Figure 3.
Doxycycline inducible lines for the production of MKs. (A) Scheme illustrating the targeting, clone selection, and culture of inducible cell lines. We simultaneously targeted the Rosa 26 (pRosa_neo CAG-rtTA expressing the protein rtTA) and the AAVS1 (pAAVS1-puro TRE-PC3 expressing GFP and the 3TFs) loci using nucleofection followed by simultaneous neomycin and puromycin selection. Upon doxycycline addition to the culture medium, GFP and the 3TFs are expressed as individual proteins; each TF was preceded by a self-cleaving 2A oligopeptide. Cells are cultured in the optimized media AMK with a 3-day mesoderm inducible step allowing for the slower dynamic of differentiation compared with lentiviral transduction. (B) Genome-editing efficiency. We show the outcome of the single-step targeting strategy for 5 independent lines (4× hiPSC and 1× hESC) from which a total of 14 clones were screened. The panel at the top indicates state of the ROSA (pink bars) and AAVS1(blue bars) alleles in the various clones. HET signifies that only 1 allele was correctly targeted whereas HOM denotes correct targeting on both alleles. (C) GFP expression as a proxy for the polycistronic cassette expression was measured by flow cytometry. Overlaid histograms are shown for a range of doxycycline concentrations for 1 HOM-HOM clone (iDELTA-3.7). The inset line graph shows the percentage of GFP expression at 36 hours reaches a plateau at 0.25 μg/mL doxycycline for both iDELTA-3.7 (solid line) and iLIPSC-GR1.1.2 (dotted line). Mean ± 1× SEM; n = 3. (D) Single protein transcripts for GATA1, TAL1, and FLI1 are produced from the polycistronic cassette. Western blots for 2 inducible lines iLIPSC-GR1.1.2 and iDELTA-3.7 at increasing doxycycline doses. The 0 μg/mL doxycycline dose is a 50:50 mix of both lines. Lysates were harvested on day 3 of the differentiation protocol. Individual proteins are seen for each of the 3TFs in the inducible cell line sample, however, a small proportion of the total TF protein is expressed as a “fusion” protein, due to incomplete cleavage at the 2A sequences. (E-F) The inducible cell lines iLIPSC-GR1.1.2 and iDELTA-3.7 differentiate into MKs readily upon addition of doxycycline. Bar graphs showing MK yield expressed per 1.00E+05 undifferentiated starting cells (E) and purity (percentage of CD41+CD42+ MKs) (F) at day 20 of differentiation. Mean ± 1× SEM; n = 10 iLIPSC-GR1.1.2; n = 15 iDELTA-3.7. (G-H) Testing GMP-grade culture components. Bar graphs showing the number (G) and the percentage (H) of viable CD41+CD42+ MKs at day 20 postinduction for iLIPSC-GR1.1.2 cultured with GMP (blue)-grade reagents expressed relative to laboratory-grade reagents. Mean ± 1× SEM, n = 3. Dox, doxycycline; E2A, equine rhinitis A virus; Neo, neomycin; P2A, porcine teschovirus-1; Puro, puromycin; T2A, Thoseaasigna virus are the self-cleaving oligopepetides; both TAL1 and FLI1 are codon optimized; TRE 3GV, third-generation Tet response element; .

Doxycycline inducible lines for the production of MKs. (A) Scheme illustrating the targeting, clone selection, and culture of inducible cell lines. We simultaneously targeted the Rosa 26 (pRosa_neo CAG-rtTA expressing the protein rtTA) and the AAVS1 (pAAVS1-puro TRE-PC3 expressing GFP and the 3TFs) loci using nucleofection followed by simultaneous neomycin and puromycin selection. Upon doxycycline addition to the culture medium, GFP and the 3TFs are expressed as individual proteins; each TF was preceded by a self-cleaving 2A oligopeptide. Cells are cultured in the optimized media AMK with a 3-day mesoderm inducible step allowing for the slower dynamic of differentiation compared with lentiviral transduction. (B) Genome-editing efficiency. We show the outcome of the single-step targeting strategy for 5 independent lines (4× hiPSC and 1× hESC) from which a total of 14 clones were screened. The panel at the top indicates state of the ROSA (pink bars) and AAVS1(blue bars) alleles in the various clones. HET signifies that only 1 allele was correctly targeted whereas HOM denotes correct targeting on both alleles. (C) GFP expression as a proxy for the polycistronic cassette expression was measured by flow cytometry. Overlaid histograms are shown for a range of doxycycline concentrations for 1 HOM-HOM clone (iDELTA-3.7). The inset line graph shows the percentage of GFP expression at 36 hours reaches a plateau at 0.25 μg/mL doxycycline for both iDELTA-3.7 (solid line) and iLIPSC-GR1.1.2 (dotted line). Mean ± 1× SEM; n = 3. (D) Single protein transcripts for GATA1, TAL1, and FLI1 are produced from the polycistronic cassette. Western blots for 2 inducible lines iLIPSC-GR1.1.2 and iDELTA-3.7 at increasing doxycycline doses. The 0 μg/mL doxycycline dose is a 50:50 mix of both lines. Lysates were harvested on day 3 of the differentiation protocol. Individual proteins are seen for each of the 3TFs in the inducible cell line sample, however, a small proportion of the total TF protein is expressed as a “fusion” protein, due to incomplete cleavage at the 2A sequences. (E-F) The inducible cell lines iLIPSC-GR1.1.2 and iDELTA-3.7 differentiate into MKs readily upon addition of doxycycline. Bar graphs showing MK yield expressed per 1.00E+05 undifferentiated starting cells (E) and purity (percentage of CD41+CD42+ MKs) (F) at day 20 of differentiation. Mean ± 1× SEM; n = 10 iLIPSC-GR1.1.2; n = 15 iDELTA-3.7. (G-H) Testing GMP-grade culture components. Bar graphs showing the number (G) and the percentage (H) of viable CD41+CD42+ MKs at day 20 postinduction for iLIPSC-GR1.1.2 cultured with GMP (blue)-grade reagents expressed relative to laboratory-grade reagents. Mean ± 1× SEM, n = 3. Dox, doxycycline; E2A, equine rhinitis A virus; Neo, neomycin; P2A, porcine teschovirus-1; Puro, puromycin; T2A, Thoseaasigna virus are the self-cleaving oligopepetides; both TAL1 and FLI1 are codon optimized; TRE 3GV, third-generation Tet response element; .

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