A Chemically Defined Feeder-free System for the Establishment and Maintenance of the Human Naive Pluripotent State

Abstract

The distinct states of pluripotency in the pre- and post-implantation embryo can be captured in vitro as naive and primed pluripotent stem cell cultures, respectively. The study and application of the naive state remains hampered, particularly in humans, partially due to current culture protocols relying on extraneous undefined factors such as feeders. Here we performed a small-molecule screen to identify compounds that facilitate chemically defined establishment and maintenance of human feeder-independent naive embryonic (FINE) stem cells. The expression profile in genic and repetitive elements of FINE cells resembles the 8-cell-to-morula stage in vivo, and only differs from feeder-dependent naive cells in genes involved in cell-cell/cell-matrix interactions. FINE cells offer several technical advantages, such as increased amenability to transfection and a longer period of genomic stability, compared with feeder-dependent cells. Thus, FINE cells will serve as an accessible and useful system for scientific and translational applications of naïve pluripotent stem cells.

Keywords: AZD5438; BCR-ABL and SRC inhibitor; CDK1/2/9 inhibitor; dasatinib; endogenous retroviral element; feeder-independent and chemically defined culture; naive human pluripotent cell state; small-molecule screens.

Graphical abstract

Figure 1

Small-Molecule Screen for Feeder-free Maintenance of Naive hESCs (A) Schematic of high-throughput screen performed to identify compounds supporting feeder-free culture of naive hESCs. Dot plot presents mean Z scores for LTR7Y-zsGreen intensity results from the screen. The gray line indicates a cutoff of Z scores ≥2. Small molecules achieving this cutoff in at least two replicates were considered as hits (blue). Other samples (orange) and DMSO controls (red) did not pass this cutoff. A full list of scores is given in Table S1. (B) Summary table with hits from the small-molecule screen. Asterisks denote compounds targeting pathways not previously demonstrated to play a role in establishment/maintenance of naive pluripotency. (C) Representative images of LTR7Y-zsGreen cells after treatment with small-molecule hits. Scale bars, 50 μm. (D) Fluorescence-activated cell sorting (FACS) quantification of LTR7Y-zsGreen signal after treatment with dasatinib, crenolanib, and AZD5438.

Figure 2

Optimization and Establishment of FINE Culture Conditions (A and B) Gene expression analysis for naive markers in (A) 3iL cultured cells and (B) 4iLA cultured cells supplemented with small molecules. Mean ± SD of three independent experiments. RNA was collected after 6 days (3iL) or 12 days (4iLA) in culture without feeders. AZD, AZD5438; CHIR, CHIR-99021; Dasa, dasatinib; Sara, saracatinib; Creno, crenolanib. (C) Relative survival of hESC culture under 4iLA supplemented with different chemical combination conditions (C1–C21) over nine passages without feeders. 4iLA was included as control. When cells appear highly differentiated morphologically or when very few cells remain after passaging, the condition is dropped off; only cells cultured in C18–C21 4iLA medium supplemented with AZD5438 and dasatinib (in green) remain after nine passages. Detailed conditions are provided in Table S2. (D) Heatmap presenting gene expression of naive pluripotency-associated markers in cells at passage 4 during adaptation to naive feeder-free conditions (C1–C21). Mean of two biological replicates is shown. Euclidean distance from 4iLA⁺ feeder across all the genes tested was calculated for each condition and represented as the bar chart. (E) Schematic showing the process of adapting primed hESCs into FINE. (F) Gene expression in hESCs throughout the course of adaptation from mTeSR1 to FINE up to passage 5. Mean ± SD of two independent experiments.

Figure 3

FINE Cells Display Hallmarks of Naive Pluripotency (A) Bright-field images of hESCs cultured in 4iLA (with and without feeders) and FINE at passage 8. Scale bars, 50 μm. (B) Expression of blastocyst-associated transcripts in hESCs cultured under mTeSR1, 4iLA⁺ feeder, and FINE conditions. Mean ± SD of three independent experiments. (C) Immunofluorescence staining of pluripotency and blastocyst-associated proteins in hESCs under mTeSR1 and FINE conditions. Scale bars, 50 μm. (D) FACS quantification of hESCs expressing naive surface markers under mTeSR1, 4iLA⁺ feeder, and FINE conditions. (E) qPCR analysis of LTR7Y and HERVH transcripts in hESCs cultured under mTeSR1, 4iLA⁺ feeder, and FINE conditions. Mean ± SD of three independent experiments. (F) Measurement of cell numbers cultured in FINE conditions at day 0 (D0) and 4 days post seeding (D4). Mean ± SD of three independent experiments. (G) Representative RNA fluorescence in situ hybridization (FISH) images detecting HUWE1 (subject of X chromosome inactivation) and XACT control (escaping X chromosome inactivation) in mTeSR1, 4iLA⁺ feeder, and FINE cells. Scale bars, 10 μm. (H) Immunofluorescence staining of H3K9me3 in hESCs under mTeSR1 and FINE conditions (left). Scale bars, 50 μm. Intensity of H3K9me3 was quantified through a line (red) randomly drawn across images (right).

Figure 4

FINE Cells Are Dependent on Both Dasatinib and AZD5438 (A) Bright-field and immunofluorescence staining of KLF4, TFE3, and KLF17 in FINE culture after withdrawal of AZD, dasatinib (Dasa), or both for three passages. Scale bars, 50 μm. (B) Quantification of fraction of nuclei positive for naive-associated transcription factors in hESCs cultured in FINE after withdrawal of AZD, Dasa, or both for three passages. Mean ± SD of three independent experiments. (C) Expression of blastocyst-associated transcripts in hESCs cultured in FINE after withdrawal of AZD, Dasa, or both for two passages. Mean ± SD of three independent qPCR experiments. (D and E) Expression of blastocyst-associated transcripts in hESCs cultured in FINE after (D) replacement of Dasa with other Src and Bcr-Abl inhibitors or (E) replacement of AZD with dinaciclib (DINA) (in H9 line) for two passages. IMA, imatinib; NILO, nilotinib. Mean ± SD of three independent qPCR experiments.

Figure 5

Transcriptomic Profile of FINE Resembles the In Vivo Pre-implantation Blastocyst (A) Principal component (PC) analysis based on top 1,000 differentially expressed genes between mTeSR1, 4iLA⁺ feeder, and FINE cultured cells. (B) Heatmap of top 1,000 differentially expressed genes between mTeSR1, 4iLA⁺ feeder, and FINE cultured cells. Six main clusters were defined by dendrogram (left). Representative genes from two main clusters (naive-specific genes, primed-specific genes) are presented in smaller heatmaps (right). (C) Scatterplots showing significantly upregulated (in red) and downregulated (in blue) genes between: mTeSR versus 4iLA⁺ feeder, mTeSR1 versus FINE, and 4iLA⁺ feeder versus FINE conditions. Genes not differentially expressed are presented in black. (D) Correspondence between gene expression (left) or transposable element (TE) expression (right) between our naive/primed ESCs and single-cell human embryonic stages from Yan et al. (2013). For each embryonic stage, the percentage of genes/TEs with expression upregulated in FINE (green), upregulated in mTeSR1 (dark gray) or unchanged between FINE and mTESR1 (light gray) is shown. (E) PC analysis plot based on the top 2,540 repeat elements differentially expressed across conditions. Single-cell in vivo embryonic data (Yan et al., 2013) are represented as squares, while FINE, 4iLA⁺ feeder, and mTeSR1 from our bulk RNA-seq data are drawn as circles. (F) Box plots representing mean normalized expression of different TEs in mTeSR1, 4iLA⁺ feeder, and FINE cultured cells. (G) Percentage of members in each TE family with expression upregulated in FINE (green), upregulated in mTeSR1 (dark gray), or unchanged between FINE and mTESR1 (light gray). TE families were ranked as specific to FINE conditions on the left and specific to mTeSR1 conditions on the right.

Figure 6

Global DNA Methylation Profile of FINE Confirms Equivalence to Feeder-Dependent Naive Pluripotent hESCs (A) Per-chromosome comparison of CG methylation fraction between mTeSR1, 4iLA⁺ feeder, and FINE conditions. (B) Relative methylation tracks of chromosome 4 under mTeSR1, 4iLA⁺ feeder, and FINE conditions. (C) Correlation plot of methylated sites in FINE versus either mTeSR1 or 4iLA⁺ feeder. Red line represents fit based on linear regression modeling (off-center best fit indicates lower correlation); blue line is based on LOESS weighted regression modeling (curved best-fit line indicates non-linear correlation). (D) Box plots (top) for CG methylation fraction at select loci representing naive-, differentiation-, 8C-, and morula-associated genes, as well as relative methylation tracks of one representative gene per group (bottom). Differential peaks are highlighted in yellow for ZSCAN4 and DNAJC15.

Figure 7

FINE Cells Offer Advantages over Other Naive Culture Conditions (A) Representative images (left) and FACS quantification (right) of cells in FINE and 4iLA⁺ feeder culture conditions after transfection with mCherry-containing plasmids gRNA 1 (targeting EGFR) and gRNA 2 (targeting STAG2). Quantification was performed after staining with an anti-CD75 antibody to account for feeders; mean ± SD of two independent experiments. Scale bar, 400 μm. (B) Summary of cytogenetic analysis of H9 cells (top) under various naive culture conditions (rows) and passage numbers (columns). Representative karyotypes at various passage numbers in FINE (bottom). (C) qPCR analysis of naive-associated transcripts in H1 hESCs cultured under mTeSR1, RSeT, and FINE conditions. Mean ± SD of three independent experiments. (D) Heatmap showing rlog values for expression of 8-cell- and morula-stage-associated genes in mTeSR1, 4iL⁺ feeder, and FINE cultures based on RNA-seq.