Different transcriptional profiles of human embryonic stem cells grown in a feeder-free culture system and on human foreskin fibroblast feeder layers

The culture systems maintain the pluripotency of hESCs

Alkaline phosphatase staining (Figure 1A), karyotype analysis (Figure 1B) and immunofluorescence staining with pluripotent markers (OCT4, Nanog, TRA-1-60 and TRA-1-181) (Figure 2) were used to characterize 3 hESC cell lines (NF4, NF5 and P096) derived from human pre-implanted embryos. The above hESC lines were positive in both AP and immunofluorescence staining, indicating that they had the characteristics of hESCs. The results of karyotype analysis showed that these hESCs exhibited normal karyotype patterns. To illustrate the differentiation potential of NF4, NF5 and P096 in feeder-free culture systems or on HFF feeder layers, teratoma formation was performed in vivo, meanwhile H & E staining of teratoma sections indicated that the NF4, NF5 and P096 cells could generate teratoma containing trilineage tissues (Figure 3).

The profiles of mRNA and lncRNA expression

Next, we performed a gene profiling analysis as well as mRNA and lncRNA microarrays for these three hESC lines cultured in a feeder-free culture system or on HFF feeder layers. The changes in the expression were defined significant when the fold change was ≥ 2.0 with a P value ≤0.05. The results are summarized in Table 1. The mRNA and lncRNA expression data was then clustered using Cluster 3.0. Using dendrogram-based methods, the samples were further clustered into two subgroups through hierarchical clustering based on similar expression patterns. The results indicated that the expression of these genes and lncRNA was significantly different in 2 different culture systems. A total of 8 genes (ABI3BP, LEFTY2, LEFTY1, KLF4, FGF4, DUSP10, MFAP5 and CABP7) were upregulated and 7 others (SUPT3H, LRAT, SLC4A5, ADAMTS4, GCNT4, ENST00000418165 and ENST00000503568) were downregulated in the feeder-free culture system compared to those on HFF feeder layers (Figure 4). Also, compared with HFF feeder layers, 6 lncRNA (ENSG00000250337.1, ENSG00000250337.1, uc.332-, XLOC_004323 and XLOC_008995) were upregulated in a feeder-free culture system, while 17 others (ENSG00000224750.2, ENSG00000172965.7, ENSG00000245526.3, ENSG00000268364.1, ENSG00000259116.1, LOC100996319, ENSG00000236373.1, DQ266889, ENSG00000272033.1, ENSG00000255506.1, ENSG00000248329.1, ENSG00000268873.1, BC069804, uc.356-, ASO1967 and ENSG00000237993.1) were downregulated in a feeder-free culture system (Figure 5). Based on the genomic relationship between the lncRNAs and the protein-coding genes, lncRNA was classified as intergenic, sense, antisense, intronic and divergent lncRNA. 23 differently-expressed lncRNA was classified as 10 intergenic, 7 antisense, 3 intronic and 3 unknown lncRNAs.

Gene ontology and pathway analysis

A GO analysis was performed to investigate the biological processes, cellular components and molecular functions of those differentially-expressed genes. Compared with the hESCs grown on the HFF feeder layers, the differentially-expressed genes in the top 7 GO terms during the biological process were those involved in response to growth factor stimulus (P=6.165E-05), organ morphogenesis (P=0.0004), transforming growth factor-beta (P=0.0007), cell growth (P= 0.0007), stem cell maintenance (P=0.0009), inter-male aggressive behavior (P=0.0006) and the negative regulation of chemokine (C-X-C motif) ligand 2 production (P=0.0006) (Figure 6).

Moreover, a KEGG pathway analysis performed with genes differentially expressed in differently-cultured stem cells showed the enrichment of gene categories. These target genes were significantly enriched in 3 different pathways (Table 2), of which the ‘TGF-beta signaling pathway’ was the most significant (P= 0.002). Next, correlated expression networks were constructed to explore the potential relationship between the differentially-expressed lncRNA and genes (Figure 7A). Uc.356-, ENST00000504246.1, uc003iol.3 and ENST00000607611.1 were identified as the node attributes of the networks, which correlated with the expression of LEFTY2, ABI3BP, MFAP5, KLF4 and FGF4 genes (Figure 7B).

Validation of array-based gene expression profiles through real-time RT-PCR

To validate the changes mentioned above in gene expression obtained through microarray analyses, we selected 3 core pluripotent transcript factors (SOX2, Nanog and OCT4) and 6 differentially-expressed genes (FGF4, LEFTY2, KLF4, MFAP5, LRAT and GCNT4) for qRT-PCR validation. As is shown in Figure 8, 3 core pluripotent transcript factors showed no significant differences in the expression of the feeder-free culture system or the HFF feeder layers. The RT-qPCR confirmed the changes in the expression of those 6 genes (FGF4, LEFTY2, KLF4, MFAP5, LRAT and GCNT4) observed in the microarray analyses.

Derivation and culture of hESCs

The hESCs derived from human pre-implanted embryos and HFF cells were performed in compliance with the protocols approved by the Reproductive Study Ethics Committee of Nanfang Hospital, Southern Medical University (Research License AF/SC-08/03.2). Embryos were obtained from the donor couples undergoing in vitro fertilization (IVF) at the Center of Reproductive Medicine of Nanfang Hospital. Informed consent forms were signed for the participation of those couples in this study.

The blastocysts discarded in a routine IVF program were used to derive the hESC lines. The inner cell mass (ICM) was mechanically isolated with an insulin syringe needle through a stereoscope, which was then cultured on HFF feeder layers that had been mitotically inactivated with 10 μg/ml mitomycin C (M4287, Sigma, Beijing, China). HFFs were isolated from children’s foreskin after circumcision. The procedure was approved by the Reproductive Study Ethics Committee of Southern Medical University Nanfang Hospital (Research License AF/SC-08/03.2). An informed consent from the legal guardians was obtained with signed forms. Briefly, the foreskin was cut into small pieces and digested with 0.25% trypsin (Invitrogen, Beijing, China). After digestion, cells were placed into tissue culture flasks in 90% Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, China) supplied with 10% HS and 1% penicillin–streptomycin (PS; HyClone Laboratories, Inc., Logan, UT, USA). The medium was changed daily. HFFs were passaged through trypsin at a split ratio of 1:6 every 2-3 days.

The primary hESC colonies were sliced into equal-sized pieces and removed from the dish mechanically with a syringe needle through a stereoscope. The cell clusters were then transferred into new dishes containing fresh feeder cells. The hESC colonies were passed to new dishes every 4-5 days. The culture medium of hESCs was mTeSR1 (Stem Cell Technologies, Vancouver, Canada), which was replenished daily. At ten passages, 3 hESC lines (NF4, NF5 and P096) were chosen to be cultured in a feeder-dependent (HFF feeder layer) and a feeder-free culture system (matrigel-coated dish) with mTeSR1 as the culture medium (Stem Cell Technologies, Vancouver, Canada). The cells were maintained in a 37° C incubator with 5% CO₂.

Alkaline phosphatase staining, karyotype analysis and immunofluorescence staining

AP staining, karyotype analysis and immunofluorescence staining were performed on hESCs with a passage between 6 and 8. AP staining was carried out using an AP detection kit (Millipore, Beijing, China) according to the manufacturer’s protocol. Briefly, cells were fixed in 4% paraformaldehyde for 20 min at room temperature. After washing thrice with 1×PBS, cells were treated in the ALP buffer for 5 min and stained in BCIP (5-bromo-4-chloro-3-indolyl-phosphate) as well as NBT (nitro-blue-tetrazolium) for another 15 min. The images of AP staining were captured with a Zeiss microscope.

For karyotype analysis, cells were first treated with 0.2 μg/ml colcemid (Gibco Invitrogen, Beijing, China) for 130 min. The hESC colonies were mechanically removed from the feeder layers through a stereoscope after washing twice in DMEM/F12. The cells were incubated in 0.4% kalium chloratum for 20 min at 37° C and then fixed in methanol: acetic acid (3:1, v/v) for 3 min, followed by Giemsa staining. 20 cells per group were randomly selected for the karyotype analysis.

For immunofluorescence staining, cells were washed in 1×PBS (Gibco) twice before and after being fixed in 4% paraformaldehyde for 20 min at room temperature. Cells were then permeabilized in 0.3% triton X-100 (Sigma, Beijing, China) for 1 h, followed by a block in 10% goat serum in PBS. After then, cells were incubated with primary antibodies of interest (anti-OCT4 antibody, CST; anti-Nanog antibody, CST; anti-TRA-1-60 antibody, CST; anti-TRA-1-181 antibody, CST) at 4° C overnight, followed by another incubation with second antibodies (Goat anti-Mouse, Alexa Fluor 568, Abcam) at room temperature for 1 h. DAPI was used for nuclear staining. Immunostaining images were captured with a Leica DMI6000B microscope (Leica Microsystems GmbH, Germany).

Teratoma formation assay

About 3×10⁶ hESCs were suspended in 25% matrigel with DMEM/F12 (1:3), and then injected subcutaneously into immunodeficient nude mice, which were humanely sacrificed 8 weeks after injection. The teratomas were excised and processed for hematoxylin-eosin staining (H & E). Tissues were fixed in 4% paraformaldehyde for 24 h, followed by dehydration in gradient ethanol (70%, 85%, 95% and 100%) and paraffin embedding. For H&E staining, 6 micrometer sections were deparaffinized twice in xylene (each for 5 min), rehydrated in gradient ethanol (100%, 90% and 70%), stained with hematoxylin for 4 min, washed in ddH₂O for 5 min, treated with 1.5% hydrochloric acid-75% ethanol for 10 s and washed in ddH₂O for 5 min, which was followed by PBS for 2 min, they were then stained with eosin for about 20 s and dehydrated in gradient ethanol as well as xylene, after which they were placed in xylene and neutral resin mounting medium. All animal protocols were approved by the ethics committee at Hainan Medical University.

Sample collection and RNA extraction

The hESCs on a HFF feeder layer with a passage of 3 (totally 13 passages) were mechanically removed from feeder layers through a stereoscope after washing twice in DMEM/F12, after which they were collected concurrently in a trizol (Invitrogen, Beijing, China) and stored at -80° C. Total RNA was extracted from the frozen trizol block and purified using NucleoSpin RNA clean-up (Macherey-Nagel, Duren, Germany). The concentration and purity of RNA were determined by a NanoDrop ND-2000 spectrophotometer (NanoDrop Technologies, Thermo Scientifics, USA), and its integrity was evaluated through formaldehyde denaturing gel electrophoresis. Only the RNA samples with a total RNA quantity ≥ 1 μg and an A260/ 280 ratio ≥ 2.0 showing clear bands of 28 s as well as 18 s rRNA with a brightness ratio of near 2:1 in the formaldehyde denaturing gel were used for the microarray analysis.

Microarray and analysis of gene expression

A total 200 ng of RNA was amplified and reversely transcribed to double-stranded cDNA and subsequently labelled through fluorescent dye (Cy3) according to the manufacturer’s protocol for CapitalBio cDNA Amplification and Labeling Kit (CapitalBio, Beijing, China). The labelled cDNA was then purified and hybridized to Agilent Human lncRNA + mRNA Array V3.0 (Agilent, USA). Images were scanned with an Agilent microarray scanner (G2565CA) and analyzed using Agilent feature extraction (v10.7). The raw data was summarized and normalized using GeneSpring (Agilent V12.0). After completing quantile normalization of the raw data, differentially expressed lncRNA and genes were identified using threshold values of ≥2- and ≤−2-fold changes and a Benjamini-Hochberg corrected P value of ≤0.05. Next, the Molecular Annotation System (CB-MAS) V3.0 (http://bioinfo.capitalbio.com/mas3/) was used to perform gene ontology and pathway analysis. The GO terms consisted of three families: biological process (BP), cellular component (CC) and molecular function (MF).

In addition, signaling pathway analysis of these genes was performed according to KEGG PATHWAY Database (http://www.kegg.jp/kegg/pathway.html), which provided information about the significant enrichment of genes with differential expression. Finally, the differentially-expressed lncRNA and genes with a Pearson correlation coefficient no. < 0.99 were chosen to draw a co-expression network. The target prediction was performed based on their close correlations (the minimum Pearson’s correlation coefficient was -0.7) to a group of differentially-expressed protein-coding genes.

Real-time PCR

The total RNA of these 3 hESC lines was isolated using a RNeasy mini kit (QIAGEN, Beijing, China) and was reversely transcribed using a reverse transcription system kit (Promega, USA) in accordance with the manufacturer’s instructions. Real-time PCR was performed with the oligos for the selected genes with Brilliant III Ultra-Fast qPCR Master Mix (Agilent Technologies, USA). The real-time PCR conditions are shown below: 95° C for 2 min; 30s at 95° C and 1 min at 60° C with a total of 45 cycles; melt curve stage (95° C for 15 s, 55° C for 1 min and 90° C for30 s). GAPDH was used as an internal control, and the 2^−ΔΔCT method was used to calculate the mRNA fold changes (feeder-free cultured hESCs/feeder-dependent cultured hESCs). The primers used in this study are listed in Table 3.

Statistical analysis

Results were presented as mean ± SD with at least 3 biological repeats using a GraphPad prism. Significance level was determined by unpaired two-tailed students’ t tests, and P< 0.05 was considered statistically significant.