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
- Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, New Zealand
- School of Biological Sciences, University of Auckland, New Zealand
- Maurice Wilkins Centre, University of Auckland, New Zealand
Figures
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 …
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 …
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 …
Figure 4 with 2 supplements
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) …
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), …
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.
Tables
Table 1
Antigens included in Panel One and their functional roles with respect to the villous core.
Antigens are grouped by cell type (grey and white boxes).
| Antigen | Full name/s | Functional capacity/relevance | References |
|---|---|---|---|
| ITGβ4/CD104 | β4 integrin | Cell adhesion molecule that uniquely identifies cytotrophoblasts in the placenta. | James et al., 2015 |
| CD45 | Protein tyrosine phosphatase, receptor type, C. lymphocyte common antigen | Expressed on all nucleated haematopoietic cells. | Hermiston et al., 2003 |
| HLA-DR | Human Leukocyte Antigen – DR isotype | Expressed by antigen presenting cells i.e. macrophages, MHC class II cell surface receptor, involved in antigen presentation and adaptive immunity, | Cruz-Tapias et al., 2013 |
| CD235a/GPA | Glycophorin A | Red blood cell marker, identifies early emerging RBCs/erythroblasts from haemogenic endothelium | Garcia-Alegria et al., 2018; Mao et al., 2016 |
| CD41 | Integrin alpha-IIb | Platelet marker. Expressed by earliest emerging haematopoietic cells from haemogenic endothelium | Garcia-Alegria et al., 2018; Li et al., 2005 |
| CD117/cKIT | Mast/stem cell growth factor receptor | Receptor expressed on haematopoietic stem cells and involved in their differentiation | Rönnstrand, 2004 |
| CD144 /VE-cadherin | Vascular endothelial cadherin | Endothelial cell-cell adherens junctional marker, stabilises vessels, inhibits vascular growth, regulates vascular permeability. | Giannotta et al., 2013 |
| CD34 | Haematopoietic Progenitor Cell Antigen | Expressed by haematopoietic and vascular progenitors and adipose-derived MSCs. Transmembrane phosphoglycoprotein, thought to identify early placental progenitors | Brooks et al., 2020; Sidney et al., 2014; Yoder, 2009 |
| CD31/PECAM1 | Platelet endothelial cell adhesion molecule | Expressed by endothelial cells, platelets, macrophages and Kupffer cells, granulocytes, lymphocytes, megakaryocytes. Adhesion molecule found at endothelial intercellular junctions. | Marelli-Berg et al., 2013 |
| VEGFR2/KDR | Vascular Endothelial Growth Factor Receptor 2/Kinase Insert Domain Receptor | Expressed by endothelial cells and thought to identify placental core progenitors. Receptor for angiogenic VEGF, involved in vasculogenesis and angiogenesis. | Demir et al., 2007 |
| CD54/ICAM1 | Intercellular Adhesion Molecule 1 | Expressed at low levels on endothelial cells, monocytes and lymphocytes. Increased expression in response to inflammatory cytokines. | Hubbard and Rothlein, 2000 |
| CD36/FAT | Platelet glycoprotein 4/fatty acid translocase | Expressed by microvascular endothelial cells, and fibroblasts. Has an anti-angiogenic effect via binding Thrombospondin 1. Involved in fatty acid uptake. | Dye et al., 2001; Heinzelmann et al., 2018; Silverstein and Febbraio, 2009 |
| CD90 /Thy-1 | Thymocyte differentiation antigen 1 | Expressed by MSCs, haematopoietic stem cells, fibroblasts, myofibroblasts. | Viswanathan et al., 2019 |
| CD73 /NT5E | Ecto-5′-nucleotidase | Expressed by MSCs and endothelial cells. Works with CD39 to convert extracellular ATP to adenosine to create immunosuppressive effect. | Roh et al., 2020; Viswanathan et al., 2019 |
| CD39 /NTPDase | Ectonucleoside triphosphate diphosphohydrolase-1 | Upregulated by MSCs to suppress lymphocyte activation. Immunosuppressive actions via the conversion of extracellular ATP (inflammatory) into adenosine (anti-inflammatory) | Saldanha-Araujo et al., 2011; Zhao et al., 2017 |
| CD55/DAF | Decay-accelerating factor | Expressed by MSCs. Complement regulatory protein, inhibits C3 convertases thereby creating a threshold for complement activation, increased expression correlated with evasion of innate immune system | Ruiz-Argüelles and Llorente, 2007; Soland et al., 2013 |
| CD271 /NGFR / p75NTR | Low-affinity Nerve Growth Factor Receptor | Used in MSC and pericyte identification. Hypothesised to identify “stem-cell” or progenitor populations with superior differentiation and colony forming capacity. | Barilani et al., 2018; Kumar et al., 2017 |
| CD146/MCAM | Melanoma cell adhesion molecule | Adhesion molecule expressed by pericytes, endothelial cells and smooth muscle cells. Involved in the regulation of angiogenesis and vessel permeability. | Crisan et al., 2009; Leroyer et al., 2019 |
| CD248 | Endosialin/ tumor endothelial marker 1 | Pericyte and stromal cell marker. Involved in cell–cell adhesion, and host defence. | Lax et al., 2010; Tomkowicz et al., 2010 |
| CD142/TF | Tissue Factor/ thromboplastin | Expression correlated with pericytes, smooth muscle and fibroblasts. Activates blood clotting after injury, located outside the vasculature, | Abe et al., 1999; Morrissey, 2004 |
| CD26/DDP4 | Dipeptidyl peptidase-4, adenosine deaminase complexing protein 2 | Expressed by many tissues; T-cells, epithelial cells, ESCs, progenitor cells, placental myofibroblasts. Serine protease that cleaves a range of chemokines. Downregulation is correlated with increased stromal/myofibroblast proliferation. | Kohnen et al., 1996; Mezawa et al., 2019; Ou et al., 2013 |
| PDPN | Podoplanin | Lymphatic vascular marker. Expression is correlated with increased fibroblast migration. Binds to CLEC-2 receptor on platelets. | Astarita et al., 2012; Suchanski et al., 2017 |
Table 2
Primary conjugated antibodies used to confirm specificity of cytotrophoblast or endothelial markers.
| Antigen | Fluorophore | Clone | Dilution | Supplier |
|---|---|---|---|---|
| β4 integrin | FITC/APC | 450-9D | 1:200 | Thermofisher |
| CD144 | BV421 | 55–7 H1 | 1:200 | BD |
| CD31 | BV480 | WM59 | 1:200 | BD |
Table 3
Panel One designed to assess villous core cells on a 3 L Cytek Aurora.
| Antibody | Fluorophore | Clone | Flow cytometry Dose (μL) | Reference control | Supplier |
|---|---|---|---|---|---|
| CD55 | BB515 | IA10 | 0.6 | Villous core cells | Beckman Coulter |
| β4 integrin | FITC | 450-9D | 0.6 | Villous core cells | Thermofisher |
| CD34 | PerCP | 581 | 0.3 | Villous core cells | BD |
| CD36 | PerCpVio700 | REA760 | 0.6 | Villous core cells | Miltenyi Biotec |
| VEGFR2 | PE | 7D4-6 | 0.6 | Villous core cells | Biolegend |
| CD271 | PE-CF594 | C40-1457 | 0.6 | Stromal vascular fraction | Biolegend |
| CD142 | BB700 | HTF-1 | 1.25 | Villous core cells | BD |
| CD26 | PE/Cy5 | BA5b | 0.15 | Villous core cells | BD |
| PDPN | PE/Cy7 | NC-08 | 0.3 | Villous core cells | BD |
| CD248 | Alexa Fluor 647 | B1/35 | 0.6 | Stromal vascular fraction | BD |
| CD41 | APC | HIP8 | 0.3 | Beads | BD |
| CD90 | Alexa700 | 5E10 | 0.6 | Villous core cells | Biolegend |
| CD39 | BUV737 | TU66 | 1.25 | Beads | BD |
| ICAM1 | APC/Fire750 | HA58 | 2.5 | Stromal vascular fraction | Biolegend |
| CD144 | BV421 | 55–7 H1 | 1.25 | Villous core cells | BD |
| CD235a | Pacific Blue | H1264 | 0.3 | Villous core cells | BD |
| CD31 | BV480 | WM59 | 0.3 | Villous core cells | BD |
| CD45 | Krome-Orange | B61840 | 0.3 | Villous core cells | Beckman Coulter |
| CD146 | BV605 | PIH12 | 0.3 | Villous core cells | BD |
| CD117 | BV650 | 104D2 | 0.3 | Beads | BD |
| HLADR | BV750 | L243 | 0.3 | Villous core cells | BD |
| CD73 | BV785 | AD2 | 0.3 | Villous core cells | BD |
-
Table 3—source data 1
All antibodies used in Panel One were titrated on placental villous core digest cells.
- https://cdn.elifesciences.org/articles/76622/elife-76622-table3-data1-v2.pdf
-
Table 3—source data 2
Titration of additional antibodies, not contained in Panel One, required for the FACS sorting with Panel Two.
- https://cdn.elifesciences.org/articles/76622/elife-76622-table3-data2-v2.pdf
-
Table 3—source data 3
Representative images depicting how forward scatter (FSC) (A) or addition of cell-specific antibodies improved detection of appropriate doses for specific placental populations.
(B) β4 integrin+ cells were negative for podoplanin but demonstrated an unspecific shift in expression at higher doses. (C) Removal of β4 integrin+ improved detection of the optimal podoplanin dose.
- https://cdn.elifesciences.org/articles/76622/elife-76622-table3-data3-v2.pdf
Table 4
Composition of Panel Two.
This panel was developed to sort CD73 + CD90 + and podoplanin + CD36 + cells from placental villous core using a BD FACS Aria II.
| Antibody | Fluorophore | Clone | Dose (μL) | Supplier |
|---|---|---|---|---|
| CD45 | FITC | HI30 | 0.3 | BD |
| PDPN | PE | NC-08 | 0.3 | Biolegend |
| CD26 | PE/Cy7 | BA5b | 0.6 | BD |
| CD271 | PE/Dazzle 594 | C40-1457 | 0.6 | BD |
| CD144 | PerCP-5.5 | 55–7 H1 | 0.6 | BD |
| CD90 | Alexa700 | 5E10 | 0.6 | Biolegend |
| CD36 | APC-Cy7 | 5–271 | 0.6 | Biolegend |
| β4 integrin | APC | 450-9D | 0.6 | Thermofisher |
| CD31 | BV480 | WM59 | 0.3 | BD |
| CD73 | BV785 | AD2 | 0.3 | BD |
| CD34 | BUV395 | 581 | 1.25 | BD |
Table 5
Composition of Panel Three.
This panel was designed to assess the phenotype of placental populations after culture in vitro using a three laser Cytek Aurora.
| Antibody | Fluorophore | Clone | Dose | Supplier |
|---|---|---|---|---|
| CD34 | PerCP | 581 | 0.3 | BD |
| CD36 | PerCpVio700 | REA760 | 0.6 | Miltenyi Biotec |
| VEGFR2 | PE | 7D4-6 | 0.6 | Biolegend |
| CD271 | PE/Dazzle 594 | C40-1457 | 0.6 | Biolegend |
| CD142 | BB700 | HTF-1 | 1.25 | BD |
| CD26 | PE/Cy5 | BA5b | 0.15 | BD |
| PDPN | PE/Cy7 | NC-08 | 0.3 | BD |
| CD90 | Alexa700 | 5E10 | 0.6 | Biolegend |
| CD144 | BV421 | 55–7 H1 | 1.25 | BD |
| CD31 | BV480 | WM59 | 0.3 | BD |
| CD45 | Krome-Orange | B61840 | 0.3 | Beckman Coulter |
| CD146 | BV605 | PIH12 | 0.3 | BD |
| HLADR | BV750 | L243 | 0.3 | BD |
| CD73 | BV785 | AD2 | 0.3 | BD |
