Stroke pathology was compared between (1) wildtype (WT) C57BL/J mice with three immunosuppressant mouse models (2): WT C57BL/J mice that were immunosuppressed with tacrolimus (3) Rag2^-/- mice and (4) NSG mice. All mice received a large photothrombotic stroke to the right sensorimotor cortex and underwent regular behavioral tests at baseline, 3, 7, 14, 21 days after stroke induction. At 21 days after stroke, mice from each group were divided into two subgroups: Brains from the first subgroup were collected, fixed and histologically analysed; brains from the other group were microdissected around the stroke region and prepared for RNAseq analysis. We chose the time point based on previous findings showing functional differences starting from three weeks following regenerative therapies (13). To test the effects of delayed tacrolimus treatment, we compared between (1) wildtype C57BL/J mice with (2) WT C57BL/J mice that were acutely immunosuppressed mice with tacrolimus (0 dpi) and (3) WT C57BL/J mice that received tacrolimus delayed at 7 dpi. The brains were treated equivalently to the previous set-up to obtain comparable histology and RNAseq data.

Long-term survival and successful immunosuppression were tested by local intraparenchymal transplantation of human NPCs to 1) WT C57BL/J mice (2) WT C57BL/J mice that were immunosuppressed with tacrolimus (3) Rag2^-/- mice and (4) NSG mice. Luciferase expressing NPCs were tracked over 35 days using in vivo bioluminescence imaging and brain tissue was collected to identify the transplanted cells in brain sections.

All procedures were conducted in accordance with governmental, institutional (University of Zurich), and ARRIVE guidelines and had been approved by the Veterinarian Office of the Canton of Zurich (license: 209/2019). In total, 20 WT (WT) mice (histology: 7, RNAseq: 8, cell therapy: 5) with C57BL/6 background mice, 17 tacrolimus treated WT mice (histology: 8, RNAseq: 4, cell therapy: 5), 19 Rag2^-/- mice (histology: 5, RNAseq: 9, cell therapy: 5) and 19 NSG mice (histology: 10, RNAseq: 4, cell therapy: 5) were used. Mice that were used for histology and RNAseq both performed the behavioral tasks. We used both sex female and male and mice were 3 months of age. Breeding of Rag2^-/- mice and NSG mice was performed at Laboratory Animal Services Center (LASC) in Schlieren, CH. WT animals were provided by Charles River Laboratories, Germany. All animals were housed in standard type II/III cages on a 12h day/light cycle (6:00 A.M. lights on) with food and water ad libitum. All mice were acclimatized for at least a week to environmental conditions before set into experiment.

Anaesthesia was performed using isoflurane (5% induction, 1.5-2% maintenance, Attane, Provet AG) and adequate sedation was confirmed by tail pinch. Novalgin (1mg/ml) was applied via drinking water; 24 h prior to the procedure and for three consecutive days directly after stroke surgery. Cerebral ischemia was induced by photothrombotic stroke surgery as previously described (13, 14). Briefly, animals were fixed in a stereotactic frame (David Kopf Instruments), the surgical area was sanitized, and the skull was exposed through a cut along the midline. A cold light source (Olympus KL 1,500LCS, 150W, 3,000K) was positioned over the right forebrain cortex (anterior/posterior: −1.5–+1.5 mm and medial/lateral 0 mm to +2 mm relative to Bregma). Rose Bengal (15 mg/ml, in 0.9% NaCl, Sigma) was injected intraperitoneally 5 min prior to illumination and the region of interest was subsequently illuminated through the intact skull for 12 min. To restrict the illuminated area, an opaque template with an opening of 3 × 4 mm was placed directly on the skull. The wound was closed using a 6/0 silk suture and animals were allowed to recover.

Cortical perfusion was evaluated using Laser Doppler Imaging (Moor Instruments, MOORLDI2-IR). Briefly, animals were fixed in a stereotactic frame and the region of interest was shaved and sanitized. To expose the skull, a cut was made along the midline and the brain was scanned using the repeat image measurement mode. All data were exported and quantified in terms of total flux in the ROI using Fiji (ImageJ).

For pharmacological immunosuppression, Alzet osmotic pumps (Model 1004, Cupertino, CA, USA) filled with 100µl tacrolimus solved in a mixture of DMSO and PEG 300 (50mg/ml; 7.56mg/ml concentration) were implanted subcutaneously on the back according to the manufacturer’s protocol. Briefly, animals were anesthetized using isoflurane (5% induction, 1.5-2% maintenance, Attane, Provet AG), the surgical area was sanitized and shaved. The implantation site was exposed through a mid-scapular incision. An implantation pocket was created using a hemostat. The pump was inserted into the pocket and the wound was closed with sutures.

Animals were euthanized using pentobarbital (i.p, 150 mg/kg body weight, Streuli Pharma AG) and perfused transcardially with Ringer solution (containing 5 ml/l Heparin, B. Braun) followed by paraformaldehyde (PFA, 4%, in 0.2 M phosphate buffer, pH 7). Brain tissue was collected and post-fixed for 6 h in 4% PFA. For cryoprotection, tissue was transferred to 30% sucrose and stored at 4°C. Coronal sections were cut at a thickness of 40 µm using a sliding microtome (Microm HM430, Leica), collected, and stored as free-floating sections in cryoprotectant solution at −20°C.

For immunostaining, brain sections were blocked with 5% normal donkey serum for 1 h at room temperature and incubated with primary antibodies (rabbit anti-GFAP 1:200, Dako, #GA524; goat anti-Iba1, 1:500 Wako, #011-27991; NeuroTrace™ 1:200, Thermo Fischer; mouse anti-NeuN Antibody 1:500, Merck, #MAB377; rabbit anti-Neurofilament 200 antibody 1:200, Merck, #N4142; guinea pig anti-Neurofilament L, 1:200, Synaptic Systems, rat anti-CD31 antibody 1:50, BD Biosciences, #MEC13.3; goat anti-CD13, 1:200; R&D Systems, #AF2335) overnight at 4°C. The next day, sections were incubated with corresponding secondary antibodies (1:500, Thermo Fischer Scientific). Nuclei were counterstained with DAPI (1:2,000 in 0.1 M PB, Sigma). Mounting was performed using Mowiol.

Vascular parameters (vessel area fraction, length, branching and nearest distance) were identified using an automated ImageJ script as previously described for brain and retinal vasculature (15, 16). Briefly, for total vessel area fraction, the area covered by anti-CD31 staining was quantified using ImageJ after applying a constant threshold. The vascular length was evaluated using the “skeleton length” tool and number of branches was calculated with the “analyse skeleton” tool. Results were normalized per mm² of brain tissue for vascular length and number of branches.

5-ethynyl-2’-deoxyuridine (EdU, 50 mg/kg body weight, ThermoFischer) was applied intraperitoneally on day 7 after stroke to label proliferating vascular endothelial cells. EdU incorporation was detected 21 days after stroke using the Click-it EdU Alexa Fluor 647 Imaging Kit (ThermoFischer) on 40 µm coronal sections.

Blood was collected through the tail-vein at baseline, 7- and 21-days post injury. Blood plasma cytokines levels (of IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, and TNF-α) were measured using the Proinflammatory Panel 1 Mouse kit, a chemiluminescence-based assays from Meso Scale Discovery (MSD, Gaithersburg, MD, USA) according to the manufacturer’s protocol. Analyses were done using a QuickPlex SQ 120 instrument (MSD) and DISCOVERY WORKBENCH^® 4.0 software.

Total RNA of stroked cortical tissue and the corresponding contralesional site was isolated using TRIzol extraction followed by the RNeasy RNA isolation kit (Qiagen) including a DNase treatment to digest residual genomic DNA. All samples had a RIN value of >8.5.

Library preparation, sequencing, read processing, read alignment, and read counting was performed at the Functional Genomics Center Zurich (FGCZ) core facility. For library preparation, the TruSeq stranded RNA kit (Illumina Inc.) was used according to the manufacturer’s protocol. The mRNA was purified by polyA selection, chemically fragmented and transcribed into cDNA before adapter ligation. Analysis of RNA sequencing data and gene set enrichment analysis was performed using EdgeR (17) and clusterProfiler 4.0 (18).

Transcriptomic raw data are available to readers via Gene Expression Omnibus with identifier GSE208605.

Animals were tested on the (1) runway (2), ladder rung test and (3) the rotarod test. All animals were tested 3 days before surgery to establish baseline performance and 3, 7, 14 and 21 days after stroke induction. Performance on the runway and the ladder rung was evaluated using a recently established deep learning-based protocol (19).

A runway walk was performed to assess whole body coordination during overground locomotion as described previously (18). Briefly, mice were recorded crossing a runway with a high-definition video camera (GoPro Hero 7) at a resolution of 4000 × 3000 and a rate of 60 frames per second from three perspectives. In a first step, the animals were acclimatized to the apparatus in daily training sessions until they crossed the runway at a constant speed and voluntarily (without external intervention). Then, each animal was placed individually on one end of the transparent plexiglas basin and was allowed to walk for 3 minutes.

The ladder rung test was performed using the same set-up as for the runway to assess skilled locomotion; with the only difference that the runway was replaced with a horizontal ladder on which the spacing of the rungs is variable. The animals were trained to cross the ladder without external reinforcement. A total of at least three runs per animal and per session were recorded. A step was defined as misstep when the toe tips of the animal reached a threshold of >0.5 cm below the ladder height.

The rotarod test is a standard sensory-motor test to investigate the animals’ ability to stay and run on an accelerated rod (Ugo Basile, Gemonio, Italy). All animals were pre-trained to stay on the accelerating rotarod (slowly increasing from 5 to 50 rpm in 300s) until they could remain on the rod for > 60 s. During the performance, the time and speed was measured until the animals fell or started to rotate with the rod without running. The test was always performed three times and means were used for statistical analysis. The recovery phase between the trials was at least 10 min.

Induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) were generated as previously described (20). In brief, NPCs at passage number < 11 were used in all experiments. Intraparenchymal cell transplantation was performed as previously described (21). In brief eGFP⁺/Luc⁺ NPCs were thawed, washed, and diluted to a final concentration of 8 × 10⁴ cells/μL in sterile 1x PBS (pH 7.4, without calcium or magnesium; Thermo Fisher Scientific). Cells were stored on ice until transplantation. Mice were anesthetized using isoflurane (5% induction, 1.5% maintenance: Attane, Provet AG). Analgesic (Rimadyl; Pfizer) was administered subcutaneously prior to surgery (5 mg/kg body weight). Animals were fixed in a stereotactic frame (David Kopf Instruments), the surgical area was sanitized, and the skull was exposed through a midline skin incision to reveal lambda and bregma points. Coordinates were calculated (the coordinates of interest chosen for this protocol were: AP: + 0.5, ML: + 1.5, DV: −0.8 relative to bregma) and a hole was drilled using a surgical dental drill (Foredom, Bethel CT) (20). Mice were injected with 1.6 × 10⁵ cells (2μL/injection) using a 10-μL Hamilton microsyringe (33-gauge) and a micropump system with a flow rate of 0.3 μL/min (injection) and 1.5 μl/min (withdrawal). The wound was closed with suture.

Bioluminescence imaging (BLI) experiments were performed with the IVIS Spectrum CT (PerkinElmer). Animals were imaged in regular intervals starting 2h after transplantation for up to 35 days. 30mg/ml D-luciferin potassium salt (PerkinElmer) was dissolved in 1x PBS (pH 7.4, without calcium or magnesium; Thermo Fisher Scientific) and sterilized through a 0.22 μm syringe filter. Luciferin was injected intraperitoneally with a final dose of 300 mg/kg body weight before isoflurane anaesthesia (5% induction, 1.5% maintenance; Attane, Provet AG). The head region was shaved before imaging. Image acquisition was performed for 20 min using the following settings: Field of View: A, Subject height: 1.5 cm, Binning: 16, F/Stop: 2. Exposure time (ranging from 1s to 60s) was set automatically by the system to reach the most sensitive setting. Imaging parameters and measurement procedures were kept consistent between mice.

The regions of interest (ROIs) were manually drawn based on anatomical landmarks (eyes, ears and snout) on each image. Plotting and statistical analysis was performed using RStudio. The brain-specific signal was calculated and corrected for nonspecific signal taken from a ROI on the skin of the animal’s back and for noise taken from a ROI outside the mouse. Signal-to-noise ratio (SNR) was calculated by dividing the total photon flux (ph/s/cm²/sr) by the standard deviation of the noise. Bioluminescent signal was calculated by subtracting the background flux from the total photon flux per ROI.

Statistical analysis was performed using RStudio (4.04 (2021-02-15). Sample sizes were designed with adequate power according to our previous studies (13, 22, 23) and to the literature (3, 24). All data were tested for normal distribution by using the Shapiro-Wilk test. Multiple comparisons (NSG, Rag2^-/-, WT-Tacr, WT) were initially tested for normal distribution with the Shapiro-Wilk test. The significance of mean differences between normally distributed multiple comparisons was assessed using repeated measures ANOVA with post-hoc analysis (p adjustment method = holm). Normally distributed data (WT-Tacr_acute vs. WT-Tacr_delayed) were tested for differences with a two-tailed unpaired one-sample t-test to compare changes between the two groups. Variables exhibiting a skewed distribution were transformed, using natural logarithms before the tests to satisfy the prerequisite assumptions of normality. Data are expressed as means ± SD, and statistical significance was defined as ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

To identify stroke-related changes in immunodeficient mouse models, we induced a photothrombotic stroke in the right sensorimotor cortex in (1) C57BL/6J wildtype (WT) mice (2) C57BL/6J WT mice that were continuously immunosuppressed with tacrolimus (WT-Tacr); as well as genetically immunodeficient (3) Rag2^-/- and (4) NSG mice ( Figure 1A ). A successful stroke was confirmed in all groups by ≈ 70% reduction of cerebral blood flow in the lesioned right hemisphere 24h after stroke induction (WT = −72%; WT-Tacr = −76%; Rag2^-/- = -69% NSG = −74%, all p > 0.5, Figures 1B, C ). Twenty-one days post injury (dpi), brain tissue was collected and revealed comparable stroke volumes and a near complete overlap (> 99%) of affected brain areas in mice from all groups (WT = 2.8 ± 0.7 mm; WT-Tacr = 2.7 ± 0.7 mm³; Rag2^-/- = 2.6 ± 0.4 mm³; NSG = 2.4 ± 0.3 mm³, all p = 1, Figures 1D, E ; Supplementary Figure 1 ). To identify stroke-related tissue responses such as inflammation, scarring and neuronal remodelling, we quantified the fluorescence intensities in the contralesional cortex as well as 300-µm cortical peri-infarct regions adjacent to the ipsilesional stroke core three weeks after injury. As expected, inflammatory as well as scarring signals were elevated in the ipsilesional compared to the contralesional cortex ( Figures 1F–H ). In the peri-infarct areas, microglial activation/macrophage infiltration (Iba1⁺) and glial scarring (GFAP⁺) signals were reduced in all immunosuppressed mouse models compared to WT controls (Iba1⁺: WT-Tacr: −84%, Rag2^-/-: −82%, NSG: −74%, all p < 0.001; GFAP⁺: WT-Tacr: −60%, p = 0.002; Rag2^-/-: −32%, p = 0.015, NSG: −55%; p = 0.002, Figures 1I, J ). On the unaffected contralesional side however, no significant differences were detected. The quantity of mature neurons indicated by NeuN⁺ expression was reduced in the lesioned regions in all groups by 50-60% but no changes have been observed in NeuN⁺ expression between the groups ( Figure 1K ). The expression of neurofilament light and heavy chain, major proteins of the neuronal cytoskeleton, was also altered after stroke ( Figure 1L ). Interestingly, we observed a significantly lower loss of Neurofilament H in WT-Tacr mice (WT-Tacr: +66%, p = 0.02) but no differences in the other immunosuppressed Rag2^-/- and NSG mice (Rag2^-/-: +39%, p = 0.37; NSG = +10%, p = 0.95) compared to WT control ( Figure 1M ). No changes have also been observed between the groups for Neurofilament L expression in the injured regions (WT-Tacr: +33%, Rag2^-/-: +23%, NSG: +2%, all p > 0.5, Figure 1M ).

Next, we asked if vascular remodelling after stroke may be altered in immunosuppressed mice, because inflammatory responses are known to affect angiogenesis (25). We quantified the vascular density and maturation as well as identified newly formed vessels in the contralesional and ipsilesional cortex three weeks after injury. The overall vascular density, number of branches and total length of the vascular network was increased in the peri-infarct areas in all immunodeficient groups compared to the WT control ( Figures 2A–C ). Most prominent, Rag2^-/- mice had an increase of +70% (p < 0.001) in vascular network density, +215% (p < 0.001) in the number of branches and +79% (p < 0.001) in the vascular length compared to WT mice. Importantly, no vascular differences between the groups were observed in the contralesional brain tissue (all p >0.05, Figure 2D ). To confirm that the blood vessels in the ischemic border zone were newly formed, the nucleotide analogue 5-ethynyl-2′-deoxyuridine (EdU) was systemically applied at the peak of poststroke angiogenesis at day 7 ( Figures 2E–G ). NSG mice had an increase in EdU⁺ blood vessels per mm² in the ischemic border zone compared to WT controls (NSG: 109.4 ± 32.4 mm^-2; WT: 39.2 ± 46.9 mm^-2; p = 0.03, Figure 2G ). As newly formed blood vessels after stroke may be associated with immature vessels that lack stabilizing pericytes (CD13⁺) we investigated whether CD31 and CD13 markers were colocalized ( Figures 2H, I ). Pericyte coverage in the ipsilesional site was ≈ 70% lower compared to the contralesional site in all groups but was comparable between the groups in the peri-infarct areas as indicated by the ratio of pericyte covered vasculature (CD13⁺CD31⁺) to total vasculature (CD31⁺) (WT: 0.36 ± 0.15, WT-Tacr: 0.15 ± 0.14, Rag2^-/-: 0.23 ± 0.12, NSG = 0.23 ± 0.1; all p > 0.4, Figure 2J ).

To test if also systemic inflammatory responses were altered following stroke, we measured serum cytokine levels at baseline, 3 and 21 days after stroke using MSD multiplex assays ( Figure 2K ). We identified that all immunosuppressed mice showed a changed cytokine signature compared to WT mice. The strongest deviation from WT was present in cytokine levels of NSG mice, particularly in the reduced levels of IFNy, IL-2, IL-5 and increased Il-1b, Il-4 ( Figure 2K ).

Large damage of the sensorimotor cortex by photothrombotic stroke causes long-term impairment in fore and hindlimb function and locomotion ^13,23. To test whether the anatomical and systemic changes following immunosuppression may affect functional recovery, motor performance and gait analysis was performed at baseline and at repeated intervals after stroke for 21 days ( Figure 3A ).

We determined in all groups, except of NSG mice, an acute deficit in the rotarod performance indicated by a ≈ 40% shorter time spent on the rod acutely after stroke (3dpi to bl: WT: −48%, p = 0.020, WT-Tacr: −35%, p = 0.048; Rag2^-/- = −43%, p = 0.037; NSG = −22%, p = 0.549) with a gradual recovery ( Figure 3B ). NSG mice had also a striking higher intra-group variability between individual animals in the rotarod task ( Figure 3B ).

Next, we applied a recently established deep learning algorithm to videos of mice during a voluntary run to identify specific gait changes after stroke (19, 26). We confirmed that the body parts of interest including tail base, iliac crest, hip, back ankles, back toe, shoulder, wrist, elbow, front toe, and head could be reliably detected from three perspectives ( Figures 3C, D ). The ratio of confident labels (>95% likelihood of detection) to total labels ranged from 93-99% ( Figure 3E ). Data points that did not pass the likelihood of detection of 95% were excluded. We confirmed that previously identified key parameters (19) were altered in all groups of animals after stroke. For instance, an asymmetric stride pattern describing the non-simultaneous placement of opposed front and back toes, was observed acutely after stroke in all groups (3dpi: all p < 0.05) ( Figure 3F ). The asymmetric stride pattern reversed to baseline with time in all groups ( Figure 3F ). Also, we detected comparable irreversible gait changes in all groups such as a reduced range of motion in the contralesional left front paw (3dpi to bl: WT: −1.2 ± 1.4 mm, WT-Tacr: −0.9 ± 0.5 mm, Rag2^-/-: −0.8 ± 0.6 mm, NSG: −0.7 ± 0.3 mm, all p < 0.05) that gradually recovered but did not reach baseline levels throughout the time course (21dpi to bl: WT: −1.0 ± 0.8 mm, WT-Tacr: −0.5 ± 0.3 mm, Rag2^-/-: −0.5 ± 0.4 mm, NSG: −0.4± 0.3 mm, all p < 0.05, Figure 3G ).

To detect fine motor deficits, we additionally performed a deep learning assisted irregular ladder rung test and quantified the ratio of stepping errors (19, 27). We achieved a high ratio of confident labels of 92-93% ( Figure 3H ) and excluded all data points that did not pass the confidence threshold. Errors were defined if the front or back toe dropped lower than the ladder height due to a misstep or a slip ( Figure 3I ). All groups of mice showed acutely increased error rates that were most pronounced in the contralateral left back (3dpi: WT: +20%, WT-Tacr: +20%, Rag2^-/-: +18%, NSG: +50%, all p < 0.05) and left front toe (3dpi: WT: +20%, WT-Tacr: +10%, Rag2^-/-: +18%, NSG: +18%, all p < 0.05) compared to baseline misstep rates ( Figure 3J ). NSG mice tend to have a higher overall error rate, most prominent in the left back toe after injury ( Figure 3J ).

In sum, the motor performance and gait alterations between immunosuppressed and WT mice were largely comparable. NSG mice deviated in few behavioral readouts from the functional deficits in WT mice. However, the data indicates that the observed anatomical and systemic changes did not lead to major changes in functional recovery between the groups in a time course of three weeks.

To further dissect molecular changes in the ischemic brain, we performed RNA sequencing of ischemic brain tissue from immunodeficient and WT mice ( Figure 4A ). Three weeks after stroke, the majority of the significantly altered genes were upregulated in all groups of mice ( Figure 4B ). Principal component analysis and a heatmap of the most differentially expressed genes demonstrate a clear separation between naïve mice from their stroked littermates ( Figures 4B, C ). As expected, we also observed a strong separation between non-stroked and stroked NSG mice to the other groups presumably because of their different background (NSG mice have a NOD-background; all other groups of mice had a C57Bl/J background, Supplementary Figures 2A, B ). Most enriched gene sets between non-stroked NSG and WT mice affected pathways related to the adaptive immune system ( Supplementary Figure 3 ).

After stroke, there was an overlap of 32% of the top₁₀₀₀ upregulated genes but only 3% of the top₁₀₀₀ downregulated genes in all groups of mice compared to their respective non-stroke controls ( Figure 4D ). WT-Tacr mice showed the closest gene expression profile to WT; and NSG mice deviated most from WT gene expression after stroke. Still, many of the top₃₀ upregulated genes were present in all groups of animals such as Mmp12, CD5l, Mmp3, C3, Apoc4, Tgm1, Clec7a, Lcn2 and Lilr4b. ( Figure 4E ). However, no top₃₀ downregulated genes were present in all groups ( Supplementary Figure 4 ).

The majority of the enriched pathways in all groups after stroke were related to the innate and adaptive immune system e.g., leukocyte migration, positive regulation of cytokine production, and regulation of immune effector process ( Figures 5A, B ). While 80% of the top 10 enriched pathways after stroke overlapped between WT-Tacr, Rag2^-/- and WT, only 40% of pathways were identical between NSG and WT ( Figures 5A, B ).

Next, we analysed gene expression differences among the immunosuppressed stroked groups that were normalized for their respective non-stroked genotype to WT stroked mice ( Figure 6A ). The majority of differentially expressed genes in the different immunosuppressed stroked mice were unique ( Figure 6B ). As expected, the enriched pathways were mostly associated with inflammatory responses such as, innate immune response, regulation of cytokine production and lymphocyte activation ( Supplementary Figures 5A–C ).

Interestingly, a second cluster of pathways that was enriched in immunosuppressed stroked mice compared to WT stroked mice was linked to angiogenic responses ( Figures 6C, D ). Surprisingly, these pathways were highly positively enriched in NSG and Rag2^-/- mice ( Figure 6D ) but negatively enriched in WT-Tacr mice at 3 weeks after injury ( Figure 6D ), indicating a reduced angiogenic response at 21 dpi in tacrolimus immunosuppressed mice.

In sum, we observed distinct gene expression and pathway enrichment changes between the immunosuppressed and WT mice three weeks after stroke. The strongest deviation was observed in NSG mice. Apart from inflammatory responses, angiogenesis related genes were most significantly altered.

Transient angiogenic responses have been previously reported after stroke (28) and may explain the enhanced vascular repair mediated by tacrolimus with concomitant downregulation of angiogenesis related genes and pathways three weeks after stroke. We therefore asked if the anatomical and gene expression differences in the stroke pathology between tacrolimus treated and WT mice could be reduced by delaying the tacrolimus administration by one week. This time window was chosen to mimic a clinically relevant scenario. In most studies, cell transplantation usually happens around one week after injury to avoid the very acute and hostile phase; and immunosuppression will be applied shortly before delivery (29). To test this hypothesis, we induced a photothrombotic stroke in (1) WT (2), acute (0 dpi) tacrolimus immunosuppressed mice (WT-Tacr_acute) and (3) delayed (7 dpi) tacrolimus immunosuppressed mice (WT-Tacr_delayed, Figure 7A ).

First, we performed brain tissue analysis of inflammatory and scarring responses at 21 dpi. ( Figure 7B ). Histological analysis in the peri-infarct region revealed an increased glial scar signal (GFAP⁺) in WT-Tacr_delayed (+21% to WT) compared to WT-Tacr_acute (−58% to WT) mice (p = 0.025, Figure 7C ). Inflammatory Iba1⁺ levels were also amplified in WT-Tacr_delayed (−25% to WT) compared to WT-Tacr_acute (−60% to WT) mice (p = 0.04, Figure 7D ). The number of newly formed Iba1⁺ cells (Iba1⁺EdU⁺) around the stroke was increased between Tacr_delayed (+1% to WT) compared to WT-Tacr_acute (−59% to WT) mice (p = 0.001, Figure 7E ), indicating overall that post-stroke scarring and inflammation in WT-Tacr_delayed mice is closer to WT tissue responses.

No differences have been observed in the stroke volumes between the groups (WT-Tacr_acute = 2.85 ± 0.62 mm³, WT-Tacr_delayed = 3.31 ± 0.61 mm³, WT = 3.30 ± 0.71 mm³, p >0.05, Figure 7F ). Additionally, delayed tacrolimus administration reduced the vascular density (−35%, p = 0.048) and number of branches (−60%, p = 0.032) but did not influence the number of newly formed blood vessels compared to WT-Tacr_acute (CD31⁺EDU⁺: WT-Tacr_acute = 56.3 ± 3.2 cells/mm², WT-Tacr_delayed = 59.3± 8.1 cells/mm², p > 0.05, Figures 7G–I ). The ratio of pericyte coverage of total vasculature remained unchanged in the peri-infarct region between the groups (WT-Tacr_acute = 0.25 ± 0.15, WT-Tacr_delayed = 0.29 ± 0.08, p > 0.05, Figures 7J ).

Interestingly, gene expression analysis of ischemic brain tissue reveals an increased gene set enrichment for cytokine production and pro-angiogenic responses in WT-Tacr_delayed mice ( Figures 7K–M , Supplementary Figure 6 ). This may indicate that the increased angiogenic responses in WT-Tacr_acute mice may be transient and/or that delayed immunosuppression shifts the angiogenic response towards a later timepoint after stroke.

These data indicate that a 7-day delayed pharmacological immunosuppression with tacrolimus shifts the course of stroke more towards the WT pathology.

Next, we evaluated long-term survival of intracerebrally transplanted grafts in (1) WT mice (2) WT mice that were continuously immunosuppressed with tacrolimus (WT-Tacr); and genetically immunodeficient (3) Rag2^-/- and (4) NSG mice ( Figure 8A ). As a cell source, we used recently generated human neural progenitor cells (NPCs) derived from induced pluripotent stem cells (iPSCs) (20). The NPCs were transduced with a dual reporter consisting of a bioluminescent firefly luciferase (rFluc) and a fluorescent eGFP. A successful transplantation in all groups of mice was confirmed by comparable levels of luciferase signal directly after transplantation ( Figures 8B, C ). As expected, we observed the absence of the graft in WT mice 14 days after transplantation presumably due to xenogenic immune rejection. In contrast, the immunosuppressed mice showed all a prolonged graft survival. However, only in Rag2^-/- and NSG mice, we observed a strong bioluminescence signal during the entire time course of more than 1 month after transplantation ( Figures 8B, C ). After 35 days, surviving graft cells were identified using human-specific antibodies in brain sections of NSG and Rag2^-/- mice but were not detectable in WT and WT-Tacr mice ( Figure 8D ).

These data suggests that genetic immunosuppression is more reliable for graft survival, especially in long-term studies.