Engineered neuroprotective stem-cell exosomes for in utero spina bifida therapy

摘要 脊髓脊膜膨出（MMC）是脊柱裂（SB）最严重的形式，也是最常见的先天性原因 在美国，每天大约有四个孩子出生时患有这种毁灭性的瘫痪， 疾病MMC是由于神经管的不完全闭合和缺少上覆脊柱， 脊髓暴露于子宫内机械和化学创伤。这种创伤会导致终身瘫痪 肠和膀胱功能障碍，肌肉骨骼畸形，和认知障碍，由于后脑 脑疝子宫内手术修复可改善发病率，但功能恢复不完全， 孩子们还不能独立行走。我们开发了一种治疗MMC的方法， 子宫内MMC手术修复，胎盘间充质基质/干细胞（PMSC）。我们发现 在子宫内修复期间，用PMSC治疗可防止发育良好的胎羊后肢瘫痪 MMC模型，由于PMSC旁分泌神经保护因子。然而，我们确认，私营军保公司 未长期植入和治疗的羔羊出现严重脊柱后凸，导致脊髓压迫， 由于缺乏骨和相邻椎旁肌肉而导致的栓系，这与人类MMC一致 肌肉骨骼畸形为了满足MMC长期治疗的需要，我们探索了使用 生物工程多功能组合支架。外泌体是细胞外囊泡， 在细胞间通讯中的作用。我们证实，PMSC分泌的外泌体（PMSC-外泌体）发挥 这些细胞具有显著的神经保护功能，在数量上与它们所来源的活的PMSC相似。在这 研究，我们建议开发一种工程水凝胶系统，将允许局部和持续释放 PMSC-外泌体的脊髓，这反过来将提供持续的神经保护，以治疗MMC之前， 出生此外，我们的目标是通过用一种 生物材料为基础的骨支架，以提供结构和功能支持。我们计划优化 生物支架的神经保护和再生功能使用我们良好建立的胎啮齿动物和兔 MMC的模型。我们将在我们的金标准中评估最终的组合多功能生物支架产品 MMC的胎羊模型。这种治疗剂将是无细胞的，现成的，并且易于使用。如果成功，这 一种新的方法将用于在出生前治疗MMC，由于其再生特性，治疗将 改善这些患者的生活质量，并显著降低与之相关的医疗费用。 目前的治疗。

ABSTRACT Myelomeningocele (MMC) is the most severe form of spina bifida (SB) and the most common congenital cause of lifelong paralysis in the United States, where approximately four children are born daily with this devastating disease. MMC results from the incomplete closure of the neural tube and absent overlying spine leaving the spinal cord exposed to intrauterine mechanical and chemical trauma. This trauma results in lifelong paralysis, bowel and bladder dysfunction, musculoskeletal deformities, and cognitive disabilities due to hindbrain herniation. In utero surgical repair improves morbidity, but functional recovery is incomplete and the majority of children are still unable to walk independently. We developed a treatment for MMC that augments the standard of care, in utero MMC surgical repair, with placental mesenchymal stromal/stem cells (PMSCs). We found that treatment with PMSCs, during in utero repair, prevents hind limb paralysis in the well-established fetal ovine model of MMC, due to PMSC paracrine secretion of neuroprotective factors. However, we confirmed that PMSCs did not engraft long-term and treated lambs developed severe kyphosis causing spinal cord compression and tethering due to the lack of bone and adjacent paraspinal muscles, which is consistent with human MMC musculoskeletal deformities. To meet this need for a long-lasting therapy for MMC, we explored the use of bioengineered multifunctional combination scaffolds. Exosomes are extracellular vesicles that play significant roles in cell-to cell communication. We confirmed that exosomes secreted by PMSCs (PMSC-exosomes) exert significant neuroprotective functions, similar in magnitude to the live PMSCs from which they are derived. In this study, we propose to develop an engineered hydrogel system that will allow for both local and sustained release of PMSC-exosomes to the spinal cord, which in turn will provide sustained neuroprotection to treat MMC before birth. In addition, we aim to increase the longevity of the in utero treatment by covering the defect with a biomaterial-based bony scaffold to provide structural and functional support. We plan to optimize the neuroprotective and regenerative functions of the bioscaffold using our well-established fetal rodent and rabbit models of MMC. We will evaluate the final combination multifunctional bioscaffold product in our gold-standard fetal ovine model of MMC. This therapeutic will be cell-free, off-the-shelf, and easy-to-use. If successful, this novel approach will be used to treat MMC before birth, and due to its regenerative qualities, the treatment will improve the quality of life of these patients, as well as significantly lower the healthcare costs associated with the current treatment.