Transplanting rejuvenated blood stem cells extends lifespan of aged immunocompromised mice.

"all data and codes are deposited under the repository 10 34810/data163 scrna-seq data are deposited at geo (accession number gse197070). a total of 15856 lsk cells were obtained after the quality control and filtering (supplementary dataset 1 ) which divided into 13 clusters by using seurat's functions (fig 5a ).; the cell clusters were annotated based on previously reported marker genes (fig 5a b supplementary fig 6b-e supplementary dataset 2 and "methods" section).; fig 5 a umap with clusters obtained for lsk cells of young aged and casin mice (defined in supplementary dataset 2) b expression levels of several marker genes c density in the umap for the young aged and casin cells d venn diagram of the common de genes in the three pairwise comparisons e single-cell expression levels of the top genes that are de between casin and aged samples in clusters hsc-1 hsc-2 and hsc-3 f gene ontology (go) terms obtained in the gsea of the de genes.; only connection values >0 4 are represented j expression pattern over pseudotime of several genes (supplementary dataset 10 ) involved in the indicated go processes.; data are provided in supplementary dataset 1 - 10 .; interestingly only the hsc-1 cluster which is characterized by the most dormant gene signature showed a significant cell number increase in both the aged and casin groups compared to the young samples while cell number in lmpps cluster is not significantly reduced in both aged and casin samples (fig 5c supplementary fig 6f and supplementary dataset 3 ).; interestingly we measured a significant increase in the proportion of cells in g0 in casin samples for all hsc clusters supporting that casin treatment preserves quiescence of aged stem cells a characteristic in general associated with higher regenerative capacity (supplementary fig 6g and supplementary dataset 3 ).; we obtained a total of 648 de genes between aged and young samples and 210 de genes between casin and aged samples (|log2fc|> 0 25 and tippett's combined p-value < 0 05 for wilcoxon rank sum test; fig 5d e supplementary fig 7 and supplementary dataset 4 ).; focusing on the de genes between casin and aged control mice that revert to youthful level nr4a1 and nr4a2 were downregulated in eight and four of our clusters respectively including all hsc clusters (fig 5e supplementary fig 7 and supplementary dataset 4 ).; dnajb1 hspa5 and edem2 which are involved in the folding and assembly of proteins and the degradation of misfolded proteins in the endoplasmic reticulum and are activated under conditions of stress were also downregulated after casin treatment in clusters hsc-2 and hsc-3 (fig 5e supplementary fig 7 and supplementary dataset 4 ).; and cell junction assembly in cluster hsc-1 and the regulation of protein localization in cluster hsc-2 (fig 5f and supplementary dataset 5 ).; we identified a total of 1659 and 903 genes with differential variability in one or more hsc clusters between young and aged samples and between casin and aged samples respectively (absolute residual dispersion difference >0 5 and posterior probability's expected fdr < 0 05; supplementary dataset 6 ).; by calculating the difference in the residual dispersions obtained for the aged and the young cells we determined that in all clusters the number of genes becoming less variable with age (negative difference) was higher than the number of genes with increased variability (positive difference; 70 7% 58 9% and 77% showed a negative difference of the total number of genes with significant differences in variability for hsc-1 hsc-2 and hsc-3 respectively; fig 5g supplementary fig 8a and supplementary dataset 6 ).; we observed a general and strong decrease in the connections between the clusters in the aged samples compared to the young (fig 5i and supplementary dataset 7 ).; interestingly in casin samples clusters showed a general increase in the connection values compared to the aged group (fig 5i and supplementary dataset 7 ) with the connection values among hscs increasing between 14 and 45% and the connections between hsc-1 and progenitors increasing of 115%.; next we identified the genes driving the changes in the connection values after casin treatment by selecting those with significantly different patterns of expression along trajectories in aged and casin samples using tradeseq (supplementary dataset 8 ) followed by gsea (supplementary dataset 9 ).; data revealed the expression of genes involved in myeloid cell differentiation pathways to be increased in the transitions from hsc-1 to hsc-2 hsc-1 to mpp1 and hsc-2 to mpp1 (fig 5j supplementary fig 8d and supplementary dataset 10 ) with casin samples resulting in a youthful expression pattern for these pathways.; similar results were observed for lymphocyte differentiation and b cell activation in the transitions from hsc-1 to hsc-2 hsc-3 and mpp1 (fig 5j supplementary fig 8d and supplementary dataset 10 ).; signaling remained sustained in the aged clusters (supplementary fig 8d and supplementary dataset 10 ).; more information on the number of cells genes and umi counts for every sample can be found at supplementary dataset 1 .; genes associated to hematopoietic stem cells (hsc) like procr fgd5 sult1a1 and nupr1 were mainly expressed in the three clusters at the left side of the umap consequently labeled as hsc-1 hsc-2 and hsc-3 (fig 5a b and supplementary dataset 2 ).; the expression of these genes is especially high in hsc-1 which also shows a very low expression of cdk6 and cd34 and higher expression of meg3 (fig 5b supplementary fig 6b and supplementary dataset 2 ) a signature that was described to be characteristic of dormant hscs .; hsc-2 cluster is characterized by a high expression of the ap-1 transcription factor complex genes like fos and jun (fig 5b supplementary fig 6b and supplementary dataset 2 ) that are known to act in response to stress and whose high expression has been suggested to be associated with an increased capacity of hscs for differentiation and commitment .; interestingly hsc-3 cluster is enriched in cd74 and other major histocompatibility complex ii (mhc ii) genes like h2-aa h2-eb1 and h2-ab1 (fig 5b supplementary fig 6b and supplementary dataset 2 ).; this cluster also presents a high expression of several differentiation-associated lncrnas like malat1 and neat1 (supplementary fig 6b and supplementary dataset 2 ) mpp2 (cd150 + cd34 + cd48 + flt3 - ) is characterized by the expression of itga2b and pf4 (supplementary fig 6b and supplementary dataset 2 ) which are markers of megakaryocytes and supporting that this might be a megakaryocyte-biased cluster.; the cluster mpp3 (cd150 - cd34 + cd48 + flt3 - ) is expected to be biased towards the granulocyte/macrophage fate although it shows very few specific marker genes (supplementary fig 6b and supplementary dataset 2 ).; finally the lymphoid multipotent progenitor (lmpp) cluster (cd150 - cd34 + cd48 + flt3 + ) presents a high expression of dntt and satb1 known to be characteristic of lymphoid biased progenitors (supplementary fig 6b and supplementary dataset 2 ).; the rest of the clusters were annotated as cycling cells (cc) for showing high expression of cell cycle genes (supplementary fig 6b and supplementary dataset 2 ) and a large proportion of cells in g2/m and m phases (supplementary fig 6e ); myeloid progenitors (mp) expressing genes like rsad2 ifit1 lgals1 irf8 elane ctsg and mpo (supplementary fig 6b and supplementary dataset 2 ); and other primed progenitors (pp) that did not express clear marker genes.; a summary of the main marker genes gates and references used for annotation can be found in supplementary dataset 2 .; supplementary figures1-8 reporting summary supplementary dataset 1 supplementary dataset 2 supplementary dataset 3 supplementary dataset 4 supplementary dataset 5 supplementary dataset 6 supplementary dataset 7 supplementary dataset 8 supplementary dataset 9 supplementary dataset 10 supplementary movie 1 supplementary movie 2 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."

"Competing interests Yi Zheng and Hartmut Geiger disclose financial interest in Mogling Bio."

"We acknowledge support from core facilities at Ulm University: Angelika Rück (Microscope Facility) for support with confocal and 2-photon microscopy, the Cell Sorting Facility, and the Tierforschungszentrum (Animal Facility). We thank support from Dr. Mercè Martì Gaudes, Technical Facilities at IDIBELL together with Andres Vaquero (IDIBELL FACS and Flow cytometry), Antoni Ventura (IDIBELL Mouse Facility). We thank Benjamin Torrejon (University of Barcelona, Confocal Miscroscopy facility). We thank CERCA Program/Generalitat de Catalunya for institutional support. We acknowledge the funding sources: European Research Council (ERC) grant 101002453 (MCF), Spanish Ministry of Science, Innovation and University grants RYC2018-025979-I (MCF), and RYC2018-024564-I (MP), and Deutsche Forschungsgemeinschaft INST 40/662, INST 40/667, SFB1506 (HG). Author contributions: Conceptualization: N.J.A., Y.Z., H.G., and M.C.F. Methodology: N.J.A., G.M., K.S., V.S., A.V., L.M., J.-P.M., M.S., F.M., S.M.-V., M.P., T.Z., J.L., and E.M.-R. Investigation: N.J.A., F.M., S.M.-V., M.P., J.L., E.M.-R., and F.M.C. Visualization: S.M.-V., N.J.A., F.M., and M.S. Funding acquisition: H.G. and M.C.F. Project administration: M.C.F. Supervision: M.P. and M.C.F. Writing – original draft: S.M.-V., F.M., and M.C.F. Writing – review & editing: Y.Z., M.P., H.G., and M.C.F."