Journal: Cell Reports Methods

Article Title: Efficient and scalable generation of primordial germ cells in 2D culture using basement membrane extract overlay

doi: 10.1016/j.crmeth.2023.100488

Figure Lengend Snippet: BMEx overlay differentiation promotes differentiation to hPGCLCs alongside amniotic ectoderm- and mesoderm-like cells (A) Uniform manifold approximation and projection (UMAP) plots showing cluster identification (ID) (left) and time period in days (right) using single-cell transcriptomics of several hPSC lines undergoing differentiation with BMEx overlay. (B) Heatmap showing expression levels of the top 12 DEGs of each cluster. (C) Expression of signature genes of cell types of interest on the UMAP plot from (A). (D) UMAP showing integrated single-cell transcriptomics data from EB differentiation method (UCLA2 from Chen et al. ) and BMEx overlay method, highlighting the cells of EB differentiation method (left) and the BMEx overlay method (right). (E) Expression of signature genes of cell types of interest on the UMAP plot from (D). (F) Immunofluorescence for TFAP2A, TFAP2C, and GATA6 (top left); GATA3, SOX17, and TFAP2C (top right); and TFAP2A, GATA6, and PDGFRA (bottom) at day 5 with BMEx overlay. In the top panels, the dashed box is magnified (right), showing separated channels. The bottom panels depict a maximum intensity projection (MIP) and render image with a digital cross section showing separated channels (bottom part). Scale bars: 50 μm. (G) Whole-mount immunofluorescence for TFAP2A, GATA6, and PDGFRA in human WG9 amnion. In the top panels, a digital cross section (right) shows separated channels. The bottom panels depict a MIP and render image, also shown from the side (bottom). Scale bars: 50 μm. AELC, amniotic ectoderm-like cell; AMLC, amniotic mesoderm-like cell; iMeLCs, incipient mesoderm-like cells; PELC, primitive endoderm-like cell; Progen, progenitor cells. See also Figure S2 .

Article Snippet: Mouse anti-AP2a (TFAP2A) (1:200) , Santa Cruz Biotechnology , Cat# sc-12726; RRID: AB_667767.

Techniques: Single-cell Transcriptomics, Expressing, Immunofluorescence

Journal: Cell Reports Methods

Article Title: Efficient and scalable generation of primordial germ cells in 2D culture using basement membrane extract overlay

doi: 10.1016/j.crmeth.2023.100488

Figure Lengend Snippet: BMEx overlay potentiates BMP4 signaling and increases expression of critical PGC specification factors (A) Volcano plot showing DEGs between hPSCs at day 0 and 2-differentiated progenitors with BMEx overlay. (B) Immunofluorescence for TFAP2A, SOX17, and PRDM1 at days 2, 3, and 5 with BMEx overlay in line M54. TFAP2A is shown on top as a single channel. Dashed box is magnified (below), showing separate channels. Scale bars: 50 μm. (C) Immunofluorescence for pSMAD1/5/9, GATA3, and SOX17 at days 2, 3, and 5 with or without BMEx overlay in line M54. pSMAD1/5/9 is shown on top as a single channel. Scale bars: 50 μm. (D) Immunofluorescence for TFAP2A, EOMES, and SOX2 in line F99 at day 2 without (a) or with (b) BMEx overlay or with addition of 10 ng/mL activin A (C) or 10 μM SB431542 (D). Scale bars: 50 μm. (E) Violin plots depict the quantification of the images in (D) as the mean fluorescence intensity in arbitrary units (A.U.) of TFAP2A (left) and EOMES (middle) in DAPI segmented areas (normalized to 1) per cell at day 2. The correlation between these two values per cell per condition was visualized in a scatterplot (right). (F) Cartoon summarizing the hPGCLC differentiation progression in the BMEx overlay method as well as the perturbations tested (from D and E), with key analyzed markers depicted. See also Figure S4 .

Article Snippet: Mouse anti-AP2a (TFAP2A) (1:200) , Santa Cruz Biotechnology , Cat# sc-12726; RRID: AB_667767.

Techniques: Expressing, Immunofluorescence, Fluorescence

Journal: Cell Reports Methods

Article Title: Efficient and scalable generation of primordial germ cells in 2D culture using basement membrane extract overlay

doi: 10.1016/j.crmeth.2023.100488

Figure Lengend Snippet:

Article Snippet: Mouse anti-AP2a (TFAP2A) (1:200) , Santa Cruz Biotechnology , Cat# sc-12726; RRID: AB_667767.

Techniques: Recombinant, Staining, Membrane, Transfection, Bicinchoninic Acid Protein Assay, Software, Microscopy

Journal: Biochemical and biophysical research communications

Article Title: Glycosaminoglycans promote osteogenesis from human induced pluripotent stem cells via neural crest induction.

doi: 10.1016/j.bbrc.2022.03.002

Figure Lengend Snippet: Fig. 1. Neural crest and mesenchymal stromal lineage differentiation from hiPSCs. (A) Schematic representation of stepwise differentiation into mature osteoblasts from hiPSCs via neural crest induction. (B) Induced NCLCs at day 10 expressed neural crest cell markers (AP2a, SOX10, NESTIN, and P75), but NCLCs did not express any pluripotent stem cell markers (NANOG, OCT3/4), as confirmed by immunofluorescence staining. (C) Induction efficiency of NCLCs was analyzed by counting the number of AP2a, SOX10 positive cells relative to the total number of nuclei in an average of five randomly selected images from three replicates. Data are represented as mean ± SD. (D) Mesenchymal cell markers CD29, CD44, CD73, and CD90 were detected in NCMCs at day 17 by flow cytometry analysis. Blue histogram; isotype control. (E) Expression of a cranial marker OTX2 in NCLCs and NCMCs, as observed by immunofluorescence staining. The cell nuclei were counterstained with Hoechst. Scale bars, 100 mm.

Article Snippet: Cells were then treated with 0.3% Triton X-100 and blocking buffer (Nacalai Tesque) at room temperature for 1 h, followed by overnight incubation in primary antibody solution at 4 C. The following primary antibodies were used: goat polyclonal anti-human SOX10 (1:200, R&D Systems), rabbit monoclonal anti-human AP2a (1:100, C83E10; Cell Signaling Technology), rabbit polyclonal anti-human NESTIN (1:200, Eurofins genomics), rabbit polyclonal anti-human P75 (1:200, Millipore), rabbit polyclonal anti-human NANOG (1:200, ReproCELL), mouse monoclonal anti-human OCT3/4 (1:200, C-10; Santa Cruz), goat polyclonal anti-human OTX2 (1:100, R&D Systems), rabbit polyclonal anti-human OPN (1:200, Abcam), and mouse polyclonal anti-human SOST (1:20, R&D Systems).

Techniques: Staining, Cytometry, Control, Expressing, Marker

Journal: Scientific Reports

Article Title: SPECC1L deficiency results in increased adherens junction stability and reduced cranial neural crest cell delamination

doi: 10.1038/srep17735

Figure Lengend Snippet: ( A ) Schematic representation of murine Specc1l gene indicating insertion of genetrap vectors in ES cell clones DTM096 (intron 1) and RRH048 (intron 15). ( B–D) Heterozygous Specc1l DTM096 embryos stained for lacZ , representing Specc1l expression, from E8.5 to E10.5. NE = neuroectoderm, NF = neural folds, PA1 = 1 st pharyngeal arch. ( E–P) Co-immunostaining of SPECC1L with NCC markers AP2A and SOX10 in E8.5 neural folds (NF; E–J ) and in E9.5 cranial sections ( K–P ). SPECC1L staining is broadly observed in the E8.5 neural folds ( E,H ; arrows), including in cells marked by AP2A (F,G; arrows) and SOX10 ( I,J ; arrows). At E9.5, SPECC1L strongly stains migrating CNCCs ( K,N ; arrows) marked by AP2A ( L,M ; arrows) and SOX10 ( O,P ; arrows).

Article Snippet: Antibodies against β-catenin (rabbit; 1:1000; Santa Cruz, Dallas TX) (mouse; 1:1000; Cell Signaling Technology, Danvers, MA), Myosin IIb (1:1000; Sigma-Aldrich, St. Louis, MO), E-cadherin (1:1000; Abcam, Cambridge, MA), AP2A (1:1000; Novus Biologicals, Littleton, CO), SOX10 (1:1000; Aviva Systems Biology, San Diego, CA), DLX2 (1:1000; Abcam, Cambridge, MA), phospho-Ser473-AKT (1:1000; Cell Signaling Technology, Danvers, MA), pan-AKT (1:1000; ThermoFisher Scientific, Waltham, MA), KI67 (1:1000; Cell Signaling Technology, Danvers MA), cleaved Caspase 3 (1:1000; Cell Signaling Technology, Danvers, MA), and β-actin (1:2500; Sigma-Aldrich, St. Louis, MO) were used as described.

Techniques: Clone Assay, Staining, Expressing, Immunostaining

Journal: Scientific Reports

Article Title: SPECC1L deficiency results in increased adherens junction stability and reduced cranial neural crest cell delamination

doi: 10.1038/srep17735

Figure Lengend Snippet: ( A,B’) E9.5 WT ( A ) embryo with migratory cranial neural crest cells (CNCCs) marked with Wnt1 -Cre ( A ’). In contrast, Specc1l mutant embryo shows open neural folds ( B ), arrow) with CNCCs that have still not migrated ( B ’, arrow). ( C,D’) Brightfield images ( C,D ) and CNCC marker DLX2 immuno-stain ( C’,D’ ) of E10.5 WT ( C,C’ ) and Specc1l mutant ( D,D’ ) embryos. In E10.5 WT embryo, DLX2 positive CNCCs populate the branchial arches ( C’ , arrowhead), while in the mutant significant staining remains in open neural folds ( D’ , arrow) with some staining in the 1 st pharyngeal arch ( D’ , arrowhead), indicating poor CNCC delamination and migration. E-R) Sections from WT and Specc1l mutant embryos at E8.5 ( E–L ) and at E9.5 ( M–R ) were stained with NCC markers SOX10 ( E,F,M,N ), AP2A ( G,H,O,P ), and DLX2 ( I,J,Q,R ). At E8.5, NCC staining is observed in the neural folds (NF) of WT and mutant sections. Co-staining of SOX10 and β-catenin in E8.5 WT ( K ) and mutant ( L ) shows increased β-catenin staining at cell boundaries in the neural folds. At E9.5, migratory CNCC staining is observed in WT ( M,O,Q ), while in mutants undelaminated CNCCs stain the open neural folds ( N,P,R ). ( S–Z) Analysis of AJ markers in vivo in WT and Specc1l DTM096/RRH048 mutant E9.5 embryo coronal sections. The approximate plane of section is indicated in the top-right corner. Increased F-actin ( S,T ) and Myosin IIb ( U,V ) staining is observed in mutant tissue sections. Similarly to in vitro results in , expanded β-catenin ( W,X ) and E-cadherin ( Y,Z ) membrane staining is observed in vivo in mutant embryos. ( AA-BB) Electron micrograph of WT embryo section looking at apico-basal cell boundary shows a distinct electron-dense region indicating adherens junction ( AA , arrowhead). In contrast, the entire apico-basal boundary appears electron-dense in Specc1l mutant embryo section ( BB , arrowheads), suggesting increased density and dispersion of adherens junctions.

Article Snippet: Antibodies against β-catenin (rabbit; 1:1000; Santa Cruz, Dallas TX) (mouse; 1:1000; Cell Signaling Technology, Danvers, MA), Myosin IIb (1:1000; Sigma-Aldrich, St. Louis, MO), E-cadherin (1:1000; Abcam, Cambridge, MA), AP2A (1:1000; Novus Biologicals, Littleton, CO), SOX10 (1:1000; Aviva Systems Biology, San Diego, CA), DLX2 (1:1000; Abcam, Cambridge, MA), phospho-Ser473-AKT (1:1000; Cell Signaling Technology, Danvers, MA), pan-AKT (1:1000; ThermoFisher Scientific, Waltham, MA), KI67 (1:1000; Cell Signaling Technology, Danvers MA), cleaved Caspase 3 (1:1000; Cell Signaling Technology, Danvers, MA), and β-actin (1:2500; Sigma-Aldrich, St. Louis, MO) were used as described.

Techniques: Mutagenesis, Marker, Immunostaining, Staining, Migration, In Vivo, In Vitro, Membrane, Dispersion

Journal: bioRxiv

Article Title: DNA-guided transcription factor cooperativity shapes face and limb mesenchyme

doi: 10.1101/2023.05.29.541540

Figure Lengend Snippet: A . Schematic of endogenous tagging TFs with the FKBP12 F36V degron and V5 epitope tag in human embryonic stem cells (hESC) followed by differentiation into cranial neural crest cells (hCNCC) and treatment with or without dTAG V -1. The ALX4 gene was instead knocked out by a frameshift mutation. B . Confirmation of TF tagging and depletion upon dTAG V -1 addition by Western blot for V5 epitope, with CTCF as a loading control. IB, immunoblot. C . Confirmation of ALX4 knockout in three independent clones by Western blot, with HSP90 as a loading control. D . Homeodomain TFs bind DNA at TWIST1 bound sites at variable occupancies. Heatmap shows promoter-distal binding sites for TWIST1 and/or AP2a. Assay indicates whether data shown is from ChIP, CUT&RUN (C&R), or ATAC, and whether an endogenous or V5 antibody was used. Rows are ranked by the sum of the homeodomain signals from undepleted cells. In the scale bar, units are reads per genome coverage, except for ATAC data, which is in signal per million reads. E . Homeodomain TFs bind the Coordinator motif. The top enriched motif (by AME) is shown for each TF, with p-values in parentheses.

Article Snippet: Antibodies used include TWIST1 (Abcam, ab50887), V5 (Abcam, ab15828), H3K27ac (Active Motif, 39133), Flag (Sigma-Aldrich, F1804), AP2a (Cell Signaling, 3215), AP2a (Novus Bio, NB100-74359).

Techniques: Mutagenesis, Western Blot, Control, Knock-Out, Clone Assay, Binding Assay