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Abstract: Here, we report in vitro generation of Math1+ cerebellar granule cell precursors and Purkinje cells from ES cells by using soluble patterning signals. When neural progenitors induced from ES cells in a serum-free suspension culture are subsequently treated with BMP4 and Wnt3a, a significant proportion of these neural cells become Math1+. The induced Math1+ cells are mitotically active and express markers characteristic of granule cell precursors (Pax6, Zic1, and Zipro1). After purification by FACS and coculture with postnatal cerebellar neurons, ES cell-derived Math1+ cells exhibit typical features of neurons of the external granule cell layer, including extensive motility and a T-shaped morphology. Interestingly, differentiation of L7+/Calbindin-D28K+ neurons (characteristic of Purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by Fgf8 rather than by Wnt3a. This is the first report of in vitro recapitulation of early differentiation of cerebellar neurons by using the ES cell system. [Copyright &y& Elsevier]

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 (O 2 ) as in ambient air (20% O 2 ), 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. [ABSTRACT FROM AUTHOR]

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. • Developmental mechanisms of the cerebellum have been revealed in model animals. • 3D self-organizing culture of PSCs recapitulates cerebellar tissue formation. • PSC-derived cerebellum can be used for mechanistic study of human cerebellum. • Spinocerebellar ataxia (SCA) is a disease with neurodegeneration of the cerebellum. • SCA patient iPSC-derived cerebellum enables pathological study and drug screening. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • iPS cell-derived cerebellar cells are grafted into Purkinje cell-degenerative mice. • Grafted cells express Purkinje cell markers and extend processes in host cerebellum. • Differentiated organoid, KIRREL2+ cell sorting and DAPT suppress hyperproliferation. [ABSTRACT FROM AUTHOR]

Abstract: 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-cadherinhigh population in the embryonic cerebellum defined PC progenitors while progenitors for Pax2+ interneurons were enriched in the Neph3+ E-cadherinlow 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. [Copyright &y& Elsevier]