction during early development, particularly cleavage, proceeds without a GI phase in the
cell cycle. This implies that regulation of
rates of cell division develop gradually during tissue differentiation. Although we do
not know whether or not early mesodermal
cells in man lack a GI phase, it is clear
that human limb mesenchyme cells prior
to cartilage or muscle differentiation possess demonstrable GI, S and G2+M phases
in their cell cycle. Hopefully, we have established a system by which changes in
morphology, growth and cycle characteristics of human mesenchyme induced by
altered experimental conditions can be
explored.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Marvin
Van Dilla for helpful discussions and use
of the Los Alamos Flow Microfluorometer,
Drs. Robert Munsick and Lewis Koplik for
the procurement of material, Dr. Leonard
Napolitano for critical reading of the
manuscript, Mrs. Joan Ivey for assistance
in manuscript preparation, and the United
States Public Health Service for grant support (HD06177 and a Research Career Development Award HD70407 to the principal author).
LITERATURE CITED
Comings, D. E., and T. A. Okada 1970 Electron
microscopy of human fibroblasts in tissue culture during logarithmic and comquent stages of
growth. Exptl. Cell Res., 61: 295-301.
Dalcq, A., and J. Pasteels 1955 DBtermination
photomktrique de la teneur relative e n DNA
des noyaux dans les serifs en segmentation du
r a t et de la souris. Exptl. Cell Res. Suppl., 3:
72-97.
661
Gamow, E. I., and D. M. Prescott
1970 The
cell life cycle during early embryogenesis of the
mouse. Exptl. Cell Res., 59: 117-123.
Goldman, R. D., and E. A. C. Follett 1969 The
structure of the major cell processes of isolated BHK21 fibroblasts. Exptl. Cell Res., 57:
263-276.
Hombruch, A., and L. Wolpert 1970 Cell division in the early growth and morphogenesis of
the chick limb. Nature (London), 226: 764-766.
Janners, M., and R. L. Searls 1970 Changes in
rate of cellular proliferation during the differentiation of cartilage and muscle in the mesenchyme of the embryonic chick wing. Devel.
Biol., 23: 136-168.
Kelley, R. 0. 1970a An electron microscopic
study of mesenchyme during development of
interdigital spaces i n man. Anat. Rec., 168:
43-54.
1970b Fine structure of apical, digital
and interdigital cells during limb morphogenesis in man. In: Proceedings of the VIIth International Congress of Electron Microscopy.
Vol. 111: 831-832.
Kraemer, P. M., L. L. Deaven, H. A. Crissman
and M. A. Van Dilla 1972 DNA constancy despite variability in chromosome number. In:
Advances in Cell and Molecular Biology. Vol. 2.
E. J. Dufraw, ed. Academic Press, New York,
pp. 47-108.
Milaire, J. 1965 Aspects of limb morphogenesis
in mammals. In: Organogenesis. R. L. DeHaan
and H. Ursprung, eds. Holt, Rinehart and
Winston, New York, N.Y., pp. 283-300.
McLaren, L. C., J. J. Holland and S. T. Sylverton
1959 The mammalian cell-virus relationship.
I. Attachment of poliovirus to cultivated cells
of primate and non-primate origin. J. Exp. Med.,
109: 475-504.
Okada, S. 1967 A simple graphical method of
computing the parameters of the life cycle of
cultured mammalian cells in the exponential
growth phase. J. Cell Biol., 34: 915-916.
O'Rahilly, R.,E. Gardner and D. J. Gray 1956
The ectodermal thickening and ridge in the
limbs of staged human embryos. J. Embryol.
exp. Morph., 4: 254-264.
Rubin, H. 1971 pH and population density i n
the regulation of animal cell multiplication. J.
Cell Biol., 5 1 : 686-702.
Saunders, J. W. 1948 The proximo-distal sequence of origin of the parts of the chick wing
and the role of the ectoderm. J. Exp. Zool., 108:
363403.
Searls, R. L., and M. Y. Janners 1971 The initiation of limb bud outgrowth in the embryonic
chick. Devel. Biol., 24: 198-213.
Streeter, G. L. 1945 Developmental horizons in
human embryos. Description of age group XIII,
embryos about 4 or 5 millimeters long, and age
group XIV, period of indentation of the lens
vesicle. Carnegie Cont. to Emb., 31: 27-63.
1948 Developmental horizons in human embryos. Description of age groups XV,
XVI, XVII and XVIII being the third issue of a
survey of the Carnegie Collection. Carnegie
Cont. to Emb., 32: 133-204.
662
R. 0. KELLEY, T. I. BAKER, H. A. CRISSMAN AND C. A. HENDERSON
Trujillo, T. T., and M. A. Van Dilla 1972 Adaptation of the fluorescent Feulgen reaction to
cells in suspension for flow microfluorometry.
Acta Cytologica, 16: 26-30.
Wimber, D. E., and L. F. Lamerton 1965 Cell
cycle of mouse embryonic tissue under continuous gamma-irradiation. Nature, 207: 432433.
PLATE 1
EXPLANATZON OF FIGURES
1
Low magnification electron micrograph of HLM15 cells fixed in situ.
Arrows indicate extracellular matrix on cell surface formerly adjacent
to substrate. Note thin peripheral heterochromatin adjacent to inner
leaflet of nuclear envelope. g, Golgi center; ger, granular endoplasmic
reticulum; m, mitochondrion; n, nucleus. x 10,000.
2
Perinuclear cytoplasm containing cisternae of granular endoplasmic
reticulum, ger, and autophagic vacuoles, av, adjacent to nucleus, n.
Arrows indicate extracellular matrix. x 33,000.
3
Cytoplasmic process with medium-coated surface illustrated a t top
of micrograph. Bundles of oriented microfilaments, mf, subjacent to
the cell surface, follow the long axis of the process. Cisterna of granular endoplasmic reticulum, ger, is filled with flocculent precipitate.
Arrows indicate extracellular matrix. x 55,000.
4
Cytoplasm between nucleus and substrate surface. Note bundles of
microfilaments, mf, subjacent to nucleus, microtubules, mt, and
granular endoplasmic reticulum, ger. x 37,000.
HUMAN LIMB MESENCHYME IN VITRO
R. 0.Kelley, T. I. Baker, H. A. Crissman and C. A. Henderson
PLATE 1
PLATE 2
EXPLANATION OF FIGURES
664
5
HLM15 cell removed from substrate with trypsin-EDTA prior to h a tion. Note indented nuclear borders and perinuclear bundles of disoriented microfilaments, mf. av, autophagic vacuole; ger, granular
endoplasmic reticulum; n, nucleus. x 11,000.
6
Peripheral nucleolus adjacent to inner leaflet of nuclear envelope.
x 12,000.
7
Disoriented microfilaments in perinuclear zone of
treated HLM15 cell. x 40,000.
8
Golgi centers, g, in perinuclear cytoplasm. Arrow denotes microtubule
in Golgi zone. x 26,000.
9
Centriole, c, i n perinuclear cytoplasm. ger, granular endoplasmic reticulum; n, nucleus; v, vesicles. x 12,000.
trypsin-EDTA
HUMAN LIMB MESENCHYME I N VITRO
R. 0. Kelley, T. I. Baker, H. A. Crissman and C. A. Henderson
PLATE 2
665
PLATE 3
EXPLANATION O F FIGURE
10 Abscissa: time in hours from inoculation of culture; Ordinate: number of cells/bottle x lo5 and 106 on a logarithmic scale. Relationship
between hours of growth and number of cells illustrating a population doubling time of 24 hours during logarithmic growth.
666
HUMAN LIMB MESENCHYME I N VITRO
R. 0. Kelley, T. I. Baker, H. A. Crissman and C. A. Henderson
PLATE 3
3
2
I
3
2
20
40
60
80
667
PLATE 4
EXPLANATION OF FIGURE
11
668
DNA distribution patterns of the various HLM cell populations: ( A )
HLM cells in exponential growth phase in cell culture as described in
the text. (B) HLM cells near stationary growth phase in cell culture.
(C)HLM cells prepared by the disaggregation technique described i n
the text. The number of cells examined in ( A ) , (B) and ( C ) were
41,298, 38,182 and 35,604, respectively. Broken lines represent values
for GI and G,+M modal channels as compared to HLM cells in exponential growth phase.
HUMAN LIMB MESENCHYME I N VITRO
R. 0. Kelley, T.
PLATE 4
I. Baker, H. A. Crissman and C. A. Henderson
8000
-
I
I
I I
' ' I '
1
I
I
I
I
I
I
I
6000
I
I
20
40
I
I
A
C
60
RELATIVE DNA CONTENT
80
HUMAN LIMB MESENCHYME IN VITRO
R. 0 . Kelley, T. I. Baker, H.A. Crissman and C. A. Henderson
PLATE 5
( H L M 15) EXPONENT/AL GROWTH
GI = 42.6
O/o
s
=45.9%
G,+M =1185Oo/
'G,= 8 , 4 h
100
t
S = 12.2 h
MITOTIC INDEX = ,032
%LABELED CELLS. 44,4To
of 50cells
'0
2
4
6
8
10
12
14
16
18
20
22
24
HOURS
EXPLANATION OF FIGURE
12 FMF distribution data for HLMl5 in exponential growth applied to a graphic plot after
Okada ('67). Traverse times through GI, S, Gz and M are 8.4 h, 12.2 h, 2.6 h and 0.8 h
re spectively
.
670
HUMAN LIMB MESENCHYME IN VITRO
R. 0. Kelley, T. I. Baker, H. A. Crissman and C. A. Henderson
PLATE 6
EXPLANATION OF FIGURE
13 HLMl5 cell karyotype analysis visualized in 97% of 63 metaphase configurations.
671
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