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中起着关键作用。 内皮细胞和壁细胞构成了血管的主要结构和功能元件。 脉管系统位于内皮细胞外的壁细胞对内皮细胞的发育至关重要。 血脑屏障和调节血流响应神经元活动。但大多数 脑血管的研究使用成年小鼠，因此对发育中的 血管或人脑血管的任何年龄。这些知识差距优先影响 早产儿，谁是倾向于发展脑血管疾病，特别是位于大脑区域称为 神经节隆起（GE）。这些脑损伤会带来永久性脑损伤的高风险， 我们没有治疗方法 从分子水平确定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的不同亚型的出现。 引人注目的是，这一策略还发现了一种新的祖细胞群体， 神经干细胞标志物，表明一个共同的神经血管祖细胞，有助于两者 血管生成和神经发生。在GE中，免疫组化显示了假定的神经血管 祖细胞标记显示这些细胞位于脑室周围和血管周围， 典型的神经干细胞。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。综上所述，上述实验使我 假设壁细胞有助于血管生成和神经发生， 发育和未成熟阶段的壁细胞在GE中占主导地位 有助于出血的发病机制。K08的短期目标 如下：1.构建发育过程中人脑壁细胞图谱， scRNAseq和死后人体组织。2.开发类器官、小鼠移植和CRISPR 工具来操纵和询问的作用，具体的信号通路的壁细胞在人类 大脑发育这些结果将推动我到R01。作为一名新生儿医学科学家， 围产期医学，我是唯一适合推进我们对脑血管的理解 开发和应用这些知识对未来的治疗。

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.