Isolation and characterization of canine perivascular stem/stromal cells for bone tissue engineering

Abstract

For over 15 years, human subcutaneous adipose tissue has been recognized as a rich source of tissue resident mesenchymal stem/stromal cells (MSC). The isolation of perivascular progenitor cells from human adipose tissue by a cell sorting strategy was first published in 2008. Since this time, the interest in using pericytes and related perivascular stem/stromal cell (PSC) populations for tissue engineering has significantly increased. Here, we describe a set of experiments identifying, isolating and characterizing PSC from canine tissue (N = 12 canine adipose tissue samples). Results showed that the same antibodies used for human PSC identification and isolation are cross-reactive with canine tissue (CD45, CD146, CD34). Like their human correlate, canine PSC demonstrate characteristics of MSC including cell surface marker expression, colony forming unit-fibroblast (CFU-F) inclusion, and osteogenic differentiation potential. As well, canine PSC respond to osteoinductive signals in a similar fashion as do human PSC, such as the secreted differentiation factor NEL-Like Molecule-1 (NELL-1). Nevertheless, important differences exist between human and canine PSC, including differences in baseline osteogenic potential. In summary, canine PSC represent a multipotent mesenchymogenic cell source for future translational efforts in tissue engineering.

Mesenchymal Stromal/Stem Cells in Regenerative Medicine and Tissue Engineering

“…For example, locationally and transcriptionally distinct subpopulations of CD34 + /CD146 − “adventitial MSCs” and CD34 − /CD146 + “pericyte-like MSCs” have been found to reside in human adipose tissue, a commonly used cell source for regenerative medicine [66]. Similar findings have also been noted in horses and canines, suggesting these dual perivascular subpopulations are conserved in mammals [67, 68]. Interestingly, both equine and human adipose-derived CD34 − /CD146 + MSCs display greater angiogenicity compared to CD34 + /CD146 − MSCs indicative of a relatively conserved functional phenotype as well, possibly due to their pericyte-like differentiation state [67, 69].…”

Perivascular Mesenchymal Progenitors for Bone Regeneration

“…Such selected, non‐cultured autologous osteogenic progenitors could then be used for therapeutic bone formation/regeneration, thus avoiding the biologic and regulatory hurdles associated with prolonged cell culture. Data collated in this review support such a strategy and tactics, including those obtained in non‐rodent larger mammals …”

“…Data collated in this review support such a strategy and tactics, including those obtained in non-rodent larger mammals. 19…”

“…Here, CD146 knockdown among human pericytes significantly impaired pericyte-endothelium spatial interaction and vascularization in a subcutaneous implant model. 18 Supporting a contact-independent role for induction of vascularization, PSC transcribe and secrete greater amounts of pro-vasculogenic growth factors including fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) 17,19 among others. In addition, other investigators have found analogous provasculogenic effects among CD146 + pericytes or PSC in non-orthopaedic tissue engineering efforts.…”

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.…”