Emergence of nociceptive functionality and opioid signaling in human induced pluripotent stem cell-derived sensory neurons.

ABSTRACT: Induced pluripotent stem cells (iPSCs) have enabled the generation of various difficult-to-access cell types such as human nociceptors. A key challenge associated with human iPSC-derived nociceptors (hiPSCdNs) is their prolonged functional maturation. While numerous studies have addressed the expression of classic neuronal markers and ion channels in hiPSCdNs, the temporal development of key signaling cascades regulating nociceptor activity has remained largely unexplored. In this study, we used an immunocytochemical high-content imaging approach alongside electrophysiological staging to assess metabotropic and ionotropic signaling of large scale-generated hiPSCdNs across 70 days of in vitro differentiation. During this period, the resting membrane potential became more hyperpolarized, while rheobase, action potential peak amplitude, and membrane capacitance increased. After 70 days, hiPSCdNs exhibited robust physiological responses induced by GABA, pH shift, ATP, and capsaicin. Direct activation of protein kinase A type II (PKA-II) through adenylyl cyclase stimulation with forskolin resulted in PKA-II activation at all time points. Depolarization-induced activation of PKA-II emerged after 35 days of differentiation. However, effective inhibition of forskolin-induced PKA-II activation by opioid receptor agonists required 70 days of in vitro differentiation. Our results identify a pronounced time difference between early expression of functionally important ion channels and emergence of regulatory metabotropic sensitizing and desensitizing signaling only at advanced stages of in vitro cultivation, suggesting an independent regulation of ionotropic and metabotropic signaling. These data are relevant for devising future studies into the development and regulation of human nociceptor function and for defining time windows suitable for hiPSCdN-based drug discovery.

High-content phenotyping of Parkinson's disease patient stem cell-derived midbrain dopaminergic neurons using machine learning classification.

Combining multiple Parkinson's disease (PD) relevant cellular phenotypes might increase the accuracy of midbrain dopaminergic neuron (mDAN) in vitro models. We differentiated patient-derived induced pluripotent stem cells (iPSCs) with a LRRK2 G2019S mutation, isogenic control, and genetically unrelated iPSCs into mDANs. Using automated fluorescence microscopy in 384-well-plate format, we identified elevated levels of α-synuclein (αSyn) and serine 129 phosphorylation, reduced dendritic complexity, and mitochondrial dysfunction. Next, we measured additional image-based phenotypes and used machine learning (ML) to accurately classify mDANs according to their genotype. Additionally, we show that chemical compound treatments, targeting LRRK2 kinase activity or αSyn levels, are detectable when using ML classification based on multiple image-based phenotypes. We validated our approach using a second isogenic patient-derived SNCA gene triplication mDAN model which overexpresses αSyn. This phenotyping and classification strategy improves the practical exploitability of mDANs for disease modeling and the identification of novel LRRK2-associated drug targets.

Dynamics of Metabolic Pathways and Stress Response Patterns during Human Neural Stem Cell Proliferation and Differentiation.

Understanding early nervous system stress response mechanisms is crucial for studying developmental neurotoxicity and devising neuroprotective treatments. We used hiPSC-derived long-term self-renewing neuroepithelial stem (lt-NES) cells differentiated for up to 12 weeks as an in vitro model of human neural development. Following a transcriptome analysis to identify pathway alterations, we induced acute oxidative stress (OS) using tert-butyl hydroperoxide (TBHP) and assessed cell viability at different stages of neural differentiation. We studied NRF2 activation, autophagy, and proteasomal function to explore the contribution and interplay of these pathways in the acute stress response. With increasing differentiation, lt-NES cells showed changes in the expression of metabolic pathway-associated genes with engagement of the pentose phosphate pathway after 6 weeks, this was accompanied by a decreased susceptibility to TBHP-induced stress. Microarray analysis revealed upregulation of target genes of the antioxidant response KEAP1-NRF2-ARE pathway after 6 weeks of differentiation. Pharmacological inhibition of NRF2 confirmed its vital role in the increased resistance to stress. While autophagy was upregulated alongside differentiation, it was not further increased upon oxidative stress and had no effect on stress-induced cell loss and the activation of NRF2 downstream genes. In contrast, proteasome inhibition led to the aggravation of the stress response resulting in decreased cell viability, derangement of NRF2 and KEAP1 protein levels, and lacking NRF2-pathway activation. Our data provide detailed insight into the dynamic regulation and interaction of pathways involved in modulating stress responses across defined time points of neural differentiation.

Suppression of kindling epileptogenesis by adenosine releasing stem cell-derived brain implants.

Epilepsy therapy is largely symptomatic and no effective therapy is available to prevent epileptogenesis. We therefore analysed the potential of stem cell-derived brain implants and of paracrine adenosine release to suppress the progressive development of seizures in the rat kindling-model. Embryonic stem (ES) cells, engineered to release the inhibitory neuromodulator adenosine by biallelic genetic disruption of the adenosine kinase gene (Adk-/-), and respective wild-type (wt) cells, were differentiated into neural precursor cells (NPs) and injected into the hippocampus of rats prior to kindling. Therapeutic effects of NP-derived brain implants were compared with those of wt baby hamster kidney cells (BHK) and adenosine releasing BHK cell implants (BHK-AK2), which were previously shown to suppress seizures by paracrine adenosine release. Wild-type NP-graft recipients were characterized by an initial delay of seizure development, while recipients of adenosine releasing NPs displayed sustained protection from developing generalized seizures. In contrast, recipients of wt BHK cells failed to display any effects on kindling development, while recipients of BHK-AK2 cells were only moderately protected from seizure development. The therapeutic effect of Adk(-/-)-NPs was due to graft-mediated adenosine release, since seizures could transiently be provoked after blocking adenosine A1 receptors. Histological analysis of NP-implants at day 26 revealed cell clusters within the infrahippocampal cleft as well as intrahippocampal location of graft-derived cells expressing mature neuronal markers. In contrast, BHK and BHK-AK2 cell implants only formed cell clusters within the infrahippocampal cleft. We conclude that ES cell-derived adenosine releasing brain implants are superior to paracrine adenosine release from BHK-AK2 cell implants in suppressing seizure progression in the rat kindling-model. These findings may indicate a potential antiepileptogenic function of stem cell-mediated adenosine delivery.

Neural conversion of human embryonic stem cell colonies in the presence of fibroblast growth factor-2.

Pluripotency and the capability for unlimited self-renewal make human embryonic stem cells a promising tool for studying development and new cell replacement strategies. Here, we present a simple differentiation protocol, which permits the direct conversion of human embryonic stem cells into neurogenic precursors without formation of embryoid bodies or coculture with other cell types. In this protocol, human embryonic stem cells propagated as adherent cultures are induced to differentiate into the neural lineage in media containing fibroblast growth factor-2. The adherent cells are proliferated to form detaching neurospheres. Upon plating, these neurospheres give rise to a homogenous population of neural precursors capable of generating neurons, astrocytes and oligodendrocytes. Our findings suggest that fibroblast growth factor-2 exposure alone suffices to promote neural conversion of adherently growing human embryonic stem cell cultures.