Chemical labelling for visualizing native AMPA receptors in live neurons

The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders

Controlling the Regional Identity of hPSC-Derived Neurons to Uncover Neuronal Subtype Specificity of Neurological Disease Phenotypes

The CNS contains many diverse neuronal subtypes, and most neurological diseases target specific subtypes. However, the mechanism of neuronal subtype specificity of disease phenotypes remains elusive. Although in vitro disease models employing human pluripotent stem cells (PSCs) have great potential to clarify the association of neuronal subtypes with disease, it is currently difficult to compare various PSC-derived subtypes. This is due to the limited number of subtypes whose induction is established, and different cultivation protocols for each subtype. Here, we report a culture system to control the regional identity of PSC-derived neurons along the anteroposterior (A-P) and dorsoventral (D-V) axes. This system was successfully used to obtain various neuronal subtypes based on the same protocol. Furthermore, we reproduced subtype-specific phenotypes of amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD) by comparing the obtained subtypes. Therefore, our culture system provides new opportunities for modeling neurological diseases with PSCs

Efficient derivation of multipotent neural stem/progenitor cells from non-human primate embryonic stem cells.

The common marmoset (Callithrix jacchus) is a small New World primate that has been used as a non-human primate model for various biomedical studies. We previously demonstrated that transplantation of neural stem/progenitor cells (NS/PCs) derived from mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) promote functional locomotor recovery of mouse spinal cord injury models. However, for the clinical application of such a therapeutic approach, we need to evaluate the efficacy and safety of pluripotent stem cell-derived NS/PCs not only by xenotransplantation, but also allotransplantation using non-human primate models to assess immunological rejection and tumorigenicity. In the present study, we established a culture method to efficiently derive NS/PCs as neurospheres from common marmoset ESCs. Marmoset ESC-derived neurospheres could be passaged repeatedly and showed sequential generation of neurons and astrocytes, similar to that of mouse ESC-derived NS/PCs, and gave rise to functional neurons as indicated by calcium imaging. Although marmoset ESC-derived NS/PCs could not differentiate into oligodendrocytes under default culture conditions, these cells could abundantly generate oligodendrocytes by incorporating additional signals that recapitulate in vivo neural development. Moreover, principal component analysis of microarray data demonstrated that marmoset ESC-derived NS/PCs acquired similar gene expression profiles to those of fetal brain-derived NS/PCs by repeated passaging. Therefore, marmoset ESC-derived NS/PCs may be useful not only for accurate evaluation by allotransplantation of NS/PCs into non-human primate models, but are also applicable to analysis of iPSCs established from transgenic disease model marmosets

Expression of cell type-specific markers at each differentiation step.

<p>qRT-PCR analysis of the expression of pluripotency markers (A: <i>Oct3/4</i> and <i>Nanog</i>), endodermal and mesodermal markers (B: <i>Afp</i> and <i>Gata4</i>, respectively), and neural markers (C: <i>Sox1</i> and <i>Pax6</i>) in ESCs, EBs and ESC-derived neurospheres. Fetal cell-derived (ganglionic eminence (GE), cortex and spinal cord) neurospheres and fetal tissues (heart and liver) were used as positive and negative controls, respectively. Data are presented as the means ± SEM (<i>n</i> = 3).</p

Efficient derivation of oligodendrocytes from marmoset ESCs.

<p>(<b>A</b>) Protocol for derivation of NS/PCs from marmoset ESCs, which efficiently generate oligodendrocytes. EBs were cultured in the presence of 1×10⁻⁶ M RA and 2 µM purmorphamine for 2 weeks. RA and purmorphamine were added on day 5 and 7 of EB formation, respectively. EBs were then dissociated and cultured in suspension for 8–10 days to form neurospheres in MHM medium containing 2% B27, 20 ng/ml FGF-2, 20 ng/ml EGF, 1 µM purmorphamine and an oligodendrocyte cocktail. Primary neurospheres were dissociated and cultured under the same condition to form secondary neurospheres. (<b>B</b>) Differentiation of neurospheres derived from EBs treated with RA and purmorphamine. Primary and secondary neurospheres were dissociated and differentiated on poly-L-ornithine/laminin-coated coverslips at a density of 2×10⁴ cells/ml in MHM medium containing 2% B27 and an oligodendrocyte cocktail for 30–35 days, followed by immunocytochemical analysis of βIII-tubulin (neurons), GFAP (astrocytes) and O4 (oligodendrocytes). The obtained NS/PCs could efficiently give rise to oligodendrocytes. Scale bars, 100 µm. (<b>C</b>) The proportions of cells positive for each cell type-specific marker in differentiated secondary neurospheres, which could generate oligodendrocytes, are presented as the percentage of total cells counted by Hoechst 33258-stained nuclei. Data are presented as the means ± SEM (<i>n</i> = 3). (<b>D</b>) An enlarged image of βIII-tubulin-positive neurons, GFAP-positive astrocytes and O4-positive oligodendrocytes differentiated from secondary neurospheres. Scale bars, 100 µm. (<b>E</b>) Immunocytochemical analysis of secondary neurospheres for various markers of oligodendrocyte lineages, including PDGFR (OPCs), CNPase (oligodendrocytes) and MBP (mature oligodendrocytes). Scale bars, 50 µm.</p

Differentiation potentials of marmoset ESC-derived NS/PCs.

<p>(<b>A</b>) Marmoset ESC-derived primary and secondary neurospheres were dissociated and allowed to differentiate for 10 days, followed by immunocytochemical analysis of βIII-tubulin (neurons), GFAP (astrocytes), CNPase (oligodendrocytes) and Nestin (undifferentiated neural cells). Scale bars, 50 µm. (<b>B</b>) The proportions of cells positive for each cell type-specific marker are presented as the percentage of total cells counted by Hoechst 33258-stained nuclei. Data are presented as the means ± SEM (<i>n</i> = 3).</p