Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs.

Validation of the ROSA26 and AAVS1 loci as bona fide genomic safe harbors. (A) Experimental approach behind the generation of genomic safe harbor (GSH) EGFP reporter hPSCs to test GSH expression during differentiation. Neurons, oligodendrocytes and astrocytes were obtained in bulk cultures containing a mixture of these cell lineages, whereas all other cell types were individually generated. (B) ROSA26 and AAVS1 EGFP reporter transgenic alleles. R26-prom, ROSA26 locus promoter; AAV-prom, AAVS1 locus promoter; 5′-HAR/3′-HAR, upstream/downstream homology arm; SA, splice acceptor; T2A, self-cleaving T2A peptide; Neo, neomycin resistance; Puro, puromycin resistance; pA, polyadenylation signal; CAG, CAG promoter. (C) Summary of EGFP flow cytometry quantification experiments in the indicated cell types generated from GSH EGFP reporter hPSCs (abbreviations indicate the lineages described in A). The percentage of EGFP-positive cells and the EGFP median fluorescence intensity (MFI) are reported. Wild-type hESCs (H9) were used as negative controls, and results are from two independent cultures per lineage. (D) Representative immunofluorescent stainings for lineage-specific markers in three of the mature cell types analyzed. EGFP fluorescence from the reporter lines is in green, and DAPI (blue) shows nuclear staining. Scale bars: 200 μm.

Development of an optimized inducible knockdown system (OPTiKD) based on dual GSH targeting of hPSCs. (A) Experimental approach for the generation of inducible knockdown (iKD) hPSCs. H1, H1 promoter; TO, tet operon; tetR, tetracycline-controlled repressor; ZFN, zinc-finger nuclease. (B) Transgenic alleles generated to obtain hESCs expressing an EGFP reporter transgene that could be silenced using an inducible EGFP shRNA. (C) EGFP expression in the absence or presence of tetracycline for 5 days in hESCs targeted with the indicated combinations of inducible EGFP shRNA and tetR [wild-type standard tetR (STDtetR) or codon-optimized tetR (OPTtetR)]. Double-targeted hESCs that did not carry the EGFP shRNA were used as negative controls. Results are from two or three individual lines per condition (see Table S1). n.s., P>0.05 (non-significant), **P<0.01, ***P<0.001 versus the same tetR line no tet and no shRNA (ANOVA with post-hoc Holm-Sidak comparisons). (D) Representative western blot for tetR in ROSA26-targeted hESCs expressing STDtetR or OPTtetR. HET, heterozygous targeting; HOM, homozygous targeting. hESCs with STDtetR random integration (Rand. Int.) are shown as a positive reference, while wild-type H9 hESCs are negative controls. Various amounts of protein were loaded to facilitate semi-quantitative comparison. TUBA4A (α-tubulin) provided a loading control. (E) EGFP knockdown and rescue kinetics in EGFP OPTiKD hESCs measured by flow cytometry (MFI) and qPCR (mRNA). Results are from two independent cultures per time point. (F) Tetracycline dose-response curve for EGFP knockdown in EGFP OPTiKD hESCs. The half-maximal inhibitory concentration (IC50) is reported. Results are from two independent cultures per dose, and the mean is shown. (G,H) qPCR analysis of OCT4 OPTiKD hESCs in the absence of tetracycline, or following treatment with different doses of tetracycline for 5 days. (H) Genes are clustered by complete Euclidean distance, and genes specific for pluripotency or for the primary germ layers are in color-coded boxes: no color, hPSCs; red, mesoderm; green, endoderm; blue, neuroectoderm. Z-scores indicate differential expression measured in the number of standard deviations from the average level. Results are from three independent cultures.

Single-step optimized inducible knockdown (sOPTiKD) of individual and multiple genes in hESCs and hiPSCs. (A) The transgenic allele behind the single-step generation of OPTiKD hPSCs. (B,C) qPCR of OCT4 and B2M sOPTiKD hESCs and hiPSCs in the absence (CTR) or presence of tetracycline for 5 days (TET). Individual clonal lines were analyzed in duplicate. (D) Heatmap summarizing qPCR analysis of cells treated as in B,C. Results are from three clonal lines per condition. Samples and genes were clustered by complete Euclidean distance, and Z-scores indicate differential expression measured in number of standard deviations from the average level. (E) The transgenic allele behind the generation of hPSCs with inducible knockdown of multiple genes. (F) qPCR analysis of sOPTiKD hESCs for individual or multiple cyclin D genes (D1, D2 and D3 in the key indicating inducible shRNAs against CCND1, CCND2 and CCND3, respectively). Cells were analyzed in the absence or presence of tetracycline for 10 days, and sOPTiKD hESCs carrying one, two or three copies of a scrambled shRNA (SCR, 2X SCR and 3X SCR, respectively) were used as negative controls. For each condition, results are from two clonal pools obtained after gene targeting. (G,H) Flow cytometry quantifications (G) and representative immunostainings (H) for the pluripotency marker OCT4 and the definitive endoderm marker SOX17 in cells treated as in F. *P<0.05, ***P<0.001 versus CTR in the same line (ANOVA with post-hoc Holm-Sidak comparisons). DAPI shows nuclear staining. Scale bars: 100 μm.

Validation of the optimized inducible knockdown platforms following hPSC differentiation. (A) EGFP expression measured by qPCR in the absence (CTR) or presence of tetracycline for 5 days (TET) in the indicated cell types derived from EGFP OPTiKD and sOPTiKD hESCs. EGFP levels are reported relative to control conditions in the same line for each individual lineage. Abbreviations indicate the lineages described in Fig. 1A (pluri indicates undifferentiated). Results are from two independent cultures per condition. (B) Representative immunofluorescent stainings for lineage-specific markers in the three germ layers and in the indicated mature cell types derived from EGFP sOPTiKD hESCs and treated as in A. EGFP fluorescence is in green, and DAPI shows nuclear staining. Similar results supporting the same conclusions were obtained for EGFP OPTiKD hESCs (data not shown). Scale bars: 100 μm for intestinal epithelium and cholangiocytes; 200 μm for all other lineages.

Functional analysis of T (brachyury) during mesendoderm specification of hPSCs. (A) The experimental approach. T knockdown was induced for 2 days in pluripotent cells and maintained throughout differentiation. PS, primitive streak-like cells; i-, inhibitor; a-, activator; CHIR, CHIR99021; LDN, LDN193189; SB, SB431542; PurM, purmorphamine. (B) qPCR for T in the indicated lineages derived from T or B2M (control) sOPTiKD hESCs as described in A. Ant/Post/Late PS refer to anterior/posterior/late primitive streak; DE, definitive endoderm; CM, cardiac mesoderm; LPM, lateral plate mesoderm; PSM, presomitic mesoderm. (C) Representative immunofluorescent staining demonstrating inducible knockdown of T in late primitive streak cells expressing the marker CDX2. DAPI shows nuclear staining. (D) Heatmap summarizing qPCR results for various mesendoderm markers in primitive streak cells from the experiment described in A,B. Genes were clustered by complete Euclidean distance, and Z-scores indicate differential expression measured in number of standard deviations from the average level. (E) qPCR results for lineage-specific markers in presomitic mesoderm cells from the experiment described in A,B. (F,G) Representative Alcian Blue staining (F) and quantification of Alcian Blue release (G) in chondrocytes differentiated from presomitic mesoderm cells generated as described in A,B. (B,E,G) *P<0.05, **P<0.01, ***P<0.001 versus B2M in the same condition (two-way ANOVA with post-hoc Sidak comparisons), and results are from three independent clonal lines per condition. Scale bars: 100 μm in C; 4 mm in F.

Functional analysis of DPY30 during hPSC differentiation. (A) The experimental design to investigate the role of DPY30 at various stages of hPSC differentiation. (B) Representative immunofluorescent staining for DPY30 and the pluripotency marker OCT4 in undifferentiated DPY30 OPTiKD hESCs in the absence (CTR) or presence of tetracycline for 10 days (TET). DAPI shows nuclear staining. Scale bars: 200 μm. (C,D) qPCR-based analyses of DPY30 (blue) and B2M (orange) OPTiKD hESCs after differentiation into mature lineages. CTR, no knockdown; KD ind/spec/mat refer to knockdown from induction/specification/maturation, respectively. Results are from three independent cultures per condition. *P<0.05 versus B2M in the same condition (two-way ANOVA with post-hoc Sidak comparisons). (E) Summary of the lineage- and stage-specific phenotypic effects following DPY30 knockdown during hESC differentiation.

Development of an optimized inducible CRISPR/Cas9 knockout platform in hPSCs. (A) Experimental approach for the generation of inducible knockout (iKO) hPSCs. (B) The cloning procedure to generate AAVS1 targeting vectors with an inducible gRNA cassette. The arrows indicate the DNA cut sites induced by digestion with AarI. (C) Transgenic alleles generated to obtain hESCs expressing an EGFPd2 reporter transgene that could be knocked out by CRISPR/Cas9 using an inducible EGFP gRNA (EGFP sOPTiKO hESCs). Bsd, blasticidin resistance; EGFPd2, destabilized EGFP. (D) Representative immunofluorescent stainings for Cas9 in EGFPd2 homozygous sOPTiKO hESCs. Wild-type hESCs (H9) were analyzed as a negative control. Cells were co-stained for the pluripotency factor NANOG, and DAPI shows nuclear staining. (E) Representative images depicting EGFPd2 fluorescence in EGFPd2 homozygous sOPTiKO hESCs in the absence (CTR) or presence of tetracycline (TET) for the indicated number of passages (P; cells were split every 5 days). (F) Representative flow cytometry for EGFPd2 expression in EGFPd2 homozygous sOPTiKO hESCs (iKO) following three passages in the presence of tetracycline. EGFPd2 homozygous cells that do not carry the inducible CRISPR/Cas9 system (EGFPd2 HOM) and wild-type hESCs were analyzed as positive and negative controls for EGFPd2 expression, respectively. The gate used to define EGFPd2-positive cells (EGFP+) is shown, and the percentage of EGFP+ cells and their MFI are reported. (G) As in F, but EGFPd2 homozygous sOPTiKO hESCs were analyzed following ten passages in the absence of tetracycline. (H,I) As in F, but EGFPd2 homozygous sOPTiKO hESCs were generated using an AAVS1 targeting vector carrying two or three copies of the inducible EGFP gRNA cassette (2× and 3× gRNA, respectively). All results in this figure were obtained using EGFP gRNA 1. Scale bars: 100 μm.

Validation of the optimized inducible CRISPR/Cas9 platform following hPSC differentiation. (A-F) Representative immunofluorescent stainings for the indicated lineage-specific markers in cells derived from EGFPd2 sOPTiKO hESCs carrying a single EGFP inducible gRNA (gRNA 1). EGFPd2 knockout was induced with tetracycline for 6 days for the germ layers (A-C) and for 10 days for the mature cells (D-F). EGFPd2 fluorescence in control conditions (CTR) or after knockout (TET) is in green, and DAPI shows nuclear staining. Merged images of the EGFPd2 and lineage-specific markers are shown. Scale bars: 100 μm. (G) Summary of the experimental strategy behind the generation and application of inducible knockdown or knockout hPSCs using the sOPTiKD or sOPTiKO platforms.