http://orcid.org/0000-0001-6188-7758
Figures
Abstract
Green fluorescent protein (GFP) reporters controlled by the regulatory region of OCT4 and NANOG—two master regulators for pluripotency are widely used in studies of pluripotent stem cell establishment and embryo development. Alongside the challenge in establishing bovine pluripotent stem cells, the application of bovine-specific gene reporters has rarely been explored.
Using lentivirus-based GFP reporter, we investigated the upstream regulatory regions of bovine OCT4 and NANOG. These reporters show activity in both naïve- and primed-state pluripotency when infected into mouse and human embryonic stem cells (ESCs), respectively. Consistent with what is found in humans and mice, the bovine OCT4-distal enhancer (bOCT4-DE) but not the proximal enhancer (bOCT4-PE) region is preferentially activated in naïve-state pluripotency. Furthermore, the bOCT4-DE region is silenced upon conversion of naive-state ESCs into primed-state epiblast stem cells (EpiSCs). Co-infection of mouse fibroblasts with the reprograming factors for induced pluripotent stem cell (iPSC) induction leads to the generation of GFP positive colonies, demonstrating that these GFP reporters can serve as live indicators for induced pluripotent cell establishment. We further proved that the bovine OCT4 distal enhancer is active in bovine blastocysts. We established the lentiviral-based fluorescent reporters controlled by bovine OCT4 and NANOG enhancer sequences. These reporter constructs show activity in naïve- and primed-pluripotent states. These reporters may serve as versatile tools for bovine ESC/iPSC generation and identification, as well as for developmental studies of bovine embryos.
Citation: Huang D, Wang L, Talbot NC, Huang C, Pu L, Zhao X, et al. (2018) Analyzing bovine OCT4 and NANOG enhancer activity in pluripotent stem cells using fluorescent protein reporters. PLoS ONE 13(10): e0203923. https://doi.org/10.1371/journal.pone.0203923
Editor: Peter Hohenstein, The Roslin Institute, UNITED KINGDOM
Received: November 3, 2017; Accepted: August 30, 2018; Published: October 5, 2018
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the United States Department of Agriculture (USDA) W2171 regional project to Y.T., the Agriculture and Food Research Initiative Competitive Grant no. 2016-67016-24894 to Y.T. from the USDA National Institute of Food and Agriculture (https://nifa.usda.gov/), and the Chinese Scholarship Council for Study Abroad for D.H. and M.Z. (https://www.csc.edu.cn/chuguo).
Competing interests: The authors have declared that no competing interests exist.
Introduction
Embryonic stem cells (ESCs) have infinite proliferation potential and are pluripotent. The induced pluripotent stem cells (iPSCs) resemble ESCs in pluripotency but are derived from somatic cells by overexpressing “reprogramming factors”, including OCT4, KLF4, SOX2, and c-MYC (OKSM), or OCT4, SOX2, NANOG, and LIN28[1, 2]. Mouse ESCs represent the pre-implantation stage “naïve” pluripotent state[3], with dome-shaped colony morphology, dependence on leukemia inhibitory factor (LIF) signaling for self-renewal, and their ability for single cell colonization[4, 5]. Conversely, post-implantation stage “primed” state pluripotency is represented by mouse epiblast stem cells (EpiSCs) and human ESCs, which are flat in colony morphology, dependent on basic fibroblast growth factor (bFGF)/Activin A signaling for pluripotency maintenance, and are refractory to single cell colonization[6–8]. Though both are considered pluripotent, the primed-state pluripotent stem cells exhibit limited chimera-forming capacity compared with cells of the naïve-state[6, 8, 9].
The naïve- or primed-state pluripotent ESCs/iPSCs derived from farm animals will be important cell resources for studies of livestock embryo development, animal reproduction, and human disease modeling. However, compared with other specie, ESC and iPSC development in bovine has been challenging. The effort of generating bovine ESCs started more than 21 years ago[10]. The reported putative bovine ESCs are very difficult to culture and usually differentiate within a few passages[11]. The limited propagation capacity is commonly seen in reported bovine iPSCs also[12–15]. These bovine iPSCs are not completely reprogrammed as shown by their dependence on the continued expression of the viral transgenes for self-renewal[12–15]. Also, there is a profound trophectoderm lineage generation or differentiation during bovine iPSC induction and ESC derivation[16, 17]. These problems negatively impact the practical applications of bovine ESCs/iPSCs. Tools are needed to help optimizing the conditions for bovine pluripotent stem cell development.
The use of green fluorescence protein (GFP) reporters controlled by the upstream regulatory regions of OCT4 and NANOG has greatly facilitated mouse and human ESC/iPSC studies[18–20]. The transcription factors OCT4 (also known as POU5F1) and NANOG are master pluripotency regulators essential for pluripotency maintenance and abundantly expressed in mammalian ESCs[21, 22]. They are also activated at the late reprogramming stage of iPSC induction[23]. The GFP reporters controlled by the enhancer regions of OCT4 and NANOG offer non-invasive, convenient monitoring of the transcription activity for these genes to study pluripotency regulation and reprogramming kinetics in live cells. Also, in naïve-state pluripotency, the expression of human and mouse OCT4 genes is preferentially regulated by their distal enhancers (OCT4-DE), which is silenced in primed-state mouse EpiSCs and human ESCs/iPSCs that preferentially use the OCT4 proximal enhancer (OCT4-PE)[6, 24, 25] (S1 Fig). GFP reporters using this unique activity of OCT4-DE have been successfully applied to help generate naïve pluripotent human iPSCs[25–27].
Although the regulatory regions of OCT4 and NANOG are highly conserved across humans, bovine, and mice[28, 29], study of the bovine-specific gene regulatory regions using fluorescent protein reporters has been scarce. A plasmid-based bovine OCT4-GFP reporter was shown to be active in mouse embryos[30]. Another plasmid-based GFP reporter controlled by a bovine NANOG regulatory fragment also showed activity in mouse ESCs[31]. However, plasmid-based reporters have low transfection efficiency and require lengthy selection and expansion for the creation of stably integrated cells. This can be cumbersome for iPSC induction that requires a large quantity of primary cells or secondary cells with low passage numbers (within P3-P5)[2, 32]. In this study, we utilized the high transduction efficiency of lentivirus-based GFP reporter vector to study the bovine-specific OCT4 and NANOG regulatory regions. We show that these reporters are active in both mouse and human ESCs. We further show that the bovine OCT4-DE region is specifically activated in naïve-state pluripotent cells but not in primed-state pluripotent cells. We also tested these lentiviral reporters in mouse iPSC generation and show that they can serve as faithful indicators for pluripotency establishment. We also showed the activity of bovine OCT4-DE in bovine blastocysts from in vitro fertilization. The reporters we generated here will be valuable tools for the study of bovine ESC/iPSC generation, maintenance, regulation and differentiation, and in the study of bovine embryo development.
Materials and methods
Chemicals and constructs
Doxycycline (Dox) was purchased from Sigma-Aldrich (St. Louis, MO). PD0325901 and CHIR99021 were obtained from SelleckChem (Houston, TX). Polybrene was purchased from AmericanBIO (Natick, MA). Dox-inducible lentiviral FUW-TetO-hOKSM and FUW-M2rtTA plasmids were obtained from Addgene (Cambridge, MA). The human OCT4 enhancer controlled pGreenFire Lenti-Reporter was from System Biosciences (Palo Alto, CA). Genomic DNA was extracted from bovine Wharton’s jelly cells using DNAzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Two bovine OCT4 full-length enhancer fragments, two distal enhancers, one proximal enhancer and one Nanog enhancer were PCR amplified based on the published bovine OCT4 gene sequence[28] and genome sequence Bos_Taurus_UMD3.1 from Ensemble[33]. The full-length OCT4 enhancer fragments were designated as bOCT4-GFP1 (-2851 to -1 bp relative to the ATG start codon) and bOCT4-GFP2 (-3851 to -1 bp, see GeneBank access number MF664108 for sequence information of bovine OCT4), respectively.
For the OCT4 distal enhancer region, bOct4-DE was from -3,851 to -1644 bp and bOCT4-DE2 was from -3,851 to -1644bp plus the OCT4 minimal promoter (-260 to -1 bp). The OCT4 proximal enhancer was amplified from -1645 to -1 bp. The Nanog enhancer was cloned from -1096 bp to -1 bp. For cloning primer sequence information see S1 Table. All the fragments were inserted into the pGreenFire Lenti_Reporter Vector (System Biosciences) using the In-Fusion kit (Clontech, Mountain View, CA). All the DNA constructs were verified by direct DNA sequencing.
Lentivirus preparation
The verified lentiviral bOCT4 or bNANOG reporter constructs, the FUW-TetO-hOKSM or FUW-M2rtTA were co-transfected with the lentiviral packaging plasmids pCMV-V-SVG and psPAX2 (Addgene, Cambridge, CA) into 293T cells using Fugene 6 reagent (Promega, Madison, WI) based on the viral packaging protocol provided from Addgene.com. The virus-containing supernatant was harvested 48 h and 72 h post-transfection and filtered with 0.8 μm sterile filters (Corning Inc., Corning, NY).
Cell culture, viral transduction and mouse embryonic fibroblast (MEF) reprogramming
Mouse R1-ESCs were cultured in 2i/LIF medium[34] on mitomycin C-treated MEF feeders. Human H9-ESCs were cultured in feeder-free mTeSR1 (Stemcell Technologies, Cambridge, MA) medium on matrigel (Corning). MEFs were cultured in medium consisting of DMEM (Invitrogen) with 10% fetal bovine serum (FBS) (Rocky Mountain Biologicals, Inc., Missoula, MT). Lentiviruses of different reporters were transduced into mouse or human ESCs with polybrene. For transduction of R1-ESCs, the next day after seeded as single cells by trypsinization, the cells were incubated with viruses and polybrene containing medium overnight. For transduction of human ESCs, cells were digested with 1 mg/ml Dispase into cell aggregates and seeded on day 0. Two lentiviral infections were performed on day 1 and day 2, respectively. The mouse and human ESCs were then maintained in their respective medium for the appearance of GFP signal after transduction.
For reprogramming, 1 million B6/129 MEFs at passage 2 were seeded on a gelatin coated six-well-plate on day -1. On day 0, lentiviruses of different reporters were co-transduced into MEFs together with the Dox-inducible polycistronic FUW-TetO-hOKSM and FUW-M2rtTA in the presence of polybrene. On day 1, the medium was replaced to MEF medium with 1μg/ml Dox. The infected MEFs were passaged onto MEF-feeders on day 2. Starting from day 3, reprogramming medium containing 1μg/ml Dox was used for continued reprogramming. The reprogramming medium was a 1:1 mixture of the KSR-ESC medium containing 20% Knock-out serum replacement (KSR), 1 x MEM non-essential amino acids, 1 x GlutaMax, 0.5 x penicillin/streptomycin, 1 x 2-mercaptoethanol and 1,000 U/mL mLIF in Knock-out DMEM (all from Invitrogen), and the serum-containing ES medium containing 20% ES-FBS, 1 x MEM non-essential amino acids, 1 x GlutaMax, 0.5 x penicillin/streptomycin, 1 x 2-mercaptoethanol and 1,000 U/ml mLIF in DMEM (all from Invitrogen). The GFP positive mouse iPSC colonies were picked and expanded in 2i/LIF medium without Dox.
Embryoid body (EB) formation
The mouse ESCs carrying reporters were dissociated with 0.05% trypsin (Invitrogen), and then plated into 60 mm tissue culture dishes for 20 min to allow MEF-feeders to attach. The ESCs remaining in the medium were transferred to 60 mm low adhesive Petri dishes for differentiation in DMEM (Invitrogen) plus 10% FBS (Rocky Mountain Biologicals, Inc.) without LIF. After 1 wk of differentiation, EBs were either subjected to RNA extraction or transferred to cell culture dishes coated with 0.1% gelatin (Merck Millipore, Billerica, MA) for further differentiation before being subjected to RNA extraction.
Epiblast stem cell (EpiSC) differentiation
EpiSC differentiation was performed based on a previous study[35]. Briefly, mouse R1-ESCs infected with reporters were seeded at a density of 1×105 per well in a 6-well plate in which MEF-feeders had been plated beforehand. Twenty-four hours after plating, the medium was changed to EpiSC medium which consists of DMEM-F12, 15% KSR, 1 x GlutaMax, 1 x MEM non-essential amino acids (all from Invitrogen), 1 x 2-mercaptoethanol (Merck Millipore, MA), 12 ng/mL FGF2 (R&D Systems, Minneapolis, MN), 20 ng/mL Activin A (R&D Systems) and 1 x penicillin/streptomycin. Thereafter, cells were maintained in EpiSC medium and passaged every 3–4 days using 1 mg/mL collagenase type IV. After 4 passages, EpiSC colonies were used for flow cytometry and other analyses.
Immunostaining
Alkaline phosphatase staining was performed with the Vector Red Alkaline Phosphate Substrate Kit (Vector Laboratories, Burlingame, CA). For immunostaining, cells were fixed in 4% paraformaldehyde with 1% sucrose in phosphate buffered saline (PBS) (Invitrogen) for 15 min at room temperature. The cell membranes were then permeabilized with 0.5% TX-100 in PBS-T (Sigma-Aldrich), blocked in donkey serum (Sigma-Aldrich) and incubated with donkey serum containing primary antibodies including rabbit anti-OCT4 (Merck Millipore), rabbit anti-SOX2 (Abcam, San Francisco, CA) and rabbit anti-NANOG (Merck Millipore) at 1:100 dilutions, washed in PBS-T, and then incubated with Alexa Fluor 594 conjugated goat anti-rabbit secondary antibody (Cell Signaling Technology, Danvers, MA) at a 1:500 dilution. The cell nuclei were counterstained with DAPI. Fluorescent images were taken using a Zeiss or Nikon fluorescence microscope.
Fluorescence flow cytometry analysis
The infected mouse R1-ES cells, EpiSCs, and EBs for flow cytometry analysis were dissociated with 0.05% trypsin and resuspended in PBS containing 2% FBS. 10,000 events of cell counts were analyzed for each sample. The dead cells were distinguished with propidium iodide (PI) staining. The live cell populations were analyzed for their GFP fluorescence intensity. A BD Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA) and FlowJo software were used for the measurement and data analysis, respectively.
Quantitative real time—PCR (qRT-PCR)
Total RNA was extracted with Trizol (Invitrogen) based on the manufacturer’s instruction. One μg of the total RNA was reverse-transcribed into cDNA with an All-In-One cDNA Synthesis kit (Bimake, Houston, TX). Real-time PCR reactions were performed with the SYBR Green qPCR Master Mix (Bimake) and an ABI 7500 Fast platform (Thermo Fisher Scientific). The 7500 software v2.3 was used for data analysis, with values normalized to GAPDH.
In-vitro fertilization (IVF) and cytoplasmic injection
Oocytes were collected from Bubalus bubalis ovaries obtained from a local slaughter house. All procedures involving animal treatment and collection of ovaries in the study were based on the Guiding Principles for animal use as described by the Council for Institutional Animal Care and Use Committee (IACUC) and approved by the Animal Experimentation Ethics Committee of Guangxi University, Nanning, China. The IVF procedure was performed as described previously[36]. Briefly, IVF were performed after oocyte maturation for 24 hours in the medium containing TCM-199, 10% Oestrus Calf Serum (OCS), 0.5 mg/mL Follicle-Stimulating Hormone (FSH), 5 mg/mL Luteinizing Hormone (LH) and 1 mg/mL estradiol-17β at 38.5°C under 5% CO2 atmosphere with maximum relative humidity. Following IVF, the presumptive zygotes were cultured in IVF medium (modified Tyrode's medium supplemented with 36% TCM-199, 10% fetal bovine serum, 0.06 mg/ml penicillin and 0.1 mg/mL streptomycin) at 38.5°C under 5% CO2 atmosphere with maximum relative humidity. Cytoplasmic injection was carried out 7 hours after IVF with IVF medium containing 0.02μl/ml cytochalasin-b with a Nikon ECLIPSE Ti micromanipulator. The reporter plasmid concentration was 30ng/μl. After cytoplasmic injection, the embryos were monitored daily with an OLYMPUS DP73 fluorescent microscope until hatching.
Statistics
Each experiment was repeated at least three times (n ≥ 3). Quantification of GFP fluorescence intensity from flow cytometry analysis and the relative gene expression from qRT-PCR analysis was presented as the mean ± SD (standard deviation). Data were processed using One-Way ANOVA with Tukey's multiple comparisons, or the Student’s T-test. A p-value < 0.05 was considered statistically significant.
Results
Reporters controlled by bovine OCT4 and NANOG enhancers are active in primed- and naïve-state pluripotent stem cells
The ~2.9 kb bovine OCT4 gene 5’ upstream regulatory region[28] share 53.43% and 64.2% sequence identity with mouse and human OCT4 upstream regulatory regions, respectively. In addition, this upstream regulatory region contains four highly conserved regions (CR1-4)(Fig 1A and 1B) across the three species[29]. To investigate the bovine OCT4 enhancer activity, we cloned the bovine OCT4 upstream regulatory regions comprising 2,851 and 3,851 nucleotides from the translational start codon into the lentiviral pGreenFire Lenti-Reporter (bOCT4-GFP1 and–GFP2, respectively, Fig 1A). We first asked whether these reporters would work in the naïve pluripotent mouse and primed pluripotent human ESCs, respectively. Transduction of the lentiviral bOCT4-GFP to single cells of mouse R1-ESCs yielded a high percentage of GFP positive (GFP+) colonies after one wk in culture in 2i/LIF medium, known to be restrictive to ground state pluripotency[34]. No GFP+ colony was observed in control vector infection (Fig 1D). Infection of the bOCT4-GFP lentivirus into mouse embryonic fibroblasts (MEFs) generated no GFP+ signal (Fig 1D). These results indicated that the bovine OCT4-enhancer could be activated in naïve pluripotent mouse ESCs. We then infected the lentiviral bOCT4-GFP into human H9 ESCs. These primed-state ESCs can only be passaged through incomplete digestion into small clumps of cells, therefore the infection efficiency was not high. Nevertheless, we observed scattered GFP+ cells within individual colonies at one wk after infection (Fig 1E).
A: Schematic representation of bovine OCT4 and NANOG (bOCT4, bNANOG) regulatory regions upstream of ATG start codon and cloning into the pGreenFire Lenti-Reporter. The conserved-regions (CR) of OCT4 and NANOG regulatory regions are also shown. The bOCT4 distal and proximal enhancers and proximal promoter are shown based on sequence homology to the human and mouse OCT4. B. Sequence homology (left) and phylogenetic tree (right) of OCT4 upstream regulatory regions. Multiple sequence alignment was performed with Clustal Omega. C. Sequence homology (left) and phylogenetic tree (right) of NANOG upstream regulatory regions. Multiple sequence alignment was performed with Clustal Omega. D: Upper: bOCT4-GFP lentiviral reporters transduced into single cell mouse R1 ESCs and cultured for 7 days in 2i/LIF medium. pGreenFire reporter vector and the human OCT4 enhancer controlled GFP reporter (hOCT4-GFP) were used as negative and positive controls, respectively. Lower: bOCT4- and bNANOG-GFP lentiviruses infected into MEFs for 7 days. pGreenFire reporter vector was used as control. Bar = 250 μm. E: bOCT4-GFP lentivirus was infected into human H9 ESCs and cultured for 7 d. pGreenFire reporter vector and the hOCT4-GFP reporter were used as negative and positive controls, respectively. Bar = 250 μm. F: bNANOG-GFP lentiviral reporter expresses in single cells of mouse R1 ESCs and cultured for 7 days in 2i/LIF medium. The human NANOG enhancer controlled GFP reporter (hNANOG-GFP) was used as a positive control. Bar = 250 μm. G: Left: bNANOG-GFP lentivirus was infected into human H9 ESCs and cultured for 7 d. Middle: enrichment of positive bNANOG-GFP expressing cells in H9 ESC colonies after passaging (P8). Right: hNANOG-GFP lentivirus used as a positive control. Bar = 250 μm. H. bOCT4- and bNANOG-GFP lentiviruses infected into bovine fibroblast cells. pGreenFire reporter vector was used as control. Bar = 250 μm.
We also cloned a 1,095 nucleotide bovine NANOG 5’ regulatory region, based on a previous study[31], into the pGreenFire Lenti-Reporter (bNANOG-GFP, Fig 1A). This bovine NANOG upstream regulatory region shares 43.49% and 50.26% sequence identity with mouse and human NANOG regulatory regions, respectively (Fig 1C). The bovine NANOG regulatory region also contains an across-species conserved region[31], as shown in Fig 1A. We observed activation of this reporter in mouse ESCs (Fig 1F), and, similar to our results with bOCT4-GFP, scattered GFP+ cells in individual colonies upon infection into human H9 ESCs, which could be further enriched through subculturing (Fig 1G). We also transduced these bovine OCT4- and NANOG-GFP lentiviral reporters into bovine fibroblast cells, no fluorescence was observed (Fig 1H). Thus, these bovine OCT4- and NANOG-GFP lentiviral reporters were specifically active in both naïve- and primed-state pluripotent ESCs.
Bovine OCT4 distal and proximal enhancers exhibit different activities in naïve pluripotent stem cells
The human OCT4-DE (from CR3 to ~3.8 kb upstream of the translation start site) is preferentially activated in the naïve-state, while the OCT4-PE (nucleotides -1,835 to -681 bp) is activated in primed-state pluripotent cells[25, 26]. Based on the high sequence homology between human and bovine OCT4, we asked if the bovine OCT4 DE and PE regions (bOCT4-DE and bOCT4-DE) would act in a similar way. We removed the bovine OCT4 PE region (nucleotides -1,643- to -602 bp) out of the 3,851 nucleotides bOCT4 regulatory sequence (bOCT4-DE), or with an additional deletion of the proximal promoter (nucleotides -601 to -1) (bOCT4–DE2), based on the sequence homology between bovine and human OCT4[29] (Fig 2A). We cloned these bOCT4 regulatory regions into the pGreenFire Lenti-Reporter and evaluated their activity in mouse R1-ESCs. Fluorescence flow cytometry analysis and microscopy observation revealed that both bOCT4-DE and–DE2 fragments were active in naïve-state ESCs, while the bOCT4-PE was not (Fig 2B and 2C). Interestingly, the bOCT4-DE-GFP showed significantly stronger GFP intensity than that of bOCT4-GFP1 in R1-ESCs (Fig 2B). Thus, similar to their human and mouse OCT4 counterparts, the bovine OCT4 distal, but not proximal regulatory region, was active in naïve-state pluripotent ESCs.
A: Schematic representation of bovine OCT4 proximal and distal regulatory regions upstream of ATG start codon cloning into the pGreenFire Lenti-Reporter. B: Left: Fluorescence flow cytometry analysis of bOCT4-DE-GFP, bOCT4-DE2-GFP, bOCT4-GFP1, and bOCT4-PE-GFP expression in R1-ESCs. Non-infected R1-ESCs served as the negative control (Ctl). Right: GFP intensity measured by flow cytometry. Bars represent mean±sd, **: p < 0.01, n = 4.C: Expression of bOCT4-DE-GFP and bOCT4-PE-GFP in R1-ESCs. Bar = 250 μm.D: Left: Cloned bOCT4-DE-GFP positive ESCs and the EBs formed at 1-wk upon differentiation. Bar = 250 μm. Right: Fluorescence flow cytometry analysis of bOCT4-DE-GFP positive ESCs and their differentiated cells at 1-wk of differentiation. GFP intensity represents mean±sd, n = 4.E: Left: Cloned bOCT4-DE2-GFP positive ESCs and the EBs formed at 1-wk upon differentiation. Bar = 250 μm. Right: Fluorescence flow cytometry analysis of bOCT4-DE2-GFP positive ESCs and the differentiated cells at 1-wk of differentiation. GFP intensity represents mean±sd, n = 3.F: qRT-PCR analysis of endogenous Oct4 and differentiation marker genes at 1-wk of EB differentiation from the bOCT4-DE-GFP+ ESCs. Values are normalized to GAPDH and compared with the undifferentiated ESCs. Bars represent mean±sd, **: p < 0.01, n = 3.
We then asked whether the bOCT4-DE-GFP reporter would be silenced after the differentiation of R1-ESCs. We observed marked decrease in GFP expression after 1-wk of EB differentiation upon LIF removal from the mouse ESCs (Fig 2D). The decrease in GFP intensity was confirmed by flow cytometry analysis (Fig 2D). The case was similar for the EB differentiation from bOCT4-DE2-GFP+ mouse ESCs (Fig 2E). qRT-PCR analysis further verified that the decreased GFP expression correlates with a reduction of endogenous Oct4 expression and increased expression of commitment genes in the differentiated cells (Fig 2F).
Bovine OCT4 distal enhancer is silenced in primed-state pluripotent stem cells
We further asked whether the bOCT4 distal enhancer region would be silenced in primed pluripotent state cells. We converted the bOCT4-DE-GFP+ naïve-state R1-ESCs to primed-state EpiSCs by culturing them in Activin A and bFGF containing medium[35]. Upon 2–3 passages, these cells formed flat monolayer EpiSC-like colonies resembling the typical primed-pluripotent state morphology, and they exhibited diminished GFP expression (Fig 3A). Compared with their parental naïve-state ESCs, these cells displayed markedly suppressed expression of naïve-specific markers (Dppa3, Rex1, Tbx3), and an increased profile of EpiSC-specific markers including Cer1, Fgf5, and T (Fig 3B). The expression of pluripotency markers including Oct4, Nanog, and Sox2 remained similar or slightly lower in EpiSCs than their parental ESCs (Fig 3B and 3C). Unlike the naïve-state ESCs, EpiSCs exhibit little alkaline phosphatase (AP) activity[8]. This was also demonstrated in these converted EpiSCs (Fig 3D). The decreased bOCT4-DE-GFP expression in these EpiSCs was further confirmed with fluorescence flow cytometry (Fig 3E). Thus, the bovine OCT4 distal enhancer was inactivated in primed-state pluripotency.
A: The parental bOCT4-DE-GFP+ ESCs and their EpiSC colonies formed after subculturing (P4) in EpiSC medium. Bar = 120 μm (left) or 120 μm (right).B: qRT-PCR gene expression ratio for primed- and naïve-state pluripotency markers between the converted EpiSCs (p4) and their parent ESCs. n = 3.C: Left: Immunostaining of pluripotent markers in ESCs and the converted EpiSCs. Bar = 60 μm. Right: Quantification of fluorescence intensity from immunostaining. Values are arbitrary unit (AU) normalized with DAPI Stain. **: p < 0.01, n = 5.D: AP-staining of EpiSCs and their parental ESC colonies. Bar = 250 μm.E: Fluorescence flow cytometry analysis of bOCT4-DE-GFP positive ESCs and their converted EpiSCs (p4). GFP intensity represents mean±sd, n = 3.F: bOCT4-DE-GFP negative EpiSC colonies (left) were further expanded and developed into GFP+ ESC-like colonies (right) upon culture in 2i/LIF medium and passaged for 3 times (P3). Bar = 250 μm.
Primed-state EpiSCs can be successfully converted to naïve-state ESCs when directly cultured in the naïve-selective 2i/LIF medium, although at low efficiency[37–39]. We wondered whether the GFP-negative EpiSCs we generated here could also be converted back to ESC-like cells and become GFP+ again. To avoid any remaining naïve GFP+ cells in the EpiSC culture, single GFP- negative EpiSC colonies were picked and expanded further through passaging. After EpiSC expansion, we cultured the expanded bOCT4-DE-GFP negative EpiSC colonies in 2i/LIF medium. Significant cell death and differentiation occurred to these cells upon switching to 2i/LIF medium similarly as described[35]. However, after 3 passages, we observed the emergence of several colonies with typical dome-shaped naïve ESC-like morphology, which were GFP+ (Fig 3F). Thus, the bovine OCT4 distal enhancer is specifically activated in naïve-state pluripotent cells.
Reporters controlled by bovine OCT4 enhancer are activated during somatic cell reprogramming
We wondered whether the bOCT4-GFP reporters we generated might be used as live indicators in reprogramming cells for iPSC generation. To test this we used the MEF reprogramming system with doxycycline (Dox)-inducible OKSM expression[40] as bovine iPSC generation remains a challenge[16]. B6/129 MEFs were infected with lentiviral OKSM plus lentivirus of empty pGreenFire vector, bOCT4-GFP1, bOCT4-DE-GFP, or bOCT4-DE2-GFP. The infected MEFs were then passaged onto CD1-feeders and cultured in reprogramming medium. We started to observe GFP fluorescence on day 11 after viral infection from colonies of bOCT4-GFP1, bOCT4-DE-GFP, and bOCT4-DE2-GFP, but not the control vector reporter infected reprogramming conditions. We picked the GFP+ colonies carrying different reporters at reprogramming wk 2 to 3. The picked colonies can be cultured in Dox-free ESC culture medium and remain GFP positive (Fig 4A). We further expanded the OCT4-DE-GFP colonies and qRT-PCR analysis showed that these cells had significantly activated endogenous pluripotent genes such as Oct4, Nanog, Sox2, and Rex1, etc., that are comparable to R1-ESCs (Fig 4B). Furthermore, these iPSC colonies showed silenced transgene expression compared with those still remained in Dox-containing medium (Fig 4C). Thus, our results indicate that these bovine-specific OCT4-GFP reporters can be used as real-time indicators for successful reprogramming.
A: Picked GFP+ putative iPSC colonies infected with bOCT4-GFP1, bOCT4-DE2-GFP, and bOCT4-DE-GFP and cultured in DOX-free ESC culture medium (P2). Bar = 120 μm.B: qRT-PCR for endogenous pluripotent gene expression from three expanded iPSC lines positive for bOCT4-DE-GFP (P2). R1-ESCs and MEFs were used as the positive and negative control, respectively. C: qRT-PCR for transgene expression from three expanded iPSC lines positive for bOCT4-DE-GFP (P2). R1-ESCs and MEFs were used as negative controls, and bOCT4-DE-GFP positive iPSCs cultured in Dox-containing medium are used as positive control.
Bovine OCT4 distal enhancer is active in bovine blastocysts
To test if our bovine OCT4-DE-GFP reporter constructs can be activated in bovine embryonic-stage cells, we microinjected the plasmids (GFP Control and bovine OCT4-DE-GFP vectors) into bovine in vitro fertilized (IVF) zygotes. Despite the challenges that only cytoplasmic injection was performed due to the invisibility of bovine pronuclei in the zygotes and that blastocyst development rate are low (~ 10%) after plasmid DNA injection, we observed GFP fluorescence in day 9 bovine blastocyst cells injected with bovine OCT4-DE-GFP (Fig 5). Interestingly, this reporter is active in both the inner cell mass (ICM) cells and trophoblast cells in the bovine embryos (Fig 5). This agrees with the previously report that mouse Oct4-DE-GFP reporter is activated in both cell types after injection into bovine embryos, and that the bovine OCT4-regulatory region is activated in ICM of mouse blastocysts[30]. Thus, our bovine OCT4-DE-GFP reporter can be activated in bovine blastocysts.
pGreenFire reporter vector was used as a negative control.
Discussion
The bovine OCT4 gene upstream regulatory region shares significant sequence homology with those of the humans and mice. However, despite the widespread usage of the fluorescence/luminescence reporters controlled by mouse and human OCT4 enhancers in embryo and ESC/iPSC studies, research using bovine-specific gene reporters has been rare. Here we established lentiviral GFP-reporters controlled by bovine OCT4 and NANOG enhancer regions. We showed that these reporters can be activated once infected into human and mouse ESCs, which represent primed- and naïve-state pluripotency, respectively. We further demonstrated that similar to human and mouse OCT4, the distal but not proximal enhancer of bovine OCT4 is active in naïve-state pluripotent stem cells. Using the mouse iPSC induction system, we showed that these lentiviral OCT4 reporters can serve as live indicators in iPSC induction for successful reprogramming. To our knowledge, this is the first study of bovine OCT4 enhancer activity in different pluripotent stem cells using fluorescent reporters.
GFP-reporters controlled by OCT4 or NANOG enhancer regions have greatly facilitated studies of stem cell biology and reprogramming of human and mouse cells. One interesting phenomenon found in human and mouse OCT4 upstream regulatory sequences is the pluripotent state-specific activation for their DE and PE regions. CR4 and CR3 form part of the DE and are required for Oct4 expression in the inner cell mass (ICM) of mouse embryos and primordial germ cells[24, 41]. Similar observations were reported for the human OCT4 DE region in naïve-state human iPSCs[25, 26]. It has been shown that the primed-state EpiSCs are less compatible with germ-line chimera formation than naïve-state mouse ESCs[6, 8]. Also, the naïve-state human iPSCs demonstrated interspecies chimera capacity while the primed-state human ESCs could not[9]. Therefore, the generation of naive-state pluripotent bovine iPSCs will be important for study of bovine early embryo development and reproduction. Using mouse ESCs and EpiSCs, we show that as with human and mouse ESCs, the bovine OCT4 DE region is specifically active in naïve ESCs but becomes silenced once converted to primed EpiSCs. Thus, the lentiviral GFP-reporters controlled by bovine OCT4-DE region can be valuable tools to help optimize and facilitate the generation and isolation of naïve-state, bona fide bovine iPSCs.
Conclusion
We generated bovine specific OCT4 and NANOG enhancer-controlled lentiviral GFP reporters. These reporters show activity in mouse and human ESCs, with reporters regulated by bovine OCT4-DE region active preferentially in naïve- but not primed-state pluripotent cells. The OCT4-DE region is silenced upon conversion from naïve- to primed-state, and can be reactivated when converted back from primed- to naïve-state pluripotency. Co-infection of mouse embryonic fibroblasts with the OKSM factors for iPSC induction leads to the generation of GFP+ colonies, demonstrating that these GFP reporters can serve as live indicators for induced pluripotent cell establishment. Furthurmore, the bovine OCT4-DE regions are active in bovine early embryos. Thus, these reporters may serve as versatile tools for completely reprogrammed bovine ESC/iPSC generation, as well as for early bovine embryo development studies.
Supporting information
S1 Fig. Schematic representation of OCT4 regulatory region upstream of the ATG codon of humans, cattle, and mice.
The four conserved regions (CR1-4) among the three species, and the reported distal and proximal enhancer regions for human and mouse OCT4 are shown.
https://doi.org/10.1371/journal.pone.0203923.s001
(PDF)
Acknowledgments
This work was supported by the USDA W2171 regional project to Y.T., the Agriculture and Food Research Initiative Competitive Grant no. 2016-67016-24894 to Y.T. from the USDA National Institute of Food and Agriculture, and the Chinese Scholarship Council for Study Abroad for D.H. and M.Z..
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