Journal: Stem Cells Translational Medicine

Article Title: Precision installation of a highly efficient suicide gene safety switch in human induced pluripotent stem cells

doi: 10.1002/sctm.20-0007

Figure Lengend Snippet: Design and AAVS1 targeting for installation of iCASP9 safety switch in human induced pluripotent stem cells (iPSCs). A, Components of the iCASP9 system and dimerization of iCASP9 induced by AP1903 activates mitochondrial apoptosis pathway (caspase 3 and 7), leading to apoptotic cell death. B, The AAVS1 locus is located in the first intron of the PPP1R12C gene in human chromosome 19. TALENs are used to create DNA double‐strand breaks for inserting iCASP9 transgenes into the genome via homologous recombination. A total of six donor repair templates were constructed as follows. 1G: Puro‐EF1α‐iCASP9‐2A‐GFP; 2G: SA‐2A‐iCASP9‐2A‐GFP‐Puro; 3G: Puro‐CMV‐EF1α‐iCASP9‐2A‐GFP; 4G: Puro‐CAG‐iCASP9‐2A‐GFP; 4‐p: Puro‐CAG‐iCASP9; and 4‐n: Neo‐CAG‐iCASP9. EF1α, EF1α core promoter; HA‐L and HA‐R, left and right homology arms. SV40 late poly(A) was used in all the constructs and located right after the last transgene; KS, Kozak sequence; CMV, CMV enhancer; SA, splicing acceptor. PuroR or NeoR spanned by two loxP sites were used for puromycin or G418 selection during clonal line establishment. The expression of PuroR and NeoR were driven by the SV40 promoter and poly(A) tail was the synthetic poly(A). The GFP versions of the donor templates were used to quickly assess iCASP9 expression levels based on GFP levels. C, The sequences of the left and right AAVS1 TALENs and their target site in the AAVS1 locus. D, Clonal line production: from nucleofection to characterization

Article Snippet: The human iPSC clone containing iCASP9 (4‐pn‐45) was differentiated into mesenchymal stem cells (MSCs) using Stem Cell Technologies Stemdiff Mesenchymal Progenitor Kit according to the manufacturer protocol. iCASP9 MSCs were expanded on Cellstart CTS (Gibco) coated flasks at 1:100 dilution and Mesencult ACF Plus (Stem Cell Technologies).

Techniques: TALENs, Homologous Recombination, Construct, Sequencing, Selection, Expressing

Journal: Stem Cells Translational Medicine

Article Title: Precision installation of a highly efficient suicide gene safety switch in human induced pluripotent stem cells

doi: 10.1002/sctm.20-0007

Figure Lengend Snippet: AP1903 rapidly induces apoptosis of the CAG‐iCASP9 induced pluripotent stem cells (iPSCs) in vitro. A, CAG promoter induced uniform, strong and stable GFP expression in the established clones. Representative images from two clones at day 7 post‐nucleofection are shown. B, The number of clones harboring one or two copies of iCASP9 out of the total clones picked in three different targeting groups. C, Karyotypes were normal (46, XY) in all 20 metaphase cells analyzed for each 11 out of total 13 clones examined. D, Representative time course images show AP1903 (10 nM) completely killed CAG‐iCASP9 iPSCs within 24 hours with no cell regrowth. E, Representative time course FACS analyses of cell size and cell death (Annexin V and 7‐AAD) during 10 nM AP1903 treatment. F, Quantification of FACS data from three different iCASP9 iPSC clones. Data are presented as mean ± SD (n = 3‐4). Two‐way ANOVA followed by multiple comparison with Tukey correction was used and P < .0001 for all the time points when compared with time 0 minute. The dead cells were quantified using 100% minus the percent AnV − /7‐AAD − cells in the FSC/SSC gated region after doublet removal. Therefore, the real percentage of total dead cells in the AP1903 treated condition at some time points could be even higher since majority of the cells became debris and were not included in calculation

Article Snippet: The human iPSC clone containing iCASP9 (4‐pn‐45) was differentiated into mesenchymal stem cells (MSCs) using Stem Cell Technologies Stemdiff Mesenchymal Progenitor Kit according to the manufacturer protocol. iCASP9 MSCs were expanded on Cellstart CTS (Gibco) coated flasks at 1:100 dilution and Mesencult ACF Plus (Stem Cell Technologies).

Techniques: In Vitro, Expressing, Clone Assay, Comparison

Journal: Stem Cells Translational Medicine

Article Title: Precision installation of a highly efficient suicide gene safety switch in human induced pluripotent stem cells

doi: 10.1002/sctm.20-0007

Figure Lengend Snippet: CMV‐EF1α promoter is gradually silenced in the established iCASP9 induced pluripotent stem cells (iPSC) clones. A, Representative images on day 10 after nucleofection: puromycin‐resistant CMV‐EF1α‐iCASP9‐2A‐GFP (Donor 3G) iPSC clones appeared. Clones expressed GFP but some of the cells within the same clone did not express GFP. B, After colony picking, more GFP negative cells appeared. C, Representative images after 24 hours exposure to AP1903 (50 nM) failed to eliminate all cells in the established clones. D, After 72 hours of puromycin reselection, the remaining cells were much more susceptible to AP1903 (50 nM) than before reselection shown in C

Article Snippet: The human iPSC clone containing iCASP9 (4‐pn‐45) was differentiated into mesenchymal stem cells (MSCs) using Stem Cell Technologies Stemdiff Mesenchymal Progenitor Kit according to the manufacturer protocol. iCASP9 MSCs were expanded on Cellstart CTS (Gibco) coated flasks at 1:100 dilution and Mesencult ACF Plus (Stem Cell Technologies).

Techniques: Clone Assay

Journal: Stem Cells Translational Medicine

Article Title: Precision installation of a highly efficient suicide gene safety switch in human induced pluripotent stem cells

doi: 10.1002/sctm.20-0007

Figure Lengend Snippet: AP1903 effectively eliminates CAG‐iCASP9 induced pluripotent stem cells (iPSC)‐derived mesenchymal stem cells (MSCs). A, Flow cytometry for marker expression in CAG‐iCASP9 iPSC‐derived MSCs. B, Representative cell morphologies of CAG‐iCASP9 MSCs after treatment with AP1903. A blow‐up image showing no survival cells left after 24‐hours treatment. C, Quantifications of killing efficiency. At 24 hours, the viability is significantly different compared with other time points

Article Snippet: The human iPSC clone containing iCASP9 (4‐pn‐45) was differentiated into mesenchymal stem cells (MSCs) using Stem Cell Technologies Stemdiff Mesenchymal Progenitor Kit according to the manufacturer protocol. iCASP9 MSCs were expanded on Cellstart CTS (Gibco) coated flasks at 1:100 dilution and Mesencult ACF Plus (Stem Cell Technologies).

Techniques: Derivative Assay, Flow Cytometry, Marker, Expressing

Journal: Stem Cells Translational Medicine

Article Title: Precision installation of a highly efficient suicide gene safety switch in human induced pluripotent stem cells

doi: 10.1002/sctm.20-0007

Figure Lengend Snippet: AP1903 effectively eliminates CAG‐iCASP9 induced pluripotent stem cells (iPSC)‐derived chondrocytes. A, Alcian blue staining on micromass chondrogenic differentiation from mesenchymal stem cells (MSCs). B, Representative cell morphologies of CAG‐iCASP9 chondrocytes after treatment with AP1903 at different time points. C, Quantifications of killing efficiency. At 24 and 48 hours, the viabilities are significantly different compared with other time points

Article Snippet: The human iPSC clone containing iCASP9 (4‐pn‐45) was differentiated into mesenchymal stem cells (MSCs) using Stem Cell Technologies Stemdiff Mesenchymal Progenitor Kit according to the manufacturer protocol. iCASP9 MSCs were expanded on Cellstart CTS (Gibco) coated flasks at 1:100 dilution and Mesencult ACF Plus (Stem Cell Technologies).

Techniques: Derivative Assay, Staining

Journal: Stem Cells Translational Medicine

Article Title: Precision installation of a highly efficient suicide gene safety switch in human induced pluripotent stem cells

doi: 10.1002/sctm.20-0007

Figure Lengend Snippet: AP1903 rapidly clears CAG‐iCASP9 induced pluripotent stem cells (iPSCs)‐derived teratomas in vivo. A, Percentage of bioluminescence imaging intensity (BLI) change of tumors after AP1903 (in DPT) administration. Starting on day 62, the animals were injected with AP1903 in DPT. Both IP or IT AP1903 administration led to a significant shrinkage of teratomas as assessed by BLI change. By contrast, teratomas in animals dosed with vehicle only continued to increase in size. B, Survival curve. Mice were euthanized when tumor size reached 2000 mm 3 . One mouse was dead on day 9 in the AP1903 in DPT ‐ IP group, cause undermined. All other mice were euthanized at the termination of the experiment on day 83. DPT, 50% N,N‐dimethylacetamide/50% (90% PEG‐400/10% Tween 80); IP, intraperitoneal; IT, intratumoral

Article Snippet: The human iPSC clone containing iCASP9 (4‐pn‐45) was differentiated into mesenchymal stem cells (MSCs) using Stem Cell Technologies Stemdiff Mesenchymal Progenitor Kit according to the manufacturer protocol. iCASP9 MSCs were expanded on Cellstart CTS (Gibco) coated flasks at 1:100 dilution and Mesencult ACF Plus (Stem Cell Technologies).

Techniques: Derivative Assay, In Vivo, Imaging, Injection

Journal: The Journal of investigative dermatology

Article Title: Base editor correction of COL7A1 in recessive dystrophic epidermolysis bullosa patient-derived fibroblasts and iPSCs

doi: 10.1016/j.jid.2019.07.701

Figure Lengend Snippet: a and b iPSC editing. Sanger chromatogram of uncorrected c.553 C>T; R553X iPSC and a representative base edited clone are shown with arrow showing the mutant/target base. B Pluripotency immunofluorescence marker analysis. Antibodies against the pluripotency markers: podocalyxin TRA-1–60/TRA-1–81, Stage-specific embryonic antigen 4 (SSEA4), SSEA3, Nanog and OCT3/4 were used to detect expression levels. c Mesenchymal stromal cell characterization. Adult bone marrow from normal donors or iPSC derived MSCs were analyzed for the cell surface markers CD73, CD90, and CD105. The isotype staining control peaks are shown in pink. d iPSC derivative MSC Western blot analysis. MSCs derived from uncorrected c.553 C>T iPSCs were compared with those corrected by ABE in a Western blot using a polyclonal anti-C7 antibody. A ~290 kD band is shown with a 42 kD actin loading control below. e-g C7 immunostaining of chamber slides containing e wild type, bone marrow derived MSCs, f unedited 553 patient RDEB iPSC-derived MSCs, and g ABE edited 553 iPSC derivative MSCs. All cells were stained at the same time with the equivalent amount of polyclonal anti-C7 primary and secondary antibodies. Scale bar=50 μm.

Article Snippet: MSCs were differentiated from iPSC using the STEMdiff™ Mesenchymal Progenitor Kit (STEMCELL Technologies, Cambridge, MA) following the manufacturer’s instructions and stained at day 28 with mouse anti-human CD73, CD90 (BD Pharmingen, San Jose, CA) and CD105 antibodies (ThermoFisher, Waltham, MA) and data was acquired on a BD FACSAria (San Jose, CA). iPSC were injected into the flank of immune deficient NSG mice and teratomas were harvested for C7 immunofluorescence as above.

Techniques: Mutagenesis, Immunofluorescence, Marker, Expressing, Derivative Assay, Staining, Western Blot, Immunostaining