Efficient isolation of uterine mesothelial cells (UtMCs) by gentle trypsinization of uterine cords.

<p>(<b>A</b>) 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 CD54^bright UtMCs (spot) on their surface. Enlarged spot area shows CD54^bright UtMCs clearly distinguishable from CD54^low/negative submesothelial cells (dashed white area). (<b>B</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. (<b>C</b>) 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. (<b>A</b>, <b>B</b>) Nuclei are counterstained in blue with Hoechst 33342.</p

Immunofluorescence analysis of the vasculogenic differentiation of UtMCs cultured in SMDM.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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.</p

Functional Vascular Smooth Muscle-like Cells Derived from Adult Mouse Uterine Mesothelial Cells

<div><p>In mammalian visceral organs, vascular smooth muscle cells (VSMCs) originate from an epithelial-to-mesenchymal transition (EMT) of embryonic mesothelial cells (MCs). The ability of adult MCs to recapitulate EMT and to acquire smooth muscle (SM) markers upon provasculogenic culture suggested they might retain embryonic vasculogenic differentiation potential. However, it remains unknown whether adult MCs-derived SM-like cells may acquire specific vascular SM lineage markers and the functionality of differentiated contractile VSMCs. Here, we describe how a gentle trypsinization of adult mouse uterine cords could selectively detach their outermost uterine mesothelial layer cells. As other MCs; uterine MCs (UtMCs) uniformly expressed the epithelial markers β-catenin, ZO-1, E-cadherin, CD54, CD29, and CK18. When cultured in a modified SM differentiation media (SMDM) UtMCs initiated a loss of epithelial characteristics and gained markers expression of EMT (Twist, Snail, and Slug), stem and progenitor (Nanog, Sox2, C-kit, Gata-4, Isl-1, and nestin), SM (α-SMA, calponin, caldesmon, SM22α, desmin, SM-MHC, and smoothelin-B) and cardiac (BMP2, BMP4, ACTC1, sACTN, cTnI, cTnT, ANF, Cx43, and MLC2a). UtMCs repeatedly subcultured in SMDM acquired differentiated VSM-like characteristics and expressed smoothelin-B in the typical stress-fiber pattern expression of contractile VSMCs. Relevantly, UtMCs-derived VSM-like cells could generate <em>“mechanical force”</em> to compact collagen lattices and displayed in diverse degree voltage (K⁺) and receptor (endothelin-1, oxytocin, norepinephrine, carbachol and vasopressin)-induced [Ca²⁺]<em>_i</em> rises and contraction. Thus, we show for the first time that UtMCs could recapitulate in vitro differentiative events of early cardiovascular differentiation and transdifferentiate in cells exhibiting molecular and functional characteristics of VSMCs.</p> </div

UtMCs cultured in SMDM undergo EMT and express stem and progenitors markers.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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. (<b>D</b>) 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. (<b>A–B</b>) Nuclei are counterstained with Hoechst 33342.</p

UtMCs cultured in SMDM acquire VSMCs markers expression.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) Immunofluorescence detection of smoothelin-B and PDGFR-β in UtMCs cultured for the indicated time periods in SMDM. (<b>D</b>) 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. (<b>E</b>) 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. (<b>C</b>, <b>D</b>), nuclei are counterstained in blue with Hoechst 33342.</p

Summary of immunofluorescence.

<p>UtMCs (freshly isolated UtMCs); EMT (UtMCs cultured in SMDM for 5 days); UtMCs-derived VSM-like cells (UtMCs cultured for 15 days in SMDM). Antigen expression was evaluated as being negative (–), very weak (+*), weak (+), intermediate (++) or strong (+++).</p><p>Alpha smooth muscle actin (α-SMA); Atrial natriuretic factor (ANF); Cytokeratin 18 CK18); Cytokeratin 19 (CK19); Cardiac troponin T (cTnT); Embryonic stem cells (ESCs); Muscarinic acetylcholine receptors (mAChR); Mesenchymal stem cells (MSCs); Platelet derived growth factor receptor beta (PDGFR-β); Sarcomeric alpha actinin (sACTN); Wilms tumor protein 1 (WT1); Zona Occludens-1 (ZO-1).</p

UtMCs cultured in SMDM express cardiac lineage markers.

<p>(<b>A</b>) 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>B</b>) Immunofluorescence expression of Gata-4, Isl1, sACTN and cTnT in UtMCs cultured for 6 h and 5, 10 and 15 days in SMDM (<b>C</b>) 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). (<b>D</b>) Double immunofluorescence detection of smoothelin-B with either sACTN or cTnT in UtMCs cultured 10 days in SMDM. (<b>B</b>, <b>D</b>) Nuclei are counterstained in blue with Hoechst 33342.</p

Recapitulation of pancreas organogenesis for efficient hESCs differentiation.

<p><b>A</b>: Schematic representation of the steps involved in ESCs differentiation toward a β-cell fate and the factors and signaling pathways involved in this process. ESC, embryonic stem cells; ME, mesendoderm; DE, definitive endoderm; PG, primitive gut; PF, posterior foregut; PE, pancreatic endoderm; EP, endocrine precursors; BC, β-cells. <b>B</b>: Schematic representation summary of the step-wise differentiation protocol used to obtain hESC-derived insulin-producing cells. RA, retinoic acid; Fib, fibronectin; ITS, insulin-transferrin-selenium.</p

Characterization of the differentiated islet-like clusters obtained at the end of the differentiation protocol.

<p><b>A</b>: RT-PCR detection of definitive endoderm, endocrine precursors and islet cells specific markers. β-actin was used as the input control. SD: hESCs spontaneously differentiated; -RSV: hESCs subjected to our differentiation protocol without RSV addition; +RSV: hESCs subjected to our differentiation protocol with RSV addition. H Pancreas: human pancreas as positive control. <b>B</b>: Immunofluorescence analysis of PDX1, GCG, INS and C-PEP expression in spontaneous differentiated cells (SD) and in differentiated β-cell-like cells without (-RSV) or with RSV addition (+RSV). Nuclei are stained with Hoechst (<i>blue</i>). Scale bar 50 μm. <b>C</b>: RT-PCR detection of definitive endoderm, endocrine precursors and islet cells specific markers of hiPSCs-derived β-cell-like cells. β-actin was used as the input control. <b>D</b>: q-PCR comparison of the levels of expression of some β-markers in β-cell-like cells derived from hiPSCs (<i>red bars</i>) or hESCs (<i>blue bars</i>) without or with RSV treatment (-RSV, +RSV).</p

In vitro and in vivo studies of hESCs-derived insulin producing cells functionality.

<p><b>A</b>: Immunohistochemical analyses of pancreas sections from control mice (Pancreas CTR) and STZ-induced diabetic mice (Pancreas STZ). Upper panel shows hematoxylin and eosin stain (H&E stain) of Langerhans Islets, which are completely compromised in diabetic mice. Lower panel shows the complete loss of insulin stain (<i>red</i>) in diabetic pancreas. <b>B</b>: Nonfasting blood glucose measurements of control mice (<i>blue line</i>, n = 4), STZ-induced diabetic mice (<i>red line</i>, n = 3) and STZ-induced diabetic mice transplanted (Tx) with 2000 islet-like clusters derived from hESCs differentiation (<i>green line</i>, n = 3). <b>C</b>: Percentage of body weight variations in control mice (<i>blue line</i>, n = 4), STZ-induced diabetic mice (<i>red line</i>, n = 3) and STZ-induced diabetic mice transplanted with 2000 islet-like clusters derived from hESCs differentiation (<i>green line</i>, n = 3). <b>D</b>: Immunohistochemical analyses of a transplanted kidney showing H&E stain of the engraftment and a dispersed insulin staining (<i>red</i>) throughout the renal parenchyma. Scale bar 75 μm and 25 μm respectively.</p