Masahiro Otsu

Proteomic identification of differentially expressed genes in mouse neural stem cells and neurons differentiated from embryonic stem cells in vitro

Embryonic stem (ES) cells are pluripotent stem cells and give rise to a variety of differentiated cell types including neurons. To study a molecular basis for differentiation from ES cells to neural cells, we searched for proteins involved in mouse neurogenesis from ES cells to neural stem (NS) cells and neurons by two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting, using highly homogeneous cells differentiated from ES cells in vitro. We newly identified seven proteins with increased expression and one protein with decreased expression from ES cells to NS cells, and eight proteins with decreased expression from NS cells to neurons. Western blot analysis confirmed that a tumor-specific transplantation antigen, HS90B, decreased, and an extracellular matrix and membrane glycoprotein (such as laminin)-binding protein, galectin 1 (LEG1), increased in NS cells, and LEG1 and a cell adhesion receptor, laminin receptor (RSSA), decreased in neurons. The results of RT-PCR showed that mRNA of LEG1 was also up-regulated in NS cells and down-regulated in neurons, implying an important role of LEG1 in regulating the differentiation. The differentially expressed proteins identified here provide insight into the molecular basis of neurogenesis from ES cells to NS cells and neurons.

Proliferation and differentiation of neural stem cells irradiated with X-rays in logarithmic growth phase.

Exposure of the fetal brain to ionizing radiation causes congenital brain abnormalities. Normal brain formation requires regionally and temporally appropriate proliferation and differentiation of neural stem cells (NSCs) into neurons and glia. Here, we investigated the effects of X-irradiation on proliferating homogenous NSCs prepared from mouse ES cells. Cells irradiated with X-rays at a dose of 1Gy maintained the capabilities for proliferation and differentiation but stopped proliferation temporarily. In contrast, the cells ceased proliferation following irradiation at a dose of >5Gy. These results suggest that irradiation of the fetal brain at relatively low doses may cause congenital brain abnormalities as with relatively high doses.

Proteomic identification of differentially expressed genes in neural stem cells and neurons differentiated from embryonic stem cells of cynomolgus monkey (Macaca fascicularis) in vitro.

Understanding neurogenesis is valuable for the treatment of nervous system disorders. However, there is currently limited information about the molecular events associated with the transition from primate ES cells to neural cells. We therefore sought to identify the proteins involved in neurogenesis, from Macaca fascicularis ES cells (CMK6 cell line) to neural stem (NS) cells to neurons using two-dimensional gel electrophoresis (2-DE), peptide mass fingerprinting (PMF), and liquid chromatography-tandem mass spectrometry (LC-MS-MS). During the differentiation of highly homogeneous ES cells to NS cells, we identified 17 proteins with increased expression, including fatty acid binding protein 7 (FABP7), collapsin response mediator protein 2 (CRMP2), and cellular retinoic acid binding protein 1 (CRABP1), and seven proteins with decreased expression. In the differentiation of NS cells to neurons, we identified three proteins with increased expression, including CRMP2, and 10 proteins with decreased expression. Of these proteins, FABP7 is a marker of NS cells, CRMP2 is involved in axon guidance, and CRABP1 is thought to regulate retinoic acid access to its nuclear receptors. Western blot analysis confirmed the upregulation of FABP7 and CRABP1 in NS cells, and the upregulation of CRMP2 in NS cells and neurons. RT-PCR results showed that CRMP2 and FABP7 mRNAs were also upregulated in NS cells, while CRABP1 mRNA was unchanged. These results provide insight into the molecular basis of monkey neural differentiation.

Uni-directional differentiation of mouse embryonic stem cells into neurons by the neural stem sphere method.

We previously showed that our neural stem sphere (NSS) method promotes the neuronal differentiation of mouse, monkey and human embryonic stem (ES) cells. Here we analyzed changes in expression of marker genes and proteins during neuronal differentiation. When cultured in astrocyte-conditioned medium (ACM) under free-floating conditions, colonies of ES cells formed floating cell spheres, which, within 4 days, gave rise to NSSs. In the spheres, the expression of ES cell marker genes was consistently down-regulated, while expression of an epiblast marker was transiently up-regulated, beginning on day 2, and the expression of neuroectoderm, neural stem cell and neuron markers was up-regulated, beginning on days 3, 4 and 6, respectively. The expression of the marker genes was consistent with that of marker proteins. The time course of expression of these markers in the spheres resembled that of neuronal differentiation from the inner cell mass (ICM) cells of blastula. In contrast, the expression of endoderm, mesoderm, epidermis, astrocyte and oligodendrocyte markers was low and not up-regulated during differentiation. Only a small number of apoptotic cells were present in the spheres. These results suggest that mouse ES cells uni-directionally differentiate into neurons via epiblast cells, neuroectodermal cells and neural stem cells.

Pluripotent stem cell-derived neural stem cells: From basic research to applications.

Basic research on pluripotent stem cells is designed to enhance understanding of embryogenesis, whereas applied research is designed to develop novel therapies and prevent diseases. Attainment of these goals has been enhanced by the establishment of embryonic stem cell lines, the technological development of genomic reprogramming to generate induced-pluripotent stem cells, and improvements in vitro techniques to manipulate stem cells. This review summarizes the techniques required to generate neural cells from pluripotent stem cells. In particular, this review describes current research applications of a simple neural differentiation method, the neural stem sphere method, which we developed.

Proteomic identification of differentially expressed genes during differentiation of cynomolgus monkey (Macaca fascicularis) embryonic stem cells to astrocyte progenitor cells in vitro

Understanding astrocytogenesis is valuable for the treatment of nervous system disorders, as astrocytes provide structural, metabolic and defense support to neurons, and regulate neurons actively. However, there is limited information about the molecular events associated with the differentiation from primate ES cells to astrocytes. We therefore investigated the differentially expressed proteins in early astrocytogenesis, from cynomolgus monkey ES cells (CMK6 cell line) into astrocyte progenitor (AstP) cells via the formation of primitive neural stem spheres (Day 4), mature neural stem spheres (NSS), and neural stem (NS) cells in vitro, using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). We identified 66 differentially expressed proteins involved in these five differentiation stages. Together with the results of Western blotting, RT-PCR, and a search of metabolic pathways related to the identified proteins, these results indicated that collapsin response mediator protein 2 (CRMP2), its phosphorylated forms, and cellular retinoic acid binding protein 1 (CRABP1) were upregulated from ES cells to Day 4 and NSS cells, to which differentiation stages apoptosis-associated proteins such as caspases were possibly related; Phosphorylated CRMP2s were further upregulated but CRABP1 was downregulated from NSS cells to NS cells, during which differentiation stage considerable axon guidance proteins for development of growth cones, axon attraction, and repulsion were possibly readied; Nonphosphorylated CRMP2 was downregulated but CRABP1 was re-upregulated from NS cells to AstP cells, in which differentiation stage reorganization of actin cytoskeleton linked to focal adhesion was possibly accompanied. These results provide insight into the molecular basis of early astrocytogenesis in monkey.

Uni-directional Neuronal Differentiation of Embryonic Stem Cells by the Neural Stem Sphere Method

Embryonic stem (ES) cells have two characteristics, pluripotency and self-renewal. ES cells can differentiate into all other cell types, including germ cells, indicating that they may be a limitless source of functional cells for stem cell applications. We have formulated a method, the neural stem sphere (NSS) method, to efficiently obtain functional cells from these sources. The NSS method is a simple method of quickly and efficiently generating numerous neural stem cells and neurons from mouse, monkey and human ES cells. Analysis of marker gene expression during the neurogenesis of mouse ES cells induced by the NSS method demonstrated that ES cells uni-directionally differentiate into neurons via epiblasts, neuroectodermal cells and neural stem cells. This process of neuronal differentiation resembles, in part, that of neurogenesis in early embryos, suggesting that the NSS method may provide a potentially powerful tool for elucidating the mechanism underlying the efficient neural differentiation of ES cells and for assessing drugs, chemical compounds and physical stimuli that may cause neurodevelopmental impairments.

Neural Stem Cells Differentiated from Embryonic Stem Cells: Proteomic Identification of Expressed Genes

Embryonic stem (ES) cells can self-renew in culture and are pluripotent, giving rise to a variety of differentiated cell types. More complete utilization of this potential, however, requires more efficient differentiation of ES cells into specific lineages, including neural stem (NS) cells, which can generate functioning cells suitable for the functional recovery of damaged tissues, including neurons. The development of methods to effectively differentiate ES cells into highly homogeneous NS cells via NS spheres, and of proteomic methods of protein identification may allow the elucidation of differentially expressed genes in NS cells, thus providing insights into the molecular events associated with the transition from ES cells to NS cells. This in turn will help facilitate clinical applications of these ES cell-derived NS cells to treat neurological diseases.

Effects of mitogens on proliferation of mouse embryonic stem cell-derived neural stem cells

proliferating NSCs were irradiated with 1 Gy, the cells stopped proliferation but started to proliferate exponentially with a lag time of about 1 day. The cells expressed nestin and maintained potential of proliferation and differentiation like non-irradiated cells. When the NSCs were irradiated with 5 Gy, the cells stopped proliferation and became larger in size. The cells expressed nestin, however, lost potential of proliferation and differentiation. These results suggest that radiation-induced damage can be repaired in the NSCs irradiated with 1 Gy but not in the cells with 5 Gy and over.

Changes in proliferation and gene expression of mouse ES cell-derived neural stem cells after heat shock

proliferating NSCs were irradiated with 1 Gy, the cells stopped proliferation but started to proliferate exponentially with a lag time of about 1 day. The cells expressed nestin and maintained potential of proliferation and differentiation like non-irradiated cells. When the NSCs were irradiated with 5 Gy, the cells stopped proliferation and became larger in size. The cells expressed nestin, however, lost potential of proliferation and differentiation. These results suggest that radiation-induced damage can be repaired in the NSCs irradiated with 1 Gy but not in the cells with 5 Gy and over.