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Department of Medical Physiology, University of Groningen, Groningen, The Netherlands4 W* K3 c3 |. y2 i
5 q: }" B, w! D# U7 ^Key Words. Neural stem cells ? Oligodendrocyte progenitor cells ? Embryonic ? Olig ? Mouse; A: H- I+ \% \6 h; B- B
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Correspondence: Sjef Copray, Ph.D., Department of Medical Physiology, University of Groningen,A. Deusinglaan 1, 9713 AV Groningen, The Netherlands. Telephone: 31-50-3632785; Fax: 31-50-3632751; e-mail: j.c.v.m.copray@med.rug.nl8 l0 e: S- A/ z; R, n# F
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ABSTRACT$ f/ L% ]1 k! ]$ U4 K7 C
. _: h# n) p M5 G! nRecently, embryonic, neural, and bone marrow stem cells have been tested as sources for oligodendrocyte progenitor cells (OPCs) to be used as transplants in a novel therapeutic approach for demyelinating diseases involving the replacement of lost or nonfunctional oligodendrocytes, such as multiple sclerosis. To that purpose, stem cells have been injected either stereotactically or intravenously in animals with experimentally induced demyelination lesions . In most of these studies, stem cells were administered without prior differentiation to OPCs, leaving the direction of differentiation to the largely unspecified conditions of the microenvironment at the site of stem cell settlement. Although these studies could demonstrate the presence of a few donor OPCs at the lesion sites as well as their contribution in remyelination, the use of undifferentiated stem cells introduces a considerable risk: undifferentiated stem cells may eventually encounter conditions that enable unrestricted proliferation leading to tumor formation throughout the body. In vitro predifferentiation into OPCs is considered a prerequisite for the safe in vivo application of stem cells.7 ^) k! P: y, s y G
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Strategies to induce the in vitro differentiation of neural stem cells (NSCs) into OPCs so far have involved the supplementation of the differentiation medium with a variety of induction and growth factors, such as Sonic Hedgehog (Shh), fibroblast growth factor-2 (FGF-2), and platelet-derived growth factor (PDGF), frequently in combination with serum. In general, this approach yields a low and highly variable percentage of OPCs .
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In addition to exposure to these extrinsic factors, we aim to induce OPC differentiation of NSCs intrinsically by transfection of genes encoding for transcription factors known to be crucial in the development of oligodendrocyte lineage during embryogenesis . Besides Sox10, the most prominent oligodendrogenic transcription factors are the basic-helix-loop-helix proteins Olig1 and Olig2 , whose essential involvement in oligodendrocyte lineage formation and in the survival and maturation of OPCs has been demonstrated. The specific temporal-spatial expression of Olig2 in the developing spinal cord (pMN domain) induces the formation of a motoneuron-oligodendrocyte lineage . Activation by Olig2 of the Ngn1 and Ngn2 genes leads to spinal motoneuron differentiation, whereas coactivation of Olig2 and Nkx2.2 provokes the formation of OPCs in the spinal cord; Olig1 is thought to promote the survival and maturation of the developing spinal OPCs. In the brain, on the contrary, the temporal-spatial expression of Olig1 is thought to be the key factor for the induction of cortical OPC formation, whereas Olig2 seems to be involved in survival and maturation of the newly formed OPCs .
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In the present study, we aim to induce the in vitro OPC differentiation of brain-derived NSCs by imitat-ing embryonic development and provoking temporal expresion of Olig1 by means of nonviral transfection of the Olig1 gene.
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, z) F$ d) o2 C5 a. G- p4 C$ Z# CMATERIALS AND METHODS
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& z/ {9 Y- x- R1 zCulturing NSCs in basic medium (Neurobasal plus B27) resulted in the differentiation of the NSCs into astrocytes and neurons at a ratio of 3:1. However, differentiation into oligodendrocytes was only sporadically observed in less than 0.1% of the cells. After 10 days in culture, these oligodendrocytes could be stained for PDGF receptor,A2B5, and O4, and some were GAL-C positive. When using the oligodendrocyte-specific SATO medium supplemented with the oligodendrogenic growth factors Shh, FGF-2, and PDGF, between 10% and 15% of the NSCs differentiated into oligodendrocytes (Fig. 1A). Most of these oligodendrocytes had the same appearance as those in the basal medium, although some of them showed more extensive branching (Fig. 1B).
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Figure 1. Culture of mouse embryonic NSCs in growth factor–supplemented SATO medium. (A): Hoechst nuclear staining (blue) reveals NSCs that have migrated from the neurosphere (left) during 10 days of culture; among these cells, two O4-immunopositive OPCs (red, arrows) can be seen. (B): In a detail of this culture double-immunostained for O4 (red) and GFAP (green), a mature OPC with elaborate extensions can be seen located next to a GFAP-positive astrocyte. (C): Culturing NSCs transfected with Olig1 resulted in the differentiation of approximately 50% into O4-immunopositive OPCs (red) after 7 days of culture. (D): A magnification of the culture depicted in (C), double immunolabeled for O4 (red) and GFAP (green), shows several immature, bipolar, and round OPCs (red) as well as some GFAP-positive astrocytes. Examples of the immature appearance of the OPCs derived from Olig1-transfected NSCs are depicted in (E) (O4 staining) and (F) (PDGF receptor immunostaining) (bars = 50 μm).Abbreviations: GFAP, Glial fibrillary acidic protein; NSC, neural stem cell; OPC, oligodendrocyte progenitor cell.
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- c) ]5 A2 [9 `( y3 pGene transfection using the Amaxa nucleofection system resulted in a transfection efficiency of approximately 60%. The expression of the transfected genes, as registered by direct fluorescence (in case of eGFP gene transfection) or indirectly by immunohistochemistry (Olig1 gene transfection), gradually diminished and was undetectable after 10 days. The Olig1 expression in undifferentiated NSCs was below the level of detection. After transfection, NSCs were cultured in SATO medium with the supplements described above. Whereas control transfection with the eGFP gene did not alter the yield of OPCs, Olig1 gene transfection significantly increased the number of OPCs with almost a factor 4, up to 55% of the NSCs (Figs. 1C, 1D, 2). These OPCs had the typical appearance of the immature OPC stage and could be stained for the PDGF receptor,A2B5, and O4. However, these OPCs did not differentiate beyond the O4 stage; no immuno-reactivity for MBP and GAL-C could be detected (data not shown), and only an immature morphology and appearance could be observed (Figs. 1D, 1E). The lack of appropriate extrinsic or intrinsic differentiation signals leading to an additional transcription of downstream differentiation genes apparently restrained additional differentiation and subsequently resulted in cell death at approximately day 8.3 s' w) w/ I4 l% B4 C& F4 R
7 ~4 z/ _$ ]* M) J, o6 BFigure 2. Graph showing the percentage of O4-positive OPCs obtained after 7 days of culture in growth factor–enriched SATO medium after transfection with either the Olig1 gene or a control gene encoding for green fluorescent protein (GFP). The exogenously introduced expression of Olig1 in the neural stem cells resulted in a significant increase (approximately four times) in the percentage of OPCs compared with control transfection. Abbreviation: OPC, oligodendrocyte progenitor cell.; S! L% n, R* O# z) a3 E- ~
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发表于 2009-3-5 10:36
发表于 2015-5-31 16:52
