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Establishment of bovine expanded potential stem cells
Thursday, 2021/04/22 | 08:39:21

Lixia Zhao, Xuefei Gao, Yuxuan Zheng, Zixin Wang, Gaoping Zhao, Jie Ren, Jia Zhang, Jian Wu, Baojiang Wu, Yanglin Chen, Wei Sun, Yunxia Li, Jie Su, Yulin Ding, Yuan Gao, Moning Liu, Xiaochun Bai, Liangzhong Sun, Guifang Cao, Fuchou Tang, Siqin Bao, Pentao Liu, and Xihe Li

PNAS April 13, 2021 118 (15) e2018505118

Significance

Bovine embryonic stem cells and pluripotent stem cells hold the potential to substantially advance biotechnology and agriculture. We report the establishment of bovine expanded potential stem cells (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer (SCNT). EPSCs have broader developmental potential to generate embryonic and extraembryonic cell lineages. bEPSCs express high levels of pluripotency genes, propagate robustly in single cell passaging, are genetically stable, and permit efficient precise gene editing. They differentiate in vitro and in chimeras to both the embryonic and extraembryonic cell lineages. Importantly, genetically modified bEPSCs can be used as donors in SCNT or cloning.

Abstract

Embryonic stem cells (ESCs) and induced pluripotent stem cells have the potential to differentiate to all cell types of an adult individual and are useful for studying development and for translational research. However, extrapolation of mouse and human ESC knowledge to deriving stable ESC lines of domestic ungulates and large livestock species has been challenging. In contrast to ESCs that are usually established from the blastocyst, mouse expanded potential stem cells (EPSCs) are derived from four-cell and eight-cell embryos. We have recently used the EPSC approach and established stem cells from porcine and human preimplantation embryos. EPSCs are molecularly similar across species and have broader developmental potential to generate embryonic and extraembryonic cell lineages. We further explore the EPSC technology for mammalian species refractory to the standard ESC approaches and report here the successful establishment of bovine EPSCs (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer. bEPSCs express high levels of pluripotency genes, propagate robustly in feeder-free culture, and are genetically stable in long-term culture. bEPSCs have enriched transcriptomic features of early preimplantation embryos and differentiate in vitro to cells of the three somatic germ layers and, in chimeras, contribute to both the embryonic (fetal) and extraembryonic cell lineages. Importantly, precise gene editing is efficiently achieved in bEPSCs, and genetically modified bEPSCs can be used as donors in somatic cell nuclear transfer. bEPSCs therefore hold the potential to substantially advance biotechnology and agriculture.

 

See: https://www.pnas.org/content/118/15/e2018505118

Figure 1:

Reprogramming BFFs to Dox-inducible iPSCs for testing bovine stem cell culture condition. (A) Schematic illustration of reprogramming BFFs to iPSCs. PB-8F: bOMSK+ pN–hLIN + hRL. bOMSK (bovine OCT4, MYC, SOX2, and KLF4 cDNAs), pN–hLIN (porcine NANOG and human LIN28 cDNAs); hRL (human RARG and LRH1 cDNAs). BFFs: bovine fetal fibroblasts; Dox: doxycycline. (Scale bar, 50 μm.) (B) Coexpression of LIN28 (L), NANOG (N), LRH1, and RARG (LR) along with four Yamanaka factors substantially increased the reprogrammed colony numbers. (C) Relative expression of key endogenous pluripotency genes in two iPSC lines cultured in bEPSCM, bEPSCiPS-Q36, and bEPSCiPS-Q99. Data represent the mean ± SD, n = 3 independent experiments. (D) Expression of lineage genes in RT-qPCR of iPSC lines in the presence or absence of Dox in M15 medium. Q36: biPS-Q36 with Dox, Q99: biPS-Q99 with Dox. Data represent the mean ± SD, n = 3 independent experiments. (E) No detectable leaky expression of the exogenous reprogramming factors in iPSCs in RT-qPCR. (F and G) RT-qPCR analysis of pluripotency (F) and lineage genes (G) in bovine iPSCs under several culture conditions in the absence of Dox. These conditions include 2i/LIF, t2iL+Gӧ, and 5i/L/A on day 8; CTFR medium (passage 4); and pEPSCM (cells of passage 2 and passage 8 for analyzing pluripotency genes, and cells of passage 8 for analyzing lineage genes). Cells cultured in bEPSCM for passage 36 were used in the analysis. pEPSCM: porcine expanded potential stem cells medium, bEPSCM: bovine expanded potential stem cells medium, CTFRM: custom TeSR1 base medium supplemented with FGF2 and IWR1. Data represent the mean ± SD, n = 3 independent experiments. (H) Immunostaining of NANOG, OCT4, SOX2, and E-CADHERIN in bovine bEPSCiPS. (Scale bar, 100 μm.) (I) The morphology and alkaline phosphatase (AP) staining of bEPSCiPS-Q36 on feeder cells (Upper) or feeder free (Lower). (Scale bar, 50 μm.)

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