Vinblastine treatment decreases the undifferentiated cell contamination of human iPSC-derived intestinal epithelial-like cells

Abstract

Human induced pluripotent stem cell-derived intestinal epithelial cells (hiPSC-IECs) are expected to be utilized in regenerative medicine. To perform a safe transplantation without the risk of tumor formation, residual undifferentiated hiPSCs must be removed from hiPSC-IECs. In this study, we examined whether vinblastine (a multiple drug resistance 1 [MDR1] substrate) could remove residual undifferentiated hiPSCs in hiPSC-IECs and attempted to generate hiPSC-IECs applicable to transplantation medicine. We found that the expression levels of pluripotent markers were largely decreased and those of intestinal markers were increased by vinblastine treatment. The treatment of undifferentiated hiPSCs with vinblastine significantly decreased their viability. These results suggested that undifferentiated hiPSCs can be eliminated from hiPSC-IECs by vinblastine treatment. We hypothesized that MDR1-negative cells (such as undifferentiated hiPSCs) die upon vinblastine treatment because they are unable to excrete vinblastine. As expected, the cell viability of MDR1-knockout hiPSC-IECs was significantly decreased by vinblastine treatment. Furthermore, teratomas were formed by subcutaneous transplantation of hiPSC-IECs mixed with undifferentiated hiPSCs into mice, but they were not observed when the transplanted cells were pre-treated with vinblastine. Vinblastine-treated hiPSC-IECs would be an effective cell source for safe regenerative medicine.

Keywords: differentiation; iPS cell; intestinal epithelial cells; regenerative medicine; vinblastine.

Graphical abstract

Figure 1

Removal of residual undifferentiated hiPSCs by vinblastine treatment (A) The schematic overview shows the protocol of intestinal differentiation of hiPSCs. Details are described in materials and methods section. (B) The hiPSC-IECs (YOW) were treated with 10 nM vinblastine during the intestinal maturation step (from day 17 to 27). The gene expression levels of pluripotent markers (NANOG, OCT3/4, and ESG1) and definitive endoderm (DE) markers (FOXA2, SOX17, and HEX) were examined by real-time RT-PCR. The gene expression levels in the DMSO-treated cells were taken as 1.0. The results are represented as means ± SD (n ≥ 3, technical replicate). (C) The undifferentiated hiPSCs, DE cells, intestinal progenitor cells, and hiPSC-IECs were treated with 10 nM vinblastine for 3 days, and the cell viability was measured. The cell viability of DMSO-treated cells was taken as 100%. The results are represented as means ± SD (n ≥ 3, technical replicate). (D) The percentage of TRA1-81-positive cells and CXCR4-positive cells in DMSO- or vinblastine (10 nM)-treated hiPSC-IECs was examined by flow cytometry. The results are represented as means ± SD (n = 3, technical replicate). (E) To estimate the number of residual undifferentiated cells in DMSO- or vinblastine (10 nM)-treated hiPSC-IECs, a colony formation assay was performed. DMSO- or vinblastine (10 nM)-treated hiPSC-IECs were cultured with AK02N medium on iMatrix-511 for 7 days. The cells were stained by alkaline phosphatase (ALP), and the number of ALP-positive colonies was counted. The results are represented as means ± SD (n = 3, technical replicate). Statistical analyses were performed using the unpaired two-tailed Student’s t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 2

Elimination of MDR1-negative cells by vinblastine treatment (A) The hiPSCs (YOW) were differentiated into IECs. The gene expression levels of MDR1 in undifferentiated hiPSCs (day 0), DE cells (day 3), intestinal progenitor cells (day 7), and hiPSC-IECs (day 28) were examined by real-time RT-PCR. The results are represented as means ± SD (n ≥ 3, technical replicate). (B) After 10 nM vinblastine treatment, the cell viability of wild-type (WT) hiPSC-IECs and MDR1-knockout hiPSC-IECs was examined. The cell viability of DMSO-treated WT hiPSC-IECs was taken as 100%. (C) The hiPSCs were transduced with 1,000 VP/mL of Ad-LacZ or Ad-MDR1 for 90 min. At 48 h post transduction, hiPSC-IECs were treated with DMSO or vinblastine (10 nM) for 10 days, and the cell viability was examined. The cell viability of the Ad-LacZ-transduced hiPSCs that were treated with DMSO was taken as 100%. The data of (B) and (C) are represented as means ± SD (n = 3, technical replicate). Statistical analyses were performed using the unpaired two-tailed Student’s t test (∗∗∗p < 0.001).

Figure 3

Enhancement of intestinal functions of hiPSC-IECs by vinblastine treatment The hiPSCs (YOW) were differentiated into IECs. During the intestinal maturation step (from day 18 to 28), the hiPSC-IECs were treated with 10 nM vinblastine. (A) The gene expression levels of intestinal markers (VIL1, SI, and FABP2) and drug-metabolizing enzyme and transporters (CYP3A4, MDR1, BCRP, and PEPT1) in hiPSC-IECs were examined by real-time RT-PCR. The gene expression levels in the human small intestine were taken as 1.0. The results are represented as means ± SD (n = 3, technical replicate). (B) The protein expression levels of CYP3A4 in DMSO- or vinblastine (1, 3, 10 nM)-treated hiPSC-IECs were examined by western blotting. (C) The CYP3A4 activities in DMSO- or vinblastine (1, 3, 10 nM)-treated hiPSC-IECs were examined. (D) The ALP staining was performed in DMSO- or vinblastine (10 nM)-treated hiPSC-IECs. (E) The VIL1 and E-cadherin expression levels were examined by immunostaining analysis. Nuclei were counterstained with DAPI. Scale bars, 50 μm. (F) Transepithelial electrical resistance (TEER) values of the DMSO- or vinblastine (10 nM)-treated hiPSC-IEC monolayers were measured by Millicell-ERS2. The results are represented as means ± SD (n ≥ 10, technical replicate). (G) Apical-to-basolateral permeability of FD4 (FITC-dextran 4 kDa) across the DMSO- or vinblastine (10 nM)-treated hiPSC-IEC monolayers was measured. The results are represented as means ± SD (n = 3, technical replicate). Statistical significance was evaluated by one-way ANOVA followed by Dunnett’s post hoc test (∗p < 0.05, ∗∗p < 0.01, compared with control).

Figure 4

Safety assessment of hiPSC-IECs (A and B) Dorsal and ventral view of the same mouse transplanted with DMSO-treated hiPSC-IECs (YOW). To perform the teratoma assay, a mixture of undifferentiated hiPSCs (5 × 10⁶ cells/mouse) and hiPSC-IECs (5 × 10⁶ cells/mouse) was subcutaneously transplanted after the DMSO or vinblastine (10 nM) treatment. At 8 weeks from the transplantation, we determined the presence or absence of teratomas. (C) H&E staining of teratoma sections. Scale bars, 200 μm.