{"id":602651,"date":"2024-05-29T20:00:00","date_gmt":"2024-05-30T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/alternative-platelet-differentiation-pathways-initiated-by-nonhierarchically-related-hematopoietic-stem-cells-nature-immunology\/"},"modified":"2024-05-31T17:01:58","modified_gmt":"2024-05-31T21:01:58","slug":"alternative-platelet-differentiation-pathways-initiated-by-nonhierarchically-related-hematopoietic-stem-cells-nature-immunology","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/alternative-platelet-differentiation-pathways-initiated-by-nonhierarchically-related-hematopoietic-stem-cells-nature-immunology\/","title":{"rendered":"Alternative platelet differentiation pathways initiated by nonhierarchically related hematopoietic stem cells – Nature Immunology","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
Animal experiments performed at the University of Oxford were approved by the Oxford Clinical Medicine Ethical Review Committee, and those performed at the Karolinska Institutet were approved by the regional review committee for animal ethics (Stockholms djurf\u00f6rs\u00f6ksetiska n\u00e4mnd). All experimental and mouse breeding procedures were performed in accordance with the UK Home Office and Swedish Jordbruksverket regulations.<\/p>\n
Young adult (7\u201314 weeks old) Vwf<\/i>-tdTomato\/Gata1<\/i>-eGFP mice9<\/a>,17<\/a><\/sup> (Vwf<\/i>-tdTomatotg\/+<\/sup> Gata1<\/i>-eGFPtg\/+<\/sup>) and Flt3<\/i>Cre\/Rosa26<\/i>tdTomato(Ai9)\/Vwf<\/i>-eGFP\/Gata1<\/i>-eGFP mice8<\/a>,17<\/a>,59<\/a><\/sup> (Flt3<\/i>Cretg\/+<\/sup> R26<\/i>Tom\/+<\/sup> Vwf<\/i>-eGFPtg\/+<\/sup> Gata1<\/i>-eGFPtg\/+<\/sup>) on a C57BL\/6OlaHsd (University of Oxford) or C57BL\/6JrJ (Karolinska Institutet) background were used as BM donors in single HSC transplantations. Seven- to 18-week-old wild-type CD45.1 B6.SJL-Ptprc<\/i>a<\/i><\/sup> Pepc<\/i>b<\/i><\/sup>\/BoyJ (University of Oxford) and B6.SJL-Ptprc<\/i>a<\/i><\/sup> Pepc<\/i>b<\/i><\/sup>\/BoyCrl (Karolinska Institutet) mice were used as recipients in primary and secondary transplantations, as donors of unfractionated BM competitor cells, and for BM analysis after anti-CD42b antibody treatment. Recipient mice that did not survive or had to be killed before 16\u201318 weeks after the primary or secondary transplantation were excluded from analyses. Flt3<\/i>Cre\/Rosa26<\/i>tdTomato(Ai14) mice (Flt3<\/i>Cretg\/+<\/sup> R26<\/i>Tom\/+<\/sup>; 7\u201311 weeks old) and Vav<\/i>Cre\/Rosa26<\/i>tdTomato(Ai14) mice51<\/a><\/sup> (Vav<\/i>Cretg\/+<\/sup> R26<\/i>Tom\/+<\/sup>; 8\u201323 weeks old) on a C57BL\/6JrJ background were used for fate-mapping and CP and 5FU treatment experiments. Flt3<\/i>Cre\/Rosa26<\/i>tdTomato(Ai9)\/Vwf<\/i>-eGFP\/Gata1<\/i>-eGFP (Flt3<\/i>Cretg\/+<\/sup> R26<\/i>Tom\/+<\/sup> Vwf<\/i>-eGFPtg\/+<\/sup> Gata1<\/i>-eGFPtg\/+<\/sup>; 8\u201313 weeks old) mice on a C57BL\/6OlaHsd background were also used for steady-state fate-mapping and anti-CD42b treatment experiments. Mice were housed in individually ventilated cages at the Oxford JR facility (12\/12\u2009h light\/dark cycle, 19\u201324\u2009\u00b0C and 45\u201365% humidity) and the Karolinska Institutet KM facility (12\/12\u2009h light\/dark cycle, 22\u2009\u00b1\u20091\u2009\u00b0C and 50% humidity).<\/p>\n Single-cell sorting of adult BM HSCs was performed using a FACSAria II or FACSAria Fusion cell sorter (BD Biosciences), prepared by crushing pelvic and leg bones (and optionally also sternum and spine bones) into PBS with 5% fetal calf serum (FCS; Sigma-Aldrich) and 2\u2009mM EDTA (Sigma-Aldrich). Single phenotypically defined HSCs (Extended Data Fig. 1a<\/a>) were sorted from Vwf<\/i>-tdTomatotg\/+<\/sup> Gata1<\/i>-eGFPtg\/+<\/sup> mice (Vwf-<\/i>tdTomato+<\/sup> and Vwf<\/i>-tdTomato\u2212<\/sup> fractions of LSKGata1<\/i>-eGFP\u2212<\/sup>CD34\u2212<\/sup>CD150+<\/sup>CD48\u2212<\/sup> or LSKGata1<\/i>-eGFP\u2212<\/sup>CD34\u2212<\/sup>CD150+<\/sup>CD48\u2212<\/sup>CD201+<\/sup> cells) and Flt3<\/i>Cretg\/+<\/sup> R26<\/i>Tom\/+<\/sup> Vwf<\/i>-eGFPtg\/+<\/sup> Gata1<\/i>-eGFPtg\/+<\/sup> mice (Vwf\/Gata1<\/i>-eGFP+<\/sup> and Vwf\/Gata1<\/i>-eGFP\u2212<\/sup> fractions of LSKCD34\u2212<\/sup>CD150+<\/sup>CD48\u2212<\/sup> or LSKCD34\u2212<\/sup>CD150+<\/sup>CD48\u2212<\/sup>CD201+<\/sup> cells).<\/p>\n Comparison of single-cell expression and coexpression of Vwf<\/i>-eGFP and Gata1<\/i>-eGFP in BM LSKCD34\u2212<\/sup>CD150+<\/sup>CD48\u2212<\/sup> cells showed that <10% express Gata1<\/i>-eGFP (Extended Data Fig. 7c<\/a>). Moreover, Gata1<\/i>-eGFP and CD201 are mutually exclusive in LSKCD34\u2212<\/sup>CD150+<\/sup>CD48\u2212<\/sup> cells, indicating that eGFP expression in LSKCD34\u2212<\/sup>CD150+<\/sup>CD48\u2212<\/sup>CD201+<\/sup> cells mainly reflects Vwf<\/i>-eGFP+<\/sup> HSCs (Extended Data Fig. 7c<\/a>). Regardless of their cell-surface phenotype, P-HSCs and multi-HSCs were defined based on their long-term lineage replenishment pattern as established by blood lineage analysis at multiple time points (see the next sections).<\/p>\n In experiments with mice that coexpress Vwf<\/i>-eGFP and Gata1<\/i>-eGFP, eGFP was used for sorting of single eGFP+<\/sup> and eGFP\u2212<\/sup> HSCs for transplantation and to identify donor-derived blood platelets (which express both Vwf<\/i> and Gata1<\/i>) and erythrocytes (which express Gata1<\/i>) in the transplantation recipients.<\/p>\n Single HSCs were sorted by an automated cell deposition unit, refrigerated at 4\u2009\u00b0C, into 96-well round-bottom plates (Corning) with 100\u2009\u03bcl per well of Iscove\u2019s modified Dulbecco\u2019s medium (IMDM, Gibco) with 20% BIT-9500 serum substitute (Stem Cell Technologies), 100\u2009U\u2009ml\u22121<\/sup> penicillin and 0.1\u2009mg\u2009ml\u22121<\/sup> streptomycin (100\u00d7 Pen\/Strep, Hyclone), 2\u2009mM l<\/span>-glutamine (Gibco) and 0.1\u2009mM 2-mercaptoethanol (Sigma-Aldrich). Single index-sorted HSCs were mixed with 2\u20133\u2009\u00d7\u2009105<\/sup> wild-type CD45.1 unfractionated BM competitor cells (100\u2009\u03bcl per well) and transplanted by intravenous lateral tail-vein injection into lethally irradiated CD45.1 mice (10\u201310.5\u2009Gy, cesium-137 or X-ray). BM cell counts were measured manually with a hemacytometer and\/or an automated cell counter (Sysmex XP-300 or ABX Pentra ES 60).<\/p>\n Peripheral blood was collected from a lateral tail vein into lithium\u2013heparin or K3 EDTA microvettes (Sarstedt). The platelet supernatant was collected after centrifugation of blood samples at 100g<\/i> for 10\u2009min at room temperature. Then, it was mixed with a small fraction (0.5\u20131\u2009\u03bcl) of red precipitate for combined analysis of platelets and erythrocytes. The remaining precipitate was incubated 1:1 with dextran (Sigma-Aldrich, M<\/i>r<\/sub> 450,000\u2013650,000) 2% w\/v in PBS for 20\u201330\u2009min at 37\u2009\u00b0C. Erythrocytes were lysed by incubation in ammonium chloride solution (Stem Cell Technologies) for 2\u2009min at room temperature. Leukocyte samples were incubated with purified CD16\/32 (Fc-block) for 10\u201315\u2009min at 4\u2009\u00b0C. Then, they were stained with anti-mouse antibodies for 15\u201320\u2009min at 4\u2009\u00b0C in PBS with 1\u20135% FCS and 2\u2009mM EDTA. Samples were analyzed using LSRII and Fortessa cytometers (BD Biosciences). See Supplementary Table 5<\/a> for antibody details.<\/p>\n Donor-derived platelets were defined as follows: CD150+<\/sup>CD41+<\/sup>TER119\u2212<\/sup>Vwf<\/i>-tdTomato+<\/sup>Gata1<\/i>-eGFP+<\/sup> for Vwf<\/i>-tdTomatotg\/+<\/sup> Gata1<\/i>-eGFPtg\/+<\/sup> donors and CD150+<\/sup>CD41+<\/sup>TER119\u2212<\/sup>Vwf\/Gata1<\/i>-eGFP+<\/sup> for Flt3<\/i>Cretg\/+<\/sup> R26<\/i>Tom\/+<\/sup> Vwf<\/i>-eGFPtg\/+<\/sup> Gata1<\/i>-eGFPtg\/+<\/sup> donors. Donor-derived erythrocytes: TER119+<\/sup>CD150\u2212<\/sup>CD41\u2212<\/sup>Vwf<\/i>-tdTomato\u2212<\/sup>Gata1<\/i>-eGFP+<\/sup> for Vwf<\/i>-tdTomatotg\/+<\/sup> Gata1<\/i>-eGFPtg\/+<\/sup> donors and TER119+<\/sup>CD150\u2212<\/sup>CD41\u2212<\/sup>Vwf\/Gata1<\/i>-eGFP+<\/sup> for Flt3<\/i>Cretg\/+<\/sup> R26<\/i>Tom\/+<\/sup> Vwf<\/i>-eGFPtg\/+<\/sup> Gata1<\/i>-eGFPtg\/+<\/sup> donors. Donor-derived myeloid (granulocyte\/monocyte) cells: CD11b+<\/sup>NK1.1\u2212<\/sup>CD19\u2212<\/sup>CD4\/CD8a\u2212<\/sup>CD45.1\u2212<\/sup>CD45.2+<\/sup>. Donor-derived B cells: CD19+<\/sup>NK1.1\u2212<\/sup>CD4\/CD8a\u2212<\/sup>CD11b\u2212<\/sup>CD45.1\u2212<\/sup>CD45.2+<\/sup>. Donor-derived T cells: CD4\/CD8a+<\/sup>NK1.1\u2212<\/sup>CD11b\u2212<\/sup>CD19\u2212<\/sup>CD45.1\u2212<\/sup>CD45.2+<\/sup>. The granulocyte\/monocyte identity of CD11b+<\/sup>NK1.1\u2212<\/sup>CD19\u2212<\/sup>CD4\/CD8a\u2212<\/sup>CD45.1\u2212<\/sup>CD45.2+<\/sup> cells from reconstituted recipient mice was confirmed by cytospins stained with eosin Y\/azure A\/methylene blue (Richard-Allan Scientific Three-Step Stain Set, Thermo Fisher Scientific) (Supplementary Fig. 4<\/a>).<\/p>\n All five mature blood cell lineages (platelets, erythrocytes, myeloid cells, B cells and T cells) were considered. We considered primary and secondary recipients to be reconstituted by HSCs if the donor contribution to platelets was \u22650.1% at \u226516\u201318 weeks after transplantation9<\/a><\/sup>. Mice reconstituted by single Vwf<\/i>+<\/sup> P-HSCs and Vwf<\/i>\u2212<\/sup> multi-HSCs were defined at \u226516\u201318 weeks after transplantation as follows. Vwf<\/i>+<\/sup> P-HSC: donor platelets \u22650.1%; donor platelet percentage \u226550-fold higher than donor erythrocytes and myeloid cells; and donor B and T cells undetectable (\u22640.01%). Where indicated (Fig. 7b<\/a> and Extended Data Fig. 7e,f<\/a>), the stricter group of platelet-restricted HSCs was considered, in which donor erythrocyte, myeloid cell, B cell and T cell lineages were all below the detection level (\u22640.01%). Vwf<\/i>\u2212<\/i><\/sup> multi-HSC: donor platelets \u22650.1%; donor erythrocytes, myeloid cells, B cells and T cells all >0.01%; and donor platelets, erythrocytes and myeloid cells all \u22642-fold higher than B and T cells. Mice reconstituted with multilineage patterns with \u22652-fold platelet, platelet\u2013erythroid and platelet\u2013erythroid\u2013myeloid bias were excluded from the Vwf<\/i>\u2212<\/sup> multi-HSC group, as such biases are typical of Vwf<\/i>+<\/i><\/sup> multi-HSCs9<\/a><\/sup>.<\/p>\n BM HSPC reconstitution analysis was performed using FACSAria Fusion, LSRII and LSR Fortessa flow cytometers (BD Biosciences) after crushing pelvic and leg bones (and optionally also sternum and spine bones) into PBS with 5% FCS and 2\u2009mM EDTA, followed by cKIT enrichment according to the manufacturer\u2019s instructions (CD117 MicroBeads and magnetic activated cell sorting (MACS) LS columns, Miltenyi Biotec). cKIT-enriched BM cells were incubated with purified CD16\/32 (Fc-block) for 15\u201320\u2009min at 4\u2009\u00b0C, followed by anti-mouse antibody staining for 15\u201320\u2009min at 4\u2009\u00b0C. For the myeloid progenitor panel, cells were incubated with fluorophore-conjugated CD16\/32 before further staining. See Supplementary Table 5<\/a> for antibody details.<\/p>\n Phenotypic BM populations were defined as follows: LSK, LIN\u2212<\/sup>SCA1+<\/sup>cKIT+<\/sup>; LK, LIN\u2212<\/sup>SCA1\u2212<\/sup>cKIT+<\/sup>; LT-HSC, LSKFLT3\u2212<\/sup>CD150+<\/sup>CD48\u2212<\/sup>; ST-HSC, LSKFLT3\u2212<\/sup>CD150\u2212<\/sup>CD48\u2212<\/sup>; MPP2, LSKFLT3\u2212<\/sup>CD150+<\/sup>CD48+<\/sup>; MPP3, LSKFLT3\u2212<\/sup>CD150\u2212<\/sup>CD48+<\/sup>; MPP4, LSKFLT3+<\/sup>; MkP, LKCD150+<\/sup>CD41+<\/sup>; preMegE progenitor, LKCD41\u2212<\/sup>CD16\/32Single-cell transplantations<\/h3>\n
Blood reconstitution analysis<\/h3>\n
Categorization of reconstitution patterns<\/h3>\n
Reconstitution analysis of HSPCs<\/h3>\n