Umbilical cord-derived mesenchymal stem cells (UC-MSCs) were seeded at a density of 3 × 10⁵ cells per well in a commercially available fibronectin-coated 6-well plate. Before differentiation was induced, the cells displayed the cells exhibited a homogeneous distribution and morphology across the entire well surface.

Osteogenic differentiation was initiated by replacing the standard culture medium with osteogenic induction medium. Significantly, by two days of induction, morphological distinctions began to appear between the upper and lower sections of the wells. Cells in the upper region of the wells exhibited initial indicators of osteogenic differentiation, including alterations in morphology and matrix deposition, whereas cells in the bottom region maintained their original morphology and displayed no discernible evidence of differentiation over the observation period.

These data indicate regional variation in the differentiation response, despite the initially homogenous cell seeding and surface coating. Representative phase contrast images depicting the observed differences among the regions are presented (see attached).

And the question is, what could be the reason for this.

Thank you in advance!