Creation of new tools to study human microglia using blood cells

项目摘要/摘要 小胶质细胞是大脑的常驻免疫细胞，对正常的大脑发育和功能至关重要，可能与抑郁症和精神分裂症等精神疾病有关。由于几乎不可能获得合适的人脑标本，我们在研究这些疾病中的小胶质细胞功能方面受到了严重限制，特别是在体内。这项由本·巴雷斯博士指导的斯坦福大学精神病学和神经生物学系临床医生兼科学家颁发的指导职业发展奖，将通过相关的基础和翻译研究目标，使精神疾病中人类小胶质细胞的研究成为现实。基本的研究目标是通过比较小胶质细胞和疾病期间进入大脑的血液来源的“小胶质细胞样细胞”(MLCs)的转录本来推进我们对小胶质细胞生物学的理解。翻译的目标是利用这些结果从血细胞中产生人类小胶质细胞，从而能够在没有人脑组织的情况下进行小胶质细胞的研究。这份提案概述了一项为期5年的有指导的职业发展计划，该计划利用斯坦福大学丰富的教育和科学资源来实现下文所述的研究目标，同时为应聘者提供作为独立调查员所需的培训。 初步数据显示，当由于CSF1受体(CSF1R)敲除而缺乏小胶质细胞的小鼠被移植到野生型(WT)骨髓(BM)时，供者来源的细胞以与WT小胶质细胞几乎相同的模式填充大脑，并表达几乎所有小胶质细胞命运的特异性标记。通过表达高度特异的小鼠和人小胶质细胞标记物Tem119，可以纯化这些植入的MCs。RNAseq数据显示，MLCs与WT小胶质细胞高度相似。这项拟议的研究将扩展这些有趣的发现，以更好地了解骨髓间充质干细胞和小胶质细胞之间的差异，并为体内研究骨髓来源的骨髓间充质干细胞创造一个可靠的系统。目标1将确定MLCs与WT小胶质细胞的相似程度，以及它们在哪些方面不同。目标2将鉴定能够产生骨髓间充质干细胞的特定造血细胞类型。AIM 3将翻译这些发现用于人类血细胞，为高纯度的静息人类小胶质细胞生成RNAseq图谱，并将它们与将人骨髓移植到免疫耐受CSF1R基因敲除小鼠所产生的MLCs进行比较。这项工作将促进我们对小胶质细胞和MLCs之间的核心差异的理解，鉴定纯人小胶质细胞的转录特征，并创建一个易于处理的系统来使用患者来源的造血细胞来研究小胶质细胞，同时培养以小胶质细胞在心理健康中的作用为中心的独立研究事业。

PROJECT SUMMARY/ABSTRACT Microglia, the brain’s resident immune cells, are essential for normal brain development and function, and likely involved in mental illnesses such as depression and schizophrenia. Because it is nearly impossible to obtain suitable human brain specimens, we are severely restricted from studying microglial function in these diseases, especially in vivo. This Mentored Career Development Award, by a clinician-scientist in the Departments of Psychiatry and Neurobiology at Stanford University, mentored by Dr. Ben Barres, will make the study of human microglia in mental illness a reality through related basic and translational research goals. The basic research goal is to advance our understanding of microglial biology by comparing the transcriptomes of microglia and blood-derived “Microglia Like Cells” (MLCs) that enter the brain during disease. The translational goal is to use these results to generate human microglia from blood cells, allowing study of microglia without human brain tissue. This proposal outlines a 5 year mentored career development plan that harnesses the abundant educational and scientific resources at Stanford to accomplish the research aims described below while providing the candidate with needed training to function as an independent investigator. Preliminary data show that when mice lacking microglia due to knock-out of the CSF1 receptor (CSF1R) are transplanted with wild type (WT) bone marrow (BM), donor-derived cells fill the brain in a near-identical pattern to WT microglia, and express nearly all specific markers of microglial fate. These engrafted MLCs can be purified based on expression of Tmem119, a highly specific mouse and human microglial marker. RNAseq data shows that MLCs are highly similar to WT microglia. The proposed research will expand on these intriguing findings to better understand differences between MLCs and microglia, and create a reliable system for studying bone marrow-derived MLCs in vivo. Aim 1 will determine how closely MLCs resemble WT microglia, and in what ways they are different. Aim 2 will identify the specific hematopoietic cell types capable of generating MLCs. Aim 3 will translate these findings for use with human blood cells, by generating RNAseq profiles for highly pure resting human microglia, and comparing them to MLCs generated by transplantation of human BM into immune tolerant CSF1R knockout mice. This work will advance our understanding of core differences between microglia and MLCs, identify the transcriptomic signature of pure human microglia, and create a tractable system to study microglia using patient-derived hematopoietic cells, while fostering an independent research career centered around the role of microglia in mental health.