| 1. |
- Delsing, Louise, et al.
(författare)
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Barrier properties and transcriptome expression in human iPSC-derived models of the blood-brain barrier
- 2018
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Ingår i: Stem Cells. - : AlphaMed Press, Inc.. - 1066-5099 .- 1549-4918. ; 36:12, s. 1816-1827
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Tidskriftsartikel (refereegranskat)abstract
- Cell-based models of the blood-brain barrier (BBB) are important for increasing the knowledge of BBB formation, degradation and brain exposure of drug substances. Human models are preferred over animal models because of inter-species differences in BBB structure and function. However, access to human primary BBB tissue is limited and has shown degeneration of BBB functions in vitro. Human induced pluripotent stem cells (iPSCs) can be used to generate relevant cell types to model the BBB with human tissue. We generated a human iPSC-derived model of the BBB that includes endothelial cells in co-culture with pericytes, astrocytes and neurons. Evaluation of barrier properties showed that the endothelial cells in our co-culture model have high transendothelial electrical resistance, functional efflux and ability to discriminate between CNS permeable and non-permeable substances. Whole genome expression profiling revealed transcriptional changes that occur in co-culture, including upregulation of tight junction proteins such as claudins and neurotransmitter transporters. Pathway analysis implicated changes in the WNT, TNF and PI3K-Akt pathways upon co-culture. Our data suggests that co-culture of iPSC-derived endothelial cells promotes barrier formation on a functional and transcriptional level. The information about gene expression changes in co-culture can be used to further improve iPSC-derived BBB models through selective pathway manipulation.
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| 2. |
- Engdahl, Elin, et al.
(författare)
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Bisphenol A Inhibits the Transporter Function of the Blood-Brain Barrier by Directly Interacting with the ABC Transporter Breast Cancer Resistance Protein (BCRP)
- 2021
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 22:11
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Tidskriftsartikel (refereegranskat)abstract
- The breast cancer resistance protein (BCRP) is an important efflux transporter in the blood-brain barrier (BBB), protecting the brain from a wide range of substances. In this study, we investigated if BCRP function is affected by bisphenol A (BPA), a high production volume chemical used in common consumer products, as well as by bisphenol F (BPF) and bisphenol S (BPS), which are used to substitute BPA. We employed a transwell-based in vitro cell model of iPSC-derived brain microvascular endothelial cells, where BCRP function was assessed by measuring the intracellular accumulation of its substrate Hoechst 33342. Additionally, we used in silico modelling to predict if the bisphenols could directly interact with BCRP. Our results showed that BPA significantly inhibits the transport function of BCRP. Additionally, BPA was predicted to bind to the cavity that is targeted by known BCRP inhibitors. Taken together, our findings demonstrate that BPA inhibits BCRP function in vitro, probably by direct interaction with the transporter. This effect might contribute to BPA's known impact on neurodevelopment.
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| 4. |
- Hernando, Sara, et al.
(författare)
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Dual effect of TAT functionalized DHAH lipid nanoparticles with neurotrophic factors in human BBB and microglia cultures
- 2022
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Ingår i: Fluids and Barriers of the CNS. - : Springer Nature. - 2045-8118. ; 19:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundNeurodegenerative diseases (NDs) are an accelerating global health problem. Nevertheless, the stronghold of the brain- the blood–brain barrier (BBB) prevents drug penetrance and dwindles effective treatments. Therefore, it is crucial to identify Trojan horse-like drug carriers that can effectively cross the blood–brain barrier and reach the brain tissue. We have previously developed polyunsaturated fatty acids (PUFA)-based nanostructured lipid carriers (NLC), namely DHAH-NLC. These carriers are modulated with BBB-permeating compounds such as chitosan (CS) and trans-activating transcriptional activator (TAT) from HIV-1 that can entrap neurotrophic factors (NTF) serving as nanocarriers for NDs treatment. Moreover, microglia are suggested as a key causative factor of the undergoing neuroinflammation of NDs. In this work, we used in vitro models to investigate whether DHAH-NLCs can enter the brain via the BBB and investigate the therapeutic effect of NTF-containing DHAH-NLC and DHAH-NLC itself on lipopolysaccharide-challenged microglia.MethodsWe employed human induced pluripotent stem cell-derived brain microvascular endothelial cells (BMECs) to capitalize on the in vivo-like TEER of this BBB model and quantitatively assessed the permeability of DHAH-NLCs. We also used the HMC3 microglia cell line to assess the therapeutic effect of NTF-containing DHAH-NLC upon LPS challenge.ResultsTAT-functionalized DHAH-NLCs successfully crossed the in vitro BBB model, which exhibited high transendothelial electrical resistance (TEER) values (≈3000 Ω*cm2). Specifically, the TAT-functionalized DHAH-NLCs showed a permeability of up to 0.4% of the dose. Furthermore, using human microglia (HMC3), we demonstrate that DHAH-NLCs successfully counteracted the inflammatory response in our cultures after LPS challenge. Moreover, the encapsulation of glial cell-derived neurotrophic factor (GNDF)-containing DHAH-NLCs (DHAH-NLC-GNDF) activated the Nrf2/HO-1 pathway, suggesting the triggering of the endogenous anti-oxidative system present in microglia.ConclusionsOverall, this work shows that the TAT-functionalized DHAH-NLCs can cross the BBB, modulate immune responses, and serve as cargo carriers for growth factors; thus, constituting an attractive and promising novel drug delivery approach for the transport of therapeutics through the BBB into the brain.
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| 5. |
- Jain, Saumey, et al.
(författare)
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On-Chip Neural Induction Boosts Neural Stem Cell Commitment : Toward a Pipeline for iPSC-Based Therapies
- 2024
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Ingår i: Advanced science (Weinheim, Baden-Wurttemberg, Germany). - : Wiley-VCH Verlagsgesellschaft. - 2198-3844.
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Tidskriftsartikel (refereegranskat)abstract
- The clinical translation of induced pluripotent stem cells (iPSCs) holds great potential for personalized therapeutics. However, one of the main obstacles is that the current workflow to generate iPSCs is expensive, time-consuming, and requires standardization. A simplified and cost-effective microfluidic approach is presented for reprogramming fibroblasts into iPSCs and their subsequent differentiation into neural stem cells (NSCs). This method exploits microphysiological technology, providing a 100-fold reduction in reagents for reprogramming and a ninefold reduction in number of input cells. The iPSCs generated from microfluidic reprogramming of fibroblasts show upregulation of pluripotency markers and downregulation of fibroblast markers, on par with those reprogrammed in standard well-conditions. The NSCs differentiated in microfluidic chips show upregulation of neuroectodermal markers (ZIC1, PAX6, SOX1), highlighting their propensity for nervous system development. Cells obtained on conventional well plates and microfluidic chips are compared for reprogramming and neural induction by bulk RNA sequencing. Pathway enrichment analysis of NSCs from chip showed neural stem cell development enrichment and boosted commitment to neural stem cell lineage in initial phases of neural induction, attributed to a confined environment in a microfluidic chip. This method provides a cost-effective pipeline to reprogram and differentiate iPSCs for therapeutics compliant with current good manufacturing practices.
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| 6. |
- Matthiesen, Isabelle, et al.
(författare)
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Continuous Monitoring Reveals Protective Effects of N‐Acetylcysteine Amide on an Isogenic Microphysiological Model of the Neurovascular Unit
- 2021
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Ingår i: Small. - : Wiley. - 1613-6810 .- 1613-6829. ; 17:32, s. 2101785-
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Tidskriftsartikel (refereegranskat)abstract
- Microphysiological systems mimic the in vivo cellular ensemble and microenvironment with the goal of providing more human-like models for biopharmaceutical research. In this study, the first such model of the blood-brain barrier (BBB-on-chip) featuring both isogenic human induced pluripotent stem cell (hiPSC)-derived cells and continuous barrier integrity monitoring with <2 min temporal resolution is reported. Its capabilities are showcased in the first microphysiological study of nitrosative stress and antioxidant prophylaxis. Relying on off-stoichiometry thiol–ene–epoxy (OSTE+) for fabrication greatly facilitates assembly and sensor integration compared to the prevalent polydimethylsiloxane devices. The integrated cell–substrate endothelial resistance monitoring allows for capturing the formation and breakdown of the BBB model, which consists of cocultured hiPSC-derived endothelial-like and astrocyte-like cells. Clear cellular disruption is observed when exposing the BBB-on-chip to the nitrosative stressor linsidomine, and the barrier permeability and barrier-protective effects of the antioxidant N-acetylcysteine amide are reported. Using metabolomic network analysis reveals further drug-induced changes consistent with prior literature regarding, e.g., cysteine and glutathione involvement. A model like this opens new possibilities for drug screening studies and personalized medicine, relying solely on isogenic human-derived cells and providing high-resolution temporal readouts that can help in pharmacodynamic studies.
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| 7. |
- Nikolakopoulou, Polyxeni, et al.
(författare)
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Recent progress in translational engineered in vitro models of the central nervous system
- 2020
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Ingår i: Brain. - : Oxford University Press (OUP). - 0006-8950 .- 1460-2156. ; 143:11, s. 3181-3213
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Tidskriftsartikel (refereegranskat)abstract
- The complexity of the human brain poses a substantial challenge for the development of models of the CNS. Current animal models lack many essential human characteristics (in addition to raising operational challenges and ethical concerns), and conventional in vitro models, in turn, are limited in their capacity to provide information regarding many functional and systemic responses. Indeed, these challenges may underlie the notoriously low success rates of CNS drug development efforts. During the past 5 years, there has been a leap in the complexity and functionality of in vitro systems of the CNS, which have the potential to overcome many of the limitations of traditional model systems. The availability of human-derived induced pluripotent stem cell technology has further increased the translational potential of these systems. Yet, the adoption of state-of-the-art in vitro platforms within the CNS research community is limited. This may be attributable to the high costs or the immaturity of the systems. Nevertheless, the costs of fabrication have decreased, and there are tremendous ongoing efforts to improve the quality of cell differentiation. Herein, we aim to raise awareness of the capabilities and accessibility of advanced in vitro CNS technologies. We provide an overview of some of the main recent developments (since 2015) in in vitro CNS models. In particular, we focus on engineered in vitro models based on cell culture systems combined with microfluidic platforms (e.g. 'organ-on-a-chip' systems). We delve into the fundamental principles underlying these systems and review several applications of these platforms for the study of the CNS in health and disease. Our discussion further addresses the challenges that hinder the implementation of advanced in vitro platforms in personalized medicine or in large-scale industrial settings, and outlines the existing differentiation protocols and industrial cell sources. We conclude by providing practical guidelines for laboratories that are considering adopting organ-on-a-chip technologies.
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| 8. |
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| 9. |
- Voulgaris, Dimitrios, 1990-, et al.
(författare)
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Generation of Human iPSC-Derived Astrocytes with a mature star-shaped phenotype for CNS modeling
- 2022
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Ingår i: Stem Cell Reviews and Reports. - : Springer Nature. - 2629-3269 .- 2629-3277.
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Tidskriftsartikel (refereegranskat)abstract
- The generation of astrocytes from human induced pluripotent stem cells has been hampered by either prolonged diferentiation—spanning over two months—or by shorter protocols that generate immature astrocytes, devoid of salient matureastrocytic traits pivotal for central nervous system (CNS) modeling. We directed stable hiPSC-derived neuroepithelial stemcells to human iPSC-derived Astrocytes (hiAstrocytes) with a high percentage of star-shaped cells by orchestrating anastrocytic-tuned culturing environment in 28 days. We employed RT-qPCR and ICC to validate the astrocytic commitmentof the neuroepithelial stem cells. To evaluate the infammatory phenotype, we challenged the hiAstrocytes with the proinfammatory cytokine IL-1β (interleukin 1 beta) and quantitatively assessed the secretion profle of astrocyte-associatedcytokines and the expression of intercellular adhesion molecule 1 (ICAM-1). Finally, we quantitatively assessed the capacityof hiAstrocytes to synthesize and export the antioxidant glutathione. In under 28 days, the generated cells express canonicaland mature astrocytic markers, denoted by the expression of GFAP, AQP4 and ALDH1L1. In addition, the notion of a maturephenotype is reinforced by the expression of both astrocytic glutamate transporters EAAT1 and EAAT2. Thus, hiAstrocyteshave a mature phenotype that encompasses traits critical in CNS modeling, including glutathione synthesis and secretion,upregulation of ICAM-1 and a cytokine secretion profle on a par with human fetal astrocytes. This protocol generates amultifaceted astrocytic model suitable for in vitro CNS disease modeling and personalized medicine.
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| 10. |
- Voulgaris, Dimitrios, 1990-
(författare)
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Human iPSC-based models of theCNS: attaining cellular biofidelitythrough conventional and advancedculture systems
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Brain development is a highly orchestrated process that entails changes in microenvironmental cues and growth factor gradients, which set the tempo for proper development of the rudimentary structures of the brain and the generation of neurons,astrocytes, and oligodendrocytes. Another intricate feature of the brain is the bloodbrain barrier (BBB). The BBB consists of specialized endothelial cells that form asemipermeable barrier between the blood and the brain; hence, the BBB plays animportant part in protecting the brain from blood-borne pathogens. In vitro modeling is inherently limiting, an artificial microenvironment that is usually not in tunewith in vivo conditions. Thus, understanding these cues and growth factor conditions is pivotal for proper in vitro modeling and achieving cell biomimicry in vitro.Stem cell differentiation is highly amenable to growth factors and microenvironmental cues that can alter the expression of proteins. Advanced in vitro culturingconsiders microenvironmental cues and applies a more holistic aspect to in vitromodeling. This thesis evaluates microenvironmental cues in neural stem cell generation and astrocyte generation by employing induced pluripotent stem cells (iPSC).This thesis introduces a new protocol for generating human iPSC-derived astrocytesin under 28 days. By creating an astrocytogenic milieu, neural stem cells give riseto star-shaped astrocytes that encompass many traits previously unmet in iPSC-derived astrocytes, namely, ICAM-1 expression under inflammatory stimulation, glutathione synthesis and secretion. A follow-up study in this thesis presents a proteomic analysis between primary fetal astrocytes and iPSC-derived astrocytes. Microphysiological systems impart a more appropriate culturing microenvironment andinfluence cell fate and functionality. Another study of this thesis focuses on thedifferences between conventional and microphysiological culture systems in iPSCreprogramming and the generation of neural stem cells. Lastly, in vitro modeling ofthe blood-brain barrier (BBB) is also investigated. Specifically, 1) a human iPSCBBB-like model is used to evaluate the permeability of a drug delivery system basedon nanostructured lipid carriers and 2) a vessel-like structure with a 3D gliomamodel.
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| 11. |
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| 12. |
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| 13. |
- Winkler, Thomas, Ph.D., et al.
(författare)
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Continuous monitoring of isogenic blood-brain barrier integrity in a pdms-free microphysiological system
- 2020
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Ingår i: MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. ; , s. 963-964
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Konferensbidrag (refereegranskat)abstract
- We present a microphysiological system (MPS) of the human blood-brain barrier (BBB) that uniquely combines three key advantages - continuous monitoring, PDMS-free fabrication, and cellular relevance - over existing MPS, and we demonstrate BBB formation, breakdown, and rescue. Specifically, we model nitrosative stress - strongly implicated in brain-related disorders from stroke to Alzheimer's - and its prevention using a BBB-permeable antioxidant. This kind of MPS paves the way toward patient-specific BBB modeling for time-resolved studies of drug kinetics and/or side effects towards personalized treatment planning.
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