Supplementary MaterialsS1 Fig: Characterization of PEG plates

Supplementary MaterialsS1 Fig: Characterization of PEG plates. experimental set up. (DCI) Representative images acquired on PEG plates for multiple cell types. (D) MEFs stained for -actin in green and DAPI in blue. (E) Hemogenic endothelial cells stained for VECAD in green and DAPI in blue. (F) Main human being keratinocytes stained for Keratin 14 in green and Involucrin in reddish. (G) Mouse embryonic stem cells stained for OCT4 in green. (H) BMP4 treated human being induced pluripotent stem cells stained for SOX17 in reddish. (I) Human being endodermal progenitor cells allowed to generate outgrowths stained for NKX6.1 in red and PDX1 in green. Underlying numerical data for this figure can be found in BMP4, bone morphogenetic protein 4; C1, carbon 1; MEF, Mouse Embryonic Fibroblasts; NKX6.1, NK6 homeobox 1; OCT4, octamer-binding transcription element 4; PDX1, pancreatic and duodenal homeobox 1; PEG, Polyethylene Glycol; PLL-g-PEG, Poly-L-Lysine-grafted-Polyethylene Glycol; SOX17, SRY-Box 17; VECAD, vascular endothelial cadherin.(TIF) pbio.3000081.s001.tif (2.3M) GUID:?9CE8F576-00DC-4B22-929A-A62635619F34 S2 Fig: Validation of hPSC patterning in PEG plates. (A) Overview of a previously explained micro-patterning centered hPSC differentiation assay [30] using OCT4 and SOX2 manifestation levels as signals of early fate choices to compare PEG and CP plates. (B) Quantified compartments of early fate choices as defined in panel A, in both PEG and CP plates. The press conditions tested were NSCNutristem, Apel (vehicle for the following), BMP (BMP4), BA (BMP4+ActivinA), FSB (bFGF+SB431542; observe Materials and methods for concentration details). Data displayed as mean (+ SD) of 4 self-employed replicates. The fate choice reactions of hPSCs on both the plates were highly correlated (R2 0.9). (C) Representative immunofluorescent pictures of hPSC colonies stained for OCT4 and SOX2 in the various media conditions examined. Scale bars suggest 500 m. (DCE) Evaluation of patterning response on PEG plates versus CP plates. (D) Variety of colonies discovered per well between PEG and CP plates. Each dot represents the amount of colonies discovered per well for 120 arbitrarily chosen wells between your 4 replicates of PEG versus CP plates. Variety of cells discovered per colony between PEG and CP plates. Each dot represents the common variety of cells per colony for 120 arbitrarily chosen wells between your 4 replicates of PEG Lenalidomide-C5-NH2 versus CP plates. (E) Consultant pictures of hPSCs micropatterned in 96-well plates using PEG-based technique versus CP. Root numerical data because of this figure are available in hPSC, individual pluripotent stem cell; OCT4, octamer-binding transcription aspect 4; PEG, Polyethylene Glycol; SOX2, SRY-box 2; CP, micro-contact printing.(TIF) pbio.3000081.s002.tif (2.2M) GUID:?6CC9845A-8413-4D30-9BC1-6F9E6AB0D604 S3 Fig: Beginning populations of test hPSC lines show high expression of pluripotency associated proteins. (A) FACS plots Rabbit Polyclonal to ADA2L of OCT4-, SOX2-, and NANOG-expressing cells in the beginning populations of H9-1, H9-2, HES2, MEL1, and HES3-1. Secondary-only recognizes the non-specific labelling observed because of the supplementary antibody. (B) Consultant immunofluorescent pictures from Fig 2 shown with corresponding DAPI staining. FACS, Fluorescence-activated cell sorting; hPSC, individual pluripotent stem cell; NANOG, homeobox proteins NANOG; OCT4, octamer-binding transcription aspect 4; SOX2, SRY-box 2.(TIF) pbio.3000081.s003.tif (2.7M) GUID:?6662C91B-9E20-4DD3-Stomach35-90B8F0D4B5AD S4 Fig: Nodal appearance dynamics in EB assay of Lenalidomide-C5-NH2 hPSC series -panel. Temporal dynamics of Nodal for the check hPSC lines proven for the 3 clusters of Nodal-Strong, Nodal-Intermediate, and Lenalidomide-C5-NH2 Nodal-weak (Fig 3Bii). Each dot represents the discovered appearance level for the biological replicate. Club plots represent mean SD. Root numerical data for this figure can be Lenalidomide-C5-NH2 found in EB, embryoid body; hPSC, human being pluripotent stem cell.(TIF) pbio.3000081.s004.tif (492K) GUID:?2B663A3F-919B-4883-90E7-B6DC256F9113 S5 Fig: MIXL1 and EOMES dynamics during EB assay predict endoderm differentiation propensity of hPSC lines. (A) Panel of hPSC lines clustered into 3 groups of Strong, Medium, and Weak responders for (i) MIXL1 and (ii) EOMES from Fig 3Bii. The manifestation levels of MIXL1 and EOMES in the pluripotent state (Day time 0) demonstrated in the boxes adjacent to the heat maps. (B) Overview of the protocol for directed differentiation towards definitive endoderm. The cells were treated with Wnt3a from 0 h to 24 h and Wnt3a+ActivinA from 24 h to 72 h. (CCD) Effectiveness of SOX17 induction in the test hPSCs using the protocol in panel B. (C) Black dash denotes the mean of 3 self-employed replicates represented from the dots. (D) FACS plots for individual replicates from panel C. (E) FACS plots showing the effectiveness of induction of pancreatic progenitors as indicated from the manifestation of PDX1 and NKX6.1 for candidate hPSC lines from your Strong and Weak clusters from panel A. The differentiation was performed using a previously explained protocol [77]. The data are from 3 self-employed wells; the experiment was performed twice. The gating was performed using the cells stained with only the secondary antibodies (demonstrated in blue). The samples stained for PDX1 and NKX6.1 are.