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Decoding pluripotency: Genetic screens to interrogate the acquisition, maintenance, and exit of pluripotency

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Abstract Pluripotent stem cells have the ability to unlimitedly self‐renew and differentiate to any somatic cell lineage. A number of systems biology approaches have been used to define this pluripotent state. Complementary to systems level characterization, genetic screens offer a unique avenue to functionally interrogate the pluripotent state and identify the key players in pluripotency acquisition and maintenance, exit of pluripotency, and lineage differentiation. Here we review how genetic screens have helped us decode pluripotency regulation. We will summarize results from RNA interference (RNAi) based screens, discuss recent advances in CRISPR/Cas‐based genetic perturbation methods, and how these advances have made it possible to more comprehensively interrogate pluripotency and differentiation through genetic screens. Such investigations will not only provide a better understanding of this unique developmental state, but may enhance our ability to use pluripotent stem cells as an experimental model to study human development and disease progression. Functional interrogation of pluripotency also provides a valuable roadmap for utilizing genetic perturbation to gain systems level understanding of additional cellular states, from later stages of development to pathological disease states. This article is categorized under: Developmental Biology > Stem Cell Biology and Regeneration Developmental Biology > Developmental Processes in Health and Disease Biological Mechanisms > Cell Fates
Pluripotency and its regulation. Pluripotent stem cells have both the ability to self‐renew to maintain the pluripotent state and the potential to differentiate to all three somatic lineages (endoderm, mesoderm and ectoderm). During lineage differentiation, pluripotent stem cells undergo the process of exiting pluripotency in which they transition identity from pluripotent stem cells to a particular somatic cell lineage. Differentiated somatic cells can acquire pluripotency through the process of reprogramming by overexpression of pluripotency inducing factors. The state of pluripotency itself exists on a spectrum encompassing the naïve and primed states, which utilize different mechanisms to achieve self‐renewal and pluripotency maintenance
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Schematic of arrayed and pooled genetic screens. Reverse transfection/infection approach is well suited for high‐throughput arrayed screens. Synthetic siRNA, gRNA or lentiviral particles are pre‐distributed in 96/384‐well plates. After reagent reconstitution according to manufacturer's instruction, cells are distributed to each well by using an automatic liquid dispensing instrument and mixed with RNAi or gRNA reagents. A forward transfection/infection approach can be used as an alternative solution if cells need to be pre‐seeded before transfection/infection. After the transfection/infection step, cells are cultured with media for normal cell growth, drug selection or differentiation. The cellular phenotype of interest (overgrowth, death, differentiation) can be evaluated by high‐content imaging based on the quantitative signals from a reporter cell line (e.g., GFP) or immunostaining. In a typical pooled screen, a lentivirus shRNA or CRISPR gRNA library is first prepared. With a low multiplicity of infection, most of the infected cells will carry one shRNA or gRNA integration. The infected cells will then be subject to positive selection, negative selection, or marker selection. For positive and negative cell selection, the target population is the population that survives the selection, and the control population can be the infected cells before selection or cultured in parallel without selection. For marker selection, the target and control populations can be the population with or without the marker expression, respectively. Deep sequencing and sequence deconvolution can be performed to quantify the degree of enrichment or depletion of each shRNA or gRNA in the target population compared to the control population
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Biological Mechanisms > Cell Fates
Developmental Biology > Developmental Processes in Health and Disease
Developmental Biology > Stem Cell Biology and Regeneration

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