Symptoms of Parkinson's disease have been improved by transplantation of fetal dopamine neurons recovered from aborted fetal tissue, but tissue recovery is difficult. Human embryonic stem cells may provide unlimited cells for transplantation if they can be converted to dopamine neurons and survive transplantation into brain. We have found that the bone morphogenic protein antagonist Noggin increased the number of dopamine neurons generated in vitro from human and mouse embryonic stem cells differentiated on mouse PA6 stromal cells. Noggin effects were seen with either early (for mouse, days 0 -7, and for human, days 0 -9) or continuous treatment. After transplant into cyclosporinimmunosuppressed rats, human dopamine neurons improved apomorphine circling in direct relation to the number of surviving dopamine neurons, which was fivefold greater after Noggin treatment than with control human embryonic stem cell transplants differentiated only on PA6 cells. We conclude that Noggin promotes dopamine neuron differentiation and survival from human and mouse embryonic stem cells.
Embryonic stem (ES) cells are expected to be a potential donor source for neural transplantation. We have obtained motoneuron-enriched neural progenitor cells by culturing mouse ES cells with retinoic acid (RA). The cells also expressed mRNA of a neurotrophic factor, neurotrophin-3 (NT-3). The left motor cortex area of mice was damaged by cryogenic brain injury, and the neural cells were transplanted underneath the injured motor cortex, neighboring to the paraventricular region. We found that the cells expressing neuronal phenotypes not only remained close to the implantation site, but also exhibited substantial migration penetrating into the damaged lesion, in a seemingly directed manner up to cortical region. We found that some of the neural cells differentiated into Islet1-positive motoneurons. It seems likely that the ability of the ES cellderived neural progenitor cells to respond in vivo to guidance cues and signals that can direct their migration and differentiation may contribute to functional recovery of the recipient mice. We found that an "island of the mature neuronal cells" of recipient origin emerged in the damaged motor cortex. This may be associated with the neuroprotective effects of the ES cell-derived neural cells. The ES cells differentiated into CD31+ vasculoendothelial cells with the RA treatment in vitro. Furthermore, the grafted cells may provide sufficient neurotrophic factors such as NT-3 for neuroprotection and regeneration. The grafted neural cells that migrated into residual cortex and differentiated into neurons had purposefully elongated axons that were stained with anti-neurofilament middle chain (NFM) antibody. Our study suggests that motoneurons can be induced from ES cells, and ES cells become virtually an unlimited source of cells for experimental and clinical neural cell transplantation.
We developed neural tube-like structures accompanying neural crest-like cells by treating embryonic stem (ES) cells with retinoic acid. The structures contained pseudostratified Nestin+Vimentin+ neuroepithelial cells surrounded by Masson staining+ basement membrane. betaIIItubulin+Synaptophysin+ mature neurons and glial fibrillary acidic protein (GFAP)+ glial cells dispersed outside of the membrane. Addition of Noggin to the culture induced prominent proliferation of the neuroepithelial cells, leading to epithelial hyperstratification of the structures. mRNAs of transcription factors essential for forebrain development such as Emx1/2 and Pax6 were specifically expressed and Islet1+Lim1/2- motoneurons appeared by the addition of Noggin. In contrast, basic fibroblast growth factor (bFGF) promoted enlargement of central lumen and elongation of the structures. mRNAs of caudal markers, Gbx2, Cdx2 and Hoxb4/9 were expressed and Lim1/2+ spinal motoneurons appeared by the addition of bFGF. Addition of BMP-4 similarly brought about mild enlargement of central lumen of the structures. Interestingly, the addition of BMP-4 induced Slug+ neural crest-like cells surrounding the tube-like structures. mRNAs of Snail and dHand, other markers for neural crest cells, were also expressed by the addition of BMP-4. These results suggest that Noggin lead the neural-tube like structures to forebrain fate, whereas bFGF was involved in the caudalization. BMP-4 was implicated in emergence of the neural crest-like cells. Differentiation of ES cells by the present methods may mimic neurulation and subsequent neural development of early embryos, and elucidates the opposite effects of Noggin and bFGF for the neural tube development.
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common non-skin cancer worldwide. Despite improvement in therapeutic strategies, the prognosis of advanced HNSCC remains poor. The extacellular lipid mediators known as lysophosphatidic acids (LPAs) have been implicated in tumorigenesis of HNSCC. LPAs activate G-protein-coupled receptors not only in the endothelial differentiation gene (Edg) family (LPA1, LPA2, LPA3) but also in the phylogenetically distant non-Edg family (LPA4, LPA5, LPA6). The distinct roles of these receptor isoforms in HNSCC tumorigenesis have not been clarified. In the present study, we investigated the effect of ectopic expression of LPA4 in SQ-20B, an HNSCC cell line, expressing a trivial level of endogenous LPA4. LPA (18:1) stimulated proliferation of SQ-20B cells, but did not affect proliferation of HEp-2, an SCC cell line expressing higher levels of LPA4, comparable to those of with LPA1. LPA-stimulated proliferation of SQ-20B cells was attenuated by Ki16425 and Rac1 inhibitor, but not by Y-27632. Infection with doxycycline-regulatable adenovirus vector expressing green fluorescent protein-tagged LPA4 (AdvLPA4G) abolished LPA-stimulated proliferation in SQ-20B cells with the accumulation of G2/M-phasic cells. Ectopic LPA4 induction further downregulated proliferation of Ki16425-treated SQ-20B cells, of which downregulation was partially recovered by LPA. Ectopic LPA4 induction also downregulated proliferation of Rac1 inhibitor-treated SQ-20B cells, however, LPA no longer recovered it. Finally, LPA-induced cell motility was suppressed by ectopic LPA4 expression as well as by Ki16425, Rac1 inhibitor or Y-27632. Our data suggest that LPA4 signaling potentially modulates malignant behavior of SQ-20B cells. LPA signaling, which is mediated by both Edg and non-Edg receptors, may be a determinant of malignant behavior of HNSCC and could therefore be a promising therapeutic target.
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