Immunologic targeting of CD30 eliminates tumourigenic human pluripotent stem cells, allowing safer clinical application of hiPSC-based cell therapy

Abstract

Induced pluripotent stem cells (iPSCs) are promising candidate cells for cardiomyogenesis in the failing heart. However, teratoma/tumour formation originating from undifferentiated iPSCs contaminating the graft is a critical concern for clinical application. Here, we hypothesized that brentuximab vedotin, which targets CD30, induces apoptosis in tumourigenic cells, thus increasing the safety of iPSC therapy for heart failure. Flow cytometry analysis identified consistent expression of CD30 in undifferentiated human iPSCs. Addition of brentuximab vedotin in vitro for 72 h efficiently induced cell death in human iPSCs, associated with a significant increase in G2/M phase cells. Brentuximab vedotin significantly reduced Lin28 expression in cardiomyogenically differentiated human iPSCs. Transplantation of human iPSC-derived cardiomyocytes (CMs) without treatment into NOG mice consistently induced teratoma/tumour formation, with a substantial number of Ki-67-positive cells in the graft at 4 months post-transplant, whereas iPSC-derived CMs treated with brentuximab vedotin prior to the transplantation did not show teratoma/tumour formation, which was associated with absence of Ki-67-positive cells in the graft over the same period. These findings suggest that in vitro treatment with brentuximab vedotin, targeting the CD30-positive iPSC fraction, reduced tumourigenicity in human iPSC-derived CMs, potentially providing enhanced safety for iPSC-based cardiomyogenesis therapy in clinical scenarios.

Figure 1

Expression of CD30 on hiPSCs (a) Expression of surface markers was compared between hiPSCs and hiPSC derived-CMs. The data are representative of at least three independent experiments. (b) Expression of CD30 on several cell surfaces. The number in the histograms indicates the percentage of CD30-positive cells. The data are representative of at least three independent experiments. (c) Expression of CD30 on hiPSC-derived CMs. The number in the histograms indicates the percentage of CD30-positive cells. The data are representative of at least three independent experiments. (d) Down-regulation of CD30 expression during hiPSC differentiation into CMs. The data are representative of at least three independent experiments.

Figure 2

Effect of brentuximab vedotin on viability of hiPSCs and differentiated cells (a) Ff-I14 cells, (b) Ff-I01 cells, (c) 253G1 cells, (d) 201B7 cells, (e) NHDFs, and (f) iPSC-derived CMs were treated with brentuximab vedotin at 0, or 5 μg/ml for 24, 48, and 72 h. Panels are representative phase-contrast images of cells. The relative viabilities of cells in response to brentuximab vedotin treatment were determined using CCK-8 solution. Graph data were repeated at least three times independently.

Figure 3

Effect of brentuximab vedotin on cell cycle (a) 253G1 cells, (b) 201B7 cells, (c) Ff-I14 cells, (d) Ff-I01 cells, and (e) NHDFs were treated with increasing concentrations of brentuximab vedotin. At 10 h after treatment, cells were stained with propidium iodide (PI) to detect DNA content and analysed by flow cytometry. Bar graphs indicate the percentage of cells in different phases of the cell cycle. Data were collected from at least three independent experiments. *p < 0.05 vs. 0 μg/ml.

Figure 4

Effect of brentuximab vedotin on apoptosis of hiPSCs (a) 253G1 cells, (b) 201B7 cells, (c) Ff-I14 cells, (d) Ff-I01 cells, (e) NHDFs, and (f) iPSC-derived CMs were treated with increasing concentrations of brentuximab vedotin. At 48 h after treatment, apoptotic cells were determined by flow cytometry following annexin-V staining and PI staining. Bar graphs indicate the percentage of annexin-V–positive cells and PI-positive cells. Data were collected from at least three independent experiments. *p < 0.05 vs. 0 μg/ml.

Figure 5

Effect of brentuximab vedotin on the expression of Lin28. Expression of Lin28 in hiPSC-derived CMs after brentuximab vedotin treatment was determined by qRT-PCR analysis. hiPSC-derived CMs were treated with brentuximab vedotin at the indicated doses for 72 and 96 h. Total RNA was isolated from the cells. Y-axis indicates relative gene expression compared with non-treated hiPSC-derived CMs for 72 h. Data were collected from at least three independent experiments. *p < 0.05 vs. 72 h, 0 μg/ml.

Figure 6

Cytotoxicity of brentuximab vedotin to hiPSC-derived CMs (a) Cytotoxicity of brentuximab vedotin in hiPSC-derived CMs was assessed by LDH assay. Y-axis indicates relative cytotoxicity compared with non-treated hiPSC-derived CMs for 72 h. Experiments were repeated at least thrice independently. *p < 0.01 vs. 72 h, 0 μg/ml. (b) Contraction and relaxation velocity of hiPSC-derived CMs was assessed by motion analyser system. Y-axis indicates relative change compared to pre-treatment hiPSC-derived CMs. (c) The percentage cTnT-expressing hiPSC-derived CMs and the relative number of hiPSC-derived CMs were assessed after they were cultured in normal medium for a week. *p < 0.05 vs. 72 h, 0 μg/ml. (d) Effect of brentuximab vedotin on cell sheet formation. At 72 h after starting treatment of hiPSC-derived CMs with brentuximab vedotin in a temperature-responsive plate, monolayered cell sheets were fabricated using cells treated with brentuximab vedotin at 10 μg/ml (left) and 20 μg/ml (right). Panels are representative macroscopic images of monolayered cell sheets.

Figure 7

Effect of brentuximab vedotin on teratoma/tumour formation. Teratoma formation in NOG mice 4 months after transplantation of hiPSC-derived CMs with or without brentuximab vedotin treatment. (a) Representative images of subcutaneous tissue stained with antibodies against human-specific lamin and cardiac troponin T. Scale bars, 20 μm. Positive cells are seen as dark brown. (b) Representative images of cTnT-positive cells. Scale bars, 50 μm. Positive cells are seen as green. (c) Representative images of subcutaneous tissue stained with antibody against Ki-67. Scale bars, 20 μm. Positive cells are seen as dark brown. The number of Ki-67–positive cells in the subcutaneous tissue was calculated. *p < 0.01 vs. non-treated group (d) Representative images of heart tissue stained with antibody against Ki-67. Scale bars, 20 μm. Positive cells are seen as dark brown. The number of Ki-67 positive cells in the heart was calculated. *p < 0.01 vs. non-treated group (e) Representative images of tumour formation in the heart of NOG mice. hiPSC-derived CM sheets with or without brentuximab vedotin treatment were used.