human basic fibroblast growth factor (bfgf, peprotech, #100-18b-500)  (PeproTech)

Journal: bioRxiv

Article Title: High resolution multi-scale profiling of embryonic germ cell-like cells derivation reveals pluripotent state transitions in humans

doi: 10.1101/2025.01.14.632914

Figure Lengend Snippet: (A) Steps to differentiate hPGCLCs from hPSCs and reprogram them into hEGCLCs and schematic of omics assays performed. hPSCs are differentiated in 2 days into iMeLCs, and further into hPGCLCs, within 3D aggregates. hPGCLCs aggregates are dissociated and hPGCLCs sorted (10 cells for each well of 96 multiwell) and grown for 2 days in a hEGCLC induction medium, containing hLIF, CHIR, forskolin, bFGF, hSCF and Retinoic Acid. After 2 days, hEGCLC induction medium is gradually switched into conventional hPSCs culture media, allowing the reprogramming of hPGCLCs into hEGCLCs. hEGCLCs are able to differentiate into the three germ layers (ectoderm, mesoderm, endoderm) and into hPGCLC (B) Phase contrast images exemplifying the key steps and cellular states of the differentiation protocol (scale bar: 250μm; 25μm for hPGCLC-hEGCLC image) (C) Representative flow-cytometry density plot showing the percentage of hPGCLCs within hPGCLC aggregates (double positive cells for: BLIMP-tdTomato and AP2γ-GFP, BTAG reporter line, left panel; EpCAM-FITC and CD49f-PE, right panel) (D) Representative immunofluorescence images of hEGCLCs colonies (passage 5) stained for pluripotency markers: SOX2, OCT4; nuclei stained with Hoechst (scale bar: 150μm) (E) hEGCLC tri-germ layer differentiation: representative immunofluorescence staining for lineage specification markers: SOX17 (endoderm), α-SMA (mesoderm), MAP2 (ectoderm); nuclei stained with Hoechst (scale bar: 150 μm) (F) On the left: representative phase contrast images of day6 hPGCLC aggregates differentiated in parallel from CTL08A hiPSCs (top panel) and hEGCLCs (bottom panel); scale bar 250 μm. On the right: corresponding flow cytometry density plots of CD49f (Integrin-α6, Y-axis) and EpCAM (X-axis) surface markers. Percentages of double-positive cells (i.e. hPGCLCs) are shown in the red gate (G) Principal Component Analysis (PCA) of bulk RNASeq profiling of the samples indicated in the legend (hiPSCs, day 4 hPGCLCs, hEGCLCs passage zero [P0, 14 days after EGC induction], hEGCLCs P3 and hEGCLC P5) (H) MA-plot showing the mean normalized counts (X-axis) and shrunken log2Fold-Change values of expressed genes, for the following pair-wise comparison: hEGCLC P0 vs hiPSC; hEGCLC P3 vs hiPSC; hEGCLC P5 vs hiPSC (from left to right respectively). Differentially expressed genes (DEGs) after p-value adjustment (q-value<0.01) are displayed in red (upregulated) or in blue (downregulated)

Article Snippet: To reprogram hPGCLCs into hEGCLCs, sorted day 4 or day 6 hPGCLCs were plated directly into 96 multi-well flat bottom (10 cells/well), pre-coated with Geltrex (Thermo Scientific, #A1413201), according to the manufacturer’s instructions, and containing 100 ul/well of hEGCLC induction medium [i.e. N2B27 medium 3 containing 10 ng/ml human leukemia inhibitory factor (hLIF, PeproTech, #300-05), 3 μM of the GSK3 inhibitor CHIR99021 (CH, Sigma-Aldrich, #SML1046-5MG), 10 μM Forskolin (FK, Sigma-Aldrich, #F3917-25MG), 25ng/ml basic fibroblast growth factor (bFGF, Preprotech, #100-18B), 100 ng/ml human stem cell factor (hSCF, PeproTech, #300-07) and 2 μM retinoic acid (RA, Sigma-Aldrich, #R2625)] and antibiotics (penicillin 100 U/mL and streptomycin 100 μg/mL, ThermoFisher, #15140122).

Techniques: Flow Cytometry, Immunofluorescence, Staining, Comparison