| 1. |
- Aldrin-Kirk, Patrick, et al.
(författare)
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A novel two-factor monosynaptic TRIO tracing method for assessment of circuit integration of hESC-derived dopamine transplants
- 2022
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Ingår i: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 17:1, s. 159-172
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Tidskriftsartikel (refereegranskat)abstract
- Transplantation in Parkinson's disease using human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons is a promising future treatment option. However, many of the mechanisms that govern their differentiation, maturation, and integration into the host circuitry remain elusive. Here, we engrafted hESCs differentiated toward a ventral midbrain DA phenotype into the midbrain of a preclinical rodent model of Parkinson's disease. We then injected a novel DA-neurotropic retrograde MNM008 adeno-associated virus vector capsid, into specific DA target regions to generate starter cells based on their axonal projections. Using monosynaptic rabies-based tracing, we demonstrated for the first time that grafted hESC-derived DA neurons receive distinctly different afferent inputs depending on their projections. The similarities to the host DA system suggest a previously unknown directed circuit integration. By evaluating the differential host-to-graft connectivity based on projection patterns, this novel approach offers a tool to answer outstanding questions regarding the integration of grafted hESC-derived DA neurons.
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| 2. |
- Drouin-Ouellet, Janelle, et al.
(författare)
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Age-related pathological impairments in directly reprogrammed dopaminergic neurons derived from patients with idiopathic Parkinson's disease
- 2022
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Ingår i: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 17:10, s. 2203-2219
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Tidskriftsartikel (refereegranskat)abstract
- We have developed an efficient approach to generate functional induced dopaminergic (DA) neurons from adult human dermal fibroblasts. When performing DA neuronal conversion of patient fibroblasts with idiopathic Parkinson's disease (PD), we could specifically detect disease-relevant pathology in these cells. We show that the patient-derived neurons maintain age-related properties of the donor and exhibit lower basal chaperone-mediated autophagy compared with healthy donors. Furthermore, stress-induced autophagy resulted in an age-dependent accumulation of macroautophagic structures. Finally, we show that these impairments in patient-derived DA neurons leads to an accumulation of phosphorylated alpha-synuclein, the classical hallmark of PD pathology. This pathological phenotype is absent in neurons generated from induced pluripotent stem cells from the same patients. Taken together, our results show that direct neural reprogramming can be used for obtaining patient-derived DA neurons, which uniquely function as a cellular model to study age-related pathology relevant to idiopathic PD.
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| 3. |
- Johansson, Pia A, et al.
(författare)
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A cis-acting structural variation at the ZNF558 locus controls a gene regulatory network in human brain development
- 2022
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Ingår i: Cell Stem Cell. - : Elsevier BV. - 1934-5909 .- 1875-9777. ; 29:1, s. 8-69
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Tidskriftsartikel (refereegranskat)abstract
- The human forebrain has expanded in size and complexity compared to chimpanzees despite limited changes in protein-coding genes, suggesting that gene expression regulation is an important driver of brain evolution. Here, we identify a KRAB-ZFP transcription factor, ZNF558, that is expressed in human but not chimpanzee forebrain neural progenitor cells. ZNF558 evolved as a suppressor of LINE-1 transposons but has been co-opted to regulate a single target, the mitophagy gene SPATA18. ZNF558 plays a role in mitochondrial homeostasis, and loss-of-function experiments in cerebral organoids suggests that ZNF558 influences developmental timing during early human brain development. Expression of ZNF558 is controlled by the size of a variable number tandem repeat that is longer in chimpanzees compared to humans, and variable in the human population. Thus, this work provides mechanistic insight into how a cis-acting structural variation establishes a regulatory network that affects human brain evolution.
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| 4. |
- Adler, Andrew, et al.
(författare)
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Transsynaptic tracing and its emerging use to assess graftreconstructed neural circuits
- 2020
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 38:6, s. 716-726
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Tidskriftsartikel (refereegranskat)abstract
- Fetal neural progenitor grafts have been evaluated in preclinical animal models of spinal cord injury and Parkinson’s disease for decades, but the initial reliance on primary tissue as a cell source limited the scale of their clinical translatability. With the development of robust methods to differentiate human pluripotent stem cells to specific neural subtypes, cell replacement therapy holds renewed promise to treat a variety of neurodegenerative diseases and injuries at scale. As these cell sources are evaluated in preclinical models, new transsynaptic tracing methods are making it possible to study the connectivity between host and graft neurons with greater speed and detail than was previously possible. To date, these studies have revealed that widespread, long-lasting, and anatomically-appropriate synaptic contacts are established between host and graft neurons, as well as new aspects of host-graft connectivity which may be relevant to clinical cell replacement therapy. It is not yet clear, however, whether the synaptic connectivity between graft and host neurons is as celltype specific as it is in the endogenous nervous system, or whether that connectivity is responsible for the functional efficacy of cell replacement therapy. Here, we review evidence suggesting that the new contacts established between host and graft neuronsmay indeed be cell-type specific, and how transsynaptic tracing can be used inthe future to further elucidate the mechanisms of graft-mediated functional recovery in spinal cord injury and Parkinson’s disease.
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| 5. |
- Alekseenko, Zhanna, et al.
(författare)
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Robust derivation of transplantable dopamine neurons from human pluripotent stem cells by timed retinoic acid delivery
- 2022
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Stem cell therapies for Parkinson’s disease (PD) have entered first-in-human clinical trials using a set of technically related methods to produce mesencephalic dopamine (mDA) neurons from human pluripotent stem cells (hPSCs). Here, we outline an approach for high-yield derivation of mDA neurons that principally differs from alternative technologies by utilizing retinoic acid (RA) signaling, instead of WNT and FGF8 signaling, to specify mesencephalic fate. Unlike most morphogen signals, where precise concentration determines cell fate, it is the duration of RA exposure that is the key-parameter for mesencephalic specification. This concentration-insensitive patterning approach provides robustness and reduces the need for protocol-adjustments between hPSC-lines. RA-specified progenitors promptly differentiate into functional mDA neurons in vitro, and successfully engraft and relieve motor deficits after transplantation in a rat PD model. Our study provides a potential alternative route for cell therapy and disease modelling that due to its robustness could be particularly expedient when use of autologous- or immunologically matched cells is considered.
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| 6. |
- Besusso, Dario, et al.
(författare)
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Stem Cell-Derived Human Striatal Progenitors Innervate Striatal Targets and Alleviate Sensorimotor Deficit in a Rat Model of Huntington Disease
- 2020
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Ingår i: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 14:5, s. 876-891
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Tidskriftsartikel (refereegranskat)abstract
- Huntington disease (HD) is an inherited late-onset neurological disorder characterized by progressive neuronal loss and disruption of cortical and basal ganglia circuits. Cell replacement using human embryonic stem cells may offer the opportunity to repair the damaged circuits and significantly ameliorate disease conditions. Here, we showed that in-vitro-differentiated human striatal progenitors undergo maturation and integrate into host circuits upon intra-striatal transplantation in a rat model of HD. By combining graft-specific immunohistochemistry, rabies virus-mediated synaptic tracing, and ex vivo electrophysiology, we showed that grafts can extend projections to the appropriate target structures, including the globus pallidus, the subthalamic nucleus, and the substantia nigra, and receive synaptic contact from both host and graft cells with 6.6 ± 1.6 inputs cell per transplanted neuron. We have also shown that transplants elicited a significant improvement in sensory-motor tasks up to 2 months post-transplant further supporting the therapeutic potential of this approach.
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| 7. |
- Birtele, Marcella, et al.
(författare)
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Single-cell transcriptional and functional analysis of dopaminergic neurons in organoid-like cultures derived from human fetal midbrain
- 2022
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Ingår i: Development: For advances in developmental biology and stem cells. - : The Company of Biologists. - 1477-9129. ; 149:23
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Tidskriftsartikel (refereegranskat)abstract
- Significant efforts are ongoing to develop refined differentiation protocols to generate midbrain dopamine (DA) neurons from pluripotent stem cells (PSCs) for application in disease modeling, diagnostics, drug screening, and cell-based therapies for Parkinson's Disease (PD). An increased understanding of the timing and molecular mechanisms promoting the generation of distinct subtypes of human midbrain DA during development will be essential for guiding future efforts to generate molecularly defined and subtype-specific DA neurons from PSCs. Here, we used droplet-based single-cell RNA sequencing to transcriptionally profile the developing human ventral midbrain (VM) when the DA neurons are generated (6-11 weeks post-conception) and their subsequent differentiation into functional mature DA neurons in primary fetal 3D organoid-like cultures. This approach revealed that 3D cultures are superior to monolayer conditions for their ability to generate and maintain mature DA neurons; hence they have the potential to be used for studying human VM development. These results provide a unique transcriptional profile of the developing human fetal VM and functionally mature human DA neurons, which can be used to guide stem cell-based therapies and disease modeling approaches in PD.
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| 8. |
- Björklund, Anders, et al.
(författare)
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A Combined α-Synuclein/Fibril (SynFib) Model of Parkinson-Like Synucleinopathy Targeting the Nigrostriatal Dopamine System
- 2022
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Ingår i: Journal of Parkinson's Disease. - 1877-718X. ; 12:8, s. 2307-2320
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Forskningsöversikt (refereegranskat)abstract
- Injections of pre-formed α-synuclein fibrils (PFFs) or overexpression of α-synuclein using AAV vectors are commonly used as models of Parkinson-like synucleinopathy in rats and mice. In the modified method reviewed here, the "SynFib" model, the PFFs and the AAV vector are administered together unilaterally into the substantia nigra. This approach combines the key features of these two models, i.e., the generation of toxic α-synuclein aggregates and Lewy body-like inclusions, in combination with the increased vulnerability caused by increased cellular levels of α-synuclein. The combined AAV/PFF delivery offers several advantages over the standard PFF model due to the enhanced and accelerated α-synuclein pathology and microglial response induced by the PFF seeds in the presence of an elevated α-synuclein level. Injection of the AAV/PFF mixture into the substantia nigra makes it possible to target a larger proportion of the nigral dopamine neurons and obtain a level of dopamine cell loss (>60%) needed to induce significant impairments in drug-induced and spontaneous motor tests. The SynFib model shares attractive features of the standard 6-OHDA lesion model: a single unilateral stereotaxic intervention; pathology and cell loss developing over a short time span; and the possibility to monitor the degenerative changes using tests of motor behavior.
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| 9. |
- Björklund, Anders, et al.
(författare)
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Dopamine Cell Therapy : From Cell Replacement to Circuitry Repair
- 2021
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Ingår i: Journal of Parkinson's Disease. - 1877-718X. ; 11:s2, s. 159-165
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Forskningsöversikt (refereegranskat)abstract
- Cell therapy for Parkinson's disease (PD) is aimed to replace the degenerated midbrain dopamine (mDA) neurons and restore DA neurotransmission in the denervated forebrain targets. A limitation of the intrastriatal grafting approach, which is currently used in clinical trials, is that the mDA neurons are implanted into the target area, in most cases the putamen, and not in the ventral midbrain where they normally reside. This ectopic location of the cells may limit their functionality due to the lack of appropriate afferent regulation from the host. Homotopic transplantation, into the substantia nigra, is now being pursued in rodent PD models as a way to achieve more complete circuitry repair. Intranigral grafts of mDA neurons, derived from human embryonic stem cells, have the capacity to re-establish the nigrostriatal and mesolimbic pathways in their entirety and restore dense functional innervations in striatal, limbic and cortical areas. Tracing of host afferent inputs using the rabies tracing technique shows that the afferent connectivity of grafts implanted in the nigra matches closely that of the intrinsic mDA system, suggesting a degree of circuitry reconstruction that exceeds what has been achieved before. This approach holds great promise, but to match the larger size of the human brain, and the 10 times greater distance between substantia nigra and its forebrain targets, it may be necessary to find ways to improve the growth capacity of the grafted mDA neurons, pointing to a combined approach where growth promoting factors are used to enhance the performance of mDA neuron grafts.
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| 10. |
- Björklund, Anders, et al.
(författare)
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Neuronal Replacement as a Tool for Basal Ganglia Circuitry Repair : 40 Years in Perspective
- 2020
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Ingår i: Frontiers in Cellular Neuroscience. - : Frontiers Media SA. - 1662-5102. ; 14, s. 146-146
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Forskningsöversikt (refereegranskat)abstract
- The ability of new neurons to promote repair of brain circuitry depends on their capacity to re-establish afferent and efferent connections with the host. In this review article, we give an overview of past and current efforts to restore damaged connectivity in the adult mammalian brain using implants of fetal neuroblasts or stem cell-derived neuronal precursors, with a focus on strategies aimed to repair damaged basal ganglia circuitry induced by lesions that mimic the pathology seen in humans affected by Parkinson's or Huntington's disease. Early work performed in rodents showed that neuroblasts obtained from striatal primordia or fetal ventral mesencephalon can become anatomically and functionally integrated into lesioned striatal and nigral circuitry, establish afferent and efferent connections with the lesioned host, and reverse the lesion-induced behavioral impairments. Recent progress in the generation of striatal and nigral progenitors from pluripotent stem cells have provided compelling evidence that they can survive and mature in the lesioned brain and re-establish afferent and efferent axonal connectivity with a remarkable degree of specificity. The studies of cell-based circuitry repair are now entering a new phase. The introduction of genetic and virus-based techniques for brain connectomics has opened entirely new possibilities for studies of graft-host integration and connectivity, and the access to more refined experimental techniques, such as chemo- and optogenetics, has provided new powerful tools to study the capacity of grafted neurons to impact the function of the host brain. Progress in this field will help to guide the efforts to develop therapeutic strategies for cell-based repair in Huntington's and Parkinson's disease and other neurodegenerative conditions involving damage to basal ganglia circuitry.
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| 11. |
- Brambach, Max, et al.
(författare)
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Neural tube patterning : from a minimal model for rostrocaudal patterning toward an integrated 3D model
- 2021
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Ingår i: iScience. - : Elsevier BV. - 2589-0042. ; 24:6
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Tidskriftsartikel (refereegranskat)abstract
- Rostrocaudal patterning of the neural tube is a defining event in vertebrate brain development. This process is driven by morphogen gradients which specify the fate of neural progenitor cells, leading to the partitioning of the tube. Although this is extensively studied experimentally, an integrated view of the genetic circuitry is lacking. Here, we present a minimal gene regulatory model for rostrocaudal patterning, whose tristable topology was determined in a data-driven way. Using this model, we identified the repression of hindbrain fate as promising strategy for the improvement of current protocols for the generation of dopaminergic neurons. Furthermore, we combined our model with an established minimal model for dorsoventral patterning on a realistic 3D neural tube and found that key features of neural tube patterning could be recapitulated. Doing so, we demonstrate how data and models from different sources can be combined to simulate complex in vivo processes.
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| 12. |
- Elabi, Osama F, et al.
(författare)
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Human Embryonic Stem Cell-Derived Dopaminergic Grafts Alleviate L-DOPA Induced Dyskinesia
- 2022
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Ingår i: Journal of Parkinson's Disease. - 1877-718X. ; 12:6, s. 1881-1896
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: First-in-human studies to test the efficacy and safety of human embryonic stem cells (hESC)-derived dopaminergic cells in the treatment of Parkinson's disease (PD) are imminent. Pre-clinical studies using hESC-derived dopamine neuron transplants in rat models have indicated that the benefits parallel those shown with fetal tissue but have thus far failed to consider how ongoing L-DOPA administration might impact on the graft.OBJECTIVE: To determine whether L-DOPA impacts on survival and functional recovery following grafting of hESC-derived dopaminergic neurons.METHODS: Unilateral 6-OHDA lesioned rats were administered with either saline or L-DOPA prior to, and for 18 weeks following surgical implantation of dopaminergic neural progenitors derived from RC17 hESCs according to two distinct protocols in independent laboratories.RESULTS: Grafts from both protocols elicited reduction in amphetamine-induced rotations. Reduced L-DOPA-induced dyskinesia preceded the improvement in amphetamine-induced rotations. Furthermore, L-DOPA had no effect on overall survival (HuNu) or dopaminergic neuron content of the graft (TH positive cells) but did lead to an increase in the number of GIRK2 positive neurons.CONCLUSION: Critically, we found that L-DOPA was not detrimental to graft function, potentially enhancing graft maturation and promoting an A9 phenotype. Early improvement of L-DOPA-induced dyskinesia suggests that grafts may support the handling of exogenously supplied dopamine earlier than improvements in amphetamine-induced behaviours indicate. Given that one of the protocols will be employed in the production of cells for the European STEM-PD clinical trial, this is vital information for the management of patients and achieving optimal outcomes following transplantation of hESC-derived grafts for PD.
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| 13. |
- Fiorenzano, Alessandro, et al.
(författare)
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Dopamine Neuron Diversity : Recent Advances and Current Challenges in Human Stem Cell Models and Single Cell Sequencing
- 2021
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Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 10:6
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Forskningsöversikt (refereegranskat)abstract
- Human midbrain dopamine (DA) neurons are a heterogeneous group of cells that share a common neurotransmitter phenotype and are in close anatomical proximity but display different functions, sensitivity to degeneration, and axonal innervation targets. The A9 DA neuron subtype controls motor function and is primarily degenerated in Parkinson's disease (PD), whereas A10 neurons are largely unaffected by the condition, and their dysfunction is associated with neuropsychiatric disorders. Currently, DA neurons can only be reliably classified on the basis of topographical features, including anatomical location in the midbrain and projection targets in the forebrain. No systematic molecular classification at the genome-wide level has been proposed to date. Although many years of scientific efforts in embryonic and adult mouse brain have positioned us to better understand the complexity of DA neuron biology, many biological phenomena specific to humans are not amenable to being reproduced in animal models. The establishment of human cell-based systems combined with advanced computational single-cell transcriptomics holds great promise for decoding the mechanisms underlying maturation and diversification of human DA neurons, and linking their molecular heterogeneity to functions in the midbrain. Human pluripotent stem cells have emerged as a useful tool to recapitulate key molecular features of mature DA neuron subtypes. Here, we review some of the most recent advances and discuss the current challenges in using stem cells, to model human DA biology. We also describe how single cell RNA sequencing may provide key insights into the molecular programs driving DA progenitor specification into mature DA neuron subtypes. Exploiting the state-of-the-art approaches will lead to a better understanding of stem cell-derived DA neurons and their use in disease modeling and regenerative medicine.
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| 14. |
- Fiorenzano, Alessandro, et al.
(författare)
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Evaluation of TH-Cre knock-in cell lines for detection and specific targeting of stem cell-derived dopaminergic neurons
- 2021
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Ingår i: Heliyon. - : Elsevier BV. - 2405-8440. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- The focal and progressive degeneration of dopaminergic (DA) neurons in ventral midbrain has made Parkinson's disease (PD) a particularly interesting target of cell-based therapies. However, ethical issues and limited tissue availability have so far hindered the widespread use of human fetal tissue in cell-replacement therapy. DA neurons derived from human pluripotent stem cells (hPSCs) offer unprecedented opportunities to access a renewable source of cells suitable for PD therapeutic applications. To better understand the development and functional properties of stem-cell derived DA neurons, we generated targeted hPSC lines with the gene coding for Cre recombinase knocked into the TH locus. When combined with flexed GFP, they serve as reporter cell lines able to identify and isolate TH+ neurons in vitro and after transplantation in vivo. These TH-Cre lines provide a valuable genetic tool to manipulate DA neurons useful for the design of more precise DA differentiation protocols and the study of these cells after transplantation in pre-clinical animal models of PD.
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| 15. |
- Fiorenzano, Alessandro, et al.
(författare)
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Single-cell transcriptomics captures features of human midbrain development and dopamine neuron diversity in brain organoids
- 2021
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Three-dimensional brain organoids have emerged as a valuable model system for studies of human brain development and pathology. Here we establish a midbrain organoid culture system to study the developmental trajectory from pluripotent stem cells to mature dopamine neurons. Using single cell RNA sequencing, we identify the presence of three molecularly distinct subtypes of human dopamine neurons with high similarity to those in developing and adult human midbrain. However, despite significant advancements in the field, the use of brain organoids can be limited by issues of reproducibility and incomplete maturation which was also observed in this study. We therefore designed bioengineered ventral midbrain organoids supported by recombinant spider-silk microfibers functionalized with full-length human laminin. We show that silk organoids reproduce key molecular aspects of dopamine neurogenesis and reduce inter-organoid variability in terms of cell type composition and dopamine neuron formation.
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| 16. |
- Giacomoni, Jessica, et al.
(författare)
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Protocol for optical clearing and imaging of fluorescently labeled ex vivo rat brain slices
- 2023
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Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 4:1
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Tidskriftsartikel (refereegranskat)abstract
- Tissue clearing is commonly used for whole-brain imaging but seldom used for brain slices. Here, we present a simple protocol to slice, immunostain, and clear sections of adult rat brains for subsequent high-resolution confocal imaging. The protocol does not require toxic reagents or specialized equipment. We also provide instructions for culturing of rat brain slices free floating on permeable culture inserts, maintained in regular CO2 incubators, and handled only at media change.
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| 17. |
- Grassi, Daniela A., et al.
(författare)
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Profiling of lincRNAs in human pluripotent stem cell derived forebrain neural progenitor cells
- 2020
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Ingår i: Heliyon. - : Elsevier BV. - 2405-8440. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can be differentiated into many different cell types of the central nervous system. One challenge when using pluripotent stem cells is to develop robust and efficient differentiation protocols that result in homogenous cultures of the desired cell type. Here, we have utilized the SMAD-inhibitors SB431542 and Noggin in a fully defined monolayer culture model to differentiate human pluripotent cells into homogenous forebrain neural progenitors. Temporal fate analysis revealed that this protocol results in forebrain-patterned neural progenitor cells that start to express early neuronal markers after two weeks of differentiation, allowing for the analysis of gene expression changes during neurogenesis. Using this system, we were able to identify many previously uncharacterized long intergenic non-coding RNAs that display dynamic expression during human forebrain neurogenesis. Cell biology; Genetics; Neuroscience; Developmental genetics; Cellular neuroscience; lincRNAs; Forebrain development; Induced pluripotent stem cells; Neural progenitor cells; Differentiation
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| 18. |
- Hoban, Deirdre B, et al.
(författare)
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Impact of α-synuclein pathology on transplanted hESC-derived dopaminergic neurons in a humanized α-synuclein rat model of PD
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490. ; 117:26, s. 15209-15220
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Tidskriftsartikel (refereegranskat)abstract
- Preclinical assessment of the therapeutic potential of dopamine (DA) neuron replacement in Parkinson's disease (PD) has primarily been performed in the 6-hydroxydopamine toxin model. While this is a good model to assess graft function, it does not reflect the pathological features or progressive nature of the disease. In this study, we establish a humanized transplantation model of PD that better recapitulates the main disease features, obtained by coinjection of preformed human α-synuclein (α-syn) fibrils and adeno-associated virus (AAV) expressing human wild-type α-syn unilaterally into the rat substantia nigra (SN). This model gives rise to DA neuron dysfunction and progressive loss of DA neurons from the SN and terminals in the striatum, accompanied by extensive α-syn pathology and a prominent inflammatory response, making it an interesting and relevant model in which to examine long-term function and integrity of transplanted neurons in a PD-like brain. We transplanted DA neurons derived from human embryonic stem cells (hESCs) into the striatum and assessed their survival, growth, and function over 6 to 18 wk. We show that the transplanted cells, even in the presence of ongoing pathology, are capable of innervating the DA-depleted striatum. However, on closer examination of the grafts, we found evidence of α-syn pathology in the form of inclusions of phosphorylated α-syn in a small fraction of the grafted DA neurons, indicating host-to-graft transfer of α-syn pathology, a phenomenon that has previously been observed in PD patients receiving fetal tissue grafts but has not been possible to demonstrate and study in toxin-based animal models.
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| 19. |
- Kajtez, Janko, et al.
(författare)
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3D biomaterial models of human brain disease
- 2021
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Ingår i: Neurochemistry International. - : Elsevier BV. - 0197-0186. ; 147
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Tidskriftsartikel (refereegranskat)abstract
- Inherent limitations of the traditional approaches to study brain function and disease, such as rodent models and 2D cell culture platforms, have led to the development of 3D in vitro cell culture systems. These systems, products of multidisciplinary efforts encompassing stem cell biology, materials engineering, and biofabrication, have quickly shown great potential to mimic biochemical composition, structural properties, and cellular morphology and diversity found in the native brain tissue. Crucial to these developments have been the advancements in stem cell technology and cell reprogramming protocols that allow reproducible generation of human subtype-specific neurons and glia in laboratory conditions. At the same time, biomaterials have been designed to provide cells in 3D with a microenvironment that mimics functional and structural aspects of the native extracellular matrix with increasing fidelity. In this article, we review the use of biomaterials in 3D in vitro models of neurological disorders with focus on hydrogel technology and with biochemical composition and physical properties of the in vivo environment as reference.
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| 20. |
- Kajtez, Janko, et al.
(författare)
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3D-Printed Soft Lithography for Complex Compartmentalized Microfluidic Neural Devices
- 2020
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Ingår i: Advanced Science. - : Wiley. - 2198-3844. ; 7:16
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Tidskriftsartikel (refereegranskat)abstract
- Compartmentalized microfluidic platforms are an invaluable tool in neuroscience research. However, harnessing the full potential of this technology remains hindered by the lack of a simple fabrication approach for the creation of intricate device architectures with high-aspect ratio features. Here, a hybrid additive manufacturing approach is presented for the fabrication of open-well compartmentalized neural devices that provides larger freedom of device design, removes the need for manual postprocessing, and allows an increase in the biocompatibility of the system. Suitability of the method for multimaterial integration allows to tailor the device architecture for the long-term maintenance of healthy human stem-cell derived neurons and astrocytes, spanning at least 40 days. Leveraging fast-prototyping capabilities at both micro and macroscale, a proof-of-principle human in vitro model of the nigrostriatal pathway is created. By presenting a route for novel materials and unique architectures in microfluidic systems, the method provides new possibilities in biological research beyond neuroscience applications.
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| 21. |
- Kajtez, Janko, et al.
(författare)
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Embedded 3D Printing in Self-Healing Annealable Composites for Precise Patterning of Functionally Mature Human Neural Constructs
- 2022
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Ingår i: Advanced science (Weinheim, Baden-Wurttemberg, Germany). - : Wiley. - 2198-3844. ; 9:25
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Tidskriftsartikel (refereegranskat)abstract
- Human in vitro models of neural tissue with tunable microenvironment and defined spatial arrangement are needed to facilitate studies of brain development and disease. Towards this end, embedded printing inside granular gels holds great promise as it allows precise patterning of extremely soft tissue constructs. However, granular printing support formulations are restricted to only a handful of materials. Therefore, there has been a need for novel materials that take advantage of versatile biomimicry of bulk hydrogels while providing high-fidelity support for embedded printing akin to granular gels. To address this need, Authors present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside Self-Healing Annealable Particle-Extracellular matrix (SHAPE) composites. SHAPE composites consist of soft microgels immersed in viscous extracellular-matrix solution to enable precise and programmable patterning of human stem cells and consequent generation mature subtype-specific neurons that extend projections into the volume of the annealed support. The developed approach further allows multi-ink deposition, live spatial and temporal monitoring of oxygen levels, as well as creation of vascular-like channels. Due to its modularity and versatility, SHAPE biomanufacturing toolbox has potential to be used in applications beyond functional modeling of mechanically sensitive neural constructs.
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| 22. |
- Kirkeby, Agnete, et al.
(författare)
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Preclinical quality, safety, and efficacy of a human embryonic stem cell-derived product for the treatment of Parkinson's disease, STEM-PD
- 2023
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Ingår i: Cell Stem Cell. - 1934-5909. ; 30:10, s. 1299-1314
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Tidskriftsartikel (refereegranskat)abstract
- Cell replacement therapies for Parkinson's disease (PD) based on transplantation of pluripotent stem cell-derived dopaminergic neurons are now entering clinical trials. Here, we present quality, safety, and efficacy data supporting the first-in-human STEM-PD phase I/IIa clinical trial along with the trial design. The STEM-PD product was manufactured under GMP and quality tested in vitro and in vivo to meet regulatory requirements. Importantly, no adverse effects were observed upon testing of the product in a 39-week rat GLP safety study for toxicity, tumorigenicity, and biodistribution, and a non-GLP efficacy study confirmed that the transplanted cells mediated full functional recovery in a pre-clinical rat model of PD. We further observed highly comparable efficacy results between two different GMP batches, verifying that the product can be serially manufactured. A fully in vivo-tested batch of STEM-PD is now being used in a clinical trial of 8 patients with moderate PD, initiated in 2022.
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| 23. |
- Mattsson, Bengt, et al.
(författare)
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Athymic Nude Rat Brain Atlas
- 2023
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Bok (refereegranskat)abstract
- Athymic Nude Rat Brain Atlas will provide the first stereotaxic brain atlas of the athymic nude rat (Crl:NIH-Foxn1rnu), a T-cell deficient rat model commonly used in experimental studies and pre-clinical safety and efficacy studies. This 2D vector-based atlas contains coronal, sagittal, and horizontal brain sections of an athymic rat brain rendered from a single cleared specimen, placed in a computerized 3D environment. The maps enable readers to better calculate co-ordinates to target specific structures for toxin, virus, or cell delivery using stereotaxic surgery. This atlas will be a valuable resource for any neuroscientist who wishes to work with nude rats in experimental and pre-clinical studies.
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| 24. |
- Nilsson, Fredrik, et al.
(författare)
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Single-Cell Profiling of Coding and Noncoding Genes in Human Dopamine Neuron Differentiation
- 2021
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Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Dopaminergic (DA) neurons derived from human pluripotent stem cells (hPSCs) represent a renewable and available source of cells useful for understanding development, developing disease models, and stem-cell therapies for Parkinson's disease (PD). To assess the utility of stem cell cultures as an in vitro model system of human DA neurogenesis, we performed high-throughput transcriptional profiling of ~20,000 ventral midbrain (VM)-patterned stem cells at different stages of maturation using droplet-based single-cell RNA sequencing (scRNAseq). Using this dataset, we defined the cellular composition of human VM cultures at different timepoints and found high purity DA progenitor formation at an early stage of differentiation. DA neurons sharing similar molecular identities to those found in authentic DA neurons derived from human fetal VM were the major cell type after two months in culture. We also developed a bioinformatic pipeline that provided a comprehensive long noncoding RNA landscape based on temporal and cell-type specificity, which may contribute to unraveling the intricate regulatory network of coding and noncoding genes in DA neuron differentiation. Our findings serve as a valuable resource to elucidate the molecular steps of development, maturation, and function of human DA neurons, and to identify novel candidate coding and noncoding genes driving specification of progenitors into functionally mature DA neurons.
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| 25. |
- Nolbrant, Sara, et al.
(författare)
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Direct Reprogramming of Human Fetal- and Stem Cell-Derived Glial Progenitor Cells into Midbrain Dopaminergic Neurons
- 2020
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Ingår i: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 15:4, s. 869-882
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Tidskriftsartikel (refereegranskat)abstract
- Human glial progenitor cells (hGPCs) are promising cellular substrates to explore for the in situ production of new neurons for brain repair. Proof of concept for direct neuronal reprogramming of glial progenitors has been obtained in mouse models in vivo, but conversion using human cells has not yet been demonstrated. Such studies have been difficult to perform since hGPCs are born late during human fetal development, with limited accessibility for in vitro culture. In this study, we show proof of concept of hGPC conversion using fetal cells and also establish a renewable and reproducible stem cell-based hGPC system for direct neural conversion in vitro. Using this system, we have identified optimal combinations of fate determinants for the efficient dopaminergic (DA) conversion of hGPCs, thereby yielding a therapeutically relevant cell type that selectively degenerates in Parkinson's disease. The induced DA neurons show a progressive, subtype-specific phenotypic maturation and acquire functional electrophysiological properties indicative of DA phenotype.
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