Marmoset iPSCs were established according to the methods in Dr Peter Hornsby's laboratory (Department of Physiology and The Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, TX 78245, USA) and maintained in our laboratory in the Department of Biochemistry and Molecular Biology, College of Life Science, Zhejiang Sci-Tech University (Hangzhou, China). All animal procedures, husbandry and housing were in accordance with the Animal Care and Use Committee requirements of Southwest National Primate Research Center (San Antonio, TX, USA (7). The cells were cultured in 5% CO₂ with 95% humidity at 37°C in human ESC medium TeSR-E8 (cat. no. 05840; StemCell Technologies, Inc., Vancouver, BC, Canada) supplemented with 10% ESC qualified FBS (cat no. SH30406.02E; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml of penicillin, 100 mg/ml of streptomycin (cat. no. 15070-063; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 ng/ml of bFGF (cat. no. F0291; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 10 µM Rho-associated kinase (ROCK) inhibitor (Y-27632, cat no. Y0503-1MG, Sigma-Aldrich; Merck KGaA) on Matrigel-coated dishes. The iPSCs were passaged when they reached 80–90% confluency. For passaging, the cells were incubated with Accutase (cat no. 07920, StemCell Technologies, Inc.) to a single-cell mass and replated on Matrigel-coated dishes according to the appropriate split ratio. The medium was replaced daily. To investigate the effect of LIF, the treatment group was supplemented with 1,000 U/ml of mouse LIF (cat no. ESG1106; EMD Millipore), whereas the control cells were cultured in the absence of LIF.

An MTT assay (Sigma-Aldrich; Merck KGaA) was performed to assess cell proliferation. Briefly, the cells were seeded onto 96-well plates at a density of 5×10³. After 24 h, 20 µl of MTT solution [0.5 mg/ml dissolved in phosphate-buffered saline (PBS)] was added to each test well, comprising the LIF group, the absence of LIF control group and a blank control group. The cells were then incubated for 3 h at 37°C. The produced formazan precipitate was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA). The absorbance was measured at 495 nm using a multi-mode microplate reader. The optical density (OD) values represented the proliferation or the survival of the marmoset iPSCs. Each experiment was repeated three times, followed by calculation of the standard error of the mean.

Cell apoptosis was measured according to the manufacturer's protocol with the FITC Annexin V Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, the cells were harvested by Accutase, washed twice with PBS and then resuspended in binding buffer. Subsequently, the cells were incubated with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) for 15 min at 37°C. The samples were analyzed using the FACSAria system (BD Biosciences). Cell apoptosis was analyzed using FlowJo version 10 software (BD Biosciences).

The marmoset iPSCs were seeded in a 35-cm² cell culture dish (~10⁴ cells) and cultured in the presence or absence of LIF (100 U/ml) at 37°C, 5% CO₂ for 24 h. The samples were treated in triplicate. For the DGE analysis (Beijing Genomics Institute, Shenzhen, China), total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was treated with DNase I, and poly-(A) mRNA was enriched with magnetic oligo (dT) beads. The RNA quality and quantity were verified using an Agilent 2100 Bioanalyzer and the ABI StepOnePlus Real-Time PCR system. The mRNA was mixed with fragmentation buffer (10X Fragmentation Reagent, Ambion; Thermo Fisher Scientific, Inc.; cat. no. AM8740) to create short fragments. These were used as templates for first-strand cDNA synthesis with random hexamer primers.

Second-strand cDNA was synthesized according to the manufacturer's protocol using a reaction system of buffer, dNTPs, RNaseH and DNA polymerase I included in the SuperScript^® Double-Stranded cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. 11917-010). Short fragments were purified with the QiaQuick PCR extraction kit (Qiagen GmbH, Hilden, Germany; cat. no. 28104) and resolved with EB buffer (Qiagen GmbH; cat. no. 19086) for end reparation and poly (A) addition. These were then connected with adapters and suitable fragments were selected as templates for PCR amplification by using Platinum™ Pfx DNA Polymerase (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. 11708013), to create the final cDNA library. The following thermocycling conditions were used for the PCR: 94°C for 2 min; 13 cycles of 94°C for 15 sec, 55°C for 30 sec, 68°C for 30 sec; and final extension at 68°C for 5 min. Primer sequences were as follows: Forward, 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGA-3; reverse, 5-CAAGCAGAAGACGGCATACGAGAT-3.

The fragments were purified by agarose gel electrophoresis and enriched by PCR amplification. The library products were then ready for solexa sequencing via the Illumina HiSeq 2000 sequencing platform (21), followed by bioinformatics analysis. The transcriptome sequence was assembled into distinct contigs using the short reads with SOAPdenovo software (version 2.21; http://soap.genomics.org.cn). The genome sequence of marmoset was obtained from the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov) database. GO term annotation (molecular function, biological process and cellular component) and enrichment analysis was conducted using the Blast2GO software (version 2.2.5) (22).

Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. For RT-qPCR analysis, 1 µg of RNA was used for cDNA synthesis in a reverse transcription reaction using the Prime Script Reagent kit (Takara Bio, Inc., Otsu, Japan). The reaction was incubated at 37°C for 15 min anf at 85°C for 5 sec and then amplified by using gene specific primers for qPCR. The qPCR analysis was set up in duplicate with SYBR Premix Ex Taq (Takara Bio, Inc.) and performed using the 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR reactions of 20 µl for each sample were made and consisted of cDNA (200 µg cDNA after dilution), 2X SYBR Premix Ex Taq, 50X ROX Reference Dye, dH₂O and 10 µM of each gene specific primer. After initial denaturation at 95°C for 30 sec, cDNA was amplified in 40 cycles. Amplification conditions were: 5 sec of denaturation at 95°C, 34 sec of annealing at 60°C and 30 sec of extension at 72°C, followed by a final extension step at 72°C for 10 min. Using the 2^−ΔΔCq method (23), the analysis of each sample was repeated three times, and β-actin was used as the housekeeping gene for internal normalization (24). The primers were designed using Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA). The sequences of the primers are listed in Table I.

The cells were washed twice in PBS, collected, lysed and subsequently boiled in loading buffer at 100°C for 10 min. Protein concentration was determined by the Quick Start Bradford Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal quantities of total protein (20 µg) were separated on a 12% SDS-PAGE gel at 120 V for 1 h and subsequently blotted onto a nitrocellulose membrane (EMD Millipore) at 100 V for 75 min at 4°C. The membranes were then blocked in 5% nonfat dried milk in PBST [10 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.1% Tween-20] at room temperature for 1 h according to the antibody manufacturer's protocol. Following a brief wash in PBST (0.1% Tween 20 in PBS), the membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies included anti-β-actin (cat. no. bs-0061R, BIOSS, Beijing, China), anti-PI3K (85α) antibody (cat. no. 60225-1-Ig; Full), anti-AKT (cat. no. 9272; Cell Signaling Technology, Danvers. MA, USA), anti-p-AKT (cat. no. 9271; Cell Signaling Technology), anti-STAT3 (cat. no. 10253-2-AP; ProteinTech, Rosemont, IL, USA), anti-Tbx-3 (cat. no. 16741-1-AP; ProteinTech) and anti-NANOG (cat. no. 14295-1-AP; ProteinTech) primary antibodies at a dilution of 1:1,000. The immunoreactive bands were then incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary anti-rabbit IgG-HRP antibodies (cat. no. A0208; 1:1,000; Beyotime Institute of Biotechnology, Haimen, China), or anti-mouse IgG-HRP antibodies (1:1,000, cat. no. A0216; Beyotime Institute of Biotechnology) for 1 h at room temperature. The protein bands were visualized using enhanced chemiluminescence (ECL) reagent (Amersham; GE Healthcare Life Sciences, Piscataway, NJ, USA) and images were captured by the ECL Tanon 5500 system (Tanon Science and Technology Co., Ltd., Shanghai, China).

LY294002, a synthetic selective inhibitor of PI3K, was dissolved in DMSO at a concentration of 10 mg/ml based on the manufacturer's protocol. To confirm the involvement of PI3K/Akt signaling on the effects of LIF on marmoset iPSCs, 2×10⁶ cells were treated with the indicated concentration (20 µM) of LY294002 or an equal volume of DMSO and cultured for 24 h at 37°C. Cell proliferation assays and western blot analysis were then performed, as described above.

The data are presented as the mean ± standard deviation of triplicate experiments (n=3). Student's t-test (two-tailed unpaired) was used to evaluate the significant differences between the control and treated groups. All graphs were plotted using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

In the present study, the effects of LIF on maintaining marmoset iPSCs were investigated. First, the morphology and proliferation of marmoset iPSC clones were compared when cultured in media containing different growth factor combinations either in the presence of LIF (LIF⁺/bFGF⁺) or in the absence of LIF (LIF⁻/bFGF⁺) for 24 h. The morphology of the marmoset iPSCs remained unchanged, whereas the cloning efficiency of the marmoset iPSCs was visibly increased in the presence of LIF (Fig. 1A). To further characterize the ability of LIF to support the proliferation of marmoset iPSCs, an MTT assay was performed. The results suggested that LIF significantly increased the cell viability ratio, compared with that in the control (P<0.01; Fig. 1B).

Subsequently, the effects of LIF on cell apoptosis were evaluated. To assess cell apoptosis, the samples were subjected to flow cytometric analysis. The results showed that the apoptotic rate in the treatment group was 4.81±1.51% and that in the control group was 4.25±1.01%. No significant difference was found in the cell apoptotic rate between the treatment group and control group (Fig. 2A and B). These results revealed that the presence of LIF had no effect on apoptosis.

The present study analyzed the differences between the stages of pluripotency in the presence and absence of LIF through DGE analysis. Two DGE libraries were constructed from the control group and LIF-treated group using Solexa technology. Of the total tags, 99.42 and 99.40% of the tags in each group, respectively, were clean tags, and were used for the analysis (Fig. 3A). The results suggested that the sequences were sufficiently reliable to meet the requirements for the experiment. A total of 15,421 genes were shared between the two groups (Fig. 3B). In the common set of genes, a total of 459 differentially expressed candidate genes were detected between the two groups, among which 151 genes were upregulated and 308 genes were downregulated (Fig. 4A and B). To determine the functions of these differentially expressed genes in inducing proliferation of marmoset iPSCs, a functional enrichment analysis was performed, including all of the differentially expressed pluripotency-associated genes. The 1.0-fold upregulated genes in the samples were defined as significantly differentially expressed genes or regulator genes following the addition of LIF. The analyses indicated that the expression levels of Tbx-3 and PI3K were significantly higher in the LIF-treated group, compared with those in the control group (Table II). The expression levels of PI3K and Tbx-3 were significantly increased, whereas those of others, including NANOG and SOX2, did not show a significant increase. To confirm the DGE results, RT-qPCR analysis was performed to confirm the expression levels of pluripotency-associated genes. The results showed a marked upregulation in the expression of Tbx-3 and PI3K (P<0.01; Fig. 5), therefore, the results were generally consistent with the DGE data.

As LIF has previously been demonstrated to integrate the Jak/Stat3 and PI3K/Akt signaling pathways to maintain the pluripotency and survival of mESCs (15,16,18,25,26), it is reasonable to hypothesize that one or both of these signaling processes may be key in the activation of pluripotency-associated genes to maintain marmoset iPSCs. To confirm this hypothesis, the cells were treated with or without LIF for 24 h, and the protein levels of STAT3, PI3K, AKT, p-AKT, Tbx-3 and NANOG were assessed by western blot analysis. Compared with the control group, treatment with LIF resulted in a significant increase in the levels of p-AKT and Tbx-3, whereas minimal change in the level of STAT3 was observed (Fig. 6), suggesting that the LIF-induced upregulation of Tbx-3 was potentially mediated by the PI3K/Akt pathway, but not the Jak/Stat3 pathway.

To confirm the above hypothesis, the present study analyzed marmoset iPSCs cultured in the presence of LY294002, a PI3K inhibitor. The results of the cell proliferation assay demonstrated that inhibition of the PI3K/Akt pathway significantly suppressed cell viability, even in the presence of LIF (P<0.01; Fig. 7A). Additionally, western blot analysis was performed to detect the levels of p-AKT and Tbx-3. The results revealed that the upregulation of p-AKT and Tbx-3 in marmoset iPSCs by LIF was significantly impaired following culture of the marmoset iPSCs in the presence of LY294002 (Fig. 7B). Taken together, these results indicated that LIF promoted the expression of the pluripotency-associated gene Tbx-3 by activating the PI3K/Akt signaling pathway (Fig. 8).