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High purity of human oligodendrocyte progenitor cells obtained from neural stem cells: Suitable for clinical application
Journal of Neuroscience Methods 240 (2015) 61–66
Contents lists available at ScienceDirect
Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth
Basic Neuroscience Short communication
High purity of human oligodendrocyte progenitor cells obtained from neural stem cells: Suitable for clinical application Caiying Wang a,b,1 , Zuo Luan a,∗,1 , Yinxiang Yang a , Zhaoyan Wang a , Qian Wang a , Yabin Lu a , Qingan Du a a b
Department of Pediatrics, Navy General Hospital, NO. 6, Fucheng Road, Beijing, China Department of Pediatrics, Beijing Ditan Hospital, NO. 8, Jingshun East Street, Beijing, China
a r t i c l e
i n f o
Article history: Received 21 July 2014 Received in revised form 19 October 2014 Accepted 21 October 2014 Available online 8 November 2014 Keywords: Oligodendrocyte progenitor cell Neural stem cells Clinical application
a b s t r a c t Background: Recent studies have suggested that the transplantation of oligodendrocyte progenitor cells (OPCs) may be a promising potential therapeutic strategy for a broad range of diseases affecting myelin, such as multiple sclerosis, periventricular leukomalacia, and spinal cord injury. Clinical interest arose from the potential of human stem cells to be directed to OPCs for the clinical application of treating these diseases since large quantities of high quality OPCs are needed. However, to date, there have been precious few studies about OPC induction from human neural stem cells (NSCs). New method: Here we successfully directed human fetal NSCs into highly pure OPCs using a cocktail of basic fibroblast growth factor, platelet-derived growth factor, and neurotrophic factor-3. Results: These cells had typical morphology of OPCs, and 80–90% of them expressed specific OPC markers such as A2B5, O4, Sox10 and PDGF-␣R. When exposed to differentiation medium, 90% of the cells differentiated into oligodendrocytes. The OPCs could be amplified in our culture medium and passaged at least 10 times. Comparison with a existing method: Compared to a recent published method, this protocol had much higher stability and repeatability, and OPCs could be obtained from NSCs from passage 5 to 38. It also obtained more highly pure OPCs (80–90%) via simpler and more convenient manipulation. Conclusions: This study provided an easy and efficient method to obtain large quantities of high-quality human OPCs to meet clinical demand. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Demyelination or myelination delay, which leads to severe functional disorders, contributes to the pathological process of a broad range of diseases, including periventricular leukomalacia, pediatric leukodystrophies, multiple sclerosis, and spinal cord injury, for which there is no effective treatment (Deng et al., 2008; Amin-Mansour, 2012). Recent studies have shown that grafted oligodendrocyte progenitor cells (OPCs) can differentiate into oligodendrocytes and develop compact myelin supplies to repair the injured white matter of rodents to an extent, which shines light on the treatment of these diseases (Cavazzin et al., 2006; Windrem et al., 2002, 2004; Webber et al., 2009).
∗ Corresponding author. Tel.: +86 13381207228; fax: +86 10 66958303. E-mail address: [email protected] (Z. Luan). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.jneumeth.2014.10.017 0165-0270/© 2014 Elsevier B.V. All rights reserved.
Human OPCs have historically been induced from embryonic stem cells (ESCs) or obtained by immunomagnetic sorting by their specific surface markers from donor brain tissues (Hu et al., 2009; Windrem et al., 2004). However, the former might be contaminated by rudimentary ESCs, which have strong potential tumorigenicity (Ben-David and Benvenisty, 2011), while the latter required large amounts of donor brain tissues to obtain adequate cells. As such, neither could meet clinical demand, which requires both large quantity and high quality. To date, NSCs have safely been used to treat patients with stroke, Parkinson’s disease, and cerebral palsy. They also have the capacity to proliferate robustly and differentiate multi-directionally, which may make them an ideal source for human OPCs for clinical use. Studies have shown that, in contrast to their rodent counterparts, human neural stem cells (hNSCs) give rise to small numbers of oligodendrocytes both in vitro and after transplantation in vivo (Wright et al., 2006; Neri et al., 2010); as a result, it is very difficult to obtain OPCs from hNSCs.
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Fig. 1. (A) Human neural stem cells (NSCs) formed neurospheres floating in the medium and tested positive for the NSC markers nestin (B) and Musashi (C). When growth factors were removed from the medium, they differentiated into Tuj1-positive neurons (D) and glial fibrillary acidic protein–positive astrocytes (E). The cells stained green were positive, while the cell nuclei were stained with 4,6-diamidino-2-phenylindle (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Monaco et al. (2012) recently reported that human neural progenitors could be induced to differentiate through many of the stages of oligodendrocyte lineage development; in their culture system, the majority of cells expressed OPC markers. In this study, we established a method that could obtain higher-purity OPCs from hNSCs with much higher stability and repeatability via simpler manipulation that could provide an adequate source of cells for future clinical use. 2. Materials and methods 2.1. Materials and Reagents The detailed information of the materials and reagents used in this study was provided in supplemental Table 1. 2.2. Culture of hNSCs We cultured hNSCs as previously described (Jordan et al., 2008). An aborted human fetus aged 11 weeks post-conception was obtained from a woman at the Department of Obstetrics and Gynecology of the Navy General Hospital, Beijing, China, who had requested to terminate gestation and consented to donate the aborted fetus after being fully informed of the study according to the guidelines approved by the hospital’s ethics committee. The brain was extracted and placed in cold phosphate-buffered saline (PBS). The brain was mechanically dissociated into a suspension of single cells by repetitive blowing using a 200-L pipette. The cells were collected by centrifugation (1400 rpm for 5 min), the supernatant was discarded, and the cell pellet was resuspended with Dulbecco’s modified Eagle medium (DMEM) mixed with F12 medium (3:1) supplemented with 15 mM HEPES, 0.15% d-glucose, 100 g/mL transferrin, 20 nM progesterone, 60 M putrescine, 30 nM sodium selenite, 5 g/mL insulin, 5 g/mL heparin, 1% l-glutamine, 20 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor (EGF), 10 ng/mL
leukemia inhibitory factor (LIF), and 100 U/mL penicillin and streptomycin. All of the reagents were purchased from Sigma unless stated otherwise. The cells were counted using a blood cell counting plate and seeded into T25 cell culture bottles in 5 mL of culture media (2 × 106 cells/bottle). Cells were maintained at 37 ◦ C in a humidified atmosphere with 8.5% CO2 in an incubator. The medium was replenished (half and half) every 4 days. The expanded cells formed colonies and were passaged every 7 days. During passaging, the cells were centrifuged (1400 rpm for 5 min), the supernatant was collected as conditioned medium, and the cell pellet was resuspended in 1 mL of 0.025% trypsin diluted in PBS and incubated at 37 ◦ C until the neurospheres were unconsolidated, after which 100 L of trypsin inhibitor was added to terminate the digestion. The spheres were gently dissociated into single cells, 15 mL of conditioned medium was added, and the cells were centrifuged, collected, and counted as described above. The cells were then seeded into T25 cell culture bottles in 5 mL of medium containing 2/3 fresh medium and 1/3 conditioned medium. For immunocytochemistry, some cells were plated onto 24-well plates pre-coated with 0.01% poly-d-lysine and 3.3 g/mL laminin, and incubated for 2 h before being fixed with 4% paraformaldehyde at room temperature for 15 min for immunocytochemistry. 2.3. Induction and amplification of OPCs The NSCs were cultured for 10 days and the 10-day-old neurospheres were used for this experiment. The neurospheres were dissociated into single cells as described above. The cells were collected by centrifugation, the supernatant was discarded, and the cell pellet was counted and resuspended at a density of 4 × 105 /mL in 5 mL of DMEM-F12 medium (3:1) supplemented with 2% B27, 5 g/mL transferrin, 10 nM progesterone, 30 M putrescine, 15 nM sodium selenite, 5 g/mL insulin, 5 g/mL heparin, 5 mM lactate, 5 ng/mL bFGF, 10 ng/mL platelet-derived growth factor, 10 ng/mL neurotrophic factor-3 (NT3), and 100 U/mL
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Fig. 2. (A) At 48 h after exposure to the induction medium, cells spheres attached to the bottom of the dish. (B) On the 4th day, a few cells migrated out of the attached spheres. (C) Subsequently, increasing numbers of cells migrated out. (D) On the 7th to 9th day, the cells reached 90% confluence and most had the morphology of early oligodendrocyte progenitor cells (OPCs). (E and F) Cells passaged five (E) and 10 (F) times retained the typical OPC morphology.
penicillin and streptomycin. The cells were maintained in the above medium at 37 ◦ C in a humidified atmosphere with 5% CO2 for 7–9 days. The cells formed spheres and attached to the bottom of the dish, and many single cells migrated out of the spheres. When the cells reached 90% confluence, they were blown up by pipette and re-plated onto T25 culture bottles at a density of 1 × 105 cells/bottle. For immunocytochemistry, these cells were plated onto 24-well plates pre-coated with poly-dlysine and laminin in DMEM-F12 supplemented with 2% B27. After 2 days, the cells were fixed with 4% paraformaldehyde for immunocytochemistry. Some cells were induced to differentiate by exposure to differentiation medium for 7 days prior to the immunocytochemistry to detect the expression of GalC or exposure to OPC differentiation medium (OPCDM) which was purchased from Sciencell Research Laboratories of USA.
2.4. Immunocytochemistry Immunocytochemistry was conducted as described previously (Wang et al., 2011). The NSCs were identified using monoclonal mouse anti-nestin (1:500) and anti-Musashi (1:500) antibodies. Mouse anti-Tuj1 (1:500) and rabbit anti-glial fibrillary acidic protein (GFAP, 1:50) antibodies were used to detect neurons and astrocytes. OPCs were detected using monoclonal mouse antiA2B5 (1:50), mouse anti-Sox10 (1:50), mouse anti-O4 (1:250), and mouse anti-PDGF receptor-␣ (PDGF-␣R, 1:250) antibodies, while the oligodendrocytes were detected using mouse anti-GalC (5 g/mL). Alexa 488 – conjugated goat anti-mouse and Alexa 594 – conjugated goat anti-rabbit secondary antibodies were used at 1:800 and 1:500, respectively. Cell nuclei were labeled with 4,6diamidino-2-phenylindle (DAPI). Wide-field microscopic images were acquired using a fluorescence microscope equipped with a digital camera (Olympus, Japan).
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Fig. 3. (A–L) These cells were positive for A2B5 (A–C), O4 (D–F), Sox10 (G–I), and platelet-derived growth factor receptor-␣ (PDGF-␣R) (J–L), the cells stained green were positive and the cell nuclei were stained with 4,6-diamidino-2-phenylindle (blue), the percentages of positive cells were 80.5 ± 2.1% (A2B5), 85.4 ± 3.9% (O4), 92.5 ± 3.7% (Sox10), and 93.2 ± 1.7% (PDGF-␣R). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
2.5. Data collection
3. Results
The percentage of positive cells was obtained by counting the number of DAPI+ cells and the number of cells stained positively by each antibody in 15 randomly selected fields from three wells using a 40× objective lens. The results shown are the average ± SEM of the data from five experiments.
3.1. NSC amplification and identification The NSCs in this study formed neurospheres floating in the NSC culture medium (Fig. 1A). They could be passaged at least 40 times in vitro, suggesting that they had the capacity for robust
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proliferation. These cells expressed the NSC markers nestin and Musashi (Fig. 1B and C). When neural growth factors were removed, the cells differentiated into neurons and glia (Fig. 1D and E), suggesting that they had the potential for multi-directional differentiation. 3.2. OPC induction, amplification, and identification NSCs from passages 5–38 were used in this experiment. The cells formed small spheres after exposure to medium supplemented with bFGF, PDGF, and NT3; 48 h later, these spheres were attached to the bottom of the dish (Fig. 2A). On the 3rd or 4th day, a few cells migrated out of the attached spheres. These cells had round, small, and bright cell bodies and two or three short processes, just like the morphology of early stage OPCs (Fig. 2B). Increasingly more cells migrated out from the spheres over the following few days (Fig. 2C). On the 7th to 9th days, the cells reached 90% confluence, most had morphology similar to that of early stage OPCs, and some cells in high-density areas had ramified processes similar those seen in medium-stage OPC morphology (Fig. 2D). The cells were blown up and counted, and the results showed that approximately 7 × 105 OPCs were generated from 2 × 106 NSCs. Moreover, these OPCs could be amplified in the present culture system and were passaged 10 times in this study. After passaging, the cells retained the primary morphology and then proliferated and reached 90% confluence in 7–9 days, and the cell numbers increased threefold at each passage (Fig. 2E and F). Two days after being plated onto 24 wells pre-treated with PDLlaminin, the cells were fixed and identified using the OPC-specific markers A2B5, O4, Sox10, and PDGF-␣R by immunocytochemistry. Under a fluorescence microscope, the number of positive cells and the total number of cells per randomly selected field were counted. The average number of 15 randomly selected fields from three wells was calculated, after which the percentage of positive cells was calculated. The average ± SEM values of data from five experiments showed that 80.5 ± 2.1% of the cells expressed A2B5, 85.4 ± 3.9% expressed O4, 92.5 ± 3.7% expressed Sox10, and 93.2 ± 1.7% expressed PDGF-␣R (Fig. 3A–L). The cells were also stained with the neuronal marker Tuj1 and the astrocytic marker GFAP, and the results showed that only 5.6 ± 1.6% and 1.2 ± 0.5% of the cells expressed Tuj1 and GFAP, respectively (Fig. 4). After being exposed to differentiation medium, 90% of the cells differentiated into GalC-expressing oligodendrocytes (Fig. 5A and B). Moreover, when the cells were exposed to OPCDM,
Fig. 4. The chart shows that most cells expressed the oligodendrocyte progenitor cell (OPC) markers A2B5, O4, Sox10 and platelet-derived growth factor receptor␣ (PDGF-␣R), while only a very small proportion of them expressed the neuronal marker Tuj1 and the astrocytic marker glial fibrillary acidic protein (GFAP).
they differentiated into oligodendrocytes with more mature morphology and more complex processes (Fig. 5C), suggesting that, under the proper conditions, they could differentiate into mature oligodendrocytes. 4. Discussion Recent studies have shown that the transplantation of OPCs, myelin-forming cells, could repair and prevent the progression of demyelinating lesions, and alleviate functional disorders in animal models, suggesting that cell therapy might be an effective treatment strategy (Cavazzin et al., 2006; Windrem et al., 2002, 2004; Webber et al., 2009). The purpose of this study was to obtain human OPCs that met clinical treatment demands. There were several points that made the cells obtained in this study suitable for future clinical applications. First, NSCs have a much lower risk of potential tumorgenicity than ESCs. Residual stem cells might exist in NSC- or ESC-derived cell populations. In this sense, OPCs from NSCs were
Fig. 5. When the cells were exposed to differentiation medium, they differentiated into oligodendrocytes (A) that expressed the specific marker GalC (B, green) at a rate of 91.3 ± 3.3%. When the cells were allowed to differentiate in OPCDM, they developed into oligodendrocytes with more mature morphology (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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much safer than those from ESCs. Second, the cells had higher purity as evidenced by their morphological homogeneity, and 80–90% of them expressed markers specific for OPCs, suggesting that they had a strong capacity to repair injured myelin structures because the abundant cells could differentiate into myelin-forming oligodendrocytes. Finally, human fetal NSCs have the capacity for unlimited proliferation, and the OPCs derived from NSCs in our culture system could be passaged at least 10 times, so large amounts of OPCs could be easily obtained using this method, which could meet the clinical demand. We noted a recently published method that directed hNSCs into OPCs (Monaco et al., 2012) that was similar to this protocol. Compared to that method, ours had several advantages. First, we could obtain OPCs with 80–90% purity from hNSCs in only 7 days compared to 70% purity in 21 days. As such, our protocol was time-saving, which meant a lower chance of potential heteromorphosis and microbial contamination. Second, compared with the NSCs of passages <11 in their study, NSCs of higher passages could be used for the OPC induction in our method. In this study, highly pure OPCs were successfully obtained from NSCs at passages 5–38. This may mean that our protocol was more stable and effective, but the possibility that the NSCs in our culture system were simply more stable could not be ruled out. In brief, NSCs of higher passages that can be used for OPC induction could guarantee an adequate cell source, avoiding the need for a large amount of human donor brain tissue. In addition, we obtained similar results in three NSC cell lines (supplemental Table 2), suggesting the high stability and repeatability of this protocol. Finally, previous studies, including that of Monaco MC et al., showed that sonic hedgehog (SHH) was important for human OPC generation (Hu et al., 2009); however, the present study showed that bFGF, PDGF, and NT3 were enough to direct NSCs into OPCs and that SHH was not necessary. The shorter induction period and fewer growth factors made our protocol low-cost. In conclusion, here we successfully obtained human OPCs that met the clinical demands of high quality and quantity using a quick, stable, and economic method.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. jneumeth.2014.10.017. References Amin-Mansour B. Multiple sclerosis (MS) as a demyelinating disease: a review on neurosurgical approaches. J Inj Violence Res 2012;4(3 Suppl. 1), Paper No. 31. Ben-David U, Benvenisty N. The tumorigenicity of human embryonic and induced pluripotent stem cells. Nat Rev Cancer 2011;11(4):268–77. Cavazzin C, Ferrari D, Facchetti F, Russignan A, Vescovi AL, La Porta CA, et al. Unique expression and localization of aquaporin-4 and aquaporin-9 in murine and human neural stem cells and in their glial progeny. Glia 2006;53(2):167–81. Deng W, Pleasure J, Pleasure D. Progress in periventricular leukomalacia. Arch Neurol 2008;65(10):1291–5. Hu BY, Du ZW, Li XJ, Ayala M, Zhang SC. Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects. Development 2009;136(9):1443–52. Jordan PM, Cain LD, Wu P. Astrocytes enhance long-term survival of cholinergic neurons differentiated from human fetal neural stem cells. J Neurosci Res 2008;86(1):35–47. Monaco MC, Maric D, Bandeian A, Leibovitch E, Yang W, Major EO. Progenitorderived oligodendrocyte culture system from human fetal brain. J Vis Exp 2012;70:e4274, http://dx.doi.org/10.3791/4274. Neri M, Maderna C, Ferrari D, Cavazzin C, Vescovi AL, Gritti A. Robust generation of oligodendrocyte progenitors from human neural stem cells and engraftment in experimental demyelination models in mice. PLoS ONE 2010;5(4):e10145. Wang C, Luan Z, Yang Y, Wang Z, Cui Y, Gu G. Valproic acid induces apoptosis in differentiating neurons by the release of tumor necrosis factor-␣ from activated astrocytes. Neurosci Lett 2011;497(2):122–7. Webber DJ, van Blitterswijk M, Chandran S. Neuroprotective effect of oligodendrocyte precursor cell transplantation in a long-term model of periventricular leukomalacia. Am J Pathol 2009;175(6):2332–42. Windrem MS, Roy NS, Wang J, Nunes M, Benraiss A, Goodman R, et al. Progenitor cells derived from the adult human subcortical white matter disperse and differentiate as oligodendrocytes within demyelinated lesions of the rat brain. J Neurosci Res 2002;69:966–75. Windrem MS, Nunes MC, Rashbaum WK, Schwartz TH, Goodman RA, McKhann G 2nd, et al. Fetal and adult human oligodendrocyte progenitor cell isolates myelinate the congenitally dysmyelinated brain. Nat Med 2004;10(1):93–7. Wright LS, Prowse KR, Wallace K, Linskens MH, Svendsen CN. Human progenitor cells isolated from the developing cortex undergo decreased neurogenesis and eventual senescence following expansion in vitro. Exp Cell Res 2006;312: 2107–20.
Contents lists available at ScienceDirect
Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth
Basic Neuroscience Short communication
High purity of human oligodendrocyte progenitor cells obtained from neural stem cells: Suitable for clinical application Caiying Wang a,b,1 , Zuo Luan a,∗,1 , Yinxiang Yang a , Zhaoyan Wang a , Qian Wang a , Yabin Lu a , Qingan Du a a b
Department of Pediatrics, Navy General Hospital, NO. 6, Fucheng Road, Beijing, China Department of Pediatrics, Beijing Ditan Hospital, NO. 8, Jingshun East Street, Beijing, China
a r t i c l e
i n f o
Article history: Received 21 July 2014 Received in revised form 19 October 2014 Accepted 21 October 2014 Available online 8 November 2014 Keywords: Oligodendrocyte progenitor cell Neural stem cells Clinical application
a b s t r a c t Background: Recent studies have suggested that the transplantation of oligodendrocyte progenitor cells (OPCs) may be a promising potential therapeutic strategy for a broad range of diseases affecting myelin, such as multiple sclerosis, periventricular leukomalacia, and spinal cord injury. Clinical interest arose from the potential of human stem cells to be directed to OPCs for the clinical application of treating these diseases since large quantities of high quality OPCs are needed. However, to date, there have been precious few studies about OPC induction from human neural stem cells (NSCs). New method: Here we successfully directed human fetal NSCs into highly pure OPCs using a cocktail of basic fibroblast growth factor, platelet-derived growth factor, and neurotrophic factor-3. Results: These cells had typical morphology of OPCs, and 80–90% of them expressed specific OPC markers such as A2B5, O4, Sox10 and PDGF-␣R. When exposed to differentiation medium, 90% of the cells differentiated into oligodendrocytes. The OPCs could be amplified in our culture medium and passaged at least 10 times. Comparison with a existing method: Compared to a recent published method, this protocol had much higher stability and repeatability, and OPCs could be obtained from NSCs from passage 5 to 38. It also obtained more highly pure OPCs (80–90%) via simpler and more convenient manipulation. Conclusions: This study provided an easy and efficient method to obtain large quantities of high-quality human OPCs to meet clinical demand. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Demyelination or myelination delay, which leads to severe functional disorders, contributes to the pathological process of a broad range of diseases, including periventricular leukomalacia, pediatric leukodystrophies, multiple sclerosis, and spinal cord injury, for which there is no effective treatment (Deng et al., 2008; Amin-Mansour, 2012). Recent studies have shown that grafted oligodendrocyte progenitor cells (OPCs) can differentiate into oligodendrocytes and develop compact myelin supplies to repair the injured white matter of rodents to an extent, which shines light on the treatment of these diseases (Cavazzin et al., 2006; Windrem et al., 2002, 2004; Webber et al., 2009).
∗ Corresponding author. Tel.: +86 13381207228; fax: +86 10 66958303. E-mail address: [email protected] (Z. Luan). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.jneumeth.2014.10.017 0165-0270/© 2014 Elsevier B.V. All rights reserved.
Human OPCs have historically been induced from embryonic stem cells (ESCs) or obtained by immunomagnetic sorting by their specific surface markers from donor brain tissues (Hu et al., 2009; Windrem et al., 2004). However, the former might be contaminated by rudimentary ESCs, which have strong potential tumorigenicity (Ben-David and Benvenisty, 2011), while the latter required large amounts of donor brain tissues to obtain adequate cells. As such, neither could meet clinical demand, which requires both large quantity and high quality. To date, NSCs have safely been used to treat patients with stroke, Parkinson’s disease, and cerebral palsy. They also have the capacity to proliferate robustly and differentiate multi-directionally, which may make them an ideal source for human OPCs for clinical use. Studies have shown that, in contrast to their rodent counterparts, human neural stem cells (hNSCs) give rise to small numbers of oligodendrocytes both in vitro and after transplantation in vivo (Wright et al., 2006; Neri et al., 2010); as a result, it is very difficult to obtain OPCs from hNSCs.
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Fig. 1. (A) Human neural stem cells (NSCs) formed neurospheres floating in the medium and tested positive for the NSC markers nestin (B) and Musashi (C). When growth factors were removed from the medium, they differentiated into Tuj1-positive neurons (D) and glial fibrillary acidic protein–positive astrocytes (E). The cells stained green were positive, while the cell nuclei were stained with 4,6-diamidino-2-phenylindle (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Monaco et al. (2012) recently reported that human neural progenitors could be induced to differentiate through many of the stages of oligodendrocyte lineage development; in their culture system, the majority of cells expressed OPC markers. In this study, we established a method that could obtain higher-purity OPCs from hNSCs with much higher stability and repeatability via simpler manipulation that could provide an adequate source of cells for future clinical use. 2. Materials and methods 2.1. Materials and Reagents The detailed information of the materials and reagents used in this study was provided in supplemental Table 1. 2.2. Culture of hNSCs We cultured hNSCs as previously described (Jordan et al., 2008). An aborted human fetus aged 11 weeks post-conception was obtained from a woman at the Department of Obstetrics and Gynecology of the Navy General Hospital, Beijing, China, who had requested to terminate gestation and consented to donate the aborted fetus after being fully informed of the study according to the guidelines approved by the hospital’s ethics committee. The brain was extracted and placed in cold phosphate-buffered saline (PBS). The brain was mechanically dissociated into a suspension of single cells by repetitive blowing using a 200-L pipette. The cells were collected by centrifugation (1400 rpm for 5 min), the supernatant was discarded, and the cell pellet was resuspended with Dulbecco’s modified Eagle medium (DMEM) mixed with F12 medium (3:1) supplemented with 15 mM HEPES, 0.15% d-glucose, 100 g/mL transferrin, 20 nM progesterone, 60 M putrescine, 30 nM sodium selenite, 5 g/mL insulin, 5 g/mL heparin, 1% l-glutamine, 20 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor (EGF), 10 ng/mL
leukemia inhibitory factor (LIF), and 100 U/mL penicillin and streptomycin. All of the reagents were purchased from Sigma unless stated otherwise. The cells were counted using a blood cell counting plate and seeded into T25 cell culture bottles in 5 mL of culture media (2 × 106 cells/bottle). Cells were maintained at 37 ◦ C in a humidified atmosphere with 8.5% CO2 in an incubator. The medium was replenished (half and half) every 4 days. The expanded cells formed colonies and were passaged every 7 days. During passaging, the cells were centrifuged (1400 rpm for 5 min), the supernatant was collected as conditioned medium, and the cell pellet was resuspended in 1 mL of 0.025% trypsin diluted in PBS and incubated at 37 ◦ C until the neurospheres were unconsolidated, after which 100 L of trypsin inhibitor was added to terminate the digestion. The spheres were gently dissociated into single cells, 15 mL of conditioned medium was added, and the cells were centrifuged, collected, and counted as described above. The cells were then seeded into T25 cell culture bottles in 5 mL of medium containing 2/3 fresh medium and 1/3 conditioned medium. For immunocytochemistry, some cells were plated onto 24-well plates pre-coated with 0.01% poly-d-lysine and 3.3 g/mL laminin, and incubated for 2 h before being fixed with 4% paraformaldehyde at room temperature for 15 min for immunocytochemistry. 2.3. Induction and amplification of OPCs The NSCs were cultured for 10 days and the 10-day-old neurospheres were used for this experiment. The neurospheres were dissociated into single cells as described above. The cells were collected by centrifugation, the supernatant was discarded, and the cell pellet was counted and resuspended at a density of 4 × 105 /mL in 5 mL of DMEM-F12 medium (3:1) supplemented with 2% B27, 5 g/mL transferrin, 10 nM progesterone, 30 M putrescine, 15 nM sodium selenite, 5 g/mL insulin, 5 g/mL heparin, 5 mM lactate, 5 ng/mL bFGF, 10 ng/mL platelet-derived growth factor, 10 ng/mL neurotrophic factor-3 (NT3), and 100 U/mL
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Fig. 2. (A) At 48 h after exposure to the induction medium, cells spheres attached to the bottom of the dish. (B) On the 4th day, a few cells migrated out of the attached spheres. (C) Subsequently, increasing numbers of cells migrated out. (D) On the 7th to 9th day, the cells reached 90% confluence and most had the morphology of early oligodendrocyte progenitor cells (OPCs). (E and F) Cells passaged five (E) and 10 (F) times retained the typical OPC morphology.
penicillin and streptomycin. The cells were maintained in the above medium at 37 ◦ C in a humidified atmosphere with 5% CO2 for 7–9 days. The cells formed spheres and attached to the bottom of the dish, and many single cells migrated out of the spheres. When the cells reached 90% confluence, they were blown up by pipette and re-plated onto T25 culture bottles at a density of 1 × 105 cells/bottle. For immunocytochemistry, these cells were plated onto 24-well plates pre-coated with poly-dlysine and laminin in DMEM-F12 supplemented with 2% B27. After 2 days, the cells were fixed with 4% paraformaldehyde for immunocytochemistry. Some cells were induced to differentiate by exposure to differentiation medium for 7 days prior to the immunocytochemistry to detect the expression of GalC or exposure to OPC differentiation medium (OPCDM) which was purchased from Sciencell Research Laboratories of USA.
2.4. Immunocytochemistry Immunocytochemistry was conducted as described previously (Wang et al., 2011). The NSCs were identified using monoclonal mouse anti-nestin (1:500) and anti-Musashi (1:500) antibodies. Mouse anti-Tuj1 (1:500) and rabbit anti-glial fibrillary acidic protein (GFAP, 1:50) antibodies were used to detect neurons and astrocytes. OPCs were detected using monoclonal mouse antiA2B5 (1:50), mouse anti-Sox10 (1:50), mouse anti-O4 (1:250), and mouse anti-PDGF receptor-␣ (PDGF-␣R, 1:250) antibodies, while the oligodendrocytes were detected using mouse anti-GalC (5 g/mL). Alexa 488 – conjugated goat anti-mouse and Alexa 594 – conjugated goat anti-rabbit secondary antibodies were used at 1:800 and 1:500, respectively. Cell nuclei were labeled with 4,6diamidino-2-phenylindle (DAPI). Wide-field microscopic images were acquired using a fluorescence microscope equipped with a digital camera (Olympus, Japan).
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Fig. 3. (A–L) These cells were positive for A2B5 (A–C), O4 (D–F), Sox10 (G–I), and platelet-derived growth factor receptor-␣ (PDGF-␣R) (J–L), the cells stained green were positive and the cell nuclei were stained with 4,6-diamidino-2-phenylindle (blue), the percentages of positive cells were 80.5 ± 2.1% (A2B5), 85.4 ± 3.9% (O4), 92.5 ± 3.7% (Sox10), and 93.2 ± 1.7% (PDGF-␣R). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
2.5. Data collection
3. Results
The percentage of positive cells was obtained by counting the number of DAPI+ cells and the number of cells stained positively by each antibody in 15 randomly selected fields from three wells using a 40× objective lens. The results shown are the average ± SEM of the data from five experiments.
3.1. NSC amplification and identification The NSCs in this study formed neurospheres floating in the NSC culture medium (Fig. 1A). They could be passaged at least 40 times in vitro, suggesting that they had the capacity for robust
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proliferation. These cells expressed the NSC markers nestin and Musashi (Fig. 1B and C). When neural growth factors were removed, the cells differentiated into neurons and glia (Fig. 1D and E), suggesting that they had the potential for multi-directional differentiation. 3.2. OPC induction, amplification, and identification NSCs from passages 5–38 were used in this experiment. The cells formed small spheres after exposure to medium supplemented with bFGF, PDGF, and NT3; 48 h later, these spheres were attached to the bottom of the dish (Fig. 2A). On the 3rd or 4th day, a few cells migrated out of the attached spheres. These cells had round, small, and bright cell bodies and two or three short processes, just like the morphology of early stage OPCs (Fig. 2B). Increasingly more cells migrated out from the spheres over the following few days (Fig. 2C). On the 7th to 9th days, the cells reached 90% confluence, most had morphology similar to that of early stage OPCs, and some cells in high-density areas had ramified processes similar those seen in medium-stage OPC morphology (Fig. 2D). The cells were blown up and counted, and the results showed that approximately 7 × 105 OPCs were generated from 2 × 106 NSCs. Moreover, these OPCs could be amplified in the present culture system and were passaged 10 times in this study. After passaging, the cells retained the primary morphology and then proliferated and reached 90% confluence in 7–9 days, and the cell numbers increased threefold at each passage (Fig. 2E and F). Two days after being plated onto 24 wells pre-treated with PDLlaminin, the cells were fixed and identified using the OPC-specific markers A2B5, O4, Sox10, and PDGF-␣R by immunocytochemistry. Under a fluorescence microscope, the number of positive cells and the total number of cells per randomly selected field were counted. The average number of 15 randomly selected fields from three wells was calculated, after which the percentage of positive cells was calculated. The average ± SEM values of data from five experiments showed that 80.5 ± 2.1% of the cells expressed A2B5, 85.4 ± 3.9% expressed O4, 92.5 ± 3.7% expressed Sox10, and 93.2 ± 1.7% expressed PDGF-␣R (Fig. 3A–L). The cells were also stained with the neuronal marker Tuj1 and the astrocytic marker GFAP, and the results showed that only 5.6 ± 1.6% and 1.2 ± 0.5% of the cells expressed Tuj1 and GFAP, respectively (Fig. 4). After being exposed to differentiation medium, 90% of the cells differentiated into GalC-expressing oligodendrocytes (Fig. 5A and B). Moreover, when the cells were exposed to OPCDM,
Fig. 4. The chart shows that most cells expressed the oligodendrocyte progenitor cell (OPC) markers A2B5, O4, Sox10 and platelet-derived growth factor receptor␣ (PDGF-␣R), while only a very small proportion of them expressed the neuronal marker Tuj1 and the astrocytic marker glial fibrillary acidic protein (GFAP).
they differentiated into oligodendrocytes with more mature morphology and more complex processes (Fig. 5C), suggesting that, under the proper conditions, they could differentiate into mature oligodendrocytes. 4. Discussion Recent studies have shown that the transplantation of OPCs, myelin-forming cells, could repair and prevent the progression of demyelinating lesions, and alleviate functional disorders in animal models, suggesting that cell therapy might be an effective treatment strategy (Cavazzin et al., 2006; Windrem et al., 2002, 2004; Webber et al., 2009). The purpose of this study was to obtain human OPCs that met clinical treatment demands. There were several points that made the cells obtained in this study suitable for future clinical applications. First, NSCs have a much lower risk of potential tumorgenicity than ESCs. Residual stem cells might exist in NSC- or ESC-derived cell populations. In this sense, OPCs from NSCs were
Fig. 5. When the cells were exposed to differentiation medium, they differentiated into oligodendrocytes (A) that expressed the specific marker GalC (B, green) at a rate of 91.3 ± 3.3%. When the cells were allowed to differentiate in OPCDM, they developed into oligodendrocytes with more mature morphology (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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much safer than those from ESCs. Second, the cells had higher purity as evidenced by their morphological homogeneity, and 80–90% of them expressed markers specific for OPCs, suggesting that they had a strong capacity to repair injured myelin structures because the abundant cells could differentiate into myelin-forming oligodendrocytes. Finally, human fetal NSCs have the capacity for unlimited proliferation, and the OPCs derived from NSCs in our culture system could be passaged at least 10 times, so large amounts of OPCs could be easily obtained using this method, which could meet the clinical demand. We noted a recently published method that directed hNSCs into OPCs (Monaco et al., 2012) that was similar to this protocol. Compared to that method, ours had several advantages. First, we could obtain OPCs with 80–90% purity from hNSCs in only 7 days compared to 70% purity in 21 days. As such, our protocol was time-saving, which meant a lower chance of potential heteromorphosis and microbial contamination. Second, compared with the NSCs of passages <11 in their study, NSCs of higher passages could be used for the OPC induction in our method. In this study, highly pure OPCs were successfully obtained from NSCs at passages 5–38. This may mean that our protocol was more stable and effective, but the possibility that the NSCs in our culture system were simply more stable could not be ruled out. In brief, NSCs of higher passages that can be used for OPC induction could guarantee an adequate cell source, avoiding the need for a large amount of human donor brain tissue. In addition, we obtained similar results in three NSC cell lines (supplemental Table 2), suggesting the high stability and repeatability of this protocol. Finally, previous studies, including that of Monaco MC et al., showed that sonic hedgehog (SHH) was important for human OPC generation (Hu et al., 2009); however, the present study showed that bFGF, PDGF, and NT3 were enough to direct NSCs into OPCs and that SHH was not necessary. The shorter induction period and fewer growth factors made our protocol low-cost. In conclusion, here we successfully obtained human OPCs that met the clinical demands of high quality and quantity using a quick, stable, and economic method.
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