Generation of iPSC lines (KAIMRCi003A, KAIMRCi003B) from a Saudi patient with Dravet syndrome carrying homozygous mutation in the CPLX1 gene and heterozygous mutation in SCN9A

The most prevalent form of epileptic encephalopathy is Dravet syndrome (DRVT), which is triggered by the pathogenic variant SCN1A in 80% of cases. iPSCs with different SCN1A mutations have been constructed by several groups to model DRVT syndrome. However, no studies involving DRVT-iPSCs with rare genetic variants have been conducted. Here, we established two DRVT-iPSC lines harboring a homozygous mutation in the CPLX1 gene and heterozygous mutation in SCN9A gene. Therefore, the derivation of these iPSC lines provides a unique cellular platform to dissect the molecular mechanisms underlying the cellular dysfunctions consequent to CPLX1 and SCN9A mutations. Supplementary Information The online version contains supplementary material available at 10.1007/s13577-023-01016-z.

Although the molecular pathway underpinning SCN1A mutations may be well studied, our knowledge of the pathophysiology of other genes in DRVT remains elusive.One of the main hurdles in understanding the mechanism of epileptic encephalopathies is the lack of an in vitro model that accurately recapitulate the complexity of these disorders, including DRVT [22].Epilepsy patient-specificinduced pluripotent stem cells (iPSCs) represent a distinctive biological platform to study disease onset and progression [22,23].Several groups have modeled DRVT in vitro by reprogramming patients' fibroblasts into iPSCs carrying distinct mutations in SCN1A [24][25][26].Transcriptomic profiling from differentiated DRVT-iPSCs into neural progenitor cells and GABAergic cells represented gene expression signatures of "Prefrontal Cortex," "Fetal Brain," "Dentate Gyrus," and "Superficial Dorsofrontal Area," brain regions that are correlated with clinical features in DRVT [25].Furthermore, electrophysiological aberrations in DRVT-iPSC GABA lines are consistent with reduced sodium current density and decreased excitability of inhibitory interneurons, as previously reported in a DRVT animal model [25,27].These combined results indicate that DRVT-iPSC lines are appropriate model for studying the neuro-pathophysiological processes underlying DRVT.
In this study, we generated iPSCs from DRVT patient with homozygous mutation in the CPLX1 gene and heterozygous mutation in SCN9A using non-integrating episomal expression of the reprogramming factors OTC3/4, L-MYC, LIN28, SOX2, KLF4, and mp53DD.Our established DRVT-iPSC lines possess bona fide characteristics of embryonic stem cells and constitute a primary cellular model to interrogate the molecular basis of rare genetic variants in DRVT.

Patient recruitment and ethical approval
This study was approved by the institutional review board (IRB) and research ethics committee of KAIMRC (NRJ22J/060/03).The patient is a 7-year-old female diagnosed with DRVT syndrome carrying C.4G > A (P.Glu2Lys) homozygous mutation in CPLX1 and C.3332-3346del (P.Ser1111_Glu1115del) heterozygous mutation in SCN9A identified by whole exome sequencing (WES).The patient's parents gave their consent via an informed consent form (ICF), which was used to collect and process the patient's sample.

PBMCs isolation and enrichment of erythroid progenitors
According to the manufacturer's instructions, peripheral blood from Saudi patients was drawn into a blood collection tube containing EDTA

Transfection of erythroid progenitor cells
Episomal iPSC Reprogramming Kit (Thermofisher Catalog #A15960) was used to reprogram expanded erythroid progenitor cells.Three pulses at 1650 V and a pulse width of 10 ms were used with 1 μg of each episomal vector (Neon Transfection System, Thermofisher).The emerging ESClike colonies were then manually selected for transfer into 48-well plates coated with Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Thermofisher Catalog #A1413201) with mTeSR™ Plus media at 37 °C with 5% CO 2 and 20% O 2 .iPSCs were dissociated using Gentle Cell Dissociation Reagent (Stem Cell Technologies Catalog #100-0485) and passaged using 1:20-1:30 splitting ratio.

Immunocytochemistry
Cells were first fixed for 15 min in 4% (w/v) paraformaldehyde, then permeabilized for 10 min in PBS containing 0.1% (v/v) Triton X-100 and blocked for 45 min in PBS containing 1% Gelatin.Primary antibodies were incubated overnight at 4 °C before they were probed with the proper secondary antibodies for one hour at room temperature (Table 1).Primary and secondary antibodies were reconstituted in PBS containing 0.2% gelatin.DAPI nuclear staining at 1 μg/mL was used to stain the nuclei.The staining was visualized under Zeiss LSM 880 Airyscan confocal laser scanning microscope using a 20× oil objective (Zeiss).

Quantitative reverse transcription PCR (RT-qPCR)
RNeasy Kit (Qiagen Catalog #74104) was used to extract total RNA, and SuperScript™ III First-Strand Synthesis (Thermo Fisher Scientific Catalog #18080051) was used to reverse-transcribe the extracted total RNA.As previously explained [28], the RT-qPCR test was performed using Fast-Start SYBR Green Master Mix (ROCHE).

In vitro differentiation
The STEMdiff™ Trilineage Differentiation Kit (Stem Cell Technologies Catalog #05230) was used to differentiate the generated DRVT-iPSCs into three germ layers.

Flow cytometry analysis
The BD IntraSure™ Kit was used for permeabilizing and staining for Intracellular Markers (BD Biosciences Catalog #641778).Briefly, 4 × 10 5 cells were fixed using Reagent A for 10 min.Primary antibodies (Table 1) were diluted in Reagent B and incubated on ice for 30 min.Secondary antibodies were diluted with PBS and incubated for 30 min at room temperature.FACS samples were analyzed on BD FACS ARIA cell sorter.FITC-positive cells were measured in stained vs unstained cells.

Karyotype analyses
For G-banding karyotyping, iPSC lines were treated with 0.3 g/mL KaryoMAX™ Colcemid™ for 15 min, dissociated by TrypLE, and incubated in hypotonic solution (75 mM potassium chloride) at 37 °C for 20 min.After that, iPSCs were preserved at 4 °C after being fixed in a methanol/ glacial acetic acid 3:1 mixture.Pathology and laboratory medicine (Ministry of the National Guard-Health Affairs) performed karyotyping on at least 50 metaphases.

Plasmids screening
Utilizing the All Prep DNA/RNA/Mini Kit (Qiagen Catalog #80204), DNA was extracted according to manufacturer instructions.PCR was carried out using EBNA-1 primers that identify all five episomal plasmids (expected size 666 bp) (Thermo Fisher Scientific Catalog #A15960).

Short tandem repeat (STR)
Following the manufacturer's instructions, genomic DNA was extracted from PBMCs and DRVT-iPSC lines DNeasy Blood&Tissue kit and AllPrep DNA/RNA/Mini Kit, respectively.The PowerPlex ® 16 System (Promega) kit was used to amplify 15 STR loci and Amelogenin.On a 3130 Genetic Analyzer from Applied Biosystems, PCR amplicons were resolved.GeneMapper ID-X Software, version 1.4 was used to gather and evaluate the data.

Statistical analysis
RT-qPCR data are represented as mean ± standard deviation (SD).Statistical significance was determined in Student's t-test (unpaired; two-tailed).A Bonferroni correction was applied to the p value from multiple comparisons.*p < 0.05.

Clinical information and mutation analysis
The 7-year-old female patient was diagnosed with global developmental delay, seizers since in infancy, and choreoathetotic movements.Molecular genetic analysis for whole exome sequencing (WES) showed positive family history suggestive of an autosomal recessive pattern of inheritance.
In addition, WES of the patient blood sample identified heterozygous mutation c.3332_3346del p.(Ser1111_Glu-1115del) in SCN9A gene.This mutation leads to an in-frame deletion of 15 bps in exon 18 (NM_002977.3),which causes the loss of 5 amino acid residues.Further analysis unraveled homozygous variant c.4G > A p.(Glu2Lys) in the CPLX1 gene.This variant leads to an amino acid exchange in exon 1 (NM_006651.4).Both mutations were confirmed in the patient's peripheral blood cells as well as in the DRVT-iPSC lines by Sanger sequencing (Fig. 1D).

The generation of integration-free DRVT-iPSC lines
Initial phone contact with the donor's parent resulted in the scheduling of an in-person interview after receiving approval.Following the signature on the informed consent form, a sample of 10 mL peripheral blood was drawn and erythroid progenitor cells (EPCs) were cultured for eight days (Fig. 1A).Due to their lack of genetic mutations and genomic structural variation, including the absence of TCR/BCR gene recombination found in T cells, EPCs were selected for reprogramming [40].We, therefore, established two DRVT-iPSC lines using a non-integrative and virusfree reprogramming approach as previously described [29].Briefly, the episomal vectors encoding OCT4, SOX2, KLF4, L-MYC, LIN28A, dominant-negative form of TP53, and EBNA1 were delivered to EPCs by electroporation (Fig. 1B).We identified emerging embryonic stem cell (ESC)-like colonies with typical ESC morphological characteristics (i.e., distinct borders, bright centers, tight-packed cells, and a high nucleus-to-cytoplasm ratio) after around 20 days (Fig. 1C).
The derived iPSC lines were picked, expanded in feederfree condition, and cryopreserved in KAIMRC facility.We thawed two DRVT-iPS clones for downstream testing.We evaluated the genomic integrity and confirmed the genetic compatibility of the generated DRVT-iPSCs and EPCs.A normal female chromosomal number and structure has been shown by G-banding analysis (Fig. 1E).The matched identities of the isolated iPS lines and the donor EPCs were validated by a short tandem repeats (STR) assay (Fig. S1B).Moreover, mycoplasma testing showed that the generated iPSC lines are mycoplasma-free (Fig. S1A).

Characterization of self-renewal and pluripotency
The slow removal of cellular episomal vectors from DRVT-iPSC lines was achieved after culturing the cells for nine passages (Fig. 2A).As a result, we rigorously evaluated the pluripotency using multiple approaches.We assessed the endogenous expression of pluripotency markers of OCT4, NANOG, and SOX2, by immunofluorescence and RT-qPCR (Fig. 2B; Fig. 2D).Flow cytometry histograms demonstrated that > 97% of cells stained positively for OCT4 and > 98% for NANOG (Fig. 2C).Direct in vitro differentiation to the three germ layers, mesoderm, endoderm, and ectoderm was used to demonstrate the tri-lineage differentiation capacity.We observed a down-regulation of OCT4 and NANOG and an upregulation of germ layer-specific markers (Fig. 2E).The positive expression of the neural progenitor markers of the central nervous system NESTIN and PAX6 indicated ectodermal differentiation.We demonstrated an upregulation of Brachyury, a member of the T-box family, and CDX2, a caudal-type homeobox protein 2, which indicated an early determination of mesoderm.We further examined the presence of the endodermal marker SRY-Box Transcription Factor 17 SOX17 and zinc-finger transcription factor GATA4 in DRVT-iPSC lines and H1 hESC positive control (Fig. 2E).However, the fold change in NANOG is not substantial during endoderm lineage.Teo et al. 2011 demonstrated that while OCT4 and SOX2 prevent DE differentiation of hESCs, NANOG is required to initiate EOMESODERMIN (EOMES) expression, which then interacts with SMAD2/3 to activate the transcriptional network that directs endoderm formation [45].

Discussion
In the last decade, the seminal discovery of cellular reprogramming and the generation of iPSCs have been widely utilized to model diseases "in a dish" and hold promise for applied biology and regenerative medicine [23,30].The characteristics of iPSCs resemble those of embryonic stem cells, including their morphology, self-renewal, gene expression, and the capacity to differentiate into virtually any cell type of the body [23].These changes are accompanied by transient expression of the pluripotency transcription factors NANOG, OCT3/4, SOX2, KLF4, c-MYC, and LIN28 [31][32][33][34][35][36][37].These factors exert a dual role by promoting the expression of pluripotency-associated genes in a self-regulatory loop and silence somatic genes [38,39].Although iPSCs can be derived from multiple sources of somatic cells, EPCs are chosen for their lack of chromosomal aberrations and genomic DNA mutations [40].We found that eight days of expansion in erythroid expansion medium, yielded 69% of cells positive for CD71 + CD235a + erythroid cell surface markers [41].The non-viral, non-integrating episomal plasmid-based reprogramming technique is practically applicable for generating clinical grade-iPSCs [42].Vectors containing oriP and EBNA-1, based on the Epstein-Barr Nuclear Antigen-1, have shown the ability to create iPSCs highly effectively with a single transfection [43].
Even though multiple groups have generated iPSCs with various SCN1A mutations to model DRVT syndrome, no studies have been conducted employing uncommon genetic variants [24][25][26].Pathogenic variants in the SCN9A gene have been associated with several autosomal dominant conditions, including familial febrile seizures 3B (613863) and generalized epilepsy with febrile seizures plus type 7 [16].Moreover, mutation in CPLX1 gene is causative for autosomal recessive developmental and epileptic encephalopathy 63.DEE63 is a neurologic disorder characterized by earlyonset refractory infantile spasms and myoclonic seizures in the first months to years of life [21,44].
Intriguingly, the differentiation of DRVT-iPSC into neuronal subtypes has yielded important mechanistic understandings of the disorder.For example, studies have shown that DRVT-iPSC-derived medial ganglionic eminence (MGE)-like inhibitory neuron reduced the action potential frequency compared to those in controls [24].In addition, the transcriptome analysis of DRVT-iPSC-derived NPCs and GABA cells compared to controls, identified unique dysregulations of genes for chromatin structure, mitotic progression, neuronal plasticity, and excitability [25].Therefore, future research involving the differentiation of DRVT-iPSC#1 and iPSC#2 into neural cells, gene expression profiling, and functional prosperity, will provide valuable insights into the disorder.In parallel, we will pursue an isogenic design of SCN9A and CPLX1 knockouts in H9 hESC using CRISPR/Cas9 to validate results obtained from DRVT-derived neural cells.In this isogenic setting, DRVT transcriptional alterations will be corroborated with dysregulated genes putatively attributable to SCN9A and CPLX1 deficiencies in disease-relevant tissues.Hence, their usefulness extends from in vitro disease modeling to drug screening, paving the way for unraveling disease mechanisms and accelerating the discovery of novel therapeutic targets for the treatment of Dravet Syndrome.
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Fig. 1 Fig. 2
Fig. 1 Cellular reprogramming and Generation of DRVT-iPSCs.A Collecting a sample of 10 ml peripheral blood from DRVT syndrome patient and expanding erythroid progenitor cells (EPCs) for eight days.B ReproTeSR™ and the episomal reprogramming process are represented schematically.During reprogramming (days 11-28), phase-contrast pictures of the mesenchymal-epithelial transition and the emergence of colonies were captured.C. Representative images of DRVT-iPSC clones show defined borders and compact morphology.D Sanger sequencing confirms the mutations in SCN9A and CPLX1 in the patient's peripheral blood cells as well as in the DRVT-iPSC lines.E Karyotypes for DRVT-iPSCs exhibit normal chromosomal content 46, XX by representative G-banded karyotype analyses ◂

Table 1
List of antibodies and primers used in this studyAntibodies and stains used for immunocytochemistry/flow-cytometry