Insulin Growth Factor and Epidermal Growth Factor
Trigger Mitosis in Lenses Cultured in a
Serum-free Medium
John R. Reddan and Dorothy Wilson-Dziedzic
The mitogenicity of insulin, insulin growth factor (IGF), and epidermal growth factor (EGF) was
evaluated on rabbit lenses cultured in medium KEI-4. IGF, the most highly purified of the insulinlike growth factors was a potent mitogen for mammalian lens epithelial cells. IGF and EGF triggered
cell proliferation throughout the normally amitotic central and pre-equatorial region of the epithelium.
The mitotic response elicited by IGF and EGF was dose dependent, was preceded by DNA synthesis,
exceeded that engendered by equimolar insulin, and exhibited a chronology identical to that brought
about by crystalline insulin. Lenses cultured in KEI-4 alone or in KEI-4 supplemented with growth
hormone, proinsulin, the A and/or B chain of insulin, or MSA, another of the insulin-like growth
factors belonging to the somatomedin family, did not show a mitotic response. Simultaneous exposure
of the lens to IGF and EGF resulted in an increase in the total number of mitotic figures over that
obtained with equimolar concentrations of IGF and EGF. Our results suggest that IGF or other
insulin-like growth factors may be capable of regulating cell division in the mammalian lens in vivo.
That the lens epithelium responded to IGF and EGF may indicate that lens epithelial cells are subject
to multiple hormonal interaction. Since growth factors appear to be cell type specific, information
obtained from the rabbit lens epithelium should be useful in delineating the factors and conditions
required for the growth of cultured human lens cells. Invest Ophthalmol Vis Sci 24:409-416, 1983
Investigations on the process of mitosis indicate
that environmental factors modulate the entrance of
cells into the cell cycle. This study is part of a continuing effort aimed at elucidating the environmental
factors that are capable of regulating cell division in
the mammalian lens.1
Mitosis in the lens in vivo is normally confined to
the peripheral region of the lens referred to as the
germinative zone. However, as a result of mechanical,
chemical, or immunologic insult or following repeated paracentesis, cells throughout the normally
amitotic central region of the epithelium enter mitosis.1'2 A stimulation of cell division is also realized
if the lens is cultured in a medium supplemented with
serum3 or plasmoid aqueous humor,4 situations that
expose the lens to complex mixtures of growth-promoting and -inhibiting factors. Insulin can initiate
DNA synthesis and mitosis in lenses cultured in a
completely defined serum-free medium.5 However,
the concentration of insulin exceeded the level of the
hormone in either primary or plasmoid aqueous humor.6 This suggests that peptides with structural and
functional homologies to insulin could be more mitogenic toward the lens than insulin itself and might
represent the type of peptide capable of regulating
mitosis in the mammalian lens.7 Such a possibility
is explored in the present study and is supported by
the finding that mitosis in the amphibian lens in vivo
is abolished by procedures that decrease the level of
circulating insulin-like growth factors and is reinstated by the systemic administration of such peptides.8
Certain polypeptides that exhibit insulin-like activity stimulate growth of mammalian cells in tissue
culture.910 The insulin growth factors, originally
termed nonsuppressible insulin-like activity, belong
to a family of polypeptides called somatomedins that
are ancestrally related to proinsulin." They are immunologically distinct from insulin, exhibit structural
and functional homologies to insulin, bind to insulin
receptors,12 and show extensive cross-immunologic
reactivity with certain of the somatomedins.13 The
complete amino acid sequence of human insulin
growth factor (IGF) has been reported.1415 Since certain lines of lens epithelial cells have receptors for
From the Department of Biological Sciences, Oakland University, Rochester, Michigan.
Supported by Grant EY-00362 from the National Eye Institute.
Submitted for publication February 1, 1982.
Reprint requests: Dr. John R. Reddan, Department of Biological
Sciences, Oakland University, Rochester, MI 48063.
0146-0404/83/0400/409/$ 1.20 © Association for Research in Vision and Ophthalmology
409
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / April 1983
Vol. 24
insulin, epidermal growth factor (EGF), and fibroblast growth factor (FGF)1617 and exhibit enhanced
growth if these factors are present in a serum-containing medium, we also examined the mitogenicity
of these peptides on lenses cultured in the absence of
serum.
We report that IGF and EGF at nanogram concentrations stimulate cell division in lenses cultured
in a serum-free medium. The response is dose-dependent, and each factor is more mitogenic toward
the lens than crystalline insulin.
growth factor or were exposed to the factor for the
initial hour of culture, whereupon the medium was
changed to KEI-4 alone for the remainder of the culture period. Lenses were fixed in 3:1 ethanol:acetic
acid. Certain lenses were exposed to [3H]thymidine
(VCi/ml; 6Ci/mM) at 30 hrs of culture and fixed at
36 hrs, a time of intensive DNA synthesis in lenses
cultured in an insulin-containing medium.5 Whole
mounts18 of the epithelial layer were prepared and
processed for autoradiography,19 or the total number
of mitotic figures per preparation was counted.
Materials and Methods
Results
New Zealand White rabbits 10-12 weeks old were
killed, the eyes enucleated, lenses isolated, and placed
in Merriam-Kinsey tubes as previously described.5
Lenses were cultured in medium KEI-4 (see ref. 5 for
formulation), or in KEI-4 containing one or more
polypeptides. Gentamicin (Schering Corp., Kenilworth, NJ) was present at 0.05 mg/ml. KEI-4 is a
serum-free medium that mimics the composition of
rabbit aqueous humor. The effect of IGF was also
determined on lenses cultured in minimal essential
medium (MEM) or in medium 199 (Cat. No. 4001100) (GIBCO, Grand Island, NY). The growth factors or peptides evaluated included: crystalline porcine insulin, porcine proinsulin, the A- or B-chain of
porcine insulin (gifts of Dr. Ronald Chance, Lilly
Research Laboratories, Indianapolis, IN); human
IGF (36mU/ml), a mixture of IGF-I and IGF-II (a
gift of Dr. Rene Humbel, Biochemisches Institut der
Universitat Zurich, Zurich, Switzerland); bovine
growth hormone (Miles Laboratories, Elkhart, IN
[0.92 IU/mg]); human growth hormone (Calbiochem, La Jolla, CA) (22 IU/mg); and culture or receptor grade EGF, FGF, or multiplication stimulating
activity (MSA) (Collaborative Research, Boston, MA).
Reference preparations of EGF and FGF were provided by Drs. Stanley Cohen, Vanderbilt University,
Nashville, TN and Denis Gospodarowicz, University
of California, San Francisco, CA, respectively. The
insulin, proinsulin, and A- or B-chain were prepared
as previously described.5 The IGF stock was prepared
in 0.5 M acetic acid and stored at 4 C. For working
solutions, the IGF was diluted further with Earle's
balanced salt solution (GIBCO, Grand Island, NY)
containing 2 mg/ml rabbit serum albumin (Sigma,
St. Louis, MO). Lenses were also cultured in medium
KEI-4 containing rabbit serum albumin at 10 ng/vaX
of culture medium, ie, the level of rabbit serum albumin present when IGF was used at 8.8 X 10"10 M.
Growth hormone was diluted in medium KEI-4 prior
to use. All peptides were present at 8.8 X 10~'°M
unless otherwise noted.
The lenses were either continuously exposed to the
Mitotic activity in lenses cultured in KEI-4 was
principally confined to the peripheral region of the
epithelium, which is thought to correspond to the site
of cell division in the lens in vivo. A low level of
mitosis was noted in the equatorial region of all the
cultured lenses. Cells residing in the pre-equatorial
and central region of lenses cultured in KEI-4 were
essentially amitotic (Fig. 1A). In contrast, epithelia
from lenses cultured in KEI-4 plus IGF exhibited
DNA synthesis and mitosis throughout the preequatorial and central region of the epithelium
(Figs. IB, D).
The magnitude of the proliferative response engendered by IGF is shown in Figure ID which depicts
the. number of nuclei that have incorporated
[3H]thymidine following a 6-hr exposure to the isotope. Approximately 50% of the nuclei are labeled.
In marked contrast, cells residing in the pre-equatorial and central regions of lenses cultured in KEI-4
alone (Fig. 1C) did not enter DNA synthesis. The
effect of various concentrations of IGF on the magnitude of cell division in the cultured lens is shown
in Figure 2. IGF at 8.8 X 10"12 M was not mitogenic,
however, at all concentrations above 4.8 X 10"" M
the proliferative response was dose dependent. IGF
was mitogenic toward the lens epithelium when present at 8.8 X 10"" M (0.66 ng/ml).
In order to gain some understanding of the specificity of IGF-induced mitosis, lenses were cultured
in medium KEI-4 plus compounds that are related
structurally or functionally to the insulin-like growth
factors. Since the production of IGF in vivo is regulated partially by somatotrophin, it was also evaluated. The addition of proinsulin, the A- and/or Bchain of insulin, or MSA to medium KEI-4, all at 8.8
X 10"10 M, did not stimulate mitosis; neither human
nor bovine growth hormone induced a program of
cell proliferation. Indeed, whole mount preparations
from lenses that had been cultured for 52 hrs in medium KEI-4 plus proinsulin, the A- and/or B-chain
of insulin, MSA, or human or bovine growth hormones were essentially amitotic throughout the entire
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411
IGF AND EGF / Reddan and Wilson-Dziedzic
8-8«lCria4-B«10r" 8-8*10~" 8-8«lCTw 4 4 * 1 0 *
Molarity of
IGF
Fig. 2. Lenses were cultured in KEI-4 containing various concentrations of IGF, fixed at 52 hrs, whole mounts of epithelium
were prepared, and the total number of mitoticfiguresper lens was
counted. Values represent the mean ± standard deviation (n =. 3).
Fig. 1. Photomicrographs from the central region of the epithelium of the cultured rabbit lens. Lenses were cultured in medium
KEI-4 or in KEI-4 containing IGF at 8.8 X 10"10 M. Lenses were
fixed at 52 hrs and examined for mitosis; or were exposed to [3H]thymidine from 30-36 hrs of culture,fixedat 36 hrs, and prepared
for autoradiography. Whole mount preparations of a lens cultured
in KEI-4 alone (A) or in KEI-4 plus IGF (B). Autoradiogram from
a lens cultured in KEI-4 alone (C). Autoradiogram from a lens
cultured in KEI-4 plus IGF (D). (Bar = 75 urn.)
central and pre-equatorial regions of the epithelium
and resembled preparations from lenses cultured in
KEI-4 alone. At least five lenses were examined for
each peptide. A mitotic activation was not obtained
in lenses cultured for 52 hrs in medium KEI-4 containing 10 Mg/ml rabbit serum albumin (RSA). FGF
at 8.8 X 10~10 M did not elicit a reproducible mitotic
response.
The chronology and magnitude of cell division in
lenses exposed to IGF is shown in Figure 3. Lenses
cultured in medium KEI-4 or in KEI-4 supplemented
with IGF showed a low level of mitosis during the
initial 40 hrs of culture. Lenses cultured in KEI-4
plus IGF for 52 hrs exhibited mitosis throughout the
central and pre-equatorial regions of the epithelium,
a situation not realized in lenses cultured in KEI-4
alone for 52 hrs (Figs. 1, 3). Lenses cultured in MEM
plus IGF or in medium 199 plus IGF also exhibited
an activation of mitosis throughout the central region
of the lens epithelium.
Since insulin is known to stimulate cell proliferation in lens epithelial cells, the mitogenicity of insulin
was compared to that of IGF. IGF was more mitogenic toward the lens epithelium than equimolar in-
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / April 1980
Vol. 24
disorganization of the epithelial monolayer (Fig. 4 A).
The disorganization, which was more pronounced in
the cells in the preequatorial region of the lens, is
thought to be a result of cellular migration. In contrast, the proliferation induced by IGF was not accompanied by marked cellular disorganization. The
mitotic response triggered by EGF was preceded by
DNA synthesis (Figs. 4B, C), was dose dependent
(Fig. 5), and exhibited a time course identical to that
of IGF (Fig. 3). EGF was mitogenic toward the lens
when present at 0.067 ng/ml (10~n M).
b
•S* as
to
20
30
10
40
Culture Time (hours)
Fig. 3. Rabbit lenses were cultured in medium KEI-4 alone
(O
O) or in K.E1-4 containing IGF at 8.8 X 10~'°M
(•
• ) . Lenses were fixed at 0, 3, 7, 24, 40, and 52 hrs of
culture, whole mounts were prepared, and the total number of
mitotic figures per lens was counted. Values represent the mean
± standard deviation (n = 4).
sulin (Table 1). This result does not appear to be
attributable to the presence of RSA since the mitogenicity of insulin was not increased by the presence
of 10ng/ml RSA.
Epidermal growth factor was also a potent mitogen;
culture and receptor grades were equally mitogenic.
The addition of EGF to medium KEI-4 engendered
a mitotic response that was accompanied by a slight
Table 1. Comparison of the effect of insulin and
insulin growth factor on cell division in the
cultured rabbit lens
Total number of mitotic figures
Lens pairs
Insulin
Insulin growth factor
1
2
3
4
2,028
1,837
2,161
1,594
8,816
7,936
9,724
7,916
Lenses were cultured in KEI-4 containing insulin or insulin growth factor
at 8.8 X IO"'°M. Lenses were fixed after 52 h of culture and the total number
of mitotic figures per whole mount was counted.
Fig. 4. Photomicrographs from the central region of the epithelium of the cultured lens. Lenses were cultured in medium K.EI4 or in KEI-4 plus EGF at 8.8 X 1O~10 M. Lenses were fixed at 52
hrs; or were exposed to [3H]-thymidine from 30-36 hrs of culture,
fixed at 36 hrs, and processed for autoradiography. A, whole mount
preparation of a lens cultured in KEI-4 plus EGF for 52 hrs; B,
autoradiogram from a lens cultured in KEI-4 alone; C, Autoradiogram from a lens cultured in KEI-4 plus EGF. (Bar = 75 /im)
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IGF AND EGF / Reddon and Wilson-Dziedzic
No. 4
413
In the preceding experiments, the lenses were exposed to medium KEI-4 containing IGF or EGF for
the entire culture period. We next determined if the
activation of mitosis brought about by EGF or IGF
was dependent upon the continued presence of the
growth factor. If lenses were placed in medium KEI4 containing EGF for the initial hour of culture and
then cultured for the remaining 51 hrs in KEI-4 alone,
the number of mitoticfiguresper whole mount preparation paralleled that obtained for lenses continuously exposed to EGF (Fig. 6). Lenses cultured in
KEI-4 plus IGF for the initial hour of culture and
examined for mitosis at 52 hrs showed a significant
increase in the number of mitotic figures per whole
mount preparation over lenses cultured for 52 hrs in
KEI-4 alone. The magnitude of the mitotic response
realized in lenses cultured in KEI-4 plus IGF for the
first hour of culture, however, was reduced relative
to that found in lenses continuously exposed to IGF
(Fig. 6). Moreover, the mitotic response noted in
lenses exposed to EGF for the initial 60 min of culture
exceeded that brought about by an initial 60-min exposure to IGF.
In many culture systems, cells are thought to respond to sets of mitogenic factors. In view of the
mitogenicity of IGF and EGF on the lens epithelium,
Length of Exposure
Fig. 6. Lenses were cultured in KEI-4 or in KEI-4 containing
either EGF or IGF at 8.8 X 10~10 M. The lenses were exposed to
the growth factors for the entire culture period (52 hrs) or for only
the initial hour (1 hr) of culture. Lenses exposed to growth factors
for 1 hr were cultured in KEI-4 alone for the remainder of the
culture period. Whole mounts of the epithelia were prepared following 52 hrs of culture and the total number of mitotic figures
per lens was counted. Values represent the mean ± standard deviation (n = 5).
we determined the magnitude of the proliferative response in lenses exposed to both mitogens (Table 2).
Simultaneous exposure of lenses to EGF and IGF
resulted in an increase in the number of mitotic figures per lens that exceeded that obtained with equimolar concentrations of EGF (Table 2) or IGF.
Table 2. Effect of IGF and EGF on cell division
in the cultured rabbit lens
Total number of mitotic figures
8-8x10''
1-8x10.-9
Molarity of EGF
Fig. 5. Lenses were cultured in KEI-4 containing various concentrations of EGF. Lenses were fixed at 52 hrs, whole mounts of
the epithelium were prepared and the total number of mitotic figures per lens was counted. Values represent the mean ± standard
deviation (n = 6).
Lens pairs
2 X [EGF]
EGF + IGF
1
2
3
7,127
3,910
6,183
17,195
13,864
16,768
Lenses were cultured in KEI-4 containing EGF and IGF with both factors
at 8.8 X 10~10 M, or in KEI-4 containing EGF at 1.8 X 10"9 M (2 X EGF).
Lenses were fixed after 52 h of culture and the total number of mitotic figures
per whole mount preparation was counted.
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1983
Lenses cultured for 52 hrs in medium KEI-4 containing IGF at 1.8 X 10"9 M (n = 3) had a total of
4,794 ± 1,124 mitotic figures, which is substantially
below the value obtained for lenses cultured in the
presence of both mitogens at 8.8 X 10~10 M (Table
2). Whole mount preparations from lenses exposed
to both mitogens exhibited cellular disorganization
similar to that depicted in Figure 3.
Discussion
Earlier we demonstrated that insulin triggered cell
division in the cultured lens.5 Since receptors for certain of the somatomedins interact with insulin receptors, high levels of insulin could bring about physiologic changes most properly attributed to insulin
growth factors. The results presented here establish
that IGF, the most highly purified of the somatomedins has a direct mitogenic effect on mammalian
lens epithelial cells. The time course of DNA synthesis and mitosis in lenses exposed to IGF agrees
with that noted in lenses cultured in medium supplemented with insulin,5 serum,3 or thrombin.20
The IGF used was a mixture of IGF-I and IGF-II
and gives no indication of the relative mitogenicity
of each insulin-like growth factor. However, studies
in our laboratory indicate that IGF-I and IGF-II are
equipotent in stimulating mitosis in the rabbit lens
in organ culture, a situation that is also obtained in
lenses exposed to a relatively pure preparation of somatomedian C.' The mitotic response elicited by IGF
is dose dependent and exceeded that engendered by
equimolar insulin. The data suggest that IGF or other
insulin-like growth factors might represent the in vivo
analog of insulin. The activation of cell division occurred with concentrations of IGF that appear to be
within the physiologic range. As a point of reference,
the concentration of insulin-like growth factors in
human10 and rat serum21 is 300 and 180 ng/ml respectively. Relative to other model systems, IGF stimulates DNA synthesis in cultured chick embryo fibroblasts10 and induces proliferation and differentiation in rat myoblasts,22 Other reports indicate that
human embryofibroblasts23and other cultured mammalian cells24 do not undergo mitosis in response to
somatomedin C alone.25 These investigators conclude
that somatomedin C is not capable of triggering the
initial sequence of events leading to division in certain
mammalian cells, but is required for their progression
into DNA synthesis.25 Our report clearly establishes
that an insulin-like peptide can initiate cell division
in cultured mammalian cells.
Lenses cultured in KEI-4 supplemented with MSA,
a molecule closely related to IGF,21 did not show a
mitotic activation. In view of the high concentration
Vol. 24
of MSA in fetal rat serum relative to adult rat serum,
Moses et al21 suggested a role for MSA in fetal growth.
Among other possibilities, our results suggest that
MSA, a factor produced by fetal rat liver cells, is unable to trigger mitosis in rabbit lens epithelial cells,
or that the response of the lens to specific insulin-like
growth factors varies as a function of age. In this
respect, it is known that donor age is a crucial variable
for the establishment and growth of lens epithelial
cell lines.26 The influence of insulin-like growth factors, including MSA on lens epithelial cells from different aged animals is under investigation.
Relative to the control of cell division in the lens
in vivo, it is known that hypophysectomy abolishes
cell proliferation in the frog lens.27 The pituitary is
thought to facilitate growth by mediating the synthesis and/or release of insulin-like growth factors from
other tissues.13 The most salient finding implicating
insulin-like growth factor in the control of cell division in the lens in vivo is the demonstration that
administration of somatomedin C to the hypophysectomized frog restores cell proliferation in the lens.8
Whether somatomedin C acts directly or indirectly
on the amphibian lens in vivo, or singly or in concert
with other factors to mediate the initiation or progression of lens cells into mitosis is unknown.
Pituitary ablation brings about a slight decrease in
the growth of the rat lens.28 An increase in the number
of dividing cells and a concommitant decrease in the
number of colchicine arrested mitoses occurs in the
lens of the hypophysectomized rat.29 These findings
might indicate that insulin-like growth factors do not
influence cell division in the mammalian lens. However, the level of insulin-like growth factors in mammals is not determined solely by the pituitary but is
subject to regulation by both insulin and adequate
levels of nutrition.13 Although cultured mammalian
lens epithelia divide in response to insulin-like growth
factors, the role of these factors on the mammalian
lens in vivo remains to be demonstrated.
In other ocular tissues, several reports, summarized
by Reddan1 indicate that hypophysectomy, a procedure known to lower the plasma level of insulin-like
growth factors, results in an actual regression of new
blood vessel formation associated with diabetic retinopathy.
Fibroblast growth factor did not bring about a reproducible stimulation of mitosis in the cultured lens.
Other investigators reported that FGF is mitogenic
toward cultured lens epithelia when added to medium
containing fetal calf serum.17 The FGF used in this
investigation was mitogenic toward rabbit lens cells
cultured in a serum-containing medium.3
Epidermal growth factor stimulates DNA synthesis
and mitosis in lenses cultured in a serum-free me-
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No. 4
IGF AND EGF / Reddan and Wilson-Dziedzic
dium and in rabbit lens epithelia cultured in a medium supplemented with fetal calf serum.16 The values for EGF in mouse and human plasma are 1 and
2-4 ng/ml, respectively.30 The chronology of cell proliferation in EGF-treated lenses is identical to that
noted with IGF. Although EGF is a potent mitogen,
the physiologic significance of this peptide remains
to be established.30 In apparent contrast to the present
findings, Gospodarowicz et al17 reported that EGF
did not stimulate proliferation of cultured bovine or
rabbit corneal or lens epithelial cells, but did initiate
mitosis if the corneal epithelium was combined with
the corneal stroma. As shown in the present study,
the epithelial cells of the lens in organ culture,
wherein the cells lie immediately subjacent to their
natural basement membrane, entered mitosis upon
exposure to EGF. Rabbit lens cells cultured on laminin or fibronectin, components of the extracellular
matrix, increase in number in a serum-free medium
supplemented with EGF.131 These findings indicate
that the response of lens cells to mitogens can be
regulated in part by components of the extracellular
matrix.
In most culture systems, EGF must be present for
6 to 8 hrs to stimulate DNA synthesis.30 In the present
study, a 1-hr exposure of the lens to EGF or IGF
resulted in an increase in mitotic activity. It would
appear that the sequence of events triggered by EGF
is completed within the initial hour of culture. Additional studies using EGF antibodies should provide
information on these possibilities. Although a 1 hour
exposure of the lens to IGF resulted in an increase
in mitosis, the magnitude of the response was decidedly lower than that brought about by a 1-hr exposure
of the lens to EGF. The cellular mechanism responsible for this remains to be determined. Of additional
interest is the finding that simultaneous exposure of
the lens to IGF and EGF evoked a mitotic response
substantially greater than that obtained by either peptide alone. Since we have no evidence indicating that
the time on onset of DNA synthesis and mitosis is
different in the presence of both mitogens, the increase in the number of mitotic figures has been tentatively ascribed to an increase in the number of cells
that have been stimulated to enter the cell cycle. Since
the cells respond to more than one peptide, lens epithelia, like their counterparts in other tissues, may
be subject to multiple hormonal interactions.32
Through a judicious selection of growth factors and
substrata it should be possible to establish long-term
cultures of lens epithelial cells in a serum-free medium. Since a single peptide can trigger mitosis in
lenses cultured in a serum-free medium, this system
is suitable for determining the growth limiting components in the medium that act in concert with spe-
415
cific polypeptides to initiate the mitotic response.
Moreover, since growth factors appear to be cell type
specific,32 information obtained from rabbit lens epithelia should be useful in delineating the factors and
conditions required for the establishment of continuous lines of human lens epithelial cells. Such studies
should permit a further understanding of the extracellular and intracellular events regulating growth,
development, repair, and senescence in mammalian
lens epithelia.
Key words: lens, mitosis, epithelial cells, insulin growth factor, somatomedins, epidermal growth factor, organ culture,
senim-free medium
References
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16. Hollenberg MD: Receptors for insulin and epidermal growth
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20. Reddan JR, Dziedzic DC, and McGee SJ: Thrombin induces
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