Recent advances have demonstrated the promising therapeutic role of MSCs in AD [12, 17]. Because AD remains a major cause of morbidity and mortality, significant effort has been directed toward Aβ removal via stem cell transplantation [13, 28]. The therapeutic properties of MSCs are largely related to their anti-apoptotic and anti-inflammatory abilities, which have been confirmed both in vivo and in vitro [13, 29, 30]. However, the low survival rates of MSCs in vivo are a challenge and the benefits of MSCs are mediated by undefined mechanisms [31–33].
Various modifications of MSCs have been attempted to improve their survival rates and therapeutic efficacy [31, 32, 34, 35]. Attempts to improve stem cell survival, metabolism, or migration ability have focused on genetic modifications to knock-out or knock-in specific genes [36–38]. However, the clinical application of genetically-modified MSCs is associated with the risk of unexpected genetic mutations resulting in tumor formation [39]. In another approach, biocompatible scaffolds as an alternative to encapsulated MSCs have been developed to improve the survival and engraftment rates [40]. This method facilitated clinical application but did not improve the efficacy of MSCs.
In recent years, pre-conditioning methods that attempted to improve the efficacy of MSCs have also been in the spotlight [41–43]. Pre-conditioning aims to promote cell proliferation [43], improve migratory ability [43], and enhance protein secretion [44]. Unlike genetic modifications, pre-conditioning can be achieved by exposing MSCs to specific microenvironments. Compared with genetic modifications, pre-conditioning enhanced therapeutic efficacy while maintaining the genotype of the cells [45]. A number of approaches have been proposed to make pre-conditioned MSCs. Pre-conditioning by hypoxia [46], inflammatory stimuli [42, 45], or other factors [42] are strategies designed to enhance the survival and effectiveness of MSCs post-transplantation. In this study, we pre-conditioned MSCs using Aβ, the most important hallmark of Alzheimer's disease and used H4SW cells for pre-conditioning through endogenous Aβ.
H4SW cells are a stable cell line whereby the amyloid precursor protein (APP) Swedish mutation was introduced into a human glioblastoma cell line. APP is an integral membrane protein of neuronal cells involved in synaptic formation, synpatic plasticity, and ion export. APP, expressed in cell membranes, is usually cleavaged byα-secretase. However, mutations in APP protein or PSEN1/PSEN2 increases the change for APP to be cleavaged by β-, and γ-secretase, resulting in high levels of Aβ production in the brain. The Aβ produced is considered the causative substance of Alzheimer's disease, as it forms oligomer aggregates and Aβ plaques, resulting in neuronal toxicity and ultimately, the death of neuronal cells. APP Swedish, which is adjacent to the β-secretase site in APP, is one of the well-known genetic mutations in familial Alzheimer’s disease, resulting in increased total Aβ production [47–49]. Therefore, the research model for AD with an APP Swedish mutation is now widely used [50–53], and the H4SW cell line is called the AD in vitro model [20]. Moreover, Aβ accumulated in the brain of AD patients activates glia cells, which are known to eliminate Aβ and have neuroprotective effects [54–57]. MSCs do not remove Aβ itself when exposed to AD but secrete proteins that can stimulate neurons or glial cells through paracrine action [58]. Therefore, we propose that the H4SW cell line was suitable for this study because the therapeutic efficacy of MSCs can be evaluated by measuring the reduction in Aβ deposits by stimulated H4SW cells.
When H4SW cells were co-cultured with primed MSCs, decreases in the level of Aβ and ubiquitin conjugates were observed in the H4SW cells (Figs. 2 and 3). In addition, when primed MSCs were administered directly into the brain of 5xFAD mice, an AD in vivo model, primed MSCs showed the therapeutic effects of suppressing neuronal death and promoting Aβ clearance (Fig. 4). Messenger RNA sequencing confirmed that SGRN secreted by H4SW cells promoted TGF-β secretion by MSCs, TGF-β protein had the same anti-cell death and anti-Aβ effects as primed MSCs, and SRGN-treated MSCs showed anti-cell death effects (Figs. 5, 6, 7, 8). It is known that the secretion of SRGN is increased when an inflammatory reaction occurs [59]. Heparin sulfate proteoglycan, which contains SRGN, was responsible for promoting the fibrillization of Aβ and tau proteins [60]. Interestingly, it was also reported that SRGN gene expression and protein expression were significantly increased in AD patients compared to normal controls [61]. Thus, SRGN may be thought of as a possible biomarker for AD, suggesting that the pre-conditioning of SRGN in MSCs may be a possible to generate enhance MSC for AD treatment. Additionally, TGF-β is highly expressed in primed MSCs or SRGN-treated MSCs, and the signaling pathway associated with TGF-β is impaired in AD [62] and TGF-β itself showed neuroprotective effects [63]. Therefore, the results of this and previous studies suggest that TGF-β, highly secreted by primed MSCs, can have therapeutic efficacy in AD.
A particularly noteworthy finding is that when MSCs were exposed to an AD microenvironment, SRGN secreted locally in the Alzheimer’s brain was recognized by the MSCs, which were induced to increase the expression of TGF-β, promoting therapeutic efficacy. As far as we know, this is the first study to generate pre-conditioned MSC using a possible biomarker for the target disease. Like the concept of vaccination, we can make MSCs in a ready-to-fight state, promoting the secretion of effective proteins by exposing them to the target disease microenvironment in advance.
Our study had several limitations. First of all, the exact mechanism of action of SGRN, MSC, and TGF-β was not elucidated. Second, the recovery of cognition in the AD in vivo model was not studied. After the injection of primed MSCs, TGF-β, or SRGN MSCs, a long-term follow-up must be observed. Finally, the optimization of signaling factors and their combinations used in MSC preconditioning requires further investigation. Studies based on preconditioned MSCs should be conducted to enhance the therapeutic capacity of MSCs and expand the platform developed in this study.