A xeno-free culturing protocol for pluripotent stem cell-derived oligodendrocyte precursor cell production
Currently, effective treatments for spinal cord injury (SCI) are under investigation. Previously it has been demonstrated that oligodendrocyte precursor cells (OPCs) derived from human embryonic stem cells (hESCs) can enhance remyelination of damaged neurons and restore the locomotor function of SCI animals [1]. Therefore, several methods have been developed to produce myelinating cells from pluripotent stem cells for treatment of SCI [2–6]. Pluripotent hESCs have efficient proliferation and differ-entiation capacity in vitro and in vivo [7]. Thus, these cells are an ideal source for oligoden-drocyte progenitor cells and oligodendrocyte differentiation [2–6].
Regenerative medicine research aims to transfer the stem cell products from bench to bedside. However, several important aspects must be taken into account prior to entering clinical use. For example, the safety risks of stem cell therapies must be reduced. Production of hESC-derived neural cell grafts for clinical use requires the cell differentiation methods to be performed according to the guidelines of the
US FDA, European Medicines Agency and other country-specific agencies [8–9]. In addi-tion, the manufacturing process of stem cell products must be performed according to good manufactory practice (GMP) instructions. All culturing components should be traceable in terms of their origins, purity, quality control and sterility [9]. Furthermore, the need for purifica-tion processes for heterogeneous hESC-derived populations and elimination of pluripotent stem cells should be taken into account to avoid the presence of tumorigenic cells in grafts [6,10–12]. In addition, with respect to methodology, the use of xeno-free (XF; free from xenogenic substances) differentiation protocols would diminish the risk of pathogen cross-transfer or changes to cell surfaces in cell grafts [13,14]. Currently existing differentiation methods for hESC-derived oligodendrocytes include the use of xenogenic components, such as Matrigel™ (BD Biosciences, USA) and B27 supplement including bovine serum albumin (BSA) [2–4]. Although the differentiation and culture condi-tions do not have to be entirely XF, the minimal
Aim: To show that human embryonic stem cells (hESCs) can be efficiently differentiated into oligodendrocyte precursor cells (OPCs) in a xeno-free medium with a specific medium supplement and specific human recombinant growth factors. Materials & methods: The xeno-free OPC-differentiation medium for pluripotent stem cells was developed by using StemPro® neural stem cell xeno-free medium supplement together with human recombinant growth factors SHH, PDGF-AA, IGF-1, EGF, basic FGF and CNTF, in addition to RA, T3, human laminin and ascorbic acid. We analyzed the differentiated hESC-derived OPCs and oligodendrocytes with quantitative real-time (RT) -PCR, RT-PCR, flow cytometry and immunocytochemistry, and we performed NG2-positive selection for OPC cultures with fluorescence-activated cell sorting. Results: Based on quantitative RT-PCR ana lysis, OPCs after 9 weeks of differentiation in xeno-free medium expressed OLIG2, SOX10 and NKX2.2 at elevated levels compared with control conditions. According to the flow cytometric analysis, the cells expressed A2B5 (>70%) and NG2 (40–60%) at 5 weeks time point whereas maturing oligodendrocytes expressed O4 (60-80%) at 11 weeks time point. In addition, hESC-derived OPC populations were purified based on NG2-positive selection using fluorescence-activated cell sorting. NG2-positive OPC populations survived and differentiated further into O4 expressing oligodendrocytes in xeno-free medium, and the sorted cell populations were free from pluripotent Tra1–81 and Oct-4 -positive cells. Conclusions: This study confirms that the xeno-free culturing method can support the differentiation and purification of hESC-derived OPC populations and provides an initial step toward safe cell graft production for the future clinical applications.
KEYWORDS: human embryonic stem cell-derived oligodendrocytes n human recombinant growth factors n oligodendrocyte precursor cells n pluripotent stem cells n xeno-free
Maria Sundberg1, Anu Hyysalo1, Heli Skottman1, Soojung Shin2, Mohan Vemuri2, Riitta Suuronen1,3,4 & Susanna Narkilahti†1
1Regea – Institute for Regenerative Medicine, University of Tampere & Tampere University Hospital, Tampere, Finland 2Life Technologies, Primary & Stem Cells, Frederick MD 21704, USA 3Department of Eye, Ear & Oral Diseases, Tampere University Hospital, Tampere, Finland 4Department of Biomedical Engineering, Tampere University of Technology, Tampere, Finland †Author for correspondence:Tel.: +358 335 514 123 Fax: +358 335 518 498 [email protected]
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use of animal-derived components diminishes the risks of unidentified components affect-ing grafted cells and inducing host immuno-rejection. Therefore, development of XF differ-entiation protocols for hESC-derived OPCs is important for safe cell graft production for the treatment of SCI.
In this study, we aimed to develop XF cul-turing protocol for the efficient differentiation of hESC- and induced pluripotent stem cell-derived OPCs. We tested XF medium supple-ment (StemPro® neural stem cell medium [NSC]-XF, Invitrogen Life Technologies, MN, USA) in different culturing conditions and com-pared these media with the culturing conditions modified from our previously published proto-col [6]. We were able to show that optimized XF culturing conditions supported the growth and differentiation of hESC-derived OPCs as well as induced pluripotent stem cell-derived OPCs. We believe that this protocol may provide a basis for GMP-level graft production.
Materials & methods n hESC culturing
In this study we used two hESC lines derived at the Regea – Institute for Regenerative Medicine, Regea06/040 (passage 44 and pas-sage 49) and Regea08/023 (passage 38 and pas-sage 43) [15]. Both of these cell lines are listed in the European Human Embryonic Stem Cell Registry [101]. hESCs were cultured as previously described [11,16]. Briefly, hESCs were cultured on top of mitotically inactivated human foreskin fibroblasts (CRL-2429, ATCC, Manassas, VA, USA) in hESC medium containing Dulbecco’s modified Eagle’s medium (DMEM; Gibco® Invitrogen, Carlsbad, CA, USA) supple-mented with 20% knockout serum replace-ment (Gibco Invitrogen), 2 mM GlutaMax (Gibco Invitrogen), 0.1 mM minimum essen-tial medium nonessential amino acids (Cambrex BioScience, Karlskoga, Sweden), 0.1 mM b-mercaptoethanol (Gibco Invitrogen), 50 U/ml penicillin/streptomycin (Cambrex) and 8 ng/ml basic FGF (bFGF; R&D Systems, Minneapolis, MN, USA). hESC passaging was performed by mechanical splitting of colonies at 5–7-day intervals, and passaged cells were replated on fresh human foreskin fibroblasts. The hESC karyotype normality was routinely tested.
n OPC differentiationHuman embryonic stem cells were differenti-ated into OPCs in a suspension culture in ultra-low cell cluster six-well plates (Costar, Corning
Inc., Corning, NY, USA). In this study we tested six different combinations of growth factors either in basic neural stem cell medium (NS) or XF media. The basal NS medium con-tained DMEM/F-12 supplemented with 1 × N2 supplement and 2 mM GlutaMax (both from Gibco Invitrogen), 0.6% glucose, 5 mM Hepes, 2 µg/ml heparin (all from Sigma, St Louis, MO, USA), and 25 U/ml penicillin and streptomy-cin as described earlier [11]. The XF-medium contained DMEM/F12, XF-NSC-supplement StemPro, 2 mM GlutaMax (Gibco Invitrogen) and 25 U/ml penicillin and streptomycin. The protocol contained three different stages:
� Stage 1, neural induction of hESCs: hESC colonies were mechanically isolated from the feeder cells into small clusters and directly transferred into neural media. Differentiating neural cell aggregates were cultured for 4 weeks in stage 1 NS1, NS2, NS3, XF1, XF2 or XF3 media;
� Stage 2, OPC production: cell spheres were cultured in stage 2 for 3 weeks to induce the OPC development in NS1, NS2 NS3, XF1, XF2 or XF3 media in suspension culture;
� Stage 3, OPC maturation: OPCs were dif-ferentiated by withdrawal of the proliferating growth factors from the media in stage 3, after which the cells were cultured for 2–4 weeks in NS1, NS2 NS3, XF1, XF2 or XF3 media.
Table 1 describes the detailed growth factor compositions included in different media.
n RNA extraction & cDNA synthesisRNA was extracted using NucleoSpin® RNA II kit (Machenery-Nagel GmbH & Co, Düren, Germany). For quantitative real-time (qRT)-PCR a total of 200 ng of RNA was used for each cDNA reaction performed with the High Capacity cDNA kit (Applied Biosystems, Foster City, CA, USA). For real-time (RT)-PCR a total of 50 ng of RNA was used for each cDNA reaction.
n RT-PCREach PCR reaction contained 1 µl of cDNA, 0.25 µM of forward and reverse primers, 1 × Taq-buffer (-MgCl, +KCl, Fermentas, Leon-Rot, Germany), 2.5 mM deoxyribo-nucleotide triphosphate (Fermentas), 25 mM MgCl (Fermentas), dH
2O and 0.6 U Taq-
DNA-polymerase enzyme (Fermentas). PCR program parameters were: denaturation at 95°C
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for 3 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 1 min; and a final extension at 72°C for 5 min.
n Quantative RT-PCRQuantitative RT-PCR was performed with a TaqMan® assay (Applied Biosystems) for OLIG2, NKX2.2, SHH, SOX10 and GAPHD using the Applied Biosystems 7300 Real-Time PCR System. Quantative PCR was performed with the following program: 2 min at 50°C, 10 min at 95°C, 40 cycles of alternating 95°C for 15 s and 60°C for 1 min, followed by a melting curve ana lysis for specificity control. Undifferentiated hESCs were used as a reference sample and GAPDH as a reference gene. Three parallel samples for each medium and time point were analyzed. The Ct values and relative quantita-tion ana lysis were calculated with the Sequence Detecting Software, 7300 Fast System version 1.3.1 (Applied Biosystems). Data ana lysis was performed with Microsoft Excel.
n Flow cytometryFlow cytometric ana lysis of differentiating OPCs was performed at stage 2 (5 weeks), stage 2 (8 weeks) and stage 3 (11 weeks) (Figure 1). Sample preparation was performed using a previously published method with modifica-tions [6,11]. Briefly, cells were trypsinized with
0.05 × trypsin-EDTA for 5 min at 37°C. Trypsin was then inactivated with 5% human serum (HS) in phosphate-buffered saline (PBS). Cells were centrifuged at 1900 rpm for 4 min at 4°C and resuspended in 2% HS in PBS. Aliquots of 100,000 viable cells per sample were used for flow cytometric ana lysis. Cells were incubated with either rabbit anti-NG2 (1:200, Chemicon, MA, USA), mouse anti-O4 (1:100, R&D Systems) or mouse anti-Tra1–81 (1:200, Santa Cruz Biotechnology, CA, USA) antibodies, which did not have fluorochrome conjugates, for 30 min at room temperature. This was fol-lowed by incubation with anti-rabbit Alexa 488 (Molecular Probes, Invitrogen) or anti-mouse PE (Calteg Laboratories, UK) antibodies for 20 min at room temperature. For detection of A2B5-APC, CD44-PE, CD56-PE, CD133-PE, CD140a-PE and CD326-APC, antibodies were incubated with cells for 20 min at room tempera-ture in the dark; the dilution of each antibody was optimized according to the manufacturers’ instructions (BD Biosciences, Franklin Lakes, NJ, USA). After labeling, the cells were washed twice with 2% HS in PBS and centrifuged at 1900 rpm (Multifuge 1S-R Heraeus, Siehe Rotor, Kendro Laboratory Products, Germany) for 4 min at 4°C. Cells were suspended in 2% HS in PBS and analyzed using a FACSAria (BD Biosciences). During ana lysis, forward and side scatter parameters were used to determine and
Table 1. Growth factor combinations in neural stem cell and xeno-free media during oligodendrocyte precursor cell differentiation
Growth factor concentrations in NS and XF media were: 20 ng/ml of EGF, 20 ng/ml (in stage 1) and 10 ng/ml (in stage 2) of bFGF, 10 ng/ml of CNTF, 100 ng/ml of SHH (all from R&D Systems, Minneapolis, MN, USA), 1× B27 (Gibco®, Invitrogen, CA, USA), 10 µM of RA, 40 ng/ml of T3, 200 µM of AA, 100 ng/ml of IGF-1, 1 µg/ml of laminin (all from Sigma, St Louis, MO, USA) and 20 ng/ml of PDGF-AA (Peprotech, NJ, USA). bFGF: Basic FGF; NS: Neural stem cell medium; XF: Xeno-free.
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gate the cell population of interest. Background fluorescence was excluded using unlabeled cells or cells incubated with only secondary anti-bodies. For each sample, 20,000 events were recorded and analyzed.
n SortingFor the purification of the hESC-derived OPC population, the OPCs were sorted with NG2-positive using fl uorescence-activated cell sort-using fluorescence-activated cell sort-ing, and plated in 48-well culture plates (Nunc., Denmark), coated as previously described in OPC medium (XF2) [6]. After 10 days of subcul-turing, NG2-positive cells were live-stained with O4 antibody (R&D Systems), and fixed and stained with NG2 (Chemicon), Tra1–81 (Santa Cruz Biotechnology) and Oct-4 (R&D Systems).
n ImmunocytochemistryDifferentiated oligodendrocytes in adherent cultures from stages 2 (5 and 8 weeks) and 3 (10 weeks) were characterized using immu-nocytochemistry. Briefly, cells were fixed with 4% paraformaldehyde for 10 min at room tem-perature and washed twice with PBS. Normal donkey serum (NDS; 10%) and BSA (1%) in
a PBS solution (containing 0.1% Triton X-100 for detection of intracellular proteins) was used for blocking. Primary antibodies were diluted in 1% BSA and 1% NDS in PBS (containing 0.1% Triton X-100) and incubated with cells overnight at 4°C. Secondary antibodies were diluted in 1% BSA in PBS and incubated with cells for 1 h at room temperature in the dark. Cells were then washed with PBS and mounted using Vectashield® mounting medium, containing 4 ,́6-diamid-ino-2-phenylindole for detection of cell nuclei (Vector Laboratories Inc., Burlinge, CA, USA). The primary antibodies used were GalC (1:200, Millipore, MA, USA), GFAP (1:800, R&D Systems), MAP-2 (1:600, Millipore), Nestin (1:200, Millipore), Oct-4 (1:100, R&D Systems), OLIG2 (1:200, Millipore), Sox2 (1:200, Santa Cruz Biotechnology) and Tra1–81 (1:200, Santa Cruz Biotechnology). The secondary antibod-ies used were anti-rabbit Alexa488 anti-mouse Alexa568, and anti-sheep Alexa568 (1:400, all from Molecular Probes, Invitrogen).
n Live stainingFor the staining of living cells, cells were incu-bated with an O4 antibody (1:100, R&D
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Figure 1. Human embryonic stem cell-derived oligodendrocyte precursor cell differentiation protocol. Differentiation protocol included three different stages: stage 1, neural induction of hESCs; stage 2, OPC production; stage 3, OPC differentiation. qRT-PCR, RT-PCR, FACS and ICC sample collection time points are presented in the timeline as well as different growth factors included in the media. bFGF: Basic FGF; FACS: Fluorescence-activated cell sorting; hESC: Human embryonic stem cell; ICC: Immunocytochemistry; OPC: Oligodendrocyte precursor cell; qRT-PCR: Quantitative real-time PCR.
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Systems) for 30 min at 37°C and then washed once with PBS. Cells were then incubated for 30 min with an anti-mouse Alexa568 second-ary antibody (Molecular Probes, Invitrogen),
washed with PBS and observed using an Olympus IX51 phase-contrast microscope equipped with f luorescence optics and an Olympus DP71 camera (Olympus, Finland).
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Figure 2. Relative quantitation of SHH, OLIG2, SOX10 and NKX2.2 expression during human embryonic stem cell-derived oligodendrocyte precursor cells differentiation at 4-, 5-, 7- and 9-week time points. Cells were cultured in NS1, XF1, NS2, XF2, NS3 and XF3 media. Results are presented in the logarithmic scale of relative quantitation, analyzed with AB 7300 Sequencing detection software, version 1.4 (Applied Biosystems, Foster City, CA, USA), undifferentiated human embryonic stem cells (represented by 0 on the y-axis = Log10 relative quantitation) were used as a control sample and the endogenous control gene for each sample was GAPDH. The standard error was less than 0.03 between three parallel samples analyzed for each time point, and the results represents means of three sample values analyzed. hESC lines Regea08/023 and Regea06/040 were used in this ana lysis. hESC: Human embryonic stem cell; NS: Neural stem cell medium; XF: Xeno-free.
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ResultsFigure 1 presents the outline of the XF differ-entiation protocol for pluripotent stem cell-derived OPCs and oligodendrocytes, and the sample collection time points. This developed differentiation protocol was based on the use of a XF medium supplement StemPro NSC-XF. The NS medium used for control conditions included commercially available neural supple-ments N2 and B27. The combinations of differ-ent growth factors used for each medium com-position were modified according to previously published protocols [6]. Detailed medium com-positions are presented in Table 1. This protocol differs from a previously published protocol [6] by its length, which was 4 weeks shorter for the initial neural induction, and by the addition of SHH, RA, T3 and AA to the medium to induce oligodendrocyte differentiation.
XF medium induce SHH, SOX10, OLIG2 & NKX2.2 expression in hESC-derived OPCsAccording to the qRT-PCR ana lysis, during oligodendrocyte differentiation the expres-sion of SHH was elevated after 4 weeks and remained stable thereafter. However, in OPCs differentiated in NS2 medium, (hESC line Regea06/040), the expression of SHH was increased at the 4-, 5- and 9-week time points compared with expression in XF
medium (Figure 2). During oligodendrocyte dif-ferentiation, the expression of OLIG2 increased gradually in all the culture conditions tested in both hESC lines. In particular, cells cultured in the XF media (XF2 and XF3) had increased OLIG2 expression at the 7- and 9-week time points (Figure 2). In addition, the expression of SOX10 was upregulated in XF2 and XF3 media-treated cells at the 9-week time point (Figure 2). In addition, the expression of NKX2.2 was increased during OPC differentiation, and the cells differentiated in XF2 and XF3 media had elevated expression of NKX2.2 compared with cells cultured in other media (hESC line Regea08/023). In addition, OPCs differentiated from the Regea06/040 cell line had increased expression of NKX2.2 in XF2 medium at the 9-week time point (Figure 2). In summary, during OPC differentiation SHH expression remained stable in all culture media in both hESC lines, and expression of OLIG2, SOX10 and NKX2.2 increased during differentiation in XF2 and XF3 media.
According to semiquantitative RT-PCR ana-lysis, expression of the pluripotency marker OCT-4 was decreased in hESC-derived OPCs after 4 weeks of differentiation. Expression of the NSC marker NESTIN was constant dur-ing differentiation in both cell groups cul-tured in NS2 and XF2 media, whereas PAX-6 expression was higher in cells differentiated in XF2 medium. Expression of the OPC mark-ers PDGFR and NG2 was higher in cells dif-ferentiated in NS2 medium, whereas SOX10 expression was increased in cells differentiated in XF2 medium. In addition, the expression of the oligodendrocyte-specific markers NKX2.2, NKX6.2 and OLIG2 appeared to be greater in cells differentiated in XF2 medium at the 9-week time point (Figure 3).
n hESC-derived OPCs in XF medium expressed NG2 & O4Flow cytometry ana lysis of hESC-derived OPCs cultured in XF media revealed that the expression of A2B5, NG2, O4, CD140a, CD44, CD133 and Tra-1–81 was similar to cells cul-tured in control conditions (Figures 4 & 5). By contrast, NS1 and XF1 media did not induce NG2 upregulation during OPC development after 8 weeks. In comparison, cells cultured in NS2, XF2, NS3 and XF3 media expressed NG2 at 50–70% (Figure 4). In addition, after the last stage of differentiation at the 11-week time point, the cells differentiated in NS2 and XF2 media had increased expression of
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Figure 3. Reverse-transcriptase PCR results of human embryonic stem cell-derived oligodendrocyte precursor cells after 4, 5, 7 and 9 weeks of differentiation in neural stem cell 2 or xeno-free 2 medium. Results are presented for the human embryonic stem cell line Regea06/040. White histogram presents background fluorescence and gray histogram presents stained sample. NS: Neural stem cell medium; XF: Xeno-free.
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O4 (70–80%) compared with cells cultured in other conditions (Figure 4). At the 11-week time point, NS1 and XF1 media did not sup-port the upregulation of O4 expression, which varied from 20 to 60% in both hESC lines. A detailed comparison of protein expression levels during OPC differentiation in NS2 and XF2 medium is presented in Figure 5. In addition, the specificity of O4 staining in flow cytomet-ric ana lysis was confirmed by live cell immu-nostaining, which showed the morphology of maturing O4-expressing oligodendrocytes after 11 weeks of differentiation XF2 media (Figure 6). Immunocytochemical ana lysis of hESC-derived OPCs in XF2 medium revealed that after the neural induction phase (stage 1,
5 weeks), the cells expressed the NSC markers Sox2 and Nestin. After the OPC production stage (stage 2, 8 weeks) the cells expressed the OPC and oligodendrocyte-specific markers OLIG2 and GalC. After the final differentia-tion stage (stage 3, 11 weeks) the morphology of GalC-positive cells in XF2 medium resembled mature oligodendrocytes. During differentia-tion Stages 1–3, few GFAP-positive astrocytes were detected, whereas MAP-2-positive neu-rons were only detected after 5 weeks of dif-ferentiation (stage 1) (Figure 7). These results also show the hetero genecity of pluripotent stem cell-derived OPC populations, which fur-ther indicate the need for enrichment of pure OPC populations.
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Figure 4. Flow cytometric ana lysis of A2B5, NG2, O4, CD140a, CD44, CD133 and Tra-1–81 expressions at 5-, 8- and 11-week time points during human embryonic stem cell-derived oligodendrocyte precursor cell differentiation. Differentiated cells were cultured in NS1, XF1, NS2, XF2, NS3 and XF3 media. hESC-lines Regea08/023 and Regea06/040 were used in this ana lysis. hESC: Human embryonic stem cell; NS: Neural stem cell medium; XF: Xeno-free.
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n Sorting of hESC-derived OPCs in XF mediumThe sorting of NG2-positive OPCs from the hESC-derived cell population differentiated in XF2 medium revealed that 10 days after sorting, the cells morphologically resembled OPCs and stained positive for NG2 (>98%).
In addition, few mature O4-positive oligoden-drocytes were detected after 10 days of sorting with live cell immunostaining (Figure 8), and the cells were negative for pluripotency markers Tra-1–81 and Oct-4 (Figure 8). After the sort-ing step nestin-positive cells were not detected (data not shown). This demonstrates that it is
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Figure 5. Histograms of flow cytometric data during differentiation stages 1–3, (5, 8 and 11 weeks, respectively). Cells were differentiated either in NS2 or XF2 media where white histogram presents background fluorescence and gray histogram presents stained sample. Results are presented from human embryonic stem cell-line Regea08/023. NS: Neural stem cell medium; XF: Xeno-free; w: Weeks.
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Figure 6. Live cell staining of O4 expression in cells differentiated in xeno-free 2 medium. Bright field images of cells (A & D), cells stained with O4 (red) (B & E) and the merged figures (C & F). Results are presented from human embryonic stem cell-line Regea08/023.
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possible to subculture purified NG2-positive OPCs in XF conditions as has been demon-strated with hESC-derived OPCs in xenogenic conditions [6].
DiscussionIn this study we developed a protocol for the production of OPCs and oligodendrocytes from hESCs for potential therapeutic applica-tions. Since efficient XF methods for genera-tion of hESCs already exist [17], it is possible to combine these methods and modify the OPC differentiation protocol to meet GMP stand-ards. Our differentiation method described here is divided into three different stages: neural induction of hESCs, induction and
propagation of OPCs and maturation of the OPCs to oligodendrocytes. Each differentia-tion stage includes human recombinant growth factors in XF medium; therefore, the method will be easily transferrable into GMP condi-tions. OPCs and oligodendrocytes generated with this protocol were analyzed in vitro for the expression of OPC and oligodendrocyte-spe-cific markers using qRT-PCR, RT-PCR, flow cyto metry, cell sorting and immunocytochem-istry. Current hESC-derived OPC and oligo-Current hESC-derived OPC and oligo-dendrocyte differentiation protocols, including animal-derived substances [1,4], are used for the graft production by the Geron Company (CA, USA). The hESC-lines that are used for these cell grafts production have not been derived in
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Figure 7. Immunocytochemical ana lysis of human embryonic stem cell-derived oligodendrocyte progenitor cells’ protein expression after 5, 8 and 11 weeks of differentiation. Cells were differentiated either in NS2 or XF2 media. Human embryonic stem cell-line Regea08/023 was used for this ana lysis. NS: Neural stem cell medium; NSC: Neural stem cell; OPC: Oligodendrocyte progenitor cell; XF: Xeno-free.
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XF conditions during the cell line establish-ment [4]. This requires extra efforts for the safety testing of cell grafts, since it has been shown that exposure to animal proteins may change the carbohydrate modifications of cell surfaces, which may lead to immunological responses or even rejection events after transplantation [13,14]. In addition, the glial differentiation medium used for OPC differentiation contains the neural supplement B27, which includes BSA [2–4]. It has been demonstrated that the quality of B27 may vary considerably between different manufacturers owing to the differ-ences in BSA quality, which causes difficulties for replicating differentiation protocols [18]. In addition, several OPC differentiation pro-tocols include use of Matrigel, which affects the differentiation process; however, Matrigel is an animal derived-component and contains substances that have not been identified thor-oughly [3,4]. Although FDA regulations do not require cells to be cultured in completely XF conditions for graft production, we believe that animal-derived substances may pose risks for xeno-contamination or rejection events when cells are engrafted into patients. Therefore, it is important to develop XF protocols for hESC-derived graft production.
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Figure 8. Sorting of human embryonic stem cell-derived oligodendrocyte precursor cells. Dot plot shows gating of oligodendrocyte precursor cell population (A). Histogram shows that 86% of cells were NG2-positive prior to sorting and 10 days after sorting cells were positive for NG2 and few cells expressed O4. (White histogram presents background fluorescence and gray histogram presents stained sample.) (B). Live cell staining with O4 shows that 10 days after sorting the O4-positive cells morphologically resembled mature oligodendrocytes. Bright field figure of oligodendrocytes (scale bar: 50 µm) (C), stained for O4 (red; scale bar: 50 µm) (D), and shown in merged figure (scale bar: 50 µm) (E). Tra1–81- (red) and Oct-4- (green) positive cells were not detected in the sorted cell population (scale bar: 100 µm) (F). The human embryonic stem cell line Regea08/023 was used for the ana lysis.
Compared with previous studies performed in the presence of xenogenic ingredients, we optimized OPC differentiation protocols for pluripotent stem cells in XF medium in the presence of several growth factors that induce OPC differentiation [2–6]. A previously pub-lished method for hESC-derived OPC produc-tion described the importance of conserved SHH signaling during OPC development [2]. According to our qRT-PCR ana lysis, SHH expression was constant during differentiation and differences were not significant between media. The elevated expression of SHH in hESC-derived OPCs is important because it affects to the sequential activation of SOX10, OLIG2 and NKX2.2, which are crucial tran-scription factors affecting oligodendrocyte development [2]. In addition, we demonstrated that SOX10, OLIG2, and NKX2.2 expression was increased during differentiation in XF2 medium. Importantly, compared with control conditions (NS1–3 media) the expression of these genes was elevated at the 9-week time point in the cells cultured in XF2 medium.
Interestingly, according to flow cytometric ana lysis, the expression of the OPC marker NG2 [19]was increased in the cells cultured in NS2, NS3, XF2 and XF3 media (50–70%) and
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last stage of differentiation at the 11-week time point in the cells cultured in XF2 or XF3 media (>60–80%). Furthermore, the expression of maturing oligodendrocyte marker O4 [20] increased after the last stage of differentia-tion at the 11-week time point in the cells cul-tured in XF2 or XF3 media (being 60–80%). Cells cultured in control conditions (NS2 and NS3 media) had 50–80% expression of O4. Therefore, XF2 and XF3 media supported hESC-derived oligodendrocyte differentiation as effectively as the control media (NS2 and NS3). Furthermore, our protocol produced O4-positive cells more effectively than previ-ously published hESC-derived oligodendro-cyte differentiation methods, which include xenogenic products and resulted in >40% O4-positive cell production [3]. As cells cultured in NS1 or XF1 showed lower NG2 (stage 2) and O4 expression levels (stage 3) during the dif-ferentiation protocol, we concluded that this was caused by the lack of important growth factors, such as SHH, RA, T3 and AA, in NS1 and XF1 media. These factors were included in XF2–3 and NS2–3 media and stimulated the oligodendrocyte differentiation of hESCs in a similar to previously shown [2]. Although we have previously used the same growth factor combinations as in NS1 medium for hESC-derived OPC production [6] we conclude that a shorter protocol for neural induction (4 vs 8 weeks) requires use of additional growth fac-tors in media for efficient neural patterning and oligodendrocyte differentiation.
Furthermore, we were able to show that cells cultured in XF2 could be sorted with NG2-positive selection, and after subcultur--positive selection, and after subcultur-selection, and after subcultur-ing in XF medium the cells differentiated into O4-positive cells. This population did not con-tain pluripotent stem cells. We suggest that the XF medium XF2 supports effective enrichment and a subculturing of hESC-derived NG2-positive OPC population. This possibility for enrichment of a pure human OPC population is
important for production of safe cell grafts for human patients that are free from tumorgenic pluripotent stem cells [12,21–24].
In summary, we have demonstrated that OPCs and oligodendrocytes derived from hESCs can be differentiated and cultured in XF medium and purified with fluorescence-acti-vated cell sorting using NG2-positive selection. The StemPro NSC-XF supplement supported the differentiation of cells in a similar manner to two known medium supplements, N2 and B27, in the control conditions. This XF dif-ferentiation protocol provides a beginning for future preclinical studies and scalable GMP-applicable processes and for the generation of pluripotent stem cell-derived OPCs and oligo-dendrocytes for CNS regeneration.
AcknowledgementsThe authors wish to thank the personnel of Regea – Institute for Regenerative Medicine, for their support and assistance in the stem cell research.
Financial & competing interests disclosureThis study was supported by the Academy of Finland, The Finnish Funding Agency for Technology and Innovation TEKES, The Competitive Research Fund of the Pirkanmaa Hospital District, the Finnish cultural foundation, the Swedish cultural foundation and the Science foundation of City of Tampere. The authors have no other relevant af� li-The authors have no other relevant af�li-ations or �nancial involvement with any organization or entity with a �nancial interest in or �nancial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate insti-tutional review board approval or have followed the princi-ples outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investi gations involving human subjects, informed consent has been obtained from the participants involved.
Executive summary
� Human embryonic stem cells (hESCs) can be differentiated efficiently into oligodendrocyte precursor cells (OPCs) and oligodendocytes in xeno-free medium.
� According to quantative real-time PCR ana lysis, the hESC-derived OPCs differentiated in xeno-free medium had elevated expression of SOX10, OLIG2 and NKX2.2 compared with cells differentiated in xenogenic conditions.
� hESC-derived glial precursor cells expressed A2B5 (>80%) and PDGF receptor (>38%), hESC-derived OPCs expressed NG2 (>60%), and maturing oligodendrocytes expressed O4 (>85%) and GalC (80%) when cultured in xeno-free medium.
� Xeno-free medium supported the purification and subculturing of hESC-derived OPC populations with fluorescence-activated cell sorting and NG2-positive selection.
� In the future, this xeno-free method is scalable for generation of human pluripotent stem cell-derived OPCs and oligodendrocytes for regenerative medicine.
ReseaRch aRticle Sundberg, Hyysalo, Skottman et al.
Regen. Med. (2011) 6(4)460 future science group
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