Santaniello, Sara (2013) Studio dei meccanismi colecolari che regolano la crescita e la plasticità di cellule staminali: identificazione di nuove strategie molecolari di riprogrammazione di cellule umane adulte a scopo terapeutico. Doctoral Thesis.
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Aim: Cellular reprogramming of somatic cells into induced pluripotent stem cells (iPS) and subsequent differentiation to mature cells, has opened up new possibilities for tissue regeneration and repair. In the present work we exposed mouse embryonic stem cells (ESC) and human skin-derived fibroblasts (hSF) to a Radio Electric Asymmetric Conveyer (REAC), an innovative device delivering radio electric conveyed fields of 2.4 GHz. We than evaluated the effect of this stimulus on cellular reprogramming of mouse embryonic stem cells and hSF toward a pluripotent state or different mature cell types.
Methods: We analyzed the expression of genes controlling stem cell pluripotency and genes orchestrating cell commitment toward specific phenotypes by real-time PCR. We than evaluated also the appearance of specific tissue-restricted marker proteins by immunofluorescence and western blot analysis.
Results: In ESC REAC primed the transcription of genes involved in cardiac, skeletal muscle and neuronal commitment, followed by an enhanced expression of the corresponding lineage-restricted marker proteins, while contemporary down-regulating the self renewal/pluripotency-associated genes Sox2, Oct4 and Nanog. In hSF REAC treatment elicited a biphasic effect on a number of stemness-related genes, leading to early transcriptional increase of Oct4, Sox2, cMyc, Nanog, and Klf4 within 6-20 hours, eliciting a persistent reprogramming towards an induced pluripotent stem cell-like state. On the other hand stimulation hSF for longer periods (24-72 hours) induced the transcription of tissue-restricted genes.
Conclusion: The REAC stimulation provided a “physical milieu” optimizing expression of pluripotentiality and the attainment of three major target lineages for regenerative medicine, without using chemical agonists or vector-mediated gene delivery.
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