Highly efficient platelet generation in lung vasculature reproduced by microfluidics

Abstract

Platelets, small hemostatic blood cells, are derived from megakaryocytes. Both bone marrow and lung are principal sites of thrombopoiesis although underlying mechanisms remain unclear. Outside the body, however, our ability to generate large number of functional platelets is poor. Here we show that perfusion of megakaryocytes ex vivo through the mouse lung vasculature generates substantial platelet numbers, up to 3000 per megakaryocyte. Despite their large size, megakaryocytes are able repeatedly to passage through the lung vasculature, leading to enucleation and subsequent platelet generation intravascularly. Using ex vivo lung and an in vitro microfluidic chamber we determine how oxygenation, ventilation, healthy pulmonary endothelium and the microvascular structure support thrombopoiesis. We also show a critical role for the actin regulator Tropomyosin 4 in the final steps of platelet formation in lung vasculature. This work reveals the mechanisms of thrombopoiesis in lung vasculature and informs approaches to large-scale generation of platelets.

Fig. 1. Mouse platelets are generated from megakaryocytes passaged multiple times through mouse pulmonary vasculature ex vivo.

Mouse megakaryocytes (MKs), labelled with CD41-PE or CD41-FITC antibodies, were passaged repeatedly through the pulmonary vasculature ex vivo. Lungs were ventilated with air throughout (b, d–g). a Diagram illustrating the approach to generating mouse platelets. End-expiratory positive pressure was applied to prevent lung collapse. b Intact MKs from perfusates after passaging the indicated number of times through lung vasculature. Quantification was from ≥250 fields of view, counting ≥230 cells in total and displayed as a percentage of total number of cells. Numbers above each column indicate significant differences to other passages. N = 7, 5, 6, 9, 8 and 6 independent experiments for passage numbers 0, 1, 2, 3, 6 & 9. Error bars are mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. c In vivo demonstration that intact mouse MKs pass through the pulmonary vasculature. Mouse MKs were stained with CellTracker™ Red CMTPX dye (red) and Hoechst 33342 (blue) prior to injection into the right external jugular vein of an anaesthetized recipient C57BL/6 mouse. Blood was collected from the left common carotid artery and cells were imaged by confocal fluorescence microscopy. Images shown are representative of n = 4 independent experiments. Scale bar: 10 µm. d Gating strategy for quantification of generated platelets. The number of generated platelets in the perfusate collected after the 18th passage was determined by the number of CD41(+) events in gate P1. e Events in P1 gate (from the experiment shown in Fig. 1d) are defined as generated platelets (indicated by the red arrow), with higher mean fluorescence compared to those derived from control IgG-PE- treated MKs. Gate P1 also captures CD41-negative cells, which include stem cells and host-derived platelets. Viability of generated platelets, and whether they contain DNA, were checked by Calcein-AM and DRAQ5 dyes, respectively. f Mitochondrial membrane potential in generated and control platelets was determined by Tetramethyl rhodamine methyl ester (TMRM) accumulation in active mitochondria and measured by FACS. g TMRM signals from (f) were quantified and displayed as mean ± SEM. N = 5 independent experiments; two-tailed unpaired t test. Source data are provided in the Source Data file.

Fig. 2. Quantification of platelets generated by passage of megakaryocytes through mouse pulmonary vasculature ex vivo.

Mouse megakaryocytes (MKs), labelled with CD41-PE or CD41-FITC antibodies, were passaged repeatedly through the pulmonary vasculature ex vivo. Lungs were ventilated with air, pure nitrogen or without ventilation. a The number of generated platelets per MK present in the perfusates from different passage numbers in lungs either ventilated with air (black circles), pure nitrogen (red triangles), or without ventilation (blue squares) were measured by FACS. N = 5 (air-ventilated lung or unventilated lung) and 4 (nitrogen-ventilated lung) independent experiments. Data are mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. b Stained MKs (CD41-FITC, green) were passaged through pulmonary vasculature ex vivo 18 times, and lung tissue was fixed and sliced followed by visualization of 20 stacked focal planes by two-photon microscopy. Lungs were either ventilated with air or pure nitrogen, or were not ventilated, as indicated. Mouse lung without MKs passed through served as control. Images shown are representative of n ≥ 4 independent experiments. scale bar: 20 µm. c Numbers of platelets generated per MK in perfusate and retained in mouse lung under air ventilation, were calculated and displayed as mean ± SEM. As a control, MKs passed through 21 G needles 18 times generated no platelets. N = 6 (air-ventilated lung) and 4 (passing through needles) independent experiments. Source data are provided in the Source Data file.

Fig. 3. Role of pulmonary endothelial cell health and microvascular structure on platelet generation.

a, b Pulmonary endothelial cells (ECs) were isolated from perfused lungs under air- (black) or pure nitrogen-ventilation (red) or without ventilation (blue) for ~2 h. ECs from fresh lung tissue served as control (grey). ECs were stained with FITC-conjugated anti-CD31/PECAM-1 or anti-CD102/ICAM-2 antibodies. N = 5 independent experiments for each group. Two-tailed Mann–Whitney test. a Viability of pulmonary ECs were determined by Calcein Deep Red retention and displayed as mean ± SEM. b Mitochondrial membrane potential was determined by accumulation of Tetramethyl rhodamine methyl ester (TMRM) in active mitochondria and displayed as mean fluorescence intensity ± SEM. Air-ventilated lung vs unventilated lung: p = 0.0159 and 0.0159 for CD102 and CD31, respectively. Air-ventilated lung vs nitrogen-ventilated lung: p = 0.0317 and 0.0317 for CD102 and CD31, respectively. c Design of microfluidic chamber simulating a physiological pulmonary vascular system (details shown in Methods), including a photomicrograph of the smallest channels in the system and indication of the flow direction by arrows (red). Dimensions indicated on the figures are width of channels. d, e Mouse megakaryocytes (MKs) prelabelled with CD41-PE were repeatedly pumped through the microfluidic chamber. d The viability of generated platelets from the microfluidic chamber was determined by Calcein AM staining (Generated platelets: CD41+/Calcein AM+ in upper right quadrant, Q3 in green). All generated platelets identified in this way showed no DNA content (DRAQ5 -ve staining). e Quantification of generated platelets per MK in perfusates under air (purple circles) or pure nitrogen conditions (green circles), measured by FACS. For comparison, numbers of platelets generated in the unventilated lung-heart system (blue squares), from Fig. 2a, are shown. N = 4 (for air-chamber or nitrogen-chamber) and 5 (for unventilated lung-heart model) independent experiments. Data are mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. f Mouse MKs prelabelled with CD41-FITC were repeatedly pumped through the microfluidic chamber under air 18 times. Generated platelets were washed and then integrin αIIbβ3 activation and P-selectin expression, induced by 2 U/mL thrombin (Thr) or 5 µg/mL CRP-XL, were measured by FACS. N = 10 independent experiments and data are mean ± SEM. Two-tailed Mann–Whitney test, p < 0.0001. Source data are provided in the Source Data file.

Fig. 4. Generated platelets demonstrate typical physical features comparable to control platelets.

a, b Mouse megakaryocytes (MKs), prelabelled with CD41-FITC (green), were passaged repeatedly through the pulmonary vasculature ex vivo. Lungs were ventilated with air throughout. a Perfusates from ex vivo heart-lung preparation, containing both generated platelets (white arrow) and host platelets (blue arrow), were stained for α-tubulin (magenta), and confocal images shown as a mixed population in the top panels. More detailed images of a-tubulin rings are shown in the magnified images in the middle panel (generated platelets) and bottom panel (control platelets). Images are representative of n = 3 independent experiments. Scale bars: 2 µm. b The diameter of platelets (40 platelets from n = 3 independent experiments) from a was measured using Fiji ImageJ-Win64, with diameters of generated platelets: 3.6 ± 0.2 µm vs control platelets: 1.9 ± 0.05 µm. In contrast to control platelets, there were two subpopulations of generated platelets based on their diameter ranges: ~33% of generated platelets (diameter range: 1.7–2.4 µm) have sizes similar to control platelets (diameter range: 1.2–2.4 µm) and 67% of generated platelets (diameter ranges: 3.7–5.6 µm) are significantly larger than control platelets. Data are mean ± SEM. Two-tailed Mann–Whitney test, P < 0.0001. c Ultrastructures of generated platelets and control platelets visualized by transmission electron microscopy. Host platelets were depleted by intraperitoneal administration of anti-GPIbα antibody R300 prior to perfusing MKs through the heart-lung preparation under air-ventilation. Subcellular structures are shown and annotated as abbreviations, in the high magnification images: α-G, α-granules; σ-G, σ-granules or dense bodies; Mit, mitochondria; OCS, open canalicular system; MTC, microtubule coils; RBC, red blood cells. Scale bars: 2 µm in the images with low magnification, 500 nm in the images with high magnification. Images shown are representative of n = 5 independent experiments. Source data are provided in the Source Data file.

Fig. 5. Generated platelets are functionality comparable to control platelets.

Mouse megakaryocytes (MKs), labelled with CD41-FITC or CD41-PE antibody or DiOC6 dye, were passaged through pulmonary vasculature ex vivo 18 times. Lungs were ventilated with air throughout. a Mean fluorescence intensity (MFI), measured by FACS, of integrin αIIbβ3 activation and P-selectin expression induced by 2 U/mL thrombin or 5 µg/mL CRP-XL in washed generated platelets (CD41-FITC) compared to washed control platelets. Data from generated platelets were pooled as total generated platelets (black dots), and segregated by platelet size (diameter <2.4 µm as pink dots, diameter 3.7–5.6 µm as light brown dots), compared to control platelets (blue dots). Generated platelets sizes were estimated using Particle Size Standard Kit (Cat. PPS-6). N = 10 (generated platelets) and 6 (control platelets) independent experiments and data are mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test, p-values indicated in the figure. b Surface glycoproteins were measured by FACS. Generated platelets were defined by staining with anti-CD41-PE antibody. Surface glycoproteins were stained with different FITC-conjugated antibodies as indicated. N = 6 independent experiments and data are mean ± SEM of FITC intensities. Two-tailed unpaired t-test, p = 0.02 for CD 42d, p = 0.0043 for CD49b and p = 0.0008 for GPVI. c Images of representative platelet-rich thrombus. Generated platelets (showing as blue in colour, stained with both DiOC6 (cyan) and CellTracker™ Red CMTPX dye (magenta)) occupied all levels of the thrombus while host platelets (stained with CellTracker™ Red CMTPX dye alone, magenta) were mainly situated on top of thrombus. Images are representative of n = 5 independent experiments. Scale bars as indicated. d, e MFI profiles of R1 and R2 (Region 1 and Region 2 from Fig. 5c) along the z-axis. d MFI profile of R1 along the z-axis. In R1, generated platelets occupied the lower part of the thrombus up to ~12 µm (both cyan and magenta signals increased simultaneously), whilst beyond this point host platelets were predominant (as magenta signals were stronger than green beyond 12 µm). e MFI profile of R2 along the z-axis. In R2, this part of thrombus was composed only of generated platelets as both cyan and magenta signals changed simultaneously along z-axis. Source data are provided in the Source Data file.

Fig. 6. Megakaryocytes show nuclear marginalization and enucleation prior to fragmentation.

Megakaryocytes (MKs) from C57BL/6 mice, labelled with CD41-PE antibody (red) and Hoechst 33342 (blue), were passaged repeatedly through the pulmonary vasculature of a C57BL/6 mouse ex vivo. Lungs were ventilated with air throughout. a Representative images of MKs derivatives during platelet generation: nuclear polarization and enucleation, where the nucleus is marginalized, of irregular shape or in the process of ejection from the cell; naked nuclei, where the ejected nucleus is >20 µm in diameter and free from the parent cell and/or partially encased in thin/patchy plasma membrane; MKs without nucleus, where the MKs have an approximately circular shape but without nuclei; and large anuclear objects, where ghost cells are of irregular shape and >10 µm in their longer axis. Scale bar: 5 µm. b Cells were imaged from samples of perfusates after passage numbers 1, 2, 3, 6 and 9. Five subgroups of MKs and their derivatives, as described above, were quantified as a percentage of total number of cells. Quantification was from at least 250 fields of view, counting at least 230 cells in total for each of the subgroups. N = 7, 5, 6, 9, 8 and 6 independent experiments for passage numbers 0, 1, 2, 3, 6 & 9. Data are mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01,***p < 0.001 and ****p < 0.0001. c–g Nuclear lobes of MKs fragment into small condensed sub-nuclei. c Representative images, from n = 3 independent experiments, of naked nuclei generated after multiple passages (as indicated) of mouse MKs through pulmonary vasculature. Scale bars: 5 µm. d–g The depth (d), aspect ratio (e), major axis (f) and minor axis (g) of sub-nuclei decreased substantially with increasing passages. These parameters were measured from after 3 passages to after 18 passages: depth 8.9–5.5 µm, aspect ratio 1.8–1.1, major axis 14.1–6.5 µm, minor axis 8.1–5.7 µm. Numbers above each column indicate significant difference to other passages. 19 sub-nuclei from n = 3 independent experiments were collected. Data are mean ± SEM. Two-tailed Mann–Whitney test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Source data are provided in the Source Data file.

Fig. 7. Tropomyosin 4 is required for the final steps in platelet generation in the pulmonary vasculature.

Megakaryocytes (MKs) from Tropomyosin4^−/− (Tpm4^−/−) mice labelled with FITC-conjugated anti-CD41 antibody, were passaged repeatedly through the pulmonary vasculature of a C57BL/6 mouse ex vivo. For controls, wild-type (WT) MKs stained either with FITC-conjugated anti-CD41 or isotype antibodies, were passaged repeatedly through the pulmonary vasculature of a C57BL/6 mouse ex vivo. Lungs were ventilated with air throughout. a Representative FACS dot plot images are shown for generated platelets (CD41-FITC positive events are within the red square). b Numbers of generated platelets per Tpm4^−/− MKs, or control WT MKs, in perfusates after different passage numbers through WT lung, were quantified by FACS. Tpm4^−/− platelets were consistently undetectable after up to 18 passages, in the perfusate. Data shown are platelets generated per MK from either Tpm4^−/− MKs or control WT MKs and displayed as mean ± SEM. N = 5 independent experiments for each group. Two-way ANOVA with Šídák’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. c Cells were imaged from samples of perfusates after passage numbers 0, 1, 2, 3, 6 and 9 through murine lung vasculature ex vivo. Cells were morphologically classified as 5 subgroups: intact MKs (as per Fig. 1b) and MK derivatives (shown in Fig. 6a) and quantified as a percentage of total number of cells. Quantification was from at least 150 fields of view, counting at least 170 cells in total for each of the subgroups. N = 4 independent experiments and data are mean ± SEM. Numbers above each column indicate significant difference to other passages within each group. Two-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Dollar sign ($) above columns represents significant difference to corresponding wild-type column in Fig. 6b. Two-tailed unpaired t test (passages 1, 2, 6 and 9) or Mann–Whitney test (passage 3), *p < 0.05, **p < 0.01. d Abundant fluorescent objects, ~10 µm diameter, were visible in sections of mouse lung after 18 passages of stained Tpm4^−/− MKs, as shown in extended focus stacks of 20 continuous two-photon planes of lung. Images shown are representative of n = 3 independent experiments. Scale bar: 20 µm. Source data are provided in the Source Data file.

Fig. 8. Intravital two-photon microscopy of bone marrow megakaryocytes in live mouse calvarium.

Bone marrow vasculature was visualized by intravenous injection of anti-CD105-AlexaFluor 546 antibody and AlexaFluor 546-labelled BSA (red). Megakaryocytes (MKs) and their derivatives were stained intravenously with anti-GPIX-AlexaFluor 488 antibody (cyan). Both wild-type (WT) and Tropomyosin 4^−/− (Tpm4^−/−) MKs were generally seen in close contact with the bone marrow sinusoidal walls. Images were taken from n = 6 biologically independent mice for each group (WT and Tpm4^−/−). Scale bars, 50 µm. a Representative images of large fragments of WT and Tpm4^−/− MKs (white arrows) within sinusoidal vessels releasing heterogeneous structures in the direction of blood flow, at time points indicated. b Representative images of WT and Tpm4^−/− MKs, with cell bodies within the marrow space, producing extensions into sinusoids (yellow arrowheads). c Representative images of WT and Tpm4^−/− MKs within the sinusoid, showing cellular extensions (white circles).

Fig. 9. Schematic diagram of the steps in platelet generation from mature megakaryocytes.

Diagram showing platelet generation pathway from intact mature megakaryocytes (MKs) to final platelet formation, by repeated passage of MKs through the pulmonary vasculature. The process involved nuclear polarization, enucleation, gradual cytoplasmic fragmentation into platelets (with the final steps dependent on Tropomyosin 4 (TPM4)) and nuclear fragmentation and condensation.