Mural stem cells in normal development and germinal matrix hemorrhage in the preterm human brain

项目摘要/总结 脑血管细胞在生理和病理状态下都发挥着关键作用。 内皮细胞和壁细胞构成了人体的主要结构和功能元素 脉管系统。壁细胞位于内皮细胞之外，对于细胞的发育至关重要 血脑屏障并根据神经元活动调节血流。然而，大多数 脑血管研究使用成年小鼠，因此对发育中的脑血管知之甚少 任何年龄的脉管系统或人脑血管。这些知识差距优先影响 早产儿，他们容易出现出血，特别是位于称为 神经节隆起（GE）。这些出血会带来永久性脑损伤的高风险， 我们没有治疗方法。 从分子水平上定义发育中的人类壁细胞的阶段和亚型 大脑，我开发了一种荧光激活细胞分选（FACS）策略，然后是单细胞 RNA 测序 (scRNAseq)。这些数据揭示了壁细胞发育的不同阶段 从 15GW 的相对不成熟阶段到 23GW 不同亚型的出现。 引人注目的是，这一策略还发现了一种新的祖先群体，它既表达壁画又表达 神经干细胞标记物，表明有一个共同的神经血管祖细胞对两者都有贡献 血管生成和神经发生。在 GE 中，免疫组织化学与假定的神经血管 祖细胞标记显示这些细胞位于心室周围和血管周围位置 典型的神经干细胞之一。与这些结果一致，FACS 纯化的壁细胞 在体外产生血管和神经谱系细胞。总之，上述实验引导我 假设壁细胞有助于血管生成和神经发生 GE 中壁细胞的未成熟阶段占主导地位 有助于出血的发病机制。我对拟议的 K08 的短期目标 具体如下： 1. 使用以下方法构建开发过程中的人脑壁细胞图谱： scRNAseq 和死后人体组织。 2. 开发类器官、小鼠移植和 CRISPR 操纵和询问人体壁细胞特定信号通路作用的工具 大脑发育。这些结果将推动我达到 R01。作为一名新生儿医学科学家 围产期医学，我非常适合增进我们对脑血管的理解 这些知识的发展和应用在未来的治疗中。

PROJECT ABSTRACT/SUMMARY Brain blood vessel cells play key roles in both physiological and pathological states. Endothelial and mural cells compose the main structural and functional elements of the vasculature. Mural cells, which lie outside of endothelial cells, are critical for the development of the blood brain barrier and regulate blood flow in response to neuronal activity. However, most studies of brain blood vessels use adult mice and therefore little is known about the developing vasculature or human brain blood vessels at any age. These knowledge gaps preferentially affect preterm infants, who are prone to develop hemorrhages specifically located in brain regions called the ganglionic eminences (GE). These hemorrhages confer high risk for permanent brain injury, and we have no treatments. To molecularly define the stages and subtypes of mural cells in the developing human brain, I developed a Fluorescence Activated Cell Sorting (FACS) strategy followed by single cell RNA sequencing (scRNAseq). These data revealed distinct stages of mural cell development from a relatively immature stage at 15GW to the emergence of distinct subtypes at 23 GW. Strikingly, this strategy also uncovered a novel progenitor population that express both mural and neural stem cell markers, suggesting a common neurovascular progenitor that contributes to both angiogenesis and neurogenesis. In the GE, immunohistochemistry with putative neurovascular progenitor markers revealed these cells in a periventricular and perivascular location situated amongst canonical neural stem cells. Consistent with these results, FACS-purified mural cells produce both vascular and neural lineage cells in vitro. In sum, the above experiments lead me to hypothesize that mural cells contribute to both angiogenesis and neurogenesis during development and that a predominance of immature stages of mural cells in the GE contributes to the pathogenesis of hemorrhage. My short-term goals for the proposed K08 are the following: 1. Construct an atlas of human brain mural cells during development using scRNAseq and postmortem human tissue. 2. Develop organoid, mouse transplant, and CRISPR tools to manipulate and interrogate the role of specific signaling pathways of mural cells in human brain development. These results will propel me to an R01. As a physician scientist in Neonatal- Perinatal Medicine, I am uniquely suited to advance our understanding of brain vascular development and the application of this knowledge towards future therapy.