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The generation of dopamine (DA) neurons from stem cells holds great promise in the treatment of Parkinson’s disease and other neural disease associated with dysfunction of DA neurons. Mesenchymal stem cells (MSCs) derived from the adult bone marrow show plasticity with regards to generating cells of other germ layers. In addition to reduced ethical concerns, MSCs could be transplanted across allogeneic barriers, making them desirable stem cells for clinical applications. We have reported on the generation of DA cells from human MSCs using sonic hedgehog (SHH), fibroblast growth factor 8 and basic fibroblast growth factor. Despite the secretion of DA, the cells did not show evidence of functional neurons, and were therefore designated DA progenitors. Here, we report on the role of brain-derived neurotrophic factor (BDNF) in the maturation of the MSC-derived DA progenitors. 9-day induced MSCs show significant tropomyosin-receptor-kinase B expression, which correlate with its ligand, BDNF, being able to induce functional maturation. The latter was based on Ca2+ imaging analyses and electrophysiology. BDNF-treated cells showed the following: increases in intracellular Ca2+ upon depolarization and after stimulation with the neurotransmitters acetylcholine and GABA and, post-synaptic currents by electrophysiological analyses. In addition, BDNF induced increased DA release upon depolarization. Taken together, these results demonstrate the crucial role for BDNF in the functional maturation of MSC-derived DA progenitors.
Human stem cells show therapeutic potential for a number of neural disorders (Miller and Bai 2007). Moreover, stem cell therapy has been suggested to be an effective treatment for neurodegenerative disorders such as Parkinson’s disease, which is caused by the degeneration of dopamine (DA) neurons in the substantia nigra (Trzaska and Rameshwar 2007a). DA neurons derived stem cells are significant to cell replacement therapy for degenerated DA neurons of the nigro-striatal system, and also as a model system to study drug reaction and DA developmental processes.
We have reported on the induction of a DA phenotype from adult human MSCs with an induction cocktail that comprises sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8) and basic FGF (bFGF) (Trzaska et al. 2007b). The MSC-derived DA cells express DA-specific markers, tyrosine hydroxylase, DA transporter, Nurr1, and Pitx3. However, despite the expression of these DA-specific genes and the synthesis and secretion of DA, they did show characteristics of mature neurons, in particular electrophysiological properties. Thus, the induced cells were considered to be at an immature state and were designated DA neuronal progenitors. The DA progenitors expressed the repressor of neural genes, REST, although at levels lower than the uninduced MSCs (Trzaska et al. 2008). Knockdown of REST led to the generation of electrophysiologically functional neurons (Trzaska et al. 2008).
As MSCs can form functional DA neurons, we sought for exogenous factors that can transition the DA progenitors to functional neurons. In this study, we examine the role of neurotrophins (NTs) in further differentiation of the MSC-derived DA progenitors to functional neurons. We hypothesize that the DA progenitors express receptors for neurotrophic factors and these factors can induce maturation of the cells. The goal was to identify what neurotrophic factor(s) can promote maturation to functional DA neurons. Temporal expression profile of NT receptors showed an increase in the expression of tropomyosin-receptor-kinase (Trk) B at day 9 induction. As brain-derived neurotrophic factor (BDNF) can bind to TrkB, we studied its role in the function of the DA progenitors, by Ca2+ imaging analyses electrophysiology.
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- Materials and methods
This report provides a novel finding showing BDNF promotes functional maturity in MSC-derived DA cells. We have previously reported on an induction cocktail comprising SHH, FGF8, and bFGF with ∼70% efficiency in the transdifferentiation of MSCs to DA-producing cells, based on tyrosine hydroxylase expression (Trzaska et al. 2007b). Among the induction was > 90% that expressed NeuN and beta-III-tubulin. Despite the markers of the latter neuronal markers, the cells were functionally immature, based on electrophysiology. In this study, we show that BDNF addition to 9-day induced MSCs results in the development of cells which elicit sPSCs and secrete DA in response to Ca2+-dependent depolarization. This is a significant finding that mimics the physiology of native neurons and has yet to be reported in MSC-derived DA cells. Furthermore, BDNF treatment results in KCl-induced depolarization and enhanced DA release (Figs 3 and 7). The ability of the induced cells to respond to KCl is a hallmark of excitable cells and the enhanced release of DA by the BDNF-treated cells indicate dopaminergic type.
As compared to other protocols in the literature, we did not change the media during the induction for two main reasons. Firstly, the evidence indicates that several unknown (or possibly neural inductive) factors are secreted by the cells during differentiation. The initial studies were performed with intermittent change in media and observed halting of differentiation with cell death (unpublished). This led us to surmise that change of media will remove key endogenously produced factors that are important for maturation. As neuronal induction is very complex and requires a multitude of factors, it is possible that the initial induction leads to the production of other secretory factors that might facilitate differentiation although autrocrine mechanism. It is also possible that addition of exogenous BDNF synergizes with factors secreted by the cells to induce differentiation. Secondly, SHH is tumorigenic (Yue et al. 2008). During induction, a key repressor of neuronal gene expression, REST, is down-regulated (Trzaska et al. 2008). As REST is a tumor suppressor gene (Coulson 2005; Reddy et al. 2009), its decrease, with the presence of the oncogeneic SHH could cause tumor formation. Thus, avoiding re-addition of SHH would be safer as we progress to apply the reach. These questions are follow-up to the present report. Also, we were trying to induce terminal differentiation and limit proliferation.
Neurotrophins, such as GDNF, BDNF, and NT-3, have an important role in the maintenance, development, and survival of DA neurons (Zhou et al. 1996; Erickson et al. 2001; Baquet et al. 2005; von Bohlen und Halbach et al. 2005). GDNF binds two receptors, GFRα1 and c-ret, and has been described as a potent trophic factor for DA neurons (Love et al. 2005). Although we detected GFRα1 on the induced cells, although with lower percentages (60–70% vs. 90% for TrkB), we focused on TrkB. We performed Ca2+ imaging experiments in which the cells were treated with combinations of BDNF and GDNF. The results were similar to studies with BDNF alone (not shown), suggesting that BDNF is the only contributor. This was a surprise as we observed GFRα1. The explanations for these observations could be explained by non-functional GFRα1 receptor or perhaps, another role for GDNF in the DA neurons. We plan to continue these studies in future experiments using knockdown of the various neurotrophic receptors.
Nerve growth factor, BDNF and NT-3 act through the Trk receptor tyrosine kinase family and regulate nervous system development (Bibel and Barde 2000; Chao 2003). NGF is the ligand for TrkA, whereas BDNF binds to TrkB and NT-3 binds to TrkC. BDNF and NT-3 have recognized roles in the differentiation and survival of DA neurons (Baquet et al. 2005). NGF has been implicated in the differentiation of a wide variety of neuronal types and particularly causes neurite outgrowth and increases Na+ voltage-gated channel expression (Kalman et al. 1990; Chao 2003). Thus, we were interested in the role that these NTs could have in further maturation of MSC-derived DA cells. The premise was that initial inductive stimuli would up-regulate receptors for additional factors required for maturation of the MSC-derived DA cells. These factors could be either too low or missing in our in vitro system and if the newly up-regulated receptors are not stimulated at the appropriate time points, the cells might fail to progress to the next stage of development. Analysis of receptor expression revealed up-regulation of TrkB and GFRα1 by day 9 of induction (Fig. 1). Interestingly, the expression of TrkA and TrkC did not change significantly during the course of differentiation (Fig. 1a). This was not surprising as MSCs have been reported to be neuroprotective and have low expression of Trk receptors (Pisati et al. 2007). Due to the high expression of TrkB (Fig. 1), we added BDNF to cells induced for 9 days and observed up-regulation of D1 receptor, indicating responsiveness to BDNF through TrkB signaling (Fig. 2) (Do et al. 2007). Thus, we hypothesized that addition of BDNF at initial up-regulation might be necessary for further maturation and functionality.
The hallmark of functional neurons is the presence of excitable properties and formation of synapses. We found that addition of BDNF to MSCs induced for 9 days facilitated further maturation evident by the intracellular Ca2+ increase upon depolarization, which was prevented in Ca2+-free external solution (Fig. 3b and c). This indicates that the cells express voltage-gated Ca2+ channels similar to native neurons. In contrast, cells stimulated with other neurotrophic factors, NGF, NT-3, or GDNF did not respond to K+-induced depolarization (Fig. 3e). The data suggest that depolarization causes direct activation of voltage-gated Ca2+ channels or activating voltage-gated Na+ channels, which then indirectly open Ca2+ channels in the 9 day MSCs treated with BDNF.
Dopamine neurons have been found to respond to neurotransmitters, ACh, l-glut, and GABA (Raye et al. 2007). In comparison to the control 12-day induced MSCs, 55 ± 13% of the cells treated with BDNF responded to ACh (Fig. 4a and c). ACh receptors are readily found on DA neurons and ACh transmission is important in striatal DA release and nerve development (Zhou et al. 2004; Calabresi et al. 2006; Raye et al. 2007). As DA neurons express both ACh nicotinic and muscarinic receptors (Calabresi et al. 2006), Ca2+ release could be mediated through excitatory nicotinic receptors or by the release of Ca2+ from intracellular stores via muscarinic receptors. Future studies are warranted to test this hypothesis. Surprisingly, the BDNF-treated cells did not respond to the other excitatory neurotransmitter, l-glut (Fig. 4d). This suggests that glutamatergic neurotransmission is not yet present in these cells. However, we found that GABA, which is an inhibitory neurotransmitter, caused an increase in intracellular Ca2+ levels upon stimulation in both the control 12-day induced cells (52% ± 15%) and the BDNF treated cells (71 ± 10%), albeit at different intensities (Fig. 5e). This may be due to the presence of higher numbers of GABAA receptors or Ca2+ voltage-gated channels in the BDNF treated cells. These observations show that the cells respond in an opposite, excitatory manner to GABA. This activity has been reported previously in developing neurons, in which GABA induces excitatory neurotransmission before the appearance of glutamatergic neurotransmission (Ben-Ari et al. 2007; Zhao et al. 2008). It was expected that the control 12-day induced MSCs would respond to GABA in an excitatory manner, as they are in an immature state, as determined by our previous studies (Trzaska et al. 2007b). However, the BDNF treated cells responded similarly, indicating that the cells are still developing and GABAergic neurotransmission has not yet shifted to inhibition. This is consistent with the lack of responses to l-glut (Fig. 4b and d), as GABA is excitatory before the presence of glutamatergic neurotransmission (Ben-Ari et al. 2007). This pattern allows for proper neuronal growth and synapse formation in developing neurons that have few synapses (Ben-Ari 2002). Moreover, this excitatory action of GABA occurs through the activation of ionotropic GABAA receptors (Maric et al. 2001). Our observations are consistent with previous reports, wherein the depolarizing response of GABA is abolished without extracellular Ca2+, indicating presence of GABAA ionotropic receptors (Fig. 5b and d). Taken together, the data indicate that the MSC-derived DA cells treated with BDNF are still developing mature synaptic connections. At this time we do not have any explanation why the BDNF-treated cells do not show fully functional neurons with regards to inhibitory and excitatory functions. Similar results were observed when the 3-day BDNF treatment was extended up to 1 week (data not shown). This suggests that another factor is required for glutamergic and GABAergic DA neurons. Further studies are planned to identify factor(s) that could show fully functional neurons.
Functional neuronal networks communicate through synaptic activity. Our data show that the BDNF-treated cells elicit inward sPSCs (Fig. 6). In comparison to the sPSCs recorded from MSCs treated with retinoic acid in our laboratories previous studies (Cho et al. 2005; Greco et al. 2007), the BDNF-treated cells exhibited kinetic properties of sPSCs that are closer to the sPSCs of those recorded from mammalian CNS neurons in brain slices (Xiao et al. 2007). Although future studies are necessary to characterize these responses, it is possible that the sPSCs could be GABAergic, judging by the time scale, rise and decay times, and recording conditions (Fig. 6b). Additionally, the sPSCs could be mediated by P2 receptor channels, based on our studies with ATP (Fig. 7a) (Safiulina et al. 2005; Saitow et al. 2005; Trzaska et al. 2007b).
Our observations on the maturation properties of BDNF are consistent with other reports. BDNF is an important regulator of synaptic transmission and addition of exogenous BDNF was found to enhance synaptic efficiency and accelerate synapse maturation (Bibel and Barde 2000; Bolton et al. 2000; Yamada et al. 2002; Gubellini et al. 2005; Carrasco et al. 2007). In addition, BDNF can enhance GABAergic post-synaptic currents in immature neurons, but had the reverse effect in mature neurons (Tanaka et al. 1997; Mizoguchi et al. 2003). Our observations are comparable with these previously reported studies, showing that BDNF promotes a maturation response in the MSC-derived DA cells.
In mature neuronal cells, the release of neurotransmitters is dependent on Ca2+ influx from the extracellular microenvironment. Our previous studies showed that the MSC-derived DA cells constitutively secrete DA that was not affected by K+-induced depolarization and not prevented by removal of extracellular Ca2+ (Trzaska et al. 2007b). We found that the basal release of DA was mediated by spontaneous ATP, as the ATP antagonist suramin, significantly decreased DA release (Fig. 7a). Conversely, this was not observed by scopolamine, an antagonist for ACh muscarinic receptors. The cells treated with BDNF showed a significant increase in DA release upon depolarization, which was abolished in a Ca2+-free extracellular solution (Fig. 7b). These results mimic the physiology of neurotransmitter release in native neurons which is not observed in the control 12-day MSCs. This indicates that day 9 of induction of MSCs is a critical point in the regulation of DA release, which is modulated by exogenous BDNF.
In summary, we have demonstrated that MSCs induced with our induction cocktail show robust up-regulation of TrkB, and addition of BDNF promotes functional maturation. The cells elicit sPSCs – an important finding that has yet to be reported in MSC-derived DA cells. The cells also secrete DA in manner similar to native neurons. Although the BDNF-treated cells exhibit many characteristics of neurons, they are not fully developed with regard to mature glutamateric and GABAergic transmissions (Figs 4 and 5). Altogether, these observations demonstrate the crucial function of TrkB and its ligand, BDNF, in the development of functional DA neurons from human MSCs.