The gene expression profiles of induced pluripotent stem cells from individuals with childhood cerebral adrenoleukodystrophy are consistent with proposed mechanisms of pathogenesis - PubMed (original) (raw)

The gene expression profiles of induced pluripotent stem cells from individuals with childhood cerebral adrenoleukodystrophy are consistent with proposed mechanisms of pathogenesis

Xiao-Ming Wang et al. Stem Cell Res Ther. 2012.

Abstract

Introduction: X-linked adrenoleukodystrophy (X-ALD) is a complex disorder with variable expressivity that affects the nervous, adrenocortical and male reproductive systems. Although ABCD1 mutations are known to provide the genetic basis for X-ALD, its pathogenesis is not fully elucidated. While elevated very long chain fatty acid (VLCFA) levels in blood and reduced VLCFA catabolic activity in cultured fibroblasts are biomarkers used to identify ABCD1 mutation carriers, the roles peroxisomal lipid metabolism play in disease etiology are unknown.

Methods: Primary skin fibroblasts from two male patients with the childhood cerebral form of the disease (CCALD) caused by ABCD1 frameshift or missense mutations and three healthy donors were transduced with retroviral vectors expressing the OCT4, SOX2, KLF4 and c-MYC factors. Candidate induced pluripotent stem cells (iPSCs) were subject to global gene expression, DNA methylation, DNA copy number variation, and genotyping analysis and tested for pluripotency through in vitro differentiation and teratoma formation. Saturated VLCFA (sVLCFA) and plasmalogen levels in primary fibroblasts and iPSCs from healthy donors as well as CCALD patients were determined through mass spectroscopy.

Results: Skin fibroblasts from CCALD patients and healthy donors were reprogrammed into validated iPSCs. Unlike fibroblasts, CCALD patient iPSCs show differentially expressed genes (DEGs) relevant to both peroxisome abundance and neuroinflammation. Also, in contrast to fibroblasts, iPSCs from patients showed no significant difference in sVLCFA levels relative to those from controls. In all cell types, the plasmalogen levels tested did not correlate with ABCD1 mutation status.

Conclusion: Normal ABCD1 gene function is not required for reprogramming skin fibroblasts into iPSCs or maintaining pluripotency. Relative to DEGs found in fibroblasts, DEGs uncovered in comparisons of CCALD patient and control iPSCs are more consistent with major hypotheses regarding disease pathogenesis. These DEGs were independent of differences in sVLCFA levels, which did not vary according to ABCD1 mutation status. The highlighted genes provide new leads for pathogenic mechanisms that can be explored in animal models and human tissue specimens. We suggest that these iPSC resources will have applications that include assisting efforts to identify genetic and environmental modifiers and screening for therapeutic interventions tailored towards affected cell populations and patient genotypes.

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Figures

Figure 1

Figure 1

Characterization of iPSCs from patient with CCALD. (A) Representative images of alkaline phosphatase (AP) staining and pluripotency protein biomarker immunostaining analysis of childhood cerebral adrenoleukodystrophy (CCALD) patient induced pluripotent stem cell (iPSCs) are presented. (B) Representative CCALD iPSCs were subject to in vitro differentiation (Materials and methods) and cell lineages derived from all three germ layers were identified by immunostaining, as depicted. Antibodies against Tuj1, αSMA and AFP were used to identify cells of ectodermal, mesodermal, and endodermal origin, respectively. (C) Histological analysis of teratomas derived from representative CCALD iPSC clones from two patients is provided. Evidence of cell lineages derived from all three germ layers is provided. C, Cartilage tissue; G, Glandular tissue; N, Neural rosettes; P, pigment neuroepithelium. Scale bar = 50 μm.

Figure 2

Figure 2

The gene expression and DNA methylation profiles of fibroblasts and iPSCs. (A) A dendrogram generated based on the unsupervised hierarchical clustering analysis of gene expression data from childhood cerebral adrenoleukodystrophy (CCALD) patient and control fibroblasts and induced pluripotent stem cells (iPSCs) is provided. The analysis was based on data from 672 probe sets producing log2-tranformed gene expression scores with coefficient of variation (CV) > 0.25 and conducted using average linkage and Euclidean distance; (B) A dendrogram generated based on the unsupervised hierarchical clustering analysis of DNA methylation data from CCALD patient and control fibroblasts and iPSCs is provided. Data from 7,493 DNA methylation assays interrogating autosomal CpG loci with CV > 0.5 and the fifth largest and smallest β-values being > 0.6 and < 0.4, respectively, were used in this analysis in order to represent the most variable loci. Clustering was performed using average linkage and the Pearson dissimilarity distance.

Figure 3

Figure 3

Comparison of saturated very long chain fatty acid (sVLCFA) and plasmalogen levels in patient and healthy control fibroblasts and iPSCs. (A) Relative sVLCFA levels in patient and healthy control fibroblasts grown in fibroblast media or iPSC media, as represented by the C26:0/C22:0 LPC (lysophosphatidylcholine) ratio; (B) Relative sVLCFA levels in patient and healthy control iPSC clones grown in induced pluripotent stem cell (iPSC) media, again represented by the C26:0/C22:0 LPC ratio; (C) Relative plasmalogen level in patient and healthy control fibroblasts grown in fibroblast media and iPSC media, as represented by the total phosphatidyl ethanolamine (PE) plasmalogen/C22:0 lysophosphatidylcholine (LPC) ratio; (D) Relative plasmalogen in patient and healthy control iPSC clones grown in iPSC media, again represented by the total PE plasmalogen/C22:0 LPC ratio.

Figure 4

Figure 4

qRT-PCR confirmation of PEX11B gene expression in patient and healthy control iPSCs. Data from the analysis of PEX11B gene expression levels are provided. The error bars represent one standard deviation. The expression was lower in patients relative to control cells (two-tailed Student's _t_-test _P_-value < 0.5).

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