Selective Inhibition of ADAM17 Efficiently Mediates Glycoprotein Ibα Retention During Ex Vivo Generation of Human Induced Pluripotent Stem Cell-Derived Platelets

Abstract

Donor-independent platelet concentrates for transfusion can be produced in vitro from induced pluripotent stem cells (iPSCs). However, culture at 37°C induces ectodomain shedding on platelets of glycoprotein Ibα (GPIbα), the von Willebrand factor receptor critical for adhesive function and platelet lifetime in vivo, through temperature-dependent activation of a disintegrin and metalloproteinase 17 (ADAM17). The shedding can be suppressed by using inhibitors of panmetalloproteinases and possibly of the upstream regulator p38 mitogen-activated protein kinase (p38 MAPK), but residues of these inhibitors in the final platelet products may be accompanied by harmful risks that prevent clinical application. Here, we optimized the culture conditions for generating human iPSC-derived GPIbα⁺ platelets, focusing on culture temperature and additives, by comparing a new and safe selective ADAM17 inhibitor, KP-457, with previous inhibitors. Because cultivation at 24°C (at which conventional platelet concentrates are stored) markedly diminished the yield of platelets with high expression of platelet receptors, 37°C was requisite for normal platelet production from iPSCs. KP-457 blocked GPIbα shedding from iPSC platelets at a lower half-maximal inhibitory concentration than panmetalloproteinase inhibitor GM-6001, whereas p38 MAPK inhibitors did not. iPSC platelets generated in the presence of KP-457 exhibited improved GPIbα-dependent aggregation not inferior to human fresh platelets. A thrombus formation model using immunodeficient mice after platelet transfusion revealed that iPSC platelets generated with KP-457 exerted better hemostatic function in vivo. Our findings suggest that KP-457, unlike GM-6001 or p38 MAPK inhibitors, effectively enhances the production of functional human iPSC-derived platelets at 37°C, which is an important step toward their clinical application. Stem Cells Translational Medicine 2017;6:720-730.

Keywords: Cell culture; Cell transplantation; Induced pluripotent stem cell; Megakaryocyte; Mitogen-activated protein kinase; Thrombopoiesis.

Figure 1

Cultivation at 37°C was necessary for efficient megakaryopoiesis and thrombopoiesis of human iPSC‐derived HPCs. (A): Schematic diagram of the in vitro differentiation protocol. To generate CD41a⁺ MKs and CD41a⁺ GPIX (CD42a)⁺ GPIbα (CD42b)⁺ platelets, human iPS‐sac‐derived HPCs were incubated with SCF, TPO, and heparin on C3H10T1/2 feeder cells at 24°C or 37°C during the MK differentiation phase (days 14–20) and platelet production phase (days 20–24). The numbers of MKs (B) and platelets (C) derived from iPSCs were measured under the different temperature conditions. ∗, p < .05 vs. 37°C culture condition on days 14–24 by Dunnett’s test, n ≥ 3. Abbreviations: GP, glycoprotein; HPC, hematopoietic progenitor cell; iPS, induced pluripotent stem; iPSC, induced pluripotent stem cell; MK, megakaryocyte; SCF, stem cell factor; TPO, thrombopoietin, VEGF, vascular endothelial growth factor.

Figure 2

CCCP‐induced GPIbα shedding was completely blocked by the ADAM17 inhibitor KP‐457 but not by p38 MAPK inhibitors. (A): Structure of KP‐457. (B): Inhibitory effects of KP‐457 and GM‐6001 on ADAM17 enzymatic activity under cell‐free conditions. n = 3. (C): Representative FACS dot plots showing the inhibitory effect of KP‐457 (15 µmol/l) and BIRB796 (10 µmol/l) on CCCP‐induced GPIbα shedding from human washed platelets. Washed platelets were incubated with 100 µmol/l CCCP in the presence of inhibitors for 24 hours at 37°C. The GPIbα⁺ fraction among CD41a⁺ platelets was measured. (D): Dose‐dependent effects of the indicated inhibitors on GPIbα retention by platelets treated with CCCP. n ≥ 3. Abbreviations: ADAM, a disintegrin and metalloproteinase; CCCP, carbonyl cyanide m‐chlorophenylhydrazone; FACS, fluorescence‐activated cell sorting; GP, glycoprotein; MAPK, mitogen‐activated protein kinase.

Figure 3

GPIbα shedding from platelets generated in vitro from iPSC‐MKs was effectively suppressed by KP‐457 but not by p38 MAPK inhibitors. (A) Numbers of CD41a⁺ iPSC‐derived MKs. Human HPCs were incubated for 8–10 days in differentiation medium supplemented with stem cell factor, thrombopoietin, heparin, and/or inhibitors at 37°C, followed by flow cytometric analysis. p38 MAPK inhibitors increased MK yields, whereas KP‐457 and GM‐6001 had only minor effects. (B) Numbers of CD41a⁺ iPSC platelets. (C) Representative FACS dot plots of platelets generated from iPSC‐derived HPCs in the presence of KP‐457 or BIRB796. (D) Percentages of the GPIbα⁺ population in CD41a⁺ platelets. Metalloproteinase inhibitors, including KP‐457, but not p38 MAPK inhibitors, enabled GPIbα retention by iPSC‐derived platelets. ∗, p < .05 vs. vehicle group by Dunnett’s test, n ≥ 4. Abbreviations: FACS, fluorescence‐activated cell sorting; GP, glycoprotein; HPC, hematopoietic progenitor cell; iPSC, induced pluripotent stem cell; MAPK, mitogen‐activated protein kinase; MK, megakaryocyte.

Figure 4

Combination of KP‐457 and BIRB796 exerted an additive effect on GPIbα⁺ platelets yields. Percentages of the GPIbα⁺ population in CD41a⁺ platelets (A) and numbers of CD41a⁺ GPIbα⁺ platelets generated from human iPSCs (B) with or without KP‐457 (15 µmol/l) and BIRB796 (10 µmol/l). ∗, p < .05 vs. vehicle group by Dunnett’s test, n = 4. Abbreviations: GP, glycoprotein; HPC, hematopoietic progenitor cell; iPSC, induced pluripotent stem cell.

Figure 5

Direct inhibition of ADAM17 more effectively sustained GPIbα retention by cultured iPSC‐derived platelets than suppression of potential upstream activators of ADAM17. Percentages of the GPIbα⁺ population in CD41a⁺ iPSC‐derived platelets treated with the indicated inhibitors during the platelet production phase (days 21–23 or 24). KP‐457 (15 μmol/l), BIRB796 (10 μmol/l), MAPK/extracellular signal‐regulated kinase kinase inhibitor U0126 (10 μmol/l), IkB kinase β inhibitor BMS345541 (10 μmol/l), caspase inhibitor Z‐VAD (10 μmol/l) and protein kinase C inhibitor Ro31‐8220 (3 μmol/l) were used. Inhibition of potential ADAM17 activator molecules failed to prevent GPIbα shedding evoked by cultivation at 37°C. ∗, p < .05 versus vehicle group by Dunnett's test, n ≥ 3. Abbreviations: ADAM, a disintegrin and metalloproteinase; GP, glycoprotein; iPSC, induced pluripotent stem cell.

Figure 6

GPIbα‐retaining iPSC platelets generated in the presence of KP‐457 exhibited an improved response to vWF in vitro and hemostatic function in vivo. (A): Transmission electron micrographs of imMKCL platelets produced with or without KP‐457. Scale bar, 2 µm. (B): Percentage of double‐colored events served as an aggregation index in a flow cytometry‐based platelet aggregation assay using peripheral blood‐derived platelets or iPSC‐derived platelets generated with or without 15 µmol/l KP‐457. A mixture of APC‐ and V450‐labeled platelets was stimulated using 2 mg/ml ristocetin to evoke vWF/GPIbα‐dependent platelet aggregation. ∗, p < .05 vs. without KP‐457, n ≥ 4. (C): Representative sequential images of GPIbα⁺ iPSC platelets generated with KP‐457 in mouse thrombus formation model. A combination of FITC‐dextran (green), Hoechst33342 (blue), and equal numbers of tetramethylrhodamine‐labeled iPSC platelets (1 × 10⁷ CD41a⁺ platelets, red) generated with or without KP‐457 were transfused into irradiated NOG mice. Hematoporphyrin was also administered to induce thrombus formation by chemical reaction after laser‐induced injury. Mesenteric capillaries were visualized using a confocal laser scanning microscope. Attachment of platelets including tetramethylrhodamine‐positive iPSC platelets on injured blood vessels and subsequent thrombus formation were observed after the injury. White arrow, direction of blood flow; pink arrow, site of thrombus formation. Scale bar, 10 µm. (D): The number of iPSC platelets in thrombus formation was estimated using the images of 30 vessels from two mice of the same group in two independent experiments. GPIbα‐retained iPSC‐derived platelets with KP‐457 exerted more effective hemostatic function in vivo. ∗, p = .0556 vs. vehicle group, n = 30. Abbreviations: APC, allophycocyanin; GP, glycoprotein; imMKCL, immortalized megakaryocyte progenitor cell line; iPSC, induced pluripotent stem cell; vWF, von Willebrand factor.