Induced pluripotent stem cells (iPSCs) as game-changing tools in the treatment of neurodegenerative disease: Mirage or reality?

4.1 AD

Alois Alzheimer first observed that the disease which was later named Alzheimer's disease (AD) has separate and discernible neuropathological symptoms (Tanzi & Bertram, 2005). After that, our understanding about the neuropathological lesions caused by this condition was improved. Nowadays, we know that the dysregulation of the amyloid-beta (Aβ) levels induce senile plaque development entailing Aβ depositions in AD (Small & Cappai, 2006). The swift progression of the iPSCs method improves iPSCs utilization in AD. Since 2008, various experimental teams have described iPSCs application in AD modeling, preparing proof-of-principle for modeling patient-specific AD pathology through iPSCs utilization and investigating various pathological characteristics of AD in the laboratory (J. Yang et al., 2017). Yagi et al. (2011) primarily established iPSCs from AD patients with PSEN1 (A246E) and PSEN2 (N141I) mutations and showed that these familial AD (FAD)-derived iPSCs had a promoted Aβ42 construction and an enhanced ratio of Aβ42/Aβ40. Later, another experimental team created iPSCs lines from two sporadic AD (SAD) as well as two FAD patients carrying a duplication of the amyloid precursor protein (APPDp; Israel et al., 2012). Investigations revealed that neurons derived have meaningfully higher rates of Aβ40 and enhanced phosphorylation of tau protein (at Thr 231) in association with an increased rate of active glycogen synthase kinase-3β (aGSK-3β; Cataldo et al., 2000). Intriguingly, differentiated-neurons' exposure to β-secretase inhibitors (BSI) attenuated phospho-tau (Thr 231) and aGSK-3β levels, whereas γ-secretase inhibitors only lessened the Aβ40 level. These results supported this hypothesis that APP proteolytic processing had a tight association with not only GSK-3β activation but also tau phosphorylation in neuronal tissue (Nieweg, Andreyeva, van Stegen, Tanriöver, & Gottmann, 2015). In another study, Auboyer and his colleagues could generate from skin fibroblasts of AD patients with G217D causal mutation on presenilin 1 (PSEN1). The supporting analysis verified the existence of the G217D mutation in the generated iPSC line and confirmed these cells' in vitro potential to explore human AD pathology (Auboyer et al., 2019). Another study revealed that nobiletin attenuated the Aβ cellular level in AD pathological conditions in the iPSC-derived AD model (Kimura et al., 2018). Furthermore, enhancement of TAU protein phosphorylation in neurons from either FAD or SAD patients was found following the performance of a comprehensive study on the iPSC-derived AD model. On the other hand, extracellular amyloid-β 1-40 (Aβ1-40) and amyloid-β 1-42 (Aβ1-42) levels and susceptibility to oxidative stress were increased in both FAD and SAD models compared with control neurons (Ochalek et al., 2017). Armijo et al. (2017) reported that neurons from AD patients carrying the A246E mutation in PSEN1 have special intrinsic features that enhance their susceptibility to the poisonous effect of the Aβ1-42 oligomers in the AD.

4.2 HD

Huntington's disease (HD) is an autosomal dominant neurological disorders characterized by emotional, cerebral, behavioral, and motor dysfunctions (Bora, Velakoulis, & Walterfang, 2016). In HD, nevertheless the most mutual zone of neuron loss is the striatal part of the basal ganglia; primary deficit and late death of the cortical neurons have been evidenced. HD commences with an increase in the number of CAG repeats in the HD gene encoding Huntington protein producing abnormally long polyglutamine districts in the protein N-terminus (Zühlke, Rless, Bockel, Lange, & Thies, 1993). HD modeling includes the differentiation of the patient-specific iPSCs into GABAergic neurons (Garcia et al., 2019; Machiela et al., 2019). Recently, generated iPSC lines from HD patients commonly demonstrated strong pathologic phenotypes and exposed various charcateristics of HD (Table 1). First, Park et al. reprogramed HD-patient fibroblasts into iPSCs (An et al., 2014). Based on proteomic analysis, oxidative stress-related proteins upregulation in association with cytoskeleton-related proteins downregulation was recognized in HD-patients derived iPSCs (Chae et al., 2012). Moreover, increased DNA damage-mediated apoptosis and attenuated differentiation potentials were found in HD-patients derived iPSC lines compared with control neurons (Chae et al., 2012; Kedaigle et al., 2019). Additionally, MAPK and Wnt signaling pathways dysregulation in association with changed expression of p53 commonly characterized in HD patients were also identified in HD-patients derived iPSCs (Szlachcic, Switonski, Krzyzosiak, Figlerowicz, & Figiel, 2015). After that, for the first time, established iPSCs from two homozygous HD patients with 39-44 CAG repeats in HTT were reported by Camnasio, Delli Carri, et al., 2012).

According to a consortium study on HD-patient derived iPSC, broad types of the transcriptional variations participating in signaling, cell cycle, axonal guidance, and neuronal development were found and their roles in HD pathogenesis were revealed (Consortium, 2012). In another study, Grigor'eva et al. (2019) reported a successful establishment of the iPSC line from peripheral blood mononuclear cells (PBMC) of HD patient with 47 CAG repeats in HTT using episomal vectors. Similarly, Bidollari and colleagues were the other experimental teams that generated human iPS cell lines from skin fibroblasts of HD patients by episomal vectors (Bidollari et al., 2018).

4.3 PD

The pathophysiology of Parkinson's disease (PD) is the loss of dopaminergic (DA) neurons upon revision in the brain PD-related biological functions, inducing degeneration of the DA neurons of the substantia nigra pars compacta (SNpc; Mercado, Valdés, & Hetz, 2013). Examinations have suggested that protein accretion in Lewy bodies, and impairment of autophagy, cell metabolism and mitochondrial function, concomitant with neuroinflammation and blood–brain barrier disturbance can promote PD occurrence and progression (Chung, Kim, & Jin, 2010). The use of PD-patient-derived iPSCs will help researchers to recognize the involved genes and mutations in the PD pathogenesis and subsequently identify the molecular mechanisms that participate in PD progression (Stoddard-Bennett & Pera, 2020). In the last years, synuclein α (SNCA), β-glucosidase (GBA), leucine-rich repeat kinase 2 (LRPK2), parkin RBR E3 ubiquitin-protein ligase (PARK2) and PTEN-induced kinase 1 (PINK1) mutations have frequently been explored in genetic PD iPSCs (Guhathakurta et al., 2020; Kaavya et al., 2020; Kang, Tang, & Guo, 2016). Investigators try to clarify the pathological character of not only dopaminergic neurons but also other genetic PD iPSCs-derived neuronal cells. Generally, researchers believe that the loss of function (LOF) mutation, triggering aberrant aggregates of inactive proteins, occur in these genes (Schulte & Gasser, 2011). The SNCA protein assembly, encoded by the SNCA gene, is a key characteristic of PD and the SNCA A53T mutant in association with SNCA gene triplication belonging to the familial PD SNCA mutants. In 2011, the first successful generation of the dopaminergic neurons from SNCA mutant iPSCs was reported by Soldner et al. (2011). After that, various iPSC lines were generated from PD-patients with the SNCA A53T mutant or SNCA gene triplication mutant generated, and boosted cellular rates of the SNCA were identified in these iPSC-derived dopaminergic neurons (Kouroupi et al., 2017). Interestingly, induction of forebrain cortical neurons, neural precursor cells (NPCs), and GABAergic neurons has been reported from SNCA mutant iPSCs, and elevated levels of ROS have been shown in these differentiated cells (Lin et al., 2016).

Besides this, Bouhouche et al. (2017) showed that the LRRK2 G2019S mutation is the most common genetic mutation in familial PD. A large number of studies have highlighted the importance of the LOF mutation of the LRRK2 mutant in diminishing neurite outgrowth, numbers, as well as process complexity (Fernández-Santiago et al., 2015). On the other hand, an increased level of DNA damage-mediated apoptosis has been indicated in dopaminergic neurons- derived from patients with LRRK2 G2019S mutation (Weykopf et al., 2019). Other reports presented LRRK2's significant role in neural development because of the observation of an impairment in the self-renewal potential of neural stem cells of PD patients carrying the G2019S mutation (G.-H. Liu et al., 2012; Walter et al., 2019). The β-glucocerebrosidase (GBA) gene mutations are other types of mutation that participate in sporadic PD incidence and progression. Some investigation on dopaminergic neurons differentiated from iPSC of PD patients carrying GBA1 mutations have demonstrated that there exist correlations between GBA1 and increased SNCA cellular levels and autophagic and lysosomal defects (Aflaki et al., 2016, 2020).

4.4 ALS

Amyotrophic lateral sclerosis (ALS) or motor neuron (MN) diseases, is a clinical syndrome entailing MNs continuing degradation in the spinal anterior horn and motor cortex and axon loss in the lateral columns of the spinal cord (Cova & Silani, 2010). The MNs are nerve cells stretching from the brain to the spinal cord and muscles contributing to the induction and transmission of an essential association between the brain and the voluntary muscles (Birger, Ottolenghi, Perez, Reubinoff, & Behar, 2018). Research has shown transactive response (TAR) DNA binding protein with Mr 43 kDa (TDP-43) as the chief pathological protein participating in sporadic and familial ALS. However, the existence of a tight relation between a variety of mutations in superoxide dismutase 1 (SOD1), RNA binding protein FUS, and C9orf72 and ALS with molecular and neuropathological symptoms has been supported (Kirby et al., 2010). In 2014, the first in vitro models for ALS was established by using iPSC-derived MN from patients with superoxide dismutase A4V (SOD1A4V) and superoxide dismutase D90A (SOD1D90A) mutations (H. Chen et al., 2014; Kiskinis et al., 2014). These studies demonstrated a notable proportion of electrophysiologically active MN derived from these patients’ generated iPSCs. Moreover, differentiated MN demonstrated spontaneous and advanced decrease in cell survival the same as exhibited in ALS patients (Kiskinis et al., 2014). Besides this, ALS-associated morphological alterations were shown in generated ALS models, in particular, attenuation in soma size as well as a change of dendrite length (H. Chen et al., 2014). It has already been demonstrated that the observed phenotype is due to neurofilament dysregulation and accumulation, triggering neuronal tissue cell-death (Campos-Melo, Hawley, & Strong, 2018; H. Chen et al., 2014). Studies showed that there were differences in mitochondrial activity, protein translation and cytoskeletal structure between iPSC-MN and control MN (Kiskinis et al., 2014). In 2017, continuous hyperexcitability of differentiated MN from iPSC lines with the SOD1A4V mutation was demonstrated by Guo, Fumagalli, Prior, and Van Den Bosch (2017); on the other hand, hypoexcitability in association with lower proportion of Na⁺/K⁺ has been detected in MN from an ALS patient expressing SOD1D90A mutation (Hawrot, Imhof, & Wainger, 2020; Naujock et al., 2016). Although there is an argument whether hyper- or hypoexcitability participates in the pathology of ALS, it is obvious that electrophysiological variations contain a minimal phenotype in ALS models (Hawrot et al., 2020). Recently, Ustyantseva et al. (2020) generated an iPSC line from PBMC of the patient with SOD1 Asp90Ala mutation utilizing episomal vectors, and Birger et al. (2019) obtained iPSC-derived astrocytes from ALS patients with C9orf72 mutations. Birger et al. found that a reduction in the secretion of various antioxidant proteins in MNs due to soluble factors secreted by C9-mutated astrocytes played a key role in MNs apoptosis in ALA patients. The results show that oxidative stress resulting from any impairment in C9-astrocytes has a toxic effect on MNs that possibly leads to neurodegeneration. Importantly, the findings show that therapeutic approaches in familial ALS in addition to MNs can target astrocytes to decrease nervous system damage (Birger et al., 2019). In another study, Bursch et al. (2019) showed that iPSC-derived MNs from ALS patients carrying a mutation in C9orf72, SOD1, and TDP43 demonstrate some mutation-specific modifications in glutamate receptor features in combination with calcium dynamics, supporting ALS pathogenesis.

5.1 AD

\As cited, the iPSCs have notable capabilities in cell transplantation therapy of AD. Klincumhom and his colleagues described that transplantable NPCs or neurons can be obtained from iPSCs (Klincumhom et al., 2012; Sareen et al., 2014). NPCs derived from iPSCs have promising potential to differentiate into neurons, astrocytes as well as oligodendrocytes, with good prospects for AD cell transplantation therapy. The neurogenesis dysfunction in AD animal models demonstrates that a lack of a favorable balance between neurogenesis and neuronal death takes place in AD incidence and development (Haughey, Liu, Nath, Borchard, & Mattson, 2002). As verified, the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus are common sites of neurogenesis in adults. It has already been shown that that AD transgenic mouse harboring PSEN1P117L demonstrated reduced viability of NPCs, supporting a significant reduction in the generation of new neurons. Furthermore, the existence of a relation between attenuated adult neurogenesis in DG with diminished contextual fear conditioning in mouse has been reported, according to a large number of studies (Hallett et al., 2015; R. Wang, Dineley, Sweatt, & Zheng, 2004). In APP V717F mice, Donovan et al. (2006) found an age-associated reduction of cell growth in SGZ of DG of the hippocampus. Research shows that neurogenesis is stimulated as a healing mechanism to compensate for neurodegenerative neuronal cell death; but the viability of freshly established neurons was diminished after neurodegeneration development (Shohayeb, Diab, Ahmed, & Ng, 2018). Although there are differences in the molecular composition and morphological structure of the SVZ of mammals and rodents (Berger, Lee, & Thuret, 2020), a reduction of NPCs progression and migration in the SVZ of young APP transgenic mice was evidenced (Veeraraghavalu, Choi, Zhang, & Sisodia, 2010).

In a study, Yahata et al. differentiated hiPSCs cells into neuronal cells presenting a forebrain marker Forkhead box protein G1 (Foxg1), as well as neocortical markers such as cut like homeobox 1 (Cux1), Special AT-rich sequence-binding protein 2 (SATB2), chicken ovalbumin upstream promoter transcription factor-interacting proteins 1 (CTIP1), and T-box brain transcription factor 1 (Tbr1). Furthermore, the expression of the APP, β-secretase, and γ-secretase agents in combination with secreting Aβ into the conditioned media were iPSC-derived neuronal cells. At the early differentiation stage, they found that exposure with the γ-secretase inhibitor (GSI) led to a swift upsurge at a lower dose (Aβ surge) and a severe decrease of Aβ production (Yahata et al., 2011). In 2018, C. Wang et al. obtained hiPSCs from skin fibroblasts of patients with apolipoprotein E4 (ApoE4) modification, and subsequently generated ApoE4-expressing neurons as the main risk factor for AD. This study suggested that ApoE4 is the cause of the pathogenic phenotype of AD, supporting the probability of treatment using an ApoE4 pathogenic conformation-specific corrector (C. Wang, Najm, et al., 2018). Interestingly, it has been found that in vivo use of the glial cells derived from iPSCs-generated from mouse skin fibroblasts induced a reduction in plaque aggregation in the 5XFAD transgenic AD mouse model, as well as resolved cognitive dysfunction, partially (Cha et al., 2017). Finally, Fujiwara and his colleagues showed an improvement in memory impairments of the transgenic mice with severe amyloid β aggregation and advanced spatial memory dysfunction following transplantation of cholinergic neurons derived from human iPSCs into the hippocampus of these mice (Fujiwara et al., 2013).

5.2 HD

Positive interventions in the progressive stages of HD can reduce neural celleloss and also replace dead nerve cells. Therefore, today the use of cell therapy has attracted considerable attention around the world (Hamilton et al., 2019; Rosser & Svendsen, 2014). In 1997, the first clinical study in this field by Philpott et al. (1997) confirmed short-term motor and cognitive improvements in HD patients following transplantation of fetal striatal tissues into their striatum, supporting allograft transplantation potential for HD treatment. Restrictions on the source of fetal cells and continued immunosuppression, as well as ethical concerns, limit these cells' application (Eguizabal et al., 2019). In recent years, researchers have been trying to access the sources of the pluripotent cells that greatly reduce the cited problems. Today, we know that iPSCs have promising potential to differentiate into the various types of cell lineage affected in disorders, giving rise a dependable and accessible source of graft material (Moradi et al., 2019). Importantly, iPSCs can establish patient-specific NPCs, removing immune rejection risk (Gorecka et al., 2019). Still, due to the existence of the causative mutation that leads to the generation of lethal mutant proteins in HD-iPSC-derived cells, use of the emerging gene therapies including RNA interference (RNAi), antisense oligonucleotide (ASO), and genome-editing methods is necessary for mHTT gene silencing or repairing (Godinho, Malhotra, O'Driscoll, & Cryan, 2015; Wiatr, Szlachcic, Trzeciak, Figlerowicz, & Figiel, 2018). The researchers believe that the use of these cells after genomic correction triggers damaged and dead cell replacement and improves functional deficiencies (Cho, Hunter, Ye, Pongos, & Chan, 2019a; Henriques, Moreira, Schwamborn, Pereira de Almeida, & Mendonca, 2019). However, iPSCs transplantation conducted in animal models of HD and a few human iPSC transplantations in rodent HD models have been successful (Aubry et al., 2008; H. Ma et al., 2012). In a study, researchers used a novel differentiation protocol, inducing normal neurodevelopment of the ventral telencephalon to promote iPSCs differentiation into NPCs (Delli Carri et al., 2013). Moreover, NPCs were differentiated into GABAergic neurons that not only expressed typical striatal medium spiny neurons (MSN) marker but also exhibited dopamine and adenosine receptors (Delli Carri et al., 2013). Also, it has already been found that grafted rodent iPSC has the potential to differentiate into DARPP-32 +neurons in the damaged striatum and improve striatal atrophy in HD animals (Delli Carri et al., 2013; Mu et al., 2014). In another significant study, Cho, Hunter, Ye, Pongos, and Chan (2019b) generated transgenic HD monkey NPCs from their iPSCs and confirmed the efficacy of the combined use of the obtained NPCs and gene therapy in a transgenic HD mouse model. On the other hand, G. Liu et al. (2014) found that the combination of iPSCs and trophic factors, in particular brain-derived neurotrophic factor (BDNF), has the potential to stimulate neurogenesis and enhance preservation of neuronal structure and/or function, providing promising potential in disease-modifying treatment of HD (G. Liu et al., 2014). According to other reports, iPSC-derived NPCs transplantations diminished behavioral deficits and improved neuropathological variations in HD mouse models (Al-Gharaibeh et al., 2017). Results indicated an enhancement in BDNF and tropomyosin receptor kinase B (TrkB) rates in striata of transplanted HD mice in comparison with control mice (Al-Gharaibeh et al., 2017).

5.3 PD

Based on reports, patient-specific iPSCs have great potential for exploring the pathogenicity and cell-based therapy of PD (Amin et al., 2019; Aoki, Sato, TAKIZAWA, Turner, & Shabanpoor, 2019). As described, one fundamental advantage of using iPSC is its ability to originate from the patient's cells, attenuating the risk of immunological rejection. This specific chracteristic of iPSCs supposedly eliminates immunological rejection risk and boosts their integration into the brain tissues of PD patients (Stoddard-Bennett & Reijo Pera, 2019). After generation of PD patient-specific iPSC, these cells' exposure to specific factors supports their differentiation into NSCs and DA neurons (Stoddard-Bennett & Reijo Pera, 2019). A large number of studies have been performed to advance iPSC generation, differentiation, and clinical applications. In this context, the primary goal is acquiring good manufacturing practice (GMP) standards for the treatment of PD (Jung, Bauer, & Nolta, 2012).

In 2012, to explore the iPSCs-derived cells' clinical ability, the therapeutic potential of mouse iPSCs was evaluated through their transplantation into the brain of a rat model of PD (Jung et al., 2012). Jung et al. (2012) identified behavioral developments in rat PD models following differentiation of the grafted iPSCs into midbrain-like DA neurons. Similarly, hiPSCs-derived neurons or NSCs demonstrated favorable therapeutic potential in rat and monkey PD models (Ross & Akimov, 2014; Sundberg et al., 2013). Although several types of research confirmed the fact that iPSCs stimulated the progression of motor function in animal PD models, these effects have not been supported by any clinical trial. Dopamine neurons from the iPSCs carrying the LRRK2 G2019S mutation were shown to be susceptible to ROS and have enhanced levels of SNCA aggregates (Ysselstein et al., 2019). Attenuation of the degeneration of DA neurons following correction of the LRRK2 G2019S mutation in iPSCs confirmed the LRRK2 mutation's significant role in the pathogenesis of PD (Marrone et al., 2018; Reinhardt et al., 2013). In another study, it was found that transplanted iPSC-derived NPCs into the brain of PD monkeys were able to differentiate into neurons, astrocytes, as well as myelinating oligodendrocytes (Reinhardt et al., 2013). In 2015, Wang et al. evaluated the potential of iPSC-derived DA neuron cells for the treatment of the monkey PD model. After the generation of iPSCs from mesenchymal stem cells of the monkey PD model and their differentiation into DA neurons, these cells were transplanted into the brain of a monkey PD model. Interestingly, they found dependable behavioral development in a transplanted monkey in comparison with the control monkey (S. Wang et al., 2015).

5.4 ALS

Phase I clinical trials to test the safety of the intraspinal transplantation of NSCs on ALS patients have been approved by the Food and Drug Administration (FDA; Gowing & Svendsen, 2011). Use of glial precursor cells (GPCs) or mature astrocytes is another practical strategy to reinforce neuroprotection and trophic effects to support damaged MNs (Robberecht & Philips, 2013). As neurodegenerative diseases are commonly identified in elderly patients, there exist some concerns about the quality and differentiation potential of generated iPSCs from their somatic cells for cell replacement, neuroprotection and neuroregeneration purposes (Forostyak & Sykova, 2017). In 2018, Dimos and his colleagues found that the obtained iPSCs from an 82-year-old woman with a familial ALS (FALS) have specific charcateristics like ESC and are able to differentiate into MN (Dimos et al., 2008). Of course, because in most studies viral methods are used to generate iPSCs, it is difficult to estimate the levels of genetic alterations and abnormalities in transplanted cells (Berry, Smith, Young, & Mack, 2018); on the other hand, there is another concern about the route of engraftment and the iPSCs developmental stage (Funakoshi et al., 2016). In this context, researchers believe that it is difficult to manipulate fully differentiated cells, suggesting precursor cells as more appropriate candidates for transplantation. Based on existing information, some clinical trials are ongoing to evaluate NSCs' ability to treat ALS patients (Glass et al., 2012). In a preclinical study, Kondo et al. obtained glial-GPCs from hiPSCs and transplanted them into the lumbar spinal cord of ALS mouse models. They observed that the life span of the transplanted mice was longer than the control group (Kondo et al., 2014). In another research study, the therapeutic potential of iPSC-derived NSCs on the mice model of ALS after intrathecal or intravenous injections was explored by Nizzardo et al. Nizzardo et al. (2013) showed that treated ALS mice demonstrated enhanced neuromuscular activity and motor unit pathology and robustly improved the life span in comparison with the control group. Nizzardo et al. (2016) in another research study found that iPSC-derived NSCs, specifically LewisX+CXCR4 + β1-integrin+  NSCs, boost neuronal viability and axonal development of human ALS-derived MNs cocultured with toxic ALS astrocytes. Nowadays, scientists are trying to develop novel therapeutic approaches based on the combination of cell therapy and gene therapy to rescue mitochondrial and MN function in ALS patients (Lorenz et al., 2017).