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. 2011 Mar;21(3):530-45.
doi: 10.1038/cr.2011.8. Epub 2011 Jan 11.

Platelets Generated From Human Embryonic Stem Cells Are Functional in Vitro and in the Microcirculation of Living Mice

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Free PMC article

Platelets Generated From Human Embryonic Stem Cells Are Functional in Vitro and in the Microcirculation of Living Mice

Shi-Jiang Lu et al. Cell Res. .
Free PMC article

Abstract

Platelets play an essential role in hemostasis and atherothrombosis. Owing to their short storage time, there is constant demand for this life-saving blood component. In this study, we report that it is feasible to generate functional megakaryocytes and platelets from human embryonic stem cells (hESCs) on a large scale. Differential-interference contrast and electron microscopy analyses showed that ultrastructural and morphological features of hESC-derived platelets were indistinguishable from those of normal blood platelets. In functional assays, hESC-derived platelets responded to thrombin stimulation, formed microaggregates, and facilitated clot formation/retraction in vitro. Live cell microscopy demonstrated that hESC-platelets formed lamellipodia and filopodia in response to thrombin activation, and tethered to each other as observed in normal blood. Using real-time intravital imaging with high-speed video microscopy, we have also shown that hESC-derived platelets contribute to developing thrombi at sites of laser-induced vascular injury in mice, providing the first evidence for in vivo functionality of hESC-derived platelets. These results represent an important step toward generating an unlimited supply of platelets for transfusion. Since platelets contain no genetic material, they are ideal candidates for early clinical translation involving human pluripotent stem cells.

Figures

Figure 1
Figure 1
Generation and characterization of megakaryocytes (MKs) derived from human embryonic stem cells (hESCs). (A) Numbers of CD41a+ cells generated from 8 × 104 hemangioblasts/blast cells derived from hESCs after 4-6 day culture in Stemline II medium supplemented with TPO (50 ng/ml) or TPO (50 ng/ml) plus SCF (20 ng/ml). ***P < 0.001, n = 4. (B) Average numbers of CD41a+ cells generated at different days. Blast cells were plated in Stemline II medium supplemented with TPO (50 ng/ml), SCF (20 ng/ml) and IL-11 (20 ng/ml), CD41a+ cells from four experiments were analyzed by flow cytometry. (C) Flow cytometry analyses of blast cells (day 0 MK culture, left panel), and cells from day 4 MK cultures show the expression of CD41a and CD235a (middle panel), and CD41a and CD42b (right panel) antigens. (D) Average size (diameter) of cells at the beginning (19.0 ± 4.2 μm, n = 111) and 6 days (25.6 ± 12.5 μm, n = 124) after MK culture. Cells were measured from digital images of Giemsa-stained cells. ***P < 0.001. (E) DNA ploidy analysis by flow cytometry of CD41a+ cells. DNA ploidy up to 32N was observed in these cells. (F) Giemsa staining of cells from day 6 MK culture. (G) Immunofluorescence of von Willebrand factor (vWF, red) and CD41 (green) proteins in cells from day 6 MK culture. vWF is localized in the cytoplasm in a punctate pattern, which is typical for MKs. CD41 is expressed on the surface. DAPI (blue) stain shows polynuclei (polyploidy). (H) A representative phase contrast image shows the pro-platelet forming MKs in day 4 cultures. Bars = 20 μm.
Figure 2
Figure 2
Characterization of platelets generated from human embryonic stem cells (hESC-PLTs). (A) The two panels show flow cytometry profiles of forward scatter (FSC) and side scatter (SSC; left), and CD41a and CD42b expression (right) on blood platelets. Approximately 80% of the gated particles express CD41a (y-axis) and CD42b (x-axis, top right). (B) The two panels show a flow cytometry FSC vs SSC profile (left), and CD41a vs CD42b expression (right) on hESC-PLTs. Approximately 30% of hESC-PLTs derived from OP9 feeder co-culture express both CD41a (y-axis) and CD42b (x-axis, top right). (C) Representative microscopic fields of hESC-PLT (top left) and blood platelet (top right) populations examined by differential interference contrast microscopy. Bar = 5 μm. hESc-PLTs are discoid in shape. Thin-section transmission electron microscopy of hESC-PLTs (lower left) and blood platelets (lower right). Bar = 1 μm. (D) Expression of P-selectin (PS) and platelet factor 4 (PF-4) on α-granules of blood platelets and hESC-PLTs. Immunogold electron microscopy of ultrathin cryosections showing the localization of P-selectin and PF-4 in resting blood platelets and hESC-PLTs. Bar = 1 μm.
Figure 3
Figure 3
Tubulin and filamentous actin staining in resting blood platelets and hESC-PLTs. Tubulin (green) and filamentous actin staining (red) of resting, blood platelets (A) and hESC-PLTs (B). Images are presented as light differential-interference contrast, anti-β1-tubulin staining, phalloidin staining, and an overlay. Plot profiles show distance in microns on the x-axis and fluorescence intensity on the y-axis. Bar = 5 μm.
Figure 4
Figure 4
Functional characterization of hESC-PLTs in vitro. (A) Human blood platelets and (B) hESC-PLTs spread on fibrinogen surface: microtiter chamber slides were coated with 100 μg/ml fibrinogen and human blood platelets treated with vehicle (CON, top left), 1 mM RGDS peptide (+RGDS, top right), 20 μM ADP (+ADP, bottom left) or 1 U/ml of thrombin (+TH, bottom right), were plated and incubated for 90 min. Adherent platelets were stained with Alexa Fluor 568 phalloidin (red), FITC conjugated anti-human CD41a (green) antibodies, and DAPI (blue), and photographed under a fluorescence microscope. Bar = 10 μm. (C) Representative images are shown at four time points after thrombin stimulation. Arrows indicate hESC-PLTs spreading, lamellipodia and filopodia formation, membrane ruffling, and some of hESC-PLTs were tethered together. (D) The left two panels are representative dot plots for blood platelets binding to FITC-conjugated PAC-1 antibody in the absence (left) and presence (right) of thrombin (1 U/ml). A dramatic increase of FITC-conjugated PAC-1 antibody binding (right shift) is observed for thrombin-treated human platelets as compared with resting controls. The right two panels are representative dot plots for hESC-PLTs binding to FITC-conjugated PAC-1 antibody in the absence (left) and presence (right) of thrombin (1 U/ml). A moderate increase in FITC-conjugated PAC-1 antibody binding (right shift) is observed for thrombin treated hESC-PLTs as compared with resting controls.
Figure 5
Figure 5
Functional characterization of hESC-PLTs in vitro. (A and B) Aggregation assay: 3 × 105 PKH67 (green) labeled blood platelets (A) or 3 × 105 PKH67 labeled hESC-PLTs (B) were mixed with unlabeled blood platelets (6 × 107) and thrombin (0.5 U/ml), then stirred to trigger platelet aggregation. Phase contrast (left) and fluorescent images (center) were merged (right) to demonstrate the participation of labeled blood platelets or hESC-PLTs into micro-aggregates. In control experiments, RGDS peptide was added before thrombin stimulation and no aggregation was observed. (C and D) Clot formation and retraction: (C) platelet-depleted human plasma was added with (+PLTs) or without (−PLTs) hESC-PLTs (1.5 × 107/ml), and (D) platelet-depleted human plasma was added with human blood platelets (1.5 × 107/ml). Thrombin (2 U/ml) and CaCl2 (10 mM) were then added to the suspensions to induce clot formation/retraction (C and D, left panels). Clot cryo-sections (C and D, right panels) were stained with anti-human CD41 (red) and anti-human fibrin (green) antibodies. Images were taken under a fluorescence microscope. No clot formation/retraction was observed without addition of hESC-PLTs (C, left panel −PLTs). Bar = 50 μm.
Figure 6
Figure 6
Incorporation of hESC-PLTs into developing mouse platelet thrombi at sites of laser-induced arteriolar injury in living mice. Calcein AM-labeled human blood platelets or hESC-derived platelets, 50-100 μl (5-10 × 105 platelets), were infused through a femoral artery cannulus immediately after laser-induced vascular injury. The developing mouse platelet thrombus was monitored by infusion of Dylight 649-labeled anti-mouse CD42 (0.05 μg/g body weight). After generation of two to three thrombi, the labeled platelets were pretreated with ReoPro, 20 μg for 2 × 106 human platelets in 200 μl, and infused after new vessel injury in the same mouse. Another two to three thrombi were generated to examine incorporation of ReoPro-treated platelets. Data were collected for 3 min following vessel injury. (A) Representative fluorescence images are shown at three time points (0, 1/2 Tmax, and Tmax) following vascular injury. Magnified images of the area within the white rectangle are shown at the bottom. Bar = 10 μm. (B and C) The number of labeled human platelets circulating into the microvessel (B) and incorporating into the developing mouse platelet thrombus at the site of vessel injury (C) was counted over 3 min after vascular injury. Data represent mean ± S.E.M. (n = 5-8 thrombi in three mice). *P < 0.05 and **P < 0.01 vs a control, Student's t-test.

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