| 1. |
- Birtele, Marcella, et al.
(author)
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Dual modulation of neuron-specific microRNAs and the REST complex promotes functional maturation of human adult induced neurons
- 2019
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In: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 593:23, s. 3370-3380
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Journal article (peer-reviewed)abstract
- Direct neuronal reprogramming can be achieved using different approaches: by expressing neuronal transcription factors or microRNAs; and by knocking down neuronal repressive elements. However, there still exists a high variability in terms of the quality and maturity of the induced neurons obtained, depending on the reprogramming strategy employed. Here, we evaluate different long-term culture conditions and study the effect of expressing the neuronal-specific microRNAs, miR124 and miR9/9*, while reprogramming with forced expression of the transcription factors Ascl1, Brn2, and knockdown of the neuronal repressor REST. We show that the addition of microRNAs supports neuronal maturation in terms of gene and protein expression, as well as in terms of electrophysiological properties.
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| 2. |
- Birtele, Marcella
(author)
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Functional and Transcriptional Studies of Human Dopaminergic Neurons
- 2020
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Doctoral thesis (other academic/artistic)abstract
- Parkinson’s Disease (PD) is the most common movement disorder and second most common neurodegenerative disease. The principal hallmark of the pathology is represented by a loss of mesencephalic Dopaminergic neurons (mesDA) that reside in the Substantia Nigra pars compacta (SNpc). Another feature of the disease is represented by formation of abnormal protein aggregates, known as Lewy Bodies (LBs), mainly composed by the a-synuclein protein. The etiology of mesDA death is still unknown, however LBs formation could represent one of the factor contributing to neuronal mesDA death and PD progression.Cell Replacement Therapy for PD aims at restoring the function of the dopaminergic neurons through the transplantation of the lost cells in the brain. Recently, cell sources derived from stem cells such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC) have been investigated and implicated in clinical trials for PD. Another route for generating neurons is represented by the direct reprogramming of terminally differentiated cells. With the overexpression of specific transcription factors (TFs) and/or micro RNA (miRNA) is possible to target somatic cells in vitro or resident brain cells in vivo for reprogramming into mesDA neurons.The overall aim of my thesis has been to study functional and transcriptional profile of newly generated mesDA neurons in vitro and in vivo for cell-based therapies of PD. Indeed the transplantation outcome depends on the ability to generate mesDA neurons that are as similar as possible to the endogenous DA neurons. However, our knowledge of human DA neurons is limited by the inaccessibility of developing and adult brain tissues. In the first part of my thesis I focused on studying the properties of directly reprogrammed cells to determine their phenotypic and functional profile. In the second part of this thesis, I performed an extensive molecular, transcriptional and functional analysis of human fetal mesDA neurons to increase our understanding of DA neurons. Lastly, I focused on establishing a stem cell derived organoid system that allowed for the generation of authentic human DA neurons.
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| 3. |
- Birtele, Marcella, et al.
(author)
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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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In: Development: For advances in developmental biology and stem cells. - : The Company of Biologists. - 1477-9129. ; 149:23
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Journal article (peer-reviewed)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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| 4. |
- Drouin-Ouellet, Janelle, et al.
(author)
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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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In: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 17:10, s. 2203-2219
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Journal article (peer-reviewed)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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| 5. |
- Fiorenzano, Alessandro, et al.
(author)
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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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In: Heliyon. - : Elsevier BV. - 2405-8440. ; 7:1
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Journal article (peer-reviewed)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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| 6. |
- Fiorenzano, Alessandro, et al.
(author)
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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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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 12:1
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Journal article (peer-reviewed)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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| 7. |
- Kajtez, Janko, et al.
(author)
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3D-Printed Soft Lithography for Complex Compartmentalized Microfluidic Neural Devices
- 2020
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In: Advanced Science. - : Wiley. - 2198-3844. ; 7:16
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Journal article (peer-reviewed)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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| 8. |
- Kajtez, Janko, et al.
(author)
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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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In: Advanced science (Weinheim, Baden-Wurttemberg, Germany). - : Wiley. - 2198-3844. ; 9:25
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Journal article (peer-reviewed)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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| 9. |
- Nilsson, Fredrik, et al.
(author)
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Single-Cell Profiling of Coding and Noncoding Genes in Human Dopamine Neuron Differentiation
- 2021
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In: Cells. - : MDPI AG. - 2073-4409. ; 10:1
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Journal article (peer-reviewed)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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| 10. |
- Nolbrant, Sara, et al.
(author)
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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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In: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 15:4, s. 869-882
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Journal article (peer-reviewed)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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