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Recent advancements in protocols to differentiate human pluripotent stem cells into midbrain dopaminergic (mDA) neurons has improved the ability to model Parkinson’s disease (PD) in a dish, and has provided a scalable source of donor cells for emerging PD cell replacement therapy (CRT). However, to facilitate reproducibility, collaboration, and clinical trials it would be highly beneficial to cryopreserve committed mDA neural precursors cells in a ready-to-use format. In terms of cell manufacturing for PD CRT trials, a cryopreserved transplantation-ready mDA cell product would provide a critical opportunity for quality control, efficacy testing, and safety assessments. To address this challenge, we have compared six (6) different clinical-grade cryopreservation media and different freezing conditions for mDA neural precursor cells differentiated from two human embryonic stem cell (ESC) lines, MasterShef7 and RC17. Significant differences in cell viability were observed at 24h post-thawing, but no differences were observed immediately upon thawing. This highlights the need to check cell viability over the first 24h after thawing, and that viability of freshly thawed cells is insufficient to gauge the success of a cryopreservation protocol. Considerable apoptosis occurs in the first 24h post-thawing, and significant differences between cryopreservation procedures were only revealed during this time period. The presence of ROCK inhibitors improved cell viability at 24h for all conditions tested. A faster cooling rate of 1-2°C/min was significantly better than 0.5°C/min for all conditions tested, while rapid thawing at 37°C was not always superior to slow thawing at 4°C. Indeed, the optimal cryopreservation and thawing conditions in this study, as determined by 24h post-thaw viability, were cells frozen in PSC Cryopreservation medium at a cooling rate of 1°C/min and slow thawing at 4°C. These conditions permitted recovery of 60%-70% live cells at 24h with respect to the starting number of cryopreserved cells. Importantly, cryopreservation of mDA neural precursor cells did not alter their potential to resume differentiation into mDA neurons.HighlightsFirst systematic comparison of multiple clinical-grade cryopreservation media for human ESC-derived mDA neural precursor cellsDifferences in cell viability were observed at 24h after thawing, but not immediately upon thawingCooling rates of 1°C/min or 2°C/min were significantly better than 0.5°C/min for all cryopreservation conditions testedA slow thawing condition at 4°C was significantly better than quick thawing at 37°C for cells frozen in PSC Cryopreservation mediumCryopreservation of mDA cells does not significantly alter their potential to differentiate into mDA neurons

Excessive reactive oxygen species (ROS) can damage proteins, lipids, and DNA, which result in cell damage and death. The outcomes can be acute, as seen in stroke, or more chronic as observed in age-related diseases such as Parkinson’s disease. Here we investigate the antioxidant ability of a novel synthetic flavonoid, Proxison (7-decyl-3-hydroxy-2-(3,4,5-trihydroxyphenyl)-4-chromenone), using a range ofin vitroandin vivoapproaches. We show that, while it has radical scavenging ability on par with other flavonoids in a cell-free system, Proxison is orders of magnitude more potent than natural flavonoids at protecting neural cells against oxidative stress and is capable of rescuing damaged cells. The unique combination of a lipophilic hydrocarbon tail with a modified polyphenolic head group promotes efficient cellular uptake and mitochondrial localisation of Proxison. Importantly,in vivoadministration of Proxison demonstrated effective and well tolerated neuroprotection against oxidative stress in a zebrafish model of dopaminergic neuronal loss.

Excessive reactive oxygen species (ROS) can damage proteins, lipids, and DNA, which result in cell damage and death. The outcomes can be acute, as seen in stroke, or more chronic as observed in age-related diseases such as Parkinson’s disease. Here we investigate the antioxidant ability of a novel synthetic flavonoid, Proxison (7-decyl-3-hydroxy-2-(3,4,5-trihydroxyphenyl)-4-chromenone), using a range of in vitro and in vivo approaches. We show that, while it has radical scavenging ability on par with other flavonoids in a cell-free system, Proxison is orders of magnitude more potent than natural flavonoids at protecting neural cells against oxidative stress and is capable of rescuing damaged cells. The unique combination of a lipophilic hydrocarbon tail with a modified polyphenolic head group promotes efficient cellular uptake and moderate mitochondrial enrichment of Proxison. Importantly, in vivo administration of Proxison demonstrated effective and well tolerated neuroprotection against cell loss in a zebrafish model of dopaminergic neurodegeneration.

Real-time monitoring of stem cells (SCs) differentiation will be critical to scale-up SC technologies, while label-free techniques will be desirable to quality-control SCs without precluding their therapeutic potential. We cultured adipose-derived stem cells (ADSCs) on top of multielectrode arrays and measured variations in the complex impedance Z* throughout induction of ADSCs toward osteoblasts and adipocytes. Z* was measured up to 17 d, every 180 s, over a 62.5–64kHz frequency range with an ECIS Zθ instrument. We found that osteogenesis and adipogenesis were characterized by distinct Z* time-courses. Significant differences were found ( P = 0.007) as soon as 12 h post induction. An increase in the barrier resistance (Rb) up to 1.7 ohm·cm 2 was associated with early osteo-induction, whereas Rb peaked at 0.63 ohm·cm 2 for adipo-induced cells before falling to zero at t = 129 h. Dissimilarities in Z* throughout early induction (2 ) was significantly different from that of adipo-induced cells (Cm = 2.25 ± 0.27 μF/cm 2 ), indicating that Cm could be used as an early marker of differentiation. Finally, we demonstrated long-term monitoring and measured a shift in the complex plane in the middle frequency range (1 kHz to 8 kHz) between early ( t = 100 h) and late induction ( t = 380 h). This study demonstrated that the osteoblast and adipocyte lineages have distinct dielectric properties and that such differences can be used to perform real-time label-free quantitative monitoring of adult stem cell differentiation with impedance sensing.

A major barrier to research on Parkinson's disease is inaccessibility of diseased tissue for study. One solution is to derive induced pluripotent stem cells from patients and differentiate them into neurons affected by disease. Triplication of SNCA, encoding α-synuclein, causes a fully penetrant, aggressive form of Parkinson's disease with dementia. α-Synuclein dysfunction is the critical pathogenic event in Parkinson's disease, multiple system atrophy and dementia with Lewy bodies. Here we produce multiple induced pluripotent stem cell lines from an SNCA triplication patient and an unaffected first-degree relative. When these cells are differentiated into midbrain dopaminergic neurons, those from the patient produce double the amount of α-synuclein protein as neurons from the unaffected relative, precisely recapitulating the cause of Parkinson's disease in these individuals. This model represents a new experimental system to identify compounds that reduce levels of α-synuclein, and to investigate the mechanistic basis of neurodegeneration caused by α-synuclein dysfunction., Pluripotent stem cells can be generated from the somatic cells of humans and are a useful model to study disease. Here, pluripotent stem cells are made from a patient with familial Parkinson's disease, and the resulting neurons exhibit elevated levels of α-synuclein, recapitulating the molecular features of the patient's disease.

Cell viability assays are essential tools for cell biology. They assess healthy cells in a sample and enable the quantification of cellular responses to reagents of interest. Noninvasive and label-free assays are desirable in two-dimensional (2D) and three-dimensional (3D) cell culture to facilitate time-course viability studies. Cellular micromotion, emanating from cell to substrate distance variations, has been demonstrated as a marker of cell viability with electric cell-substrate impedance sensing (ECIS). In this study we investigated if optical coherence phase microscopy (OCPM) was able to report phase fluctuations of adult stem cells in 2D and 3D that could be associated with cellular micromotion. An OCPM has been developed around a Thorlabs engine (λo = 930 nm) and integrated in an inverted microscope with a custom scanning head. Human adipose derived stem cells (ADSCs, Invitrogen) were cultured in Mesenpro RS medium and seeded either on ECIS arrays, 2D cell culture dishes, or in 3D highly porous microplotted polymeric scaffolds. ADSC micromotion was confirmed by ECIS analysis. Live and fixed ADSCs were then investigated in 2D and 3D with OCPM. Significant differences were found in phase fluctuations between the different conditions. This study indicated that OCPM could potentially assess cell vitality in 2D and in 3D microstructures.