Platelet-rich plasma and ovarian quiescence: a bovine in vitro model for regeneration of the ovary.

CONTEXT: Ovarian quiescence can be due to hormonal deficiency usually caused by apoptosis of granulosa cells responsible for oestrogen synthesis. AIM: This study evaluated the regenerative effect of platelet rich plasma (PRP) on bovine in vitro models to understand its effect on granulosa cells. METHODS: Quiescent and healthy ovarian sections were cultured in the presence/absence of PRP for 72h and, at different times (0, 24, 48 and 72h), hematoxylin-eosin and immunohistochemical detection of Ki-67 were performed. Additionally, granulosa cells collected from healthy bovine ovaries were stressed with 100ng/mL of lipopolysaccharide (LPS) in presence/absence of PRP and evaluated at 0, 4, 8 and 24h for apoptosis by acridine orange and propidium iodide staining. Enzyme-linked immunosorbent assay tests were performed to evaluate oestrogen (E2) and anti-Müllerian hormone (AMH) concentrations on cultures of ovarian slices and granulosa cells. KEY RESULTS: In slides of quiescent ovaries treated with PRP, a marked and widespread positivity to Ki-67 was expressed by 40-60% of the follicular wall cells at 48h of culture. Levels of E2 and AMH were significantly higher compared to untreated quiescent samples reaching the levels of healthy control samples. PRP counteracted the LPS effect and apoptosis (at 24h, there were 93.44±3.51% live cells with LPS+PRP compared to 37±1.32% with LPS) and significantly increased concentrations of E2 and AMH. CONCLUSIONS: PRP can stimulate granulosa cell proliferation and counteract inflammatory processes in vitro . IMPLICATIONS: This treatment could improve the reproductive ability of quiescent females.

Application of Perinatal Derivatives in Ovarian Diseases.

Reproductive diseases could lead to infertility and have implications for overall health, most importantly due to psychological, medical and socio-economic consequences for individuals and society. Furthermore, economical losses also occur in animal husbandry. In both human and veterinary medicine, hormonal and surgical treatments, as well as assisted reproductive technologies are used to cure reproductive disorders, however they do not improve fertility. With ovarian disorders being the main reproductive pathology in human and bovine, over the past 2 decades research has approached regenerative medicine in animal model to restore normal function. Ovarian pathologies are characterized by granulosa cell and oocyte apoptosis, follicular atresia, decrease in oocyte quality and embryonic development potential, oxidative stress and mitochondrial abnormalities, ultimately leading to a decrease in fertility. At current, application of mesenchymal stromal cells or derivatives thereof represents a valid strategy for regenerative purposes. Considering their paracrine/autocrine mode of actions that are able to regenerate injured tissues, trophic support, preventing apoptosis and fibrosis, promoting angiogenesis, stimulating the function and differentiation of endogenous stem cells and even reducing the immune response, are all important players in their future therapeutic success. Nevertheless, obtaining mesenchymal stromal cells (MSC) from adult tissues requires invasive procedures and implicates decreased cell proliferation and a reduced differentiation capacity with age. Alternatively, the use of embryonic stem cells as source of cellular therapeutic encountered several ethical concerns, as well as the risk of teratoma formation. Therefore, several studies have recently focussed on perinatal derivatives (PnD) that can be collected non-invasively and, most importantly, display similar characteristics in terms of regenerating-inducing properties, immune-modulating properties and hypo-immunogenicity. This review will provide an overview of the current knowledge and future perspectives of PnD application in the treatment of ovarian hypofunction.

Case Report: Use of Amniotic Microvesicles for Regenerative Medicine Treatment of a Mare With Chronic Endometritis.

Chronic endometritis is an inflammation in the inner layer of uterine mucosa, with or without an infectious process, which affects the animal's fertility but not its general health. A variety of treatments has been adopted over the years but to date, no effective cures have been able to renew the injured tissue. Since the defects in the fetal-maternal communication are caused by degenerative changes due to chronic endometrial inflammation, our working hypothesis was a new approach to this disease by the regenerative medicine using amniotic derived microvesicles (MVs) for their anti-inflammatory and regenerative effects. The MVs are responsible for horizontal transfer of genetic materials, including microRNA (miRNAs) that are involved in paracrine communication between origin cells and target cells. Thus, intrauterine MV infusion may be beneficial in degenerative chronic endometritis and in the fetal-maternal talk. The selected mare was an 11-year-old Friesian, with a history of failed pregnancies despite numerous insemination attempts. Punctual and evident heats characterized the reproductive history, but no insemination attempts had been made for many years. The first (failed) insemination was when the mare was 9-years-old. In the next two reproductive seasons, other attempts were made at regular intervals but none was successful. After a final insemination attempt using a stallion of proven fertility, the collection of an 8-day old embryo suggested that the mare was affected by implantation failure related to endometritis. The mare was treated with two cycles of intrauterine administration of amniotic-derived MVs. The success of the intrauterine administration of MVs was demonstrated by an improvement in the classification of endometritis and in a successful artificial insemination (AI) with implantation of an embryo, as detected at day 14 and with a pregnancy that is still ongoing. Probably, MVs were able to restore the injured endometrium and re-establish the proper communication for a successful embryo implantation.

Priming with inflammatory cytokines is not a prerequisite to increase immune-suppressive effects and responsiveness of equine amniotic mesenchymal stromal cells.

BACKGROUND: Equine amniotic mesenchymal stromal cells (AMSCs) and their conditioned medium (CM) were evaluated for their ability to inhibit in vitro proliferation of peripheral blood mononuclear cells (PBMCs) with and without priming. Additionally, AMSC immunogenicity was assessed by expression of MHCI and MHCII and their ability to counteract the in vitro inflammatory process. METHODS: Horse PBMC proliferation was induced with phytohemagglutinin. AMSC priming was performed with 10 ng/ml of TNF-α, 100 ng/ml of IFN-γ, and a combination of 5 ng/ml of TNF-α and 50 ng/ml of IFN-γ. The CM generated from naïve unprimed and primed AMSCs was also tested to evaluate its effects on equine endometrial cells in an in vitro inflammatory model induced by LPS. Immunogenicity marker expression (MHCI and II) was evaluated by qRT-PCR and by flow cytometry. RESULTS: Priming does not increase MHCI and II expression. Furthermore, the inhibition of PBMC proliferation was comparable between naïve and conditioned cells, with the exception of AMSCs primed with both TNF-α and IFN-γ that had a reduced capacity to inhibit T cell proliferation. However, AMSC viability was lower after priming than under other experimental conditions. CM from naïve and primed AMSCs strongly inhibited PBMC proliferation and counteracted the inflammatory process, rescuing about 65% of endometrial cells treated by LPS. CONCLUSION: AMSCs and their CM have a strong capacity to inhibit PBMC proliferation, and priming is not necessary to improve their immunosuppressive activity or reactivity in an inflammatory in vitro model.

Insights into animal models for cell-based therapies in translational studies of lung diseases: Is the horse with naturally occurring asthma the right choice?

Human asthma is a widespread disease associated with chronic inflammation of the airways, leading to loss of quality of life, disability and death. Corticosteroid administration is the mainstream treatment for asthmatic patients. Corticosteroids reduce airway obstruction and improve quality of life, although symptoms persist despite treatment in many patients. Moreover, available therapies failed to reverse the lung pathology present in asthma. Animal models, mostly rats and mice, in which the disease is experimentally induced, have been studied to identify new therapeutic targets for human asthma. Alternative animal models could include horses in which naturally occurring asthma could represent an important step to test therapies, potentially designed around mouse studies, before being translated to human testing. Horses naturally suffer from asthma, which has striking parallels with human asthma. Severe equine asthma (SEA) is characterized by reversible bronchospasms and neutrophil accumulation in the lungs immunologically mediated mainly by Th2. Moreover, the pulmonary remodelling that occurs in SEA closely resembles that of human asthma, making the equine model unique for investigation of tissue repair and new therapies. Cell therapy, consisting on mesenchymal stromal cells (MSCs) and derivatives (conditioned medium and extracellular vesicles), could represent a novel therapeutic contribution for tissue regeneration. Cell therapy may prove advantageous over conventional therapy in that it may repair or regenerate the site of injury and reduce the reaction to allergens, rather than simply modulating the inflammatory process.

Glycan Profiling Analysis of Equine Amniotic Progenitor Mesenchymal Cells and Their Derived Extracellular Microvesicles.

Equine amniotic mesenchymal cells (eAMCs) are involved in many mechanisms in tissue regenerative processes. Their secreted vesicles are important effectors in a wide array of biological processes, and contribute to in vivo healing of equine tendon lesions and endometrial inflammation. Glycoconjugates are involved in cellular recognition and in the efficient uptake of extracellular vesicles (EVs) by recipient cells. In this study, we evaluated the surface glycosylation pattern of eAMCs and their EVs from the eAMCs released in conditioned medium. We used a microarray procedure in which eAMCs and eAMC-EVs were spotted on microarray slides, and incubated with a panel of 14 biotinylated lectins and Cy3-conjugated streptavidin. Signal intensity was detected using a microarray scanner. Both eAMC and eAMC-EV microarrays interacted with all the lectins, indicating the presence of N- and O-linked glycans. With respect to eAMCs, eAMC-EVs, were found to be (1) enriched in Galß1,3GalNAc terminating O-glycans, α2,3-linked sialoglycans, and high-mannose N-glycans (Con A); (2) diminished in N-acetyllactosamine, GalNAc, Gal, GlcNAc, and fucose terminating glycans; and (3) unchanged in α2,6 linked sialoglycans content. These results suggest that eAMC-EVs emerge from a specific eAMC microdomain, and that the high simultaneous presence of Galß1,3GalNAc, α2,3 sialic acid, and high-mannose N-linked glycans may constitute markers of the eAMC-EVs. The role of these sugars in equine regenerative medicine requires further investigation.

Different Culture Times Affect MicroRNA Cargo in Equine Amniotic Mesenchymal Cells and Their Microvesicles.

Conditioned medium (CM) and microvesicles (MVs) are produced using different protocols: CM is collected following 12-96 h of cell culture without renewal of tissue culture medium, while MVs are collected after overnight cell culture. For future comparative studies in regenerative medicine looking at the efficacy of CM and MVs, it is important to understand how the quality of cell secretions is affected by culture. The aim of this study was to evaluate whether the duration of culturing influences the micro-RNAs (miRNAs) cargo of equine amniotic mesenchymal cells (AMCs) and their MVs. The analysis identified 990 miRNAs. After one night, there were 347 differently expressed (DE)-miRNAs between MVs and cells, whereas after four nights there were 359. About 58.3% of the DE-miRNAs were shared between samples produced under the two conditions. The comparison between miRNA content in AMC cells cultured for one night versus four nights showed eight DE-Equus caballus (eca)-miRNAs, which target genes were involved in immune response to external stimulus, inflammatory response, and production of reactive oxygen species. Comparing MVs isolated from one or four nights, four DE-miRNAs that target genes regulating cell cycle progression and production of reactive oxygen species were found, but only eca-miR-214 was enriched in the MVs after four nights. In conclusion, after 4 days of cell culture, the profile of AMC miRNAs was altered, indicating a probable phenotypic transition versus a new cell culture environment and aging. After this time, MVs accumulated eca-miR-214, which may help cells survive or adapt to new culture conditions.

Isolation, molecular characterization, and in vitro differentiation of bovine Wharton jelly-derived multipotent mesenchymal cells.

Extrafetal tissues are a noncontroversial and inexhaustible source of mesenchymal stem cells that can be harvested noninvasively at low cost. In the veterinary field, as in man, stem cells derived from extrafetal tissues express plasticity, reduced immunogenicity, and have high anti-inflammatory potential making them promising candidates for treatment of many diseases. Umbilical cord mesenchymal cells have been isolated and characterized in different species and have recently been investigated as potential candidates in regenerative medicine. In this study, cells derived from bovine Wharton jelly (WJ) were isolated for the first time by enzymatic methods, frozen/thawed, cultivated for at least 10 passages, and characterized. Wharton jelly-derived cells readily attached to plastic culture dishes displaying typical fibroblast-like morphology and, although their proliferative capacity decreased to the seventh passage, these cells showed a mean doubling time of 34.55 ± 6.33 hours and a mean frequency of one colony-forming unit fibroblast like for every 221.68 plated cells. The results of molecular biology studies and flow cytometry analyses revealed that WJ-derived cells showed the typical antigen profile of mesenchymal stem cells and were positive for CD29, CD44, CD105, CD166, Oct-4, and c-Myc. They were negative for CD34 and CD14. Remarkably, WJ-derived cells showed differentiation ability. After culture in induced media, WJ-derived cells were able to differentiate into osteogenic, adipogenic, chondrogenic, and neurogenic lines as shown by positive staining and expression of specific markers. On polymerase chain reaction analysis, these cells were negative for MHC-II and positive for MHC-I, thus reinforcing the role of extrafetal tissue as an allogenic source for bovine cell-based therapies. These results provide evidence that bovine WJ-derived cells may have the potential to differentiate to repair damaged tissues and reinforce the importance of extrafetal tissues as stem cell sources in veterinary regenerative medicine. A more detailed evaluation of their immunologic properties is necessary to better understand their potential role in cellular therapy.

Microvesicles secreted from equine amniotic-derived cells and their potential role in reducing inflammation in endometrial cells in an in-vitro model.

BACKGROUND: It is known that a paracrine mechanism exists between mesenchymal stem cells and target cells. This process may involve microvesicles (MVs) as an integral component of cell-to-cell communication. METHODS: In this context, this study aims to understand the efficacy of MVs in in-vitro endometrial stressed cells in view of potential healing in in-vivo studies. For this purpose, the presence and type of MVs secreted by amniotic mesenchymal stem cells (AMCs) were investigated and the response of endometrial cells to MVs was studied using a dose-response curve at different concentrations and times. Moreover, the ability of MVs to counteract the in vitro stress in endometrial cells induced by lipopolysaccharide was studied by measuring the rate of apoptosis and cell proliferation, the expression of some pro-inflammatory genes such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin 1ß (IL-1ß), and metalloproteinases (MMP) 1 and 13, and the release of some pro- or anti-inflammatory cytokines. RESULTS: MVs secreted by the AMCs ranged in size from 100 to 200 nm. The incorporation of MVs was gradual over time and peaked at 72 h. MVs reduced the apoptosis rate, increased cell proliferation values, downregulated pro-inflammatory gene expression, and decreased the secretion of pro-inflammatory cytokines. CONCLUSION: Our data suggest that some microRNAs could contribute to counteracting in-vivo inflammation of endometrial tissue.

Effects of platelet-rich plasma in a model of bovine endometrial inflammation in vitro.

BACKGROUND: Endometritis reduces fertility and is responsible for major economic losses in beef and dairy industries. The aim of this study was to evaluate an alternative therapy using platelet-rich plasma (PRP). PRP was tested in vivo, after bovine intrauterine administration, and in vitro on endometrial cells. METHODS: Bovine endometrial cells were cultured until passage (P) 10 with 5 % or 10 % PRP. Effect of PRP on endometrial cell proliferation and on the expression of genes [prostaglandin-endoperoxide synthase 2 (COX2), tumor protein p53 (TP53), oestrogen receptors (ER-α and ER-ß), progesterone receptor (PR) and c-Myc] involved in the regulation of oestrus cycle and fetal-maternal interaction were evaluated. Moreover, to evaluate the ability of PRP to counteract inflammation, 10 and 100 ng/ml of bacterial endotoxin lipopolysaccharide (LPS) were used to inflame endometrial cells in vitro for 1, 6, 12, 24 and 48 h. The expression of genes such as interleukin 1ß (IL-1ß), interleukin-8 (IL-8), inducible nitric oxide synthase (iNOS), prostaglandin-endoperoxide synthase 2 (COX2/PTGS2), and the release of PGE-2, IL-1ß and IL-8 were evaluated. RESULTS: In vivo treatment with PRP increased the detection of PR. In vitro, 5 % PRP at passage 5 increased proliferation rate and induced a significant increase in the expression of all studied genes. Furthermore, the results revealed that 10 ng/ml of LPS is the most effective dose to obtain an inflammatory response, and that PRP treatment significantly down regulated the expression of pro-inflammatory genes. CONCLUSION: This study lays the foundations for the potential treatment of endometritis with PRP in vivo.

Evaluation of amniotic mesenchymal cell derivatives on cytokine production in equine alveolar macrophages: an in vitro approach to lung inflammation.

BACKGROUND: Data obtained in both animal models and clinical trials suggest that cell-based therapies represent a potential therapeutic strategy for lung repair and remodeling. Recently, new therapeutic approaches based on the use of stem cell derivatives (e.g., conditioned medium (CM) and microvesicles (MVs)) to regenerate tissues and improve their functions were proposed. The aim of this study was to investigate the immunomodulatory effects of equine amniotic mesenchymal cell derivatives on lipopolysaccharide (LPS)-induced cytokine production in equine alveolar macrophages, which may be beneficial in lung inflammatory disorders such as recurrent airway obstruction (RAO) in horses. RAO shares many features with human asthma, including an increased number of cells expressing mRNA for interleukin (IL)-4 and IL-5 and a decreased expression of IFN-γ in bronchoalveolar lavage fluid (BALF) of affected horses. METHODS: The release of TNF-α, IL-6, and TGF-ß1 at different time points (1, 24, 48, and 72 h) was measured in equine alveolar macrophages stimulated or not with LPS (10 and 100 ng/mL) in the presence or absence of 10 % CM or 50 × 10(6) MVs/mL. Cytokines were measured using commercially available ELISA kits. For multiple comparisons, analysis of variance was used with Tukey post-hoc test. Differences were considered significant at p ≤ 0.05. RESULTS: Significant modulatory effects of CM on LPS-induced TNF-α release at 24 h, and of both CM and MVs on TNF-α release at 48 h were observed. A trend toward a modulatory effect of both CM and MVs on the release of TGF-ß and possibly IL-6 was visible over time. CONCLUSIONS: Results support the potential use of CM and MVs in lung regenerative medicine, especially in situations in which TGF-ß may be detrimental, such as respiratory allergy. Further studies should evaluate the potential clinical applications of CM and MVs in equine lung diseases, such as RAO and other inflammatory disorders.

Does the Bovine Pre-Ovulatory Follicle Harbor Progenitor Stem Cells?

Recent studies have revealed the presence of a mesenchymal stem cell (MSC) population in human and in gilt granulosa cells (GCs), thus increasing the interest in identifying the same population in the bovine species. We first isolated GCs by scraping from bovine preovulatory follicles and then tested several different media to define the ideal conditions to select granulosa-derived stem cells. Although expressing MSC-associated markers, none of the media tested proven to be efficient in selecting MSC-like cells that were able to differentiate into mesodermic or ectodermic lineages. We performed another experimental approach exposing cells to a chemical stress, such as lowering of pH, as a system to select a more plastic population. Following the treatment, granulosa-specific granulose markers [follicle-stimulating hormone receptor (FSHR), follistatin (FST), and leukemia inhibitory factor receptor (LIFR)] were lost in bovine GCs, whereas an increase in multi- (CD29, CD44, CD73) and pluripotent (Oct-4 and c-Myc) genes was noticed. The stress allowed up-regulation of tumor necrosis factor-α and interleukin-1ß expression and the dedifferentiation of GCs, which was demonstrated by differentiation studies. Indeed, pH-treated cells were able to differentiate into the mesodermic and ectodermic lineages, thus suggesting that the chemical stress allows for the selection of cells that are more prone to adjust and respond to the environmental changes.

Equine Amniotic Microvesicles and Their Anti-Inflammatory Potential in a Tenocyte Model In Vitro.

Administration of horse amniotic mesenchymal cells (AMCs) and their conditioned medium (AMC-CM) improves the in vivo recovery of spontaneous equine tendon lesions and inhibits in vitro proliferation of peripheral blood mononuclear cells (PBMC). This process may involve microvesicles (MVs) as an integral component of cell-to-cell communication during tissue regeneration. In this study, the presence and type of MVs secreted by AMCs were investigated and the response of equine tendon cells to MVs was studied using a dose-response curve at different concentrations and times. Moreover, the ability of MVs to counteract in vitro inflammation of tendon cells induced by lipopolysaccharide was studied through the expression of some proinflammatory genes such as metallopeptidase (MPP) 1, 9, and 13 and tumor necrosis factor-α (TNFα), and expression of transforming growth factor-ß (TGF-ß). Lastly, the immunomodulatory potential of MVs was investigated. Results show that AMCs secrete MVs ranging in size from 100 to 200 nm. An inverse relationship between concentration and time was found in their uptake by tendon cells: the maximal uptake occurred after 72 h at a concentration of 40 × 10(6) MVs/mL. MVs induced a downregulation of MMP1, MMP9, MMP13, and TNFα expression without affecting PBMC proliferation, contrary to CM and supernatant. Our data suggest that MVs contribute to in vivo healing of tendon lesions, alongside soluble factors in AMC-CM.

Leptin and leptin receptor are detectable in equine spermatozoa but are not involved in in vitro fertilisation.

In human and swine, leptin (OB) has been identified in seminal plasma and leptin receptors (OB-R) on the cell surface of spermatozoa, indicating that spermatozoa are a target for OB. This hormone has also been detected in follicular fluid (FF) in women and mares, although its role requires further study. The aims of this study were to investigate the immunolocalisation and the expression of OB and OB-R in equine spermatozoa and to evaluate the involvement of OB in equine in vitro fertilisation (IVF). Since progesterone (P) and OB are both found in FF, the individual and combined effects of these two hormones were studied in equine IVF and compared with the results obtained from the use of FF for in vitro sperm preparation. For the first time, we were able to identify OB and OB-R mRNA and their corresponding proteins in equine spermatozoa. When spermatozoa were treated with OB, there was a decrease in the three motility parameters VSL, STR and LIN, commonly associated with hyperactivation, whilst the acrosome reaction rate increased (P<0.05). The fertilisation rate was 51% with FF, 46.15% with P, 43.64% with P+OB and 0% with OB alone. The percentage of eight-cell stage embryos was 18.7% with FF, 17.1% with P and 16.7% with OB+P. OB alone did not permit oocyte fertilisation, indicating that, in the horse, OB is involved in capacitation and hyperactivation but not in sperm penetration.

Peculiarity of Porcine Amniotic Membrane and Its Derived Cells: A Contribution to the Study of Cell Therapy from a Large Animal Model.

The aim of this work was to provide, for the first time, a protocol for isolation and characterization of stem cells from porcine amniotic membrane in view of their potential uses in regenerative medicine. From three samples of allanto-amnion recovered at delivery, the amniotic membrane was stripped from overlying allantois and digested with trypsin and collagenase to isolate epithelial (amniotic epithelial cells [AECs]) and mesenchymal cells, respectively. Proliferation, differentiation, and characterization studies by molecular biology and flow cytometry were performed. Histological examination revealed very few mesenchymal cells in the stromal layer, and a cellular yield of AECs of 10 × 10(6)/gram of digested tissue was achieved. AECs readily attached to plastic culture dishes displaying typical cuboidal morphology and, although their proliferative capacity decreased to the fifth passage, AECs showed a mean doubling time of 24.77 ± 6 h and a mean frequency of one fibroblast colony-forming unit (CFU-F) for every 116.75 plated cells. AECs expressed mesenchymal stem cell (MSC) mRNA markers (CD29, CD166, CD90, CD73, CD117) and pluripotent markers (Nanog and Oct 4), whereas they were negative for CD34 and MHCII. Mesodermic, ectodermic, and endodermic differentiation was confirmed by staining and expression of specific markers. We conclude that porcine amniotic membrane can provide an attractive source of stem cells that may be a useful tool for biomedical research.

Amniotic membrane-derived mesenchymal cells and their conditioned media: potential candidates for uterine regenerative therapy in the horse.

Amniotic membrane-derived mesenchymal cells (AMCs) are considered suitable candidates for a variety of cell-based applications. In view of cell therapy application in uterine pathologies, we studied AMCs in comparison to cells isolated from the endometrium of mares at diestrus (EDCs) being the endometrium during diestrus and early pregnancy similar from a hormonal standpoint. In particular, we demonstrated that amnion tissue fragments (AM) shares the same transcriptional profile with endometrial tissue fragments (ED), expressing genes involved in early pregnancy (AbdB-like Hoxa genes), pre-implantation conceptus development (Erα, PR, PGRMC1 and mPR) and their regulators (Wnt7a, Wnt4a). Soon after the isolation, only AMCs express Wnt4a and Wnt7a. Interestingly, the expression levels of prostaglandin-endoperoxide synthase 2 (PTGS2) were found greater in AM and AMCs than their endometrial counterparts thus confirming the role of AMCs as mediators of inflammation. The expression of nuclear progesterone receptor (PR), membrane-bound intracellular progesterone receptor component 1 (PGRMC1) and membrane-bound intracellular progesterone receptor (mPR), known to lead to improved endometrial receptivity, was maintained in AMCs over 5 passages in vitro when the media was supplemented with progesterone. To further explore the potential of AMCs in endometrial regeneration, their capacity to support resident cell proliferation was assessed by co-culturing them with EDCs in a transwell system or culturing in the presence of AMC-conditioned medium (AMC-CM). A significant increase in EDC proliferation rate exhibited the crucial role of soluble factors as mediators of stem cells action. The present investigation revealed that AMCs, as well as their derived conditioned media, have the potential to improve endometrial cell replenishment when low proliferation is associated to pregnancy failure. These findings make AMCs suitable candidates for the treatment of endometrosis in mares.

Cell Surface Glycan Changes in the Spontaneous Epithelial-Mesenchymal Transition of Equine Amniotic Multipotent Progenitor Cells.

Amniotic epithelial cells (AECs) spontaneously transform into amniotic mesenchymal cells (AMCs) in vitro during cell culture. Glycocalyx was analyzed to identify the glycan pattern in AECs, AMCs and epithelial-mesenchymal transdifferentiated cells (EMTCs). Pure cell cultures were derived using cloned AEC and AMC cell lines obtained by the dilution technique from amniotic membranes. Mesenchymal cells generated by differentiation of clonal epithelial cells were considered transdifferentiated. Immunocytoscreen, in vitro multipotent differentiation and molecular characterization of EMTCs were performed. In combination with saponification and sialidase digestion, a panel of 12 lectins was used to analyze the glycan pattern of AEC, AMC and EMTC glycocalyx. Cytokeratin cell markers were lost in EMTCs and typical mesenchymal markers, such as vimentin, appeared. These cells retained their differentiation potential. Lectin histochemistry revealed a cell-specific glycan profile. Galactose (Gal)ß1,4GlcNAc, Neu5Acα2,6Gal/GalNAc and N-acetyl neuraminic (sialic) acid (NeuNAc)α2,3Galß1,3(±NeuNAcα2,6)GalNAc were highly expressed on the surface of all the amniotic cell cultures. AECs expressed asialoglycans with terminal GalNAc and GlcNAc. More highly mannosylated N-linked glycans and NeuNAcα2,3Galß1,3GalNAc in O-linked glycans were expressed by EMTCs, but these cells had fewer glycans ending with fucose (Fuc), Gal, GlcNAc and GalNAc than AECs. GlcNAc- and GalNAc-terminating glycans were similarly expressed on the glycocalyx of the mesenchymal cell populations (EMTCs and AMCs). These results demonstrate for the first time that the spontaneous epithelial-mesenchymal transition (EMT) of equine amnion cells is characterized by cell surface glycan remodeling and that glycosylation changes result in a cell type-specific glycan profile. The glycopattern of equine amnion spontaneous EMTCs differs from EMT of tumoral cells.

Conditioned medium from horse amniotic membrane-derived multipotent progenitor cells: immunomodulatory activity in vitro and first clinical application in tendon and ligament injuries in vivo.

We have recently demonstrated that heterologous transplantation of horse amniotic membrane-derived mesenchymal cells (AMCs) can be useful for cell therapy applications in tendon diseases, and hypothesized that these cells may promote tendon repair via paracrine-acting molecules targeting inflammatory processes. To test this hypothesis, here we examined the immunomodulatory characteristics of AMCs and of their conditioned medium (AMC-CM) in vitro, and studied the potential therapeutic effect of AMC-CM in thirteen different spontaneous horse tendon and ligament injuries in vivo. Our results demonstrate that AMCs are capable of inhibiting peripheral blood mononuclear cell (PBMC) proliferation after allogenic stimulation either when cocultured in cell-to-cell contact, or when the two cell types are physically separated by a transwell membrane, suggesting that soluble factors are implicated in this phenomenon. Our hypothesis is further supported by the demonstration that PBMC proliferation is inhibited by AMC-CM. In our in vivo studies, no significant adverse effects were observed in treated tendons, and clinical and ultrasonographical evaluation did not reveal evidence of inappropriate tissue or tumor formation. Clinical outcomes were favorable and the significantly lower rate (15.38%) of reinjuries observed compared to untreated animals, suggests that treatment with AMC-CM is very efficacious. In conclusion, this study identifies AMC-CM as a novel therapeutic biological cell-free product for treating horse tendon and ligament diseases.

Investigating the efficacy of amnion-derived compared with bone marrow-derived mesenchymal stromal cells in equine tendon and ligament injuries.

BACKGROUND AIMS: This is the first study to compare the treatment of horse tendon and ligament injuries with the use of mesenchymal stromal cells (MSCs) obtained from two different sources: amniotic membrane (AMSCs) and bone marrow (BM-MSCs). The objective was to prove the ability of AMSCs to exert beneficial effects in vivo. METHODS: Five million allogeneic frozen-thawed AMSCs or autologous fresh BM-MSCs were injected intralesionally in horses belonging to group A (51 horses) and group B (44 horses). The interval lesion/implantation was of 6-15 days for the AMSCs and 16-35 days for the BM-MSCs. Healing was assessed clinically and ultrasonographically. Follow-up was monitored for 2 further years from return to full work. RESULTS: No significant adverse effects after MSCs treatment were seen in any of the horses studied, independent of the type of stromal cell implanted. All animals belonging to group A resumed their activities between 4-5 months after treatment, whereas animals of group B resumed their activities after 4-12 months. The rate of re-injury in horses treated with AMSCs is lower (4.00%) compared with the average observed when horses were treated with BM-MSCs (23.08%). CONCLUSIONS: The possibility to inject allogeneic AMSCs in real time, before any ultrasonographic change occurs within the injured tendon and ligament, together with the higher plasticity and proliferative capacity of these cells compared with BM-MSCs, represents the main features of interest for this novel approach for the treatment of equine tendon diseases. An obvious active proliferative healing in the area injected with AMSCs makes these cells more effective than BM-MSCs.

Molecular characterization and in vitro differentiation of feline progenitor-like amniotic epithelial cells.

INTRODUCTION: While amniotic mesenchymal cells have been isolated and characterized in different species, amniotic epithelial cells (AECs) have been found only in humans and horses and are recently considered valid candidates in regenerative medicine. The aim of this work is to obtain and characterize, for the first time in the feline species, presumptive stem cells from the epithelial portion of the amnion (AECs) to be used for clinical applications. METHODS: In our study, we molecularly characterized and induced in vitro differentiation of feline AECs, obtained after enzymatic digestion of amnion. RESULTS: AECs displayed a polygonal morphology and the mean doubling time value was 1.94 ± 0.04 days demonstrating the high proliferating capacity of these cells. By RT-PCR, AECs expressed pluripotent (Oct4, Nanog) and some mesenchymal markers (CD166, CD44) suggesting that an epithelial-mesenchymal transition may occur in these cells that lack the hematopoietic marker CD34. Cells also showed the expression of embryonic marker SSEA-4, but not SSEA-3, as demonstrated by immunocytochemistry and flow cytometry. Moreover, the possibility to use feline AECs in cell therapies resides in their low immunogenicity, due to the absence of MHC-II antigen expression. After induction, AECs differentiated into the mesodermic and ectodermic lineages, demonstrating high plasticity. CONCLUSIONS: In conclusion, feline AECs appear to be a readily obtainable, highly proliferative, multipotent and non-immunogenic cell line from a source that may represent a good model system for stem cell biology and be useful in allogenic cell-based therapies in order to treat tissue lesions, especially with loss of substance.