COLLEONI S., PONDERATO N., DUCHI R., GALLI C., LAZZARI G. Laboratorio di Tecnologie della Riproduzione Istituto Sperimentale Italiano Lazzaro Spallanzani, CIZ srl Via Porcellasco 7/F, 26100 Cremona, Italy. Recent studies have determined that stem cells exist in most tissue. To date, in several species, it has been detected that bone marrow is a rich source of stem cells, in particular hematopoietic and mesenchymal stem cells that are capable of differentiating in multiple lineages. Mesenchymal stem cells have been studied in human, mouse, rat, and only occasionally in sheep, horse, dog, cat and pig. They can proliferate in vitro for many passages displaying a stable fibroblast-like phenotype and they can be differentiated not only to classical mesenchymal derivatives such as bone, fat, cartilage, muscle and cardiomyocytes, but also to other lineages such as oval hepatocyte and neurons. The aim of this study was to isolate and to characterise bovine and swine mesenchymal stem cells lines potentially useful for therapy, transgenesis or cloning. Bovine bone marrow cells were aspirated from the iliac crest of two one-month old calves, immediately after slaughter, using an 11g bone marrow biopsy/aspiration needle. Twenty ml syringes containing 3 ml of aspiration medium (TCM199/DMEM) with 5000 IU of heparin were used and about 40 ml of bone marrow were removed from each animal. Swine cells were collected flushing femurs and humerus of two one-week old slaughtered piglets with the same medium as above. After multiple PBS washings the cell suspension was loaded onto 70% Percoll gradient and centrifuged at 1100g for 30 min. Cells were collected from the interface and washed; residual erythrocytes were removed resuspending the cell pellet for 1 min in 1 ml of a solution 0,83% NH 4 Cl in Tris HCl 10 mM, then 9 ml of culture medium were added and the suspension was washed three times. Following counting, cells were plated at 200.000 cells/cm2 and cultured in M199/DMEM with 10% FBS. Medium was replaced at 24 and 72 hours and then twice weekly. Cells were subsequently subcultured at 5000 cell/cm2 for at least 15 passages; part of cells from first passage was expanded and frozen. Ten days after plating both bovine and swine cells acquired a fibroblastic shape; when they reached 90% of confluence, they were subcultured until exhaustion of growth potential. As described in previous studies performed in others species, they grew with regular speed until passage 10, than they started to slow down and they arrested growth about at passage 15 (about 50 population doublings for bovine and 40 for pig). Slowdown of growth was accompanied by increasing of cell size and by changing of morphology from spindle-shaped to flat. For induction of differentiation cells from all passages (fresh or frozen-thawed) where replated at 3000 cells/cm2 and treated with osteogenic medium containing dexamethasone 100 nM, ascorbic acid 0,25 mM, βglycerolphosphate 10 mM. Medium was changed tree At eight to twelve days after the start of the culture in osteogenic medium the cells were stained with BCIP/NBT (LabVision) to detect alkaline phosphatase activity and they appeared always positive. At the end of the culture, after 21 days, the cells resulted positive at Von Kossa staining with silver nitrate showing the accumulation of calcium. Furthermore, undifferentiated bovine cells from passage 2 were treated with 3 µM 5- azacytidine for 24 hours for myogenic and adipogenic induction, then the medium was removed and replaced with culture medium. Cultures were maintained under observation for at least five weeks changing medium twice weekly. When lipid vacuoles in the cytoplasm were detected, cells were stained positive with Red Oil O. Gene expression of undifferentiated cells from early passages and of differentiated cells was analysed by RT-PCR. Data obtained on bovine undifferentiated cells from passage 2 showed that they express collagen type X, characteristic of cartilage, the neuronal marker vimentin, the germinal marker protamine, gp130 and LIF receptor. When treated with osteogenic medium they became positive for ostepontin. Cells examined 60 days after treatment with 5-azacytidine resulted positive for myosin HC. Preliminary data obtained on swine undifferentiated cells indicate that they express myosin HC and cartilage markers collagene type X and decorin. The results of our work are in agreement with previous studies in different species and confirms that in bone marrow there are multipotent cells that have multiple capacity to differentiate. However it is still unclear whether these cells are really undifferentiated or only “multidifferentiated” given that they have relatively reduced capacity of self-renewal and their profile of gene expression is not standardised.

Source: http://www.istitutospallanzani.it/doc/193.pdf


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