Additional file 1 of Chick fetal organ spheroids as a model to study development and disease
Creators: name: Dakhel, Soran
name: Davies, Wayne I. L.
name: Joseph, Justin V.
name: Tomar, Tushar
name: Remeseiro, Silvia
name: Gunhaga, Lena
Publisher: name: figshare
Year: 2021
Subjects: subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Biotechnology
subject: Immunology
subject: FOS: Clinical medicine
subject: 69999 Biological Sciences not elsewhere classified
subject: FOS: Biological sciences
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Fig. S1. Schematic overview illustrating the generation of chick FOS. (a) Five samples of brain, heart, liver, stomach and epidermis were collected from E12, and (b) five samples of lung and intestine E14 chick embryos. (c) FOSs workflow (steps 1–4), from harvesting organs to seeding cells in culture flasks. (d) A table indicating the duration of Accutase incubation required to dissociate specific organs into a cell suspension depending on the tissue structure (step 3 in c). Scale bars: 5 mm (white), 2 mm (black). Fig. S2. Day 1 of FOSs cultures. (a-g) Cells from all fetal organs; brain, lung, heart, liver, stomach, intestine and epidermis, formed spheroid-like structures after 1 day in culture. Scale bar: 100 μm. Fig. S3. Sox2 expression analyzed by immunocytochemistry in day 8 in FOSs. (a-c) No expression of Sox2 was detected in day 8 fetal heart, intestinal or epidermal spheroids (n = 7 for all). DAPI indicates nuclei. Scale bar: 100 μm. Fig. S4. Expression of cleaved (c) Caspase3 by immunocytochemistry in day 8 FOSs. (a-g) Only a few cCasp3+ cells, indicative of apoptotic cells, were observed in FOSs derived from the seven analyzed organs (n = 5 for all FOSs). DAPI indicates nuclei. Scale bar: 100 μm. Fig. S5. Full-length gel images of gene expression in seven different FOSs and their corresponding chick embryo in vivo organs. (a-i) RT-PCR of day 8 FOSs revealed that mRNA expression of typical organ characteristic genes are enriched in the different FOSs. (a) GFAP expression in brain, but not in heart or liver FOSs/organs. (b) SP-C expression in lung, but not in heart or stomach FOSs/organs. (c) TNNT2 expression in heart, but not in brain or stomach FOSs/organs. (d) Alb expression in liver, but not in lung or heart FOSs/organs. (e) Barx1 expression in stomach, but not in brain or lung FOSs/organs. (f) SI expression in intestine, but not in brain or heart FOSs/organs. (g) EDMTFH expression in epidermis, but not in lung or liver FOSs/organs. (h, i) The house-keeping gene GAPDH was used as a reference gene. (a-i) The first and last lane of each gel image show the ladder, and an amplicon size of 1500 base pairs (bp) are indicated. Fig. S6. Full-length gel images of RT-PCR Vimentin expression in seven different FOSs and their corresponding chick embryo in vivo organs. (a, b) RT-PCR revealed mRNA expression of Vimentin (Vim) in all day 8 FOSs (a), and in their corresponding embryonic in vivo organs (b); E12 brain, heart, liver, stomach and epidermis, and E14 lung and intestine. The house-keeping gene GAPDH was used as a reference. The first lane of each gel image shows the ladder, and an amplicon size of 1500 base pairs (bp) is indicated. Table S1. Perimeter of fetal organ spheroids at culture day 4, 8 and 12. All measurements are expressed in mm and stated as mean ± standard error of the mean (SEM). Δp = fold change of the mean perimeter between FOSs measured on the stated days. Table S2. Genes detected by RT-PCR. Sequences for each primer pair (forward, F, reverse, R), with indicated amplicon sizes (base pairs, bp) and annealing temperatures (T; °C).