Breaking Down Pluripotency in the Porcine Embryo Reveals Both a Premature and Reticent Stem Cell State in the Inner Cell Mass and Unique Expression Profiles of the Naive and Primed Stem Cell States

Abstract

To date, it has been difficult to establish bona fide porcine embryonic stem cells (pESC) and stable induced pluripotent stem cells. Reasons for this remain unclear, but they may depend on inappropriate culture conditions. This study reports the most insights to date on genes expressed in the pluripotent cells of the porcine embryo, namely the inner cell mass (ICM), the trophectoderm-covered epiblast (EPI), and the embryonic disc epiblast (ED). Specifically, we reveal that the early porcine ICM represents a premature state of pluripotency due to lack of translation of key pluripotent proteins, and the late ICM enters a transient, reticent pluripotent state which lacks expression of most genes associated with pluripotency. We describe a unique expression profile of the porcine EPI, reflecting the naive stem cell state, including expression of OCT4, NANOG, CRIPTO, and SSEA-1; weak expression of NrOB1 and REX1; but very limited expression of genes in classical pathways involved in regulating pluripotency. The porcine ED, reflecting the primed stem cell state, can be characterized by the expression of OCT4, NANOG, SOX2, KLF4, cMYC, REX1, CRIPTO, and KLF2. Further cell culture experiments using inhibitors against FGF, JAK/STAT, BMP, WNT, and NODAL pathways on cell cultures derived from day 5 and 10 embryos reveal the importance of FGF, JAK/STAT, and BMP signaling in maintaining cell proliferation of pESCs in vitro. Together, this article provides new insights into the regulation of pluripotency, revealing unique stem cell states in the different porcine stem cell populations derived from the early developing embryo.

Introduction

Embryonic stem cells (ESCs) have been established and well characterized in mouse [1], human [2], and monkey [3]; however, the establishment in other species, such as the pig, has been a particularly difficult task [4,5]. Reasons for this difficulty could relate to species-specific differences [6], or be due to inappropriate culture conditions. Culture conditions play a particularly important role in regulating pluripotency, in mouse ESCs (mESC), human ESCs (hESC), and mouse EPI stem cells in vitro, which has been eloquently shown recently by the ability to convert stem cells states by simply conversion of media [7 –9]. Furthermore, the stem cell state in vitro appears to alternate transiently, even in stable culture conditions. Heterogenous expression of Nanog and Stella has been described in mESC [10,11] and the recent discovery of the ability of mESC to form a totipotent cell type in vitro, which is devoid of Oct4, Nanog, and Sox2 expression [12], shows just how intermittent the stem cell state is within culture. It was only recently that scientists were able to successfully derive rat ESC by blocking differentiation events which are induced in the culture medium [13]. Induced pluripotent stem cells (iPSC) can also be maintained in similar culture conditions to ESC; however, despite research showing that porcine iPSC (piPSC) can progress at least to a certain stage [14 –17], these cells are not stable in culture when the transgenes are silenced or removed [17,18]. Therefore, given the difficulties in maintaining porcine ESCs (pESC) in vitro, it appears that the culture conditions for maintenance of pluripotency in this species are not fully optimized.

Supplementation of varying growth factors has improved and enables the sustained culture of pluripotent stem cells in mouse and human. In the case of mESC, addition of leukemia inhibitory factor (LIF) helps sustain the growth and proliferation of these cells in vitro, given these cells are dependent on LIF-activated JAK/STAT signaling, LIF-activated phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), and LIF-activated Src homology 2 domain-containing tyrosine phosphatase 2/mitogen-activated protein kinase (SHP2/MAPK) pathways [19]. In contrast, hESC growth in vitro is dependent on supplementation of FGF2 to the media, which activates the FGF signaling pathway and TGFβ/ACTIVIN/NODAL signaling [20] and can be further enhanced with supplementation of Activin A in the absence of serum or feeder cells [21]. Supplementation of LIF, FGF2, or a combination of both has been attempted to sustain pluripotency in pESC and has resulted in short-term cultures of cells [22 –26]. Strikingly, one research group produced chimeric piglets after an injection of early-passage pESC into embryos; however, the rate of chimerism was reported as low [26]. This suggests that potentially unknown cell signaling pathways may be more important in regulation of pluripotency in this species.

The genes expressed in the different stem cell states in the pig are currently unknown. The naive stem cell state in mESC can be currently characterized by the expression of Rex1, Nrob1, and Fgf4 and pluripotent markers such as Oct4, Nanog Sox2, Klf2, and Klf4 [27]. Whether these same markers hold true for the naive state in the pig is unknown, but preliminary investigations already reveal some differences. Cell signaling in the porcine pluripotent cell populations of the preimplantation blastocyst reveals some differences in this species compared with mouse. In the case of the key transcription factors known to regulate pluripotency, the porcine inner cell mass (ICM) lacks expression of SOX2 and NANOG [28], differing from both the mouse and human ICM. Furthermore, OCT4 is observed in both the ICM and the trophectoderm (TE), which has also been observed in human and primate ICM [29,30]. It is only in the TE-covered epiblast (EPI) and the embryonic disc epiblast (ED) of the porcine embryo that OCT4, NANOG, and SOX2 are exclusively expressed [28,31]. Expression of NANOG has even been observed in the underlying hypoblast [32]. The specific genes governing the naive state have not yet been characterized in the pig.

Absence of the LIFR has also been demonstrated in the porcine ICM and EPI, which indicate that LIF may not have an important role in cell signaling in this species [28]. However, the downstream activator of JAK/STAT signaling, STAT3 has been observed in the EPI [28], which may indicate that the JAK/STAT pathway may be activated by another, yet unidentified factor. The presence of FGF receptor 1 (FGFR1) has also been shown to be exclusively expressed in the porcine EPI [28], and may give some preliminary evidence for the presence of FGF signaling. Despite this research, there are very few indications of which pathways may be present in the porcine pluripotent ICM and EPI.

Other pathways have also been shown to play a role in the regulation of mouse and hESCs, in particular, BMP signaling, which plays a role in both ESC regulation and stem cell fate [33] and WNT signaling is also important, particularly in ESC regulation, cell fate, and also for somatic cell reprogramming [34]. Both of these pathways warrant further investigation in the porcine embryo to determine whether they may, indeed, play a role in the regulation of the stem cell state. A deeper understanding of the genes expressed and pathways involved in regulating embryonic pluripotency could help improve culture conditions of porcine pluripotent stem cells.

One way to determine which pathways may be important is through the use of chemical inhibitors that block particular cell pathways. Chemical inhibitors have been successfully used to block differentiation of ESC, which has resulted in the successful derivation of rat ESC [13]. In this study mentioned earlier, a combination of two different inhibitors (termed 2i) blocked Erk1/2 and Gsk3 signaling and enabled maintenance of pluripotency. In hESC, inhibition of Erk1/2 can improve culture in a chemically defined medium, however, only in the presence of FGF2 and Activin A, and in the absence of BMP4 [35]. It seems that blocking the TGFβ pathway with a targeted chemical inhibitor in hESC and human iPSC (hiPSC) can also induce neural differentiation [36]. In the pig, neural differentiation appears to be the default differentiation pathway when culturing ESCs in vitro, and, therefore, investigation of factors or inhibitors that may block this affect could also be of interest. Using an inhibitor approach is particularly useful for determining which pathways are crucial for the growth and maintenance of ESC in vitro. Very few studies have used chemical inhibitors to modulate the culture of porcine pluripotent stem cells; however, one recent paper performed on piPSC was able to establish a naive-like state of piPSC by culturing with an MEK inhibitor (MEKi), a GSK3b inhibitor, and LIF [37]. This induced the expression of STELLA and REX1, and these cells were able to form embryoid bodies expressing genes associated with the three embryonic germ layers. This may suggest that MEK signaling and WNT signaling are important in establishment of the naive state; however, detailed research on this has not yet been performed.

In this study, we investigate cell surface markers and pluripotency markers associated with pluripotency in four distinct stages of development, including the early blastocyst, the expanded late blastocyst, the EPI isolated from the hatched embryo, and the ED isolated from the expanded and elongating embryo, to give a clear overview of the transient change in the expression of genes in the different stem cell populations of the developing embryo. The porcine embryo is unique in comparison to human and mouse, due to its extended preimplantation development. This unique state means that the pluripotent cell populations reside and divide in vivo for a longer period compared with murine and human embryos which is reflected in the larger size of the porcine ED compared with mouse and human [38], which may have implications related to pluripotency and pluripotency-related genes. We also investigate genes associated with FGF, JAK/STAT, BMP, WNT, and CRIPTO/NODAL signaling in the porcine developing embryo to further delineate differences in expression between the pig and other species and to assess the importance of these pathways by culturing the porcine ICM and EPI in vitro along with a number of different inhibitors associated with the different cell signaling pathways and also an inhibitor that regulates the mesoderm to neural switch.

Materials and Methods

Collection of in vivo embryos

“Sows slaughtered for research purposes were performed under strict conditions of Danish Slaughterhouse practices. Approval for slaughter of sows for the purpose of collection of embryos for research purposes was obtained from Danish Crown.” Danish Landrace×Yorkshire sows were artificially inseminated with Duroc boar sperm twice over 48 h, starting at 4 days after weaning. Sows were first slaughtered and uteri were collected on day (D) 6, D8, D10, and D12 after the first date of insemination, meaning that embryos were D5/6, D7/8, D9/10, and D11/12 in age, respectively. Embryos were flushed through the uterine horns using transfer medium [phosphate-buffered saline (PBS) containing 1% fetal bovine serum]. Embryos were selected based on their age and their morphology and defined into four different groups for comparisons. Group 1 included nonexpanded blastocysts that were zona-enclosed (defined as D5/6). These embryos contain the early ICM. Group 2 included expanded and early hatched blastocysts and contained the late ICM (defined as D7/8). Group 3 included hatched, expanded embryos containing a well-formed EPI that had not protruded, or only partially protruded through the Rauber's layer (defined as D9/10). Group 4 included elongating embryos that had a large oval-shaped ED, which had fully protruded through the Rauber's layer (defined as D11/12).

Immunocytochemistry

For assessment of cell surface marker expression in porcine embryos, fluorescence immunocytochemistry was performed on D5/6 blastocysts (containing the ICM) and D9/10 embryos (containing the TE-covered EPI) to evaluate the expression of SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Furthermore, whole D5/6 and D7/8 embryos and D11/12 embryos containing the ED were analyzed for expression of KLF4, cMYC, BMPR-1, and pSMAD2. D7/8 embryos were also analyzed for expression of OCT4 and NANOG. In addition, D5/6, D9/10, and D11/12 embryos were analyzed for expression of REX1, NrOB1, CRIPTO, and LIFr. In vivo embryos were collected from sows as described earlier and fixed in 4% paraformaldehyde for 15 min at room temperature. A minimum of three embryos was analyzed per antibody and per embryonic stage. Embryos were stored in PBS at 4°C until analysis. The embryos were permeabilized in 1% Triton-X 100 (Sigma Aldrich) for 1 h at room temperature. Cells were washed twice with PBS and then incubated in 5% normal donkey serum (Sigma Aldrich) in PBS (blocking buffer) for 1 h at room temperature. The embryos were then incubated overnight in diluted primary antibodies at 4°C. Primary antibodies were diluted in blocking buffer at the following dilutions: goat polyclonal OCT4a isoform 1:250 (Santa Cruz), rabbit polyclonal NANOG 1:500 (Peprotech), goat polyclonal KLF4 1:400 (R&D Systems), mouse monoclonal cMYC 1:100 (Abcam), rabbit polyclonal BMPR-1 1:100 (Santa Cruz) and mouse monoclonal pSMAD2 (Cell Signaling Technology), rat monoclonal SSEA-3 1:25 (Biolegend), mouse monoclonal SSEA-4 1:100 (Biolegend), mouse monoclonal TRA-1-60R 1:400 (Biolegend), mouse monoclonal TRA-1-81 1:100 (Biolegend), rabbit polyclonal LIFR 1:100 (Santa Cruz), goat polyclonal REX1 1:100 (Santa Cruz), mouse monoclonal CRIPTO 1:100 (Santa Cruz), and rabbit polyclonal NrOB1 (also known as DAX1) 1:50 (Santa Cruz). Secondary antibody-negative controls were performed by omission of primary antibodies. In addition, isotype controls were performed to rule out nonspecific binding of primary antibody to other antigens. For positive control tissues for the REX1, NrOB1, and CRIPTO antibodies, we used piPSC, which were found to positively express these proteins (data not shown). A commercial hiPSC line (System Biosciences) was used as a positive control for SSEA-3, SSEA-4, TRA-1-61, and TRA-1-81 (data not shown). Embryos were washed twice with PBS and then incubated in either FITC- or Cy3 donkey-conjugated secondary antibodies raised in the appropriate species (ie, mouse, rabbit, or goat) (Jackson Immunoresearch), which were diluted at 1:200 in blocking buffer. Embryos were washed twice in PBS and incubated in 0.1 μg/mL Hoechst 33342 (Sigma Aldrich). Embryos were mounted on glass slides using Fluorescent mounting medium (Dako) and observed by immunofluorescence imaging using a DMRB fluorescent microscope (Leica Microsystems) and a TCS SPE confocal microscope (Leica Microsystems). Images were captured using Leica Application suite (2.8.1) capture or MM-AF software (Leica Microsystems).

Culture of ICM-derived and EPI-derived stem cells

A total of 41×D5/6, 7×D7/8, and 62×D9/10 embryos were collected for in vitro culture studies. The zona pellucida of the zona-enclosed blastocysts was digested using 0.5% pronase, and whole embryos were mechanically attached to a feeder layer of mitomycin-C (Sigma Aldrich)-treated CF-1 mouse embryonic fibroblasts (MEFs) using insulin needles; whereas hatched D7/8 embryos were cultured as whole embryos. In the case of the later D9–D12 embryos, the EPIs and the EDs were mechanically isolated from the embryo and attached to the MEFs using insulin needles. All embryos were cultured individually. The embryonic tissue was grown in stem cell media [Knock Out^™ Dulbecco's modified Eagle's medium (Invitrogen/Gibco) plus 15% knockout serum replacement (Invitrogen/Gibco) either with or without 20 ng/mL human bFGF (R&D Systems) 1× nonessential amino acids (Sigma), 1× penicillin/streptomycin, 1× GlutaMAX supplement (Invitrogen), and 100 mM beta-mercaptoethanol (Invitrogen)]. Embryos that formed outgrowth colonies were designated as passage 0. Medium was changed every 2 days, and the cells were passaged mechanically using ultrasharp splitting blades (Bioniche) every 4–5 days onto new MEF plates. Cells were grown until they differentiated or terminated when a loss of pluripotent features was observed. Embryos were cultured in the presence of cytokines and/or chemical inhibitors. In the case of the chemical inhibitors, the small chemicals were tested, in addition to 20 ng/mL bFGF in the medium, including 2 mM MEKi PD0325901 (Selleck Chemicals), FGFR inhibitor (FGFRi) 20 ng/mL AZD4547 (Chemitek), 5 μM BMP inhibitor (BMPi), Dorsomorphin (Biomol International/ENZO Life Sciences), 40 μM JAK/STAT inhibitor (JAK/STATi) (AUH-6-96) (kind gift from Gyeong-Hun Baeg, New York Medical College), 2 μM of a WNT inhibitor (WNTi) (IWR-1) (kind gift from Lawrence Lum, University of Texas), or 5 μM of a neural inhibitor (NEURALi) retinoic acid receptor-selective retinoid (AGN193109) (kind gift of Pelle Serup, University of Copenhagen). Culture of D5/6 and D9/10 embryos was also performed in stem cell medium, including recombinant human bFGF, recombinant mouse LIF, and also in stem cell medium minus bFGF, but in the presence of 50 ng/mL porcine interleukin 6 (IL6) (Prospec) and 30 ng/mL human recombinant IL6 receptor (IL6R) (Prospec) or human recombinant IL11 (Prospec). A minimum of four embryos per treatment was performed for each cytokine/inhibitor treatment and for each stage evaluated.

Semiquantitative reverse transcription–polymerase chain reaction

In order to examine gene expression, embryos were collected for reverse transcription–polymerase chain reaction (PCR) as follows. Embryos were collected, lysed in 50 μL RLT buffer (Qiagen) containing beta-mercaptoethanol (Sigma Aldrich), snap frozen in liquid nitrogen, and stored at −80°C until required for RNA extraction. In the case of embryo staging, whole blastocysts (D5/6) (pool of 5) and (D7/8) (pool of 10) whole were snap frozen in buffer as described earlier. The EPI was isolated mechanically from D9/10 embryos from the TE and underlying hypoblast using insulin needles and separately snap frozen in buffer (pool of 5). The ED was also isolated from D11/12 embryos and snap frozen (pool of 3). Total RNA was extracted using the RNeasy micro kit (Qiagen) according to the manufacturer's instructions and included a DNA digest step with DNaseI (Qiagen). RNA was amplified and then converted into cDNA using the Qiagen QuantiTect^® whole transcriptome kit (Qiagen) using the long amplification cycle according to the manufacturer's instructions. PCR was performed with cDNA using a 25 μL mastermix that was composed of 5× reaction buffer (Fermentas/Thermo Scientific), 2 mM dNTPs (Fermentas/Thermo Scientific), 1.5 mM MgCl₂, (Fermentas/Thermo Scientific), 10 pmol/μL of both sense and antisense oligos (TAGC Oligos), and 200 U/μL HotStart Taq polymerase (Fermentas) in RNAse-free water. PCR conditions were as follows: initial denaturation of 95°C for 4 min followed by 35 cycles of 95°C for 1 min, 55°C for 1 min, and 70°C for 1 min. A further extension was performed at 70°C for 15 min. Porcine-specific primers used for evaluation of expression of genes are listed in Table 1. PCR products were run on agarose gels and visualized using the geldoc system (Biorad).

Table 1.

Porcine-Specific Primers Used for Semiquantitative Reverse Transcription–Polymerase Chain Reaction

Gene	Sequences 5′- 3′	PCR product size (bp)	Intron spanning	Reference no. Genbank NCBI
OCT4a	5′-AGGTGTTCAGCCAAACGACC-3′	374	Yes	NM_001113060.1
	3′-TGATCGTTTGCCCTTCTGGC-5′
NANOG	5′-CGAATGAAATGTAAGAGGT-3′	162	Yes	DQ447201
	5′-CCAGCTCTGATTACCCCAC-3′
SOX2	5′-GCAACTCTACTGCTGCGGCG-3′	351	No	NM_001123197.1
	3′-GCCATGCTGTTGCCTCC-5′
KLF4	5′-CTCCTCTTCGTCGTCGCCGT-3′	521	No	GI2825099
	3′-CAGCGACGCCTTCAGCACGA-5′
cMYC	5′-TCGGACTCTCTGCTCTCCTC-3′	261	Yes	TC238599
	3′-CTGCATAATTGTGCTGGTGC-5′
REX1	5′-TCAACCAAGTGTCGGACAGA-3′	244	No	GI298162945
	3′-GGGACTCTGAACGGAGAGA-5′
NROB1	5′-CACGGCAGAGTGGCATCCT-3′	319	No	NW_003612745.1
	3′-CCAAGGTCTCCACCGTCT-5′
KLF2	5′-CCACCTCTTCTAACTCAG-3′	189	No	NW_003609596.1
	3′-CTCTTGAACCGACTCAGCCT-5′
FGFR1	5′-CACCTACTTCTCCGTCAAC-3′	127	Yes	NW_003612200.1
	3′-GGTTGATGCTGCCATACT-5′
FGFR2	5′-CCTGTTGCGAGATGCC-3′	407	Yes	NW_003538149.1
	3′-GCCTCCAATGCGATGTTCCT-5′
MEK	5′-CAGAGCGGGAGAATCACA-3′	274	Yes	NW_003612416.1
	3′-TGGTGTTACGAGATGGAG-5′
cFOS	5′-GACTACGAGGCGTCATCAT-3′	179	Yes	AJ132510
	3′-CGAGATAGCAGTCACCGT-5′
LIFr	5′-CTCATCCCAGTGGCAGTG-3′	213	Yes	U91518.1
	3′-CCAGAACCTCAACATTAT-5′
GP130	5′-GTCGTCCCTGTGTGTTTA-3′	230	Yes	TC281458
	3′-GCTTCTATTTCCACAACA-5′
JAK1	5′-GCAGTACGATTTGGTCA-3′	459	Yes	NW_003535085.2
	3′-CAGTCACCATCACCTC-5′
STAT3	5′-CGTCATCCCAGATTACAT-3′	251	Yes	NM_001044580
	5′-GAGTCGAAGCTCTGCGGAT-3′
BMPr1a	5′-CAGATACAGATGGTTCGGCAA-3′	381	Yes	NW_003612038.1
	5′-GTGGAGATGGCACAGACC-3′
SMAD1	5′-GCATCAACCCCTACCACTA-3′	194	Yes	AY245888
	5′-GAGGAAACGGATGGCTGT-3′
SMAD2	5′-CACCAAATACGATAGAT-3′	254	Yes	Genomic DNA Ch2. (CU062419.13)
	5′-CTGGTGTCTCAACTCTCT-3′
SMAD3	5′-GCTGTGTGAGTTCGCCTT-3′	154	Yes	Genomic DNA Ch1. (FP236622.1)
	5′-GGAATGGCTGTAGTCGTC-3′
SMAD4	5′-GGCTTCAGGTGGCTGGTCGGA-3′	223	Yes	AB053483
	5′-ACCTGATGGAGCATTACT-3′
SMAD5	5′-GGATTCACAGACCCTTCAA-3′	203	No	NM_005903.5
	5′-GACAGTGGTAGGATGGAA-3′
SFRP1	5′-GGTCTTCCTCTGTTCGCTCT-3′	219	Yes	XM_001926524
	5′-CGTGCCTTGGGGCTTGGA-3
GSK3β	5′-GCAGAGACAAGGATGGCA-3′	123	Yes	EU586114
	5′-ACCACACCAAATGACCCA-3′
CRIPTO	5′ TCCCAGTTTGTACCATCCAC-3′	116	Yes	TC313920
	5′-TAGAAGGAAGGAGGGCAAGC-3′
GAPDH	5′-TCGGAGTGAACGGATTTG-3′	219	Yes	AF107079
	5′-CCTGGAAGATGGTGATGG-3′

bp, base pair; PCR, polymerase chain reaction.

Comparative real-time PCR

The cDNA obtained from pooled embryos, EPIs, and EDs (as described earlier) was analyzed for the expression of OCT4a and NANOG. The housekeeping gene used was GAPDH, and the reference sample used was pooled D11/12 EDs. A total of 50 ng cDNA was loaded for each 10 μL reaction containing LightCycler^® 480 SYBR Green I mastermix along with 0.5 μM primers. Samples were performed in triplicate (Roche Diagnostics) and run using standard conditions on a Roche LightCycler 480 (Roche Diagnostics).

Statistical analyses

Kaplan–Meier survival curves were performed to analyze survival times of cultured embryos using the Prism 6.0 statistic workpackage. Statistical analyses of differences in survival curves between different treatments were performed using the Log-rank (Mantel–Cox) test, and P values were obtained using the chi-square test (df=1). Significance was achieved when P≤0.05. Fold change was evaluated from comparative real-time PCR analyses using the formula, 2^−(ΔΔCT). Error bars represented in the quantitative PCR graphs show the standard deviation.

Results

Pluripotent markers of the naive and primed state differ in porcine pluripotent stem cells

In order to evaluate the expression of varying stages of pluripotency, four stages of embryos were evaluated. cDNA was produced from RNA using a transcription amplification kit from whole pooled (n=5) D5/6 embryos (containing early ICM), pooled isolated D7/8 embryos (containing the late ICM), pooled isolated D9/10 EPIs, and pooled isolated D11/12 EDs. These stages were selected to evaluate the transient changes in expression of the blastocyst containing the early and late pluripotent ICM (D5/6 and D7/8), the pluripotent EPI (D9/10), and the pluripotent ED (D11/12). We investigated the expression of several transcription factors associated with pluripotency, including OCT4, NANOG, SOX2, KLF4, and cMYC, and found that all of these were expressed in the early D5/6 blastocyst, although expression of NANOG seemed very weak (Fig. 1A). Several of these genes became downregulated in the D7/8 blastocyst, as only weak OCT4a (the stem cell-specific isoform [39]) and cMYC were detectable at this stage (Fig. 1A). OCT4a and NANOG were the only two markers expressed in the D9/10 EPI. All the genes were upregulated again in the D11/12 ED; however, KLF4 appeared to be very weak (Fig. 1A). This indicates that pluripotency genes are expressed predominantly in the D5/6 early embryo and D11/12 ED, whereas the stages in between are devoid of many of the markers. To confirm the observed changes in expression, we performed comparative real-time PCR for OCT4a and NANOG (Fig. 1B). We observed that expression levels varied between the embryo stages, mimicking the results observed in the semiquantitative PCRs. We were surprised to see a reduction in the expression of OCT4a in the porcine D7/8 pooled blastocysts, so we performed immunocytochemical analyses at this embryonic stage. We found that expression in the TE was reduced at this stage, but remained high in the ICM (Fig. 1C). Expression of NANOG was also performed in D7/8 embryos and confirmed that protein expression was absent (Fig. 1C). We then performed immunocytochemical analyses on D5/6 and D7/8 porcine in vivo embryos and in D11/12 EDs to analyze expression of both KLF4 and cMYC (Fig. 1D). Interestingly, KLF4 protein expression was absent from D5/6 embryos, but nuclear and cytoplasmic, weak expression was detected in the late ICM and TE of D7/8 embryos. KLF4 was largely absent in the D11/12 ED but found to be cytoplasmically located in a few cells of the EPI (Fig. 1D). cMYC was found to be nuclear and cytoplasmic in both D5/6 and D7/8 embryos, but it was mosaic in expression in the D7/8 embryos (Fig. 1D). The ED showed only a few positively labeled cells, and expression of the protein was only cytoplasmic.

FIG. 1.

Expression of pluripotency genes in the developing porcine embryo. Expression of pluripotency markers in pooled whole D5/6 blastocysts, whole D7/8 blastocysts, isolated D9/10 epiblasts (EPIs), and isolated D11/12 embryonic disc epiblasts (EDs) (A). Comparative reverse transcription–polymerase chain reaction reveals varying expression of OCT4a and NANOG during preimplantation development in whole embryos and isolated EPIs and EDs (B). Expression of OCT4a is reduced in trophectoderm (TE) of D7/8 blastocysts, and NANOG is absent (C). Expression of pluripotent-associated proteins KLF4, cMYC, REX1, and NROB1 reveals distinct patterns of expression (D). A small subset of positive cells are identified and shown by white arrows for KLF4 and REX1 (D). Expression of SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 reveals that expression is absent in blastocysts (D5/6) and limited to TE in D9/10 embryos (E). Scale bars represent 100 μM. The EPIs and EDs are delineated by white dotted lines. Color images available online at www.liebertpub.com/scd

In the mouse, the EPI reflects the naive stem cell state and can be determined by the expression of specific markers, including, Rex1, Nr0b1, and Klf2 [27]; whereas primed stem cells lack the expression of these genes. We evaluated the expression of these three markers in the four different stem cell stages of development to determine when they are expressed in the porcine embryo. In this study, we found REX1 to be expressed in all stages examined (Fig. 1A). Considering this protein is found in the nucleus and cytoplasm of both the ICM and TE in the mouse [40], we performed immunocytochemistry on different staged embryos and confirmed that REX1 was weakly expressed in both the ICM and TE of D5/6 embryos, particularly in the cytoplasm (Fig. 1D). Expression could be determined in the TE of D9/10 embryos, and only weak expression was detected in the D9/10 EPI. In the ED of D11/12 embryos, the cells were largely REX1 negative with the exception of a single layer underling the EPI (not shown) and a few scattered cells throughout the ED (Fig. 1D). Cytoplasmic staining of REX1 was also observed in some regions of the ED (Fig. 1D). Weak nuclear and cytoplasmic expression was also observed in the TE of these embryos (not shown). REX1, therefore, marked only a few cells of the ED and may be considered a better marker of proliferation, which is not exclusive to the pluripotent stem cells. Next, we investigated the expression of the naive marker NROB1 (also known as DAX1). We found that gene expression was detected only in the D9/10 embryos (Fig. 1A); however, three different-sized PCR product bands could be detected in the D5/6 embryos (data not shown). We decided to perform immunocytochemistry on embryos of different stages and identified that expression of NROB1 could be found in the cytoplasm of both porcine D5/6 embryos and D9/10 embryos (Fig. 1D). Expression was found in both the ICM and TE at D5/6, whereas NrOB1 was weak in both the EPI and the TE of the D9/10 blastocysts. Expression was nuclear in the D9/10 EPI and both nuclear and cytoplasmic in the TE (Fig. 1D). Detection of NROB1 in the D5/6 blastocyst surprised us due to the lack of identifying the corresponding transcript; however, we speculate that this may be due to splice variants or isoforms that have at least been identified in human [41]. The expression of NROB1 was not detectable in D11/12 embryos. We can, therefore, conclude that NROB1 may be a useful marker for identifying the ICM and EPI pluripotent cell populations in the pig; however, this marker is not exclusive to the stem cell niche. On analyzing KLF2 expression, we found that this gene could only be determined at a weak level in the D11/12 ED and at an almost undetectable level in the EPI, which suggests that KLF2 is not strongly expressed in the pig. In sum, we can see that there was no obvious overlap in the expression of REX1, NrOB1, and KLF2. Therefore, it would be difficult to use these genes for marking the naive state in the pig. The EPI expressed both REX1 and NROB1, but the detection was weak by immunocytochemistry compared with the surrounding TE that these cannot justify use for identification of the pluripotent cells within this species.

Cell surface marker expression differs between mouse and hESC/iPSC presumably due to differences in the pluripotent state (naive vs. primed state). Expression studies in embryos, however, are limited and have only been performed in expanded and hatching mouse and human blastocysts containing the presumed ICM and/or EPI by one research group [42]. The data indicate that mESC and mouse iPSC express SSEA-1; whereas hESC and hiPSC express SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 [43]. In order to determine whether porcine embryos reflect these differences in cell surface marker expression at different stages of development, we decided to evaluate the expression of SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 in embryos collected at D5/6 containing the early ICM and D9/10 embryos containing the EPI. We have also previously investigated the expression of SSEA-1 and found it to be localized to the complete ICM, mosaic in the EPI, and absent in the ED [44]. In the present study, we found that the blastocysts (D5/6) did not express any of the cell surface markers evaluated (Fig. 1E). We found that, in addition, the porcine EPI (D9/10) did not express any of the surface markers, but all markers were detected in the surrounding TE (Fig. 1E). This appears to corroborate with the expression profile of the naive state in mESC/iPSC that express the cell surface marker, SSEA-1.

We have identified markers associated with the porcine naive and primed state that are unique to this species. The porcine early blastocyst (D5/6) expresses several markers of pluripotency. The proteins found at this stage include OCT4a, cMYC, and SSEA-1 and weak expression of NrOB1 and REX1. The expanded blastocyst (D7/8) containing the late ICM expresses OCT4a, weak KLF4, and mosaic expression of cMYC. The EPI can be characterized by the expression of OCT4a; weak expression of NANOG, SOX2, REX1, and NROB1; and mosaic expression of SSEA-1. Finally, the D11/12 primed ED can be characterized by the expression of OCT4, NANOG, and SOX2; mosaic expression of KLF4 and cMYC; and expression of a very few cells for REX1. These markers obviously differ compared with other species and are temporally up and downregulated.

MEK signaling and JAK/STAT signaling pathways are important in both the early ICM and embryonic disc

Regulation of pluripotency has remained a topic of interest in the case of porcine stem cell research, due to the difficulties in establishment of bona fide stem cells using culture media with supplementation of either LIF or bFGF [45,46]. We have previously shown that the LIFr is absent from the porcine blastocyst (D6) and EPI (D11) as well as from downstream components, including JAK1 and STAT3 [27]. Furthermore, FGFr1 and FGFr2 are active in the EPI, suggestive that FGF signaling is present in porcine pluripotent stem cells. However, further detailed analyses of these pathways are lacking and there are some conflicting reports regarding the expression of STAT3, as exogenous LIF has been shown to activate STAT in piPSC [47]. Therefore, we evaluated both pathways more thoroughly in all four embryonic stages (D5/6, D7/8, D9/10, and D11/12). First, we evaluated genes involved in FGF signaling, including FGFr1, FGFr2, MEK, and cFOS. In concordance with our previous research, we detected FGFr1 and FGFr2 expression in the D11/12 ED and only very weak expression of MEK and cFOS (Fig. 2A). The EPI (D9/10) and expanded blastocyst (D7/8) failed to express these genes, with the exception of a weak detectable cFOS band in the D7/8 blastocysts (Fig. 2A). Interestingly, both MEK and cFOS were expressed in the early D5/6 blastocysts but FGFr1 was absent and FGFr2 was barely detectable. We, therefore, show here that FGF signaling genes are present within the D5/6 blastocyst, but which receptors are important for signaling remain unclear.

FIG. 2.

Expression and regulation of FGF and leukemia inhibitory factor (LIF) signaling in both in vivo and in vitro cultured porcine embryos. Expression of genes are analyzed in pooled whole D5/6 blastocysts, whole D7/8 blastocysts, isolated D9/10 early EPI, and isolated D11/12 embryonic disc epiblasts (ED). FGF receptors (FGFRs) and downstream genes of FGF signaling are present in the ED, whereas the receptors are absent in earlier-stage embryos (A). LIF signaling in the preimplantation porcine embryo is well defined in the D11/12 ED, and components of JAK/STAT signaling are expressed in the D5/6 embryo; however, LIFr is absent (B). LIFR protein is absent in the D5/6 blastocyst but present in a small population of cells in the D11/12 ED and the TE (C). Scale bars represent 100 μM. Kaplan–Meier survival curves show that cultured D5/6 embryos and D9/10 early EPIs cannot be grown for long in cell culture without the presence of any cytokines (D). However, bFGF supports proliferation of cells to a maximum of six passages in vitro (E); whereas LIF in D5 cultured embryos and LIF in the presence of bFGF does not support extended culture in vitro (F, G). Cultured inner cell mass (ICMs) and EPIs are also unable to survive in the long term in the presence of an MEKi (PD0325901) (H) or in bFGF and an FGFR inhibitor (FGFRi) (I). Culture in the presence of a JAK/STAT inhibitor (JAK/STATi) (AUH-6-96) did not increase the length of culture in vitro (J). Supplementation of interleukin 6 (IL6) and IL6 receptor (IL6R) (K) and IL11 (L) in D5/6 and D9/10 cultures was also unable to extend culture of cells beyond passage 4 and 3, respectively. Color images available online at www.liebertpub.com/scd

In the case of JAK/STAT signaling, we found that the early blastocyst expressed members of the pathway, including GP130, JAK1, and STAT3, but were negative for LIFr (Fig. 2B). We did not see the expression of any of these genes in the late blastocyst (D7/8) and EPI (D9/10), which corroborates our previous findings [28]. We did, however, see expression of LIFr and weak expression of GP130 and STAT3 upregulated in the ED (D11/12). To investigate these findings further, we performed immunocytochemistry on the D11/12 ED and also included early D5/6 blastocysts, which have not been previously evaluated. We found that the predominant D11/12 ED did not express LIFr with the exception of a very small population of cells present (Fig. 2C). The fact that there was only a small subpopulation of cells expressing LIFr may explain the weak gene expression detected by PCR. Interestingly, this subpopulation had nuclear expression, in contrast to the plasma membrane expression observed in the TE. The early blastocyst was also negative for LIFr (Fig. 2C). This suggests that JAK/STAT signaling is present in the early blastocyst but is unlikely to be regulated by LIFr. In addition, the porcine primed ED (D11/12) may, indeed, harvest a very small population of cells that are involved in JAK/STAT signaling. These data suggest that FGF signaling may be the predominant pathway in the primed ED; however, a small subpopulation of cells also exists that express the LIFr.

To test this theory, we cultured in vivo embryos collected at different stages on MEFs in stem cell medium containing either human recombinant bFGF or mouse recombinant LIF. Whole embryos (D5 and D8) and isolated EPIs (D10) were cultured independently on inactivated MEFS and mechanically passaged every 4–5 days until these cells either stopped proliferating or differentiated. Kaplan–Meyer survival curves were generated and analyzed to determine significant differences both within and between treatments (See Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/scd). The results indicated that recombinant bFGF alone could significantly prolong the lifespan of the cultures in comparison to culture in no cytokines, LIF, or bFGF in combination with LIF (bFGF+LIF) (Fig. 2D–G). To further test whether cell proliferation, indeed, was dependent on FGF signaling and JAK/STAT signaling, we analyzed the affect of three inhibitors, an inhibitor of MEK signaling (PD0325901), an inhibitor of FGFR signaling (AZD4547), and an inhibitor of JAK/STAT signaling (AUH-6-96), in cultured embryos from D5/6 and D9/10 of development with medium containing bFGF. We found there was a trend for embryo-derived cells to arrest earlier in the presence of either the MEKi (Fig. 2F) or the JAK/STATi compared with culture with bFGF, and this arrest was significantly earlier in the case of FGFRi (Fig. 2H–J and Supplementary Table S1), indicating that these inhibitors play a role in proliferation of porcine embryo-derived cells in vitro. Considering the FGFR1 is absent and 2 is only weakly detectable in the early ICM, we can only speculate that MEK signaling via FGFRs is more important in vitro. Alternately, FGFR3 may be important, as the FGFRi AZD4547 is known to antagonize FGFRs 1–3 [48]. With regard to the lack of the effect of LIF in the embryonic cultures, but the importance of the pathway in proliferation, we speculate, therefore, that JAK/STAT signaling may be regulated, at least in the early blastocyst by another cytokine than LIFr, of which several are known. We, therefore, cultured whole D5/6 embryos and isolated D9/10 EPIs in the presence of either IL6 and IL6R or IL11. We found, however, that embryo cultures were arrested by passages 4 and 3, respectively (Fig. 2K, L), which was significantly later than with treatment with no cytokines alone, but not significantly different from culture in bFGF, indicating that triggering JAK/STAT signaling via IL6 and IL11 signaling has only a limited effect in vitro. Therefore, we have shown that both FGF signaling and JAK/STAT signaling are critical for the proliferation of ESCs; however, the cytokines bFGF, LIF, and a combination of IL6+IL6R or IL11 are unable to support the long-term proliferation of these cells.

BMP signaling but not WNT signaling is indispensible for maintaining pluripotency

Alternate cell signaling pathways to bFGF and JAK/STAT pathways, which are involved in pluripotency, have been poorly characterized in the porcine embryo and its counterpart in vitro derived lines. Therefore, we decided to analyze genes involved in both WNT and BMP signaling further in the four different embryonic stages. These pathways are known to regulate pluripotency primarily in mESCs [33,49 –51]. In the case of BMP signaling, we analyzed the expression of BMPr, SMAD1, SMAD5, and SMAD4. We detected the expression of these genes primarily in the early blastocyst (D5/6) and in the porcine ED (D11/12), with the exception of SMAD5 in D5/6 embryos (Fig. 3A). We then analyzed the expression of BMPR-1 in D5/6, D7/8, and D11/12 embryos and detected the expression of BMPR-1 (Fig. 3B). Surface membrane expression was detected in both the ICM and TE of D5/6 embryos, and expression was slightly stronger in the ICM (Fig. 3B). Expression of BMPR was weak in the TE of D7/8 embryos and slightly stronger in the late ICM (Fig. 3B). Expression was also detected in the cell membrane of cells within the ED of D11/12 embryos (Fig. 3B). This is suggestive that BMP signaling is important in both the ICM and the ED. In order to test this, we cultured D5/6 whole embryos, isolated D9/10 EPIs in the presence of a BMPi (dorsomorphin) which targets BMP type 1 receptors ALK2, ALK3, and ALK6 [52], and found that at both stages, cell proliferation was lost in early passages (Fig. 3C). We attribute that BMP signaling is, therefore, important for porcine pluripotent cell survival in vitro. We also assessed a number of genes associated with WNT signaling, namely secreted frizzled-like protein 1 (SFRP1), GSK3b, and b-catenin. SFRP1 expression was detectable only in the ED (D11/12), whereas both GSK3b and b-catenin were detectable from the EPI (D9/10) until the ED (D11/12), with the exception of a loss of GSK3b expression in the EPI (Fig. 3D). To test whether WNT signaling was important in the regulation of porcine pluripotency, we again cultured both D5/6 and D9/10 embryos in vitro in the presence of a WNTi (IWR-1). This inhibitor indirectly targets b-catenin by inducing AXIN expression, which is a member of the b-catenin destruction complex [53]. After the addition of this inhibitor, we found that the D10 EPIs had a significantly poorer outcome in their cell survival compared with the D5 embryos (Fig. 3E and Supplementary Table S1). This indicated that WNT signaling and the blockage of b-catenin signaling may be crucial for cell proliferation of porcine EPIs in vitro.

FIG. 3.

BMP and WNT gene expression and regulation in cultured in vivo porcine embryos. Expression of genes is analyzed in pooled whole D5/6 blastocysts, whole D7/8 blastocysts, isolated D9/10 early EPI, and isolated D11/12 embryonic disc epiblasts (EDs). Genes associated with BMP signaling are expressed in D5/6 embryos and the D11/12 ED (A). Expression of BMPR-1 reveals expression in the ICM and TE of D5/6 and D7/8 and in D11/12 EDs (B). Cell survival curves of individually cultured D5 embryos and D10 EPIs in the presence of a BMP inhibitor (dorsomorphin) reveal that cells can only be cultured for a short time in vitro (C). Expression of WNT genes is found in the ICM, EPI, and ED (D); however, inhibition of b-catenin by addition of the WNT inhibitor (WNTi) (IWR-1) shows that this pathway may not be crucial for cell proliferation of porcine embryonic stem cells in vitro (E). Genes associated with CRIPTO signaling are expressed in the porcine embryo, with SMAD genes detectable in both the D5/6 embryo and the D11/12 ED (F). Expression of pSMAD2 shows that this protein is active in D5/6 and D7/8 embryos, and D11/12 EDs and CRIPTO reveals weak expression exclusively in the porcine ICM, and expression in D9/10 and D11/12 embryos (G). Scale bars represent 100 μM. Cell survival curve of cultured D10 EPIs in the presence of a NEURAL inhibitor (NEURALi) (AGN193109) indicates cells can be maintained for approximately a maximum of five passages in an undifferentiated state (H). Color images available online at www.liebertpub.com/scd

NODAL/CRIPTO signaling-related proteins are expressed in the pluripotent stem cell populations of the porcine embryo

Finally, we considered addressing NODAL/CRIPTO signaling in the embryos because CRIPTO expression is found in the murine ICM, EPI, and developing mesoderm and also plays a role in early differentiation events [54]. We analyzed the expression of CRIPTO, SMAD2, and SMAD4 in the four embryonic stages. These latter genes are important for forming the SMAD2/4 complex, which is then shuttled into the nucleus. Here, we found that CRIPTO was expressed from the early blastocyst (D5/6) until the ED (D11/12) (Fig. 3F). Expression of SMAD2 and SMAD4 was also present in the early blastocyst but was down-regulated in D7/8 ICM and EPI (D9/10) and again upregulated in the ED (D11/12; Fig. 3F). This indicates that the pathway is transiently expressed across the different pluripotent cell populations. In order to gain more clarity over the localization of expression, we performed immunocytochemical staining on D5/6, D7/8, and D11/12 embryos for pSMAD2 and on D5/6, D9/10, and D11/12 embryos for CRIPTO. We identified that pSMAD2 was not only nuclear specific and upregulated in the D5/6 ICM, but also detectable in the TE (Fig. 3G). Expression of pSMAD2 was also observed in the late ICM of D7/8 embryos but again not specific and although detectable in the TE, it was more weakly expressed (Fig. 3G). We also observed nuclear expression of pSMAD2 within the ED (Fig. 3G). We discovered that weak CRIPTO nuclear and cytoplasmic expression could be observed specifically in the ICM of D5/6 embryos (Fig. 3G). Stronger cytoplasmic staining was also observed in both the EPI and TE the D9/10 embryos and the ED and TE of the D11/12 embryos (Fig. 3G). Thus, CRIPTO was expressed in the pluripotent cells of the early porcine embryo and may be considered a good marker for use in identifying porcine pluripotent stem cells in vitro. The expression profiles also indicate that this signaling pathway is likely active throughout the developing D5-D12 embryo.

Finally, to identify whether this neural differentiation may be blocked in vitro, we tested a NEURALi, specifically a retinoic acid receptor-selective retinoid (AGN193109) known to switch the neural cell fate of ESCs to the mesoderm cell fate [55] to cultured D9/10 isolated EPIs, and monitored their proliferation activity over time. This chemical is also known to indirectly modulate the NODAL/WNT signaling pathway [55]; therefore, we thought it would be interesting to evaluate it in our cultured cells. Culture of the cells in the presence of the NEURALi, however, did not significantly disrupt the culture outcomes of the EPIs (Fig. 3H). Therefore, we consider that this chemical may not be able to extend cell proliferation or prevent the differentiation outcome of the cultured cells.

Discussion

Maintenance of pluripotent stem cells in mouse and man is critically dependent on a stable culture system that is supplemented with appropriate growth factors which drive cell signaling events associated with cell renewal. Given that bona fide pESC have not yet been established and that piPSC are also not stable in culture in the absence of the reprogramming transgenes, this suggests that culture conditions are not yet optimized and may either induce differentiation events or fail to support pluripotency. Here, we provide detailed insights into the cell signaling events that regulate pluripotency in the developing porcine embryo and evaluated key signaling pathways, which are important in vitro in the establishment and proliferation of pESCs. Along with our previously published findings on the expression of OCT4, NANOG, SOX2, and SSEA-1 in the porcine embryo [28,44] and our ultrastructure studies on cell characteristics of the porcine pluripotent cells [44], we have characterized the pluripotency markers of the different stem cell populations in the developing porcine embryo and suggest two new states of pluripotency (Fig. 4).

FIG. 4.

Overview of genes and proteins expressed in the pluripotent cells of the developing embryo reveals that distinct signatures exist in the porcine embryo compared with other species. Expression profiles are based on findings from this article and our previous published expression and ultrastructural studies. The ICM of the D5/6 embryo is composed of two types of cells, the inner and the outer cells. Both have a large nuclear-to-cytoplasmic ratio and are characterized by small and immature mitochondria (depicted by blue organelles in cells). They contain lipid drops (white organelles) and display a few mitoses. They express most genes associated with pluripotency but do not translate NANOG and SOX2. The D7/8 embryo containing the late ICM expresses fewer genes than the D5/6 embryo and appears to lie in a transient reticent state of pluripotency. The early EPI of the hatched blastocyst (D7/8) lies above the hypoblast and contains larger mitochondria and fewer lipid droplets. The primed embryonic disc epiblast (ED) from the D11/12 embryo has broken through the Rauber's layer and has developed into an epithelium, which can be seen by the formation of a basal membrane and the apical/basal elongated cell form. Microvilli can be identified on the outer surface of the apical membrane, mitochondria are more mature and abundant, and many free ribosomes and abundant endoplasmic reticulum can be observed. The ED from the D11/12 embryo expresses many genes related to pluripotency, similar to the early porcine ICM. Genes are denoted as uppercase italics and proteins are indicated as uppercase. Color images available online at www.liebertpub.com/scd

Here, we report that the initial formed blastocyst containing the ICM expresses many genes associated with a naive state of pluripotency, although interestingly, not all these are translated to a degree enabling immunocytochemical detection. In many respects, the expression profile of mRNA at this state is very similar to the expression at the naive mouse stem cell state; however, translation of NANOG, SOX2, and KLF4 is not observed. We are confident that this is not due to species-specific issues with the antibodies used, as clear expression of NANOG, SOX2 using the same antibodies has previously been observed in D11/12 porcine ED [28]. Furthermore, we see expression of KLF4 in the D11/12 ED in this study. In the case of NANOG and SOX2, we were able to detect weak gene expression of NANOG and also expression of SOX2 at this stage, although translation of these two genes was not detected (shown in previous research [28]). In the mouse E3.5 embryo, protein expression of Oct4 and Sox2 is detectable in both the ICM and TE [56,57] and Nanog is expressed exclusively in the ICM [58]. The early human blastocyst also appears to mirror the expression and localization pattern of these three genes in the mouse [29,59,60]. This highlights the fact that expression of proteins related to pluripotency differs in the pig ICM and that although the transcripts are present, not all of these are translated. OCT4 and cMYC are, therefore, the only two pluripotent markers expressed both as transcripts and as proteins. A weak expression of REX1 and NROB1 was also observed, but this was not exclusive to the ICM. Our previous research shows that the ICM expresses SSEA-1 [44], and this study highlighted the absence of SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Together, these findings illustrate a premature state of pluripotency in the early formed ICM, thereby precluding an active state of pluripotency. We denote this to be an early premature state of pluripotency in the developing pig embryo, where several genes of pluripotency are not translated.

Evaluation of pluripotency signaling pathways of cultured ICMs revealed that MEK, JAK/STAT, and BMP pathways may be important for continued cell proliferation and survival of the D5/6 cultured pluripotent cells. Addition of FGF could prolong the culture of the cells in vitro, and blockage of FGFR1/2/3 using the FGFRi resulted in poor survival of the cells, indicating that FGF signaling is important in maintaining self-renewal. In the case of LIF, we found that the addition of this cytokine was unable to support the growth of these cells in the long term, either alone or in combination with bFGF (as others have also previously described [22 –24]). Along with the lack of expression of the LIFR at this stage of development, this suggests that this cytokine alone is not important for regulation of downstream STAT3. STAT3 proteins can be activated by a wide range of cytokines [61] and growth factors, typically through the JAK family of kinases, and can also be activated by cytoplasmic Src kinases [62] and by the tyrosine kinase of various TKR growth factor receptors. In our study, the addition of recombinant porcine IL6 or human IL11 improved culture conditions above that of the no-cytokine treatment; however, it failed to extend culture of the cells beyond passage 4. It is quite possible that the inhibitors may potentially impact the function of the underlying MEFS, which may influence the outcome of the results. However, there is very little that can be down about this potential effect, as we have had previous difficulties in establishing cell cultures in feeder-free conditions. Therefore, we have not yet been able to identify which cytokine or growth factor may be important for JAK/STAT regulation. Future studies are, therefore, required in order to investigate what cytokine/s may be crucial for maintaining JAK/STAT signaling in these cells.

In progression from the D5/6 embryo to the D7/8 embryo, we observed that some mRNA transcripts were either reduced in abundancy or even lost. This was the case for OCT4a, SOX2, KLF4, and cMYC. In evaluating the protein expression of the late ICM, we detected OCT4a and only weak expression of KLF4 and cMYC. The OCT4a isoform has been attributed to be responsible for maintaining ESC potency [39], so detecting a weak mRNA transcript to us was surprising; however, we observed a lower expression of OCT4a in the TE, explaining the reduction in expression in the pooled embryo cDNA. The loss of SOX2 and very weak KLF4 transcripts was also a surprising feature. Our previous research on ultrastructure of the porcine ICM has revealed that only a few ribosomes, immature mitochondria, large lipid stores, and very few mitoses can be observed in these cells [44]. Together, these findings suggest that the late porcine ICM may represent a transient, reticent state where only a few genes and proteins associated with pluripotency are expressed. This state of development may be reminiscent of a transient state of diapause, which has previously been observed in rodent, bear, mustelid, and marsupial embryos [63]; however, further studies are required to investigate this notion. What remains unknown at present is what the cell signaling cues may be in the pig that trigger the activation of the other pluripotency genes and proteins. We could see activation of CRIPTO/NODAL signaling; however, this was not exclusive the ICM. This, however, points to the potential importance of this pathway. Previous research has shown CRIPTO mRNA expression in both porcine ICM and EPI in vitro cultures [64]. Expression of Cripto in mouse embryos also appears to be analogous to the expression observed in the porcine embryos. Mouse embryos express Cripto in both the ICM and TE of the blastocyst, as well as in D6.5 EPIs and in the primitive streak within the forming mesoderm [65]. Cripto signaling appears to be important for mesodermal differentiation in the mouse [54] and can negatively regulate neural differentiation [66]. To conclude, there is downregulation of several pluripotent transcripts and lack of expression of NANOG and SOX2. We, therefore, suggest this state to be “reticent”; that is, poised to become the naive state.

In this study, we have been able to demonstrate that the porcine EPI assumes characteristics resembling the naive stem cell state in the mouse. Rex1, Nrob1, and Klf2 are some associated genes of the naive state [27]; however, we observed that only weak expression of REX1 and NROB1 and KLF2 expression was lacking. Furthermore, expression of REX1 and NROB1 was not exclusive to the EPI or to this stage of development, as we could detect weak expression of NROB1 and REX1 in the ICM and localized expression of REX1 in the ED. In the porcine naive state, we see colocalization of OCT4, NANOG, and SOX2, but interestingly, our previous research showed that these latter two pluripotent markers were only weakly expressed [28]. Whether this general weak expression is an underlying factor for the difficulties in culturing pESC is unknown, but is, nonetheless, intriguing. With regard to regulation of the porcine naive state, such as the porcine late blastocyst, we are left with very little clues as to which pathways may regulate pluripotency. We did see expression of CRIPTO in the EPI but failed to see expression of SMAD2 and SMAD4. NODAL mRNA expression, however, has previously been detected in cultured EPIs [67]. Furthermore, in Alberio et al., culture of the EPIs in an ALK5 inhibitor (which inhibits this receptor and that is involved in ACTIVIN/NODAL signaling) led to neural differentiation of cultured EPIs [67]. Further research, however, is required to determine whether CRIPTO/NODAL signaling is important in the EPI. It may be difficult, therefore, in producing porcine pluripotent cells in vitro that are naive like when we do not know how this state may be regulated. Whole transcriptome profiling of porcine EPI cells may perhaps be the next required step for determining the regulatory pathways of this state of pluripotency.

The porcine ED, on the other hand, assumes characteristics resembling the primed stem cell state in mouse; expresses OCT4, NANOG, SOX2 (previous research), and KLF2. Only a small subset of cells expressed KLF4, and cMYC expression was mosaic; however, this was the only state where all five pluripotent markers could be observed. We were surprised that both REX1 and KLF2, which are considered markers of the naive state in mouse embryos [27], are expressed at this stage. REX1 expression has been shown to be heterogenous in mESCs, which is presumed to be due to different differentiation capabilities of subpopulations in undifferentiated ESC cultures [68]. In our study, we observed some positive cells scattered throughout the ED, indicating that a subpopulation of REX1-positive cells exist at this developmental stage, which could not be definitively identified in the EPI. Further studies investigating this heterogenous population of REX1 in the porcine ED may help uncover whether these cells also hold different differentiation capabilities. In sum, we envisage that this cohort of genes may help researchers more adequately define the stem cell states of porcine pluripotent stem cells cultured in vitro.

Our study highlighted that a number of different pathways associated with regulation of pluripotency in both mouse and hESCs appear to be coexpressed in the porcine ED. Furthermore, several pathways were found to be important for culture of pESC in vitro derived from D9/10 embryos; namely the FGF, JAK/STAT, and WNT pathways. The importance of JAK/STAT signaling was particularly striking considering that only a small subpopulation of cells expressed LIFr in the ED. We have previously investigated expression of LIFr in the ED and failed to find positive cells [28]. This may be due to the fact that only D11 embryos were used for the previous study (whereas in this study we also included D12 embryos), and that these cells may be particularly rare. This small subpopulation appears to be indispensible for the establishment of pluripotent ESCs in vitro due to the lack of effect of recombinant LIF, although we cannot discount that the LIFr-negative population may be dependent on JAK/STAT signaling which is activated by another cytokine. The importance of FGF signaling via FGFR and MEK in our cell culture studies was also striking, as addition of recombinant FGF could not sustain pluripotency (also shown by others [22]). The WNT pathway seems to be particularly important for culture of D10 embryos, which were inhibited in growth by the WNTi. It may, therefore, be interesting to include agonists of the WNT pathway in culture of porcine EPIs in the future. We were unable to block neural differentiation or extend culture of the pESC with the use of the NEURALi (AGN193109), which has previously been shown to be important for a neural to mesoderm switch in hESCs [55]. Finally, we revealed that CRIPTO is expressed in the stem cell populations of the embryo as well as in pSMAD2. In the case of hESC and mouse EPI stem cells, SMAD2/Smad2 has been shown to be critical for maintaining the primed cell state [69]. Smad2 mRNA has also been ubiquitously expressed in pregastrulation embryos [70]. Further investigation into agonists/antagonists of CRIPTO/NODAL signaling will be important to verify the role of this particular pathway in regulating porcine pluripotency. Therefore, we conclude that the use of several cytokines and inhibitors simultaneously may be necessary in order to sustain proliferation. These are the studies that are currently being pursued.

In summary, this research brings forward new data regarding expression of genes and proteins in the developing porcine preimplantation embryo and insights into differences in the expression profiles of the different pluripotent cell niches. By the use of small chemical inhibitors, our study also demonstrates that FGF and JAK STAT signaling is critical for the maintenance and cell proliferation of porcine pluripotent cells in vitro and that WNT signaling is also important in the culture of EPIs. Determination of the cell signaling cascades that govern pluripotent stem cell renewal will not only aim at improving culture conditions that could result in establishment of bona fide porcine pluripotent cells, but would also significantly advance the research in this field. We have discovered that a premature state of pluripotency may exist in the early porcine ICM and that a reticent state exists in the late porcine ICM. In addition, the presumptive naive EPI expresses a few genes governing pluripotency, whereas the presumptive primed ED expresses all the proteins associated with pluripotency. Together, this research illustrates the unique status of the pig in relation to pluripotency compared with the well-studied ESC models produced from murine and human embryos. Most importantly, they provide an important benchmark for researchers in identifying the counterpart stem cell state in in vitro porcine pluripotent stem cell cultures that may help elucidate whether cultured iPSC or ESC are in a naive or primed stem cell state.

Footnotes

Acknowledgments

The authors are grateful to Gyeong-Hun Baeg (New York Medical College) for the kind donation of the chemical inhibitor AUH-6-96 (JAK/STATi); to Lawrence Lum (University of Texas) for the kind gift of the chemical inhibitor, IWR-1 (WNTi); and to Pelle Serup for the kind gift of AGN193109 (NEURALi). This research was also financially supported by the EU FP7 projects PartnErS, PIAP-GA-2008-218205, and PluriSys, HEALTH-2007-B-223485, as well as by the Danish National Advanced Technology Foundation, Pigs, and Health Project, and by the Danish Council for Independent Research, Technology, and Production Science in Denmark.

Part of the enclosed study has been included in the following published abstract: Hall VJ, Nielsen J and Hyttel P. Attempt to establish porcine embryonic stem cell lines using defined medium and cell signaling inhibitors. International Society of Stem Cell Research (ISSCR) 8th Annual Meeting, June 16–19, 2010, San Francisco, California.

Part of the enclosed study was also presented at an oral talk at the 2013 International Embryo Transfer Society conference (IETS), Hannover, Germany entitled “Early development of the porcine embryo—the importance of cell signaling in development of pluripotent cell lines.”

Author Disclosure Statement

There are no commercial associations that might create a conflict of interest in connection with this article. Vanessa Jane Hall is employed as an assistant professor at the University of Copenhagen and Poul Hyttel is employed as a professor at the University of Copenhagen. Both authors do not participate in any activities or financial activities that might add to any conflict of interest to this article. No competing financial interests exist.

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