Functional Comparison of Induced Pluripotent Stem Cell- and Blood-Derived GPIIbIIIa Deficient Platelets

Human induced pluripotent stem cells (hiPSCs) represent a versatile tool to model genetic diseases and are a potential source for cell transfusion therapies. However, it remains elusive to which extent patient-specific hiPSC-derived cells functionally resemble their native counterparts. Here, we generated a hiPSC model of the primary platelet disease Glanzmann thrombasthenia (GT), characterized by dysfunction of the integrin receptor GPIIbIIIa, and compared side-by-side healthy and diseased hiPSC-derived platelets with peripheral blood platelets. Both GT-hiPSC-derived platelets and their peripheral blood equivalents showed absence of membrane expression of GPIIbIIIa, a reduction of PAC-1 binding, surface spreading and adherence to fibrinogen. We demonstrated that GT-hiPSC-derived platelets recapitulate molecular and functional aspects of the disease and show comparable behavior to their native counterparts encouraging the further use of hiPSC-based disease models as well as the transition towards a clinical application

Functional comparison of induced pluripotent stem cell- and blood-derived GPIIbIIIa deficient platelets.

Human induced pluripotent stem cells (hiPSCs) represent a versatile tool to model genetic diseases and are a potential source for cell transfusion therapies. However, it remains elusive to which extent patient-specific hiPSC-derived cells functionally resemble their native counterparts. Here, we generated a hiPSC model of the primary platelet disease Glanzmann thrombasthenia (GT), characterized by dysfunction of the integrin receptor GPIIbIIIa, and compared side-by-side healthy and diseased hiPSC-derived platelets with peripheral blood platelets. Both GT-hiPSC-derived platelets and their peripheral blood equivalents showed absence of membrane expression of GPIIbIIIa, a reduction of PAC-1 binding, surface spreading and adherence to fibrinogen. We demonstrated that GT-hiPSC-derived platelets recapitulate molecular and functional aspects of the disease and show comparable behavior to their native counterparts encouraging the further use of hiPSC-based disease models as well as the transition towards a clinical application

Flow cytometry of PAC-1 binding and integrin surface expression after platelet activation.

<p>(A) Cells were stained with PAC-1 (x-axis) and anti-CD42b (y-axis) antibodies in the absence (top) or presence (bottom) of ADP/TXA₂. (B) Flow cytometry of CD41 (GPIIb) and CD61 (GPIIIa) surface expression on hiPSC-derived and peripheral blood platelets in the presence of ADP/TXA₂. After activation with ADP/TXA₂, cells were stained with anti-CD42b, anti-CD41/CD61, anti-CD41 and anti-CD61 antibodies. The FSC/SSC log gate of peripheral platelets was applied and further gated for CD42b+ cells.</p

Surface spreading, adhesion and cytoskeleton rearrangement of peripheral blood and hiPSC-derived platelets.

<p>(A) Platelets spreading on fibrinogen-coated wells were imaged with DICM in the presence of ADP/TXA₂ at the indicated time points. For complete movie sequence see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115978#pone.0115978.s012" target="_blank">S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115978#pone.0115978.s015" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115978#pone.0115978.s016" target="_blank">S5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115978#pone.0115978.s018" target="_blank">S7</a> Movies. (B) Quantification of fibrinogen binding of MKs and platelets. The percentages of attached cells are shown as scatter columns with horizontal bars representing the mean. In the hiPSC-derived groups, red and black symbols indicate results from the two hiPSC clones. A total mean of 1991±262.6 cells per sample was counted before washing. P-values from unpaired t-tests; peripheral blood platelets from GT (n = 10) and CTR (n = 10); hiPSC-derived platelets from GT (n = 5) and CTR (n = 6). (C) Rhodamine-conjugated phalloidin was used to stain actin filaments after permeabilization of platelets spreading on fibrinogen. All cells shown stained positive for CD42b and negative for DAPI. All scale bars represent 10 μm. Representative images for each sample.</p

Generation and integrin surface expression of hiPSC-derived platelets from GT and CTR.

<p>(A) Immunofluorescence (top) and flow cytometry (bottom) of hiPSC-derived and peripheral blood platelets. The cells were stained with anti-CD42b (red / y-axis), anti-CD41/CD61 (green / x-axis) antibodies and DAPI (blue). (B) Flow cytometry of CD41 (GPIIb) and CD61 (GPIIIb) surface expression on hiPSC-derived and peripheral blood platelets. The cells were stained with anti-CD42b, anti-CD41/CD61, anti-CD41 and anti-CD61 antibodies. The FSC/SSC log gate of peripheral platelets was applied and further gated for CD42b+ cells. All scale bars represent 10 μm. Representative images for each sample.</p