A cytokine screen using CRISPR-Cas9 knock-in reporter pig iPS cells reveals that Activin A regulates NANOG

Xu, Junjun; Zhang, Zheng; Du, Xuguang; Shi, Bingbo; Wang, Jichang; Gao, Dengfeng; Zhu, Qianqian; Chen, Xinze; Han, Jianyong

doi:10.1186/s13287-020-1588-z

Cited by 7 publications

(4 citation statements)

References 57 publications

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“…Second, to obtain NANOG-tdTomato knock-in cell lines, we constructed a NANOG DNA donor vector with four fragments, Backbone, Left Homology Arm-3× Flag, 3× Flag-P2A-tdTomato-Loxp-Puro-Loxp and Right Homology Arm, using NEBuilder ® HiFi DNA Assembly Master Mix (NEB, E2621X) as previously described. 89 The NANOG sgRNA targeted the sequence before the stop codon site to knock in the donor fragment as a reporter. The annealed sgRNA sequence was cloned into the Bsa I-digested pGL3-U6-sgRNA-PGK-puromycin vector (Addgene, 51133).…”

Section: Methodsmentioning

confidence: 99%

Generation and characterization of stable pig pregastrulation epiblast stem cell lines

et al. 2021

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Pig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.

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Section: Methodsmentioning

confidence: 99%

Generation and characterization of stable pig pregastrulation epiblast stem cell lines

et al. 2021

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“…All piPSCs can exhibit terminal differentiation and generation of teratoma in vivo , but none can produce chimeric offspring and germline transmission, suggesting that their pluripotency may be defective ( Cheng et al, 2012 ; Esteban et al, 2009 ; Ezashi et al, 2009 ; Xue et al, 2016 ; Zhang et al, 2015 , 2017 ). In the past several years, various laboratories have obtained piPSCs and modified the culture system ( Cheng et al, 2012 ; Haraguchi et al, 2012 ; Hou et al, 2016 ; Xu et al, 2020 ; Xue et al, 2016 ; Zhang et al, 2015 , 2017 ). Here, we attempted to obtain naïve piPSCs by modifying the patterns of gene expression.…”

Section: Discussionmentioning

confidence: 99%

<i>LIN28A</i> inhibits <i>DUSP</i> family phosphatases and activates MAPK signaling pathway to maintain pluripotency in porcine induced pluripotent stem cells

Zhu

Xiao

et al. 2021

Zoological Research

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LIN28A, an RNA-binding protein, plays an important role in porcine induced pluripotent stem cells (piPSCs). However, the molecular mechanism underlying the function of LIN28A in the maintenance of pluripotency in piPSCs remains unclear. Here, we explored the function of LIN28A in piPSCs based on its overexpression and knockdown. We performed total RNA sequencing (RNA-seq) of piPSCs and detected the expression levels of relevant genes by quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis, and immunofluorescence staining. Results indicated that piPSC proliferation ability decreased following LIN28A knockdown. Furthermore, when LIN28A expression in the sh LIN28A2 group was lower (by 20%) than that in the negative control knockdown group ( sh NC), the pluripotency of piPSCs disappeared and they differentiated into neuroectoderm cells. Results also showed that LIN28A overexpression inhibited the expression of DUSP (dual-specificity phosphatases) family phosphatases and activated the mitogen-activated protein kinase (MAPK) signaling pathway. Thus, LIN28A appears to activate the MAPK signaling pathway to maintain the pluripotency and proliferation ability of piPSCs. Our study provides a new resource for exploring the functions of LIN28A in piPSCs.

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“…These cell lines displayed conventional PSC properties and could be differentiated into three germ layers in vitro and form teratomas. Integrative reprogramming strategies have proven effective for efficiently making piPSC-like colonies from porcine somatic cells and have been used for many applications related to xenotransplantation and immunogenicity ( Park et al, 2013 ; Liu et al, 2013 ), understanding key developmental signaling ( Arai et al, 2013 ; Xu et al, 2020 ; Yang et al, 2022 ; Yuan et al, 2019 ), and deriving disease-relevant cell types ( Gu et al, 2012 ; Aravalli, Cressman, and Steer, 2012 ; Yang et al, 2013 ; Park et al, 2016 ; Liao et al, 2018 ; Yu et al, 2022 ; Liao et al, 2023 ) ( Table 1 ). However, an inevitable drawback of using integrating methods for introducing reprogramming factors is that they compromise the integrity of the host cell genome, raising their oncogenic potential ( Prigione et al, 2011 ; Chen et al, 2014 ) and limiting their translational applications ( Fan et al, 2013 ; Kang et al, 2015 ).…”

Section: Technical Challenges In the Derivation Of Transgene-free Pipscsmentioning

confidence: 99%

The progress of induced pluripotent stem cells derived from pigs: a mini review of recent advances

Neira,

Conrad,

Rusteika

et al. 2024

Front. Cell Dev. Biol.

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Pigs (Sus scrofa) are widely acknowledged as an important large mammalian animal model due to their similarity to human physiology, genetics, and immunology. Leveraging the full potential of this model presents significant opportunities for major advancements in the fields of comparative biology, disease modeling, and regenerative medicine. Thus, the derivation of pluripotent stem cells from this species can offer new tools for disease modeling and serve as a stepping stone to test future autologous or allogeneic cell-based therapies. Over the past few decades, great progress has been made in establishing porcine pluripotent stem cells (pPSCs), including embryonic stem cells (pESCs) derived from pre- and peri-implantation embryos, and porcine induced pluripotent stem cells (piPSCs) using a variety of cellular reprogramming strategies. However, the stabilization of pPSCs was not as straightforward as directly applying the culture conditions developed and optimized for murine or primate PSCs. Therefore, it has historically been challenging to establish stable pPSC lines that could pass stringent pluripotency tests. Here, we review recent advances in the establishment of stable porcine PSCs. We focus on the evolving derivation methods that eventually led to the establishment of pESCs and transgene-free piPSCs, as well as current challenges and opportunities in this rapidly advancing field.

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A cytokine screen using CRISPR-Cas9 knock-in reporter pig iPS cells reveals that Activin A regulates NANOG

Cited by 7 publications

References 57 publications

Generation and characterization of stable pig pregastrulation epiblast stem cell lines

Generation and characterization of stable pig pregastrulation epiblast stem cell lines

<i>LIN28A</i> inhibits <i>DUSP</i> family phosphatases and activates MAPK signaling pathway to maintain pluripotency in porcine induced pluripotent stem cells

The progress of induced pluripotent stem cells derived from pigs: a mini review of recent advances

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