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Therapeutic genetic restoration through allogeneic brain microglia replacement

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Abstract

Migration of transplanted allogeneic myeloid cells into the brain following systemic haematopoietic stem and progenitor cell transplantation (HCT) holds great promise as a therapeutic modality to correct genetic deficiencies in the brain such as lysosomal storage diseases1,2,3. However, the toxic myeloablation required for allogeneic HCT can cause serious, life-threatening side effects, limiting its applicability. Moreover, transplanted allogeneic myeloid cells are highly vulnerable to rejection even in an immune-privileged organ like the brain. Here we report a brain-restricted, high-efficiency microglia replacement approach without myeloablative preconditioning. Contrary to previous assumptions, we found that haematopoietic stem cells are not required to repopulate the myeloid compartment of the brain environment, and Sca1− committed progenitor cells were highly efficient in replacing microglia following intracerebral injection. This finding enabled the development of brain-restricted preconditioning and avoided long-term peripheral engraftment, thus eliminating complications such as graft-versus-host disease. Evaluating its therapeutic potential, we found that our allogeneic microglia replacement method rescued the mouse model of Sandhoff disease, a lysosomal storage disease caused by hexosaminidase B deficiency. In support of the translational relevance of this approach, we discovered that human embryonic stem cell-derived myeloid progenitor cells display a similar engraftment potential following brain-restricted conditioning. Our results overcome current limitations of conventional HCT and may pave the way for the development of allogeneic microglial cell therapies for the brain.

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Fig. 1: A high-efficiency non-myeloablative microglia replacement approach.
Fig. 2: HSCs are not required for CNS myeloid niche repopulation.
Fig. 3: Co-stimulation blockade enables allogeneic myeloid cell engraftment in the CNS.
Fig. 4: Genetic rescue of Hexb deficiency through allogeneic microglia replacement.
Fig. 5: Replacement of microglia with human induced myeloid progenitor cells.

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Data availability

The supporting data for all graphs are provided along with this publication. If additional information for replication of this study is required, it will be available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

Computational and statistical analyses were performed using freely available software packages. If additional information for replication of this study is required, it will be available from the corresponding author on reasonable request.

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Acknowledgements

We would like to thank all members of the Wernig laboratory, as well as A. Wilkinson, H. Nakauchi, M. Miyauchi and K. Niizuma, for helpful discussions throughout the project. We thank A. Lang and M. Vangipuram for administrative support, and we thank the FACS core at the Institute for Stem Cell Biology and Regenerative Medicine, especially C. Crumpton and C. Pan, and the Stanford Neuroscience Microscopy Service (supported by NIH NS069375) and G. Wang for technical support. We thank T. Südhof and Z. Liu for training and access to equipment for behavioural studies. We thank E. E. Graves for advice regarding small animal irradiation and calibration of the irradiation restrainer. Csf1r−/− (FVB.129×1-Csf1rtm1Ers) mice were a gift from R. Stanley (Albert Einstein College of Medicine). B6.KUO mice were generated at the University of Tokyo and were a gift from H. Nakauchi. This work was supported by the Helen C. and Robert J. Kleberg Foundation and a DISC-0 award from the California Institute for Regenerative Medicine (DISC0-13875). 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). T.U. was supported by the Wu Tsai Neurosciences Institute Knight Initiative for Brain Resilience Scholar Award.

Author information

Author notes

  1. Marius Marc-Daniel Mader

    Present address: Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

  2. Marius Marc-Daniel Mader

    Present address: Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

  3. Marius Marc-Daniel Mader

    Present address: Research Institute Children’s Cancer Center, Hamburg, Germany

Authors and Affiliations

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

    Marius Marc-Daniel Mader, Alexa Scavetti, Yongjin Yoo, Aaron Tianyue Chai, Takeshi Uenaka & Marius Wernig

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

    Marius Marc-Daniel Mader, Alexa Scavetti, Yongjin Yoo, Aaron Tianyue Chai, Takeshi Uenaka & Marius Wernig

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

    Marius Marc-Daniel Mader, Alexa Scavetti, Yongjin Yoo, Aaron Tianyue Chai, Takeshi Uenaka & Marius Wernig

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Contributions

Study concept and design: M.M.-D.M. and M.W. Animal experiments: M.M.-D.M., A.S. and A.T.C. Histological analyses: M.M.-D.M., A.S. and A.T.C. Cell culture: M.M.-D.M., Y.Y., A.S. and T.U. Flow cytometry experiments: M.M.-D.M. and A.S. 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.

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Competing interests

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

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Extended data figures and tables

Extended Data Fig. 1 Additional evaluation of the effects of CSF1R inhibitors and irradiation dose.

a, Experimental design. The last injection of busulfan was applied one day before transplantation, and PLX5622 diet was withdrawn on the day of transplantation. 200 ×103 B6.UBC-GFP graft cells were transplanted per recipient animal. iv, intravenous. b, Representative flow cytometry plots and gating strategy. c, Quantification of donor derived GFP+ chimerism in the peripheral blood and myeloid compartment of the contralateral brain hemisphere based on flow cytometry. n = 4, 4, 3, and 4 animals per group, respectively (from left to right). Two-sided Welch t-test. d, Evaluation of microglia depletion. Representative flow cytometry plots and gating strategy are shown for two female animals. e, Flow cytometric quantification. The brain was analyzed one day after the last PLX5622 injection. The drug was administered daily over 4 days. n = 3 animals per group. Two-sided Welch t-test. f, Flow cytometric quantification. The brain was analyzed one day after the last PLX3397 injection. The drug was administered daily over 3 days. n = 3 animals per group. Two-sided Welch t-test. g, Experimental design. 200 ×103 B6.UBC-GFP HSPC were transplanted per recipient animal. Numbers (n) represent assigned and survived animals per group. ip, intraperitoneal. h, Representative flow cytometry plots and gating strategy per experimental group. i, Flow cytometric quantification (contralateral brain hemisphere). n = 5, 4, and 1 animals per group, respectively (from left to right). Two-sided Welch t-test. j, Experimental outline. 200 ×103 B6.UBC-GFP HSPC were transplanted per recipient animal. n = 3 animals per group. k, Flow cytometric quantification (contralateral brain hemisphere). l, Immunofluorescent staining for GFP of sagittal brain sections of the ipsilateral hemisphere of two animals with irradiation+PLX3397 treatment. Shown are cortical areas of reduced donor cell engraftment density. Purple square illustrates magnified area of panel m. Scale bar = 200 µm. m, Immunofluorescent stain shows repopulated endogenous GFP− microglia and grafted GFP+ cells. Demonstrated is the border between high-density donor cell repopulated brain and an area with low chimerism. Scale bar = 50 µm. n, Experimental outline. 200 ×103 B6.UBC-GFP HSPC were transplanted per recipient animal. n = 3 animals per group. o, Flow cytometric quantification (contralateral brain hemisphere). Two-sided Welch t-test. p, Flow cytometric quantification of myeloid donor chimerism in the brain and blood 33 weeks after ICV transplantation and preconditioning with PLX3397 and 10 Gy head irradiation. n = 4 animals. Box-plot elements in Extended Data Fig. 1 represent median (center line), first and third quartiles (lower and upper hinges) and smallest/highest value with at most 1.5*IQR (inter-quartile range) from the hinge (whiskers).

Source data

Extended Data Fig. 2 Expression of microglial homeostatic marker proteins on transplanted myeloid cells.

Representative immunofluorescent stain shows the expression of IBA1, P2RY12, and TMEM119 in endogenous GFP− microglia and grafted GFP+ cells in the cortex. Grafted cells were evaluated 25 days after transplantation. Samples of three different animals per group were examined with comparable results. Scale bar = 30 µm.

Extended Data Fig. 3 Flow cytometric gating strategies for hematopoietic stem and progenitor cells.

Flow cytometry plots and gating strategies are provided for the sorting of primary bone marrow cells (a) and ex vivo expanded HSPCs (c), as well as for the quantification of GFP+ or KUO+ fractions in myeloid cells in vitro (b, d, e). FMO, fluorescence minus one.

Extended Data Fig. 4 Flow cytometric gating strategy for peripheral blood evaluation.

Flow cytometry plots and gating strategy for the evaluation of peripheral blood cells in the context of graft rejection. BC, B cells; NK, natural killer cells; TC, T cells; TCM, central memory T cells; TEM, effector memory T cells; Tnaive, naïve T cells; Treg, regulatory T cells.

Extended Data Fig. 5 Additional characterization of allogeneic microglia replacement as a cell therapy.

a, Engraftment of GFP+ donor cells in the cerebrum ~12 weeks after transplantation. Shown as total density and relative engrafted brain area. n = 12 CT animals. Regression line based on linear model (shaded area = confidence interval); r = Pearson correlation coefficient. Two-sided test. b, Representative GFP immunofluorescence image of a sagittal brain section. Dotted line indicates replicate in panel a. Scale bar = 500 µm. c, Body weight and conditioning score as well as GvHD score demonstrated for live animals of the experimental groups. A late increase in GvHD score especially in the KO and Mock groups coincided with neurological decline and was primarily based on weight loss and hunching. No occurrences of skin fibrosis or diarrhea were detected in this cohort. Lines represent the mean with 95% confidence interval. n = 61 animals total (WT = 19, KO = 12, Mock = 10, CT = 20). d, Representative images of in situ β-hexosaminidase determination in the cortex. A total of 27 animals were evaluated. Scale bar = 200 µm. e, Quantification of the β-hexosaminidase positive area in the whole brain. A total of 27 animals were evaluated. Two-sided Welch t-test. f, Quantification of relative time spent in the inner zone of an open field setup over 5 min. Deceased animals were excluded from the analysis. n = 19, 12, 10, and 20 animals (13 weeks) and 19, 10, 7, and 18 animals (17 weeks) in the WT, KO, Mock, and CT group, respectively. Two-sided Mann–Whitney U test. g, Correlation between brain area of engrafted cells and clinical variables. In two animals with mortality before week 18, engraftment was evaluated in necropsy tissue, and no GFP+ cells were detected (see panel h). n = 14 CT animals. Regression line based on linear model (shaded area = confidence interval); r = Pearson correlation coefficient. Two-sided test. h, Immunofluorescent images of necropsy brain tissue. Positive sample represents a CT animal with unclear mortality on day 114. Negative samples 1 and 2 represent CT animals with mortality due to neurological decline before 18 weeks of age, and no detected GFP+ myeloid cells in the brain. Red arrows illustrate present or absent colocalization of Iba1 and GFP. Scale bar = 50 µm. i, Neuro Score displayed for WT (n = 19 animals up to week 18, then 11 animals), KO (n = 12), and long-term CT (n = 5) animals. Deceased animals received a maximum score of 9. Lines represent the mean with 95% confidence interval. j, Quantification of locomotor activity in an open field setup over 5 min (performed at 13, 17, 22, 26, and 30 weeks of age). Displayed for WT (n = 19 animals up to week 17, then 11 animals), KO (n = 10 and 12 animals at weeks 17 and 13, respectively) and long-term CT (n = 5 animals up to week 17, then 4 animals) animals. Deceased animals were excluded from the analysis. Lines represent the mean with 95% confidence interval. k, Flow cytometric quantification of myeloid donor chimerism in the peripheral blood after bone marrow transplantation (BMT). syngeneic control n = 4 animals (6 weeks after BMT). allogeneic with co-stimulation blockade n = 12 animals (7×6 weeks and 5×12 weeks after BMT). l, Body weight and GvHD score. Lines represent the mean with 95% confidence interval. n = 12 animals per group. Box-plot elements in Extended Data Fig. 5 represent median (center line), first and third quartiles (lower and upper hinges) and smallest/highest value with at most 1.5*IQR (inter-quartile range) from the hinge (whiskers).

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Mader, M.MD., Scavetti, A., Yoo, Y. et al. Therapeutic genetic restoration through allogeneic brain microglia replacement. Nature (2025). https://doi.org/10.1038/s41586-025-09461-6

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