Establishing a robust analysis workflow for studying hiPSC shapes in 3D lumenoids

Cells exhibit collective behaviors and diverse morphologies within 3D spatial configurations in morphogenesis. Toward a more complete understanding of these phenomena, we aim to establish robust analysis workflows to quantify cell shapes within 3D arrangements. Previously, we studied the morphology of human induced pluripotent stem cells (hiPSCs) grown in flat colonies with a natural basal-down/apical-up biological axis in the microscope coordinate system (MCS). We extend this work to hiPSC-derived 3D lumenoids that are hollow, closed-spheres with apical-inward/basal-outward polarity and use spherical harmonics to parameterize and study individual cell shapes. Shape registration is a critical step that precedes morphological analysis but remains subjective. Here, we assess how deliberate registration choices with respect to cell-intrinsic or colony-specific geometry may reveal biologically meaningful shape variation. For instance, we orient the 3D lumenoid cells along their apical–basal axis, aligning the basal surface normal with the negative z-direction of the MCS, and then examine the effects of enforcing positive shape skewness along the MCS x-axis. Spherical harmonic fitting and principal component analysis reveal that decisions made in shape alignment profoundly affect symmetry and physical interpretation of shape modes, underscoring the need for standardized and biologically appropriate registration in morphological analysis.