Cryopreservation of Human Midbrain Dopaminergic Neural Progenitor Cells Poised for Neuronal Differentiation

Drummond, Nicola J.; Dolt, Karamjit Singh; Canham, Maurice A.; Kilbride, Peter; Morris, G.J.; Kunath, Tilo

doi:10.3389/fcell.2020.578907

Cited by 17 publications

(8 citation statements)

References 54 publications

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“…SNCA -/and SNCA +/+ human embryonic stem cells (hESCs) were generated and differentiated towards midbrain dopaminergic neural progenitors to day 16 by the Kunath laboratory as previously described 13 . Following this, the cells were cryopreserved as described 14 and shipped to the Beckham laboratory. Cells were then thawed and plated in Laminin-111(BioLamina)-coated 48-well plates at a density of 800,000 cells/cm 2 in neuronal differentiation media consisting of Neurobasal Media (Thermo Fisher Scientific) + B27 supplement (without Vitamin A, 1:50, Thermo Fisher Scientific) + l-Glutamine (2 mM, Thermo Fisher Scientific) supplemented with ascorbic acid (AA, 0.2 mM, Sigma), brain-derived neurotrophic factor (BDNF, 20 ng/ml, Peprotech), glial cell line-derived neurotrophic factor (GDNF, 10 ng/ml, Peprotech), dibutyryl cyclic AMP (dbcAMP, 0.5 mM, Sigma), and DAPT (1 μM, Tocris).…”

Section: Cell Culturementioning

confidence: 99%

Alpha-synuclein supports type 1 interferon signalling in neurons and brain tissue

Monogue

Chen

Sparks

et al. 2022

Brain

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The protein alpha-synuclein is predominantly expressed in neurons and is associated with neurodegenerative diseases like Parkinson’s disease and dementia with Lewy bodies. However, the normal function of alpha-synuclein in neurons is not clearly defined. We have previously shown that mice lacking alpha-synuclein expression exhibit markedly increased viral growth in the brain, increased mortality and increased neuronal cell death, implicating alpha-synuclein in the neuronal innate immune response. To investigate the mechanism of alpha-synuclein-induced immune responses to viral infections in the brain, we challenged alpha-synuclein knockout mice and human alpha-synuclein knockout dopaminergic neurons with RNA virus infection and discovered that alpha-synuclein is required for neuronal expression of interferon-stimulated genes. Furthermore, human alpha-synuclein knockout neurons treated with type 1 interferon failed to induce a broad range of interferon stimulated genes, implying that alpha-synuclein interacts with type 1 interferon signalling. We next found that alpha-synuclein accumulates in the nucleus of interferon-treated human neurons after interferon treatment and we demonstrated that interferon-mediated phosphorylation of STAT2 is dependent on alpha-synuclein expression in human neurons. Next, we found that activated STAT2 co-localizes with alpha-synuclein following type 1 interferon stimulation in neurons. Finally, we found that brain tissue from patients with viral encephalitis expresses increased levels of phospho-serine129 alpha-synuclein in neurons. Taken together, our results show that alpha-synuclein expression supports neuron-specific interferon responses by localizing to the nucleus, supporting STAT2 activation, co-localizing with phosphorylated STAT2 in neurons and supporting expression of interferon-stimulated genes. These data provide a novel mechanism that links interferon activation and alpha-synuclein function in neurons.

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Section: Cell Culturementioning

confidence: 99%

Alpha-synuclein supports type 1 interferon signalling in neurons and brain tissue

Monogue

Chen

Sparks

et al. 2022

Brain

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“…We have cultured our midbrain organoids up to D200 without any significant hypoxia or necrosis in the core, and longer culture times are likely to be as successful, especially since DNs are generally congregated on the organoid surface where there is more access to nutrients and oxygen. In addition, other published midbrain differentiation protocols have shown successful cryopreservation of DNs at different stages of differentiation ( Drummond et al., 2020 ; Gantner et al., 2020 ; Wakeman et al., 2017 ). Although not yet tested with our protocol, we expect that neurons from dissociated midbrain organoids can be cryopreserved for future use.…”

Section: Expected Outcomesmentioning

confidence: 99%

High-throughput generation of midbrain dopaminergic neuron organoids from reporter human pluripotent stem cells

et al. 2021

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Summary Here, we describe a high-throughput 3D differentiation protocol for deriving midbrain dopaminergic neurons from human pluripotent stem cells. The use of organoids has become prevalent in disease modeling, but there is a high demand for more homogeneous cultures. Our approach is advantageous for large-scale production of uniform midbrain organoids that can be maintained in diverse formats, and our reporters allow for sorting of dopaminergic neurons. The maturing long-term organoid cultures can be used as a model for the entire midbrain. For complete details on the use and execution of this protocol, please refer to Ahfeldt et al. (2020) .

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“…Some cells died during the freeze/thaw procedure due to apoptosis and necrosis [ 21 ], but the survived cells started to proliferate within 24 to 48 h after thawing [ 22–24 ]. Previous report indicated that measuring cell viability at 24 h post-thawing is essential to evaluate the quality and efficiency of a cryopreservation process [ 25 ]. Therefore, we examined the percentage of viable cells (viability) at 24 h, finding that BBK (63±4%) gave a significantly higher percentage than STEM-CELL BANKER DMSO free (SCB DMSO-free; 21±7%) or CryoStor CS5 (CS5; 16±6%) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Cryopreservation of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurospheres for Clinical Application

Hiramatsu

Morizane

Kikuchi

et al. 2022

JPD

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Background: Pluripotent stem cell (PSC)-derived dopaminergic (DA) neurons are an expected source of cell therapy for Parkinson’s disease. The transplantation of cell aggregates or neurospheres, instead of a single cell suspension has several advantages, such as keeping the 3D structure of the donor cells and ease of handling. For this PSC-based therapy to become a widely available treatment, cryopreservation of the final product is critical in the manufacturing process. However, cryopreserving cell aggregates is more complicated than cryopreserving single cell suspensions. Previous studies showed poor survival of the DA neurons after the transplantation of cryopreserved fetal ventral-mesencephalic tissues. Objective: To achieve the cryopreservation of induced pluripotent stem cell (iPSC)-derived DA neurospheres toward clinical application. Methods: We cryopreserved iPSC-derived DA neurospheres in various clinically applicable cryopreservation media and freezing protocols and assessed viability and neurite extension. We evaluated the population and neuronal function of cryopreserved cells by the selected method in vitro. We also injected the cells into 6-hydroxydopamine (6-OHDA) lesioned rats, and assessed their survival, maturation and function in vivo. Results: The iPSC-derived DA neurospheres cryopreserved by Proton Freezer in the cryopreservation medium Bambanker hRM (BBK) showed favorable viability after thawing and had equivalent expression of DA-specific markers, dopamine secretion, and electrophysiological activity as fresh spheres. When transplanted into 6-OHDA-lesioned rats, the cryopreserved cells survived and differentiated into mature DA neurons, resulting in improved abnormal rotational behavior. Conclusion: These results show that the combination of BBK and Proton Freezer is suitable for the cryopreservation of iPSC-derived DA neurospheres.

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Cryopreservation of Human Midbrain Dopaminergic Neural Progenitor Cells Poised for Neuronal Differentiation

Cited by 17 publications

References 54 publications

Alpha-synuclein supports type 1 interferon signalling in neurons and brain tissue

Alpha-synuclein supports type 1 interferon signalling in neurons and brain tissue

High-throughput generation of midbrain dopaminergic neuron organoids from reporter human pluripotent stem cells

Cryopreservation of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurospheres for Clinical Application

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