To delve into whether the extra chromosome 21 affects the maintenance of pluripotency of DS hiPSCs, we primarily checked the cell morphology of DS hiPSCs and normal hiPSCs. The results showed that the DS hiPSCs exhibited pluripotent stem cells' typical characteristics with a large nucleus and compact clone (Figure 1A). A strong positive expression for alkaline phosphatase staining (Figure 1B) and the results of cell immunofluorescence assays (Figure 1C) showed no significant difference in cell morphology and surface marker expression between DS hiPSCs and normal hiPSCs. Overall, these results indicated that the maintenance of pluripotency of DS hiPSCs was similar to that of normal hiPSCs and was not significantly affected by the redundant chromosome 21.

As it is known that trisomy 21 causes alterations to both stem and precursor cells (Liu et al., 2015), it is also possible that the alteration of the proliferative ability of DS hiPSCs is caused by the differences in expression of genes and the aberrant AS events. To determine if this is the case, we used the GeneChip Human Transcriptome Array 2.0 (HTA 2.0) for the global gene analysis with hiPSCs from DS and healthy individuals. We examined differentially expressed genes (DEGs) in these groups focusing on specific transcripts with AS events. The quality control analyses of the HTA 2.0 data highlight the correct segregation of samples from each cell line (Figure 2).

A more detailed analysis of HTA 2.0 datasets revealed, the total differentially expressed genes are 466 up-regulated and 722 down-regulated genes (in total 1,188 significantly differentially expressed genes) in DS hiPSCs, compared with normal hiPSCs (Figure 3). By summarizing the distribution of differentially expressed genes on each chromosome and the proportion of coding genes in the chromosome, we found that the proportion of up-regulated genes on chromosome 21 (5.20%) was significantly higher than that on the other chromosomes (0–1.79%), which showed a gene dosage effect of the genes on chromosome 21. This result is consistent with previous studies of DS somatic cells that demonstrated that the redundant chromosome 21 leads to gene dosage effects (Moldrich et al., 2007; Nawa et al., 2019).

Tables 1 and 2 list the top 10 up-regulated and down-regulated genes in DS hiPSCs sorted by the P-value, respectively. The up-regulated H1-6 is a member of the histone H1 family. The linker histone, H1, interacts with linker DNA between nucleosomes and functions in chromatin compaction into higher-order structures. The down-regulated H3C11 and H4C13 are members of the histone H3 and H4 families, respectively, which are essential nuclear proteins responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. The up- and down-regulation of these genes may affect the compaction of chromatin and the higher-order structures of chromosomes in DS hiPSCs, leading to the instability of chromosomes and alternative modifications of histones resulting in the aberrant regulation of gene expression. DYRK1A, a gene coding for a kinase known to regulate splicing factors that maps to chromosome 21 (Qian et al., 2011), was identified as an up-regulated gene in DS hiPSCs. The aberrant expression of this regulator of splicing factors may lead to splicing changes in the trisomic cells.

We tested multiple splicing algorithms to identify differences in splicing between DS and normal hiPSCs and chose EventPointer (see Methods). Using this algorithm with the filter criteria: Exon Splicing Index > 2 or < −2 and Exon P-value < 0.01, we identified 1,862 annotated genes with splicing changes when comparing DS with normal hiPSCs, half of which are the Cassette Exon events and more than one third are Alternative 5′ Donor Site events and the Alternative 3′ Acceptor Site (Figure 4A).

To explore the effect of splicing changes on the DS hiPSCs, we performed the gene ontology (GO) and KEGG signaling pathway analysis on all selected genes with alternative splicing events (Figure 4B). The results showed that 1,593 genes (87.8% of 1,862 annotated genes with splicing changes) are enriched in 188 biological processes, while 1,675 genes (92.3%) were enriched in 84 cellular components. We observed enrichment for 1,601 genes (88.2%) in 75 molecular functions, as well as 707 genes (39%) in 49 KEGG signaling pathways. The top 10 enrichment items are listed in Figure 4B. Fifty-six genes with splicing changes are enriched in the cell division process; this might be the driving force behind the phenomenon that the proliferative ability of DS hiPSCs and normal hiPSCs is different.

Figure 5 demonstrates the visualization of three genes as examples of splicing changes, namely the gene RPL39L (Ribosomal Protein L39 Like, OMIM: 607547), PARP2 (Poly(ADP-Ribose) Polymerase 2, OMIM: 607725), and FN1 (Fibronectin 1, OMIM: 135600).

To explore the effect of differentially expressed genes on the DS hiPSCs, we further performed GO functional enrichment and KEGG signaling pathway analysis of 466 up-regulated and 722 down-regulated genes, respectively. The results show that 466 up-regulated genes are enriched in 64 biological processes, 34 cellular components, 18 molecular functions, and 10 KEGG signaling pathways. The 722 down-regulated genes are enriched in 50 biological processes, 27 cellular components, 16 molecular functions, and 8 KEGG signaling pathways. The top 10 enrichment items in up- and down-regulated genes are listed in Tables 3 and 4, respectively. Eight up-regulated genes were significantly enriched in the negative regulation of growth, which may be related to somatic cells' lower proliferative ability and stem cells from patients with DS (Kimura et al., 2005). Overexpression of genes related to negative growth regulation may inhibit cell proliferation, suggesting that the proliferation of DS hiPSCs may also be impaired. Nine up-regulated genes are significantly enriched in proteins targeting the mitochondria, which may affect ATP synthesis by damaging mitochondrial function, resulting in the inhibition of select cell functions (Valenti et al., 2018). Fifteen up-regulated genes were significantly enriched in nucleosome assembly, implying that the redundant chromosome 21 may increase chromosome synthesis and assembly stress. Twenty-seven down-regulated genes were significantly enriched in cell adhesion. The impaired cell adhesion ability affects cell growth and neural cell migration during embryonic development in DS patients (Huo et al., 2018). In nervous system development there were 17 down-regulated genes that were enriched, suggesting that the molecular regulation might be abnormal before the differentiation of DS neurocytes (Liu et al., 2015). Moreover, in focal adhesion there were 16 down-regulated genes that were significantly enriched. This is consistent with the enrichment in cell adhesion of the GO analysis. Another 16 down-regulated genes were enriched in the PI3K Akt signaling pathway. The hyperactivity of this pathway promotes carcinogenesis (Yang et al., 2019). The silencing of this signaling pathway in DS hiPSCs may be the critical cause of the low incidence of solid tumors in DS patients. The disorder of the whole-genome expression profile indicates that other biological characteristics of DS hiPSCs, such as proliferation and cell adhesion, are also affected.

To test the reliability of gene expression microarray results, several genes were selected and were validated by real time qPCR. The relative quantifications of the expression of these genes in DS hiPSCs and normal hiPSCs are represented in Figure 6.

In compare with normal hiPSCs, DS hiPSCs exhibited a 2.1-fold increase of H1-6 (Figure 6A), a linker histone, which interacts with DNA between nucleosomes and plays an important role in the compaction of chromatin into higher order structures. In DS hiPSCs, the expressions of FN1 (Figure 6B) and H4C13 (Figure 6C) were reduced 4- and 2.5-fold, respectively. The expression of the gene RPL13 (Figure 6D) was not significant changed in DS hiPSCs in comparison with the normal hiPSCs. All of these results are consistent with the DEG analysis of the microarray dataset.

DS hiPSC (ATCC® ACS-1003™) and normal hiPSC (ATCC® ACS-1011™) were from the ATCC (American Type Culture Collection) cell bank. HiPSCs were cultured using Gibco's StemFlex™ medium (ThermoFisher Scientific, USA). The petri dishes were pre-treated with Matrigel, and the medium was changed daily. After culturing for 4–5 days, the iPSCs were digested, and the suspended cell clusters were collected and subcultured at a ratio of 1:6 at 37°C, 5% CO₂ (Aalders et al., 2019).

After removing the culture medium, hiPSCs were washed twice with PBS, adding blocking solution (PBS solution containing 0.6% BSA) within 30 min, a membrane breaker (PBS solution containing 0.02% Triton X-100) was added to break the cell membrane. The diluted primary antibody (1:250) [TRA1-60 primary antibody (ThermoFisher Scientific, USA) and LIN28A primary antibody (Cell Signaling, USA)] were added and incubated at 4°C overnight. The cells were washed twice with PBS the next day and then incubated with the fluorescent secondary antibody (1:500) [FITC-labeled goat anti-rabbit IgG (H + L), Cy3 labeled goat anti-mouse IgG (H + L)], respectively, at room temperature, avoiding light for 2 h. Finally, the anti-fluorescence quenching mount solution (containing DAPI) was added and incubated at room temperature for 10 min before the cells were observed under a fluorescence microscope (Weltner et al., 2018).

According to the instructions of the BCIP/NBT alkaline phosphatase staining kit's manufacturer (Beyotime Biotechnology, Shanghai, China), the protocol involved mixing 3 mL of alkaline phosphatase staining buffer with 10 μL of BCIP solution (300×) and 20 μL of NBT solution (150×) to prepare the working solution. Cells in 6-well plate were washed twice with PBS, then an appropriate amount of working solution was added to a single well of the 6-well plate while avoiding light exposure for 5–30 min until the color displayed to the expected depth. The working solution was then removed, and the cells were washed twice with distilled water to stop coloring. Before the photographs were taken, the stained cells were dried at room temperature and keep away from light.

DS hiPSC and normal hiPSC were seeded in a 6-well plate, each cultured for 3 days, and then digested to harvest about 5 × 10⁵ cells. The experiment was repeated three times. The total RNA was extracted with TRIzol™ reagent (ThermoFisher Scientific, USA) and was first detected by the Agilent 2100 bioanalyzer with the Agilent RNA 6000 Nano Kit (Agilent Technologies, Waldbronn, Germany) according to the instruction of the manufacture. Total RNAs with an RNA integrity index (RIN) >7.0 were used for subsequent experiments.

By using WT Amplification Kit Module 1 and WT Amplification Kit Module 2 in the GeneChip™ WT PLUS Kit (ThermoFisher Scientific, USA), 100 ng of total RNA as input was performed in vitro transcription (IVT) to synthesize cRNA, and then reverse transcription and purification to obtain single-stranded cDNA (sscDNA). The GeneChip™ WT end labeling kit was used to fragment and label sscDNA, which was hybridized with the human transcriptome array (HTA 2.0), and the hybridization signals were detected on a chip scanner to obtain CEL files for DS and normal hiPSCs.

The SuperScript™ IV reverse transcriptase (Invitrogen™, USA) was used for the synthesis of first stand cDNA from the isolated RNA. 1 μg of total RNAs were reverse transcribed according to the manufacturer's instructions. cDNA was real time polymerase chain (PCR) amplified in a LightCycler® 96 System (Roche, USA) using the FastStart Essential DNA Green Master (Roche, USA). The LightCycler® 96 SW 1.1 software was used for raw data collection and gene expression comparisons (2^−ΔΔCT method). The R packages “Rmisc” and “multcomp” are used for the data analysis and “ggplot2” for the visualization of the results.