Whole-Cell Dissociated Suspension Analysis in Human Brain Neurodegenerative Disease: A Pilot Study

Biochemical analysis of human brain tissue is typically done by homogenizing whole pieces of brain and separately characterizing the proteins, RNA, DNA, and other macromolecules within. While this has been sufficient to identify substantial changes, there is little ability to identify small changes or alterations that may occur in subsets of cells. To effectively investigate the biochemistry of disease in the brain, with its different cell types, we must first separate the cells and study them as phenotypically defined populations or even as individuals. In this project, we developed a new method for the generation of whole-cell-dissociated-suspensions (WCDS) in fresh human brain tissue that could be shared as a resource with scientists to study single human cells or populations. Characterization of WCDS was done in paraffin-embedded sections stained with H&E, and by phenotyping with antibodies using immunohistochemistry and fluorescence-activated cell sorting (FACS). Additionally, we compared extracted RNA from WCDS with RNA from adjacent intact cortical tissue, using RT-qPCR for cell-type-specific RNA for the same markers as well as whole transcriptome sequencing. More than 11,626 gene transcripts were successfully sequenced and classified using an external database either as being mainly expressed in neurons, astrocytes, microglia, oligodendrocytes, endothelial cells, or mixed (in two or more cell types). This demonstrates that we are currently capable of producing WCDS with a full representation of different brain cell types combined with RNA quality suitable for use in biochemical analysis.

[1]  Melanie A. Huntley,et al.  Alzheimer’s Patient Microglia Exhibit Enhanced Aging and Unique Transcriptional Activation , 2020, Cell reports.

[2]  I. Cobos,et al.  Single-soma transcriptomics of tangle-bearing neurons in Alzheimer’s disease reveals the signatures of tau-associated synaptic dysfunction , 2020, bioRxiv.

[3]  A. Myers,et al.  ESHRD: deconvolution of brain homogenate RNA expression data to identify cell-type-specific alterations in Alzheimer’s disease , 2020, Aging.

[4]  D. Holtzman,et al.  Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and TREM2-independent cellular responses in Alzheimer’s disease , 2020, Nature Medicine.

[5]  Kieran R. Campbell,et al.  Dissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses , 2019, Genome Biology.

[6]  Manolis Kellis,et al.  Single-cell transcriptomic analysis of Alzheimer’s disease , 2019, Nature.

[7]  Trygve E Bakken,et al.  Single-nucleus and single-cell transcriptomes compared in matched cortical cell types , 2018, PloS one.

[8]  S. Linnarsson,et al.  A comparative strategy for single-nucleus and single-cell transcriptomes confirms accuracy in predicted cell-type expression from nuclear RNA , 2017, Scientific Reports.

[9]  Salah Ayoub,et al.  Cell fixation and preservation for droplet-based single-cell transcriptomics , 2017, BMC Biology.

[10]  M. Ronaghi,et al.  Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain , 2016, Science.

[11]  Zvi Ram,et al.  A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells , 2016, BMC Neuroscience.

[12]  E. Chang,et al.  Purification and Characterization of Progenitor and Mature Human Astrocytes Reveals Transcriptional and Functional Differences with Mouse , 2016, Neuron.

[13]  H. Shill,et al.  Arizona Study of Aging and Neurodegenerative Disorders and Brain and Body Donation Program , 2015, Neuropathology : official journal of the Japanese Society of Neuropathology.

[14]  S. Quake,et al.  A survey of human brain transcriptome diversity at the single cell level , 2015, Proceedings of the National Academy of Sciences.

[15]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[16]  K. Becker,et al.  FACS purification of immunolabeled cell types from adult rat brain , 2012, Journal of Neuroscience Methods.

[17]  D. Connor,et al.  The Sun Health Research Institute Brain Donation Program: Description and Eexperience, 1987–2007 , 2007, Cell and Tissue Banking.

[18]  Jun Fujita,et al.  Invited review: Effects of heat and cold stress on mammalian gene expression. , 2002, Journal of applied physiology.

[19]  J. Fujita.  Cold shock response in mammalian cells. , 1999, Journal of molecular microbiology and biotechnology.

[20]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .