Hoxa9 and EGFP reporter expression in human Embryonic Stem Cells (hESC) as useful tools for studying human development

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.

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“…We differentiated pluripotent human ESC H9-GFP 18 expressing OCT4 and SOX2 into mDA (Fig. 1 a), using a reported floor-plate protocol 12 .…”

Section: Resultsmentioning

confidence: 99%

“…H9 human embryonic stem cells (WA09) of female origin were purchased from WiCell (Madison, WI, USA), transduced with lentiviral vectors to express GFP as described 18 and cultured until 75% confluency. These GFP-expressing cells are useful for transplantation studies 13 and behave as its parental H9 hESC.…”

Section: Methodsmentioning

confidence: 99%

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

Meléndez-Ramírez

Duran

Barrios-García

et al. 2021

Sci Rep

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“…25 HOXB13-AS1 is a 564 nucleotide-lncRNA and situates in the homeobox (HOX) gene locus on chromosome 17, which has been identified to function critical roles in embryo development and numerous cellular processes. [26][27][28][29] A substantial body of scientific evidence reported that aberrations in HOX gene expression associated with many different types of malignancies. Recently, certain kinds of lncRNAs have been reported to be located on HOX gene clusters, including HOXB13-AS1.…”

mentioning

confidence: 99%

Long noncoding RNA HOXB13‐AS1 regulates HOXB13 gene methylation by interacting with EZH2 in glioma

Xiong

Guo

et al. 2018

Cancer Medicine

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Dysregulation of long noncoding RNAs (lncRNAs) has been implicated in human diseases, in particular, cancers. In this study, we determined the expression of an lncRNA, HOXB13‐AS1, involving in glioma. We showed that HOXB13‐AS1 was significantly upregulated in glioma tissues and cells and was negatively correlated with its surrounding gene HOXB13 levels. Functional experiments in vitro and in vivo revealed that high level of HOXB13‐AS1 increased cell proliferation and tumor growth by promoting cell cycle progression. Conversely, knockdown of HOXB13‐AS1 resulted in decreased cell proliferation and tumor growth. Mechanistically, we showed that HOXB13‐AS1 overexpression increased DNMT3B‐mediated methylation of adjacent gene HOXB13 promoter by binding with the enhancer of zeste homolog 2 (EZH2) using bisulfite sequencing PCR (BSP), epigenetically suppressing HOXB13 expression. Additionally, the interaction between HOXB13‐AS1 and HOXB13 was validated by RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP) assays using antibody against to EZH2. Taken together, our study indicated that HOXB13‐AS1 could regulate HOXB13 gene expression by methylation HOXB13 promoter and acts as an epigenetic oncogenic in glioma.

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“…Enhanced Green fluorescent protein (EGFP) expressing hESCs were employed [19]. We first corroborated the expression of pluripotency markers in H9 wild-type hESCS by detecting OCT4, SOX2, NANOG, and SSEA4 (Figure S1A).…”

Section: Dopaminergic Neuron Differentiationmentioning

confidence: 87%

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

López-Ornelas,

Escobedo-Avila,

Ramírez-García

et al. 2023

Cells

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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.

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Hoxa9 and EGFP reporter expression in human Embryonic Stem Cells (hESC) as useful tools for studying human development

Cited by 8 publications

References 5 publications

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

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

Long noncoding RNA HOXB13‐AS1 regulates HOXB13 gene methylation by interacting with EZH2 in glioma

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

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