Abstract
BackgroundArticular 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.ResultsNo 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.ConclusionsThis 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.
Highlights
IntroductionEmbryonic stem cells (ESCs) are a source of pluripotent cells for cartilage regeneration
Several studies focusing on allogeneic models of mouse ES cell transplantation have shown that as few as 400–500 mouse ES cells can lead to teratoma formation in immunodeficient mice and between 50,000 and 100,000 cells are required for tumour formation in immunocompetent animals [8]
In vitro embryo production (IVP) and sexing, isolation of embryonic stem-like (ES-like) cells, culture and characterization As previously described [10], 80% of vitrified embryos successfully expanded after warming and about 50% were found to be male by sexing PCR, with 2 bands corresponding to the sex-determining region Y-linked gene (SRY) and to the sheep Sheep SAT DNA repeat unit sequence (SAT) 1114 DNA repeat unit (SAT) sequences, while female embryos showed only the band corresponding to the autosomal sequence SAT [10]
Summary
Embryonic stem cells (ESCs) are a source of pluripotent cells for cartilage regeneration. Their use, 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. Pilichi et al BMC Veterinary Research (2018) 14:213 source for tissue regeneration because of their ability to self-renew and pluripotency. Their use raises several concerns related to immunologic incompatibility and possible development of teratoma [2,3,4]. Several studies focusing on allogeneic models of mouse ES cell transplantation have shown that as few as 400–500 mouse ES cells can lead to teratoma formation in immunodeficient mice and between 50,000 and 100,000 cells are required for tumour formation in immunocompetent animals [8]
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