Efficient isolation of embryonic stem (ES) cells from pre-implantation porcine embryos has remained a challenge. Here, we describe the derivation of porcine embryonic stem-like cells (pESLCs) by seeding the isolated inner cell mass (ICM) from in vitro-produced porcine blastocyst into α-MEM with basic fibroblast growth factor (bFGF). The pESL cells kept the normal karyotype and displayed flatten clones, similar in phenotype to human embryonic stem cells (hES cells) and rodent epiblast stem cells. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers such as OCT4, NANOG, SOX2, SSEA-4, TRA-1-60, and TRA-1-81 as determined by both immunofluorescence and RT-PCR. Additionally, these cells formed embryoid body (EB), teratomas and also differentiated into 3 germ layers in vitro and in vivo. Microarray analysis showed the expression of the pluripotency markers, PODXL, REX1, SOX2, KLF5 and NR6A1, was significantly higher compared with porcine embryonic fibroblasts (PEF), but expression of OCT4, TBX3, REX1, LIN28A and DPPA5, was lower compared to the whole blastocysts or ICM of blastocyst. Our results showed that porcine embryonic stem-like cells can be established from in vitro-produced blastocyst-stage embryos, which promote porcine naive ES cells to be established.

SIX1 and SIX4 genes play critical roles in kidney development. We evaluated the effect of these genes on pig kidney development by generating SIX1 −/− and SIX1 −/− /SIX4 −/− pig foetuses using CRISPR/Cas9 and somatic cell nuclear transfer. We obtained 3 SIX1 −/− foetuses and 16 SIX1 −/− /SIX4 −/− foetuses at different developmental stages. The SIX1 −/− foetuses showed a migration block of the left kidney and a smaller size for both kidneys. The ureteric bud failed to form the normal branching and collecting system. Abnormal expressions of kidney development-related genes (downregulation of PAX2, PAX8, and BMP4 and upregulation of EYA1 and SALL1) were also observed in SIX1 −/− foetal kidneys and confirmed in vitro in porcine kidney epithelial cells (PK15) following SIX1 gene deletion. The SIX1 −/− /SIX4 −/− foetuses exhibited more severe phenotypes, with most foetuses showing retarded development at early stages of gestation. The kidney developed only to the initial stage of metanephros formation. These results demonstrated that SIX1 and SIX4 are key genes for porcine metanephros development. The creation of kidney-deficient porcine foetuses provides a platform for generating human kidneys inside pigs using blastocyst complementation. K E Y W O R D S blastocyst complementation, CRISPR/Cas9, kidney development, pig, SIX1, SIX4

The establishment of ungulate embryonic stem cells (ESCs) has been notoriously difficult via a conventional approach. We combined a traditional ESC culture method with reprogramming factors to assist the establishment of porcine naive‐like ESCs (nESCs). Pig embryonic fibroblasts were transfected with a tetracycline‐inducible vector carrying 4 classic mouse reprogramming factors, followed by somatic cell nuclear transfer and culturing to the blastocyst stage. Then, the inner cell mass was isolated and seeded in culture medium. The naive‐like ESCs had characteristic verys similar to those of mouse ESCs and showed no signs of altered morphology or differentiation, even after 130 passages. They depended on leukemia inhibitory factor signals for maintenance of pluripotency, and the female cell lines had low expression of the X‐inactive specific transcript gene and no histone H3 lysine 27 trimethylation spot. Notably, the ESCs differentiated into 3 germ layers in vitro and could be induced to undergo directional neural and kidney precursor differentiation under defined conditions, and the ESCs could keep proliferating after doxycycline was removed. nESCs can be established, and the well‐characterized ESC lines will be useful for the research of transgenic pig models for human disease.—Zhang, M., Wang, C., Jiang, H., Liu, M., Yang, N., Zhao, L., Hou, D., Jin, Y., Chen, Q., Chen, Y., Wang, J., Dai, Y., Li, R. Derivation of novel naive‐like porcine embryonic stem cells by a reprogramming factor–assisted strategy. FASEB J. 33, 9350–9361 (2019). http://www.fasebj.org

Pluripotent stem cells (PSCs) generated from somatic cells via ectopic expression of specific transcription factors provide an unlimited cell resource for regenerative medicine and transgenic breeding. Here, we describe the successful generation of bovine induced PSCs (biPSCs) from foetal fibroblasts by lentivirus-mediated delivery of bovine pluripotency reprogramming factors (PRFs) OCT3/4, SOX2, KLF4, c-MYC, NANOG and LIN28. The generated biPSCs resembled embryonic stem cells (ESCs) in their gene expression profiles, self-renewal capabilities and proliferation, as well as maintenance of a normal karyotype and differentiation into diverse cell types of all three germ layers both in vitro and in vivo. Qualitative phosphoproteomics of biPSCs revealed a large number of phosphorylated proteins, which might be related to the control of biPSCs status. The successful generation of biPSCs and the analysis of their phosphoproteome would further our understanding of the epigenetic mechanisms underlying iPSC pluripotency, thus promoting their application in bovine transgenic breeding and marking avenues for future research.

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