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Isolation and culture of human oligodendrocyte precursor cells from neurospheres
Accepted Manuscript Title: Isolation and culture of human oligodendrocyte precursor cells from neurospheres Author: Yabin Lu Yinxiang Yang Zhaoyan Wang Caiying Wang Qingan Du Qian Wang Zuo Luan PII: DOI: Reference:
S0361-9230(15)30026-5 http://dx.doi.org/doi:10.1016/j.brainresbull.2015.08.008 BRB 8886
To appear in:
Brain Research Bulletin
Received date: Revised date: Accepted date:
4-4-2015 6-8-2015 24-8-2015
Please cite this article as: Yabin Lu, Yinxiang Yang, Zhaoyan Wang, Caiying Wang, Qingan Du, Qian Wang, Zuo Luan, Isolation and culture of human oligodendrocyte precursor cells from neurospheres, Brain Research Bulletin http://dx.doi.org/10.1016/j.brainresbull.2015.08.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Isolation and culture of human oligodendrocyte precursor cells from neurospheres Yabin Lua,b1, Yinxiang Yanga1, Zhaoyan Wanga, Caiying Wanga, Qingan Dua, Qian Wanga, Zuo Luana* [email protected]
a
Department of Pediatrics, Navy General Hospital, No. 6, Fucheng Road, Beijing,
100048, China b
Department of Biochemistry and Molecular Biology, Capital Medical University,
Beijing, 100069, China
*
Corresponding author: Tel.: +86 13381207228; fax: +86 10 66958303.
1
These authors contributed equally to this work.
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Highlights We developed a reproducible, simple, and economical method of obtaining human OPCs from neurospheres. Isolated human OPCs have high purity and robust proliferative capacity. After induction, the OPCs can differentiate into oligodendrocytes.
2
Abstract Culture of human oligodendrocyte precursor cells (OPCs) can help understand the regulatory mechanism of differentiation and myelination of oligodendrocytes. However, existing culture methods have limitations, particularly the lack of a source of human donor tissue and high cost. We sorted cells with the A2B5+PSA-NCAM– phenotype
from
neurospheres
instead
of
human
donor
tissues
through
immunomagnetic sorting and subsequently cultured the isolated cells in OPC medium. Of all the isolated cells, 15.69% were of the A2B5+PSA-NCAM– phenotype. More than 90% of the isolated OPCs expressed the OPC-specific markers O4, PDGFαR, and Sox10, and less than 5% of cells expressed GFAP and Tuj-1. After induction, the isolated cells had the capacity to differentiate into oligodendrocytes. Furthermore, the OPCs could be stably passaged in vitro for at least four generations and all the cells had high expression levels of O4 and Sox10 and very low expression levels of GFAP and Tuj-1; moreover, the cells had the capacity to differentiate into oligodendrocytes. After four passages, OPCs can proliferate at least 14 times above. In addition, in the presence of B27, only one cytokine, namely, bFGF, was sufficient to maintain proliferation, and this greatly reduced the experimental cost. Cells of the A2B5+PSA-NCAM– phenotype have already been identified as OPCs. We developed and characterized a reproducible, simple, and economical method for the isolation and culture of human OPCs. This method will contribute to studying the function of OPCs in development, disease, and treatment. Keywords:
Oligodendrocyte
precursor
cells;
Neurosphere;
Proliferation;
Differentiation; Oligodendrocyte. 3
1. Introduction Oligodendrocytes (OLs) are cells derived from oligodendrocyte precursor cells (OPCs) and occur in the central nervous system (CNS). They play a crucial role in myelin sheath formation and facilitation of rapid conduction of neuronal action potentials [17]. OPCs differentiate through a series of developmental stages, such as pro-OLs, immature OLs, and mature OLs [23]. If OPCs are injured during development or remyelination, myelination of brain white matter is delayed or damaged, which can lead to nerve function deficit [15, 20]. OPC differentiation has been found to have important significance in the promotion of myelin repair [2, 30]. Therefore, understanding the mechanism by which OPC differentiation is regulated would provide insights into the process of myelin-associated diseases. It is important to establish a method for the in vitro isolation, amplification, and differentiation of OPCs. The specific immunophenotype of OPCs is distinct from those of the human fetal CNS tissue and adult human white matter. Several methods have been described for the isolation of human OPCs from the CNS, such as fluorescence-activated cell sorting (FACS) by exploiting cell surface-specific antigens, immunomagnetic sorting, or a combination of the two [9, 29]. Because the source of human donor tissue is very limited, it is difficult to obtain a large number of cells for research. Another method for obtaining human OPCs is by neural stem cell- or embryonic stem cell-induced directional differentiation. This method often needs complicated procedures of induction. Furthermore, obtaining OPCs of high purity via this method takes at least
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21 days [18]; therefore, the experimental cost increases, limiting the potential productivity of OPCs. Therefore, a simple and economical method for the isolation and culture of human OPCs is needed to overcome these limitations. In this study, we described a simple and reproducible method for the isolation and culture of human OPCs. OPCs were isolated from neurospheres derived from human fetal brains by immunomagnetic sorting. Neurospheres were selected as the source of OPCs because they can be stably passaged in vitro; thus, they can serve as a stable source of sorting cells. First, we used microbeads to deplete polysialylated neural cell adhesion molecule (PSA-NCAM)+ neurons. Then, A2B5+ cells were isolated from the larger PSA-NCAM– cell population via magnetic sorting. Previous studies have reported subpopulations of A2B5+PSA-NCAM– marker cells in brain tissues, and this cell subpopulation has been identified to be OPCs, which exhibit extensive myelin production in shiverer mice [24, 29]. After sorting from neurospheres, the cells showed stably high expression levels of O4, NG2, and Sox10 and very low expression levels of GFAP and Tuj-1. After induction, the cells differentiated into oligodendrocytes, indicating the oligodendrocytic bias of the sorted cells. Furthermore, these cells can be stably cultured and passaged at least four times and proliferate at least 14 times in vitro. Therefore, we can use this reproducible and simple method to stably isolate and culture human OPCs in vitro. 2 Materials and methods 2.1 Reagents and supplies All reagents and supplies were obtained commercially. Dulbecco’s modified Eagle’s
5
medium/F12 (DMEM/F12; Gibco, C11330500BT), Neurobasal-A medium (Gibco, 10888-022), N2 supplement (Gibco, 17502-048), B27 supplement (Gibco, 17504-044), recombinant human FGF-basic (PeproTech, AF-100-18B), recombinant human
EGF
(PeproTech,
AF-100-15),
heparin
(Sigma,
H3149),
penicillin/streptomycin (Invitrogen, 15140), 0.25% trypsin (Gibco, 15050-065), trypsin
inhibitor
(Sigma,
T6522),
L-glutamine
(Gibco,
35050-061),
ethylenediaminetetraacetic acid (EDTA) disodium salt dehydrate (Amresco, 6381-92-6), Poly-D-lysine hydrobromide (Sigma, p6407), laminin (Invitrogen, 23017-015), OPC differentiation medium (OPCDM; ScienCell, 1631), cell strainer (BD Falcon, 352340), MS Column (Miltenyi Biotec, 130-091-506), fetal bovine serum (FBS; Gibco, 10099-141), anti-PSA-NCAM microbeads (Miltenyi Biotec, 130-092-966),
anti-PSA-NCAM
antibodies
(Miltenyi
Biotec,
130-093-273),
anti-A2B5 microbeads (Miltenyi Biotec, 130-093-388), Anti-A2B5 antibodies (Miltenyi Biotec, 130-093-581), mouse anti-O4 antibodies (1:300 dilution; R&D, MAB1326), mouse anti-Sox10 (R&D, MAB2864, 1:50), rabbit anti-GFAP (Invitrogen, 18-0063, 1:80), mouse anti-Tuj-1 (1:500 dilution; Abcam, ab7751), rabbit anti-Galc (Millipore, AB142, 1:50), rabbit anti-PDGFαR antibodies (1:400 dilution; Cell Signaling, 5241), Alexa Fluor 488-AffiniPure Goat Anti-Mouse IgG + IgM (H+L) (1:800 dilution; Jackson Immunoresearch, 111-545-042), and Alexa Fluor 594-AffiniPure goat anti-rabbit IgG (H + L, 1:800 dilution; Jackson Immunoresearch, 111-585-144).
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2.2 Neurosphere culture Neurospheres were cultured and passaged using the methods described previously [28]. Human fetal CNS tissues obtained from 10- to 13-week-old embryos were provided by Navy General Hospital, Beijing, China. All women had requested to terminate their pregnancy. After being fully informed according to the guidelines approved by the Ethics Committee of the hospital, they consented to donate the aborted fetuses. Surface regions of the cortex were randomly chosen. Cortex tissues were dissected and mechanically dissociated by repetitive blowing into a single-cell suspension. Primary cells were cultured at a density of 1 × 106 cells/mL in primary culture medium containing DMEM/F12, 1% L-glutamine, 1% N2 supplement, 2% B27 supplement, 20 ng/mL bFGF, 20 ng/mL EGF, 5 µg/mL heparin, and 1% penicillin/streptomycin. Every three to four days, two-thirds of the medium was removed and replaced with fresh primary culture medium. The expanded cells formed colonies and were cultured as free-floating “neurospheres” after 5–7 days of culture. As not all the cells in the tissues had survived, the cells that remained alive after dissection and mechanical dissociation could form neurospheres. The neurospheres were passaged every 10–12 days using 0.025% trypsin. The reaction was stopped by using 1.2 mg/mL of trypsin inhibitor. Neurospheres were repeatedly blown in order to obtain single-cell suspensions. The resultant cells were seeded into a flask, and the medium from passage 1 was used but without B27 supplement. 2.3 Magnetic cell sorting (MACS) for OPC Cells were sorted according to the instructions provided by microbeads. Neurospheres
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were digested into single cells using 0.025% trypsin solution. Cells were passed through a 40-μm nylon mesh to remove cell clumps, which may clog the column. The cell number was then determined, and the cell suspension was centrifuged at 300 g for 10 min. The supernatant was completely aspirated, and 107 cells were resuspended in 60 μL of MACS buffer, which consisted of phosphate-buffered saline (PBS; pH 7.2), 0.5% FBS, and 2 mM EDTA. The suspension was mixed well and incubated for 10 minutes in the refrigerator (2–8°C). Then, 20 μL of anti-PSA-NCAM microbeads were added to the cell suspension containing 107 cells, mixed well, and incubated for 15 minutes in the refrigerator. The cells were washed by adding 1–2 mL of MACS buffer and centrifuged at 300 g for 10 min. During centrifugation, the MS column was placed in the magnetic field of a suitable MACS Separator and equilibrated with 0.5 mL of MACS buffer. The supernatant was completely aspirated and the cells (up to 108 cells) were resuspended in 500 μL of MACS buffer. The cell suspension was applied on to the column. Unlabeled cells that passed through the column were collected, and the column was washed with the appropriate amount of buffer. The total effluent, i.e., the unlabeled cell fraction, was then collected from the column. Washing was carried out three times by adding 500 μL of MACS buffer. The cell suspension was centrifuged at 300 g for 10 min. The supernatant was completely aspirated, and the cell pellet (107 cells) was resuspended in 60 μL of MACS buffer. Then, 20 μL of anti-A2B5 microbeads were added for 107 cells and the mixture was incubated for 15 min in the refrigerator. This mixture was passed through the MS column and washed thrice according to the same procedure. The column was then
8
removed from the separator, placed on a suitable collection tube, and 1 mL of buffer was pipetted on to the column. The magnetically labeled cells were immediately flushed by firmly pushing the plunger into the column. The cell suspension was then centrifuged at 300 g for 10 min to obtain the A2B5+PSA-NCAM– cell population. 2.4 Culture, passage, and differentiation of OPCs OPCs were plated into a flask at a density of 4 × 104 cells/cm2 and cultured in OPC medium for propagation. OPC medium was prepared by adding 2% B27 supplement (2%), 20 ng/mL bFGF, 1% penicillin/streptomycin, 5 μg/mL heparin, and 2 mM L-glutamine
to Neurobasal-A medium. Every three days, two-thirds of the medium
was removed and replaced with fresh OPC medium. After 7–10 days of proliferation, the OPCs reached 80–90% confluence. They were gently blown with a pipette and plated into a flask. For differentiation, the OPCs were plated on to PDL/laminin-coated well plates at a density of 4 × 104 cells/cm2 and cultured in OPC differentiation medium (OPCDM, ScienCell). Two-thirds of the medium was replaced with fresh medium every 2–3 days. The cells were cultured for 14–21 days. After incubation of the plates for 24 h, the OPCs were fixed with 4% paraformaldehyde for 20 min at room temperature (RT). The cells were then blocked with 5% normal goat serum for 60 min and incubated with the following primary antibodies: mouse anti-O4 antibody (R&D, 1:300), mouse anti-Sox10 (R&D, 1:50), rabbit anti-GFAP (Invitrogen, 1:80), mouse anti-Tuj-1 (Abcam, 1:500), rabbit anti-PDGFαR antibody (Cell Signaling, 1:200), and rabbit anti-Galc (Millipore, 1:50)
9
at 4°C overnight. After rinsing with 1× PBS three times, fluorescence-labeled secondary antibodies (Jackson Immunoresearch) were applied for 45 min at RT. Cell nuclei were stained with DAPI (1 μg/mL in PBS) for 5 min[5]. 2.5 OPC analysis by FACS Cells were double stained with anti-PSA-NCAM and anti-A2B5 antibodies (Miltenyi Biotec) to identify the purity of OPCs. According to the manufacturer’s instructions, up to 106 nucleated cells was resuspended in 100 μL of buffer. To this, 10 μL of anti-PSA-NCAM and anti-A2B5 antibodies were added, mixed well, and incubated for 10 min in the dark in the refrigerator. The cells were washed by adding 1−2 mL of MACS buffer and centrifuged at 300 g for 10 min. The supernatant was completely aspirated, and the cell pellet was resuspended in a suitable amount of buffer for flow cytometry analysis. Each experiment was independently repeated in triplicate. Data are presented as means ± standard deviation. 2.6 Cell proliferation assay The cell proliferation rate was measured at 7- or 10-day intervals (at each passage, for a total of four generations) by counting the number of viable cells. Cells were counted when OPCs were passaged. In brief, 1.5 × 105 cells were seeded into 24-well plates and designated as P0. After 10 days, cells were gently blown and the number of OPCs were counted, and designated as P1. After 7 days, the cells were spread to two wells to calculate the number of cells in the two wells, i.e., P2. Similarly, the number of cells for P3 and P4 were counted. Each experiment was carried out in triplicate, and all assays were done in triplicate. Data are presented as means ± standard deviation.
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Each experiment was independently repeated in triplicate. 2.7 Data collection 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 10 randomly selected fields from three wells using a 40× objective lens. Data are presented as means ± standard deviation. Each experiment was independently repeated in triplicate. 3 Results 3.1 Identification of cells isolated from neurospheres Of the cells isolated from neurospheres within the tenth generation, 15.69 ± 2.23% (n = 5) were of the A2B5+PSA-NCAM– phenotype, as determined by flow cytometry (Fig.1A). After immunomagnetic sorting, the cells were immediately examined to determine the purity of the populations, and 85.51% of the cells were found to be of the A2B5+PSA-NCAM– phenotype (Fig.1B). If the quantity of the initial human tissue cell was 106, we can get 107 neural stem cells after culturing 10-12 days. Then we used the 107 cells for sorting, we can approximate get 1.2×106 OPCs. Three days after plating, the cells gradually adhered to culture plates and had typical bipolar and/or tripolar morphology (Fig.1C). Cells were plated onto PDL-laminin-coated well plates. Three days later, 90.24 ± 1.04% of the cells expressed O4 (the OPC markers), 91.99 ± 1.32% expressed PDGFαR (the OPC markers), and 95.75 ± 1.52% expressed Sox10 (the OPC markers), and none of the cells were Galc+ (markers of oligodendrocytes). 4.63 ± 0.78% of the cells expressed GFAP (an astrocyte marker) and 4.68 ± 0.51% expressed Tuj-1 (a neuron marker) (Fig.1D-O). These data suggested that early stage
11
OPCs can be obtained with high purity by MACS of neurospheres. 3.2 First generation of OPCs differentiated into oligodendrocytes In order to confirm whether the sorted cells can differentiate into oligodendrocytes, OPCs were plated onto PDL/laminin-coated well plates and cultured in OPC differentiation medium. Fourteen days after plating, the cells cultured in the absence of serum formed more complex arborization patterns with more ramified, longer, and multiple processes and most of them were immature oligodendrocytes (Fig.2). These cells expressed antigens that could be recognized by the anti-O4 and anti-Galc antibodies with a multipolar morphology. Furthermore, some cells differentiated into astrocytes expressing GFAP. When cultured in the presence of serum, nearly all the cells were astrocytes. These data suggested that the OPCs isolated from neurospheres had the capacity to differentiate into oligodendrocytes. 3.3 In vitro amplification of OPCs sorted from neurospheres We obtained OPCs by sorting neurospheres, and these OPCs could be stably passaged for at least four generations in vitro and retained their original morphology and capacity. In this period, four passages of cells had typical bipolar and/or multipolar morphology, continuously expressed O4 at high levels, and most of them were in the early stage (Figs. 1C, 1F, and 3A). From the first generation to the fourth generation, O4+ % was respectively 90.24 ± 1.04%, 91.63 ± 1.23%, 92.06 ± 2.11% and 91.62 ± 0.82% (Fig.3D). There was no significant statistical difference between these generations. At the fourth passage of OPCs, over 90% of the cells expressed Sox10, and GFAP and Tuj-1 were expressed at very low levels, similar to the cells from the
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first passage of OPCs (Fig.3C). At the same time, the cells had a strong proliferative capacity. The cell growth curve was drawn according to the number of viable cultured OPCs, which were counted at each passage. After four generations of in vitro culture, OPCs could proliferate at least 14 times above (Fig.3B). After induction, different passages of OPCs also differentiated into oligodendrocytes (data not shown). Therefore, OPCs isolated from neurospheres can be stably passaged, and the cells can proliferate and retain their morphology and capacity even after in vitro culture. 3.4 OPCs can be isolated from different passages of neurospheres We obtained OPCs from neurospheres within tenth generation. Neurospheres can be stably passaged and amplified many times in vitro; therefore, we tried to isolate OPCs from neurospheres of higher passages. We found that OPCs could be also isolated by MACS of neurospheres of passage 30. OPCs derived from thirtieth generation neurospheres were cultured in OPC medium for proliferation. These cells displayed the typical morphology of OPCs with bipolar and/or tripolar processes (Fig.4A). Immunofluorescence staining showed that 90.84 ± 3.68% of cells expressed O4, 90.56 ± 1.08% expressed PDGFαR, 93.7 ± 1.87% expressed Sox10, 5.3 ± 0.33% expressed GFAP and 4.62 ± 0.47% of the cells expressed Tuj-1. There was no significant statistical difference between early and late passages (Fig.4B–M). The cells were cultured in OPC differentiation medium to determine whether the cells had the capacity to differentiate into oligodendrocytes, and a subpopulation of the cells were O4+ and Galc+ with a multipolar morphology. Other cells expressing GFAP were astrocytes (Fig.4N and O). These data suggested that OPCs of high purity can be
13
obtained by magnetic bead sorting of late passage neurospheres, similar to that of early passage neurospheres and that the OPCs obtained from late passage neurospheres had the capacity to differentiate into oligodendrocytes. 4 Discussion OPCs play an important role not only as a major glial population in the CNS but also as an active participant in CNS signaling [15]. Devising a method for culturing human OPCs is essential to understanding the differentiation and myelination of oligodendrocytes. This will contribute to promoting myelin repair and understanding the process of myelin-associated diseases. OPCs are a model for understanding the regulatory mechanism of OL differentiation and myelination; therefore, research requires a large number of cells. If OPCs cannot be cultured in vitro, it will increase the cost of the research. If OPCs can stably proliferate in vitro, the procedures will be simplified and the cost of the research can be reduced. Therefore, it is necessary to devise a simple method for generating large quantities of highly purified OPCs. 4.1 Select the A2B5+PSA-NCAM- as the sorting marker We sorted cells with the A2B5+PSA-NCAM– phenotype (positive for the early oligodendrocyte marker A2B5 and negative for PSA-NCAM) from neurospheres. Previously, human OPCs obtained by sorting have been derived from fetal or adult tissues [1, 3, 6, 22]. However, human donor tissues are difficult to obtain, and ethical issues are involved in obtaining tissues. Therefore, the source of cells that can be used to obtain OPCs is limited. Sorting requires higher activity of cells in tissues. These aspects limited the use and yield of OPCs.
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4.2 Advantages of choosing neurospheres as a source of OPCs sorting OPCs were sorted from neurospheres. The neurospheres were cultured from the brains of aborted human fetuses. These neurospheres survived passage for many generations. Therefore, we obtained a large number of neurospheres, which served as an abundant source of OPCs. Neurospheres cultured in vitro had the capacity to differentiate into neurons, astrocytes, and oligodendrocytes [16]. We speculated that neurospheres cultured in vitro may contain OPCs. Indeed, 15.69 ± 2.23% of the neurospheres expressed the A2B5+PSA-NCAM– phenotype. This cell subpopulation showed high expression levels of O4, PDGFαR and Sox10 (more than 90%) and very low expression levels of GFAP and Tuj-1 (less than 5%). After induction, the cells can differentiate into oligodendrocytes, suggesting that the cells isolated from neurospheres were OPCs, similar to those isolated from brain tissues, exhibiting the various features of OPCs. Therefore, the most important innovation in this study is the use of neurospheres as a source of OPCs. Our OPCs were directly sorted from neurospheres without any induction. Sorting from neurospheres has many advantages. First, the use of neurospheres as a source of OPCs can avoid ethical issues. Only first generation neurospheres were cultured from discarded human fetal brains. Once neurospheres were formed, they can be passaged for many generations. Thus, the use of human tissues can be avoided. Second, the methods of culturing and passaging neurospheres have been relatively mature. Neurospheres can be cryopreserved and thawed when required, and stably passaged and amplified in vitro to obtain a sufficient number of cells. Third, OPCs also can be isolated from 25–30 generations
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of neurospheres, and have the same characteristics as the OPCs isolated from early passages such as passages 1, 3, 5, and 7. The capability to produce OPCs and ologodendrocytes is not altered by passaging neurospheres. As a large number of neurospheres can be obtained from one fetal brain tissue; theoretically, OPCs can be obtained as long as neurospheres can be passaged. Thus, a single fetal brain tissue sample can yield a large number of neurospheres within 30 generations. In addition, cultured neurospheres can be easily digested into single cells using trypsin, and these cells were found to have very high activity of more than 95% in vitro. Thus, neurospheres are a very good source of OPCs. 4.3 Only one cytokine – bFGF is sufficient to stably maintain growth and proliferation of OPCs. Previous studies have demonstrated that long-term culture and expansion of human OPCs after sorting from brain tissues in vitro require at least three or four cytokines such as bFGF, T3, PDGF-AA, and NT3 [7, 22]. Cells can be maintained for 1–7 days using N1 and bFGF [3, 29]. Apart from sorting, stem cell induction is another way to harvest human OPCs, but this method also requires a series of cytokines. Human OPCs obtained by neurosphere-induced directional differentiation also require a combination of many different cytokines such as bFGF, PDGF-AA, Shh, NT-3, and T3 [18-19]. OPCs derived from human embryonic stem cells require more cytokines, complicated procedures, and longer time, and the experimental cost involved consequently increases [11, 13, 21, 25]. Furthermore, human embryonic stem cells have potential tumorigenicity [4, 12, 26]. These disadvantages limit the potential
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application and productivity of OPCs. In our study, we devised a simple and economic system for the culture of human OPCs. In the presence of B27, only one cytokine, namely, bFGF, is sufficient to stably maintain the growth and proliferation of OPCs. This will greatly reduce the experimental cost. Previous studies have demonstrated that medium supplemented with N1 and bFGF could maintain OPCs for only a short period of time. In our study, we used B27 instead of N1, and found that OPCs had higher activity in medium containing B27 compared to medium containing N1 (data not shown). Furthermore, our medium greatly enhanced the growth of OPCs, and the OPCs proliferated more than 14 times after four generations. B27 has been reported to enhance the survival of OPCs, especially at low plating densities [14, 27, 31], and bFGF is known to stimulate the proliferation of cultured cells [8, 10]. Thus, B27 could improve the activity of OPCs. Therefore, the use of B27 and bFGF helped simplify the procedure, reduce the costs involved, and increase cell viability. 4.4 OPCs can be sorted and cultured stably in vitro. The OPCs in this study, regardless of whether they were sourced from early or late passage neurospheres or cultured for different generations after sorting, were found to have high stable expression levels of O4, PDGFαR, and Sox10 (more than 90%), and very low expression levels of GFAP and Tuj-1 (less than 5%), indicating that OPCs isolated from neurospheres can be stably cultured and proliferated. The isolated OPCs can be stably cultured for at least two months in vitro. In addition, when cultured in OPC differentiation medium, some subpopulations of OPCs can differentiate into
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oligodendrocytes, indicating that the method of culturing OPCs is stable. Using this method, large numbers of human OPCs of high purity can be obtained by a simple and economical sorting and culture method. The human OPCs isolated in this way can not only be stably cultured and proliferated, but they also have the capacity to differentiate into oligodendrocytes. And the capability to produce OPCs and ologodendrocytes is not altered by passaging neurospheres or purified OPCs. Thus, we can use this simple, economical, and reproducible method to stably obtain a large number of human OPCs from neurospheres and culture the OPCs in vitro. This method will contribute to studying the function of OPCs in the development and treatment of myelin-associated disease.
Acknowledgements This work was supported by the International Science & Technology Cooperation Program of China (ISTCP, No. 2012DFA30880).
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demyelination models in mice, PLoS One, 5 (2010) e10145. [20] A. Nishiyama, Glial progenitor cells in normal and pathological states, Keio J Med, 47 (1998) 205-208. [21] G.I. Nistor, M.O. Totoiu, N. Haque, M.K. Carpenter, H.S. Keirstead, Human embryonic stem cells differentiate into oligodendrocytes in high purity and myelinate after spinal cord transplantation, Glia, 49 (2005) 385-396. [22] F. Ruffini, N. Arbour, M. Blain, A. Olivier, J.P. Antel, Distinctive properties of human adult brain-derived myelin progenitor cells, Am J Pathol, 165 (2004) 2167-2175. [23] M. Stangel, H.P. Hartung, Remyelinating strategies for the treatment of multiple sclerosis, Prog Neurobiol, 68 (2002) 361-376. [24] F.G. Strathmann, X. Wang, M. Mayer-Proschel, Identification of two novel glial-restricted cell populations in the embryonic telencephalon arising from unique origins, BMC Dev Biol, 7 (2007) 33. [25] M. Sundberg, H. Skottman, R. Suuronen, S. Narkilahti, Production and isolation of NG2+ oligodendrocyte precursors from human embryonic stem cells in defined serum-free medium, Stem Cell Res, 5 (2010) 91-103. [26] D.E. Suzuki, A.M. Nakahata, O.K. Okamoto, Knockdown of E2F2 inhibits tumorigenicity, but preserves stemness of human embryonic stem cells, Stem Cells Dev, 23 (2014) 1266-1274. [27] C.N. Svendsen, J.W. Fawcett, C. Bentlage, S.B. Dunnett, Increased survival of rat EGF-generated CNS precursor cells using B27 supplemented medium, Exp Brain Res, 102 (1995) 407-414. [28] Y.I. Tarasenko, Y. Yu, P.M. Jordan, J. Bottenstein, P. Wu, Effect of growth factors on proliferation and phenotypic differentiation of human fetal neural stem cells, J Neurosci Res, 78 (2004) 625-636. [29] M.S. Windrem, M.C. Nunes, W.K. Rashbaum, T.H. Schwartz, R.A. Goodman, G. McKhann, 2nd, N.S. Roy, S.A. Goldman, Fetal and adult human oligodendrocyte progenitor cell isolates myelinate the congenitally dysmyelinated brain, Nat Med, 10 (2004) 93-97. [30] B. Wu, X. Ren, Promoting axonal myelination for improving neurological recovery in spinal cord injury, J Neurotrauma, 26 (2009) 1847-1856. [31] Z. Yang, M. Watanabe, A. Nishiyama, Optimization of oligodendrocyte progenitor cell culture method for enhanced survival, J Neurosci Methods, 149 (2005) 50-56.
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Figure legends Fig.1 Isolated cells expressing multiple OPC-specific markers. (A) Flow cytometry analysis of neurospheres within the tenth generation. (B) Flow cytometry analysis of cells isolated from third generation neurospheres. (C) After isolation for three days, the cells displayed typical OPC morphology with bipolar and/or tripolar processes. (D–F) Isolated cells express the OPC surface markers O4. (G–I) Isolated cells express the OPC surface marker PDGFαR. (J–L) Isolated cells express the OPC marker Sox10. (M) Astrocyte marker GFAP. (N) Neural cell marker Tuj-1. (O) The percentage of cells expressing O4, PDGFαR, Sox10, GFAP and Tuj-1. All cell nuclei were labeled with DAPI (blue). The red arrows indicated the negative cells. Scale bars: 50 μm (C–L, N), 100 μm (M). Fig.2 The first generation of OPCs differentiated into oligodendrocytes. (A, D) Cells exhibit ramified, longer, multiple processes. (B) Immunofluorescence staining of O4 (green), GFAP (red). (E) Cells were O4+. (C, F) Cells were Galc+. All cell nuclei were labeled with DAPI (blue). Scale bars: 50 μm (A–C), 25 μm (D–F). Fig.3 Isolated OPCs cultured for four cell passages. (A) Cells of passages 2–4 displayed the typical morphological characteristics of OPCs with bipolar and/or tripolar processes and the surface maker O4+. Nuclei were labeled with DAPI. Scale bars = 50 μm. (B) The cell growth curve was drawn according to the numbers of viable OPCs, which were counted at each passage (passages 1–4). OPCs (1.5 × 105) cells were seeded into the wells of a 24-well plate, and designated as P0. Data are shown as means ± SD, n = 3. (C) OPCs of passage 4 expressed Sox10, GFAP, and 21
Tuj-1. All cell nuclei were labeled with DAPI (blue). The red arrows indicated the negative cells. Scale bars: 50 μm (Sox10, DAPI, and Tuj-1), 100 μm (GFAP). (D) OPCs of passage1-4 expressed O4. Fig.4 Cells isolated from neurospheres of passage 30 expressed multiple OPC-specific markers. (A) OPCs displayed typical OPC morphology with bipolar and/or tripolar processes. (B) Astrocyte marker GFAP. (C) Neural cell marker Tuj-1. (D–F) Isolated cells express the OPC surface marker O4. (G–I) Isolated cells express the OPC surface marker PDGFαR. (J-L) Isolated cells express Sox10. (M) Isolated cells obtained from neurospheres of early and late passages expressed O4, PDGFαR, Sox10, GFAP and Tuj-1. (N–O) OPCs differentiated into oligodendrocytes. Immunofluorescence staining of O4 (green, N) and GFAP (red, N). Oligodendrocytes were highly arborized and Galc+ (O). All cell nuclei were labeled with DAPI (blue). The red arrows indicated the negative cells. Scale bars: 50 μm.
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Figure 2
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Figure 3
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S0361-9230(15)30026-5 http://dx.doi.org/doi:10.1016/j.brainresbull.2015.08.008 BRB 8886
To appear in:
Brain Research Bulletin
Received date: Revised date: Accepted date:
4-4-2015 6-8-2015 24-8-2015
Please cite this article as: Yabin Lu, Yinxiang Yang, Zhaoyan Wang, Caiying Wang, Qingan Du, Qian Wang, Zuo Luan, Isolation and culture of human oligodendrocyte precursor cells from neurospheres, Brain Research Bulletin http://dx.doi.org/10.1016/j.brainresbull.2015.08.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Isolation and culture of human oligodendrocyte precursor cells from neurospheres Yabin Lua,b1, Yinxiang Yanga1, Zhaoyan Wanga, Caiying Wanga, Qingan Dua, Qian Wanga, Zuo Luana* [email protected]
a
Department of Pediatrics, Navy General Hospital, No. 6, Fucheng Road, Beijing,
100048, China b
Department of Biochemistry and Molecular Biology, Capital Medical University,
Beijing, 100069, China
*
Corresponding author: Tel.: +86 13381207228; fax: +86 10 66958303.
1
These authors contributed equally to this work.
1
Highlights We developed a reproducible, simple, and economical method of obtaining human OPCs from neurospheres. Isolated human OPCs have high purity and robust proliferative capacity. After induction, the OPCs can differentiate into oligodendrocytes.
2
Abstract Culture of human oligodendrocyte precursor cells (OPCs) can help understand the regulatory mechanism of differentiation and myelination of oligodendrocytes. However, existing culture methods have limitations, particularly the lack of a source of human donor tissue and high cost. We sorted cells with the A2B5+PSA-NCAM– phenotype
from
neurospheres
instead
of
human
donor
tissues
through
immunomagnetic sorting and subsequently cultured the isolated cells in OPC medium. Of all the isolated cells, 15.69% were of the A2B5+PSA-NCAM– phenotype. More than 90% of the isolated OPCs expressed the OPC-specific markers O4, PDGFαR, and Sox10, and less than 5% of cells expressed GFAP and Tuj-1. After induction, the isolated cells had the capacity to differentiate into oligodendrocytes. Furthermore, the OPCs could be stably passaged in vitro for at least four generations and all the cells had high expression levels of O4 and Sox10 and very low expression levels of GFAP and Tuj-1; moreover, the cells had the capacity to differentiate into oligodendrocytes. After four passages, OPCs can proliferate at least 14 times above. In addition, in the presence of B27, only one cytokine, namely, bFGF, was sufficient to maintain proliferation, and this greatly reduced the experimental cost. Cells of the A2B5+PSA-NCAM– phenotype have already been identified as OPCs. We developed and characterized a reproducible, simple, and economical method for the isolation and culture of human OPCs. This method will contribute to studying the function of OPCs in development, disease, and treatment. Keywords:
Oligodendrocyte
precursor
cells;
Neurosphere;
Proliferation;
Differentiation; Oligodendrocyte. 3
1. Introduction Oligodendrocytes (OLs) are cells derived from oligodendrocyte precursor cells (OPCs) and occur in the central nervous system (CNS). They play a crucial role in myelin sheath formation and facilitation of rapid conduction of neuronal action potentials [17]. OPCs differentiate through a series of developmental stages, such as pro-OLs, immature OLs, and mature OLs [23]. If OPCs are injured during development or remyelination, myelination of brain white matter is delayed or damaged, which can lead to nerve function deficit [15, 20]. OPC differentiation has been found to have important significance in the promotion of myelin repair [2, 30]. Therefore, understanding the mechanism by which OPC differentiation is regulated would provide insights into the process of myelin-associated diseases. It is important to establish a method for the in vitro isolation, amplification, and differentiation of OPCs. The specific immunophenotype of OPCs is distinct from those of the human fetal CNS tissue and adult human white matter. Several methods have been described for the isolation of human OPCs from the CNS, such as fluorescence-activated cell sorting (FACS) by exploiting cell surface-specific antigens, immunomagnetic sorting, or a combination of the two [9, 29]. Because the source of human donor tissue is very limited, it is difficult to obtain a large number of cells for research. Another method for obtaining human OPCs is by neural stem cell- or embryonic stem cell-induced directional differentiation. This method often needs complicated procedures of induction. Furthermore, obtaining OPCs of high purity via this method takes at least
4
21 days [18]; therefore, the experimental cost increases, limiting the potential productivity of OPCs. Therefore, a simple and economical method for the isolation and culture of human OPCs is needed to overcome these limitations. In this study, we described a simple and reproducible method for the isolation and culture of human OPCs. OPCs were isolated from neurospheres derived from human fetal brains by immunomagnetic sorting. Neurospheres were selected as the source of OPCs because they can be stably passaged in vitro; thus, they can serve as a stable source of sorting cells. First, we used microbeads to deplete polysialylated neural cell adhesion molecule (PSA-NCAM)+ neurons. Then, A2B5+ cells were isolated from the larger PSA-NCAM– cell population via magnetic sorting. Previous studies have reported subpopulations of A2B5+PSA-NCAM– marker cells in brain tissues, and this cell subpopulation has been identified to be OPCs, which exhibit extensive myelin production in shiverer mice [24, 29]. After sorting from neurospheres, the cells showed stably high expression levels of O4, NG2, and Sox10 and very low expression levels of GFAP and Tuj-1. After induction, the cells differentiated into oligodendrocytes, indicating the oligodendrocytic bias of the sorted cells. Furthermore, these cells can be stably cultured and passaged at least four times and proliferate at least 14 times in vitro. Therefore, we can use this reproducible and simple method to stably isolate and culture human OPCs in vitro. 2 Materials and methods 2.1 Reagents and supplies All reagents and supplies were obtained commercially. Dulbecco’s modified Eagle’s
5
medium/F12 (DMEM/F12; Gibco, C11330500BT), Neurobasal-A medium (Gibco, 10888-022), N2 supplement (Gibco, 17502-048), B27 supplement (Gibco, 17504-044), recombinant human FGF-basic (PeproTech, AF-100-18B), recombinant human
EGF
(PeproTech,
AF-100-15),
heparin
(Sigma,
H3149),
penicillin/streptomycin (Invitrogen, 15140), 0.25% trypsin (Gibco, 15050-065), trypsin
inhibitor
(Sigma,
T6522),
L-glutamine
(Gibco,
35050-061),
ethylenediaminetetraacetic acid (EDTA) disodium salt dehydrate (Amresco, 6381-92-6), Poly-D-lysine hydrobromide (Sigma, p6407), laminin (Invitrogen, 23017-015), OPC differentiation medium (OPCDM; ScienCell, 1631), cell strainer (BD Falcon, 352340), MS Column (Miltenyi Biotec, 130-091-506), fetal bovine serum (FBS; Gibco, 10099-141), anti-PSA-NCAM microbeads (Miltenyi Biotec, 130-092-966),
anti-PSA-NCAM
antibodies
(Miltenyi
Biotec,
130-093-273),
anti-A2B5 microbeads (Miltenyi Biotec, 130-093-388), Anti-A2B5 antibodies (Miltenyi Biotec, 130-093-581), mouse anti-O4 antibodies (1:300 dilution; R&D, MAB1326), mouse anti-Sox10 (R&D, MAB2864, 1:50), rabbit anti-GFAP (Invitrogen, 18-0063, 1:80), mouse anti-Tuj-1 (1:500 dilution; Abcam, ab7751), rabbit anti-Galc (Millipore, AB142, 1:50), rabbit anti-PDGFαR antibodies (1:400 dilution; Cell Signaling, 5241), Alexa Fluor 488-AffiniPure Goat Anti-Mouse IgG + IgM (H+L) (1:800 dilution; Jackson Immunoresearch, 111-545-042), and Alexa Fluor 594-AffiniPure goat anti-rabbit IgG (H + L, 1:800 dilution; Jackson Immunoresearch, 111-585-144).
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2.2 Neurosphere culture Neurospheres were cultured and passaged using the methods described previously [28]. Human fetal CNS tissues obtained from 10- to 13-week-old embryos were provided by Navy General Hospital, Beijing, China. All women had requested to terminate their pregnancy. After being fully informed according to the guidelines approved by the Ethics Committee of the hospital, they consented to donate the aborted fetuses. Surface regions of the cortex were randomly chosen. Cortex tissues were dissected and mechanically dissociated by repetitive blowing into a single-cell suspension. Primary cells were cultured at a density of 1 × 106 cells/mL in primary culture medium containing DMEM/F12, 1% L-glutamine, 1% N2 supplement, 2% B27 supplement, 20 ng/mL bFGF, 20 ng/mL EGF, 5 µg/mL heparin, and 1% penicillin/streptomycin. Every three to four days, two-thirds of the medium was removed and replaced with fresh primary culture medium. The expanded cells formed colonies and were cultured as free-floating “neurospheres” after 5–7 days of culture. As not all the cells in the tissues had survived, the cells that remained alive after dissection and mechanical dissociation could form neurospheres. The neurospheres were passaged every 10–12 days using 0.025% trypsin. The reaction was stopped by using 1.2 mg/mL of trypsin inhibitor. Neurospheres were repeatedly blown in order to obtain single-cell suspensions. The resultant cells were seeded into a flask, and the medium from passage 1 was used but without B27 supplement. 2.3 Magnetic cell sorting (MACS) for OPC Cells were sorted according to the instructions provided by microbeads. Neurospheres
7
were digested into single cells using 0.025% trypsin solution. Cells were passed through a 40-μm nylon mesh to remove cell clumps, which may clog the column. The cell number was then determined, and the cell suspension was centrifuged at 300 g for 10 min. The supernatant was completely aspirated, and 107 cells were resuspended in 60 μL of MACS buffer, which consisted of phosphate-buffered saline (PBS; pH 7.2), 0.5% FBS, and 2 mM EDTA. The suspension was mixed well and incubated for 10 minutes in the refrigerator (2–8°C). Then, 20 μL of anti-PSA-NCAM microbeads were added to the cell suspension containing 107 cells, mixed well, and incubated for 15 minutes in the refrigerator. The cells were washed by adding 1–2 mL of MACS buffer and centrifuged at 300 g for 10 min. During centrifugation, the MS column was placed in the magnetic field of a suitable MACS Separator and equilibrated with 0.5 mL of MACS buffer. The supernatant was completely aspirated and the cells (up to 108 cells) were resuspended in 500 μL of MACS buffer. The cell suspension was applied on to the column. Unlabeled cells that passed through the column were collected, and the column was washed with the appropriate amount of buffer. The total effluent, i.e., the unlabeled cell fraction, was then collected from the column. Washing was carried out three times by adding 500 μL of MACS buffer. The cell suspension was centrifuged at 300 g for 10 min. The supernatant was completely aspirated, and the cell pellet (107 cells) was resuspended in 60 μL of MACS buffer. Then, 20 μL of anti-A2B5 microbeads were added for 107 cells and the mixture was incubated for 15 min in the refrigerator. This mixture was passed through the MS column and washed thrice according to the same procedure. The column was then
8
removed from the separator, placed on a suitable collection tube, and 1 mL of buffer was pipetted on to the column. The magnetically labeled cells were immediately flushed by firmly pushing the plunger into the column. The cell suspension was then centrifuged at 300 g for 10 min to obtain the A2B5+PSA-NCAM– cell population. 2.4 Culture, passage, and differentiation of OPCs OPCs were plated into a flask at a density of 4 × 104 cells/cm2 and cultured in OPC medium for propagation. OPC medium was prepared by adding 2% B27 supplement (2%), 20 ng/mL bFGF, 1% penicillin/streptomycin, 5 μg/mL heparin, and 2 mM L-glutamine
to Neurobasal-A medium. Every three days, two-thirds of the medium
was removed and replaced with fresh OPC medium. After 7–10 days of proliferation, the OPCs reached 80–90% confluence. They were gently blown with a pipette and plated into a flask. For differentiation, the OPCs were plated on to PDL/laminin-coated well plates at a density of 4 × 104 cells/cm2 and cultured in OPC differentiation medium (OPCDM, ScienCell). Two-thirds of the medium was replaced with fresh medium every 2–3 days. The cells were cultured for 14–21 days. After incubation of the plates for 24 h, the OPCs were fixed with 4% paraformaldehyde for 20 min at room temperature (RT). The cells were then blocked with 5% normal goat serum for 60 min and incubated with the following primary antibodies: mouse anti-O4 antibody (R&D, 1:300), mouse anti-Sox10 (R&D, 1:50), rabbit anti-GFAP (Invitrogen, 1:80), mouse anti-Tuj-1 (Abcam, 1:500), rabbit anti-PDGFαR antibody (Cell Signaling, 1:200), and rabbit anti-Galc (Millipore, 1:50)
9
at 4°C overnight. After rinsing with 1× PBS three times, fluorescence-labeled secondary antibodies (Jackson Immunoresearch) were applied for 45 min at RT. Cell nuclei were stained with DAPI (1 μg/mL in PBS) for 5 min[5]. 2.5 OPC analysis by FACS Cells were double stained with anti-PSA-NCAM and anti-A2B5 antibodies (Miltenyi Biotec) to identify the purity of OPCs. According to the manufacturer’s instructions, up to 106 nucleated cells was resuspended in 100 μL of buffer. To this, 10 μL of anti-PSA-NCAM and anti-A2B5 antibodies were added, mixed well, and incubated for 10 min in the dark in the refrigerator. The cells were washed by adding 1−2 mL of MACS buffer and centrifuged at 300 g for 10 min. The supernatant was completely aspirated, and the cell pellet was resuspended in a suitable amount of buffer for flow cytometry analysis. Each experiment was independently repeated in triplicate. Data are presented as means ± standard deviation. 2.6 Cell proliferation assay The cell proliferation rate was measured at 7- or 10-day intervals (at each passage, for a total of four generations) by counting the number of viable cells. Cells were counted when OPCs were passaged. In brief, 1.5 × 105 cells were seeded into 24-well plates and designated as P0. After 10 days, cells were gently blown and the number of OPCs were counted, and designated as P1. After 7 days, the cells were spread to two wells to calculate the number of cells in the two wells, i.e., P2. Similarly, the number of cells for P3 and P4 were counted. Each experiment was carried out in triplicate, and all assays were done in triplicate. Data are presented as means ± standard deviation.
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Each experiment was independently repeated in triplicate. 2.7 Data collection 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 10 randomly selected fields from three wells using a 40× objective lens. Data are presented as means ± standard deviation. Each experiment was independently repeated in triplicate. 3 Results 3.1 Identification of cells isolated from neurospheres Of the cells isolated from neurospheres within the tenth generation, 15.69 ± 2.23% (n = 5) were of the A2B5+PSA-NCAM– phenotype, as determined by flow cytometry (Fig.1A). After immunomagnetic sorting, the cells were immediately examined to determine the purity of the populations, and 85.51% of the cells were found to be of the A2B5+PSA-NCAM– phenotype (Fig.1B). If the quantity of the initial human tissue cell was 106, we can get 107 neural stem cells after culturing 10-12 days. Then we used the 107 cells for sorting, we can approximate get 1.2×106 OPCs. Three days after plating, the cells gradually adhered to culture plates and had typical bipolar and/or tripolar morphology (Fig.1C). Cells were plated onto PDL-laminin-coated well plates. Three days later, 90.24 ± 1.04% of the cells expressed O4 (the OPC markers), 91.99 ± 1.32% expressed PDGFαR (the OPC markers), and 95.75 ± 1.52% expressed Sox10 (the OPC markers), and none of the cells were Galc+ (markers of oligodendrocytes). 4.63 ± 0.78% of the cells expressed GFAP (an astrocyte marker) and 4.68 ± 0.51% expressed Tuj-1 (a neuron marker) (Fig.1D-O). These data suggested that early stage
11
OPCs can be obtained with high purity by MACS of neurospheres. 3.2 First generation of OPCs differentiated into oligodendrocytes In order to confirm whether the sorted cells can differentiate into oligodendrocytes, OPCs were plated onto PDL/laminin-coated well plates and cultured in OPC differentiation medium. Fourteen days after plating, the cells cultured in the absence of serum formed more complex arborization patterns with more ramified, longer, and multiple processes and most of them were immature oligodendrocytes (Fig.2). These cells expressed antigens that could be recognized by the anti-O4 and anti-Galc antibodies with a multipolar morphology. Furthermore, some cells differentiated into astrocytes expressing GFAP. When cultured in the presence of serum, nearly all the cells were astrocytes. These data suggested that the OPCs isolated from neurospheres had the capacity to differentiate into oligodendrocytes. 3.3 In vitro amplification of OPCs sorted from neurospheres We obtained OPCs by sorting neurospheres, and these OPCs could be stably passaged for at least four generations in vitro and retained their original morphology and capacity. In this period, four passages of cells had typical bipolar and/or multipolar morphology, continuously expressed O4 at high levels, and most of them were in the early stage (Figs. 1C, 1F, and 3A). From the first generation to the fourth generation, O4+ % was respectively 90.24 ± 1.04%, 91.63 ± 1.23%, 92.06 ± 2.11% and 91.62 ± 0.82% (Fig.3D). There was no significant statistical difference between these generations. At the fourth passage of OPCs, over 90% of the cells expressed Sox10, and GFAP and Tuj-1 were expressed at very low levels, similar to the cells from the
12
first passage of OPCs (Fig.3C). At the same time, the cells had a strong proliferative capacity. The cell growth curve was drawn according to the number of viable cultured OPCs, which were counted at each passage. After four generations of in vitro culture, OPCs could proliferate at least 14 times above (Fig.3B). After induction, different passages of OPCs also differentiated into oligodendrocytes (data not shown). Therefore, OPCs isolated from neurospheres can be stably passaged, and the cells can proliferate and retain their morphology and capacity even after in vitro culture. 3.4 OPCs can be isolated from different passages of neurospheres We obtained OPCs from neurospheres within tenth generation. Neurospheres can be stably passaged and amplified many times in vitro; therefore, we tried to isolate OPCs from neurospheres of higher passages. We found that OPCs could be also isolated by MACS of neurospheres of passage 30. OPCs derived from thirtieth generation neurospheres were cultured in OPC medium for proliferation. These cells displayed the typical morphology of OPCs with bipolar and/or tripolar processes (Fig.4A). Immunofluorescence staining showed that 90.84 ± 3.68% of cells expressed O4, 90.56 ± 1.08% expressed PDGFαR, 93.7 ± 1.87% expressed Sox10, 5.3 ± 0.33% expressed GFAP and 4.62 ± 0.47% of the cells expressed Tuj-1. There was no significant statistical difference between early and late passages (Fig.4B–M). The cells were cultured in OPC differentiation medium to determine whether the cells had the capacity to differentiate into oligodendrocytes, and a subpopulation of the cells were O4+ and Galc+ with a multipolar morphology. Other cells expressing GFAP were astrocytes (Fig.4N and O). These data suggested that OPCs of high purity can be
13
obtained by magnetic bead sorting of late passage neurospheres, similar to that of early passage neurospheres and that the OPCs obtained from late passage neurospheres had the capacity to differentiate into oligodendrocytes. 4 Discussion OPCs play an important role not only as a major glial population in the CNS but also as an active participant in CNS signaling [15]. Devising a method for culturing human OPCs is essential to understanding the differentiation and myelination of oligodendrocytes. This will contribute to promoting myelin repair and understanding the process of myelin-associated diseases. OPCs are a model for understanding the regulatory mechanism of OL differentiation and myelination; therefore, research requires a large number of cells. If OPCs cannot be cultured in vitro, it will increase the cost of the research. If OPCs can stably proliferate in vitro, the procedures will be simplified and the cost of the research can be reduced. Therefore, it is necessary to devise a simple method for generating large quantities of highly purified OPCs. 4.1 Select the A2B5+PSA-NCAM- as the sorting marker We sorted cells with the A2B5+PSA-NCAM– phenotype (positive for the early oligodendrocyte marker A2B5 and negative for PSA-NCAM) from neurospheres. Previously, human OPCs obtained by sorting have been derived from fetal or adult tissues [1, 3, 6, 22]. However, human donor tissues are difficult to obtain, and ethical issues are involved in obtaining tissues. Therefore, the source of cells that can be used to obtain OPCs is limited. Sorting requires higher activity of cells in tissues. These aspects limited the use and yield of OPCs.
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4.2 Advantages of choosing neurospheres as a source of OPCs sorting OPCs were sorted from neurospheres. The neurospheres were cultured from the brains of aborted human fetuses. These neurospheres survived passage for many generations. Therefore, we obtained a large number of neurospheres, which served as an abundant source of OPCs. Neurospheres cultured in vitro had the capacity to differentiate into neurons, astrocytes, and oligodendrocytes [16]. We speculated that neurospheres cultured in vitro may contain OPCs. Indeed, 15.69 ± 2.23% of the neurospheres expressed the A2B5+PSA-NCAM– phenotype. This cell subpopulation showed high expression levels of O4, PDGFαR and Sox10 (more than 90%) and very low expression levels of GFAP and Tuj-1 (less than 5%). After induction, the cells can differentiate into oligodendrocytes, suggesting that the cells isolated from neurospheres were OPCs, similar to those isolated from brain tissues, exhibiting the various features of OPCs. Therefore, the most important innovation in this study is the use of neurospheres as a source of OPCs. Our OPCs were directly sorted from neurospheres without any induction. Sorting from neurospheres has many advantages. First, the use of neurospheres as a source of OPCs can avoid ethical issues. Only first generation neurospheres were cultured from discarded human fetal brains. Once neurospheres were formed, they can be passaged for many generations. Thus, the use of human tissues can be avoided. Second, the methods of culturing and passaging neurospheres have been relatively mature. Neurospheres can be cryopreserved and thawed when required, and stably passaged and amplified in vitro to obtain a sufficient number of cells. Third, OPCs also can be isolated from 25–30 generations
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of neurospheres, and have the same characteristics as the OPCs isolated from early passages such as passages 1, 3, 5, and 7. The capability to produce OPCs and ologodendrocytes is not altered by passaging neurospheres. As a large number of neurospheres can be obtained from one fetal brain tissue; theoretically, OPCs can be obtained as long as neurospheres can be passaged. Thus, a single fetal brain tissue sample can yield a large number of neurospheres within 30 generations. In addition, cultured neurospheres can be easily digested into single cells using trypsin, and these cells were found to have very high activity of more than 95% in vitro. Thus, neurospheres are a very good source of OPCs. 4.3 Only one cytokine – bFGF is sufficient to stably maintain growth and proliferation of OPCs. Previous studies have demonstrated that long-term culture and expansion of human OPCs after sorting from brain tissues in vitro require at least three or four cytokines such as bFGF, T3, PDGF-AA, and NT3 [7, 22]. Cells can be maintained for 1–7 days using N1 and bFGF [3, 29]. Apart from sorting, stem cell induction is another way to harvest human OPCs, but this method also requires a series of cytokines. Human OPCs obtained by neurosphere-induced directional differentiation also require a combination of many different cytokines such as bFGF, PDGF-AA, Shh, NT-3, and T3 [18-19]. OPCs derived from human embryonic stem cells require more cytokines, complicated procedures, and longer time, and the experimental cost involved consequently increases [11, 13, 21, 25]. Furthermore, human embryonic stem cells have potential tumorigenicity [4, 12, 26]. These disadvantages limit the potential
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application and productivity of OPCs. In our study, we devised a simple and economic system for the culture of human OPCs. In the presence of B27, only one cytokine, namely, bFGF, is sufficient to stably maintain the growth and proliferation of OPCs. This will greatly reduce the experimental cost. Previous studies have demonstrated that medium supplemented with N1 and bFGF could maintain OPCs for only a short period of time. In our study, we used B27 instead of N1, and found that OPCs had higher activity in medium containing B27 compared to medium containing N1 (data not shown). Furthermore, our medium greatly enhanced the growth of OPCs, and the OPCs proliferated more than 14 times after four generations. B27 has been reported to enhance the survival of OPCs, especially at low plating densities [14, 27, 31], and bFGF is known to stimulate the proliferation of cultured cells [8, 10]. Thus, B27 could improve the activity of OPCs. Therefore, the use of B27 and bFGF helped simplify the procedure, reduce the costs involved, and increase cell viability. 4.4 OPCs can be sorted and cultured stably in vitro. The OPCs in this study, regardless of whether they were sourced from early or late passage neurospheres or cultured for different generations after sorting, were found to have high stable expression levels of O4, PDGFαR, and Sox10 (more than 90%), and very low expression levels of GFAP and Tuj-1 (less than 5%), indicating that OPCs isolated from neurospheres can be stably cultured and proliferated. The isolated OPCs can be stably cultured for at least two months in vitro. In addition, when cultured in OPC differentiation medium, some subpopulations of OPCs can differentiate into
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oligodendrocytes, indicating that the method of culturing OPCs is stable. Using this method, large numbers of human OPCs of high purity can be obtained by a simple and economical sorting and culture method. The human OPCs isolated in this way can not only be stably cultured and proliferated, but they also have the capacity to differentiate into oligodendrocytes. And the capability to produce OPCs and ologodendrocytes is not altered by passaging neurospheres or purified OPCs. Thus, we can use this simple, economical, and reproducible method to stably obtain a large number of human OPCs from neurospheres and culture the OPCs in vitro. This method will contribute to studying the function of OPCs in the development and treatment of myelin-associated disease.
Acknowledgements This work was supported by the International Science & Technology Cooperation Program of China (ISTCP, No. 2012DFA30880).
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Figure legends Fig.1 Isolated cells expressing multiple OPC-specific markers. (A) Flow cytometry analysis of neurospheres within the tenth generation. (B) Flow cytometry analysis of cells isolated from third generation neurospheres. (C) After isolation for three days, the cells displayed typical OPC morphology with bipolar and/or tripolar processes. (D–F) Isolated cells express the OPC surface markers O4. (G–I) Isolated cells express the OPC surface marker PDGFαR. (J–L) Isolated cells express the OPC marker Sox10. (M) Astrocyte marker GFAP. (N) Neural cell marker Tuj-1. (O) The percentage of cells expressing O4, PDGFαR, Sox10, GFAP and Tuj-1. All cell nuclei were labeled with DAPI (blue). The red arrows indicated the negative cells. Scale bars: 50 μm (C–L, N), 100 μm (M). Fig.2 The first generation of OPCs differentiated into oligodendrocytes. (A, D) Cells exhibit ramified, longer, multiple processes. (B) Immunofluorescence staining of O4 (green), GFAP (red). (E) Cells were O4+. (C, F) Cells were Galc+. All cell nuclei were labeled with DAPI (blue). Scale bars: 50 μm (A–C), 25 μm (D–F). Fig.3 Isolated OPCs cultured for four cell passages. (A) Cells of passages 2–4 displayed the typical morphological characteristics of OPCs with bipolar and/or tripolar processes and the surface maker O4+. Nuclei were labeled with DAPI. Scale bars = 50 μm. (B) The cell growth curve was drawn according to the numbers of viable OPCs, which were counted at each passage (passages 1–4). OPCs (1.5 × 105) cells were seeded into the wells of a 24-well plate, and designated as P0. Data are shown as means ± SD, n = 3. (C) OPCs of passage 4 expressed Sox10, GFAP, and 21
Tuj-1. All cell nuclei were labeled with DAPI (blue). The red arrows indicated the negative cells. Scale bars: 50 μm (Sox10, DAPI, and Tuj-1), 100 μm (GFAP). (D) OPCs of passage1-4 expressed O4. Fig.4 Cells isolated from neurospheres of passage 30 expressed multiple OPC-specific markers. (A) OPCs displayed typical OPC morphology with bipolar and/or tripolar processes. (B) Astrocyte marker GFAP. (C) Neural cell marker Tuj-1. (D–F) Isolated cells express the OPC surface marker O4. (G–I) Isolated cells express the OPC surface marker PDGFαR. (J-L) Isolated cells express Sox10. (M) Isolated cells obtained from neurospheres of early and late passages expressed O4, PDGFαR, Sox10, GFAP and Tuj-1. (N–O) OPCs differentiated into oligodendrocytes. Immunofluorescence staining of O4 (green, N) and GFAP (red, N). Oligodendrocytes were highly arborized and Galc+ (O). All cell nuclei were labeled with DAPI (blue). The red arrows indicated the negative cells. Scale bars: 50 μm.
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