52 Autofluorescence and metabolic signatures in a pig model of differentiation based on induced pluripotent cells and embryonic bodies A.C. Croce, 1 G. Bottiroli, 1 G. Santin, 1 G. Pacchiana, 2 P. Vezzoni, 2 E. Di Pasquale 2 1 IGM-CNR and Department of Biology and Biotechnology, University Pavia, Pavia 27100, Italy 2 Humanitas Clinical and Research Center and IRGB/CNR, UOS of Milan, Rozzano 20089 (MI), Italy Corresponding author: Anna Cleta Croce Istituto di Genetica Molecolare del CNR c/o Dipartimento di Biologia e Biotecnologie “L. Spallanzani”, via Ferrata, 9 27100, Pavia, Italy Tel. +39.0382.986428 - Fax +39.0382.986430 E-mail: croce@igm.cnr.it Summary Besides several genetic and molecular markers, stem cells undergoing differentiation show changes in their metabolic engagement, such as a switch from anaerobic to aerobic energetic metabolism and decreased autophagic activity, representing metabolic sig- natures of the stemness status. These activities involve biomolecules - respectively NAD(P)H and flavins, and lipofuscin-like lipopigments- that give rise to autofluorescence (AF) emission under suitable excitation light conditions. Since these endogenous fluorophores can be exploited as cell intrinsic biomarkers, attention is currently deserved to assess the potential of AF analysis to monitor the cell stemness status or maturation degree into a desired phenotype. In our work, we employed pig induced pluripotent stem (iPS) cell as a model of differentiation to compare their AF properties in living conditions with those of embryoid bodies (EBs) derived from them, by means of imaging, microspectrofluorometric techniques and spectral fitting analysis. The AF results indicate that reprogramming of suine iPS cells do not lead to typical metabolic functions of the undifferentiated status. These findings sup- port the value of AF analysis as a tool for a comprehensive assessment of the actual stem cells differentiation degree in the devel- opment of regenerative medicine strategies, and for application to animal species of interest in veterinary, zootechnology and, in general, to the species for which immunomarkers are not yet available. Key words: aerobic/anaerobic metabolism, lipofuscin-like lipopigments, microspectrofluorometry, stem cells. Introduction The natural presence of biomolecules acting as endogenous fluorophores is responsible for the autofluorescence (AF) emission rising from cells and tissues when submitted to a suitable excita- tion light. The strict involvement of endogenous fluorophores in metabolic reactions influences their amount and physicochemical state, in turn affecting their emission properties. As a conse- quence, the overall cell AF emission signal carries multiple information, making endogenous fluoro- phores to act as intrinsic biomarkers of the cell functional engagement in the absence of exoge- nous labeling agents (Ramanujam, 2000; Bottiroli and Croce, 2004). Besides several genetic and molecular biomar- kers, cells undergoing differentiation or repro- gramming processes are characterized by a meta- bolic plasticity based on activities involving endo- genous fluorophores and likely affecting AF emis- sion properties (Folmes et al., 2012; Santin et al., 2013) Figure 1. Mitochondria, in particular, play an essential role in the maintenance of stem cell homeostasis, consistently both with the switch from anaerobic to aerobic respiration during diffe- rentiation and with the autophagic processes, pro- viding for the removal of undesired cell compo- nents to maintain the pluripotent status, the oppo- site occurring during the somatic to pluripotent cell reprogramming (Hernebring et al., 2006; Wang et al., 2011; Rehman et al., 2010; Chen et al., 2012). The consequent changes in the cell engagement in energetic metabolism influence the amount, redox state and bound-free condition of the coenzymes NAD(P)H and flavins (Croce et al., 1999; Salmon et al., 1982; Kunz et al., 1986). The AF emission properties of these endogenous fluorophores up CONTRIBUTI SCIENTIFICI CONTRIBUTI SCIENTIFICI microscopie Marzo 2014