Ilaria Bronzini

Canine adipose-derived-mesenchymal stem cells do not lose stem features after a long-term cryopreservation.

Adult stem cells are nowadays used for treating several pathologies. A putative stem cell population was found in the adipose tissue of mammals and canine adipose tissue-derived-mesenchymal stem cells (cA-MSC) have been shown to possess the capacity to differentiate into several lineages. The main goal of our research was to fully characterize cA-MSC and examine the effects of cryopreservation on their stemness features. Each sample of cA-MSC was analyzed immediately and then again after being frozen in liquid nitrogen for one year. After the cryopreservation period cells conserved their fibroblast-like morphology, alkaline phosphatase positivity and CD expression but showed a lower proliferation ratio and a lower telomerase activity in comparison with fresh cells. Finally, the cryopreservation protocol did not change the cA-MSC adipogenic, osteogenic and myogenic differentiative potential. Our data demonstrate that stored cA-MSC might represent a promising type of progenitor cell for autologous cellular-based therapies in veterinary medicine.

Successful recellularization of human tendon scaffolds using adipose-derived mesenchymal stem cells and collagen gel.

The major goal of regenerative medicine is to determine experimental techniques that take maximal advantage of reparative processes that occur naturally in the animal body. Injection of mesenchymal stem cells into the core of a damaged tendon represents such an approach. Decellularization of native tendons as potential targets and seeding protocols are currently under investigation. The aim of our study was to manufacture a recellularized biocompatible scaffold from cadaveric tissue for use in total or partial tendon injuries. Results showed that it was possible to introduce proliferating cells into the core of a decellularized tendon to treat the scaffold with a collagen gel. The method was effective in maintaining scaffold extracellular matrix and for expressing collagen type I and cartilage oligomeric matrix protein by injecting mesenchymal stem cells. Copyright

Effects of in vivo applications of peripheral blood-derived mesenchymal stromal cells (PB-MSCs) and platlet-rich plasma (PRP) on experimentally injured deep digital flexor tendons of sheep.

Tendon injuries, degenerative tendinopathies, and overuse tendinitis are common in races horses. Novel therapies aim to restore tendon functionality by means of cell‐based therapy, growth factor delivery, and tissue engineering approaches. This study examined the use of autologous mesenchymal stromal cells derived from peripheral blood (PB‐MSCs), platelet‐rich plasma (PRP) and a combination of both for ameliorating experimental lesions on deep digital flexor tendons (DDFT) of Bergamasca sheep. In particular, testing the combination of blood‐derived MSCs and PRP in an experimental animal model represents one of the few studies exploring a putative synergistic action of these treatments. Effectiveness of treatments was evaluated at 30 and 120 days comparing clinical, ultrasonographic, and histological features together with immunohistochemical expression of collagen types 1 and 3, and cartilage oligomeric matrix protein (COMP). Significant differences were found between treated groups and their corresponding controls (placebo) regarding tendon morphology and extracellular matrix (ECM) composition. However, our results indicate that the combined use of PRP and MSCs did not produce an additive or synergistic regenerative response and highlighted the predominant effect of MSCs on tendon healing, enhanced tissue remodeling and improved structural organization.

Influence of temperature, time and different media on mesenchymal stromal cells shipped for clinical application.

Cell-based therapies, such as the use of mesenchymal stromal cells (MSCs), are becoming popular in veterinary medicine. When MSCs are not cryopreserved, they are shipped in suspension, but no previous studies have analyzed MSC viability during delivery. Here, the impact of several experimental shipping conditions on the number of equine blood-derived (ePB-MSC) and canine adipose-derived (cA-MSC) MSCs were evaluated. Among the different parameters tested, only time and temperature influenced MSC number during the experimental shipping conditions. Cells were monitored over different time intervals for gene expression of typical MSC markers and to evaluate acquired resistance to apoptosis and beta-galactosidase activity. Overall, these results indicate that ePB-MSC and cA-MSC should be delivered in phosphate buffered saline at room temperature and within 9-12 h.

Refinement of IKZF1 status in pediatric Philadelphia-positive acute lymphoblastic leukemia

Refinement of IKZF1 status in pediatric Philadelphia-positive acute lymphoblastic leukemia

Fine tuning of surface CRLF2 expression and its associated signaling profile in childhood B-cell precursor acute lymphoblastic leukemia

Genomic rearrangements of the cytokine receptor-like factor 2 (CRLF2) gene,[1][1],[2][2] which is part of the thymic stromal lymphopoietin receptor (TSLPR), result in overexpression of CRLF2 itself leading to JAK2-mediated activation of STAT5, which regulates cell proliferation, survival, and

DNA methylation and targeted sequencing of methyltransferases family genes in canine acute myeloid leukaemia, modelling human myeloid leukaemia

Tumours shows aberrant DNA methylation patterns, being hypermethylated or hypomethylated compared with normal tissues. In human acute myeloid leukaemia (hAML) mutations in DNA methyltransferase (DNMT3A) are associated to a more aggressive tumour behaviour. As AML is lethal in dogs, we defined global DNA methylation content, and screened the C-terminal domain of DNMT3 family of genes for sequence variants in 39 canine acute myeloid leukaemia (cAML) cases. A heterogeneous pattern of DNA methylation was found among cAML samples, with subsets of cases being hypermethylated or hypomethylated compared with healthy controls; four recurrent single nucleotide variations (SNVs) were found in DNMT3L gene. Although SNVs were not directly correlated to whole genome DNA methylation levels, all hypomethylated cAML cases were homozygous for the deleterious mutation at p.Arg222Trp. This study contributes to understand genetic modifications of cAML, leading up to studies that will elucidate the role of methylome alterations in the pathogenesis of AML in dogs.

CRLF2 over-expression is a poor prognostic marker in children with high risk T-cell acute lymphoblastic leukemia

Pediatric T-ALL patients have a worse outcome compared to BCP-ALL patients and they could benefit from new prognostic marker identification. Alteration of CRLF2 gene, a hallmark correlated with poor outcome in BCP-ALL, has not been reported in T-ALL. We analyzed CRLF2 expression in 212 T-ALL pediatric patients enrolled in AIEOP-BFM ALL2000 study in Italian and German centers. Seventeen out of 120 (14.2%) Italian patients presented CRLF2 mRNA expression 5 times higher than the median (CRLF2-high); they had a significantly inferior event-free survival (41.2%±11.9 vs. 68.9%±4.6, p=0.006) and overall survival (47.1%±12.1 vs. 73.8%±4.3, p=0.009) and an increased cumulative incidence of relapse/resistance (52.9%±12.1 vs. 26.2%±4.3, p=0.007) compared to CRLF2-low patients. The prognostic value of CRLF2 over-expression was validated in the German cohort. Of note, CRLF2 over-expression was associated with poor prognosis in the high risk (HR) subgroup where CRLF2-high patients were more frequently allocated. Interestingly, although in T-ALL CRLF2 protein was localized mainly in the cytoplasm, in CRLF2-high blasts we found a trend towards a stronger TSLP-induced pSTAT5 response, sensitive to the JAK inhibitor Ruxolitinib. In conclusion, CRLF2 over-expression is a poor prognostic marker identifying a subset of HR T-ALL patients that could benefit from alternative therapy, potentially targeting the CRLF2 pathway.

In Vivo Applications of Mesenchymal Stem Cells and Platelet-Rich Plasma to Improve Tendon Regeneration in Sheep

The “restitutio ad integrum” pursue in the treatment of tenodesmic lesions might represents a tangible target thanks to the increased number of novel cellular-based therapies. In this work, we evaluated the efficacy of the application of platelet-rich plasma (PRP), mesenchymal stem cells (MSCs), and PRP + MSCs to experimentally injured sheep deep digital flexor tendon (DM n° 97/2010-B). Our results indicate that the in vivo integration of injected MSCs was successful as verified by the presence of green fluorescent protein GFP-positive cells. Tissue architecture and the tendon linear fiber pattern were significantly improved on histologic sections, especially after the use of MSCs, and the clinic evaluation was also satisfactory after the use of PRP.

Comparison in vivo applications between peripheral blood-derived mesenchymal stromal cells (PB-MSCs) and platlet-rich plasma (PRP) in injured tendons of sheep

T properties of DLC films, which are similar to those of diamond, are attractive because of their extreme mechanical hardness, high electrical resistivity, low friction, stability against acids, uniformly flat surface and optical transparency over a wide area. DLC films are deposited on several kinds of substrates at room temperature. These advantages of the DLC films lead to many application in mechanical, electrical and medical fields. In this study, the cytocompatibility of amorphous hydrogenated carbon-a-C:H:N films on polymeric fibrous materials has been investigated to apply to the biodevices. The a-C:H:N films was deposited on fibrous scaffolds by decomposed source methane gas in addition with N2 gas as dopant. The deposited a-C:H:N films were characterized for their chemical, optical, structural and electrical properties using X-ray photoelectron spectroscopy, contact angle measurement, Raman spectroscopy, and cellular affinity. We have found that the cell culture of a-C:H:N films increased monotonously with the increase of nitrogen concentration up to 40% of N2 concentration. The introduction of nitrogen gas leaded the addition of nitrogen atoms into the films. The cytocompatibility of a-C:H films was improved by the formation of C=N arrangement on the surface.