Perivascular Mesenchymal Stem Cells in Sheep: Characterization and Autologous Transplantation in a Model of Articular Cartilage Repair

Abstract

Publisher Rights Statement:This is the final peer-reviewed manuscript as accepted for publication General rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Immunohistochemistry and fluorescence-activated cell sorting (FACS) were used to ascertain the reactivity of anti-human and anti-ovine antibodies, which were combined and used to identify and isolate pericytes (CD34-CD45-CD146+) and adventitial cells (CD34+CD45-CD146-). The purified cells demonstrated osteogenic, adipogenic and chondrogenic potential in culture.Autologous ovine PSCs (oPSCs) were isolated, cultured and efficiently transfected using a GFP encoding lentivirus. The cells were implanted into articular cartilage defects on the medial femoral condyle using a hydrogel and collagen membranes.Four weeks following implantation the condyle was explanted and confocal laser scanning microscopy demonstrated the presence of oPSCs in the defect repaired with the hydrogel.These data suggest the testability in a large animal of native mesenchymal stem cell autologous grafting, thus avoiding possible biases associated with xenotransplantation.Such a setting will be used in priority for indications in orthopaedics, at first to model articular cartilage repair.

Pericytes and their potential in regenerative medicine across species

“…More definite proof of a perivascular origin of MSCs was provided by seminal studies showing that pericytes in different human tissues stained for classical MSC markers and, on in vitro expansion, displayed an MSC phenotype while maintaining the expression of pericyte markers (CD146, PDGFRβ, NG2, αSMA). These findings have more recently extended to horse, dog and sheep , overall indicating that pericytes are in vivo counterparts of culture‐expanded MSCs in different species . Some of the previous studies also identified a population of adventitial cells (marked by the expression of CD34) as a perivascular cell type (in addition to pericytes) with MSC properties, and proposed these adventitial cells to be precursors of pericyte .…”

“…Given the promising results obtained using human pericytes in animal models of tissue regeneration, significant interest has also arisen in the isolation and characterization of pericytes from veterinary species, either for the purpose of developing improved regenerative therapies for companion animals or use in preclinical models of human regenerative medicine. Isolation and culture of pericytes from species other than human and rodents has been particularly challenging due to the lack of specific antibodies, and only very recently were the first successful attempts reported (in sheep, horse, and dog) (Table ).…”

“…Nonetheless, subsequent experiments in a canine model of stroke concluded that intra‐arterial delivery of autologous PDGFRβ+ cells following hyperosmolar mannitol administration was a feasible and safe therapeutic approach. In a second study, IHC was performed to identify CD146+ cells in adipose tissue followed by isolation from SVF by FACS using a panel of antibodies, as previously described in other species , to isolate CD146+/CD45−/CD34− (pericytes) and CD146−/CD45−/CD34+ (adventitial cells) fractions. Characterization of each separate fraction was not reported but rather the two combined fractions were shown to be osteogenic in vitro and to produce trophic factors, VEGF, FGF‐2 and PDGF, which expression was increased when cells were treated with the osteoinducer protein, NELL‐1.…”

“…The sheep is a recognized orthopedic research model and in that regard ovine pericytes may be particularly useful for preclinical testing of novel musculoskeletal therapies. Péault's group isolated pericytes (CD146+/CD45−/CD34−) from reportedly different ovine tissues, namely placenta, myocardium and adipose tissue, including infrapatellar fat pads . Unlike in some of the previous studies in humans and horse , depletion of endothelial cells was not performed due to lack of a suitable antibody, and no attempts were made to assess the level of endothelial contamination in the preparations obtained.…”

Pericytes for Therapeutic Bone Repair

“…The immunophenotype CD146+CD34-CD31-CD45-CD56- successfully isolates pericytes in multiple tissues including the skeletal muscle, bone marrow, white adipose tissue, placenta, pancreas, umbilical cord, heart, kidneys, infrapatellar fat pad, and liver [6, 9, 14, 18, 29, 30, 35–38, 39–41]. Of note, the same immunophenotype has been used to isolate pericytes from other mammalian species, including dog [42], sheep [43], and horse [44, 45], offering large animal models of perivascular cell-mediated tissue regeneration.…”