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• W4285086384 abstract "Article13 July 2022Open Access Transparent process Genetically modified macrophages accelerate myelin repair Marie-Stéphane Aigrot Marie-Stéphane Aigrot orcid.org/0000-0003-0307-2774 INSERM UMR1127 Sorbonne Université, Paris Brain Institute (ICM), Paris, France Contribution: Data curation, Formal analysis, ​Investigation Search for more papers by this author Clara Barthelemy Clara Barthelemy orcid.org/0000-0003-4658-6169 INSERM UMR1127 Sorbonne Université, Paris Brain Institute (ICM), Paris, France Contribution: Data curation, ​Investigation Search for more papers by this author Sarah Moyon Sarah Moyon orcid.org/0000-0002-6142-614X NYU Langone Health, Neuroscience Institute, New York City, NY, USA Contribution: Data curation, Formal analysis, Validation, ​Investigation Search for more papers by this author Gaelle Dufayet-Chaffaud Gaelle Dufayet-Chaffaud INSERM UMR1127 Sorbonne Université, Paris Brain Institute (ICM), Paris, France Contribution: Data curation, Formal analysis Search for more papers by this author Leire Izagirre-Urizar Leire Izagirre-Urizar Achucarro Basque Center for Neuroscience/Department of Neuroscience, School of Medicine University of the Basque Country, Leioa, Spain Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Contribution: Data curation, ​Investigation Search for more papers by this author Beatrix Gillet-Legrand Beatrix Gillet-Legrand orcid.org/0000-0002-1159-5949 INSERM UMR1127 Sorbonne Université, Paris Brain Institute (ICM), Paris, France Contribution: Data curation, Formal analysis Search for more papers by this author Satoru Tada Satoru Tada orcid.org/0000-0002-0323-5644 INSERM UMR1127 Sorbonne Université, Paris Brain Institute (ICM), Paris, France Contribution: Formal analysis, ​Investigation Search for more papers by this author Laura Bayón-Cordero Laura Bayón-Cordero orcid.org/0000-0001-5551-3290 Achucarro Basque Center for Neuroscience/Department of Neuroscience, School of Medicine University of the Basque Country, Leioa, Spain Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Contribution: Data curation, Formal analysis Search for more papers by this author Juan-Carlos Chara Juan-Carlos Chara Achucarro Basque Center for Neuroscience/Department of Neuroscience, School of Medicine University of the Basque Country, Leioa, Spain Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Contribution: Data curation Search for more papers by this author Carlos Matute Carlos Matute orcid.org/0000-0001-8672-711X Achucarro Basque Center for Neuroscience/Department of Neuroscience, School of Medicine University of the Basque Country, Leioa, Spain Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Contribution: Funding acquisition Search for more papers by this author Nathalie Cartier Corresponding Author Nathalie Cartier [email protected] orcid.org/0000-0003-2298-5261 Asklepios Biopharmaceutical, Inc., Institut du Cerveau (ICM), Paris, France Contribution: Conceptualization, Supervision, Funding acquisition, Project administration Search for more papers by this author Catherine Lubetzki Corresponding Author Catherine Lubetzki [email protected] orcid.org/0000-0001-7164-3175 INSERM UMR1127 Sorbonne Université, Paris Brain Institute (ICM), Paris, France AP-HP, Sorbonne Université, Hôpital Pitié-Salpêtrière, Paris, France Contribution: Conceptualization, Supervision, Funding acquisition Search for more papers by this author Vanja Tepavčević Corresponding Author Vanja Tepavčević [email protected] orcid.org/0000-0003-3230-4888 Achucarro Basque Center for Neuroscience/Department of Neuroscience, School of Medicine University of the Basque Country, Leioa, Spain Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, ​Investigation, Methodology Search for more papers by this author Marie-Stéphane Aigrot Marie-Stéphane Aigrot orcid.org/0000-0003-0307-2774 INSERM UMR1127 Sorbonne Université, Paris Brain Institute (ICM), Paris, France Contribution: Data curation, Formal analysis, ​Investigation Search for more papers by this author Clara Barthelemy Clara Barthelemy orcid.org/0000-0003-4658-6169 INSERM UMR1127 Sorbonne Université, Paris Brain Institute (ICM), Paris, France Contribution: Data curation, ​Investigation Search for more papers by this author Sarah Moyon Sarah Moyon orcid.org/0000-0002-6142-614X NYU Langone Health, Neuroscience Institute, New York City, NY, USA Contribution: Data curation, Formal analysis, Validation, ​Investigation Search for more papers by this author Gaelle Dufayet-Chaffaud Gaelle Dufayet-Chaffaud INSERM UMR1127 Sorbonne Université, Paris Brain Institute (ICM), Paris, France Contribution: Data curation, Formal analysis Search for more papers by this author Leire Izagirre-Urizar Leire Izagirre-Urizar Achucarro Basque Center for Neuroscience/Department of Neuroscience, School of Medicine University of the Basque Country, Leioa, Spain Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Contribution: Data curation, ​Investigation Search for more papers by this author Beatrix Gillet-Legrand Beatrix Gillet-Legrand orcid.org/0000-0002-1159-5949 INSERM UMR1127 Sorbonne Université, Paris Brain Institute (ICM), Paris, France Contribution: Data curation, Formal analysis Search for more papers by this author Satoru Tada Satoru Tada orcid.org/0000-0002-0323-5644 INSERM UMR1127 Sorbonne Université, Paris Brain Institute (ICM), Paris, France Contribution: Formal analysis, ​Investigation Search for more papers by this author Laura Bayón-Cordero Laura Bayón-Cordero orcid.org/0000-0001-5551-3290 Achucarro Basque Center for Neuroscience/Department of Neuroscience, School of Medicine University of the Basque Country, Leioa, Spain Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Contribution: Data curation, Formal analysis Search for more papers by this author Juan-Carlos Chara Juan-Carlos Chara Achucarro Basque Center for Neuroscience/Department of Neuroscience, School of Medicine University of the Basque Country, Leioa, Spain Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Contribution: Data curation Search for more papers by this author Carlos Matute Carlos Matute orcid.org/0000-0001-8672-711X Achucarro Basque Center for Neuroscience/Department of Neuroscience, School of Medicine University of the Basque Country, Leioa, Spain Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Contribution: Funding acquisition Search for more papers by this author Nathalie Cartier Corresponding Author Nathalie Cartier [email protected] orcid.org/0000-0003-2298-5261 Asklepios Biopharmaceutical, Inc., Institut du Cerveau (ICM), Paris, France Contribution: Conceptualization, Supervision, Funding acquisition, Project administration Search for more papers by this author Catherine Lubetzki Corresponding Author Catherine Lubetzki [email protected] orcid.org/0000-0001-7164-3175 INSERM UMR1127 Sorbonne Université, Paris Brain Institute (ICM), Paris, France AP-HP, Sorbonne Université, Hôpital Pitié-Salpêtrière, Paris, France Contribution: Conceptualization, Supervision, Funding acquisition Search for more papers by this author Vanja Tepavčević Corresponding Author Vanja Tepavčević [email protected] orcid.org/0000-0003-3230-4888 Achucarro Basque Center for Neuroscience/Department of Neuroscience, School of Medicine University of the Basque Country, Leioa, Spain Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, ​Investigation, Methodology Search for more papers by this author Author Information Marie-Stéphane Aigrot1, Clara Barthelemy1, Sarah Moyon2, Gaelle Dufayet-Chaffaud1, Leire Izagirre-Urizar3,4, Beatrix Gillet-Legrand1, Satoru Tada1,7, Laura Bayón-Cordero3,4, Juan-Carlos Chara3,4, Carlos Matute3,4, Nathalie Cartier *,5, Catherine Lubetzki *,1,6 and Vanja Tepavčević *,3 1INSERM UMR1127 Sorbonne Université, Paris Brain Institute (ICM), Paris, France 2NYU Langone Health, Neuroscience Institute, New York City, NY, USA 3Achucarro Basque Center for Neuroscience/Department of Neuroscience, School of Medicine University of the Basque Country, Leioa, Spain 4Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain 5Asklepios Biopharmaceutical, Inc., Institut du Cerveau (ICM), Paris, France 6AP-HP, Sorbonne Université, Hôpital Pitié-Salpêtrière, Paris, France 7Present address: Department of Neurology, Osaka University Graduate School of Medicine, Osaka, Japan *Corresponding author. Tel: +33 157274604; E-mail: [email protected] author. Tel: +33 157274465; E-mail: [email protected] author. Tel: +34 94 601 8284; E-mail: [email protected] EMBO Mol Med (2022)14:e14759https://doi.org/10.15252/emmm.202114759 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Preventing neurodegeneration-associated disability progression in patients with multiple sclerosis (MS) remains an unmet therapeutic need. As remyelination prevents axonal degeneration, promoting this process in patients might enhance neuroprotection. In demyelinating mouse lesions, local overexpression of semaphorin 3F (Sema3F), an oligodendrocyte progenitor cell (OPC) attractant, increases remyelination. However, molecular targeting to MS lesions is a challenge. A clinically relevant paradigm for delivering Sema3F to demyelinating lesions could be to use blood-derived macrophages as vehicles. Thus, we chose transplantation of genetically modified hematopoietic stem cells (HSCs) as means of obtaining chimeric mice with circulating Sema3F-overexpressing monocytes. We demonstrated that Sema3F-transduced HSCs stimulate OPC migration in a neuropilin 2 (Nrp2, Sema3F receptor)-dependent fashion, which was conserved in middle-aged OPCs. While demyelinating lesions induced in mice with Sema3F-expressing blood cells showed no changes in inflammation and OPC survival, OPC recruitment was enhanced which accelerated the onset of remyelination. Our results provide a proof of concept that blood cells, particularly monocytes/macrophages, can be used to deliver pro-remyelinating agents “at the right time and place,” suggesting novel means for remyelination-promoting strategies in MS. Synopsis While several pro-remyelinating molecules have been identified, targeting these to demyelinated MS lesions, disseminated in the CNS, is a major challenge. This study shows that blood–macrophages can be used as efficient molecular deliverers to demyelinating CNS lesions to increase remyelination. Transplanted hematopoietic stem cells give rise to macrophages that infiltrate CNS lesions shortly after demyelination. HSCs genetically modified to overexpress Sema3F secrete Sema3F that attracts both young and middle-aged OPCs in vitro. Lesions in chimeric mice with Sema3F-overexpressing hematopoietic cells show increased OPC recruitment, followed by accelerated generation of new oligodendrocytes and remyelination. Introduction Halting the progression of neurological disability in patients with multiple sclerosis (MS) is a major challenge. MS frequently starts as a relapsing–remitting disease (RRMS), with neurological symptoms alternating with episodes of recovery. RRMS eventually evolves into progressive disease, characterized by the development of permanent neurological handicaps. A subset of patients directly enters the progressive phase (Lassmann, 2018). Currently used treatments for MS are mostly immunomodulators and immunosuppressants. These reduce inflammation and related relapses but are largely ineffective in preventing transition into progressive disease (Lassmann, 2018) where accumulation of disability is the consequence of neuronal/axonal loss, likely triggered by increased vulnerability of demyelinated axons (Kornek et al, 2000). Remyelination (myelin regeneration) of demyelinated axons protects these from degeneration, as shown by neuropathological studies of MS tissue (Kornek et al, 2000), experiments using a model of demyelination (Irvine & Blakemore, 2008), and myelin PET imaging of MS patients (Bodini et al, 2016). Hence, developing remyelination-promoting strategies is a major therapeutic goal to prevent progression in patients with MS (Lubetzki et al, 2020). In a subset of MS lesions, remyelination failure is associated with depletion/low numbers of oligodendroglial cells (Boyd et al, 2013; Moll et al, 2013; Tepavčević et al, 2014; Heß et al, 2020). In these, stimulating oligodendrocyte progenitor cell (OPC) repopulation/recruitment to demyelinated areas appears crucial to enhance remyelination and achieve neuroprotection. OPC recruitment in experimental lesions can be achieved by locally injecting a viral vector overexpressing a guidance molecule semaphorin 3F (Sema3F), an OPC attractant (Piaton et al, 2011; Boyd et al, 2013). Moreover, we previously showed that OPCs in MS lesions express Sema3F receptor, neuropilin 2 (Nrp2), and that while Sema3F is detected in early lesions, it is not expressed in chronically demyelinated lesions (Williams et al, 2007). Thus, these data suggest that OPC recruitment in MS lesions may be enhanced by increasing intralesional Sema3F expression. However, the viral vector injection paradigm is not applicable in the patient setting for a number of reasons, one of them being that MS lesions are disseminated within the CNS. This question of how to optimally deliver therapeutic molecules to demyelinating MS lesions applies not only to strategies to increase OPC recruitment but also to other neuroprotective approaches such as delivering neurotrophic or antioxidative molecules to protect the axons, and in general, to any attempts to modify intralesional events in order to stop the progression of the pathology. In leukodystrophies, a group of diseases in which a molecular defect in glial cells leads to anomalies in white matter formation and/or maintenance, intraparenchymal delivery of therapeutic proteins has been achieved using transplantation of autologous genetically modified hematopoietic stem/progenitor cells (HSCs). Using this strategy, blood-born cells engineered to overexpress the missing brain protein were reported to infiltrate the brain, which raised parenchymal levels of the protein in question and corrected, at least in part, the associated myelin pathology. This approach has been successful in both mouse models and human leukodystrophy patients (Biffi et al, 2004, 2006, 2013; Cartier et al, 2009; Sessa et al, 2016; Eichler et al, 2017). We hypothesized that intralesional targeting of therapeutic molecules using blood-born cells could be a clinically relevant strategy to stimulate remyelination in demyelinating CNS diseases, such as MS, by means of targeting the expression of Sema3F to demyelinating lesions and increasing OPC recruitment. We first showed that monocytes/macrophages derived from transplanted HSCs efficiently infiltrate demyelinating mouse lesions. We then designed lentiviral vectors that allowed us to genetically modify HSCs to overexpress Sema3F and investigated whether these cells can stimulate OPC migration in vitro, as well as OPC recruitment to demyelinating lesions and remyelination in vivo. We showed that the supernatant of Sema3F-transduced HSCs increases migration of young and middle-aged OPCs in vitro in Nrp2-dependent manner, and that chimeric mice with Sema3F-overexpressing blood cells show increased OPC recruitment and accelerated onset of remyelination. Thus, we conclude that genetically modified blood cells, particularly macrophages, can be used to efficiently and quickly express pro-remyelinating molecules in demyelinating lesions and promptly stimulate myelin repair. Our results represent a proof of concept that genetically- = modified monocytes/macrophages can be used to enhance CNS remyelination, which provides novel cues for therapies to prevent/diminish neurological disability in patients with MS. Results Donor-derived macrophages infiltrate demyelinating lesions To investigate whether transplant-derived cells target demyelinating CNS lesions, we injected preconditioned mice with whole bone marrow (WBM) isolated from actin-GFP mice (Fig 1A). At 2 months post-transplant, 72.6 ± 5.8% of blood cells were GFP+. Moreover, 87.07 ± 9.7% of blood CD11b+ cells (monocytes) were GFP+. Thus, monocytes in chimeric mice derive mostly from transplanted cells (Fig 1B). Figure 1. Graft-derived macrophages in demyelinating lesions A. Bone marrow isolated from actin-GFP mice was transplanted into preconditioned recipients. B. Flow cytometry analyses of recipient blood 2 months post-transplant. (Bi) Transplanted cells detected by GFP fluorescence. (Bii) CD11b labeling shows a large proportion of monocytes expressing GFP. C. Demyelinating lesions in chimeric mice at 3 dpl. (Ci) Demyelination was induced by injecting LPC in the spinal cord white matter. (Cii–Civ) Co-immunolabeling for GFP, MBP, and a mixture of CD11b/CD68, showing graft-derived macrophages in the lesion (identified by lack of MBP staining). White lines indicate lesion borders. Scale bar 20 μm. MBP—myelin basic protein; GFP—green fluorescent protein. Source data are available online for this figure. Source Data for Figure 1 [emmm202114759-sup-0003-SDataFig1.zip] Download figure Download PowerPoint We next induced demyelination in the spinal cord of chimeric mice by injecting lysolecithin (LPC) (Fig 1Ci). At both 3 and 7 days post-lesion (dpl) (ongoing OPC recruitment), GFP+ cells were found in demyelinated areas, and were CD11b/CD68+ (macrophage marker) (Fig 1Cii–Civ). Quantification revealed that 91.5 ± 1.6% of GFP+ cells were macrophages. Moreover, 47.5 ± 4.6% of macrophages in the lesion were GFP+. Because 96.35% (median) of circulating blood monocytes were GFP+, we assume that within lesions, all blood-derived cells are GFP+, while resident microglia/macrophages are GFP−, and thus conclude that both resident and blood-derived macrophages are recruited early to LPC lesions. Therefore, graft-derived macrophages reliably target CNS lesions early after demyelination and hence represent appropriate molecular vehicles for stimulating OPC repopulation/myelin repair. Genetically modified HSCs secrete Sema3F that increases OPC migration Next, we generated lentiviral vectors to overexpress Sema3F. We used a bicistronic construction with self-cleaving T2A sequence inserted between Sema3F- and GFP-encoding sequences, all under PGK promoter (PGK-GFP-T2A-SemaF; Appendix Fig S1). Thus, Sema3F and GFP should be produced as separate proteins. Next, we genetically modified HSCs to overexpress Sema3F. HSCs were isolated, transduced with PGK-GFP-T2A-Sema3F or PGK-GFP lentiviral vectors, and expanded for 5 days (Fig 2A). 35.8 ± 5.3% of PGK-GFP-T2A-Sema3F- and 76.7 ± 4.8% of PGK-GFP-transduced cells were GFP+ (Fig 2B). This difference in transduction efficiency can be explained by a 30% greater size of Sema3F-GFP transgene (Appendix Fig S1). Figure 2. Genetically engineered HSCs secrete Sema3F that attracts OPCs in vitro A–C. Transduction of HSCs. (A) Mouse bone marrow was isolated and differentiated cells (Lin+) were removed using MACS. Non-differentiated cells were transduced and expanded for 5 days. (B) Lower numbers of GFP+ cells, verified using flow cytometry, after transduction with Sema3F vector, ***P < 0.0001, Student's t-test, n = 8 independent experiments. Mean ± SEM. (C) Western blot showing GFP expression in cells transduced with both vectors and Sema3F expression in the supernatant of Sema3F-transduced cells only. D. OPCs isolated from adult PDGFRα-GFP mice using FACS were subjected to transwell-chamber migration assay in response to supernatants from non-transduced (NT), GFP-transduced (GFP), or Sema3F-transduced (Sema3f-GFP) HSCs. E. Fold increase in cells that crossed the membrane insert compared to NT. ***P < 0.0001. One-way ANOVA followed by Bonferroni's multiple-comparison test. n = 1–2 normalized replicates from 3 to 5 independent experiments. Mean ± SEM. ***P < 0.0001. R. = recombinant. Super = supernatant. Anti-Nrp2 = Neuropilin2 function-blocking antibody. Source data are available online for this figure. Source Data for Figure 2 [emmm202114759-sup-0004-SDataFig2.zip] Download figure Download PowerPoint Using western blot, we detected GFP expression in the lysates of both control and Sema3F-transduced cells, although to a lesser extent in the latter (Fig 2C), consistent with lower transduction efficiency of PGK-GFP-T2A-Sema3F. Sema3F was detected exclusively in the supernatant of PGK-GFP-T2A-Sema3F-transduced cells (Fig 2C). Cell viability (propidium iodide and Viobility Dye labeling) and the pattern of colony formation (proliferation/differentiation potential) were similar between GFP- and Sema3F-transduced HSC preparations (Appendix Fig S2). To verify the functionality of Sema3F secreted by HSCs, we used transwell chamber assay and quantified migration of adult OPCs in response to the supernatant from non-transduced (NT)-, PGK-GFP- (GFP), or PGK-GFP-T2A-Sema3F-transduced (Sema3F-GFP) HSCs (Fig 2D). Migration was twofold higher in Sema3F-GFP versus GFP (Fig 2E), which was similar to the increase in migration induced by a recombinant Sema3F at 500 ng/ml (2.2-fold compared to control, Fig 2E). An increase in OPC migration was also observed in experiments using supernatants from PGK-GFP-T2A-Sema3F- or PGK-GFP-transduced HEK cells. The increase in migration induced by the supernatant from Sema3F-GFP-transduced HSCs was abrogated in the presence of anti-Nrp2 function-blocking antibody. Similar findings were obtained for recombinant Sema3F (Fig 2E). Thus, Sema3F-GFP-transduced HSC supernatant increases OPC migration in vitro in Nrp2-dependent manner. Therefore, while transduction with PGK-GFP-T2A-Sema3F does not affect HSC viability or differentiation in vitro, it leads to secretion of Sema3F that increases OPC migration, via Nrp2. OPCs from both young and middle-aged mice are attracted by Sema3F Next, we investigated whether the responsiveness of OPCs to Sema3F decreases with age. We first checked whether aged OPCs retain Nrp2 expression. Both microarray data on FACS-sorted OPCs from the brains of PDGFRα-GFP mice (Fig 3A) and RNA-sequencing data on oligodendroglial spinal cord cells (Fig 3B) demonstrated a lack of statistically significant changes in Nrp2 mRNA expression between 2-month-old (young) and 12-month-old (middle-aged) mice. Interestingly, immunohistochemical analyses of Sema3F expression in LPC lesions at 4 dpl (Fig 3C–E) and 7 dpl showed a significant decrease in Sema3F-expressing cells in lesions induced in middle-aged and old (18-month-old) mice at 4 dpl (onset of OPC recruitment), as compared to young mice (Fig 3F), which was partially due to a decrease in microglial/macrophage Sema3F expression (Fig 3G). We then evaluated whether recombinant Sema3F or the supernatant from PGK-GFP-T2A-Sema3F-transduced cells retains its capacity to attract OPCs with age. We exposed OPCs FACS sorted from the brains of young versus middle-aged PDGFRα-GFP mice to recombinant Sema3F- or PGK-GFP-T2A-Sema3F-transduced cell culture supernatant in a Boyden transwell chamber for 24 h. Our results show that OPCs isolated from middle-aged mice are mobilized by both recombinant Sema3F and supernatant-derived PGK-GFP-T2A-Sema3F-transduced cell supernatant (Fig 3H). We have not performed migration experiments with OPCs isolated from old (18 months of age) mice because, using FACS sorting, we obtain very low numbers of cells, insufficient for migration experiments. Figure 3. Middle-age OPCs maintain Nrp2 expression and responsiveness to Sema3F A, B. Expression of Nrp2 gene by OPCs is maintained in middle-age (12-month (m)-old) mice. (A) Microarray analyses results for Nrp2 expression by OPCs purified using FACS from the brains of 2-month versus 12-month-old PDGFRα–GFP mice (n = 3–4 independent experiments). (B) RNA-sequencing results for Nrp2 expression by oligodendroglia-enriched preparations isolated from the spinal cords of 2-month versus 12-month old mice. (n = 3–4 independent experiments). Mean ± SEM. C–G. Sema3F expression in demyelinating lesions at 4 dpl (onset of OPC recruitment) is decreased in middle-aged (12-m-old) and old (18-m-old) mice compared to young (2-m-old) mice. (C–E) Co-labelings for Sema3F and Iba1 (macrophage marker) in demyelinating lesions at 4 dpl in 2-m (C), 12-m (D), and 18-m-old mice (E). Arrowheads point to double-labeled cells. (F) Quantification of Sema3F-expressing cells in the lesion. (G) Percentage of Iba1+ cells positive for Sema3F. NWM—normal white matter. Dpl—days post-lesion. n = 4–8 mice/group. Mean ± SEM two-way ANOVA followed by Tukey's multiple-comparison test. *P < 0.05, **P < 0.01, ***P < 0.0001. H. Transwell chamber assay was used to evaluate migration of young versus middle-age OPCs in response to recombinant Sema3F and supernatant from PGK-GFP-T2A-Sema3F-transduced cells. OPCs of both ages migrate in response to recombinant Sema3F- and PGK-GFP-T2A-Sema3F-transduced cell supernatant. n = 1–2 normalized replicates from 2 to 5 independent experiments. Mean ± SEM. One-way ANOVA followed by Bonferroni's multiple-comparison test. *P < 0.05, **P < 0.01. Scale bar 50 μm. Source data are available online for this figure. Source Data for Figure 3 [emmm202114759-sup-0005-SDataFig3.zip] Download figure Download PowerPoint Thus, OPCs from middle-aged mice retain the expression of Nrp2 gene and are attracted by Sema3F. Hematopoietic reconstitution by transduced HSCs We transplanted female GFP and Sema3F-transduced HSCs (a mixture of transduced and non-transduced cells) to preconditioned male mice (GFP and Sema3F mice; Fig 4A). At 2 months post-grafting, qPCR for sex-containing region of the Y chromosome showed extensive reconstitution by transplanted (female) cells in both groups (87.96 ± 4.05% GFP and 88.68 ± 2.41% Sema3F) (Fig 4B). Flow cytometry analyses of GFP expression revealed 73.8 ± 3.1% of GFP+ (transgene-carrying) cells in GFP and 22.9 ± 2.1% in Sema3F group (Fig 4C), consistent with lower transduction efficiency of PGK-GFP-T2A-Sema3F vector mentioned above. Figure 4. Generation of Sema3F chimeras A. Transduced female HSCs were injected retro-orbitally into preconditioned male mice. Blood was analyzed 2 months post-transplant. B. Percentage of transplanted (female) cells in the blood. n = 18–19 mice/group. Mean ± SEM. C. Percentage of transduced (GFP+) cells in the blood. Mean ± SEM. ***P < 0.0001. n = 22–25 mice/group. D. Percentages of GFP+ cells labeled with CD11b, CD19, and CD3. The proportion of GFP+ cells labeled with CD11b is increased in Sema3F mice (P = 0.01), while that of CD19-labeled cells is decreased (P = 0.0003). Mean ± SEM. Unpaired Student's t-test. n = 9 mice/group. *P < 0.05, ***P < 0.001. E. Percentages of total blood cells expressing CD11b, CD19, and CD3. Red dashed lines indicate the percentage expression of these antigens in blood cells of control mice. n = 9 mice/group. Mean ± SEM. Source data are available online for this figure. Source Data for Figure 4 [emmm202114759-sup-0006-SDataFig4.zip] Download figure Download PowerPoint Using flow cytometry, we analyzed differentiation of transduced (GFP+) cells into monocyte (CD11b+), B-cell (CD19+), and T-cell (CD3+) lineages. Interestingly, the percentage of GFP cells co-expressing CD11b was higher in Sema3F mice, while that co-expressing CD19 was lower (Fig 4D). However, this difference did not affect the blood cell composition in chimeric mice as the percentages of total CD11b+, CD19+, and CD3+ cells in the blood were similar between the two groups and comparable to that of control (non-treated) mice (Fig 4E). Therefore, Sema3F mice show normal blood composition. We then analyzed whether Sema3F modifies GFP+ cell numbers in the heart, liver, lung, and spleen. We detected numerous GFP+ cells in the spleen, as expected. We also detected GFP+ cells in the lungs, albeit at lower numbers as compared to the spleen. Correlation analyses revealed that these numbers were determined by the GFP chimerism in the blood (r = 0.84 spleen, r = 0.94 lung), which indicated that Sema3F expression did not alter GFP+ cell homing and maintenance in these organs. Sema3F-carrying cells successfully target demyelinating lesions and are predominantly macrophages We investigated the response of transgene-carrying cells to LPC-induced spinal cord demyelination. GFP+ cells were detected at 7 and 10 dpl in GFP and Sema3F mice with less cells in the latter, although this difference was not statistically significant (Fig 5A and B, D and E, G). Using Prism, we detected a strong correlation between the percentage of GFP+ cells in the blood and numbers of GFP+ cells in lesions at both 7 (r = 0.88) and 10 dpl (r = 0.81). This indicates that numbers of GFP+ cells in the lesions are determined by GFP blood chimerism, itself dependent on transduction efficiency (lower for PGK" @default.
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• W4285086384 title "Genetically modified macrophages accelerate myelin repair" @default.
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