fcell-09-713503 July 31, 2021 Time: 12:43 # 1 ORIGINAL RESEARCH published: 05 August 2021 doi: 10.3389/fcell.2021.713503 Establishment of Mouse Primed Stem Cells by Combination of Activin and LIF Signaling Mengyi Wei1,2†, Yanglin Chen1,2,3†, Chaoyue Zhao1,2, Li Zheng1,2, Baojiang Wu1,2, Chen Chen1,2, Xihe Li1,2,4 and Siqin Bao1,2* 1 State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, Hohhot, China, 2 Institute of Animal Genetic Research of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China, 3 School of Basic Medical Sciences, Southern Medical University, Guangzhou, China, 4 Inner Mongolia Saikexing Institute of Breeding and Reproductive Biotechnology in Domestic Animal, Hohhot, China In mice, embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) are established from pre- and post-implantation embryos and represent the naive and primed state, respectively. Herein we used mouse leukemia inhibitory factor (LIF), which supports Edited by: Wei Jiang, ESCs self-renewal and Activin A (Act A), which is the main factor in maintaining EpiSCs Wuhan University, China in post-implantation epiblast cultures, to derive a primed stem cell line named ALSCs. Reviewed by: Like EpiSCs, ALSCs express key pluripotent genes Oct4, Sox2, and Nanog; one X Tetsuya S. Tanaka, Elixirgen Scientific, Inc., United States chromosome was inactivated; and the cells failed to contribute to chimera formation Fabiana Passaro, in vivo. Notably, compared to EpiSCs, ALSCs efficiently reversed to ESCs (rESCs) University of Naples Federico II, Italy on activation of Wnt signaling. Moreover, we also discovered that culturing EpiSCs in *Correspondence: AL medium for several passages favored Wnt signaling-driven naive pluripotency. Our Siqin Bao [email protected] results show that ALSCs is a primed state stem cell and represents a simple model to †These authors have contributed study the control of pluripotency fate and conversion from the primed to the naive state. equally to this work and share first authorship Keywords: primed stem cells, embryonic stem cells, conversion, Activin A, LIF Specialty section: This article was submitted to INTRODUCTION Stem Cell Research, a section of the journal Embryonic stem cells (ESCs) are known for their potential of self-renewal and differentiating into Frontiers in Cell and Developmental different embryonic tissues (Evans and Kaufman, 1981). Pluripotency is temporary and transient Biology in vivo, whereas in vitro many different states of pluripotent stem cells have been established to Received: 23 May 2021 model the embryonic stem cells of early embryos (Brons et al., 2007; Tesar et al., 2007; Ying et al., Accepted: 09 July 2021 2008; Joo et al., 2014; Ohinata and Tsukiyama, 2014; Yang J. et al., 2017; Yang Y. et al., 2017). Published: 05 August 2021 Using leukemia inhibitory factor (LIF) and two inhibitors, Ying et al.(2008) established mouse Citation: embryonic stem cells, which are defined as “naive.” These naive mESCs are in a ground state Wei M, Chen Y, Zhao C, Zheng L, of pluripotency and display a distinct morphology and a more uniform gene expression profile Wu B, Chen C, Li X and Bao S (2021) than conventional mESCs maintained in cultures supplemented with serum/LIF and, are capable Establishment of Mouse Primed Stem Cells by Combination of Activin of producing chimera and germline offspring (Ying et al., 2008). In contrast, human pluripotent and LIF Signaling. stem cells or mouse epiblast stem cells, derived from culture medium containing Activin A (Act A) Front. Cell Dev. Biol. 9:713503. and bFGF and defined as “primed,”fail to contribute to blastocyst chimera formation although they doi: 10.3389/fcell.2021.713503 have the ability to form teratoma (Brons et al., 2007; Tesar et al., 2007). Frontiers in Cell and Developmental Biology| www.frontiersin.org 1 August 2021| Volume 9| Article 713503 fcell-09-713503 July 31, 2021 Time: 12:43 # 2 Wei et al. Mouse Primed Stem Cells Extensive efforts have been made to identify approaches able AL medium consisted of Act A (20 ng/ml, R&D systems) to reverse the two states of pluripotent stem cells and mainly and LIF (1,000 U/ml, Millipore) added into a basic N2B27 involve either specific culture conditions with different factors medium including 50% Neurobasal (Gibco), 50% DMEM/F12 or forced “naive” gene expression (Bao et al., 2009; Guo et al., (Gibco), 2 mM GlutaMax (Gibco), 1 × non-essential amino acids 2009; Guo and Smith, 2010; Okashita et al., 2016; Chen et al., (NEAA, Gibco), 1 × penicillin/streptomycin (Gibco), 0.1 mM 2018; Du et al., 2018; Pastor et al., 2018; Qiu et al., 2015; b-mercaptoethanol (Gibco), and 0.005% (25 mg) bovine serum Rathjen et al., 1999; Zhou et al., 2010; Tai and Ying, 2013; Yu albumin (BSA; Gibco) supplemented with 0.5 × N2 (Gibco), and S. et al., 2020). D’Aniello et al.(2017) proposed the pivotal 0.5 × B27 (Gibco). All culture dishes were coated with fibronectin roles of vitamin C and L-proline in controlling the pluripotency [1 mg/ml in phosphate-buffered saline (PBS), Millipore] for at continuum from naive to primed states by affecting global DNA least 0.5 hour (h) before use. methylation, transcriptional profile, and energy metabolism. Recently, Yu S. et al.(2020) proposed that BMP4 plays an Conversion of ALSCs to rESCs and essential role in primed-to-naive transition (PNT) by opening up chromatin loci to activate critical regulators of PNT. These EpiSCs to epiALSCs reports demonstrate that the state of pluripotent stem cells can To convert ALSCs to rESCs, ALSCs were dissociated into single be reversed to some extent by factors in their culture conditions. cells using Accutase (Invitrogen) and were plated in CL medium Two recent studies have suggested that there is a “formative” state or 2iL medium. The CL medium consisted of N2B27 medium in ESCs which is between the “naive” and “primed” state and supplemented with 3 mM CHIR99021 and LIF (1,000 U/ml), also presents formative features of human stem cells and horse while the 2iL medium consisted of N2B27 supplemented with stem cells (Kinoshita et al., 2020; Yu L. et al., 2020). These two 3 mM CHIR99021, 1 mM PD0325901, and LIF (1,000 U/ml). studies describe different culture systems. The first involves the To transform EpiSCs to epiALSCs, EpiSCs were cultured in inhibition of the Wnt signaling pathway and the cells named FS AF medium supplemented with Act A (20 ng/ml) and bFGF cells, while the other culture system is dependent on the Wnt (12 ng/ml, R&D systems), then dissociated with Accutase, and signaling pathway and the cells named XPSCs; however, both plated in AL medium. LDN193189 (100 nM, Selleckchem) cell lines exhibit formative pluripotency features (Kinoshita et al., and SB431542 (10 mM, Selleckchem) were also added into the 2020; Yu L. et al., 2020). All intermediate stem cells, including the AL medium and the CL medium to inhibit BMP4 and Act formative stem cells (FS cells), XPSCs and rosette-like stem cells A, respectively. (RSCs), encode a higher pluripotent gene expression than EpiSCs and contribute to chimera formation; however, different culture AP Staining conditions and the unique properties of stem cells still require Before staining, cells were placed in four-well plates, washed further exploration (Kinoshita et al., 2020; Neagu et al., 2020; Yu with 1 × PBS, and then fixed in 4% paraformaldehyde at room L. et al., 2020). temperature for 30 min. The cells were washed with 1 × PBS In this study, we investigated which factors play important again, followed by the addition of AP staining solution. The roles for establishing pluripotent stem cells from mouse post- AP staining solution was prepared as follows: 50 ml sodium implantation embryos. Using a chemically defined medium nitrite solution was gently mixed with 50 ml FRV-alkaline N2B27 supplemented with Act A and LIF, we successfully derived ◦ solution and incubated at 37 C for 3 min; next, 2.25 ml H2O primed pluripotent stem cells. These pluripotent stem cells were and 50 ml naphthol-As-BI alkaline solution were added to the named as ALSCs, like EpiSCs, which expressed pluripotent mixture. The staining solution with fixed cells was incubated in genes Oct4, Sox2, Nanog, and one inactive X chromosome the dark overnight. and contributed to multiple tissues in teratoma but failed to contribute to chimera in vivo. The ALSCs were in a primed state, closed to EpiSCs, and able to reverse to naive state with high Karyotype efficiency by activating Wnt signaling. The tested cells were incubated with 0.2 mg/ml colchicine supplemented to the culture medium for 2 h, followed by cell dissociation using Accutase; the suspensions were centrifuged at MATERIALS AND METHODS 1,500 rpm for 5 min to collect the tested cells. The cell pellets were gently resuspended in 8 ml 0.075 mol/L KCL (Sigma) and Derivation of ALSCs incubated at 37◦C in a water bath for 40 min for hypotonic Mouse gastrulas were collected from E6.5 pregnant female treatment. Fixative liquid (methanol/glacial acetic acid = 3:1) of ICR mice mated with GOF/GFP transgenic male mice with a 1 ml was subsequently added to the resuspended cells and mixed mixed background. The isolate epiblasts (E6.5) were obtained gently, and the solution was then centrifuged at 1,000 rpm for from gastrulas using a glass needle and were cultured in AL 10 min. After discarding the supernatant, the cells were mixed medium. After 5–10 days, outgrowths were minced into several gently in 8 ml fixative solution and incubated in 37◦C water bath smaller pieces using a glass needle and moved into a fresh AL for 30 min for cell fixation, which was repeated twice.