We also observed the alteration of

We also observed the alteration of proteomic networks related to the apoptotic and proliferative pathways. Senescence and apoptosis are essential mechanisms for ionomycin to respond to the changing environment. Apoptosis involves intricate proteomic networks and Table 7 of S1 in Ref. Mindaye et al., (submitted for publication) summarizes modulated proteins in hBMSCs that are linked in prior studies with apoptosis (IKDB). Some of these are pro- and others are anti-apoptotic proteins. Stress-related heat shock proteins have been markedly reduced at P7, observations consistent with expectations (Madeira et al., 2012). GAPDH is another important pro-apoptotic protein whose expression level almost quadrupled at P7. During apoptosis, its expression and mitochondrial accumulation increases and this leads to increased membrane permeability with subsequent release of pro-apoptotic proteins (Tarze et al., 2007). Based on systematic IPA analysis, apoptosis has an overall activation z score of 1.6 (the fold change that this biological function increased at P7 as compared to P3). Other cellular events with similar biological tendencies are cell death and necrosis, each with activation z-scores of 2.1 and 1.8, respectively. Moreover, proteins involved in cell growth and proliferation have also experienced passage-dependent expression patterns (p value 6.31E−8). The overall decline in proliferation capacity has −0.60 z-score, indicating P7 cells become somewhat less proliferative than P3. These results correlate with the significant reduction in the frequency of CFUs or increase in time to 80% confluence of all hBMSC samples with passaging except 8F3560, which had fairly constant CFUs across passaging (Lo Surdo et al., 2013). According to the IKDB, some of the proliferation-associated proteins are pro-proliferative (e.g., nucleolin); however, most are anti-proliferative (e.g., galectin 1 and annexin 1), and the effect on proliferation for some proteins is cell context-dependent (e.g., annexin 2). Taken together, hBMSC-based therapy and research depend highly on the isolation and manufacturing of cells with predictable biological functions in large numbers. We extensively explored the underpinning molecular factors that influence the age-associated phenotype changes in hBMSCs. Using a robust label-free quantitative proteomic approach, the study mapped the expression changes associated with long-term passaging in five hBMSC cultures derived from different human donors. Over 1700 proteins were quantified at three passages and a differentially expressed protein list was identified. Bioinformatic-based network analysis and term enrichment helped to identify altered biological functions. We documented that culturing stimulates extensive proteomic alterations in functional categories including apoptosis, ER-based protein processing and sorting, and metabolic pathways. Identification of these affected biological functions together with the underlying molecular networks tremendously benefit the effort to uncovering targets that are not just used to monitor cell fitness but that can also be employed to slowdown the in vitro aging process in hBMSCs and hence ensure manufacturing of cells with known quality, efficacy and stability.
Author contributions
Introduction Two hypotheses have been proposed to explain how stem cells maintain the corneal epithelium. The limbal epithelial stem cell (LESC) hypothesis proposes that all the stem cells are in the basal epithelial layer of the limbus, which lies between the cornea and conjunctiva (Schermer et al., 1986). The LESCs remain in the limbus, where they replace themselves and generate transient (or transit) amplifying cells (TACs) in the basal layer. The TACs divide, move into the cornea and then move centripetally across the corneal radius. They also produce more differentiated cells, which do not divide but leave the basal layer, move vertically through the suprabasal layers and are shed from the surface.