CRISPR-Cas9 mediated deletion of RMST in human pluripotent stem cells (hPSCs)
We hypothesized that loss of RMST function leads to impaired function or development of hypothalamic neurons. To test this hypothesis, we sought to generate hypothalamic neurons from RMST-deleted hPSCs. To this end we used CRISPR-Cas9 genome editing to delete RMST in H9 wild-type (H9WT) hPSCs (WA09, WiCell Research Institute). A pair of sgRNAs targeted RMST upstream of exon 3 and downstream exon 8 (Fig. 1A). Nonhomologous end-joining repair of the two double-strand breaks created by sgRNA-targeted Cas9 were expected to introduce a major deletion in the RMST gene. After nucleofection and single cell plating, multiple clones were obtained with heterozygous or homozygous deletion (Fig. S1A). Two clones, named clone-24 and clone-38 (C-24 and C-38), were further characterized as having homozygous deletion of 41,540 bp and 41,548 bp respectively. Deletion in RMST was confirmed by DNA sequencing (Fig. 1B) and gel electrophoresis of PCR-amplified genomic DNA (Fig. 1C). Primer pair (FP1 and RP2) amplify the entire region spanning exon 3 to exon 8 (Fig. 1A) consisting of 42,802 bp, and such a large sequence prevents PCR amplification from wild-type genomes. However, in RMST-deleted clones, intron 2 and intron 8 are conjoined such that the primer sites come into proximity yielding a 902 bp amplicon (Fig. 1B, C). The use of primer pairs (FP1 and RP1) and (FP2 and RP2) resulted in expected amplification products in wild-type hPSCs, whereas in clones C-24 and C-38 which lack binding sites for FP2 and RP1 primers after deletion, no PCR amplification was observed (Fig. 1C). These results confirmed the homozygous deletion of the RMST gene in C-24 and C-38 hPSCs.
A Schematic illustration of RMST gene locus (GRCh38/hg38, chr12:97,430,884-97,565,035) and targeting strategy for CRISPR-Cas9 mediated editing. RMST exons are indicated by black rectangles and black arrows indicate single guide RNA (sgRNA) targeting sites. sgRNA1 and sgRNA2 (Table S1) targeted upstream of exon 3 and downstream of exon 8, respectively. Forward primers (FP1 and FP2) and reverse primers (RP1 and RP2) shown in blue arrows were used for genotyping and sequencing. Primer sequences are listed in Table S1. B Sanger sequencing of PCR products in single cell-derived clones showing deletion in RMST gene following CRISPR-Cas9 editing. Clone-24 (C-24) has a 41.540 kb deletion and clone-38 (C-38) has a 41.548 kb deletion. C Agarose gel electrophoresis of PCR-amplified genomic DNA for the validation of genetic deletion of RMST with indicated forward primers (FP1 and FP2) and reverse primers (RP1 and RP2). D RT-PCR of RMST cDNA in hPSCs. In H9WT cell, RMST transcript amplification yielded an amplicon length of 1269 bp with the primers listed in Table S2, whereas RMST transcript is truncated in C-24 and C-38 clones yielding an amplicon of 384 bp. E Bright field morphology of H9WT, C-24, and C-38 hPSCs. Scale, 100 µm. F qPCR for the validation of pluripotency markers OCT4, NAN, SOX2, KLF4 and C-MYC in RMST-deleted hPSCs.
We assessed the expression of RMST transcripts in C-24 and C-38 and wild-type cells. Deletion of RMST was confirmed by RT-PCR using the forward primer binding to exon 1 and exon 2 junction and reverse primer binding to exon 10 (listed in Table S2). The PCR amplification of RMST cDNA in wild-type hPSCs resulted in an amplicon of 1269 bp, whereas in C-24 and C-38, the RMST transcript was truncated as indicated by an amplicon of 384 bp (Fig. 1D). This result indicates that genomic deletion in RMST results in expression of substantially truncated transcripts.
The C-24 and C-38 hPSCs lines showed typical stem cell morphology indistinguishable from wild type (Fig. 1E) and normal karyotype (Fig. S1B). We also found that pluripotency-related genes OCT4, NANOG, KLF4, and C-MYC were equivalently expressed in all the lines (Fig. 1F), indicating that deletion in RMST is dispensable for maintaining hPSCs pluripotency. In conclusion, we demonstrated successful generation of stable hPSCs lines harboring large genomic deletions in the RMST gene.
Directed differentiation of hPSCs into GnRH neurons
Next, we generated hypothalamic neurons that included GnRH-expressing neurons from wild-type and RMST-deleted hPSCs using a published protocol [23, 24]. A schematic illustration of hPSCs differentiation into GnRH neurons is presented in Fig. 2A. For GnRH neurons differentiation, hPSCs were treated for 12 days with dual SMAD inhibitors SB431542 and dorsomorphin to block TGF-b/activin and BMP signaling pathways, respectively [25]. This was followed by treatment with FGF8, which functions as a key growth factor in the development of GnRH neurons. On day 20 of differentiation, the cells organized in neuronal rosettes and immunostaining confirmed the homogenous expression of neuroectodermal markers SOX2, PAX6 and NESTIN, indicating efficient neural conversion (Fig. 2B). The anterior fate of the cells was confirmed by staining with FOXG1 and OTX2 (Fig. 2B). No differences in the expression of progenitor markers were observed in control and RMST-deleted hPSCs-derived NPCs using RT-qPCR (Fig. S2A).
A Schematic representation of the protocol depicting the stepwise differentiation of hPSCs into GnRH neurons. For the first 12 days, cells were treated with dual SMAD inhibitors, SB431442 and Dorsomorphin. At day 12 of differentiation, FGF8 was added and from day 21 onwards, FGF8 and notch inhibitor DAPT were added to the culture medium. Arrows indicate the time of cell splitting. B Immunostaining of cells on day 20 of differentiation showing the expression of progenitor markers SOX2, PAX6, OTX2, FOXG1, and Nestin. C Immunostaining of mature GnRH neurons showing the expression of MAP2, GnRH, and FOXG1 on day 28 of differentiation. D High-resolution images of GnRH neurons showing the expression and punctuate staining of GnRH. Arrows indicate the GnRH containing vesicles. Cell nuclei were stained with DAPI (blue). E qPCR of GnRH1 and KISS1R in differentiated GnRH neurons. Graphs show mean with ±SEM of 3–4 independent biological replicates and the data were analyzed using unpaired student t-test. Primer sequences are listed in Table S2. B, C Scale, 100 µm. D Scale, 50 µm.
To induce differentiation of NPCs into GnRH neurons, cells were treated with FGF8 and Notch inhibitor DAPT for one week. The neuronal identity of differentiated cells was confirmed by immunostaining with MAP2 and FOXG1 (Fig. 2C). Immunofluorescence showed the presence of GnRH-positive cells and most of the cells were expressing FOXG1 (Fig. 2C). The number of proliferating (Ki67 positive) cells was very low, indicating that the cells were terminally differentiated and exited the cell cycle (Fig. S2B). High-magnification images of GnRH-expressing cells showed a punctate staining pattern in most cells, indicating vesicular packaging of GnRH decapeptide (Fig. 2D). The neurons express kisspeptin receptor (KISS1R) which binds kisspeptin, a neuropeptide triggering the release of GnRH and there were no significant differences in GnRH and KISS1R transcripts level among the lines (Fig. 2E). In conclusion, hPSCs were efficiently differentiated into mature GnRH neurons and no significant differences in differentiation potential were observed between the control and RMST-deleted cells.
Physiological characterization of hPSCs-derived GnRH neurons
We investigated the functional properties of RMST-deleted neurons by measuring their action potentials (AP) and calcium influx. We used whole-cell patch-clamp recordings to assess the AP firing patterns of in vitro-generated neurons. We found that both control (H9WT) and mutant neurons (C-24, C-38) generated multiple and repetitive AP (Fig. 3A, C). Notably, deletion in RMST significantly increased the AP frequency compared to control hPSCs-derived neurons (Fig. 3B). In addition, the proportion of neurons that generated multiple AP was higher in mutant neurons than in control (57%); C-24 (90.9%), C-38 (90%) (Fig. 3C).
A Whole-cell patch-clamp recording to monitor action potential (AP) generated by injection of current pulses in a current-clamp mode; no AP, single AP, or multiple and repetitive AP. B AP frequency of control and mutant neurons after one week differentiation. Data are means ± SEM from 3 independent differentiations: n = 14 (control), 11 (C-24), 10 (C-38). One-way ANOVA with Tukey’s multiple comparisons was used. *, p < 0.05. **p < 0.01. C Distributions of AP generation; no AP, single AP, or multiple/repetitive AP in neurons differentiated for one week. D Representative traces of intracellular calcium ions (Fura-2 F340/F380 ratio) in neurons stimulated by 50 mM KCl. E Net changes of calcium increase by 50 mM KCl. Data are means ± SEM from 3 independent differentiation: n = 449 (control), 426 (C-24), 399 (C-38). One-way ANOVA with Tukey’s multiple comparisons was used. ****p < 0.0001. F Percentage of neurons that evoke calcium influx by 50 mM KCl. The number of cells tested are shown from 3 independent differentiation.
Single-cell calcium imaging was performed to further assess the functional activity of RMST-deleted neurons (Fig. 3D, F). Calcium influx through voltage-gated calcium ion channels (VGCC) is essential for synaptic transmission and plasticity. We stimulated neurons with 50 mM KCl and measured the calcium influx through VGCC. We observed that RMST deletion enhanced the calcium influx (Fig. 3E), and the percentage of neurons that responded to KCl also increased (Fig. 3F); control (81%), C-24 (98%), and C-38 (89%). These results suggest that neurons derived from RMST-deleted cells are functionally mature and electro-physiologically more active than control neurons.
Gene expression analysis of GnRH neurons
To understand transcriptional changes in GnRH neurons derived from WT and RMST-deleted hPSCs, we performed global gene expression analysis of GnRH neurons in three independent differentiation replicates. RNA was collected from neurons and analyzed by RNA sequencing (RNA-seq) to gain a comprehensive view of transcriptional differences between the neurons derived from WT and RMST-deleted cells (Fig. 4). Principle component analysis demonstrated good reproducibility of the experimental replicates (Fig. S3A). Comparison of controls and RMST-deleted neurons identified 1423 differentially expressed genes (DEGs) (adjusted p-value < 0.05), of which 717 were upregulated [fold change (FC) > 1.5] and 706 were downregulated [fold change (FC) < 0.5] (Fig. 4A, B). We performed Gene Ontology (GO) enrichment analysis (cut-off criteria of adjusted p value < 0.05) to identify biological processes/molecular functions associated with DEGs. The key biological process terms for upregulated genes showed their roles in nervous system development, cell-cell signaling, neurogenesis, generation of neurons, regulation of transport, neurons projection development, synaptic signaling, neurotransmitter transport and secretion (Fig. 4C). The key biological terms for downregulated genes showed their role in system development, cellular developmental process, cell differentiation, tissue development, movement of cell or subcellular component, cilium organization, cilium movement, and axoneme assembly (Fig. 4D). The molecular functions for the upregulated genes showed enrichment for GO terms including transporter activity, ion channel activity, cation channel activity, voltage-gated ion channel activity, potassium channel activity, voltage-gated potassium channel activity etc. (Fig. 4E), while the downregulated genes showed enrichment for GO terms including cytoskeletal protein binding, extracellular matrix structural constituent, tubulin binding, glycosaminoglycan binding, heparin binding, growth factors binding, and extracellular matrix structural constituent conferring tensile strength Fig. S3B). These results demonstrate that RMST deletion results in altered expression of genes involved in neurogenesis, neurotransmitter transport, synapse organization, cilium assembly and organization, epithelium development, and channel activity. Several of these dysregulated genes have previously been implicated to function in nervous system and hypothalamus development (GAS1, GPR139, MAGED1, NNAT, NTRK2, POU6F2, BHMT, FGF13, TCEAL5, TMOD1, TNR), ion channels proteins (ASIC4, CACHD1, CACNA1B, CLCN5, GABRB2, GLRA2, KCNA2, KCNC1, KCND3, KCNH4, KCNJ3, KCNJ12, KCNJ13, SCN7A, SCN8A) and cell adhesion proteins (CDH8, DCHS2, PCDH7, PCDH10, PCDH15, PCDHA4) (Fig. 4F). To validate the RNA-seq data, we performed qPCR analysis for several genes showing differential expression in neurons derived from RMST-deleted hPSCs compared to control (Fig. 4G). In conclusion, these results indicate that deletion in RMST caused altered expression of key genes involved in the development of hypothalamus and neuronal development, ion channels and cell adhesion proteins.
A Heatmap of all differentially expressed genes (DEGs) in RMST-deleted hPSCs-derived GnRH neurons compare to control H9WT. Three independent biological replicates from each sample were analyzed (P-value < 0.05 and 1.5-fold change). Expression data have been standardized as z-scores for each mRNA. B Volcano plot showing the log2 fold change and the adjusted P-value for all the detected transcripts; upregulated (green), downregulated (red), unchanged (black). C, D Gene ontology (GO) enrichment analysis for biological processes of upregulated and downregulated genes in hPSCs-derived GnRH neurons. E GO enrichment analysis for molecular function of upregulated genes. The GO cut-off criteria included q (adjusted p value) < 0.05. F Sub-heatmap of differentially expressed genes associated with neuronal development, ion channels and cell adhesion. G qPCR validation of genes showing differential expression in neurons derived from RMST-deleted hPSCs compared to control. Graphs show mean with ±SEM of 3–4 independent biological replicates and the data were analyzed using unpaired student t-test. Primer sequences are listed in Table S2. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Differential DNA methylation analysis
DNA methylation is an important epigenetic process that cells use to regulate gene expression and emerging evidence indicates intricate regulatory connections between lncRNA and DNA methylation [26]. To understand the effect of RMST deletion on DNA methylation, we performed DNA methylation analyses on WT and C-38 hPSCs-derived neurons using whole genome bisulfite sequencing. In total, 1759 differentially methylated regions (DMRs) were detected (q value < 0.05), out of which 669 regions were hypomethylated and 1090 were hypermethylated in C-38 hPSCs-derived neurons. These regions are distributed across the genome (Fig. 5A) in 5′ UTR, promoter regions, exons, introns and 3′ UTR (Fig. 5B). For hypermethylated DMRs, 2.5% occurred at the core promoter, 2.4% at the proximal promoter, 4.5% at the 3′untranslated regions (3’UTR), 2.9% at the 5′UTR, 8.2% at exons, 28% at introns, and 51.4% in intergenic regions. Whereas 16.7% of hypomethylated DMRs occurred at the core promoter, 4.4% at the proximal promoter region, 3.3% at the 3′UTR, 9.3% at the 5’UTR, 10% at exons, 12.2% at introns, and 44% at intergenic regions (Fig. 5B).
Distribution pattern of differential methylation regions (DMRs) across the genome (A) and distinct genomic element types (B): promoter, 5′-UTS, exon, intron, intergenic and 3’-UTR. (C) The DMR enrichment analyses using the GREAT annotation tool. (D) Sub-heatmap of RNA-seq results showing upregulation and downregulation of genes that colocalized with these DMRs. (E) qPCR validation of genes expression associated with DMRs. Graphs show mean ± SEM of 3–4 independent biological replicates and the data were analyzed using unpaired student t-test. Primer sequences are listed in Table S2. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
We checked whether DMRs associated with RMST loss were linked to specific biological and molecular processes. The DMR enrichment analyses were performed using the GREAT annotation tool for the hypomethylated and hypermethylated regions, independently. Our findings revealed that hypermethylated targets following RMST deletion are enriched for diverse biological processes, including detection of chemical stimulus involved in sensory perception of smell, sensory perception of smell, regulation of ossification, opsonization, central nervous system neuron development (Fig. 5C). The molecular processes affected by these DMRs were olfactory receptor activity, odorant binding, and G protein-coupled receptor activity (Fig. 5C). The hypomethylated targets following RMST deletion are enriched only for anterior/posterior pattern specification (Fig. S3C). Our RNA-seq results show that several of these DMRs corresponded with the upregulation and downregulation expression of colocalized genes (Fig. 5D), which were confirmed by qPCR for several examples (Fig. 5E). In conclusion, these results demonstrate that RMST lncRNA deletion altered genomic DNA methylation in hPSCs-derived neurons.
- SEO Powered Content & PR Distribution. Get Amplified Today.
- PlatoData.Network Vertical Generative Ai. Empower Yourself. Access Here.
- PlatoAiStream. Web3 Intelligence. Knowledge Amplified. Access Here.
- PlatoESG. Carbon, CleanTech, Energy, Environment, Solar, Waste Management. Access Here.
- PlatoHealth. Biotech and Clinical Trials Intelligence. Access Here.
- Source: https://www.nature.com/articles/s41420-024-02074-4