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.

Keywords: cell biology; flow cytometry; human; mesenchymal stromal cells; placental mesenchymal heterogeneity; regenerative medicine; stem cells.

Figure 1.. Placental villus structure and specificity of markers used to exclude unwanted cell populations.

(A) Placental villous morphology and plane of section, (B) haematoxylin and eosin staining of a thin section though a placental villus (7.1 weeks), localisation of (C) β4 integrin (red) to cytotrophoblasts and (E) CD144 (red) and CD31 (green) to blood vessels (white arrows) in placental villus sections confirmed antibody specificity. No fluorescence is seen in negative IgG controls (D, F) run simultaneously. Nuclei are counterstained with DAPI (blue). Scale bar = 100 µm. Rendered images in (A) have been acquired from Biorender.com.

Figure 2.. Categorisation of placental villus core subsets using Panel One markers.

(A) Samples were gated to exclude debris, doublets, dead cells, hematopoietic cells (CD45⁺ and CD235a⁺), and cytotrophoblasts (β4 integrin). (B) Marker expression was used to categorise five subsets that were overlaid onto viSNE plots generated in Cytobank. (C) The average percentage contribution of each subset is presented as a pie chart. (D) A scatter plot with bars depicting the mean percentage of CD73⁺CD90⁺ cells from villous core cells across first trimester analysed on an Aria II (n=24, black) or an Aurora spectral analyser (n=5, red). Error bars represent the standard deviation of the mean.

Figure 3.. Phenotypic characterisation of villous core subsets.

(A) The gating strategy used to identify subsets (CD31⁺CD34⁺, CD73⁺CD90⁺, perivascular cells, podoplanin⁺CD36⁺ and CD26⁺CD90⁺), and (B) heat maps comparing the expression of specific antigens between subsets (n=5).

Figure 4.. In vitro culture of CD73⁺CD90⁺ and podoplanin⁺CD36⁺ cells.

(A) FACS sorting was used to isolate CD73⁺CD90⁺ and podoplanin⁺CD36⁺ cells from placental villous core cells (n=3). (B) Morphology and phenotype of CD73⁺CD90⁺ cells after 7 days in culture. (C) Phenotype of podoplanin⁺CD36⁺ cells after 7 days in culture. (D) CD146 and CD142 expression on CD73⁺CD90⁺ analysed with Panel One (day 0, n=5) or at 7 days after culture (n=3). (E) CD146 and CD142 expression on podoplanin⁺CD36⁺ cells analysed with Panel One (day 0, n=5) or at 7 days after culture (n=3). (F) Isolation of explant-derived pMSCs, and morphology and phenotype of passaged pMSCs (n=3). Error bars = standard error of the mean and scale bar = 100 µm.

Figure 4—figure supplement 1.. Representative 2-dimensional flow cytometry plots displaying the phenotype of FACS sorted CD73⁺CD90⁺ and podoplanin⁺CD36⁺ cells after 7 days culture in vitro (n=3), and explant isolated pMSCs after culture in vitro (n=3, p2-6).

Figure 4—figure supplement 2.. Flow cytometry histograms displaying the phenotype of FACS sorted CD73⁺CD90⁺ and podoplanin⁺CD36⁺ cells after 7 days culture in vitro (n=3), and explant isolated pMSCs after culture in vitro (n=3, p2-6).

Figure 5.. CD73⁺CD90⁺ and podoplanin⁺CD36⁺ cells upregulate markers consistent with contractile cells.

(A) FACS sorting was used to isolate CD73⁺CD90⁺ and podoplanin⁺CD36⁺ cells from placental villous core digests (n=3). CD73⁺CD90⁺ (B) and podoplanin⁺CD36⁺ (I) cell expression of αSMA (C, F, J, M), calponin (D, G, K, H) or MYH-11 (E, H, L, O) following 7 days of culture in advanced-DMEM/F12 or EGM-2. Irrelevant mouse IgG (P) and rabbit IgG (Q) were used as negative controls. Decidual sections containing spiral arteries with intact smooth muscle layers were used as positive controls for staining (R–T). Scale bar = 100 µm.

Figure 6.. Schematic diagram demonstrating the enzymatic digestion process used to obtain a single-cell suspension of placental villous core cells for flow cytometry analysis.

Figure 7.. Representative 2-dimensional flow cytometry plots displaying the gating strategy used to assess the proportion of extravillous trophoblasts (HLAG +cells) in villous core digests.

(A) Villous core cells and, (B) cells from the first digest washing steps known to contain extravillous trophoblasts were used as a positive control.