Postmortem Human Dura Mater Cells Exhibit Phenotypic, Transcriptomic and Genetic Abnormalities that Impact their Use for Disease Modeling

Abstract

Patient-derived cells hold great promise for precision medicine approaches in human health. Human dermal fibroblasts have been a major source of cells for reprogramming and differentiating into specific cell types for disease modeling. Postmortem human dura mater has been suggested as a primary source of fibroblasts for in vitro modeling of neurodegenerative diseases. Although fibroblast-like cells from human and mouse dura mater have been previously described, their utility for reprogramming and direct differentiation protocols has not been fully established. In this study, cells derived from postmortem dura mater are directly compared to those from dermal biopsies of living subjects. In two instances, we have isolated and compared dermal and dural cell lines from the same subject. Notably, striking differences were observed between cells of dermal and dural origin. Compared to dermal fibroblasts, postmortem dura mater-derived cells demonstrated different morphology, slower growth rates, and a higher rate of karyotype abnormality. Dura mater-derived cells also failed to express fibroblast protein markers. When dermal fibroblasts and dura mater-derived cells from the same subject were compared, they exhibited highly divergent gene expression profiles that suggest dura mater cells originated from a mixed mural lineage. Given their postmortem origin, somatic mutation signatures of dura mater-derived cells were assessed and suggest defective DNA damage repair. This study argues for rigorous karyotyping of postmortem derived cell lines and highlights limitations of postmortem human dura mater-derived cells for modeling normal biology or disease-associated pathobiology.

Keywords: Biobanking; Chromosomal karyotype; Dermal epithelium; Dermal fibroblasts; Dural cells; Human dura mater; Loss of Y chromosome; Mural cells; Neurodegenerative disease; Postmortem tissue.

Fig. 1

Timeline in days for the isolation, expansion, and banking of dermal cells from fresh dermis and dural cells from fresh and frozen dura mater. Cell outgrowth, proliferation, and banking from fresh and frozen dural tissue were delayed compared to dermal tissue. Successfully banked cell lines were karyotyped and tested for mycoplasma

Fig. 2

Dural cells displayed reduced cell outgrowth with larger cell body morphology than cultured dermal cells. A Dermal cells exhibited classical fibroblast morphology. In contrast, dural cells were enlarged, variable, and had disorganized alignment. Representative brightfield images are shown for each cell type and condition with a 5x objective B Cells were stained with rhodamine phalloidin (red) and DAPI (blue), reflecting differences in actin filament arrangement between dermal and dural cells. Representative immunofluorescent images are shown for each cell type and condition with a 10x objective, performed in triplicates

Fig. 3

Differences in cell proliferation were observed between cells derived from dermis and dura mater. A The total number of cells were counted for a span of six days for four dermal cell lines (black), four fresh dural cell lines (solid blue), and two frozen dural cell lines (dotted blue), performed in triplicates. B Cell proliferation rates for dermal and dural cell lines are shown. There was a significant decrease in cell proliferation in both frozen and fresh dural cell lines compared to the dermal cell lines (One-way ANOVA, * p < 0.05). C Proliferation assay with Click-it EdU (green) and DAPI (blue) for paired dermal (left) and dural (right) cell lines from the same subject, performed in quadruplicates. D Percentage of EdU positive cells of the total DAPI positive cells. There was a significant decrease in cell proliferation in the dural cell line compared to the paired dermal cell line (unpaired t-test, *** p < 0.0005)

Fig. 4

Protein expression differences in fibroblast markers were observed between dermal and dural cell lines. A Cell lysates from five dermal and six dural cell lines were immunoblotted against Vimentin and S100A4. Tissue indicates from which source the cells were derived from, storage indicates whether tissue underwent frozen or fresh storage conditions before processing, and paired indicates cell lines derived from the same subject (2 cases, lanes 1A/1B and 2A/2B). rVimentin and rS100A4 were recombinant proteins that were used as positive controls for their respective antibodies. B There was a significant decrease in vimentin protein levels in dural cell lines relative to the dermal cell lines (normalized to actin, unpaired t-test, * p < 0.05). C There was a significant decrease in S100A4 protein levels in dural cell lines relative to the dermal cell lines (normalized to actin, unpaired t-test, *** p < 0.0005). D Immunofluorescence for PDE3A (green, top panel), CD146 (green, bottom panel), and DAPI (blue) for paired dermal (left) and dural (right) cell lines from the same subject, performed in quadruplicates. E Quantification of mean PDE3A fluorescence intensity with a significant increase in the dural cell line compared to the paired dermal cell line (unpaired t-test, ** p < 0.005). F Quantification of mean CD146 fluorescence intensity in the paired dermal and dural cell lines

Fig. 5

Cells cultured from human postmortem dura mater adopt mural cell gene expression patterns. A Volcano plot of differentially expressed genes. Red dots denote differentially expressed genes, blue dots denote genes with expression differences of -Log₁₀P between −1 and 1, and the green dots denote non-significantly expressed genes. B GO Biological Processes significantly enriched amongst genes upregulated in dural cells. C Dural cells enriched cell types from the mouse Allen Brain Atlas. Top enriched pathways and cell types with a false discovery rate (FDR) <0.05 are shown (Benjamini-Hochberg correction). D UMAP feature plot mapping the combined expression of the top 50 genes upregulated in dural cells compared to vascular-enriched nuclei from human postmortem brains. E Example of 15 genes from (D), demonstrating human brain mural-specific expression of genes upregulated in dural cells

Fig. 6

Cells cultured from human postmortem dura mater exhibit enhanced numbers and a differential pattern of somatic mutations compared to dermal cells. A VAF distribution of discovered mutations from dermal and dural cell lines from the same subject. The red dashed line represents the median VAF and the black dashed line represents 50% VAF. B The frequency of each of the 6 pyrimidine SNVs and their complements called in each cell line. C The frequency of the pyrimidine SNVs that are separated into 96 possible combinations (4 starting nucleotides × 6 pyrimidine variants × 4 ending nucleotides). COSMIC SBS signatures are identified using these 96 different contexts. D COSMIC SBS signatures that are decomposed using contexts in (C). Bar graph shows how many mutation calls in each cell line that contribute to each of the decomposed COSMIC signatures