Stem Cells. RapidCommunication. Transient Expression of Olig1 Initiates the Differentiation of Neural Stem Cells into Oligodendrocyte Progenitor Cells

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1 Stem Cells RapidCommunication Transient Expression of Olig1 Initiates the Differentiation of Neural Stem Cells into Oligodendrocyte Progenitor Cells Veerakumar Balasubramaniyan, Nienke Timmer, Britta Kust, Erik Boddeke, Sjef Copray Department of Medical Physiology, University of Groningen, Groningen, The Netherlands Key Words. Neural stem cells Oligodendrocyte progenitor cells Embryonic Olig Mouse Abstract In order to develop an efficient strategy to induce the in vitro differentiation of neural stem cells (NSCs) into oligodendrocyte progenitor cells (OPCs), NSCs were isolated from E14 mice and grown in medium containing epidermal growth factor and fibroblast growth factor (FGF). Besides supplementing the medium with oligodendrogenic factors such as Sonic Hedgehog (Shh), FGF- 2, and PDGF, we attempted to initiate the gene transcription program for OPC differentiation by transfection of the Olig1 gene, a transcription factor known to be involved in the induction of oligodendrocyte lineage formation during embryogenesis. Whereas addition of Shh, FGF-2, and PDGF could induce OPC differentiation in 12% of the NSCs, the transient expression of Olig1 by use of Nucleofector gene transfection initiated OPC differentiation in 55% of the NSCs. Our results show that nonviral transfection of genes encoding for oligodendrogenic transcription factors may be an efficient way to initiate the in vitro differentiation of NSCs into OPCs. Stem Cells 2004;22: Introduction Recently, 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 [1 8]. 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. 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 Correspondence: Sjef Copray, Ph.D., Department of Medical Physiology, University of Groningen,A. Deusinglaan 1, 9713 AV Groningen, The Netherlands. Telephone: ; Fax: ; j.c.v.m.copray@med.rug.nl Received January 29, 2004; accepted for publication June 15, AlphaMed Press /2004/$12.00/0 STEM CELLS 2004;22:

2 Balasubramaniyan, Timmer, Kust et al. 879 serum. In general, this approach yields a low and highly variable percentage of OPCs [5, 9 13]. 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 [12, 14 17]. Besides Sox10, the most prominent oligodendrogenic transcription factors are the basic-helix-loop-helix proteins Olig1 and Olig2 [12, 14, 16, 18 24], 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 [20, 25]. 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 [18, 26]. 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. Materials and Methods Neural Stem Cell Isolation and Culture NSCs were obtained from C57/Bl6 mouse embryos at E14. The whole embryonic brain was dissected and dissociated as described previously [27]. Briefly, the brain was cut into small pieces, incubated with 0.05% trypsin EDTA for 30 minutes at room temperature, and mechanically triturated through a 26-gauge needle. Cells were cultured in serum-free Neurobasal medium (Invitrogen, Breda, The Netherlands) supplemented with human recombinant epidermal growth factor (20 ng/ml, Invitrogen), basic fibroblast growth factor (20 ng/ml, Invitrogen), B27 (Invitrogen), penicillin-streptomycin 1% (Sigma-Aldrich, Zwijndrecht, The Netherlands), L-glutamine 1%, and glutamax 1% in T25 (Nunc, Roskilda, Denmark) culture flasks in a humidified 5% CO 2 /95% air incubator at 37 C. Within 5 7 days, the cells grew as freefloating neurospheres and were passaged after mechanical dissociation through a fire-polished Pasteur pipette after 3 days. After two passages, neurospheres were used for the OPC differentiation induction experiments. These neurospheres were directly plated or dissociated (after a short exposure [5 minutes] to 0.1 mm ethylene glycol tetra-acetic acid) through a fire-polished Pasteur pipette and subsequently cultured in poly-l-lysin-laminin coated chamber slides (Nunc), approximately 104 cells per well. Three differentiation conditions were evaluated. The first condition was cultured in the serum-free Neurobasal medium supplemented with B27, L-glutamine 1%, glutamax 1%, and penicillin-streptomycin 1% (Sigma-Aldrich). The second condition was cultured in the oligodendrocyte-specific Sato medium, consisting of Dulbecco s modified Eagle s medium with additives glutamax 1%, L-glutamine 1%, penicillinstreptomycin 1%, putrescine (16 mg/ml, Sigma), thyroxine (400 mg/ml, Sigma), triiodothyroxine (400 mg/ml, Sigma), progesterone (6.2 ng/ml, Sigma), sodium selenite (5 ng/ml, Sigma), bovine serum albumin (100 mg/ml, Sigma), insulin (5 mg/ml, Sigma), and transferrin (50 mg/ml, Sigma). During the first 2 days of culture, the Sato medium was supplemented with Shh (100 ng/ml; R&D Systems,Abington, UK), FGF-2 (10 ng/ml; R&D Systems), and PDGF-aa (10 ng/ml; R&D Systems). The third condition was cultured in the same Sato medium as described in the second condition, but after previous transfection of the NSCs with the Olig1 gene. Gene Transfection Gene transfection was done using Nucleofector (Amaxa GmbH, Cologna, Germany) with a protocol specifically designed for the transfection of NSCs by Amaxa. The plasmid used for transfection was the expression vector pires (Clontech, Palo Alto, CA) containing the Olig1 gene (735 bp) (kindly donated by Dr. J. Cai, Louisville, KY). NSCs transfected with the gene encoding for green fluorescent protein (egfp) served as a transfection control. Neurospheres (in total, approximately cells) were transfected with 10 µg of the vector. After transfection, the neurospheres were kept overnight at 37 C in proliferation medium. Subsequently, the neurospheres were dissociated, and before culturing, the transfection efficiency was determined either by counting the green fluorescent cells (after egfp transfection) or by counting Olig1-immunopositive cells after Olig1 gene transfection. Immunohistochemistry After various culture periods up to 14 days, cell cultures were fixated with 4% paraformaldehyde and immunostained after permeabilization in 0.3% Triton X-100 to identify the differentiated neural cell types. To avoid nonspecific membrane staining, immunostaining for O4 and A2B5 was performed on nonfixated cell cultures placed on ice without prior permeabilization. The following antibodies were used to identify neuronal cells: anti-map2 (1:500, Chemicon, Hamp-

3 880 Oligodendrocyte Differentiation of Neural Stem Cells shire, UK) and anti-β-tubulin III (1:200, Sigma-Aldrich). To identify astrocytes, we used anti glial fibrillary acidic protein (GFAP) (1:200, Chemicon). To identify oligodendrocytes at different developmental stages, the following antibodies were used: anti-o4 and anti-a2b5 (1:100, both generously donated by W. Baron), anti-pdgf receptor (1:200, Santa Cruz), anti-myelin basic protein (MBP) (1:500, Chemicon), and anti Galactosidase-C (GAL-C) (1:500, Chemicon). Undifferentiated NSCs were identified with anti-nestin antibody (1:200, Chemicon), and the expression of the Olig1 transcription factor was verified using anti-olig1 (1:200, Chemicon). Immunolabeled cells were detected using fluorescently labeled secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA). To identify individual viable cells, Hoechst nuclear staining was applied. Results Culturing 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). Gene 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 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.

4 Balasubramaniyan, Timmer, Kust et al. 881 Figure 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. 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 immunoreactivity 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. Discussion Our results show that the induction of the expression of Olig1 in NSCs by nonviral gene transfection of mouse embryonic NSCs is a far more efficient way to initiate their differentiation toward OPCs than by external induction factors such as Shh in combination with FGF and PDGF. Although Shh has been suggested to induce the expression of Olig transcription factors in the developing spinal cord [14, 16], it is still unclear whether this is a direct effect of Shh or mediated via other factors. Our data indicate that Shh can induce OPC differentiation in only a very small percentage of the NSCs, likely due to the absence of other essential mediators or an inappropriate receptor repertoire of the NSCs. Various analyses of Olig knockout animals have clearly demonstrated the involvement of these transcriptions factors in the generation and maturation of oligodendrocytes during embryogenesis, but it has been debated whether expression of Olig1 or Olig2 itself is sufficient to induce the oligodendrocyte lineage formation in neural stem cells. Whereas Zhou et al. [23] were unable to generate oligodendrocytes from spinal cord neural stem cells by retroviral transfection of Olig 1 or 2, Lu et al. [18] demonstrated that cortical neural stem cell cultures infected with an Olig1 expressing adeno-virus yielded a substantial amount of OPCs. Although the experimental setup and staining procedures are difficult to compare, our findings seem to be in accordance with those of Lu et al. However, in our in vitro setup, oligodendrocyte differentiation initiated by Olig1 gene transfection did not proceed to full maturation and stability. Various explanations for that can be forwarded. For a smooth progress of oligodendrocyte differentiation, in particular the step toward maturation, during embryonic development, the transcription factor Nkx2.2 has to be expressed at an appropriate time point after Olig1 [14]. It is possible that our culture conditions did not provide the proper stimuli to trigger Nkx2.2 expression. Transplantation experiments will have to reveal whether in vivo conditions may be sufficient to induce Nkx2.2 expression and so additionally support the Olig1-initiated oligodendrocyte development of NSCs. Besides Nkx2.2, Olig2 and Sox10 may also be involved in oligodendrocyte maturation, and cotransfection experiments will have to elucidate the role of these transcription factors in that. In addition, the absence of full maturation of the Olig1-induced oligodendrocytes may have included the absence of functional integrin receptors. These receptors, in particular the β1-subunit, have been shown essential for oligodendrocyte survival and maintenance (besides myelination induction) [28]. The lack of a functional integrin receptor may have resulted in the death of the Olig1-induced oligodendrocytes derived from NSCs. References 1 Akiyama Y, Radtke C, Kocsis JD. Remyelination of the rat spinal cord by transplantation of identified bone marrow stromal cells. J Neurosci 2002; 22: Akiyama Y, Radtke C, Honmou O et al. Remyelination of the spinal cord following intravenous delivery of bone marrow cells. Glia 2002; 39: Brustle O, Jones KN, Learish RD et al. Embryonic stem cell-derived glial precursors: a source of myelinating transplants. Science 1999; 285: Chopp M, Zhang XH, Li Y et al. Spinal cord injury in rat: treatment with bone marrow stromal cell transplantation. Neuroreport 2000; 11:

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