Preferred mitotic orientation in pattern formation by vascular mesenchymal cells

Area of Science:

• Cellular biology
• Developmental biology
• Tissue engineering

Background:

• Cellular self-organization is fundamental for developing physiological tissues and organs.
• Vascular mesenchymal cells, a multipotent subpopulation of aortic smooth muscle cells, exhibit self-organization into macroscopic, periodic patterns in culture.
• These patterns manifest as raised aggregates, specifically nodules or ridges.

Purpose of the Study:

• To investigate whether oriented cell division or postmitotic cell relocation drives the self-organization of vascular mesenchymal cells into macroscopic patterns.
• To analyze the orientation of cell divisions and cell movement during the self-organization process.

Main Methods:

• Time-lapse videomicrography (phase-contrast and fluorescence) was used to capture cell behavior during self-organization.
• Cell division events were analyzed for the orientation of daughter cells relative to local cell alignment.
• Statistical methods (histographic and bin-free) were employed to analyze the distribution of mitotic angles.
• Pharmacological inhibitors (blebbistatin, Rho kinase inhibitor, mitomycin C) were used to probe the roles of myosin II, stress fibers, and cell division.

Main Results:

• A statistically significant preferential orientation of daughter cells along the local cell alignment axis was observed as early as day 8, preceding aggregate formation.
• This alignment of mitotic axes remained significant during aggregate development (day 11) and after pattern completion (day 15).
• Blebbistatin treatment attenuated mitotic orientation alignment, while Rho kinase inhibition disrupted local cell alignment, indicating the involvement of stress fibers.
• Inhibition of cell division with mitomycin C reduced macroscopic pattern formation.
• Time-lapse monitoring revealed postmitotic migration of cells into neighboring aggregates.

Conclusions:

• Polarization of mitoses (oriented cell division) is a key contributor to the self-organization of vascular stem cells into periodic patterns.
• Postmitotic cell migration also plays a significant role in the formation of these macroscopic patterns.
• The findings elucidate the mechanisms underlying vascular stem cell self-organization, with implications for tissue engineering and regenerative medicine.