CGG repeat expansion in LRP12 in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of motor neurons. Although repeat expansion in C9orf72 is its most common cause, the pathogenesis of ALS isn't fully clear. In this study, we show that repeat expansion in LRP12, a causative variant of oculopharyngodistal myopathy type 1 (OPDM1), is a cause of ALS. We identify CGG repeat expansion in LRP12 in five families and two simplex individuals. These ALS individuals (LRP12-ALS) have 61-100 repeats, which contrasts with most OPDM individuals with repeat expansion in LRP12 (LRP12-OPDM), who have 100-200 repeats. Phosphorylated TDP-43 is present in the cytoplasm of iPS cell-derived motor neurons (iPSMNs) in LRP12-ALS, a finding that reproduces the pathological hallmark of ALS. RNA foci are more prominent in muscle and iPSMNs in LRP12-ALS than in LRP12-OPDM. Muscleblind-like 1 aggregates are observed only in OPDM muscle. In conclusion, CGG repeat expansions in LRP12 cause ALS and OPDM, depending on the length of the repeat. Our findings provide insight into the repeat length-dependent switching of phenotypes.

Investigating developmental and disease mechanisms of the cerebellum with pluripotent stem cells.

The cerebellum is a brain region located in the dorsal part of the anterior hindbrain, composed of a highly stereotyped neural circuit structure with small sets of neurons. The cerebellum is involved in a wide variety of functions such as motor control, learning, cognition and others. Damage to the cerebellum often leads to impairments in motor skills (cerebellar ataxia). Cerebellar ataxia can occur as a result of neurodegenerative diseases such as spinocerebellar ataxia. Recent advances in technologies related to pluripotent stem cells and their neural differentiation has enabled researchers to investigate the mechanisms of development and of disease in the human brain. Here, we review recent applications of leading-edge stem cell technologies to the mechanistic investigation of human cerebellar development and neurological diseases affecting the cerebellum.

Generation of thalamic neurons from mouse embryonic stem cells.

The thalamus is a diencephalic structure that plays crucial roles in relaying and modulating sensory and motor information to the neocortex. The thalamus develops in the dorsal part of the neural tube at the level of the caudal forebrain. However, the molecular mechanisms that are essential for thalamic differentiation are still unknown. Here, we have succeeded in generating thalamic neurons from mouse embryonic stem cells (mESCs) by modifying the default method that induces the most-anterior neural type in self-organizing culture. A low concentration of the caudalizing factor insulin and a MAPK/ERK kinase inhibitor enhanced the expression of the caudal forebrain markers Otx2 and Pax6. BMP7 promoted an increase in thalamic precursors such as Tcf7l2+/Gbx2+ and Tcf7l2+/Olig3+ cells. mESC thalamic precursors began to express the glutamate transporter vGlut2 and the axon-specific marker VGF, similar to mature projection neurons. The mESC thalamic neurons extended their axons to cortical layers in both organotypic culture and subcortical transplantation. Thus, we have identified the minimum elements sufficient for in vitro generation of thalamic neurons. These findings expand our knowledge of thalamic development.

Self-organizing cortex generated from human iPSCs with combination of FGF2 and ambient oxygen.

Human brain development has generally been studied through the analysis of postmortem tissues because of limited access to fetal brain tissues. This approach, however, only provides information from the perspective of long-term development. To investigate the pathophysiology of neurodevelopmental disorders, it is necessary to understand the detailed mechanisms of human brain development. Recent advances in pluripotent stem cell (PSC) technologies enable us to establish in vitro brain models from human induced PSCs (hiPSCs), which can be used to examine the pathophysiological mechanisms of neurodevelopmental disorders. We previously demonstrated that self-organized cerebral tissues can be generated from human PSCs in a three-dimensional (3D) culture system. Here, we describe the cerebral tissues differentiated from hiPSCs in a further-optimized 3D culture. We found that treatment with FGF2 is helpful to form iPSC aggregates with efficiency. Neuroepithelial structures spontaneously formed with apico-basal polarity in the aggregates expressing forebrain marker FOXG1. The neuroepithelium self-forms a multilayered structure including progenitor zones (ventricular and subventricular zones) and neuronal zone (cortical plate). Furthermore, with the same level of oxygen (O2) as in ambient air (20% O2), we found that self-formation of cortical structures lasted for 70 days in culture. Thus, our optimized 3D culture for the generation of cortical structure from hiPSCs is a simple yet effective method.

Self-Organized Cerebellar Tissue from Human Pluripotent Stem Cells and Disease Modeling with Patient-Derived iPSCs.

Recent advances in the techniques that differentiate induced pluripotent stem cells (iPSCs) into specific types of cells enabled us to establish in vitro cell-based models as a platform for drug discovery. iPSC-derived disease models are advantageous to generation of a large number of cells required for high-throughput screening. Furthermore, disease-relevant cells differentiated from patient-derived iPSCs are expected to recapitulate the disorder-specific pathogenesis and physiology in vitro. Such disease-relevant cells will be useful for developing effective therapies. We demonstrated that cerebellar tissues are generated from human PSCs (hPSCs) in 3D culture systems that recapitulate the in vivo microenvironments associated with the isthmic organizer. Recently, we have succeeded in generation of spinocerebellar ataxia (SCA) patient-derived Purkinje cells by combining the iPSC technology and the self-organizing stem cell 3D culture technology. We demonstrated that SCA6-derived Purkinje cells exhibit vulnerability to triiodothyronine depletion, which is suppressed by treatment with thyrotropin-releasing hormone and Riluzole. We further discuss applications of patient-specific iPSCs to intractable cerebellar disease.

Self-formation of functional adenohypophysis in three-dimensional culture.

The adenohypophysis (anterior pituitary) is a major centre for systemic hormones. At present, no efficient stem-cell culture for its generation is available, partly because of insufficient knowledge about how the pituitary primordium (Rathke's pouch) is induced in the embryonic head ectoderm. Here we report efficient self-formation of three-dimensional adenohypophysis tissues in an aggregate culture of mouse embryonic stem (ES) cells. ES cells were stimulated to differentiate into non-neural head ectoderm and hypothalamic neuroectoderm in adjacent layers within the aggregate, and treated with hedgehog signalling. Self-organization of Rathke's-pouch-like three-dimensional structures occurred at the interface of these two epithelia, as seen in vivo, and various endocrine cells including corticotrophs and somatotrophs were subsequently produced. The corticotrophs efficiently secreted adrenocorticotropic hormone in response to corticotrophin releasing hormone and, when grafted in vivo, these cells rescued the systemic glucocorticoid level in hypopituitary mice. Thus, functional anterior pituitary tissue self-forms in ES cell culture, recapitulating local tissue interactions.

Deciphering potential vascularization factors of on-chip co-cultured hiPSC-derived cerebral organoids.

The lack of functional vascular system in stem cell-derived cerebral organoids (COs) limits their utility in modeling developmental processes and disease pathologies. Unlike other organs, brain vascularization is poorly understood, which makes it particularly difficult to mimic in vitro. Although several attempts have been made to vascularize COs, complete vascularization leading to functional capillary network development has only been achieved via transplantation into a mouse brain. Understanding the cues governing neurovascular communication is therefore imperative for establishing an efficient in vitro system for vascularized cerebral organoids that can emulate human brain development. Here, we used a multidisciplinary approach combining microfluidics, organoids, and transcriptomics to identify molecular changes in angiogenic programs that impede the successful in vitro vascularization of human induced pluripotent stem cell (iPSC)-derived COs. First, we established a microfluidic cerebral organoid (CO)-vascular bed (VB) co-culture system and conducted transcriptome analysis on the outermost cell layer of COs cultured on the preformed VB. Results revealed coordinated regulation of multiple pro-angiogenic factors and their downstream targets. The VEGF-HIF1A-AKT network was identified as a central pathway involved in the angiogenic response of cerebral organoids to the preformed VB. Among the 324 regulated genes associated with angiogenesis, six transcripts represented significantly regulated growth factors with the capacity to influence angiogenic activity during co-culture. Subsequent on-chip experiments demonstrated the angiogenic and vasculogenic potential of cysteine-rich angiogenic inducer 61 (CYR61) and hepatoma-derived growth factor (HDGF) as potential enhancers of organoid vascularization. Our study provides the first global analysis of cerebral organoid response to three-dimensional microvasculature for in vitro vascularization.

Phototoxicity avoidance is a potential therapeutic approach for retinal dystrophy caused by EYS dysfunction.

Inherited retinal dystrophies (IRDs) are progressive diseases leading to vision loss. Mutation in the eyes shut homolog (EYS) gene is one of the most frequent causes of IRD. However, the mechanism of photoreceptor cell degeneration by mutant EYS has not been fully elucidated. Here, we generated retinal organoids from induced pluripotent stem cells (iPSCs) derived from patients with EYS-associated retinal dystrophy (EYS-RD). In photoreceptor cells of RD organoids, both EYS and G protein-coupled receptor kinase 7 (GRK7), one of the proteins handling phototoxicity, were not in the outer segment, where they are physiologically present. Furthermore, photoreceptor cells in RD organoids were vulnerable to light stimuli, and especially to blue light. Mislocalization of GRK7, which was also observed in eys-knockout zebrafish, was reversed by delivering control EYS into photoreceptor cells of RD organoids. These findings suggest that avoiding phototoxicity would be a potential therapeutic approach for EYS-RD.

[iPS cell technologies toward overcoming neurological diseases].

Neurological diseases are often life threatening, with severely affecting an individual's quality of life. However, the disease mechanisms are still less understood, mainly because of lacking good disease models. Over the past decades, researchers developed many models using cell lines or animals, but most of them did not faithfully recapitulate the disease phenotypes. In particular, it is almost impossible to create animal models for multifactorial diseases or sporadic cases of unknown etiology. In these circumstances, it has come to be expected that induced pluripotent stem cells (iPSCs) can revolutionize neurological disease research as they retain patient's genetic information and provide an expandable source of disease-relevant neurons and glial cells. iPSC technologies are now widely used for disease modeling, and further for drug discovery and regenerative medicine. They are also enabling previously infeasible studies such as those uncovering how disease-associated single nucleotide polymorphism (SNP) and genetic variants increase the disease risk. This review describes a variety of iPSC technologies to produce various types of neurons and brain-like tissues (brain organoids) and summarize recent trends in iPSC technology-based neurological disease research. We also discuss the remaining challenges for understanding and overcoming brain disorders.

Survival and process outgrowth of human iPSC-derived cells expressing Purkinje cell markers in a mouse model for spinocerebellar degenerative disease.

Purkinje cells are the sole output neurons of the cerebellar cortex and play central roles in the integration of cerebellum-related motor coordination and memory. The loss or dysfunction of Purkinje cells due to cerebellar atrophy leads to severe ataxia. Here we used in vivo transplantation to examine the function of human iPS cell-derived cerebellar progenitors in adult transgenic mice in which Purkinje-specific cell death occurs due to cytotoxicity of polyglutamines. Transplantation using cerebellar organoids (42-48 days in culture), which are rich in neural progenitors, showed a viability of >50% 4 weeks after transplantation. STEM121+ grafted cells extended their processes toward the deep cerebellar nuclei, superior cerebellar peduncle, and vestibulocerebellar nuclei. The transplanted cells were mostly located in the white matter, and they were not found in the Purkinje cell layer. MAP2-positive fibers seen in the molecular layer of cerebellar cortex received VGluT2 inputs from climbing fibers. Transplanted neural progenitors overgrew in the host cerebellum but were suppressed by pretreatment with the γ-secretase inhibitor DAPT. Hyperproliferation was also suppressed by transplantation with more differentiated organoids (86 days in culture) or KIRREL2-positive cells purified by FACS sorting. Transplanted cells expressed Purkinje cell markers, GABA, CALB1 and L7, though they did not show fan-shaped morphology. We attempted to improve neuronal integration of stem cell-derived cerebellar progenitors by transplantation into the adult mouse, but this was not successfully achieved. Our findings in the present study contribute to regenerative medical application for cerebellar degeneration and provide new insights into cerebellar development in future.

Subregional specification of embryonic stem cell-derived ventral telencephalic tissues by timed and combinatory treatment with extrinsic signals.

During early telencephalic development, the major portion of the ventral telencephalic (subpallial) region becomes subdivided into three regions, the lateral (LGE), medial (MGE), and caudal (CGE) ganglionic eminences. In this study, we systematically recapitulated subpallial patterning in mouse embryonic stem cell (ESC) cultures and investigated temporal and combinatory actions of patterning signals. In serum-free floating culture, the dorsal-ventral specification of ESC-derived telencephalic neuroectoderm is dose-dependently directed by Sonic hedgehog (Shh) signaling. Early Shh treatment, even before the expression onset of Foxg1 (also Bf1; earliest marker of the telencephalic lineage), is critical for efficiently generating LGE progenitors, and continuous Shh signaling until day 9 is necessary to commit these cells to the LGE lineage. When induced under these conditions and purified by fluorescence-activated cell sorter, telencephalic cells efficiently differentiated into Nolz1(+)/Ctip2(+) LGE neuronal precursors and subsequently, both in culture and after in vivo grafting, into DARPP32(+) medium-sized spiny neurons. Purified telencephalic progenitors treated with high doses of the Hedgehog (Hh) agonist SAG (Smoothened agonist) differentiated into MGE- and CGE-like tissues. Interestingly, in addition to strong Hh signaling, the efficient specification of MGE cells requires Fgf8 signaling but is inhibited by treatment with Fgf15/19. In contrast, CGE differentiation is promoted by Fgf15/19 but suppressed by Fgf8, suggesting that specific Fgf signals play different, critical roles in the positional specification of ESC-derived ventral subpallial tissues. We discuss a model of the antagonistic Fgf8 and Fgf15/19 signaling in rostral-caudal subpallial patterning and compare it with the roles of these molecules in cortical patterning.

In vitro recapitulation of neural development using embryonic stem cells: from neurogenesis to histogenesis.

Embryonic stem (ES) cells have been successfully used over the past decade to generate specific types of neuronal cells. In addition to its value for regenerative medicine, ES cell culture also provides versatile experimental systems for analyzing early neural development. These systems are complimentary to conventional animal models, particularly because they allow unique constructive (synthetic) approaches, for example, step-wise addition of components. Here we review the ability of ES cells to generate not only specific neuronal populations but also functional neural tissues by recapitulating microenvironments in early mammalian development. In particular, we focus on cerebellar neurogenesis from mouse ES cells, and explain the basic ideas for positional information and self-formation of polarized neuroepithelium. Basic research on developmental signals has fundamentally contributed to substantial progress in stem cell technology. We also discuss how in vitro model systems using ES cells can shed new light on the mechanistic understanding of organogenesis, taking an example of recent progress in self-organizing histogenesis.

Differentiation of human induced pluripotent stem cells into hypothalamic vasopressin neurons with minimal exogenous signals and partial conversion to the naive state.

Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disease of vasopressin (AVP) neurons. Studies in mouse in vivo models indicate that accumulation of mutant AVP prehormone is associated with FNDI pathology. However, studying human FNDI pathology in vivo is technically challenging. Therefore, an in vitro human model needs to be developed. When exogenous signals are minimized in the early phase of differentiation in vitro, mouse embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) differentiate into AVP neurons, whereas human ESCs/iPSCs die. Human ESCs/iPSCs are generally more similar to mouse epiblast stem cells (mEpiSCs) compared to mouse ESCs. In this study, we converted human FNDI-specific iPSCs by the naive conversion kit. Although the conversion was partial, we found improved cell survival under minimal exogenous signals and differentiation into rostral hypothalamic organoids. Overall, this method provides a simple and straightforward differentiation direction, which may improve the efficiency of hypothalamic differentiation.

Purkinje cells originate from cerebellar ventricular zone progenitors positive for Neph3 and E-cadherin.

GABAergic Purkinje cells (PCs) provide the primary output from the cerebellar cortex, which controls movement and posture. Although the mechanisms of PC differentiation have been well studied, the precise origin and initial specification mechanism of PCs remain to be clarified. Here, we identified a cerebellar and spinal cord GABAergic progenitor-selective cell surface marker, Neph3, which is a direct downstream target gene of Ptf1a, an essential regulator of GABAergic neuron development. Using FACS, Neph3(+) GABAergic progenitors were sorted from the embryonic cerebellum, and the cell fate of this population was mapped by culturing in vitro. We found that most of the Neph3(+) populations sorted from the mouse E12.5 cerebellum were fated to differentiate into PCs while the remaining small fraction of Neph3(+) cells were progenitors for Pax2(+) interneurons, which are likely to be deep cerebellar nuclei GABAergic neurons. These results were confirmed by short-term in vivo lineage-tracing experiments using transgenic mice expressing Neph3 promoter-driven GFP. In addition, we identified E-cadherin as a marker selectively expressed by a dorsally localized subset of cerebellar Neph3(+) cells. Sorting experiments revealed that the Neph3(+) E-cadherin(high) population in the embryonic cerebellum defined PC progenitors while progenitors for Pax2(+) interneurons were enriched in the Neph3(+) E-cadherin(low) population. Taken together, our results identify two spatially demarcated subregions that generate distinct cerebellar GABAergic subtypes and reveal the origin of PCs in the ventricular zone of the cerebellar primordium.

Minimization of exogenous signals in ES cell culture induces rostral hypothalamic differentiation.

Embryonic stem (ES) cells differentiate into neuroectodermal progenitors when cultured as floating aggregates in serum-free conditions. Here, we show that strict removal of exogenous patterning factors during early differentiation steps induces efficient generation of rostral hypothalamic-like progenitors (Rax(+)/Six3(+)/Vax1(+)) in mouse ES cell-derived neuroectodermal cells. The use of growth factor-free chemically defined medium is critical and even the presence of exogenous insulin, which is commonly used in cell culture, strongly inhibits the differentiation via the Akt-dependent pathway. The ES cell-derived Rax(+) progenitors generate Otp(+)/Brn2(+) neuronal precursors (characteristic of rostral-dorsal hypothalamic neurons) and subsequently magnocellular vasopressinergic neurons that efficiently release the hormone upon stimulation. Differentiation markers of rostral-ventral hypothalamic precursors and neurons are induced from ES cell-derived Rax(+) progenitors by treatment with Shh. Thus, in the absence of exogenous growth factors in medium, the ES cell-derived neuroectodermal cells spontaneously differentiate into rostral (particularly rostral-dorsal) hypothalamic-like progenitors, which generate characteristic hypothalamic neuroendocrine neurons in a stepwise fashion, as observed in vivo. These findings indicate that, instead of the addition of inductive signals, minimization of exogenous patterning signaling plays a key role in rostral hypothalamic specification of neural progenitors derived from pluripotent cells.

Restoration of the defect in radial glial fiber migration and cortical plate organization in a brain organoid model of Fukuyama muscular dystrophy.

Fukuyama congenital muscular dystrophy (FCMD) is a severe, intractable genetic disease that affects the skeletal muscle, eyes, and brain and is attributed to a defect in alpha dystroglycan (αDG) O-mannosyl glycosylation. We previously established disease models of FCMD; however, they did not fully recapitulate the phenotypes observed in human patients. In this study, we generated induced pluripotent stem cells (iPSCs) from a human FCMD patient and differentiated these cells into three-dimensional brain organoids and skeletal muscle. The brain organoids successfully mimicked patient phenotypes not reliably reproduced by existing models, including decreased αDG glycosylation and abnormal radial glial (RG) fiber migration. The basic polycyclic compound Mannan-007 (Mn007) restored αDG glycosylation in the brain and muscle models tested and partially rescued the abnormal RG fiber migration observed in cortical organoids. Therefore, our study underscores the importance of αDG O-mannosyl glycans for normal RG fiber architecture and proper neuronal migration in corticogenesis.

A ROCK inhibitor permits survival of dissociated human embryonic stem cells.

Poor survival of human embryonic stem (hES) cells after cell dissociation is an obstacle to research, hindering manipulations such as subcloning. Here we show that application of a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, to hES cells markedly diminishes dissociation-induced apoptosis, increases cloning efficiency (from approximately 1% to approximately 27%) and facilitates subcloning after gene transfer. Furthermore, dissociated hES cells treated with Y-27632 are protected from apoptosis even in serum-free suspension (SFEB) culture and form floating aggregates. We demonstrate that the protective ability of Y-27632 enables SFEB-cultured hES cells to survive and differentiate into Bf1(+) cortical and basal telencephalic progenitors, as do SFEB-cultured mouse ES cells.

Generation of a human induced pluripotent stem cell line, BRCi005-A, derived from a Best disease patient with BEST1 mutations.

Best Disease is an inherited retinal dystrophy that results in progressive and irreversible central vision loss caused by mutations of BESTROPHIN1 (BEST1). We established human induced pluripotent stem cells (iPSCs) from a Best disease patient with mutations R218H and A357V in the BEST1 gene. The generated iPSCs showed pluripotency markers and three-germ layer differentiation ability in vitro. A genetic analysis revealed mutations of R218H and A357V in the iPSCs. This iPSC line will be useful for elucidating the pathomechanisms of and drug discovery for Best disease.

Generation of a human induced pluripotent stem cell line, BRCi004-A, derived from a patient with age-related macular degeneration.

Age-related macular degeneration (AMD) is a late-onset progressive blinding disease. We established human induced pluripotent stem cells (iPSCs) from an AMD patient. The generated iPSC line showed pluripotency markers and three-germ layer differentiation ability in vitro. This iPSC line will be useful for elucidating the pathomechanisms of and drug discovery for AMD.

Congenital pituitary hypoplasia model demonstrates hypothalamic OTX2 regulation of pituitary progenitor cells.

Pituitary develops from oral ectoderm in contact with adjacent ventral hypothalamus. Impairment in this process results in congenital pituitary hypoplasia (CPH); however, there have been no human disease models for CPH thus far, prohibiting the elucidation of the underlying mechanisms. In this study, we established a disease model of CPH using patient-derived induced pluripotent stem cells (iPSCs) and 3D organoid technique, in which oral ectoderm and hypothalamus develop simultaneously. Interestingly, patient iPSCs with a heterozygous mutation in the orthodenticle homeobox 2 (OTX2) gene showed increased apoptosis in the pituitary progenitor cells, and the differentiation into pituitary hormone-producing cells was severely impaired. As an underlying mechanism, OTX2 in hypothalamus, not in oral ectoderm, was essential for progenitor cell maintenance by regulating LHX3 expression in oral ectoderm via FGF10 expression in the hypothalamus. Convincingly, the phenotype was reversed by the correction of the mutation, and the haploinsufficiency of OTX2 in control iPSCs revealed a similar phenotype, demonstrating that this mutation was responsible. Thus, we established an iPSC-based congenital pituitary disease model, which recapitulated interaction between hypothalamus and oral ectoderm and demonstrated the essential role of hypothalamic OTX2.