Type of stem cells

Various stem cells are being investigated for their potential in stroke treatment, including embryonic stem cells (ESCs), neural stem cells (NSCs), mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs). Despite the potential of these stem cells, several obstacles remain in their clinical application for stroke treatment, such as ethical and teratoma risk of ESC, tumorigenicity potential of iPSC and low cell replacement of MSCs, as well as extraction issues, especially when derived from bone marrow.

In addition to the previously mentioned stem cell types, neural progenitor cells (NPCs) are particularly promising for stroke treatment due to their capacity to differentiate into the necessary neural lineages for repairing damaged brain tissue. Animal models have demonstrated that new neurons generated through NPC transplantation can anatomically and functionally integrate with host neural circuits, facilitating the establishment of novel neuronal relays across the injured site.

While they are one of the few cell types that can functionally recover and generate new neurons, they pose some challenges like sourcing, immunogenicity and engraftment rate.

Cell source

Neural progenitor cells can be obtained from aborted fetal tissues or derived from human embryonic stem cells (hESCs); however, ethical concerns and the risk of immune rejection limit their clinical use. The introduction of human-induced pluripotent stem cells (hiPSCs) has opened new avenues for treatment by allowing the use of a patient’s own somatic cells, thereby avoiding immune rejection. Despite this promise, producing autologous hiPSC-derived NPCs for personalized treatment is costly and labor-intensive. Additionally, these cells must meet regulatory approval for each use, and hiPSCs derived from patients with genetic disorders may carry the same defects, potentially diminishing their therapeutic effectiveness. Consequently, while the concept is appealing, generating autologous hiPSC-NPCs for every patient is impractical for widespread clinical application, and it also presents 2 major limitations:

1. Immunogenicity

Previous efforts have focused on generating HLA-homozygous hiPSCs for biobanking. In Spain, the Blood and Tissue Bank (BST) has recently established a clinical-grade HLA-homozygous iPSC line bank that covers 20% of the Spanish population. Initially, the plan was to leverage this technology to create a hiPSC-NPC HLA biobank. Upon patient arrival, HLA testing would be conducted, and the appropriate cell type would be thawed for that patient.

However, this approach proved to be highly unfeasible due to the significant storage space required for the cells, increased costs associated with HLA testing, and inefficiencies in scaling up the process. Furthermore, considering that current stroke treatments, such as mechanical thrombectomy and thrombolysis, are only available for 15-20% of patients, there is a clear need to increase accessibility to effective treatments.

Inspired by mesenchymal cells, research led to the discovery of a concept relatively unknown to many: cell universalization. By adapting CRISPR technology, it is possible to modify hiPSC-derived NPCs to make them immunocompatible by knocking out key HLA class I and II genes (B2M, CIITA), thereby minimizing T-cell recognition. At the same time, retaining HLA-C expression helps avoid triggering natural killer (NK) cell responses. Using advanced CRISPR base editing ensures precise and safe modifications, allowing for the creation of a universal, off-the-shelf NPC line that minimizes the risk of immune rejection. Dr. Arístides López, a researcher at the University of Barcelona, provided valuable guidance on designing this approach and optimizing its implementation.

2. Teratoma potential

iPSCs have long been associated with a heightened risk of teratoma formation, which limits their clinical applicability [3]. To address this issue, we developed a co-transfection cassette that expresses a synthetic membrane marker under a NPC-specific promoter. This allows us to utilize flow cytometry to selectively isolate cells expressing the marker, significantly reducing the likelihood of errors over two or three rounds. Furthermore, the cell priming protocol we employ boasts a 100% success rate, ensuring the reliability of our selected NPCs.

Neurotrophic factor

One key mechanism of action for stem cell therapies is the release of neurotrophic factors, either systemically or directly at the infarct site. Major neurotrophic factors secreted by stem cells include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), and hepatocyte growth factor (HGF) [4]. Among these, BDNF is particularly notable for its role in neurogenesis and promoting recovery outcomes [5].

BDNF, part of the neurotrophin family, which includes NGF, neurotrophin 3 (NT-3), and neurotrophins 4/5 (NT-4/5), is naturally upregulated following ischaemic brain injury as part of the body’s neuroprotective response [6]. Studies have shown that administering BDNF intravenously before an ischaemic event, delivering it intracranially after ischaemia, or transplanting BDNF-overexpressing mesenchymal stem cells all significantly reduce infarct size and improve behavioural recovery [7].

However, BDNF expression in certain cell types may be insufficient to produce strong therapeutic effects. Additionally, a major challenge in stem cell therapies is the low survival rate of transplanted cells in ischaemic environments, where most fail to persist beyond a few days [8]. Nevertheless, research suggests that BDNF enhances both cell engraftment and survival [9], making its overexpression a promising strategy to address the current limitations of NPC-based therapies. As a validation, our laboratory results indicate that BDNF-transfected NPCs exhibit improved integration into brain tissue, providing a promising pathway for subsequent in vivo models. For detailed findings, please refer to the results section.

Expression system

Nevertheless, research on neurotrophic factors uncovered a significant limitation: their toxicity is dose-dependent, and prolonged exposure could be detrimental to cells. As a result, we prioritized limiting BDNF overexpression to the early stages of therapy. To do this, we have also developed a novel BDNF expression system to ensure its expression only the first 6 weeks pots-transplant.

Due to the biosafety limitations of lentiviruses (LVs) in the iGEM competition and their potential for random DNA integration, we opted for adeno-associated viruses (AAVs) to transfect our cells.

AAVs facilitate episomal genetic modifications, allowing transfected DNA to remain in the cytoplasm without integrating into the cell's genome. Consequently, this DNA is diluted with each cell division, leading to its eventual disappearance.

To sum up, by utilizing AAV with our patent-pending expression system, we have 2 safety measures to ensure there is no toxic exposure of the neurotrophic factors.

Administration route

During discussions with stroke experts and hospitals, we identified significant safety concerns regarding cell administration methods. Many experts preferred intravenous (IV) or intra-arterial (IA) administration, believing these methods could provide systemic effects through the secretion of neurotrophic factors benefiting multiple organs. However, two key limitations were recognized: systemic administration of iPSC-derived NPCs increased the risk of teratoma formation in non-target tissues and diminished the cells' regenerative potential.



In light of these challenges, we explored intracranial (IC) administration, which offers significant advantages in precision, efficacy, and neural regeneration. Many leading experts agree that IC delivery is the optimal choice for our therapy.

To sum up, by utilizing AAV with our patent-pending expression system, we have 2 safety measures to ensure there is no toxic exposure of the neurotrophic factors.

Therapeutic window

A critical component of our therapeutic design is determining the optimal therapeutic window for intervention. Ischaemic stroke can be divided into three chronological phases: acute (< 24 hours), subacute (<6 months), and chronic.

Drawing on preclinical data and established stroke recovery patterns, we have identified a three-week therapeutic window that commences five days post-stroke. This timing allows us to target the subacute phase of recovery, which is crucial for effective intervention. During the acute stage, the patient's condition is often very unstable, increasing the risk of complications and rendering them too fragile to undergo surgical procedures safely.

By strategically waiting until the subacute phase, we enable the patient to stabilize, ensuring that the intervention can be performed with minimal risk to their health. This approach not only prioritizes patient safety but also aligns with the brain's natural healing processes. The subacute phase is characterized by an enhanced capacity for repair and neuroplasticity, making it an ideal period for administering cell therapies aimed at promoting regeneration and recovery.

However, delaying intervention beyond this therapeutic window can significantly diminish the likelihood of successful recovery. As time progresses, the potential for neuroplasticity and repair decreases, and the window for effective treatment narrows. However, recent studies indicate that human brain tissue, even at the stroke cavity area, may maintain its regenerative capacity for 2 or more years after stroke, and implantation of neural stem cells may trigger the release of such regenerative capacity [13].

Research also indicates that during the subacute phase, inflammatory responses shift from a pro-inflammatory to an anti-inflammatory profile [14], facilitating tissue healing. Additionally, interventions during this period can stimulate endogenous stem cell activity, neurogenesis, and angiogenesis, further supporting the recovery process. By intervening on this critical therapeutic window, our therapy is designed to maximize efficacy and improve outcomes for patients recovering from stroke.

Dosage

A crucial aspect of designing this therapy is determining the appropriate dosage and frequency of interventions. Currently, the optimal dose of stem cells remains largely undefined in the literature. Therefore, additional studies are necessary to address the significant discrepancies between preclinical and clinical trials and to conduct Phase 3 clinical trials with robust controls over study characteristics and outcomes.

Dosages in existing studies range from 1 million to 70 million cells. However, it is evident that intracranial dosages should be substantially lower than those administered through other routes, which can be as high as 1.2 billion cells [15]. In light of this, we propose an initial delivery of 1 to 10 million NPCs per intervention, drawing on insights from other human trials. The exact dosage will be refined following preclinical studies. Regarding frequency, we plan for a single intervention, with potential adjustments based on the patient’s progress and treatment response. A proposed protocol may also include administration of NPC-derived extracellular vesicles post intervention, which could further enhance the therapeutic effects of the therapy [16]. Nevertheless, our primary focus will be on a single intervention, as this approach is the most extensively studied and has already demonstrated effectiveness.