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. 2023 Jun 7;10(7):1239–1253. doi: 10.1002/acn3.51820

Figure 1.

Figure 1

Engineered Extracellular Matrix fabrication and neural differentiation. (A) Fabrication of Engineered Extracellular Matrices (EECMs) used for seeding ESCs and downstream organoid generation. 3D jet writing is used to produce poly(d,l‐lactide‐co‐glycolide, PLGA) scaffolds, as previously described. 72 Polymer PLGA scaffolds are mounted onto medical‐grade stainless steel and then this structure is placed into a fibronectin solution that undergoes rotation to induce fibrillogenesis and deposit an insoluble fibronectin (FN) matrix. 20 , 22 Human ESCs are then seeded onto EECMs. (B) Scanning electron micrograph (SEM) of the PLGA scaffold structure (scale bar is 200 μm). (C) Deposited fibronectin matrix is stained using an amine‐reactive fluorophore (red channel; scale bar is 500 μm). (D) SEM of confluent embryonic stem cells (ESCs; H9) cultured on the EECM (scale bar is 100 μm). (E) Timeline for brain organoid production using a commercial media kit. (F) A confluent stem cell layer is generated prior to Neural Induction Medium (NIM), stained for pluripotency markers SOX2 (green channel), OCT4 (red channel), and NANOG (yellow channel) plus merged image (top; scale bar is 30 μm). (G) EECMs with differentiated H9 ESCs after NIM, stained for ZO‐1 (pink channel; scale bar is 200 μm), SOX1 (yellow channel), and beta‐catenin (red channel) markers (scale bar in insets is 100 μm), identifying neural progenitor cells and neural rosette formation. (H) Neuronal populations stained for neuronal markers TUJ1 (green channel), OTX2 (purple channel), and synaptophysin (red channel), plus merged image, at the end of maturation (scale bar is 20 μm). Panels F–H are all counterstained with DAPI (cyan channel). (I) qPCR results showing the fold‐change of expression for neuronal differentiation markers of EECM organoids at D45 of culture relative to day 0 (undifferentiated cells); n ≥ 3 biological replicates, that is, three distinct batches of brain organoids, for each gene of interest. All genes are normalized to GAPDH expression levels. (J) Western blot showing protein expression for hPSCs before differentiation (D0) and EECM organoids at D45 of culture.