Specific Marker Expression and Cell State of Schwann Cells during Culture In Vitro

SC isolation purification and enrichment

Newborn mice were sacrificed by decapitation, and their sciatic nerve (SN) segments were harvested aseptically under a dissecting microscope. Nerve segments were digested by an enzymatic solution containing Collagenase NB 4 (0.26 U/ml, Serva, Germany) and Dispase II(neutral protease, grade II, 0.94 U/ml, Roche, USA), which were both dissolved into DMEM (Thermo, USA) at a concentration of 0.2%.

To digest tissues, the enzymatic solution (50 μl per segment) was added and incubated at 37°C for 80 min within a cell incubator (Thermo, USA). The mixture was then centrifuged at 600×g for 10 min. After removal of the supernatant, the cell pellet was resuspended in Schwann cell culture medium (SCCM) consisting of DMEM medium supplemented with b-FGF 0.1 mg/ml (Peprotech, USA), heregulin-β-1 0.1 mg/ml (Peprotech, USA), 2 μM forskolin (Cayman,USA), 10% fetal bovine serum (FBS) (Hyclone, Australia), Penicillin-Streptomycin (Gibco, USA) [ 30 , 31 ]. The cells were cultured in a cell incubator with a humidified atmosphere of 5% carbon dioxide at 37°C.

After 48 hours, SCs were purified and enriched as described [ 31 ]. In short, after cultured 48 hours, the culture medium was replaced with 0.2% Dispase II diluted in DMEM with a volume of 0.1 ml/m ² . After incubation at 37°C for 20 mins, the flasks were kept shaking horizontally for 1–3 min to release detaching cells. The suspended cells were then collected into a 15-ml centrifuge tube(BD Falcon, USA) and centrifuged at 600×g for 5min. After removal of supernatant, the pellet was resuspended in SCCM and plated onto flasks at a density of 2–2.5 × 10 ⁴ /cm ² . This purification and enrichment was repeated every 48 hours until the Schwann cells were cultured for a total of 8 days in vitro .

Tissue Preparation

Mice were anesthetized with pentobarbitone and perfused through the left ventricle with 4% (w/v) paraformaldehyde (PFA, Sigma, USA) in phosphate-buffered saline (PBS) (pH 7.4). Unfixed tissue was used for real-time quantitative RT-PCR. The SN tissue was dissected, postfixed in 4% PFA and 10% (w/v) sucrose at 4°C for 2 hours, cryo-preserved in 30% (w/v) sucrose at 4°C overnight, embedded in O.C.T. compound, sectioned in a cryotome (10 μm). Longitudinal cryo-sections were thaw-mounted onto slides and stored at -20°C.

Immunohistochemistry

Frozen sections, after several rinses in PBS, were permeabilized and blocked with PBS containing 0.3% (v/v) Triton X-100 and 10% (v/v) goat (or rabbit) serum for 30 mins at 37°C, then incubated at 4°C overnight with primary antibodies(without primary antibodies as blank control) followed by incubation with secondary antibodies for 40 mins at 37°C. Primary antibodies were diluted in PBS containing 0.1% Triton X-100,and secondary antibodies were diluted in PBS. Cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI, Invitrogen, USA) diluted in PBS(1:500). Slides were observed under a confocal laser scanning microscope (TCS SP5, Leica, Germany).

Immunocytochemistry

Cells cultured for 2 days (at the primary passage, P0) and 8 days (at the third passage, P3) were fixed with 4% PFA in PBS at 37°C for 15 mins. Unfixed cells were used for Western blotting and real-time quantitative RT-PCR. Fixed cells, after several rinses in PBS, were permeabilized and blocked with PBS containing 0.3% Triton X-100 and 10% goat (or rabbit) serum in PBS for 30 mins at 37°C, then incubated at 4°C overnight with primary antibodies(without primary antibodies as blank control) followed by incubation with fluorophore-conjugated secondary antibodies for 30 mins at 37°C. Primary and secondary antibodies were diluted in PBS. Cell nuclei were counterstained with DAPI diluted in PBS(1:500). Cells were observed under a fluorescence microscope (Olympus, Japan).

Western blotting

For Western immunoblotting, cultured cells were homogenized in lysis buffer (0.25 M Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 10% mercaptoethanol). The protein lysates were fractionated on a SDS-PAGE gel and transferred to nitrocellulose membrane (Millipore, USA). The blotted membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 1 hour, and the membranes were incubated overnight at 4°C with primary antibodies diluted in prepared TBST containing 3% nonfat milk. After 3 washes in TBST, the blots were reacted with horseradish peroxidase-conjugated secondary antibodies (Abmart, China) for 1 hour at room temperature and then washed again with TBST. For detection, an enhanced chemiluminescence-Western blot system (Amersham, Piscataway, USA) was used. For quantification, the X-ray films were put through a scanner (Samsung, Seoul, Korea) and analyzed with LAS image analysis system (Fujifilm, Tokyo, Japan). The picture were analysed with software ImageJ (National institutes of Health, USA) to obtain gray value of each band. Then relative gray value of each marker(gray value of special marker / gray value of β-actin)were calculated and analysed.

Primary and secondary antibodies

The following primary antibodies were used: anti-MBP (rabbit 1:250) (Abcam, UK), anti-Sox10 (rabbit 1:200) (Abcam, UK), anti-MPZ (rabbit 1:250) (Abcam, UK), anti-GAP43 (rabbit 1:250) (Abcam, UK), anti-P75 NGF Receptor (rabbit 1:500, (Abcam, UK), anti-Neural Cell Adhesion Molecule (rabbit 1:500) (Millipore, USA),anti-S100 (rabbit 1:500) (DakoCytomation, USA), Krox20 (rabbit 1:100) (Covance, USA), anti-Sox2 (rabbit 1:200) (Epitomics,USA), anti-Oct6 (goat 1:100) (Santa Cruz, USA), anti-β-actin (Abmart, China). For immunohistochemistry and immunocytochemistry tests, the secondary antibodies were conjugated with Alexa 488 and Alexa 555 (goat and rabbit 1:500) (Invitrogen, USA).

Positive rate of SC markers

After immunostaining with antibodies, SCs and fibroblasts were identified based on their morphologies. Cells with a bipolar or tripolar shape were recognized as SCs, whereas flat or polygonal cells were recognized as fibroblasts [ 32 , 33 ]. The positive rate of SCs markers was derived from the calculation of the percentage of marker-positive SCs with respect to the total number of counted SCs:

We counted at least 100 SCs from at least 3 random photo-areas at 100x magnification for each experiment. Three independent experiments were repeated and Schwann cells in each experiment were harvested from a single isolation respectively.

Real-Time quantitavtive RT-PCR

Total mRNAs using Trizol (Invitrogen, USA) were extracted from cultured cells and sciatic nerve tissue. Real-time quantitative RT-PCR was performed on the StepOnePlus instrument (Applied Biosystems, USA), using Promega M-MLV kit (Promega, USA) and SYBR green method. We set 40 cycles as a cycle cut-off point for no detection.

The primer sequences (designed by Invitrogen, USA) are listed below: -->

The in vitro expression profile of SC-specific markers

After checking the in vivo expression of SC markers in SNs, we digested SNs harvested from the newborn mice by enzyme solution, and isolated cells for in vitro culture. We observed two kinds of distinct cell morphology under light microscopy: some cells had a bipolar or tripolar spindle shape with a small cytoplasm / nuclear ratio and a strong refraction rate (Fig ​ (Fig2a 2a and ​ and2c, 2c , arrow, arrowhead), which are consistent with the morphology of SCs [ 35 ]. In contrast, other cells were flat and irregularly shaped with a large cytoplasm / nuclear ratio and a poor refraction rate (Fig ​ (Fig2a 2a and ​ and2c 2c asterisk), which were recognized as fibroblasts [ 35 ]. After three rounds of purification and culture, the obtained SCs had very little contamination from fibroblasts ( Fig 2E ).

Fig 2

The expression of S100 and P75 ^NTR in in vitro cultured SCs.

The in vitro expression of S100 and P75 ^NTR , classic SC markers

We first checked the expression levels of S100 and P75 ^NTR , the most widely used SC markers in the field of neuron development and regeneration, in in vitro cultured SCs. Immunofluorescence staining data showed that in the early stage of SC culture SCs didn’t totally express S100 and P75 ^NTR ( Fig 2a – 2d arrow), and some SCs could not express either marker ( Fig 2a – 2d , arrowhead), which show a staining pattern similar to fibroblasts ( Fig 2a – 2d , asterisks). After three passage culture, all SCs expressed S100 and P75 ^NTR ( Fig 2E – 2H ). In P0 SCs, the early stage of cultured cells, the positive rates for S100 and P75 ^NTR were 85.84% ± 1.86% and 93.57% ± 3.40%, respectively. Interestingly, the positive rates for both markers reached 100% in P3 cultured SC ( Fig 2I ). After extracting total proteins from SCs at different time points and assaying by Western blot, we found that the protein levels of S100 in P3 SCs were higher than those in P0 SCs ( Fig 2J ). The grey value of S100 in P0 SCs and P3 SCs were 0.540 ± 0.014 and 0.596 ± 0.012. While the protein levels of P75 ^NTR in P3 SCs were significantly higher than those in P0 SCs, which grey value in P0 SCs and P3 SCs were 0.435 ± 0.020 and 0.738 ± 0.023, respectively. Real-time quantitative RT-PCR data showed that the mRNA level of S100 in P0 SCs was significantly higher than that in P3 SCs, while the opposite trend was detected in the mRNA level of P75 ^NTR ( Fig 2K ).

The expression profile of transcription factors Sox10, Sox2, Oct6 and Krox20

We also detected the expression of Sox10, Sox2, Oct6 and Krox20, four transcription factors involved in regulating the development, differentiation, and myelination of SCs [ 1 , 19 , 36 , 37 ]. We found that Sox10 and Sox2 are constitutively expressed in all SCs with 100% expression rate ( Fig 3a – 3d arrow, Fig 3I ), but not in fibroblasts ( Fig 3a – 3d asterisk). The protein levels of Sox10 and Sox2 were higher in P3 SCs than those in P0 SCs ( Fig 3J ). The grey value of Sox10 in P0 SCs and P3 SCs were 0.738 ± 0.013 and 0.863 ± 0.011, and the grey value of Sox2 were 0.098 ± 0.012 and 0.341 ± 0.013.

Fig 3

The expression of Sox10 and Sox2 in cultured SCs.

The mRNA level of Sox10 in P3 SCs was higher than that in P0 SCs, while no significant difference in the mRNA level of Sox2 was detected at different culture times ( Fig 3K ). Surprisingly, the transcription factor Oct6 were not expressed in all P0 and P3 SCs ( Fig 4 ), and the positive rate was 82.59% ± 4.13% and 92.64% ± 3.49%, respectively ( Fig 4K ). To further verify this result, we did Sox10 and Oct6 double staining in P3 SCs (Fig ​ (Fig4E, 4E , ​ ,4F, 4F , ​ ,4a 4a – 4d ) and found that most SCs were Sox10 ⁺ / Oct6 ⁺ , while the rest part of SCs were Sox10 ⁺ / Oct6 ^- . In contrast, fibroblasts were all Sox10 ^- / Oct6 ^- (Fig ​ (Fig4E 4E and ​ and4F 4F ).

Fig 4

The expression of Oct6 and Krox20 in in vitro cultured SCs.

Furthermore, the expression of Krox20, the downstream transcription factor of Oct6, was similar to that of S100. It is not expressed in all in early in vitro cultured SCs (Fig ​ (Fig4e 4e and ​ and4f 4f arrowhead), and the positive expression rate is approximately 91.67% ± 1.71% ( Fig 4K ). After cultured for 8 days, Krox20 is expressed in all SCs. The protein levels of Oct6 and Krox20 in P3 SCs was higher than those in P0 SCs ( Fig 4L ), and the grey value of Oct6 in P0 SCs and P3 SCs were 0.269 ± 0.011 and 0.501 ± 0.014, and the grey value of Krox20 were 0.428 ± 0.025and 0.644 ± 0.012. And the similar pattern was observed in the mRNA content of these two transcription factors ( Fig 4M ).

The expression of myelin-associated protein MBP and MPZ

Transcription factor Oct6 interacts with transcription factor Krox20 to regulate myelination and affects the expression of myelin-specific proteins MBP and MPZ in mSC [ 5 , 38 – 41 ]. Therefore, we further examined the expression of MBP and MPZ in in vitro cultured SCs. At the early stage of cultured SCs, the positive expression rates of MBP and MPZ were 86.74% ± 2.66% and 91.95% ± 2.25%, respectively, and both of them reached 100% in P3 SCs ( Fig 5I ). In addition, we observed that MBP-positive SCs or MPZ-positive SCs were generally highly translucent, and those cells are transparent when observed under the light microscope ( Fig 5a – 5d arrow). However, SCs with negative or low expression of MBP or MPZ had poor transparency, dark cell bodies and visible nuclei. Halos appear at the outer edge of the membranes of these cells ( Fig 5a – 5d arrowhead), which differs from the typical morphology of fibroblasts (Fig ​ (Fig5a 5a and ​ and5b 5b asterisk).

Fig 5

The expression of MBP and MPZ in cultured SCs.

The expression of NCAM and GAP43

Besides mSC, iSC can differentiate into nmSC as well. The in vivo markers of iSC and nmSC are NCAM and GAP43, which are very similar [ 6 , 19 , 42 ]. Through our immunofluorescence experiment, we found that most of the early culture SCs expressed NCAM at the positive rate of 94.75% ± 1.19% ( Fig 6I ). Interestingly, although GAP43 is expressed in iSC and nmSC in vivo , its in vitro expression pattern was similar to Oct6’s expression, which was not fully expressed in all in vitro SCs. GAP43 showed the positive rate of 66.60% ± 7.30% and 94.06% ± 2.62% at early and later culture time points, respectively ( Fig 6I ).

Fig 6

The expression of NCAM and GAP43 in cultured SCs.

Potential multiple SC stages in newborn mice

By immunofluorescence, we detected nine SC markers, except for Sox2, expressed in SN in newborn mice. For technical reasons, we could not examine the expression rate of these SC markers in vivo in the present study. However, previous studies have demonstrated that transcription factor Sox2 is only transiently expressed in SCPs and iSCs of mouse embryos before myelination, and its expression level rapidly decreases after birth [ 5 , 28 , 29 ]. That may be the reason that Sox2 was not detected in SN harvested from either newborn mice or adult mice. Another report showed that the expression of transcription factor Oct6 peaks on day 0 after birth, followed by gradual decreasing, and no expression in mature mSCs [ 37 , 43 ]. Consistent with these previous reports, we detected expression of Oct6 in the newborn mice, but not in the adult mice. Based on our finding and previous reports, we speculated that in newborn mice SC might exist as iSC, pro-mSC, mSC and nmSC; while in adult mice SCs exist as the two major mature stages, which are nmSC and mSC.

S100 is not a reliable pan-SC marker

S100 is the most commonly used SC marker, and prior studies suggest that S100 is expressed in iSC, pro-mSC, mSC and nmSC in vivo , but not in SCP [ 37 , 44 – 46 ]. Therefore, we first examined the expression of S100 in in vitro cultured SC. We found that, in early culture, only about 85% of the SCs expressed S100, while all SCs cultured for eight days expressed S100. Some studies show that S100 is closely related to the maturation of SC [ 45 , 47 ], and therefore we interpreted our data as reflecting that in SN of newborn mice, a considerable portion of SCs are in SCP stage, which do not express S100. Then, after a period of time in vitro , the environment and various cytokines secreted by SCs, such as BDNF and GDNF, may promote SCP to differentiate into iSC [ 48 , 49 ].

Real-time quantitative RT-PCR results showed that, in the late stage of in vitro cultured SCs, the mRNA level of S100 was unexpectedly lower than that in the earlier cultures with unknown reason. We hypothesized that these SCs may be in an immature stage, and S100 expression in these cells was negatively regulated, which led to a low mRNA level.

Because S100 is not fully expressed in the early cultured SCs, and because a variety of other cells, such as chondrocytes, adipocytes, cardiomyocytes, skeletal muscle cells, and melanoma cells, also express S100 in vivo [ 45 , 50 , 51 ], we believe that S100 alone cannot be used as a marker to identify cultured SCs. Moreover, S100 expression in induced SCP is negative, but S100 expression can be positive if the induced cells contain other S100-positive cells. Thus, S100 cannot be used singly to identify SC-like cells derived from various types of stem cells either.

Sox10 expression is necessary for SC identification

Transcription factor Sox10 is a specific marker for neural crest-derived cells, and it is expressed in neural crest cells and various types of differentiated cells, including all types of SCs. Sox10, a key transcription factor, is essential for the development of SCs [ 52 ], and is involved in the regulation of myelination [ 19 ]. In this study, in vitro cultured SCs always express Sox10, which is similar to the in vivo expression pattern. Possibly the expression of Sox10 is not easily affected by environmental changes. These results suggest that Sox10 may be used to identify in vitro cultured SCs. However, Sox10 alone cannot distinguish SCs in different types or different developmental stages, and it cannot distinguish between other cell types derived from neural crest cells, which suggests that it cannot be used singly to identify SC-like cells after stem-cell induction. Since Sox10 is stably expressed in vitro and in vivo , it could serve as a necessary, but not sufficient, marker for identifying SC and SC-like cells.