Rational design and optimization of synthetic gene switches for controlling cell-fate decisions in pluripotent stem cells

Data underlying the figures in the publication “Rational Design and Optimization of Synthetic Gene Switches for Controlling Cell-fate Decisions in Pluripotent Stem Cells”, published in Metabolic Engineering, 2021, 65, 99-110. https://doi.org/10.1016/j.ymben.2021.03.009

Table of contents:

1. Dataset 1; Excel file with the curated data underlying the figures.

Figure 1. Comparative analysis of synthetic gene switches in HEK-293T and hiPSCs. (A) Erythromycin-responsive synthetic gene switch consisting of a constitutively expressed erythromycin-responsive transactivator (pVH620; PmPGK1-E-VP16-pA) and an inducible Nluc expression vector controlled by an operator containing two erythromycin transcription response (ETR) sites (pVH619; OETR2-PCMVmin-2-Nluc-pA), assessed in terms of production of secreted Nluc 24 h after transfection. EM indicates addition of erythromycin (2.0 µg/mL) to the culture medium. (B) Phloretin-responsive synthetic gene switch consisting of a constitutively expressed phloretin-responsive transactivator (pVH655; PmPGK1-TtgR-VP16-pA) and an inducible Nluc expression vector controlled by an operator containing two TtgR binding sites (pVH654; OTtgO2-PCMVmin-2-Nluc-pA), assessed in terms of production of secreted Nluc 24 h after transfection. Phlo indicates addition of phloretin (50 µM) to the culture medium. (C) Vanillic acid-responsive synthetic gene switch consisting of a constitutively expressed vanillic acid-responsive transactivator (pVH238-1; PmPGK1-VanR-VP16-pA) and an inducible Nluc expression vector controlled by an operator containing two VanR binding sites (pVH294-1.3; OVanO2-PCMVmin-2-Nluc-pA), assessed in terms of production of secreted Nluc 24 h after transfection. VA indicates addition of vanillic acid (500 µM) to the culture medium.

Figure 2. Rational design and assembly of a vanillic acid-controlled synthetic gene switch in hiPSCs. (B) Combinatorial analysis of constitutive promoters in hiPSCs for the expression of the VanR-VP16 trans-activator (pVH238-1; PmPGK1-VanR-VP16-pA, pVH238-2; PhEF1a-VanR-VP16-pA) and inducible expression cassette minimal promoters (pVH294-1.1; OVanO2-Pmin-Nluc-pA, pVH294-1.3; OVanO2-PCMVmin-2-Nluc-pA, pVH294-1.2; OVanO2-PCMVmin-1-Nluc-pA), assessed in terms of the production of secreted Nluc 24 h after transfection. (C) Tuning the induction level of the inducible expression cassette in hiPSCs by increasing the number of VanR binding sites within the operator (pVH294-1.1; OVanO2-Pmin-Nluc-pA, pVH294-2.1; OVanO4-Pmin-Nluc-pA, pVH294-3.1; OVanO6-Pmin-Nluc-pA), assessed in terms of the production of secreted Nluc 24 h after co-transfection with PmPGK1-driven VanR-VP16 (pVH238-1; PmPGK1-VanR-VP16-pA). (D) Assessment of the structural assembly of the VanR-VP16 transactivator in hiPSCs, comparing direct fusion of VanR and VP16 (pVH238-1; PmPGK1-VanR-VP16-pA), fusion with a short flexible (pVH261; PmPGK1-VanR-FL1-VP16-pA, FL1; GGGGS) or rigid (pVH263; PmPGK1-VanR-RL1-VP16-pA, RL1; EAAAK) linker, and fusion with a longer flexible (pVH262; PmPGK1-VanR-FL2-VP16-pA, FL2; GGGGSGGGGS) or rigid (pVH264; PmPGK1-VanR-RL2-VP16-pA, RL2; EAAAKEAAAK) linker, assessed in terms of the production of secreted Nluc 24 h after co-transfection with a Pmin-driven reporter construct controlled by four VanR binding sites (pVH294-2.1; OVanO4-Pmin-Nluc-pA). (E) Tuning the induction level of the VA-controlled synthetic gene switch in hiPSCs by promoting active nuclear translocation of VanR-VP16 (pVH238-1; PmPGK1-VanR-VP16-pA) with importin-alpha (pVH250; PmPGK1-NLSSV40-VanR-VP16-pA) or importin-beta (pVH252; PmPGK1-NLSG46-VanR-VP16-pA)-recognized NLS peptide, assessed in terms of the production of secreted Nluc 24 h after co-transfection with a Pmin-driven reporter construct controlled by four VanR binding sites (pVH294-2.1; OVanO4-Pmin-Nluc-pA). (F) Combinatorial analysis for the final assembly of the VA-controlled synthetic gene switch in hiPSCs using a Pmin-driven reporter construct controlled by either four (pVH294-2.1; OVanO4-Pmin-Nluc-pA) or six (pVH294-3.1; OVanO4-Pmin-Nluc-pA) VanR binding sites and different combinations of selected components from previous characterization steps to assemble the transactivator (pVH238-1; PmPGK1-VanR-VP16-pA, pVH263; PmPGK1-VanR-RL1-VP16-pA, pVH262; PmPGK1-VanR-FL2-VP16-pA, pVH250; PmPGK1-NLSSV40-VanR-VP16-pA, pVH252; PmPGK1-NLSG46-VanR-VP16-pA, pVH629; PmPGK1-NLSSV40-VanR-RL1-VP16-pA, pVH628; PmPGK1-NLSSV40-VanR-FL2-VP16-pA, pVH300; PmPGK1-NLSG46-VanR-RL1-VP16-pA, pVH624; PmPGK1-NLSG46-VanR-FL2-VP16-pA), assessed in terms of the production of secreted Nluc 24 h after transfection. (G) Dose-response curves in hiPSCs of the PmPGK1-driven NLSG46-VanR-VP16 (pVH252; PmPGK1-NLSG46-VanR-VP16-pA) and the basic VanR-VP16 (pVH238-1; PmPGK1- VanR-VP16-pA) fusion constructs using the Pmin-driven reporter construct controlled by four VanR binding sites (pVH294-1.2; OVanO4-Pmin-Nluc-pA), assessed in terms of the production of secreted Nluc 24 h after transfection. (B-F) VA indicates addition of vanillic acid (500 µM) to the culture medium.

Figure 3. Regulation of the VanA system during the generation of the three germ layers. hiPSCs transiently transfected with the complete VA-controlled expression vector (pVH438; 5’ITR-OVanO4-Pmin-Nluc-pA-PmPGK1-NLSG46-VanR-VP16-2A-NeoR-pA-3’ITR) were specified for 3 days into (A) DE or (B) ME, and for 5 days into (C) EC. The VanA system was maintained in its repressed state by the addition of VA (500 µM) and induced by the removal of VA from the differentiation medium on the last day of differentiation. Induction of the VanA system was assessed in terms of the intracellular production of non-secreted Nluc.

Figure 4. Synthetic gene-switch-guided posterior foregut lineage specification. (C) The mRNA expression levels of HHEX and TGIF2 in iHHEX/iTGIF2 hiPSCs cultured for 24 h in the presence of different combinations of VA and DOX were determined by means of qRT-PCR. VA indicates addition of vanillic acid (500 µM) and DOX indicates addition of doxycycline (0.75 µg/mL) to the culture medium. Values were normalized to those in the VA (+)/DOX(-) condition. (G-H) iHHEX/iTGIF2 hiPSC-derived DE cells were differentiated towards PG with FGF7 (25 ng/mL) for 3 days, and subsequently towards PFG with FGF7 (25 ng/mL), noggin (50 ng/mL), and retinoic acid (RA; 2 µM) for 1 day. During differentiation HHEX and TGIF2 was maintained uninduced/repressed (VA on D0-7, No DOX), or HHEX was induced on day 2-3 by the addition of DOX and TGIF2 was induced on day 3-7 by removal of VA from the differentiation medium (VA on D3-7, DOX on D2-3). VA indicates addition of vanillic acid (500 µM) and DOX indicates addition of doxycycline (0.75 µg/mL) to the culture medium. (G) Quantification of PDX1-expressing PFG cells. Flow cytometry results quantification of PDX1-expressing PFG cells differentiated using growth-factor-directed (VA on D0-7, No DOX) and TGIF2/HHEX-guided (VA on D0-3, DOX on D2-3) differentiation protocols. (H) Relative mRNA expression levels for PP (PDX1), PFG (SOX9, HNF6), HP (ALB), MHG (CDX2), and AFG (OTX2) genes on day 7 in iHHEX/iTGIF2 hiPSC-derived PFG cells generated with the indicated HHEX and TGIF2 induction conditions were determined by means of qRT-PCR. Values were normalized to those in day 0 iHHEX/iTGIF2 hiPSCs.

Figure S1. Effect of constitutive promoter expression strength (pVH238-1; PmPGK1-VanR-VP16-pA, pVH238-2; PhEF1a-VanR-VP16-pA) on the basal expression levels of inducible vectors containing different minimal promoters (pVH294-1.1; OVanO2-Pmin-Nluc-pA, pVH294-1.3; OVanO2-PCMVmin-2-Nluc-pA, pVH294-1.2; OVanO2-PCMVmin-1-Nluc-pA) relative to mock transfection (pColaDuet-1) in hiPSCs, assessed in terms of the production of secreted Nluc 24 h after transfection. VA indicates addition of vanillic acid (500 µM) to the culture medium.

Figure S2. Assessment of optimal fusion configurations for NLS peptides. (A) NLSSV40 or (B) NLSG46 was fused to the N-terminus of VanR-VP16 (pVH250; PmPGK1-NLSSV40VanR-VP16-pA, pVH252; PmPGK1-NLSG46-VanR-VP16-pA), in between VanR and VP16 as a linker (pVH260; PmPGK1-VanR-NLSSV40-VP16-pA, pVH622; PmPGK1-VanR-NLSG46-VP16-pA), or to the C-terminus (pVH251; PmPGK1-VanR-VP16-NLSSV40-pA, pVH623; PmPGK1-VanR-VP16-NLSG46-pA) to identify the best conformation in hiPSCs, assessed in terms of the production of secreted Nluc 24 h after co-transfection with a Pmin-driven reporter construct controlled by four VanR binding sites (pVH294-2.1; OVanO4-Pmin-Nluc-pA). (A-B) VA indicates addition of vanillic acid (500 µM) to the culture medium.

Figure S3. Complete expression vector design for stable integration encoding OVanO4-Pmin-driven Nluc expression cassette and PmPGK1-driven NLSG46-VanR-VP16 (pVH438; 5’ITR-OVanO4-Pmin-Nluc-pA-PmPGK1-NLSG46-VanR-VP16-2A-NeoR-pA-3’ITR). (B) Transient performance characterization in hiPSCs assessed in terms of the production of intracellular Nluc 24 h after transfection. VA indicates addition of vanillic acid (500 µM) to the culture medium.

Figure S4. Generation of the three germ layers from hiPSCs transfected with complete VA-controlled expression vector (pVH438; 5’ITR-OVanO4-Pmin-Nluc-pA-PmPGK1-NLSG46-VanR-VP16-2A-NeoR-pA-3’ITR). (A) Relative mRNA expression levels of definitive endoderm (DE) genes (EOMES, SOX17, FOXA2, CXCR4) in cells transfected with the complete VA system expression vector after 3 days of DE specification with VA. (B) Relative mRNA expression levels of mesoderm (ME) genes (T, PDGFRa, TBX6, HAND1) in cells transfected with the complete VA system expression vector after 3 days of ME specification with VA. (C) Relative mRNA expression levels of ectoderm (EC) genes (PAX6, NES, OTX2, SOX10) in cells transfected with the complete VA system expression vector after 5 days of EC specification with VA. (A-C) Values were normalized to those of day 0 hiPSCs. VA indicates the addition of vanillic acid (500 µM) to the differentiation medium.

Figure S5. Performance comparison and multiplexing of the VanA and rtTA systems in hIPSCs. (A) Comparison of selected inducible expression cassette containing four VanR response elements (pVH294-2.1; OVanO4-Pmin-Nluc-pA) in combination with PmPGK1-driven NLSG46-VanR-VP16 (pVH252; PmPGK1-NLSG46-VanR-VP16-pA) fusion construct relative to the DOX-controlled rtTA system (pVH627; OTetO7-Pmin-Nluc-pA / pVH235; PmPGK1-rtTA-pA) in hiPSCs assessed in terms of the production of secreted Nluc 24 h after transfection. (B) Assessment of multiplexing potential of the VA system (pVH425; OVanO4-Pmin-Nluc-pA / pVH252; PmPGK1-NLSG46-VanR-VP16-pA) and the DOX-controlled rtTA system (pVH424; OTetO7-Pmin-Fluc-pA / pVH235; PmPGK1-rtTA-pA) in hiPSCs assessed in terms of the production of intracellular Nluc and Fluc 48 h after transfection. (A-B) VA indicates addition of vanillic acid (500 µM) and DOX indicates addition of doxycycline (1 µg/mL) to the culture medium.

Figure S6. (C) iHHEX/iTGIF2 hiPSCs were differentiated towards anterior primitive streak (APS) and DE using the standard combination of exogenous Wnt (GSK3 inhibition CHIR99021 [CHIR]; 3 µM) and TGF-β (Activin A [AA]; 100 ng/mL) induction. During differentiation, TGIF2 was maintained repressed by the addition of VA (VA on D0-3) and HHEX was maintained uninduced (No DOX). VA indicates addition of vanillic acid (500 µM) and DOX indicates addition of doxycycline (0.75 µg/mL) to the culture medium. Relative mRNA expression levels for pluripotency markers (POU5F1, NANOG, SOX2), and definitive endoderm (DE) markers (EOMES, SOX17, FOXA2, CXCR4) on day 3 in iHHEX/iTGIF2 hiPSC-derived DE cells were determined by means of qRT-PCR. Values were normalized to those of day 0 iHHEX/iTGIF2 hiPSCs. (E) iHHEX/iTGIF2 hiPSC-derived DE cells were differentiation towards PG for 1 day with FGF7 (25 ng/mL). During PG specification, TGIF2 was induced alone (VA on D0-3, No DOX), HHEX was induced alone (VA on D0-4, No DOX), or TGIF2 and HHEX were both maintained uninduced (VA on D0-4, No DOX) on day 3-4 by removal or addition of VA and DOX as specified. VA indicates addition of vanillic acid (500 µM) and DOX indicates addition of doxycycline (0.75 µg/mL) to the culture medium. The expression levels for HHEX and TGIF2 on day 4 during PG differentiation of iHHEX/iTGIF2 hiPSC-derived DE cells were determined by means of qRT-PCR. Values were normalized to GAPDH and TBP expression levels in day 4 iHHEX/iTGIF2 hiPSCs.

Figure S7. (B) iHHEX/iTGIF2 hiPSC-derived DE cells were differentiated towards PG with FGF7 (25 ng/mL) for 3 days, and subsequently towards PFG with FGF7 (25 ng/mL), noggin (50 ng/mL), and retinoic acid (RA; 2 µM) for 1 day. During differentiation TGIF2 was maintained repressed under all conditions by the addition of VA to the differentiation media on day 0-7 (VA on D0-7). HHEX was either maintained uninduced (No DOX) or induced on day 2-3, 2-4, or 2-5 by the addition of DOX to the differentiation media (DOX on D2-3, D2-4, or D2-5). VA indicates addition of vanillic acid (500 µM) and DOX indicates addition of doxycycline (0.75 µg/mL) to the culture medium. Relative mRNA expression levels for PP (PDX1), HP (ALB), and PFG (SOX9, HNF6) genes on day 7 in iTGIF2/iHHEX hiPSC-derived PFG cells generated with the indicated differentiation protocol and HHEX induction conditions were determined by means of qRT-PCR. Values were normalized to those of day 0 iHHEX/iTGIF2 hiPSCs. (D) iHHEX/iTGIF2 hiPSC-derived DE cells were differentiated towards PG with FGF7 (25 ng/mL) for 3 days, and subsequently towards PFG with FGF7 (25 ng/mL), noggin (50 ng/mL), and retinoic acid (RA; 2 µM) for 1 day. During differentiation HHEX was maintained uninduced under all conditions (No DOX). TGIF2 was either maintained uninduced by the addition of VA (VA on D0-7) or induced on day 5-7, 4-7, or 3-7 by removal of VA from the differentiation medium (VA on D0-5, D0-4, or D0-3). VA indicates addition of vanillic acid (500 µM) and DOX indicates addition of doxycycline (0.75 µg/mL) to the culture medium. Relative mRNA expression levels for PP (PDX1), HP (ALB), and PFG (SOX9, HNF6) genes on day 7 in iHHEX/iTGIF2 hiPSC-derived PFG cells generated with the indicated differentiation protocol and TGIF2 induction conditions were determined by means of qRT-PCR. Values were normalized to those of day 0 iHHEX/iTGIF2 hiPSCs.