Mami Yamasaki

Transplantation of human neural stem cells for spinal cord injury in primates

Recent studies have shown that delayed transplantation of neural stem/progenitor cells (NSPCs) into the injured spinal cord can promote functional recovery in adult rats. Preclinical studies using nonhuman primates, however, are necessary before NSPCs can be used in clinical trials to treat human patients with spinal cord injury (SCI). Cervical contusion SCIs were induced in 10 adult common marmosets using a stereotaxic device. Nine days after injury, in vitro‐expanded human NSPCs were transplanted into the spinal cord of five randomly selected animals, and the other sham‐operated control animals received culture medium alone. Motor functions were evaluated through measurements of bar grip power and spontaneous motor activity, and temporal changes in the intramedullary signals were monitored by magnetic resonance imaging. Eight weeks after transplantation, all animals were sacrificed. Histologic analysis revealed that the grafted human NSPCs survived and differentiated into neurons, astrocytes, and oligodendrocytes, and that the cavities were smaller than those in sham‐operated control animals. The bar grip power and the spontaneous motor activity of the transplanted animals were significantly higher than those of sham‐operated control animals. These findings show that NSPC transplantation was effective for SCI in primates and suggest that human NSPC transplantation could be a feasible treatment for human SCI.

Human neural stem/progenitor cells, expanded in long‐term neurosphere culture, promote functional recovery after focal ischemia in Mongolian gerbils

Transplantation of human neural stem cells (NSCs) is a promising potential therapy for neurologic dysfunctions after the hyperacute stage of stroke in humans, but large amounts of human NSCs must be expanded in long‐term culture for such therapy. To determine their possible therapeutic potential for human stroke, human fetal neural stem/progenitor cells (NSPCs) (i.e., neurosphere‐forming cells) were isolated originally from forebrain tissues of one human fetus, and expanded in long‐term neurosphere culture (exceeding 24 weeks), then xenografted into the lesioned areas in the brains of Mongolian gerbils 4 days after focal ischemia. Sensorimotor and cognitive functions were evaluated during the 4 weeks after transplantation. The total infarction volume in the NSPC‐grafted animals was significantly lower than that in controls. Approximately 8% of the grafted NSPCs survived, mainly in areas of selective neuronal death, and were costained with antibodies against neuronal nuclei antibody (NeuN), microtubule associated protein (MAP‐2), glial fibrillary acidic protein (GFAP), and anti‐2′3′ cyclic nucleotide 3′‐phosphodiesterase (CNPase). Synaptic structures between NSPCs‐derived neurons and host neurons were observed. Furthermore, gradual improvement of neurologic functions was observed clearly in the NSPC‐grafted animals, compared to that in controls. Human NSPCs, even from long‐term culture, remarkably improved neurologic functions after focal ischemia in the Mongolian gerbil, and maintained their abilities to migrate around the infarction, differentiate into mature neurons, and form synapses with host neuronal circuits. These results indicate that in vitro‐expanded human neurosphere cells are a potential source for transplantable material for treatment of stroke.

Evaluation of in vitro proliferative activity of human fetal neural stem/progenitor cells using indirect measurements of viable cells based on cellular metabolic activity

To scale up human neural stem/progenitor cell (NSPC) cultures for clinical use, we need to know how long these cells can live ex vivo without losing their ability to proliferate and differentiate; thus, a convenient method is needed to estimate the proliferative activity of human NSPCs grown in neurosphere cultures, as direct cell counting is laborious and potentially inaccurate. Here, we isolated NSPCs from human fetal forebrain and prepared neurosphere cultures. We determined the number of viable cells and estimated their proliferative activity in long‐term culture using two methods that measure viable cell numbers indirectly, based on their metabolic activity: the WST‐8 assay, in which a formazan dye is produced upon reduction of the water‐soluble tetrazolium salt WST‐8 by dehydrogenase activity, and the ATP assay, which measures the ATP content of the total cell plasma. We compared the results of these assays with the proliferative activity estimated by DNA synthesis using the 5‐bromo‐2′‐deoxyuridine incorporation assay. We found the numbers of viable human NSPCs to be directly proportional to the metabolic reaction products obtained in the WST‐8 and ATP assays. Both methods yielded identical cell growth curves, showing an exponentially proliferative phase and a change in the population doubling time in long‐term culture. They also showed that human NSPCs could be expanded for up to 200 days ex vivo without losing their ability to proliferate and differentiate. Our findings indicated that indirect measurements of viable cells based on metabolic activity, especially the ATP assay, are very effective and reproducible ways to determine the numbers of viable human NSPCs in intact neurospheres.

Effect of neurosphere size on the growth rate of human neural stem/progenitor cells

Neural stem/progenitor cells (NSPCs) proliferate as aggregates in vitro, but the mechanism of aggregation is not fully understood. Here, we report that aggregation promotes the proliferation of NSPCs. We found that the proliferation rate was linear and depended on the size of the aggregate; that is, the population doubling time of the NSPCs gradually decreased as the diameter approached 250 μm and flattened to a nearly constant value beyond this diameter. Given this finding, and with the intent of enhancing the efficiency of human NSPC expansion, we induced the NSPCs to form aggregates close to 250 μm in diameter quickly by culturing them in plates with U‐bottomed wells. The NSPCs formed aggregates effectively in the U‐bottomed wells, with cell numbers approximately 1.5 times greater than those in the aggregates that formed spontaneously in flat‐bottomed wells. In addition, this effect of aggregation involved cell–cell signaling molecules of the Notch1 pathway. In the U‐bottomed wells, Hes1 and Hes5, which are target genes of the Notch signal, were expressed at higher levels than in the control, flat‐bottomed wells. The amount of cleaved Notch1 was also higher in the cells cultured in the U‐bottomed wells. The addition of γ‐secretase inhibitor, which blocks Notch signaling, suppressed cell proliferation in the U‐bottomed wells. These results suggest that the three‐dimensional architecture of NSPC aggregates would create a microenvironment that promotes the proliferation of human NSPCs.

Phenotypic Spectrum of COL4A1 Mutations: Porencephaly to Schizencephaly

Recently, COL4A1 mutations have been reported in porencephaly and other cerebral vascular diseases, often associated with ocular, renal, and muscular features. In this study, we aimed to clarify the phenotypic spectrum and incidence of COL4A1 mutations.

Functional expression of ABCG2 transporter in human neural stem/progenitor cells

We have studied the expression, localization, and function of the ABCG2 transporter, a universal stem cell marker, at the protein level in human cultured neural stem/progenitor cells (hNSPCs) using immunoblotting, immunofluorescence, and ATPase assays. Human NSPCs were isolated from human fetal brain and propagated in vitro as neurospheres. Both the cells in neurospheres and single cells dissociated from neurospheres showed high levels of ABCG2, and about 63% of the cells in neurospheres were ABCG2-positive, similar to the proportion of nestin-positive cells, and in most cases the ABCG2 and nestin staining co-localized in the same cells. Both the three-dimensional structure of single hNSPCs stained with anti-ABCG2 antibodies and an examination using a biochemical marker for the plasma membrane indicated that ABCG2 was localized to the plasma membrane of hNSPCs. The ABCG2 expressed in hNSPCs had prazosin-sensitive ATP hydrolysis activity, and the ABCG2 level was sharply down-regulated during hNSPC differentiation. All these results suggested that ABCG2, was functionally expressed in hNSPCs. ABCG2 might play a significant role in maintaining human neural stem cells in an undifferentiated state and in protecting hNSPCs from xenobiotics or other toxic substances in vivo.

Isolation and expression analysis of a novel human homologue of the Drosophila glial cells missing (gcm) gene

A novel human homologue (GCMB) of the Drosophila glial cells missing gene (dGCM) was isolated using RACE. GCMB contained a gcm motif sequence and a nuclear targeting sequence similar to that of dGCM and mouse GCMb. Homology searches indicated that GCMB was located within chromosome 6p24.2. Transcripts of GCMB were detected by means of RT‐PCR in fetal brain, normal adult kidney, 3/3 medulloblastomas, 1/3 gliomas and 4/8 non‐neuroepithelial tumor cell lines. Our data suggest that humans have two homologues of gcm like mice and that human gcm genes form a novel family which may function not only during fetal development but also in the postnatal or pathological stage.

Hydrocephalus and Hirschsprung’s disease with a mutation of L1CAM

AbstractAbnormalities of the L1CAM gene, a member of the immunoglobulin gene superfamily of neural-cell adhesion molecules, are associated with X-linked hydrocephalus and some allelic disorders. Hirschsprungs disease (HSCR) is characterized by the absence of ganglion cells and the presence of hypertrophic nerve trunks in the distal bowel. There have been three reports of patients with X-linked hydrocephalus and HSCR with a mutation in the L1CAM gene. We report three more patients with similar conditions. We suspect that decreased L1CAM may be a modifying factor in the development of HSCR.

Isolation and cellular properties of mesenchymal cells derived from the decidua of human term placenta

The clinical promise of cell-based therapies is generally recognized, and has driven an intense search for good cell sources. In this study, we isolated plastic-adherent cells from human term decidua vera, called decidua-derived-mesenchymal cells (DMCs), and compared their properties with those of bone marrow-derived-mesenchymal stem cells (BM-MSCs). The DMCs strongly expressed the mesenchymal cell marker vimentin, but not cytokeratin 19 or HLA-G, and had a high proliferative potential. That is, they exhibited a typical fibroblast-like morphology for over 30 population doublings. Cells phenotypically identical to the DMCs were identified in the decidua vera, and genotyping confirmed that the DMCs were derived from the maternal components of the fetal adnexa. Flow cytometry analysis showed that the expression pattern of CD antigens on the DMCs was almost identical to that on BM-MSCs, but some DMCs expressed the CD45 antigen, and over 50% of them also expressed anti-fibroblast antigen. In vitro, the DMCs showed good differentiation into chondrocytes and moderate differentiation into adipocytes, but scant evidence of osteogenesis, compared with the BM-MSCs. Gene expression analysis showed that, compared with BM-MSCs, the DMCs expressed higher levels of TWIST2 and RUNX2 (which are associated with early mesenchymal development and/or proliferative capacity), several matrix metalloproteinases (MMP1, 3, 10, and 12), and cytokines (BMP2 and TGFB2), and lower levels of MSX2, interleukin 26, and HGF. Although DMCs did not show the full multipotency of BM-MSCs, their higher proliferative ability indicates that their cultivation would require less maintenance. Furthermore, the use of DMCs avoids the ethical concerns associated with the use of embryonic tissues, because they are derived from the maternal portion of the placenta, which is otherwise discarded. Thus, the unique properties of DMCs give them several advantages for clinical use, making them an interesting and attractive alternative to MSCs for regenerative medicine.

Characterization of ABC transporter ABCB1 expressed in human neural stem/progenitor cells

We investigated the localization and functional expression of the ABC transporter ABCB1 in human fetal neural stem/progenitor cells (hNSPCs). RT‐PCR analysis revealed ABCB1 gene expression in hNSPCs. We found a single band in immunoblotted hNSPCs lysates probed with ABCB1 antibody, and detected ABCB1 at the hNSPCs cell membrane by immunocytochemistry and subcellular fractionation. ABCB1 inhibitors and substrate, and ATP‐depleting agents enhanced hNSPCs’ rhodamine 123 accumulation, and hNSPCs microsomes had vanadate‐sensitive ATPase activity. ABCB1 and nestin expression decreased during hNSPCs differentiation, while the astroglial marker GFAP increased. ABCB1 may maintain hNSPCs in an undifferentiated state and could be a neural stem/progenitor marker.