Intracerebroventricular 2-hydroxypropyl-γ-cyclodextrin alleviates hepatic manifestations without distributing to the liver in a murine model of Niemann-pick disease type C.

Niemann-Pick disease type C (NPC) is a lysosomal lipid storage disorder characterized by progressive neurodegeneration and hepatic dysfunction. A cyclic heptasaccharide, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), is currently under clinical investigation for NPC, but its adverse events remain problematic. We previously identified that a cyclic octasaccharide, 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD), also ameliorated NPC manifestations with higher biocompatibility than HP-ß-CD. However, preclinical studies describing the associations between the biodistribution and pharmacodynamics of these compounds, which are essential for clinical application, are still lacking. Here, we investigated these properties of HP-γ-CD by measuring its organ biodistribution and therapeutic effect after systemic and central administration. The effect of HP-γ-CD on disturbed cholesterol homeostasis appeared within several hours after exposure and persisted for several days in NPC model cells and mice. Tissue distribution indicated that only a small fraction of subcutaneously administered HP-γ-CD rapidly distributed to peripheral organs and contributed to disease amelioration. We found that a subcutaneous dose of HP-γ-CD negligibly ameliorated neurological characteristics because it has limited penetration of the blood-brain barrier; however, an intracerebroventricular microdose unexpectedly attenuated hepatic dysfunction without the detection of HP-γ-CD in the liver. These results demonstrate that central administration of HP-γ-CD can indirectly attenuate peripheral manifestations of NPC.

Characterization of heterozygous ATTR Tyr114Cys amyloidosis-specific induced pluripotent stem cells.

Hereditary transthyretin (TTR) amyloidosis (ATTRv amyloidosis) is autosomal dominant and caused by mutation of TTR gene. Heterozygous ATTR Tyr114Cys (p.Tyr134Cys) amyloidosis is a lethal disease with a life expectancy of about 10 years after onset of the disease. However, the molecular pathogenesis of ATTR Tyr114Cys amyloidosis is still largely unknown. In this study, we took advantage of disease-specific induced pluripotent stem (iPS) cells and generated & characterized the heterozygous ATTR Tyr114Cys amyloidosis-specific iPS cells (Y114C iPS cells), to determine whether Y114C iPS cells could be useful for elucidating the pathogenesis of ATTR Tyr114Cys amyloidosis. We successfully differentiated heterozygous Y114C iPS cells into hepatocyte like cells (HLCs) mainly producing TTR protein. On day 27 after differentiation, the expression of hepatocyte maker albumin was detected, and TTR expression was significantly increased in HLCs differentiated from Y114C iPS cells. LC-MS/MS analysis showed that both WT TTR & ATTR Y114C protein were indeed expressed in the HLCs differentiated from Y114C iPS cells. Notably, the number of detected peptides derived from ATTR Y114C protein was lower than that of WT TTR protein, indeed indicating the clinical phenotype of ATTR Tyr114Cys amyloidosis. Taken together, we first reported the heterozygous Y114C iPS cells generated from patient with ATTR Tyr114Cys amyloidosis, and suggested that Y114C iPS cells could be a potential pathological tool, which may contribute to elucidating the molecular pathogenesis of heterozygous ATTR Tyr114Cys amyloidosis.

Pathophysiological Investigation of Skeletal Deformities of Musculocontractural Ehlers-Danlos Syndrome Using Induced Pluripotent Stem Cells.

Musculocontractural Ehlers-Danlos syndrome caused by mutations in the carbohydrate sulfotransferase 14 gene (mcEDS-CHST14) is a heritable connective tissue disorder characterized by multiple congenital malformations and progressive connective tissue fragility-related manifestations in the cutaneous, skeletal, cardiovascular, visceral, and ocular systems. Progressive skeletal deformities are among the most frequent and serious complications affecting the quality of life and activities of daily living in patients. After establishing induced pluripotent stem cells (iPSCs) from cultured skin fibroblasts of three patients with mcEDS-CHST14, we generated a patient iPSC-based human osteogenesis model and performed an in vitro assessment of the phenotype and pathophysiology of skeletal deformities. Patient-derived iPSCs presented with remarkable downregulation of osteogenic-specific gene expression, less alizarin red staining, and reduced calcium deposition compared with wild-type iPSCs at each stage of osteogenic differentiation, including osteoprogenitor cells, osteoblasts, and osteocytes. These findings indicated that osteogenesis was impaired in mcEDS-CHST14 iPSCs. Moreover, the decrease in decorin (DCN) expression and increase in collagen (COL12A1) expression in patient-derived iPSCs elucidated the contribution of CHST14 dysfunction to skeletal deformities in mcEDS-CHST14. In conclusion, this disease-in-a-dish model provides new insight into the pathophysiology of EDS and may have the potential for personalized gene or drug therapy.

Lactose-Appended Hydroxypropyl-ß-Cyclodextrin Lowers Cholesterol Accumulation and Alleviates Motor Dysfunction in Niemann-Pick Type C Disease Model Mice.

Niemann-Pick disease type C (NPC) is characterized by the accumulation of glycolipids such as free cholesterol, sphingomyelin, and gangliosides in late endosomes/lysosomes (endolysosomes) due to abnormalities in the membrane proteins NPC1 or NPC2. The main symptoms of NPC caused by free cholesterol accumulation in various tissues vary depending on the time of onset, but hepatosplenomegaly and neurological symptoms accompanied by decreased motor, cognitive, and mental functions are observed in all age groups. However, the efficacy of NPC treatment remains limited. Herein, we have fabricated lactose-appended hydroxypropyl-ß-cyclodextrin (Lac-HPßCD) and evaluated its lowering effects on cholesterol accumulation in NPC model mice. We reveal that Lac-HPßCD lowers cholesterol accumulation in the liver and spleen by reducing the amount of free cholesterol. Moreover, Lac-HPßCD reduces the amount of free cholesterol in the cerebrum and slightly alleviates motor dysfunction. These results suggest that Lac-HPßCD has potential for the treatment of NPC.

Movements of Ancient Human Endogenous Retroviruses Detected in SOX2-Expressing Cells.

Human endogenous retroviruses (HERVs) occupy approximately 8% of the human genome. HERVs, transcribed in early embryos, are epigenetically silenced in somatic cells, except under pathological conditions. HERV-K is thought to protect embryos from exogenous viral infection. However, uncontrolled HERV-K expression in somatic cells has been implicated in several diseases. Here, we show that SOX2, which plays a key role in maintaining the pluripotency of stem cells, is critical for HERV-K LTR5Hs. HERV-K undergoes retrotransposition within producer cells in the absence of Env expression. Furthermore, we identified new HERV-K integration sites in long-term culture of induced pluripotent stem cells that express SOX2. These results suggest that the strict dependence of HERV-K on SOX2 has allowed HERV-K to protect early embryos during evolution while limiting the potentially harmful effects of HERV-K retrotransposition on host genome integrity in these early embryos. IMPORTANCE Human endogenous retroviruses (HERVs) account for approximately 8% of the human genome; however, the physiological role of HERV-K remains unknown. This study found that HERV-K LTR5Hs and LTR5B were transactivated by SOX2, which is essential for maintaining and reestablishing pluripotency. HERV-K can undergo retrotransposition within producer cells without env expression, and new integration sites may affect cell proliferation. In induced pluripotent stem cells (iPSCs), genomic impairment due to HERV-K retrotransposition has been identified, but it is a rare event. Considering the retention of SOX2-responsive elements in the HERV-K long terminal repeat (LTR) for over 20 million years, we conclude that HERV-K may play important physiological roles in SOX2-expressing cells.

ATP citrate lyase controls hematopoietic stem cell fate and supports bone marrow regeneration.

In order to support bone marrow regeneration after myeloablation, hematopoietic stem cells (HSCs) actively divide to provide both stem and progenitor cells. However, the mechanisms regulating HSC function and cell fate choice during hematopoietic recovery remain unclear. We herein provide novel insights into HSC regulation during regeneration by focusing on mitochondrial metabolism and ATP citrate lyase (ACLY). After 5-fluorouracil-induced myeloablation, HSCs highly expressing endothelial protein C receptor (EPCRhigh ) were enriched within the stem cell fraction at the expense of more proliferative EPCRLow HSCs. These EPCRHigh HSCs were initially more primitive than EPCRLow HSCs and enabled stem cell expansion by enhancing histone acetylation, due to increased activity of ACLY in the early phase of hematopoietic regeneration. In the late phase of recovery, HSCs enhanced differentiation potential by increasing the accessibility of cis-regulatory elements in progenitor cell-related genes, such as CD48. In conditions of reduced mitochondrial metabolism and ACLY activity, these HSCs maintained stem cell phenotypes, while ACLY-dependent histone acetylation promoted differentiation into CD48+ progenitor cells. Collectively, these results indicate that the dynamic control of ACLY-dependent metabolism and epigenetic alterations is essential for HSC regulation during hematopoietic regeneration.

Analysis of induced pluripotent stem cell clones derived from a patient with mosaic neurofibromatosis type 2.

The diagnosis of mosaicism is challenging in patients with neurofibromatosis type 2 (NF2) subset due to low variant allele frequency. In this study, we generated induced pluripotent stem cells (iPSCs) were generated from a patient clinically diagnosed with NF2 based on multiple schwannomas, including bilateral vestibular schwannomas and meningiomas. Genetic analysis of the patient's mononuclear cells (MNCs) from peripheral blood failed to detect NF2 alteration but successfully found p.Q65X (c.193C>T) mutation in all separate tumors with three intracranial meningiomas and one intraorbital schwannoma, and confirming mosaicism diagnosis in NF2 alteration using deep sequencing. Five different clones with patient-derived iPSCs were established from MNCs in peripheral blood, which showed sufficient expression of pluripotent markers. Genetic analysis showed that one of five generated iPSC lines from MNCs had the same p.Q65X mutation as that found in NF2. There was no significant difference in the expression of genes related to NF2 between iPSC clones with the wild-type and mutant NF2. In this case, clonal expansion of mononuclear bone marrow-derived stem cells recapitulated mosaicism's genetic alteration in NF2. Patient-derived iPSCs from mosaic NF2 would contribute to further functional research of NF2 alteration.

Fate of adipocyte progenitors during adipogenesis in mice fed a high-fat diet.

OBJECTIVE: Expansion of adipose tissue during obesity through the recruitment of newly generated adipocytes (hyperplasia) is metabolically healthy, whereas that through the enlargement of pre-existing adipocytes (hypertrophy) leads to metabolic complications. Accumulating evidence from genetic fate mapping studies suggests that in animal models receiving a high-fat diet (HFD), only adipocyte progenitors (APs) in gonadal white adipose tissue (gWAT) have proliferative potential. However, the proliferative potential and differentiating capacity of APs in the inguinal WAT (iWAT) of male mice remains controversial. The objective of this study was to investigate the proliferative and adipogenic potential of APs in the iWAT of HFD-fed male mice. METHODS: We generated PDGFRα-GFP-Cre-ERT2/tdTomato (KI/td) mice and traced PDGFRα-positive APs in male mice fed HFD for 8 weeks. We performed a comprehensive phenotypic analysis, including the histology, immunohistochemistry, flow cytometry, and gene expression analysis, of KI/td mice fed HFD. RESULTS: Contrary to the findings of others, we found an increased number of newly generated tdTomato+ adipocytes in the iWAT of male mice, which was smaller than that observed in the gWAT. We found that in male mice, the iWAT has more proliferating tdTomato+ APs than the gWAT. We also found that tdTomato+ APs showed a higher expression of Dpp4 and Pi16 than tdTomato- APs, and the expression of these genes was significantly higher in the iWAT than in the gWAT of mice fed HFD for 8 weeks. Collectively, our results reveal that HFD feeding induces the proliferation of tdTomato+ APs in the iWAT of male mice. CONCLUSION: In male mice, compared with gWAT, iWAT undergoes hyperplasia in response to 8 weeks of HFD feeding through the recruitment of newly generated adipocytes due to an abundance of APs with a high potential for proliferation and differentiation.

A neuropathological cell model derived from Niemann-Pick disease type C patient-specific iPSCs shows disruption of the p62/SQSTM1-KEAP1-NRF2 Axis and impaired formation of neuronal networks.

Niemann-Pick disease type C (NPC) is a rare neurodegenerative disorder caused by a recessive mutation in the NPC1 or NPC2 gene, in which patients exhibit lysosomal accumulation of unesterified cholesterol and glycolipids. Most of the research on NPC has been done in patient-derived skin fibroblasts or mouse models. Therefore, we developed NPC patient neurons derived from induced pluripotent stem cells (iPSCs) to investigate the neuropathological cause of the disease. Although an accumulation of cholesterol and glycolipids, which is characteristic of NPC, was observed in both undifferentiated iPSCs and derived neural stem cells (NSCs), we could not observed the abnormalities in differentiation potential and autophagic activity in such immature cells. However, definite neuropathological features were detected in mature neuronal cells generated from NPC patient-derived iPSCs. Abnormal accumulation of cholesterol and other lipids identified by lipid droplets and number of enlarged lysosomes was more prominent in mature neuronal cells rather than in iPSCs and/or NSCs. Thin-sectioning electron microscopic analysis also demonstrated numerous typical membranous cytoplasmic bodies in mature neuronal cells. Furthermore, TUJ1-positive neurite density was significantly reduced in NPC patient-derived neuronal cells. In addition, disruption of the p62/SQSTM1-KEAP1-NRF2 axis occurred in neurons differentiated from NPC patient-derived iPSCs. These data indicate the impairment of neuronal network formation associated with neurodegeneration in mature neuronal cells derived from patients with NPC.

Differential mode of cholesterol inclusion with 2-hydroxypropyl-cyclodextrins increases safety margin in treatment of Niemann-Pick disease type C.

BACKGROUND AND PURPOSE: Niemann-Pick disease type C (NPC) is a lysosomal storage disorder with disrupted intracellular cholesterol trafficking. A cyclic heptasaccharide, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), is a cholesterol solubilizer that is being developed to treat NPC, but its ototoxicity and pulmonary toxicity remain important issues. We have characterized 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD), a cyclic octasaccharide with a larger cavity than HP-ß-CD, as a candidate drug to treat NPC. However, the molecular target of HP-γ-CD with respect to NPC and its potential for clinical application are still unclear. EXPERIMENTAL APPROACH: We investigated the mode of interaction between HP-γ-CD and cholesterol by phase-solubility analysis, proton NMR spectroscopy and molecular dynamics simulations. We then evaluated the therapeutic effects of HP-γ-CD compared with HP-ß-CD using cellular and murine NPC models. Mouse auditory and pulmonary function tests were also conducted. KEY RESULTS: HP-γ-CD solely formed a 1:1 inclusion complex with cholesterol with an affinity similar to that of HP-ß-CD. In vitro, HP-γ-CD and HP-ß-CD amelioration of NPC-related manifestations was almost equivalent at lower concentrations. However, at higher concentrations, the cholesterol inclusion mode of HP-ß-CD shifted to the highly soluble 2:1 complex whereas that of HP-γ-CD maintained solely the 1:1 complex. The constant lower cholesterol solubilizing ability of HP-γ-CD conferred it with significantly reduced toxicity compared with HP-ß-CD, but equal efficacy in treating a mouse model of NPC. CONCLUSIONS AND IMPLICATIONS: HP-γ-CD can serve as a fine-tuned cholesterol solubilizer for the treatment of NPC with a wider safety margin than HP-ß-CD in terms of ototoxicity and pulmonary toxicity.

Taurine rescues mitochondria-related metabolic impairments in the patient-derived induced pluripotent stem cells and epithelial-mesenchymal transition in the retinal pigment epithelium.

Mitochondria participate in various metabolic pathways, and their dysregulation results in multiple disorders, including aging-related diseases. However, the metabolic changes and mechanisms of mitochondrial disorders are not fully understood. Here, we found that induced pluripotent stem cells (iPSCs) from a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) showed attenuated proliferation and survival when glycolysis was inhibited. These deficits were rescued by taurine administration. Metabolomic analyses showed that the ratio of the reduced (GSH) to oxidized glutathione (GSSG) was decreased; whereas the levels of cysteine, a substrate of GSH, and oxidative stress markers were upregulated in MELAS iPSCs. Taurine normalized these changes, suggesting that MELAS iPSCs were affected by the oxidative stress and taurine reduced its influence. We also analyzed the retinal pigment epithelium (RPE) differentiated from MELAS iPSCs by using a three-dimensional culture system and found that it showed epithelial mesenchymal transition (EMT), which was suppressed by taurine. Therefore, mitochondrial dysfunction caused metabolic changes, accumulation of oxidative stress that depleted GSH, and EMT in the RPE that could be involved in retinal pathogenesis. Because all these phenomena were sensitive to taurine treatment, we conclude that administration of taurine may be a potential new therapeutic approach for mitochondria-related retinal diseases.

Rapid and Simplified Induction of Neural Stem/Progenitor Cells (NSCs/NPCs) and Neurons from Human Induced Pluripotent Stem Cells (hiPSCs).

Human induced pluripotent stem cells (iPSCs) and their progeny displaying tissue-specific characteristics have paved the way for regenerative medicine and research in various fields such as the elucidation of the pathological mechanism of diseases and the discovery of drug candidates. iPSC-derived neurons are particularly valuable as it is difficult to analyze neural cells obtained from the central nervous system in humans. For neuronal induction with iPSCs, one of the commonly used approaches is the isolation and expansion of neural rosettes, following the formation of embryonic bodies (EBs). However, this process is laborious, inefficient, and requires further purification of the cells. To overcome these limitations, we have developed an efficient neural induction method that allows for the generation of neural stem/progenitor cells (NSCs/NPCs) from iPSCs within 7 days and of functional mature neurons. Our method yields a PAX6-positive homogeneous cell population, a cortical NSCs/NPCs, and the resultant NSCs/NPCs can be cryopreserved, expanded, and differentiated into functional mature neurons. Moreover, our protocol will be less expensive than other methods since the protocol requires fewer neural supplements during neural induction. This article also presents the FM1-43 imaging assay, which is useful for the presynaptic assessment of the iPSCs-derived human neurons. This protocol provides a quick and simplified way to generate NSCs/NPCs and neurons, enabling researchers to establish in vitro cellular models to study brain disease pathology.

Impaired NEPHRIN localization in kidney organoids derived from nephrotic patient iPS cells.

Mutations in the NPHS1 gene, which encodes NEPHRIN, cause congenital nephrotic syndrome, resulting from impaired slit diaphragm (SD) formation in glomerular podocytes. We previously reported NEPHRIN and SD abnormalities in the podocytes of kidney organoids generated from patient-derived induced pluripotent stem cells (iPSCs) with an NPHS1 missense mutation (E725D). However, the mechanisms underlying the disease may vary depending on the mutations involved, and thus generation of iPSCs from multiple patients is warranted. Here we established iPSCs from two additional patients with different NPHS1 mutations and examined the podocyte abnormalities in kidney organoids derived from these cells. One patient had truncating mutations, and NEPHRIN was undetectable in the resulting organoids. The other patient had a missense mutation (R460Q), and the mutant NEPHRIN in the organoids failed to accumulate on the podocyte surface to form SD precursors. However, the same mutant protein behaved normally when overexpressed in heterologous cells, suggesting that NEPHRIN localization is cell context-dependent. The localization of another SD-associated protein, PODOCIN, was impaired in both types of mutant organoids in a cell domain-specific manner. Thus, the new iPSC lines and resultant kidney organoids will be useful resources for dissecting the disease mechanisms, as well as for drug development for therapies.

Vulnerability to shear stress caused by altered peri-endothelial matrix is a key feature of Moyamoya disease.

Moyamoya disease (MMD) is characterized by progressive bilateral stenotic changes in the terminal portion of the internal carotid arteries. Although RNF213 was identified as a susceptibility gene for MMD, the exact pathogenesis remains unknown. Immunohistochemical analysis of autopsy specimens from a patient with MMD revealed marked accumulation of hyaluronan and chondroitin sulfate (CS) in the thickened intima of occlusive lesions of MMD. Hyaluronan synthase 2 was strongly expressed in endothelial progenitor cells in the thickened intima. Furthermore, MMD lesions showed minimal staining for CS and hyaluronan in the endothelium, in contrast to control endothelium showing positive staining for both. Glycosaminoglycans of endothelial cells derived from MMD and control induced pluripotent stem cells demonstrated a decreased amount of CS, especially sulfated CS, in MMD. A computational fluid dynamics model showed highest wall shear stress values in the terminal portion of the internal carotid artery, which is the predisposing region in MMD. Because the peri-endothelial extracellular matrix plays an important role in protection, cell adhesion and migration, an altered peri-endothelial matrix in MMD may contribute to endothelial vulnerability to wall shear stress. Invading endothelial progenitor cells repairing endothelial injury would produce excessive hyaluronan and CS in the intima, and cause vascular stenosis.

An iPSC-based neural model of sialidosis uncovers glycolytic impairment-causing presynaptic dysfunction and deregulation of Ca2+ dynamics.

Sialidosis is a neuropathic lysosomal storage disease caused by a deficiency in the NEU1 gene-encoding lysosomal neuraminidase and characterized by abnormal accumulation of undigested sialyl-oligoconjugates in systemic organs including brain. Although patients exhibit neurological symptoms, the underlying neuropathological mechanism remains unclear. Here, we generated induced pluripotent stem cells (iPSCs) from skin fibroblasts with sialidosis and induced the differentiation into neural progenitor cells (NPCs) and neurons. Sialidosis NPCs and neurons mimicked the disease-like phenotypes including reduced neuraminidase activity, accumulation of sialyl-oligoconjugates and lysosomal expansions. Functional analysis also revealed that sialidosis neurons displayed two distinct abnormalities, defective exocytotic glutamate release and augmented α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR)-mediated Ca2+ influx. These abnormalities were restored by overexpression of the wild-type NEU1 gene, demonstrating causative role of neuraminidase deficiency in functional impairments of disease neurons. Comprehensive proteomics analysis revealed the significant reduction of SNARE proteins and glycolytic enzymes in synaptosomal fraction, with downregulation of ATP production. Bypassing the glycolysis by treatment of pyruvate, which is final metabolite of glycolysis pathway, improved both the synaptsomal ATP production and the exocytotic function. We also found that upregulation of AMPAR and L-type voltage dependent Ca2+ channel (VDCC) subunits in disease neurons, with the restoration of AMPAR-mediated Ca2+ over-load by treatment of antagonists for the AMPAR and L-type VDCC. Our present study provides new insights into both the neuronal pathophysiology and potential therapeutic strategy for sialidosis.

Generation of a human induced pluripotent stem cell line, BRCi009-A, derived from a patient with glycogen storage disease type 1a.

Glycogen storage disease type 1a (GSD1a) is an autosomal recessive disorder caused by mutations of the glucose-6-phosphatase (G6PC) gene. Mutations of the G6PC gene lead to excessive accumulation of glycogen in the liver, kidney, and intestinal mucosa due to the deficiency of microsomal glucose-6-phosphatase. Human induced pluripotent stem cells (iPSCs) enable the production of patient-derived hepatocytes in culture and are therefore a promising tool for modeling GSD1a. Here, we report the establishment of human iPSCs from a GSD1a patient carrying a G6PC mutation (c.648G > T; p.Leu216 = ).

Rapid repair of human disease-specific single-nucleotide variants by One-SHOT genome editing.

Many human diseases ranging from cancer to hereditary disorders are caused by single-nucleotide mutations in critical genes. Repairing these mutations would significantly improve the quality of life for patients with hereditary diseases. However, current procedures for repairing deleterious single-nucleotide mutations are not straightforward, requiring multiple steps and taking several months to complete. In the current study, we aimed to repair pathogenic allele-specific single-nucleotide mutations using a single round of genome editing. Using high-fidelity, site-specific nuclease AsCas12a/Cpf1, we attempted to repair pathogenic single-nucleotide variants (SNVs) in disease-specific induced pluripotent stem cells. As a result, we achieved repair of the Met918Thr SNV in human oncogene RET with the inclusion of a single-nucleotide marker, followed by absolute markerless, scarless repair of the RET SNV with no detected off-target effects. The markerless method was then confirmed in human type VII collagen-encoding gene COL7A1. Thus, using this One-SHOT method, we successfully reduced the number of genetic manipulations required for genome repair from two consecutive events to one, resulting in allele-specific repair that can be completed within 3 weeks, with or without a single-nucleotide marker. Our findings suggest that One-SHOT can be used to repair other types of mutations, with potential beyond human medicine.

Role of induced pluripotent stem cells in lysosomal storage diseases.

Lysosomal storage diseases (LSDs) are a group of metabolism inborn errors caused by defective enzymes in the lysosome, resulting in the accumulation of undegraded substrates. Many characteristic cell features have been revealed in LSDs, including abnormal autophagy and mitochondrial dysfunction. The development of induced pluripotent stem cells (iPSCs) dramatically boosted research on LSDs, particularly regarding novel opportunities to clarify the disease etiology based on the storage of macromolecules, such as sphingolipids in lysosomes. iPSCs made from LSD patients (LSD-iPSCs) have been differentiated into neurons, endothelial cells, cardiomyocytes, hepatocytes, and macrophages, with each cell type closely resembling the primary disease phenotypes, providing new tools to probe the disease pathogenesis and to test therapeutic strategies. Abnormally accumulated substrates impaired autophagy and mitochondrial and synapse functions in LSD-iPSC-derived neurons. Reducing the accumulation with the treatment of drug candidates improved LSD-iPSC-derived neuron functions. Additionally, iPSC technology can help probe the gene expressions, proteomics, and metabolomics of LSDs. Further, gene repair and the generation of new mutations in causative genes in LSD-iPSCs can be used to understand both the specific roles of causative genes and the contributions of other genetic factors to these phenotypes. Moreover, the development of iPSC-derived organoids as disease models has bridged the gap between studies using cell lines and in vivo animal models. There are some reproducibility issues in iPSC research, however, including genetic and epigenetic abnormalities, such as chromosomal abnormalities, DNA mutations, and gene modifications via methylation. In this review, we present the disease and treatment concepts gathered using selected LSD-iPSCs, discuss iPSC research limitations, and set our future research visions. Such studies are expected to further inform and generate insights into LSDs and are important in research and clinical practice.

Novel Drug Candidates Improve Ganglioside Accumulation and Neural Dysfunction in GM1 Gangliosidosis Models with Autophagy Activation.

GM1 gangliosidosis is a lysosomal storage disease caused by loss of lysosomal ß-galactosidase activity and characterized by progressive neurodegeneration due to massive accumulation of GM1 ganglioside in the brain. Here, we generated induced pluripotent stem cells (iPSCs) derived from patients with GM1 gangliosidosis, and the resultant neurons showed impaired neurotransmitter release as a presynaptic function and accumulation of GM1 ganglioside. Treatment of normal neurons with GM1 ganglioside also disturbed presynaptic function. A high-content drug-screening system was then established and identified two compounds as drug candidates for GM1 gangliosidosis. Treatment of the patient-derived neurons with the candidate agents activated autophagy pathways, reducing GM1 ganglioside accumulation in vitro and in vivo, and restoring the presynaptic dysfunction. Our findings thus demonstrated the potential value of patient-derived iPSC lines as cellular models of GM1 gangliosidosis and revealed two potential therapeutic agents for future clinical application.

Activin Is Superior to BMP7 for Efficient Maintenance of Human iPSC-Derived Nephron Progenitors.

Kidney formation is regulated by the balance between maintenance and differentiation of nephron progenitor cells (NPCs). Now that directed differentiation of NPCs from human induced pluripotent stem cells (iPSCs) can be achieved, maintenance and propagation of NPCs in vitro should be beneficial for regenerative medicine. Although WNT and FGF signals were previously shown to be essential for NPC propagation, the requirement for BMP/TGFß signaling remains controversial. Here we reveal that activin has superior effects to BMP7 on maintenance efficiency of human iPSC-derived NPCs. Activin expanded ITGA8+/PDGFRA-/SIX2-GFP+ NPCs by 5-fold per week at 80%-90% efficiency, and the propagated cells possessed robust capacity for nephron formation both in vitro and in vivo. The expanded cells also maintained their nephron-forming potential after freezing. Furthermore, the protocol was applicable to multiple non-GFP-tagged iPSC lines. Thus, our activin-based protocol will be applicable to a variety of research fields including disease modeling and drug screening.