The typical morphology of the maturing (differentiating) human
epithelium from which conditioned media were collected is demonstrated
in
Figure 1 . Photographs in
Figures 1A through 1E show
the development of the cultures from cells that had just attained
confluence to mature epithelium. Photographs of the differentiating
cultures were taken at the end of the 16-hour serum-starvation period,
and morphology did not change throughout the duration of the collection
periods. Longer periods of collection (more than 24 hours) resulted in
breakdown of epithelial cell–cell contacts and deterioration of
morphology (data not shown). By day 2
(Fig. 1A) the cultures started to
take on the appearance of the early stages of differentiation,
indicated by diminishing intercellular spaces (onset of gradual
reduction in bright halos between cell boundaries), and the onset of
cell enlargement in small areas. More patches of large differentiated
cells started to appear by day 7 (
Fig. 1B , arrow) and subsequently
increased throughout the cultures with time (
Fig. 1C , arrow). By day 22
areas of differentiated cells started to whorl
(Fig. 1D) in a manner
similar to that described during in vivo
re-epithelialization.
6 Conditioned medium was not
collected beyond 28 days when the majority of the visible cells had
taken on a large, fully differentiated morphology
(Fig. 1E) . However,
the cultures remained intact with good morphology until they were
discarded after 35 days. From approximately 14 days onward many cells
were sloughing from the maturing culture indicating a healthy
self-renewing epithelium. In addition, the photographs in
Figure 2B confirm that the number of human epithelial cells expressing
cytokeratin 3 increased with time, indicating differentiating
epithelium. Photographs in
Figures 2A 2C and 2E show negative
controls for cultures on days 2, 7, and 14, respectively. On day 2
(Fig. 2B) several areas contained enlarging cells with cytokeratin 3
expression (arrowhead). By day 7
(Fig. 2C) cells were sloughing from
the culture surface (arrowhead), and approximately 40% of the cells
were expressing cytokeratin 3
(Fig. 2D) . By day 14 sloughing continued
(Fig. 2E) , and the majority of the cells expressed cytokeratin 3
(Fig. 2F) .