Figure 1.
Efficient isolation of uterine mesothelial cells (UtMCs) by gentle trypsinization of uterine cords.
(A) Left photograph shows representative aspect of uterine cords after their enzymatic digestion. Scale bar is 1 cm. Right panel shows CD54 immunofluorescence labelling of trypsinized uterine cords. Note that trypsinized uterine cords still retain CD54bright UtMCs (spot) on their surface. Enlarged spot area shows CD54bright UtMCs clearly distinguishable from CD54low/negative submesothelial cells (dashed white area). (B) Trypsin isolated UtMCs widely immunoexpress β-catenin, ZO-1, E-cadherin, CD29, CD54 and vimentin, but are immunonegative against CD44 and α-SMA. Scale bar is 50 µm. (C) Flow cytometry analysis of trypsin isolated UtMCs indicating their expression (pink histograms) of CD54, CD29, β-catenin, E-cadherin and ZO-1 and lack of expression of CD45, CD11b, CD44, Sca1, CD106 and CD117. Green histograms are cells labelled with fluorescent-conjugated isotype-matched antibodies. (A, B) Nuclei are counterstained in blue with Hoechst 33342.
Figure 2.
UtMCs cultured in SMDM undergo EMT and express stem and progenitors markers.
(A) Top panel, UtMCs cultured for 36 hours in SMDM exhibit cobblestone morphology (phase contrast) and express CK18. β-catenin, E-cadherin and ZO-1 were mostly expressed at intercellular contacts (arrowheads). Bottom panel, UtMCs cultured for 72 h in SMDM display irregular intercellular expression of E-cadherin (arrowheads) and strong nuclear expression of WT1 (arrowhead). Majority of UtMCs expressed displayed nuclei positive for Nanog (arrowheads). Sox2 was expressed in less extent in part of the UtMCs examined (arrowheads). UtMCs lacked nuclear immunoreactivity against Oct3/4. (B) Time-course immunofluorescence expression of β-catenin (green staining) and α-SMA (red staining) in UtMCs cultured in SMDM for the indicated time periods. Scale bar is 25 µm. (C) RT-PCR analysis of mesothelial (CK18, WT1, E-cadherin), EMT (Twist, Snail, Slug) and pluripotent (Oct 3/4, Sox2, Nanog) markers expression in freshly isolated UtMCs (0 h) and UtMCs cultured in SMDM for the indicated time periods. B, are RNAs isolated from adult mouse bladders. FH, are RNAs isolated from E17 mouse fetal hearts. ESCs, are RNAs isolated from mouse D3 ESCs. (D) Western blot analysis of freshly isolated UtMCs (0 h) and SMDM cultured UtMCs. For markers labelled with *, control (Ctrl) proteins were isolated from E17 mouse fetal visceral organs (heart, lung, and peritoneal organs). For markers labelled with **, Ctrl proteins were extracted from D3 mouse ESCs. For β-catenin and α-SMA, Ctrl proteins were extracted from adult mouse bladder. (A–B) Nuclei are counterstained with Hoechst 33342.
Table 1.
Summary of immunofluorescence.
Figure 3.
Immunofluorescence analysis of the vasculogenic differentiation of UtMCs cultured in SMDM.
(A) Left images show phase-contrast pictures of UtMCs cultured in SMDM for the indicated time periods. Scale bar is 50 µm. Right images show corresponding immunofluorescence expression of E-cadherin, CK18, α-SMA and nestin. Nuclei are counterstained in blue with Hoechst 33342. (B) Summary of MetaMorph-based fluorescence signals quantification of E-cadherin, CK18, nestin and α-SMA. Data are shown as marker fluorescence ± s.d., as deduced from immunofluorescent images performed onto 3 independent cultures. (C) Expansion of UtMCs during 15 days of culture in SMDM as deduced after calculation of cumulating numbers of cells generated after 10 and 15 days of culture.
Figure 4.
UtMCs cultured in SMDM acquire VSMCs markers expression.
(A) RT-PCR analysis of SM genes expression in freshly isolated UtMCs (0 h) and UtMCs cultured in SMDM for the indicated time periods. FH, are RNAs isolated from E17 mouse fetal hearts. (B) Western blot analysis of α-SMA, smoothelin-B and PDGFR-β expression in freshly isolated UtMCs (0 h) and UtMCs cultured in SMDM for the indicated time periods. B, are proteins extracted from adult mouse bladders. Note that smoothelin-B antibody (H-300) also detected an intermediate smtn-C isoform in SMDM cultured. (C) Immunofluorescence detection of smoothelin-B and PDGFR-β in UtMCs cultured for the indicated time periods in SMDM. (D) Immunofluorescence and intracellular flow cytometric expression of SM markers in UtMCs cultured for 15 days in SMDM (d15 SMDM cultured UtMCs). Red histograms are cells incubated with primary and secondary antibodies. Black line histograms are cells incubated with secondary antibody alone. (E) Summary results of intracellular flow cytometric quantification of SM markers positive cells in d15 SMDM cultured UtMCs and A7r5 VSMCs. Results are represented as mean percentages of cells marker positive ± s.d. from 3 independent cultures. (C, D), nuclei are counterstained in blue with Hoechst 33342.
Figure 5.
UtMCs cultured in SMDM express cardiac lineage markers.
(A) RT-PCR analysis of cardiac genes expression in freshly isolated UtMCs (0 h) and UtMCs cultured for 3, 5, 10 and 15 days in SMDM. FH, are RNAs isolated from E17 mouse fetal hearts. (B) Immunofluorescence expression of Gata-4, Isl1, sACTN and cTnT in UtMCs cultured for 6 h and 5, 10 and 15 days in SMDM (C) Western blot analysis of Isl-1, GATA-4, sACTN and cTnT expression in freshly isolated UtMCs (0 h) and UtMCs cultured for 3, 5, 10 and 15 days in SMDM. FVO, are proteins extracted from E17 mouse fetal visceral organs (heart, lung, and peritoneal organs). (D) Double immunofluorescence detection of smoothelin-B with either sACTN or cTnT in UtMCs cultured 10 days in SMDM. (B, D) Nuclei are counterstained in blue with Hoechst 33342.
Figure 6.
UtMCs-derived VSM-like cells display
[Ca2+]i rises against several vasoactive agonists. (A–C) SMDM cultured UtMCs acquire functional muscarinic receptors (mAChRs). (A) Representative immunoexpression of M2 and M3 mAChRs in UtMCs cultured in SMDM for 18 h, 5 days (undergoing EMT) and 15 days (UtMCs-derived VSM-like cells). Nuclei are counterstained in blue with Hoechst 33342. Scale bar is 50 µm. (B) Representative traces of cytosolic Ca2+ concentrations (represented as fura-2 ratio, F340/F380) recorded in UtMCs cultured for 5 days in SMDM (induced EMT) or in serum-free media (mesothelial phenotype) after their sequential exposure to 1mM carbachol and 5 µM adenosine triphosphate (ATP). (C1–C2) UtMCs cultured in SMDM exhibit an atropine-sensitive Ca2+ response to carbachol (C1) Representative traces of [Ca2+]i recorded in UtMCs cultured for 5 and 15 days in SMDM that were preincubated or not with 1 mM atropine (1 mM) and then challenged against 1 mM carbachol. (C2) Summary data of [Ca2+]i rises (Δratio ± s.d.) calculated from indicated number (n) of cells tested. (D1–D2) UtMCs-derived VSM-like cells exhibit [Ca2+]i rises to other several vasoactive agonists. (D1) Representative traces of [Ca2+]i recorded in UtMCs-derived VSM-like cells that were incubated against 60 mM high K+ (KCl), 50 nM endothelin 1 (ET1), 10 µM angiotensin II (Ang II), 1 mM carbachol (CCh), 10 µM vasopressin (VAP), 10 µM oxytocin (OXY),10 µM noradrenaline (NE), 10 µM Oxotremorine (OXO) and 10 µM acetylcholine (ACh). (D2) Summary results of vasoactive agonists-induced [Ca2+]i rises (Δratio ± s.d.) calculated from indicated number (n) of cells tested (*, indicates p<0.05 and **p<0.001 by comparison to basal Δratio).
Figure 7.
UtMCs-derived VSM-like cells display a contractile phenotype.
(A1–A2) Cells-collagen gel lattices contraction assay. (A1) Shows picture of collagen gel lattices lacking cells (w/o cells) or seeded with MS1 endothelial cells, freshly isolated UtMCs (UtMCs) and UtMCs-derived VSM-like cells after 0, 24 and 48 hours (h) of their initial release from the well. (A2) Summary of the collagen gel lattices contraction assay. Data indicate the mean percentile reduction in lattices area ± s.d. from 3 independent experiments (*, p<0.001 compared to initial area). (B1–B2) UtMCs-derived VSM-like cells display vasoactive agonists-induced contractile responses. (B1) Schematic protocol used to generate UtMCs-derived VSM-like cells spheroids. Right images, spheroids adhered for 4 hours (spreading) and 24 hours (multilayered culture). Multilayered UtMCs-derived VSM-like cells cultures retain SM22α and smoothelin-B immunofluorescence expression. (B2) Shows summary results of vasoactive agonists-induced contractile responses displayed by UtMCs-derived VSM-like cells. Contractile responses displayed by primary VSMCs to several vasoactive agonists are shown for comparison. (n), number of independent cultures tested.