Short preconditioning with TGFß of equine adipose tissue-derived mesenchymal stem cells predisposes towards an anti-fibrotic secretory phenotype: A possible tool for treatment of endometrosis in mares.

Endometrosis in mares is a disease resulting from chronic inflammation characterized by peri glandular fibrosis. There is no effective treatment so far, which opens the door for exploring the use of stem cells as a candidate. Transforming growth factor beta (TGFß) is crucial for the establishment and progression of fibrosis in mare's endometrosis. We aimed to develop regenerative approaches to treat endometrosis by using mesenchymal stem cells (MSC), for which understanding the effect of TGFß on exogenous MSC is crucial. We isolated and characterized equine adipose MSC from six donors. Cells were pooled and exposed to 10 ng/ml of TGFß for 0, 4, and 24 h, after which cells were analyzed for proliferation, migration, mesodermal differentiation, expression of fibrosis-related mRNAs, and prostaglandin E2 secretion. At 24 h of exposition to TGFß, there was a progressive increase in the contraction of the monolayer, leading to nodular structures, while cell viability did not change. Exposure to TGFß impaired adipogenic and osteogenic differentiation after 4 h of treatment, which was more marked at 24 h, represented by a decrease in Oil red and Alizarin red staining, as well as a significant drop (p < 0.05) in the expression of key gene regulators of differentiation processes (PPARG for adipose and RUNX2 for osteogenic differentiation). TGFß increased chondrogenic differentiation as shown by the upsurge in size of the resulting 3D cell pellet and intensity of Alcian Blue staining, as well as the significant up-regulation of SOX9 expression (p < 0.05) at 4 h, which reached a maximum peak at 24 h (p < 0.01), indicative of up-regulation of glycosaminoglycan synthesis. Preconditioning MSC with TGFß led to a significant increase (p < 0.05) in the expression of myofibroblast gene markers aSMA, COL1A1, and TGFß at 24 h exposition time. In contrast, the expression of COL3A1 did not change with respect to the control but registered a significant downregulation compared to 4 h (p < 0.05). TGFß also affected the expression of genes involved in PGE2 synthesis and function; COX2, PTGES, and the PGE2 receptor EP4 were all significantly upregulated early at 4 h (p < 0.05). Cells exposed to TGFß showed a significant upregulation of PGE2 secretion at 4 h compared to untreated cells (p < 0.05); conversely, at 24 h, the PGE2 values decreased significantly compared to control cells (p < 0.05). Preconditioning MSC for 4 h led to an anti-fibrotic secretory phenotype, while a longer period (24 h) led to a pro-fibrotic one. It is tempting to propose a 4-h preconditioning of exogenous MSC with TGFß to drive them towards an anti-fibrotic phenotype for cellular and cell-free therapies in fibrotic diseases such as endometrosis of mares.

DNA Content in Embryonic Extracellular Vesicles Is Independent of the Apoptotic Rate in Bovine Embryos Produced In Vitro.

Pre-implantation embryos release extracellular vesicles containing different molecules, including DNA. The presence of embryonic DNA in E-EVs released into the culture medium during in vitro embryo production could be useful for genetic diagnosis. However, the vesicles containing DNA might be derived from embryos suffering from apoptosis, i.e., embryos of bad quality. This work intended to confirm that embryos release DNA that is useful for genotyping by evaluating the effect of embryonic apoptosis on DNA content in E-EVs. Bovine embryos were produced by parthenogenesis and in vitro fertilization (IVF). On Day 5, morulae were transferred to individual cultures in an EV-depleted SOF medium. On Day 7, embryos were used to evaluate cellular apoptosis, and each culture medium was collected to evaluate E-EV concentration, characterization, and DNA quantification. While no effect of the origin of the embryo on the apoptotic rate was found, arrested morulae had a higher apoptotic rate. E-EVs containing DNA were identified in all samples, and the concentration of those vesicles was not affected by the origin or quality of the embryos. However, the concentration of DNA was higher in EVs released by the arrested parthenogenetic embryos. There was a correlation between the concentration of E-EVs, the concentration of DNA-positive E-EVs, and the concentration of DNA. There was no negative effect of apoptotic rate on DNA-positive E-EVs and DNA concentration; however, embryos of the best quality with a low apoptotic rate still released EVs containing DNA. This study confirms that the presence of DNA in E-EVs is independent of embryo quality. Therefore, E-EVs could be used in liquid biopsy for noninvasive genetic diagnosis.

Proteomic Analysis of Domestic Cat Blastocysts and Their Secretome Produced in an In Vitro Culture System without the Presence of the Zona Pellucida.

Domestic cat blastocysts cultured without the zona pellucida exhibit reduced implantation capacity. However, the protein expression profile has not been evaluated in these embryos. The objective of this study was to evaluate the protein expression profile of domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were generated: (1) domestic cat embryos generated by IVF and cultured in vitro (zona intact, (ZI)) and (2) domestic cat embryos cultured in vitro without the zona pellucida (zona-free (ZF group)). The cleavage, morula, and blastocyst rates were estimated at days 2, 5 and 7, respectively. Day 7 blastocysts and their culture media were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). The UniProt Felis catus database was used to identify the standard proteome. No significant differences were found in the cleavage, morula, or blastocyst rates between the ZI and ZF groups (p > 0.05). Proteomic analysis revealed 22 upregulated and 20 downregulated proteins in the ZF blastocysts. Furthermore, 14 proteins involved in embryo development and implantation were present exclusively in the culture medium of the ZI blastocysts. In conclusion, embryo culture without the zona pellucida did not affect in vitro development, but altered the protein expression profile and release of domestic cat blastocysts.

Mare stromal endometrial cells differentially modulate inflammation depending on oestrus cycle status: an in vitro study.

The modulation of inflammation is pivotal for uterine homeostasis. Here we evaluated the effect of the oestrus cycle on the expression of pro-inflammatory and anti-inflammatory markers in a cellular model of induced fibrosis. Mare endometrial stromal cells isolated from follicular or mid-luteal phase were primed with 10 ng/mL of TGFß alone or in combination with either IL1ß, IL6, or TNFα (10 ng/mL each) or all together for 24 h. Control cells were not primed. Messenger and miRNA expression were analyzed using real-time quantitative PCR (RT-qPCR). Cells in the follicular phase primed with pro-inflammatory cytokines showed higher expression of collagen-related genes (CTGF, COL1A1, COL3A1, and TIMP1) and mesenchymal marker (SLUG, VIM, CDH2, and CDH11) genes; p < 0.05. Cells primed during the mid-luteal overexpressed genes associated with extracellular matrix, processing, and prostaglandin E synthase (MMP2, MMP9, PGR, TIMP2, and PTGES; p < 0.05). There was a notable upregulation of pro-fibrotic miRNAs (miR17, miR21, and miR433) in the follicular phase when the cells were exposed to TGFß + IL1ß, TGFß + IL6 or TGFß + IL1ß + IL6 + TNFα. Conversely, in cells from the mid-luteal phase, the treatments either did not or diminished the expression of the same miRNAs. On the contrary, the anti-fibrotic miRNAs (miR26a, miR29b, miR29c, miR145, miR378, and mir488) were not upregulated with treatments in the follicular phase. Rather, they were overexpressed in cells from the mid-luteal phase, with the highest regulation observed in TGFß + IL1ß + IL6 + TNFα treatment groups. These miRNAs were also analyzed in the extracellular vesicles secreted by the cells. A similar trend as seen with cellular miRNAs was noted, where anti-fibrotic miRNAs were downregulated in the follicular phase, while notably elevated pro-fibrotic miRNAs were observed in extracellular vesicles originating from the follicular phase. Pro-inflammatory cytokines may amplify the TGFß signal in the follicular phase resulting in significant upregulation of extracellular matrix-related genes, an imbalance in the metalloproteinases, downregulation of estrogen receptors, and upregulation of pro-fibrotic factors. Conversely, in the luteal phase, there is a protective role mediated primarily through an increase in anti-fibrotic miRNAs, a decrease in SMAD2 phosphorylation, and reduced expression of fibrosis-related genes.

Zona pellucida removal modifies the expression and release of specific microRNAs in domestic cat blastocysts.

The in vitro culture of domestic cat embryos without the zona pellucida affects their implantation capacity. MicroRNAs (miRNAs) have an important role in embryo-maternal communication and implantation. The objective of this study was to evaluate the expression of specific miRNAs in domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were done: (1) domestic cat embryos cultured with the zona pellucida (zona intact control group, ZI); and (2) cultured without the zona pellucida (zona free group, ZF). The cleavage, morula and blastocyst rates were evaluated. The blastocysts and their spent medium were used for miRNA expression analysis using RT-qPCR (miR-21, miR-24, mi25, miR-29, miR-96, miR-98, miR-103, miR-191, miR-196, miR-199, miR-130, miR-155 and miR-302). The pre-mature microRNAs (pre-miRNAs) and miRNAs were evaluated in the blastocysts and only miRNAs were evaluated in the spent medium. No differences were observed in the cleavage, morula and blastocyst rates between the ZF and ZI groups (P > 0.05). For miRNAs analysis, miR-103 and miR-191 had the most stable expression and were selected as internal controls. ZF blastocysts had a higher expression of miR-21, miR-25, miR-29 and miR-199 and a lower expression of miR-96 than their ZI counterparts (P < 0.05). Furthermore, higher levels of miR-21, miR-25 and miR-98 were detected in the spent medium of ZF blastocysts (P < 0.05). In conclusion, in vitro culture of domestic cat embryos without the zona pellucida modifies the expression of miR-21, miR-25, miR-29, miR-199 and miR-96 at the blastocyst stage and the release of miR-21, miR-25 and miR-98.

Extracellular Vesicles Secreted by Pre-Hatching Bovine Embryos Produced In Vitro and In Vivo Alter the Expression of IFNtau-Stimulated Genes in Bovine Endometrial Cells.

The embryo-maternal interaction occurs during the early stages of embryo development and is essential for the implantation and full-term development of the embryo. In bovines, the secretion of interferon Tau (IFNT) during elongation is the main signal for pregnancy recognition, but its expression starts around the blastocyst stage. Embryos release extracellular vesicles (EVs) as an alternative mechanism of embryo-maternal communication. The aim of the study was to determine whether EVs secreted by bovine embryos during blastulation (D5-D7) could induce transcriptomic modifications, activating IFNT signaling in endometrial cells. Additionally, it aims to assess whether the EVs secreted by embryos produced in vivo (EVs-IVV) or in vitro (EVs-IVP) have different effects on the transcriptomic profiles of the endometrial cells. In vitro- and in vivo-produced bovine morulae were selected and individually cultured for 48 h to collect embryonic EVs (E-EVs) secreted during blastulation. E-EVs stained with PKH67 were added to in vitro-cultured bovine endometrial cells to assess EV internalization. The effect of EVs on the transcriptomic profile of endometrial cells was determined by RNA sequencing. EVs from both types of embryos induced several classical and non-classical IFNT-stimulated genes (ISGs) and other pathways related to endometrial function in epithelial endometrial cells. Higher numbers of differentially expressed genes (3552) were induced by EVs released by IVP embryos compared to EVs from IVV (1838). Gene ontology analysis showed that EVs-IVP/IVV induced the upregulation of the extracellular exosome pathway, the cellular response to stimulus, and the protein modification processes. This work provides evidence regarding the effect of embryo origin (in vivo or in vitro) on the early embryo-maternal interaction mediated by extracellular vesicles.

Analysis of trophectoderm markers in domestic cat blastocysts cultured without zona pellucida.

Domestic cat embryos generated by in vitro fertilization (IVF) and cultured without the zona pellucida have a reduced implantation capacity after embryo transfer at the blastocyst stage. The objective of this study was to evaluate the expression of trophectoderm markers in domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were selected: (1) domestic cat embryos generated by IVF and cultured in vitro normally (zona intact group, ZI); and (2) domestic cat embryos generated by IVF and cultured in vitro without a zona pellucida (zona-free group, ZF). In the ZF group, the zona pellucida of the presumptive zygote was removed and these were cultured using the well of the well (WOW) system. In vitro culture was carried out for 7 days. The cleavage, morula and blastocyst rates were estimated. Finally, the relative expression levels of the trophectoderm markers TEAD4, YAP1, CDX2 and EOMES, the cell adhesion marker E-cadherin and the apoptosis marker CASP3 were evaluated by RT-qPCR in the blastocysts. The Wilcoxon test was used to evaluate differences (P < 0.05). No differences were observed in the cleavage, morula and blastocyst rates between the ZF and ZI groups. No differences were found in the expression of TEAD4, CDX2, E-cadherin and CASP3 between groups. The expression of YAP1 and EOMES was higher in ZF blastocysts than in ZI blastocysts. In conclusion, the in vitro culture without the zona pellucida generates an overexpression of YAP1 and EOMES in the domestic cat blastocysts. More studies are needed to confirm if this overexpression might affect in vivo development.

Disruption of the Blood-Brain Barrier by Extracellular Vesicles From Preeclampsia Plasma and Hypoxic Placentae: Attenuation by Magnesium Sulfate.

[Figure: see text].

Domestic cat embryos generated without zona pellucida are capable of developing in vitro but exhibit abnormal gene expression and a decreased implantation rate.

The removal of the zona pellucida has been used to improve the in vitro development of domestic cat embryos generated by IVF and SCNT. However, the in vivo development of domestic cat embryos generated without the zona pellucida has not been evaluated. The objective of this study was to evaluate the effects of zona pellucida removal on the in vitro and in vivo development of domestic cat embryos generated by IVF. For this purpose, two experimental groups were created: 1) domestic cat embryos cultured in vitro (Zona-intact group, ZI) and 2) domestic cat embryos cultured in vitro without the zona pellucida (Zona-free group, ZF). Domestic cat embryos were generated by IVF and cultured in vitro for 8 days. In the ZF group, the zona pellucida was removed after IVF, and embryos were cultured using the well of the well system (WOW). Cleavage, morula and blastocyst rates were evaluated in both groups. The diameter and total cell number of blastocysts were assessed. Relative expression of pluripotency (OCT4, SOX2 and NANOG), differentiation (CDX2 and GATA6) and apoptotic markers (BAX and BCL2) was evaluated in blastocysts. Finally, to evaluate in vivo development, embryos at days 5, 6 and 7 of development were transferred into recipient domestic cats, and ultrasonography was performed to evaluate implantation. No differences were observed in the cleavage, morula or blastocyst rates between embryos from the ZI and ZF groups. The diameter (mean ± SD) of blastocysts from the ZF group was greater (253.4 ± 83.3 µm) than that from the ZI group (210.5 ± 78.5 µm). No differences were observed in the relative expression of OCT4, CDX2 or GATA6. However, the relative expression of SOX2 and NANOG was significantly reduced in ZF blastocysts compared to ZI blastocysts. Furthermore, the relative expression of BAX was higher in ZF blastocysts than in ZI blastocysts. Finally, four pregnancies were confirmed after the transfer of ZI embryos (n = 110). However, no pregnancies were observed after the transfer of ZF embryos at the morula or blastocyst stage (n = 56). In conclusion, domestic cat embryos cultured without the zona pellucida were able to develop in vitro until the blastocyst stage. However, the removal of the zona pellucida negatively affected the gene expression of pluripotency and apoptosis markers, and ZF embryos were unable to implant. This might indicate that the removal of the zona pellucida is detrimental for the implantation and in vivo development of domestic cat embryos.

Advantages in Wound Healing Process in Female Mice Require Upregulation A2A-Mediated Angiogenesis under the Stimulation of 17ß-Estradiol.

Estrogenic steroids and adenosine A2A receptors promote the wound healing and angiogenesis processes. However, so far, it is unclear whether estrogen may regulate the expression and pro-angiogenic activity of A2A receptors. Using in vivo analyses, we showed that female wild type (WT) mice have a more rapid wound healing process than female or male A2A-deficient mice (A2AKO) mice. We also found that pulmonary endothelial cells (mPEC) isolated from female WT mice showed higher expression of A2A receptor than mPEC from male WT mice. mPEC from female WT mice were more sensitive to A2A-mediated pro-angiogenic response, suggesting an ER and A2A crosstalk, which was confirmed using cells isolated from A2AKO. In those female cells, 17ß-estradiol potentiated A2A-mediated cell proliferation, an effect that was inhibited by selective antagonists of estrogen receptors (ER), ERα, and ERß. Therefore, estrogen regulates the expression and/or pro-angiogenic activity of A2A adenosine receptors, likely involving activation of ERα and ERß receptors. Sexual dimorphism in wound healing observed in the A2AKO mice process reinforces the functional crosstalk between ER and A2A receptors.

Distinctive Cellular Transcriptomic Signature and MicroRNA Cargo of Extracellular Vesicles of Horse Adipose and Endometrial Mesenchymal Stem Cells from the Same Donors.

Equine endometrial and adipose mesenchymal stem cells (eMSCs and aMSCs, respectively) were isolated from the same donors of thoroughbred mares. The cells displayed characteristic features of MSCs, including trilineage mesodermal and also neurogenic differentiation. We evaluated the influence of cellular origin on their transcriptome profile. Cellular RNA was isolated and sequenced and extracellular vesicles (EVs) were obtained from conditioned medium of cells cultured in medium depleted of EVs, and their microRNA (miRNA) cargo analyzed by sequencing. Differential expression of mRNAs and EV-miRNA was analyzed, as well as pathways and processes most represented in each cell origin. mRNA reads from all expressed genes clustered according to the cellular origin. A total of 125 up- and 51 downregulated genes were identified and 31 differentially expressed miRNAs. Based on mRNA sequencing, endometrial MSCs strongly upregulated genes involved in the Hippo, transforming growth factor beta, and pluripotency signaling pathways. Alongside with this, pathways involved in extracellular matrix reorganization were the most represented in the miRNA cargo of EVs secreted by eMSCs. The niche from which MSCs originated defined the transcriptomic signature of the cells, including the secretion of lineage-specific loaded EV to ensure proper communication and homeostasis. Identification and testing their biological functions can provide new tools for the therapeutic use of horse MSC.

Embryo aggregation allows the production of kodkod (Leopardus guigna) blastocysts after interspecific SCNT.

The kodkod (Leopardus guigna) is a small felid endemic of Chile and is considered a vulnerable species. Domestic cat oocytes have been successfully used as recipient cytoplast to reprogram somatic cells from different felids by interspecific somatic cell nuclear transfer (iSCNT). The developmental competence of felid embryos generated by iSCNT can be improved by the aggregation method using a zona-free culture system. The objective of this research was to evaluate the developmental competence of kodkod embryos generated by iSCNT using domestic cat oocytes and the aggregation method. For this purpose, five experimental group were done: (1) cat embryos generated by IVF, (2) cat embryos generated by SCNT (Ca1x), (3) aggregated cat embryos generated by SCNT (Ca2x), (4) kodkod embryos generated by iSCNT (K1x) and (5) aggregated kodkod embryos generated by iSCNT (K2x). Cleavage, morulae and blastocyst rates were estimated. The blastocyst diameter was evaluated. The gene expression level of pluripotency (OCT4, SOX2 and NANOG) and differentiation markers (CDX2 and GATA6) was analyzed in blastocysts. Morulae rate was higher in the IVF group and when cloned embryos were cultured in aggregates (IVF: 68.2%, Ca2x: 58.0% and K2x: 62.4%) compared to individually cultured kodkod embryos (K1x: 37.0%) (P < 0.05). Embryo aggregation increased blastocysts formation in the Ca2x group (30.9%) to a similar rate compared to the IVF group (44.5%) (P > 0.05). No blastocysts were generated in the K1x group, whereas blastocysts formation was obtained in K2x group (5.9%). The diameter of blastocysts from the K2x group (172.8 µm) was significantly lower than blastocysts from the Ca2x group (P < 0.05). The relative expression of OCT4 was lower in blastocysts from Ca1x than in blastocysts from IVF (P < 0.05). Furthermore, CDX2 expression was lower in blastocysts from Ca2x than in blastocysts from Ca1x and IVF groups (P < 0.05). In kodkod embryos, only one blastocyst from the K2x group expressed OCT4. No expression of SOX2, NANOG, CDX2 and GATA6 was detected in kodkod blastocysts. In conclusion, after iSCNT, domestic cat oocytes support the development of kodkod embryos until the morula stage. The aggregation method increases the morulae rate of kodkod cloned embryos and allows blastocysts formation. However, kodkod blastocysts have a poor morphological quality and a lacking expression of pluripotency and differentiation markers, probably caused by an incomplete nuclear reprogramming.

Characterization of mesenchymal stem cells derived from adipose tissue of a cougar (Puma concolor).

Adipose derived mesenchymal stem cells (AMSCs) have been isolated from domestic and wild cats. For wild cats, the isolation of AMSCs has been reported in the black-footed cats (Felis nigripes) and guigna (Leopardus guigna). Stromal vascular fraction (SVF) isolated from cougar adipose tissue have been used to restore elbow functionality in the cougar (Puma concolor) but multipotent characteristics of these cells have not been described. The present study describes for the first time the isolation and characterization of mesenchymal stem cells derived from adipose tissue of cougar. AMSCs and fibroblasts from six months female cougar were isolated and cultured in DMEM/F12, supplemented with FBS 10% + 1% Antibiotic/Antifungal + 2.4 mM L-Glutamine + 2.4 mM pyruvate up to passage 5. Expression of pluripotent and surface marker genes was evaluated at mRNA level. Mesodermal differentiation (adipogenic, osteogenic and chondrogenic) was described. AMSCs expressed mRNA of pluripotent genes Oct4, Nanog, Sox2 and Klf4 and surface markers Cd44, Cd90, Cd105 and MHCII. Fibroblasts showed similar mRNA expression with the exception of Sox2. AMSCs obtained from cougar exhibit multipotency features similar to domestic cats MSC, nevertheless, other analyses are required. AMSCs from cougar could be a source of interest for treatment of individuals that remain in captivity or arrive to wildlife rehabilitation centers.

Assessment of the anti-inflammatory and engraftment potential of horse endometrial and adipose mesenchymal stem cells in an in vivo model of post breeding induced endometritis.

Horse mesenchymal stem cells (MSC) are potential anti-inflammatory tools for post-breeding induced endometritis (PBIE). In this research MSCs isolated from the endometrium or subcutaneous fat of the same donors were infused iu into mares with PBIE for assessment of their anti-inflammatory action and engraftment. PBIE was induced in nine gynecologically healthy mares by iu infusion of 500 million dead sperm in saline. Inflammatory markers were analyzed in uterine lavages and biopsies immediately before (phase I) and 3 h after infusion of sperm (phase II). Measurements: polymorph nuclear cells (PMN), proteins IL-6 and TNFα (ELISA in the lavages) and immunostaining in biopsies, transcripts of IL-1α, 6, 8, 10, TNFα and COX2 (qPCR of pelleted lavages). At 24 h after sperm deposition (phase III), mares were instilled iu with 20 ml of saline containing 2 × 107 adipose MSCs (n = 3, group 1) or endometrial MSCs (n = 3, group 2). Cells were labeled previously with carboxyfluorescein diacetate succinimidyl ester (CFDA SE). A third group (n = 3) received 20 mL of sterile saline alone. After 48 h another biopsy/lavage were done and the same parameters analyzed. For engraftment, additional biopsies were taken at days 10 and 30 of sperm infusion and analyzed by confocal microscopy. Dead sperm in saline markedly increased PMNs counts, IL-6 and TNFα expression in the ELISA (p < 0.05) and immunostaining. In phase III a significant reduction (p < 0.0001) of PMN was found in all samples, including control mares. A decrease (p < 0.05) of IL-6 and TNF-α was detected by ELISA, in the groups that received MSC, but not in control group. In the aMSC-treated group, a significant decrease was found in the expression of (IL1α, p = 0.0003; IL-6 p 0.04; IL-8, p = 0.006, TNFα p = 0.004). Expression of IL-10 and COX2 remained unchanged (p = 0.08). In the mares that received eMSC, IL-6 and 8 decreased significantly (p = 0.01), IL-10 increased (p = 0.009), while TNFα, COX2 and IL1α did not significantly change their expression. In the engraftment experiment CFDA label was found sparingly in all the samples analyzed until day 30, mainly at the stromal compartment of the endometrium. No differences in the engraftment pattern was found among cell origins. We conclude that inoculation of MSCs significantly reduced inflammation independently of the origin of the cells and that cells perform limited engraftment detectable after one month of infusion. These findings can be of help for the design of new anti-inflammatory therapies of uterine diseases in mares.

Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem Cells.

In mesenchymal stem cells (MSCs), it has been reported that prostaglandin E2 (PGE2) stimulation of EP2 and EP4 receptors triggers processes such as migration, self-renewal, survival, and proliferation, and their activation is involved in homing. The aim of this work was to establish a genetically modified adipose (aMSC) model in which receptor genes EP2 and EP4 were edited separately using the CRISPR/Cas9 system. After edition, the genes were evaluated as to if the expression of MSC surface markers was affected, as well as the migration capacity in vitro of the generated cells. Adipose MSCs were obtained from Chilean breed horses and cultured in DMEM High Glucose with 10% fetal bovine serum (FBS). sgRNA were cloned into a linearized LentiCRISPRv2GFP vector and transfected into HEK293FT cells for producing viral particles that were used to transduce aMSCs. GFP-expressing cells were separated by sorting to obtain individual clones. Genomic DNA was amplified, and the site-directed mutation frequency was assessed by T7E1, followed by Sanger sequencing. We selected 11 clones of EP2 and 10 clones of EP4, and by Sanger sequencing we confirmed 1 clone knock-out to aMSC/EP2 and one heterozygous mutant clone of aMSC/EP4. Both edited cells had decreased expression of EP2 and EP4 receptors when compared to the wild type, and the edition of EP2 and EP4 did not affect the expression of MSC surface markers, showing the same pattern in filling the scratch. We can conclude that the edition of these receptors in aMSCs does not affect their surface marker phenotype and migration ability when compared to wild-type cells.

Effect of BMP15 and/or AMH during in vitro maturation of oocytes from involuntarily culled dairy cows.

The high metabolic activity to which the dairy cattle are exposed to maintain milk production altered steroid metabolism that affects reproductive physiology and reduce oocyte competence. Our aims were (a) to characterize the competence of immature oocytes collected from dairy cattle based on the expression of genes in cumulus cells (CCs) and (b) to improve oocyte competence to support preimplantation embryo development by the supplementation of maturation medium with bone morphogenetic protein 15 (BMP15) and/or anti-mullerian hormone (AMH). Oocyte donors were identified at the moment of ovary collection and grouped by involuntarily culled dairy cows (Holstein breed) or beef cattle. The embryo development speed to blastocyst of the cull dairy cattle versus beef cattle (control group) was lower. Besides, <10% of oocytes (with CC biopsies) derived from dairy cattle were able to develop to the blastocyst stage. In addition, a higher level of expression and a positive correlation were observed in the expression of most of the genes evaluated (LUM, KRT18, KRT8, CLIC3, BMPR1B, and SLC38A3) in the cumulus-oocyte complexes that produced blastocysts versus those which did not develop correctly (arrested development). Further, use of BMP15 in the maturation of oocytes from dairy cattle seems to increase competence, modulating the expression of OCT4, SOX2, CDX2, GATA6, and TP1 in resulting blastocysts.

Endometrial Stem Cells in Farm Animals: Potential Role in Uterine Physiology and Pathology.

The endometrium is an accessible source of mesenchymal stem cells. Most investigations of endometrial mesenchymal stem cells (eMSCs) have been conducted in humans. In animals, particularly in livestock, eMSC research is scarce. Such cells have been described in the bovine, ovine, caprine, porcine, and equine endometrium. Here we provide the state of the art of eMSCs in farm animals with a focus on the bovine species. In bovines, eMSCs have been identified during the phases of the estrous cycle, during which their functionality and the presence of eMSC-specific markers has been shown to change. Moreover, postpartum inflammation related to endometritis affects the presence and functionality of eMSCs, and prostaglandin E2 (PGE2) may be the mediator of such changes. We demonstrated that exposure to PGE2 in vitro modifies the transcriptomic profile of eMSCs, showing its potential role in the fate of stem cell activation, migration, and homing during pathological uterine inflammation in endometritis and in healthy puerperal endometrium. Farm animal research on eMSCs can be of great value in translational research for certain uterine pathologies and for immunomodulation of local responses to pathogens, hormones, and other substances. Further research is necessary in areas such as in vivo location of the niches and their immunomodulatory and anti-infective properties.

Endometritis and In Vitro PGE2 Challenge Modify Properties of Cattle Endometrial Mesenchymal Stem Cells and Their Transcriptomic Profile.

Mesenchymal stem cells (MSCs) were isolated and characterized from postpartum bovine endometrium of animals with subclinical (n = 5) and clinical endometritis (n = 3) and healthy puerperal females (n = 5). Cells isolated displayed mean morphological features of MSCs and underwent osteogenic, chondrogenic, and adipogenic differentiation after induction (healthy and subclinical). Cells from cows with clinical endometritis did not undergo adipogenic differentiation. All cells expressed mRNAs for selected MSC markers. Endometrial MSCs were challenged in vitro with PGE2 at concentrations of 0, 1, 3, and 10 µM, and their global transcriptomic profile was studied. Overall, 1127 genes were differentially expressed between unchallenged cells and cells treated with PGE2 at all concentrations (763 up- and 364 downregulated, fold change > 2, and P < 0.05). The pathways affected the most by the PGE2 challenge were immune response, angiogenesis, and cell proliferation. In conclusion, we demonstrated that healthy puerperal bovine endometrium contains MSCs and that endometritis modifies and limits some functional characteristics of these cells, such as their ability to proceed to adipogenic differentiation. Also, PGE2, an inflammatory mediator of endometritis, modifies the transcriptomic profile of endometrial MSCs. A similar situation may occur during inflammation associated with endometritis, therefore affecting the main properties of endometrial MSCs.

Transient Expression of Functional Glucocerebrosidase for Treatment of Gaucher's Disease in the Goat Mammary Gland.

Gaucher disease (GD) is an orphan disease characterized by the lack or incapacity of glucocerebrosidase (hGCase) to properly process glucosylceramide, resulting in its accumulation in vital structures of the human body. Enzyme replacement therapy supplies hGCase to GD patients with a high-cost recombinant enzyme produced in vitro in mammalian or plant cell culture. In this study, we produced hGCase through the direct injection of recombinant adenovirus in the mammary gland of a non-transgenic goat. The enzyme was secreted in the milk during six days at a level up to 111.1 ± 8.1 mg/L, as identified by mass spectrometry, showing high in vitro activity. The milk-produced hGCase presented a mass correspondent to the intermediary high-mannose glycosylated protein, which could facilitate its delivery to macrophages through the macrophage mannose receptor. Further studies are underway to determine the in vivo delivery capacity of milk-hGCase, but results from this study paves the way toward the generation of transgenic goats constitutively expressing hGCase in the milk.

Constitutive expression of the embryonic stem cell marker OCT4 in bovine somatic donor cells influences blastocysts rate and quality after nucleus transfer.

Nuclear transfer (NT) is associated with epigenetic reprogramming of donor cells. Expression of certain genes in these cells might facilitate their expression in the NT embryo. This research was aimed to investigate the effect of constitutive expression of OCT4 in bovine somatic cells used for NT on the developmental potential of derived cloned embryos as well as in the expression of pluripotency markers in the Day-7 resulting embryos. Cloned blastocysts were generated from five cell lines that expressed OCT4. Pools of blastocysts were screened to detect OCT4, SOX2, and NANOG by qPCR. In vitro-fertilized time-matched blastocysts were used as controls. The development potential was assessed on the basis of blastocysts rate; grading and total cell counts at Day 7. OCT4 expression in the cell lines positively correlates with blastocysts rate (r = 0.92; p = 0.02), number of grade I blastocysts (r = 0.96; p = 0.01), and total cell number (r = 0.98; p = 0.002). The high expression of OCT4 in the cell line did not improve the final outcome of cloning. Somatic expression of OCT4 lead to increased expression of OCT4 and SOX2 in cloned grade I blastocysts; however, there was a bigger variability in OCT4 and SOX2 (p = 0.03; p = 0.02) expression in the embryos generated from cells expressing highest levels of OCT4. Probably the higher variability in OCT4 expression in cloned embryos is due to incorrect reprogramming and incapability of the oocyte to correct for higher OCT4 levels. For that reason, we concluded that OCT4 expression in somatic cells is not a good prognosis marker for selecting cell lines.