H1 hESCs were kindly provided by Dr. Donghui Zhang (Hubei University, Wuhan, China), and the peripheral blood mononuclear cells (PBMCs)-derived hiPSCs were purchased from CELLAPY Bio (Beijing, China). Cells were cultured using the mTeSR™ 1 Complete Kit (STEMCELL, 85,850) in a shaking incubator set at 80rpm, at 37°C with 5% CO2. The hPL-treated group was supplemented with 0.3% hPL (SETXON, PL-NH-100). Passaging is performed every three to 4 days. To passage the hPSCs, cell aggregates were collected, centrifuged at 100 g for 3 min, incubated with ACCUTASE (STEMCELL, 07,920) at 37°C, and dissociated into single cells. Cells were then centrifuged at 300 g for 5 min, and resuspended in mTeSR™ 1 medium with 10 μM Y-27632 (Sellseck, S1049) in the presence or absence of hPL. Cells were seeded at the density of 10^5 cells/mL in 6 well cell culture plates. Daily half medium change was performed until passaging.

Cell viability was determined with the CCK-8 assay. One day after passaging, hPL-treated and hPL-free cell aggregates were collected and centrifuge at 100 g for 3 min. Cells were then resuspended in culture medium containing 10% CCK-8 (Solarbio, CA1210-100) and incubated at 37°C for 4 h. Absorbance at 450 nm was then measured.

10^6 single cells were collected, washed twice with 1 mL pre-cooled PBS, and then resuspended in 300 μL PBS. After that, 700 μL pre-cooled absolute ethanol was added to fix the cells at 4°C overnight. The next day, cells were washed with PBS, and incubated with 250 μL PI staining solution (containing RNase) at room temperature in the dark for 15 min. Then, flow cytometry analysis was performed on LSRFortessa X-20 (BD Biosciences).

Cell aggregates were dissociated with ACCUTASE (STEMCELL, 07,920), wash once, resuspended in 450 uL FACS buffer (PBS with 1% FBS and 8 μM EDTA). Then 100ul FACS buffer containing antibodies was added to each sample and incubated on ice for 20 min. After incubation, cells were washed twice, and resuspended in 200 ul FACS buffer containing DAPI. Flow cytometry was performed on LSRFortessa X-20 (BD Biosciences), and all flow cytometry data were analyzed with FlowJo (Tree Star).

To analyze the stem cell markers of the cultured cells, we used the following antibodies purchased from Biolegend: FITC anti-human SSEA-4 Antibody; PE anti-human TRA-1-60 Antibody; FITC Mouse IgG3, Isotype Ctrl Antibody; PE Mouse IgM, Isotype Ctrl Antibody.

The cells were cultured to the 10th passage and then sent to Feifan Testing Co., Ltd, for chromosome G-banding analysis.

The cultured cells were evaluated for their trilineage (ectoderm, mesoderm, and endoderm) differentiation potential utilizing the STEMdiff™ Trilineage Differentiation Kit (STEMCELL, 05230). Cells from both the fourth and 10th passages were chosen for the analysis, following the manufacturer’s instructions.

5000 cells were transferred to one well of a 24-well plate pre-coated with Matrigel (Corning, 354277). After 2–3 days, cells were fixed with 4% paraformaldehyde at 4°C for 10 min, washed twice with PBS, permeabilized with 0.1% Triton X-100 for 10 min and blocked with PBS containing 1% FBS for 30 min. The cells were then incubated with primary antibodies added at a 500-fold dilution at 4°C overnight. After several washes with PBS, cells were incubated with secondary antibodies added at a 500-fold dilution at room temperature in the dark for 1 h. Cells were finally incubated with DAPI at room temperature for 10 min, and observed under a fluorescence microscope.

To analyze the stem cell markers of the cultured cells, we used the following primary antibodies (all purchased from Abcam): Anti-SOX2 antibody (ab92494); Anti-OCT4 antibody (ab181557); Anti-TRA-1-60 antibody (ab16288); Anti-SSEA4 antibody (ab16287); Anti-NANOG antibody (ab109250). Secondary antibodies were purchased from Thermo Fisher Scientific: Donkey anti-Mouse IgG (A32744); Donkey anti-Rabbit IgG (A32754); Goat anti-Mouse IgG (A32723); Goat anti-Rabbit IgG (A32731).

RNA was extracted from the fourth generation of cell aggregates and sent to Suzhou GENEWIZ Technology Co., Ltd for RNA-sequencing analysis. QC assessment was performed by FastQC (V0.10.1). Different expression gene analysis was performed by DESeq2 (V1.6.3). GO enrichment analysis was performed by TopGO (V2.18.0).

The RNA-seq dataset generated during this study has been deposited to NCBI Trace Archive NCBI Sequence Read Archive, this data can be found here: https://www.ncbi.nlm.nih.gov/sra/PRJNA1019100.

Data are presented as the mean plus or minus standard deviation. The significance of differences between groups was determined by a two-tailed unpaired t-test. p-values <0.05 were considered statistically significant. Statistical analysis was performed with GraphPad Prism 9.

To evaluate the effect of hPL on hPSC suspension culture, we supplemented hPL in the 3D culture system. We observed substantial proliferation benefit in hPL-treated H1 hESCs (Figures 1A,B) and PBMC-derived hiPSCs (Supplementary Figures S1A,B) throughout the 3D culture process, with as low as 0.3% hPL supplementation. Interestingly, while PBMC-derived hiPSCs showed normal expansion and passaging phenotype in both conditions (Supplementary Figures S1A–C), H1 hESCs grown in the absence of hPL exhibited noticeable morphological changes as early as the fifth generation (after being passaged five times in the 3D culture system), with a reduction in the diameter of cell aggregates (Figures 1A, E). By the sixth generation, no cell aggregates were formed in the hPL-free culture system. However, in the hPL-supplemented culture system, H1 cells were able to be passaged normally and form normal-size cell aggregates even up to the 20th generation (Figure 1A).

We also monitored the viable cell number and cell viability at each passage and found that the supplementation of hPL in H1 hESC culture increased the viable cell number by over 60% comparing to hPL-free group and maintained cell viability above 90%. In contrast, the viability of H1 cells cultured without hPL gradually declined to below 50% (Figures 1B,C). hiPSCs cultured with hPL also showed increased cell number although the viability is similar to the culture condition without hPL (Supplementary Figures S1B,C). In addition, hPL supplementary was also able to maintain higher than 90% viability for both hESCs and hiPSCs following the freeze/thaw cycle (Supplementary Figure S1D).

We compared the viability and cell cycle phase distribution between the hPL-treated H1 at the 10th generation and untreated H1 cells at fourth generation by CCK8 assay. Our findings revealed that hPL-treated H1 exhibited higher cell viability and an increased number of cells in the G2 and M phases, suggesting heightened cellular division activity (Figures 1D, F).

These results suggest that hPL supplement increased the proliferation and viability of hPSCs in the 3D culture system.

To investigate whether hPL treatment affected the pluripotency of hPSCs, we performed flow cytometry analysis, immunofluorescence staining, and trilineage differentiation assay in hPL-treated H1 cells. Remarkably, more than 98% of hPL-treated H1 cells at the 10th passage stained positive for the cell surface markers SSEA-4 and TRA-1-60 and transcription factors NANOG and OCT4 (Figures 2A,B), suggesting that these cells maintained the undifferentiated state in aggregated culture. Furthermore, the trilineage differentiation assay demonstrated the hPL-treated H1 at the 10th generation retained the capability to differentiate into cells of all three germ layers (Figure 2C). In addition, these cells exhibited normal diploid karyotype even after extended culture (Figure 2D). Similar findings were observed for hiPSCs (Supplementary Figures S1F–G).

Taken together, these findings indicate that the effectiveness of hPL supplementation within the 3D culture environment in preserving the characteristic features of hPSCs, validating the viability of incorporating hPL as a supplement within the hPSC culture system.

In our pursuit of understanding the mechanism associated with hPL-driven proliferation in hPSCs within the 3D culture setup, we conducted RNA-seq analysis to capture the transcriptome profiles of hPL-treated and control H1 cells under the 3D culture system at the fourth generation.

Principal component analysis (PCA) revealed distinct clustering and discernible group formations for hPL-treated cells in the principal component space, whereas the distribution of hPL-free cells was more scattered (Figure 3A). This highlights that hPL supplementation mitigated variances across different cell batches.

Differences in the expression profile between untreated H1 and hPL-treated H1 are shown in the heat map of whole-genome gene expression (Figure 3B). Bioinformatics analysis revealed that 364 transcripts were upregulated (p < 0.05 and FC ≥ 2) and 575 transcripts were downregulated (p < 0.05 and FC ≤ 2) in hPL-treated hPSCs compared to hPL-free hPSCs (Figures 3C,D). Furthermore, we subjected the upregulated and downregulated genes to Gene Ontology (GO) analysis containing three sub-ontologies (BP, biological process; MF, molecular function; CC, cellular component) (Supplementary Figure S2). Interestingly, these upregulated genes were involved in several important biologic processes including positive regulation of gene expression and stem cell proliferation (Figure 3E). This finding aligns with our earlier experimental results, reaffirming that hPL bolsters hPSC proliferation within the 3D environment. Additionally, we found that a majority of genes associated with cyclins and cyclin-dependent kinases (CDKs) showed higher expression in hPL-treated cells. This observation suggests that hPL-treated cells under 3D culture system enhanced its cell cycle regulatory mechanisms, thereby enabling cells to undergo division and proliferation more efficiently, which is also consistent with our previous findings (Figure 3F).

Taken together, the analysis of transcriptome profile suggests that hPL-treated cells exhibit reduced intercellular variability. Additionally, the supplementation of hPL enhances cell cycle regulatory mechanisms, thereby promoting proliferation.