Perivascular human endometrial mesenchymal stem cells express pathways relevant to self-renewal, lineage specification, and functional phenotype

Abstract

Human endometrium regenerates on a cyclic basis from candidate stem/progenitors whose genetic programs are yet to be determined. A subpopulation of endometrial stromal cells, displaying key properties of mesenchymal stem cells (MSCs), has been characterized. The endometrial MSC (eMSC) is likely the precursor of the endometrial stromal fibroblast. The goal of this study was to determine the transcriptome and signaling pathways in the eMSC to understand its functional phenotype. Endometrial stromal cells from oocyte donors (n = 20) and patients undergoing benign gynecologic surgery (n = 7) were fluorescence-activated cell sorted into MCAM (CD146)(+)/PDGFRB(+) (eMSC), MCAM (CD146)(-)/PDGFRB(+) (fibroblast), and MCAM (CD146)(+)/PDGFRB(-) (endothelial) populations. The eMSC population contained clonogenic cells with a mesenchymal phenotype differentiating into adipocytes when cultured in adipogenic medium. Gene expression profiling using Affymetrix Human Gene 1.0 ST arrays revealed 762 and 1518 significantly differentially expressed genes in eMSCs vs. stromal fibroblasts and eMSCs vs. endothelial cells, respectively. By principal component and hierarchical clustering analyses, eMSCs clustered with fibroblasts and distinctly from endothelial cells. Endometrial MSCs expressed pericyte markers and were localized by immunofluorescence to the perivascular space of endometrial small vessels. Endometrial MSCs also expressed genes involved in angiogenesis/vasculogenesis, steroid hormone/hypoxia responses, inflammation, immunomodulation, cell communication, and proteolysis/inhibition, and exhibited increased Notch, TGFB, IGF, Hedgehog, and G-protein-coupled receptor signaling pathways, characteristic of adult tissue MSC self-renewal and multipotency. Overall, the data support the eMSC as a clonogenic, multipotent pericyte that displays pathways of self-renewal and lineage specification, the potential to respond to conditions during endometrial desquamation and regeneration, and a genetic program predictive of its differentiated lineage, the stromal fibroblast.

FIG. 1.

Isolation and characteristics of MCAM (CD146)⁺/PDGFRB⁺ cells from human endometrium. A–E) FACS analysis of endometrial stromal cells obtained by enzymatic dissociation of endometrial tissue labeled with DAPI and antibodies to CD45, EPCAM⁺, MCAM (CD146), and PDGFRB, showing single (B), viable (C), and CD45⁻ and EPCAM⁻ (D) cells sorted into three populations according to MCAM (CD146) and PDGFRB expression (E). F) Colony derived from FACS-sorted MCAM (CD146)⁺/PDGFRB⁺ stromal cells. G) Vimentin-positive cells derived from FACS-sorted MCAM (CD146)⁺/PDGFRB⁺ eMSC. Inset: vimentin-positive endometrial stromal fibroblasts (positive control). H) Oil red O-positive eMSC differentiated in vitro to adipogenic lineage. Inset: eMSC not cultured in adipogenic medium (negative control). Original magnifications ×40 (F) and ×100 (G, H, and insets in G and H).

FIG. 2.

Clustering analysis of eMSCs. Principal component analysis clustering: eMSC (red), endometrial stromal fibroblast (brown), and endothelial cell (blue) populations.

FIG. 3.

Hierarchical clustering. Complete hierarchical clustering analysis between eMSC, endometrial stromal fibroblast, and endothelial cell populations (left). Magnified inset of most differentially expressed genes (right) taken from the complete clustering analysis.

FIG. 4.

Colocalization of MCAM (CD146) and PDGFRB in human endometrium. Sections of full-thickness human endometrium were dual labeled for MCAM (CD146) and PDGFRB. MCAM (CD146) was localized to all endothelial cells and some perivascular cells in the basalis (A and D), whereas PDGFRB showed a wider distribution on endometrial stromal cells as well as some perivascular cells (B and E), but was absent in glandular epithelium (B and E). Colocalization (arrow) of MCAM (CD146) and PDGFRB was observed in a perivascular location (C and F). Nuclei in C, F, and I are stained with DAPI. Negative IgG controls for MCAM (CD146) (mouse; G) and PDGFRB (rabbit; H) and merged images (I) are shown. GE, glandular epithelium; BV, blood vessel; ST, stroma. Original magnification ×100.