The goal of the present study is to identify the differential expression of circular RNA (circRNA), miRNA, and piwi-interacting RNA (piRNA) after lineage commitment towards osteo- and chondrogenesis of human bone marrow mesenchymal stromal cells (hMSCs). The cells were maintained for 7 days in either osteogenic or chondrogenic medium. RNA sequencing was performed to assess the expression of miRNA and piRNA, while RNA hybridization arrays were used to identify which circRNA were differentially expressed. qPCR validation of a selection of targets for both osteogenic and chondrogenic differentiation was carried out. The differential expression of several circRNA, miRNA, and piRNA was identified and validated. The expression of total and circular isoforms of FKBP5 was upregulated both in osteo- and chondrogenesis and it was influenced by the presence of dexamethasone. ZEB1, FADS2, and SMYD3 were also identified as regulated in differentiation and/or by dexamethasone. In conclusion, we have identified a set of different non-coding RNAs that are differentially regulated in early osteogenic and chondrogenic differentiation, paving the way for further investigation to understand how dexamethasone controls the expression of those genes and what their function is in MSC differentiation.

Adipose-derived stem cells (ADSCs) represent one of the cellular populations resident in adipose tissue. They can be recruited under certain stimuli and committed to become preadipocytes, and then mature adipocytes. Controlling stem cell differentiation towards the adipogenic phenotype could have a great impact on future drug development aimed at counteracting fat depots. Stem cell commitment can be influenced by different molecules, such as melatonin, which we have previously shown to be an osteogenic inducer. Here, we aimed at evaluating the effects elicited by melatonin, even in the presence of vitamin D, on ADSC adipogenesis assessed in a specific medium. The transcription of specific adipogenesis orchestrating genes, such as aP2, peroxisome proliferator-activated receptor γ (PPAR-γ), and that of adipocyte-specific genes, including lipoprotein lipase (LPL) and acyl-CoA thioesterase 2 (ACOT2), was significantly inhibited in cells that had been treated in the presence of melatonin and vitamin D, alone or in combination. Protein content and lipid accumulation confirmed a reduction in adipogenesis in ADSCs that had been grown in adipogenic conditions, but in the presence of melatonin and/or vitamin D. Our findings indicate the role of melatonin and vitamin D in deciding stem cell fate, and disclose novel therapeutic approaches against fat depots.

Despite the huge body of research on osteogenic differentiation and bone tissue engineering, the translation potential of in vitro results still does not match the effort employed. One reason might be that the protocols used for in vitro research have inherent pitfalls. The synthetic glucocorticoid dexamethasone is commonly used in protocols for trilineage differentiation of human bone marrow mesenchymal stromal cells (hBMSCs). However, in the case of osteogenic commitment, dexamethasone has the main pitfall of inhibiting terminal osteoblast differentiation, and its pro-adipogenic effect is well known. In this work, we aimed to clarify the role of dexamethasone in the osteogenesis of hBMSCs, with a particular focus on off-target differentiation. The results showed that dexamethasone does induce osteogenic differentiation by inhibiting SOX9 expression, but not directly through RUNX2 upregulation as it is commonly thought. Rather, PPARG is concomitantly and strongly upregulated, leading to the formation of adipocyte-like cells within osteogenic cultures. Limiting the exposure to dexamethasone to the first week of differentiation did not affect the mineralization potential. Gene expression levels of RUNX2, SOX9, and PPARG were simulated using approximate Bayesian computation based on a simplified theoretical model, which was able to reproduce the observed experimental trends but with a different range of responses, indicating that other factors should be integrated to fully understand how dexamethasone influences cell fate. In summary, this work provides evidence that current in vitro differentiation protocols based on dexamethasone do not represent a good model, and further research is warranted in this field.

Decline in the gene expression of senescence repressor Bmi1 and telomerase, together with telomere shortening, underlay senescence of stem cells cultured for multiple passages. Here, we investigated whether the impairment of senescence preventing mechanisms can be efficiently counteracted by exposure of human adipose-derived stem cells to radio electric asymmetrically conveyed fields by an innovative technology, named Radio Electric Asymmetric Conveyer (REAC). Due to REAC exposure, the number of stem cells positively stained for senescence associated β-galactosidase was significantly reduced along multiple culturing passages. After a 90-day culture, REAC-treated cells exhibited significantly higher transcription of Bmi1 and enhanced expression of other stem cell pluripotency genes and related proteins, compared to unexposed cells. Transcription of the catalytic telomerase subunit (TERT) was also increased in REAC-treated cells at all passages. Moreover, while telomere shortening occurred at early passages in both REAC-treated and untreated cells, a significant rescue of telomere length could be observed at late passages only in REAC-exposed cells. Thus, REAC-asymmetrically conveyed radio electric fields acted on a gene and protein expression program of both telomerase-independent and telomerase-dependent patterning to optimize stem cell ability to cope with senescence progression.

In this review, we described different factors that modulate pluripotency in stem cells, in particular we aimed at following the steps of two large families of miRNAs: the miR-200 family and the miR-302 family. We analyzed some factors tuning stem cells behavior as TGF-β, which plays a pivotal role in pluripotency inhibition together with specific miRNAs, reactive oxygen species (ROS), but also hypoxia, and physical stimuli, such as ad hoc conveyed electromagnetic fields. TGF-β plays a crucial role in the suppression of pluripotency thus influencing the achievement of a specific phenotype. ROS concentration can modulate TGF-β activation that in turns down regulates miR-200 and miR-302. These two miRNAs are usually requested to maintain pluripotency, while they are down-regulated during the acquirement of a specific cellular phenotype. Moreover, also physical stimuli, such as extremely-low frequency electromagnetic fields or high-frequency electromagnetic fields conveyed with a radioelectric asymmetric conveyer (REAC), and hypoxia can deeply influence stem cell behavior by inducing the appearance of specific phenotypes, as well as a direct reprogramming of somatic cells. Unraveling the molecular mechanisms underlying the complex interplay between externally applied stimuli and epigenetic events could disclose novel target molecules to commit stem cell fate.

Research for the use of physical means, in order to induce cell differentiation for new therapeutic strategies, is one of the most interesting challenges in the field of regenerative medicine, and then in the treatment of neurodegenerative diseases, Parkinson's disease (PD) included. The aim of this work is to verify the effect of the radio electric asymmetric conveyer (REAC) technology on the PC12 rat adrenal pheochromocytoma cell line, as they display metabolic features of PD. PC12 cells were cultured with a REAC regenerative tissue optimization treatment (TO-RGN) for a period ranging between 24 and 192 hours. Gene expression analysis of specific neurogenic genes, as neurogenin-1, beta3-tubulin and Nerve growth factor, together with the immunostaining analysis of the specific neuronal protein beta3-tubulin and tyrosine hydroxylase, shows that the number of cells committed toward the neurogenic phenotype was significantly higher in REAC treated cultures, as compared to control untreated cells. Moreover, MTT and Trypan blue proliferation assays highlighted that cell proliferation was significantly reduced in REAC TO-RGN treated cells. These results open new perspectives in neurodegenerative diseases treatment, particularly in PD. Further studies will be needed to better address the therapeutic potential of the REAC technology.