Supervised by: Amanda Liu, BEng, MSc. Amanda spent her undergraduate years studying Biochemical Engineering at UCL (University College London), where she was awarded First Class Honours. She then completed her Master’s degree in Clinical and Therapeutic Neuroscience at the University of Oxford. She is currently studying Medicine (Graduate Entry) at the University of Cambridge.
Stem cells are a novel treatment that functions by regrowing the parts of the body which have broken down. This ability has been a factor in starting many investigations into the ability of stem cells to heal different diseases, one of which is epilepsy. This paper examines the use of neural, induced pluripotent, and mesenchymal stem cells to treat epilepsy, their effectiveness when compared to current treatments (anti-epileptic drugs and surgery), and current progress with treatment options through the use of stem cells for modelling epilepsy (3D organoids).
Keywords: Epilepsy, Neural stem cells, Induced pluripotent stem cell models, Mesenchymal stem cells, Dravet Syndrome, Cell therapy, Anti-epileptic drugs, 3D organoid
Causes and effects of epilepsy
Epilepsy is a common and serious brain disorder that affects over 70 million people of varying ages and causes seizures (1). Epilepsy can be broadly categorised, by seizure type, into focal seizures (one section or hemisphere of the brain is affected) and generalised seizures (both hemispheres are affected), of which there are a vast number of subcategories. Epilepsy has multiple causes, such as injury, infection, or other diseases such as auto-immune disorders, but they all have a strong genetic basis. Epileptic seizures can have significant effects on the lives of those with the disease and their families but can also affect the morphology of the brain, causing abnormal development of basal dendrites that extend toward the hilus and incorrect migration and differentiation of cells in the granule cell layer which combine to have drastic effects with respect to the brain’s function (2).
- Anti-epileptic drugs
The current treatments for epilepsy remain widely therapeutic, with patients taking antiepileptic drugs (AEDs), which are seen to control seizures well (3).
Factors that need to be taken into consideration during epilepsy care are age (taking care of persons between birth to 16 years of age and persons aged 65 and over), gender (taking care of women) and taking care of those with developmental delays (3). 30% of patients are not well-controlled on AEDs and thus often receive multiple AEDs. When two or more AEDs are taken, with no effect of controlling (reducing or stopping) seizures, the epilepsy the patient has is reclassified as drug-resistant epilepsy. AEDs seem to aggravate certain types of epilepsy in a minority of cases, with the main culprits being carbamazepine, vigabatrin, tiagabine and gabapentin (phenytoin is reported to be less aggravating, but logically, this does not disprove aggravation via this AED) (4).
Resection surgery is available to cut away the parts of the brain that have been causing seizures or reduce the occurrence and severity of seizures. Possible resection surgery solutions are temporal lobectomy which cuts away brain tissue in the temporal lobe; extra-temporal resection, lesionectomy, which cuts away abscesses, tumours and any similar damage and hemispherectomy, where the cerebral hemisphere is either removed or functionally disconnected, which is the predominant treatment for drug-resistant epilepsy and frequently utilised in paediatric cases due to the promise of low risk and high efficacy (5). Other surgical solutions disconnecting both parts of the brain from each other are the corpus callosotomy, which aims at stopping generalised seizures by disconnecting both cerebral hemispheres, and is a palliative surgery (6), and multiple subpial transections where tangential intracortical fibres are severed, whilst preserving any penetrating vertical blood vessels and the vertical fibre connections of incoming and outgoing blood vessels (7). Neurosurgery does not cause pain to the patient and so can be seen as a less inconvenient procedure than having to continuously take AEDs; however, the process is invasive, and patients may be averse to it.
- Stem cells
Stem cells have the potential to play a vital role in regard to treatment options for epileptic disorders, including the generation of 3D organoids, cell therapies and evaluation of current AEDs. This paper will focus mainly on 3 different types of stem cells which may be used in stem cell therapies for epilepsy: neural, induced pluripotent and mesenchymal stem cells.
Neural stem cells (NSC) are multipotent cells found in certain zones of the adult mammalian brain, including the subgranular zone in the hippocampal senate gyrus, the hypothalamus and in the subventricular zone around the lateral ventricles. Neural stem cells have the ability for self-renewal and the infinite ability for proliferation to produce neural progenitor cells. Neural progenitor cells may be unipotent, bipotent or multipotent as they have the capacity to proliferate and terminally differentiate to form oligodendrocytes, astrocytes and neurons.
Induced pluripotent stem cells are pluripotent cells generated from genetically modified somatic cells. Through the use of the TALEN (transcription activator-like effector nuclease) or the CRISPR/Cas 9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9 nucleases) systems, dermal fibroblasts from skin or blood-derived hematopoietic cells undergo genome editing. These cells have the ability of infinite self-renewal due to a high expression of telomerase and have the ability to differentiate into any somatic cell (9). Induced pluripotent stem cells may be used in the treatment of epilepsy through cell therapies and transplants as well as the creation of 2D and 3D models, which allow for the development of novel anti-epileptic drugs as well as an increased understanding of the pathophysiology of epileptic disorders.
Finally, Mesenchymal stem cells (MSC) are multipotent adult stem cells which may be isolated from a selection of specialised tissues in the body and possess the capacity for self-renewal and multilineage differentiation. Current sources for mesenchymal stem cells include the menses blood, bone marrow, umbilical cord, endometrial polyps, endometrium and adipose tissue. Due to the widespread availability of these cells, they are easy to harvest in large quantities and provide a practical source for experimental and clinical applications (8).
Neural Stem Cells
Due to neural stem cells’ properties of self-renewal, plasticity, and ease of integration, they are promising tools for many neuro-restorative therapies for several disorders, including epilepsy (2). There are several ways that stem cells may be used to help treat epilepsy, such as delivering neurotransmitters to act as growth factors or else dampening the inflammatory response. Still, currently, the most investigated application is the use of neural stem cells to create new neurons which are integrated into the neural network (2). These neurons would then replace the damaged or faulty ones. In rats, embryonic neural stem cells were transplanted and grew into new neurons. The newly generated neurons did not migrate far from the implantation site. Nonetheless, they were able to send projections into the host brain tissue, suggesting that they could still control brain function (2). In another experiment, this time with foetal cells implanted into rats, the cells once again survived and differentiated well but again failed to migrate. The newly developed neurons did not cure the seizures altogether but reduced their occurrence by 69-87% (2). A later experiment involving mice resulted in most of the stem cells turning into benign tumours (2). Cannabis and anti-epileptic drugs combined reduced the occurrence of seizures by 50-90% (10). These results show great potential for neural stem cell therapies for epilepsy in the future; however, due to some of the side effects, neural stem cells are not yet ready for clinical trials.
Induced Pluripotent Stem Cells
This section will summarise and discuss a study conducted by Hirugashi et al. (11) in 2013, which evaluated the use of iPSCs as a model for Dravet Syndrome and provided a scientific basis for many future studies relating to the use of iPSCs with regard to treating epilepsy. Induced pluripotent stem cells have the potential to treat epileptic disorders through a variety of methods, including providing a patient-specific model through which disorders may be studied (as discussed in detail below), generating 3D organoids, regenerative medicine and cell therapies, and evaluating AEDs. Currently, modelling is the most advanced use of iPSCs technologies, being a cogent first step toward a possible treatment for epilepsy.
1. Dravet Syndrome
Dravet syndrome (DS) is an epileptic encephalopathy condition characterised by prolonged and/or frequent generalised tonic-clonic seizures, which are refractory to all current treatments and frequently develop into status epilepticus (12, 13). Dravet syndrome is a severe form of infantile-onset epilepsy, the symptoms of which include cognitive impairment as well as developmental delays in language, motor function, learning and social skills (12, 13).
Over 80% of DS patients have been found to carry de novo heterozygous dominant mutations in the SCN1A gene. This gene encodes the α-subunit of the voltage-gated Na+ channel 1.1 (Nav 1.1) (12, 13). Currently, over 100 mutations (including missense and nonsense mutations, frameshifts, insertions, deletions and microdeletions) of the relevant allele have been reported. The SCN1A mutations generate impaired Na+ channel function as well as decreased neuronal excitability (12, 13).
1.2 Mice Models
Initial studies of DS in mouse models produced conflicting results. Certain studies concluded that the Nav 1.1 subunit demonstrates increased expression in GABAergic inhibitory neurons, the decreased excitability of which results in an excitation/inhibition unbalance which triggers epileptic seizures (13, 14, 15). This was observed particularly in the axon initial segment of a parvalbumin (PV) – positive subgroup, where Nav 1.1 haploinsufficiency directly impacted action potential generation (11). However, a study conducted by Favero et al. (16, 13) demonstrated the presence of parvalbumin interneurons present hypo-excitability only for a restricted period of time during young mice development, the activity of which is normalised in later adulthood, suggesting this class of neurons is not responsible for the chronic epilepsy presented by DS patients.
The direct sequencing of blood-leukocyte-extracted genomic DNA revealed an SCN1A point mutation in the DS patient from which the skin fibroblasts (iPSCs) were obtained (11). This mutation was expected to prematurely truncate the Nav 1.1 protein in the fourth homologous domain.
Skin fibroblasts were obtained from a skin punch biopsy specimen and allowed for the establishment of 2 lines of patient-derived iPSCs (D1-1 and D1-6) (11). The iPSC line 201B7 was developed and used in the control experiment. The fibroblasts were cultured in DMEM (Dulbecco’s Modified Eagle Medium) containing: 10% foetal bovine serum, 50 IU/mL penicillin and 50 mg/mL streptomycin (11).
The fibroblasts were lentivirus transduced with Slc7al, plated (at a density of 3.5 x 105 cells/ 60 mm dish), and the following day four reprogramming factors (Sox2, Lkf4, Oct3/4 and c-Myc) were transduced using retroviruses. Seven days later, the fibroblasts were replated (at a density of 5 x 10³ -5 x 105 cells/ 100 mm dish) along with a mitomycin C-treated SNL feeder layer. The following day the medium was substituted with a human iPS medium (DMEM/F12) which contained: 20% knockout serum replacement, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 0.1 mM 2-mercaptoethanol, 4 µg/mL penicillin, and 50 mg/mL streptomycin. 24-28 days after the transduction of the programming factors, the iPSC colonies were isolated; up until this point, the medium was replaced daily / every other day (11).
All iPSC colonies presented the conventional human embryonic stem cell morphology: a round shape, tightly packed cells and a defined border. The expression of pluripotency markers was confirmed through the formation of teratomas. In support of the iPSCs’ undifferentiated state (confirmed using immunochemistry) as well as pluripotency, further analysis revealed that the resulting teratomas consisted of tridermic tissues (11). The silencing of reprogramming transgenes (using real-time PCR), the presence of the SCN1A mutation and normal karyotype were confirmed (11). To maintain the iPSC cultures, the cells were passaged every 4-7 days (depending on colony size) and the medium was refreshed daily. The iPSCs which were passaged fewer than 32 times were selected to be used for neural induction (11). iPSC colonies were then detached from the feeder layers and cultured in bacteriological dishes in suspension as embryoid bodies (EB) for 30 days. The EBs were then enzymatically dissociated, and the individual cells were cultured in suspension in the serum-free neurosphere medium (media-hormone-mix) for 10-14 days to allow neurosphere formation. Neurospheres which were passaged 1-3 times (using the dissociation procedures described above) were utilised for analysis (11).
In some preliminary assets, the medium was supplemented during the later phase of embryoid body and/or neurosphere formation with 5 or 30 nM of sonic hedgehog or 1µM of purmorphamine for ventilation of neuronal properties (11). Then the neurospheres were plated into poly-L-ornithine / fibronectin-coated coverslips with B27 supplements to allow terminal differentiation. The dissociated cells were then plated at a density of 1 x 105 cells/cm² (11). The differentiation medium was then supplemented with the following compounds: 10 ng/mL rhBDNF and rhGDNF and 200 µg/mL L-ascorbic acid to promote neuronal maturation and enhance cell viability (11). During neuronal induction, all clones effectively generated neurospheres. Adherent cells were differentiated from the neurospheres and presented the expression of neuron and astrocyte markers. Staining for an oligodendrocyte marker (CNPase) was negative across all cell lines (11). Wild type status was revealed for all examined sequence regions following direct sequencing of additional sodium channel genes, SCN2A and the genes for the subunits β1 and β2 (SCN1B and SCN2B) (11). Real-time PCR targeting genes (SCN1A, SCN2A, SCN3A, SCN8A) on iPSC-derived neurons 30 days after differentiation were used to establish expression levels for the voltage-gated sodium channels. Across all cell lines expression of SCN2A was the highest (then SCN1A, SCN3A and finally SCN8A) (11). The LightCycler 480 System ll with the SYBR Premix Ex Taq was used to perform real-time PCR (11).
After the expression levels across the lines were normalised to each other, it was discovered that SCN1A expression was higher within patient neurons as opposed to control neurons. It was also confirmed that SCN1A mRNA translated from the mutated allele was present in patient neurons, suggesting that the mutated mRNA escaped nonsense-mediated decay (11). TRIZOL Reagent, RNase-Free DNase set, and RNeasy Mini / Micro kits were used to extract total cellular RNA. Complementary DNA synthesis was executed using the SuperScript lll First-Strand Synthesis System for RT-PCR and oligo-dT primers from 0.2 – 1.0 µg or total RNA (11). The relative expression of different mRNAs was analysed by normalising the amount of cDNA to ᵝ-actin mRNA expression. At least 3 different cultivated samples were used to determine the mRNA expression levels in iPSC-derived neurons (11).
Next, a polyclonal antibody targeting the D1-D2 linker was used to examine Nav 1.1 expression at the protein level. Epitope peptide pre-treatment was used to confirm the antibody’s specificity (11). 4% paraformaldehyde was used to fix the cells on coverslips for 10-30 minutes at room temperature. Next, the cells were washed 3 times with PBS, then incubated with blocking buffer (PBS containing 5% normal goat / foetal calf serum as well as 0.1 – 0.3% triton X-100) and lastly incubated overnight at 4°C with primary antibodies diluted with the blocking buffer (11). The cells were then washed 3 times with PBS, then incubated with secondary antibodies conjugated with Alexa Fluor 488 / 555 and Hoechst33342 for 1 hour at room temperature. Next, the cells were washed 3 times with PBS, then once with distilled water and mounted on slides with FluorSave Reagent (11).
Nav 1.1 immunostaining was apparent in cell bodies, dendrites and axons, especially within neurons with well-developed axons. The intense expression of Nav channels (PAN-Nav) in the axon initial segment became evident after several weeks of in vitro differentiation. It has been suggested that this spatial and temporal expression pattern is vital in action potential generation (11). The use of GAD67 staining confirmed that the majority of the Nav 1.1 positive control and patient-derived neurons were GABAergic in nature ( 58.3% in 201B7, 52.6% in D1-6, 54.8% in D1-1 ) (11). Subsequently, Nav 1.1 expression variations among the GABAergic neuron subtypes were examined according to co-expression of PV, calretinin, or somatostatin. Several calretinin positive 2017B neurons were produced, which stained for Nav 1.1 as well after 33 days of differentiation. However, in all cases, somatostatin-positive neurons presented with indistinct or negligible Nav 1.1-staining. PV expression was imperceptible, despite treatment (with sonic-hedgehog or purmorphamine for ventralization and/ or BMP4) (11).
Nav 1.1 gene expression comparisons were made with 1-way ANOVA (between Nav channel genes) and 2-way ANOVA (between the iPSC clones) (11). However, PV mRNA along with mRNA for Nkx 2.1 (a medial ganglionic eminence neuron marker) were detected, indicating the probable presence of PV-neuron precursors. However, the culture conditions of the experiment may have prevented further maturation (11).
Technical difficulties arose within the study with regards to distinguishing neuronal subtypes other than GABAergic amongst the Nav 1.1 positive neurons. Nevertheless, glutamatergic neurons were established as a minor population as some cells were positive for VGlut1 (as a marker for glutamatergic neurons). Occasionally these neurons co-localised with SCN1A Venus fluorescence (11).
The upstream genomic sequence of a SCN1A’5 untranslated exon (obtained from the patient’s genomic DNA) was used as the SCN1A promoter sequence. The untranslated exon connected with the 5’-end of the first coding exon (obtained from D1-1 iPSC-derived neuronal cDNA) via PCR, then transferred into the pSIN-Venus vector. The pSIN-Venus vector served as a cloning site connected to Venus cDNA (11). The pSIn construct, pLP1, pLP2 and pLP/VSVG plasmids were combined and transfected into 293FT cells using CalPhos Mammalian Transfection Kit or Lipofectamine 2000 Reagent to produce the lentivirus. The medium was replaced the subsequent day. After 48 hours, the virus-containing medium was collected, filtered and ultracentrifuged (at 25 000 rpm with an SW 28 Rotor) at 4°C for 90 minutes. The viral pellet was then resuspended in 1/200 of the original medium volume along with media-hormone-mix, which was aliquoted and stored at -80°C until use (11).
The SCN1A-Venus construct was utilised to infect freshly plated cells from dissociated neurospheres. SCN1A-Venus fluorescence developed in a few neurons after a few days of differentiation and continued to increase in both Venus-positive neuron number and fluorescence intensity as neural differentiation continued (11). The coexistence of Nav 1.1 and the Venus protein in the same neuron was confirmed through the use of immunostaining. The majority of the Venus-positive neurons expressed the Nav 1.1 protein as well (81% in 201B7 neurons, 90.1% in D1-1 neurons and 78.8% in D1-6 neurons). Many of the SCN1-Venus-positive neurons also tested positive for GABA, indicating GABAergic neurons (79.3% in 201B7 neurons, 83% in D1-1 neurons and 70.3% in D1-6 neurons) (11). A confocal laser-scanning microscope (FV100-D) was used to acquire images, with observations made through x20 objective to determine whether Nav 1.1-positive neurons were positive for GAD67, calretinin or GFP as well (for the detection of Venus) (11).
Current clamp experiments on both patient and control-derived neurons were carried out 22-50 days into neuronal differentiation to examine the electrophysiological behaviour of the cells. Through the use of an upright microscope (BX51WI – Olympus Melville, NY) equipped with a CMOS image sensor camera, ORCA-Flash 2.8 cell micrographs were generated. It was found that more succinct differentiation periods produced unreliable results, which may have been the result of incomplete neuronal maturation (11).
The following factors were taken into consideration to determine which neurons to select for electrophysiological analyses:
- Clear SCN1A-Venus fluorescence,
- Mature neuronal morphology (with large and complex cell body and growth of over 4 neurites),
- A membrane capacitance exceeding 30 pF,
- A resting membrane potential more negative than or equal to -30 mV.
A 40x water-immersion objective (LUMPlanFl/IR2 – Olympus) with a U-MGFPHQ cube (excitation: 460-480 nm, dichroic mirror: 485 nm, emission: 495-540 nm – Olympus) was used to detect reporter fluorescence, Venus. The images were then processed with Aqua Cosmos software. The extracellular solution was made up of the following compounds: 150 mM NaCl, 4 mM KCl, 2 mM CaCl2, 2 mM MgCl2.6 H2O, 10 mM HEPES, and 10 mM glucose (adjusted to pH 7 with NaOH). Patch pipettes were made from borosilicate glass with filament and pulled to resistances of 2-4 MΩ when filled with 0.22-µm filtered intracellular solution. The intracellular solution contained: 117 mM K-methanesulfonate, 9 mM EGTA, 9 mM HEPES, 1.8 mM Tris-GTP and 5 mM KCl (adjusted to pH 7.3 using KOH) (11).
An Axopatch 700B Amplified and pCLAMP 10 software was utilised to carry out whole-cell patch clamp recordings. The signals were low-pass Bessel filtered at 10 kHz and sampled at 50 kHz using an Axon Digidata 1440A digitizer (11). Integrating the capacitive current evoked by a 10-mV depolarizing pulse from a holding potential of -65 mV was the method used to calculate cell capacitance (11).
Resting membrane potential was calculated using the mean potential throughout a 10-second continuous recording in zero-current clamp mode (11). Cell capacitance, resting membrane potential, action potential firing threshold, peak voltage, action potential decrement and area under the input-output relationship curve were compared amongst the clones using one-way ANOVA and/or the Kruskal-Wallis test (11).
48 neurons were recruited from the D1-1 patient-derived cell line. 27 neurons were recruited from the D1-6 patient-derived cell line. 33 neurons were recruited from the 201B7 control cell line (11). In an effort to minimise the inclusion of potentially inappropriate cell responses, the cell capacitance and resting membrane potential for all cells were established as indicators for neuron maturity. Resting membrane potential averaged between -40 and -45 mV for both the patient-derived as well as the control neurons. Most of the neurons had a membrane capacitance of up to 70 pF, and some anomalies of 100+ pF were present; however, they were removed from the experiment as the excessive current injection was required to generate action potentials (11).
Next action potential generation was examined in the current clamp configuration using 10-ms depolarising current injections from a holding potential of -70 mV. No statistical variations were found between the patient-derived and control neurons with regard to firing threshold and peak voltage (11). During current-clamp experiments, constant current injections were used as needed to maintain the cells at -70 mV (11). Determining the firing thresholds and peak voltages of the neurons was achieved through the generation of single action potentials (operationally defined to minimally reach 0mV) by current injections (10 ms). The injection current amplitude was increased in 10 -pA increments from sub- to supra thresholds (11).
The input-output relationship was determined by triggering action potentials using sustained 500-ms injections of depolarising current. For all neurons, the number of action potentials per 500-ms stimulation period increased as the intensity of the injected current did. It was found that amplitude attenuation became apparent as current injection intensified. Amplitude attenuation intensified up to a certain current injection level, at which action potentials began to obviously decline in amplitude and number, eventually stopping completely (11). Sustained depolarising currents (500 ms) were injected, and the current amplitude was increased from 5 to 100 pA in 5-pA increments to investigate the input-output relationship (11).
The Wilcoxon rank-sum test was used to compare the action potential number of each injection level in the input-output relationship between the clones. ANCOVA was used to compare the gradient of the number of action potentials vs injected current (11). The action potential number of each injection level in the input-output relationship was compared between the clones using the Wilcoxon rank-sum test. The slope of the number of action potentials vs injected current was compared using ANCOVA (11). It was suspected that electrically-immature neurons were plentiful amongst the cells nominated for analysis as depolarisation blocks were common. As a result, only neurons which produced 10 or more action potentials were admitted for further electrophysiological characterisation (11). 12 neurons in D1-1, 15 neurons in D1-6 and 16 neurons in 201B7 were used in electrophysiological comparisons between the cell lines. It was found that capacitance, action potential threshold, resting membrane potential, and action potential peak voltage was indistinguishable between control and patient-derived neurons (11).
In the input-output relationship, however, both patient-derived neuron lines frequently produced marked amplitude attenuation, something which was not observed in the control neurons. Another observation made in the patient-derived cell lines only was a reduction in action potential firing at once the current exceeded 50 pA (11). A higher number of D1-1 neurons (33.3%) and D1-6 neurons (46.7%) reached their peak output prior to reaching a current of 100 pA compared to 201B7 neurons (12.5%). These observational differences support theories of functional impairment (reduced output capacity during intense stimulation) in the patient-derived GABAergic neurons (11). Final data was obtained from the neurons on at least 8 coverslips of minimum of 4 separately cultivated samples in each clone. All data analyses were performed using SAS Software Package (11).
a. Discussion of results from the research paper
The study successfully generated neurons from DS patient iPSCs where neurons of identical character were obtained from the control and two patient cell lines (as demonstrated by gene expression and immunocytochemical analyses). However, compared to the control neurons, the patient-derived cells presented impairments in action potential generation in response to sustained current injection, producing fewer action potentials with attenuated amplitudes and earlier depolarisation block. This demonstrates that neurons with DS pathophysiology have an inability to adequately respond to high-intensity stimulation, as previously seen in neurons isolated from rodent epilepsy models (11).
It is mentioned that difficulties were encountered when attempting to conclusively determine whether the Nav 1.1 -positive neurons were GABAergic or glutamatergic; these challenges were thought to be the result of cell population heterogeneity and low marker protein expression. However, data obtained from immunocytochemical analyses strongly suggested most Nav 1.1-positive neurons were GABAergic. GABA immunostaining was also present in the majority of SCN1A Venus-positive neurons, indicating that the neurons undergoing electrophysiological analysis were phenotypically homogenous (11).
The study did not include the investigation of glutamatergic neurons, as the culture conditions did not permit ready differentiation into this neuronal subtype. As a result, the number of glutamatergic neurons present was below what is usable for functional analyses (11). The results of this research paper (11) are in agreement with the mice models discussed above, where lower Na+ currents and hypo-excitability in iPSC-derived GABAergic interneurons, due to a functional decline in GABAergic neuron activity, carrying SCN1A mutation was reported (13, 12). This may indicate that the pathophysiology of human and mouse DS employs similar mechanisms (11).
However, several key differences exist between human and rodent brains with regards to Nav 1.1 expression: in the rodent cerebral cortex Nav 1.1 is predominantly expressed in the axon initial segment of GABAergic interneurons (and not in calretinin and somatostatin-positive neurons), with minor Nav 1.1 expression levels observed in pyramidal neurons, whereas in humans Nav 1.1 shows somatodendritic localisation and expression in pyramidal neurons (specifically in cortical layer V and in the hippocampus) (11). It was then concluded that the underlying mechanism behind DS is resultant of the imbalance between inhibitory and excitatory systems (12); however, the involvement of other neuron types cannot be excluded, and further characterisation of non-GABAergic neurons must be evaluated and studied in the future in further our understanding of this DS model (11).
b. Contrasting findings
In 2013, two other groups (17, 18) conducted similar investigations as the one described in this paper. However, contrasting results were obtained (12). The first of these groups (17) observed hyperexcitability of disease-specific iPSC-derived glutamatergic neurons. In contrast, the second (18) demonstrated hyperexcitability of both glutamatergic and GABAergic neurons derived from patient iPSC lines (12); in the investigation discussed in this paper, however, less information was reported on excitatory neurons (11, 13).
All 3 groups (17, 18, 11) used similar experimental protocols with regard to the use of iPSCs generated from DS patients, the fibroblast reprogramming method as well as electrophysiological analyses using whole-cell patch clumping (12). However, a few essential differences were present in the methods used: (17) made use of a direct conversion method to differentiate the iPSCs into neurons, whereas (11) and (18) made use of indirect differentiation methods via embryonic bodies (12).
The contradicting findings and phenotypic variability may have been caused by the types and locations of the SCN1A mutations used, the Nav 1.1 functional aspects which were evaluated in vitro, differentiation protocols, the generation of inhibitory neurons at different stages of maturation or the subtypes and origin of the derived GABAergic neurons (12) (13). These differing findings provided essential information concerning caveats when dealing with iPSC, specifically the importance of the careful definition of neuronal lineage, subtype, differentiation and maturation (12).
2. Generation of 3D Neuronal Organoids
iPSCs also demonstrate the ability to differentiate to form cerebral organoids: 3D self-assembled structures which exhibit functional behaviour and generate complex neuronal networks, providing a promising model to study genetic epilepsies (13). For example, this model has permitted an increased understanding of the ‘second hit’ in Tuberous sclerosis complex (13, 19), as well as the study of the Miller-Dieker syndrome (13, 20), and holds great potential for future research and the development of personalised treatment for epileptic conditions. Cortical organoids demonstrate an enhanced and constant increase of electrical activity (including mean firing rate, burst frequency and synchrony) over time (10 months) compared to iPSC 2D culture models, as well as the generation of nested oscillatory network dynamics which are produced and maintained by the involvement of GABAergic and glutamatergic neurons (13). These findings provide the first evidence of the development of complex and functional neuronal networks by cerebral organoids, indicating that the organoids allow for studying the physiology of brain network formation as well as neurological disorders, including epilepsy (13).
3. Evaluation of AEDs and Drug Discovery
Disease-specific iPSCs derived from human somatic cells may also be used to develop and evaluate the anti-epileptic pharmacological actions of empirically developed and herbal medicines in vitro, as opposed to ‘animal seizure models’ which were used previously (12). An example of this is cannabidiol (CBD), which has been used in combination with other AEDs to effectively ameliorate seizure activity in refractory epilepsies, including Dravet syndrome (12), by compensation for the characteristic inhibitory/excitatory imbalance caused by SCN1A mutations (12, 21).
Furthermore, the use of iPSC-derived 2D and 3D epilepsy models effectively recapitulate epilepsy pathogenesis allowing for drug repurposing, which may facilitate the discovery of new and successful AEDs (12). Recent developments in MEA technology enable the analysis of the electrophysiological activities of iPSC-derived epilepsy models, allowing for the evaluation of anticonvulsant toxicity and screening of epilepsy drugs (12, 22).
Mesenchymal Stem Cells
The successful in vitro model developed by Hiroshima et al. (as described above) has since been used in combination with human umbilical cord mesenchymal stem cells in a study conducted by Zhao et al. to develop potential treatments for the generalised tonic-clonic seizures, which are characteristic of Dravet Syndrome. These frequent and severe seizures may cause neuroinflammatory reactions and oxidative stress, which may result in neuronal toxicity and dysfunction, hippocampal inflammation, which may contribute to the generation of individual seizures and cell death, and blood-brain barrier disruption (23).
The study successfully created a human umbilical cord mesenchymal stem cell culture medium (HMSC-CM) which was introduced to the patient-derived neural cells differentiated from iPSCs. The presence of HUMSC-CM in the patient-derived iPSC model allowed for an increase in superoxide dismutase 1 and 2 (SOD1 and SOD2), glutathione peroxidase and glutathione levels in the differentiated neurons. These increases were shown to contribute to a reduction in reactive oxygen species levels (ROS) caused by oxidative stress, which, when too high, may damage DNA, RNA and may lead to the activation of cell death processes. The presence of HUMSC-CM also resulted in decreased inflammation of the DS patient-derived neuronal cells due to an increased expression of anti-inflammatory cytokines (TGF-β, IL-6, and IL-10) as well as significant downregulation of the expression of tumour necrosis factor-α ( an inflammatory cytokine responsible for a range of signalling responses within cells leading to aptosis or necrosis) and interleukin-1β (a pro-inflammatory cytokine). Furthermore, the HUMCs decreased intracellular calcium concentration, malondialdehyde (a highly toxic molecule which is the principal product of polyunsaturated fatty acid peroxidation and may cause oxidative stress) and ROS levels in the patient-derived neurons exhibiting the DS pathophysiology. Finally, the study reported an enhanced action potential firing ability in the patient-derived neurons as a result of HUMSC-CM (23).
As of the present stage in stem cell treatment, there are no ethical issues.
Limitations and Challenges
One of the main challenges which persist within epilepsy studies is how closely iPSCs and MSCs can model primary neural cells with regard to functionality, immunochemistry and morphology in both 2D and 3D cultures (24). The extent to which 3D cultures are able to recapitulate the neuronal networks and complexity of the brain poses a further challenge; however, recent advancements have been made allowing for prolonged culture periods, facilitating cell maturity, as well as intrinsic differentiation into various specific subtypes of neurons and glial cells (25). Furthermore, the low reproducibility of 3D organoids poses another challenge to their application to disease modelling as well as large scale drug testing (13). However, the use of a feeder and xeno-free method has been developed, which may be able to circumvent this issue (13, 26). Finally, the absence of a vascular system within the organoids further hinders their effectiveness in regards to modelling epileptic disorders (13). This limits the study of epileptogenesis in later stages of neurodevelopment as long-term organoids frequently exhibit apoptotic cell death in their inner regions.
Another challenge faced when modelling epileptic disorders using iPSCs, is the potential dissimilarity between the iPSC cultures and primary cells in vivo (24, 25, 12). A persistent challenge faced in vitro is the lingering of the cells in the maturation state, resembling foetal or neonatal cells despite the differentiation method used. This may be the result of the unknown in vitro factors present in the environment during neural development as well as the limited cultivation time (24, 27, 25). This creates a shortage of mature iPSC-derived neural cells which may be used, hindering the progress of epilepsy studies.
Furthermore, the reprogramming processes used to create iPSCs may allow for the rise of genetic alterations, creating genetic variability among clones obtained from a single donor (12).
Another factor to be considered when using iPSCs or MSCs is the potential for non-negligible variations and alterations in genetic identity and the extent of maturation, which arises from the current limitations in differentiation techniques as well as other unique factors which the cells may conceive (24). A study conducted by Hu et al. demonstrated a decreased differentiation ability of iPSC to produce PAX61 neural progenitors as compared to embryonic stem cells (28), which is a crucial initial step to allowing the differentiation into many CNS neuronal subtypes (9, 28). This decreased ability may be the result of epigenetic differences from the donor somatic cell, which are retained throughout the reprogramming process (9). A final challenge which may arise is the ability to fully differentiate iPSC-derived neurons (9). This is demonstrated by reports of depolarised resting potentials as well as small percentages of neurons which undergo evoked firing in patch-clamp recordings obtained from secondary neuronal cultures (9). This may be overcome through the recent development of a cell culture medium which stimulates the maturation and supports the electrophysiological function of iPSC-derived neurons (9, 29).
A further cause of iPSC variabilities, including growth and the expression of specific genes, maybe the specific genotype of the donor, as well as genetic differences between donors, which may influence the reprogramming efficacy in iPSC generation (24). These genetic variations may be minimised through the use of genome editing techniques to generate isogenic iPSCs (comparison between a virtual patient and healthy individual iPSCs). Furthermore, epigenetic variations of donors may impact gene expression and influence the reprogramming efficacy in iPSC generation (24).
With regards to the use of IPSCs, MSCs and NSCs in regenerative medicine to treat epilepsy, challenges regarding transplantation arise (12). Due to the pluripotency of iPSCs, tumorigenicity must be prevented as well as other factors which may cause heterogeneity including immunogenic potential, maturation tendency, differentiation potential and epigenetic status (24). A further challenge preventing these stem cells from being used in transplantation is the high probability of immune rejection when donor cells are used due to genetic variations. These obstacles may be overcome through current clinical trials as well as the development of novel technology (27).
Another critical challenge to consider with the use of iPSC-derived neurons to study epileptic diseases is the time required for electrophysiological properties and synaptic connections to mature in culture (9). Certain neurotrophic factors (brain-derived, glial-derived, nerve growth factor and neurotrophic-3) may have the potential to promote neuronal survival and maturation (9). The rate of maturation may be increased through the use of certain compounds (e.g. gamma-secretase inhibitors) and by co-culturing iPSC-derived neural progenitors with human or rodent astrocytes (9). Furthermore, due to the heterogeneity of epilepsy and its diverse pathogenic and genetic causes, it is impossible to address and discuss all epileptic disorders in this research paper.
Finally, the practical implications and restrictions of using iPSCs, NSCs and MSCs must be considered, including the cost of research as well as time constraints. Research, the development of clones from multiple patients and donors, gene editing and differentiation techniques, as well as costly culture supplementations must all be taken into consideration. It may exponentially slow the development of regenerative and cell therapies for epileptic disorders (12).
Future research into the human in vitro disease models, using different sources of pluripotent stem cells, is therefore required to overcome these obstacles. A particularly promising area for future research may be Nestin-expressing hair follicle stem cells as they are easily accessible, can be utilised without any genetic manipulation and have the potential to differentiate into neurons (11).
Through continual technological and scientific advancements which are being made in the fields of stem cell research as well as epilepsy, the treatments discussed above, including cell therapy, AEDs, resection surgery and the generation of 3D organoids, stem cells provide great promise with regards to advancing our knowledge and therapies for epileptic disorders as they resolve a number of the compromises made with AEDs and surgery, such as giving patients with drug-resistant epilepsy more treatment options and minimising the number of treatments that only reduce or control the impact/occurrence/both of seizures, with the possibility of better long-term outcomes for patients. Perpetual amelioration and enhancement of various epileptic disease models through the use of iPSCs, MSCs and NSCs, allow for the study of the pathophysiology of numerous epileptic conditions as well as the development of patient-specific treatments through the use of stem cells derived from the patient themselves, bringing the medical field ever closer to treating epilepsy, taking further into account the homogeneity of cases. Many challenges and difficulties such as conflicting findings continue to arise within this field; however, continued progress, as well as the rapid development of solutions, are possible in the future with adequate and dedicated research.
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