BIOLOGY OF REPRODUCTION 64, 1487–1493 (2001)
Effect of Fibroblast Donor Cell Age and Cell Cycle on Development of Bovine
Nuclear Transfer Embryos In Vitro1
Poothapillai Kasinathan, Jason G. Knott, Pedro N. Moreira, Amy S. Burnside, D. Joseph Jerry,
and James M. Robl2
Department of Veterinary and Animal Science, University of Massachusetts, Amherst, Massachusetts 01003
ABSTRACT
The effects of cell cycle stage and the age of the cell donor
animal on in vitro development of bovine nuclear transfer embryos were investigated. Cultures of primary bovine fibroblasts
were established from animals of various ages, and the in vitro
life span of these cell lines was analyzed. Fibroblasts from both
fetuses and calves had similar in vitro life spans of approximately
30 population doublings (PDs) compared with 20 PDs in fibroblasts obtained from adult animals. When fibroblasts from both
fetuses and adult animals were cultured as a population, the
percentage of cells in G1 increased linearly with time, whereas
the percentage of S-phase cells decreased proportionately. Furthermore, the percentage of cells in G1 at a given time was
higher in adult fibroblasts than in fetal fibroblasts. To study the
individual cells from a population, a shake-off method was developed to isolate cells in G1 stage of the cell cycle and evaluate
the cell cycle characteristics of both fetal and adult fibroblasts
from either 25% or 100% confluent cultures. Irrespective of the
age, the mean cell cycle length in isolated cells was shorter (9.6–
15.5 h) than that observed for cells cultured as a population.
Likewise, the length of the G1 stage in these isolated cells, as
indicated by 5-bromo-deoxyuridine labeling, lasted only about
2–3 h. There were no differences in either the number of cells
in blastocysts or the percentage of blastocysts between the embryos reconstructed with G1 cells from 25% or 100% confluent
cultures of fetal or adult cell lines. This study suggests that there
are substantial differences in cell cycle characteristics in cells
derived from animals of different ages or cultured at different
levels of confluence. However, these factors had no effect on in
vitro development of nuclear transfer embryos.
developmental biology, implantation/early development
INTRODUCTION
The cloning of mammalian embryos by fusing somatic
cells to oocyte cytoplasts has been achieved in several laboratories [1–10]. This technique is of significantly greater
commercial and research importance than the previous
well-established technique of cloning from embryonic cells
[11]. One great advantage of cloning from somatic cells is
that specific types of somatic cells are easily propagated in
culture. Propagation in culture provides many millions of
cells that can be used either to produce large numbers of
identical offspring or for genetic modification and production of transgenic animals. A second advantage is that somatic cells can be recovered from adult animals and have
This research was supported in part by USDA grant 97-35205-5132 to
D.J.J. and J.M.R.
2
Correspondence. FAX: 413 545 6326; e-mail: [email protected]
1
Received: 15 September 2000.
First decision: 16 October 2000.
Accepted: 28 December 2000.
Q 2001 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
been successfully used to make genetically identical copies
of existing animals.
Success in cloning from embryonic cells has been improved by information on compatibility between the nuclear donor cell cycle and the recipient cytoplast cell cycle
[11–17]. Primarily, two types of recipient cytoplasts have
been used: the enucleated and activated metaphase II oocyte cytoplast and the enucleated but not activated oocyte
cytoplast [11, 14]. In the activated oocyte, the cytoplast is
in an interphase state and has a low level of histone H1
kinase activity. In the inactivated oocyte, the cytoplast is in
a metaphase state and has a high level of histone H1 kinase
activity [11]. The response of the donor cell nucleus to
these two types of cytoplasts is quite different.
In the activated cytoplast, little change is noted in the
transplanted nucleus. DNA synthesis, if initiated, is completed or, if not initiated, progresses normally. This type of
recipient is compatible with both G1 and S-phase stages of
the donor cell cycle [13]. Morphologically, little change is
observed in the nucleus following transfer to the activated
cytoplast. In the metaphase oocyte cytoplast, the donor nuclear chromatin immediately condenses, in conjunction
with a breakdown of the nuclear envelope, following contact with the recipient cytoplasm [18]. Spindle microtubules
form in association with the condensed chromatin [19].
When the nuclear transfer embryo is activated, causing a
decrease in histone H1 kinase activity, a nucleus forms and
acquires the morphology of a large pronucleus [18]. If
DNA synthesis has commenced prior to nuclear envelope
breakdown, then DNA rereplication will take place, resulting in a defective embryo. Furthermore, the progression of
DNA synthesis in the donor nucleus is not compatible with
normal chromatin condensation, which also results in defective embryos. Consequently, only nuclei that have not
entered S-phase (G1 stage of the cell cycle) produce normal
embryos when transplanted to a nonactivated oocyte cytoplast [13]. Although the nonactivated recipient cytoplast is
only compatible with G1 stage nuclei, it is capable of inducing morphological reprogramming of the donor nucleus
[18]. Although not proven, it is believed that reprogramming is important for success in somatic cell nuclear transfer [20]; therefore, a G1 stage donor cell is preferred.
Research is routinely done with a wide variety of somatic cells in culture, and some information is available on
the relative proportions of the cell population in the various
stages of the cell cycle [21]. For example, cultures of human fibroblast cells consist of 63% cells with a G1/G0
DNA content, 25% in S-phase, and 12% in G2/M phase
during logarithmic growth [22]. For bovine fibroblasts,
which have been successfully used to produce nuclear
transplant offspring, essentially no information is available
on the characteristics of the cells in culture. For nuclear
transfer, it is important to identify and to be able to precisely control the stage of the cell cycle at the time of donor
and recipient cell fusion. Therefore, one objective of this
1487
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KASINATHAN ET AL.
study was to evaluate the cell cycle distribution in bovine
fibroblasts from various sources either as a population in
standard cultures or as individual cells. Furthermore, a
method was developed for obtaining synchronized populations of cells in G1 stage. G1 cells were also evaluated
as nuclear donors.
A further concern with nuclear transplantation is both
the age of the cell donor animal and the number of doublings a cell population undergoes in culture [22, 23]. Primary cells are only capable of undergoing a finite number
of population doublings (PDs) in culture before becoming
senescent [23, 24]. This senescence model is well established. Information is also available to indicate that senescence is related to the number of divisions a cell has undergone and that in vitro cell life span decreases with the
age of the donor individual [22].
Cell senescence may be a limiting factor in the application of nuclear transfer in animals. In agriculture, it
would be advantageous to use cells from progeny-tested
animals for multiplication of superior genotypes. Some agricultural and biomedical applications would require genetic modification of the donor cells for the production of
transgenic animals [2, 25]. Inserting genes into cells in culture requires selection of transgenic cells from nontransgenic cells and usually clonal derivation of cell lines [2].
These manipulations require a significant number of PDs
before cells can be used as nuclear donors. Therefore, a
second objective of this study was to determine the life
span, in culture, of fibroblast cells derived from fetuses and
animals of various ages. Furthermore, the effect of age of
the cell donor on cell cycle characteristics and nuclear
transfer success in culture was determined.
tissue culture plates and covered with a glass slide to prevent floating in the culture medium. Ten milliliters of aMEM 1 FCS was then added to the dish, and the dish was
incubated at 398C in an atmosphere of 5% CO2 in air. After
removal of the tissue samples on Day 10, monolayers of
cells were harvested using trypsin-EDTA in PBS, counted,
and seeded in 100-mm tissue culture plates.
Establishment of Mouse Fetal Fibroblast Feeder Layers
Mouse fetal fibroblasts were produced from Day 13 fetuses using the protocol described for the preparation of
bovine fetal fibroblasts. Confluent cultures of mouse fibroblasts were mitotically inactivated by g-ray irradiation
(2956 rads). After inactivation, 5.0 3 105 cells were plated
in four-well plates (Nunc, VWR), and after 24 h the medium was changed and the monolayer was used for embryo
culture.
Determination of PDs and Cell Counts
After the initial seeding, cells were counted when they
were 95% confluent. Cells were removed from plates by
the standard trypsinization procedure described above. Harvested cells were spun at 1000 3 g for 5 min, the pellet
was dissolved in 10 ml of a-MEM 1 FCS, and a sample
was counted. These cells were then seeded again and allowed to reach 95% confluence. The cells were then harvested and counted, and the number of PDs was calculated.
The procedure was repeated until the cells became senescent.
MATERIALS AND METHODS
Isolation of G1 Cells
Establishment of Fetal Fibroblast Cell Lines
Twenty-four hours prior to isolation, 5.0 3 105 cells
were plated onto 100-mm tissue culture plates containing
10 ml of a-MEM 1 FCS. The following day, plates were
washed with PBS and the culture medium was replaced for
1–2 h before isolation. The plates were then shaken for 30–
60 sec at medium speed (Vortex-Genie 2; Fisher Scientific,
Houston, TX), the medium was removed and spun at 1000
3 g for 5 min, and the pellet was resuspended in 250 ml
of a-MEM 1 FCS. Newly divided cell doublets attached
by a cytoplasmic bridge were then selected because these
cells are in early G1. This isolation procedure is referred
to as the shake-off method.
Bovine fetal cell lines were developed using a modification of a method described earlier [26]. Bovine fetuses
were obtained from the slaughterhouse and transported to
the laboratory in Dulbecco’s PBS (DPBS) with 16 ml/ml of
antibiotic-antimycotic (Gibco, Grand Island, NY), 4 ml/ml
tylosin tartrate (Sigma, St. Louis, MO), and 8 ml/ml fungizone (Gibco). Fetuses were rinsed in DPBS, the head and
internal organs were removed, and remaining tissues were
finely chopped into pieces with a scalpel blade. The fibroblasts were separated from the tissue pieces by a standard
trypsinization procedure (described elsewhere [26]) using
0.08% trypsin and 0.02% EDTA in PBS (trypsin-EDTA).
The cells were seeded onto 100-mm tissue culture plates
(Corning, VWR, Chicago, IL) in a minimal essential medium (a-MEM; Gibco) supplemented with 10% fetal calf
serum (FCS; Hyclone, Logan, UT), 0.15 g/ml glutamine
(Sigma), 0.003% b-mercaptoethanol (Gibco), and antibiotic-antimycotic (Gibco) (a-MEM 1 FCS). On Day 3 of
seeding, the cells were harvested using DPBS with trypsinEDTA solution and were counted. One million cells were
reseeded onto 100-mm tissue culture plates, and the remaining cells were frozen in a-MEM with 10% FCS and
dimethyl sulfoxide (Sigma).
Establishment of Calf and Adult Fibroblast Cell Lines
Bovine ear biopsies were taken after clipping the hair
and washing with disinfectant. The samples were washed
three times in DPBS, and the cartilage was removed and
discarded. Whole tissue samples were placed in 100-mm
Cell Cycle Analysis
The cell cycle of bovine fetal and adult fibroblasts was
analyzed using a fluorescent activated cell sorter (FACS;
Becton Dickinson, San Jose, CA). Cells (5.0 3 105) were
seeded onto 100-mm tissue culture plates, grown to the
desired confluence, and harvested by the standard trypsinization procedure at different time points. The cell suspension was centrifuged at 300 3 g and resuspended in chilled
(248C) 70% ethanol with 5% glycerol in water while shaking the sample at low speed. After overnight incubation of
the suspension at 248C, cells were washed twice with
chilled DPBS. The pellet was resuspended in 500 ml of
DPBS with 16 mg of RNAseA (Worthington Biochemicals,
Lake Wood, NJ). The suspension was incubated in a water
bath for 3–4 h. Twenty-five to 50 mg of propidium iodide
was then added to the suspension, and 10 000 events were
analyzed per treatment.
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FIBROBLAST CELL CYCLE IN NUCLEAR TRANSFER
Preparation of Cells for Nuclear Transplant
from Confluent Fibroblast Cultures
Twenty-four hours prior to nuclear transplantation, 1 3
106 cells at the desired PD were plated onto 35-mm tissue
culture plates containing 4 ml of a-MEM 1 FCS. The
plates were washed on the following day and trypsinized
by the standard procedure. The suspension was then centrifuged at 300 3 g for 5 min, and the pellet was resuspended in 500 ml of a-MEM 1 FCS. Cells from this suspension were used as nuclear transplant donor cells from
confluent cultures.
5-Bromo-Deoxyuridine Labeling of G1 Cells
Cells in G1 stage were placed in four-well tissue culture
plates containing 250 ml of a-MEM 1 FCS supplemented
with 25 m g 5-bromo-deoxyuridine (BrdU; Boehringer
Mannheim, Indianapolis, IN). At 0, 2, 4, and 7 h, cells were
fixed with 70% ethanol (in 50 mM glycine buffer, pH 2.0)
for 20 min. Following fixation, cells were washed and incubated with mouse anti-BrdU IgG (Sigma) for 30 min at
378C. After 30 min, cells were washed, antimouse Ig-fluorescein (Sigma) was added, and cells were then incubated
again for an additional 30 min at 378C. Following the second incubation, fixed cells were washed and mounted with
glycerol. The percentage of cells in S-phase was assessed
with an epifluororescent microscope (Nikon, Garden City,
NY).
TABLE 1. Effect of donor age on life span and growth rate in primary
bovine fibroblast cultures.
Donor typea
No.
animals
Mean (SEM) PD no.
PDs/day
5
9
6
31 (1.2)
32 (0.6)
18 (3.4)b
1.30b
0.85
0.80
Fetuses
Calves
Adults
a Fetuses were 45 days old, calves ranged from newborn to 13 mo, and
adults were from 2 to 15 yr old.
b Different from other donor ages within column (P , 0.05).
bryo culture medium covered with 0.2 ml of embryo-tested
mineral oil (Sigma). Twenty-five to 50 embryos were
placed in each well and incubated at 38.58C in an atmosphere of 5% CO2 in air. On Day 4, 10% FCS was added
to the culture medium. On Days 7 and 8, development to
the blastocyst stage was recorded. Cell numbers in blastocysts were assessed by mounting with 1% bis-benzimide in
glycerol.
Statistical Analysis
Cells in G1 stage were transferred into 50-ml drops of
a-MEM 1 FCS derived from actively dividing cultures of
fibroblasts (conditioned medium) using a micromanipulator
and were observed every 2 h for 24 h for cell division.
Mean cell cycle length was calculated from cells dividing
within the first 24 h.
Cell life span (total number of PDs to senescence) and
the mean PDs/day were compared among three ages of cell
donors (fetuses, 0- to 13-mo calves, and 2- to 15-yr adults)
by analysis of variance (ANOVA) and regression analysis.
Length of the cell cycle was compared in isolated cells,
either isolated in G1 from low-confluence cultures or isolated directly from high-confluence cultures, from two fetal
and two adult cell lines by the Student t-test. FACS and PD
results from fetal and adult fibroblasts were analyzed at
various times after plating by regression analysis. The percentage of embryos developing to the blastocyst stage and
total cell number were compared among different ages of
animals and between G1 cells isolated from low-PD cultures and confluent cultures by ANOVA.
Nuclear Transplantation, Activation, and Embryo Culture
RESULTS
The nuclear transfer procedure was essentially as described previously [2]. In vitro matured oocytes were enucleated about 18–20 h postmaturation (hpm), and chromosome removal was confirmed by bis-benzimide (Hoechst
33342, Sigma) labeling under ultraviolet light. These cytoplast-donor cell couplets were fused by application of a
single electrical pulse of 2.4 kV/cm for 20 msec (Electrocell
manipulator 200; Genetronics, San Diego, CA). After 3–4
h, a random subset of 25% of the total transferred couplets
was removed, and fusion was confirmed by bis-benzimide
labeling of the transferred nucleus. At 30 hpm, reconstructed oocytes and controls were activated with calcium ionophore (5 mM) for 4 min (Cal Biochem, San Diego, CA)
and 10 mg cycloheximide and 2.5 mg cytochalasin D (Sigma) in ACM culture medium (100 mM NaCl, 3 mM KCl,
0.27 mM CaCl2, 25 mM NaHCO3, 1 mM sodium lactate,
0.4 mM pyruvate, 1 mM L-glutamine, 3 mg/ml BSA (fatty
acid free), 1% basal minimum essential (BME) amino acids, and 1% MEM nonessential amino acids; all chemicals
obtained from Sigma) for 6 h as described previously [27,
28]. After activation, eggs were washed in Hepes-buffered
hamster embryo culture medium (114 mM NaCl, 3.2 mM
KCl, 2 mM CaCl2, 10 mM sodium lactate, 0.1 mM sodium
pyruvate, 2 mM NaHCO3, 10 mM Hepes, and 1% BME
amino acids; all chemicals obtained from Sigma) five times
and placed in culture in four-well tissue culture plates containing irradiated mouse fetal fibroblasts and 0.5 ml of em-
Effect of Donor Age on Cell Proliferation
Assessment of Cell Cycle Length
Cultures of primary bovine fibroblasts were established
from animals ranging in age from gestational Day 45 fetuses to 15-yr-old adults (n 5 20). Each cell line was passaged to senescence, and total PDs and PDs/day were calculated (Table 1). Fibroblast cell lines derived from fetuses
and calves had similar in vitro life spans of approximately
30 PDs compared with 20 PDs in fibroblasts obtained from
adult animals. Cell lines derived from fetuses differed from
those derived from calves and adults with respect to growth
kinetics. Upon initial plating, fetal fibroblasts grew much
faster than did calf and adult fibroblasts. However, growth
rate of fetal fibroblasts declined more rapidly than did that
of calf and adult fibroblasts (P , 0.05).
Effect of Donor Age and Culture Density on Cell
Cycle Distribution
FACS analysis was used to assess the cell cycle distribution of both fetal and adult fibroblasts as a population in
culture. Data were collected at different time points following initial seeding and continued for 76 h. Cell counts were
taken at each time point, and PDs were calculated (Fig. 1c).
The percentage of S-phase cells decreased with time in culture (Fig. 1a), whereas the percentage of G1-phase cells
increased over time in culture for both fetal and adult cell
lines (Fig. 1b). The rate of increase in the proportion of
1490
KASINATHAN ET AL.
FIG. 1. PD and cell cycle analysis of fetal and adult cell lines. a) Mean percentage of cells in S-phase at various time points in culture after passage.
b) Mean percentage of cells in G1 phase at various time points in culture after passage. c) PDs/day at various time points in culture after passage
(v 5 fetus, m 5 adult).
cells in G1 was similar for both fetal and adult cell lines,
and the rate of decrease in cell division rate (PDs/day) was
similar (Fig. 1c). However, the rate of cell division for all
time points combined was lower for adult cells than for
fetal cells (P , 0.05).
TABLE 2. Effect of cell donor age and culture confluence on cell cycle
length in isolated pairs of bovine fibroblasts.
Donor age
45-day
45-day
45-day
45-day
4 yr
4 yr
15 yr
15 yr
a
fetus
fetus
fetus
fetus
%
Confluence
No. cells
25
100
25
100
25
100
25
100
39
36
28
30
37
31
35
34
% Cells
divided in Mean (SEM) cell
24 h
cycle lengtha (h)
97
95
98
96
97
95
92
94
13.1
13.6
13.1
13.3
10.1
9.5
14.2
15.3
(0.5)
(0.5)
(0.7)
(0.6)
(0.7)
(0.3)
(0.5)
(0.6)
Calculated only on those cells that divided within the first 24 h. Average
cell cycle length was shorter for the cells from the 4-yr-old animal than
for cells from other donor animals (P , 0.05). No differences were observed between low and high percentage confluence or between fetal
and adult animals.
Length of Cell Cycle of Isolated Early G1
Bovine Fibroblasts
To obtain a population of actively dividing G1 cells a
shake-off method was developed. This method was also
used to evaluate differences among individual cells in a
population and to evaluate the cell cycle characteristics of
isolated cells in culture. Cells obtained by the shake-off
method were cultured individually in microdrops at 38.58C
in a 5% CO2 atmosphere and observed every 2 h for 24 h.
Cells were isolated from 25% and 100% confluent plates
from each of two fetus- and two adult-derived cultures. Between 92% and 98% of all cells divided within 24 h. The
mean length of the cell cycle for each of the treatments
ranged from 9.6 to 15.5 h and was similar among fetusand adult-derived cell lines and for cells isolated from either 25% or 100% confluent plates (Table 2).
Length of G1 in Isolated Fibroblasts
Because isolated cells in culture have an extremely short
cell cycle, it was of interest to determine the length of G1
so that we could be assured that donor cells would be in
G1 at the time of fusion with recipient cytoplasts. To as-
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FIBROBLAST CELL CYCLE IN NUCLEAR TRANSFER
TABLE 3. Length of G1 stage in isolated pairs of bovine fetal fibroblasts.
Doublets from shake
Hours after
isolation
No. cells
2
3
4
7
a
30
14
28
28
offa
Random
No. (%) cells
labeled with
BrdU
No. cells
0
8 (57)
15 (53)
22 (79)
45
45
30
46
cellsa
No. (%) cells
labeled with
BrdU
23
25
19
29
(51)
(55)
(63)
(63)
Cells from 25% confluence culture.
certain the length of G1 in fibroblasts isolated by the shakeoff method, incorporation of BrdU into DNA was evaluated
at various times after cell isolation. The results indicate that
these cells enter S-phase between 2 and 3 h after isolation
(Table 3). Therefore, fibroblasts isolated in G1 must be
fused to recipient cytoplasts within 2 h following isolation.
Development of Nuclear Transfer Embryos Produced
from Isolated G1 Fibroblasts
The G1 fibroblasts isolated from cultures at either 25%
or 100% confluence were transferred to enucleated metaphase II oocytes and fused. In 25% confluent cultures, cells
were isolated by the shake-off method. In 100% confluent
cultures, random cells were used because nearly all cells
were in G1 and thus the shake-off method could not be
used. After activation, nuclear transfer embryos were cultured for 8 days, and the extent of development to the blastocyst stage and total cell number was compared (Table 4).
There was no difference in either the number of cells in
blastocysts or the percentage of blastocysts between G1 fibroblasts from 25% and 100% confluence cultures. The percentage of blastocyst development among individual cells
lines was different (P , 0.05); however, the number of cells
in blastocysts did not differ among cultures from individual
animals.
DISCUSSION
One factor that may affect the successful development
of nuclear transfer embryos is cell cycle stage of the donor
cell. At the time of nuclear transfer, the preferred phase of
the cell cycle for the donor nucleus is G1 if a metaphase
II oocyte is used as a recipient because chromosome abnormalities and DNA rereplication may occur when S-phase
chromatin is forced to condense into metaphase chromosomes [11, 13]. The percentage of blastocysts has been
shown to decrease when mid to late S-phase donors were
fused to metaphase II oocytes [13, 29].
To obtain somatic cells in the G1 stage of the cell cycle,
several methods have been employed. Nocadozole and colcemid, inhibitors of microtubules, have both been used to
synchronize cells in metaphase, and following removal of
the drugs, a subpopulation of cells divides and enters G1
[30]. Aphidicolin, a DNA synthesis inhibitor, has been used
in combination with microtubule inhibitors to arrest cells
at the G1/S-phase boundary [13]. Other chemicals have
been used to directly arrest cells in G1 by interfering with
regulatory protein kinases [13, 31]. Chemical intervention
may be avoided by using cells derived from cultures at high
confluence [21, 30].
In this study, both adult and fetal bovine fibroblast cell
lines were analyzed by FACS analysis to determine the relative proportions of cells in different phases of the cell
cycle, particularly G1. Our results indicate that the per-
TABLE 4. Effect of cell donor age and culture confluence on rate of development to blastocyst and blastocyst cell number in nuclear transplant
embryos.
%
ConNo.
fluencea oocytesb
Donor
age
45-day
45-day
45-day
45-day
4 yr
4 yr
15 yr
15 yr
fetus
fetus
fetus
fetus
25
100
25
100
25
100
25
100
9
9
15
29
11
9
26
18
No.
fused
40
54
55
43
63
42
67
73
Mean
no. (%)
blastocystsc
9
9
15
29
11
9
26
18
(23)
(17)
(27)
(67)
(18)
(21)
(39)
(25
Mean (SEM)
no. cellsd
82
95
83
118
104
97
125
107
(15)
(24)
(9)
(11)
(16)
(15)
(11)
(15)
a Cells from 25% confluence cultures were isolated as G1 pairs. Random
cells were used from 100% confluence cultures.
b Total number of oocytes from three replicates.
c Blastocyst development rates were adjusted for fusion rates each day.
Fusion rates were observed 3 h after the electrical pulse.
d Cell number was determined on Day 8. No differences were observed
for cell number among treatments.
centage of cells in G1, in both adult and fetal fibroblasts,
increases linearly with time in culture after seeding. Furthermore, adult fibroblast cultures contain a higher percentage of cells in G1 throughout the culture period than
do fetal cell cultures, indicating that adult cells may be
more sensitive than fetal cells to contact inhibition. The
higher percentage of cells in G1 suggests that the adult cells
contain a longer cell cycle than the fetal cells. This result
was verified directly by evaluating the average number of
PDs/day either for the entire life span of the culture or
between passages. Overall, a high proportion of cells with
a G1 DNA content can be obtained by growing cultures to
confluence in either fetal or adult fibroblast cultures.
Growing cultures to confluence cannot be used as a
method for separating cells in G1 and capable of active
division from cells that have stopped dividing because they
have become senescent or they are dying or differentiating.
Therefore, a shake-off method was developed to obtain a
synchronous group of dividing cells in early G1. This method was used to observe the variation in cell cycle length
within a population of cells and to evaluate cell cycle characteristics in isolated cells. The results indicated that isolated cells have a very short cell cycle and that a high
proportion of cells within a population have this characteristic.
Mean cell cycle length in isolated cells ranged from 9.6
to 15.5 h, considerably shorter than expected and shorter
than that observed for cells cultured as a population, even
at low confluence (22 h at 25% confluence). This result
indicates that fibroblasts, and possibly other cells, have an
inherent cell cycle length similar to that of embryonic cells.
Furthermore, this result indicates that cell cycle length is
primarily determined by the degree of contact inhibition in
culture.
In comparing the combined data from adults and the
combined data from fetal cells, no differences were observed in cell cycle length when cells were cultured individually. However, adult cells appear to be more responsive
than fetal cells to contact inhibition, as indicated by the
slower rate of division, when grown as a population. Contact inhibition is readily reversible; cells recovered from
either low- or high-confluence cultures have similar cell
cycle lengths when cultured individually. Furthermore, passaging of cells from confluent cultures results in immediate
resumption of more rapid cell cycles. These results have
1492
KASINATHAN ET AL.
implications for the use of G1 cells in nuclear transfer and
in controlling for stage of the cell cycle when making treatment comparisons with nuclear transfer.
Differences in cell cycle length in isolated cells were not
related to the age of the donor animal nor were they related
to the confluence of the culture from which they were derived. However, cells from a 4-yr-old animal had a cell
cycle length of about 10 h, whereas cells from a 15-yr-old
animal had a cell cycle length of about 15 h. The two fetal
cell lines were similar, and cells divided about every 13.3
h. The marked difference between the two adult cell lines
could be due to spontaneous transformation of the cell line
derived from the 4-yr-old animal. Following the experiments in this study, the cell line from the 4-yr-old animal
continued to grow to at least 50 PDs in culture without
becoming senescent.
One concern about the life span of donor cells in culture
is related to the use of these cells for genetic modifications.
Transgenic sheep and cattle have been produced by transfection of fibroblast cells followed by nuclear transplantation [2, 32]. In both of these studies, fibroblast cells were
recovered from fetuses to ensure that the cell lines would
have as long a life span as possible. Genetic modification
of cells involves establishment of the cell line, transfection,
and then clonal propagation under antibiotic selection.
Clonal propagation to a colony of 500 000 cells requires 20
PDs. As demonstrated in other species [22, 23, 33], the age
of a cell donor is correlated with the in vitro life span of a
primary cell line derived from the individual. In this study,
cells from fetuses and young adult animals had a similar in
vitro life span of about 31 PDs, whereas cells from older
adult animals had a shortened life span of about 18 PDs.
These results indicate that adult cells are likely to become
senescent before selection for transgenic clonal lines would
be complete. Fetal cells and cells from young animals can
be transfected and selected as shown previously [2]; however, it is unlikely that the cells would continue to divide
for a sufficient length of time to allow for more than one
genetic modification to be completed. Regeneration and rejuvenation of a cell line would be necessary for multiple
genetic modifications.
A second concern about the limited life span of nuclear
donor cells is that cloning efficiency may decrease as cells
progress towards senescence. The results in this study indicate that cloning efficiency would decrease more quickly
in cells from adults than in cells from calves or fetuses. It
is clear from previous studies [5, 34] that cells cultured for
an extended time can produce offspring. In one study [5],
developmental potential of nuclear transfer embryos was
shown to be similar for cells from low-passage and those
from high-passage cultures. In another study, cells expressing markers of senescence, EPC-1 and reduced telomere
length [34], and a senescent morphology produced offspring following nuclear transfer. More information is required before conclusions can be drawn concerning differences in efficiency of development to term between senscent cells and cells from early PD cultures.
In in vitro development, both percentage of blastocysts
and cell number for nuclear transfer embryos derived from
actively dividing G1 cells or cells from high-confluence
cultures was similar. Furthermore, no difference was observed in development between embryos derived from fetal
fibroblasts and those from adult fibroblasts when comparing
combined data.
The results of this study indicate that differences in cell
cycle characteristics do exist in cells derived from animals
of different ages. Although the age of the animal appears
to have no impact on the inherent length of the cell cycle
in isolated cells, cells from calves and adults do divide
more slowly in culture as a result of a greater sensitivity to
contact inhibition when compared with fetal cells. Age of
the donor animal had no impact on in vitro development
of nuclear transplant embryos. The shake-off method can
be used to isolate cells synchronized in G1 from an actively
dividing population. Cells isolated by shake off, however,
did not support a higher rate of development of nuclear
transfer embryos to the blastocyst stage over that of cells
from a confluent culture.
ACKNOWLEDGMENTS
The authors thank John Balise for his technical assistance and Grant
Morgan for his helpful discussions on the cell cycle.
REFERENCES
1. Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM,
Cammuso C, Williams JL, Nims SD, Porter CA, Midura P, Palacios
MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM,
Godke RA, Gavin WG, Overstorm EW, Echelard Y. Production of
goats by somatic cell nuclear transfer. Nat Biotechnol 1999; 17:456–
461.
2. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce
de Leon FA, Robl JM. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 1998; 280:1256–1258.
3. Galli C, Duchi R, Moor RM, Lazzari G. Mammalian leukocytes contain all the genetic information necessary for the development of a
new individual. Cloning 1999; 1:161–170.
4. Kato Y, Tani T, Sotomaru Y, Kurokama K, Kato J, Doguchi H, Yasue H. Eight calves cloned from somatic cells of single adult. Science
1998; 282:2095–2098.
5. Kubota C, Yamakuchi H, Todoroki J, Mizoshita K, Tabara N, Barber
M, Yang X. Six cloned calves produced from adult fibroblast cells
after long term culture. Proc Natl Acad Sci U S A 2000; 973:990–
995.
6. Vignon X, Chesne P, LeBourhis D, Flechon JE, Hayman Y, Renard JP.
Developmental potential of bovine embryos reconstructed from enucleated matured oocytes fused with cultured somatic cells. Life Sci
1998; 321:735–745.
7. Wakayama T, Perry AC, Zuccotti M, Johnson KR, Yanagimachi R.
Full term development of mice from enucleated oocytes injected with
cumulus cell nuclei. Nature 1998; 394:369–374.
8. Wells DN, Misica PM, Tervit HR. Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. Biol
Reprod 1999; 60:996–1005.
9. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH. Viable
offspring derived from fetal and adult mammalian cells [published
erratum appears in Nature 1997; 386:200]. Nature 1997; 385:810–
813.
10. Zakhartchenko V, Alberio R, Stojkovic M, Prelle K, Schernthaner W,
Stojkovic P, Wenigerkind H, Wanke R, Duchler M, Steinborn R,
Mueller M, Brem G, Wolf E. Adult cloning in cattle: potential of
nuclei from a permanent cell line and from primary cultures. Mol
Reprod Dev 1999; 54:264–272.
11. Campbell KH, Loi P, Cappai P, Wilmut I. Cell cycle coordination in
embryo cloning by nuclear transfer. Rev Reprod 1996; 1:40–46.
12. Campbell KH, Ritchie WA, Wilmut I. Nuclear-cytoplasmic interactions during the first cell cycle of nuclear transfer reconstructed bovine
embryos: implications for deoxyribonucleic acid replication and development. Biol Reprod 1993; 49:933–942.
13. Collas P, Balise JJ, Robl JM. Influence of cell cycle stage of the donor
nucleus on development of nuclear transplant rabbit embryos. Biol
Reprod 1992; 46:492–500.
14. Kono T. Nuclear transfer and reprogramming. Rev Reprod 1997; 2:
74–80.
15. Liu L, Dai Y, Moor RM. Nuclear transfer in sheep embryos: the effect
of cell cycle coordination between nucleus and cytoplasm and the use
of in vitro matured oocytes. Mol Reprod Dev 1997; 47:255–264.
16. Prather RS, Boquest AC, Day BN. Cell cycle analysis of cultured
porcine mammary cells. Cloning 1999; 1:17–24.
17. Robl JM, Gilligan B, Crister ES, First NL. Nuclear transplantation in
FIBROBLAST CELL CYCLE IN NUCLEAR TRANSFER
18.
19.
20.
21.
22.
23.
24.
25.
26.
mouse embryos: assessment of recipient cell stage. Biol Reprod 1986;
34:733–739.
Collas P, Robl JM. Relationship between nuclear remodeling and development in nuclear transplant rabbit embryos. Biol Reprod 1991;
45:455–465.
Pinto-Correia C, Collas P, Ponce de Leon FA, Robl JM. Chromatin
and microtubule organization in the first cell cycle in rabbit parthenotes and nuclear transplant embryos. Mol Reprod Dev 1993; 34:33–
42.
Wolf DP, Meng L, Ouhibi N, Zelinski-Wooten M. Nuclear transfer in
rhesus monkey: practical and basic implications. Biol Reprod 1999;
60:199–204.
Tobey RJ, Valdez JA, Crissman M. Synchronization of human diploid
fibroblasts at multiple stages of the cell cycle. Exp Cell Res 1988;
179:400–416.
Cristofalo VJ, Allen RG, Pignalo RJ, Martin BG, Beck JC. Relationship between donor age and replicative lifespan of human cells in
culture: a reevaluation. Proc Natl Acad Sci U S A 1998; 95:10614–
10619.
Pignalo RJ, Masoro EJ, Nicholas WW, Bradt CI, Cristofalo VJ. Skin
fibroblasts from Fischer 344 rats undergo similar changes in replicative lifespan but not immortalization with caloric restriction of donors.
Exp Cell Res 1992; 201:16–22.
Hyflick L, Moorhead PS. The serial cultivation of human diploid cell
strains. Exp Cell Res 1961; 25:585–621.
Wall RJ. Transgenic livestock: progress and prospects for the future.
Theriogenology 1996; 38:337–357.
Freshney IR. Culture of Animal Cells, 3rd ed. New York: Wiley-Liss,
Inc.; 1994: 310–348.
1493
27. Liu L, Ju J, Yang X. Parthenogenetic development and protein patterns of newly matured bovine oocytes after chemical activation. Mol
Reprod Dev 1998; 49:298–307.
28. Presicce GA, Yang X. Parthenogenetic development of bovine oocytes
matured in vitro for 24hr and activated by ethanol and cycloheximide.
Mol Reprod Dev 1994; 38:380–385.
29. Campbell KH, Loi P, Cappai P, Wilmut I. Improved development to
blastocyst of bovine nuclear transfer embryos reconstructed during the
presumptive S-phase of enucleated activated oocytes. Biol Reprod
1994; 50:1385–1393.
30. Johnson RT, Downs CS, Meyn RE. Synchronization of mammalian
cells. In: The Cell Cycle: A Practical Approach. New York: Oxford
University Press; 1993: 1–22.
31. Gadbois DM, Crissman HA, Tobey RJ, Bradbury EM. Multiple kinase
arrest points in the G1 phase of non-transformed mammalian cells are
absent in transformed cells. Proc Natl Acad Sci U S A 1992; 89:
8626–8630.
32. Schnieke AE, Kind AJ, Ritchie WA, Mycock K, Scott AR, Ritchie
M, Wilmut I, Colman A, Campbell KH. Human factor IX transgenic
sheep produced by transfer of nuclei from transfected fetal fibroblasts.
Science 1997; 278:2130–2133.
33. Campisi J, Dimri G, Hara E. Control of Replicative Senescence. In:
Handbook of Biology of Aging. San Diego: Academic Press, Inc.;
1996: 121–149.
34. Lanza RP, Cibelli JB, Blackwell C, Cristofalo VJ, Francis MK, Baerlocher GM, Mak J, Schertzer M, Chavez EA, Sawyer N, Lansdorp
PM, West MD. Extension of cell life-span and telomere length in
animals cloned from senescent somatic cells [see comments]. Science
2000; 288:665–669.