Journal: iScience
Article Title: A microfluidic platform for anterior-posterior human endoderm patterning via countervailing morphogen gradients in vitro
doi: 10.1016/j.isci.2025.111744
Figure Lengend Snippet: A microfluidic platform models anterior-posterior endoderm patterning (A) Overview of morphogen signals used to direct hPSC differentiation into anterior foregut (AFG) and midgut/hindgut (MHG) over the course of six days, with schematics depicting cross-sections of the microfluidic device corresponding to three stages of stem cell differentiation. FOXA2 expression indicates definitive endoderm, OTX2 expression indicates anterior foregut, and CDX2 expression indicates mid/hindgut. (B) Photo of a microfluidic device with side reservoirs filled with red and blue food coloring. Cells are seeded into the central cell culture channel. Scale bar, 1 cm. (C) Dynamics of concentration gradients in the cell channel of microfluidic devices over the course of 24 h, as modeled using finite element analysis (COMSOL). Blue lines represent small molecule morphogens (TGFβi A8301, BMPi LDN193189) with the left reservoir as source. Green lines represent protein morphogens (FGF2, BMP4) with the right reservoir as source, and the pink line represents small molecule morphogens (Wnt, CHIR99021) with the right reservoir as source. (D) Representative image of protein-level immunostaining for OTX2, CDX2, and FOXA2, showing localized AFG and MHG populations and FOXA2 staining throughout. Scale bar, 100 μm. (E) Representative image of mRNA in situ hybridization, showing OTX2 and CDX2 mRNA transcripts localized to their respective morphogen sources and FOXA2 transcripts present throughout. Scale bar, 100 μm. (F) Average OTX2 and CDX2 intensities (protein-level) as a function of distance across the cell culture channel in the patterned cultures. Intensity is normalized to Hoechst and the highest level of cell expression; N = 15 across 3 biological replicates, error bar = SEM. (G) Scatterplot showing protein-level CDX2 vs. OTX2 intensity for every cell in the patterned cultures. (H) Overlapping scatterplots showing CDX2 vs. OTX2 intensity for every cell exposed to uniform discrete morphogen concentrations that replicate those found at specific positions within the cell culture channel (x = 0, 250, 500, 750, 1000 μm).
Article Snippet: For anterior foregut induction, CDM2 with an added 1 μM of A8301 (Tocris 2939) TGFβ inhibitor and 250nM of LDN193189 was used following Loh and Ang et al.. For posterior endoderm induction, CDM2 with an added 100 ng/mL FGF2, 10 ng/mL of BMP4, and 3 μM of CHIR99201 was used following Loh and Ang et al.. Media in each side reservoir was replaced once every 24 h. For rinse steps and to replace the media in each side reservoir, 15 μL of solution was added to the inlet and 15 μL of solution was removed from the outlet of each side reservoir 5–6 times.
Techniques: Cell Differentiation, Expressing, Cell Culture, Concentration Assay, Immunostaining, Staining, In Situ Hybridization
