Transcriptomic and computational analysis identified LPA metabolism, KLHL14 and KCNE3 as novel regulators of Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) is a complex biological program between physiology and pathology. Here, amniotic epithelial cells (AEC) were used as in vitro model of transiently inducible EMT in order to evaluate the transcriptional insights underlying this process. Therefore, RNA-seq was used to identify the differentially expressed genes and enrichment analyses were carried out to assess the intracellular pathways involved. As a result, molecules exclusively expressed in AEC that experienced EMT (GSTA1-1 and GSTM3) or when this process is inhibited (KLHL14 and KCNE3) were identified. Lastly, the network theory was used to obtain a computational model able to recognize putative controller genes involved in the induction and in the prevention of EMT. The results suggested an opposite role of lysophosphatidic acid (LPA) synthesis and degradation enzymes in the regulation of EMT process. In conclusion, these molecules may represent novel EMT regulators and also targets for developing new therapeutic strategies.

Fusion as the result of sperm-somatic cell interaction.

The research has been designed to investigate whether acrosome-reacted spermatozoa can fuse with somatic cells and to check whether this event may involve the molecular machinery implicated in the sperm-egg fusion. Boar spermatozoa were capacitated in vitro and then treated with A23187 to induce acrosome reaction and activate their fusogenic potential. Reacted spermatozoa, loaded with the membrane-permeant fluorescent dye calcein AM, were incubated with plated granulosa cells or cells derived from stable cell lines: CRFK, VERO, and ESK4. The fusion between spermatozoa and somatic cells was revealed by the diffusion of the fluorescent dye from the sperm to the cell as membrane fusion and cytoplasmic continuity between the two cells were established. The involvement of integrin alpha6 and tetraspanin CD9 in the process of fusion was assessed by carrying out the experiment in the presence of antibodies against these molecules. Moreover, the incidence of fusion displayed by the different cell types used was analyzed in relation to their content in the above molecules assessed by western blot and immunostaining. The role of CD9 was additionally investigated by using CD9-negative cells. The data presented demonstrate that boar spermatozoa can fuse with different somatic cell types derived from different species and the process requires the combined presence of both integrin and tetraspanin molecules on the cell plasma membrane.

Vascular supply as a discriminating factor for pig preantral follicle selection.

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.

Characterization of the methylation status of five imprinted genes in sheep gametes.

Genomic imprinting is a mammalian developmental process that uses epigenetic mechanisms to induce monoallelic and parental-specific expression of particular autosomal genes. A crucial epigenetic event consists of DNA methylation of CpG-islands, which become differentially methylated regions (DMRs) on the maternal and paternal alleles during oogenesis or spermatogenesis (germline DMRs). By contrast, somatic DMRs are acquired after fertilization. While there are several studies referring to methylation acquisition within germline DMRs in the mouse and human, a comparable methylation analysis of orthologous sequences is still lacking in sheep. To identify germline DMRs, this study analysed the methylation status of the available CpG-islands of five ovine imprinted genes (H19, IGF2R, DLK1, DIO3 and BEGAIN) in mature spermatozoa and in female gametes at different stages of their follicle growth, including in vitro matured oocytes. The 5'-end CpG-island of H19 showed a full methylation in spermatozoa and an absent methylation in growing and fully grown oocytes. The intron 2 CpG-island of IGF2R was unmethylated in male gametes, while it showed a high level of methylation in early stages of oogenesis. The promoter CpG-islands of DLK1 and DIO3 were found to be unmethylated both in spermatozoa and oocytes. Finally, the exon 9 CpG-island of BEGAIN was hypermethylated in mature male gametes, while it showed an almost complete methylation only in late stages of oocyte development. Our findings suggest that DNA methylation establishment during early stages of sheep oogenesis and subsequent in vitro maturation is gene-specific and that, of the five genes investigated, only the CpG-islands of H19 and IGF2R might represent ovine germline DMRs.

Placental Stem Cells from Domestic Animals: Translational Potential and Clinical Relevance.

The field of regenerative medicine is moving toward clinical practice in veterinary science. In this context, placenta-derived stem cells isolated from domestic animals have covered a dual role, acting both as therapies for patients and as a valuable cell source for translational models. The biological properties of placenta-derived cells, comparable among mammals, make them attractive candidates for therapeutic approaches. In particular, stemness features, low immunogenicity, immunomodulatory activity, multilineage plasticity, and their successful capacity for long-term engraftment in different host tissues after autotransplantation, allo-transplantation, or xenotransplantation have been demonstrated. Their beneficial regenerative effects in domestic animals have been proven using preclinical studies as well as clinical trials starting to define the mechanisms involved. This is, in particular, for amniotic-derived cells that have been thoroughly studied to date. The regenerative role arises from a mutual tissue-specific cell differentiation and from the paracrine secretion of bioactive molecules that ultimately drive crucial repair processes in host tissues (e.g., anti-inflammatory, antifibrotic, angiogenic, and neurogenic factors). The knowledge acquired so far on the mechanisms of placenta-derived stem cells in animal models represent the proof of concept of their successful use in some therapeutic treatments such as for musculoskeletal disorders. In the next future, legislation in veterinary regenerative medicine will be a key element in order to certify those placenta-derived cell-based protocols that have already demonstrated their safety and efficacy using rigorous approaches and to improve the degree of standardization of cell-based treatments among veterinary clinicians.

Genetic rescue of an endangered mammal by cross-species nuclear transfer using post-mortem somatic cells.

Since the advent of procedures for cloning animals, conservation biologists have proposed using this technology to preserve endangered mammals. Here we report the successful cloning of a wild endangered animal, Ovis orientalis musimon, using oocytes collected from a closely related, domesticated species, Ovis aries. We injected enucleated sheep oocytes with granulosa cells collected from two female mouflons found dead in the pasture. Blastocyst-stage cloned embryos transferred into sheep foster mothers established two pregnancies, one of which produced an apparently normal mouflon. Our findings support the use of cloning for the expansion of critically endangered populations.

Effects of 50 Hz extremely low frequency magnetic field on the morphology and function of boar spermatozoa capacitated in vitro.

The aim of this study was to evaluate the effect of an acute exposure to a sinusoidal MF-ELF (50 Hz, 1mT) on the ability of boar mature spermatozoa to acquire the fertilizing competence in vitro. The spermatozoa exposed during the 4h of incubation to the MF-ELF were evaluated for morphological (surface morphology and acrosome integrity) and functional parameters (cell viability, motility, induction of acrosomal reaction, AR, and the ability to in vitro fertilize oocytes). In parallel, the intracellular Ca(2+) levels as well as the major mechanisms of Ca(2+) clearance were assessed: (45)Ca intakes and intracellular Ca(2+) sequestration by analyzing intracellular Ca(2+) elevation induced by thapsigargin or studying mitochondrial function with Mito-Tracker. The MF-ELF exposure did not affect sperm viability and morphology during the first h of incubation when sperm Ca(2+) homeostasis were already compromised. First of all, MF-ELF treated spermatozoa showed resting intracellular Ca(2+) levels significantly lower than those recorded in controls. This result was dependent on a lower extracellular Ca(2+) intake and from the inhibitory role exerted on both intracellular Ca(2+) storages. As a consequence, after 1h of incubation MF-ELF exposed cells displayed a reduced motility, a modest reactivity when coincubated with solubilized zonae pellucidae and a reduction in oocyte penetrating ability. After 2 or 4h of incubation, in addition, signs of morphological damage appeared on plasma membrane and at acrosomal level. In conclusion, MF-ELF influence negatively spermatozoa first by impairing cell Ca(2+) homeostasis then by dramatically affecting sperm morphology and function.

Spatio-temporal analysis of vascular endothelial growth factor expression and blood vessel remodelling in pig ovarian follicles during the periovulatory period.

Vascular endothelial growth factor (VEGF) expression pattern and blood vessel remodelling were evaluated during the transition from the preovulatory follicle to the corpus luteum (CL). To this end, prepubertal gilts were treated with equine chorionic gonadotrophin (eCG) to collect preovulatory follicles (60 h after eCG) and with human chorionic gonadotrophin (hCG) to obtain periovulatory follicles 18 h and 36 h later. The VEGF mRNA content was analysed by in situ hybridization, while protein localization in follicular fluid (FF) and in granulosa and theca compartments was evaluated by ELISA, immunohistochemistry or western blot. Blood vessel architecture and vascular area (VA) were investigated using immunohistochemistry for von Willenbrand Factor, a specific endothelial marker. Vascular remodelling was finally tested using Ki-67 immunocytochemistry as a proliferation marker, or alpha-smooth muscle actin (alpha-SMA) as a specific mural cell marker. eCG-treated follicles showed high VEGF levels and two concentric blood vessel networks composed of proliferating endothelial cells without any association with mural components. hCG injection inhibited VEGF synthesis in the granulosa compartment and, as a consequence, the protein fell within the FF. In parallel, endothelial cell proliferation stopped and the VA decreased. Close to ovulation, VEGF production restarted in both follicular compartments and VEGF mRNA content significantly increased in the theca layer. Changes in follicular VEGF secretion were observed; the protein disappeared from FF and was observed in the extracellular matrix. An active angiogenesis characterized the follicle; endothelial cell proliferation was associated with a recruitment of alpha-SMA-positive mural cells. The data presented in this work showed that, in the phases preceding ovulation, a complete vascular remodelling occurs, characterized by both an evident neovascularization and the appearance of blood vessels presenting smooth musculature which could be involved in CL formation after ovulation.

Luteinizing hormone inhibits potassium outward currents in swine granulosa cells by intracellular calcium mobilization.

Potassium currents of swine granulosa cells were studied using the patch clamp technique in the whole cell configuration. Granulosa cells stepped to positive potentials (+60 mV) from -40 mV holding potential exhibit a slowly activating, noninactivating outward potassium current. Tail current reverse potential (between -90 and -100 mV) and the current inhibition brought about by the replacement of KCl with CsCl in the pipette solution indicate that this current is carried by K ions. LH was found to significantly reduce the amplitude of this current. The effect was dose and time dependent. Similar inhibition (20-30% of the initial current) was reached with doses of 1-50 micrograms/ml, but in times proportionally shorter as the dose increased (50% inhibition was reached in 170-180 and 30-40 sec with 1 and 50 micrograms LH/ml, respectively). Much longer and variable times (3-10 min) were required with lower doses (0.2 microgram/ml). The effect of LH was independent of extracellular Ca, while preexposure of cells to TMB-8, an inhibitor of intracellular Ca mobilization, completely prevented the effect of LH. Outward currents after LH treatment could be completely restored by perfusing the cells with ionomycin in Ca-free medium to facilitate calcium efflux from the cells. The present studies indicate that LH modifies the bioelectrical properties of swine granulosa cells. This effect is mediated by an elevation of intracellular calcium, probably mobilized from intracellular stores. The induced changes in K conductance may play a specific role in the transduction mechanisms for LH.

Influence of progesterone on boar sperm capacitation.

This research investigates the effect of progesterone (P4) on boar sperm capacitation. Ejaculated spermatozoa were washed and incubated under capacitating conditions with or without P4. At different times of incubation samples of sperm were exposed to solubilized zonae pellucidae (ZP) and the degree of capacitation was evaluated by the incidence of zona-induced acrosome reaction (AR). The status of the acrosome was studied by using an FITC-conjugated lectin (Pisum sativum agglutinin; FITC-PSA). The effect of P4 on the fertilizing ability of semen was then evaluated in an in vitro fertilization system by exposing in vitro matured oocytes to sperm preincubated for 2 or 4 h with or without P4, under capacitating conditions. PSA staining showed that P4 does not affect the incidence of spontaneous AR. By contrast, spermatozoa incubated with P4 showed a higher percentage of AR than controls after the exposure to solubilized ZP. This enhanced reactivity to ZP suggests a direct effect of P4 on sperm capacitation. The in vitro fertilization assay was consistent with these results demonstrating a higher fertilizing ability in sperm preincubated with P4 than in controls while the steroid was without effect when added only during the fertilization step. These results demonstrate that P4 improves the fertilizing ability of boar semen essentially by facilitating the process of capacitation.

Activation of protein kinase A and protein kinase C mediates the depolarising effect of LH in ovine cumulus-corona cells.

We have studied the changes in membrane potential induced by LH in cumulus and granulosa cells isolated from sheep antral follicles. The investigation was carried out by using a non-invasive technique based on the use of a membrane potential sensitive probe, bis-oxonol. The membrane potential of mural granulosa cells was totally unaffected by LH, while that of cumulus or corona cells showed a marked depolarisation, starting 2-3 min after the addition of the hormone and plateauing after 5-10 min. None of the cells tested reacted to FSH. In the second part of the experiment the role of protein kinase A (PKA) and protein kinase C (PKC) in mediating the effect of LH was studied. The selective activation of PKA or PKC induced in cumulus-corona cells a rapid hyperpolarisation due to increased Cl and K conductance respectively. By contrast, the simultaneous activation of the two kinases induced a rapid membrane depolarisation due to the progressive decrease in K conductance. The activation of each kinase or their combined stimulation did not induce any change in the membrane potential of mural granulosa cells. These data demonstrated that LH has a depolarising effect regionally circumscribed to cumulus-corona cells and that this depolarisation depends on a reduction of K conductance caused by the activation of PKA and PKC.

Signal transduction mechanism for LH in the cumulus-oocyte complex.

The paper reviews recently described signalling mechanisms by which cumulus cells exposed to peak levels of gonadotropins, activate oocyte maturation. Cumulus cells react to LH with a prompt Ca raise which diffuses through gap junctions in a few minutes also into the oocyte where a local amplification system spreads the signal all over the cell. Few h later, still as a consequence of LH stimulation, cumulus cells undergo aprogressive depolarisation of their plasma membrane potential. Due to the electric coupling with these cells the oocyte depolarises too and this open specific voltage gated Ca channels responsible for a second wider and more sustained intracellular Ca rise. As a result of changes throughout maturation with a consequent modification of the size and charge of the molecules that can diffuse from one cell compartment to the other. This cell to cell interaction is further modified with cumulus expansion that leads to a progressive uncoupling of outer cumulus cells while the inner cell layer, corona radiata, remains in oocyte maturation by addressing to the oocyte nutrients and instructions in a well-orchestrated sequence. The identification of these mechanisms are a fundamental prerequisite for the development of in vitro systems suitable to produce oocytes matured in vitro with normal developmental competence.

Nerve growth factor production in sheep antral follicles.

Despite their continuous turnover, sheep antral follicles are always regularly innervated. The local production of neurotrophins is probably involved in the control of ovarian innervation. In this context the present investigation was designed to evaluate the ability of sheep antral follicles to produce neurotrophic factors. In the first part of the paper neurotrophic activity was measured in follicular fluid of sheep antral follicles of different size. Using an in vitro model the effect of gonadotrophins on neurotrophin production was then evaluated. The levels of neurotrophic activity in conditioned medium or follicular fluid and the kind of neurotrophin produced were determined by using the chicken embryo dorsal root ganglia test combined with an immunoneutralization step. Follicular fluid from medium-large follicles (>4 mm) contains high levels of NGF (240-250 ng/ml), whereas the factor is nearly undetectable in small follicles (<3 mm) and in early atretic follicles. Experiments in vitro based on the culture of follicle shells for 12 hr confirmed that medium-large follicles can produce NGF. The production is strictly dependent on gonadotrophin stimulation. When gonadotrophins were not added or were added separately, no detectable levels of neurotrophic activity accumulated in medium. By contrast, in the presence of both LH and FSH the production of NGF became apparent showing a clear dose-response behavior. In addition, this production increased progressively with increasing follicle size from 4 to >5 mm up to values of about 60 ng/follicle, whereas follicles with a diameter of less than 3 mm were insensitive to gonadotrophins stimulation and did not produce significant amount of NGF. The data presented demonstrate that sheep follicles produce relevant amounts of NGF as long as the correct hormonal milieu is provided. Under these conditions the production of the NGF increases with increasing follicle size. This may be responsible for the rapid innervation of the wall of growing follicles and/or take part in other non-neural processes that are generally attributed to gonadotrophin stimulation.

Ram-induced ovulation to improve artificial insemination efficiency with frozen semen in sheep.

Ram effect, defined as shortening of seasonal anestrus in ewes by exposure to the ram, is now well recognized but the underlying mechanisms are still unclear. Little information also exists whether the ram is able to influence the estrus cycle and ovulation. Three experiments were conducted to investigate endocrine response, time of ovulation and pregnancy rate of ewes in proestrus, exposed to the ram (treated) or an adult ewe (control). In the first experiment, ewes (n = 20) were treated with fluorgestone acetate pessaries for 12 days and were given eCG and cloprostenol one day before withdrawal of pessaries. On the day after removal of the pessaries ewes in the treated group (n = 10) were exposed to the ram and those in the control group (n = 10) were exposed to an adult ewe. Blood samples were taken for LH assay every 20 min from 2 h before to 24 h after ram exposure. In the second experiment, ewes (n = 120) were induced into proestrus and on the day after removal of the pessaries were exposed to either a ram (n = 60) or a ewe (n = 60) as described above and were laparoscoped 50, 60 or 70 h after pessary withdrawal (n = 20 at each time interval). In the third experiment ewes (n = 90) were induced and exposed to the ram (n = 45) or an adult ewe (n = 45) and inseminated via a laparoscope whit frozen-thawed semen at 50 or 60 h after pessary removal, respectively. Exposure to the ram was followed in 2 h by a marked rise in LH, equivalent to a preovulatory surge in duration and amplitude. It was also followed by concentrated ovulation within 25 to 30 h and by an increased pregnancy rate in exposed ewes (73.3 vs. 53.3%).

Identification of capacitation in boar spermatozoa by chlortetracycline staining.

The functional status of boar spermatozoa undergoing capacitation in vitro was investigated. Two fluorescent stains were used: chlortetracycline (CTC) and a FITC-conjugated lectin (FITC-PSA). The first has been used for the direct identification of the capacitated boar spermatozoa, while the second, based on the identification of capacitated spermatozoa by their ability to undergo zona-induced acrosome reaction (AR), was used to confirm and validate the CTC assay in this species. Spermatozoa obtained from 5 different boars was washed and incubated under capacitating conditions. Aliquots of spermatozoa were collected at 0, 90 and 180 min of incubation and then stained with CTC or FITC-PSA. After CTC staining, 3 different fluorescent patterns were observed: Pattern A with the fluorescence uniformly distributed on the sperm head, Pattern B with the fluorescence concentrated in the post-acrosomial region, and Pattern C with the fluorescence concentrated in the acrosomial region. The percentage of spermatozoa displaying fluorescent Pattern A decreased throughout the incubation while that of spermatozoa with Pattern C showed a concomitant progressive increase. Pattern B fluorescence remained unchanged throughout the maturation period. Exposure to zonae pellucidae (ZP) brought back the levels of Pattern C fluorescence to basal values. Since only the capacitated spermatozoa are believed to react to ZP, this observation together with the rising incidence of Pattern C throughout maturation suggests that fluorescence in the acrosomial region identifies capacitated spermatozoa. The analysis of acrosome integrity carried out with FITC-PSA showed that the proportion of zona-induced AR was nearly the same as that of spermatozoa displaying Pattern C, thus confirming that CTC staining is suitable for the detection of boar sperm capacitation. In the second part of this study, CTC was used to investigate the effects of sperm origin and storage on the capacitation process. Our finding demonstrates that capacitation kinetics show wide variations in sperm samples derived from different boars; moreover, capacitation is also affected by sperm storage. While fresh semen showed a progressive increase in capacitated spermatozoa, ranging from low levels at the beginning of the culture to 46% at the end of incubation, the refrigerated semen had a relatively high percentage of capacitated spermatozoa at the beginning of culture, but this proportion increased only slightly during the following 90 to 180 min of treatment. These data indicate that CTC can be used to identify capacitated boar spermatozoa, and, because of its rapid and easy execution, it can be used routinely to identify the optimal capacitation time for different sperm samples.

Characterization, GFP gene Nucleofection, and allotransplantation in injured tendons of ovine amniotic fluid-derived stem cells.

Amniotic fluid has drawn increasing attention in the recent past as a cost-effective and accessible source of fetal stem cells. Amniotic fluid-derived mesenchymal stem cells (AFMSCs) that display high proliferation rate, large spectrum of differentiation potential, and immunosuppressive features are considered optimal candidates for allogeneic repair of mesenchymal damaged tissues. In this study, ovine AFMSCs (oAFMSCs) isolated from 3-month-old sheep fetuses were characterized for their proliferation rate, specific surface antigen and pluripotency marker expression, genomic stability, and mesenchymal lineage differentiation during their in vitro expansion (12 passages) and after nucleofection. The high proliferation rate of oAFMSCs gradually decreased during the first six subculture passages while the expression of surface molecules (CD29, CD58, CD166) and of pluripotency-associated markers (OCT4, TERT, NANOG, SOX2), the in vitro osteogenic differentiation potential, and a normal karyotype were maintained. Afterwards, oAFMSCs were nucleofected with a selectable plasmid coding for green fluorescent protein (GFP) using two different programs, U23 and C17, previously optimized for human mesenchymal stem cells. Transfection efficiencies were ∼63% and ∼37%, while cell recoveries were ∼10% and ∼22%, respectively. Nucleofected oAFMSCs expressing the GFP transgene conserved their pluripotency marker profile and retained a normal karyotype and the osteogenic differentiation ability. Seven single clones with a GFP expression ranging from 80% to 97% were then isolated and expanded over 1 month, thus providing stably transfected cells with long-term therapeutic potential. The in vivo behavior of GFP-labeled oAFMSCs was tested on a previously validated preclinical model of experimentally induced Achille's tendon defect. The allotransplanted oAFMSCs were able to survive within the host tissue for 1 month enhancing the early phase of tendon healing as indicated by morphological and biomechanical results. Altogether these data suggest that genetically modified oAFMSCs might represent a valuable tool for in vivo preclinical studies in a highly valid translational model.

Ovine amniotic epithelial cells: in vitro characterization and transplantation into equine superficial digital flexor tendon spontaneous defects.

In vitro expanded and frosted ovine amniotic epithelial cells (oAECs) were evaluated for their phenotype, stemness and attitude to differentiate into tenocytes. Fifteen horses with acute tendon lesions were treated with one intralesional injection of oAECs. Tendon recovery under controlled training was monitored. In vitro expanded oAECs showed a constant proliferative ability, a conserved phenotype and stable expression profile of stemness markers. Differentiation into tenocytes was also regularly documented. US controls showed the infilling of the defect and early good alignment of the fibers and 12 horses resumed their previous activity. Histological and immunohistochemical examinations in an explanted tendon demonstrated the low immunogenicity of oAECs that were able to survive in the healing site. In addition, oAECs supported the regenerative process producing ovine collagen type I amongst the equine collagen fibers. Considering our results, oAECs can be proposed as a new approach for the treatment of spontaneous equine tendon injuries.

Osteo-regenerative potential of ovarian granulosa cells: an in vitro and in vivo study.

Granulosa cells (GC) express stemness markers and can differentiate into cell types not present within the follicles. Given that follicles at different stages of development populate the ovary, we undertook this research in the pig model to identify the stage of follicle, growing or luteinizing, from which GC with the best regenerative potential can be retrieved. Growing follicles were isolated from prepubertal gilts 50 h after equine chorionic gonadotropin (eCG) (1,200 IU) administration. Luteinizing follicles were obtained from prepubertal gilts treated with eCG (1,200 IU) followed, 60 h later, by hCG (500 IU). The follicles were isolated 30 h after hCG. The GC isolated from growing (GGC) and from luteinizing (LGC) follicles were expanded in vitro for three passages and exposed to osteogenic medium to trigger differentiation. The GC incorporated in PLGA scaffolds were cultured in osteogenic medium for 2 wks and then implanted subcutaneously in the dorsal region of SCID mice to assess their osteogenic potential in vivo. In addition to the typical granulosa cells characteristics (inhibin, progesterone and estrogen production and FSH receptors), GGC and LGC showed a diffused expression of the stemness markers Sox2, Nanog and TERT immediately after isolation. Expansion caused in both cell types a rapid disappearance of granulosa cell characters while it did not modify stemness marker expression. Osteogenic medium induced a marked extracellular matrix mineralization and alkaline phosphatase activation in LGC, clearly detectable after two wks, while the process was much lighter in GGC, where it became evident after 3 wks. Osteocalcin and Runx2 expressions were upregulated and stemness markers downregulated by osteogenic medium. The GC loaded implants, retrieved 8 wks after transplantation, had viable GC surrounding the several nodules of calcifications recorded. Similar effects were induced by GGC and LGC while calcification nodules were not recorded when scaffolds without cells were implanted. These data confirm that GC, expanded in vitro undergo progressive de-differentiation retaining their plasticity and demonstrate that both GGC and LGC have osteogenic potential, luteinizing cells being more efficient. Transplanted in SCID mice, GC participate in new bone formation, thus confirming their therapeutic potential.

Achilles tendon regeneration can be improved by amniotic epithelial cell allotransplantation.

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.