Isolation and characterization of minipig perivascular stem cells for bone tissue engineering

Human subcutaneous adipose tissue has been recognized as a rich source of tissue resident mesenchymal stem/stromal cells (MSC) in recent years. The current study was designed to sort the minipig (mp) perivascular stem cells (PSCs) and investigate the osteogenic potential. Purification of human PSCs was achieved via fluorescence‑activated cell sorting (FACS) from human liposuction samples [cluster of differentiation (CD)45‑CD34‑CD146+ perithelial cells and CD45‑CD34+CD146‑ adventitial cells]. Subsequently, PSCs were isolated from mp adipose tissue samples (n=9), characterized and, using purified mpPSCs (obtained by FACS, which is used in human PSC purification), the mpPSC osteogenic and adipogenic potential was evaluated by Alizarin Red S and Oil Red O staining in vitro, respectively. The cell morphometry was observed following cell isolation and culture, and hematoxylin and eosin staining was performed to identify the fat tissue structure and vascular distribution. Osteogenic and adipogenic differentiation‑associated gene expression levels were analyzed by reverse transcription‑quantitative polymerase chain reaction. The results demonstrated that the same antigens used for human PSC identification and isolation were working in mp tissue (CD45, CD146 and CD34). The two cell groups: CD45‑CD34‑CD146+ pericytes and CD45‑CD34+CD146‑ adventitial cells were successfully isolated from the subcutaneous fat in the posterior neck of mps, mpPSCs accounted for 8.6% of the stromal vascular fraction (SVF) with 1.4% pericytes and 7.2% adventitial cells. mpPSCs demonstrated characteristics of MSCs, including cell surface marker expression, colony forming unit‑fibroblast inclusion, and the stronger osteogenic and adipogenic differentiation potential than that of the non‑selected vascular stromal cells. The mRNA expression levels of osteocalcin, collagen, type I, α1 and peroxisome proliferator‑activated receptor‑γ in the mpPSCs group were significantly higher than those of the unsorted pSVF group (P<0.05). Thus, the current study successfully isolated and cultured CD146+ and CD34+ cell populations from mp tissues, characterized the cells' PSC‑like phenotype and identified their distinctly osteogenic and adipogenic potential.