Induction of Oligodendrocytes From Adult Human Olfactory Epithelial-Derived Progenitors by Transcription Factors
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《干细胞学杂志》
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@gwise.louisville.edu
ABSTRACT
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 . Dissociated cultures of adult olfactory neuroepithelium isolated from cadavers , or patients undergoing endoscopic nasal sinus surgery , 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 .
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 . 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 under the influence of the ventral midline signal sonic hedgehog . 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 . Molecular and genetic studies have demonstrated that expression of the Olig genes is required for oligodendrocyte lineage determination in vivo . 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 and the homeodomain transcription factor Nkx2.2 . 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 . 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 .
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
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 ?-tubulin III and peripherin (Figs. 1I, 1J); 49.1 ± 2.9% were positive for nestin (Figs. 1E, 1M) in the absence of triton; 26.1 ± 1.7% were positive for A2B5 (Fig. 1J); and 40.5 ± 2.3% were positive for neuronal cell adhesion molecule (NCAM) (Fig. 1K). 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
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% ?-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, 1L, 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–2C). 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. 1D). 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, 3B). 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. 4).
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.
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.
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. 2B). 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 2). Similarly, no detectable lineage-restricted changes were observed in NSFCs transfected with Nkx2.2, Sox10 cDNA, or control vectors alone (Table 2).
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. 5A). 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–5D; Table 2). In addition, the transfected cells coexpressed GalC and CNP (Fig. 5E) as well as RIP and MBP (Fig. 5F). 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 2).
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, 6E). 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, 5F, 6C, 6E).
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 . 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, 1L); 49% of the NSFCs expressed nestin, which is expressed in neural stem cells, progenitors, or proliferating oligodendrocyte progenitors ; and > 97% of the NSFCs were peripherin-positive (Fig. 1E) before transfection (0 DIV). After transfection with Olig2-Nkx2.2, NSFCs formed elaborate compound processes (Figs. 1B–1D) and gradually lost their expression of nestin (Fig. 1M); 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, 1M). It was not until 9 DIV that > 85% of the cells expressed the more mature oligodendrocyte markers RIP and MBP (Figs. 1G, 1M). At no time point was expression of NeuN, GFAP (Figs. 1H, 1M), O4, and OX 42 (Fig. 1M) 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 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 2). In contrast, 25.5 ± 1.8% of the NSFCs transfected with both Olig2 and Nkx2.2 expressed Sox10 (Table 2). 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, 7B; Table 2). However, when Sox 10 and Nkx2.2 were introduced into the cells simultaneously, the cells assumed the characteristic phenotype of the oligodendrocytes (Fig. 7C; Table 2).
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, 6E). 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. 6D; Table 2). In addition, several bands of smaller sizes were detected, which were probably degradation products of the Sox10 protein (Fig. 6D).
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, 8B). The transfected cells were MBP-positive. Pilot ultrastructural analysis demonstrated the early stages of axonal ensheathment by processes from the transfected NSFCs (Figs. 9A–9C). Frequent regions of subplasmalemmal densities were observed at the contact sites between axons and the NSFC processes, perhaps reflecting the initial development of mesaxons.
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.
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
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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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@gwise.louisville.edu
ABSTRACT
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 . Dissociated cultures of adult olfactory neuroepithelium isolated from cadavers , or patients undergoing endoscopic nasal sinus surgery , 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 .
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 . 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 under the influence of the ventral midline signal sonic hedgehog . 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 . Molecular and genetic studies have demonstrated that expression of the Olig genes is required for oligodendrocyte lineage determination in vivo . 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 and the homeodomain transcription factor Nkx2.2 . 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 . 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 .
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
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 ?-tubulin III and peripherin (Figs. 1I, 1J); 49.1 ± 2.9% were positive for nestin (Figs. 1E, 1M) in the absence of triton; 26.1 ± 1.7% were positive for A2B5 (Fig. 1J); and 40.5 ± 2.3% were positive for neuronal cell adhesion molecule (NCAM) (Fig. 1K). 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
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% ?-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, 1L, 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–2C). 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. 1D). 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, 3B). 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. 4).
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.
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.
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. 2B). 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 2). Similarly, no detectable lineage-restricted changes were observed in NSFCs transfected with Nkx2.2, Sox10 cDNA, or control vectors alone (Table 2).
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. 5A). 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–5D; Table 2). In addition, the transfected cells coexpressed GalC and CNP (Fig. 5E) as well as RIP and MBP (Fig. 5F). 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 2).
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, 6E). 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, 5F, 6C, 6E).
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 . 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, 1L); 49% of the NSFCs expressed nestin, which is expressed in neural stem cells, progenitors, or proliferating oligodendrocyte progenitors ; and > 97% of the NSFCs were peripherin-positive (Fig. 1E) before transfection (0 DIV). After transfection with Olig2-Nkx2.2, NSFCs formed elaborate compound processes (Figs. 1B–1D) and gradually lost their expression of nestin (Fig. 1M); 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, 1M). It was not until 9 DIV that > 85% of the cells expressed the more mature oligodendrocyte markers RIP and MBP (Figs. 1G, 1M). At no time point was expression of NeuN, GFAP (Figs. 1H, 1M), O4, and OX 42 (Fig. 1M) 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 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 2). In contrast, 25.5 ± 1.8% of the NSFCs transfected with both Olig2 and Nkx2.2 expressed Sox10 (Table 2). 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, 7B; Table 2). However, when Sox 10 and Nkx2.2 were introduced into the cells simultaneously, the cells assumed the characteristic phenotype of the oligodendrocytes (Fig. 7C; Table 2).
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, 6E). 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. 6D; Table 2). In addition, several bands of smaller sizes were detected, which were probably degradation products of the Sox10 protein (Fig. 6D).
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, 8B). The transfected cells were MBP-positive. Pilot ultrastructural analysis demonstrated the early stages of axonal ensheathment by processes from the transfected NSFCs (Figs. 9A–9C). Frequent regions of subplasmalemmal densities were observed at the contact sites between axons and the NSFC processes, perhaps reflecting the initial development of mesaxons.
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.
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
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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