Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype

Abstract

Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in equine AT and colocalized around blood vessels. Importantly, as assessed by flow cytometry, both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD44, CD90, and CD105) markers were present in a majority (≥90%) of cells in cultures of AT-MSCs and BM-MSCs; however, levels of pericyte markers were variable within each of those populations. Moreover, the expression of pericyte markers was maintained for at least eight passages in both AT-MSCs and BM-MSCs. Hematopoietic (CD45) and endothelial (CD144) markers were also detected at low levels in MSCs by quantitative polymerase chain reaction (qPCR). Finally, in coculture experiments, AT-MSCs closely associated with networks produced by endothelial cells, resembling the natural perivascular location of pericytes in vivo. Our results indicate that equine MSCs originate from perivascular cells and moreover maintain a pericyte-like phenotype in culture. Therefore, we suggest that, in addition to classical MSC markers, pericyte markers such as CD146 could be used when assessing and characterizing equine MSCs.

Pericytes and their potential in regenerative medicine across species

“…The widespread use of cell therapies in the horse has fostered great interest in the characterization of MSCs with the ultimate aim of improving their therapeutic efficacy. Although a large number of studies have been devoted to this subject, no attempts had been reported until recently to characterize different cell subpopulations with the aim of identifying subsets with optimum therapeutic properties. Earlier PCR and flow cytometry studies already reported the expression of the pericyte marker, CD146, in MSC preparations.…”

“…More recently, we confirmed that indeed multiple pericyte markers (CD146, NG2, αSMA, PDGFRβ) are expressed by culture‐expanded MSCs at least up to passage 8 (50). In addition, similar to results in humans, pericyte and MSC markers co‐localized in different equine tissue as well as in cultured MSCs , overall indicating that pericytes give rise to equine MSCs in culture. Following on these results, we used a FACS procedure similar to that described for isolation of human perivascular cells to separately isolate pericytes (CD146+/CD144−/CD45−/CD34−) and adventitial cells (CD146−/CD45−/CD144−/CD34+) from equine adipose SVF .…”

“…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 .…”

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.…”

Hematopoietic stem cells can differentiate into pericytes and myofibroblast in wound healing, not bone Mesenchymal stem cells

“…In short, the origin and differentiation of pericytes still have a lot of controversy. Although bone marrow is the exact source of pericytes in the angiogenesis process of wound healing [10][11][12]. The pluripotent stem cells originated in the bone marrow have hematopoietic stem cells and marrow mesenchymal stem cells [13].…”