CDK8/19 inhibition plays an important role in pancreatic β-cell induction from human iPSCs

Abstract

Background: Transplantation of differentiated cells from human-induced pluripotent stem cells (hiPSCs) holds great promise for clinical treatments. Eliminating the risk factor of malignant cell transformation is essential for ensuring the safety of such cells. This study was aimed at assessing and mitigating mutagenicity that may arise during the cell culture process in the protocol of pancreatic islet cell (iPIC) differentiation from hiPSCs.

Methods: We evaluated the mutagenicity of differentiation factors used for hiPSC-derived pancreatic islet-like cells (iPICs). We employed Ames mutagenicity assay, flow cytometry analysis, immunostaining, time-resolved fluorescence resonance energy transfer-based (TR-FRET) cell-free dose-response assays, single-cell RNA-sequencing and in vivo efficacy study.

Results: We observed a mutagenic effect of activin receptor-like kinase 5 inhibitor II (ALK5iII). ALK5iII is a widely used β-cell inducer but no other tested ALK5 inhibitors induced β-cells. We obtained kinase inhibition profiles and found that only ALK5iII inhibited cyclin-dependent kinases 8 and 19 (CDK8/19) among all ALK5 inhibitors tested. Consistently, CDK8/19 inhibitors efficiently induced β-cells in the absence of ALK5iII. A combination treatment with non-mutagenic ALK5 inhibitor SB431542 and CDK8/19 inhibitor senexin B afforded generation of iPICs with in vitro cellular composition and in vivo efficacy comparable to those observed with ALK5iII.

Conclusion: Our findings suggest a new risk mitigation approach for cell therapy and advance our understanding of the β-cell differentiation mechanism.

Keywords: Activin receptor-like kinase 5 inhibitor II; CDK8/19 inhibitors; Human-induced pluripotent stem cells; Mutagenicity; Pancreatic islet cell.

Fig. 1

Mutagenicity of the naphthyridine derivative and endocrine cell inducer ALK5 inhibitor II in the Ames test. a Schematic representation of the protocol for iPIC differentiation from hiPSCs. Small-molecule compounds (14 reagents; 11 widely used reagents plus 3 original ones in italic) are highlighted in bold. Act A, activin A; CHIR, CHIR99021; K-CYC, KAAD-cyclopamine; TTNPB, 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl] benzoic acid; ROCKi, TR05991851; PDBu, phorbol 12, 13-dibutyrate; SANT, SANT-1; ALK5iII, ALK5 inhibitor II; LDN, LDN-193189; XAV, XAV939; Y, Y-27632; GSI, RO4929097; PD, PD-166866; R428, bemcentinib; and TR, TR06141363. b Flowchart of mutagenicity evaluation of the 14 compounds used in the iPIC differentiation protocol. Red and blue arrows indicate positive and negative results, respectively. Cpds, compounds. c Positive results (more than twofold increase above the value in the concurrent vehicle control) in the bacterial reverse mutation assay in the absence of rat liver S9 fraction. 9-AA, 9-aminoacridine; ICR, ICR 191. d Summary of the mutagenicity evaluation results for ALK5iII, a non-ALK5 inhibitor structurally related to ALK5iII (TR04411299), and other ALK5 inhibitors (SB525334 and SB431542)

Fig. 2

CDK8/19 are the primary candidates for the off-target action of ALK5iII during β-cell induction. a, b Representative screening results for an alternative ALK5 inhibitor to induce iPICs. Here, we selected the well-characterized ALK5 inhibitors, SB431542 and SB525334 plus randomly selected two compounds IN1130 and EW-7197, as representative compounds out of twenty. Representative dot plots (left, 10 μM SB525334; 10 μM SB431542; 1 μM IN1130; and 1 μM EW-7197) and average proportions of β-cells (INSULIN⁺NKX6.1⁺) (right) (a) and cell yields per aggregate (b). Data are shown as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 versus cells that were not treated by an ALK5 inhibitor; Dunnett’s test. c The list of kinases inhibited by ALK5iII with estimated pIC₅₀ values > 6.00 in Axcelead TR-FRET-based kinase profiling assays. (ALK5iII was screened against approximately 350 kinases.) The pIC₅₀ values were extrapolated from the inhibition rate at two ALK5iII concentrations, 0.1 and 1 μM. Data are shown as the mean ± SD of three independent experiments. The values “8.00*” indicate that actual pIC₅₀ values were predicted to be > 8.00 from the extrapolated result. d Comprehensive comparison of the relative inhibition of the 11 kinases listed in c by ALK5iII and other representative ALK5 inhibitors, described in a and b, at a concentration of 1 μM. Data are shown as the mean ± SD from three independent experiments

Fig. 3

Proof of efficient β-cell induction by CDK8/19 inhibition. a Representative dot plots from the FCM analysis (left, 0.3 μM TAK-583; 3 nM BI-1347; and 0.1 μM senexin B) and dose–response relationships describing the induction of β-cells by CDK8/19 inhibitors (right). Data are shown as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 versus cells that were not treated by either ALK5iII or CDK8/19 inhibitors; Dunnett’s test. b Schematic docking model of a pair of on-target pharmacological tools, TR06096159 and TR05978156, bound to CDK8 (modified from the previous report [32]). c Proportions of β-cells differentiated with TR06096159, which binds CDK8, and TR05978156, which does not bind CDK8. Data are shown as the mean ± SD from three independent experiments. *P < 0.05 and **P < 0.01 versus untreated cells; Dunnett’s test

Fig. 4

Transcriptome dissection reveals distinctive roles of ALK5 and CDK8/19 inhibition for endocrine cell induction. a, b iPICs were differentiated with 10 µM ALK5iII or another ALK5 inhibitor, 3 µM SB431542, and/or the CDK8/19 inhibitor, 0.3 µM senexin B to replace ALK5iII. Representative dot plots from FCM analysis (a, left), average proportions of β-cells (a, right), and density of obtained cells (b) are illustrated. Data are representative of three independent experiments and presented as the mean ± SD (n = 4 technical replicates). *P < 0.05 and **P < 0.01 versus data from the ALK5iII-treated cells, Dunnett’s test. c and d Individual (c) and merged (d) distributions of cells based on their gene expression profiles, shown as uniform manifold projections for four types of iPICs treated with ALK5 and CDK8/19 inhibitors as described in a and b. e Shared nearest neighbor clustering divided iPICs into seventeen cell clusters. Superimposed annotation indicated identified cell types based on both DEG-driven enrichment analysis and expression levels of well-validated cellular markers. f The proportion of cells assigned from each sample in each cluster. Total cell numbers were uniformly targeted to 3000 cells for all four samples. For “Common clusters among ALK5iII-, Sen- and SB/Sen-cells groups,” less than 5% of cells were categorized from SB-cells. For “SB-cells dominant clusters” groups, more than 66% of cells were SB-cells. g Internal composition of cells highly expressing INS and NKX6.1 (clusters #1, 3, 6, and 12) within each sample. Pie charts describe the proportions of the cells allocated to each cluster. h Differentially gene expression analysis in iPIC samples compared to data from ALK5iII-treated cells. The number of up- and down-regulated genes (P < 0.05 and FC < 2 or >  − 2) were presented in the scatter plots in red or blue

Fig. 4

Transcriptome dissection reveals distinctive roles of ALK5 and CDK8/19 inhibition for endocrine cell induction. a, b iPICs were differentiated with 10 µM ALK5iII or another ALK5 inhibitor, 3 µM SB431542, and/or the CDK8/19 inhibitor, 0.3 µM senexin B to replace ALK5iII. Representative dot plots from FCM analysis (a, left), average proportions of β-cells (a, right), and density of obtained cells (b) are illustrated. Data are representative of three independent experiments and presented as the mean ± SD (n = 4 technical replicates). *P < 0.05 and **P < 0.01 versus data from the ALK5iII-treated cells, Dunnett’s test. c and d Individual (c) and merged (d) distributions of cells based on their gene expression profiles, shown as uniform manifold projections for four types of iPICs treated with ALK5 and CDK8/19 inhibitors as described in a and b. e Shared nearest neighbor clustering divided iPICs into seventeen cell clusters. Superimposed annotation indicated identified cell types based on both DEG-driven enrichment analysis and expression levels of well-validated cellular markers. f The proportion of cells assigned from each sample in each cluster. Total cell numbers were uniformly targeted to 3000 cells for all four samples. For “Common clusters among ALK5iII-, Sen- and SB/Sen-cells groups,” less than 5% of cells were categorized from SB-cells. For “SB-cells dominant clusters” groups, more than 66% of cells were SB-cells. g Internal composition of cells highly expressing INS and NKX6.1 (clusters #1, 3, 6, and 12) within each sample. Pie charts describe the proportions of the cells allocated to each cluster. h Differentially gene expression analysis in iPIC samples compared to data from ALK5iII-treated cells. The number of up- and down-regulated genes (P < 0.05 and FC < 2 or >  − 2) were presented in the scatter plots in red or blue

Fig. 5

Generation of iPICs using a non-mutagenic ALK5 inhibitor and CDK8/19 inhibitor instead of mutagenic ALK5iII. a, b Blood glucose (a) and human C-peptide (b) levels after ALK5iII- and SB/Sen-cells (3 × 10⁶ cells/mouse) implantation in diabetic NOD-scid mice. Data are shown as the mean ± SD (n = 4–5 for ALK5iII, n = 3–4 for SB/Sen, n = 1–5 for sham). Unexpected death occurred during the period due to both hyperglycemia and hypoglycemia. All sham mice died by 21 weeks after implantation. Black arrow indicates streptozotocin injection point. c Plasma glucose levels during an oral glucose tolerance test at 21 weeks after implantation. Data are shown as the mean ± SD (ALK5iII-cells; n = 4, SB/Sen-cells; n = 3). d Representative sectional images of grafts 6 months after implantation. Sections were stained with antibodies against insulin (INS), glucagon (GCG) (green/red), and Ki-67/HuN (green/red). Scale bars (white) indicate 0.5 mm