Current progress in regenerative medicine has proposed cell-based therapy as a novel treatment to cure ALS. Despite being in the early stages of clinical translation, numerous preclinical studies have investigated the neuroprotective mechanisms of stem cells in animal models. Most promising for this purpose are hUC-MSCs, which can survive readily after transplantation and have good migratory potential
[103][104]. In 2008, Rizvanov et al. transplanted genetically modified hUC-MSCs in transgenic G93A mice adopted as an ALS animal model. The results obtained demonstrated that transplanted cells successfully grafted into nervous tissue and could differentiate into endothelial cells, forming new blood vessels. In this report, it was hypothesized that the neuroprotective effect could be derived from the delivery of various neurotrophic factors by newly formed blood vessels
[105]. Similar data were confirmed by other groups that have focused their attention on the combined effect of the gene/ stem-cell approaches. In addition, multiple studies have labeled hUC-MSCs to detect transplanted cells in vitro and in vivo by magnetic resonance imaging (MRI) after intraspinal injection in a transgenic mouse model of ALS
[106][107][108]. In line with these promising findings, over the years, several labeling strategies have been proposed. In this regard, it was described for the first time the paramagnetic labeling of hAFCs and their subsequent long-term tracking in a murine model of ALS. Surprisingly, the presence of double tracers has not altered the survival of hAFCs but has allowed for a correlation between in vivo and ex vivo data at different moments
[109]. This study describes the therapeutic potential of hAFSCs in the treatment of ALS; however, additional preclinical trials are required to elucidate their benefits in clinical therapy for motor neuron disorders. Among the gestational stem cells, hAECs have also been proposed as an attractive source in cellular treatment for ALS. These stem cells are a heterogeneous population, containing several undifferentiated progenitor cells, which have not been extensively investigated. They constitute an ethically acceptable alternative to embryonic stem cells, with a comparable multipotentiality and a very low immunogenic response. The finding that hAFCs express and release numerous cytokines and neuro-glial factors
[110] further promotes their application in the field of NDDs. A single preclinical study has reported the beneficial effects of their transplantation in terms of extended survival, the improvement of motor function, and decrease in neuroinflammation
[111].
Although several studies have shown that MSC transplantation results in disease improvement, the mechanisms by which the beneficial effects of MSC therapy arise are not entirely understood. Several mechanisms of repair and support, including cell replacement, trophic factor or gene delivery, and immunomodulation have been observed, sometimes in tandem
[112]. Some studies have shown the ability of MSCs to differentiate into cells with neuron-like morphology, gene expression, and protein expression
[113][114]. However, this phenomenon is still controversial, mainly due to the lack of evidence of functional synapse formation between trans-differentiated MSCs, and their therapeutic contribution is still uncertain. MSCs express or can be stably transduced to overexpress trophic factors which may promote endogenous restorative or regenerative processes, such as neurogenesis, gliogenesis, and synaptogenesis
[115]. MSCs may play several immunoregulatory roles which may contribute to their beneficial effects in ALS. They reduce the proliferation of B cells, T cells, and natural killer cells, and impair the maturation of dendritic cells. Then, they can also affect immune cell function by reducing (i) antibody production by B cells, (ii) the activation of dendritic cells and T cells, and (iii) the secretion of natural killer cells
[112]. In the central nervous system, MSCs migrate to areas of inflammation, reducing it. In experimental models of ALS, MSCs attenuate microglial activation and reduce astrogliosis
[116][117][118].
6. Functional Differentiation of MSCs towards Neuronal Lineage in Neurodegenerative Diseases: An Unmet Clinical Challenge
The potential efficacy of MSCs to restore neurological functions in NDDs depends on neurogenic differentiation, cell replacement, and the secretion of neurotrophic factors
[119]. Unfortunately, the direct transplantation of MSCs at the injury site or injection into the vascular system frequently translates, within several days, into their death, due to natural senescence
[120], the hostile microenvironment, and (or) nutrient deprivation
[121]. Thus, even if the transdifferentiation of MSCs into neurons provides a practical technique for NDD treatment, it is limited by the unmet challenge of getting well-differentiated and mature neurons. The use of a single or combination of growth factors has been adopted to guide the differentiation of MSCs into a neuronal lineage. Growth factors, including Epidermal Growth Factor (EGF), Fibroblast Growth Factor, basic (bFGF), and Platelet-derived Growth Factor (PDGF), engage various cell surface receptors and a variety of signaling pathways, which often crosstalk, leading to an unexpected biological outcome
[122].
New strategies for the differentiation of MSCs into neurons, which could eventually be used to treat patients who are in need, are recently developing. These include (i) highly specific systems for MSC differentiation into neurons directed by local electrical stimuli
[123], and (ii) MSC-based gene delivery strategies
[124]. Since the inherent characteristics of neurons is to transmit electrochemical signals throughout the nervous system, electrical stimulation could significantly promote the neural differentiation of stem cells or neuron maturation
[125][126], with different potential advantages, such as rare immune response, controllable parameters, low damage, easy implementation, localized induction, and synergy with other inducers. Thus, most studies have used external electric fields generated by electrodes or large electrical signal-generating devices to directly induce stem cell differentiation
[127][128]. However, this is an invasive approach with an increased risk of wound pain and infection, unsuitable for nerve repair in humans
[129]. Thus, recently, increasing research has focused on the development of an implantable, low-cost, non-invasive wireless stimulation system
[130][131][132][133] and on the use of stimulus-responsive materials, such as graphene, which has been shown to promote MSC neural differentiation
[134].
The transfection of MSCs with genes that promote cell resistance to hypoxia/ischemia, oxidative stress, and acute or chronic inflammation or with genes enhancing neurotrophy and neuroprotection may increase cell survival in vivo and, importantly, facilitate neuronal replacement and repairing, and the reconstruction of neural circuitry, thus potentially restoring neurological function
[124]. The main tools for gene delivery include viral-based methods, which allow for the construction of stably transfected MSCs, with a more sustained gene expression time, and nonviral-based methods, always sustaining only a transient gene expression in MSCs
[124]. In addition to the stable long-term expression of integrated genes, virus-based methods for gene delivery also have the advantage of a high infection efficiency, but potential disadvantages that can also reduce safety include immunogenicity, the risk of gene integration and the insertion of mutations, and lethal and carcinogenic risks. On the other hand, non-viral-based methods, which include physical methods (such as sonotransfection and electroporation) are characterized by a high transfection efficiency but they can be associated with high cytotoxicity and a lack of targeting. Moreover, non-viral-based methods can be difficult to apply in vivo
[124].
7. Immunological Response in Cell Therapy for Neurodegenerative Diseases
The well-documented immunomodulatory and regenerative properties of MSCs are the reason why they are being used for the treatment of many diseases, including NDDs. Moreover, as reported before, they have been considered “immune-privileged cells”, as they do not activate aggressive immune responses. For this reason, MSC treatments are performed without considering the histocompatibility and without preventing possible immune rejections [135]. However, several studies have provided evidence that mismatched MSCs are immunogenic: mismatches in HLA antigens between donor and recipient lead to serious complications such as graft failure, transplant rejection, or graft versus host disease (GVHD).
Several critical factors could impact the immune response and should be taken into consideration when implanting cells to treat neurodegeneration [135]. The first is the transplantation procedures: although these are becoming minimally invasive and extremely accurate, immunosuppression is needed to overcome the inflammation and morbidity associated with the procedure. However, immunosuppression therapy could cause toxicity and worsen the clinical scenario; thus, it should be accurately selected and monitored [136][137][138]. Other factors include the cell type used [fetal tissue, ESCs, iPSCs, neural progenitor cells (NPCs), MSCs], the presence of genetic modifications, and the degree of mismatch between the donor and recipient. The compatibility of the major histocompatibility complex (MHC), known in humans as human HLA, represents an important factor: the degree of mismatch between donor and host increases the risk of immune rejection, ranging from the absence of rejection to the need for immunosuppressive therapy throughout the lifespan. MSCs seem to be more compatible with the host’s immune system due to their low levels of MHC I and the lack of MHC II molecule expression [139][140].
Figure 2 summarizes the main strategies to restrain the immunological response following cell therapy for NDD. Among them, the graft of autologous material from an identical donor twin is associated with the lowest immunogenic risk. However, currently, obtaining this type of transplantation for patients with NDD, such as PD or HD, is not easy. Possible realistic alternatives have been proposed [135]. Among them, the selection of the donor based on HLA compatibility with the host, which has to be accompanied by treatment with immunosuppressive drugs, has been proposed; in this context, the generation of cell banks could increase the availability of HLA-matched cells [141][142][143].
Figure 2. Strategies to overcome graft rejection in cell therapy for neurodegenerative diseases. Created in Biorender.com.
Tolerance induction approaches include the blockade of co-stimulatory molecules that are crucial for T-cell activation, such as CD28-CD80/86 and CD40-40L
[144]. However, the immune tolerance strategy mainly developed in mouse models must be re-evaluated in the context of the human immune system. The use of stem cells in combination with the target neuronal cells to immunomodulate the response upon grafting has also been proposed. In animal models, this approach can delay allograft rejection and preserve the functionality of the graft
[145][146][147]
In conclusion, up until now, the occurrence of the immune response when considering cell therapy for NDDs has remained an open challenge. The immune response may impair the survival of grafted cells and, therefore, their functionality. Thus, immunosuppression is needed to overcome the inflammation and morbidity associated with the procedure, and this could cause toxicity and worsen the clinical scenario. Most clinical assays in the field are not performed based on clear and feasible guidelines for monitoring the immune response.
8. Large-Scale Production of Human Mesenchymal Stem Cell Manufacturing for Clinical Uses
The clinical uses of MSCs are limited by technical problems associated with mass production, high manufacturing cost, and contamination. The production of MSCs on a large scale is further complicated by the need for manufacturing processes able to provide a high therapeutic quality and purity of cells according to the current GMP standards. Several expansion methods to obtain appropriate numbers of cells with preserved therapeutic quality have been proposed [148]. However, currently, an ideal method for the expansion of MSCs on a large scale remains an important challenge.
The most used approach for the large-scale manufacturing of MSCs for clinical use is represented by standard bioreactor systems
[149]. This automatic system of cell cultures allows for the growth of large numbers of adherent cells, providing reduced labor costs and improvements in cell quality, a central issue when scaling up the processes. Bioreactors can enable the frequent feeding of the culture; thus, they maintain the levels of metabolites necessary for cell expansion under control and allow for a faster and safer expansion of MSCs compared to conventional cultures
[150].
In this system, the main process parameters to be controlled include temperature, pH, pO2, pCO2, microcarrier suspension, and shear stresses
[148]. Therefore, it is necessary to develop online control systems that ensure that product characteristics remain unchanged. Another major limitation to the therapeutic use of MSCs is the composition of culture media, which hinders the validation of GMP-compliant processes. To date, a large number of laboratories use culture media supplemented with fetal bovine serum (FBS) to expand MSCs, but this option will be not applicable in the future. FBS has a not well-defined composition, and it may promote interspecies cross-contamination. Proposed alternatives include human platelet lysate (hPL), but the potential risk of disease transmission and its limited availability reduce its application to large-scale production. Alternatively, new GMP-compliant, commercially available, chemically well-defined xenogeneic-free media that support MSC growth would constitute a more cost-effective and risk-reduced approach. However, some changes in morphology, phenotype, potency, and cellular senescence have been reported, thus suggesting that methods for MSC culture need to be further optimized to enhance batch-to-batch consistency in the cell manufacturing process
[151].
The challenge of the future in the field of the MSC manufacturing process is to harmonize it for different clinical conditions and to work to obtain a unique ‘off-the-shelf’ MSC product. Ideally, this product should be derived from freely available tissue sources, such as umbilical cord tissue, which can be collected with non-invasive procedures. Moreover, it should be cultured in GMP- and regulation-compliant xenogeneic-free media and expanded in a closed automated bioreactor system. Then, it should be delivered ‘off-the-shelf’ as a cryobanked product suspended in a chemically defined, dimethyl sulfoxide (DMSO)-free media. DMSO is an efficacious and economical cytoprotective agent, but its use can be associated with negative effects on humans depending on concentration, administration, and dose. Finally, the final cryobanked product should not require further manipulation at the bedside. Nevertheless, there is the need to repeat pre-clinical safety and efficacy studies when changes are introduced into the bioprocess.
As reported above, in recent years, several strategies have been designed to improve the therapeutic potential of MSCs, which, in Europe, are considered advanced therapy medicinal products (ATMPs). However, the manufacture and handling of these cells for their use as ATMPs is still poorly studied, and a large part of the available data is not related to industrial processes. Up until now, the MSCs used to obtain ATMPs could only be isolated in authorized centers with processes which are standardized around the world. In contrast, the optimal protocols for culturing isolated MSCs are not standardized. This constitutes a major open challenge to improve their therapeutic properties. Cell culture conditions, such as the cell density, time of culture, and culture medium composition represent bottlenecks that need critical controls
[150].
9. Conclusions
Cell-based therapies have been proposed as a promising tool in the treatment of several human NDDs. Remarkably, preclinical studies have demonstrated encouraging results on the functional benefit of cell transplantation in the neurological field and several efforts have been undertaken to successfully apply cell therapy in NNDs. However, to date, this approach has remained experimental and most of the undertaken clinical trials have not been properly designed to assess efficacy and to confirm the promising results about the safety. One of the challenges when considering cell therapy for NNDs is the immune response, which can compromise the survival of grafted cells, impairing their integration and, therefore, their functionality. For this reason, in recent years, in vitro strategies have been developed to evaluate the potential immunogenicity of cell therapy. Funding agencies and the neuroscience community should invest in this kind of strategy to improve and standardize preclinical studies for the development of cell therapies. Good well-standardized in vitro models may provide access to understanding some mechanisms that are difficult to assess in animal models. In fact, the molecular mechanisms underlying the beneficial effect of cell therapy are still largely unidentified. There are some concrete pieces of evidence to support the hypothesis that the transplanted cells produce neurotrophic factors, enhance neuronal plasticity, and activate local progenitors and cell replacement (Figure 3).
Figure 3. Mechanisms underlying the functional benefits of MSC transplantation in the neurological field can be associated with their capacity to activate local progenitors and cell replacement, to produce neurotrophic factors, including Fibroblast growth factor (FGF), Epidermal Growth Factor (EGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), enhance neuronal plasticity and the immunomodulation mediated by several factors, including Indoleamine 2, 3-dioxygenase (IDO), prostaglandin E2 (PGE2), nitric oxide (NO), transforming growth factor (TGF)-β these limitations.