Neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), multiple sclerosis (MS), spinal cord injury (SCI), and amyotrophic lateral sclerosis (ALS), are characterized by acute or chronic progressive loss of one or several neuronal subtypes. However, despite their increasing prevalence, little progress has been made in successfully treating these diseases. Research has recently focused on neurotrophic factors (NTFs) as potential regenerative therapy for neurodegenerative diseases. Here, we discuss the current state of knowledge, challenges, and future perspectives of NTFs with a direct regenerative effect in chronic inflammatory and degenerative disorders. Various systems for delivery of NTFs, such as stem and immune cells, viral vectors, and biomaterials, have been applied to deliver exogenous NTFs to the central nervous system, with promising results. The challenges that currently need to be overcome include the amount of NTFs delivered, the invasiveness of the delivery route, the blood–brain barrier permeability, and the occurrence of side effects. Nevertheless, it is important to continue research and develop standards for clinical applications. In addition to the use of single NTFs, the complexity of chronic inflammatory and degenerative diseases may require combination therapies targeting multiple pathways or other possibilities using smaller molecules, such as NTF mimetics, for effective treatment.

Over the past two decades, significant advances have been made in the field of regenerative medicine. However, despite being of the utmost clinical urgency, there remains a paucity of therapeutic strategies for conditions with substantial neurodegeneration such as (progressive) multiple sclerosis (MS), spinal cord injury (SCI), Parkinson’s disease (PD) and Alzheimer’s disease (AD). Different cell types, such as mesenchymal stromal cells (MSC), neuronal stem cells (NSC), olfactory ensheathing cells (OEC), neurons and a variety of others, already demonstrated safety and regenerative or neuroprotective properties in the central nervous system during the preclinical phase. As a result of these promising findings, in recent years, these necessary types of cell therapies have been intensively tested in clinical trials to establish whether these results could be confirmed in patients. However, extensive research is still needed regarding elucidating the exact mechanism of action, possible immune rejection, functionality and survival of the administered cells, dose, frequency and administration route. To summarize the current state of knowledge, we conducted a systematic review with meta-analysis. A total of 27,043 records were reviewed by two independent assessors and 71 records were included in the final quantitative analysis. These results show that the overall frequency of serious adverse events was low: 0.03 (95% CI: 0.01–0.08). In addition, several trials in MS and SCI reported efficacy data, demonstrating some promising results on clinical outcomes. All randomized controlled studies were at a low risk of bias due to appropriate blinding of the treatment, including assessors and patients. In conclusion, cell-based therapies in neurodegenerative disease are safe and feasible while showing promising clinical improvements. Nevertheless, given their high heterogeneity, the results require a cautious approach. We advocate for the harmonization of study protocols of trials investigating cell-based therapies in neurodegenerative diseases, adverse event reporting and investigation of clinical outcomes.

Regulatory T cells (Tregs) are crucial in inducing and maintaining tolerance. This unique capacity of Tregs, in combination with proof-of-principle in preclinical studies, highlights the potential clinical use of Tregs for the treatment of autoimmunity and transplant rejection. Although proven to be safe and well tolerated in the first clinical trials, only modest clinical results were observed. In this regard, it has been hypothesized that current challenges lie in the development of antigen-specific Tregs. Here, we present an innovative, good manufacturing practices (GMP)-compliant manufacturing protocol for Tregs applicable in a clinical-grade setting, allowing efficient and safe redirection of Treg specificity. First, a soluble polymer conjugated with antibodies to CD3 and CD28 and high amounts of exogenous IL-2 for in vitro Treg expansion resulted in a >70-fold and 185-fold increase of a pure population of CD4⁺CD127^-CD25^hi Tregs and CD4⁺CD127^-CD25⁺CD45RA⁺ Tregs, respectively. Next, as a proof-of-principle, expanded Tregs were engineered by means of TCR-encoding mRNA electroporation to generate antigen-specific Tregs. This resulted in an expression of the newly introduced TCR in up to 85% of Tregs. Moreover, we did not observe a negative effect on the phenotype of Tregs, as demonstrated by the expression of FOXP3, Helios, CTLA-4 and CCR4, nor on the TSDR methylation status. Importantly, mRNA-engineered Tregs were still able to induce in vitro suppression of effector T cells and produced anti-inflammatory, but not pro-inflammatory, cytokines when activated. In conclusion, our findings demonstrate that high numbers of stable and functional Tregs can be obtained with high purity and successfully engineered for gain of function, in a GMP-compliant manner. We envisage that this clinical-grade protocol will provide solid basis for future clinical application of mRNA-engineered Tregs.

Multiple sclerosis (MS) is a chronic neurological disorder involving immune‐mediated demyelination and neurodegeneration in the central nervous system (CNS). Current therapies primarily target inflammation, with limited strategies to promote remyelination or neural repair. This study explores the therapeutic potential of Brain‐Derived Neurotrophic Factor (BDNF) delivered via an adeno‐associated virus (AAV) vector to enhance remyelination and improve cognitive function in a subchronic cuprizone (CPZ)‐induced demyelination mouse model. Sixty female C57BL/6 mice were used, with half receiving a 7‐week CPZ diet to induce oligodendrocyte loss. After demyelination, mice were treated with AAV‐BDNF, AAV‐eGFP, or saline injections into the corpus callosum (CC), followed by a 5‐week recovery phase. Behavioral assessments revealed improved cognitive performance with BDNF treatment, demonstrated by increased latency in passive avoidance tests. Immunofluorescence analysis showed increased proliferation and maturation of oligodendrocyte progenitor cells, with higher PDGFRα and CC1 markers, alongside elevated MBP. Transmission electron microscopy (TEM) indicated thicker myelin sheaths and a higher percentage of myelinated axons in AAV‐BDNF‐treated mice. Mitochondrial analyses revealed that BDNF treatment preserved mitochondrial integrity, with reduced swelling and improved structural regularity. Inflammatory markers showed no differences in Iba1 but indicated a trend of reduced astrocytic activation with BDNF. These results demonstrate that AAV‐BDNF therapy enhances remyelination, myelin integrity, mitochondrial structure, and cognitive function in a CPZ model, underscoring its potential for treating MS. BDNF‐based strategies may offer innovative avenues to improve neurological recovery and address unmet needs in MS management.

Genetic engineering of regulatory T cells (Tregs) presents a promising avenue for advancing immunotherapeutic strategies, particularly in autoimmune diseases and transplantation. This study explores the modification of Tregs via mRNA electroporation, investigating the influence of T‐cell activation status on transfection efficiency, phenotype, and functionality. For this CD45RA⁺ Tregs were isolated, expanded, and modified to overexpress brain‐derived neurotrophic factor (BDNF). Kinetics of BDNF expression and secretion were explored. Treg activation state was assessed by checking the expression of activation markers CD69, CD71, and CD137. Our findings show that only activated Tregs secrete BDNF post‐genetic engineering, even though both activated and resting Tregs express BDNF intracellularly. Notably, the mTOR pathway and CD137 are implicated in the regulation of protein secretion in activated Tregs, indicating a complex interplay of signalling pathways. This study contributes to understanding the mechanisms governing protein expression and secretion in engineered Tregs, offering insights for optimizing cell‐based therapies and advancing immune regulation strategies.

Neurodegenerative diseases such as multiple sclerosis (MS), Alzheimer’s disease (AD), and Parkinson’s disease (PD) pose major medical and societal challenges. They are characterized by the progressive loss of neurons and glial cells, resulting in declining motor and cognitive functions. Current treatments mainly aim to slow disease progression but fail to promote regeneration or remyelination, leaving a strong need for innovative regenerative strategies. This thesis investigates the potential of regulatory T cells (Tregs) as a novel therapeutic approach. Tregs are best known for maintaining immune tolerance and limiting inflammation, but growing evidence shows they can also contribute to tissue repair. Their dual ability to modulate neuroinflammation and stimulate regeneration makes them attractive candidates for neurodegenerative diseases. To further enhance their potential, Tregs were genetically engineered using messenger RNA (mRNA) electroporation to transiently express Brain-Derived Neurotrophic Factor (BDNF), a molecule with well-established roles in neuronal survival and myelin repair. The work presented here demonstrates that mRNA electroporation is a feasible and safe strategy to engineer Tregs, enabling them to produce and secrete BDNF for limited periods. However, experiments in both murine and human stem cell–based models revealed that even unmodified Tregs possess regenerative properties: they were able to promote differentiation and maturation of oligodendrocyte precursor cells (OPCs), a key step in remyelination. Interestingly, these effects appeared largely independent of BDNF expression, suggesting that Tregs exert intrinsic regenerative functions beyond their established immunomodulatory role. Together, these findings expand our understanding of Tregs as a versatile cell therapy platform. While transient BDNF expression may provide an additional boost in certain contexts, the intrinsic capacity of Tregs to influence repair mechanisms is a powerful discovery in itself. Translating these insights into clinical practice will require strategies to enhance Treg stability, prolong or regulate delivery of neurotrophic factors, and further uncover regenerative pathways mediated by Tregs. By combining immune regulation with regenerative support, Tregs, engineered or not, emerge as promising candidates for future therapies aimed at not only slowing but also repairing the damage caused by neurodegenerative diseases.