Transcriptional Networks in Single Perivascular Cells Sorted from Human Adipose Tissue Reveal a Hierarchy of Mesenchymal Stem Cells

Adipose tissue is a rich source of multipotent mesenchymal stem‐like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31−/CD45−/CD34+/CD146− cells (adventitial stromal/stem cells [ASCs]) and CD31−/CD45−/CD34−/CD146+ cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence‐activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH‐staining spectrum were transcriptionally profiled by Fluidigm single‐cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single‐cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single‐cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDHbrASC (most primitive); (b) ALDHdimASC; (c) ALDHbrPC; (d) ALDHdimPC (least primitive). This order was independently supported by specific combinations of class‐specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single‐cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression networks. Stem Cells 2017;35:1273–1289 A hierarchy of perivascular mesenchymal stem cells. Schematic bifurcations from arteries (A) to arterioles (B) to capillaries (C). Pericytes are sparsely distributed on capillaries, but completely ensheathe arterioles. Perivascular fibroblast like cells contribute to the outmost tunica adventitia of arteries. Both pericytes and adventitial cells yield mesenchymal stem cells following purification and culture. Hardy et al. now show, based on transcriptome analysis in single perivascular cells,