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<i>Elp1</i> is required for myeloid differentiation and brain repopulation

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Data availability

The mouse sequencing datasets generated and analysed in this study are available from the Gene Expression Omnibus repository83 under accession numbers GSE255732, GSE255735 and GSE255893. The murine reference genome was obtained from www.gencodegenes.org, and the murine reference proteome was obtained from www.uniprot.org. Gene sets were obtained from www.gsea-msigdb.org and www.geneontology.org. The MS proteomic data have been deposited to into the ProteomeXchange84 Consortium via the PRIDE85 partner repository with the dataset identifier PXD049985. Source data are provided with this paper.

Code availability

Computational analyses were performed using freely available software packages as described in detail in the Methods. If additional information for replication of this study is required, it will be available from the corresponding author upon reasonable request.

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Acknowledgements

We would like to thank all members of the Wernig Laboratory for discussions throughout the project; A. Lang and M. Vangipuram for administrative support; staff at the FACS Core at the Institute for Stem Cell Biology and Regenerative Medicine, specifically C. Crumpton and C. Pan, and staff at the Stanford Wu Tsai Neuroscience Institute Neuroscience Microscopy Service, specifically G. Wang, for technical support; R. Leib, F. Liu and N. Brown for their technical support for LC–MS/MS; and R. Stanley for the gift of Csf1r−/− (FVB.129×1-Csf1rtm1Ers) mice. This work was supported by the Vincent Coates Foundation Mass Spectrometry Laboratory, Stanford University Mass Spectrometry (RRID:SCR_017801) utilizing the Thermo Exploris 480 nanoLC/MS system (RRID:SCR_022215). This work was supported in part by NIH P30 CA124435 utilizing the Stanford Cancer Institute Proteomics/Mass Spectrometry Shared Resource.

Funding

This work was supported by the DISC0-13875 grant from the California Institute for Regenerative Medicine and by the Robert J. Kleberg, Jr and Helen C. Kleberg Foundation. Additional support was provided by the NIH/NIDDK (R01DK117473; to F.L.). M.M.-D.M. was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG, MA 8492/1-1). Y.Y. was supported by the New York Stem Cell Foundation Druckenmiller Fellowship (NYSCF-D-F74). A.C.W. acknowledges support from the Wellcome Trust (302479/Z/23/Z). W.L. was supported by the LLHF Fellowship Grant (no. 2025-A-214-FEL) from the Larry L. Hillblom Foundation.

Author information

Authors and Affiliations

  1. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Marius Marc-Daniel Mader, Wanhua Li, Alexa Scavetti, Yongjin Yoo, Gernot Neumayer, Katie Han, Yohei Shibuya, Hiromitsu Nakauchi & Marius Wernig

  2. Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA

    Marius Marc-Daniel Mader, Wanhua Li, Alexa Scavetti, Yongjin Yoo, Gernot Neumayer, Katie Han, Yohei Shibuya & Marius Wernig

  3. Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Marius Marc-Daniel Mader

  4. Research Institute Children’s Cancer Center Hamburg, Hamburg, Germany

    Marius Marc-Daniel Mader & Marius Marc-Daniel Mader

  5. Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA

    Wanhua Li, Alexa Scavetti, Yongjin Yoo, Gernot Neumayer, Katie Han, Yohei Shibuya & Marius Wernig

  6. Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Marius Marc-Daniel Mader

  7. Department of Neuroscience, Korea University College of Medicine, Seoul, Republic of Korea

    Yongjin Yoo

  8. Department of Biomedical Sciences, Korea University College of Medicine, Seoul, Republic of Korea

    Yongjin Yoo

  9. Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA

    Michael Haney & Tony Wyss-Coray

  10. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Michael Haney

  11. Department of Biological and Physical Sciences, Montana State University Billings, Billings, MT, USA

    Lynn George

  12. Department of Cell Biology and Neuroscience, Montana State University, Bozeman, MT, USA

    Frances Lefcort

  13. Tikun Therapeutics, Brookeville, MD, USA

    Frances Lefcort

  14. Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA

    Michael Bassik & Hiromitsu Nakauchi

  15. Advanced Research Initiative, Institute of Science Tokyo, Tokyo, Japan

    Hiromitsu Nakauchi

  16. Department of Haematology, Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK

    Adam C. Wilkinson

Authors

  1. Marius Marc-Daniel Mader
  2. Wanhua Li
  3. Alexa Scavetti
  4. Yongjin Yoo
  5. Gernot Neumayer
  6. Katie Han
  7. Yohei Shibuya
  8. Michael Haney
  9. Tony Wyss-Coray
  10. Lynn George
  11. Frances Lefcort
  12. Michael Bassik
  13. Hiromitsu Nakauchi
  14. Adam C. Wilkinson
  15. Marius Wernig

Contributions

Study concept and design: M.M.-D.M., M.W. and A.C.W. Animal experiments: M.M.-D.M., A.S., K.H., Y.Y. and Y.S. Cell culture: M.M.-D.M., W.L., A.C.W., Y.S. and A.S. ddPCR: M.M.-D.M. and G.N. Immunostaining, microscopy and image analyses: M.M.-D.M., A.S., W.L. and K.H. Flow cytometry: M.M.-D.M., Y.S. and A.C.W. CRISPR-KO screen: M.M.-D.M., A.C.W., M.H., M.B. and T.W.-C. RNA-seq and proteomic experiments: M.M.-D.M. and Y.S. Data analyses and interpretation: M.M.-D.M., M.W., A.C.W., W.L., F.L., L.G. and H.N. Drafting and major editing of original manuscript: M.M.-D.M. and M.W. All authors reviewed, revised and approved the final version of the paper.

Corresponding author

Correspondence to Marius Wernig.

Ethics declarations

Competing interests

M.W. is a co-founder of Neucyte, a scientific advisor for bit.bio and iPeace, co-founder and scientific advisor of Lytherian Therapeutics and Theseus Therapies. PLX5622 was provided by Plexxikon under a material-transfer agreement between Stanford University and Plexxikon. The other authors declare no competing interests.

Peer review

Peer review information

Nature thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

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Extended data figures and tables

Extended Data Fig. 1 Additional characterization of cHSPC-derived myeloid cells in the brain.

a, Representative flow cytometric gating for sorting of hematopoietic stem cells for the generation of cHSPC cultures. b, Donor-derived (GFP+) chimerism of CD45+ cells in the peripheral blood assessed via flow cytometry. n = 6 for WBM-graft recipient animals; n = 6 for cHSPC-graft recipient animals. Two-sided Mann–Whitney U test. Box plot definition (applicable to all box plots in this figure): median (center line), first and third quartiles (lower and upper hinges), smallest/highest value with at most 1.5*IQR from the hinge (whiskers). c, Representative immunofluorescence images of microglia or brain myeloid cells derived from either WBM or cHSPCs at 2 weeks after PLX5622 withdrawal. 8 animals were assessed in total. Scale bars = 30 µm. d, Morphological analysis of brain myeloid cells in cortex and hippocampus. Colors represent different animals. n = 81 cells in total. Two-sided Welch t-test. e, Expression of macrophage self-renewal regulator genes Maf and Mafb. n = 13 biological replicates total (n = 4 biological replicates in the MG group, n = 3 biological replicates per WBM, pHSC, and cHSPC group). f, Top 25 significantly (padj <0.05; based on DESeq2 DEG analysis using default two-sided Wald test with Benjamini-Hochberg adjustment)61 up & downregulated genes between MG and cHSPC-derived myeloid cells (heatmap is scaled row-wise). n = 13 biological replicates total.

Source data

Extended Data Fig. 2 Additional flow cytometric and histological characterization of in vitro cHSPC-derived myeloid cells.

a, Flow cytometric quantification of the absolute myeloid cell number per well (based on CD11b+ CD45+ gate). Cells were plated on 1.9 cm2 CellBIND plates either with or without PLX5622 pretreated C57BL/6 J glia and maintained in MG-medium supplemented with 10% FBS for 14 days with 50% medium changes twice a week. Absolute count calculation based on counting beads. n = 4 technical replicates (culture wells) per group (n = 16 total). Box plot definition (applicable to all box plots in this figure): median (center line), first and third quartiles (lower and upper hinges), smallest/highest value with at most 1.5*IQR from the hinge (whiskers). b, Flow cytometric assessment of the effects of pretreatment of glia with PLX5622 and addition of FBS on myeloid cell generation. Co-cultures were analyzed 14 days after initiation (cHSPC plating number = 0.2 × 103). n = 5 technical replicates (culture wells) per group. Two-sided Welch t-test. c, Co-cultures were analyzed either 14 days (1 × 103 plated cHSPC) or 10 days (0.2 × 103 plated cHSPC) after initiation. Surface of primary glia was 1.9 cm2. n = 3 technical replicates (culture wells) per condition. Two-sided Welch t-test. d, Flow cytometric quantification of the myeloid fraction in culture with or without plated cHSPCs (C57BL/6 J primary glia with or without PLX5622 pretreatment). Evaluated after 14 days. n = 3 technical replicates (culture wells) per condition. e, Representative immunofluorescence images of in vitro cHSPC-derived myeloid cells or primary microglia with different FBS and PLX5622 pretreatment conditions. Evaluated in 3 technical replicates (culture wells) per condition. Scale bars = 200 µm.

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Extended Data Fig. 3 Different temporal stages of myeloid differentiation in cHSPC-glia cocultures.

a, Immunofluorescence images of cHSPC-glia co-cultures 1, 3, or 7 days after initiation. Yellow circles indicate GFP+ Ki67+ cells. Blue circles indicate early processes and red circle indicate early Iba1 expression present in day-3 cHSPC-derived myeloid cells. Scale bars = 100 µm. Experiment performed with 3 technical replicates (culture wells).

Extended Data Fig. 4 Additional transcriptional characterization of in vitro cHSPC-derived myeloid cells.

a, Hierarchical clustering based on Euclidean distance using the top 2500 variant genes. n = 4 technical replicates per group. b, Venn diagrams of significantly (padj <0.05, based on DESeq2 DEG analysis using default two-sided Wald test with Benjamini-Hochberg adjustment)61 DEGs compared to cHSPCs. Upregulated: log2 fold change > 1; Downregulated: log2 fold change < −1. c, Volcano plot of genes returned by marker gene analysis for d2.HDMC with unidirectional change (log2 fold change consistently either >0 or <0 compared to all other groups). Red color indicates upregulated (139 genes; padj <0.05; log2 fold change > 1) and blue downregulated (114 genes; padj <0.05; log2 fold change < −1) genes. DESeq2-derived p values (two-sided Wald test with Benjamini-Hochberg adjustment) resulting from pairwise comparisons across conditions were combined per gene using Berger’s intersection-union test (combineMarkers, scran, pval.type = “all”; Benjamini-Hochberg adjustment). d, Expression of selected proliferation genes. n = 4 technical replicates per group. Adjusted p values derived from DESeq2 DEG analysis (two-sided Wald tests with Benjamini-Hochberg adjustment). Box plot definition: median (center line), first and third quartiles (lower and upper hinges), smallest/highest value with at most 1.5*IQR from the hinge (whiskers).

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Extended Data Fig. 5 Confirmatory experiments on the Elp1 knockout phenotype on myeloid cell repopulation in vitro and in vivo.

a, Competition assay: KUO cHSPCs were edited via RNP CRISPR-KO. Edited KUO cHSPCs were co-cultured on PLX5622 pretreated primary glia together with UBC-GFP non-edited control cHSPCs at consistent plating numbers (100 + 100 cHSPCs) for 13 days and then analyzed via flow cytometry. b, Quantification of the fraction of KUO+ (edited) cells within the overall population of grafted myeloid cells (CD45+ CD11b+ and either KUO+ or GFP+). The control group represented non-edited KUO cells. The fractions were normalized to the mean fraction of the control group. Red diamonds represent the respective indel rate per sgRNA. n = 5 technical replicates (culture wells) per group. Two-sided Welch t-test. Box plot definition (applicable to all box plots in this figure): median (center line), first and third quartiles (lower and upper hinges), smallest/highest value with at most 1.5*IQR from the hinge (whiskers). c, Elp1flox/flox;UBC-CreER+/− or UBC-GFP cHSPCs were pretreated either with 4OH-tamoxifen (T) or vehicle (V). Cells were then plated (á 300 cells per well) on CSF1R-KO glia for 10 days (serum free condition), followed by flow cytometric analysis. Fractions were normalized to the mean fraction of the vehicle/vehicle condition. n = 6 technical replicates (culture wells) per condition. Two-sided Welch t-test. d, Competitive transplantation of whole bone marrow donor cells (total of 10 million / recipient) derived from either Elp1flox/flox;UBC-CreER+/− or Elp1flox/flox;UBC-CreER−/− male mice mixed in equal numbers with UBC-GFP bone marrow (all CD45.2) after 5 day tamoxifen treatment of all donor animals into busulfan-conditioned wildtype B6-CD45.1 mice. After 1 week, recipient animals were provided with PLX3397 chow for 3 weeks. Analysis was performed 2 weeks after PLX3397 withdrawal. e, GFP+ chimerism of different CD45.2+ cell fractions based on flow cytometry 6 weeks after bone marrow transplantation. n = 3 animals per group. Two-sided Welch t-test. f, Representative flow cytometry plots for GFP+ chimerism. Illustrations in a and d adapted from refs. 3,24, Springer Nature.

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Extended Data Fig. 6 Impact of Elp1 knockout on in vivo hematopoiesis.

a, Wildtype B6-CD45.1 mice received busulfan-conditioned bone marrow transplantations with donor cells derived from Elp1flox/flox;UBC-CreER+/− (CD45.2) mice. After ~10 weeks, recipient animals were treated with or without tamoxifen and were analyzed 1 week after the last injection. n = 4 animals per group. b, Quantification of donor derived chimerism. n = 8 animals in total. Two-sided Welch t-test. Box plot definition (applicable to all box plots in this figure): median (center line), first and third quartiles (lower and upper hinges), smallest/highest value with at most 1.5*IQR from the hinge (whiskers). c, ddPCR-based quantification of Elp1 knockout efficiency in the bone marrow. n = 8 animals in total. Two-sided Welch t-test. d, Flow cytometric gating strategies for the analysis of peripheral blood and bone marrow. e, Fraction of different peripheral blood cells. n = 8 animals in total. Two-sided Welch t-test. f, Flow cytometric analysis of blood myeloid cells for Ly6C expression. n = 8 animals in total. Two-sided Welch t-test. g, Absolute donor derived cell count in the combined leg bones per animal. Based on multiplication of the absolute bone marrow cell count with the percentage of donor-derived chimerism. n = 8 animals in total. Two-sided Welch t-test. h, Relative frequency of LIN− subpopulations. n = 8 animals in total. Two-sided Welch t-test. i, Relative frequency of LIN− CD127− cKit+ Sca1− subpopulations. n = 8 animals in total. Two-sided Welch t-test. j, Flow cytometric gating strategies for the analysis of peripheral blood and bone marrow and representative flow cytometry plots for GFP+ chimerism 6 weeks after tamoxifen injections. Illustrations in a adapted from refs. 3,24, Springer Nature.

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Extended Data Fig. 7 Supporting information on the persistence of differentiated myeloid cells after Elp1 knockout.

a, Wildtype B6-CD45.1 mice received busulfan-conditioned bone marrow transplantations with donor cells derived from Elp1flox/flox;UBC-CreER+/− mice, followed by PLX3397 diet after one week for 3 weeks. ~8 months after PLX3397 withdrawal, recipient animals were treated with or without tamoxifen and analyzed after another ~4 weeks. Representative flow cytometry plots and quantification. n = 4 animals with tamoxifen, n = 3 animals with vehicle. Two-sided Welch t-test. Box plot definition (applicable to all box plots in this figure): median (center line), first and third quartiles (lower and upper hinges), smallest/highest value with at most 1.5*IQR from the hinge (whiskers). b, Elp1flox/flox;CX3CR1-CreER+/− or Elp1flox/flox;CX3CR1-CreER−/− mice were treated with tamoxifen for an initial 5 days followed by continuous injections three times weekly. Flow cytometric analysis was performed ~7 weeks after tamoxifen initiation. n = 6 animals per group (each consisting of 2 males and 4 females). Two-sided Welch t-test. c, Flow cytometric analysis of brain cells in wildtype C57BL/6 J animals treated with PLX3397 (i.p. 92 mg/kg/d for 7 days). n = 5 animals in total.

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Extended Data Fig. 8 Additional molecular characterization of early Elp1-deficient myeloid differentiation.

a, Protein expression of Actin beta and Elp1 based on LC-MS/MS. Shown are normalized iBAQ values. Missing values were set to 0. n = 4 biological replicates per group. Box plot definition (applicable to all box plots in this figure): median (center line), first and third quartiles (lower and upper hinges), smallest/highest value with at most 1.5*IQR from the hinge (whiskers). b, Number of detected proteins per sample after filtering and normalization. The dotted line shows the number of uniquely identified proteins over all samples. P value based on two-sided Welch t-test between the two experimental groups. c, Correlation between RNA (shown as FPKM values) and protein expression (shown as filtered normalized iBAQ values) for the two experimental groups. Colors represent different biological replicates. R = Pearson correlation coefficient. n = 4 biological replicates per group. Two-sided test. d, WGNCA module eigengene expression compared between the experimental conditions. n = 4 biological replicates per group. Two-sided Welch t-test with Holm p value adjustment. e, Top 30 hub genes of WGNCA module 9. Genes that are significant DEGs are labeled in red. f, Gene set enrichment for UPR. Based on genes underlying the WGNCA analysis ranked by Module Membership to module 9 (fgsea R package59, adaptive multi-level split Monte-Carlo p value estimation; two-sided test, Benjamini-Hochberg adjustment. g, Significantly differentially expressed proteins (padj <0.05; based on DEP package using a two-sided moderated t-test [limma] with FDR correction) between the experimental groups. Heatmap is scaled row-wise. Missing values have been assigned the color grey. n = 4 biological replicates per group.

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Extended Data Fig. 9 Human myeloid cell repopulation is Elp1 dependent.

a, Human CD34+ cells from either mobilized peripheral blood or cord blood were co-cultured with human iPSC-derived astrocytes. b, Representative immunofluorescence images of co-cultures after 21 days. Scale bars = 100 µm. N = 2 different peripheral blood and N = 4 different cord blood samples. c, Human cord blood CD34+ cells were edited using RNP-mediated Cas9 knockout, targeting the CLYBL or ELP1 loci. d, ELP1 indel rate. Measured on day 5 or 6 after editing. N = 4 biological replicates. Box plot definition see Fig. 1. e, Representative immunofluorescence images of co-cultures after 21 days. Scale bars = 100 µm. N = 4 different cord blood samples. f, Quantification of myeloid cells in the co-culture after 21 days. Iba1+ cells were counted in 2-3 regions of interest (each 5325 µm2) per sample and averaged. Numbers were normalized between matched samples (i.e. derived from the same cord blood) using CLYBL as the reference. N = 4 biological replicates. Two-sided Welch t-test. Illustrations in a and c adapted from refs. 3,24, Springer Nature.

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Mader, M.MD., Li, W., Scavetti, A. et al. Elp1 is required for myeloid differentiation and brain repopulation. Nature (2026). https://doi.org/10.1038/s41586-026-11112-3

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