Extracellular vesicles produced by hiPSC-derived mesenchymal stem cells (iEV) for the neuroprotection of the brain following neonatal encephalopathy.

A lack of oxygen (hypoxia) to the baby's brain can cause devastating long-term damage, called neonatal encephalopathy. There is no cure for it. Therefore, there is an urgent need to develop a treatment to protect the developing brain of affected babies.

We have discovered that the fluid surrounding the baby in the womb contains some stem cells. Because these stem cells belong to the baby, they are called fetal stem cells.

Using an experimental mouse model mimicking human neonatal encephalopathy, we have recently discovered that human fetal stem cells are very efficient at repairing the developing brain following injection directly into the brain. However, to obtain sufficient numbers of fetal stem cells for transplantation, it is necessary to let the cells multiply in the laboratory. Unfortunately, during this process, fetal stem cells age and lose their ability to repair tissues. To overcome this pitfall, we have rejuvenated the fetal stem cells to resemble embryonic stem cells, which are very primitive stem cells. This is helpful because rejuvenated cells do not age, such that we can let them multiply in vitro for a long period of time without losing their ability to protect and repair tissues. Once we obtain sufficient numbers, we then induce the rejuvenated stem cells to become less primitive and resemble the original fetal stem cells. These fantastic cells are called iMSCs.

We have then tested the ability of iMSCs to repair the brain and found that they are as efficient as the original fetal stem cells isolated from amniotic fluid. We found that iMSCs reduced the size of the lesion in the brain, decreased inflammation and prevented brain cells from dying. We also discovered that these effects could also be observed if we used the tiny sacks called extracellular vesicles (iEVs) released by the iMSCs. Extracellular vesicles cannot be rejected and do not expose the patients to the risk of having live cells in the brain. Therefore, they represent the next generation of neuroprotective agent.

From there, we want to validate iEVs as a cell-free treatment for NE. As we found that iEVs are especially rich in a factor called MFGE8, which is already known to have neuroprotective potential, we also want to determine whether enriching iEVs with MFGE8 can makes the extracellular vesicles more efficacious.

First, we will isolate iEVs and engineer them to contain either more or less MFGE8. Second, using a preclinical model of NE, we will validate the neuroprotective effects of iEVs and determine whether increasing the amount of MFGE8 in the extracellular vesicles make them more efficacious, and decreasing MFGE8 decrease their efficacy. We will also determine whether MFGE8 is still active outside the vesicles. We will analyse the short- and long-term effects of vesicle treatment on the pathology of the brain and on motor and cognitive function. Finally, we will test the efficacy of iEV treatment on human cells using mini brains cultivated in a culture dish. This will enable us to determine the effects of the vesicle treatment on the different human brain cells and determine whether engineering the vesicles to contain greater amounts of MFGE8 is beneficial.

Ultimately, we expect that our research will validate iEVs (naïve or engineered) for the treatment of neonatal encephalopathy and pave the way for clinical trials. Acellular therapy will change the way babies affected by NE are managed, ultimately leading to better paediatric health care and lower heath costs.

Neonatal encephalopathy (NE) is a devastating cause of infant mortality and neuro-developmental disability. There is robust clinical and preclinical evidence for the brain repair potential of primary mesenchymal stem cells (MSC). However, primary MSC undergo replicative senescence during in vitro expansion. This represents an issue of standardisation, as new donors need to be identified repeatedly to isolate new MSC batches, which constitutes a barrier to clinical implementation. Luckily, MSC can be derived from pluripotent progenitors (iMSCs) and replace the use of primary MSCs. We recently evidenced the neuroprotective effects of iMSC in a preclinical mouse model of NE and showed in a pilot study that intracerebral injection of the extracellular vesicles produced by iMSCs (iEVs) decreased brain lesion size and improved behavioural outcome through vesicular MFGE8.

Using a preclinical model of NE and human cerebral organoids, we aim to validate iEVs as a treatment for NE and determine whether engineering iEVs to contain higher amounts of MFGE8 confers superior neuroprotective efficacy to iEVs.

We will first engineer iEVs to enrich or deplete their vesicular MFGE8 content (iEV-MFGE8-enriched and iEV-MFGE8-depleted) in order to study the molecular function of MFGE8. We will then use the Rice-Vannucci mouse model of NE to determine the short-term and long-term (longitudinal analysis) cellular and molecular mechanisms of action of iEVs, iEV-MFGE8-enriched and iEV-MFGE8-depleted, PBS alone and human recombinant MFGE8 (hrMFGE8) following a single ipsilateral intracerebro-ventricular injection in post-natal day 10 mice (males and females). In complement, to increase the physiological relevance of our data, we will use oxygen-glucose deprived (OGD) human cerebral organoids cultivated in millifluidic conditions to determine the neuroprotective effects of these treatments on recipient human brain cell phenotype and the molecular mechanisms involved.