Evaluation of TH-Cre knock-in cell lines for detection and specific targeting of stem cell-derived dopaminergic neurons

Fiorenzano, Alessandro; Birtele, Marcella; Wahlestedt, Jenny Nelander; Parmar, Malin

doi:10.1016/j.heliyon.2021.e06006

Cited by 8 publications

(6 citation statements)

References 18 publications

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“…Detailed protocols to generate human VM organoids, however, are still lacking. Moreover, modeling human VM in vitro requires the precise balance of patterning factors that lead to the generation of DA neurons exhibiting both mature molecular features and electrophysiological properties, which is not trivial (Nolbrant, Heuer, Parmar, & Kirkeby, 2017;Tiklova et al, 2020;Fiorenzano, Birtele, Wahlestedt, & Parmar, 2021;Fiorenzano et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Generation of Human Ventral Midbrain Organoids Derived from Pluripotent Stem Cells

et al. 2022

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Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide and is caused by the degeneration and loss of dopamine (DA) neurons in the ventral midbrain (VM). The focal and progressive degeneration of DA neurons in the VM makes PD a particularly attractive target for cell‐based therapies. Human pluripotent stem cells (hPSCs) offer unprecedented opportunities to model the development and functional properties of human DA neurons in a dish. The use of human in vitro models based on hPSCs has empowered studies of VM development and provided access to neurons expressing a particular disease‐specific phenotype. Currently, hPSC differentiation is most routinely carried out in monolayer cultures, which do not properly recapitulate cell‐cell interactions and the structural complexity of the brain. Moreover, 2D cultures are challenging to maintain long term, as the cells tend to detach from the plate and lose their functional characteristics. This precludes the possibility of mimicking later phases of DA neurogenesis and recreating the complexity of functional neural circuitries. Here, we describe protocols showing how to maintain hPSCs in an undifferentiated state and how to then drive these hPSCs into 3D regionalized VM organoids. After long‐term culture, these VM organoids exhibit mature and post‐mitotic molecular features, including neuromelanin pigments similar to those released in primate VMs. We also report a protocol describing how to efficiently perform immunohistochemistry and how to detect neuromelanin‐containing DA neurons in VM organoids. Together, these protocols provide a 3D in vitro platform that can be used to better understand the molecular mechanisms underlying DA neuron function and disease and may serve as a powerful tool for designing more targeted disease‐modifying therapies. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Human pluripotent stem cell culture Basic Protocol 2: hPS cell differentiation for the generation of human ventral midbrain organoids Basic Protocol 3: Characterization of ventral midbrain organoids

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Section: Introductionmentioning

confidence: 99%

Generation of Human Ventral Midbrain Organoids Derived from Pluripotent Stem Cells

et al. 2022

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“…To test whether MISCOs could be used as a human-specific in vitro model to study aspects relevant for Parkinson's disease cell replacement therapy approaches, we injected ventral midbrain progenitors into MISCOs. We used ventral midbrain progenitors with TH-positive cells being genetically labeled with GFP using lentiviral transductions into a TH-Cre reporter line as previously described 53 , and injected 40,000 progenitors into the ventral midbrain tissue 25 (Fig. 5a and Extended Data Fig.…”

Section: Miscos As a Platform For Parkinson's Disease Cell Replacemen...mentioning

confidence: 99%

In vitro modeling of the human dopaminergic system using spatially arranged ventral midbrain–striatum–cortex assembloids

Reumann,

Krauditsch,

Novatchkova

et al. 2023

Nat Methods

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Ventral midbrain dopaminergic neurons project to the striatum as well as the cortex and are involved in movement control and reward-related cognition. In Parkinson’s disease, nigrostriatal midbrain dopaminergic neurons degenerate and cause typical Parkinson’s disease motor-related impairments, while the dysfunction of mesocorticolimbic midbrain dopaminergic neurons is implicated in addiction and neuropsychiatric disorders. Study of the development and selective neurodegeneration of the human dopaminergic system, however, has been limited due to the lack of an appropriate model and access to human material. Here, we have developed a human in vitro model that recapitulates key aspects of dopaminergic innervation of the striatum and cortex. These spatially arranged ventral midbrain–striatum–cortical organoids (MISCOs) can be used to study dopaminergic neuron maturation, innervation and function with implications for cell therapy and addiction research. We detail protocols for growing ventral midbrain, striatal and cortical organoids and describe how they fuse in a linear manner when placed in custom embedding molds. We report the formation of functional long-range dopaminergic connections to striatal and cortical tissues in MISCOs, and show that injected, ventral midbrain-patterned progenitors can mature and innervate the tissue. Using these assembloids, we examine dopaminergic circuit perturbations and show that chronic cocaine treatment causes long-lasting morphological, functional and transcriptional changes that persist upon drug withdrawal. Thus, our method opens new avenues to investigate human dopaminergic cell transplantation and circuitry reconstruction as well as the effect of drugs on the human dopaminergic system.

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“…To establish the bioengineered human brain organoid platform, we used a silk scaffold generated via a simple, easily adaptable, and reproducible approach (overall methodological concept presented in Figure 1A). The recombinant spider silk (Widhe et al, 2016) scaffold was biofunctionalized with full length human laminin 111, an important extracellular matrix protein that provides cell-adhesion motifs for stable neuronal adhesion, expansion, and maturation (Kirkeby et al, 2017;Fiorenzano et al, 2021a;Fiorenzano et al, 2021b). To form the scaffolding, air bubbles were introduced into droplets of ice-cold silk protein solution via repeated pipetting (Figures 1B,C).…”

Section: Generation Of Silk Cerebral Organoidsmentioning

confidence: 99%

Silk scaffolding drives self-assembly of functional and mature human brain organoids

Sozzi

Kajtez

Bruzelius

et al. 2022

Front. Cell Dev. Biol.

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Human pluripotent stem cells (hPSCs) are intrinsically able to self-organize into cerebral organoids that mimic features of developing human brain tissue. These three-dimensional structures provide a unique opportunity to generate cytoarchitecture and cell-cell interactions reminiscent of human brain complexity in a dish. However, current in vitro brain organoid methodologies often result in intra-organoid variability, limiting their use in recapitulating later developmental stages as well as in disease modeling and drug discovery. In addition, cell stress and hypoxia resulting from long-term culture lead to incomplete maturation and cell death within the inner core. Here, we used a recombinant silk microfiber network as a scaffold to drive hPSCs to self-arrange into engineered cerebral organoids. Silk scaffolding promoted neuroectoderm formation and reduced heterogeneity of cellular organization within individual organoids. Bulk and single cell transcriptomics confirmed that silk cerebral organoids display more homogeneous and functionally mature neuronal properties than organoids grown in the absence of silk scaffold. Furthermore, oxygen sensing analysis showed that silk scaffolds create more favorable growth and differentiation conditions by facilitating the delivery of oxygen and nutrients. The silk scaffolding strategy appears to reduce intra-organoid variability and enhances self-organization into functionally mature human brain organoids.

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Evaluation of TH-Cre knock-in cell lines for detection and specific targeting of stem cell-derived dopaminergic neurons

Cited by 8 publications

References 18 publications

Generation of Human Ventral Midbrain Organoids Derived from Pluripotent Stem Cells

Generation of Human Ventral Midbrain Organoids Derived from Pluripotent Stem Cells

In vitro modeling of the human dopaminergic system using spatially arranged ventral midbrain–striatum–cortex assembloids

Silk scaffolding drives self-assembly of functional and mature human brain organoids

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