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. 2021 May 26;11:11045. doi: 10.1038/s41598-021-90422-0

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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In vitro and in vivo differentiation of NBFR-bESCs. (a) Gene expression levels of pluripotency (OCT4, SOX2, NANOG), trophectoderm (CDX2), endoderm (FOXA2), ectoderm (PAX6), and mesoderm (MEOX1) markers in representative tissues and two independent NBFR-bESCs lines before (NBFR#A and NBFR#D at passage 10) and after adaptation to feeder-free culture (NBFR-FF #A and #D, passage 10 and 20 after adaptation, respectively). Relative expression was calculated using the comparative CT method (ΔΔCT), normalizing values to HMBS. (b) Bright field images of NBFR-bESCs and embryoid bodies obtained at different times of differentiation. RT-PCR for genes representative of different lineages (OCT4, CDX2, SOX17, PAX6, and FOXA2). HMBS: housekeeping gene. Scale bar 100 μm. (c) Teratomas obtained 12 weeks after injection of two independent NBFR-bESC lines (NBFR #D and #Y) into immunodeficient mouse. Representative histological images showing derivates of the 3 germ lineages. BFF: bovine fetal fibroblasts; Bls: blastocysts, d: days.