The first step is to carry out in vitro studies to demonstrate proof of concept of our stem cell therapy. This step includes the formation of the culture condition optimizations for NPCs, the evaluation of their differentiating potential, and a check whether they are capable of neuroprotection and stimulus repair in controlled environments. This important step will give a green light to the therapy's success and will be the preliminary stage to animal trials.

As stated above, our lab assays demonstrate that BDNF expression in iPSC-NPCs holds significant promise for neural transplants. BDNF-expressing NPCs not only differentiate more rapidly than wild-type NPCs, but also exhibit enhanced proliferation without excessive growth or increased risk of teratoma formation. Furthermore, these cells generate numerous projections, indicating their ability to integrate effectively into brain tissue, which facilitates the incorporation of transplanted cells into patient’s brains. For more detailed in vitro results, visit the Wet Lab Section.

Many therapeutics are proven to be efficacious in the petri dish, but the biggest challenge lies in optimizing them for efficacy in an in vivo context.

After successful in-vitro testing, we will be moving to preclinical studies in animals to further assess the safety, efficacy as well as the best treatment schedules. This phase will consist of the assessment of the therapeutic effects of NPC transplantation in stroke models, the detection of therapy side effects, as well as the supportive data for our clinical applications.

There are several well-established animal models for studying cerebral infarction, with two of the most widely used being the Middle Cerebral Artery Occlusion (MCAO) model and the intraluminal filament model.

• Middle Cerebral Artery Occlusion (MCAO) with craniectomy and fine suture: This method is highly effective in replicating the severe neurological deficits seen in ischaemic stroke patients [17]. By performing a craniectomy, the middle cerebral artery (MCA) is directly accessed and occluded with a fine nine-zero suture. The ischaemia induced can be either transient or permanent, depending on the specific goals of the study. This model is particularly useful for examining cortical damage and the brain’s acute response to ischaemia, making it ideal for understanding the early mechanisms of stroke [18].
• Transient ischaemia with the intraluminal filament model: This technique offers a less invasive approach. A filament is inserted into the internal carotid artery (ICA), temporarily blocking the MCA to induce transient ischaemia followed by reperfusion (restoration of blood flow) [18]. This model is especially valuable for simulating clinical stroke scenarios, as it allows for precise control of the duration of ischaemia and subsequent reperfusion. The ability to study both the damage caused by the initial ischaemic event and the effects of reperfusion injury makes it highly relevant for testing potential treatments and understanding recovery mechanisms.

For our preclinical trials, the intraluminal filament model is the preferred choice. Its ability to closely mimic clinical conditions such as transient ischaemic attacks and strokes, along with its flexibility in controlling ischaemia duration, makes it a more accurate and reliable platform. This model is also particularly suitable for evaluating neuroprotective and regenerative therapies, especially in conjunction with treatments like endovascular thrombectomy, where reperfusion plays a crucial role.

After successful preclinical studies demonstrating the therapy's viability, we will transition to large-scale production of our BNDF-NPCs under Good Manufacturing Practices (GMP). This process involves developing protocols that ensure every step, from differentiation to storage, meets rigorous quality and safety standards. We will need to optimize logistics for cell processing, storage, and transportation to guarantee consistent product quality.

Our approach utilizes iPSC-derived NPCs, which can be produced in large quantities and stored until needed. iPSCs provide a renewable source, enabling the mass production of standardized cell lines. Our differentiation and freezing protocols are optimized for scalability, allowing us to generate large batches of NPCs ready for deployment.

Nevertheless, scaling GMP-compliant cell therapies still presents several challenges [19]:

• Cost-effectiveness: Producing large quantities of adherent cells requires specialized surfaces, leading to long expansion times and limited yields, which necessitate significant resources and personnel.
• Regulatory compliance: Transitioning from research-grade to clinical-grade cells GMP-compliant processes involves complex documentation and validation, which can be resource-intensive.
• Line-to-line variability: Human embryonic stem cells (hESCs) and iPSCs exhibit variability that affects differentiation efficiency and therapeutic potential, requiring extensive pre-screening of pluripotent stem cell lines.

To address these challenges, we plan to implement advanced bioprocessing techniques, such as the Quantum Cell Expansion (QCE) system from Terumo BCT [20]. This innovative hollow fiber bioreactor system provides a compact, automated environment for cell growth, allowing continuous monitoring of key environmental factors such as gas levels, temperature, and nutrient flow

The QCE system enhances scalability by offering a large surface area for cell growth equivalent to 120 T-175 flasks, facilitating the rapid production of substantial cell quantities. In a study [21] using the Quantum Cell Expansion (QCE) system to scale up clinical-grade neural stem cells (NSCs), seeding 5.2 × 10⁷ NSCs in one unit resulted in up to 3 × 10⁹ cells within 10 days. The NSCs expanded through QCE showed genetic and functional stability comparable to those grown via traditional flask methods. By utilizing seven units simultaneously, researchers generated a pooled GMP-grade clinical lot of over 1.5 × 10¹⁰ cells in just 9 days, while CellStacks yielded 8 × 10⁹ cells over 6 weeks. This efficiency meets the increasing demands of clinical trials and commercialization.

Additionally, the QCE system allows successful lentiviral and adenoviral transduction of cells [22], producing modified cells that express therapeutic enzymes, thereby enhancing their therapeutic potential. Using this automated process to modify our NPC will minimize contamination risks and human errors, reducing production costs.

Through extensive consultations, we learn that this rigorous clinical trial design is crucial to success. Without it, we risk struggling with patient recruitment. No matter how effective our therapy might prove to be, we will never have the chance to test it. Financial support is needed, too, as these clinical trials are notoriously expensive.

The next step is conducting clinical trials in three phases. Phase I will focus on safety, testing the therapy’s tolerance in a small patient group. Phase II will evaluate the therapy’s efficacy and dosage in a larger cohort of affected patients. Phase III will involve a broader population to confirm the treatment's effectiveness, monitor side effects, and compare it to existing options.

The document below serves as a guide to understanding how a clinical trial is structured and what key factors need to be considered when designing it. It is not a definitive plan, but rather an outline to help navigate the process, covering aspects like patient recruitment, treatment protocols, follow-up schedules, and data collection to ensure the trial's scientific and ethical integrity.

Regulations are crucial at each step of the development process. Our NPC-based therapy has the ultimate goal of being admitted to the Advanced Therapy Medicinal Product (ATMP) classification as of Somatic Cell Therapy Medicinal Product. This category is parallel to the regulations enforced by both EMA (European Medicines Agency) and FDA (Food and Drug Administration) in the US. For more information on ATMP classification and regulations, visit our EP handbook.

In the case of the EU, the product will follow the regulatory pathways devised by the EMA Committee for Advanced Therapies (CAT), while in the US, it would require approval as a Biologic License Application (BLA) under the FDA Center for Biologics Evaluation and Research (CBER).

An occupational therapist confirmed that IC administration would have minimal impact on recovery if there were no hemodynamic issues. Conversations with stroke survivors indicated that most would accept IC procedures for a chance to regain critical functions, highlighting the urgency for effective treatment while minimizing tumour risks.

Ultimately, discussions confirmed that IC administration would be accepted if prior results were promising, allowing us to validate our approach and finalize safety considerations.

To minimize the risk of intracranial haemorrhage during needle insertion, which affects 5% to 15% of patients (ref), several strategies will be implemented in future trials based on recent clinical findings. First, the transplantation site will be selected with great care to ensure maximum effectiveness while minimizing risk. We will use FLAIR-high signals to identify fragile brain tissue during the subacute phase of ischaemic stroke. Additionally, antiplatelet agents will be discontinued before the procedure, keeping in mind the risk of ischaemic stroke recurrence. During the procedure, reversal agents for direct oral anticoagulants may be utilized to further reduce risks. Finally, a specially designed transplantation needle with an obtuse tip will be used to decrease the likelihood of injuring intracranial vessels (ref). These measures aim to enhance the overall safety of the transplantation procedure.

Preclinical and toxicology studies

Prior to the beginning of clinical trials, the preclinical and toxicology studies must demonstrate the safety of the therapy in animal models. The studies must follow Good Laboratory Practices (GLP) and their focus is especially on some critical points:

Once safety has been shown during the preclinical trials, then the data can be submitted to the regulatory agencies to obtain permission for human clinical trials.

Clinical trials

The first step to starting clinical trials is obtaining a Clinical Trial Authorization. This involves submitting detailed information to regulatory bodies, including a description of the manufacturing process, which outlines how NPCs are sourced, expanded, and prepared for transplantation. This covers aspects like cell sorting, cell expansion protocols, Good Manufacturing Practices, and ensuring product consistency across cell batches. The submission must also include comprehensive preclinical data obtained from GLP studies, proving the therapy's safety in animal models. Additionally, clinical protocols need to be presented, specifying the design of the clinical trial, the patient population, and safety monitoring procedures for each phase of the trial. Documents such as the Investigator’s Brochure and the Informed Consent Document must also be submitted, ensuring that patients are fully informed of the potential risks and benefits of participating in the trial. All submissions and documents must be submitted through the Clinical Trials Information System (CTIS) of the EMA (here).

We expect to achieve Orphan Drug Designation (ODD) during this stage. As an ischaemic stroke is such a serious condition with limited treatments, Reneurish's therapy could fit in with ODD, which offers benefits like market exclusivity, reduced fees, and shorter review periods that come with the incentive program. With such a feather in the cap, the development can be quite faster, as a result, patients can benefit from the therapy earlier.

Good Manufacturing Practices and product quality

To gain approval for ATMPs, adherence to Good Manufacturing Practices is essential. This ensures the product’s quality and safety during production, with a particular focus on the cell culture expansion process. Dedicated cleanroom facilities and standardized protocols will be used to maintain product consistency for each batch of cells. Every batch will be tested for cell identity, purity (to confirm that unwanted cell types or contaminants are absent), and potency (to ensure the cells survive and differentiate post-transplantation). Product tracking and traceability are also critical, as they allow for quick investigation and corrective actions in the event of safety concerns or adverse events.

Market approval

Following the successful completion of phase III clinical trials, Reneurish will submit a Biologic License Application (BLA) to the FDA and a Marketing Authorization Application (MAA) to the EMA to obtain market approval. These applications will include comprehensive clinical data demonstrating the therapy’s benefits relative to its risks. A pharmacovigilance plan will be implemented to monitor patient safety, tracking any long-term effects such as graft rejection, tumorigenicity, or delayed immune responses. A risk management plan will also outline strategies to identify, characterize, and mitigate potential risks associated with the therapy, such as intracranial haemorrhage or immune reactions.

By carefully navigating these regulatory pathways, we will ensure that our cell therapy for stroke adheres to the highest standards of safety, quality, and efficacy, paving the way for a successful transition from clinical trials to market approval

As we advance through clinical trials, our market entry strategy aims to facilitate a seamless transition from production to patient treatment. This is achieved through strategic partnerships, scalable manufacturing, and a highly efficient supply chain. Our approach not only guarantees a steady supply for stroke patients but also lays the groundwork for potential scalability and acquisition by a larger pharmaceutical company following Phase 2, as outlined in our exit strategy.

Production and quality assurance

Reneurish cells will initially be produced centrally in our laboratory, adhering to strict GMP to ensure the highest quality and consistency. As we prepare for larger-scale production post-clinical trials, we will integrate partner production facilities into our supply chain to meet increasing demand. These partners will be meticulously selected to uphold the same high standards of manufacturing excellence, positioning Reneurish for a smooth scale-up process after Phase 2, which is crucial in the event of an acquisition by a larger entity.


To enhance our production process, we will infect our cells with AAV during the critical priming phase, occurring after thawing and before transplantation. This phase is essential for differentiation and mandatory safety testing. This innovative approach was developed through consultations with five directors of Stroke Units from leading hospitals, the Catalan Blood and Tissue Bank, and the Catalan Department of Health.

Leveraging Spain's transplant system

After production, Reneurish cells will be cryopreserved and stored under ultra-low temperature conditions to maintain viability during transport. The prepared cells will be delivered to hospitals much like organs for transplant, benefiting from Spain’s globally recognized leadership in organ donation and transplant logistics. This system ensures that the therapy can be delivered safely, reliably, and efficiently to stroke patients, while also meeting the stringent regulatory and safety requirements for cell therapies.

By centralizing production, we ensure that each batch of cells is identical and prepared under the same conditions.

M&A strategy and scalability post-Phase 2:

With a robust production and supply chain in place, Reneurish will be well-positioned for strategic acquisition after Phase 2 clinical trials. At this stage, our therapy will have demonstrated both safety and efficacy, and our scalable supply chain will be appealing to pharmaceutical companies looking to expand their pipeline in regenerative medicine. The priming and infection of cells at external facilities, integrated with Spain’s efficient transplant infrastructure, guarantees a seamless transition to large-scale production.

In the event of an acquisition, the acquiring company may either integrate our production into their own facilities or continue to leverage our established partner network for national and global distribution. This flexibility ensures a smooth transition from a clinical-stage therapy to a commercially viable product.