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Research Interests:
Research Interests:
Research Interests:
Preclinical studies with osteoprogenitor cells derived from human embryonic stem cells (hESC) do not lead to substantial bone regeneration in vivo. The degree of survival following implantation might play a role in their long term... more
Preclinical studies with osteoprogenitor cells derived from human embryonic stem cells (hESC) do not lead to substantial bone regeneration in vivo. The degree of survival following implantation might play a role in their long term efficiency. We investigated the initial engraftment of hESCs-derived cells during two weeks post-implantation and compared it to such response for adult bone marrow stromal cells (hBMSC)-derived osteoprogenitor cells. hBMSC and H9-hES cells pre-treated with osteogenic factors were implanted into a calvarial defect in both adult WT and nude rats. At days 7 and 14 post-implantation, samples were analysed for persistence of implanted cells, initiation of regeneration of host bone, angiogenesis and apoptosis. At day 7, hESC and hBMSC were detected within defects in both rat strains. By day 14 human cells were only detected in immune-deficient rats whilst still maintaining an osteoblastic phenotype and engendered a significant increase in bone formation. In WT ...
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The aim of this study was to evaluate the viability (percentage of dead cells) and the incidence of DNA fragmentation of horse embryos after storage in three different media at 5 degrees C for 6 and 24 h. Forty embryos were stored in... more
The aim of this study was to evaluate the viability (percentage of dead cells) and the incidence of DNA fragmentation of horse embryos after storage in three different media at 5 degrees C for 6 and 24 h. Forty embryos were stored in Emcare Holding Solution for 6 and 24 h, in Hams'F10 or Vigro Holding Plus for 24 h at 5 degrees C (n = 9-10 per group) and 10 embryos were evaluated immediately after collection. First, embryos were stained, immediately after collection or following storage, to detect dead cells (DAPI) and, subsequently, DAPI-stained embryos were fixed and stained to detect DNA fragmentation (TUNEL). Finally, all the fixed embryos were re-stained with DAPI to determine the total number of cells. The percentage of cells stained with both TUNEL and DAPI or TUNEL-only or DAPI-only were determined. The percent of dead cells (DAPI-labelled) per embryo increased with duration of storage, but no differences were detected between the storage media. The percentage of early apoptotic cells (TUNEL+/DAPI-) in fresh and stored embryo for 6 h or 24 h did not differ significantly (P > 0.05). There was a significant correlation between the percentage of cells labelled by TUNEL and DAPI (R = 0.87) (P < 0.001). These results suggest that cooled storage increases cell death but this does not appear to occur by induction of apoptosis and that DAPI staining proves to be a quick and reliable method for assessing embryo viability.
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It has been suggested that preculturing immature oocytes in a manner that maintains them in meiotic arrest may improve cytoplasmic maturation and, thereby, the eventual developmental competence of oocytes matured in vitro. This study... more
It has been suggested that preculturing immature oocytes in a manner that maintains them in meiotic arrest may improve cytoplasmic maturation and, thereby, the eventual developmental competence of oocytes matured in vitro. This study examined the ability of follicular cells to maintain meiotic arrest in equine oocytes. Cumulus-oocyte complexes (COCs) recovered from dead mares were cultured for 38 h in M199 either attached to, or together with, different follicle wall components, as follows: (1) attached to the follicle wall, (2) cocultured with separated follicle wall, (3) attached to membrana granulosa (COCG), (4) COCGs cocultured with sheets of theca cells, (5) COCGs cultured in theca-cell conditioned medium, and (6) control COCs without any follicle wall components. When oocytes were cultured attached to their follicle wall, 79% remained in the GV stage throughout the 38 h incubation. However, when oocytes were cocultured with separate pieces of follicle wall, meiosis resumed and a similar proportion of oocytes progressed to metaphase II (79%) as under control conditions (84%). Only 16% of oocytes cultured while still attached to the membrana granulosa (COCGs) maintained the GV stage, whereas when COCGs were cocultured with theca cells or in theca-cell conditioned medium, significantly more oocytes remained in the GV stage (64 and 52%, respectively), indicating that theca cells secrete a meiosis-inhibiting factor. The effect of FSH on the meiosis-inhibiting activity of follicular cells was investigated by culturing COCs attached to the follicle wall and COCGs in the presence or absence of theca cells in medium containing FSH. Addition of 0.05 iu recombinant human FSH ml(-1) to the culture medium did not affect nuclear maturation and failed to overcome the suppressive effect exerted by the follicle wall or by theca cells, despite the fact that mRNA for the FSH receptor was found using RT-PCR in both cumulus and granulosa cells. These results demonstrate that the maintenance of meiotic arrest in equine oocytes during culture can be promoted by theca cells, which appear to act via a secreted inhibitory factor that cannot be suppressed or counteracted by FSH.
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The blood-brain barrier (BBB), a critical guardian of communication between the periphery and the brain, is frequently compromised in neurological diseases such as multiple sclerosis (MS), resulting in the inappropriate passage of... more
The blood-brain barrier (BBB), a critical guardian of communication between the periphery and the brain, is frequently compromised in neurological diseases such as multiple sclerosis (MS), resulting in the inappropriate passage of molecules and leukocytes into the brain. Here we show that the glucocorticoid anti-inflammatory messenger annexin A1 (ANXA1) is expressed in brain microvascular endothelial cells, where it regulates BBB integrity. In particular, ANXA1(-/-) mice exhibit significantly increased BBB permeability as a result of disrupted interendothelial cell tight junctions, essentially related to changes in the actin cytoskeleton, which stabilizes tight and adherens junctions. This situation is reminiscent of early MS pathology, a relationship confirmed by our detection of a selective loss of ANXA1 in the plasma and cerebrovascular endothelium of patients with MS. Importantly, this loss is swiftly restored by i.v. administration of human recombinant ANXA1. Analysis in vitro confirms that treatment of cerebrovascular endothelial cells with recombinant ANXA1 restores cell polarity, cytoskeleton integrity, and paracellular permeability through inhibition of the small G protein RhoA. We thus propose ANXA1 as a critical physiological regulator of BBB integrity and suggest it may have utility in the treatment of MS, correcting BBB function and hence ameliorating disease.
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Research Interests:
The nuclear factor (NF)-κB pathway is involved in arterial inflammation. Although the signaling pathways that regulate transcriptional activation of NF-κB are defined, the mechanisms that regulate the expression levels of NF-κB... more
The nuclear factor (NF)-κB pathway is involved in arterial inflammation. Although the signaling pathways that regulate transcriptional activation of NF-κB are defined, the mechanisms that regulate the expression levels of NF-κB transcription factors are uncertain. We studied the signaling mechanisms that regulate RelA NF-κB subunit expression in endothelial cells (ECs) and their role in arterial inflammation. Gene silencing and chromatin immunoprecipitation revealed that RelA expression was positively regulated by c-Jun N-terminal kinase (JNK) and the downstream transcription factor ATF2 in ECs. We concluded that this pathway promotes focal arterial inflammation as genetic deletion of JNK1 reduced NF-κB expression and macrophage accumulation at an atherosusceptible site. We hypothesized that JNK signaling to NF-κB may be controlled by mechanical forces because atherosusceptibility is associated with exposure to disturbed blood flow. This was assessed by positron emission tomography imaging of carotid arteries modified with a constrictive cuff, a method that was developed to study the effects of disturbed flow on vascular physiology in vivo. This approach coupled to en face staining revealed that disturbed flow elevates NF-κB expression and inflammation in murine carotid arteries via JNK1. We demonstrate that disturbed blood flow promotes arterial inflammation by inducing NF-κB expression in endothelial cells via JNK-ATF2 signaling. Thus, our findings illuminate a novel form of JNK-NF-κB crosstalk that may determine the focal nature of arterial inflammation and atherosclerosis.
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Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the... more
Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the osteogenic differentiation potential of human embryonic stem cells (hESC) with human adult-derived stem cells in vivo. hESC lines H7, H9, the HEF-1 mesenchymal-like, telomerized H1 derivative, the human embryonic kidney epithelial cell line HEK293 (negative control), and adult human mesenchymal stem cells (hMSC) were either used untreated or treated with osteogenic factors for 4 days prior to injection into diffusion chambers and implantation into nude mice. After 11 weeks in vivo chambers were removed, frozen, and analyzed for evidence of bone, cartilage, and adipose tissue formation. All hESCs, when pretreated with osteogenic (OS) factors gave rise exclusively to bone in the chambers. In contrast, untreated hESCs (H9) formed both bone and cartilage in vivo. Untreated hMSCs did not give rise to bone, cartilage, or adipose tissue in vivo, while pretreatment with OS factors engendered both bone and adipose tissue. These data demonstrate that hESCs exposed to OS factors in vitro undergo directed differentiation toward the osteogenic lineage in vivo in a similar fashion to that produced by hMSCs. These findings support the potential future use of hESC-derived cells in regenerative medicine applications.