Full spectrum flow cytometry reveals mesenchymal heterogeneity in first trimester placentae and phenotypic convergence in culture, providing insight into the origins of placental mesenchymal stromal cells

Abstract

Single-cell technologies (RNA-sequencing, flow cytometry) are critical tools to reveal how cell heterogeneity impacts developmental pathways. The placenta is a fetal exchange organ, containing a heterogeneous mix of mesenchymal cells (fibroblasts, myofibroblasts, perivascular, and progenitor cells) . Placental mesenchymal stromal cells (pMSC) are also routinely isolated, for therapeutic and research purposes. However, our understanding of the diverse phenotypes of placental mesenchymal lineages, and their relationships remain unclear. We designed a 23-colour flow cytometry panel to assess mesenchymal heterogeneity in first-trimester human placentae. . Four distinct mesenchymal subsets were identified; CD73+CD90+ mesenchymal cells, CD146+CD271+ perivascular cells, podoplanin+CD36+ stromal cells, and CD26+CD90+ myofibroblasts. CD73+CD90+ and podoplanin+CD36+ cells expressed markers consistent with cultured pMSCs, and were explored further. Despite their distinct ex-vivo phenotype, in culture CD73+CD90+ cells and podoplanin+CD36+ cells underwent phenotypic convergence, losing CD271 or CD36 expression respectively, and homogenously exhibiting a basic MSC phenotype (CD73+CD90+CD31-CD144-CD45-). However, some markers (CD26, CD146) were not impacted, or differentially impacted by culture in different populations. Comparisons of cultured phenotypes to pMSCs further suggested cultured pMSCs originate from podoplanin+CD36+ cells. This highlights the importance of detailed cell phenotyping to optimise therapeutic capacity, and ensure use of relevant cells in functional assays.

Characterization of adult human skeletal cells in different tissues reveals a CD90+CD34+ periosteal stem cell population

“…Many studies on SSPCs use plastic adherence, or adherence to fibronectin-coated plates for chondroprogenitors, but numerous studies have shown that the cell surface phenotype of various mesenchymal progenitor populations is altered by attachment, in vitro culture conditions, and passaging (32)(33)(34)(35)(36)(37). Therefore, it is important to identify functionally different SSPCs and define their origins in vivo.…”

“…We used Panel One reported in Boss et al (37) and similar panels used for the analysis of adipose tissue as the backbone of our stain. The panel is shown in Table 1.…”

A live-cell platform to isolate phenotypically defined subpopulations for spatial multi-omic profiling

“…FACS is a technique initially developed for immune cell classification, and now is utilized in a variety of fields to identify subpopulation heterogeneity within a larger population [65][66][67] . Traditional approaches utilizing FACS often isolate subpopulations with known markers, making it difficult to identify novel subpopulations utilizing FACS alone.…”

Human placental vascular and perivascular cell heterogeneity differs between first trimester and term, and in pregnancies affected by foetal growth restriction

“…Placental villous tissue was carefully dissected away from the foetal membranes and washed thoroughly in PBS to remove maternal blood and debris. Placental explants (∼1 cm 2 ) were largely denuded of trophoblasts (the epithelial cells that are present around the outside of placental villi) by digestion in 10 ml/1 g tissue of Enzyme Digest Solution (1 mg/ml Dispase II (Roche, Mannheim, Germany), 0.5 mg/ml Collagenase A (Roche), and 1.5 mg/ml DNAse l (Sigma-Aldrich, Saint Louis, MO, USA)) in advanced-DMEM/F12 (Gibco, Grand Island, NY, USA) for 10 min at 37 °C as previously described ( Boss et al , 2022 ). Explants were washed repeatedly in PBS and then placed in a 90 mm petri dish and finely macerated with two sterile disposable scalpels (with additional maceration applied to term explants to ensure complete digestion of this more fibrous tissue).…”

“…Despite a recent flurry of studies employing single-cell RNA sequencing to investigate placental cell populations ( Suryawanshi et al , 2018 ; Vento-Tormo et al , 2018 ), a method that has revolutionized our understanding of cell heterogeneity, RNA changes are not always representative of protein changes and studies often fail to report on the stromal/endothelial lineages of the placenta ( Liu et al , 2018 ; Vento-Tormo et al , 2018 ; Pique-Regi et al , 2019 ). Recently, we developed a high-dimensional flow cytometry panel to explore the extent of cellular heterogeneity within the first trimester villous core, with a focus on stromal cell populations ( Boss et al , 2022 ). Here, we capitalize on this advance in resolving the placental villous core to better understand how endothelial and perivascular heterogeneity and abundance vary both across gestation and in FGR.…”