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

婴儿大脑的缺氧(缺氧)会导致破坏性的长期损害，称为新生儿脑病。这是无法治愈的。因此，迫切需要开发一种治疗方法来保护受影响婴儿的发育中的大脑。我们发现，子宫中婴儿周围的液体中含有一些干细胞。因为这些干细胞属于婴儿，所以它们被称为胎儿干细胞。使用模拟人类新生儿脑病的实验性小鼠模型，我们最近发现，人类胚胎干细胞在直接注射到大脑中后，在修复发育中的大脑方面非常有效。然而，为了获得足够数量的胎儿干细胞进行移植，有必要让细胞在实验室中繁殖。不幸的是，在这个过程中，胎儿干细胞会老化，失去修复组织的能力。为了克服这一缺陷，我们将胎儿干细胞恢复活力，使其类似于胚胎干细胞，这是非常原始的干细胞。这是有帮助的，因为恢复活力的细胞不会老化，这样我们就可以让它们在体外长时间繁殖，而不会失去保护和修复组织的能力。一旦我们获得了足够的数量，我们就会诱导恢复活力的干细胞变得不那么原始，与原始的胎儿干细胞相似。这些神奇的细胞被称为间充质干细胞。然后，我们测试了iMSCs修复大脑的能力，发现它们与从羊水中分离出的原始胎儿干细胞一样有效。我们发现，iMSCs缩小了脑部病变的大小，减少了炎症，防止了脑细胞死亡。我们还发现，如果我们使用iMSCs释放的称为细胞外小泡(IEV)的微小麻袋，也可以观察到这些影响。细胞外小泡不能被排斥，也不会使患者面临大脑中存在活细胞的风险。因此，它们代表了下一代神经保护剂。从那时起，我们想要验证iEV作为一种无细胞治疗NE的方法。由于我们发现iEV特别富含一种名为MFGE8的因子，这种因子已经被认为具有神经保护潜力，我们还想确定用MFGE8丰富iEV是否可以使细胞外小泡更有效。首先，我们将隔离iEV并对其进行工程，使其包含或多或少的MFGE8。其次，利用去甲肾上腺素的临床前模型，我们将验证iEV的神经保护作用，并确定是否增加细胞外小泡中MFGE8的含量会使其更有效，而减少MFGE8会降低其疗效。我们还将确定MFGE8是否仍然活跃在小泡外。我们将分析水泡治疗对大脑病理以及对运动和认知功能的短期和长期影响。最后，我们将使用培养皿中培养的迷你大脑来测试IEV对人类细胞的治疗效果。这将使我们能够确定囊泡治疗对不同人脑细胞的影响，并确定改造囊泡以包含更多MFGE8是否有益。最终，我们预计我们的研究将验证iEV(幼稚的或经过改造的)用于治疗新生儿脑病，并为临床试验铺平道路。去细胞疗法将改变受NE影响的婴儿的管理方式，最终导致更好的儿科保健和更低的医疗成本。

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.