Effect of BMSCs on remyelination in the spinal cord of HD-exposed rats
Our earlier report observed that BMSCs transplantation alleviated motor neuron dysfunction and pathological damage in HD-intoxicated rats . To investigate the underlying mechanism associated with this observation, the present study employed a SD rat model administrated with HD (400 mg/kg/day, i.p) for 5 weeks (intoxication period) and then treated with either BMSCs transplantation (HD+BMSCs group, 5×107cells/kg suspended in saline) or normal saline (HD+NS group, equal amount of saline used as control) by tail vein injection for another 5 weeks (recovery period). Two groups of mice receiving no treatment (Control group) or only HD treatment (HD only group) were necropsied at the end of intoxication period and three groups of mice receiving BMSC only treatment, HD + BMSCs treatment and HD + saline treatment were necropsied at the end of recovery period. The animal experiment was designed as being diagramed in Fig 1A.
In order to examine whether the demyelinating damage caused by HD had been recovered by BMSC transplantation, spinal cords were harvested and subjected to ultrastructure analysis using transmission electron microscopy. As shown in Fig. 1B, comparing to the control group or the BMSC group with intact myelinated nerves (yellow arrow), HD group and HD+NS group demonstrated similar demyelinating structure of spinal cord axons featuring large swollen axons surrounded with disintegrated, thinner and split myelin sheaths (red arrow). In contrast, remyelination was observed in HD+BMSCs group showing reconstructed structure with more compact and thicker myelin sheaths (green arrow). Furthermore, the changes of myelin structure were also assessed using Luxol-fast blue (LFB) staining (Fig. 1C, D), which is a frequently used procedure for the demonstration of myelin structure in nervous tissue. Comparing to the fainter and patchy depigmenting staining feature in the HD group and HD+NS group, HD+BMSCs group showed noticeably increased LFB staining with more refined substructure (Fig. 1 C, D). Together, these results suggested that HD caused severe demyelination in rat spinal cords, spontaneous remyelination without therapeutical intervention played a limited role to repair the damage but BMSC transplantation could markedly promote regrowth of myelin sheath upon the injured nerve fibers.
BMSCs promote OPCs differentiation to mature OLs in vivo and in vitro
Next, we sought to understand the associated mechanism(s) responsible for BMSC-promoted remyelination. Since it has been reported that the differentiation of OPCs to mature OLs is a rate-limiting step of remyelination , experiments were carried out to compare the number of immature OPCs and mature OLs in spinal cord sections from HD-intoxicated rats, using NG2 and MBP as immunoblotting markers, respectively. As shown in Fig. 2A, B, HD treatment was associated with significantly reduced number of NG2 as well as MBP positive cells, whereas BMSC grafting significantly recreated the number of myelinating oligodendrocytes in the spinal cord tissues. Such an observation was also confirmed by Western blot using whole tissue lysate of spinal cords (Fig. 2C). These results indicated that BMSCs exerted certain impact increasing the number of available OPCs and OLs in the spinal cords after HD-induced neurotoxicity in vivo.
To further determine the regulatory factors in BMSCs’ impact on oligodendrocyte differentiation, in vitro experiments were conducted to replicate the aforementioned observations using cultured primary cells. Cultured primary OPCs were treated with 25 mM HD or vehicle for 24 h and then incubated with BMSC-derived conditioned medium (BMSC-CM), the acellular medium harvested from BMSCs culture, for additional 2 days, as being described in the Materials & Methods section. The cells were subjected to morphometric analysis to distinguish and quantify the different oligodendrocyte population. Importantly, our in vitro results also found that BMSC-CM treatment significantly increased the number of differentiating immature OLs (cyan arrows) and mature OLs (green arrows), comparing with HD group (Fig. 2D, E). Elevated level of MBP protein were detected in the BMSC-CM-treated cells (Fig.2F), suggesting BMSCs might promote oligodendrocyte maturation in HD-intoxicated OPCs.
BMSCs promote OPCs differentiation via downregulating Hes1
Some studies have reported the inhibitory effects of hairy-enhancer-of-split (Hes) family members, such as Hes1 that is highly expressed by OPCs, on oligodendrogenesis [25-27]. To determine whether Hes1 participated in the effect of BMSCs on OPC differentiation, the mRNA and protein levels of Hes1 in spinal cords were measured and compared among different HD/BMSCs treatment groups. The results showed that HD intoxication increased while BMSCs transplantation decreased mRNA and protein expression of Hes1, respectively (Fig. 3A and 3B). In line with this, immunostaining demonstrated significant elevation of Hes1 positive cells in the spinal cords of HD group rats and reduction of Hes1 positive cells in those rats grafted with BMSCs (Fig. 3C and 3D).
Similar to the in vivo findings, western blot analysis of the OPCs cultured in BMSC-CM also showed that Hes1 level was increased by HD and decreased by BMSC-CM treatment (Fig. 3E). Next, we overexpressed Hes1 gene in cultured OPCs (Hes1+ OPCs) and confirmed that the Hes1 mRNA and protein expression were higher (Fig 3F and 3G, respectively). Meeting our expectation, as shown in Fig. 3H and 3I, morphological analysis revealed that in the Hes1-overexpressing OPCs, the beneficial effect of BMSC-CM was abolished, with more mature OLs (green arrows) presenting in HD-treated OPCs cultured in BMSC-CM but less mature OLs being identified under the condition that Hes1 was overexpressed. Consistent with this, immunostaining and western blot results (Figure 3J-K and 3L, respectively) also revealed that the change of MBP in response to BMSC-CM was cancelled after Hes1 overexpression. Together, these results indicated that BMSCs promoted OPC differentiation into mature OLs in a Hes1-dependent manner.
BMSCs regulate Hes1 expression in OPCs independent of Notch1 signaling
Earlier studies have implied that Hes1 expression is commonly regulated by Notch1 signaling in adult OPCs . Therefore, our work continued by comparing the expressional changes of several key Notch1 signal transducers in the spinal cords of HD-intoxicated rats with or without BMSCs transplantation. As shown in Fig.4, no significant difference on mRNA level and protein level were observed in Jagged1 (Fig. 4A-B), Notch1 (Fig. 4E-F), NICD (Fig. 4I-J) or RBPJ (Fig. 4M-N) expressions in spinal cords comparing among the HD group, HD + NS group and HD + BMSC group. Further analyses were also carried out to detect these Notch1 players in HD-dosed OPCs +/- BMSC-CM and failed to evidence any expressional difference (Fig. 4C-D for Jagged1, 4G-H for Notch1, 4K-L for NICD, 4O-P for RBPJ, respectively). Collectively, these in vivo and in vitro data strongly argued that in our scenario, Hes1 expression in OPCs was regulated by BMSCs in a Notch1-independent manner.
The regulation of Hes1 by BMSCs in oligodendrogenesis is associated with TNFα
Some studies provided another clue that Hes1 expression could also be modulated by the inflammatory cytokine TNFα [29-31]. To clarify whether TNFα plays a role in BMSCs-induced remyelination, experiments were carried out to compare the mRNA and protein levels of TNFα among different treatment groups in our animal and cell culture models. As a result, RT-PCR (Fig. 5A) and western blot (Fig. 5B) showed that HD intoxication increased TNFα production in rat spinal cord and BMSC transplantation, in turn, attenuated the increase of TNFα. The changes of TNFα protein expression in in vitro OPCs followed the same pattern (Fig. 5C). Therefore, it seemed that TNFα participated in the regulation of oligodendrogenesis in our BMSC-OPC model.
The regulatory role of TNFα was studied by challenging the cultured OPCs in the presence or absence of HD/BMSC-CM with recombinant rat TNFα (10 ng/mL). Using morphological analysis to distinguish oligodendrocyte subpopulations, while BMSCs treatment was found to increase the proportion of mature OLs, supplementation with TNFα to the BMSC-CM-treated cells markedly blocked the increase of OLs (Fig 5D and 5E). Similarly, measurement of myelin sheath marker MBP using immunofluorescence (Fig. 5F and 5G) or western blot (Fig. 5H) identified increased MBP expression upon BMSC-CM treatment and yet no such increase after cells being co-treated with TNFα. In addition, western blot experiment also showed that Hes1, as the negative regulator of oligodendrocyte differentiation, was increased by HD, decreased by BMSC-CM and re-increased by TNFα supplement (Fig. 5I). Together, our results suggested that BMSCs might repress Hes1 expression and promote oligodendrogenesis via inhibiting TNFα production in OPCs.
Hes1 is an indirect target of TNFα via RelB in BMSCs-regulated oligodendrogenesis
Since both Hes1 and TNFα seemed to play negative regulatory roles in OPC differentiation (Fig 3H-L and Fig 5D-H) and both were repressed by BMSC grafting (Fig 3A-E and Fig 5A-C), we questioned whether these factors could be biologically connected. Some studies suggested that TNFα increased expression of the noncanonical NF-κB protein RelB, which in turn potentiated Hes1 expression by binding to and promoting nuclear translocation of NICD onto the Hes1 promoter [20-33]. To examine this potential interconnection, the mRNA and protein levels of RelB were assessed using real time PCR and western blot. Fig. 6A an 6B showed that in rat spinal cords, the RelB expression was increased by HD intoxication and decrease by BMSC treatment. Fig 6C showed that the change of RelB expression also occurred in cultured OPCs receiving BMSC-CM treatment. In addition, the RelB expression was tuned up when the BMSC-CM-treated OPCs were added with recombinant TNFα (Fig 6C, the last lane).
To further prove that RelB participated in BMSCs-induced remyelination, RelB gene were overexpressed in OPCs (RelB+ OPCs) and overexpression status was evidenced (Fig 6D, E). Investigations were carried out to evaluate the effect of RelB overexpression on BMSC-promoted oligodendrocyte differentiation after HD treatment using in vitro cultured OPCs/RelB+ OPCs that were treated with BMSC-CM. As shown in Fig. 6F and 6G, BMSC-CM failed to increase the number of mature OLs under the condition of RelB overexpression. Similarly, MBP expression assessed by immunofluorescence and western blot was increased in BMSC-CM-treated, HD-exposed OPCs, but not in RelB+ OPCs (Fig. 6H-J). The finding was further supported by the result shown in Fig. 6K, which demonstrated that the decreased level of Hes1 in BMSC-CM-treated OPCs was recovered by RelB overexpression. Collectively, our results suggested that BMSCs regulated Hes1 expression and promote oligodendrogenesis in HD-intoxicated OPCs via TNFα/RelB signaling mediators.
Inhibition of TNFα production is related with NGF secretion from BMSCs
Nerve growth factor (NGF), as a secretory neutrophin functioning in a paracrine manner, could be a crucial factor involved in BMSCs’ protective effect against HD-induced damage. Several studies by us [21,24,34] and others [35,36] have shown that the remyelination-promoting effect of BMSCs appeared to be closely related with NGF. Therefore, we further tested whether there be any potential linkage between NGF secretion and TNFα production. Using in vitro cultured OPCs, it was found that the NGF expression (Fig. 7A) as well as NGF signal activation (represented by p-TrkA) (Fig. 7B) were significantly elevated in response to BMSC-CM treatment. Next, the impact of NGF on TNFα production was evaluated using NGF-neutralizing antibody or recombinant NGF in the cultured OPCs. As shown in Fig. 7C, co-treatment using anti-NGF together with BMSC-CM remarkably abolished the effect of BMSC-CM to repress TNFα production (lane 3 vs lane 4), whereas supplementation with NGF by itself decreased TNFα expression in a similar way that BMSC-CM did (lane 2 vs lane 3 vs lane 6). Whether NGF directly regulates TNFα production was further examined using k252a, an inhibitor of TrkA receptor, that blocks NGF signaling pathway. As shown in Fig. 7D, BMSC-CM decreased TNFα production in OPCs but this effect was significantly blocked by the addition of K252a. Together, our results suggested that the regulatory role of BMSCs on TNFα production is closely correlated with the secretory neurotrophin factor NGF from BMSCs.