Biomedical Engineering Reference
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FIGURE 2.1: Phase contrast time-lapse microscopy of in vitro dissociation
and migration of ARO cells, grown in 10% fetal calf serum and incubated in
the presence of 100 U/mL of hepatocyte growth factor (HGF) for 2 h, 12 h,
18 h, and 24 h. The scale bar is 25 m long.
of local, contact-dependent signals (juxtracrine). In fact, we can hypothesize
that the loss of intercellular adhesions does not cause by itself a dissociation
phenotype, since E-cadherin -/- cells may still form strong adhesive junctions.
Instead, the loss of E-cadherin signaling, related to HGF downstream signal-
ing pathways (e.g., via BCL9-2, [43]), may be responsible for this behavior
[260, 324]. The above assumptions are validated also by the fact that in the
absence of HGF the transmission of the juxtracrine is recovered so that the
dissociated and disconnected cells have a tendency to get back together and
form new compact colonies (not shown).
Another scattering example is given by cultures of MLP-29, a strongly
HGF-responsive epithelial cell line established from mouse embryonic liver.
MLP-29 are small cells with ovoidal nuclei, forming tightly packed colonies.
After the addition of nanomolar concentration of HGF, the cells start to elon-
gate, migrate, and differentiate, leading the islands to assume the charac-
teristic stellar configuration within 1 day, as shown in Figure 2.2 (the full
time-lapse movie is available at calvino.polito.it/preziosi/MLPex.avi). This
morphogenic response is dose dependent and consists of three steps:
1. Within the first 6 h, the colony borders start roughening and most of
cellular junctions (but not all) start breaking or reorganizing.
2. In the following 6 h there is a cytoskeletal reorganization followed by an
asymmetric polarization and an active migration of cells, which lead to
the formation of some sprouts.
3. Within 24 h, at the end of the branching process, the colonies assume the
characteristic stellar shape, with sprouts formed by one or few elongated
cells that keep a thin adhesion contact with the rest of the island.
None of these events is observed in control medium or after stimulation with
other growth factors: in those cases, MLP-29 cells tend to form spherical and
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