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Stem Cells 2005;23:442-453 www.StemCells.com
© 2005 AlphaMed Press

Induction of Oligodendrocytes From Adult Human Olfactory Epithelial-Derived Progenitors by Transcription Factors

Xiaodong Zhang, Jun Cai, Kathleen M. Klueber, Zhanfang Guo, Chengliang Lu, Mengsheng Qiu, Fred J. Roisen

Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, Kentucky, USA

Key Words. Oligodendrocyte • Transcription factors • Olig2 • Nkx2.2 • Sox10 • Progenitors • Adult olfactory neuroepithelium

Correspondence: Dr. Fred J. Roisen, Department of Anatomical Sciences and Neurobiology, University of Louisville, School of Medicine, 500 South Preston Street, Louisville, Kentucky 40202, USA. Telephone: 502-852-6227; Fax: 502-852-6228; e-mail: fjrois01{at}gwise.louisville.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neurosphere-forming cell (NSFC) lines have been derived from cultures of adult olfactory neuroepithelium obtained from patients and cadavers. These progenitors remain undifferentiated when maintained in minimal essential medium with 10% fetal bovine serum, but have the potential to differentiate along glial or neuronal lineages. However, few of these cells ever express mature neuronal or glial markers in defined medium. To evaluate the potential of NSFCs to form oligodendrocytes, two transcription factors, Olig2 and Nkx2.2, were introduced into NSFCs to determine whether their expression is sufficient for oligodendrocyte differentiation, as has been shown in the embryonic avian and murine central nervous systems in vivo. NSFCs transfected with Olig2 or Nkx2.2 alone exhibited no phenotypic lineage restriction. In contrast, simultaneous transfection of Olig2 and Nkx2.2 cDNA produced characteristic oligodendrocyte morphology and antigenicity, including myelin basic protein (MBP). Furthermore, a population of Olig2-expressing NSFCs also expressed Sox10. Cotransfection of NSFCs with Nkx2.2 and Sox10, but not Olig2 and Sox10, produced a MBP+ oligodendrocytic phenotype. Coculture of NSFCs transfected with Olig2 and Nkx2.2 or Nkx2.2 and Sox10 with purified sensory neurons, demonstrated frequent contacts between NSFC processes and axons, including the early stages of ensheathment. These studies demonstrate transcription factors governing early development of chick and mouse oligodendrocyte formation, also apply to human progenitors isolated from adult olfactory neuroepithelium. Our long-term goal is to develop cell populations for future studies used to determine the therapeutic utility of these olfactory-derived NSFCs for autologous transplantation into donors with central nervous system trauma or neurodegenerative diseases.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the olfactory neuroepithelium, the receptor neurons and their supporting cells arise from a population of basal stem cells, which are responsible for their lifelong replacement [1–4]. Dissociated cultures of adult olfactory neuroepithelium isolated from cadavers [5], or patients undergoing endoscopic nasal sinus surgery [6], produced neurosphere-forming cells (NSFCs) that have been used to generate approximately 60 lines. These lines may have the potential to differentiate into neurons or glia depending on their environmental signals. Thus, they may have the potential to be used therapeutically to treat neurological disorders such as the demyelinating diseases in which oligodendrocytes are selectively lost [7, 8].

Oligodendrocytes are macroglial cells that form myelin in the central nervous system (CNS), as well as modulate the activities of adjacent neurons by regulating their microenvironment [9]. The mechanisms underlying oligodendrocytic specification and differentiation from embryonic neural stem or progenitor cells are under extensive investigation. During development, oligodendrocytes arise from restricted loci of neuroepithelial precursor cells in the ventral neural tube [10–14] under the influence of the ventral midline signal sonic hedgehog [15–18]. In the early stage of oligodendrogenesis, the basic helix-loop-helix transcription factors Olig1 and Olig2 are initially expressed in oligodendrocyte-generative zones of the neuroepithelium. As oligodendrocyte progenitors leave the ventricular zone, Olig1/2 expression is retained in oligodendrocyte progenitors and persists in mature oligodendrocytes [19–21]. Molecular and genetic studies have demonstrated that expression of the Olig genes is required for oligodendrocyte lineage determination in vivo [22–24]. Interestingly, either before or after oligodendrocyte progenitors migrate into the white matter, they acquire the expression of two other transcription factors, the high-mobility transcriptional regulator Sox10 [25] and the homeodomain transcription factor Nkx2.2 [26–29]. The expression of Nkx2.2 and Sox10 seems to directly regulate myelin gene expression and oligodendrocyte differentiation; mutations of both genes result in a decreased number of mature oligodendrocytes in the CNS [21, 25]. Conversely, expression of Nkx2.2 in combination with Olig2 can induce ectopic formation of mature myelin basic protein (MBP)-positive oligodendrocytes in embryonic chicken spinal cord [28].

The role of these transcription factors in the differentiation of glial cells from human-adult derived neural stem cells has not been demonstrated. Thus, the purpose of this study was to investigate the roles of Olig2 and Nkx2.2 genes in human oligodendrocyte lineage specification and differentiation in vitro using adult human olfactory neuroepithelial progenitors. In this study we report that the simultaneous transfection of NSFCs with Olig2 and Nkx2.2 or Sox10 and Nkx2.2 can lead to oligodendroglial morphology and lineage-restricted marker expression.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
The two different NSFC lines used in this study were obtained from adult olfactory neuroepithelium, one from a cadaver of a 96-year-old man [5] and the other from a 22-year-old female patient via endoscopic biopsy [6]. Procedures for the harvest of these lines from primary cultures have been previously described [5, 6]. Frozen stock (passage 3 through 6) of each cell line was thawed rapidly, and 5 x 105 cells were placed in each flask in minimal essential medium (MEM) with 10% heat-inactivated fetal bovine serum (FBS) (GIBCO, Grand Island, NY), 10 mg/100ml gentamycin (MEM10) in flasks (25 cm2, Corning Incorporated, Corning, NY) in humidified 5% CO2/95% air (37°C) for 24 hours. The NSFCs were adapted to the absence of serum via serial dilution of serum every 2 days for 1 week until the cells were finally cultured in DFB27M (DMEM/F12 supplemented with 2% B27) and 10 mg/100ml gentamycin for 1 week [30] and then used for in vitro differentiation analyses between passages 10 and 20. Parallel experiments were preformed on NSFCs with both lines to determine if patient-specific differences were obtained. Because equivalent results were obtained with these two different lines, data from only one line has been presented.

Construction of Expression Vectors
Full-length mouse Olig2 cDNA was cloned into the pIRES2-enhanced green fluorescent protein (EGFP) expression vector (Clontech, Palo Alto, CA). The Nkx2.2 gene was isolated by screening the mouse 129Sv cDNA library and cloned into the pIRES2-EGFP expression vector. For the Olig2 and Nkx2.2 coexpression vector, Olig2 cDNA was cloned into pIRES (Clontech) between NheI and EcoRI, and Nkx2.2 cDNA was inserted between XbaI and SalI. Similarly, the chicken Sox10 cDNA was cloned into the pIRES2-EGFP expression vector. For coexpression of Sox10 and Nkx2.2, the chicken Sox10 cDNA and mouse Nkx2.2 were sequentially cloned into the pIRES expression vector. The pIRES2-EGFP and pIRES expression vectors served as controls. All expression vectors were verified by extensive DNA sequencing. A further control was provided by lipofectamine alone.

Transfection and Selection
All plasmid constructs were introduced into the NSFCs by liposomal transfection. The cells were plated on glass coverslips in six-well plates (3 x 104 cells/35-mm well) in DFB27M without antibiotics 1 day before transfection. NSFCs were transfected with each plasmid (4 3g/well) for 48 hours according to the manufacturer’s protocol (Life Technologies, Rockville, MD). Two days after transfection, the cells were fixed or fed with DFB27M supplemented with G418 (50 3g/ml; GIBCO, Grand Island, NY) for selection.

Cell Process Formation
NSFCs were plated on glass coverslips in six-well plates (3 x 104 cells/35-mm well) in DFB27M without antibiotics, transfected with each plasmid (4 3g/well) for 48 hours, and selected by G418 for 7 days. Cells were seeded on coverslips in six-well plates (1 x 103 cells/35-mm well) in DFB27M without transfection and selection for controls. On 1, 3, 5, 7, and 9 days in vitro (DIV) during transfection and selection, the number and length of processes were measured with the aid of an eyepiece retical under constant magnification with phase-contrast optics. Cells (500 to 1,000) were sampled systematically from standardized fields (total magnification, x 200). Only primary processes originating directly from the soma that were longer than the diameter of the cell body were evaluated. The cultures were coded before evaluation.

MTT (3-[4, 5-Dimtheylthiazol-2-yl]-2, 5-Diphenyl Tetrazolium Bromide) Assay
The viability of the NSFCs after 2 days of transfection and 7 days of selection in flasks (25 cm2, Corning Incorporated) was measured with MTT kit (Sigma). Cells were seeded in the flasks in DFB27M without transfection and selection as controls. Cells were plated at a density of 5 x 104 cells per well in 24-well plates (Falcon). MTT solution was added to each well. Mitochondrial dehydrogenases in living cells metabolized MTT into formazan crystals, the concentration of which was determined spectrophotometrically at a wavelength of 570 nm, as described previously [30].

Immunocytochemistry
The NSFCs (3 x 104 cells/well) were plated on 22-mm round glass coverslips in six-well plates (Falcon, Franklin Lakes, NJ) and incubated at 37°C in 5% CO2/95% air for 24 hours, transfected for 2 days, and either immediately fixed or selected for 1, 4, or 7 days before fixation for immunofluorescence. Cultures were incubated with 4',6-diamidino-2-phenylindole dihydrochloride (1:1,000, 2 mg/ml, Molecular Probes, Eugene, OR) for 30 minutes at 37°C for vital labeling of DNA when nuclear staining was desired. The coverslips were rinsed with cytoskeletal buffer (CB) (1.95 mg/ml 2-N-Morpholino ethane sulfonic acid, 8.76 mg/ml NaCl, 5 mM EGTA, 5 mM MgCl2, 0.9 mg/ml glucose; pH 6.1) twice and fixed in 3% paraformaldehyde in CB (10 minutes) when permeabilization was desired, treated with 0.2% Triton X-100 (Sigma) for 10 minutes, and incubated (1 hour) in 3% bovine serum album in Tris-buffered saline (TBS). Primary antibodies (Table 1Go) were applied overnight at 43C. After washing (1 hour) in TBS three times, the cells were incubated with the following secondary antibodies: Texas red–conjugated goat anti-rabbit immunoglobulin G (IgG), Texas red–conjugated goat anti-mouse IgG, and Cy2-conjugated goat anti-mouse IgG (all diluted 1:100, Cy2, Jackson Immunology Research Laboratories, West Grove, PA; Texas red, Molecular Probes, Eugene, OR). Experiments were preformed in triplicate; first and second antibody omission controls were performed with each experiment to ensure the specificity of staining.


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  		Table 1. Antibodies and specificity
 
Western Blot Analysis
Western blot analysis was used to support the immunofluorescence studies. Proteins from human fibroblasts (ATCC, CRL-1836), NSFCs cultured in DFB27M without selection, NSFCs transfected with control vectors, and NSFCs transfected with the vectors plus each combination of transcriptions factors (Olig2, Nkx2.2, Olig2-Nkx2.2, Sox10, and Sox10-Nkx2.2) were collected in lysis buffer (20 mM Tris-HCl containing 1% NP40, 10% glycerol, 137 mM NaCl, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 4 mM phenylmethylsulfonyl fluoride, 20 mM sodium fluoride (NaF), 1 mM Na3VO4, and 1% Triton X-100). After 10 minutes of incubation at 4°C, samples were centrifuged at 12,000g (20 minutes) and the protein concentration of the supernatant was determined. The protein samples (20 µg/well) were electrophoresed on 10% to 14% SDS-polyacrylamide gels along with standarized molecular size marker proteins in an adjacent lane (150 V, 60 to 90 minutes) and transferred (100 V, 60 to 90 minutes) from gel to nitrocellulose paper. Nonspecific binding was blocked (1 hour) with 5% nonfat dry milk in TBST buffer (5 mM Tris-HCl [pH 7.6], 136 mM NaCl, 0.1% Tween 20). Blots were incubated (4°C overnight) after the sequential addition of each of the following primary antibodies: polyclonal anti-Olig2 (1:2,000 dilution), monoclonal anti-Nkx2.2 (1:50 dilution), polyclonal anti-human MBP (1:1,000 dilution), polyclonal anti-Sox10 (1:1,000 dilution), and monoclonal anti-actin (1:2,000 dilution, Amersham Life Science). Blots were washed with TBST buffer four times for 20 minutes; then three times for 10 minutes. Washed blots were incubated for 1 hour in polyclonal horseradish peroxidase–labeled anti-rabbit IgG (1:1,000) for Olig2, human MBP, and Sox10 as well as monoclonal horseradish peroxidase–labeled anti-mouse IgG (1:1,000) for Nkx2.2 and actin. Chemiluminescence Western blotting detection (Bio-Rad, Hercules, CA) was used to identify bound antibodies. Densitometry of the protein bands was carried out on a Molecular Dynamics gel scanner (Molecular Dynamics, Sunnyvale, CA). Data were analyzed using the Image Quant software programs supplied by the manufacturer.

Coculture
Purified cultures of either EGFP mouse (Jackson Laboratory, Bar Harbor, ME) or rat dorsal root ganglia neurons (DRGNs) were established by classic techniques [31]. Briefly, DRGNs from 1- to 3-day-old (P1 to P3) GFP mice or Sprague-Dawley rats were purified and maintained in MEM10 for 4 DIV on glass coverslips in six-well plates; then in DFB27M for 3 weeks before seeding of NSFCs. The medium was supplemented with nerve growth factor (NGF) (50 ng/ml; Sigma) and dibutyryl cyclic AMP (0.5 mM; Sigma), which stimulated axonal elongation [32, 33]. On 2 to 4, 6 to 8, and 10 to 12 DIV, fluorodeoxyuridine (Sigma) was added to the media to reduce the nonneuronal (dividing) cells.

NSFCs for coculture with green fluorescent protein (GFP) mouse DRGNs or retroviral-labeled with GFP NSFCs for coculture with rat DRGNs after transfection with Olig2-Nkx2.2 or Sox10-Nkx2.2 and selection were maintained in DFB27M for 1 day and then detached to seed onto established DRGN cultures at 500 cells per well supplemented with ascorbic acid (50 3g/ml). Cocultures were maintained for 10 to 14 days before fixation for immunoflorescence or transmission electron microscopy. DRGN cultures alone and DRGNs cocutured with NSFCs without transfection served as controls.

Electron Microscopy
Cocultures (10 to 15 DIV) were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 at 4°C for 4 hours. After treatment with 1% osmium tetroxide, dehydration in an ethyl alcohol series, and embedment, selected are as we remounted, sectioned, stained with 1% uranyl acetate and lead citrate, and examined with an electron microscope [34]. The nature of the axonal or NSFCs process contacts was examined to determine if the transfected cells would ensheath the axons.

Statistics
Statistical analysis (Graph pad Prism) was carried out using ANOVA (significance level, p < .05). Cells (500 to 1,000) were sampled systematically from standardized microscopic fields (total magnification, x 200) of cells stained for each marker. The mean and standard deviation of triplicate samples repeated a minimum of three times was determined for each of the two NSF Clines. Because there were no detectable differences between the two cell lines, data have been reported without reference to which line was evaluated.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NSFC Population
The heterogeneous nature of the NSFC population just before transfection was demonstrated by immunolocalization on fixed triton-treated cells. More than 97% (n = 54 fields) of cells were positive for ß-tubulin III and peripherin (Figs. 1I, 1JGo); 49.1 ± 2.9% were positive for nestin (Figs. 1E, 1MGo) in the absence of triton; 26.1 ± 1.7% were positive for A2B5 (Fig. 1JGo); and 40.5 ± 2.3% were positive for neuronal cell adhesion molecule (NCAM) (Fig. 1KGo). In contrast, no cells were detected that were reactive for O4, galactosylceramide (GalC), oligodendrocyte specific molecule (RIP), MBP, Glial fibrillary acidic protein (GFAP), neuronal nuclear marker (NeuN), Olig2, Nkx2.2, or Sox10. No differences in phenotypic expression were detected between the two lines selected for these studies



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  		Figure 1. Time course of phenotype and lineage changes. Adult human olfactory progenitors (NSFCs, passage 12) before transfection (0 DIV) or without transfection for 9 DIV (L) did not exhibit an oligodendrocyte-like phenotype. (A): Immunolocalization demonstrated > 97% ß-tubulin III+ (green) and peripherin+ (red) (I); 26% A2B5+ (green, J); 41% NCAM+ (green, K); and 49% nestin+ (green, E) of the NSFC population (E). During transfection with Olig2-Nkx2.2 and selection, NSFCs began to form networks of extensive processes (B: 3 DIV; C: 6 DIV; D: 9 DIV). Furthermore, approximately 80% of cells gained CNP (green) and GalC (red) expression on 6 DIV (F, M), and most lost nestin expression (M); on 9 DIV, most cells (> 85%) gained RIP (green) and MBP (red) expression (G, M). There was no expression of NeuN (green), GFAP (red) (H, M), and OX 42 (M) 4', 6-diamidino-2-phenylindole dihydrochloride (blue stain for DNA). (A–D, L): Phase-contrast optics; (E–K): Confocal microscopy, differential interference contrast (DIC). Data are expressed as mean ± standard deviation, n = 54 fields. Each experiment included triplicate samples, repeated aminimum of three times. Abbreviations: CNP, 2'3'-Cyclic nucleotide-3'-phosphohydrolase; DIV, days in vitro; GalC, galactosylceramide; GFAP, Glial fibrillary acidic protein; MBP, myelin basic protein; NSFC, neurosphere-forming cell.

 
NSFCs Transfected With Olig2 and Nkx2.2 Exhibit Oligodendrocyte-Like Morphologies
To examine the phenotypic expression of NSFCs in vitro after transfection and selection, representative cultures and controls were compared. Three different NSFC morphologies were observed when nontransfected cells were maintained in DFB27 medium: round, bipolar, or multipolar with relatively few processes (0DIV, 9 DIV; Figs. 1A, 1LGo, respectively). Nontransfected NSFCs or those transfected with lipofectamine died within 1 week after selection with 50 3g/ml G418. Transfection with single genes or the control vectors resulted in no morphologic changes as indicated for Olig2, Olig2-EGFP, or Nkx2.2-EGFP alone (Figs. 2A–2CGo). In contrast, the morphology of NSFCs transfected with both Olig2 and Nkx2.2 and selection with G418 for 7 days underwent dramatic changes that resulted in a phenotype characteristic of oligodendrocytes with extensive arborization (Fig. 1DGo). Quantitative analysis demonstrated not only that NSFCs transfected with both Olig2 and Nkx2.2 had increased numbers of processes but also that the processes were longer (Figs. 3A, 3BGo). After 1 week of selection with G418, several of the cells in the transfected groups were round or had only one process and contained condensed or degraded nuclei. These cells likely reflected a population of nontransfected cells undergoing apoptosis. Because they remained attached to the surface, they were included in the process formation assay. Relatively few NSFCs (10.4 ± 0.3%, n = 54 fields) survived after transfection with vector alone, Olig2, Nkx2.2 or Olig2 and Nkx2.2, and 7-day selection. However, no difference in viability was observed between cells that survived after 2 days of transfection and 7 days of selection and controls without transfection and selection, as determined by a cytotoxicity assay (Fig. 4Go).



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  		Figure 2. NSFCs (passage 10 through 20) transfected with either Olig2 or Nkx2.2 did not exhibit oligodendrocyte-like morphology. (A): Immunolocalization demonstrated Olig2 expression (red) in 5% to 10% of the NSFCs after 2 days of transfection with Olig2. (B): After 2 days of transfection with Olig2-EGFP and 7 days of selection, the transfected NSFCs appeared similar to nontransfected controls. Immunoreactivity to nestin (red) was also similar to control populations. (C): Transfection with Nkx2.2-EGFP for 2 days followed by 7 days of selection did not alter NSFC morphology of cells reactive for Nkx2.2 (green). (Confocal microscopy, A and B, differential interference contrast (DIC). Abbreviations: DIV, days in vitro; NSFC, neurosphere-forming cell.

 


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  		Figure 3. Time-dependant changes in process number (A) and average length (B) between 1 and 9 days of transfection and selection. Transfection with both Olig2 and Nkx2.2 (Olig2-Nkx2.2) or Sox10 and Nkx2.2 (Sox10-Nkx2.2) increased the number and length of processes per cell (p < .01). The decrease in number of processes per cell in the DFB27 group reflects the high proliferative activity of NSFCs on the coverslips at 7 DIV. Values are mean ± standard deviation. **p < .01 (t-test). pIRES as the control vector, NSFCs without transfection (DFB27), transfected with Olig2 with EGFP (Olig2-EGFP), Nkx2.2 with EGFP (Nkx2.2-EGFP), and Sox10 with EGFP (Sox10-EGFP) alone. Abbreviations: DIV, days in vitro; NSFC, neurosphere-forming cell.

 


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  		Figure 4. MTT assay. NSFCs (passage 10 through 20) were cultured in DFB27M without transfection and selection (DFB27) or transfected with control vector, Olig2-EGFP, Nkx2.2-EGFP, Sox10-EGFP, Olig2-Nkx2.2, and Sox10-Nkx2.2 for 2 days, selected for 7 days, and assayed for viability using the MTT assay. No differences were observed between the groups. Data are expressed as mean ± standard deviation (n = 12). Each experiment includes triplicate samples. All experiments were repeated a minimum of three times. Abbreviations: DIV, days in vitro; NSFC, neurosphere-forming cell.

 
NSFCs Transfected With Olig2 and Nkx2.2 Express Oligodendrocyte Lineage-Restricted Markers
Immunohistochemistry of Olig2-expressing cells revealed that 48.1 ± 5.1% of the cells coexpressed nestin (Fig. 2BGo). On further analysis, no coexpression of O4, GalC, oligodendrocyte specific molecule (RIP), MBP, GFAP, neuronal nuclear marker (NeuN), or OX 42 was noted in the transfected cells (Table 2Go). Similarly, no detectable lineage-restricted changes were observed in NSFCs transfected with Nkx2.2, Sox10 cDNA, or control vectors alone (Table 2Go).


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  		Table 2. Analysis of the presence of oligodendrocyte markers in NSFCs after transfection
 
However, when the NSFCs were transfected with Olig2 and Nkx2.2 simultaneously, morphologic and lineage-restricted changes occurred. Immunohistochemistry confirmed that the cotransfected cells expressed both Olig2 and Nkx2.2 proteins (Fig. 5AGo). Interestingly, the early oligodendrocyte precursor marker O4 was not expressed in the cotransfected cells. Instead, these cotransfected cells expressed more mature oligodendrocyte markers, including 2'3'-cyclic nucleotide-3'-phosphohydrolase (CNP), GalC, RIP, and MBP (Figs. 5B–5DGo; Table 2Go). In addition, the transfected cells coexpressed GalC and CNP (Fig. 5EGo) as well as RIP and MBP (Fig. 5FGo). Thus, the presence of the transcription factors Olig2 and Nkx2.2 is sufficient to direct differentiation of the human NSFCs toward an oligodendrocytic phenotype. No mature markers for other cell lineages were detected, including GFAP (astrocyte marker), NeuN (neuronal marker), or OX 42 (microglia marker) (Table 2Go).



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  		Figure 5. Cotransfection of Olig2 and Nkx2.2 resulted in lineage change. After 2 days of simultaneous transfection with Olig2 and Nkx2.2 and 7 days of selection, NSFCs (passage 10 through 20) exhibited a characteristic oligodendrocytic phenotypic expression. These representative micrographs illustrate immunolocalization with probes for individual transcription factors (B, C, D) or both transcription factors (A) as well as for several oligodendrocyte specific lineage-restricted antigens (E, F). The specific primary antibodies have been noted on the respective micrographs. They were demonstrated with secondary antibodies labeled with CY2 (green) or Texas red (red). The confocal images included a DIC channel to demonstrate the extent of phenotypic expression. (A): NSFCs immunostained for both transcription factors, Nkx 2.2 (green) and Olig2 (red). (B): Approximately 85% of the NSFCs transfected with both transcription factors were immunoreactive for oligodendrocytic-specific antigens GalC (red), RIP (green) (C), and human MBP (red) (D). The simultaneous presence of two independent oligodendrocyte-specific antigens, CNP (green) and GalC (red) (E), as well as oligodendrocyte specific molecule (RIP) (green) and MBP (red) (F), was demonstrated in cells transfected with both transcription factors. Abbreviations: CNP, 2'3'-Cyclic nucleotide-3'-phosphohydro-lase; DIV, days in vitro; GalC, galactosylceramide; MBP, myelin basic protein; NSFC, neurosphere-forming cell.

 
The oligodendrocytic phenotype induced by the transcription factors was further confirmed by Western blot analyses with antibodies that recognize the late oligodendrocyte marker MBP. In NSFCs cultured in defined medium (DFB27) or transfected by Olig2, Nkx2.2, Sox10, or control vectors alone, MBP bands were not present (Figs. 6C, 6EGo). However, in the cells cotransfected with both Olig2 and Nkx2.2, these bands were detected, indicating that only those cells transfected with these two transcription factors produced MBP (Figs. 5D, 5FGo, 6C, 6EGo).



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  		Figure 6. Western blot assay. After 2 days of transfection and 7 days of selection, NSFCs (passage 10 through 20) were lysed in buffer; protein samples were separated on SDS-PAGE gels, and the expression of Olig2, Nkx2.2, human MBP, and Sox10 was detected. Human fibroblasts and NSFCs cultured in defined medium (DFB27) served as the controls. Actin was used as a control for variation in cell density. (A): There was endogenous Olig2 expression in NSFCs, but not human fibroblasts. (B): No endogenous Nkx2.2 expression was detected in either NSFCs or human fibroblasts. (C): NSFCs transfected with Olig2 and Nkx2.2 or Sox10 and Nkx2.2 expressed human MBP. (D): NSFCs transfected with Olig2 alone, Olig2 with Nkx2.2, Sox10 alone, or Sox10 with Nkx2.2 expressed Sox10. (E): Quantification of protein bands expressed as mean ± standard deviation. The density of the actin band was used as standard to adjust tracing quantification. The pIRES2 expression vector served as the control vector. Abbreviations: MBP, myelin basic protein; NSFC, neurosphere-forming cell.

 
Time Course Analysis of Antigen Expression
To examine the changed pattern of antigen expression by NSFCs transfected with Olig2-Nkx2.2, NSFCs before transfection (0 DIV) and after 2 days transfection and 7 days selection (3, 6, and 9 DIV) were fixed for immunohistochemistry. Although some cells within the initial population of NSFCs in DFB27M were A2B5-positive, this antigen was not examined further because it may be unreliable for specific glial restriction since it has been reported to cross-react with neurons [35]. Therefore, the expression of one early, O4, an early oligodeudrocyte marker, and several relatively mature oligodendrocyte markers, including CNP, GalC, RIP, and MBP, was examined. Most NSFCs were round, and a few were bipolar or multipolar (Figs. 1A, 1LGo); 49% of the NSFCs expressed nestin, which is expressed in neural stem cells, progenitors, or proliferating oligodendrocyte progenitors [36]; and > 97% of the NSFCs were peripherin-positive (Fig. 1EGo) before transfection (0 DIV). After transfection with Olig2-Nkx2.2, NSFCs formed elaborate compound processes (Figs. 1B–1DGo) and gradually lost their expression of nestin (Fig. 1MGo); furthermore, a few cells began to express CNP and GalC on 3 DIV. By 6 DIV, most cells were positive for these two antigens (Figs. 1F, 1MGo). It was not until 9 DIV that > 85% of the cells expressed the more mature oligodendrocyte markers RIP and MBP (Figs. 1G, 1MGo). At no time point was expression of NeuN, GFAP (Figs. 1H, 1MGo), O4, and OX 42 (Fig. 1MGo) observed (0 DIV to 9 DIV).

Sox10 Can Mimic the Effects of Olig2 in Inducing Oligodendrocyte Phenotype in Collaboration With Nkx2.2
Recent studies have suggested that Sox10 is the downstream target gene of Olig2 [17, 26] and may mediate the function of Olig2 in regulating oligodendrocyte specification and differentiation. Thus, the expression of Sox10 in NSFCs during the induction by Olig2 and Nkx2.2 was examined. When the NSFCs were transfected with Olig2 alone, 9.4 ± 0.8% of the transfected cells expressed Sox10 (Table 2Go). In contrast, 25.5 ± 1.8% of the NSFCs transfected with both Olig2 and Nkx2.2 expressed Sox10 (Table 2Go). To test the role of Sox10 in oligodendrocyte induction, NSFCs were transfected with Sox10-EGFP alone or both Sox10 and Olig2; no oligodendrocyte morphology was detected (Figs. 7A, 7BGo; Table 2Go). However, when Sox 10 and Nkx2.2 were introduced into the cells simultaneously, the cells assumed the characteristic phenotype of the oligodendrocytes (Fig. 7CGo; Table 2Go).



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  		Figure 7. Transfection with Sox10 alone or with Nkx2.2 simultaneously. The NSFCs (passage 10 through 20) after 2 days of transfection with Sox10 with EGFP alone (A), Olig2 and Sox10 with EGFP (B), or Sox10 with Nkx2.2 (C) and 7 days of selection expressed (A) Sox10 (red), (B) Olig2 (red), and (A, B) GFP (green) or (C) Nkx2.2 (green). Furthermore, NSFCs transfected simultaneously with Sox10 and Nkx2.2 (C) were immunoreactive for MBP (red) and phenotypically characterized by extensive arborization. Abbreviations: MBP, myelin basic protein; NSFC, neurosphere-forming cell.

 
The induction of Sox10 protein in the NSFCs transfected with various expression vectors was further verified by Western blot (Figs. 6D, 6EGo). When the cells were transfected with only Nkx2.2 or the control vectors or grown in DFB27M, Sox10 protein expression was not detected. However, Sox10 protein was detected in cells transfected with either Olig2 alone or with Olig2 and Nkx2.2 simultaneously and at a much higher level in cells that were transfected with Sox10 alone or in combination with Nkx2.2 (Fig. 6DGo; Table 2Go). In addition, several bands of smaller sizes were detected, which were probably degradation products of the Sox10 protein (Fig. 6DGo).

Cocultured of NSFCs Transfected With Olig2-Nkx2.2 or Sox10-Nkx2.2 With Purified Rat or GFP Mouse DRG Neurons
No direct axonal-NSFC association was observed when non-transfected NSFCs were maintained on top of an established DRG neuronal layer (controls) for 10 to 14 days. In contrast, NSFCs transfected with Olig2-Nkx2.2 or Sox10-Nkx2.2, cocultured with DRGNs for 10 to 14 days, formed multiple processes that often were observed in direct contact with the DRG neurites. As demonstrated with confocal microscopy, NSFC processes were observed, wrapping around individual regions of the DRG neurites (Figs. 8A, 8BGo). The transfected cells were MBP-positive. Pilot ultrastructural analysis demonstrated the early stages of axonal ensheathment by processes from the transfected NSFCs (Figs. 9A–9CGo). Frequent regions of subplasmalemmal densities were observed at the contact sites between axons and the NSFC processes, perhaps reflecting the initial development of mesaxons.



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  		Figure 8. Coculture studies. After 2 days of transfection (Olig2-Nkx2.2, Sox10-Nkx2.2) and 7 days of selection, NSFCs (passage 16) were maintained in DFB27M without G418 for 1 day and then seeded onto purified GFP mouse DRGNs for 11 days. The NSFCs were MBP (red) positive; their processes were frequently found surrounding individual axonal segments of DRGNs (arrow). (A): Cells transfected with Olig2-Nkx2.2; (B): Cells transfected with Sox10-Nkx2.2. Abbreviations: DRGN, dorsal root ganglia neuron; GFP, green fluorescent protein; MBP, myelin basic protein; NSFC, neurosphere-forming cell.

 


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  		Figure 9. Ultrastructural observation of transfected NSFCs cocultured with rat DRGNs. After transfection (Olig2-Nkx2.2) and selection, GFP-labeled NSFCs (passage 15) were maintained in DFB27M without G418 for 1 day and then seeded onto purified rat DRGNs for 11 days. (A): Two processes of transfected NSFCs wrapped around the axon of the DRGN. (B): One larger process of transfected NSFCs ensheathed the axon of the DRGN. (C): The processes of a transfected NSFC ensheathed a middle axonal segment. Abbreviations: DRGN, dorsal root ganglia neuron; NSFC, neurosphere-forming cell.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Neural stem cells can give rise to a variety of neuronal and glial phenotypes in culture and therefore have the potential to provide a cell population for replacement-based therapeutic treatments for various neurological diseases or injuries [37–40]. The human NSFCs from adult olfactory epithelium cultured in MEM with 10% FBS were shown to remain relatively undifferentiated despite exposure to a variety of media and trophic factors [5, 30]. In fact, this progenitor population seems to have an immature neuronal default. This suggests that the NSFCs obtained from adult human olfactory epithelium may be different from embryonic or other species of neural stem or progenitor cells [37]. In the present study, during transfection-mediated oligodendrocyte differentiation, NSFCs gradually lost their progenitor characteristics, such as reactivity to nestin as previously reported for other cells [36], and gained the properties of mature oligodendrocytes, bypassing the earliest stages of oligodendrocyte differentiation. A few CNP+ or GalC+ but MBP– oligodendrocytes were observed that exhibited two to three processes without the web-like morphology, suggesting that they were premature or immature oligodendrocytes. However, we did not detect any O4, a marker for a specific preoligodendrocyte developmental stage. One possible explanation is that overexpression of Olig2 or Nkx2.2 could modify cell surface properties or the overexpression of the factors could result in a rapid maturation and our timing for evaluation of the cells’ differentiation missed a very brief period of O4 expression. Another explanation is that NKx2.2 may directly activate MBP gene expression in collaboration with Olig2 [21] without activating O4 expression.

Neural stem cells have been shown to differentiate into neurons or glia after exposure to growth factors or mitogen withdrawal [38–40]. Recent identification of many key lineage-specific molecules has provided important insights into directing progenitors to differentiate into the desired specific cell types. Molecular and genetic studies have shown that many transcription factors, when expressed in combination, are capable of driving lineage-specific differentiation during embryonic development [41]. For instance, in the developing chick spinal cord, coelectroporation of Olig2 and Nkx2.2 promoted oligodendrocyte differentiation and maturation [28], whereas expression of either Olig2 or Nkx2.2 alone was not sufficient to cause such differentiation. In this study, Olig2 and Nkx2.2 have been used to drive adult human progenitors to differentiate along an oligodendrocyte lineage. Consistent with the previous observations in embryonic neural stem cells, neither Olig2 nor Nkx2.2 could induce NSFCs to differentiate into oligodendrocytes. However, NSFCs transfected simultaneously with Olig2 and Nkx2.2 exhibited oligodendrocyte morphology and antigen expression, resembling in vivo patterns. Western blot analysis provided parallel independent assessment to complement the immunolocalization. The two different approaches demonstrated that Olig2 and Nkx2.2 or Nkx2.2 and Sox10 but not Olig2 or Nkx2.2 possessed the capability of driving oligodendrocyte lineage-specific differentiation. Interestingly, the expression of Olig2 gene in avian olfactory epithelium from E11.5 onward has been reported [28]. However, no expression of Olig2 or Nkx2.2 was detected in the adult human olfactory epithelial-derived NSFCs by immunocytochemistry. Western blot analysis further demonstrated the absence of endogenous Nkx2.2 expression. In contrast, a low level of endogenous Olig2 expression was detected in the NSFCs. This suggests that the low level of endogenous Olig2 expression was not sufficient to cooperate with Nkx2.2 to induce NSFC differentiation into oligodendrocytes.

In the present study, the mechanism of induction of oligodendrocytes by Olig2 and Nkx2.2 was investigated. Previous studies indicated that overexpression of Olig2 alone in ovo induced the expression of Sox10 but not other oligodendrocyte-specific transcription factors, such as Nkx2.2 [19, 28, 42]. The Sox proteins represent a family of high-mobility group–containing transcription factors. So far, at least two Sox proteins are known to be involved in the development of myelin-forming oligodendrocytes. Sox9 regulates oligodendrocyte specification, whereas Sox10 is required for terminal differentiation [25, 43–45]. These findings suggest that Sox10 may function downstream of Olig2 and mediate the function of Olig2 in collaboration with Nkx2.2 to control oligodendrocyte differentiation and maturation. In agreement with this observation, NSFCs transfected with Olig2 alone expressed Sox10, although only a small percentage of transfected NSFCs were able to respond to Olig2 by the induction of Sox10. However, NSFCs transfected with Olig2 and Nkx2.2 together induced more Sox10, consistent with a previous report that an interaction between Olig2 and Nkx2.2 could promote Sox10 expression in chick neural tube–generated cells [42]. Consistent with the concept that Sox10 functions downstream of Olig2, overexpression of Sox10 and Nkx2.2 in NSFCs achieved equivalent effects compared with cells with Olig2 and Nkx2.2. Expression of Sox10 in conjunction with Nkx2.2 resulted in myelin gene expression, which is important for terminal oligodendrocyte differentiation [25, 46, 47]. NSFCs expressing Olig2 or Sox10 alone did not express the myelin gene or differentiate into oligodendrocytes, possibly because Sox10 itself is a weak transcriptional activator and needs to exert its function in concert with other transcription factors, such as Nkx2.2 [43, 48].

In vivo evidence revealed that oligodendrocyte progenitors express both Sox9 and Sox10 and can cope with loss of either protein. In Sox10 knockout mice, a few residual MBP-positive oligodendrocytes are still found in the spinal cord at birth [25, 45]. This raises the possibility that other transcription factors might be involved in the regulation of myelin gene expression as well. Interestingly, in our study, although only 25.5% of NSFCs transfected with Olig2 and Nkx2.2 induced Sox10 expression, more than 84% CNP-, GalC-, RIP-, or MBP-positive cells were found among the transfected cells. This result supports the hypothesis that other unidentified molecules might exist to regulate expression of myelin proteins cooperating with Nkx2.2 in vitro. Those unidentified factors could be the downstream proteins induced by Olig2 and possess a similar function as Sox10 in driving NSFCs towards the more mature differentiated oligodendrocyte phenotypes. Together, our results indicated that Sox10 in adult progenitors could mimic the effectsofOlig2collaborationwithNkx2.2tocontrolmyelin gene expression and oligodendrocyte differentiation.

Previously, olfactory ensheathing cells (OECs) [49], oligodendrocytes from mouse embryonic stem cells [50], and oligodendrocyte precursor cells from 10-day-old rats [51] have been reported to form myelin when cocultured with DRGNs. Others report that OECs fail to myelinate axons of DRGNs in vitro [52] but could form myelin when transplanted to the demyelinated spinal cords of rats [53]. In the present study, although NSFCs transfected with Olig2-Nkx2.2 or Sox10-Nkx2.2 gained the expression of more mature oligodendrocyte markers and in coculture were competent to initiate axonal ensheathment, they did not form myelin. This may reflect that myelination is a complicated process that requires a specific in vitro environment that was not provided by our culture conditions. This possibility is consistent with other reports [37]. Future studies will examine the ability of these transfected cells to remyelinated demyelinated regions of rat spinal cord. Furthermore, substantial evidence exists demonstrating differences between species, especially when comparisons between human and rodent cells are undertaken [37].

In summary, these studies reveal, first, that the transcription factors (Olig2 and Nkx2.2) that control oligodendrocytic development in embryonic chick and rodent CNS are able to direct adult human olfactory-derived progenitors towards oligodendrocyte lineage. Second, in this model, Olig2 and Nkx2.2 functioned cooperatively to produce oligodendrocyte differentiation. Neither Olig2 nor Nkx2.2 alone showed phenotypic changes. Third, NSFCs transfected with Olig2 or Nkx2.2 or Sox10 and Nkx2.2 expressed oligodendrocyte-specific antigens, including GalC, CNP, RIP, and human MBP. Fourth, NSFCs transfected with Olig2 and Nkx2.2 or Sox10 and Nkx2.2 and cocultured with DRGNs gained the capacity to form axonal ensheathments. Furthermore, it was particularly interesting to note that molecular mechanisms that function in early avian and rodent embryonic development, as expected, can be used to drive progenitors derived from individuals 22 or 96 years of age.

The use of transcription factors to modulate adult human olfactory epithelial-derived progenitors to differentiate into specific neuronal or glial cell types may expand the therapeutic potential of these progenitors. This is especially important, because the readily accessible location of adult olfactory neuroepithelium, which does not require highly invasive surgery for its biopsy [6], could provide an autologous progenitor source for cell replacement transplantation strategies. Furthermore, the use of these progenitors would eliminate ethical concerns related to the use of embryonic stem cells, avoid donor availability issues, and ensure complete histocompatibility, thereby negating the need for immunosuppression.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Xiaodong Zhang and Jun Cai contributed equally to this work. The authors thank George Harding for his assistance with the confocal microscopy and Cathie Caple for technical assistance with the electron microscopy. This work was supported by NIH (1920RR15576 to F.J.R.), Kentucky Spinal Cord Head Injury Research Trust (to M.Q.), and National Multiple Sclerosis Society (FA1400-A-1 to J.C.).


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received October 12, 2004; accepted for publication November 22, 2004.



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