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The Culture And Characterization Of Oligodendrocyte Precursor Cells In Vitro: A Preliminary Study Of Cell Transplantation

Bo Wu*, Shu-zhang Guo, Tao Jiang, Xian-jun Ren

*Department of Orthopedics, Xinqiao Hospital, The Third Military Medical University, Chongqing, China.

Address for Correspondence:  

Xian-jun Ren, MD;
Department of Orthopedics, Xinqiao Hospital,
The Third Military Medical University, 400037, Chongqing,

Tel: 00-86-23-68774608;




Objectives: To establish a protocol of OPCs culture for cell transplantation to treat spinal cord injury and to collect useful data about growth, differentiation, and proliferation of OPCs, which are important for their therapeutic effect of cell transplantation.

Methods: Mixed cells from cerebral cortices of neonatal rats were cultured in vitro. Later, the OPCs were separated by shaking process and differential adhesion. Then, the OPCs were cultured in the conditional medium for differentiation and proliferation. The growth pattern and differentiation of OPCs were observed by microscopy and electron microscopy. The maturation of OPCs was identified with immunocytochemical technique and the proliferative ability of OPCs was detected by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay.

Results: Around 9-10 days the distinct stratification of glial cells well developed in primary culture. Most of OPCs stayed and grew on the surface of astrocytes. The OPCs were further separated by shaking process and differential adhesion, and identified by the expression of specific marker. Furthermore, it was proved that the OPCs were able to differentiate into mature oligodendrocytes and proliferate in vitro.

Conclusion: In this study, we have established the rat OPCs culture in vitro. The OPCs stay at immature stage of development and they are able to differentiate and proliferate under certain condition in vitro, which are significant for the therapeutic action after cell transplantation.

J.Orthopaedics 2008;5(4)e3


Oligodendroglia; cell culture; differentiation; proliferation; spinal cord injury


Axonal demyelination is a common pathological change of a number of diseases in central nervous system (CNS), including brain trauma, multiple sclerosis, schizophrenia, normal aging, and spinal cord injury (SCI) as well1,2. In SCI, both initial insults and secondary injuries together cause the demyeliantion of white matter3. Recently, it becomes clear that demyelination of axons in SCI first takes place at the lesion epicenter, then it chronically progresses in the adjacent white matter fasciculus. Thus, it is proposed that intervention of axonal demyelination have important therapeutic implication in the treatment of SCI 4. 

Myelin-forming cells in CNS exclusively come from oligodendrocytes. It is likely that demyelination of axons have close relationship with the dysfunction of oligodendrocytes in SCI. Several experiments have presented convincing proofs relative to the mechanisms which result in the death and apoptosis of oligodendrocytes in wihte matter after SCI 5, which further causes axonal demyelination and impairs the functional recovery of injured spinal cord. Recently, some researches have indicated transplantation of myelin-forming cells can facilitate axonal remyelination and improve the neural function of injured spinal cord 6,7. Oligodendrocytes play a vital role in both facilitating the conduction of neural action potential and supporting axonal survival. Oligodendrocyte precursor cells (OPCs), the ancester of oligodendrocytes, can proliferate and migrate throughout CNS during the late embryonic development, which can differentiate into mature myelinating oligodendrocytes. As immature cells, OPCs stay at the early stage of development of oligodendroglial lineage cells, which have more potential to differentiate and proliferate in vivo than that of mature oligodendroctyes. Therefore, it is of great significance for the functional recovery of SCI that OPCs are transplanted to improve remyelination of survived axons and maximize the function of injured spinal cord. In this study, we aim to establish an OPCs culture in vitro to provide huge amount of myelinating cells for cell transplantation. Furthermore, we also investigate the important biological characteristics of OPCs in vitro to obtain useful data for cell transplantation in the treatment of SCI.

Material and Methods :

Animal care

All experimental animals were supplied by the experimental animal center of the Third Military Medical University. All procedures were performed according to institutional and governmental regulations, and in accordance with the policy set and delineated by the animal care committee of the University. 

The culture of OPCs

Briefly, the 48-hour-old Sprague-Dawley (SD) rats were anesthetized with an i.p. injection of 1% sodium pentobarbital (50mg/kg), sprayed with 70% ethanol and decapitated. Then, brains were removed under sterile conditions with the aid of a dissecting microscope (Leica). The basal ganglia, hippocampus, meninges and vessels were completely removed. After washed in Hanks balanced salt solution (Gibco), the meninges-free cerebral cortices were minced into 1 mm3 cubes and dissociated into cell suspension. Passing through 74 μm cell strainer, the filtrate was collected and centrifuged (1000 r/min for 10 min, 4℃). The pelleted cells were re-suspended with basic culture medium (BCM) [Dulbecco’s Modified Eagle Media(DMEM)(Gibco)supplemented with 10% fetal bovine serum (FBS) (Gibco), 0.6% glucose (Gibco), 4 mmol/L L-glutamine (Amresco), 5 mmol/L sodium pyruvate (Amresco), 50 u/ml penicillinum (Gibco) and 50 ug/ml streptomycin (Gibco)] and seeded to Poly-L-lysine (PLL) (Sigma)-coated flasks at the concentration of (1.0–2.0)×106 cells/flask. Add BCM into flasks and the flasks were transferred into a humidified incubator at 37℃ with 5% CO2. The cultures should not be disturbed in the first 3 days, with BCM fed every 2–3 days after that.

The separation of OPCs was usually carried out around 9–10 days in primary culture. Secondary to additional 24-hour incubation with fresh BCM, the flasks were placed onto a rotary shaker to remove microglia (180rpm, 37ºC, 1–2h). Pour off the medium with dislodged cells, wash flasks with phosphate-balanced saline (PBS) ( 0.01 M, pH 7.4) and add fresh BCM. In addition to 2-hour incubation, the flasks were placed back onto the shaker again for overnight shaking (200rpm, 37ºC, 18–20h). After the long-time shaking, the supernatant was poured through 74 μm strainer and the filtrate was collected and centrifuged (1000rpm, 4ºC, 10min). The pelleted cells were re-suspended with BCM and seeded onto uncoated culture dish for 1-hour incubation. Expel the medium over the surface of dish several times to remove loosely adherent cells, then collect the supernatant and centrifuge again (1000rpm, 4ºC, 10min).

Then, the isolated OPCs were re-suspended and further cultured in differentiation culture medium [DMEM supplemented with 0.5% FBS, 50 ug/ml transferrin (Sigma), 5 ug/ml insulin (Sigma), 30 nmol/L sodium selenite (Sangon), 30 nmol/L thyroxine (Sigma), 4 mmol/L L-glutamine, 5 mmol/L sodium pyruvate, 50 u/ml penicillinum and 50 ug/ml streptomycin] for differentiation or in OPCs culture medium [Differentiation culture medium supplemented with 10 nmol/L basic fibroblast growth factor (bFGF) (Peprotech) and 10 nmol/L platelet-derived growth factor-AA (PDGF-AA) (Peprotech)] for proliferation.


Observation of growth pattern and differentiation of OPCs

Microscopy and scanning electron microscopy

The growth pattern and cellular morphology of OPCs in the primary culture and in the differentiation culture were continuously investigated with phase contrast microscope and scanning electron microscope (Hitachi). For scanning electron microscopy (SEM) analysis, primary cultures were terminated when cell stratification distinctly formed in the culture. Cell cultures were rinsed with PBS, fixed in 2.5% glutaraldehyde for 2 h and postfixed in 1% osmium tetroxide for 1 h. Then, the samples were dehydrated in a graded series of ethanol and further dried in t-butyl alcohol for 5 min. After coated with gold-palladium, the specimens were viewed with S-3400N Hitachi scanning electron microscope.


Here, we identified the OPCs and their differentiation with immunocytochemistry. The separated cells were seeded onto PLL-coated coverslips and rinsed with PBS and fixed in 4% paraformaldehyde. After washed with PBS, the coverslips were treated with 0.5% Triton X-100 for 10 min. Following blockage with 5% goat serum for 15 min, the samples were incubated with rabbit anti-platelet-derived growth factor receptor alpha antibody (anti-PDGPR-α) (1:100, Santa Cruz) overnight at 4ºC. After washed in PBS, the coverslips were incubated with FITC-conjugated goat anti-rabbit IgG (1:100, Santa Cruz) for 1 h at 37ºC. The specimens were rinsed with PBS and coverslipped with mounting medium.

The differentiation of OPCs in vitro was also studied with immunocytochemical technique. After the differentiation in the conditional medium, the OPCs were immunostained with the specific antibody for mature oligodendrocytes. The cell culture were labelled with the primary antibody, myelin basic protein (MBP) (1:100, Santa Cruz), overnight at 4ºC and visualized with FITC-conjugated goat anti-rabbit IgG (1:100, Santa Cruz) for 1 h at 37ºC. The negative controls used PBS instead of primary antibodies for immunostaining. 

The proliferation of OPCs in vitro

The viability and proliferation of OPCs in vitro were also detected by MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]. After the separation, the pelleted OPCs were re-suspended with the OPCs culture medium and adjusted to the concentration of 5.0×104 cells/ml. Plate cells at 200 ul/well (~1.0×104 cells) into 96-well tissue culture plate. After incubated for 24 h, a total of 7 serial wells (each in triplicate) were further tested for the following 7 consecutive days. The reagent of 20 ul MTT (Sigma) (5mg/ml) was added to each well for color reaction, and absorbance of the soluble formazan product in wells was measured at wavelength of 492nm with 650nm as a reference, reading in a plate reader (Tecan Model). Three control wells were added with culture medium alone. The average values from the triplicate wells were determined. 

Statistical analysis

All data were expressed as mean±standard deviation. The results of MTT assay were analyzed using SPSS12.0.

Results :

The growth pattern and morphology of OPCs in primary culture

Initially, the OPCs grew scattered on the substratum of primary culture. Later on, some OPCs migrated and grew onto the surface of astrocytes. The OPCs were small in sizes, with round or oval shapes. Some of the OPCs displayed fine cell processes. Around 9–10 days in primary culture, the stratification of mixed glial cells distinctly formed. The bed layer mainly consisted of flat and confluent astrocytes (identified by immunolabelling, data not shown). Meanwhile, most of OPCs grew clustered and scattered on the surface of astrocytes (i.e. top layer), which represented the typical growth pattern of OPCs in the primary culture. At this time, most of OPCs displayed typical appearance of round or oval shapes with two or three fine processes (Fig.1A).

Moreover, the SEM clearly revealed the growth pattern of OPCs in the primary culture. The OPCs were observed resting close on the surface of confluent astrocytes. They had small and round soma, 6–10 μm in diameter. Meanwhile, their cell bodies typically had two or three fine cell processes. Otherwise, the confluent astrocytes of bed layer had large, flat cell bodies and irregular shapes (Fig.1B). Such a growth pattern of OPCs adhesive to astrocytes indicated there was close relationship between the two types of cells.

After the separation, the OPCs were further identified by immunocytochemistry. These isolated OPCs significantly expressed the PDGPR-α which is the lineage-specific marker of oligodendrocytes, and the control showed the negative result (Fig.1C).

Fig 1: The growth pattern and morphology of OPCs in culture.

(A)The stratification of mixed glial cells in primary culture distinctly formed around 9–10 days in vitro. The bed layer were confluent astrocytes and the OPCs grew clustered or scattered on the top of the astroctye layer. Scale bar=50μm.

(B)The feature of OPCs was further observed by scanning electron microscopy. The OPCs were seen resting on the top of confluent and flat astrocytes. Typically, the OPCs had small and round soma with two or three fine cell processes. This growth pattern of OPCs indicated the close relationship between the two different cell types.

(C)The OPCs were identified by immunocytochemistry. The shaken-off OPCs from mixed glial cultures were further immunostained with the specific cell marker of precursor cells, PDGPR-α. Scale bar=50μm.

The differentiation of OPCs in vitro

To study the differentiation ability, the separated OPCs were further cultured in the conditional medium for differentiation. Initially, the OPCs displayed typical appearance of precursor cells, which only had small round or oval cell bodies with few processes. Later on, the OPCs progressively differentiated into mature oligodendrocytes. After 1–2 days in the conditional culture, the OPCs had extended delicate processes without apparent branching (Fig.2A). After 3–5 days, this simple multipolar morphology of OPCs had evolved to more complex forms, characterized by the profuse outgrowth of elongated processes and extensive secondary branching. At last, the mature oligodendrocytes displayed typical appearance of “ramificated” or “cobweb-like” processes reticulating in their periphery (Fig.2B). Correspondingly, the expression of MBP, specific marker for mature oligodendrocytes, further confirmed the differentiation of OPCs in vitro (Fig.2C), and the result of control was negative. In all, these findings demonstrated that OPCs retained the ability to differentiate into mature oligodendrocytes in vitro.


Fig 2: The characteristics of OPCs differentiation in the conditional medium

(A)Following the shake-off process, the separated OPCs were further cultured for differentiation. Initially, the OPCs typically displayed small round or oval soma with few simple processes as the typical appearance of precursor cells. Scale bar=50μm.

(B)After 3–5 days in the culture, the morphology of OPCs developed into more complex forms, characterized by the pattern of “ramificated” or “cobweb-like” processes reticulating in their periphery. Scale bar=50μm.

(C)The differentiated oligodendrocytes were further identified by immunocytochemistry as the expression of specific cell marker,MBP, correspondingly. Scale bar=20μm.

The proliferation of OPCs in vitro

In this study MTT assay was performed to investigate the proliferation of OPCs. MTT assay involves the use of mitochondrial activity of live cells to convert MTT to formazan, whose concentration can be measured spectrophotometrically. The separated OPCs were plated in 96-well tissue culture plate for proliferation. These OPCs continued to grow in the OPCs culture medium. As a result, the mitochondrial activity of OPCs increased gradually in the early stages, which indicated the number of OPCs in wells rose progressively. Thereafter, the absorbance of the wells peaked on the 6th day and slightly decreased later (Fig 3). Here, the results of MTT assay revealed the OPCs also retained the reproductive activity and were able to proliferate in vitro.

Fig 3:The proliferation of OPCs in vitro detected by MTT assay.

The results of MTT assay clearly revealed that the OPCs still maintained the proliferative ability in vitro. Values represent specific absorbance (A492-A650) of the formazan product generated after incubation in MTT. Graph represents the data (mean ± standard deviation) from triplicate wells.The average absornance values were plotted on the y-axis with the days of culture on the x-axis.

Discussion :

Myelin sheath supports fast nerve conduction along axons8. Demyelination is one of the prominent pathological changes in SCI, which further interferes with nerve conduction. So, the normal function of myelin-forming cells and myelin sheath is essential for the normal function of CNS. Amelioration of axonal myelination is of great importance for functional recovery of injured spinal cord 9,10. As the unique myelinating cells in CNS, oligodendrocytes are reasonably expected to be one of the suitable candidates for cell transplantation to improve axonal myelination after SCI.

 The culture of oligodendrocytes is the prerequisite for the study of cell transplantation in vivo. There are several distinct developmental stages of cell differentiation identified for oligodendroglial lineage cells. At different stages of development, oligodendroglial lineage cells express stage-specific antigens, showing different proliferative and migrative capacities and distinct morphologies11. In this study we have developed a protocol of OPCs culture with modification and improvement of previous methods12.  Since myelination of nervous system of rats peaks around 3 weeks postnatally, the appropriate time to harvest OPCs should be prior to complete maturation in order to ensure OPCs are in active immature stages. Furthermore, the appropriate time for the purification of OPCs should be also precise. In this experiment, distinct stratification of mixed glial cells developed around 9–10 days in primary culture, which represented the very condition to separate OPCs. That the period of OPCs growth in vitro is too short or too long is unfavorable for the separation of OPCs later 12.

 As differentiation and proliferation of implanted cells are important for the therapeutic effect of cell transplantation, the characteristics of differentiation and proliferation of OPCs were also investigated in vitro to provide useful data for transplantation study in vivo. At present, differentiation of oligodendrocytes is usually induced by the low-serum or serum-free chemical conditional medium. The thyroxine in the conditional medium is vital for the survival of oligodendrocytes13. Furthermore, the trace element of selenium also plays an important role in the differentiation of oligodendrocytes from immature stages to more mature stages. It has been reported that the selenite is able to up-regulate gene expression of proteolipid protein and myelin-associated glycoprotein, which is important for myelination14.

 In this study, we induced differentiation of OPCs in the low-serum conditional medium with selenite and thyroxine, and investigated the development of OPCs into mature oligodendrocytes in vitro. After cultured in the differentiation medium, the morphology of OPCs underwent characteristic changes from the simple multipolar appearance to more complex appearance with profuse outgrowth of elongated processes and extensive secondary branching. The morphological changes correspondingly reflect the differentiation and maturation of OPCs in vitro. Moreover, these mature oligodendrocytes were also identified by immunostaining with the specific marker of MBP, which indicated their ability of myelin production. Altogether, these results demonstrated that the OPCs still maintained the ability to differentiate into mature oligodendrocytes in vitro.

 Furthermore, another important biological characteristic, the proliferative ability of OPCs, was also studied in vitro. The results of MTT assay indicated the OPCs retained the proliferative capacity in the conditional medium. The PDGF-AA and bFGF in the medium are vital trophic factors for the growth and proliferation of OPCs 15. In vivo, the PDGF-AA and bFGF are usually generated by astrocytes and neurons. As such, the survival and proliferation of OPCs in vitro also need the existence of both factors. In this experiment, the growth pattern of OPCs adhesive to the astrocytes in primary culture suggested that the astrocytes were likely to provide necessary growth substrate or cellular signals for the survival and growth of OPCs. After the separation, the PDGF-AA and bFGF were added in the conditional medium and the proliferation of OPCs in vitro was accordingly observed. Thus, in the future study of cell transplantation the survival and proliferation of implanted OPCs can be enhanced by the provision of PDGF-AA and bFGF.


Overall, in the study we have successfully established a protocol to culture the OPCs from cerebral cortices of neonatal rats. Both appropriate primary culture and timely shaking process are important for the efficient separation of OPCs. These OPCs are also proved to retain the abilities to differentiate into mature oligodendrocytes and to survive and proliferate in vitro, which are critical for the therapeutic effect of cell transplantation to treat SCI in vivo.


Reference :

1.Kövari E, Gold G, Herrmann FR, Canuto A, Hof PR, Michel JP, et al. Cortical microinfarcts and demyelination significantly affect cognition in brain aging. Stroke 2004; 35: 410-414.

2 Kakulas BA. The applied neuropathology of human spinal cord injury. Spinal Cord 1999; 37: 79-88.

3 Hulsebosch CE. Recent advances in pathophysiology and treatment of spinal cord injury. Advances in Physiology Education 2002; 26: 238-255.

4 Totoiu MO, Keirstead HS. Spinal cord injury is accompanied by chronic progressive demyelination.  Journal of Comparative Neurology 2005; 486: 373-383.

5 Casha S, Yu WR, Fehlings MG. Oligodendroglial apoptosis occurs along degenerating axons and is associated with FAS and p75 expression following spinal cord injury in the rat. Neuroscience 2001; 103: 203-218.

6 Kocsis JD, Akiyama Y, Lankford KL, Radtke C. Cell transplantation of peripheral-myelin-forming cells to repair the injured spinal cord. Journal of Rehabilitation Research & Development 2002; 39: 287-298.

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10 Utzschneider DA, Archer DR, Kocsis JD, Waxman SG, Duncan ID. Transplantation of glial cells enhances action potential conduction of amyelinated spinal cord axons in the myelin-deficient rat. The Proceedings of the National Academy of Sciences USA 1994; 91: 53-57.

11 Baumann N, Pham-Dinh D. Biology of Oligodendrocyte and Myelin in the Mammalian Central Nervous System. Physiological Reviews 2001; 81: 871-927.

12 McCarthy KD, de Vellis J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. Journal of Cell Biology 1980; 85: 890-902.

13 Jones SA, Jolson DM, Cuta KK, Mariash CN, Anderson GW. Triiodothyronine is a survival factor for developing oligodendrocytes. Molecular and Cellular Endocrinology 2003; 199: 49-60.

14 Gu J, Royland JE, Wiggins RC, Konat GW. Selenium is required for normal upregulation of myelin genes in differentiating oligodendrocytes. Journal of Neuroscience Research 1997; 47: 626-635.

15 Bögler O, Wren D, Barnett SC, Land H, Noble M. Cooperation between two growth factors promotes extended self-renewal and inhibits differentiation of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells. The Proceedings of the National Academy of Sciences USA 1990; 87: 6368-6372.


This is a peer reviewed paper 

Please cite as : Bo Wu: The Culture And Characterization Of Oligodendrocyte Precursor Cells In Vitro: A Preliminary Study Of Cell Transplantation

J.Orthopaedics 2008;5(4)e3





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