Human Embryonic Stem Cell-Derived Immature Midbrain Dopaminergic Neurons Transplanted in Parkinsonian Monkeys.

Human embryonic stem cells (hESCs) differentiate into specialized cells, including midbrain dopaminergic neurons (DANs), and Non-human primates (NHPs) injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine develop some alterations observed in Parkinson's disease (PD) patients. Here, we obtained well-characterized DANs from hESCs and transplanted them into two parkinsonian monkeys to assess their behavioral and imaging changes. DANs from hESCs expressed dopaminergic markers, generated action potentials, and released dopamine (DA) in vitro. These neurons were transplanted bilaterally into the putamen of parkinsonian NHPs, and using magnetic resonance imaging techniques, we calculated the fractional anisotropy (FA) and mean diffusivity (MD), both employed for the first time for these purposes, to detect in vivo axonal and cellular density changes in the brain. Likewise, positron-emission tomography scans were performed to evaluate grafted DANs. Histological analyses identified grafted DANs, which were quantified stereologically. After grafting, animals showed signs of partially improved motor behavior in some of the HALLWAY motor tasks. Improvement in motor evaluations was inversely correlated with increases in bilateral FA. MD did not correlate with behavior but presented a negative correlation with FA. We also found higher 11C-DTBZ binding in positron-emission tomography scans associated with grafts. Higher DA levels measured by microdialysis after stimulation with a high-potassium solution or amphetamine were present in grafted animals after ten months, which has not been previously reported. Postmortem analysis of NHP brains showed that transplanted DANs survived in the putamen long-term, without developing tumors, in immunosuppressed animals. Although these results need to be confirmed with larger groups of NHPs, our molecular, behavioral, biochemical, and imaging findings support the integration and survival of human DANs in this pre-clinical PD model.

Epigenetics of neural differentiation: Spotlight on enhancers.

Neural induction, both in vivo and in vitro, includes cellular and molecular changes that result in phenotypic specialization related to specific transcriptional patterns. These changes are achieved through the implementation of complex gene regulatory networks. Furthermore, these regulatory networks are influenced by epigenetic mechanisms that drive cell heterogeneity and cell-type specificity, in a controlled and complex manner. Epigenetic marks, such as DNA methylation and histone residue modifications, are highly dynamic and stage-specific during neurogenesis. Genome-wide assessment of these modifications has allowed the identification of distinct non-coding regulatory regions involved in neural cell differentiation, maturation, and plasticity. Enhancers are short DNA regulatory regions that bind transcription factors (TFs) and interact with gene promoters to increase transcriptional activity. They are of special interest in neuroscience because they are enriched in neurons and underlie the cell-type-specificity and dynamic gene expression profiles. Classification of the full epigenomic landscape of neural subtypes is important to better understand gene regulation in brain health and during diseases. Advances in novel next-generation high-throughput sequencing technologies, genome editing, Genome-wide association studies (GWAS), stem cell differentiation, and brain organoids are allowing researchers to study brain development and neurodegenerative diseases with an unprecedented resolution. Herein, we describe important epigenetic mechanisms related to neurogenesis in mammals. We focus on the potential roles of neural enhancers in neurogenesis, cell-fate commitment, and neuronal plasticity. We review recent findings on epigenetic regulatory mechanisms involved in neurogenesis and discuss how sequence variations within enhancers may be associated with genetic risk for neurological and psychiatric disorders.

Dynamic landscape of chromatin accessibility and transcriptomic changes during differentiation of human embryonic stem cells into dopaminergic neurons.

Chromatin architecture influences transcription by modulating the physical access of regulatory factors to DNA, playing fundamental roles in cell identity. Studies on dopaminergic differentiation have identified coding genes, but the relationship with non-coding genes or chromatin accessibility remains elusive. Using RNA-Seq and ATAC-Seq we profiled differentially expressed transcripts and open chromatin regions during early dopaminergic neuron differentiation. Hierarchical clustering of differentially expressed genes, resulted in 6 groups with unique characteristics. Surprisingly, the abundance of long non-coding RNAs (lncRNAs) was high in the most downregulated transcripts, and depicted positive correlations with target mRNAs. We observed that open chromatin regions decrease upon differentiation. Enrichment analyses of accessibility depict an association between open chromatin regions and specific functional pathways and gene-sets. A bioinformatic search for motifs allowed us to identify transcription factors and structural nuclear proteins that potentially regulate dopaminergic differentiation. Interestingly, we also found changes in protein and mRNA abundance of the CCCTC-binding factor, CTCF, which participates in genome organization and gene expression. Furthermore, assays demonstrated co-localization of CTCF with Polycomb-repressed chromatin marked by H3K27me3 in pluripotent cells, progressively decreasing in neural precursor cells and differentiated neurons. Our work provides a unique resource of transcription factors and regulatory elements, potentially involved in the acquisition of human dopaminergic neuron cell identity.