Resident angiogenic mesenchymal stem cells from multiorgan donor thoracic aortas

Stem cells are one of the most fascinating areas of biology today, and since the discover of an adult population, i.e., adult Stem Cells (aSCs), they have generated much interest especially for their application potential as a source for cell based regenerative medicine and tissue engineering. aSCs have been found in different tissues including bone marrow, skin, intestine, central nervous system, where they reside in a special microenviroment termed “niche” which regulate the homeostasis and repair of adult tissues. The arterial wall of the blood vessels is much more plastic than ever before believed. Several animal studies have demonstrated the presence of cells with stem cell characteristics within the adult vessels. Recently, it has been also hypothesized the presence of a “vasculogenic zone” in human adult arteries in which a complete hierarchy of resident stem cells and progenitors could be niched during lifetime. Accordingly, it can be speculated that in that location resident mesenchymal stem cells (MSCs) with the ability to differentiate in smooth muscle cells, surrounding pericytes and fibroblasts are present. The present research was aimed at identifying in situ and isolating MSCs from thoracic aortas of young and healthy heart-beating multiorgan donors. Immunohistochemistry performed on fresh and frozen human thoracic aortas demonstrated the presence of the vasculogenic zone between the media and the adventitial layers in which a well preserved plexus of CD34 positive cells was found. These cells expressed intensely HLA-I antigens both before and after cryopreservation and after 4 days of organ cultures remained viable. Following these preliminary results, we succeeded to isolate mesenchymal cells from multi-organ thoracic aortas using a mechanical and enzymatic combined procedure. Cells had phenotypic characteristics of MSC i.e., CD44+, CD90+, CD105+, CD166+, CD34low, CD45- and revealed a transcript expression of stem cell markers, e.g., OCT4, c-kit, BCRP-1, IL6 and BMI-1. As previously documented using bone marrow derived MSCs, resident vascular wall MSCs were able to differentiate in vitro into endothelial cells in the presence of low-serum supplemented with VEGF-A (50 ng/ml) for 7 days. Under the condition described above, cultured cells showed an increased expression of KDR and eNOS, down-regulation of the CD133 transcript, vWF expression as documented by flow cytometry, immunofluorescence, qPCR and TEM. Moreover, matrigel assay revealed that VEGF induced cells were able to form capillary-like structures within 6 hours of seeding. In summary, these findings indicate that thoracic aortas from heart-beating, multi-organ donors are highly suitable for obtaining MSCs with the ability to differentiate in vitro into endothelial cells. Even though their differentiating potential remains to be fully established, it is believed that their angiogenic ability could be a useful property for allogenic use. These cells can be expanded rapidly, providing numbers which are adequate for therapeutic neovascularization; furthermore they can be cryostored in appropriate cell banking facilities for later use.

Studio di popolazione sull'associazione tra sindrome delle gambe senza riposo ed emicrania

Introduzione: La sindrome delle gambe senza riposo (RLS) è un disturbo caratterizzato da sensazione spiacevole disestesica generalmente agli arti inferiori, che si presenta o peggiora nelle ore serali-notturne e che migliora con il movimento. Studi clinici hanno mostrato una maggiore prevalenza di RLS negli emicranici, mentre mancano studi condotti su popolazione generale non selezionata. Lo scopo di questo studio era quello di valutare la associazione tra emicrania e RLS in una popolazione italiana adulta. Inoltre è stata valutata l’associazione tra RLS e cefalea fenotipizzata attraverso metodica di principal components analysis (PCA). Materiali e metodi: la presenza di RLS e di emicrania è stata determinata attraverso questionari basati sui criteri diagnostici correnti in un campione di 1567 partecipanti di un fase preliminare di uno studio in corso sulla popolazione adulta della Val Venosta (BZ). Risultati: gli emicranici hanno presentato un significativo maggior rischio di soffrire di RLS rispetto ai non emicranici, anche dopo aggiustamento per fattori confondenti come età, sesso, depressione, ansia e qualità del sonno (p = 0.049). Questa associazione non era modificata dalla presenza di aura emicranica, di cause possibili di RLS secondaria e dalla frequenza di attacchi emicranici. Inoltre la RLS non era risultata significativamente associata alla cefalea di tipo tensivo (TTH). Dall’analisi di associazione tra RLS e cefalea fenotipizzata attraverso PCA era emerso che la componente 1, caratterizzata da sintomi di sensitivizzazione del sistema nervoso centrale (SNC), correlava significativamente con la presenza di RLS (p = 0.021). Conclusioni: RLS ed emicrania sono risultate associate nel nostro campione di popolazione adulta; inoltre la RLS ha mostrato una correlazione significativa con i sintomi di sensitivizzazione del SNC legati agli attacchi di cefalea. Questa associazione potrebbe risiedere in una possibile base patogenetica comune.

Wide-scale population genomics of Atlantic bluefin tuna (Thunnus thynnus) inferred by novel high-throughput technology

My PhD project was focused on Atlantic bluefin tuna, Thunnus thynnus, a fishery resource overexploited in the last decades. For a better management of stocks, it was necessary to improve scientific knowledge of this species and to develop novel tools to avoid collapse of this important commercial resource. To do this, we used new high throughput sequencing technologies, as Next Generation Sequencing (NGS), and markers linked to expressed genes, as SNPs (Single Nucleotide Polymorphisms). In this work we applied a combined approach: transcriptomic resources were used to build cDNA libreries from mRNA isolated by muscle, and genomic resources allowed to create a reference backbone for this species lacking of reference genome. All cDNA reads, obtained from mRNA, were mapped against this genome and, employing several bioinformatics tools and different restricted parameters, we achieved a set of contigs to detect SNPs. Once a final panel of 384 SNPs was developed, following the selection criteria, it was genotyped in 960 individuals of Atlantic bluefin tuna, including all size/age classes, from larvae to adults, collected from the entire range of the species. The analysis of obtained data was aimed to evaluate the genetic diversity and the population structure of Thunnus thynnus. We detect a low but significant signal of genetic differentiation among spawning samples, that can suggest the presence of three genetically separate reproduction areas. The adult samples resulted instead genetically undifferentiated between them and from the spawning populations, indicating a presence of panmictic population of adult bluefin tuna in the Mediterranean Sea, without different meta populations.

Assessment of the population structure and temporal changes in spatial dynamics and genetic characteristics of the Atlantic bluefin tuna under a fishery independent framework.

As a large and long-lived species with high economic value, restricted spawning areas and short spawning periods, the Atlantic bluefin tuna (BFT; Thunnus thynnus) is particularly susceptible to over-exploitation. Although BFT have been targeted by fisheries in the Mediterranean Sea for thousands of years, it has only been in these last decades that the exploitation rate has reached far beyond sustainable levels. An understanding of the population structure, spatial dynamics, exploitation rates and the environmental variables that affect BFT is crucial for the conservation of the species. The aims of this PhD project were 1) to assess the accuracy of larval identification methods, 2) determine the genetic structure of modern BFT populations, 3) assess the self-recruitment rate in the Gulf of Mexico and Mediterranean spawning areas, 4) estimate the immigration rate of BFT to feeding aggregations from the various spawning areas, and 5) develop tools capable of investigating the temporal stability of population structuring in the Mediterranean Sea. Several weaknesses in modern morphology-based taxonomy including demographic decline of expert taxonomists, flawed identification keys, reluctance of the taxonomic community to embrace advances in digital communications and a general scarcity of modern user-friendly materials are reviewed. Barcoding of scombrid larvae revealed important differences in the accuracy of the taxonomic identifications carried out by different ichthyoplanktologists following morphology-based methods. Using a Genotyping-by-Sequencing a panel of 95 SNPs was developed and used to characterize the population structuring of BFT and composition of adult feeding aggregations. Using novel molecular techniques, DNA was extracted from bluefin tuna vertebrae excavated from late iron age, ancient roman settlements Byzantine-era Constantinople and a 20th century collection. A second panel of 96 SNPs was developed to genotype historical and modern samples in order to elucidate changes in population structuring and allele frequencies of loci associated with selective traits.