Disruption of the Mouse Blood-Brain Barrier by Small Extracellular Vesicles from Hypoxic Human Placentas

Cerebrovascular complications, including cerebral edema and ischemic and hemorrhagic stroke, constitute the leading cause of maternal mortality associated ...

Universidad de Concepción, Chillan

The objective of this study was to identify microRNAs (miRNAs) expressed in bovine (Bos Taurus) cloned embryos at Day 17 of development (Day 0=day of nucleus transfer or in vitro fertilization) during elongation. Day 7 bovine expanded blastocysts produced by hand made cloning (HMC) or in vitro fertilization were bulk-transferred to synchronized recipient cattle (48 HMC embryos to 10 recipients and 28 in vitro-produced embryos to four recipients). Elongated embryos were retrieved at Day 17; miRNAs were isolated and subjected to microarray screening using custom composite slides spotted with human, mouse, and rat and in silico-predicted miRNAs. An initial profile of expressed miRNAs was determined in cloned embryos and somatic donor cells; this profile changed after somatic cell nucleus transfer, identifying differentially expressed miRNAs between cloned and in vitro-produced bovine embryos. Furthermore, microarray data were validated using a miRNA-specific quantitative reverse transcription-polymerase chain reaction (qRT-PCR) approach (miR-Q). There was an 83% correlation (P=0.01) between microarray and qPCR data. Based on qRT-PCR, correct reprogramming of some miRNAs from the donor cells was confirmed in cloned bovine embryos, whereas other somatic miRNAs were not appropriately reprogrammed. Some of the miRNAs that were equally reprogrammed clustered on the same chromosomal location in the bovine genome. In conclusion, reprogramming of miRNAs seemed to occur in cloned bovine embryos. This could have profound implications for elucidating nuclear reprogramming in somatic cloning, as well as for the role of miRNAs in preimplantation mammalian development.

MicroRNA expression profiling of elongated cloned and in vitro-fertilized bovine embryos.

Repair of injured soft and hard tissues in horses can benefit greatly from the use of regenerative therapies with mesenchymal stem cells (MSC). Vitamin-C and platelet-rich-plasma had been used for in vitro differentiation of MSC. This study was aimed to evaluate the effect of vitamin-C, platelet-rich-plasma and their combination on the in vitro differentiation of adipose horse MSC. We isolated MSC from horse fat and differentiated them in vitro into osteogenic and chondrogenic lineages, as demonstrated by specific staining and RT-qPCR of selected genes. Combining vitamin-C and plasma-rich-platelet positively affected the ability of MSC to differentiate in vitro into mesodermal lineages during 14 days of culture; this effect was not as marked when differentiation was attempted for 21 days. This provides valuable information on the effect of combined use of these molecules in regenerative therapies and their potential application along stem cells for lesions of musculoskeletal tissue in sport horses.

Combined use of platelet rich plasma and vitamin C positively affects differentiation in vitro to mesodermal lineage of adult adipose equine mesenchymal stem cells.

Adult stromal mesenchymal stem cells (MSCs) have been postulated as responsible for cell renewal in highly and continuously regenerative tissues such as the endometrium. MSCs have been identified in the endometrium of many species including humans, rodents, pets and some farm animals, but not in horses. The objective of this work was to isolate such cells from the endometrium of mares and to compare their main biological attributes with horse adipose-derived MSCs. Here we successfully isolated and characterized endometrial MSCs (eMSCs) from mares. Said cells showed fibroblast-like morphology, grew on plastic, had doubling population times of 46.4 ± 3.38 h, underwent tri-lineage (osteo, chondro and adipogenic) differentiation after appropriate inductions, migrated toward the attraction of fetal calf serum and displayed a pattern of surface markers commonly accepted for horse MSCs. All these are properties of MSCs. Some of these attributes were shared with equine adipose-derived MSCs, but the migration pattern of eMSC at 12 and 24 h after stimulation was reduced in comparison with adipose MSCs. Also, expression of CD44, CD90 and MHCI surface markers were dramatically down-regulated in eMSCs. In conclusion, equine-derived endometrial MSC share biological attributes with adipose MSC of this species, but displayed a different surface marker phenotype and an impaired migration ability. Conceivably, this phenotype is distinctive for MSC of this origin.

Equine mesenchymal stem cells derived from endometrial or adipose tissue share significant biological properties, but have distinctive pattern of surface markers and migration.

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.

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

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 × 10 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.

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.

Mesenchymal stem cells (MSC) are modern tools in regenerative therapies of humans and animals owed to their immunomodulatory properties, which are activated in a pro-inflammatory environment. Different preconditioning strategies had been devised to enhance the immunomodulatory properties of MSC. In this research, we evaluated the immunological attributes of equine adipose MSC (eAMSC) before and after preconditioning in vitro with prostaglandin E (PGE), substance P (SP), their combination and IFNγ. PGE/SP was the best combination to keep or enhance the mesodermal lineage differentiation of eAMSC. Alongside with this, preconditioning of eMSC with PGE and SP did not affect expression of stemness MSC surface phenotype: CD90, CD44, MHC class I, MHC class II and CD45, assessed by cytometry. Both naïve and preconditioned eAMSC expressed genes related with immune properties, such as MHC-I, PTGES, IL6, IL1A, TNFα and IL8 assessed by qPCR. Only TNFα was under expressed in treated cells, while the other markers were either overexpressed or not changed. In no cases MHC-II expression was detected. The antiproliferative effect of preconditioned eAMSC exposed to activated peripheral blood mononuclear cells (PBMC) showed that SP treatment significantly inhibited proliferation of LPS stimulated PBMC. When eAMSC were stimulated with Poly I:C, all the treatments significantly inhibited proliferation of stimulated PBMC (p < 0.05). Direct contact (coculture) between the preconditioned eAMSC and PBMC, induced a shift of significantly more (CD4/CD25/FOXP3) T-regulatory PBMC than naïve eAMSC. In the experiments of this research, we investigated the secreted proteomic profile of naïve and preconditioned eAMSC, 42 up-regulated and 40 down-regulated proteins were found in the proteomic assay. Our proteomic data revealed profound changes in the secretory pattern of MSC exposed to different treatments, compared to naïve eAMSC as well as among treatments. In overall, compared to naïve cells, the protein profile of preconditioned cells resembled the mesenchymal-epithelial transition (MET). Here we showed that the combined use of PGE and SP provoked in overall the highest expression of anti-inflammatory markers as well as lead to an increased acquisition of a T-regulatory phenotype in preconditioned eAMSC without affecting their "stemness".

In vitro preconditioning of equine adipose mesenchymal stem cells with prostaglandin E, substance P and their combination changes the cellular protein secretomics and improves their immunomodulatory competence without compromising stemness.

Fidel O. Castro

Laboratory of Animal Biotechnology,
Department of Animal Science,
Faculty of Veterinary Sciences,
Laboratory of Animal Biotechnology, Department of Animal Science, Faculty of Veterinary Sciences

Universidad de Concepción, Chillan

Disruption of the Mouse Blood-Brain Barrier by Small Extracellular Vesicles from Hypoxic Human Placentas

Fidel O. Castro

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Animal Biotechnology,Department of Animal Science,Faculty of Veterinary Sciences,Laboratory of Animal Biotechnology, Department of Animal Science, Faculty of Veterinary Sciences

Universidad de Concepción, Chillan

Disruption of the Mouse Blood-Brain Barrier by Small Extracellular Vesicles from Hypoxic Human Placentas

Laboratory of Animal Biotechnology,
Department of Animal Science,
Faculty of Veterinary Sciences,
Laboratory of Animal Biotechnology, Department of Animal Science, Faculty of Veterinary Sciences