Volume 24, Issue 6, Pages 685-694.e4 (June 2017) Efficient, Selective Removal of Human Pluripotent Stem Cells via Ecto-Alkaline Phosphatase-Mediated Aggregation of Synthetic Peptides  Yi Kuang, Kenji Miki, Callum J.C. Parr, Karin Hayashi, Ikue Takei, Jie Li, Mio Iwasaki, Masato Nakagawa, Yoshinori Yoshida, Hirohide Saito  Cell Chemical Biology  Volume 24, Issue 6, Pages 685-694.e4 (June 2017) DOI: 10.1016/j.chembiol.2017.04.010 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 685-694. e4DOI: (10. 1016/j. chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 The Ecto-Alkaline Phosphatase Expressed on iPSCs Mediates iPSC-Selective Cell Death (A) Illustration of the possible mechanism by which phospho-D-peptides induce iPSC-selective cell death. Placental-type alkaline phosphatase, which is highly expressed on iPSCs, induces dephosphorylation of the peptides. The spatial-temporal accumulation of dephosphorylated peptides around iPSCs generates peptide aggregates in situ that trigger stress and iPSC death. The absence or low expression of ecto-alkaline phosphatase in other cell types makes them tolerant to incubation with phospho-D-peptides. (B) Chemical structures of the phospho-D-peptide derivatives. (C) The viability of the human iPSC line 201B7 was reduced after 2 hr incubation with phospho-D-peptides (the data are presented as the mean ± SD; n = 3). See also Figure S1A. Cell Chemical Biology 2017 24, 685-694.e4DOI: (10.1016/j.chembiol.2017.04.010) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 D-3 Induces High Toxicity in Human iPSCs but Little Viability Loss in Other Cell Types (A) D-3-induced viability loss in human fibroblasts, primary human cells, and cells differentiated from 201B7 cells using different methods. Spontaneously differentiated 201B7 cells were produced by incubating 201B7 cells in the absence of bFGF for 16 days. Neurons and EGFP-positive cardiomyocytes were differentiated from 201B7 cells and purified by FACS before use (the data are presented as the mean ± SD; n = 3 for NHDFs and spontaneously differentiated 201B7 cells; n = 2 for neurons and cardiomyocytes derived from 201B7 cells; n = 3 samples in one experiment using primary hepatocytes and renal proximal tubule epithelial cells). (B) D-3 toxicity in three iPSC lines. The cells were seeded at a density of 2.6 × 104 cells/cm2 to form small colonies or a density of 5.3 × 104 cells/cm2 to form large colonies (the data are presented as the mean ± SD; n = 3). (C) Percentage of live 201B7 cells after daily treatment with D-3 compared with control cells with a daily medium change. 201B7 cells were seeded at different densities to form small colonies, large colonies, or a nearly confluent monolayer. NHDF cells (seeding density of 5.3 × 104 cells/cm2) were also treated daily as a control (the data are presented as the mean ± SD; n = 3). See also Figure S2. Cell Chemical Biology 2017 24, 685-694.e4DOI: (10.1016/j.chembiol.2017.04.010) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Ecto-Alkaline Phosphatase and Spatial-Temporal D-3 Aggregation Are Essential for Viability Loss (A) Alkaline phosphatase expression (antibody staining) and the viability (live cell count) after 1 hr treatment of D-3 at 400 μM on different types of cells (iPSCs; 201B7 and 1390C1, ESCs; khES1, mesenchymal stem cells [MSC]). For the viability test, the data are presented as the mean ± SD; n = 3. For the ALP amount, the data are presented as the mean ± SE; n = 3. (B) Congo red staining indicates aggregation of peptides after 15 min (NHDF and HeLa) or 5 min (khES1 and 201B7) treatment with 300 μM of D-3. Scale bar, 20 μm. (C) ALP activity and relative viability of 293FT cells transfected with different amounts of native placental-type ALP mRNA after 1 hr treatment with 400 μM D-3. The 293FT cells after the same medium change were used as controls. For ALP activity, the data are presented as the mean ± SD; n = 2 independent experiments each containing triplicate samples. For toxicity, the data are presented as the mean ± SD; n = 3. (D) Loss of 201B7 cell viability induced by D-3 or D-4, measured by WST1 assay (the data are presented as the mean ± SD; n = 2 independent experiments performed in triplicate). See also Figures S3 and S4. Cell Chemical Biology 2017 24, 685-694.e4DOI: (10.1016/j.chembiol.2017.04.010) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 D-3 Specifically and Efficiently Eliminates iPSCs from a Mixed Population (A and B) Flow cytometry analysis (A) and mRNA profiling analysis (B) of co-cultured 201B7 cells and 201B7-derived MHY6-EGFP-expressing cardiomyocytes with or without 1 hr incubation with 400 μM D-3. The total seeding density was 5.3 × 104 cells/cm2; the seeding ratio was 1 (201B7): 9 (cardiomyocytes). (C) Daily treatment with 400 μM D-3 for 2 hr over several days removed large 201B7 colonies. The total seeding density was 5.3 × 104 cells/cm2; the seeding ratio was 7 (201B7): 3 (cardiomyocytes). See also Figures S1 and S5. Cell Chemical Biology 2017 24, 685-694.e4DOI: (10.1016/j.chembiol.2017.04.010) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 D-3 Prevents the Formation of Residual iPSC-Induced Teratoma (A) Representative images of testes approximately 3 months after the transplantation of 201B7 cells (2 × 104 201B7 cells; n = 5), cardiomyocytes (2 × 105 cardiomyocytes; n = 6), co-culture controls (2 × 104 201B7 and 1.8 × 105 cardiomyocyte; n = 8), and co-cultures treated with 400 μM D-3 for 1 hr (2 × 104 201B7 cells and 1.8 × 105 cardiomyocytes; n = 8). Scale bar, 1 cm. (B) Tumor incidence in each group. (C) Images of H&E staining of testes after the transplantation of cardiomyocytes or co-cultured cells treated with D-3. Scale bar, 1 mm. (D) Representative H&E images of the co-culture control and 201B7-derived teratoma containing tissues from the three embryonic germ layers. Scale bar, 200 μm. Cell Chemical Biology 2017 24, 685-694.e4DOI: (10.1016/j.chembiol.2017.04.010) Copyright © 2017 Elsevier Ltd Terms and Conditions