Transplantation of sheep embrionic stem cells in cartilage lesions: preliminary observations

The aim of this study is to evaluate whether ovine embryonic stem cells transplanted in experimental lesions can differentiate into chondrocyte cells and if the new cartilage is of ialine type

Treatment with embryonic stem-like cells into osteochondral defects in sheep femoral condyles

Background Articular cartilage has poor intrinsic capacity for regeneration because of its avascularity and very slow cellular turnover. Defects deriving from trauma or joint disease tend to be repaired with fibrocartilage rather than hyaline cartilage. Consequent degenerative processes are related to the width and depth of the defect. Since mesenchymal stem cells (MSCs) deriving from patients affected by osteoarthritis have a lower proliferative and chondrogenic activity, the systemic or local delivery of heterologous cells may enhance regeneration or inhibit the progressive loss of joint tissue. Embryonic stem cells (ESCs) are very promising, since they can self-renew for prolonged periods without differentiation and can differentiate into tissues from all the 3 germ layers. To date only a few experiments have used ESCs for the study of the cartilage regeneration in animal models and most of them used laboratory animals. Sheep, due to their anatomical, physiological and immunological similarity to humans, represent a valid model for translational studies. This experiment aimed to evaluate if the local delivery of male sheep embryonic stem-like (ES-like) cells into osteochondral defects in the femoral condyles of adult sheep can enhance the regeneration of articular cartilage. Twenty-two ewes were divided into 5 groups (1, 2, 6, 12 and 24 months after surgery). Newly formed tissue was evaluated by macroscopic, histological, immunohistochemical (collagen type II) and fluorescent in situ hybridization (FISH) assays. Results Regenerated tissue was ultimately evaluated on 17 sheep. Samples engrafted with ES-like cells had significantly better histologic evidence of regeneration with respect to empty defects, used as controls, at all time periods. Conclusions Histological assessments demonstrated that the local delivery of ES-like cells into osteochondral defects in sheep femoral condyles enhances the regeneration of the articular hyaline cartilage, without signs of immune rejection or teratoma for 24 months after engraftment.</br

Oct4 expression in in-vitro-produced sheep blastocysts and embryonic-stem-like cells

Transcription factor Oct4 (octamer-binding transcription factor-4) is important in early embryonic development and differentiation. It is also required for maintenance of pluripotency of the inner cell mass, and is used as a staminality marker of embryonic stem cells. Changes in Oct4 expression during the different stages of early embryo development have been reported, and therefore we have conducted a quantitative study of Oct4 gene expression of sheep blastocysts in vitro, and of embryonic-stem-like cells at the undifferentiated stage and in the course of differentiation. To characterize embryonicstem-like cells, alkaline phosphatase activity, stage-specific embryonic surface antigens SSEA-1, SSEA-3, SSEA-4 and three specific gene markers Nanog, Sox2 and Stat3 were assayed. cDNA produced by RT (reverse transcriptase)-PCR was synthesized and amplified by PCR; sequencing gave 98, 95 and 98% homology with the bovine sequences of Oct4, Nanog and Stat3 respectively. Using the ovine sequence of 290 bp, quantitative expression of Oct4 in the inner cell mass, trophoblast and embryonic-stem-like cells was performed by qRT-PCR (quantitative real-time PCR). Oct4 was expressed in the inner cell mass, trophoblast and embryonic-stem-like cells. Expression in the inner cell mass was significantly higher than in the trophoblast. This could be useful in defining the quality of embryos produced and makes it possible to use Oct4 to detect pluripotency. In addition, the different levels of Oct4 expression between undifferentiated and differentiating embryonic-stem-like cell cultures could be used to detect this gene as a staminality marker. \ua9 The Author(s) Journal compilation. \ua9 2010 Portland Press Ltd

Sexing of in vitro produced ovine embryos by duplex PCR

The aim of this article was to develop a fast and easy duplex polymerase chain reaction (PCR) method, for sex determination of ovine in vitro produced embryos prior to implantation. We tested the approach with 107 samples of autosomal cells (oviductal sheep cells and male lamb fibroblasts), divided into three groups for each sex according to the number of cells employed (30, 5, 2, respectively). We then used the test on 21 embryos at blastocyst stage. On the same day the embryos were transferred in pairs into 11 recipient synchronized ewes. The PCR utilized two different sets of primers: the first pair recognized a bovine Y-chromosome-specific sequence (SRY), that showed 100% homology with the corresponding sequence of the ovine Y-chromosome and is amplified in males only. The second pair recognized the bovine 1.715 satellite DNA (SAT) which was amplified in all ovine samples but, when submitted to the GenBank database did not show homology with any of the reported ovine sequences. However, after sequencing, ovine amplification product showed 98% homology with the bovine specific satellite sequence. The autosomal samples were amplified with 85.0% efficiency and 91.2% accuracy, while amplification was successful with all 21 embryos (100% efficiency). Eight lambs were born and the sex as determined by PCR corresponded to the anatomical sex in seven (87.5% accuracy). These results confirm that this method can be applied in ovine breeding programs to manipulate sex ratio of offspring

Isolation, culture, and characterization of embryonic cell lines from vitrified sheep blastocysts

This study was conducted to isolate, to culture, and to characterize embryonic cell lines from in vitro produced vitrified sheep blastocysts. Embryos were produced and vitrified at the expanded blastocyst stage. Ten inner cell masses arising from day 6-7 blastocysts were isolated by immunosurgery, disaggregated, and cultured onto mitomocin-C-inactivated mouse STO fibroblasts (MIF). After 5 or 6 days of culture the primary cell colonies were disaggregated, seeded in a new MIF, and cultured for 3 or 4 days to form new colonies called Passage 1. These cells were then disaggregated and cultured for other two passages. The primary cell colonies and Passage 2 colonies expressed stage specific embryonic markers SSEA-1, SSEA-3, and SSEA-4, and were alkaline phosphatase positive. In the absence of feeder layer and human leukemia inhibitory factor (LIF), these cells differentiated into variety of cell types and formed embryoid bodies. When cultured for an extended period of time, embryoid bodies differentiated into derivatives of three embryonic germ (EG) layers. These were characterized by detection of specific markers for differentiation such early mesoderm (FE-C6), embryonic myosin (F1-652), neural precursor (FORSE-1), and endoderm (anti-cytokeratin 18). To our knowledge, this is the first time that embryonic cell lines from in vitro produced and vitrified ovine blastocysts have been isolated and examined for detection of SSEA markers, and embryoid bodies have been cultured and examined for specific cell surface markers for differentiation

Sheep embryonic stem-like cells engrafted into sheep femoral condyle osteochondral defects: 4-year follow-up

Abstract Background Articular cartilage lacks a regenerative response. Embryonic stem cells (ESCs) are a source of pluripotent cells for cartilage regeneration. Their use, however, is associated with a risk of teratoma development, which depends on multiple factors including the number of engrafted cells and their degree of histocompatibility with recipients, the immunosuppression of the host and the site of transplantation. Colonies of sheep embryonic stem-like (ES-like) cells from in vitro-produced embryos, positive for stage-specific embryonic antigens (SSEAs), alkaline phosphatase (ALP), Oct 4, Nanog, Sox 2 and Stat 3 gene expression, and forming embryoid bodies, were pooled in groups of two-three, embedded in fibrin glue and engrafted into osteochondral defects in the left medial femoral condyles of 3 allogeneic ewes (ES). Empty defects (ED) and defects filled with cell-free glue (G) in the condyles of the controlateral stifle joint served as controls. After euthanasia at 4 years post-engraftment, the regenerated tissue was evaluated by macroscopic, histological and immunohistochemical (collagen type II) examinations and fluorescent in situ hybridization (FISH) assay to prove the ES-like cells origin of the regenerated tissue. Results No teratoma occurred in any of the ES samples. No statistically significant macroscopic or histological differences were observed among the 3 treatment groups. FISH was positive in all the 3 ES samples. Conclusions This in vivo preclinical study allowed a long-term evaluation of the occurrence of teratoma in non-immunosuppressed allogeneic adult sheep engrafted with allogeneic ES-like cells, supporting the safe and reliable application of ES cells in the clinic

Additional file 2: of Sheep embryonic stem-like cells engrafted into sheep femoral condyle osteochondral defects: 4-year follow-up

Table S3. Median (IQR)* of comparison between macroscopic assessment of treatments (ES, ED and G) at 2 and 4 years. Table S4. Median (IQR)* of comparison between histological assessment of treatments (ES, ED and G) at 2 and 4 years. (DOCX 18 kb