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amaxa eNews #2
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OLIG2 Over-expression in Primary Mouse Neural Stem Cells using Nucleofection
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Sjef Copray, Veerakumar Balasubramaniyan, Josien Levenga, Jorick de Bruijn, Robert Liem and Erik Boddeke
Department of Medical Physiology, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands
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Abstract
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Neural stem cells (NSCs) are differentiated into oligodendrocytes by a complex interaction between local induction factors and intracellular transcription factors. The transcription factors Olig1 and Olig2 have been determined to be essential for oligodendrocyte lineage determination at the early stage of differentiation. In-vitro procedures to control the oligodendrocyte differentiation process have been developed to use NSCs as a source for remyelinating cell transplants in demyelinating diseases such as Multiple Sclerosis. The present study shows that the over-expression of Olig2 after nucleofection of NSCs from embryonic mouse brain with the Olig2 gene results in the development of fully maturated oligodendrocytes, expressing the transcription factor Nkx2.2 and all major myelin specific proteins.
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Introduction
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Demyelination of the axons is the major underlying factor responsible for the symptoms of multiple sclerosis (MS). The relapsing-remitting form of MS is usually characterized by acute demyelinating episodes followed by the generation of new oligodendrocytes with remyelination and recovery. Possible therapeutics include approaches to stimulate the remyelination capacity of endogenous oligodendrocytes as well as to apply exogenous cells capable of promoting remyelination.
In rat models for demyelination, various studies have reported the survival, integration and remyelinating activity of transplanted oligodendrocytes and oligodendrocyte progenitor cells (OPCs) (for a review see [1]). Bone marrow derived stem cells and neural stem cells (NSCs) are discussed as a potential source for remyelinating cell transplants [2-4]. Until now, research has concentrated on the implantation of undifferentiated labeled stem cells, presuming that the stem cells would differentiate into remyelinating cells at sites with a damaged myelin sheath. However, to promote an efficient contribution of implanted NSCs to remyelination, pre-differentiation into an oligodendrocyte cell lineage is required.
It has already been shown in vitro [5] that the first stage of oligodendrocyte differentiation, but not a complete maturation of embryonic mouse brain derived NSCs, can be triggered by transient expression of the basic helix-loop-helix (bHLH) protein Olig1 by non-viral transfection. The crucial role of Olig1 and Olig2 in in-vivo oligodendrogenesis and myelination has been confirmed by several publications [6-8]. To further elucidate the differentiation induction potential of Olig2 in oligodendrocytes and to compare its potency with Olig1 and Nkx2.2, we transfected mouse embryonic NSCs with Olig2 using amaxa's Nucleofector Technology.
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Material and Methods
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Neural stem cell isolation and culture
Neural stem cells (NSCs) were isolated from C57/Bl6 mouse embryos at E14. The cells were cultured in serum-free Neurobasal medium (Invitrogen, Breda, The Netherlands) supplemented with human recombinant EGF (20ng/ml, Invitrogen), bFGF (20ng/ml, Invitrogen), B27 (Invitrogen), penicillin-streptomycin 1% (Sigma-Aldrich, Zwijndrecht, The Netherlands), L-glutamine 1%, GlutaMAX 1% in T25 (Nunc, Roskilde, Denmark) culture flasks in a humidified 5% CO2/95% air incubator at 37° C. After 5-7 days the cells grew as free-floating neurospheres and were passaged 3 days after formation. The obtained neurospheres were used for the differentiation induction experiments after 2 passages.
Nucleofection
Transfection of the NSCs was performed using the Mouse NSC Nucleofector Kit (amaxa AG, Cologne, Germany). After careful dissociation, approximately 5x106 NSCs were transfected with 10μg of the expression vector (pIRES expression vector containing the Olig2 gene (972bp)). NSCs transfected with pmaxGFP (amaxa) were used as transfection control. After transfection, the NSCs were cultured overnight at 37°C in proliferation medium according to amaxa's protocol.
Immunohistochemistry
The cell cultures were fixed (4% formaldehyde) and immunostained after different culture periods to identify the various stages of differentiation of the developing neural cell types. Identification of oligodendrocytes at different developmental stages was performed using the following antibodies: anti-O4, anti-MBP, anti-GAL-C, antimyelin/oligodendrocyte specific protein, anti-Olig2 and anti-Nkx2.2.
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Results
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Oligodendrocyte differentiation induction in vitro
Prior to the transfection and differentiation experiments immunostaining was performed in undifferentiated mouse embryonic brain neural stem cells to assess the expression of the oligodendrogenic transcription factors Olig1, Olig2 and Nkx2.2. The neural stem cells displayed no expression of Olig1 and Nkx2.2 whereas in >95% of the NSCs expression of low levels of Olig2 could be detected. NSCs transfected with GFP or untransfected NSCs differentiated intro astrocytes (65%), neurons (26%) and approximately 10% of early stage oligodendrocytes using the oligodendrocyte-specific growth factor-enriched SATO medium (O4 staining, figure B).
The transfection efficiency in mouse NSCs was determined to be 60-80% under the described conditions and resulted in transient expression of the respective gene lasting up to 12 days [5].
A transient over-expression of Olig2 induced by nucleofection with the Olig2 gene not only induced intranuclear Olig2 expression (figure A), but led to an increase in the number of cells differentiating into an oligodendrocyte cell lineage of up to 25% (figure C). All of these oligodendrocytes developed into fully maturated oligodendrocytes after 7 days in culture, displaying elaborate myelin extensions with positive staining for major oligodendrocytic proteins, such as O4, GalC, MOG and MBP (figures C, E, F). The transcription factor Nkx2.2 could only be detected in fully maturated oligodendrocytes after transfection with Olig2 (figures D'-D''). In contrast to the transfection with Olig2, transfection with the Nkx2.2 or the Olig1 gene did not drive the differentiation of fully maturated oligodendrocytes. As previously shown [5] Olig1 gene transfection with Nucleofection could only initiate oligodendrocyte differentiation, however, the primitive immature precursors did not develop into mature oligodendrocytes and did not express the Nkx2.2 transcription factor.
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(Reproduced from Stem Cells 2006 24(4): 1001 by copyright of AlphaMed Press.) |
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Discussion
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The results clearly indicate that the differentiation of mouse embryonic neural stem cells (NSCs) can be induced by non-viral transfection with Olig2 using nucleofection. Unlike Olig1 and Nkx2.2, transfection and overexpression of Olig2 clearly promotes the differentiation of NSCs into mature oligodendrocytes using oligodendrocyte-specific, enriched SATO-medium. The experiments demonstrate that the introduction of Olig2 into neural stem cells can be used to drive their differentiation into an oligodendrocyte cell lineage. Neural stem cells derived from bone marrow, blood or skin therefore might serve as an autologous source for cell therapy approaches in demyelinating diseases.
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References
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[1] Pluchino, S., Furlan, R., Martino, G., Cell-based remyelinating therapies in multiple sclerosis: evidence from experimental studies, Curr. Opin. Neurol., 17 (2004) 247-255.
[2] Akiyama, Y., Radtke, C., Kocsis, J.D., Remyelination of the rat spinal cord by transplantation of identified bone marrow stromal cells, J. Neurosci., 22 (2002) 6623-6630.
[3] Akiyama, Y., Radtke, C., Honmou, O., Kocsis, J.D., Remyelination of the spinal cord following intravenous delivery of bone marrow cells, Glia, 39 (2002) 229-236.
[4] Brustle, O., Jones, K.N., Learish, R.D., Karram, K., Choudhary, K., Wiestler, O.D., Duncan, I.D., McKay, R.D., Embryonic stem cell-derived glial precursors: a source of myelinating transplants, Science, 285 (1999) 754-756.
[5] Balasubramaniyan, V., Timmer, N., Kust, B., Boddeke, E., Copray, S., Transient expression of Olig1 initiates the differentiation of neural stem cells into oligodendrocyte progenitor cells, Stem Cells, 22 (2004) 878-882.
[6] Grinspan, J., Cells and signaling in oligodendrocyte development, J. Neuropathol. Exp. Neurol., 61 (2002) 297-306.
[7] Lu, Q.R., Yuk, D., Alberta, J.A., Zhu, Z., Pawlitzky, I., Chan, J., McMahon, A.P., Stiles, C.D., Rowitch, D.H., Sonic hedgehog-regulated oligodendrocyte lineage genes encoding bHLH proteins in the mammalian central nervous system, Neuron, 25 (2000) 317-329.
[8] Lu, Q.R., Sun, T., Zhu,Z., Ma,N., Garcia, M., Stiles, C.D., Rowitch, D.H., Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection, Cell, 109 (2002) 75-86.
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amaxa's Nucleofection Process, Nucleofector Device and Nucleofector Solutions are covered by PCT Applications PCT/EP01/07348, PCT/DE02/01489, PCT/DE02/01483, and other pending patents, and domestic or foreign applications corresponding thereto.
amaxa, Nucleofector, Nucleofection and pmaxGFP are trademarks of amaxa AG.
GlutaMAX is a trademark of Invitrogen. Other product and company names mentioned herein are the trademarks of their respective owners.
amaxa disclaims all warranties, whether expressed or implied, including any warranty as to the quality, accuracy, safety, or suitability of the information provided in this e-newsletter for any particular purpose. Any use of the information contained on any page of this e-newsletter is evidence of agreement with these terms of use.
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