Abstract TMP9: Novel Single-cell Isolation From Cerebral Cavernous Angioma Specimens For Transcriptomic Studies

Abstract

Introduction: Cavernous angiomas (CAs) are common neurovascular lesions predisposing patients to seizures and hemorrhagic stroke. Recent evidence shows non-endothelial cell autonomous effects contribute to CA pathogenesis within the dysfunctional neurovascular units (NVUs), consisting of endothelial cells, pericytes, astrocytes, and microglia. Herein, we developed a single-cell isolation protocol to study the transcriptome of each of these cell populations. Method: CAs and control brain tissue were frozen in optimal cutting temperature compound immediately after surgical resection. Tissue was enzymatically digested with Collagenase Type IV and DNase I. The cell suspension was then filtered, washed, and pelleted. Cell debris and myelin were removed using 25% Percoll. A multispectral LED light was used for 30 minutes to reduce background autofluorescence. Cells were stained with a CD31, CD45, CD13, P2RY12, CD49f, GLAST, CD24 and CD90 antibody cocktail. Size, granularity, and antibody-specific gating were set to sort each cell population using the BD FACSymphony S6 Cell Sorter. After isolation, RNA was extracted for each cell population, and the quality was assessed. Cell-specific genes including VWF (endothelial cells), ACTA2 (pericytes ), AQP4 (astrocytes ) and IBA-1 (microglia ), as well as GAPDH were assessed with real-time qPCR (rt-qPCR) for validation. Results: Endothelial cells (CD31 + , CD13 - , CD45 - , CD49f - , GLAST - ), pericytes (CD13 + , CD31 - , CD45 - , CD49f - , GLAST - ), astrocytes (CD49f + , GLAST + ) and microglia (P2RY12 + , CD45 + , CD31 - , CD13 - , CD49f - , GLAST - ) were individually isolated from both 5 CA lesions and 5 control brain tissue using FACS sorting. RNA quantity ranged from 31 to 83 pg/μl, with RNA Integrity Number ranging from 1 to 8.7. Each cell population within the CA lesion was validated by showing differential expression of cell-specific genes. Conclusion: We have shown, for the first-time, feasibility of individual cell type isolation from frozen, surgically excised CA lesions. The RNA quantity and quality of each cell population was suitable for the establishment of cell-specific transcriptome libraries. These will help clarify individual cell type contributions to CA pathogenesis.