Exploring the heterogeneity of perivascular cells

Abstract

Perivascular cells have been identified as in vivo progenitors of mesenchymal stem cells (MSCs), these being widely used as adult stem cells in multiple clinical conditions. Indeed, pericytes can differentiate into mesodermal lineages when cultured in induction media. Compared to MSCs, pericytes represent a more refined population of cells. As the interest to identify and isolate pure populations of pericytes increased, several markers have simultaneously and independently become reference pericyte identifiers. Nevertheless, the term “pericyte” exclusively refers to cells in a specific anatomical position -cells in the perivascular area of small blood vessels and embedded within its basal lamina- and in situ can be found around multiple types of vessels, in varying morphology and immunophenotype. This heterogeneity is being explored but remains largely unknown. To investigate perivascular cell heterogeneity, we have used multiple well established pericyte markers (CD146, αSMA, NG2, leptin receptor [LepR], PDGFRβ, PDGFRα) across multiple tissues, including foetal and adult human (heart, skeletal muscle, bone marrow, subcutaneous white adipose tissue), murine (heart, skeletal muscle) and porcine tissues (heart, subcutaneous white adipose tissue). Immunohistological analysis was used to identify different pericyte subtypes, as well as their tissue distribution. Flow cytometry allowed pericyte subtype quantification and, when possible, cell sorting was performed for subsequent culture. When long-term cell cultures were established, cell lines at low passages were used in multiple in vitro assays to characterise their mesenchymal potential, including clonogenesis, growth, migration, metabolic activity or mesodermal cell differentiation ability. Analysis focused on LepR showed presence of subtypes in the foetal and adult bone marrow, but when sorted, presented donor-related differences in vitro. Foetal cardiac histological analysis revealed different distribution of perivascular layers within cardiac compartments, with homogeneous coverage of PDGFRβ + and PDGFRα + perivascular layers but significantly different for αSMA+ (p-value<0.0001). Subsets based on PDGFRβ and PDGFRα were present in both foetal human and adult murine tissues, but when sorted cell cultures were not achieved. NG2+ pericytes presented high mortality following enzymatical tissue dissociation, but were successfully isolated and sorted. In vitro, NG2 subtypes presented donor-dependent variations in multiple assays, but the NG2- subset performed better when results were homogeneous. Together, this work shows the existence of multiple subtypes within generally assumed homogeneous pericyte populations. Variability concerns tissue distribution, with respect to the specific marker used, both within a specific tissue and across different tissues, and in vitro mesenchymal developmental potentials. Also, this work highlights donor-related differences within human tissues. In addition, current practices and terminology use, or misuse, involving perivascular and mesenchymal cells isolation, cell culture and later use are discussed.