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Restoration of neuronal progenitors by partial reprogramming in the aged neurogenic niche

Nature Aging volume 4, pages 546–567 (2024)Cite this article

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Abstract

Partial reprogramming (pulsed expression of reprogramming transcription factors) improves the function of several tissues in old mice. However, it remains largely unknown how partial reprogramming impacts the old brain. Here we use single-cell transcriptomics to systematically examine how partial reprogramming influences the subventricular zone neurogenic niche in aged mouse brains. Whole-body partial reprogramming mainly improves neuroblasts (cells committed to give rise to new neurons) in the old neurogenic niche, restoring neuroblast proportion to more youthful levels. Interestingly, targeting partial reprogramming specifically to the neurogenic niche also boosts the proportion of neuroblasts and their precursors (neural stem cells) in old mice and improves several molecular signatures of aging, suggesting that the beneficial effects of reprogramming are niche intrinsic. In old neural stem cell cultures, partial reprogramming cell autonomously restores the proportion of neuroblasts during differentiation and blunts some age-related transcriptomic changes. Importantly, partial reprogramming improves the production of new neurons in vitro and in old brains. Our work suggests that partial reprogramming could be used to rejuvenate the neurogenic niche and counter brain decline in old individuals.

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Fig. 1: Effect of whole-body in vivo partial reprogramming on the old SVZ neurogenic niche by single-cell transcriptomics.
Fig. 2: Transcriptomic pathway changes in the old neurogenic niche after whole-body in vivo partial reprogramming.
Fig. 3: Validation of neuroblast proportion changes after whole-body partial reprogramming.
Fig. 4: Effect of partial reprogramming targeted to the SVZ neurogenic niche by single-cell transcriptomics.
Fig. 5: Transcriptomic pathway changes after partial reprogramming targeted to the SVZ.
Fig. 6: Impact of aging and reprogramming on primary NSC differentiation.
Fig. 7: Gene expression in primary NSCs with aging, reprogramming and differentiation.
Fig. 8: Newborn neuron production with partial reprogramming.

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Data availability

Raw sequencing reads and processed files for single-cell RNA-seq are available at the Gene Expression Omnibus under accession number GSE224438. Processed Seurat objects are available on Zenodo (https://doi.org/10.5281/zenodo.10626910)95. Raw sequencing reads and count files for bulk RNA-seq are available at the Gene Expression Omnibus under accession number GSE245385. Gene mapping was based on the mm10 mouse genome. Source data are provided with this paper. All other data supporting the conclusions of this study are available from the corresponding author upon request.

Code availability

Code used to process and analyze single-cell RNA-seq data and bulk RNA-seq data are available on GitHub (https://github.com/gitlucyxu/SVZreprogramming)96.

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Acknowledgements

We thank T. Ruetz, P. Navarro and O. Zhou for assistance with surgeries and sample processing; F. Boos for independent code checking; J. Butterfield for help with mouse husbandry and genotyping; J. Na for help with pilot experiments; the entire Brunet laboratory and in particular X. Zhao, C. Bedbrook, K. Papsdorf, P. Navarro and J. Chen for discussion and feedback on the manuscript; M. Abu-Remaileh for use of equipment; D. Schaffer for the AAV-SCH9 plasmid; and K. Shen, S. Dixon, H. Nakauchi and T. Wyss-Coray for guidance. Some illustrations were created with https://www.biorender.com. Cell sorting and flow cytometry analysis were performed on instruments in the Stanford Shared FACS Facility, including those obtained using NIH S10 Shared Instrument Grants S10RR025518-01 and S10RR027431-01. This work was supported by NIH P01 AG036695 (A.B.), the Milky Way Research Foundation (A.B.), NSF Graduate Research Fellowships (L.X. and M.T.B.) and a Stanford Graduate Fellowship (L.X.).

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Authors and Affiliations

  1. Department of Genetics, Stanford University, Stanford, CA, USA

    Lucy Xu, Julliana Ramirez-Matias, Max Hauptschein, Eric D. Sun, Judith C. Lunger, Matthew T. Buckley & Anne Brunet

  2. Department of Biology, Stanford University, Stanford, CA, USA

    Lucy Xu

  3. Department of Biomedical Data Science, Stanford University, Stanford, CA, USA

    Eric D. Sun

  4. Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA

    Anne Brunet

  5. Glenn Center for the Biology of Aging, Stanford University, Stanford, CA, USA

    Anne Brunet

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Contributions

This study was conceived by L.X. and A.B. L.X. designed and performed all experiments and analyses unless noted. J.R.-M. helped with immunofluorescence of brain sections, surgeries and sample processing for single-cell RNA-seq and performed mouse husbandry and genotyping. M.H. helped with immunofluorescence of brain sections and RT–qPCR and performed mouse husbandry and genotyping and independent code checking. E.D.S. performed analyses with transcriptomic clocks and provided independent code checking. J.C.L. helped with RT–qPCR and AAV experiments. M.T.B. helped with sample processing and analysis for single-cell RNA-seq. The manuscript was written by L.X. and A.B., and all authors provided comments.

Corresponding author

Correspondence to Anne Brunet.

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Competing interests

M.T.B. is a cofounder of Retro Biosciences. The other authors declare no competing interests.

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Peer review information

Nature Aging thanks Alejandro Ocampo, Juan Song and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Characterization of whole-body inducible iOSKM mice and NSCs.

a-b, Western blot of tissues from whole-body reprogramming iOSKM mice after treatment with doxycycline. Two different exposures of the same OCT4 blot are shown due to varying levels of transgene expression across tissues. Size in kDa is indicated on the left. Arrowheads indicate OCT4 bands. Expression differences between tissues may be due to differences in accessibility of the Col1a1 genomic locus and doxycycline bioavailability. c, Western blot of primary NSCs from iOSKM mice (n = 2 cultures, each from one mouse) treated in culture with doxycycline at different doses. For NSCs, protein concentration was equalized within each culture; total amount of protein loaded is indicated in parentheses. iPSC: induced pluripotent stem cell, as a positive control for OCT4 expression. Size in kDa is indicated on the left. Normalized densitometry quantification of OCT4 is indicated below each lane. KPNB1 is a loading control for OCT4 and a sample processing control for SOX2 and rtTA. d, Western blot of primary NSCs from iOSKM mice (n = 2 cultures, each from one mouse) treated in culture with doxycycline for the indicated amount of time. Normalized densitometry quantification of OCT4 is indicated below each lane. KPNB1 is a loading control. e, RT-qPCR of OSKM transgene expression in primary NSCs from iOSKM mice (n = 8 cultures (Days 0, 2, 4) or 4 cultures (Days 1, 3), each culture from one mouse, over 2 independent experiments) during doxycycline treatment (green bars) and withdrawal (gray bars). Each dot represents expression level from one culture. Bar plot indicates mean +/− SEM. FDR-corrected P-values are as follows, using Welch’s ANOVA with multiple comparisons, for Day 1–4 vs. Day 0: P = 0.041, 0.019, 0.017, 0.019. f, Body weight of old (24–28 months) mice during partial reprogramming. n = 10 old (5 males and 5 females) and 15 old+OSKM (6 males and 9 females) mice. Note that not every mouse was weighed at every time point. Each dot represents weight for one mouse, normalized to starting weight. Data are mean +/− SEM. All P-values > 0.2 by two-sided Wilcoxon rank sum test. g, Survival of old (18–28 months) mice during partial reprogramming. n = 40 old (25 males and 15 females) and 55 old+OSKM (36 males and 19 females) mice. P-value: Mantel-Cox test.

Source data

Extended Data Fig. 2 Single-cell transcriptomics after whole-body partial reprogramming.

a, Body weight of mice during doxycycline treatment. n = 3 old and 6 old+OSKM, all old (18–20 months) males. b, Experimental design for first single-cell RNA-sequencing experiment with old (18–20 months) whole-body iOSKM mice using 10x Genomics v3 kit. n = 2 male mice per condition. c, Example of expression of the lipid-modified oligonucleotides used for sample multiplexing of cells from six mice into one lane of a 10x Genomics chip. d-e, Dimensionality reduction of 1194 high-quality cells from single-cell RNA-seq of cohort 1, colored by celltype (d) or condition (e). f, Dimensionality reduction from single-cell RNA-seq of cohort 1, split by condition. Cell number is downsampled to 550 cells per condition. g, Dimensionality reduction from single-cell RNA-seq of cohort 2, split by condition. Cell number is downsampled to 732 cells per condition. h, Cell type proportions by single-cell RNA-seq, as a proportion of total cells recovered per mouse. Each dot represents cell proportion for one mouse. Bar plot indicates mean. Mice from both independent cohorts are combined: n = 2 young (3-4 months; 2 males), 5 old (18–20 months or 24–26 months; 4 males and 1 female), 4 old+OSKM (18–20 months or 24–26 months; 3 males and 1 female), over 2 independent experiments (see Fig. 1d, Extended Data Fig. 2b, Supplementary Table 1). P-values: two-sided Wilcoxon rank sum test. i, Cell type proportions for clusters with very few cells, as a proportion of total cells per mouse, and normalized as log2 fold change over the mean of old untreated mice within each experiment. Bar is mean, each dot is one mouse. n = 2 young, 5 old, 4 old+OSKM, over 2 independent experiments, as in (h). P-values: two-sided Wilcoxon rank sum test. j, Heatmap of the expression of the top 5 differentially expressed genes in each cell type cluster. Annotation of cell types is indicated on top. Full list of marker genes is in Supplementary Table 2. k, Performance of linear regression model trained to predict age based on cell type proportions in the SVZ from single-cell RNA-seq of 28 mice from 3–29 months old (see Methods). Scatter plot shows correlation between predicted age from using leave-one-mouse-out testing vs. actual chronological age of the mouse. Each point represents one mouse. R is Pearson’s correlation coefficient. P-value: two-sided. Blue line is linear model, gray band is 95% confidence interval. l, Difference between predicted age from cell type proportion model and actual chronological age for each mouse. Line represents median. n = 2 young, 5 old, 4 old+OSKM, over 2 independent experiments, as in (h). P-value: two-sided Wilcoxon rank sum test.

Source data

Extended Data Fig. 3 Pathways and transcriptomic clocks after whole-body partial reprogramming.

a, Differential gene expression by MAST for each cell type from single-cell RNA-seq, comparing old+OSKM vs. young untreated iOSKM mice. Each dot represents the Z-score for one gene. Positive Z-score indicates upregulation in old+OSKM. Color indicates FDR < 0.2. b, UpSet plot showing overlap between significantly changing pathways (BH-adjusted two-sided FGSEA P-value, padj < 0.05) after whole-body partial reprogramming across cell types. Purple bars at lower left indicate total number of significant pathways for each cell type. Main bar plot shows number of pathways shared between the cell types indicated in the matrix below (note that direction of change may be different). c, Cell type-specific transcriptomic aging clocks previously trained to predict chronological age using single-cell RNA-seq of the SVZ of 28 mice from 3–29 months old were applied to each cell type after partial reprogramming. Density plots show the distribution of predicted ages of each cell type from each condition. The 2 independent experiments (Fig. 1d, Extended Data Fig. 2b) are shown separately. d-g, Dot plot of gene set enrichment analysis comparing the effects of aging and reprogramming in neuroblasts (d), aNSCs-NPCs (e), mural cells (f), and endothelial cells (g). Names of gene ontology (GO) pathways are listed on the left. padj: BH-adjusted two-sided FGSEA P-value. Pathways that are significantly enriched (padj < 0.05) in at least one comparison are shown. Size of dot reflects adjusted P-value (padj). Color indicates normalized effect size (NES), where positive values (red) indicate increase with age (‘aging’) or increase with partial reprogramming (‘OSKM’).

Extended Data Fig. 4 Neuroblasts and proliferation in the intact SVZ after whole-body partial reprogramming.

a, High-magnification images of Fig. 3a. Immunofluorescence staining of the SVZ in coronal sections of brains after in vivo partial reprogramming. Dorsal is up, lateral is left. Magenta, DCX (neuroblasts); green, Ki-67 (proliferative cells). Dashed line indicates border of ventricle. One optical section (1.0 μm thick) of a confocal image acquired using 63x objective. Scale bars are 25 μm. b-e, Quantification of immunofluorescence of the SVZ in coronal brain sections, related to Fig. 3. Multiple optical sections totaling 8 μm thick were acquired for each SVZ, and both hemispheres in five coronal sections at 120 μm intervals were quantified per mouse (see Methods). n = 11 young (3-4 months; 5 males and 6 females), 8 old (20–26 months; 3 males and 5 females), and 10 old+OSKM (20–26 months; 4 males and 6 females) mice, over 3 independent experiments (for individual experiments, see Source Data Table). Each dot represents value for one mouse. Data are mean +/− SEM. P-values: two-sided Wilcoxon rank sum test. b, Same as Fig. 3e, but symbols represent sex. Neuroblast density (number of DCX+ cells per mm3) in the SVZ per mouse. c, Total DCX fluorescence intensity (intensity x area) per mouse, normalized to the mean of old control mice. d, Proportion of proliferative cells (Ki-67+ over DAPI+) in the SVZ per mouse, normalized to the mean of old control mice. e, Density of proliferative cells (number of Ki-67+ cells per mm3) in the SVZ per mouse. Right, symbols represent sex. f, Representative images for immunofluorescence staining of the SVZ. Magenta, DCX (neuroblasts); green, PSA-NCAM (neuroblasts); blue, DAPI (nuclei). Dorsal is up, lateral is right. Yellow rectangle is region shown in insets in (g). Scale bar is 100 μm. g, High-magnification images of inset region from (f). One optical section of a confocal image acquired using 63x objective. Scale bars are 25 μm. h, Quantification of immunofluorescence. Fraction of cells expressing DCX or PSA-NCAM that are positive for one or both markers. Each bar is one image of an SVZ (as shown in i), n = 20 images across 2 young mice. i-k, Quantification of immunofluorescence of the SVZ in coronal brain sections, related to Fig. 3. Multiple optical sections totaling 8 μm thick were acquired for each SVZ, and both hemispheres in five coronal sections at 120 μm intervals were quantified per mouse (see Methods). n = 9 young (3-4 months; 4 males and 5 females), 8 old (20–26 months; 3 males and 5 females), and 10 old+OSKM (20–26 months; 4 males and 6 females) mice, over 3 independent experiments (for individual experiments, see Source Data Table). Each dot represents value for one mouse. Data are mean +/− SEM. P-values: two-sided Wilcoxon rank sum test. i, Same as Fig. 3h, but symbols represent sex. Neuroblast density (number of PSA-NCAM+ cells per mm3) in the SVZ per mouse. j, Proportion of aNSCs/NPCs (EGFR+PSA-NCAM- over DAPI+) in the SVZ per mouse, normalized to the mean of old control mice. k, Density of aNSCs/NPCs (number of EGFR+PSA-NCAM- per mm3) in the SVZ per mouse. Right, symbols represent sex.

Source data

Extended Data Fig. 5 Characterization of Cre-dependent and inducible c+iOSKM mice and AAV-SCH9.

a-c, Representative images (a-b) and quantification (c) of immunofluorescence staining to detect mCherry (virus), SOX2 (NSC marker), and DCX (neuroblasts) in the SVZ, 7 days post injection of AAV into the lateral ventricle. Scale bars are 50 μm (a) and 25 μm (b). AAV-mCherry-Cre (AAV SCH9 Ef1a-mCherry-IRES-Cre) was injected into the lateral ventricle of the right hemisphere for experimental animals (pink, n = 4 mice), and ipsilateral and contralateral hemispheres relative to injection site were quantified separately. The right hemisphere was counted for uninjected control (n = 1 mouse). In (a), yellow arrows indicate mCherry+SOX2+ cells; mCherry+ population includes both NSCs (SOX2+) and non-NSC cells (SOX2-). Each dot represents cell counts for one image, shape denotes an individual mouse. Box plot indicates median (center line), upper and lower quartiles (box limits), and 1.5x interquartile range (whiskers). P-values: two-sided Wilcoxon rank sum test. d, Representative images of immunofluorescence of the olfactory bulb and cerebellum using an antibody against mCherry (virus), 4–6 days post injection of AAV-mCherry-Cre (AAV SCH9 Ef1a-mCherry-IRES-Cre) into the lateral ventricle. No mCherry signal was detected. Scale bars are 500 μm. e, Primer sites for PCR from genomic DNA to detect Cre-mediated recombination of lox-STOP-lox cassette in c+iOSKM cells. f, PCR from genomic DNA of NSCs after infection with AAV (0.125 μl to 1μl, 2-fold dilutions). AAV-mCherry: AAV SCH9 Ef1a-mCherry. AAV-Cre: AAV SCH9 Ef1a-Cre. Colored arrows indicate expected size of products with and without recombination. Equal numbers of cells were plated before infection; note that we observed a correlation between cell death and high virus doses that is reflected in the amount of PCR product (for example, see 3.2 kb band in mCherry-infected cells). Representative of n = 2 cultures. g, Scheme for in vitro AAV infection of primary NSCs isolated from c+iOSKM mice. All lots of virus were tested for infectivity/recombination in primary NSC culture by flow cytometry and/or immunofluorescence. h, Immunofluorescence staining of NSCs after infection with the indicated AAVs, showing efficient infection (Cre, mCherry) and recombination (GFP). Composite includes DAPI (blue). Scale bars are 50 μm. i, Flow cytometry of primary NSCs after infection with AAV carrying mCherry only, Cre only, or mCherry-IRES-Cre. j-k, Flow cytometry of NSCs after infection with increasing doses of AAV mCherry-IRES-Cre (j) and quantification of populations (k). l, Related to Fig. 4e. RT-qPCR in primary c+iOSKM NSCs after infection with AAV-mCherry or AAV-Cre, showing expression of the respective viral genes. n = 4 young (3-4 months; 3 males and 1 female) and 4 old (20–23 months; 3 males and 1 female) cultures for Cre, n = 3 young (3-4 months; 2 males and 1 female) and 4 old (20–23 months; 3 males and 1 female) cultures for mCherry, over 1 independent experiment. Each dot represents expression level in one culture; each culture was isolated from one mouse. Bar plot indicates mean +/− SEM. P-values: two-sided Wilcoxon rank sum test. m, RT-qPCR in quiescent NSCs, showing AAV-SCH9 infection (mCherry), Cre-mediated recombination (Egfp), OSKM transgene induction (Oct4-P2A), and total Oct4 expression (Oct4). n = 2 cultures; one independent experiment. Each dot represents expression level in one culture; each culture was isolated from one mouse. Bar plot indicates mean +/− SEM. n, Western blot of primary NSCs from c+iOSKM mice, infected in culture with AAV-mCherry (control) or AAV-Cre, then treated with doxycycline. Size in kDa is indicated on the left.

Source data

Extended Data Fig. 6 Single-cell transcriptomics after SVZ-targeted reprogramming.

a, Cell type proportions by single-cell RNA-seq, as a proportion of total cells recovered per mouse. Each dot represents cell proportion for one mouse. Bar plot indicates mean. n = 5 young, 6 old, 6 old+OSKM (SVZ), over 2 independent experiments. P-values: two-sided Wilcoxon rank sum test. b, Cell type proportions for small clusters and immune cells, as a proportion of total cells per mouse, and normalized as log2 fold change over the mean of old untreated mice. Each dot represents cell proportion for one mouse. Bar plot indicates mean. n = 5 young, 6 old, 6 old+OSKM (SVZ), over 2 independent experiments. P-values: two-sided Wilcoxon rank sum test. c, Dot plot of rtTA-IRES-Egfp expression by cell type from single-cell RNA-seq. Size of dot reflects percentage of cells within cluster with non-zero expression. Color indicates average expression level. d, Difference between predicted age from cell type proportion model and actual chronological age for each mouse. Line represents median. n = 5 young, 6 old, 6 old+OSKM (SVZ), over 2 independent experiments. P-value: two-sided Wilcoxon rank sum test. e, UpSet plot showing overlap between significantly changing pathways (BH-adjusted two-sided FGSEA P-value, padj < 0.05) after SVZ-targeted partial reprogramming across the six most abundant cell types. Purple bars at lower left indicate total number of significant pathways for each cell type. Main bar plot shows number of pathways shared between the cell types indicated in the matrix below (note that direction of change may be different). f, Dot plot of gene set enrichment analysis comparing the effects of aging and SVZ-targeted reprogramming on expression of inflammation pathways across cell types. Names of gene ontology (GO) pathways are listed on the left. padj: BH-adjusted two-sided FGSEA P-value. Size of dot reflects adjusted P-value (padj). Color indicates normalized effect size (NES), where positive values (red) indicate increase with age (‘aging’) or increase with SVZ-targeted reprogramming (‘OSKM’). g, Cell type-specific transcriptomic aging clocks previously trained to predict chronological age using single-cell RNA-seq of the SVZ of 28 mice from 3–29 months old were applied to each cell type after SVZ-targeted reprogramming. Density plots show the distribution of predicted ages of each cell type from each condition. h, Survival of old (19–28 months) mice during SVZ-targeted partial reprogramming. n = 14 old (8 males and 6 females) and 14 old+OSKM (SVZ) (7 males and 7 females) mice. P-value: Mantel-Cox test. i-j, Dot plot showing normalized enrichment score from gene set enrichment analysis in aNSCs-NPCs (f) or neuroblasts (g) after SVZ-targeted reprogramming (old+OSKM (SVZ) vs. old control). GO pathway names are listed on the left. padj: BH-adjusted two-sided FGSEA P-value. Color reflects adjusted P-value (padj). Size of dot reflects number of genes in set. Significantly enriched pathways (padj < 0.05) are shown.

Source data

Extended Data Fig. 7 Comparison of whole-body and SVZ-targeted reprogramming.

a, Scatter plots showing normalized enrichment scores from gene set enrichment analysis, comparing SVZ-targeted reprogramming (old+OSKM (SVZ) vs. old) vs. whole-body partial reprogramming (old+OSKM vs. old) for common cell types. Each point represents the normalized enrichment scores for one pathway. For all panels, padj: BH-adjusted two-sided FGSEA P-value. Pathways where adjusted P-value (padj) < 0.05 for at least one comparison are shown. Color indicates padj for SVZ-targeted reprogramming. Pathways in the upper right quadrant and lower left quadrant change in the same direction in both partial reprogramming paradigms. b, Heatmap of pathway enrichment for each cell type, comparing normalized enrichment score (NES) in whole-body (old+OSKM vs. old) and SVZ-targeted (old+OSKM (SVZ) vs. old) reprogramming. Pathways where padj < 0.05 for at least one paradigm are included. Pathways are divided into 4 clusters by k-means clustering. Color reflects NES. Size is number of pathways falling into each cluster. c, UpSet plot showing overlap between cell types of pathways increasing (top) or decreasing (bottom) in both whole-body and SVZ-targeted partial reprogramming (padj < 0.05 for at least one paradigm). Bars at lower left indicate total number of pathways meeting these criteria for each cell type. Main bar plot shows number of pathways shared between the cell types indicated in the matrix below. d-e, Heatmap showing normalized enrichment score (NES) from gene set enrichment analysis in aNSCs-NPCs (d) and neuroblasts (e), comparing whole-body and SVZ-targeted partial reprogramming. GO pathway names are listed on the right. Pathways are divided into 4 clusters by k-means clustering. Color reflects NES. Pathways where padj < 0.05 for at least one comparison are included.

Extended Data Fig. 8 Differentiation of primary NSCs in culture.

a, Representative images of immunofluorescence staining of primary NSC cultures. Primary NSCs express markers of activated NSCs (Ki-67, GFAP, EGFR, and Nestin). Scale bar is 50 μm. b, Representative images of immunofluorescence staining after in vitro differentiation of primary NSCs using markers for neurons (TUJ1, NeuN), astrocytes (GFAP), and oligodendrocytes (O4). Scale bar is 25 μm. c, Experimental schematic for immunofluorescence of primary NSC cultures from iOSKM mice with partial reprogramming, used in panels d-j. PDL: poly-D-lysine. n = 9 young (3-4 months; 4 males and 5 females) and 9 old (24–26 months; 4 males and 5 females), over 2 independent experiments. Each culture was isolated from one mouse. d-e, Representative images of immunofluorescence staining for NSC markers (panel d: EGFR, green; Nestin, magenta), proliferation markers (panel e: Ki-67, magenta; EdU, green) and neuroblast markers (panel e: DCX, red) in primary NSCs from young mice before differentiation. Scale bar is 50 μm. f-j, Quantification of immunofluorescence in primary NSCs. n = 9 young (3-4 months; 4 males and 5 females) and 9 old (24–26 months; 4 males and 5 females), over 2 independent experiments. Each dot represents the proportion of cells positive for the indicated marker in one culture, where each culture was isolated from one iOSKM mouse. Box plot indicates median (center line), upper and lower quartiles (box limits), and 1.5x interquartile range (whiskers). P-values: two-sided Wilcoxon rank sum test. k, Representative image of immunofluorescence staining for neuroblasts (PSA-NCAM, green) after in vitro differentiation for 4 days. Scale bar is 50 μm. l, Quantification of immunofluorescence staining for neuroblasts (PSA-NCAM+ over DAPI+). n = 8 young (3-4 months; 4 males and 4 females) and 8 old (24–26 months; 4 males and 4 females), over 2 independent experiments. Each dot represents neuroblast proportion for one culture, where each culture was isolated from one iOSKM mouse. Box plot indicates median (center line), upper and lower quartiles (box limits), and 1.5x interquartile range (whiskers). P-values: two-sided Wilcoxon rank sum test. m-n, Related to Fig. 6a–f. Quantification of immunofluorescence for proliferative cells (Ki-67+, m) and glia (sum of GFAP+ cells and SOX10+ cells, n) after 4 days of differentiation with partial reprogramming. n = 8 young (3-4 months; 4 males and 4 females) and 8 old (24–26 months; 4 males and 4 females), over 2 independent experiments. Each dot represents proliferative proportion for one culture, where each culture was isolated from one iOSKM mouse. Box plot indicates median (center line), upper and lower quartiles (box limits), and 1.5x interquartile range (whiskers). P-values: two-sided Wilcoxon rank sum test. o-p, Related to Fig. 6d,e. Quantification of immunofluorescence for neuroblasts (DCX+) and proliferative cells (Ki-67+) after AAV infection and 4 days of differentiation with partial reprogramming. n = 4 young (3-4 months; 3 males and 1 female) and 4 old (20–23 months; 3 males and 1 female), over 1 independent experiment. Each dot represents proliferative proportion for one culture, where each culture was isolated from one c+iOSKM mouse. Box plot indicates median (center line), upper and lower quartiles (box limits), and 1.5x interquartile range (whiskers). P-values: two-sided Wilcoxon rank sum test. q, Related to Fig. 8a–c. Quantification of immunofluorescence for total neuronal cells (sum of DCX+ cells and TUJ1+ cells, representing neuroblasts and neurons) after 8 days of differentiation with partial reprogramming. n = 8 young (3-4 months; 4 males and 4 females) and 8 old (24–26 months; 4 males and 4 females), over 2 independent experiments. Each dot represents total neuronal proportion for one culture, where each culture was isolated from one iOSKM mouse. Box plot indicates median (center line), upper and lower quartiles (box limits), and 1.5x interquartile range (whiskers). P-values: two-sided Wilcoxon rank sum test.

Source data

Extended Data Fig. 9 Bulk RNA-seq of primary NSCs with partial reprogramming and differentiation.

a, Heatmap of expression of the top variable genes across all samples from bulk RNA-seq of NSCs with reprogramming and differentiation. Color indicates expression, scaled by row. Condition and time point are indicated at the top. Sex-related genes are removed for visualization. b, Expression of selected genes that change across differentiation. Each dot represents normalized count for one culture, where each culture was isolated from one iOSKM mouse. n = 5 young (3-4 months; 2 male and 3 female) and 4 old (25 months; 2 male and 2 female) cultures. Box plot indicates median (center line), upper and lower quartiles (box limits), and 1.5x interquartile range (whiskers). P-values: two-sided Wilcoxon rank sum test. c, Expression of the reprogramming factors in old control and old+OSKM NSCs (6h time point). Each dot represents normalized count for one culture, where each culture was isolated from one iOSKM mouse. n = 4 cultures (25 months; 2 male and 2 female). Box plot indicates median (center line), upper and lower quartiles (box limits), and 1.5x interquartile range (whiskers). P-values: two-sided Wilcoxon rank sum test. d-f, Dot plot showing normalized enrichment score from gene set enrichment analysis after partial reprogramming in NSCs (old+OSKM vs. old control) at 6h (d), 18h (e), and 48h (f) time points. GO pathway names are listed on the left. padj: BH-adjusted two-sided FGSEA P-value. Color reflects padj. Size of dot reflects number of genes in set. Top and bottom 15 (by NES) significantly enriched pathways (padj < 0.05) are shown. g, Dotplots of gene set enrichment analysis comparing the effects of aging and reprogramming at each differentiation time point. Names of gene ontology (GO) pathways are listed on the left. padj: BH-adjusted two-sided FGSEA P-value. Size of dot reflects padj. Color indicates normalized effect size (NES). h, Differential gene expression by DESeq2 for each time point, comparing old vs. young (left) or old+OSKM vs. old (right). Each dot represents the Z-score for one gene. Positive Z-score indicates upregulation. Color indicates FDR < 0.1. i, Heatmap of expression of genes that are significantly differentially expressed in NSCs between old and old+OSKM cultures at the 18h time point. Color indicates expression, scaled by row. Condition and time point are indicated at the top. Genes are separated into 4 clusters by k-means clustering. j, UpSet plot showing overlap between differentially expressed genes (DEGs) after partial reprogramming (old+OSKM vs. old control) in in vitro time points (adjusted P-value < 0.2 by DESeq) and in vivo paradigms (adjusted P-value < 0.3 by MAST in aNSC-NPC, neuroblast, or astrocyte-qNSC clusters).

Extended Data Fig. 10 FACS gating and Western blots.

a, Representative gating for FACS sorting of live cells from the SVZ for single-cell RNA-seq. Population shown is listed above each plot. b, Representative gating strategy to identify live cells from primary NSCs in culture. Population shown is listed above each plot. c, Experimental schematic for flow cytometry analysis of AAV infection of primary NSCs in culture. d, Representative gating strategy for analysis of mCherry (virus) and EGFP (recombination marker). Live cells (see b) are shown. Each plot is a culture treated with the virus listed above the plot. Note that these plots are also shown in Extended Data Fig. 5i. e-j, Full Western blots. Corresponding figure panels are indicated above the blots. Dotted rectangles indicate cropped area shown in corresponding figure.

Supplementary information

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Supplementary Tables

Supplementary Tables 1–9.

Source data

Source Data for Figs. 1, 3, 4, 6 and 7 and Extended Data Figs. 1, 2, 4–6 and 8

Statistical source data including individual data point values and independent experiment numbers.

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Xu, L., Ramirez-Matias, J., Hauptschein, M. et al. Restoration of neuronal progenitors by partial reprogramming in the aged neurogenic niche. Nat Aging 4, 546–567 (2024). https://doi.org/10.1038/s43587-024-00594-3

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