Cell jamming, stratification and p63 expression in cultivated human corneal epithelial cell sheets

Abstract

Corneal limbal epithelial stem cell transplantation using cultivated human corneal epithelial cell sheets has been used successfully to treat limbal stem cell deficiencies. Here we report an investigation into the quality of cultivated human corneal epithelial cell sheets using time-lapse imaging of the cell culture process every 20 minutes over 14 days to ascertain the level of cell jamming, a phenomenon in which cells become smaller, more rounded and less actively expansive. In parallel, we also assessed the expression of p63, an important corneal epithelial stem cell marker. The occurrence of cell jamming was variable and transient, but was invariably associated with a thickening and stratification of the cell sheet. p63 was present in all expanding cell sheets in the first 9 days of culture, but it's presence did not always correlate with stratification of the cell sheet. Nor did p63 expression necessarily persist in stratified cell sheets. An assessment of cell jamming, therefore, can shed significant light on the quality and regenerative potential of cultivated human corneal epithelial cell sheets.

In airway epithelium, basal stem cells and their stress fibers remodel during the unjamming transition

“…We speculate that basal cells may have been extruded during UJT. Or these basal stem cells may be differentiating to replace any loss of apical cells (through cell death or extrusion) (Baba et al, 2020; Gudipaty and Rosenblatt, 2017; Kretschmer et al, 2017; Tadokoro et al, 2016). In monolayer cell culture, increasing cell density has been shown to decrease the speed of collective cellular migration (Angelini et al, 2011; Garcia et al, 2015; Loza et al, 2016; Saraswathibhatla and Notbohm, 2020; Tambe et al, 2011; Tlili et al, 2018).…”

Label-Free Quality Control and Identification of Human Keratinocyte Stem Cells by Deep Learning-Based Automated Cell Tracking

“…Furthermore, the trained SSD model can be easily utilized for automated nuclear recognition of other types of cells with “transfer learning.” This system can also be applied to other types of cell cultures with a smaller number of image data. Collective cell motion is observed in various cells, 38 including neural progenitor cells, 39 and human keratinocytes in epidermal 40 and corneal sheets 41 . Furthermore, cell motility is associated with the state of human embryonic stem cells (hESCs) 42 and human induced pluripotent stem cells (hiPSCs), 43 which strongly suggests that automated cell motion analysis can be also applied to quality control of human pluripotent stem cell cultures.…”

“…Collective cell motion is observed in various cells, 38 including neural progenitor cells, 39 and human keratinocytes in epidermal 40 and corneal sheets. 41 Furthermore, cell motility is associated with the state of human embryonic stem cells (hESCs) 42 and human induced pluripotent stem cells (hiPSCs), 43 which strongly suggests that automated cell motion analysis can be also applied to quality control of human pluripotent stem cell cultures. Thus, the DeepACT technology can be widely used for stem cell research and cell manufacturing for regenerative medicine.…”

Corneal Epithelial Stem Cells–Physiology, Pathophysiology and Therapeutic Options

“…The p63β and p63γ isoforms, which are absent in the resting limbus, are inclined to participate in epithelial differentiation during corneal regeneration [ 60 ]. Some scientists suggest that evaluation of p63 in cultivated limbal epithelial cell sheets is vital for assessing and selecting the quality of the cell sheet [ 61 ]. In addition, p63 knockout mice lack all stratified squamous epithelia and do not express differentiation markers [ 62 ].…”