Yoshimasa Sakamoto

Differences in neurogenic potential in floor plate cells along an anteroposterior location: midbrain dopaminergic neurons originate from mesencephalic floor plate cells

Directed differentiation and purification of mesencephalic dopaminergic (mesDA) neurons from stem cells are crucial issues for realizing safe and efficient cell transplantation therapies for Parkinsons disease. Although recent studies have identified the factors that regulate mesDA neuron development, the mechanisms underlying mesDA neuron specification are not fully understood. Recently, it has been suggested that mesencephalic floor plate (FP) cells acquire neural progenitor characteristics to generate mesDA neurons. Here, we directly examined this in a fate mapping experiment using fluorescence-activated cell sorting (FACS) with an FP cell-specific surface marker, and demonstrate that mesencephalic FP cells have neurogenic activity and generate mesDA neurons in vitro. By contrast, sorted caudal FP cells have no neurogenic potential, as previously thought. Analysis of dreher mutant mice carrying a mutation in the Lmx1a locus and transgenic mice ectopically expressing Otx2 in caudal FP cells demonstrated that Otx2 determines anterior identity that confers neurogenic activity to FP cells and specifies a mesDA fate, at least in part through the induction of Lmx1a. We further show that FACS can isolate mesDA progenitors, a suitable transplantation material, from embryonic stem cell-derived neural cells. Our data provide insights into the mechanisms of specification and generation of mesDA neurons, and illustrate a useful cell replacement approach for Parkinsons disease.

Identification of activity‐regulated proteins in the postsynaptic density fraction

Background: The postsynaptic density (PSD) at synapses is a specialized submembranous structure where neurotransmitter receptors are linked to cytoskeleton and signalling molecules. Activity‐dependent dynamic change in the components of the PSD is a mechanism of synaptic plasticity. Identification of the PSD proteins and examination of their modulations dependent on synaptic activity will be valuable for an understanding of the molecular basis of learning and memory.

Migrating postmitotic neural precursor cells in the ventricular zone extend apical processes and form adherens junctions near the ventricle in the developing spinal cord

Postmitotic neural precursors are generated in the ventricular zone (VZ) of the developing neural tube and immediately migrate to the mantle layer (ML) where they differentiate into mature neurons. Although the regulation of neuronal differentiation and migration has extensively been studied, the behavior of the early postmitotic precursors migrating toward the ML is largely unknown. In this study, we have identified Neph3 as a specific marker for early postmitotic neural precursors in the VZ of the developing spinal cord. Analysis of Neph3 localization by immunofluorescence and immunoelectron microscopy revealed that early neural precursors in the VZ possessed not only pia-connected processes but also ones that reached the ventricle. This apical extension of processes was confirmed by analyzing another early postmitotic marker, Dll1 mRNA, which was actively transported toward the ventricle and accumulated at the termini of the processes. Furthermore, adherens junctions (AJs) were formed around the apical end of processes extending from Neph3- and Dll1 mRNA-positive postmitotic precursors. Taken together, these observations suggest that migrating early postmitotic neural precursors in the VZ of the developing spinal cord form a neuroepithelial cell-like bipolar morphology and communicate with their neighboring cells through AJs.

Purification of functional human ES and iPSC-derived midbrain dopaminergic progenitors using LRTM1

Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (mDA) neurons for cell replacement therapy for Parkinsons disease (PD). However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. To eliminate these unwanted cells, cell sorting using antibodies for specific markers such as CORIN or ALCAM has been developed, but neither marker is specific for ventral midbrain. Here we employ a double selection strategy for cells expressing both CORIN and LMX1A::GFP, and report a cell surface marker to enrich mDA progenitors, LRTM1. When transplanted into 6-OHDA-lesioned rats, human iPSC-derived LRTM1+ cells survive and differentiate into mDA neurons in vivo, resulting in a significant improvement in motor behaviour without tumour formation. In addition, there was marked survival of mDA neurons following transplantation of LRTM1+ cells into the brain of an MPTP-treated monkey. Thus, LRTM1 may provide a tool for efficient and safe cell therapy for PD patients.