P. Ceccarelli

Flow cytometric characterization of culture expanded multipotent mesenchymal stromal cells (MSCs) from horse adipose tissue: towards the definition of minimal stemness criteria.

In the last decades, multipotent mesenchymal progenitor cells have been isolated from many adult tissues of different species. The International Society for Cellular Therapy (ISCT) has recently established that multipotent mesenchymal stromal cells (MSCs) is the currently recommended designation. In this study, we used flow cytometry to evaluate the expression of several molecules related to stemness (CD90, CD44, CD73 and STRO-1) in undifferentiated, early-passaged MSCs isolated from adipose tissue of four donor horses (AdMSCs). The four populations unanimously expressed high levels of CD90 and CD44. On the contrary, they were unexpectedly negative to CD73. A small percentage of the cells, finally, showed the expression of STRO-1. This last result might be due to the existence of a small subpopulation of STRO-1+ cells or to a poor cross-reactivity of the antibody. A remarkable donor-to-donor consistency and reproducibility of these findings was demonstrated. The data presented herein support the idea that equine AdMSCs may be easily isolated and selected by adherence to tissue culture plastic and exhibit a surface profile characterized by some peculiar differences in comparison to those described in other species. Continued characterization of these cells will help to clarify several aspects of their biology and may ultimately enable the isolation of specific, purified subpopulations.

Histology and Ultrastructure of the Gut of the Tilapia (Tilapia spp.), a Hybrid Teleost

The morphology of the intestine has been studied in a species of warm water fish, Tilapia spp., a hybrid teleost of notable economic importance. Light and electron microscope results show that the intestine is a relatively undifferentiated muscular tube lined with a simple columnar epithelium interspersed with goblet cells. The proximal region has a greater surface area, manifested by elongated mucosal ridges. The enterocytes are covered apically with uniform microvilli and exhibit the typical ultrastructural features of pinocytosis, namely extensive invaginations of the luminal plasma membrane and massive accumulation of vesicles in the apical cytoplasm. The distal intestine mucosa is thinner and less elaborately folded and consists of columnar cells with shorter and sparser microvilli. Their supranuclear cytoplasm contains abundant clear vacuoles. Numerous endocrine cells can also be seen. Regional cellular ultrastructural features are correlated with digestive functions.

Carbohydrate histochemistry of the alimentary canal of the shi drum, Umbrina cirrosa L.

Histochemical staining techniques, which differentiate the main categories of carbohydrates, were applied to sections from different segments of the alimentary canal of the shi drum Umbrina cirrosa L. to study patterns of distribution of epithelial glycoconjugates. In the oesophagus, mucous cells contained sulphomucins, together with a small amount of sialomucins. Stomach epithelial cells secreted neutral and acidic glycoconjugates, while gastric glands only produced small quantities of sialomucins. Goblet cells showed the presence of sialo and sulphomucins in the pyloric caeca, whereas intestinal mucous cells secreted sulphated glycoconjugates. This work serves as a baseline for further studies on carbohydrate composition of the mucosa of the shi drum digestive system.

Ultrastructural Study on the Stomach of Tilapia spp (Teleostei)

An ultrastructural study has been made of gastric mucosa of a teleostean fish, Tilapia spp. The cytological features of the surface mucous cells, mucous neck cells, glandular cells and endocrine cells are described. The surface mucous cells, identified by their superficial localization, are characterized by apical granules. The mucous neck cells are distinguished by the appearance of their mucous granules and their localization between surface mucous cells and glandular cells. The gastric glands contain only one form of cell whose fine structure is similar to cells that secrete hydrochloric acid.

Mesenchymal stromal cells primed with Paclitaxel attract and kill leukaemia cells, inhibit angiogenesis and improve survival of leukaemia‐bearing mice

Current leukaemia therapy focuses on increasing chemotherapy efficacy. Mesenchymal stromal cells (MSCs) have been proposed for carrying and delivery drugs to improve killing of cancer cells. We have shown that MSCs loaded with Paclitaxel (PTX) acquire a potent anti‐tumour activity. We investigated the effect of human MSCs (hMSCs) and mouse SR4987 loaded with PTX (hMSCsPTX and SR4987PTX) on MOLT‐4 and L1210, two leukaemia cell (LCs) lines of human and mouse origin, respectively. SR4987PTX and hMSCsPTX showed strong anti‐LC activity. hMSCsPTX, co‐injected with MOLT‐4 cells or intra‐tumour injected into established subcutaneous MOLT‐4 nodules, strongly inhibited growth and angiogenesis. In BDF1‐mice‐bearing L1210, the intraperitoneal administration of SR4987PTX doubled mouse survival time. In vitro, both hMSCs and hMSCsPTX released chemotactic factors, bound and formed rosettes with LCs. In ultrastructural analysis of rosettes, hMSCsPTX showed no morphological alterations while the attached LCs were apoptotic and necrotic. hMSCs and hMSCsPTX released molecules that reduced LC adhesion to microvascular endothelium (hMECs) and down‐modulated ICAM1 and VCAM1 on hMECs. Priming hMSCs with PTX is a simple procedure that does not require any genetic cell manipulation. Once the effectiveness of hMSCsPTX on established cancers in mice is proven, this procedure could be proposed for leukaemia therapy in humans.

Membrane vesicles mediate pro-angiogenic activity of equine adipose-derived mesenchymal stromal cells

Multipotent mesenchymal stromal cells (MSCs) have attracted a great deal of interest, due to several distinctive features, including the ability to migrate to damaged tissue and to participate in tissue regeneration. There is increasing evidence that membrane vesicles (MVs), comprising exosomes and shedding vesicles, represent a key component, responsible for many of the paracrine effects of MSCs. The aim of the present study was to establish whether equine adipose-derived MSCs (E-AdMSCs) produce MVs that are capable of influencing angiogenesis, a key step in tissue regeneration. A morphological study was performed using MSC monolayers, prepared for transmission and scanning electron microscopy and on ultracentrifuged MSC supernatants, to identify production of MVs. The ability of MVs to influence angiogenesis was evaluated by means of the rat aortic ring and scratch assays. The results demonstrated that MVs, constitutively produced by E-AdMSCs, are involved in intercellular communication with endothelial cells, stimulating angiogenesis. Although many questions remain regarding their formation, delivery, content and mechanism of action, the present study supports the concept that MVs released by MSCs have the potential to be exploited as a therapeutic tool for regenerative medicine.

Immunohistochemical evidence of Orexin-A in the pancreatic beta cells of domestic animals

A large body of information proves that Orexin-A is present in the pancreatic endocrine cells of humans and laboratory animals; more detailed studies identify Orexin-A-immunopositive cells as beta cells. Because no data have been reported on the pancreas of domestic animals, we investigated the presence and the distribution of cells containing Orexin-A in the pancreas of cattle, sheep and pigs by means of immunohistochemical techniques. Using a polyclonal antibody against Orexin-A, the immunopositive reaction was identified in the cytoplasm of many insular cells in the three species studied. Double immunohistochemical staining, using a polyclonal anti-insulin antibody, showed that Orexin-A is co-expressed with insulin. Our results, besides showing the presence of Orexin-A in the endocrine pancreas of domestic animals, together with data present in the literature, could contribute to the understanding of complex mechanisms regulating the functionality of the endocrine pancreas in domestic animals.

Immunohistochemical identification and localization of orexin A and orexin type 2 receptor in the horse gastrointestinal tract.

The aim of the present study was to investigate the presence and the distribution of cells containing orexin A and orexin type 2 receptor in the horse stomach and gut, by means of immunohistochemical techniques. Orexin A was identified in the stomach fundic and pyloric regions and in the duodenum. In the same stomach regions, a large subset of orexin A-positive cells also showed orexin type 2 receptor-like immunoreactivity. Moreover, in the duodenum, many of them, seemed to store serotonin. Characteristically, enteric neurons or ganglia also displayed orexin A and, sometimes, orexin type 2 receptor immunoreaction. Orexin A and orexin type 2 receptor immunoreactivity was also found in the nerve fibers in the enteric submucosal layer. Our results, together with data present in the literature, could contribute to the understanding of complex mechanisms regulating the horse gut functionality that are depending very likely on the consequence of the co-operation of both a central and a peripheral control.

Identification of orexin A- and orexin type 2 receptor-positive cells in the gastrointestinal tract of neonatal dogs

The presence and distribution of cells positive to orexin A (OXA) and to orexin type 2 receptor (OX2R) were investigated in the gastrointestinal tract of neonatal dogs by means of immunohistochemical techniques. The orexin A-positive cells were identified with some of the endocrine cells in the stomach and in the duodenum; they were both of the open and closed type and were lacking in the large intestine. In the stomach, a large subset of orexin A-positive cells also showed gastrin-like immunoreactivity while, in the duodenum, many of them seemed to store serotonin. The orexin type 2 receptor-positive cells were evidenced all along the gastrointestinal tract examined, also in the large intestine, and they showed the same morphological characteristics as those positive to orexin A. Moreover, the immunohistochemical techniques revealed intense positivity for both orexin A and orexin type 2 receptor in the neurons and fibers of the enteric nervous system. A large subset of orexin A-positive neurons seemed to store substance P.

The Endocrine Cells in the Gastro‐enteric Tract of Adult Fallow Deer (Dama dama L.)

Endocrine cells were detected in the gastro‐enteric tract of the fallow deer by means of immunohistochemical procedures, using antibodies against serotonin, somatostatin, gastrin, glucagon and cholecystokinin. The number of cells positive for each antiserum in each region was evaluated. Serotonin‐containing enterochromaffin (Ec) cells were present in every region investigated and were most numerous in the proximal duodenum. Cells positive for somatostatin were present in all the regions studied, with the exception of the colon, and were especially numerous in the proper gastric‐gland region. Cells that were stained by the anti‐gastrin antibody were very numerous in the pyloric‐gland region but only rare in the duodenum. Glucagon‐immunoreactive cells were only detected in the large intestine and their frequency was always less than 10/0.5 mm2. Cholecystokinin‐containing cells were scarce and restricted to the pyloric‐gland region and duodenum.