Arrayed functional genetic screenings in pluripotency reprogramming and differentiation.


Rodrigo Alexandre Panepucci


Laboratory of Functional Biology (LFBio), Center for Cell-Based Therapy (CTC), Regional Blood Center of Ribeirão Preto, Rua Tenente Catão Roxo, 2501, Ribeirão Preto, SP, CEP: 14051-140, Brazil. [Email]


Thoroughly understanding the molecular mechanisms responsible for the biological properties of pluripotent stem cells, as well as for the processes involved in reprograming, differentiation, and transition between Naïve and Primed pluripotent states, is of great interest in basic and applied research. Although pluripotent cells have been extensively characterized in terms of their transcriptome and miRNome, a comprehensive understanding of how these gene products specifically impact their biology, depends on gain- or loss-of-function experimental approaches capable to systematically interrogate their function. We review all studies carried up to date that used arrayed screening approaches to explore the function of these genetic elements on those biological contexts, using focused or genome-wide genetic libraries. We further discuss the limitations and advantages of approaches based on assays with population-level primary readouts, derived from single-parameter plate readers, or cell-level primary readouts, obtained using multiparametric flow cytometry or quantitative fluorescence microscopy (i.e., high-content screening). Finally, we discuss technical limitation and future perspectives, highlighting how the integration of screening data may lead to major advances in the field of stem cell research and therapy.


Arrayed functional screenings,Cell differentiation,Cellular reprogramming,Flow cytometry,Fluorescence microscopy,High content screening,Human embryonic stem cells,Pluripotent stem cells,RNA interference,microRNA,