Intracortical transplantation of human induced pluripotent stem cell-derived progenitors ameliorates delayed thalamic degeneration following cortical stroke

Intracortical transplantation of human induced pluripotent stem cell-derived progenitors ameliorates delayed thalamic degeneration following cortical stroke | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Intracortical transplantation of human induced pluripotent stem cell-derived progenitors ameliorates delayed thalamic degeneration following cortical stroke View ORCID Profile Sopiko Kartsivadze , Linda Jansson , Lucía Galán-Pintado , Keshav Kher , Sara Avilés , View ORCID Profile Raquel Martinez-Curiel , View ORCID Profile Olle Lindvall , View ORCID Profile Sara Palma-Tortosa , View ORCID Profile Zaal Kokaia doi: https://doi.org/10.1101/2025.03.17.643730 Sopiko Kartsivadze 1 Laboratory of Stem Cells and Restorative Neurology, Lund Stem Cell Center, Lund University , 22184 Lund, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sopiko Kartsivadze Linda Jansson 1 Laboratory of Stem Cells and Restorative Neurology, Lund Stem Cell Center, Lund University , 22184 Lund, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lucía Galán-Pintado 1 Laboratory of Stem Cells and Restorative Neurology, Lund Stem Cell Center, Lund University , 22184 Lund, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site Keshav Kher 1 Laboratory of Stem Cells and Restorative Neurology, Lund Stem Cell Center, Lund University , 22184 Lund, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sara Avilés 1 Laboratory of Stem Cells and Restorative Neurology, Lund Stem Cell Center, Lund University , 22184 Lund, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site Raquel Martinez-Curiel 1 Laboratory of Stem Cells and Restorative Neurology, Lund Stem Cell Center, Lund University , 22184 Lund, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Raquel Martinez-Curiel Olle Lindvall 1 Laboratory of Stem Cells and Restorative Neurology, Lund Stem Cell Center, Lund University , 22184 Lund, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Olle Lindvall Sara Palma-Tortosa 1 Laboratory of Stem Cells and Restorative Neurology, Lund Stem Cell Center, Lund University , 22184 Lund, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sara Palma-Tortosa Zaal Kokaia 1 Laboratory of Stem Cells and Restorative Neurology, Lund Stem Cell Center, Lund University , 22184 Lund, Sweden Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Zaal Kokaia For correspondence: zaal.kokaia{at}med.lu.se Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Ischemic stroke, a leading cause of death and disability worldwide, frequently results in cortical damage. Due to disrupted neural connectivity, cortical lesions often trigger secondary neurodegeneration in remote brain regions, such as the thalamus. This secondary thalamic injury exacerbates neurological deficits, leading to long-term sensory, motor, and cognitive impairments. Although animal models have demonstrated that secondary thalamic damage is driven by retrograde degeneration, excitotoxicity, apoptosis, blood-brain barrier disruption, and neuroinflammation, the mechanisms linking cortical stroke to thalamic degeneration remain poorly understood. In this study, we investigated the dynamics of secondary thalamic injury in a rat model of cortical stroke. We evaluated the therapeutic potential of intracortical transplantation of human induced pluripotent stem cell (iPSC)-derived neuronal progenitors. Cortical ischemic stroke was induced via distal middle cerebral artery occlusion, and animals were assessed at multiple time points post-stroke. We observed stable cortical infarcts by 2 weeks, followed by progressive thalamic degeneration, particularly in the ventral posterior nucleus (VPN), which began at 3 months and persisted up to 6 months. Neuronal loss in the VPN correlated with the size of the cortical lesion, and microglial activation in the thalamus peaked at 2-4 months, suggesting a role for neuroinflammation in secondary degeneration. Intracortical transplantation of cortically primed iPSC-derived progenitors 48 hours post-stroke did not alter the cortical infarct volume but significantly reduced thalamic neuronal loss. Grafted cells integrated into the host tissue and presumably established functional connections, potentially mitigating secondary thalamic injury. These findings highlight the therapeutic potential of stem cell-based interventions to prevent secondary neurodegeneration and improve long-term outcomes after cortical stroke. This study provides critical insights into the mechanisms of secondary thalamic injury and demonstrates the feasibility of intracortical transplantation as a strategy to enhance post-stroke recovery. INTRODUCTION Stroke is the second-leading cause of death and the third-leading cause of death and disability combined globally ( Feigin, 2021 ). Ischemic stroke is the most common type, representing over 60% of all stroke cases worldwide ( Pu et al ., 2023 ). During an ischemic stroke, the interruption of cerebral blood flow results in an infarct in the affected brain region. Most often, ischemic stroke causes damage to the cerebral cortex. Estimates suggest that cortical involvement occurs in approximately 60-70% of the cases. The middle cerebral artery (MCA) is the most frequently involved artery in acute stroke. The cortical branches of the MCA supply blood to the primary motor and somatosensory cortical areas. About 50% to 80% of stroke survivors experience somatosensory deficits, closely linked to the specific location of the brain lesion ( Kessner et al ., 2019 ). Cortical lesions often lead to secondary neurodegeneration in remote brain regions due to disrupted neural connectivity. These secondary injuries can significantly impede long-term recovery processes ( Seitz et al ., 1999 ). The somatosensory cortex has dense reciprocal connections with the thalamus, and when an ischemic lesion occurs in this cortical area, it frequently triggers neurodegeneration in the thalamus. This phenomenon is referred to as secondary thalamic injury. Such damage worsens neurological deficits, contributing to long-term sensory, motor, and cognitive impairments. Secondary thalamic injury has been observed in several animal models of stroke involving cortical lesions, including transient ( Cao et al ., 2017 ) and permanent ( Iizuka et al ., 1990 ) occlusion of MCA and photothrombotic stroke ( Jones et al ., 2018 ). The studies have demonstrated that secondary neuronal damage in the thalamus can be influenced by the location and size of the infarct. This damage is primarily attributed to retrograde degeneration, a process in which neuronal destruction spreads backward along the axon after axonal injury ( Fujie et al ., 1990 ; Iizuka et al ., 1990 ). Other mechanisms contributing to the secondary thalamic lesion, such as excitotoxicity ( Ross & Ebner, 1990 ; Paz et al ., 2010 ), apoptotic cell death ( Fujie et al ., 1990 ; Liang et al ., 2007 ), amyloid-beta (Aβ) accumulation, blood-brain barrier (BBB) disruption ( Yu et al ., 2017 ), and neuroinflammation ( Schroeter et al ., 2006 ; Holmberg et al ., 2009 ), have also been identified. It was reported that delayed secondary degeneration in the ventral posterior nucleus (VPN) of the ipsilateral thalamus following distal branch MCA occlusion in rats can be alleviated by inhibition of cathepsin B ( Zuo et al ., 2016 ). Functional behavioral tests indicate that improved long-term behavioral performance is associated with reduced secondary thalamic damage following stroke ( Villa et al ., 2007 ; Cao et al ., 2017 ). However, the precise mechanisms by which secondary thalamic injury influences long-term functional recovery and stroke outcomes remain incompletely understood. In humans, clinical studies using imaging techniques have demonstrated that cortical infarction triggers reduced cerebral blood flow, oxygen, and glucose metabolism in the ipsilateral thalamus ( Nagasawa & Kogure, 1990 ; De Reuck et al ., 1995 ; Ogawa et al ., 1997 ; Reidler et al ., 2018 ). A key unresolved question is whether these changes in the thalamus represent purely functional alterations resulting from either early functional disconnection with cortical areas or indicate neuronal degeneration. Furthermore, magnetic resonance imaging (MRI) studies in patients with cerebral infarction sparing the thalamus have identified alterations in thalamic signal intensity and diffusion properties on the side ipsilateral to the infarct. Recently, Geng et al. provided additional MRI-based evidence for secondary remote thalamic atrophy post-ischemic stroke and suggested that the effect of infarction on ipsilateral thalamic volume is associated with global post-stroke cognitive impairment ( Geng et al ., 2023 ). Understanding the cascade of secondary thalamic injury is critical for developing strategies to decrease its impact and potentially improve patient outcomes. In this study, we aimed to characterize the dynamics of cellular events involved in secondary thalamic injury using a rat model of cortical stroke and to test the hypothesis that intracortical transplantation of neuronal progenitors can reduce thalamic lesions. MATERIALS AND METHODS lt-NES cell line maintenance and cortical priming Human iPS cell-derived lt-NES cells were generated from skin fibroblasts as previously described (Grønning Hansen et al ., 2020 ) and expanded on plates coated with 0.1 mg/mL poly-L-ornithine and 10 mg/mL laminin (both from Sigma) in proliferation media (DMEM-F12 without Hepes + Glutamine, N2 at 1:100, and Glucose at 1.6 g/l), supplemented with bFGF (10 ng/mL, Peprotech), EGF (10 ng/mL, Peprotech) and B27 (1:1000, Invitrogen. The cells were passaged at a ratio of 1:2 to 1:3 every second to third day using trypsin (Sigma). For cortical priming, the growth factors (FGF, EGF) and B27 were omitted, and the cells were cultured at low density in differentiation-defined medium with bone morphogenetic protein 4 (BMP4) (10 ng/mL, R&D Systems), wingless-type MMTV integration site family, member 3A (Wnt3A) (10 ng/mL, R&D Systems), and cyclopamine (1 μM, Calbiochem) for 4 days. Animals Twenty-two Sprague Dawley rats (251-275 g) and 15 Nude Rats (Crl:NIH-Foxn1 RNU, 8 weeks old) were obtained from Charles River Laboratories. The animals were housed in individually ventilated cages under standard temperature and humidity conditions, along with a 12-hour light/dark cycle and unrestricted access to food and water. All procedures were conducted in accordance with the European Union Directive 2010/63/EU and received approval from the ethical committee for laboratory animal use at Lund University and the Swedish Department of Agriculture. Distal middle cerebral artery occlusion The focal ischemic injury was induced in 12-13 weeks-old rats in the cerebral cortex by distal MCA occlusion (dMCAO) as described by Chen et al. ( Chen et al ., 1986 ) with some modifications. Briefly, the animals were anesthetized with isoflurane mixed with air, and the temporal bone was exposed. A craniotomy of 3 mm was performed, the dura mater was carefully opened, and the cortical branch of the middle cerebral artery was permanently ligated with a suture, cauterized, and cut. Both common carotid arteries were isolated and ligated for 30 to 120 minutes. The surgical wounds were closed, and the animals were allowed to wake up during the ischemic period. Sham animals underwent the same surgery but without ligation of the MCA. Intracortical transplantation Intracortical transplantation of cortically fated lt-NES cell-derived progenitors, transduced with lentivirus carrying GFP reporter, was performed stereotaxically 48 h after dMCAO under anesthesia, as previously described ( Palma-Tortosa et al ., 2020 ). Prior to the surgery, the cortically primed cells at day 4 of differentiation were resuspended in citocon buffer at a final concentration of 100,000 cells/µL. A volume of 1 μL was injected into 2 deposits at the following coordinates (relative to bregma and brain surface): Site 1: Anterior/posterior: +1.5 mm, medial/lateral: 1.5 mm, dorsal/ventral: −2.0 mm; Site 2: Anterior/posterior: +0.5 mm, medial/lateral: 1.5 mm, dorsal/ventral: −2.5 mm. Tissue processing and immunohistochemistry Animals were sacrificed at different time points after stroke surgery by injection of pentobarbital followed by transcardial perfusion of saline and 4% paraformaldehyde (PFA) solution. Brains were extracted and left in PFA for post-fixation for 24 h, then transferred to 20% sucrose solution until saturated. Brains were cut coronally (30 μm) on a microtome (Leica SM 2000 R). Sections were stored in an anti-freezer solution until further use. Free-floating coronal sections were preincubated in a blocking solution containing 5% normal donkey serum (NDS) and 0.25% Triton X-100 in 0.1 M phosphate-buffered saline (PBS). Primary antibodies (chicken anti-GFP (Merck Millipore, 1:1000), rabbit anti-NeuN (Abcam, 1:500), and goat anti-Iba1 (Biolegend, 1:200)) were diluted in blocking solution and applied overnight at 4°C. The following day, sections were incubated with fluorophore-conjugated secondary antibodies (donkey anti-chicken A488, donkey anti-rabbit A488, or donkey anti-goat Cy3, all from Jackson Immunoresearch) for 2 h at room temperature. Nuclei were counterstained with DAPI (Sigma) for 10 minutes before mounting on gelatin-coated slides using Dabco (Sigma). Fluoro-Jade C staining Sections were mounted in coated slides, and the staining protocol was followed according to the instructions of the Fluoro-Jade C (FJC) staining kit (Biosensis). Briefly, tissue was treated with 9 parts 80% ethanol and 1 part Solution A (sodium hydroxide), submerged in a 50 mL tube for 5 min, then sequentially transferred to 70% ethanol for 2 min and distilled water for 2 min. Slides were incubated in 9 parts distilled water and 1 part Solution B (potassium permanganate) for 5 min, optimizing the initial 10 min incubation to reduce autofluorescence and enhance contrast. To improve fluorescence intensity, a staining solution of 7 parts distilled water, 2 parts Solution C (Fluoro-Jade C), and 1 part DAPI was prepared. Slides were incubated in the dark for 10 min, followed by three rinses in distilled water (1 min each) and a drying step at 50–60°C for 5 min. Slides were cleared in xylene for 3 min in the dark and coverslipped using a non-aqueous mounting medium such as DPX. Fluoro-Jade C-labeled degenerating cells were visualized under blue light excitation, with an excitation peak of 485–506 nm and an emission peak of 525–527 nm. Imaging An overview image of rat brain slices was taken using the extended focal imaging (EFI) fluorescence mode at a 20X magnification in a Virtual Slide Scanning System (VS-120-S6-W, Olympus, Germany). High-magnification images were taken using a confocal microscope (LSM780, Zeiss, Germany) and a 20X or a 40X immersion objective. Quantifications Infarct volume was quantified using NeuN-immunostained sections. The intact area, identified by NeuN+ cells in both the ipsilateral and contralateral hemispheres, was delineated and measured using ImageJ software. The infarcted area was determined by subtracting the non-lesioned (NeuN-stained) area in the affected hemisphere from that in the corresponding contralateral hemisphere. The lesion volume was then calculated by multiplying the infarcted area by the section thickness and the spacing between sections (300 μm). NeuN+ cell quantification was conducted using a deep learning-based application within Visiopharm software (version 3.5.1). The application identifies neurons stained with the NeuN antibody in the FITC channel within user-defined regions of interest (ROIs). It calculates both the quantity of NeuN+ cells and the total area within each ROI. Using a trained model, AI-driven image analysis was applied to distinguish target cells from the background. Post-processing steps - including background removal, object dilation to resolve fragmentation, and object separation - were implemented to enhance accuracy. For each animal, ROIs were delineated to distinguish between the ipsilateral and contralateral regions. Statistical analysis Statistical analysis was performed using Prism 10 software (Version 10.4.1, GraphPad). One-way ANOVA, followed by Tukey’s multiple comparisons test, was used to analyze a single variable across different time points. Two-way ANOVA followed by Sidak’s multiple comparisons test was applied when comparing two variables. Simple linear regression analysis was used to compare infarct volume with neuronal loss or affected area. Significance was set at p < 0.05. Data are presented as mean ± SD. RESULTS 1. Cortical ischemic stroke causes early and stable lesion in somatosensory cortex followed by progressive secondary thalamic injury To elucidate the timeline of lesion development in both the primary and secondary affected areas (cortex and thalamus, respectively), animals were sacrificed at different time points after stroke: 2 weeks, 3, 4, and 6 months. Sham animals, subjected to surgery but without distal MCA occlusion, were used as controls. To evaluate the cortical lesion caused by stroke, we quantified the total volume of the infarcted area using the neuronal marker NeuN ( Figure 1A-B ). We observed that the cortical damage was clearly detectable as early as 2 weeks after the insult, with no significant changes in infarct volume at later time points ( Figure 1C ). Download figure Open in new tab Figure 1. Temporal progression of the primary cortical and secondary thalamic lesions in a rat model of cortical ischemic stroke. (A) Schematic representation of the location of the cortical lesion following middle cerebral artery occlusion (dMCAO) in rats. (B) Representative brightfield images of the neuronal nuclei (NeuN)-DAB immunostaining in rat brain slices at different time points post-stroke. Scale bar, 2 mm. (C) Quantification of the infarct volume over time. (D) Schematic and overview of NeuN immunofluorescence in a caudal rat brain slice containing the ventral posterolateral nuclei (VPN). Scale bar, 2 mm. (E) Representative immunofluorescence images of NeuN staining in rat brain slices at different time points post-stroke. Ipsilateral and contralateral VPN are outlined in white. Scale bar, 500 μm. (F) Quantification of the percentage of reduction in VPN area calculated as (Area VPN ipsilateral/ Area VPN contralateral) x 100, at different time points. (G-J) Correlation between relative VPN area and cortical infarct volume at 2 weeks (G), 3 months (H), 4 months (I), and 6 months (J) post-stroke. N=5-6 number of animals per time point. *p<0.05. Data are shown as mean ± SD. To analyze the thalamic lesion, we measured the size of the VPN, a thalamic area with massive projections to the somatosensory cortex, at different times post-stroke. We observed no differences in the VPN area between contralateral and ipsilateral hemispheres in sham animals. Importantly, the contralateral VPN area of stroke-subjected animals also did not differ from their time-matching shams (Supplementary Figure 1), indicating that the effect of the cortical stroke on VPN is an ipsilateral event. Based on these findings, in all subsequent analyses, the contralateral VPN area served as control in each stroke-subjected rat, allowing us to account for variability among individual animals. Two weeks after the stroke, no changes in the ipsilateral VPN area of stroke animals were observed compared to sham animals. However, after 3 months, the ipsilateral VPN area had decreased and then remained the same size up to the latest time point examined, 6 months after the stroke ( Figure 1D-F ). Notably, from 3 months onwards, the extent of the thalamic damage, measured as the relative decrease in the ipsilateral VPN area, significantly correlated with the size of the cortical lesion ( Figure 1G-J ). 2. Neuronal loss is the primary contributor to the thalamic damage The degeneration of various brain cell types is the ultimate consequence of a series of events initiated by the brain injury, such as an ischemic stroke. To investigate tissue degeneration in both the cortex and VPN, we performed Fluoro-Jade C staining at 2 time points: 2 weeks and 4 months post-stroke. No degeneration was detected in sham animals or in the contralateral hemisphere of stroke-affected rats ( Figure 2A-B ). However, significant degeneration was observed in both the stroke-affected cortex and ipsilateral VPN at both time points ( Figure 2B ). Download figure Open in new tab Figure 2. Tissue degeneration in rats’ somatosensory cortex and the ventral posterior nuclei (VPN) 2 weeks and 4 months after a cortical ischemic stroke. (A-B) Representative image of Fluoro-Jade C (FJ) labeling in the (A) contralateral and (B) ipsilateral cortex (Ctx, Scale bar, 500 μm) and VPN (Scale bar, 50 μm). Neurons are the most vulnerable cells to ischemic and other insults causing brain tissue damage. We explored the role of neuronal loss as a contributor to the secondary thalamic lesion after cortical ischemic stroke. We observed no significant changes in the number of surviving neurons (NeuN+) cells in sham animals or in the contralateral hemisphere of stroke-affected rats (Supplementary Figure 1C). Therefore, we counted the numbers of NeuN+ cells in the ipsilateral VPN and compared them with respective non-lesioned structures. Data were presented as relative neuronal loss (percentage, using contralateral VPN as internal control). Although no differences were observed 2 weeks after the stroke, a significant neuronal loss in the VPN was detected after 3 months, which remained stable at later time points ( Figure 3A-C ). In line with the correlations observed in the analysis of VPN size, the loss of neurons in the ipsilateral VPN negatively correlated with the size of the cortical lesion starting 3 months after the onset of the stroke ( Figure 3D-G ). Download figure Open in new tab Figure 3. Progression of neuronal loss in the ventral posterior nuclei (VPN) following a cortical ischemic stroke in rats. (A) Schematic representation of the area where representative images for the neuronal nuclei (NeuN) were captured. Scale bar: 2 mm. (B) Representative immunofluorescence images of the NeuN+ cells in the ipsilateral and contralateral VPN at various time points after the stroke. Scale bar: 50 μm. (C) Quantification of the relative loss of NeuN+ cells ((NeuN+ cells in ipsilateral VPN / NeuN+ cells in contralateral VPN) x 100) in the VPN over time. (D-G) Correlation analysis of the relative number of NeuN+ cells in the VPN and the infarct volume at 2 weeks (D), 3 months (E), 4 months (F), and 6 months (G) post-stroke. N=5-6 animals per time point. *p<0.05. Data are presented as mean ± SD. 3. Cortical ischemic stroke induces microglial activation in the ipsilateral VPN Microglia activation is part of the neuroinflammatory response of the brain’s innate immune system to any type of lesion. It extends beyond the primary injury site and involves functionally connected regions (secondary inflammation). However, the timeline for this event after cortical stroke is still not clear. Here, we studied the interaction between activated microglia and neuronal loss, specifically in the ipsilateral and contralateral VPNs ( Figure 4A-B ). Download figure Open in new tab Figure 4. Time course of microglial activation in the ipsilateral ventral posterior nuclei (VPN) of a rat model of cortical ischemic stroke. (A) Schematic representation indicating where representative images of the microglial marker Iba1 were taken. Scale bar, 2 mm. (B) Representative immunofluorescence images displaying Iba1 and neuronal nuclei (NeuN) staining in the ipsilateral and contralateral VPN at various time points post-stroke. Scale bar, 50 μm. (C) High magnification images of Iba1 and NeuN at 2 weeks, where both markers were in close proximity, and at 4 months post-stroke, where colocalizations were observed. Scale bar, 20 μm. (D) Orthogonal view illustrating the colocalization of NeuN and Iba1 at 4 months post-stroke. Scale bar, 20 μm. The number of Iba1+ microglial cells in the ipsilateral VPN increased from 2 weeks post-stroke and remained elevated until 4 months. However, by 6 months post-stroke, microglial density had declined to levels comparable to those observed in sham animals ( Figure 4B ). At earlier time points (2 weeks post-stroke), Iba1+ microglia were frequently observed close to intact NeuN+ neurons, suggesting early microglial-neuronal interactions. At 3-and 4 months post-stroke, colocalization of Iba1 and NeuN staining in the same cell became evident, potentially depicting ongoing phagocytosis of damaged neurons ( Figure 4B-D ). Interestingly, at 6 months post-stroke, no colocalization between NeuN and Iba1 was detected ( Figure 4B ), which might reflect the resolution of the inflammatory response and the complete clearance of degenerated neurons ( Figure 4B ). No changes in the number or appearance of Iba1+ cells were observed in the contralateral VPN at any examined time point ( Figure 4B ). 4. Intracortical transplantation of iPS cell-derived progenitors rescues the stroke-induced neuronal loss in VPN To be able to transplant human-derived cells into rat brains (xenotransplantation) and avoid immune rejection, nude athymic rats were subjected to distal MCA occlusion to induce damage in the somatosensory cortex. Forty-eight hours later, cortically primed human lt-NES cell-derived progenitors were transplanted in close proximity to the cortical injury. Six months later, animals were sacrificed and processed for immunohistochemistry. We first analyzed whether the intracortical transplantation of human lt-NES cell-derived progenitors influenced the extent of cortical damage. No differences were observed in the infarct volume between transplanted and non-transplanted animals ( Figure 5A ), consistent with our previous findings ( Tornero et al ., 2017 ). Download figure Open in new tab Figure 5. Early intracortical transplantation of human long-term neuroepithelial stem (lt-NES) cell-derived progenitors inhibits secondary neuronal degeneration in the ipsilateral ventral posterior nuclei (VPN) of the thalamus in a rat model of cortical ischemic stroke. (A) Quantification of infarct volume 6 months post-stroke in transplanted and non-transplanted rats. Representative brightfield images of NeuN-DAB immunostaining in rat brain slices. Scale bar, 2 mm. (B) Overview of graft location in rostral and caudal brain slices. Lt-NES cells are labeled with GFP for visualization. Scale bar: 2 mm (low magnification), 500 μm (high magnification). (C) Quantification of ipsilateral and contralateral VPN area in sham, stroke, and transplanted rats. (D) The percentage of reduction in VPN area is calculated as (Ipsilateral VPN Area / Contralateral VPN Area) × 100. (E) Representative immunofluorescence images of NeuN staining in brain slices of sham, stroke, and transplanted rats. Ipsilateral and contralateral VPN are outlined in white. Scale bar, 500 μm. (F) Quantification of NeuN+ cells in the ipsilateral and contralateral VPN. (G) Quantification of the relative loss of NeuN+ cells ((NeuN+ cells in ipsilateral VPN / NeuN+ cells in contralateral VPN) x 100) in the VPN. (H) High-magnification immunofluorescence images of NeuN staining in stroke-affected rat brain slices with and without transplantation. Scale bar, 50 μm. *p<0.05. Data are shown as mean ± SD. We then studied the effect of intracortical transplantation on the stroke-induced secondary thalamic lesion. Six months after transplantation, analysis of graft location revealed that while most grafted cells remained near the transplantation site in the frontal somatosensory cortex, some graft-derived processes extended into the caudal region near the VPN ( Figure 5B ). Notably, transplanted animals exhibited a significantly larger ipsilateral VPN than non-grafted stroke-lesioned animals ( Figure 5C-E ). No significant differences were observed between the ipsilateral VPN of grafted animals and the corresponding area in sham animals. Finally, we investigated whether the preservation of VPN size following lt-NES cell-derived transplantation was associated with the increased survival of neurons in this region. To assess this, we quantified the number of NeuN+ neurons in the ipsilateral VPN of transplanted and non-transplanted stroke-lesioned animals. We observed a significantly higher number of NeuN+ cells in the VPN of the transplanted group ( Figure 5F-H ). DISCUSSION In this study, we investigated the dynamics of secondary thalamic injury following cortical stroke. We evaluated the therapeutic potential of intracortical transplantation of human iPSC-derived neuronal progenitors. Our findings demonstrate that early intracortical transplantation of these progenitors can significantly reduce thalamic neuronal loss, highlighting a promising strategy to mitigate secondary neurodegeneration and improve post-stroke recovery. Secondary thalamic injury is a well-documented phenomenon in both animal models and human patients following cortical stroke. In our rat model of distal MCA occlusion, we observed stable cortical infarcts by 2 weeks post-stroke, followed by progressive thalamic degeneration, particularly in the VPN, which began at 3 months and persisted for up to 6 months. This delayed thalamic injury aligns with previous studies indicating that secondary neurodegeneration in the thalamus can be detected as early as 3 days post-stroke and continues for months in both rodents and humans ( Fujie et al ., 1990 ; Geng et al ., 2023 ). The mechanisms behind this secondary injury are multifaceted, involving retrograde degeneration, excitotoxicity, apoptosis, blood-brain barrier disruption, and neuroinflammation ( Ross & Ebner, 1990 ; Paz et al ., 2010 ; Yu et al ., 2017 ). Neuronal death is the primary cause of the neurological deficits observed in stroke patients. In our study, we observed that neuronal loss in the VPN correlated with the size of the cortical lesion, suggesting that the extent of cortical damage directly influences the severity of secondary thalamic injury. This finding is consistent with previous reports indicating that the thalamus, which has dense reciprocal connections with the somatosensory cortex, is particularly susceptible to secondary degeneration following cortical stroke ( Seitz et al ., 1999 ; Schroeter et al ., 2006 ). It is well known that after a cortical stroke, microglial activation extends beyond the initial injury site to remote but functionally connected regions (e.g., thalamus), an event known as secondary inflammation. Microglial activation is a hallmark of secondary thalamic injury, and our findings offer additional insights into the timeline and role of microglia in this process. We observed that microglial activation in the ipsilateral VPN increased from 2 weeks post-stroke and remained elevated for 4 months, after which it declined to levels comparable to sham animals by 6 months. This temporal pattern of microglial activation indicates that neuroinflammation plays a critical role in the early stages of secondary thalamic injury, potentially contributing to neuronal loss through the phagocytosis of damaged neurons ( Holmberg et al ., 2009 ; Jones et al ., 2018 ). Interestingly, at 2 weeks post-stroke, microglia were frequently observed close to intact neurons, suggesting early microglial-neuronal interactions that may precede neuronal death. By 3-4 months, colocalization of microglial markers (Iba1) and neuronal markers (NeuN) became evident, indicating ongoing phagocytosis of degenerated neurons. However, by 6 months, no colocalization was observed, suggesting that the inflammatory response had resolved, and the remaining neurons were either fully degenerated or had been cleared by microglia. These findings highlight the dynamic nature of microglial activation in secondary thalamic injury and suggest that interventions targeting neuroinflammation could benefit the subacute phase of stroke recovery. In support of the role of neuroinflammation in the pathophysiology of secondary thalamic lesions following stroke, VX-765, a potent and selective inhibitor of caspase-1, injected seven days post-stroke, significantly reduced sensory deficits in these rats, along with a decrease in both neuronal loss and the activation of astrocytes and microglia. These results indicate that secondary damage may be managed by controlling inflammation and suppressing the production of pro-inflammatory factors generated downstream of NF- κ B signaling ( Liang et al ., 2025 ). One of the key findings of this study is that early intracortical transplantation of human iPSC-derived neuronal progenitors can significantly reduce thalamic neuronal loss after cortical stroke. The transplanted cells, which were cortically primed and transduced with a GFP reporter, integrated into the host tissue and established functional connections with thalamocortical neurons. Importantly, while the transplantation did not change the size of the cortical infarct, it effectively mitigated secondary thalamic injury, as indicated by the reduced shrinkage of the VPN and the preservation of NeuN+ neurons in the thalamus. The delayed nature of thalamic injury likely allowed sufficient time for the transplanted neurons to project to the VPN, prevent ongoing neuronal death caused by cortico-thalamic denervation, and provide trophic support. Consistent with our findings, it was recently reported that transplantation of human embryonic stem cell-derived neural progenitor cells (hESCs-NPCs) in rats subjected to stroke reduced secondary damage in the ipsilateral VPN and improved neurological functions possibly mediated by the activation of the BDNF/TrkB pathway ( Li et al ., 2023 ). Johansson and colleagues have previously demonstrated that cortical ischemic injury in rats results in a substantial lesion (around 24%) in the thalamic nuclei that project to the affected somatosensory area of the cortex ( Grabowski et al ., 1993b ). They also showed significant correlations between the size of the cortical and thalamic lesions (r=.69, P=.0001), aligning with our data and indicating a positive correlation between lesions in the somatosensory cortex and those in the VPN of the thalamus. Fetal rat frontal neocortical grafts, when placed in the cortical infarcts of adult rats, were found to develop a morphology similar to normal neocortex and to establish both afferent and efferent neural connections—albeit sparse—with the host thalamus ( Sorensen et al ., 1996 ; Mattsson et al ., 1997 ). Importantly, similar to our findings with human iPSC-derived neuronal progenitors, they observed that fetal neural grafting reduced thalamic atrophy and improved functional outcomes, although this effect was observed only in stroke-affected animals housed in enriched environments ( Mattsson et al ., 1997 ). Interestingly, when cortical ablation is performed on neonatal rats and fetal cortical tissue is immediately grafted into the cavity, thalamic atrophy can be mitigated either transiently ( Sharp & Gonzalez, 1986 ) or permanently ( Haun & Cunningham, 1984 ). In line with these studies, using the rabies virus-based trans-synaptic tracing method, we have previously shown that when the same human lt-NES cell-derived progenitors as in the present study were grafted in the stroke-lesioned cortex, they received direct synaptic inputs from host neurons of the ipsilateral thalamus ( Tornero et al ., 2017 ). We also showed, with electrophysiological and optogenetic approaches, that these thalamocortical afferent connections are synaptically functional. In the present study, we observed fibers, presumably axons of the grafted neurons, in the host thalamus. Taken together, these data indicate that grafted neurons become integrated into thalamo-cortico-thalamic neural circuitry, which could be one important mechanism involved in the decreased secondary thalamic lesion after transplantation. The ability of the grafted cells to prevent secondary thalamic injury likely also stems from their capacity to modulate the local microenvironment, which includes reducing neuroinflammation and providing trophic support to surviving neurons. Previous studies have shown that stem cell-derived progenitors can secrete neurotrophic factors, promote angiogenesis, and modulate immune responses, all of which may contribute to the observed neuroprotective effects ( Tornero et al ., 2017 ; Palma-Tortosa et al ., 2020 ). We have previously shown that cortically primed human lt-NES cell-derived progenitors become neurons and establish functional afferent connections with host thalamocortical neurons after early intracortical transplantation into the rat stroke-injured brain ( Tornero et al ., 2017 ). However, whether the grafted cells could also affect the secondary degeneration occurring in the thalamus has been unknown. Despite being distant from the primary site of cortical injury, the ipsilateral thalamus shows increasing secondary neuroinflammation even as the cortical inflammatory response related to the primary injury diminishes. This phenomenon has been observed in both rodent models and human patients and is linked to secondary thalamic neurodegeneration and neuroinflammation after a stroke ( Pappata et al ., 2000 ; Langen et al ., 2007 ; Kuchcinski et al ., 2017 ; Cao et al ., 2020 ). In our study, the transplanted cells may have decreased microglial activation and the release of pro-inflammatory cytokines, thereby creating a more favorable environment for neuronal survival. Preventing secondary thalamic injury has significant implications for stroke recovery and patient outcomes. Thalamic damage is linked to a variety of neurological deficits, including sensory loss, motor impairment, and cognitive dysfunction, which can significantly affect a patient’s quality of life ( Kessner et al ., 2019 ; Geng et al ., 2023 ). By preserving thalamic neurons, intracortical transplantation of cortically primed human lt-NES cell-derived progenitors could help reduce these deficits and improve functional recovery. In conclusion, our study demonstrates that early intracortical transplantation of human iPSC-derived neuronal progenitors can effectively reduce secondary thalamic injury following cortical stroke. The progressive nature of the thalamic injury, as evidenced by our study’s delayed neuronal loss and microglial activation, underscores the importance of developing cell transplantation-based interventions that can stop or reverse this degenerative process. By preserving thalamic neurons and modulating neuroinflammation, these interventions should be able to improve stroke patients’ long-term functional recovery and quality of life. While challenges remain in translating these findings to clinical practice, our results highlight the promise of stem cell-based therapies as a novel approach not only to reconstruct injured neural circuitries but also to prevent secondary neurodegeneration, thereby enhancing stroke recovery. Future research should focus on optimizing transplantation protocols, assessing long-term outcomes, and addressing safety concerns to pave the way for clinical applications. AUTHOR CONTRIBUTIONS S.P.-T. and Z.K. conceived the project. S.K., L.J., R.M.-C., L.G.-P., K.K., S.A. and S.P.-T. conducted the experiments and analyzed the data. S.K., S.P.-T., O.L. and Z.K. wrote the manuscript. S.K., R.M.-C., L.G.-P., and K.K. were involved in collecting and/or assembly of data, data analysis, and interpretation. All authors reviewed and edited the manuscript. CONFLICT OF INTERESTS The authors declare no competing interests. ACKNOWLEDGEMENTS This work is supported by grants from the Swedish Research Council, Swedish Brain Foundation, Swedish Stroke Foundation, Regional Research Support from the Southern Swedish Healthcare Region, and the Swedish Government Initiative for Strategic Research Areas (StemTherapy). 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Share Intracortical transplantation of human induced pluripotent stem cell-derived progenitors ameliorates delayed thalamic degeneration following cortical stroke Sopiko Kartsivadze , Linda Jansson , Lucía Galán-Pintado , Keshav Kher , Sara Avilés , Raquel Martinez-Curiel , Olle Lindvall , Sara Palma-Tortosa , Zaal Kokaia bioRxiv 2025.03.17.643730; doi: https://doi.org/10.1101/2025.03.17.643730 Share This Article: Copy Citation Tools Intracortical transplantation of human induced pluripotent stem cell-derived progenitors ameliorates delayed thalamic degeneration following cortical stroke Sopiko Kartsivadze , Linda Jansson , Lucía Galán-Pintado , Keshav Kher , Sara Avilés , Raquel Martinez-Curiel , Olle Lindvall , Sara Palma-Tortosa , Zaal Kokaia bioRxiv 2025.03.17.643730; doi: https://doi.org/10.1101/2025.03.17.643730 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42003) Biophysics (21470) Cancer Biology (18624) Cell Biology (25533) Clinical Trials (138) Developmental Biology (13390) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40432) Molecular Biology (17200) Neuroscience (88681) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4828) Physiology (7653) Plant Biology (15161) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9826) Zoology (2271)