BackgroundProgressive diversification of paralogs after gene expansion is essential to increase their functional specialization. However, mode and tempo of this divergence remain mostly unclear. Here we report the comparative analysis of PRDM genes, a family of putative transcriptional regulators involved in human tumorigenesis.ResultsOur analysis assessed that the PRDM genes originated in metazoans, expanded in vertebrates and further duplicated in primates. We experimentally showed that fast-evolving paralogs are poorly expressed, and that the most recent duplicates, such as primate-specific PRDM7, acquire tissue-specificity. PRDM7 underwent major structural rearrangements that decreased the number of encoded Zn-Fingers and modified gene splicing. Through internal duplication and activation of a non-canonical splice site (GC-AG), PRDM7 can acquire a novel intron. We also detected an alternative isoform that can retain the intron in the mature transcript and that is predominantly expressed in human melanocytes.ConclusionOur findings show that (a) molecular evolution of paralogs correlates with their expression pattern; (b) gene diversification is obtained through massive genomic rearrangements; and (c) splicing modification contributes to the functional specialization of novel genes.

Mesenchymal stem cells (MSCs) are adult multipotent cells currently employed in several clinical trials due to their immunomodulating, angiogenic and repairing features. The adipose tissue is certainly considered an eligible source of MSCs. Recently, putative adipose tissue derived MSCs (ADMSCs) have been isolated from the mediastinal depots. However, very little is known about the properties, the function and the potential of human mediastinal ADMSCs (hmADMSCs). However, the lack of standardized methodologies to culture ADMSCs prevents comparison across. Herein for the first time, we report a detailed step by step description to optimize the isolation and the expansion methodology of hmADMSCs using a virally inactivated good manufacturing practice (GMP)-grade platelet lysate, highlighting the critical aspects of the procedure and providing useful troubleshooting suggestions. Our approach offers a reproducible system which could provide standardization across laboratories. Moreover, our system is time and cost effective, and it can provide a reproducible source of adipose stem cells to enable future studies to unravel new insights regard this promising stem cell population.

Cellular senescence has been widely recognized as a tumor suppressing mechanism that acts as a barrier to cancer development after oncogenic stimuli. A prominent in vivo model of the senescence barrier is represented by nevi, which are composed of melanocytes that, after an initial phase of proliferation induced by activated oncogenes (most commonly BRAF), are blocked in a state of cellular senescence. Transformation to melanoma occurs when genes involved in controlling senescence are mutated or silenced and cells reacquire the capacity to proliferate. Pirin (PIR) is a highly conserved nuclear protein that likely functions as a transcriptional regulator whose expression levels are altered in different types of tumors. We analyzed the expression pattern of PIR in adult human tissues and found that it is expressed in melanocytes and has a complex pattern of regulation in nevi and melanoma: it is rarely detected in mature nevi, but is expressed at high levels in a subset of melanomas. Loss of function and overexpression experiments in normal and transformed melanocytic cells revealed that PIR is involved in the negative control of cellular senescence and that its expression is necessary to overcome the senescence barrier. Our results suggest that PIR may have a relevant role in melanoma progression.

Good manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon's fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2-3 cm(2) undamaged Tenon's biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon's fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon's and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes.

Nowadays, cardiac regenerative medicine is facing many limitations because of the complexity to find the most suitable stem cell source and to understand the regenerative mechanisms involved. Mesenchymal stem cells (MSCs) have shown great regenerative potential due to their intrinsic properties and ability to restore cardiac functionality, directly by transdifferentiation and indirectly by paracrine effects. Yet, how MSCs could respond to definite cardiac-committing microenvironments, such as that created by resident cardiac progenitor cells in the form of cardiospheres (CSs), has never been addressed. Recently, a putative MSC pool has been described in the mediastinal fat (hmADMSCs), but both its biology and function remain hitherto unexplored. Accordingly, we investigated the potential of hmADMSCs to be committed toward a cardiovascular lineage after preconditioning with CS-conditioned media (CCM). Results indicated that CCM affects cell proliferation. Gene expression levels of multiple cardiovascular and stemness markers (MHC, KDR, Nkx2.5, Thy-1, c-kit, SMA) are significantly modulated, and the percentage of hmADMSCs preconditioned with CCM and positive for Nkx2.5, MHC, and KDR is significantly higher relative to FBS and explant-derived cell conditioned media (EDCM, the unselected stage before CS formation). Growth factor-specific and survival signaling pathways (i.e., Erk1/2, Akt, p38, mTOR, p53) present in CCM are all equally regulated. Nonetheless, earlier BAD phosphorylation (Ser112) occurs associated with the CS microenvironment (and to a lesser extent to EDCM), whereas faster phosphorylation of PRAS40 in FBS, and of Akt (Ser473) in EDCM and 5-azacytidine occurs compared to CCM. For the first time, we demonstrated that the MSC pool held in the mediastinal fat is adequately plastic to partially differentiate in vitro toward a cardiac-like lineage. Besides, we have provided novel evidence of the potent inductive niche-like microenvironment that the CS structure can reproduce in vitro. hmADMSCs can represent an interesting tool in order to exploit their possible role in cardiovascular diseases and treatment.