Supplemental Information. Tissue Mechanics Orchestrate Wnt-Dependent. Human Embryonic Stem Cell Differentiation

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1 Cell Stem Cell, Volume 19 Supplemental Information Tissue Mechanics Orchestrate Wnt-Dependent Human Embryonic Stem Cell Differentiation Laralynne Przybyla, Johnathon N. Lakins, and Valerie M. Weaver

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8 Supplemental Figure Legends Figure S1. Related to Figure 1. Substrate stiffness does not affect hesc properties. (A) Quantification of immunofluorescent staining for cleaved Caspase 3-positive nuclei in hescs grown on the indicated stiffness. (B) Quantification of immunofluorescent staining for BrdU positive nuclei after 1 hour BrdU pulse in hescs grown on the indicated stiffness. (C) PCR analysis of mrna expression levels of self-renewal markers in hescs grown on a range of stiffnesses. (D) Oct4 staining of hescs replated from hydrogels onto MEFs and cultured for several days. (E) Gene expression panel analysis data of self-renewal and lineage marker expression in hescs grown on the indicated stiffness. Data are represented as mean of at least three independent experiments ± SD. All scale bars = 20μm. Figure S2. Related to Figure 2. Differentiation potential of hescs is altered based on mechanical conditions. (A) Gene ontology terms based on genes with the highest significant upregulation in cells differentiated on soft gels versus those differentiated on stiff gels. (B) Heatmap showing a subset of genes from RNA-seq analysis that are significantly upregulated and correspond to gene ontology designations for mesoderm, endoderm, or ectoderm differentiation. RNA levels were normalized to averaged day 0 values to reflect changes upon induction. (C) Relative mrna expression levels of self-renewal and differentiation markers in undifferentiated hescs and those induced towards mesoderm on soft and stiff hydrogels. (D) mrna expression levels of key markers of mesoderm differentiation and the associated EMT in H7 hescs differentiated on gels of the indicated stiffness. (E) Immunofluorescent staining for Ncam for H7 hescs differentiated on gels of the indicated stiffness. (F) Percentage of cells positive for phosphorylated Histone H3 in cells differentiated on soft or stiff substrates. (G) Relative mrna expression levels of key markers of mesoderm differentiation and EMT in cells induced to differentiate toward mesoderm in the indicated conditions, including up to 6 days in cells on stiff gels. (H) Flow cytometry analysis of an MSC marker (top) and non-msc marker (bottom) in undifferentiated hescs and differentiated cells replated from 400Pa gels and further induced toward an MSC fate. Inset shows phase image of confluent MSCs. (I) Immunofluorescent staining for Cardiac Troponin T and Nkx2.5 in cells replated from a self-renewing culture or from differentiated cells on soft or stiff gels onto a vitronectin-coated plate to undergo 10 days of cardiomyocyte differentiation. (J) Relative mrna expression levels of cardiomyocyte differentiation in cells replated from a selfrenewing culture or from induced cells on a 400Pa gels onto a vitronectin-coated plate to undergo 10 days of cardiomyocyte differentiation. Data are represented as mean of at least three independent experiments ± SD. =p<0.001, =p<0.05. All scale bars = 20μm. Figure S3. Related to Figure 3. Comparison of relative transcript abundance of secreted factors involved in mesoderm differentiation. (A) RPKMs generated from RNA-sequencing data across a panel of Wnt genes. (B) RPKMs generated from RNA-sequencing data across a panel of Sfrp genes. (C) Heatmap showing a subset of genes from RNA-seq analysis that correspond to secreted factors involved in mesoderm differentiation as annotated in GO. Data are gathered from three independent replicates. =p<0.001, =p<0.05. Figure S4. Related to Figure 4. Cadherins and catenins establish and reinforce strong cell-cell adhesions. (A) mrna expression levels of a panel of Wnts in hescs on soft and stiff gels relative to levels in cells differentiated on soft gels (=1). (B) Immunofluorescent staining for β-catenin in hesc-derived embryoid bodies or in hescs cultured on tissue culture plastic (TCP). (C) mrna expression level of E-cadherin in hescs with an IPTG-inducible shrna against E-cadherin. (D) mrna expression level of β-catenin in hescs with an IPTG-inducible shrna against β- catenin. (E) mrna expression level of P120-catenin in hescs with an IPTG-inducible shrna against P120- catenin. (F) Immunofluorescent staining for E-cadherin in hescs on soft gels with or without induction of the indicated shrna knockdowns. (G) Immunofluorescent staining for Ncam in cells differentiated on soft gels with or without E-cadherin knockdown. Data are represented as mean of at least three independent experiments ± SD. =p<0.05. All scale bars = 20μm.

9 Figure S5. Related to Figure 6. Proteins involved in β-catenin destabilization. (A) Western blot and quantification of total and phospho-s9 GSK3β protein levels in hescs cultured on soft and stiff substrates. (B) Quantification of nuclear β-catenin levels in cells differentiated on stiff gels with or without CHIR99021, or on soft gels, as measured by immunofluorescence. (C) Immunofluorescent staining for CBLL1 in hescs with an IPTG-inducible shrna against CBLL1. (D) Immunofluorescent staining for non-phosphorylated SFKs in hescs on soft and stiff substrates. (E) Actin organization in hescs grown on stiff gels in the indicated conditions. Data are represented as mean of two independent experiments ± SD. =p<0.05. All scale bars = 20μm. Figure S6. Related to Figure 7. Epiblast and ingressing mesoderm cells in gastrulation-stage chick embryos express markers consistent with differentiating hescs undergoing mesoderm differentiation. (A) Staining with phalloidin and DAPI to highlight actin organization in cells in the epithelial sheet of epiblast (i) and those that have ingressed below the epithelial sheet (ii). (B) Immunofluorescent staining for Ncam in top and ingressing layers of a gastrulation-stage chick embryo. (C) Immunofluorescent staining for CBLL1 and non-phosphorylated SFKs in top and ingressing layers of a gastrulation-stage chick embryo. All scale bars = 20μm. Movie S1. Related to Figure 2. Beating cardiomyocytes formed from hescs differentiated on 400Pa gels for 3.5 days then transferred to vitronectin-coated tissue culture plates and cultured for 14 days.

10 Table S1. Related to Figure 2. Relative abundance (RPKM) of genes that make up heat map in Figure 2C, listed in sequential order. Substrate stiffnesses are listed in heading, and diff refers to cells that have undergone the mesoderm differentiation protocol. GENE 400Pa hesc 60kPa hesc 400Pa diff 60kPa diff AFP EPCAM CLDN FOXD NANOG POU5F SDC SOX MMP NES CTNNB NKX CLDN CDH HLX GATA4 Not Detected PAX CLDN FN KDR TWIST MIXL LEF MESP EOMES ZEB RIPK TBX SNAI SNAI T FOXA FOXP VIM ST

11 Supplemental Experimental Procedures shrna knockdowns For shrna knockdowns, candidate shrna hairpins were cloned into a packaging vector for transfection into 293 cells and subsequent infection into hescs. E-cadherin shrna had the following sequence: 5 - GAACGAGGCTAACGTCGTAAT - 3 ; β-catenin shrna had the following sequence: 5 - GCTTGGAATGAGACTGCTGAT - 3 ; P120-catenin shrna had the following sequence: 5 - CTCCCAATGTTGCCAACAATA - 3 ; CBLL1 shrna had the following sequence: 5 - TCATATCAACCATCGCCATAT - 3. Culture media additives The following concentrations of culture media additives were used for all experiments: IWP2 2 μm (Sigma), Wnt3a 100 ng/ml (R&D Systems), CHIR μm (Millipore), aiib2 monoclonal Integrin β1 blocking antibody (1 μg/ml; prepared in house from hybridoma), MG nm (Sigma-Aldrich), Src inhibitor (PP1) 10 μm (Millipore). IPTG was added to induce shrna production in IPTG-inducible cell lines at 200 μm. Flow cytometry Cells were harvested using 0.05% trypsin (for intercellular markers) or 5mM EDTA (for cell surface markers). Flow cytometry was performed on a BD Fortessa SORP. The following antibodies were used for flow cytometry: efluor- 660-conjugated E-cadherin (ebioscience), PE-conjugated NCAM (BD Biosciences), PerCP-Cy5.5-conjugated CD73 (BD Pharmingen), PE-conjugated CD34 (BD Pharmingen). Immunofluorescence Specific antibodies used are listed in supplemental experimental procedures. Confocal images were taken using a Nikon S10 Spinning Disk Confocal scope, with z-stacks taken 1µm apart. Nuclear quantification was performed using Fiji to create a mask using DAPI images, which was then applied to a corresponding image taken with the relevant marker of interest. Imaris software was used to render z-stacks from confocal image stacks. The following antibodies were used for immunofluorescent images: Oct4 (Santa Cruz Biotechnology), phospho-histone 3 (Cell Signaling Technology), Ncam (Cell Signaling Technology), Slug (Cell Signaling Technology), Wnt3a (Abcam), Sfrp1 (Sigma-Aldrich), β-catenin (Cell Signaling Technology clone D10A8), E-cadherin (Cell Signaling Technology clone 24E10), P120-catenin (Millipore clone 15D2), activated integrin β1 (BD Pharmingen clone 9EG7), non-phosphorylated SFK (Cell Signaling Technology), phosphorylated SFK (Cell Signaling Technology), CBLL1 (Bethyl Laboratories), Kaiso (Millipore clone 6F), Cleaved Caspase 3 (Cell Signaling Technology), Cardiac troponin T (R&D Systems), Nkx2.5 (Cell Signaling Technology). Fluorescently-conjugated secondary antibodies were from Life Technologies. Actin images were taken using fluorescently conjugated phalloidin (Life Technologies). BrdU staining was performed by pulsing cells with BrdU labeling reagent for one hour before fixation and staining with fluorescein-conjugated anti-brdu antibody (Roche). Immunoblot Cell cultures were lysed and sonicated, then samples were run on a 10% polyacrylamide gel before transferring and blotting against the protein of interest. Antibodies used include β-catenin (Cell Signaling Technology clone D10A8), E-cadherin (Cell Signaling Technology clone 24E10), total GSK3 (Cell Signaling Technology), phosphorylated (S9) GSK3β (Cell Signaling Technology), and β-actin (Sigma). Blots were developed using a CCD luminescence images for membrane based ECL imaging. Quantitative gene expression analysis For RT-PCR, cells were harvested using TRIzol and total RNA was isolated according to manufacturer s instructions. RNA was converted to cdna using M-MLV reverse trascriptase (Promega) and quantitative PCR reactions were set up using the LC FastStart DNA Master SYBR Green mix (Roche). Reactions were run on an Eppendorf RealPlex machine. Primers are listed in Supplemental Experimental Procedures. For the curated gene

12 expression panel analysis, the IPS precision assay from Cellular Research, Inc. was used according to manufacturer s instructions. qrt-pcr primer sequences Gene qrt-pcr forward primer qrt-pcr reverse primer Nanog AGATGCCTCACACGGAGACT AAGTGGGTTGTTTGCCTTTG Oct4 AGTGAGAGGCAACCTGGAGA ACACTCGGACCACATCCTTC Ncam TGAGTGGAGAGCAGTTGGTG CCTTACGGCGTACGTTGTTT Brachyury CAGCAAAGTCAAGCTCACCA TGGACCCCCAACTCTCACTA Gsc TCTCAACCAGCTGCACTGTC CGTTCTCCGACTCCTCTGAT MixL1 GGTACCCCGACATCCACTT CGCCTGTTCTGGAACCATAC HLX CTCCAACCTGCAGAGGAAAG CTGGAACCACACCTTCACCT KDR TGGGGAAAGCATCGAAGTCT TTCCGGTTCCCATCCTTCAA Twist CTCGGTCTGGAGGATGGAG TCCTTCTCTGGAAACAATGACA Snai1 CATGTCCGGACCCACACT TGGTACTTCTTGACATCTGAGTGG Snai2 GCCTCCAAAAAGCCAAACTA GGTGTCAGATGGAGGAGGG PDGFRA CGGAATAACATCGGAGGAGA GCTCAGCCCTGTGAGAAGAC Ctnt TTCGACCTGCAGGAGAAGTT CGGGTCTTGGAGACTTTCTG Nkx2.5 CAAGTGTGCGTCTGCCTTT TTTTCGGCTCTAGGGTCCTT Wnt3 AGCTGCCAGGAGTGTATTCG TCCTGCTTCCCATGAGACTT Wnt3a GCCCCACTCGGATACTTCTT GAGGAATACTGTGGCCCAAC Wnt4 CCCTCATGAACCTCCACAAC ACCTCACAGGAGCCTGACAC Wnt5a TGGCTTTGGCCATATTTTTC GCAGAGAGGCTGTGCTCCTA Wnt5b AGACTGGCATCAAGGAATGC GTCTCTCGGCTGCCTATCTG Wnt6 CGAGAGTGCCAGTTCCAGTT GTCTCCCGAATGTCCTGTTG Wnt8a TGTGATGGGTCAAACAATGG TCCTTCCCCTTCTCCAAACT Wnt9b CAGCACCAAGTTTCTGAGCA GTCCTGAGGCCACTCTTCAC Wnt11 CAGGATCCCAAGCCAATAAA AGACACCCCATGGCACTTAC Sfrp1 ATCTCTGTGCCAGCGAGTTT TCAGGGGCTTCTTCTTCTTG Sfrp2 CGACATAATGGAAACGCTTTG TTGCTCTTGGTCTCCAGGAT Sfrp4 CGCTCAAGGATGATGCTTCT GAACTGTTCTCCGCTGTTCC Sfrp5 AGCAGATGTGCTCCAGTGAC CTTTTTCTGGGCTCCAATCA β-catenin GCCGGCTATTGTAGAAGCTG GAGTCCCAAGGAGACCTTCC E-cadherin GAACGCATTGCCACATACAC ATTCGGGCTTGTTGTCATTC P120-catenin CTTTTGGACGTGACCAGGAT TCCACAGGGTTCCGGTAATA Axin2 AGTGTGAGGTCCACGGAAAC TGGCTGGTGCAAAGACATAG GAPDH CAGCCTCAAGATCATCAGCA TGTGGTCATGAGTCCTTCCA RNA-sequencing Whole transcriptome mrna sequencing of barcoded samples was performed and data was processed according to the Illumina pipeline. Reads were mapped to the human reference genome and reads per kilobase of exon per million mapped reads (RPKM) values were generated. Heat map was generated using the freely downloadable software Java Treeview and Cluster 3.0. GO term enrichment results were produced by PANTHER (Mi et al., 2013). Statistical analysis All results were analyzed by student s T-test and the resulting pairwise p-values are reported. Significance was established at p<0.05, and was evaluated up to the level of p<0.001.