- Mouse embryo cells cultured in vitro in serum-supplemented media undergo growth
crisis, resulting in the loss of genomically normal cells prior to the appearance of
established, aneuploid cell lines. I used the technique of serum-free cell culture to develop
a serum-free mouse embryo (SFME) cell line in which serum was replaced by a set of
defined supplements. SFME cells, cultured in a nutrient medium supplemented with
insulin, transferrin, epidermal growth factor (EGF), high-density lipoprotein (HDL), and
fibronectin, have maintained a diploid karyotype with no detectable chromosomal
abnormalities for more than 200 generations. The cells did not undergo growth crisis and
remain in culture today. SFME cells were dependent on EGF for survival and were
reversibly growth inhibited by serum or platelet-free plasma. Treatment of SFME cells
with serum or transforming growth factor beta led to the appearance of glial fibrillary acid
protein (GFAP), a specific marker for astrocytes, identifying SFME cells as proastroblasts.
Following the derivation of SFME cells my research focussed on (1) defining more
precisely the growth response of SFME cells to medium supplements, (2) investigating the
relationship between the nonsenescent nature of SFME cells and their responses to serum
and EG1., and (3) applying the serum-free cell culture methods to the multipassage culture
of human embryonic astrocytes.
SFME cells in serum-containing medium arrested in the G1 phase of the cell cycle
with greatly reduced DNA replication activity. A portion of the inhibitory activity of serum
was extracted by charcoal, a procedure that removed steroid and thyroid hormones.
However, the effect of serum on untransformed SFME cells could not be prevented by
addition of antiglucocorticoid, and ras-transformed clones of SFME cells, which are not
growth inhibited by serum, retained inhibitory responses to glucocorticoid and thyroid
hormone T3. These results suggest that glucocorticoid or thyroid hormones may contribute
to the inhibitory activity of serum on SFME cells, but additional factors are involved.
SFME cell death resulting from EGF deprivation exhibited characteristics associated
with apoptosis or programmed cell death. Ultrastructural analysis showed cells became
small and vacuolated, with pyknotic nuclei. The cultures contained almost exclusively G1-
phase cells. Chromatin exhibited a pattern of degradation into oligonucleosome-length
fragments generating a regularly spaced ladder.
I applied the serum-free approach used to derive SFME cells to the multipassage
culture of human embryonic astrocytes. Cells were cultured in nutrient medium
supplemented with insulin, transferrin, EGF, HDL, fibronectin, basic fibroblast growth
factor and heparin. Cultures were maintained for a maximum of 70 population doublings
before proliferation ceased. The cells synthesized GFAP.