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1.
Cell Transplant ; 21(11): 2377-95, 2012.
Article in English | MEDLINE | ID: mdl-22507232

ABSTRACT

Amniotic epithelial cells (AECs) are ideal seed cells for tissue regeneration, but no research has yet been reported on their tendon regeneration potential. This study investigated the efficiency of AEC allotransplantation for tendon healing, as well as the mechanism involved. To this aim ovine AECs, characterized by specific surface and stemness markers (CD14(-), CD31(-), CD45(-), CD49f, CD29, CD166, OCT4, SOX2, NANOG, TERT), were allotransplanted into experimentally induced tissue defects in sheep Achilles tendon. In situ tissue repair revealed that AEC-treated tendons had much better structural and mechanical recoveries than control ones during the early phase of healing. Immunohistochemical and biochemical analyses indicated that extracellular matrix remodeling was more rapid and that immature collagen fibers were completely replaced by mature ones in 28 days. Moreover, spatial-temporal analysis of cellularity, proliferation index, vascular area, and leukocyte infiltration revealed that AECs induced a specific centripetal healing process that first started in the tissue closer to the healthy portion of the tendons, where AECs rapidly migrated to then progress through the core of the lesion. This peculiar healing evolution could have been induced by the growth factor stimulatory influence (TGF-ß1 and VEGF) and/or by the host progenitor cells recruitment, but also as the consequence of a direct tenogenic AEC differentiation resulting in the regeneration of new tendon matrix. These findings demonstrate that AECs can support tendon regeneration, and their effects may be used to develop future strategies to treat tendon disease characterized by a poor clinical outcome in veterinary medicine.


Subject(s)
Achilles Tendon/cytology , Achilles Tendon/physiology , Amnion/cytology , Epithelial Cells/cytology , Cells, Cultured , Epithelial Cells/transplantation , Female , Humans , Pregnancy
2.
Vet Res Commun ; 34 Suppl 1: S25-8, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20437094

ABSTRACT

Stem cell (SC) regenerative therapy represents an emerging strategy for the treatment of human diseases. Since amniotic fluid-derived cells have been recently proposed as a promising source of human SCs, the present research aimed to amplify in vitro and characterize ovine amniotic fluid-derived SCs collected from the membranes (AMSCs) or fluid (AFSCs). These cells were found to proliferate, express the pluripotent SC markers OCT-4 and TERT, and differentiate in both osteogenic and smooth muscle lineages in vitro. However, AMSCs presented an earlier down-regulation of SC markers and a faster rate of differentiation. Thus, AMSCs and AFSCs may represent sources of characterized pluripotent SCs that can be easily collected and amplified in vitro. These ovine SCs may be used in preclinical studies on large animals to develop future human therapies.


Subject(s)
Extraembryonic Membranes/cytology , Sheep/physiology , Stem Cells/cytology , Animals , Cell Culture Techniques , Cell Differentiation , Female , Pregnancy
4.
Reproduction ; 137(1): 45-58, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18840644

ABSTRACT

This research analyses how somatic and vascular compartments change during preantral follicle growth. To address this aim, theca-granulosa (somatic) proliferation indexes (PIs), proportion of proliferating endothelial cells (PE), vascular area (VA) and vascular endothelial growth factor A (VEGFA) expression were simultaneously recorded on single healthy preantral follicles, classified into six different stages on the basis of the diameter and the granulosa layers. An autonomous blood vessel network starts to appear only in class 3. Vascular remodelling requires VEGFA expression, and VEGFA mRNA and VA significantly increase between class 3 and classes 4 and 5 and, further, in class 6. In addition, a positive correlation exists between these parameters in classes 3-5. Despite variation in angiogenesis results from classes 3 to 5, the statistical analysis reveals that the vascular parameters are positively and strictly correlated with somatic PIs. Conversely, class 6, also characterized by higher values of somatic PIs, displays a stable proportion of PEs ( congruent with 40%) without showing any correlation among the different parameters analysed. To identify follicular subpopulations within different classes, a multivariate hierarchical cluster analysis was performed. This analysis reveals that the majority of classes 3 and 4 are quiescent follicles or structures that grow very slowly. Class 5 represents a transitory category, where half of the follicles maintain a low activity and the remaining express significantly higher levels of granulosa PI and VA. The follicles with this high activity are probably able to reach class 6 becoming dominant structures where somatic and vascular parameters are constantly on high levels and the VA remains the unique differentiating element.


Subject(s)
Follicular Phase/physiology , Ovarian Follicle/blood supply , Swine/physiology , Animals , Biomarkers/analysis , Blotting, Western , Cell Count , Cell Proliferation , Cluster Analysis , Endothelial Cells/cytology , Female , Granulosa Cells/cytology , Immunohistochemistry , Ki-67 Antigen/analysis , Neovascularization, Physiologic , Ovarian Follicle/growth & development , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Theca Cells/cytology , Vascular Endothelial Growth Factor A/analysis , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , von Willebrand Factor/analysis
7.
Anat Histol Embryol ; 33(1): 23-7, 2004 Feb.
Article in English | MEDLINE | ID: mdl-15027958

ABSTRACT

Apoptosis is the cellular mechanism of ovarian follicular atresia. The major downstream effector of this phenomenon in many tissues is caspase-3 but little is known about its role in pig ovarian apoptosis. In the present study, we detected the localization of caspase-3 in parallel with nuclear fragmentation (TUNEL) on healthy and early atretic antral follicles. In healthy antral follicles caspase-3 and TUNEL positivity were occasionally recorded within theca layer. The incidence of DNA fragmentation, as indicated also by the biochemical detection, increased mainly in the granulosa layer of early atretic follicles. Quantitative analysis revealed, besides, that atresia was accompanied by a higher incidence of caspase-3 (57.20 +/- 20.05 versus 3.64 +/- 0.61 positive cells in atretic versus healthy follicles, respectively; P < 0.05), of TUNEL positivity (20.13 +/- 9.33 versus 0.42 +/- 0.12; P < 0.05) and simultaneous immunostaining for caspase-3 and TUNEL (15.02 +/- 6.95 versus 0.31 +/- 0.05; P < 0.05) in the granulosa layer. In detached granulosa cells isolated from the follicular fluid of early atretic follicles a further significantly increase was recorded in the percentage of TUNEL positivity and in the incidence of cells that showed colocalization of caspase-3 activity and DNA fragmentation. Granulosa cells of early atretic follicles exhibited a higher positivity for caspase-3 localized in the cytoplasm and occasionally in the nucleus area of granulosa cells. These results indicate that capsase-3 was involved and precociously activated during the process of atresia. Finally, the progressively higher incidence of TUNEL positivity and of double immunostaining in atretic cells collected within the follicular fluid seems to indicate that proteases activity leads only tardily in a detectable DNA fragmentation.


Subject(s)
Caspases/metabolism , DNA Fragmentation , Follicular Atresia/physiology , Animals , Apoptosis/physiology , Caspase 3 , Caspases/genetics , Enzyme Activation , Female , Follicular Atresia/genetics , Follicular Atresia/metabolism , Gene Expression Regulation, Enzymologic , Granulosa Cells/chemistry , Granulosa Cells/enzymology , Immunohistochemistry , In Situ Nick-End Labeling , Ovarian Follicle/chemistry , Ovarian Follicle/enzymology , Swine , Theca Cells/chemistry , Theca Cells/enzymology
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