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. 2021 May 21;12:3020. doi: 10.1038/s41467-021-23294-7

Fig. 1. Polarized gene expression and morphology in elongating multi-lineage organized (EMLO) gastruloids.

Fig. 1

a Overview of EMLO gastruloid formation. Intact hiPSC colonies were pretreated with CHIR and FGF2 for 2 days prior to dissociation and transition to shaking culture on day 0. b Schematic representation of SOX2 (pink) and GATA6 (yellow) polarized expression in EMLOs over time. Early invagination of SOX2/GATA6 cells precedes the formation of a primitive gut tube-like structure. c Phase contrast of early EMLO shaking cultures at days 1, 2, and 4. Early compartmentalization of spinal cord (SC) neural versus mesoderm–endoderm protrusion (ME) can be visualized. d SOX2/GATA6 immunofluorescence (IF) of early EMLO aggregates. Day 4 inset depicts SOX2/GATA6 early tube formation (Z-slice). e Phase contrast of day 13 EMLO. f SOX2/GATA6 IF in day 13 EMLOs with DAPI (gray). SOX2/GATA6 colocalization persists in the primitive gut tube. Inset is GATA6 inverted LUT. H3.3.1 EMLOs were formed in N = 11 separate biological repeat experiments over the course of this study with similar results. Phase and IF data were acquired each time c–f. EMLOs from the other F3.5.2 and A2.1.1 representative lines were separately formed in N = 5 biological repeat experiments. All other lines were formed in N = 3 repeat experiments. Images are representative of the EMLO populations across repeated experiments. g Schematic representation of the EMLO size parameters. Length (L), width (W), and SC diameter (D) were measured. h Histogram of the proportion of aggregates that are elongated long (L:W ratio >3.5), elongated short (2 < L:W < 3.5), or spherical/ovoid (L:W < 2) at day 13 using nine ED-hiPSC lines. The plot corresponds to elongation efficiency. EMLO formation using all lines was performed N = 3. n = number of aggregates measured is shown for each line. i Aspect ratio over time in representative ED-hiPSC lines. Elongation after pretreatment (n = 153 per line) was compared with elongation in basal medium N2B27 (n = 138 per line). Individual scale bars provided. Data reported as (mean ± s.e.m.).