Use of Postmortem Human Dura Mater and Scalp for Deriving Human Fibroblast Cultures

Abstract

Fibroblasts can be collected from deceased individuals, grown in culture, reprogrammed into induced pluripotent stem cells (iPSCs), and then differentiated into a multitude of cell types, including neurons. Past studies have generated iPSCs from somatic cell biopsies from either animal or human subjects. Previously, fibroblasts have only been successfully cultured from postmortem human skin in two studies. Here we present data on fibroblast cell cultures generated from 146 scalp and/or 53 dura mater samples from 146 postmortem human brain donors. In our overall sample, the odds of successful dural culture was almost two-fold compared with scalp (OR = 1.95, 95% CI: [1.01, 3.9], p = 0.047). Using a paired design within subjects for whom both tissues were available for culture (n = 53), the odds of success for culture in dura was 16-fold as compared to scalp (OR = 16.0, 95% CI: [2.1–120.6], p = 0.0007). Unattended death, tissue donation source, longer postmortem interval (PMI), and higher body mass index (BMI) were associated with unsuccessful culture in scalp (all p<0.05), but not in dura. While scalp cells proliferated more and grew more rapidly than dura cells [F (1, 46) = 12.94, p<0.008], both tissues could be generated and maintained as fibroblast cell lines. Using a random sample of four cases, we found that both postmortem scalp and dura could be successfully reprogrammed into iPSC lines. Our study demonstrates that postmortem dura mater, and to a lesser extent, scalp, are viable sources of living fibroblasts for culture that can be used to generate iPSCs. These tissues may be accessible through existing brain tissue collections, which is critical for studying disorders such as neuropsychiatric diseases.

Strong Components of Epigenetic Memory in Cultured Human Fibroblasts Related to Site of Origin and Donor Age

“…The genes with differences in expression and DNAm levels by sampling location were previously implicated in processes relating to cell proliferation and apoptosis, which likely relate to the function of the fibroblasts in the primary tissue. One might have predicted this outcome, as fibroblasts in the scalp, including those that are cultured, turnover much more rapidly than those in the dura mater [ 15 ], which we confirmed here with increased FSP-1 expression in the scalp-derived fibroblasts.…”

“…Differential expression analysis of the RNA-seq data, independent of the results from the epigenetic analyses above, identified many genes that differed by the source of the primary fibroblast– 5,830 genes at FDR < 5%. Both scalp- and dura-derived fibroblasts expressed high levels of Fibroblast Specific Protein-1 (FSP-1) and this gene was more highly expressed scalp-derived fibroblasts (fold change = 5.5, FDR = 5.6x10 -6 ) in line with increased higher proliferation rates in the scalp-derived versus dura-derived fibroblasts [ 15 ]. The differentially expressed genes were strongly enriched for signaling and cell communication, cell proliferation, apoptotic processes, and epithelium development and morphogenesis via gene ontology (GO) analysis (all p < 10 −8 , S4 Table )–these gene sets were similar, and much more significant, to those identified comparing gene expression profiles across positional-identity genes in dermal fibroblasts [ 17 ].…”

“…We compared these cultured fibroblast lines to those derived from scalp samples from the same individuals, and found that the rate of culture success was higher for dura-derived fibroblasts; in some cases only the dura fibroblasts from an individual would culture. While the resulting cultured cells from these two sampling locations were largely morphologically indistinguishable (see Figure 1 in Bliss et al, 2012 [ 15 ]), we hypothesized that increased culture success might have a strong epigenetic component. Previous research has shown that dermal fibroblasts from different locations in the body have distinct gene expression profiles [ 16 ], including compared to some non-dermal sources [ 17 ], and previous reports have indicated that cultured cells have largely stable epigenomes, with the exception of a small number of loci [ 18 ].…”

“…In our previous work, we successfully cultured fibroblast lines from the dura mater of postmortem human donors, a source location largely protected from environmental insult with slowly dividing cells [ 15 ]. We compared these cultured fibroblast lines to those derived from scalp samples from the same individuals, and found that the rate of culture success was higher for dura-derived fibroblasts; in some cases only the dura fibroblasts from an individual would culture.…”

Concise Review: Modeling Central Nervous System Diseases Using Induced Pluripotent Stem Cells

“…Another advantage is the ability to derive iPSCs from different cellular sources. iPSCs can be reprogrammed from patientderived cell sources, including blood [15][16][17][18], skin fibroblasts [19,20], hair follicles [21], dental tissue [22], urine [23], and even postmortem tissue [24][25][26]. Another unique consideration is that patient cellular sources can be collected across a lifetime to examine the effects of the environment and age on the differentiation ability over time.…”