Additional file 2 of Development and evaluation of a novel xeno-free culture medium for human-induced pluripotent stem cells
Creators: name: Hua, Ying
name: Yoshimochi, Kenji
name: Li, Junjun
name: Takekita, Kazuhiro
name: Shimotsuma, Motoshi
name: Li, Lingjun
name: Qu, Xiang
name: Zhang, Jingbo
name: Sawa, Yoshiki
name: Liu, Li
name: Miyagawa, Shigeru
Publisher: name: figshare
Year: 2022
Subjects: Cell Biology
Biological Sciences not elsewhere classified
Developmental Biology

description: Additional file 2: Fig. S1. 253G1 maintained on GNF in ON2 or mTeSR1. Most of seeded single cells attached to GNF within 2 h and protruding regions were formed on the edge the clones, clinging tightly to the GNF substrate. Scale bar, 200 µm. Fig. S2. Commercial xeno-free media could not support hiPSCs on GNF. (a) Neither AK02N nor E8 could maintain the hiPSCs on GNF. Most the cells apoptosis induced from P2 even with 10 µM Rock inhibitor. On the contrary, ON2 shares equal capability as mTeSR1 for maintaining survival and adhesion of cultured hiPSCs. Scale bar, 200 µm. (b) Teratomas generated from 253G1 after maintained on GNF with mTeSR1 for over P20. Scale bar, 100 µm. Fig. S3. Optimization of coating conditions of FN and VN. (a) 253G1 cultured on FN with a coating concentration of 0.5 g/ml, 1.0 g/ml or 2.0 g/ml. Scale bar, 200 µm. (b) 253G1 maintained on VN with a coating concentration of 0.5 g/ml. Scale bar, 200 µm. Fig. S4. Characterization of 253G1 hiPSCs cultured on FN and VN in ON2. (a) 253G1 colony morphology on FN and VN at P10. Scale bar, 200 µm. (b) qRT–PCR gene expression analysis of 253G1 hiPSCs grown on FN and VN in ON2. Fold expression is compared to cells cultured on iMatrix-511 in ON2 (n = 3 biological replicates, data are mean ± SD). (c) FACS analysis of pluripotency marker-positive cells. light-gray solid histograms show the isotype control populations, and green and blue hollow histograms show the stained populations on FN and VN, respectively. The percentage of maker-positive cells is presented on each graph. Cell events were normalized to mode. (d) Immunostaining of pluripotency markers. Green: Oct3/4; Red: Nanog; Blue: DAPI. Scale bar, 100 µm. Fig. S5. 253G1 cells cultured in E8 showed abnormalities after long-term expansion. (a) Spontaneous differentiation was found in 253G1 hiPSCs maintained on iMatrix-511 with E8 medium over P20, verified by immunostaining of hiPSCs for pluripotent markers: Oct3/4 (green); Nanog (red). Nuclei were stained with DAPI (blue). Scale bar, 100 µm. (b) Karyotype abnormality of 253G1 hiPSCs on iMatrix-511 with E8 at P22, identified by G-band karyotype analysis. Fig. S6. Characterization of HLA-homo Ff-l01 hiPSCs cultured in E8. (a) Ff-l01 colonel morphology in E8 at P14. Scale bar, 200 µm. (b) Comparison of doubling times for Ff-l01 grown in E8 with those in ON2 (passage number = 15, **p &lt; 0.01, data are mean ± SD). (c) Immunostaining of pluripotency markers. Green: Oct3/4; Red: Nanog; Nuclei were stained with DAPI (blue). Scale bar, 200 µm. (d) FACS analysis of pluripotency marker-positive cells. light-gray solid histograms show the isotype control populations, and blue hollow histograms show the stained Ff-l01 populations in E8. The percentage of maker-positive cells is presented on each graph. Cell events were normalized to mode.