VINE-seq [1] (Vessel Isolation and Nuclei Extraction for Sequencing) is a method to isolate and molecularly characterize the vascular and perivascular cells of the human brain microvessels at single-nuclei resolution. This technique is achieved by combining various known laboratory-based strategies involving the mechanical dissociation of brain tissue samples into single cells, density gradient centrifugation and filtration to isolate nuclei of microvessels, fluorescence-activated cell sorting (FACs) of cellular populations and droplet-based single-nuclei RNA sequencing (drop-snRNA-seq). Altogether, this generates a single-nuclei transcriptomic profile of the various cell types present in the vasculature of the brain. Through processing and analyzing the single-nuclei transcriptomic data, the heterogeneity within and between cell types can be distinguished to construct the molecular landscape of the human brain vasculature that was not previously done before. [1]
A central feature of brain health is the maintenance of the complex vasculature that functions to transport essential metabolites, oxygen, nutrients and wastes to and from the human brain. [2] Disruption of the vasculature can lead to neurological disorders such as Alzheimer's disease and stroke. [3] The vasculature is home to a heterogeneous group of cell types including but not limited to the endothelial cells, pericytes, astrocytes, and leukocytes. [4] Although there have been many attempts to understand the cellular diversity in the vasculature over the years, methodological and technological limitations have hindered our ability to decode the complex entity of the brain. [5] Therefore, our treatment options for neurological disorders have been inadequate due to our incomplete characterization of the vascular and perivascular cells in this complex environment. [6] Through recent advances in single-cell RNA sequencing (scRNA-seq), the molecular atlas of the brain vasculature in mice has been decoded and the heterogeneous nature within the cell types found in the human brain have been slowly uncovered. [1] [6] [7] Despite these achievements, murine brain vasculature may not fully recapitulate the human brain vasculature leading to concerns of translational potential. [1] [8] Additionally, many previous studies focusing on scRNA-seq of the human brain did not characterize the cellular heterogeneity in the vasculature. [1] By introducing a novel method called VINE-seq, a research group derived a method to isolate intact nuclei from the microvessels of the brain. To accomplish this, they had to overcome the challenge of removing the extracellular matrix (ECM) that surrounds the microvessels without damaging the nuclei. By using VINE-seq on frozen brain tissues from the hippocampus and cortex, they provided the first characterization of the various cell types in the human brain vasculature at single-nuclei resolution. [1]
VINE-Seq is a novel method to detail the vasculature of the human brain. It uses a combination of laboratory methods with single-nucleotide transcriptomics in order to compose an atlas of the vascular and perivascular cell types within the brain. The following steps detail the basis of the VINE-Seq protocol:
Through their experimentation and transcriptomic analysis, the authors and creators of VINE-Seq discovered that the series of methods were able to elucidate the transcriptomic expression of the vascular endothelial and smooth muscle cells present in the arteriovenous organization in healthy human brain tissue. Additionally, VINE-seq was able to capture two novel subpopulations of pericytes called matrix pericytes and transport pericytes that may be involved in ECM modulation and transmembrane transportation, respectively. [1]
Using VINE-seq, these researchers were able to help uncover the molecular basis of the human brain vasculature in Alzheimer's disease (AD). They showed that AD-associated genes were highly expressed in the vascular cell types and that there are gene expression alterations or loss of abundance of endothelial cells, smooth muscle cells and perivascular fibroblasts cells in AD brain samples compared to normal brain samples. [1]
VINE-Seq was originally designed to map out the vasculature of the human brain, as no molecular map currently exists. Ever since the pre-print of the paper introducing VINE-seq was available in 2021, the molecular map has been recognized for its contribution in expanding our knowledge of the brain vasculature which will allow for more in-depth studies of brain diseases and potentially overcoming the challenge of drug delivery to the brain. [9] Some applications of the data generated by VINE-seq have contributed to furthering our knowledge in the pathogenesis of AD, dementia, ischemic stroke, and the effects of severe COVID-19 on the brain. [10] [11] [12] [13]
VINE-seq [1] (Vessel Isolation and Nuclei Extraction for Sequencing) is a method to isolate and molecularly characterize the vascular and perivascular cells of the human brain microvessels at single-nuclei resolution. This technique is achieved by combining various known laboratory-based strategies involving the mechanical dissociation of brain tissue samples into single cells, density gradient centrifugation and filtration to isolate nuclei of microvessels, fluorescence-activated cell sorting (FACs) of cellular populations and droplet-based single-nuclei RNA sequencing (drop-snRNA-seq). Altogether, this generates a single-nuclei transcriptomic profile of the various cell types present in the vasculature of the brain. Through processing and analyzing the single-nuclei transcriptomic data, the heterogeneity within and between cell types can be distinguished to construct the molecular landscape of the human brain vasculature that was not previously done before. [1]
A central feature of brain health is the maintenance of the complex vasculature that functions to transport essential metabolites, oxygen, nutrients and wastes to and from the human brain. [2] Disruption of the vasculature can lead to neurological disorders such as Alzheimer's disease and stroke. [3] The vasculature is home to a heterogeneous group of cell types including but not limited to the endothelial cells, pericytes, astrocytes, and leukocytes. [4] Although there have been many attempts to understand the cellular diversity in the vasculature over the years, methodological and technological limitations have hindered our ability to decode the complex entity of the brain. [5] Therefore, our treatment options for neurological disorders have been inadequate due to our incomplete characterization of the vascular and perivascular cells in this complex environment. [6] Through recent advances in single-cell RNA sequencing (scRNA-seq), the molecular atlas of the brain vasculature in mice has been decoded and the heterogeneous nature within the cell types found in the human brain have been slowly uncovered. [1] [6] [7] Despite these achievements, murine brain vasculature may not fully recapitulate the human brain vasculature leading to concerns of translational potential. [1] [8] Additionally, many previous studies focusing on scRNA-seq of the human brain did not characterize the cellular heterogeneity in the vasculature. [1] By introducing a novel method called VINE-seq, a research group derived a method to isolate intact nuclei from the microvessels of the brain. To accomplish this, they had to overcome the challenge of removing the extracellular matrix (ECM) that surrounds the microvessels without damaging the nuclei. By using VINE-seq on frozen brain tissues from the hippocampus and cortex, they provided the first characterization of the various cell types in the human brain vasculature at single-nuclei resolution. [1]
VINE-Seq is a novel method to detail the vasculature of the human brain. It uses a combination of laboratory methods with single-nucleotide transcriptomics in order to compose an atlas of the vascular and perivascular cell types within the brain. The following steps detail the basis of the VINE-Seq protocol:
Through their experimentation and transcriptomic analysis, the authors and creators of VINE-Seq discovered that the series of methods were able to elucidate the transcriptomic expression of the vascular endothelial and smooth muscle cells present in the arteriovenous organization in healthy human brain tissue. Additionally, VINE-seq was able to capture two novel subpopulations of pericytes called matrix pericytes and transport pericytes that may be involved in ECM modulation and transmembrane transportation, respectively. [1]
Using VINE-seq, these researchers were able to help uncover the molecular basis of the human brain vasculature in Alzheimer's disease (AD). They showed that AD-associated genes were highly expressed in the vascular cell types and that there are gene expression alterations or loss of abundance of endothelial cells, smooth muscle cells and perivascular fibroblasts cells in AD brain samples compared to normal brain samples. [1]
VINE-Seq was originally designed to map out the vasculature of the human brain, as no molecular map currently exists. Ever since the pre-print of the paper introducing VINE-seq was available in 2021, the molecular map has been recognized for its contribution in expanding our knowledge of the brain vasculature which will allow for more in-depth studies of brain diseases and potentially overcoming the challenge of drug delivery to the brain. [9] Some applications of the data generated by VINE-seq have contributed to furthering our knowledge in the pathogenesis of AD, dementia, ischemic stroke, and the effects of severe COVID-19 on the brain. [10] [11] [12] [13]