Journal: bioRxiv

Article Title: Transplantation of Human IPSC-derived Microglia Ameliorates Neuropathology and Circuit Dysfunction in Progranulin-Deficient Mice

doi: 10.64898/2026.01.13.699312

Figure Lengend Snippet: (A) Xenotolerant Grn -deficient mice were generated by crossing immune-intact Grn −/− mice onto the humanized, immune-deficient hCSF1 +/+ /Rag2 −/− /Il2rg −/y ( hCSF1 ) knockin background and restoring homozygosity for all four alleles. (B) Schematic illustrating the neonatal transplantation and analysis paradigm for hCSF1 and hCSF1-Grn −/− mice. (C) Representative confocal microscopy of xenografted GFP-positive (green) human microglia (Iba1, red) in 12-month-old hCSF1-Grn −/− +xMG adult mice; scale bars: 500 µm (low magnification), 50 µm (high magnification). (D, F ) Representative confocal microscopy of human xMG (GFP, green) and autofluorescent inclusions (lipofuscin, red) in the hippocampus (D) and thalamus (F) of WT and transplanted mice; scale bar, 50 µm. (E, G) Quantification of autofluorescence within the hippocampus (E) and thalamus (G) of wild-type and transplanted mice; P values from one-way ANOVA (Hippocampus: F 2,42 = 65.00; ∗∗∗∗ p < 0.0001; Thalamus: F 2,42 = 20.86; ∗∗∗∗ p < 0.0001) with Tukey’s multiple comparisons tests. Data represented as mean value ± SEM (non-injected hCSF1 littermates, n = 15; hCSF1-Grn −/− +PBS, n = 14; hCSF1-Grn −/− +xMG, n = 16) (Female, n = 8 per group; Male, n = 6–8 per group). ns, not significant; ∗∗∗∗ p < 0.0001.

Article Snippet: All animal procedures were conducted in accordance with guidelines set forth by the National Institutes of Health and the University of California, Irvine Institutional Animal Care and Use Committee, and the University of California, San Francisco Institutional Animal Care and Use Committee. hCSF1-Grn −/− mice (abbreviated as hCSF- Grn −/− ) were generated by backcrossing ( Grn-/- mice, Jackson Laboratories #029919) with xenotransplantation compatible hCSF1 mice (Jackson Laboratories #017708), harboring deletions in Rag2 and Il2rg and expressing humanized CSF-1.

Techniques: Generated, Knock-In, Transplantation Assay, Confocal Microscopy, Injection

Journal: bioRxiv

Article Title: Transplantation of Human IPSC-derived Microglia Ameliorates Neuropathology and Circuit Dysfunction in Progranulin-Deficient Mice

doi: 10.64898/2026.01.13.699312

Figure Lengend Snippet: Levels of murine progranulin within the liver (t=27.60, **** p < 0.0001) (A) , plasma (t=96.25, ****p < 0.0001) (B) , and spleen (t=7.07, ** p = 0.0021) (C) of hCSF1-WT and hCSF1-Grn −/− littermates were measured by ELISA. N = 3 mice/group, all unpaired two-tailed t-test. (D) Progranulin levels measured within the brain (t=10.31, **** p < 0.0001). N = 4–5 mice/group, respectively, unpaired two-tailed t-test. (E) Progranulin levels measured within the brain using a human-targeted ELISA, n = 5 samples/group, one-way ANOVA with Tukey’s multiple comparisons test (F 2,12 =1.803, **** p < 0.0001). (F) Progranulin levels measured within the brain using a mouse-targeted ELISA, n = 5 samples/group, one-way ANOVA with Tukey’s multiple comparisons test (F 2,12 =2.80, **** p < 0.0001).

Article Snippet: All animal procedures were conducted in accordance with guidelines set forth by the National Institutes of Health and the University of California, Irvine Institutional Animal Care and Use Committee, and the University of California, San Francisco Institutional Animal Care and Use Committee. hCSF1-Grn −/− mice (abbreviated as hCSF- Grn −/− ) were generated by backcrossing ( Grn-/- mice, Jackson Laboratories #029919) with xenotransplantation compatible hCSF1 mice (Jackson Laboratories #017708), harboring deletions in Rag2 and Il2rg and expressing humanized CSF-1.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Transplantation of Human IPSC-derived Microglia Ameliorates Neuropathology and Circuit Dysfunction in Progranulin-Deficient Mice

doi: 10.64898/2026.01.13.699312

Figure Lengend Snippet: (A) Quantification of proinflammatory IL-1 β , TNF- ⍺ , CXCL10, IL- 33, MIP-1a, and IL-10 levels in soluble brain extracts of hCSF1 littermates and hCSF1-Grn −/− + PBS and hCSF1-Grn −/− + WTxMG mice; p- values from one-way ANOVA (IL-10: F 2,27 =7.332; ** p = 0.0029) (IL-1B: F 2,27 = 7.958; ** p = 0.0019) (TNF-a: F 2,27 = 7.445; ** p = 0.0027) (IL-33 : F 2,27 = 12.78; *** p = 0.0001) (IP-10: F 2,27 = 6.734; ** p = 0.0042) (MIP-1a: F 2,27 = 18.61; **** p < 0.0001) with Tukey’s multiple comparisons tests. Data represented as average mean value ± SEM ( hCSF1 littermates, n = 10; hCSF1-Grn −/− +PBS, n = 10; hCSF1-Grn −/− +WTxMG, n = 10) (Female, n = 5 per group; Male, n = 5 per group). (B) Representative confocal microscopy of human xMG (GFP, green), microglial marker (IBA1, white), and microglial activation marker (CD68, red) in the thalamus (top) and hippocampus (bottom) of wild-type and transplanted Grn −/− mice, scale bar, 50 µm. (C) Quantification of CD68-positive immunoreactivity (total volume/FOV) within the thalamus (top) and hippocampus (bottom) of wild-type and transplanted mice p values from one-way ANOVA (Hippocampus: F 2,42 = 24.32**** p < 0.0001; Thalamus: F 2,42 = 17.43; **** p < 0.0001) data analyzed with Tukey’s multiple comparisons tests. Data are presented as mean ± SEM ( hCSF1 littermates, n = 15; hCSF1-Grn −/− +PBS, n = 14; hCSF1-Grn −/− + WTxMG, n = 16) (Female, n = 8 per group; Male, n = 6–8 per group). ns, not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Comparisons not shown are not significant.

Article Snippet: All animal procedures were conducted in accordance with guidelines set forth by the National Institutes of Health and the University of California, Irvine Institutional Animal Care and Use Committee, and the University of California, San Francisco Institutional Animal Care and Use Committee. hCSF1-Grn −/− mice (abbreviated as hCSF- Grn −/− ) were generated by backcrossing ( Grn-/- mice, Jackson Laboratories #029919) with xenotransplantation compatible hCSF1 mice (Jackson Laboratories #017708), harboring deletions in Rag2 and Il2rg and expressing humanized CSF-1.

Techniques: Confocal Microscopy, Marker, Activation Assay

Journal: bioRxiv

Article Title: Transplantation of Human IPSC-derived Microglia Ameliorates Neuropathology and Circuit Dysfunction in Progranulin-Deficient Mice

doi: 10.64898/2026.01.13.699312

Figure Lengend Snippet: A multiplex ELISA was used to concurrently examine protein levels of several murine cytokines and chemokines within the brains of hCSF1 -PBS, hCSF1-Grn −/− +PBS, and hCSF1-Grn −/− +xMG engrafted mice. For each analyte, N = 10 mice/group were compared by one-way ANOVA with Tukey’s multiple comparisons test. No significant (ns) differences in IL-6 (A) or KC/GRO (B) were detected. (C) IL-9 levels in the brain, * p = 0.018. (D) IL-15 levels (F 2,27 = 1.238, *** p = 0.0002 and **** p < 0.0001). (E) IL-17A/F levels in brain (F 2,27 = 3.026, **** p < 0.0001). (F) IP-27p/28 levels in brain (F 2,27 = 3.685, *** p = 0.0006 and **** p < 0.0001). (G) MCP-1 levels in brain, ns = nonsignificant. (H) MIP-2 levels in brain (F 2,27 = 1.12, * p = 0.0156 and ** p = 0.0083). (I) IL-2 levels in brain, ns = nonsignificant. (J) IL-12p70 levels in the brain, ns = nonsignificant. (K) IL-5 levels in the brain (F 2,27 = 2.819, * p = 0.0367).

Article Snippet: All animal procedures were conducted in accordance with guidelines set forth by the National Institutes of Health and the University of California, Irvine Institutional Animal Care and Use Committee, and the University of California, San Francisco Institutional Animal Care and Use Committee. hCSF1-Grn −/− mice (abbreviated as hCSF- Grn −/− ) were generated by backcrossing ( Grn-/- mice, Jackson Laboratories #029919) with xenotransplantation compatible hCSF1 mice (Jackson Laboratories #017708), harboring deletions in Rag2 and Il2rg and expressing humanized CSF-1.

Techniques: Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Transplantation of Human IPSC-derived Microglia Ameliorates Neuropathology and Circuit Dysfunction in Progranulin-Deficient Mice

doi: 10.64898/2026.01.13.699312

Figure Lengend Snippet: (A) Schematic of mouse strains: hCSF1 littermates (WT) and hCSF1-Grn −/− xenografted with WT (WTxMG) or KO (KOxMG) human microglia. (B) Schematic experimental timeline of microglia injected at P1, behavior at 9.5 months, electrophysiology, and snRNA-seq at 10.5–11.5 months. (C) Quantification of human progranulin ELISA showing increased progranulin levels in WTxMG mice. Unpaired t-test (t(8) = 2.647, * p = 0.0294). Data represented as mean ± SEM ( hCSF1-Grn −/− +KOxMG, n = 3; hCSF1-Grn −/− +WTxMG, n = 7). (D) Volcano plot of pseudobulk DEGs in human microglia between WTxMG and KOxMG groups, with key genes up- or downregulated. (E) Volcano plot of pseudobulk DEGs in thalamic mouse microglia between WTxMG and KOxMG groups, with key genes up- or downregulated. (F) Top predicted upstream regulators identified using Ingenuity Pathway Analysis (IPA). (G) Top predicted downregulated pathways in WTxMG v. KOxMG. (H) UMAP of human microglia by genotype, showing clustering differences between WTxMG and KOxMG groups. (I) Quantification of average cell ratios in each subcluster by group in human microglia. (J) Volcano plot of subcluster 1 DEGs in human microglia between WTxMG and KOxMG groups, with key genes up- or downregulated. (K) Volcano plot of subcluster 3 DEGs in human microglia between WTxMG and KOxMG groups, with key genes up- or downregulated. ( L ) Quantification of human C1Q ELISA showing significantly decreased levels in WTxMG injected mice. p -value from one-way ANOVA (F 2,12 =2.525, **** p < 0.0001) with Tukey’s multiple comparisons test. Data represented as mean ± SEM ( hCSF1 , n = 5 ; hCSF1-Grn-/- + KOxMG, n = 3 ; hCSF1-Grn −/− + WTxMG, n = 7). * p < 0.05 and **p < 0.01.

Article Snippet: All animal procedures were conducted in accordance with guidelines set forth by the National Institutes of Health and the University of California, Irvine Institutional Animal Care and Use Committee, and the University of California, San Francisco Institutional Animal Care and Use Committee. hCSF1-Grn −/− mice (abbreviated as hCSF- Grn −/− ) were generated by backcrossing ( Grn-/- mice, Jackson Laboratories #029919) with xenotransplantation compatible hCSF1 mice (Jackson Laboratories #017708), harboring deletions in Rag2 and Il2rg and expressing humanized CSF-1.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Transplantation of Human IPSC-derived Microglia Ameliorates Neuropathology and Circuit Dysfunction in Progranulin-Deficient Mice

doi: 10.64898/2026.01.13.699312

Figure Lengend Snippet: (A) Volcano plot of pseudobulk DEGs in thalamic excitatory neurons between WT-xMG and KO- xMG groups, showing upregulation of Cck, Asna1, Abca6, Dab2, Nwd2, Tac1, Kcnh8, Scube1, and Arxes1 and downregulation of Mri1, Podxl2, Tsnax, Rab24, Nwd1, Oser1, Vegfa, Capn11, Pdzrn3, and Pot1b. (B) Dotplot of several epilepsy-related genes, showing reduced expression in the KOxMG group and some restoration in several genes in the WTxMG group (bolded). (C) Dotplot of same epilepsy-related genes in human frontal cortex excitatory neurons of control and progranulin-FTD brains, showing a similar pattern as GRN +/+ human xMG (bolded). (D) Volcano plot of pseudobulk DEGs in thalamic inhibitory neurons between WTxMG and KOxMG groups, showing upregulation of Scg5 , Caly , Pgrmc1 , Asna1 , Nrsn2 , Sst , and Prkd3 and downregulation of Podxl2 , Pip4p1 , Map11 , Cpne7 , Plp1 , Vegfa , and Arf5 in WTxMG injected mice in comparison to those receiving KOxMG. (E) Dotplot of several epilepsy-related genes, showing increased expression in the WT group and restoration of several genes in the WTxMG group (bolded). (F) Dotplot of the same epilepsy-related genes in human frontal cortex inhibitory neurons of control and Progranulin-FTD brains, showing a similar pattern as GRN +/+ human xMG (bolded).

Article Snippet: All animal procedures were conducted in accordance with guidelines set forth by the National Institutes of Health and the University of California, Irvine Institutional Animal Care and Use Committee, and the University of California, San Francisco Institutional Animal Care and Use Committee. hCSF1-Grn −/− mice (abbreviated as hCSF- Grn −/− ) were generated by backcrossing ( Grn-/- mice, Jackson Laboratories #029919) with xenotransplantation compatible hCSF1 mice (Jackson Laboratories #017708), harboring deletions in Rag2 and Il2rg and expressing humanized CSF-1.

Techniques: Expressing, Control, Injection, Comparison

Journal: bioRxiv

Article Title: Transplantation of Human IPSC-derived Microglia Ameliorates Neuropathology and Circuit Dysfunction in Progranulin-Deficient Mice

doi: 10.64898/2026.01.13.699312

Figure Lengend Snippet: (A) Representative image of multi-unit recordings setup of ex vivo thalamic horizontal slices in a humidified, oxygenated interface chamber. (B) Schematic of multi-unit activity recordings with a Neuronexus 16-channel recording electrode positioned in the thalamus following electrical stimulation of the internal capsule. (C) Representative 3-second recording of intrathalamic multi-unit activity evoked by stimulation of the internal capsule (black dot indicates time of stimulation). Only 12 of 16 channels are shown for clarity. (D) Collapsed activity from 12 recording channels in (C). Black dot indicates time of stimulation. (E) Post-stimulus time histogram of instantaneous spiking frequency from multi-unit activity recorded across 16 channels (all 16 channels were analyzed, 12 representative channels are displayed in (C) from 15 slices from 9 hCSF1 (WT) mice, 8 slices from 7 hCSF1-Grn −/− +KOxMG mice, and 12 slices from 9 hCSF1-Grn −/− +WTxMG mice. The grey box denotes early response (0–1.5 s), and the white box denotes delayed response (1.5–3 s) after stimulation. (F) Enlarged instantaneous frequency of the delayed response (1.5–3 s) from (D). (G) Relative probability of eliciting spiking during the direct response (0–1.5 s) after stimulation; p values from Kolmogorov-Smirnov test with Bonferroni correction (* p = 0.0024). (H) Instantaneous frequency of spiking during the early response (0–1.5 s) after stimulation; Kruskal–Wallis test, H(3) = 6.767, p = 0.0797. (I) Relative probability of eliciting spiking during the delayed response (1.5–3 s) after stimulation; p values from Kolmogorov-Smirnov test with Bonferroni correction (**** p < 0.0001). (J) Instantaneous frequency of spiking during the delayed response (1.5–3 s) after stimulation; Kruskal-Wallis test, H(3)=56.6, **** p < 0.0001, and Dunn’s multiple comparisons test (**** p < 0.0001). Data are presented as mean ± SEM. Comparisons not shown are not significant.

Article Snippet: All animal procedures were conducted in accordance with guidelines set forth by the National Institutes of Health and the University of California, Irvine Institutional Animal Care and Use Committee, and the University of California, San Francisco Institutional Animal Care and Use Committee. hCSF1-Grn −/− mice (abbreviated as hCSF- Grn −/− ) were generated by backcrossing ( Grn-/- mice, Jackson Laboratories #029919) with xenotransplantation compatible hCSF1 mice (Jackson Laboratories #017708), harboring deletions in Rag2 and Il2rg and expressing humanized CSF-1.

Techniques: Ex Vivo, Activity Assay

Journal: bioRxiv

Article Title: Transplantation of Human IPSC-derived Microglia Ameliorates Neuropathology and Circuit Dysfunction in Progranulin-Deficient Mice

doi: 10.64898/2026.01.13.699312

Figure Lengend Snippet: (A) Grooming behavior apparatus consisted of a clear plexiglass cylinder placed on a transparent plexiglass bottom and covered with a white opaque plastic top with a 4k video camera fitted underneath the apparatus. (B) Grooming episodes as seen from a side view. Mice groomed both their head and body with front and hind limbs. (C) Grooming episodes snapshots as a single video frame corresponding to schematics in B. Open circles denote grooming limbs, crossed—non-grooming. (D) Representative episodes of self-grooming during 20-minute recordings in 5 mice from each genotype. (E) Quantification of total grooming time, p value from Student’s t-test (t(17) = 1.868, † p = 0.079). (F) Quantification of the number of grooming episodes, p value from Student’s t- test (t(17) = 0.1506, p = 0.8820). Data represented as mean ± SEM ( hCSF1-Grn −/− + KOxMG, n = 8; hCSF1-Grn −/− +WTxMG, n= 11). (G) Quantification of the duration of grooming episodes, p- value from Student’s t-test (t(93)=2.738, ** p = 0.0074). Data represented as mean ± SEM ( hCSF1-Grn −/− + KOxMG, n = 41 episodes from 8 mice; hCSF1-Grn −/− + WTxMG, n = 54 episodes from 11 mice). (H) Probability of grooming episode duration fitted with a nonlinear regression model of exponential plateau curves, p- value from extra sum-of-squares F-test (F 3,41 = 76.74, *** p < 0.0001). (I) (Top) Three-chamber sociability apparatus comprised of an opaque white acrylic box with two clear plastic inserts with openings with a 4k video camera fitted above the apparatus. (Bottom) Top view of the apparatus with a mouse placed in the middle chamber. (J) Interaction episodes of social approach as seen from a side view. Mice approach the pencil wire cup with a 1) snout touch, 2) snout touch and one front limb touch, and 3) rearing (both front limbs touch). (K) Interaction episodes snapshots as a single video frame corresponding to schematics in (J). Crossed circles denote snout, open—front limbs. (L) Ratio of time spent approaching empty right vs. empty left pencil wire cup during 10 minutes of habituation, p- value from Student’s t-test (t(15) = 0.7824, p = 0.6335). (M) Ratio of time spent approaching the mouse-containing vs object-containing pencil wire cup during 10 minutes of testing, p value from Student’s t-test (t(15) = 2.441, * p = 0.0275). Data represented as mean ± SEM ( hCSF1-Grn −/− + KOxMG, n = 8; hCSF1-Grn −/− +WTxMG, n = 9). * p < 0.05, ** p < 0.01. Comparisons not shown are not significant.

Article Snippet: All animal procedures were conducted in accordance with guidelines set forth by the National Institutes of Health and the University of California, Irvine Institutional Animal Care and Use Committee, and the University of California, San Francisco Institutional Animal Care and Use Committee. hCSF1-Grn −/− mice (abbreviated as hCSF- Grn −/− ) were generated by backcrossing ( Grn-/- mice, Jackson Laboratories #029919) with xenotransplantation compatible hCSF1 mice (Jackson Laboratories #017708), harboring deletions in Rag2 and Il2rg and expressing humanized CSF-1.

Techniques: