As we wanted to see the effect of constitutive expression of BDNF, to observe any potential harm BDNF-exposure might afflict on iPSC-derived NPC, we prepared three different culture batches according to the cells they contained:

• Wild-type culture (negative control).
• BDNF enriched media culture (positive control).
• BDNF-transfected culture, using lentiviral transfection and constitutive expression of BDNF under Ef1a promoter (experimental test).

Although our product is designed to be transfected with an Adeno-Associated Virus (AAV), lentivirus use was much more easily available, cheaper, and had additional benefits we needed to perform this proof of concept.

In this case, and most importantly, use of lentivirus would allow us to generate a uniform, replicating cell line, that would allow us to sow many plates for the experiment, as well as freeze some for future assays.

Additionally, as we want to study the different stages and maturity of the neurons, we needed BDNF expression to last at least 50 days, and we did not want the expression to decrease during well-passes, due to dilution of the plasmid.

One of the main reasons we use BDNF is because it improves the formation of projections by the neural cells. This is why the interactions between BDNF-free and BDNF-exposed cells was of our particular interest.

To test this, we are using two-compartment chips that establish contact with the other compartment via 450 µm long channels. We specifically chose the length of 450 µm, as that's a length axons can reach, but no dendrites can grow up to, ensuring localised synapses.

Additionally, as we want to study the different stages and maturity of the neurons, we needed BDNF expression to last at least 50 days, and we did not want the expression to decrease during well-passes, due to dilution of the plasmid.

Due to the working mechanism of the chip plates, we would sow wild type cells on one side of the channels and the cells we wanted to test on the other side of the channel. This would allow us to see how the different conditions affected non-modified cells (Wild-type), therefore simulating the effect our BDNF-producing cells would have on a brain.

These chip plates were fixed on day 31 post-differentiation, and were then stained to localise proteins that indicate the neurons' degree of maturity.

We wanted to determine our NPC's levels of BDNF expression in order to have a quantification approximation of BDNF production by Ef1a constitutive primer. This would give us an insight into the cell's ability to produce BDNF, and also compare constitutive expression to our own expression system (further described in the engineering section).
We used a qPCR assay to quantify the amount of BDNF expressed by our NPC. The substrate of the assay were extracted media samples from our cell cultures, at various differentiation timepoints, giving us an insight on how BDNF expression fluctuated between WT, transformed cells and media-enriched cells.
In order to have enough extracted media, we prepared additional cultures in ibidi plates, that have enough media volume (~1mL) for the qPCR assay. These cells were sowed, transfected and differentiated the same day as the cultures in our chip plates.

The total amount of ibidi cultures is 9, and were used for various different experiments to verify this proof of concept, such as GCaMP recordings, immunohistochemical assays, and qPCR media extraction.

Immunohistochemical stainings were performed on both chip and ibidi plates, at different timepoints. This assay allows us to loacte key neural proteins that indicate how mature the cultures are.
The immunohistochemicalstaining assays for this Proof of Concept were performed in two different batches, with different antibody stainings. Some of the proteins we considered we stained were:

Our cell line contains a Genetically Encoding Calcium Indicator (GECI). We are specifically using the GCaMP system, that reports changes in Caܑ²⁺ by fusing Calmodulin (a calcium-binding protein) to GFP. When calmodulin binds to Caܑ²⁺, there's a change in GFP's conformation, increasing its fluorescence. This system was crucial to our experiments, as it allows us to visualise individual neurons' synaptic activity at different maturation stages [2].
The recordings started on day 30 after iPSC differentiation of NPC, for which we used 6 different ibidi plates: 2 WT, 2 media-enriched BDNF, and 2 tranformed-BDNF. We performed a total of 5 GCaMP recordings: day 29, day 37, day 44, day 51 and day 55.

We used the program HoKaWo from Hamamatusu to perform the recordings, and we analysed the data generated with the program Netcal. We focused on analysing fluorescence spikes, generating and classifying impulse traces. This allows us to determine the intensity of the fluorescence, as well as obtain graphs to visualise the synaptic coordination between multiple neurons via spike interference.

Cell therapies are very strictly regulated, specially when they involve the genetic engineering of the strain. POC#3 is just the start of many future trials Reneurish needs to undergo, but it is one of the most important ones.

As we have mentioned, safety is one of the key factors that characterises Reneurish. We rely on the use of Adeno-Associated Viruses to avoid genomic modification of the patient's cells, as well as guaranteeing that BDNF-expression will dissolve over time.

During POC#3 we intend to figure out exactly how long BDNF-expression by our transfected cells can last for. We have been able to estimate that expression should not last longer than 6 months, however, having cultures during a prolonged amount of time will allow us to confirm our suspicions.

As you can find in this section, our proposed implementation for the product is for the iPSC to be defrosted for the stroke patient, transfected with our AAV, primed for differentiation, and after the priming process is complete, they will undergo a strict quality test.

We have been able to approximate how long it takes to undergo this entire process (around 4~6 days), but POC#3 will allow us to obtain an accurate estimate of this timeline.