Purified putative stem cells are plated at very low density on specific substrates and their growth observed by living cell microscopy. In a physiological situation, asymmetric division prevails with only one of the two daughter cells prosecuting with multiple rounds of divisions . The transformed stem cell, as in the case of cells derived from the ErbB2 transgenic mice which express the activated ErbB2 oncogene in the mammary epithelium, abrogates asymmetric divisions preferring to give rise to multiple actively proliferating cells with self-renewal potential.
Asymmetric mitosis possesses a specific molecular signature characterized by a polarized three-dimensional redistribution of certain segregating determinants, proteins that are differentially partitioned between the daughter cells and contributing to determine their stem cell fate. The optical sectioning ability of a confocal microscope consequently becomes fundamental to reconstruct the volumetric localization of a polarity marker as shown in Figure 2. The extremely different signal distribution of the segregating determinant Numb in normal mammary (panel A) and cancer (panel B) stem cells confirmed that asymmetric division is lost upon transformation.
The tight link existing between stem cell fate and the physiological environment surrounding them (the stem cell niche) calls for imaging cells in living organisms or in fixed thick tissue slices maintaining the native histological architecture. As a consequence, two-photon microscopy is now massively entering the field. Nonlinear fluorescence excitation contribution could also be extremely relevant in in vitro studies. Mature mammosphere dimension can exceed 100 microns in thickness, representing a relevant challenge for conventional confocal imaging.