Primed to Naive-Like Conversion of the Common Marmoset Embryonic Stem Cells

Abstract

Mammalian pluripotent stem cells are thought to exist in two states: naive and primed. Generally, unlike those in rodents, pluripotent stem cells in primates, including humans, are regarded as being in the primed pluripotent state. Recently, several groups reported the existence of naive pluripotent stem cells in humans. In this study, we report the conversion of primed state embryonic stem cells from common marmoset, a New World monkey, to the naive state using transgenes. The cells showed typical naive state features, including dome-like colony morphology, growth factor requirement, gene expression profile, X chromosome activation state, and energy metabolic status. Moreover, interspecies chimeric embryo formation ability with mouse embryos was increased in the naive state. This technique can be applied in basic medical research using nonhuman primates, such as preclinical use of naive pluripotent stem cells and generating genetically modified primates.

Introduction

Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass (ICM) of blastocyst stage embryos [1,2]. Primate and mouse ESCs exhibit distinct characteristics [3,4]. Currently, primate ESCs are considered counterparts to mouse epiblast stem cells (EpiSCs), which are derived from the postimplantation embryo [5,6]. The pluripotent state of mouse EpiSCs is known as the primed state, distinguishing it from the naive state of mouse ESCs [7]. Primed pluripotent stem cells are often problematic when they are used in differentiation experiments due to their inconsistent differentiation propensity among cell lines and different clones [8].

Naive pluripotent stem cells are thought to be the developmentally ground state and show a uniform and high differentiation ability. When introduced into early embryos, these cells can contribute to animal development and can produce chimeric animals. In contrast, although primed pluripotent stem cells can differentiate into the three germ layers and show self-renewal ability, they rarely form chimera [9]. In addition, cell features are clearly distinguished between naive and pluripotent stem cells. For instance, naive pluripotent stem cells form compact dome-like colonies, whereas primed pluripotent stem cell colonies are flat and monolayer. During growth, the self-renewal of naive pluripotent stem cells depends on leukemia inhibitory factor (LIF)/signal transducer and activator of transcription (STAT)3 signaling in contrast to primed pluripotent stem cells, which depends on fibroblast growth factor (FGF)-2 and ACTIVIN, but not LIF/STAT3 [7]. In addition, the X chromosome inactivation state in female cells is different between naive and primed pluripotent stem cells. Female naive pluripotent stem cells have two active X chromosomes, whereas primed pluripotent stem cells have an inactivated X chromosome. Since primate ESCs share the characteristics of primed pluripotency at these points mentioned above, they are regarded as primed state pluripotent stem cells.

The common marmoset, a nonhuman primate, has attracted attention as an experimental primate due to its small size, high reproductive efficiency, and genetic, as well as physiological, similarities to humans [10 –12]. In fact, genetic modification technologies, including transgenesis and genome editing, have been developed for use in common marmoset [13,14], and pluripotent stem cell lines have been established from this animal [15 –22]. Common marmoset ESCs (cjESCs) clearly show primed pluripotent characteristics similar to those of other primate pluripotent stem cells, including human ES/induced pluripotent stem cells (iPSCs) [23]. Although derivations of naive state pluripotency have been reported in humans, by several groups [24 –37], naive cjESCs (N-cjESCs) have not yet been established.

Our group recently developed a naive conversion method for human iPSCs using an original set of transgenes and culture conditions. Using ESCs from the common marmoset, we successfully converted cells from primed state to naive state pluripotency.

Materials and Methods

Animal experiments

All animal experiments were approved by The Keio University Institutional Animal Care and Use Committee or Institutional Animal Care and Use Committee of the CIEA and were carried out under the Institutional Guidelines on Animal Experimentation at Keio University and CIEA.

Cell culture and transfection

cjESC lines DSY127 and No. 40 were used in this study. Primed cjESCs (P-cjESCs) were cultured as previously reported [23]. Briefly, cjESCs were grown on irradiated ICR strain mouse embryonic fibroblasts in ESMB medium, which consists of KnockOut™ D-MEM (Thermo Fisher Scientific, Waltham, MA) supplemented with 20% Knockout Serum Replacement (KSR; Thermo Fisher Scientific), 0.1 mM MEM nonessential amino acid solution (NEAA) (Sigma-Aldrich, St. Louis, MO), 2 mM l-glutamine (Thermo Fisher Scientific), 0.1 mM β-mercaptoethanol (2-ME; Sigma-Aldrich), and 10 ng/mL basic FGF-2 (Wako Pure Chemical Industries Ltd., Osaka, Japan).

Transgene transfection was performed using Lipofectamine LTX (Thermo Fisher Scientific) as described previously [23]. The triple transgenic cell lines were obtained as shown in Fig. 1A. Briefly, the cells were selected by adding 25 μg/mL hygromycin for 12–14 days. Resistant colonies were treated with 2 μg/mL doxycycline (Dox) overnight. The next day, TdTomato-positive colonies were picked up and expanded.

FIG. 1.

Generation of naive conversion-competent cjESCs. (A) Transgene constructs and schematic representation of the establishment of triple transgenic cell lines. (B) PCR genotyping results of each transgene. No pHyPBase integration was confirmed. (C) Phase contrast and fluorescence microscopy images of TdTomato and EGFP expression of each clone. In the absence of Dox, TdTomato expression was not detected. Following Dox addition, TdTomato expression was induced. EGFP expression was constitutively expressed under control of a ubiquitous promoter (CAG promoter). Scale bar, 200 μm. cjESCs, common marmoset embryonic stem cells; Dox, doxycycline; EGFP, enhanced green fluorescent protein; PCR, polymerase chain reaction. Color figures are available online.

Naive conversion

For naive conversion of the triple transgenic cjESC lines, the cells were treated with 10 μM Y27632 (Sigma) in ESMB for 1 h. Subsequently, the cells were dissociated into single cells using 0.25% Trypsin-EDTA (Thermo Fisher Scientific). The dissociated cells were then seeded onto new mouse embryonic fibroblast feeder cells in conversion medium containing KnockOut Dulbecco's modified Eagle's medium (KO-DMEM) supplemented with 20% KSR, 1 mM l-glutamine, 1% NEAA, 0.2 mM 2-ME, 2 μg/mL Dox, 10 ng/mL human LIF (Nacalai Tesque, Kyoto, Japan), 3 μM CHIR99021 (Axon Medchem, Groningen, Netherlands), 1 μM PD0325901 (Wako), 10 μM forskolin (Sigma), and 5 μM A83–01 (Santa Cruz Biotechnology, Dallas, TX). The cells were maintained under this condition. In later studies using female cjESCs, the conversion and maintenance conditions were further optimized. NBK5 medium, which consists of a 1:1 mixture of Neurobasal medium (Thermo Fisher Scientific) and DMEM/F-12 (Wako), supplemented with 1% N2 supplement (Thermo Fisher Scientific), 2% B27 supplement (Thermo Fisher Scientific), 1 mM l-glutamine, 1% NEAA, 0.2 mM 2-ME, 2 μg/mL Dox, 10 ng/mL human LIF, 3 μM CHIR99021, 1 μM PD0325901, 10 μM forskolin, and 5 μM A83–01 was used for conversion. After 7–9 days of culture under these conditions, the concentration of CHIR99021 was decreased to 1 μM for maintenance. For transgene-independent culture, Dox was removed from the medium.

Janus kinase inhibitor treatment

For Janus kinase inhibitor (JAKi) treatment experiment, the cells were cultured in the presence or absence of 0.6 μM JAK inhibitor I (Millipore, Billerica, MA) for 6 days. The cells were collected, and RNA was extracted for quantitative reverse transcription–polymerase chain reaction (qRT-PCR). For short-term JAKi treatment, the cells were seeded onto Matrigel-coated well plates. The cells were cultured for 5 days, and then 0.6 μM JAKi was added for the indicated time.

qRT-PCR

For qRT-PCR, total RNA was isolated using an RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse-transcribed using ReverTra Ace (Toyobo, Osaka, Japan). qRT-PCR analysis was performed with SYBR Green Master Mix (Thermo Fisher Scientific) on a ViiA7 real-time PCR platform (Thermo Fisher Scientific) according to the manufacturer's instructions. GAPDH expression was used for normalization. Ct values were calculated by the relative standard curve method. The control sample is mentioned in each figure legend. The sequences of the primers used in this study are shown in Supplementary Table S1.

Immunostaining and alkaline phosphatase staining

The cells were cultured in multiwell plates or on chamber slides and fixed with 4% paraformaldehyde or 100% ethanol for 10 min at room temperature. After incubation with blocking buffer (Protein Block, Serum Free; DAKO, Glostrup, Denmark) for 30–60 min at room temperature, the cells were incubated with primary antibodies at 4°C overnight. The antibodies used in this study are listed in Supplementary Table S2. Subsequently, the cells were incubated with secondary antibodies for 30 min and then washed twice with PBS (−).

For alkaline phosphatase staining, the cells were fixed with 4% paraformaldehyde for 1 min at room temperature, washed twice with PBS (−), and stained with a SIGMAFAST BCIP/NBT Kit (Sigma-Aldrich).

Chimera formation experiment

Interspecific chimera formation with mouse embryo was performed by the 8-cell aggregation method. Briefly, 8-cell C57BL/6 mouse embryos were collected from oviducts of 2.5 days post-coitum pregnant mice, and the zona pellucida was removed using acid Tyrode solution. Two 8-cell embryos and single cell-dissociated P- or N-cjESCs were coincubated in a depression of a 35-mm culture dish made by darning needle using mWM medium at 37°C 5% CO₂. After overnight incubation, the chimeric embryos were examined by fluorescence microscopy (Olympus, Tokyo, Japan), fixed with 4% paraformaldehyde at room temperature, and washed twice with PBS (−).

For marmoset chimera formation experiments, frozen marmoset embryos were thawed and incubated before microinjection. Ten to 20 N-cjESCs were injected into the perivitelline space (morula) or blastocoel cavity (blastocyst). The embryos were cultured in Blast™ medium (Origio, Måløv, Denmark). Some embryos were fixed and immunostained with anti-NANOG and anti-enhanced green fluorescent protein (EGFP) antibodies. The remaining embryos were transferred into the uterus of the foster mother as previously described [14].

Transcriptome analysis

For cjESC transcriptome analysis, RNA extraction was performed as previously described [38]. Frozen marmoset early embryos were recovered in ISM1 medium. After removing the zona pellucida using acid Tyrode solution, ICM cells were isolated by immunosurgery except for morula stage embryos. RNA extraction, reverse transcription, library preparation, and sequencing were performed as previously described [39]. For postimplantation marmoset embryo, E25 pregnant female marmosets were anesthetized with 50 mg/kg of ketamine (Fujita Pharmaceutical, Tokyo) and 4 mg/kg xylazine (Selacter; Bayer, Leverkusen, Germany) by intramuscular injection. The anesthetized state was maintained with 1.0%–3.0% isoflurane (Pfizer, NY), while the uteri were removed. Uteri were embedded in OCT compound and immediately frozen in liquid nitrogen. The frozen uteri were then sliced to 5 μm thickness with a cryostat, and the epiblast of the implanted marmoset early embryo was isolated from the specimen by laser microdissection. RNA was extracted as described above.

Sequencing was performed using an Illumina HiSeq 2500 system (Illumina) to obtain 50- or 100-nucleotide sequences (pair-end). Poor-quality reads (score <30) were trimmed using Trim Galore! (ver.0.4.0, www.bioinformatics.babraham.ac.uk/projects/trim_galore/), and the resulting reads were aligned to the Callithrix jacchus genome (Callithrix_jacchus-3.2.1) using STAR aligner (version 2.5.3a) [40]. The mapped reads were counted using featureCounts (1.5.2) [41]. Read count normalization and differential expression of transcripts were analyzed using customized R script. RNA-seq data have been submitted in GEO database (number GSE138944).

Flux analysis

Energy metabolic profile analysis was performed using an XFe24 Analyzer (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions (XF Cell Mito Stress Test Kit and XF Glycolysis Stress Test Kit; Seahorse Bioscience, Billerica, MA). Briefly, for the XF Cell Mito Stress Test, P- and N-cjESCs were seeded onto a Matrigel-coated 24-well cell culture plate (Seahorse Bioscience) at a density of 1–2 × 10⁵ cells per well in the growth medium 20 h before the experiment. During the experiment, 2 μM oligomycin, 0.5 μM carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone, and 1 μM rotenone/antimycin were injected in turn.

For the glycolysis assay, an Agilent Seahorse XF Glycolysis Stress Test Kit was used. A total of 0.5–1 × 10⁵ cells per well of P- and N-cjESCs were plated as described above. The reagent concentrations were 10 mM glucose, 2 μM oligomycin, and 500 mM 2-deoxy-D-glucose.

Statistical analysis

All experiments were repeated at least thrice, and the data were expressed as the mean ± SEM. Data were analyzed with the two-tailed Student's t-test.

Results

Generation of naive conversion-competent cjESCs

To test the feasibility of our original naive conversion method in human induced pluripotent stem cells (hiPSCs) for cjESCs, the cells were converted to the naive state as described previously [38]. Briefly, PiggyBac transposon vectors, including pPB-Octet1, which express Dox-inducible six reprogramming factors and TdTomato and CAG-rtTAM2-iN with hyperactive PiggyBac transposase [42], were introduced into the marmoset ESC line DSY127 (Supplementary Fig. S1A). The cells were transferred into naive conversion medium containing LIF, 2i (PD0325901, CHIR99021), A83–01, forskolin, and Dox. The resulting naive-converted cells showed dome-like colony morphology similar to mouse embryonic stem cells (mESCs) and increased expression of several naive markers (Supplementary Fig. S1B, C).

Next, we conducted clonal formation analysis. For this experiment, we used pPB-Octet3, a slightly modified version of pPB-Octet1 and CAG-EGFP-iH to label the transgenic cjESCs. After transfection of the transgene set, the cells were selected by hygromycin for 12–14 days. Resistant colonies were treated with Dox. Next day, TdTomato-positive colonies were picked up and expanded under conventional conditions without Dox (Fig. 1A).

Among these triple transgenic clones, three independent clones were further analyzed (Fig. 1B). These clones showed induced expression of TdTomato after adding Dox (Fig. 1C). qRT-PCR revealed that the Dox-induced transgene expression levels varied among clones (Supplementary Fig. S2). Clone #2 showed the highest expression, clone #4 expressed low levels of transgene, and clone #1 showed medium expression.

Naive conversion of the cjESCs

Next, to induce reprogramming, clones #1, #2, and #4 were treated with Dox in medium containing 2i, A83–01, forskolin, and LIF (Fig. 2A). The resulting cells formed compact dome-like colonies (Fig. 2B). After several passages, we examined the gene expression profile of these cells by qRT-PCR (Fig. 2C). Based on primed ESC versus marmoset ICM cell microarray data (data not shown) and references using mouse pluripotent stem cells, we defined ESRRB, DPPA3, and KLF5 as naive marker genes. In addition, LEFTY1 and LEFTY2 were used as primed marker genes, and NANOG, OCT3/4, and SOX2 were used as pluripotency marker genes.

FIG. 2.

Naive conversion of cjESCs. (A) Schematic representation of naive conversion. (B) Phase contrast and fluorescent microscopy images of TdTomato and EGFP expression of resulting naive-converted cjESCs. Scale bar, 200 μm. (C) Gene expression profiles of P- and N-cjESCs. Relative expression levels to wild-type P-cjESCs are shown (n = 3; mean ± SEM; independent experiments). Normalization was performed using GAPDH expression. P-cjESCs, primed cjESCs; N-cjESCs, naive cjESCs. Color figures are available online.

After induction, the cells showed upregulation of the naive marker genes, whereas the primed marker genes were downregulated in three independent clones compared to under conventional culture conditions. Pluripotency marker genes were maintained or moderately increased.

Three germ layer differentiation potential of N-cjESCs

To evaluate whether the cells from clones #1, #2, and #4 retained their differentiation capacity as pluripotent stem cells, the cells were transferred to suspension culture in the absence of Dox, LIF, and small molecules. As a result, the cells spontaneously formed embryoid bodies (EBs; Supplementary Fig. S3A). qRT-PCR analysis revealed that the EBs were composed of three germ layers (Supplementary Fig. S3B). When the naive-converted cjESCs (clone #2) were injected into immunodeficient mice, they formed a teratoma composed of three germ layer derivatives (Supplementary Fig. S3C). Therefore, the cells retained their pluripotency.

LIF/STAT3 signal dependence in N-cjESCs

Dependency on LIF/STAT3 signaling in self-renewal is one of the major characteristics of naive pluripotent stem cells. To test the LIF/STAT3 dependency of the cells, the effect adding 0.6 μM JAKi in culture was examined (Fig. 3A). Six days after culture in the presence of JAKi, some naive marker genes and pluripotency genes were downregulated, indicating that the LIF/STAT3 signal is required for self-renewal of the cells. Furthermore, KLF4 and TFCP2L1, which are direct STAT3 targets that play an important role in maintaining naive pluripotency in mouse ESCs [43 –45], were also significantly decreased.

FIG. 3.

Effect of JAKi treatment on N-cjESCs. (A) Gene expression analysis of N-cjESCs in the presence and absence of JAKi by qRT-PCR. JAKi (−) sample is used as calibrator sample (n = 3; mean ± SEM; independent experiments; *P < 0.05; **P < 0.01; as determined by Student's t-tests). (B) Schematic diagram showing short-term JAKi treatment. (C) Effects of short-term JAKi treatment on gene expression of N-cjESCs. Wild-type P-cjESCs (0 h) are used as calibrator sample (n = 3; mean ± SEM; independent experiments). Endo, endogenous; JAKi, Janus kinase inhibitor; qRT-PCR, quantitative reverse transcription–polymerase chain reaction. Color figures are available online.

To clarify whether KLF4 and TFCP2L1 downregulation is due to the direct effect of STAT3 inhibition or indirect effect of loss of the naive state, short-term JAKi treatment experiments were carried out (Fig. 3B, C). At this time point, the naive state was maintained, as the expression of DPPA3, a naive marker, did not change. KLF4 and TFCP2L1 were decreased within 24 h in the naive state. In contrast, the expression of these genes was not affected in the conventional primed state. These results suggest that the mESC-like downstream circuit of STAT3 was formed in the naive state, but not in the primed state.

Transgene-independent maintenance of the naive state in cjESCs

Next, we examined the dependence on transgene expression in N-cjESCs. In this system, minimal transgene expression was detected in the absence of Dox. After removal of Dox, the colony morphology was maintained (Supplementary Fig. S4A, B). qRT-PCR analysis revealed that the gene expression profile was largely maintained (Supplementary Fig. S4C). However, endogenous SOX2 and NANOG were increased by Dox removal. As both genes are included in the transgenes, endogenous gene expression is thought to be upregulated to compensate for the loss of transgene-derived transcripts to maintain the naive state pluripotency. The N-cjESCs were converted to the primed state when they were transferred to primed culture conditions in the absence of Dox (Supplementary Fig. S5A, B).

ICM incorporation ability of N- and P-cjESCs

To test the interspecies chimera formation ability of the N-cjESCs, we carried out an aggregation chimera formation assay using N- and P-cjESCs with mouse 8-cell embryos (Fig. 4A). As a result, for two of three clones tested, N-cjESCs incorporated mouse ICM efficiently compared to the primed counterparts (Fig. 4B–D). In contrast, clone #4, the clone which showed the lowest level of transgene expression, did not show an increased ICM incorporation efficiency (Fig. 4C). This suggests that high transgene expression is required for sufficient naive conversion in terms of interspecies chimera formation ability, whereas other characteristics did not show obvious differences.

FIG. 4.

ICM incorporation assay. (A) Scheme of mouse-marmoset interspecies chimera generation experiment. Primed or naive EGFP-expressing cjESCs were aggregated with mouse 8-cell embryos. (B) EGFP-positive N-cjESCs were efficiently incorporated into the ICM of blastocyst stage mouse embryos. (C) Bars represent ICM incorporation efficiency determined in three independent experiments. (D) Confocal fluorescent microscopy image of the EGFP and OCT3/4 double immunostained chimeric embryo. Scale bar, 50 μm. (E) Summary of ICM incorporation assay. ICM, inner cell mass. Color figures are available online.

Establishment of naive conversion-competent female cjESC line

Next, to confirm the reproducibility of the conversion method in another cjESC line, we reestablished triple transgenic cell lines using the female cjESC line No. 40. We selected clone Ex29.3 showing the highest transgene expression (Fig. 5A). To optimize the culture conditions, we switched the basal medium to N2B27 from KO-DMEM (Fig. 5B). This change enabled stable growth while maintaining surface marker (Fig. 5C) and pluripotency-related gene expression (Fig. 5D) in the absence of Dox. As the new cell line was derived from female marmoset ESCs, the X chromosome activation state was examined by trimethylated H3K27 (H3K27me3) immunostaining (Fig. 5E). As a result, the typical condensed spot-like signal indicating inactivated X chromosome in nucleus disappeared in the naive state but not in the primed state. Furthermore, since the change in localization of TFE3, a bHLH transcription factor, is regarded as an early event of naive–primed transition, we examined the localization of TFE3 in both P- and N-cjESCs [46]. While TFE3 was exclusively expressed in the cytoplasm of P-cjESCs, the N-cjESCs expressed TFE3 in both the nucleus and cytoplasm (Fig. 5F).

FIG. 5.

Establishment of naive conversion-competent female cjESC line. (A) Phase contrast and fluorescence microscopy images of EGFP and Dox-inducible TdTomato expression of the highest transgene expression clone derived from female cjESC line. Phase contrast (left panel), TdTomato (red; middle panel), EGFP (green; right panel). Scale bar, 100 μm. (B) Phase contrast images of naive-converted cjESCs in KO-DMEM basal medium (KSR base; upper left) and under optimized conditions (N2B27 base; upper right). Bottom panel shows colony morphology (left) and AP staining (right) of N-cjESCs maintained under the optimized condition in the absence of Dox. Scale bar, 200 μm. (C) Immunostaining of pluripotency markers. Scale bar, 100 μm. (D) Gene expression profiles of naive state cjESCs in the presence or absence of Dox [passage number (P) 2–5]. Relative expression levels to wild-type P-cjESCs are shown. Data indicate the means of three technical replicates. (E) Immunostaining of trimethylated H3K27 of P- and N-cjESCs. Scale bar, 20 μm. (F) TFE3 subcellular localization of P- and N-cjESCs. Scale bar, 20 μm. AP, alkaline phosphatase; KO-DMEM, KnockOut Dulbecco's modified Eagle's medium. Color figures are available online.

Next, we examined the chimera formation ability of N-cjESCs. For this, N-cjESCs were injected into the marmoset morula or blastocyst stage embryo. Forty-eight hours after injection, the EGFP signal was observed in the marmoset ICM (Supplementary Fig. S6A). To explore the possible contribution of N-cjESCs to further embryonic development, chimeric blastocysts were transferred into foster mothers. At E60, we obtained three normally developed embryos. However, an EGFP signal was not observed in these embryos, indicating that the N-cjESC-derived cells were eliminated at this stage (Supplementary Fig. S6B, C).

Transcriptome analysis of naive and primed state of marmoset ESCs

To elucidate the developmental stage of naive state marmoset ESCs, we carried out transcriptome analysis of pre- and postimplantation stage marmoset embryos, as well as naive and primed state marmoset ESCs. As shown in Fig. 6A, a heatmap generated using stem cell-related genes indicated that N-cjESCs and preimplantation embryo epiblasts were clearly distinguished from P-cjESCs and the epiblast of postimplantation marmoset embryos. Using a set of differentially expressed genes among each developmental stage of the marmoset embryos (13,393 genes), we performed principle component analysis (Fig. 6B). As a result, the developmental timescale was observed on the principal component 1 axis. Naive and primed state marmoset ESCs formed distinct clusters and the N-cjESCs were plotted in the vicinity of the hatched blastocyst epiblast, whereas P-cjESCs were plotted nearby the epiblast of postimplanted embryos.

FIG. 6.

Transcriptome analysis and flux analysis. (A) Heatmap of the samples using a GO term stem cell population maintenance. (B) PCA analysis of marmoset early stage embryos, as well as P- and N-cjESCs. (C) Mitochondria functional analysis using a flux analyzer. OCR (pmol/min). (D) Glycolysis activity of P- and N-cjESCs determined using a flux analyzer. ECAR (mpH/min). (n = 3; mean ± SEM; independent experiments; *P < 0.05; **P < 0.01; ***P < 0.001, Student's t-tests). ECAR, extracellular acidification rate; GO, gene ontology; OCR, oxygen consumption rate. Color figures are available online.

As the switching of energy metabolism (increased mitochondrial oxidative phosphorylation in naive state) is regarded as a characteristic of naive conversion, we next examined the difference in energy metabolism between P- and N-cjESCs. We performed functional assessment of energy metabolism using a metabolic flux analysis system (Seahorse XF Analyzer) (Fig. 6C, D). The N-cjESCs showed a significantly increased spare respiratory capacity and significantly decreased glycolysis and glycolytic capacity compared to P-cjESCs. These results indicate that the N-cjESCs acquired a naive-type energy metabolic status.

Discussion

In this study, we established a method for converting cjESCs from the pluripotent state to naive state. The resulting naive state cjESCs formed a mouse ESC-like colony morphology and acquired naive state phenotypes, including their gene expression profile, LIF responsiveness, energy metabolic profile, and increased incorporation ability to mouse early embryo ICM. Honda et al. proposed an evaluation method to discriminate stem cell status using cynomolgus monkey PSCs [47], which includes colony morphology, naive-related gene expression, LIF dependency, and mitochondrial respiration. In addition to their criteria, we explored global gene expression profile by RNA-seq and identified the position in the developmental timescale by comparison with pre- and postimplantation embryos. We showed that the N-cjESCs are close to ICM of preimplantation embryo, compared with P-cjESCs.

We used the naive conversion method for human iPSCs using transgenes [38]. Using this method, the resulting N-cjESCs expressed naive marker genes, including ESRRB, which plays several essential roles in murine naive pluripotency and reprogramming. Although the importance of ESRRB in primate naive pluripotency remains unclear, cjESCs also exhibited increased expression, which is consistent with our previous study using hiPSCs [38].

As in mouse and human naive pluripotent stem cells, N-cjESCs acquired LIF dependency. Since the expression of both KLF4 and TFCP2L1, which are direct targets of Stat3 in the self-renewal in naive mouse ESCs, was decreased soon after inhibition of STAT3 with a JAKi, LIF/STAT3-dependent transcriptional circuitry can be formed in N-cjESCs similar to in mouse ESCs.

Generally, naive pluripotency in mice is thought to be maintained by specific transcriptional circuitry [41]. Two extrinsic pathways are known to activate the circuit in mouse pluripotent stem cells: LIF/STAT3 signaling and Wnt/β-catenin signaling. LIF/STAT3 signaling promotes self-renewal by activating components of the core transcriptional circuit, such as KLF4 and TFCP2L1, to maintain the pluripotent state. In contrast, Wnt/β-catenin signaling inhibits one downstream target TCF3, which functions as a transcriptional suppresser [48]. By inhibiting GSK3β, degradation of β-catenin is inhibited and then intracellular β-catenin accumulates. This accumulation suppresses TCF3 activity and induces expression of TCF3 target genes. Among the TCF3 targets, ESRRB is pivotal in maintaining the naive pluripotent state in mice [49]. Although our results suggest that LIF/STAT3 signaling has a similar function in N-cjESCs, it remains unclear whether Wnt/β-catenin signaling has conserved functions in primate naive pluripotency. Additional studies are needed to understand how naive pluripotency is maintained in primate and other species.

We demonstrated that naive-converted cjESCs, but not P-cjESCs, can contribute to the ICM of mouse embryos. Interestingly, clone #4 did not show increased ICM incorporation ability, despite the observation that other phenotypes were indistinguishable from those of other clones. This clone showed the lowest transgene expression level among those investigated, suggesting that high transgene expression is required for sufficient naive conversion. In addition, we attempted to generate chimera animals using marmoset embryos, but have not yet obtained chimeric offspring. For efficient chimera animal production, stage matching of host embryo developmental stage and pluripotent state of the cells is required [50]. In our experiment, morula and blastocyst stage embryos were used as hosts. In addition to improvement of the naive conversion protocol, optimization of chimera formation methods is important to generate chimeric marmosets.

Recent advances in transgenesis and genome editing technology have enabled genetic modification, even in nonhuman primates. However, complicated genetic modifications, such as spatiotemporal control of gene expression using Cre/LoxP or FLP/FRT systems or Dox-inducible transgenesis, remain difficult. Therefore, it is important to develop genetic modification techniques using pluripotent stem cells, as well as to understand naive and primed pluripotency from a basic research perspective. In addition to its application in the generation of genetically modified animals, further studies are important for therapeutic modeling in the common marmoset, which is used as a model animal for preclinical studies. Our approach can be applied in preclinical studies of stem cell therapy using naive pluripotent stem cells.

Footnotes

Acknowledgments

The authors thank all members of the Okano laboratory for their generous support. The piggyBac transposase expression vector was kindly provided by Dr. Kosuke Yusa (Wellcome Trust Sanger Institute). The common marmoset ESC line DSY127 is a gift from Sumitomo Dainippon Pharma Co., Ltd. The authors thank Mr. Akihiro Matsushima and Mr. Manabu Ishii for their assistance with the IT infrastructure for the data analysis. The authors are also grateful to all members of the Laboratory for Bioinformatics Research, RIKEN Center for Biosystems Dynamics Research, for their helpful advice.

Author Disclosure Statement

H.O. is a compensated scientific consultant of SanBio, Co., Ltd. and K-Pharma Inc. All other authors declare no conflicts of interest.

Funding Information

Specific results from this study were from the “Construction of System for Spread of Primate Model Animals,” which was performed under the Strategic Research Program for Brain Sciences of the MEXT, and the AMED (to H.O and S. S), Scientific Research in Innovative Areas, which is the MEXT Grant-in-Aid Project FY2014–2018: “Brain Protein Aging and Dementia Control” (to H.O.) and Program for the Advancement of Research in Core Projects in the Longevity Initiative at Keio University Global Research Institute from Keio University. This work was supported by the Projects for Technological Development, Research Center Network for Realization of Regenerative Medicine by Japan, AMED (to I.N.).

Supplementary Material

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

Supplementary Figure S5

Supplementary Figure S6

Supplementary Table S1

Supplementary Table S2

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