Delineating the Neuropathology of Lysosomal Storage Diseases Using Patient-Derived Induced Pluripotent Stem Cells

Abstract

Lysosomal storage diseases (LSDs) are inherited metabolic diseases caused by deficiency of lysosomal enzymes, essential for the normal development of the brain and other organs. Approximately two-thirds of the patients suffering from LSD exhibit neurological deficits and impose an escalating challenge to the medical and scientific field. The advent of induced pluripotent stem cell (iPSC) technology has aided researchers in efficiently generating functional neuronal and non-neuronal cells through directed differentiation protocols, as well as in decoding the cellular, subcellular, and molecular defects associated with LSDs using two-dimensional cultures and cerebral organoid models. This review highlights the information assembled from patient-derived iPSCs on neurodevelopmental and neuropathological defects identified in LSDs. Multiple studies have identified neural progenitor cell migration and differentiation defects, substrate accumulation, axon growth and myelination defects, impaired calcium homeostasis, and altered electrophysiological properties, using patient-derived iPSCs. In addition, these studies have also uncovered defective lysosomes, mitochondria, endoplasmic reticulum, Golgi complex, autophagy and vesicle trafficking and signaling pathways, oxidative stress, neuroinflammation, blood–brain barrier dysfunction, neurodegeneration, gliosis, and altered transcriptomes in LSDs. The review also discusses the therapeutic applications such as drug discovery, repurposing of drugs, synergistic effects of drugs, targeted molecular therapies, gene therapy, and transplantation applications of mutation-corrected lines identified using patient-derived iPSCs for different LSDs.

Introduction

Lysosomal storage diseases (LSDs) are inherited metabolic disorders caused by the deficiency of lysosomal enzymes, required for the healthy development of the central and peripheral nervous system and other organs. Mutation in the lysosomal enzymes, aberrant vesicular trafficking, defects in lysosomal membrane channels and transporters, and aggregation of cellular waste could interrupt the normal cellular functions [1 –5]. More than 50 LSDs have been diagnosed so far. LSDs also contribute to the cause of rare neurological disorders. Approximately two-thirds of the patients suffering from LSDs exhibit neurological deficits [6]. The pathophysiology of LSDs is multifactorial and multifaceted, with heterogeneous symptoms. Severity ranges from death in early infancy to onset of mild abnormalities in adolescence [7,8].

During the early stages of development, these manifestations comprise of developmental delays, intellectual disability, seizures, and neuromotor regressions, leading to rare neurodevelopmental disorders and neurodegeneration, whereas, in adults, the disease is associated with motor deficits, dementia, and psychological disarrays [9,10]. Various gene mutations and proteins are involved in the pathology of neurological LSD. Axonal demyelination has been observed in several neuropathic LSD [11 –13]. Moreover, microgliosis and astrocytosis, which are some of the major hallmarks of LSD neuropathology, could trigger neuroinflammation along with neurodegeneration [14 –18].

Treatment modalities such as enzyme replacement therapy (ERT), hematopoietic stem cell transplantation (HSCT), gene therapy, and substrate reduction therapy have been developed for different LSDs to improve the defective system. HSCT is based on engrafting the transplanted stem cells into the bone marrow. Some cells may cross the blood brain barrier and differentiate into microglial cells to secrete the functional lysosomal enzymes [19,20]. Gene therapy involves delivering the genetic material, specifically the functional copy of the defective gene, using viral/nonviral derived vectors [21 –23]. In contrast, in ERT, the recombinant functional enzyme administration compensates for the defective protein [24].

When released into the systemic circulation, the newly synthesized enzyme is then utilized by the neighboring cells. Consequently, through the cross-correction mechanism, organs such as the liver can produce the enzyme and deliver to the required organs through circulation [25]. Nevertheless, one of the major drawbacks is that some LSDs are due to defective membrane-bound proteins and hence cannot utilize the cross-correction mechanism [23].

Animal models have facilitated a close recapitulation of the disease pathogenesis and progression and delineated the different stages of LSDs [10,12,18]. While these animal models mimic the various mutations specific to humans, they may not faithfully reproduce all human syndrome features due to species differences. Moreover, the clinical and pathological manifestations may be milder or more severe than the human disorder due to the presence or absence of species-specific compensatory mechanisms. The skin fibroblasts from LSD patients carrying the disease mutations in genetic background may remain useful for studying some phenotypes [26 –28]. Nevertheless, these systems have limitations for performing a thorough analysis of the disease, as the patient fibroblasts are unlikely to mimic the specific phenotype of neurons under these metabolic conditions.

From these perspectives, patient-specific cell-derived induced pluripotent stem cells (patient-derived iPSCs) exquisitely aided with directed differentiation protocols for generating functional neuronal and non-neuronal cells could serve as an attractive archetype in delineating the cellular phenotypes and mechanisms in neurodevelopmental and neurological dysfunctions associated with LSDs [29 –35]. While various LSDs have been modeled in two-dimensional (2D) cultures, certain LSDs have also been studied in detail, using three-dimensional (3D) cultures [36 –38] under a controlled laboratory environment. Many studies have also incorporated gene-editing tools like CRISPR/Cas9, transcription activator-like effector nucleases (TALENs), or zinc-finger nucleases (ZFNs) [36,39,40].

This review highlights the information assembled from patient-derived iPSCs on cellular phenotypes of developing progenitor cells, neurons, and glial cells and how their normal architecture is disrupted. Specific enzyme deficiency-related accumulation of substrates in different LSDs and their supplementation on the functional restoration of multiple central nervous system (CNS) cell types have been discussed. Apart from mechanistic insights, various studies have also explored prospective applications of patient-derived iPSCs in drug discovery, repurposing of drugs, gene therapy, and cell transplantation for the LSDs (Figure 1).

FIG. 1.

Neurodevelopmental and pathological defects identified in LSDs using patient-derived iPSCs: neural progenitor cell migration and differentiation defects, substrate accumulation, axon growth and myelination defects, impaired calcium homeostasis, altered electrophysiology and network connectivity, aberrant lysosomes, mitochondria, endoplasmic reticulum and Golgi complex, defective autophagy and vesicle trafficking, altered transport and signaling pathways, oxidative stress, neuroinflammation, blood–brain barrier dysfunction, neurodegeneration, gliosis, altered transcriptomes, etc. Therapeutic applications identified using patient-derived iPSCs for LSDs: drug discovery, repurposing of drugs, synergistic effects of drugs, molecular targeted therapies, gene therapy, and transplantation applications of mutation corrected lines, etc. iPSC, induced pluripotent stem cell; LSDs, lysosomal storage diseases.

Metachromatic Leukodystrophy

Metachromatic leukodystrophy (MLD), an autosomal recessive disorder, caused due to mutations in arylsulfatase A (ARSA gene located at 22q13.33), exhibits loss of lysosomal enzyme ARSA or its activator protein saposin-B. Consequently, degradation of cerebroside-3-sulfate is hampered, leading to its accumulation in lysosomes of all cells, including glia and subtypes of neurons. Such accumulation in oligodendrocytes and Schwann cells causes white matter injury with progressive demyelination, resulting in CNS and peripheral nervous system dysfunction.

Based on the age of onset, MLD is classified as late infantile, early juvenile, late juvenile, and adult-onset type, with an overall prevalence of 1.4–1.8 per 100,000 individuals. Late infantile and early juvenile types are more common. They are exemplified by wide-ranging neurological symptoms such as intellectual disability, cognitive decline, motor deficits, blindness, deafness, seizures, and premature death [41 –46].

MLD-iPSC-derived neural stem cells display fetal phenotypes, and differentiated neurons and glia accumulate sulfatides

Neural stem cells generated from MLD patient-derived iPSCs (MLD-iPSCs) exhibited the characteristic features of human fetal neural stem cells when propagated from the diencephalic or telencephalic region of a 10.5-week gestational age fetus. These MLD-iPSC-derived neural stem cells (NSCs), when differentiated into GABA-ergic (including subtypes expressing calbindin or somatostatin), dopaminergic, and glutamatergic neurons, apart from negligible amounts of cholinergic and serotonergic neurons, astrocytes, and oligodendrocytes [32], revealed a significant increase in the composition and levels of multiple sulfatides, along with substantial enlargement of lysosomal compartments, aggravated oxidative stress, and enhanced programmed cell death. Such changes resulted in impaired neuronal and glial differentiation and altered neuronal networks [47].

Restoration of ARSA reduces sulfatide levels and reinstates normal function

Since ARSA deficiency is the primary causative factor for MLD, its supplementation could restore normal cellular physiology. A large body of research on the cross-correction and its underlying mechanisms has been carried out in animal models and human studies in the last five decades [48 –51]. Replenishment of ARSA levels in corrected MLD-iPSCs [47] refills the sulfatide levels in neurons and glia, thereby restoring normal cellular function. Expression of the human ARSA gene in MLD-iPSCs and intracerebral transplantation of NSCs derived from them into immunodeficient neonatal or adult MLD mice resulted in widespread and stable ARSA expression with reduced sulfatide storage [32]. Also, neural precursor cells (NPCs) and astroglial progenitor cells (APCs) generated from juvenile variant, ARSA-engineered MLD-iPSCs, expressed robust ARSA.

When these NPCs and APCs were transplanted into the telencephalon of postnatal day 1 ARSA-deficient mice, the NPCs were differentiated into neurons and APCs into astrocytes. Such grafting also induced a massive reduction in the brain's sulfatide storage, even in far regions containing graft-derived axonal projections and in areas containing clusters of APCs. Apart from their presence in graft-derived cells, ARSA diffused into cells in neighboring regions of the brain and reduced sulfatide storage. Overall, the study showed that autologous grafting of NPCs from patient-derived, engineered iPSCs could provide an unlimited supply of ARSA to the MLD brain to prevent the accumulation of sulfatides [52].

Thus, different studies with MLD highlight the possibility of generating bonafide cells from MLD patients and their efficient engineering to avail an autologous cell-based vehicle for continuous ARSA supply for treating MLD patients.

Mucopolysaccharidoses

Mucopolysaccharidoses (MPS) constitute a group of lysosomal storage disorders with a characteristic accumulation of partially degraded glycosaminoglycans (GAGs) in lysosomes. This accumulation is primarily due to the deficiency of different enzymes responsible for GAGs' degradation, leading to cellular and tissue dysfunction [5]. The stepwise degradation of heparin sulfate, dermatan sulfate, chondroitin, and keratan sulfate requires ten different enzymes through four different pathways. The deficiency of these enzymes results in seven diverse MPS [53], but only mucopolysaccharidosis type II (MPS II) is X-linked, while the other types are autosomal recessive disorders.

Mucopolysaccharidosis type I

Based on the severity, MPS1 is classified into three subtypes, Hurler (MPS IH), Hurler–Scheie (MPS IH/S), and Scheie (MPS IS) syndrome, with MPS-IH being the most severe form. These subtypes stem from the deficiency of enzyme α-L-iduronidase (IDUA, a gene located at 4p16.3). In MPS-IH, severe accumulation of dermatan, and heparan sulfate, results in dysfunction of multiple organs, including the brain. The phenotype is exemplified by mental retardation and death by 5–10 years of age [53,54].

Neuronal phenotypes from all subtypes of MPS I-derived iPSCs (MPS-iPSCs) have been generated and analyzed. The MPSI-iPSC-derived NSCs exhibited varying degrees of IDUA deficiency. Higher accumulation of GAGs was observed in NSCs from MPS IH, compared to iPSC-derived NSCs from MPS IH/S and MPS IS. Likewise, lysosomal enlargement was highest in NSCs from MPS IH, intermediate in MPS IH/S, and low in MPS IS. Transcriptome profiling of NSCs from all 3 subtypes and their differential expression analysis revealed that the MPS IH subtype has significant changes in the expression of 21 genes related to GAG metabolism. The severity declined in MPS IH/S, and MPS IS, with alterations in nine and four genes of GAG metabolism in NSCs. Moreover, altered gene expressions correlated with clinical symptoms found with respective MPS I subtypes.

Similarly, GAG accumulation altered the lysosomal pathway gene expression and the extracellular matrix genes in NSCs, derived from the respective subtypes of MPS I-iPSCs. Multiple autophagy genes exhibited radically higher expression in MPS-IH NSCs than other subtypes [33]. The authors developed a disease severity model based on the transcriptomic characteristics of different subtypes of MPS I-iPSC-derived NSCs and IDUA activity. While MPS IS displayed minor lysosomal accumulation, a progressive dysfunction was seen in the extracellular matrix of MPS IH/S, and significant dysfunction of autophagy was activated in the MPS IH subtype [33]. Studies with MPS iPSCs and corrected lines also revealed the migration defects in NPCs and neurite outgrowth defects due to this enzyme deficiency [55].

Mucopolysaccharidosis type II

MPS II or Hunter syndrome is an X-linked recessive disorder located at Xq28, found almost exclusively in males [56,57]. This syndrome is caused by the deficiency of the lysosomal enzyme iduronate 2-sulfatase (IDS), which is vital for degrading dermatan and heparan sulfate. Such deficiency causes the accumulation of GAGs in cells and tissues and excessive urinary excretion of dermatan and heparan sulfates. In severe cases, apart from other tissue anomalies, the patients display mental retardation and may die before 15 years of age [53].

Differentiation of MPS II-iPSCs into neurons, astrocytes, and oligodendrocytes demonstrated a deficiency of IDS with a moderate accumulation of GAGs, and structural abnormalities similar to patient tissues of MPS II. Exogenous administration of recombinant IDS to cultured neurons partially reduced the intracellular GAG accumulation [58]. Deficiency of the functional enzyme in MPS II-iPSCs resulted in significant structural changes in the Golgi apparatus and endoplasmic reticulum with intracellular accumulation of storage vesicles in differentiated cells. Lysosomal accumulation of GAGs and lysosome-associated membrane glycoprotein 2 (LAMP2) positive vacuoles was distinctly associated with astrocytic death, rather than neurodegeneration [59].

Although MPS-II manifests exclusively in males, neurons have also been generated from MPS II-iPSCs from a female patient with a heterozygous mutation in IDS gene [60] to study the disease pathology.

Mucopolysaccharidosis type IIIB

Mucopolysaccharidosis type IIIB (MPS IIIB), also called Sanfilippo syndrome type B, is caused by the deficiency of α-N-acetylglucosaminidase (NAGLU gene located in 17q21), leading to the accumulation of heparan sulfate with substantial mental deterioration and hyperactivity. Undifferentiated MPS-IIIB iPSCs showed reduced proliferation with prominent storage lesions. Neural stem cells derived from iPSCs showed alterations in multiple cell signaling pathways, accumulation of heparan sulfate-containing GAGs, but no upregulation in the expression of LAMP1 or ganglioside GM3 or storage vacuoles. However, upon differentiation, neurons generated from NSCs showed LAMP1- and GM3-positive vacuoles with severe storage lesions. For the first time, this study revealed the existence of disorganized Golgi complex in a lysosomal disorder [61].

Mucopolysaccharidosis type IIIC

Mucopolysaccharidosis type IIIC (MPS III C), also called Sanfilippo syndrome type C, is caused by deficiency of heparan acetyl-CoA: α-glucosaminide N-acetyltransferase (HGSNAT gene located at 8p11.1). This deficiency results in the accumulation of heparan sulfate, leading to clinical features such as hyperactivity and intense mental deterioration [62,63].

The neurons generated from MPS III C-iPSCs have elucidated several in vivo pathological phenotypes, including the reduced activity of acetyl-CoA α-glucosaminide N-acetyltransferase, GAGs accumulation, and several large and LAMP1-positive lysosome-derived vacuoles. These neurons also exhibited aberrant neuronal connectivity and network activity. The isogenic lines obtained from the patient-derived iPSCs were devoid of the disease phenotype [64]. The cells were genetically corrected with a lentivirus vector.

Mucopolysaccharidosis type VII

Mucopolysaccharidosis type VII (MPS type VII) is caused by a mutation in β-glucuronidase (GUSB gene, located at 7q21.11), leading to the partial degradation and accumulation of dermatan, keratan, heparan, and chondroitin sulfates [65]. The disease is characterized by cognitive and motor impairments and facial dysmorphia [66,67]. Genetically corrected NSCs, generated from MPS VII-iPSCs, could reverse the disease pathology following intraventricular grafting in neonatal NOD/SCID mice supporting gene therapy [68]. NPCs derived from healthy controls and MPS VII-iPSCs displayed comparable self-renewal, viability, and differentiation in 2D cultures, but the β-glucuronidase activity was 50-fold lower in MPS VII-iPSC-derived NPCs.

Evaluation of 3D neurospheroids on day 28 revealed lower levels of presynaptic vesicle synaptophysin and reduced GABAergic interneurons in MPS VII-iPSC-derived cultures, while the number of glutamatergic and dopaminergic neurons was unaltered. GAG-containing vesicles were 20-fold higher in MPS VII-iPSC-derived neurons with enlarged LAMP1+ lysosomal vesicles and defective endocytic compartment. Furthermore, MPS VII patient-iPSC-derived neurons showed more significant alterations in the endolysosomal pathway and neurological anomalies with aberrant neuronal activity. Only a few neurons derived from MPS VII-iPSCs showed network connectivity, while several other neurons were either weakly connected or disconnected [34].

Mucolipidosis

Mucolipidosis, like MPS, is a group of LSDs caused due to impaired functioning of lysosomal hydrolases resulting in the accumulation of GAGs. Based on the defective enzyme, mucolipidosis is classified into four types—ML I also called Sialidosis, ML II or the I-cell disease, ML-III A and IIIC, and ML IV. Unlike MPS, mucolipidosis is associated with uridine-diphosphate N-acetyl glucosamine-1-phoshotransferase (UDP-GlcNAc-1-phosphotransferase) deficiency). Sialidosis, a mutation in the sialidase gene NEU1, results in deficiency of alpha-neuraminidase activity leading to the accumulation of sialylated glycopeptides and oligosaccharides [69,70]. human induced pluripotent stem cells (HiPSCs) have been generated from sialidosis patients carrying NEU1 gene mutations [70 –72].

A recent study generated two types of iPSCs with sialidosis-specific NEU1 G227R and NEU1 V275A/R347Q mutations. These iPSCs, when differentiated into NPCs and characterized, recapitulated the disease-specific phenotypes, including abnormal differentiation of astrocytes and oligodendrocytes. However, neuronal differentiation was not affected. This is one of the first studies that used sialidosis patient-derived iPSCs as a prototype to elucidate the neurological deficits related explicitly to the defective autophagy-lysosome pathway [72].

Neuronal Ceroid Lipofuscinosis

Neuronal ceroid lipofuscinosis (NCLs) is a sporadic and fatal autosomal recessive neurodevelopmental disorder affecting 1:100,000 live births [73,74]. The disease is caused by one of 446 mutations among the 13 different (ceroid lipofuscinosis, neuronal, CLN) genes with onset at different ages [75,76]. In NCL, ceroid lipopigments accumulate in the lysosome, along with several NCL proteins. Clinical symptoms involve cognitive decline, motor deficits, seizures, visual impairment, and death.

Among NCLs, Classic Infantile (INCL, CLN1 gene), Classic Late Infantile (LINCL, CLN2 gene), and Classic Juvenile (JNCL, CLN 3 gene) are three primary genetic forms. INCL occurs due to deficiency of palmitoyl protein thioesterase 1 (PPT-1), a serine lipase present in lysosomes [77,78]. This NCL is distinguished by psychomotor retardation, muscular hypotonia, retinal degeneration, seizures, and microcephaly. The clinical features appear between 8 and 18 months, and death typically occurs by 7 years [79].

In LINCL, loss of function mutation in the tripeptidyl peptidase-1 (TPP1) gene leads to a severe deficiency of encoding lysosomal peptidase [80]. The phenotype starts to appear at 2–4 years of age with the accumulation of ceroid lipopigments in neurons and other cells. The clinical features include seizures, mental deterioration, blindness, massive neurodegeneration, motor dysfunction, and death between 6 and 15 years [81,82]. A variant of LINCL (vLINCL caused by defective CLN5) appears at 4–7 years after birth, and gradually develops motor, mental, and attention deficits, visual dysfunction, seizures, severe neurodegeneration and death between the second to fourth decades of life [83].

In JNCL, also known as Batten disease, a loss of function mutation in CLN3, encoding an endolysosomal transmembrane protein (battenin), causes blindness, seizures, and loss of motor function and cognition. The phenotype appears at the age of 5–10 years and leads to mortality in the early 20s [84,85].

Disrupted intracellular compartments in NCL-iPSC-derived cells with accumulation of ceroid lipopigments

The iPSCs from JNCL patients showed disruption in late endolysosomal compartments with up to 20 μm diameter-sized vacuoles. The NPCs generated from LINCL and JNCL patients displayed NCL-like characteristics with an accumulation of autofluorescent deposits in 5% of cells and several large ring-like structures positive for LAMP-1 extending to the cell periphery. Furthermore, the NPCs from both patient lines showed multiple ∼200–800 nm empty vacuolar structures and depleted multivesicular bodies with intraluminal vesicles. Nearly 15%–25% of mitochondria from JNCL patient-derived NPCs showed gross anomalies, including swelling with disrupted internal architecture and loss of cristae, whereas LINCL patient-derived NPCs showed dilated endoplasmic reticulum. Although with several abnormal features, functional neurons could be generated from NPCs derived from LINCL and JNCL patients [30].

In another study, NSCs differentiated from INCL, and LINCL patient-derived iPSCs showed standard self-renewable capability, but exhibited reduced PPT-1 and TPP-1, respectively. These NSCs also displayed NCL phenotypes with lysosomal enlargement and storage of subunit c and intracytoplasmic accumulation of lipid droplets [86]. Neural lineage cells from vLINCL patient-derived iPSCs also showed enlarged lysosomes and distended endoplasmic reticulum and the intracytoplasmic accumulation of subunit c of mitochondrial ATP synthase and autofluorescent storage material, which are hallmarks of this disease. Furthermore, the transport of sphingolipids from endolysosomes to the Golgi was perturbed in these neural lineage cells [87].

Restoring the TPP1 levels can rescue the NPCs from the disease phenotype

Accumulation of ceroid lipopigments was observed in both NSCs and neurons derived from LINCL and JNCL patient-derived iPSCs. Overexpression of normal CLN3 or TPP1 in respective NPCs ameliorated most of the abnormalities. Two lipid-lowering drugs enhanced TPP1 levels and enzymatic activity in these cells, highlighting the significance of NCL-iPSCs in evaluating the pharmacological potential of various drugs for treatment [30]. NSCs derived from INCL, and LINCL patient-derived iPSCs, revealed NCL phenotypes, and treatment with ERT and δ-tocopherol (DT) and hydroxypropyl-β-cyclodextrin (HPBCD), ameliorated this effect. Moreover, combined DT and HPBCD therapy was more beneficial [86].

Collectively, neural cells differentiated from NCL-iPSCs recapitulate most of the typical features of NCL. Identifying pharmacological agents capable of restoring the TPP1 levels through high-throughput drug screening using NCL-iPSC-derived neural cells would likely help develop new treatment modalities in combating NCL.

GM2 Gangliosidosis

GM2 gangliosidosis includes three disorders, namely Sandhoff disease (SD), Tay-Sachs disease (TSD), and AB variant, all related to the deficiency of hexosaminidase gene, causing abnormal lysosomal accumulation of ganglioside GM2 and neuronal degeneration. Patient-derived iPSC studies are available for SD and TSD.

Sandhoff disease

SD, one of the GM2 gangliosidoses, is a lysosomal storage disorder caused by a mutation in the HEXB gene. The disease has an incidence rate of 1:422,000 [88]. Along with β-hexosaminidase αβ (HEXAB) subunit produced by the HEXA gene and β-hexosaminidase ββ subunit (HEXB) produced by the HEXB gene, a noncatalytic GM2 activator is required for the hydrolysis of GM2 gangliosides. In Sandhoff's disease, a mutation in HEXB results in the paucity of both hexosaminidase A (HEXA) and HEXB subunits, which leads to an abnormal lysosomal accumulation of the substrate ganglioside GM2. The disease onset could be seen in infancy with traits such as developmental delay, seizures, macrocephaly, blindness, progressive loss of motor function, speech regression, and cognitive defects, leading to death by 4 years of age [89,90].

A 3D model of the disease has been created to comprehend the early neurodevelopmental processes altered by the disease. Cerebral organoids were generated from infantile SD patient-derived iPSCs and isogenic controls through CRISPR/Cas9-mediated correction of the mutant HEXB allele. Compared to the isogenic controls, organoids generated from SD-iPSCs were more extensive due to enhanced cell proliferation and displayed GM2 ganglioside accumulation. Moreover, transcript profiling analysis suggested an alteration in neuronal differentiation pathways in SD-iPSC-derived cerebral organoids [36]. The study findings open new possibilities for screening various therapeutic agents and treatment modalities in combating the SD phenotype using SD-iPSC-derived organoids.

Tay-Sachs disease

TSD is caused due to mutation in the HEXA gene that encodes β-hexosaminidase. Deficiency in the HEXA enzyme leads to the accumulation of GM2 ganglioside in the lysosomes, triggering progressive neurodegeneration [91,92]. The disease manifests at 6 months of age, leading to floppy muscle tone, motor skill deficits, progressive intellectual disability, blindness, cognitive decline, and ultimately death by the age of 5 [93,94]. So far, no curative treatment modality has been established for TSD. Like other lysosomal storage disorders, ERT and exposure to pharmacological agents have been beneficial in managing the symptoms and reducing the lysosomal GM2 accumulation.

HEXA-deficient human iPSCs have been generated from the fibroblasts of an infantile TSD patient through reprogramming using STEMCCA lentivirus [95]. Vu et al. generated human iPSCs from the dermal fibroblasts of two TSD patients and differentiated them into neural stem cells that showcased the disease phenotype such as lipid accumulation. These TSD iPSC-derived NSCs were used to study the efficacy of ERT with recombinant human HEXA protein. Moreover, small molecular compounds such as DT and HPBCD were also efficient in ameliorating the lipid accumulation and lysosomal enlargement in TSD–NSCs [94,96].

Recently, iPSCs-derived NPCs and neurons from TSD patients exhibited characteristic phenotypes, such as enlarged lysosomes, upregulation of LAMP 1, GM2 ganglioside accumulation, and decreased exocytotic activity, due to presynaptic dysfunction [97]. These studies corroborate the application of human iPSC-derived NSCs and neurons in investigating the mechanism of TSD neuropathology and evaluating drug efficacy.

Niemann-Pick Disease

Niemann-Pick disease (NPD) is a group of lysosomal storage disorders represented by abnormal lipid storage and foam cell infiltration with varying severity. NPD manifests as severe hepatosplenomegaly, pulmonary insufficiency, and rapidly progressive neurodegeneration. Based on metabolic abnormalities, NPD is classified as type A, B, and C.

Niemann-Pick disease type A and B

Niemann-Pick disease type A and B (NPA and NPB) are autosomal recessive disorders affecting 1 in 100,000 births. NPA is clinically represented by hepatosplenomegaly, severe neurological defects, pulmonary insufficiency, and a cherry-red spot of the eye, with a high mortality rate in infants not exceeding 2–3 years of age [98 –101]. NPB also exhibits hepatosplenomegaly and lung defects, but with no neurological involvement in adults [101,102]. Mutation in the gene encoding SMPD1 leads to deficiency of the enzyme acid sphingomyelinase, resulting in accumulation of sphingomyelin in the lysosomes. Pharmacological interventions with tocopherols and cyclodextrin have attenuated the lysosomal accumulation of sphingomyelin.

Application of iPSCs generated from NPA and NPB patient fibroblast in drug screening

Human iPSCs have been derived from the fibroblasts of 21-week-old female NPA patient carrying a heterozygous mutation of p.L302P variant using nonintegrating Sendai virus technique [103]. Similarly, human iPSCs have also been generated from dermal fibroblast of a 1-year-old male NPB patient carrying a mutation in SMPD1 using the same technique [104]. Recently, four lines of iPSCs have been generated from two NPA patient fibroblasts carrying fsP330 and L302P mutation. These iPSCs were differentiated into neural stem cells, demonstrating the characteristic phenotypes, and effects of α-tocopherol, DT, acid sphingomyelinase, and cyclodextrin were evaluated, signifying their suitability for drug screening [105].

Niemann-Pick disease type C

Niemann-Pick disease type C (NPC) is an autosomal recessive rare progressive, LSD primarily affecting the viscera and CNS. NPC is a result of a mutation in the NPC1 gene [106,107], which encodes NPC1 protein involved in intracellular cholesterol-trafficking pathways [3,108,109]. Such mutation causes cholesterol accumulation and other lipids in the late endosomal/lysosomal compartment of cells. As autophagy regulates lipid metabolism and maintains cellular homeostasis, impairment in autophagic flux is the major causative factor in NPC [4,110,111]. This disruption in the intracellular lipid trafficking mechanism leads to neurodegeneration and extensive loss of Purkinje cells in the cerebellum [18,112].

The disease is characterized by hepatosplenomegaly and progressive neurological impairments such as ataxia, dystonia, developmental delays, seizures, and intellectual disability, eventually incurable and lethal. The incidence rate of NPC is 1 in 92,104, whereas the late-onset NPC phenotype has an incidence rate of 1 in 19,000 to 1 in 36,000 [113].

NPC-iPSC-derived neurons exhibit abnormal cholesterol accumulation

The phenotypic and functional analysis of neuronal stem cells generated from NPC patient-derived fibroblasts showed defective self-renewal and differentiation potential with cholesterol accumulation [114]. Accumulation of cholesterol in neural progenitor cells and spontaneous action potentials and postsynaptic currents in the neurons generated from NPC patient-derived iPSCs were observed [19].

Genomic analysis revealed that NPC-iPSC-derived neurons exhibited premature cell death and disruption in calcium and WNT signaling pathways compared to controls, suggesting that altered calcium signaling and WNT pathways could contribute to the pathophysiology of the disease [115]. Glial cells differentiated from NPC patient-derived iPSCs have been shown to undergo gliosis, in addition to hypophosphorylation and abnormal assembly of GFAP and vimentin.

Induction of NPC phenotype using U18666A in control cells led to the accumulation of cholesterol in glia and gliosis. Such glial changes could be attributed to reduced PKC activity since activation of PKC using phorbol 12-myristate 13-acetate (PMA) attenuated gliosis. Thus, targeting PKC may reduce cholesterol accumulation and reactive astrocytes induced by NPC [116]. NPC1-deficient human hepatic and neural cells exhibit cholesterol accumulation, autophagic flux dysfunction, and enhanced cell death, whereas isogenic lines generated from NPC patient-derived iPSCs using TALENs rescued these defects [39].

Studies have shown that sphingosine accumulation could trigger the pathogenic mechanism in NPC. Attenuated sphingosine kinase (SphK) activity due to reduced vascular endothelial growth factor (VEGF) levels has been observed in NPC patient fibroblasts and Purkinje neurons from NPC mice. Inhibition of autophagosome-lysosome fusion due to reduced VEGF/SphK activity leads to sphingosine accumulation [117].

Functional characterization of neurons from NPC-iPSCs revealed reduced AMPA-mediated calcium influx. This reduced Ca²⁺ influx could be due to the subunit's hypophosphorylation, resulting in increased GluA2 containing Ca²⁺-impermeable AMPARs. Thus, aberrant AMPA receptor-mediated calcium signaling in NPC neurons may attribute to the pathophysiology and progressive neurodegeneration in NPC patients [118]. Also, messenger RNA (mRNA) and protein expression of NPC-iPSC-derived neurons indicated a massive reduction of catalase levels with concomitant elevation of oxidative stress, while SOD1 and SOD2 were unaltered [119]. Physiological catalase administration could help as an interventional strategy for NPC.

Application of NPC-iPSC-derived neurons in evaluating the therapeutic efficacy of drugs

NPC patient-derived iPSCs serve as a cell-based disease model for screening and evaluating the efficacy of various drugs and compounds. NPC patient-derived iPSCs typify the disease characteristics of lysosomal cholesterol accumulation. However, treatment with DT, hydroxyl-β-cyclodextrin, and methyl-β-cyclodextrin attenuated these defects. Moreover, combined cyclodextrin treatment with DT exhibited a synergistic effect compared to individual treatment [120]. NPC1-deficient human hepatic and neural cells demonstrated cholesterol accumulation, autophagic flux dysfunction, and enhanced cell death. Treatment with carbamazepine reinstated the standard autophagic flux and induced cytoprotection in NPC patient-derived iPSCs differentiated into hepatic and neuronal cells [39].

Moreover, studies using iPSC-derived human NPC neurons revealed that replenishment with VEGF ameliorated the aberrations induced by VEGF/SphK inactivity [117]. Hepatocyte-like cells and neural progenitors generated from NPC-iPSCs displayed NPC phenotypes such as cholesterol accumulation and impaired autophagy. Treatment with 2-hydroxypropyl-β-cyclodextrin normalized the cholesterol level and revived the deviant molecular and functional properties in NPC patient-specific cells [121]. Targeting PKC may help in reducing cholesterol accumulation and reactive astrocytes induced by NPC1. Attenuated gliosis observed following PKC activation through PMA further substantiated this mechanism [116].

In summary, NPC patient-derived-iPSCs have been valuable in unraveling processes involved in the disease, such as cholesterol accumulation and autophagy dysfunction. These iPSCs also offer specific cell type modeling for screening therapeutic compounds having the potential to restore physiological functions.

Gaucher's Disease

Gaucher's disease (GD) is an autosomal recessive disorder caused by a mutation in the GBA1 gene encoding glucocerebrosidase, which cleaves glucosylceramide into ceramide and glucose [122 –124]. Such defect results in the glycolipid agglomeration in macrophages and manifests in various systems leading to hepatosplenomegaly, and hematological, skeletal, and neural defects. Epidemiological studies reveal that GD incidence in the general population ranged from 0.39 to 5.80 per 100,000 [125].

Based on the severity and neurological manifestation, GD is classified into type 1 (non-neuronopathic), type 2 (acute neuronopathic), and type 3 (chronic neuronopathic) phenotypes. Type 1 involves systemic pathology, whereas type 2 and type 3 are mainly associated with neuronopathic phenotypes. Gliosis, microglial activation, macrophage accumulation, and neurodegeneration constitute the disease etiology.

GD iPSC-derived neurons and macrophages recapitulate the disease phenotype

Human iPSCs have been generated from type 1 and type 3 GD patients and differentiated into macrophages and neuronal cell types. These GD iPSC-derived macrophages recapitulated the disease phenotype. The GD iPSC-derived macrophages were phagocytic and showed elevated inflammatory mediators release when exposed to lipopolysaccharide. Pathological hallmarks such as reduced GC activity, sphingolipid accumulation, and lysosomal dysfunction, along with defects in phagocytosed red blood cells (RBC) clearance, were observed. However, treatment with recombinant GC reversed this defect, suggesting that GC deficiency leads to functional defects.

When macrophages were exposed to the chaperons isofagomine and ambroxol, the defect in RBC clearance was partially restored, and the abnormal phenotype was rectified in GD [126,127]. In another study carried out by Tiscornia et al., macrophages and neurons differentiated from GD iPSCs exhibited reduced acid-β-glucosidase activity. Treatment with compounds having acid-β-glucosidase chaperone activity restored the acid-β-glucosidase protein levels and enzyme activity. Thus, GD iPSCs can be a relevant prototype to evaluate potent chaperone compounds in rescuing the enzymatic activity [128].

In GD iPSC-derived neurons, GBA1 mutation leads to lysosomal depletion, impeded autophagic vesicle clearance, and autophagy-induced neuronal death. Moreover, GBA1 mutation and GCase deficiency were associated with reduced transcription factor EB (TFEB) expression and stability. TFEB is a master regulator of lysosomal genes and plays a crucial role in lysosomal function and neuronal survival [129,130]. Treatment with recombinant GCase normalized lysosomal levels, and autophagy block upregulated the TFEB gene expression and increased TFEB stability. This study reveals that GBA1 mutation impedes TFEB-mediated lysosomal biogenesis, and GCase controls lysosomal function using TFEB [131].

GD iPSC-derived neurons generated from GD type 2 patients display electrophysiological properties with reduced sodium and potassium currents and attenuated action potential. Similar results were obtained when control neurons were exposed to GCase inhibitors, suggesting that reduced GCase activity is responsible for the aberrant electrophysiological properties in GD neurons [132].

iPSC-derived dopaminergic neurons carrying GBA1 mutation revealed a direct link between GD and PD

Homozygous mutation in the GBA1 gene leads to GD, whereas heterozygous mutation in the GBA1 gene could increase vulnerability to Parkinson's disease and other synucleinopathies. Based on the clinical manifestation of Parkinsonism and Lewy Body in GD patients, studies have shown a direct correlation between GD and Parkinson's disease patients carrying a heterozygous mutation in the GBA1 gene [133 –137].

HiPSC-derived dopaminergic neurons generated from GD and PD patients carrying the GBA1 mutations showed pathological hallmarks such as reduced GCase activity, α-synuclein aggregation, and defects in autophagic and lysosomal functions [40,132,138 –140]. Given that GBA1 mutation is one of the significant risk factors for PD susceptibility, Aflaki et al. generated dopaminergic neurons from GD iPSCs with and without Parkinsonism. These dopaminergic neurons obtained from patients with Parkinsonism exhibited elevated levels of aggregated α-synuclein.

Treatment with novel noninhibitory glucocerebrosidase chaperone reverted the disease phenotype by reinstating the GCase activity and reducing the accumulation of glycolipids and α-synuclein. This study highlights the therapeutic efficacy of glucocerebrosidase chaperones in PD and neuronopathic GD forms [138]. Perturbations in calcium homeostasis and increased intracellular calcium levels due to high susceptibility to stress response were observed in iPSC-derived dopaminergic neurons carrying the GBA1 mutations. Correction of this mutation using ZFN-mediated homologous recombination ameliorated the disease phenotype. These findings suggest a strong association among GBA1 mutation, defective autophagic/lysosomal function, and dysregulation of calcium homeostasis that culminates in neurodegenerative disorders like PD [40].

Using iPSC-derived dopaminergic neurons of synucleinopathies, Mazzulli et al. revealed that accumulation of α-synuclein results in abnormal hydrolase trafficking leading to attenuated lysosomal degradation capacity. Furthermore, disruption in the localization of RAB1A in endoplasmic reticulum-Golgi also occurred. However, overexpression of rab1a ameliorated the hydrolase trafficking, refurbished the Golgi structure, and attenuated the α–syn aggregation in these patient-derived iPSC neurons [141]. Recent studies suggest that one possible mechanism for α-synuclein accumulation and mitochondrial dysfunction could be their interaction with the accumulated glucosylceramide [142,143]. Also, the accumulation of GlcCer, in mature GD iPSC-derived dopaminergic neurons, suggested PD-like phenotype [144].

Mechanistic studies with GD iPSC-derived neurons suggest therapeutic potential of DKK1 in GD

Canonical Wnt pathway is considered a potential therapeutic target for GBA1-associated neurodegeneration [145]. This is derived from the fact that Wnt/β-catenin pathway downregulation in GD iPSC-derived neuronal cells upregulates Dkk1, an extracellular Wnt antagonist. Detrimental effects of Wnt/β-catenin downregulation in GD may be controlled by preventing Dkk1 binding to the Wnt co-receptor LRP6, suggestive of the therapeutic potential of Dkk1 in GD [145].

Collectively, extensive studies on GD iPSCs have aided our understanding of the molecular pathophysiology and treatment modalities such as chaperone compounds and gene therapy to restore the GCase activity, as well as potential of Wnt pathway for therapeutically targeting GD. Moreover, GD iPSCs have also facilitated delineating the link between GD and PD patients carrying the GBA1 mutation.

Krabbe Disease (Globoid Cell Leukodystrophy)

Krabbe disease, also known as globoid cell leukodystrophy, is an autosomal recessive disorder caused by deficiency of the enzyme galactocerebrosidase [146 –148]. Krabbe disease is a rare lysosomal storage disorder affecting 1:100,000 live births. Mutation in chromosome 14 that codes for glucosylceramide beta hydrolase (GAL), a lysosomal hydrolase that metabolizes galactolipids, leads to the accumulation of galactosylceramide and psychosine. This aggregated psychosine in the central and peripheral nervous system triggers neurodegeneration [149,150]. It derives its name globoid cell leukodystrophy due to multinucleated globoid bodies in the brain biopsy. Based on the disease onset, Krabbe disease is classified into four subtypes: Early infantile (0–1 years), Late infantile (1–3 years), juvenile (3–16 years) and Adult type (above 16 yrs) [151 –153].

Krabbe disease patient-derived iPSCs reveal the characteristic phenotype

HiPSCs have been derived from the dermal fibroblasts of a young 13-year-old patient with Krabbe disease, carrying a mutation in the GalC gene. These generated iPSCs exhibited the characteristic features and expressed pluripotent stem cell markers [154].

Recently, iPSC-derived neural progenitors and neuronal/glial progeny obtained from two Krabbe disease patients were used to demonstrate the effect of GALC deficiency and lentiviral vector-mediated GALC overexpression. The neural progeny exhibited the disease phenotype such as psychosine accumulation, neuronal and oligodendroglial abnormalities, and early cell death. GALC reconstitution and psychosine clearance lead to partial rescue of the differentiated neurons. Moreover, supra physiological GALC levels triggered the pathological phenotype, suggesting the need for a regulated GALC expression for normal neuronal differentiation [155].

Summary, Current Challenges, and Future Perspectives

Human iPSCs have provided us with an unprecedented prospect to gain mechanistic insights to unravel the pathophysiology associated with neurodevelopment in several rare LSDs when differentiated into specific cell lineage. Patient-derived iPSCs differentiated into specific CNS cell types are pertinent models for recapitulating the disease phenotype. Clinical manifestation of the disease phenotype, ease of handiness and manipulability, and origin from human patients have undeniably made them an excellent archetype in decoding the functional impairment of specific genes involved in various LSDs.

Patient-derived iPSCs have helped identify cellular, subcellular, and molecular phenotypes associated with LSDs such as neural progenitor cell migration and differentiation defects, substrate accumulation, axon growth and myelination defects, impaired calcium homeostasis, electrophysiological properties, and network connectivity.

Moreover, such studies have also discovered defective lysosomes, mitochondria, endoplasmic reticulum and Golgi complex, defective autophagy and vesicle trafficking, altered transport and signaling pathways, oxidative stress, neuroinflammation, blood–brain barrier dysfunction, neurodegeneration, gliosis, and altered transcriptomes. Further, these cells helped explore therapeutic prospects such as drug discovery, repurposing of drugs, synergistic effects of drugs, targeted molecular therapies, gene therapy, and transplantation applications of mutation-corrected lines for different LSDs. A summary of different LSDs and disease phenotypes as demonstrated from patient-derived iPSCs is shown in Table 1.

Table 1.

Summary of Induced Pluripotent Stem Cell Model of Lysosomal Storage Diseases, the Implicated Gene/Enzyme, Their Characteristic Phenotypes Demonstrated by Induced Pluripotent Stem Cell Derived Cells, and Its Application in Therapeutic Prospects

Disease	Gene/enzyme implicated	Cell types generated from iPSCs	Disease phenotype in iPSC derived cells	Treatment modalities	References
MLD	ARSA gene Arylsulfatase A	Neural stem cells, neural and astrocyte progenitors, neurons, astrocytes, oligodendrocytes.	Multiple sulfatide accumulation, enlargement of lysosomal compartments, aggravated oxidative stress, and enhanced programmed cell death, impaired neuronal and glial differentiation, neuronal network dysfunction.	Intracerebral transplantation of progenitor cells expressing human ARSA gene in MLD-iPSCs.	[32,47,52]
MPS type 1 (Hurler, Scheie syndrome)	IDUA gene α-L-iduronidase	NSCs, neurons, astrocytes, oligodendrocytes	Neural migration defects, neurite outgrowth defects, accumulation of GAGs, lysosomal enlargement, autophagy dysfunction.	—	[33,55]
MPS-II (Hunter syndrome)	IDS gene iduronate 2-sulfatase	NSCs, neurons, astrocytes, oligodendrocytes	Structural changes in Golgi apparatus and endoplasmic reticulum with intracellular accumulation of GAGs and LAMP2-positive vacuoles distinctly associated with the astrocytic death.	Exogenous administration of recombinant IDS.	[58 –60]
MPS-III B (Sanfilippo syndrome)	NAGLU gene α-N-Acetyl glucosaminidase	NSCs, neuron	Alteration in multiple cell signaling pathways, LAMP1- and GM3-positive vacuoles, severe storage lesions. First time identified Golgi defects in LSDs.	—	[61]
MPS-IIIC	HGSNAT gene	Neurons	GAG accumulation, LAMP1+vacuoles, aberrant neuronal connectivity and activity.	—	[64]
MPS-VII (Sly syndrome)	GUSB gene β-glucuronidase	NSCs, neurons, astrocytes, neurospheroids	Lower levels of synaptophysin, enlarged LAMP1+ lysosomal vesicles, GAG accumulation, reduced GABAergic interneurons, altered endolysosomal pathway, defective neuronal activity, weak network connectivity. Increased GFAP and astrocytic reactivity.	Intraventricular grafting of genetically corrected NSCs.	[34,68]
Mucolipidosis (Sialidosis)	NEU1 gene α-neuraminidase	Neurons, astrocytes, oligodendrocytes	Abnormal differentiation of astrocytes and oligodendrocytes, defective autophagy-lysosome pathway.	Rapamycin.	[70 –72]
Neuronal Ceroid Lipofuscinoses	CLN	NSCs, neurons	Mitochondrial anomalies, dilated endoplasmic reticulum, neuronal abnormalities, accumulation of autofluorescent deposits, altered intracellular transport system.	δ-Tocopherol and hydroxypropyl-β-cyclodextrin or their combination.	[30,86,87]
Sandhoff disease	HEXB gene	Cerebral organoids	Enhanced cell proliferation, altered neuronal differentiation.	—	[36]
Tay-Sachs disease	HEXA	NSCs, neurons	Enlarged lysosomes, upregulation of LAMP 1, presynaptic dysfunction.	ERT using iPSC-NSCs. Efficacy of δ-tocopherol and hydroxypropyl-β-cyclodextrin.	[95 –97]
Niemann-Pick disease type A and B	SMPD1 Sphingomyelinase	NSCs, neurons, Glial cells	Enlarged LAMP 1 vesicles, presynaptic dysfunction.	α-Tocopherol, δ-tocopherol, acid sphingomyelinase, and cyclodextrin.	[103 –105]
Niemann–Pick disease type C	NPC1	Macrophages and neurons	Defective differentiation, cholesterol and sphingomyelin accumulation, aberrant AMPA receptor-mediated calcium signaling, autophagy dysfunction, reduced PKC activity, aberrant WNT signaling, drastic reduction in catalase levels, gliosis.	WNT pathway modulation, δ-tocopherol, hydroxyl-β-cyclodextrin, and methyl-β-cyclodextrin and synergistic effects, VEGF, catalase and carbamazepine administration, PKC activators.	[39,114 –121]
Gaucher's disease	GBA1 glucocerebrosidase	NPCs, neurons glial cells	Sphingolipid accumulation, abnormal WNT and mTOR pathway, reduced TFEB expression and stability, attenuated action potential. Golgi complex, mitochondrial and lysosomal dysfunction, defective autophagy, perturbations in calcium homeostasis, α-synuclein aggregation, high susceptibility to stress response in dopaminergic neurons, direct link between Gaucher's disease and Parkinson's disease.	Treatment with recombinant GCase and compounds with acid-β-glucosidase chaperone activity, noninhibitory glucocerebrosidase chaperones. Therapeutic potential of Dkk1 in GD.	[126 –128,131,132,137 –145]
Krabbe Disease (globoid cell leukodystrophy)	GALC Galactocerebrosidase	NPCs, neurons and glial cells	Psychosine accumulation, neuronal and oligodendroglial abnormalities and early cell death, unbalanced lipid composition.	—	[154,155]
Fabry disease	GLA α-Galactosidase A	Cardiomyocyte endothelial cells	Globotriaosylceramide accumulation, interleukin-18 upregulation, increased lysosomal storage inclusions, accumulation of LIMP-2, downregulated superoxide dismutase 2, increased reactive oxygen species production, increased AMPK activity.	Substrate reduction therapy by glucosylceramide synthase inhibition, neutralization of IL-18 with specific antibodies combined with ERT.	[163 –166]
Pompe disease	GAA Acid α-glucosidase	Cardiomyocyte skeletal muscles	Glycogen accumulation, Golgi-based glycosylation deficit, oxidative stress, reduced GAA activity, mitochondrial dysfunction, altered mTORC1 signaling.	Recombinant human acid α-glucosidase treatment, l-carnitine, and TFEB overexpression.	[167 –171]
Cystinosis	CTNS Cystinosin	Kidney cells and organoids	Increased levels of cysteine, enhanced apoptosis, and defective autophagy.	Dual treatment with cysteamine and mTOR inhibitor everolimus.	[172]

CLN, ceroid lipofuscinosis, neuronal; ERT, enzyme replacement therapy; GAGs, glycosaminoglycans; HEXA, hexosaminidase A; HGSNAT, heparan acetyl-CoA: α-glucosaminide N-acetyltransferase; IDS, iduronate 2-sulfatase; iPSC, induced pluripotent stem cell; LAMP2, lysosome-associated membrane glycoprotein 2; LSD, lysosomal storage disease; MLD, metachromatic leukodystrophy; NPCs, neural precursor cell; TFEB, transcription factor EB; VEGF, vascular endothelial growth factor.

Patient-derived iPSC modeling holds excellent promise with significant benefits. However, several challenges need to be addressed to exploit them to their full potential in clinical settings and for the effective translation of findings.

For example, the scarcity of subjects with neuronopathic LSD makes it quite challenging to develop specific patient-derived iPSCs in large cohorts. In such cases, it is feasible to use gene editing to introduce the underlying mutation/s in healthy cells (from the control population) to generate healthy and disease isogenic iPSCs as an alternative to patient-derived iPSCs for studying LSDs. Although this could help understand the functional implications of the specifically mutated gene in LSDs, patient-specific iPSCs and their corrected lines are more suitable for molecular studies and therapeutic applications due to patient-specific genetic backgrounds.

In the case of some neuronopathic LSDs with late-onset disease manifestation, human iPSC-based disease models have failed to precisely recapitulate the disease phenotype due to the incomplete maturation of iPSC-derived cells. Moreover, alteration in the epigenetic profile could lead to significant gene expression variation between the parent and the reprogrammed cells. Hence, it is imperative to be wary of the genetic alterations introduced during reprogramming and epigenetic changes due to heterogeneity in passage number and culture conditions that could lead to clonal variation during the generation of specific patient-derived iPSC lines.

Neuropathic LSDs are associated with intellectual disability, cognitive deficits, and other psychiatric disorders. Nevertheless, it is currently not possible to recapitulate behavioral phenotypes using in vitro systems. Hence, parallel studies using appropriate animal models are required for corroborating the behavioral phenotype associated with neuronopathic LSD. Also, it is quite challenging to delineate the early cortical maldevelopment due to abnormal neurogenesis and neuronal migration using 2D cultures. Hence, for such longitudinal studies, the 3D cerebral organoids could serve as a suitable archetype [36 –38] with functional vascular [156] and immune systems [157].

Therapeutic strategies that can counteract substrate accumulation in LSDs can be exploited using patient-specific iPSCs [30,38,47,64,158]. Furthermore, selected drug testing with cells derived from patient-derived iPSCs and gene-corrected cell lines could help to minimize animal experiments. Moreover, intracerebral grafting of iPSC-derived neural progenitor cells has shown functional benefits in preclinical models of neuronopathic LSDs, such as MLD and MPS VII [32,52,68]. Nevertheless, studies to assess neuronal signatures that could be used as predictive biomarkers in neuronopathic LSDs should be rigorously explored, using patient-specific iPSCs [158 –161].

Instituting cell bank repositories that ensure reproducibility of results for different LSDs would provide a powerful resource for conducting inventive studies using multiple cell lines carrying specific mutations. Genetic identification of LSDs and collecting samples for iPSC generation is a significant issue in several developing countries due to the unavailability of experts, facilities, and funds.

Enhanced research collaborations from established researchers to create networks for collecting details and samples of all such cases could help progress in cell banking and distributing such cells. Patient-derived iPSC lines in such banks should be necessarily tested for their characteristics, genetic stability and fidelity, sterility, and potency [162]. This networking, banking, and controlled distribution of generated cells to interested researchers could promote understanding of neuropathology and neurodevelopmental pathways to modulate treatment along with ERT to minimize specific enzyme deficiency-related phenotypes of LSDs.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work is supported by grants from Science and Engineering Research Board (SERB-EMR/2017/005213 to D.U.) and Intramural grant from Manipal Academy of Higher Education, Manipal (MAHE/PDF/2019 to S.K.R.).

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