Refined and benchmarked homemade media for cost-effective, weekend-free human pluripotent stem cell culture

Abstract

Background: Cost-effective, practical, and reproducible culture of human pluripotent stem cells (hPSCs) is required for basic and translational research. Basal 8 (B8) has emerged as a cost-effective solution for weekend-free and chemically-defined hPSC culture. However, the requirement to home-produce some recombinant growth factors for B8 can hinder access and reproducibility. Moreover, we found the published B8 formulation suboptimal in widely-used normoxic hPSC culture. Lastly, the performance of B8 in functional applications such as genome editing or organoid differentiation required systematic evaluation.

Methods: We formulated B8 with commercially available, growth factors and adjusted its composition to support normoxic culture of WTC11 human induced pluripotent stem cell line. We compared this formulation (B8+) with commercial Essential 8 (cE8) and a home-made, weekend-free E8 formulation (hE8). We measured pluripotency marker expression and cell cycle by flow cytometry, and investigated the transcriptional profiles by bulk and single-cell RNA sequencing. We further assessed genomic stability, genome editing efficiency, single-cell cloning, and differentiation in both monolayer and organoids. Finally, we validated key findings using male (H1) and female (H9) human embryonic stem cells.

Results: hE8 performed comparably to cE8 across most functional assays and cell lines. In contrast, cells in B8+ displayed higher NANOG expression and improved genome editing efficiency. At the same time, B8+ led to gene expression changes indicative of marked lineage priming, reflected in altered morphology and differential response to some differentiation protocols. Both weekend-free media resulted in a modest transcriptional shift towards a less metabolically active state, consistent with intermittent media starvation.

Conclusions: Homemade weekend-free media can provide a cost-effective alternative to commercial formulations. hE8, integrating some features of B8 while resembling cE8, emerges as a robust and practical option with limited compromises. B8+, though advantageous in some contexts, warrants caution due to lineage priming effects that may impact differentiation outcomes.

Keywords: hiPSC; pluripotency; culture media; thermostable FGF2; TGF beta.

Figure 1.. Evaluation of TGF-β and FGF2 sources and concentrations in B8 media.

( A– B) Variants of FGF2-G3 ( A) or TGF-β ( B) activity assay using a serum response element luciferase reporter assay in transfected HEK293T cells. Medium pre-incubated in 37 °C for 2 days was compared to the fresh medium. Firefly luciferase activity was normalised to control Renilla luciferase activity. n = 3 experiments. ( C) Schematic of B8-based media formulations with different types of FGF2 (standard versus thermostable [G3]; full length versus truncated [t]) and TGF-β-superfamily growth factor. ( D– E) Representative flow cytometry data of OCT4 and NANOG expression in hiPSCs grown in media described in panel C ( D; no data could be acquired for condition 3), or in media with 1 ng/mL TGF-β3 and the indicated concentrations of the truncated or full-length FGF2-G3 ( E).

Figure 2.. Characterization of hiPSCs adapted to weekend-free media.

( A) Final concentrations of components in the supplements for two weekend-free media compositions (both based on DMEM/F12). ( B) Weekly schedule of hiPSC culture in three different media. Days highlighted in colour involve media changes. ( C) Phase-contrast images of representative hiPSC colonies 72 h after passage. Scale bar: 200 µm. ( D) Representative flow cytometry analyses of OCT4 and NANOG expression in hiPSCs grown in different media. ( E– F) Flow cytometry quantifications of the percentage of NANOG ⁺ OCT4 ⁺ cells ( E) and the normalised median fluorescence intensity value of NANOG and OCT4 ( F). Median values were normalised through division by the median intensity value of the respective isotype control. n = 3 independent adaptations of the cells into the media. Statistical analyses by one-way ANOVA followed by Dunnet’s multiple comparisons test. ( G) Representative flow cytometry cell cycle analyses in EdU-treated cells stained for DNA content. ( H– I) Flow cytometry quantifications of the percentage of cells in G1 ( H) or S ( I) phase. Circle and triangle represent 2 independent media adaptations, while the filled/empty symbols represent 2 repetitions of the experiment. Statistical analyses by mixed effects model followed by Dunnet’s multiple comparisons test. ( J) Copy number variation (CNV) analysis of cells after 20 passages in the media. Colored bands represent the number of copies of detected CNVs (or loss of heterogeneity - LOH). n = 2 independent media adaptations.

Figure 3.. Effects of weekend-free media on gene expression.

( A– B) Pearson correlation ( A) and Principal Component Analysis (PCA; B) based on the expression of the 1000 most variable genes in bulk RNA-seq measurements of hiPSCs adapted to the indicated media. n =3 independent adaptations of the cells into the media. ( C) Volcano plots showing differentially expressed genes in cE8, hE8 or B8+ in comparison to cE8. Significantly different genes are highlighted in color. ( D) Changes in gene expression of key pluripotency genes, measured as a Z-scores of TPM normalised counts. P-values are calculated by linear model fitting and are adjusted for Benjamini-Hochberg false discovery rate. ( E) Schematic of genes involved in primed pluripotency signalling (according to KEGG pathways); those significantly upregulated in cE8 or B8+ are coloured in shades of yellow or red, respectively.

Figure 4.. Heterogeneity in hiPSCs adapted to weekend-free media.

( A) Aggregated Monocle clustering of single cell RNA-seq data from two individual media adaptations of hiPSCs to cE8, hE8 and B8+. ( B) Expression levels of representative lineage markers. ( C) Cell labeling according to previously established subpopulations of hiPSCs . ( D) Cumulative frequency distribution of cells from each cluster based on the human embryo developmental day they correspond most closely to . ( E) Expression of transcriptional signature of nascent mesoderm across the clusters. ( F) Scores based on the expression of genes characteristic for S or G2/M phase of the cell cycle. ( G- H) Gene Ontology terms enriched in genes overexpressed in iPSC-1 ( G) versus iPSC-2 ( H) clusters. ( I) Volcano plot showing differentially expressed genes in iPSC-1 versus iPSC-2 clusters Significantly different genes are highlighted in color. ( J) Cluster assignment of hiPSCs adapted to weekend-free media. ( K) Proportion of cells from the particular media assigned to the cluster. Statistical analysis by one-way ANOVA followed by a Dunnet’s multiple comparison’s test.

Figure 5.. Applications of hiPSCs adapted to weekend-free media.

( A) Representative images of GFP-positive iPSC colonies after puromycin selection of AAVS1 genome-edited cells. ( B) Quantification of the positive colonies. Circles and triangles represent 2 independent media adaptations, while the different types of symbol filling represents 3 repetitions of the experiment. Statistical analyses by mixed effects model followed by Dunnet’s multiple comparisons test. ( C) Quantification of hiPSC clonality after single cell sorting into 96-well plate. n = 2 media adaptations. ( D) Z-scores of gene expression levels in hiPSCs adapted to weekend-free media and the same cells differentiated into three germ layers, as measured by RT-qPCR. n = 2 media adaptations. ( E) Exemplary flow cytometry analyses of TNNT2 expression in hiPSCs successfully differentiated towards cardiomyocytes in monolayer ( F) Exemplary brightfield images of organoids derived from hiPSCs. Scale bar: 500 µm. ( G) Exemplary flow cytometry analyses of TNNT2 expression in hiPSCs successfully differentiated towards cardiomyocytes in organoids. ( H– I) Flow cytometry quantifications of the percentage of TNNT2-positive cells ( H) and the normalised median TNNT2 value ( I) in cardiac organoid differentiations. Median values were normalised through division by the median intensity value of the respective isotype control. Circles and triangles represent 2 independent media adaptations, while the different types of symbol filling represents 4 repetitions of the experiment. Statistical analyses by mixed effects model followed by Dunnet’s multiple comparisons test.

Figure 6.. Differentiation of hiPSCs into colon organoids.

( A) Brightfield images of budding endodermal layers derived from hiPSCs adapted to different media. ( B- C) Quantifications of number ( B) and the roundness ( C) of organoids extracted from the monolayers at day 13 of differentiation. ( D) Images of whole-mount immunostainings of mature colon organoids. Scale bar: 100 µm.

Figure 7.. Adaptation of hESC lines to weekend-free media.

( A) Flow cytometry analyses of OCT4 and NANOG expression in H1 hESCs grown in different media. The results are shown for two independent media adaptations. ( B) Flow cytometry analysis of CD43 and CD34 expression of H1 hESCs differentiated towards primitive hematopoietic stem cells. ( C) Flow cytometry analysis of OCT4 and NANOG expression in H9 hESCs grown in different media. ( D) Immunostainings of sectioned human cortical organoids derived from H9 hESCs. Scale bar: 50 µm.