Biology subjects

Tackenberg, C.

Publications and source records attributed to Tackenberg, C..

13 recordsLinked to original sources

bioRxiv2026

Neural graft elimination via dual safety switch compromises therapeutic recovery after stroke

Safety switch systems are increasingly incorporated into stem cell therapies to enable selective graft elimination in the event of adverse outcomes. Yet how removing transplanted cells affects the underlying pathology remains largely unexplored. Here, we show that iPSC-derived neural stem cells (NSCs) co-transduced with herpes simplex virus thymidine kinase (HSV-TK) and inducible caspase-9 (iC9) are more efficiently ablated by combined ganciclovir (GCV) and chemical inducer of dimerization (CID) treatment than either single switch alone, under both proliferating and differentiating conditions. Using bioluminescence imaging to track graft survival longitudinally, we demonstrate that dual safety switch NSC (DS-NSC) grafts are selectively eliminated by GCV and CID administration in immunodeficient mice following photothrombotic stroke. The combination treatment initiated on day 8 post-transplantation resulted in reduced graft volume, lower NSC proliferation, and persistently reduced bioluminescence signal compared to mice receiving only solvent. Histological analysis revealed that combination treatment was associated with larger stroke lesions, higher IBA1 fluorescence intensity, and reduced vascular density in the ischemic border zone. Mice receiving GCV and CID showed persistently elevated contralateral hindlimb error rates on a ladder walk task from day 28 post-stroke onward compared to solvent-treated mice. These findings demonstrate that while DS-NSC elimination is feasible, the pharmacological activation of the safety switch system is associated with impaired functional stroke recovery.

neuroscience↗

bioRxiv2026

Targeting integrin αvβ3 by chimeric antigen receptor neural stem cell (CAR-NSC) therapy for stroke

Stroke remains a leading cause of adult disability due to the brains limited regenerative capacity. Although stem cell therapies show favorable safety and feasibility profiles in early clinical trials, poor spatial retention and limited engagement with peri-infarct salvageable tissue constrain efficacy. Here, we engineered human induced pluripotent stem cell-derived neural stem cells (NSC) with a chimeric antigen receptor (CAR)-like architecture to enable targeted recognition of injury-associated cues. Specifically, cells were modified to express a membrane-anchored single chain variable fragment (scFv) targeting integrin v{beta}3, a receptor selectively upregulated in peri-infarct vasculature after stroke. Engineered CAR-NSC retained progenitor identity and selectively bound recombinant integrin v{beta}3 in vitro. Following focal transplantation into a photothrombotic stroke mouse model, CAR-NSC displayed broader dispersion within peri-infarct tissue and covered a greater proportion of the ischemic lesion compared to non-binding control-CAR-NSC. CAR-NSC grafts extended longer neurites that aligned more closely with the lesion border. In addition, CAR-NSC transplantation reduced microglial activation and was associated with increased vascular density and blood-brain barrier integrity in the peri-infarct zone. Together, these findings establish a CAR-like NSC strategy for stroke to direct the spatial distribution and tissue engagement of transplanted cells. Molecular targeting of injury-associated cues may improve the precision and regenerative efficacy of cell-based therapies for stroke and related neurological disorders.

neuroscience↗

bioRxiv2025

Hypoxia preconditioned neural xenografts promote repair of brain tissue after stroke

Stroke is a leading cause of long-term disability, yet no effective regenerative therapies exist. While cell-based therapies have shown promise in preclinical animal models, their clinical application remains limited due to poor survival of transplanted cells in the ischemic stroke environment. Hypoxic preconditioning has emerged as a strategy to potentially enhance graft survival, but the cellular mechanisms and translational relevance in human iPSC-derived neural progenitor cells (NPCs) are not fully understood. Here, we tested whether hypoxic preconditioning of NPCs affects their molecular and functional properties including proliferation and survival in vitro and after transplantation into a stroke mouse model. Hypoxic preconditioning enhanced proliferation and glial differentiation in vitro, improved cell survival post-transplantation, and enhanced regeneration-associated tissue responses such as vascular remodeling in the peri-infarct brain. These findings suggest that hypoxic preconditioning is a clinically translatable approach to increase the NPC graft survival in the post-stroke brain.

neuroscience↗

bioRxiv2025

APOE genotype-dependent differences in human astrocytic energy metabolism

The main genetic risk factor for Alzheimers disease (AD) is the presence of the apolipoprotein E4 (APOE4) allele. While APOE4 increases the risk of developing AD, the APOE2 allele is protective and APOE3 is risk-neutral. In the brain, APOE is primarily expressed by astrocytes and plays a key role in various processes including cholesterol and lipid transport, neuronal growth, synaptic plasticity, immune response and energy metabolism. Disruptions in brain energy metabolism are considered a major contributor to AD pathophysiology, raising a key question about how different APOE isoforms affect the energy metabolism of human astrocytes. In this study, we generated astrocytes (iAstrocytes) from APOE-isogenic human induced pluripotent stem cells (iPSCs), expressing either APOE2, APOE3, APOE4 or carrying an APOE knockout (APOE-KO), and investigated APOE genotype-dependent changes in energy metabolism. ATP Seahorse assay revealed a reduced mitochondrial and glycolytic ATP production in APOE4 iAstrocytes. In contrast, proteomic GO enrichment analysis and mitochondrial stress tests indicated increased mitochondrial respiration and activity in APOE4 iAstrocytes, accompanied with elevated proton leak, while mitochondrial fusion and fission protein levels remain unchanged. Glycolysis stress tests also demonstrated enhanced glycolysis and glycolytic capacity in APOE4 iAstrocytes while genetically encoded nanosensor-based FLIM analysis revealed that APOE does not affect lactate dynamics. Mass spectrometry-based metabolomic analysis identified various energy and glucose metabolism-related pathways that were differentially regulated in APOE4 compared to the other genotypes, including mitochondrial electron transport chain and glycolysis. In general, APOE2 and APOE-KO iAstrocytes showed a very similar phenotype in all functional assays and differences between APOE2/APOE-KO and APOE4 were stronger than between APOE3 and APOE4. Our study provides evidence for APOE genotype-dependent effects on astrocyte energy metabolism and highlights alterations in the bioenergetic processes of the brain as important pathomechanisms in AD.

neuroscience↗

bioRxiv2025

Delayed transplantation of neural stem cells improves initial graft survival following stroke

Neural stem cell therapies hold great promise for improving stroke recovery, but the hostile stroke microenvironment can hinder the initial graft survival. It has long been well documented that the microenvironment evolves over time, making it crucial to identify the optimal transplantation window to maximize therapeutic efficacy. However, it remains uncertain whether acute or delayed local cell transplantations better supports graft viability after stroke. Here, we show that delayed intracerebral transplantation of neural progenitor cells (NPCs) derived from human induced pluripotent cells (iPSCs) at 7 days post stroke significantly enhances graft proliferation and survival, compared to acute transplantation at 1 day post stroke, in a mouse model of large cortical stroke. Using in vivo bioluminescence imaging over a 6-week period post-transplantation, we observe a more than 5-fold increase in bioluminescence signal in mice that received delayed NPC therapy, compared to those that underwent acute NPC transplantation. The increased number of cell grafts in mice receiving delayed NPC transplantation was driven by increased proliferation rates early after transplantation, which subsequently declined to similarly low levels in both groups. Notably, we found that the majority of transplanted NPCs differentiated into neurons after 6 weeks, with no significant differences in the neuron-to-glia ratio between acute and delayed transplantation groups. These findings suggest that delayed NPC transplantation improves early graft survival and proliferation, which could help identify the optimal therapeutic window for maximizing the effectiveness of NPC-based therapies in stroke.

neuroscience↗

bioRxiv2024

A molecular brain atlas reveals cellular shifts during the repair phase of stroke

Ischemic stroke triggers a cascade of pathological events that affect multiple cell types and often lead to incomplete functional recovery. Despite advances in single-cell technologies, the molecular and cellular responses that contribute to long-term post-stroke impairment remain poorly understood. To gain better insight into the underlying mechanisms, we generated a single-cell transcriptomic atlas from distinct brain regions using a mouse model of permanent focal ischemia at one month post-injury. Our findings reveal cell- and region-specific changes within the stroke-injured and peri-infarct brain tissue. For instance, GABAergic and glutamatergic neurons exhibited upregulated genes in signaling pathways involved in axon guidance and synaptic plasticity, and downregulated pathways associated with aerobic metabolism. Using cell-cell communication analysis, we identified increased strength in predicted interactions within stroke tissue among both neural and non-neural cells via signaling pathways such as those involving collagen, protein tyrosine phosphatase receptor, neuronal growth regulator, laminin, and several cell adhesion molecules. Furthermore, we found a strong correlation between mouse transcriptome responses after stroke and those observed in human nonfatal brain stroke lesions. Common molecular features were linked to inflammatory responses, extracellular matrix organization, and angiogenesis. Our findings provide a detailed resource for advancing our molecular understanding of stroke pathology and for discovering therapeutic targets in the repair phase of stroke recovery.

neuroscience↗

bioRxiv2024

APOE4 increases energy metabolism in APOE-isogenic iPSC-derived neurons

The apolipoprotein E4 (APOE4) allele represents the major genetic risk factor for Alzheimers disease (AD). In contrast, APOE2 is known to lower the AD risk while APOE3 is defined as risk neutral. APOE plays a prominent role in the bioenergetic homeostasis of the brain, and early-stage metabolic changes have been detected in brains of AD patients. Although APOE is primarily expressed by astrocytes in the brain, neurons also have been shown as source for APOE. However, little is known about the differential role of the three APOE isoforms for neuronal energy homeostasis. In this study, we generated pure human neurons (iN cells) from APOE-isogenic induced pluripotent stem cells (iPSCs), expressing either APOE2, APOE3, APOE4 or carrying an APOE-knockout (KO) to investigate APOE isoform-specific effects on neuronal energy metabolism. We showed that endogenously produced APOE4 enhanced mitochondrial ATP production in APOE-isogenic iN cells but not in the corresponding iPS cell line. This effect neither correlated with the expression levels of mitochondrial fission or fusion proteins, nor with the intracellular or secreted levels of APOE, which were similar for APOE2, APOE3 and APOE4 iN cells. ATP production and basal respiration in APOE-KO iN cells strongly differed from APOE4 and more closely resembled APOE2 and APOE3 iN cells indicating a gain-of-function mechanism of APOE4 rather than a loss-of-function. Taken together, our findings in APOE isogenic iN cells reveal an APOE genotype-dependent and neuron-specific regulation of oxidative energy metabolism.

neuroscience↗

bioRxiv2024

Human iPSC-derived cell grafts promote functional recovery by molecular interaction with stroke-injured brain

Stroke is a leading cause of disability and death due to the brains limited ability to regenerate damaged neural circuits. To date, stroke patients have only few therapeutic options and are often left with considerable disabilities. Induced pluripotent stem cell (iPSC)-based therapies are emerging as a promising therapeutic approach for stroke recovery. In this study, we demonstrate that local transplantation of iPSC-derived neural progenitor cells (NPCs) improves long-term brain tissue repair responses and reduces neurological deficits after cerebral ischemia in mice. Using in vivo bioluminescence imaging and post-mortem histology, we show long-term graft survival over the course of five weeks and preferential graft differentiation into mature neurons without signs of pluripotent residuals. Transplantation of NPCs led to a set of brain tissue repair responses including increased vascular sprouting and repair, improved blood-brain barrier integrity, reduced microglial activation, and increased neurogenesis compared to littermate control animals receiving sham transplantation. Employing deep learning-assisted behavior analysis, we found that NPC-treated mice displayed improved gait performance and complete fine-motor recovery in the horizontal ladder rung walk, five weeks post-injury. To dissect the molecular graft composition and identify graft-host interactions, single nucleus profiling of the cell transplants and host stroke tissue was performed. We identified graft differentiation preferentially towards neurons with GABAergic and glutamatergic phenotypes in similar proportions, with the remaining cells acquiring astrocyte and NPC-like phenotypes. Interaction between graft and host transcriptome indicated that GABAergic cell grafts were primarily involved in graft-host communication through the regeneration-associated neurexin (NRXN), neuregulin (NRG), neural cell adhesion molecule (NCAM) and SLIT signalling pathways. In conclusion, our study reveals that transplanted iPSC-derived NPCs primarily differentiate into GABAergic neurons contributing to long-term recovery and further delineates the regenerative interactions between the graft and the stroke-injured host tissue.

neuroscience↗

bioRxiv2023

Visualization and estimation of stroke infarct volumes in rodents

Stroke volume is a key determinant of infarct severity and an important metric treatments evaluation. However, accurate estimation of stroke volume can be challenging, due to the often confined 2-dimensional nature of available data. Here, we introduce a comprehensive semi-automated toolkit to reliably estimate stroke volumes based on (1) whole brains ex-vivo magnetic resonance imaging (MRI) and (2) brain sections that underwent immunofluorescence staining. We located and quantified infarct areas from MRI three days (acute) and 28 days (chronic) after photothrombotic stroke induction in whole mouse brains. MRI Results were compared with measures obtained from immunofluorescent histologic sections of the same brains. Using our toolkit, we found that infarct volume determined by post-mortem MRI was highly correlated with a deviation of only 6.6% (acute) and 4.9% (chronic) to the measurements as determined in the histological brain sections indicating that both methods are capable of accurately assessing brain tissue damage.

neuroscience↗

bioRxiv2022

Molecular and anatomical roadmap of stroke pathology in immunodeficient mice

BackgroundStroke remains a leading cause of disability and death worldwide. It has become apparent that inflammation and immune mediators have a pre-dominant role in initial tissue damage and long-term recovery following the injury. Still, different immunosuppressed mouse models are necessary in stroke research e.g., to evaluate therapies using human cell grafts. Despite mounting evidence delineating the importance of inflammation in the stroke pathology, it is poorly described to what extent partial immune deficiency influences the overall stroke outcome. MethodsHere, we assessed the stroke pathology of popular genetic immunodeficient mouse models, i.e., NOD scid gamma (NSG) and recombination activating gene 2 (Rag2-/-) mice as well as pharmacologically immunosuppressed mice and compared them to immune competent, wildtype (WT) C57BL/6J mice up to three weeks after injury. We performed histology, gene expression profiling, serum analysis and functional behavioural tests to identify the impact of immunosuppression on the stroke progression. ResultsWe detected distinct changes in microglia infiltration, scar-forming and vascular repair in immune-suppressed mice three weeks after injury. Gene expression analysis of stroked tissue revealed the strongest deviation from immune competent mice was observed in NSG mice, for instance, affecting immunological and angiogenic pathways. Pharmacological immunosuppression resulted in the least variation in gene expression compared with the WT. Major differences have been further identified in the systemic inflammatory response following stroke acutely and three weeks following injury. These anatomical, genetic, and systemic changes did not affect functional deficits and recovery in a time course of three weeks. To determine whether the timing of immunosuppression after stroke is critical, we compared mice with acute and delayed pharmacological immunosuppression after stroke. Mice with a delayed immunosuppression (7d) after stroke showed increased inflammatory and scarring responses compared to animals acutely treated with tacrolimus, thus more closely resembling WT pathology. Transplantation of human cells in the brains of immunosuppressed mouse models led to prolonged cell survival in all immunosuppressed mouse models, which was most consistent in NSG and Rag2-/- mice. ConclusionsIn sum, we detected distinct anatomical and molecular changes in the stroke pathology between the individual immunosuppressed mouse models that should be carefully considered when selecting an appropriate mouse model for stroke research.

neuroscience↗

bioRxiv2022

Xeno-free induced pluripotent stem cell-derived neural progenitor cells for in vivo applications

Cell-based therapies are a promising treatment paradigm for neurodegenerative diseases and other brain injuries. Despite recent advances in stem cell technology, major concerns have been raised regarding the feasibility and safety of cell therapies for clinical applications. Here, we generate good manufacturing practice (GMP)-compatible neural progenitor cells (NPCs) from transgene- and xeno-free induced pluripotent stem cells (iPSCs) that can be smoothly adapted for clinical applications. The produced NPCs have a stable gene-expression over at least 15 passages and can be scaled for up to 1018 cells per initially seeded 106 cells. To ensure a pure NPC population for in vivo applications, we reduce risks of iPSC contamination using micro RNA-switch technology as a safety checkpoint. Using lentiviral transduction with a fluorescent and bioluminescent dual-reporter construct, combined with non-invasive in vivo bioluminescent imaging, we longitudinally tracked the grafted cells in healthy wild-type and genetically immunosuppressed mice as well as in a mouse model of ischemic stroke. Long term in-depth characterization revealed that transplanted cells have the capability to survive and spontaneously differentiate into functional and mature neurons throughout a time course of a month.

neuroscience↗

bioRxiv2021

Deep learning based behavioral profiling of rodent stroke recovery

Stroke research heavily relies on rodent behavior when assessing underlying disease mechanisms and treatment efficacy. Although functional motor recovery is considered the primary targeted outcome, tests in rodents are still poorly reproducible, and often unsuitable for unraveling the complex behavior after injury. Here, we provide a comprehensive 3D gait analysis of mice after focal cerebral ischemia based on the new deep learning-based software (DeepLabCut, DLC) that only requires basic behavioral equipment. We demonstrate a high precision 3D tracking of 10 body parts (including all relevant joints and reference landmarks) in several mouse strains with an accuracy of 99.4%. Building on this rigor motion tracking, a comprehensive post-analysis (with >100 parameters) unveils biologically relevant differences in locomotor profiles after a stroke over a time course of three weeks. We further refine the widely used ladder rung test using deep learning and compare its performance to human annotators. The generated DLC-assisted tests were then benchmarked to five widely used conventional behavioral set-ups (neurological scoring, rotarod, ladder rung walk, cylinder test, and single-pellet grasping) regarding sensitivity, accuracy, time use and costs. We conclude that deep learning-based motion tracking with comprehensive post-analysis provides accurate and sensitive data to describe the complex recovery of rodents following a stroke. The experimental set-up and analysis can also benefit a range of other neurological injuries that affect locomotion.

neuroscience↗

bioRxiv2021

APOE2, E3 and E4 differentially modulate cellular homeostasis, cholesterol metabolism and inflammatory response in isogenic iPSC-derived astrocytes

Apolipoprotein E (APOE) is the principal lipid carrier in the CNS and mainly expressed by astrocytes. The three different APOE alleles (E2, E3, and E4) impose differential risk to Alzheimers disease (AD); E2 is protective, E3 is defined as average risk, while E4 is the major genetic risk factor for sporadic AD. Despite recent advances, the fundamental role of different APOE alleles in brain homeostasis is still poorly understood. To uncover the functional role of APOE in human astrocytes, we differentiated human APOE-isogenic iPSCs (E4, E3, E2 and APOE-knockout (KO)) to functional astrocytes (hereafter "iAstrocytes"), with a resting, non-proliferating phenotype. Functional assays indicated that polymorphisms in APOE (APOE4>E3>E2=KO) reduced iAstrocyte metabolic and clearance functions including glutamate uptake and receptor-mediated uptake of {beta}-amyloid aggregates. We performed unlabelled mass spectrometry-based proteomic analysis of iAstrocytes at baseline and after activation with interleukin-1{beta} (IL-1{beta}) showing a reduction of cholesterol and lipid metabolic and biosynthetic pathways, and an increase of immunoregulatory pathways at baseline (E4>E3>E2). Cholesterol efflux and biosynthesis were reduced in E4 iAstrocytes, and subcellular localization of cholesterol in lysosomes was increased. In APOE-KO iAstrocytes, APOE-independent mechanisms showed to be proficient in mediating cholesterol biosynthesis and efflux. Proteomic analysis of IL-1{beta}-treated iAstrocytes showed an increase of cholesterol/lipid metabolism and biosynthesis as well as inflammatory pathways. Furthermore, cholesterol efflux, which was reduced in APOE4 iAstrocytes at baseline, was alleviated in activated E4 iAstrocytes. Inflammatory cytokine release was exacerbated upon IL-1{beta} treatment in E4 iAstrocytes (E4>E3>E2>KO), in line with the proteomic data. Taken together, we show that APOE plays a major role in several physiological and metabolic processes in human astrocytes with APOE4 pushing iAstrocytes to a disease-relevant phenotype, causing dysregulated cholesterol/lipid homeostasis, increased inflammatory signalling and reduced {beta}-amyloid uptake while APOE2 iAstrocytes show opposing effects. Our study provides a new reference for AD-relevant proteomic and metabolic changes, mediated by the three main APOE isoforms in human astrocytes.

neuroscience↗