LETTER dols10 1038/maturea2706 The lung is a site of platelet biogenesis and areservoir for haematopoietic progenitors Emma Lefrangais!, Guadalupe Ortiz-Mufioz!*, Axelle Cauctillier’, Befiat Mallavia', Fengchun Liu!, David M. Sayah?, Emily E. Thornton’, Mark B. Headley", Tovo Davids, Shaun R. Coughlin‘, Matthew F Krummel’, Andrew D. Leavitt Emmanuelle Passegue! & Mark R. Looney!* Platelets are critical for haemostasis, thrombosis, and inflammatory responses! but the events that lead to mature platelet production remain incompletely understood. The bone marrow has been proposed to be a major ste of platelet production, although there is indirect evidence that the lungs might also contribute to platelet biogenesis*’. Here, by directly imaging the lung microcirculation in mice’, we show that a large number of megakaryocytes circulate through the lungs, where they dynamically release platelets Megakaryocytes that release platelets inthe lungs originate from extrapulmonary sites such as the bone marrow; we observed large megakaryocytes migrating out of the bone marrow space. ‘The contribution of the lungs to platelet biogenesis is substantial, accounting for approximately 50% of total platelet production oF 10 million platelets per hour. Furthermore, we identified populations ‘of mature and immature megakaryocytes slong with haematopoietic progenitors in the extravascular spaces ofthe lungs. Under conditions of thrombocytopenia and relative stem cell deficiency in the bone arrow’, these progenitors can migrate out ofthe lungs repopulate the bone marrow, completely constitute blood platelet counts, and contribute to multiple haematopoietic lineages. These results identify the lungs as a primary site of terminal platelet production and an organ with considerable haematopoietic potential Platelets are released from megakaryocytes: however, even, though they were discovered in the nineteenth century, we do not completely understand the mechanisms by which platelets are produced. On the basis of previous work showing the presence of megakaryocytes in the lungs" and demonstrating that blood leaving the lungs contains more platelets and fewer megakaryocytes an blood entering the lungs*™, we hypothesized thatthe lungs could have a major role in platelet biogenesis, and directly investi gated this process using 2-photon intravital microscopy (2PIVM) of the lungs and fluorescent reporter mouse strains, We used 4-Cre x G1(ROSA)26Sor!™4AB Torn BGPP (niTmnG) (hercalter called PF4-miTmG) reporter mice, in which PF4-Cre'? drives mem: brane GFP expression in megakaryocytes and platelets, while ll other cells ar labelled with membrane tomato. We observed large irulating GED™ cells that passed through the lung microcirculation, where they produced GFP* extensions in a flow-dependent manner (Fig. 18, b and Supplementary Video 1). These events resembled proplatelet and preplatelet formation from cultured megakaryocytes" In the jungs, the duration ofthese events varied from approximately 20 0 60min (Fig. la, b and Supplementary Video 1). Many of the GEP cells contained large nuclei (more than 10 um), which appeared as unlabelled dark hoes that remained intact durin this proces (Fig. 1b and Supplementary Video 2) and resulted in naked intravascular nuclei ater platelets were released (Supplementary Video 2). We confirmed that we labelled large mobile nucleated cells by imaging the lung ierocirculation of PPA-Cre x Gr(ROSA)26Soy'i/CAG tina GHP (tnG) (hereater called PF4-1TUG) reporter mice, in which a uores cence switch allows GEP* nucei tobe tracked (Extended Data Fig 1a and Supplementary Video 3). ‘We next quantified the GFP* megakaryocytes and proplate- Jets in PFa-mitmG lungs by assigning surface volumes (Fig. 1¢ and Supplementary Video 4). The putative megakaryocytes (large GF! cells undergoing platelet release) had median volumes of 10,000)* and diameters of more than 251m (Fig 1d) whereas the putative platelets (small eiculating GEP* events) had median volumes ofbelow 10,’ and diameters of2-3}sm (Fig. 1d). These values are consistent with previous estimates of megakaryocytes and platelet sizes For each lange GEP' cell undergoing platelet release, we calculated the number of platelets that could be liberated into the lung circulation, and this ranged from fewer than 500 platelets for small megakaryocytes o pro- platelets to moe than 1,000 platelets for larger megakaryocytes (Fig 11), ‘witha median of around 500 platelets per megakaryocyte, Previous studies have produced widely varying estimates of the number of platelets produced from a single megakaryocyte (200-10,000platelets)"™"”. Our ‘method uss direct measurement foreach event, and therefore islikely to yield more accurate estimates. In total, we analysed 20h of footage from 10 mice, and observed an average of 2.20.26 (n=10) megs- karyocytes per hour in an imaged lung volume of 0.07 mm? (Fig. 1g and Supplementary Video 5). When extrapolated to the entire lung volume, this equals more than 10 millon platelets produced per hour from the lungs (Fig. th, Methods and Extended Data Table 1). Overall, ‘when adjusted for platelet lifespan and splenic sequestration, we «timate that the lung is esponsiblefor approximately 50% of total platelet production in the mouse (Fig. i, Methods and Extended DataTable 1). Blood platelet counts were unchanged after 2PIVM (Extended Data Fig. 1b, Platelet production by the lungs is also biologically tunable, asthe administration ofthe megakaryocyte growth factor thrombopoi- «tin (TPO) increased blood platelets threefold (Fig. )) and the number ‘of megakaryocytes undergoing propltelet formation observed per hhour twofold (39:4 0.38, n—9) (Fig. 1K). We conclude from these experiments that th lungs area primary site of platelet biogenesis. “To investigate the origin ofthe intravascular megakaryocytes and proplatclets in the lungs, we adoptively transferred lung resident cells sing the orthotopic single lung transplant model in mice™. We trans planted a lung from an mimG mouse (with no Cre or GFP expression) into a PF4-milimG recipient mouse and vice versa (Extended Data Fig. le-e and Supplementary Video 6). Using 2PIVM, we observed proplatclet formation from GHP* megakaryocytes i the lung vascu- Jature following transplantation of mitmG lungs to PF4-niG mice, ‘but not following the reverse transplant (PF4-miTiG lungs to miimG rice) These experiments confirmed that megakaryocytes releas ing platelets in the lung ciulation originate from outside the lungs. Sin Francie (Um, Sonate Calta 3449, 084 apa ‘edie Urey al alow Son Francs (05) So ann Clore 9103 USA Oe Pate Unies calor, Son Froese (US) Son Freese nto Hen Ura eof We, Ue of alors as gees (EL Las ogee ris 9183 USA, earl Resawch bt, nero ear ia San ric (UES) San Fre, Carn 3818, igen Pisters ies pt of Sprnge sur, Nirah reseedLETTER Figure |The lunge are an important site of megakaryocyte circulation and platelet production. a-c,Visulization of megakaryocytes and Platelet production inthe lung citculation by 2PIVM in PPA-miTmaG Inicea,b, Sequential images show a large megakaryocyte (green) inthe Tang capillaries (red) where st undergoes proplatelet formation (arrows), ’, Dark hlein the cytoplasm (dashed outline) indicates the mele Time elapsed is indicated. e~f, Characterization of PE4" events by Image analysis. Representative mage of surface analysis of the GEP channel. de, Valume distribution (d) and equivalent diameter (e) of megakaryocytes (MKs, n—35) and platelets (n—492)-f, Number of ateles produced by one megakaryocyte according to is sie: small < 500 platelets, 18), edt (300-1,000 platelets »—7) and large (1000 platelets n= 10), def, Minimum: to-maximm boxplots are presented. g-i, Quantification oflung platelet production. g, Number ff megakaryocyte releasing platelets observed per hour ia imaged lng volume 2-h movies, 10) bi, Estimation ofthe sumber(h) and the percentage of platelets produced bythe hung jk Platelet counte in the blood @) and numberof megakaryocytes releasing platelets i the Inge (k) 5 days after TPO treatment 25 mice per group. Unpaired fess *#*4P 0.0001, #97 0005, gpk, Mean Led. are presented JL, Visualization of proplatelet release (arrow) and megakaryocyte migration (ctcled) in Sone marrow (BM) sinusoids by 2P1VM in PFA-niTmG mice We hypothesized that the bone marrow", spleen, or liver could be the source ofthese intravascular megakaryocytes and proplatelets We imaged the calvarial bone marrow (Extended Data Fig. 1) and the spleen in PF4-milimG mice and observed extravascular megakar yocytes seleasing proplatelets into the sinusoids of the bone marzow (ig. 11, Extended Data Fig. 1g and Supplementary Video 7) and spleen (Extended Data Fig, and Supplementary Video 9), We lso observed large megakaryocytes exiting the bone marrow space (Fig. Im and Supplementary Video 8) Ip contrast to observations in the lung, we dil not observe any intravascular megakaryocytes undergoing pro: platelet formation. We did not observe any megakaryocytes inthe liver (Extended Data Fig. 1h), In adeition to intravascular megakaryocytes, we also observed large cells in the perivascular lung interstitium in PF4-Cre GI(ROSA)2650r"*7°40 MTonswtee ROSAI6-tdTomato) (hereafter called PF4-tomato) and PE4-mimG mice (Fig, 2a, b, Extended Data Fig. 22 and Supplementary Video 10), and in mTinG mice that had received PF4-miinG lung transplants (Extended Data Fig. 2b). These extravascular eels were sessile during our imaging (up to 4h) and con. tained large nuclei (Fig. 2c). Although they were comparatively lage, Iss He i in hl A | z “ee ah ‘igure 2 | Resident megakaryocytes are present inthe extravascular spaces of the lng. ac, Vievalizaion of resident cr state megakaryocytes inthe lang by 22LVM of P4-tomato (a), PFa-mitmG (b), and PPS: TAG mice (c).() Sze characterization of PF4~ cells (red, > 10,2) by ‘quaitative image analyse of PF4-tomato lngs (n~ 312), Minimam Co-maximum hoxplot are presented. «, Quantification of PEA” cells (red >10jim) (85), g, Comparison of nuclear sie P snd eteulait between PFS" cells green, 17) and all therleng eel by quantitative image analysis of PF&-nfaG lungs. Unpaired test stetp 0.0001 b, Representative immunofluorercence images of PEA and CD41 cells sorted from perfused PF4-tomato lungs and stained with anti-vW ancibedies (aeen) and 45-diamidino-2-phenylindole (DAPI, blue), Inteavascslar (i) or extravascular (e3) localization of PE4! snd CDAi-BIC" eal, Experimental schema and representative ‘Avorescence activated cell sorting (FACS) plots, Percentage of cells, located intravasculaly or extravasculaiy (mean of four experiments, = 8 ice). k FACS gating strategy and surface expression of nucleated PF nd CDAL" cells rom lunge of PE4-n7inG mice, m, FACS quantification of aucleated PFI" (and nudeated PR&" CD41" (my) cells in PFA-8TG whole lung (n= 28), bone marrow (BM, »—24) (two femurs, wo bias) and blood (15m, 8). eng. 1m, Meant ed presente, these extravascular cells were on average one-third of the volume and approximately half ofthe diameter of intravascular megskaryocytes (Fig, 24) and also smaller than resident megakaryocytes in the bone marrow and spleen (Extended Data Fig. cc). Using image analysis, wwe detected around 2,000 PF4-tomato cells per cubic millimetre of lung tissue or moze than 1 million cll per lung (Fig. 2). The nuclear diameters ofthese cells were significantly larger than those of non- GEP~ cells (Fig. 24) and the nuclei had more complex shapes (Fig. 2g) We used a method of intravascular labelling before lung digestion®® to determine the relative proportions of intravascular and extravascular megakaryocytes (Fig. 2) and found that 85% of PF4-tomato events were extravascular and 15% intravascular (Fig. 2), further characterize ing megakaryocytes, we sorted PFS tomato sand CD41" cells from perfused and digested lungs and stained them with the megakaryocyte and platelet marker von Willebrand factor (WE). Thelarge, PF4-tomato” CD41" cells with complex nude: also stained postive for vWF ina granular pattern, which is consistent with megakaryocytes (Fig. 2h). To avoid cell aggregation with PF4-tomato™ platelets during flow cytometry staining, we prepared digested lungs {rom PF4-ni iG mice, in which GFP is targeted tothe cll nucleus and thus does not stain anucleate platelets, We gated on nGEP~ CD41+ pina, ea ieee Figure 3 |Lung-derived progenitors reconstitute platelet counte sind haematopoietic tem col deficiency in thrombocytopenic mice, 2-f,Transplastation of PES tomato lungs to © mp" mice a, Experimental schema. b, Blood platelet counts (n~ 4-6 mice per group. « d. Percentage of donor-derved platelets analysed by counting tomato’ eventsin the CD41" ESC" gute. b, d, Mean +.em. presented. ¢,f Bone marrow cells from donor (PF4-tom). recipient (empl) or wansplanted nice ‘with 10 monthe sustained donor derived platelet production (Lung Tx) ‘were analysed, e, FACS analysis of bone marrow cells reveals Tomato cells (CD41! snd CD41- populations) Pereentage of lineage negative bone marrow cells positive lor Tomato and CD41. f Representative lmmunefluorescence image of Tomato’ cell (red) inthe hone marrow of A uansplanted mouse stained with anti-CD41 (gren) and Sytos0 nucleic acid sta (ue). gn, Transplantation of mTinG lungs into cmp!” mice Bone marrow cell from donor (D, mifimG), recipient (R, empl ).or lesnsplanted (Ts) mice wilh sustained donor derived platelet production (@ months) were analysed. g. Reprerentatve PACS analysis ofthe myeloid progenitor compartment and the MP popslation. , Percentage of the MAP population within the myeloid progenitor compartment 4), Percentage of donor origin Tomato” cells in the MAP population I Representative FACS analysis of the LSK compartment showing MPP2, MPP3/4, ST-HSC and LT-HSC population frequencies within the SK compartment I, Percentage ofthe LT HSC population within the ISK compartment. m,n, Percentage of donor origin Tomato" cell inthe UEHSC population b.jn, Mean td. are precented (= 2-4 mice per group). Unpalred test: **P-<0.0, *P<0.05;a.5, net significant events a the putative lang megakaryocyte pool (Fig. 2k and Extended Data Fig. $a) and this population stained for the megakaryocyte and platelet- specific markers glycoprotein VI (GP VI) and the TPO receptor -Mpl (Fig. 24), but did not stan for markers of other lung resident cells, such as F4/80 for macrophages (Fig, 2k and Extended Data Fig. 3a-d) confirming that these lung, derived cells were megakaryocytes, The majorly ofthe nGEP" celle i the hung were CD41 (Extended Data Fig. 3), but both CD41" and CD41~ cells co-stained for GPVI and ¢-Mpl, confirming their megakaryocyte lineage (Extended Data Fig 30). The nGEP? CD41 cells had a more immature profile with lower CD61 and CD42b expression, a smaller size and lower DNA con tent than the nGFP~ CD41” cells (Extended Data Fig. 3e-i). Overall, selon Plsters ies pat of Sprnger sure LETTER tung megakaryocytes were more immature than bone marrow mega: karyocytes (Extended Data Fig. 3)-m), bu the total number of meg- akaryocyts in the lungs was comparable to that in the Bone marrow (Fig 21,1), Wealso used RNA-sequencing (RNA-sea) to characterize Tung and bone marrow megskaryocytes (nGEP” CD41 cells) n PEA ‘TRG mice, We identified more than 700 genes that were expressed differentially inthe lungs and bone marrow (Extended Data Fig. 4a and Supplementary Table 1); many megakaryocyte and platelet path ‘ways were represented in both groups, but there was ess expression cof mature megakaryocyte markers in the lung group (Extended Data, Fig 4b, c), consistent with our profiling by immunostaining, Lung 2PIVM indicated that only intravascular megskaryocytes released platelets To test the function of extravascular megakaryocytes {nthe lungs, we used the orthotopic single-lung transplant model to aaloptively transfer lung resent cells, PF4-tomato donor lungs were extensively perfused andthe left lungs were immediately transplanted {nto wild-type or -mpl ‘thrombocytopenic recipient mice (ig. 3). ‘These transplanted mice were injected with TPO at 3 and 40 days ppost-transplantaton and bled weekly to ack the numberof platelets (Gig. 3a,b), Peripheral blood tomato" events after lang transplantation, are, by definition, of donor lung origin. In wild-type recipients, we detected low level (1-296 oftotal CD41" events) and transient produc tion of tomato” events (Fig. 3d and Extended Data Fig. si). However, {nthe majority (70%) of mpi! recipients, we detected iange and sus- tained (90 days) production of tomato” events (Fig 3, d and Extended Data Fig. 5c, d) that fall reconstituted platelet counts (Fig. 3b and Extended Data Fig 5h). We observed the same response in two out of five empl” recipients not eated with TPO alter transplantation Extended Data Fig. 5b). The tomato CD41" events were also positive for CD42, GPV! and c-Mpl (Extended Data Fig. 6a, b) and expressed CD62? when stimulated with thrombin (Extended Data Fig. 6c-e), confirming that they were platelets, Tn selected experiments, cmpl~/ lung transplant recipients were followed for up to 10 months afer transplantation. In these mice, we observed sustained production of tomato” platelets and sustained reconstitution of platelet counts (Extended Data Fig. 5j, K). Because platelet lifespan in mice is 3-5 days, the persistence of donor-origin platelets for more than 3 months suggested thatthe transplanted lungs contained a progenitor population capable of long-term reconstitu tion of mature megakaryocytes and platelets, Indeed, the fet that the extravascular megakaryocytes were smaller than the intravascular meg akaryocytes in the lungs and the extravascular megakaryocytes in the bone marraw and spleen could point tothe presence of megakaryocyte progenitors. ‘We imaged lungs 3 months after transplantation and confirmed the persistence of PF4-tomato cells (Extended Data Fig. 72) We also Aletected the presence of tomato” CD41~ cells in the bone marrow of empl” mice that had received PF4-tomato lung transplants using flow eytometry (Fig 3e) and immunofluoreseence (Fig. {and Extended Data Hig, 7b). io tst for lung megakaryocyte progenitors and to track donor eels in recipient mice, we transplanted miTsG lungs, in which ll ells and platelets are tomato", into compl” mice (Extended Data Fig. 7c-c), We next quantified the megakaryocyte progenitor (MtKP) population in the bone marrow of e-mpl-’~ mice transplanted ‘with nifinG lungs by staining myeloid progenitors (Lin~ Sca-1~ Kit") for CD41 and CD150% (Fig. 3g). We found more myeloid progenitors and MkPs inthe bone marrow ofthe lung transplant recipients than in ‘empl’ bone marrow; the numbers i the transplant recipients were similar to the numbers of myeloid progenitors and MkPs normally found in the bone maztow of wild-type (miTimG) mice (Fig. 3h and Extended Data Fig 7 g)- One-third of the MRPs in the bone marrow cof empl /~ mice transplanted with mitinG lungs expressed tomato ig. 3,9. ‘We next tested whether the haematopoietic stem cell (HSC) defi- ciency characteristic of e-mpl-’~ bone marrow? could be reversed by Tung transplantation, We gated on the bone marrow LSK (Lin~ Sea-Lpers po i toe aes ee ume eu ee a Figure 4 |The lung contains haematopoietic progenitors, including megakaryocyte progenitors» , Representative ling (a) and bone marrow (@) FACS plats of haematopoietic progenitors within the [SK and myeloid compartments, bd Call counts of haematoposetic progenitor (MPP2, MPP3/4,ST-HSC, LI-HSG, and MIP) populations In whole lungs (n=~12)(b) and bone marrow (n=) (legs d). e-Kit) population and probed for the following subsets: long-term HSCs (LT-HSCs; CD48~ CD150"), short-term HSCs (ST-HSCs; CD4s~ CD1S0-), multipotent progenitor 2 (MPP2) cells (CD48* CD150"), and MPP3/4 cells (CD48* CD150° (ig. 34). Lung trans plantation from TPO-competent (milmG) donors reversed the LSK population deficiencies (Fig. 31 and Extended Data Fig. 75-1). Lung- derived MkPs, LT-HSCs, SIUHSCs, MPP2s and MPP3/4s were also found in the bone marrow, spleen and recipient lungs (Fig. 3m, n and Extended Data Fig. 7n-p). A non-specific post-lung transplantation response was ruled out because transplantation of e-mpl"’~ Tangs into ce-mpl-/ mice did not produce increased platelet counts (Fig. 3b) or alter the bone marrow composition Extended Data Fig. 7m). Together, these results suggest that a haemalopoieic progenitor population resid ing in the langs can migrate to the bone marrow and reverse HSC Aefects and associated eytopenias ‘We tested for haematopoietic progenitors in dispersed wild-type lungs using similar gating on live CD45! Lin Sca-1! c-Kit! cells as in the bone marrow. We discovered thatthe lungs contain an array of ‘haematopoietic progenitors, including ST-HSCs, MPP2s, MP3s, myeloid progenitors and MkPs (Fig. a,b), which were morphologically indistinguishable from bone marrow LSK cells (Fig, 4e) These cells vere present at lower numbers than i the bone marrow (Fig. 4c, d) and spleen (Extended Data Fig a,b), except that there were more ST-HSCs in the langs than inthe spleen. These cells were extravascular, because hey were not removed by perfusion and were not stained by nlravas- cular CD45-APC antibodies (Extended Data Fig, 8c). To our knowl edge, this isthe first description of haematopoietic progenitor celisin the adult lungs, and we reasoned that these cells could be the source ofthe reconstituting effects of ling transplantation. To test this hypo: thesis, we isolated ISK and ST-IISCs from perfused wild-type lungs a: Repreestative nage of Wight Gems sting of LK el sorted Irom bone marrow or lung f, Experimental schema. gb, Pereentage of donor derived platelets analysed by FACS counting ofe Apl” events the CD41" FSC™ gate j, Blood platelet counts i Mean +5 4 presented (n=4-5 mice per group). Unpaired test P< 001, Seep coool, **°*P-< 00001, {and the bone marzow for comparison), injected these cll intrave ously into comp”~ recipients, and teste forthe presence of e-Mpl~ platelets in the peripheral blood Fig. Injection of ung LSK cells nd ST-HSCs increased peripheral e-Mpl” platelets and partially restored platelet counts, and injection at bone marrow cells bad a simular flect Fig 4). These results show tht the aaltlungs contain functional haematopoietic precursors capable of migration, bone smarxow engraftment, nd reconstitution of haematepoietic defect, Finally, we tested whether hung haematopoietic progenitors are capable of mult-lineage bone marrow reconstitution. We transplanted lange from miTzmG donors to allow us to tack matare lineages inthe peripheral blood and bone marrow (Extended Data Fig. 70), and detected sustained production of donor-derived (tomato CD41”) callin the peripheral blood ofthe cmpi-’- recipient mice (Extended Data Fig. 8.) The donor derived cls included platelets (Extended Data Fig 7d, c) neutrophils, and B and T cells (Extended Data 5g 81). Consiering that there are no neutrophil B cell or cel defects incompl’” mice and therefore no impets for donor-derived recon: ‘tution, these results demonstrate an important contribution ofthe lungs to overall hematopoiesis (ur results provide direct evidence that the lungs ate a major site of platelet biogenesis, which savolvesa distint mechanism of proplatelet selene from intravascular megakaryocytes (of extrapulmonary origin) inthe lung microcirculation (Extended Data Fig. 98). These result open nev lines of investigation o improve our approach to Ueating thrombocytopenia, which affect millions of patents worldwide and cates substantial morbidity and mortality We propose thatthe lungs sre an ideal bioreactor forthe production of mature platelets rom meg akaryocytes, and could advance studies ofthe treatment of thrombaey topenia with cell-based therapies. Beyond the mechanical forces thatpromote propltelet formation and extension, the lung may contain ‘unique signaling partners for megakaryocytes that promote platelet release Interactions between megakaryocytes and endothelial cells, {hough glycoprotein Ib (GPIb)-v WF signaling have been shown (0 promete proplatelet formation in vitro Considering that WWF levels ste particularly high in the pulmonary arteries, this pathway could finely regulate megakaryocytes for platelet production “The lungs ate a reservoir fr resident megakaryocytes and haemato- poietic progenitor clls (Extended Data Fig 9b), which rases questions about the factors responsible for the homing ofthese cells into and. (tof the lungs, the function ofthese cells in the kung niche, and the roles ofthese cells in host defence™. Additionally, megakaryocytes are arich source of ytokines and growth factors that have the potential to dnfluence inflammatory or fibrotic lung diseases, Our RNA-seq analysis, revealed that lung megakaryocytes were skewed towards an innate smmunity function (Extended Data Fig. 4d—f and Supplementary “Table 1), which may reflect the unique environment ofthe exposed lung, versus the bone marrow. Indeed, we detected changes inthe resident ani eculting lng megakaryocyte populations in mice with bacterial ‘pneumonia (Extended Data Fig. 4-4). Ou findings may also be appli Cable to the field of king transplantation n which poat-transplanttion chimaerism could affect acute and chronic allograft ejection. Our results add tothe growing evidence thatthe lungs area sophisticated organ that is capable of regeneration after major injury, area major site of platelet production, and have untapped potential aa contebutor to hematopoiesis ‘Online Content ethos, lng wih any aionel Extended Dat spay ems ana Source at. ae seninsie nthe ol ne wersion ol oe paper referees unsaue these secions appea only tne anne ape: Received 24 Apil 2026; accepted 14Febn Published online 22 March 2017. ry 2017. 1. Semple Wa talane 1 EJ 4 Freedman J Pte andthe immune farina es nol 1, 268274 (2011). 2. rsa, HH. Heerstrk J W.Lav M. Gera, H, Mew undarentas in homostas's Pro Re. 38, 327-356 (2013), 3. MachWs, 48. Halane, 1 Ear The ereisie journey: tom megokaryocyle development plaee' formation. / Cel Bel 202, 783-196 (2019), 4 Howell WH. & Donahue, D,D-The production of oes platelets inthe lings. AB. Mea 68, 177-203 (1837). 5. 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Cel Stem Co, azenat2 2007, 23, Pitas, £-M ak Funconaly din! subsets of tneage iased mutigetent progenitor earl Sioa praductan i normal an regenerstie canton {et tom Cel 17, 35-46 2015). 24, Dung ad 6 etal Exoesure at human megakarycytes thigh shear ates {azeleses patel! produetan. Scag 114, 1875-1883 2009), 25, Yemamet, de Waar, Pears C8 Laat, O-1 isve éstbuton nd {egulston 9! murine vor Willebran ator eve expression in vivo Band 92, 2at-z801 (980) 26, Haas Seta nfammetansncuced emergency megotaryopees even ay herstapoite stem eslike megakoryocte progentors Ce Stam Cal 1, 422-434 015), mentary Information evalabe inte onne version othe pape [Acknowledgements We hank the UCSF BID for asitance wilh 2°V¥ ane 30 Dintng: Herault E Yerovekays and 8% 2mang tem the Passage nbortory ‘orasstance with heratapoetie progenr solaion and vansplotation, tnd. Ee andthe UCSF SABRE Runctonal Genomics Fach for asistance vith the RNA'seqseneing experimen This work was sppared in prt by Nil grants 082471 tof 1L107360 ang HL130324%0 MRL. te UES [Ninalfeland Program in Ling “eat: (VL), andthe UCSF Pregiam for Brosithvough Somedal Research (ARL). [Author Contributions E, desgnestan conducted most ofthe experiments analysed te dara, and wrote te manuseriptC.0-M, designed and conducted fraeriments ang analysed the Gat AC and BM. conducted exper ments and habe data FL performed the Lng vansplantaton experiments. DIES, EXT MB are ib seston ncesgn ng ane conducting experiment SRE MEK ane AOL assed ny ceign ng uperments snd oov ded fectovial support onthe manuseripe E ass stea In designing exaeriments, Drowded technical expertise wth hermatopoietic rege nates ahd Drovded ecitort support an the manuser pt MRI designed the experiments Eoneucted experiments, analysed data and wrote the manuer pt ‘Author Information Reprints anc permissions information s aval at ‘ralurecom/eprints. The suors cedare no carpeting inane! Interests Resders are welcome a cammest on the arin version the paper Publeners noe: Springer Nature remain neta with regard‘ jorsdtions flaims in published maps andinsttuvaral alain, Corespendence ar ‘equess fr mates shoctd be adcreseed te MAL (arhoooney@uesed). Reviewer nfrmation Nature thanks &GinnouxS, Morrison 6, Zimmerman andthe otter anerymaus reviewer) fer he carrauten tothe ae revlew 162017 Maciln Pbieters iis pt of Spenger sur Niven eredLETTER METHODS ‘Mice, Mice were housed and bred under specific pathogen-free conditions at the Univers of Calfornia San Francisco (UCSF) Laborsory Animal Resazch ‘Center and ll experiments conformed to ethical principles and guidlines approved bythe UCSF Intiutional Animal Care and Use Commitee Male and female ice between 8 and 12 weeks of age were wid for experiments, CS7BLI PEU-Gre, Ron26-LSL-tdlamatoalinG, al and Pip Popo Boy) mice were ‘parchared from lsckson Laboratories. To track platelet ot megakaryocytes, EILCre expresing mice were croreed with Rosa26-LSL t€Tomato, mlm’ or nTnG reporter strains, in which the fsorophore of PFi-exprssing celle (cdfemato or GFP) isloaied tothe croplasm, the cell membrane othe nudevs, respecey compl ce (CS7BLIS background) were obtained froma Matsa Tranfer Agreement from Genentech [Lang intravital imaging, We used 2PTVM to cbervemegnkaryoeyte and plalelet ‘prodoction in rea time a mouse ngs. A mated version” of te previously published method of stabilized lung imaging was used”, Mice were anaesthsized With Ketamine ad xylazine and secured wit ape to a custom heated microscope ‘age. smal rachel cannula was inserted, sutured into place, and atached to A MiniVent mouse venilatr (Harvard Apparatus) Mice wee ventilated witha {ial volume of 1 compressed ait (21% Op per gram of mote weight asp loryrate of 130-240 breath per minute and postive-end expiratory pressure af 2-3em TO lrofurane ws delivered contineoly to maintain anaetheria and tice were injected wth 300009 saline soktonintrapentonealy every hour The mice were placed inthe right lateral decubitus postion and a stall surgical Incision was made to expose the cage. second incision wat then made ito the intercostal space between tbe 4 and, through the paletalpeur, to expose thesuface ofthe letung lobe. A flanged horace window with an 8 am coverslip teas then inserted between the two ibs and secured othe age using asetol te pies port and 29° age pot clap (Thor Las)” We applied 20-25mm Fig of suction (Amex Corporation) to gently immobilize the lg, The t- Inicroscope objective was then lowered into place over the thoracic window. selected experiments, to perm identification of the lng vascalatute, FITC desea (50) of 25 m/l Life Techsologies) was injected nzavenousy tothe ta ein belo imaging Spleen and liver intravital imaging. Mice were anaesthetized and ventlated as noted above, expose the spleen, 3 kin incision was made inthe lef lank An incision was made along the costal margin to expose and externalze the liver, Te same window as was used for lung imaging was used to facitate imaging ofthe spleen and liver. The mouse was placed on. 237°C temperature-contaled ‘ated tage forthe dation ofthe imaping and line slution was administered intrapertonesly evry hour Boas mervew inreritlienging. Mice were tntesthetized with an niin doe of [ktamine and xyanine an anaesthesia vas mainsined with wollrane delivered through a nose cone Hair and the underlying subcutaneous tissue were removed to expose the clara, The peioream war emoved using amicrosurgcl kale. Tostabiie the sal we 3D printed an apparatus that was fied tothe mouse sll with Vthend and stached to the heated stage below. The microscope objective tet then lowered nt a -mm bevelled hole filed with line, Theo-photon microscopy. intravital imaging wa performed suing a Nikon AIR “Molt: photon mieroscope equipped with a Mas Zu DeepSee IR Laser (Spectra Physic) (UCSF BID). The MaiTal laser was tuned to 920m for simultane cous extitton of GFP of FITC and wéTomato Emited ight wae detected ung 25x wate Tens (Nikon) with geen (500-880 nm) and ed (570-620) fers Tnnages were captured witha high-resolution galvan reanner (1 fame per second, 5125512 pine), The microscope was contd wing NIS Hlement AR sofware (450), Weeapred 31052): 15744 -ysrlace ea (1 6mm) and zack innages were acquired with: depths fm tal of m= depth) Weeaptured a complete image every Lm for 20min. Tnmage analysis. mages ere analysed using Inavs 761 (Biplane) or NIS-Element (Sikea) software (UCSF BID), Surface analysis was performed to quatiy and sharaerze the volume, diameter, of cculanty of eset and declaing megs Tearyocyes or platelets, Mepaaryoeyies or megakaryocyte Fragments were defined PEA evens tha diameter 153m, Platelets were defined ar PEA" events eth a diameter betveen 05 and 5m To calculate the numberof platelets leased by ‘ach amegakaryocye observed the ratio ofthe megakaryocyte to platelet volumet ‘was calculated for each ofthe 35 fragmenting megakaryocytes ot propatelets served during lng itsaging. Megas yoctes were divided into tres groups ‘cording tothe numberof platelets tal canbe producedby one megakaryocyte mall («900 platelet, n= 18), media (500-1,00 platelets, 7) and lage (1,000 patel 20. ‘Quantifying platelet production in the lng see variables in Extended Data Table 1)- For each movie (~2h), the megakaryocytes observed tobe undergoing fragmentation inthe lung (LungMKj,) were quantified: LungMRjae per hour = (LamgMKin)/(Acquistion time in min) x 60, The numberof platelets released by each megakaryocyte undergoing fragmentation nthe hung was ealeu- lated using the volume ra ofthe megakaryocyte volume tothe average platelet ‘volume: Nang — Volumes Volume. The amber of ates produced inthe Tung was thes calelated: Tungrgay Pes hour Lug Kn et HOLE Na x Lung fraction, whete the lung fraction isthe ratio of the mouse total lang volume? tothe cheerved hing vlisne- Lang fraction = Voleun/ Voki uma Final we estimated the contbution ofthe king to overall thrombopiei ang platelet production = (Lang platelets per hour x 24/(Total Platelets per day) x 100 “The total umber of platelets produced per day was calulated according to te aumber of cueuating platelets in the mouse blood divided by thee span of| platelets, nd take ato account the fac tht one-thd ofthe produced pitts re sequestered by the spleen; total platelet per day —(aigou(l-+ Plc) (fe). selected experiments, mice were ealed with recombinant rman ‘Grombepoictin ATPO, Genentech) itrapentonaly (250 gh, 5 day before Ing imaging “Lung, bone marrow, spleen and blood single-cell preparation for flow cytom- yor ell sorting. Lng digestion. Forlung HSC oF megakaryocyte cell sorting. lunge wee perfused before removal and digestion. Lungs were placed in 21 PBS with Sind Dnatel (Roche) and 0 mpl LiberaeTM (Roche), minced oth teligore in 15m tubes and digested for 30min at 37 Cheforefilration {rough 100 jum cll trainer andre blood elise. Sampler were then Eee through 24m ite and resuspended for subsequent FACS staining, Forexper- tmmente in hich vascular lcalination was teste, mice were injected intavenouhy with CD41-APC (eBioscience o CDAS-APC (eBloscence) Sin before lang allen, Bone marrow sation, Tae and fers som bot es were removed from mice folowing euthanasia Bone marrow cells were shed wilh PBS wih SMM EDTA before fitration through 70 yum cll etraine and redblond cl ye. Spleen isolation. The spleen was removed and pressed with the end ofa plunger fom a 1m eyring into Il PRS with SmiM EDTA before tation through a opm cll suainer and ted Mood ellis Flow cytometry and cell sorting. For surface staining cll or platelets were incu bred with ant e receptor antibodies (clone 2.462) sn etained with stbodies fn Hanks alfred alt elton (2185S) with 2% fetal eal serum and SMC EDTA for 30min “Antibody clones used: CDAL-APC (MWReg30,eBioscence), CD41-FITC (atwReg30, BD), CD41-BV421 (MWReg30, Biokegend), CD4l-BV570 (twRegs0, BioLegend), cxmpl-Biotin (AMD, IBL), streptavidin PE-Cy7 (2D), GPVE-RITC (TAQI, emlrt), F4/80-PB (CLAS-1, BioLegend), CD’S- APC GORI, Biokgend), CDA24-APC (102, eBiosienc), CD62P APC (Pel O25, eBiocience) HSCs were stnned with rat unconjugated Lin antibodier (Gel (RB6-8C5), Mel (91/70), 8220 (RA3-6B2) CDS (53-73), CD4 (GKL) (CDs (53-67) (Bioscience), Ter-19 (BD), CDS (17A2 Biokegend), goatant-za E-Cy5 (lnvittogen),e-Kit-APC- > B98 7 a 7 Lung waneplant experiments: PE& MTG miTmG PFimimG Peres mim Lung PIN an; z eo BE Pas Nose cone Warming pad Pr&mT mG Spl Extended Data Figure I | Megakaryocytes and proplaelets observed in Jang citculstion are from an extrapulmonary source. Lung 2P1VM, of aPFé-nTaG mouse (elear GFP), The presence of the mobile GEP nucleated cells (ircled) indicales the presence ofa nucleus in eitelating megakaryocytes. b Platelet count in the blood before and alter imaging. 1s. no significant (3), Experimental schema of transplantation lungs fem mifmG mouse (perfused donor lung) to PF&-ilinG (vecipient) mouse and vice-versa fllowed by 2PIVM. d,2PIVM ofa niTinG mouse lung showing no GEP signal ¢, 2PIVM ofan miTtnG mouse SS eds oH) n 2PM recipient mouse showing GFP recipient and platelet production in the ling. f, Bone marrow 2PIVM apparatus. g, Representative image of proplatelet eare in bone ‘arrow (BM) sinuslds (arrows) b, 2PIVM of P-mTimG mouse liver Small platelets (GEE, green) were sen i the cizculation but nether resident nor circulating megakaryocytes or propatelets were observed, £,22IVM of BF4 milmG mouse spleen. Sequential megakaryocytes (GPP, green) releasing proplatcets vasculature (in red) ges show resident ‘arrove) inthe slLing Te PF4mTm@-+mTm@ © PF&mTmGBM 2P1M Extended Data Figure 2 | Resident megakaryocytes i the lungs and other organs. a, Survey of PF4-tomato mouse lung visualized by 2P1VM. PFi-tomato-cxpressing cell (red) ae found in high numbers in the lings Lung vasculature i labelled by intravascular injection of FITC dextran, (green) A total area (1mm % 1.6mm) was imaged by Resident state) GFP! cells are found ina PF4-miTinG lungtransplanted ito an milmG mouse. c,d, 2PIVM images of bone marrow () and Pomato Long 2PM 4 Mk Diameter (a) Lung BM Spleen spleen (@) from PFé-miTisG mice. Many lage megakaryocytes (GFP, green ‘te found inthe bone marrow and spleen, e Sie charactertaaion of resident (state) GED” calls by smage analysis of PP4 msTiaG lungs (n= 16), bone marrow (12), and spleen (n= 16) Minimomto:maximum boxpots are shown’ the lie inthe mide of the boxis plotted atthe median, the box ‘xtend fom the 25th o 75th percentiles and the whiskers range from the tmnllet othe largest values, The + indicate the meanLETTER vrteg Seca eam omerers a PTO SS eS SS ‘ ated on nFPVCDA or nGFPCOS © norercosn rere coer coer ‘oe we coe eS eS eS aS i 5 ‘i ng 2 MMOH. cone oerrcowr _narpreoer " ncrprcoss _nrpvcour oa ! we] 2" | fo 8] com) | comm] 8 Wrscssct| WYrscascte bn 8] a fo er fox : oa re eens preatrosn ie cr ere i ™ nee 5 04 7 Ee ‘ie Boo 2 ‘ io © e % ‘cbs CDs a= 20 g OK zo Bo. aTom ——> cb41 ——_s © LungeM °° tung BM ‘Lung BM Extended Data Figure 3 | Surface expression of lung megakaryocytes compared to bone marrow megakaryocytes a, Flow cytometric analysis fof nGEP* cell from PP nTaG lungs. b, CD41 expression defines Wo populations of megakaryocytes: CD41” (red) and CD41” (gr «© Postive erface expression of GPVI, Mil and CD45 was detected in ‘oth populations, Unstaine cells are potted in be. Surface expression of 84/80, CD34, CDIIb, Sea-L and cKit was not detected, e-4, The CD4L population has ahigher percentage of CD61" cells (e), CD42" cells) and larger cells (g) and had higher DNA content (h), as summarized ind (a3), Flow cytometric analysis of GED ces rom PFA-aTaG. bone marrow. kom, Compared tothe Langs, the bone marrow nGFP population has higher percentage of CD41" cell (n~ 21-28) (), cells (n= 3)() and CD42b" cells n~ 3) (ma). Data are representative fof three or more replicates. Mean = ed. are presented, Unpaited ter. *P<005,"*P<001,***P <0.001, 61LETTER aoe A . FoR<005 5 B a. i 3 2 By ° Pt4Cxerd CDK2d Vit CD42a Pobp COG! CDF CO42e CDA Gp6 COSZP e 4 ite or din inflammty response cal yb imme syste process cteoosaie sgrgiten chenott spony tion [UR 4 sgn pty od engin nae oct ble og i yan postive egton of THF prdtin regain of uct sp eee ‘mceuaes oktoctere ame tes clench postive regulon of oy kine sorta rte axtophsphary atin postive relatn of 18 rode ela repo efron weenie relation of Patt sctiv an aki enponto opto ao region cl GBM aston mia el eye ‘nit ane respons nemo crag lat ton ube of genes Numba of gnes| ca FOR<0.05 17 FoRo0s g g 83 £3 zi 2? Eo E 3 Sh Ph CL SF HS oS SS — PMP LL FI FSF PS PSSM tow Hoh Pre Ling ater bce pemeig e 1 : 9.300000] _» F 200000) F rooe0 np 1 e * “ig 100) Le 2 rsooo04— «80.000 goof TL 5g % i im 3 sas age] ie 8 000004 I x = 40,000 ge §o 0 & § woo lf =) § am 88 10. es 6 o eo : °. g* 6 5° 0. mesa = MRSA MRSA = MRSA ~ MRSA Extended Data Figure 4 | See next page for caption.LETTER Extended Data Figure 4 | Gene expression analyse of lung versus ‘bone marrow megakaryocytes and bacterial pneumonia experiment reveal an immature profile and a potential role in imeaunity for lung megakaryocytes. Megakaryocyter (sGEP” CDM") were sorted fom PE&:nInG lungs and bone marrow followed by mRNA isolation and sequencing. a. Relative mRNA expression is shown ona scale from low (gzeen) to high (ed). Three independest experiments four mice cach) ‘were used for saistial analyse eatmap of ll signsicantlydiferenally expeessed genes (FDR <0.05)b, Read counts for megakaryocyte felated genes. These genes ate found in both lung and bane marzow megtharyoeytes, bul tome are underrepresented in lng megakaryocytes «4, Analysis of gene ontology biclogieal processes related t genes that, axe downregulated (¢) or upregulated (tn lung megakaryocytes. The {op 20 biological processes are shown, The vertical aie represent gene ‘ontology categories, and the horizontal axis indicates the number of genes ineach ontology category. ,£ Read counts for TLR gene pathways (e) and chemokines (0) that were overexpressed in lung megakaryocyte. PKM, Fragments perklobate of exon per milion fragments mapped. g-k, Lang megakaryocytes and progenitor populations ae altered during infection. low eytomelric anys of nGEP* celle from PF4-rnG lungs 2¢h after intratracheal administration of S. aureus (MSA, 5% 10” colosy-forming ‘units (¢£). CDAL-APC was injected intavenousy before lung digestion and staining with CD41-FITC, The numberof els n normal or infected Jungs are shows for all nGEP' cel) ess mature cells (2GFP~ CD41). snd mature eels (aGFP* CD41"; i), jk Percentage of intravascular ‘megakaryocytes () and extravascular megakaryoeytes(k) inthe matsre population (aGEP! CD41!) 4-8mice per group. (©2017 Merion Pubishers ited pa of Springer Nature A ahs esetLETTER a Without TPO. . * ‘With TPO e gi] neas gy git] mmo eee z z z z Sol fo. Ff 70% ae de i deo 3 oie tac os © cre ato 3 i ox 2% 3 308 Ew He Em. ai ° 0 0 ° Cra Wi Eee ai vo ic eae ao IS wpeetooe 1 eoniaes Rint a ener eas Tieton) Tine (4a) Tie (doy) Time aa) e Witout TPO ¢ ° win Teo h E 1000. E 1000. E 1000. E 1000. s s z : a S00. a S00. @ 500. az 500. 3 3 3 3 é é é é of of 0 ° 2m mC dat m® mm 0 » mw Time a) Time an) Tine (doy) Time ear) i 20 peer k |L_Donor Lung Recipient TPO Response Eis. Ee F sco. ~e ome om i Fo. 8 eo Prom wT foo aa E eo Ae Préiom omot® = Sustained io Pm 5 = Préiom ompt® —— Transent é é é “& Prétom comp" TPO Sustained o. . ° “* PF4tom c-mpr" TPO Transient 7. lUmlUUCUC OCC e|h[U OUT Uh OO Tee (de) “Tine ront) Tee (monte) Extended Data Figure 5 | Platelet reconstitution after lung. tuansplantation with or without TPO injection Blood was collected fom the mandibular vein every week following lng transplantation. Aiteransplantation, a group of mice (ed and orange) received TPO. injection (250.mg kg’, days 3 snd 40) The ober group was left untreated (bie and purple) Some mice in each group showed sustained platelet production for more than 3 months (blue and red), Inthe other mice (purple and orange), the platelet production waslower and transient (less than 3 months). The percentages of mice in each group are indicated. ym individual mice (a, g) or group averages (bd, h) are plotted and.ij Percentage of donor derived platelets Percentage was analysed by FACS, counting the tomato" platelets (CD41" F5Cman gat) -h.k Overall platelet counts in the peripheral blood jk, Plots fom, tnice wth 1O month of sustained platelet production &Peteentage of, tomate platelets in control lung tansplants(n 4-6). Colour code forthe diferent ung transplant groups according to lung origin (doner), recipient mouse, treatment received (with or without TPO) and observed response (sustained or transient). Unites pt of Spenger sure vans eredLETTER oma blood ee) 3 oe j j Feo Seo Sc Soe Hi 6 conto} Ti npr mouse [Ei Prom mouse HE ‘count Count Mel ———> Count bee” sc ——S Tomato >) Tomato > os eae cur ee = leer ee a ; | um emp HS tonepe ! | un 3 if 5 — ct con oon Ne ———> Lung transplant exprimons : PF&-tomato —» cxmoté Prono Patio) Cg Tcl Tow Ton ‘conor unge Recipient pulls Tom’ platoets crack eOe2P —— capeoton on Tor oa a ae Acton nth Desens Thrombin ont Gato Toe pasts PFvicmao PPasonato cap 8 ep z sme mam sen) 28 ron stnstos > >| $2 I a: 9. Bi tirombin 10 nit 2 : é fide. |S 2, Res Oo Extended Data Figure 6 | Characterization of platelets produced after Jang transplantation. ab, Flow eytomettic analysis of vomato® platelets ‘observed inthe Blood and stained with antibodies against CD41. cDazd, GPVI and €-pl. Blood from PF4-tomato mice (a) or mpi’ mice that nad received PM tomato lung transplant (b).«, Experimental schema for plalelet activation. d, low cytometric analysis of tomato” platelets after stimlation with thrombin (10nM) stained with antibody again CD62P « ecentage of CDS2P” platelets before and ater thombin activation, (Gu PE4-tomato; Lung te, PF4-tomato lungs wansplanted inte mp! tice. Mean t ad are presented (n 2-3 mice per group). Unpated tet: SP oot, **P-<0001 (© 2017 Merion Pubishers ined par of Springer Nature A ahs reseed