The differences between freshly isolated stromal cells and their
subcultures with respect to competence for collagenase synthesis might
be due to their transformation in culture, similar to the
transformation they undergo in the corneal wound. However, because
primary stromal cells undergo many replications before subculture, it
was also possible that the change was due to selection for a
subcomponent of the cell population with a growth advantage under the
culture conditions. To distinguish between these possibilities, freshly
isolated cells were plated in replicate culture wells and their
phenotype was monitored daily to determine whether transformation could
occur without subculturing. Results of a representative experiment are
depicted in
Figure 2 and summarized in
Table 1 . Rhodamine isothiocyanate (RITC)-phalloidin
staining of cells in culture for 24 hours
(Fig. 2A) revealed a poorly
developed actin cytoskeleton with a wispy, weblike structure. However,
stress fibers had begun to develop in cells viewed 2 days after plating
(Fig. 2B) and were fully developed by day 3
(Fig. 2C) . The cells had
also become spindle-shaped by day 3, appearing identical with
subcultured cells (cf.
Fig. 2C and
1A ). No further changes in the actin
cytoskeleton were observed on day 4 (data not shown). Progress of these
cells toward competence for collagenase expression in response to CB
was scored by using immunofluorescent localization to identify
individual cells synthesizing collagenase and then by quantifying the
percentage of these cells in the total population
(Table 1) . Before day
three, none of the cells scored competent in this assay; however, on
day 3, 30% of the cell population exhibited competence. Because
intracellular collagenase concentrates within secretory cell
compartments, collagenase-positive cells could be identified easily by
their brightly fluorescent perinuclear spots
(Fig. 2F) . Besides the
change in cell shape, two additional changes were correlated with the
burst of competence acquisition. The first was in cell replication: the
number of cells in a culture well doubled daily through day 3, and then
replication ceased as cells became confluent. The second involved a
change in response to CB: day 1 and 2 cultures did not collapse in
response to CB
(Figs. 2D ,
2E) , even though actin filaments were
disrupted (cf.
Fig. 2B and
2E ). In contrast, treatment of day 3
cultures caused the collapse in cell-shape characteristic of fibroblast
response to CB
(Fig. 2F) . Nevertheless, the percentage of
collagenase-positive cells was still increasing on day 4
(Table 1) ,
indicating that these changes, even if necessary, are not sufficient.
Of more importance to our purposes was the rapidity of competence
acquisition in relation to the rate of cell replication. This reveals
that competence is not acquired by selection for a specific cell type.
In fact, competence acquisition does not require that cells even be
subcultured, but instead appears to be an individual response by each
cell to being placed in serum-containing culture.