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

Abstract

IntroductionX-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.MethodsPrimary 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.ResultsSkin 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.ConclusionNormal 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.

Role of ALKBH1 in the Core Transcriptional Network of Embryonic Stem Cells

“…Notably, stem cell differentiation, epigenetic regulation and stem cell maintenance were identified as targets of ALKBH1 in ESCs. Interestingly, previously published iPSC datasets identified an increased ALKBH1 expression upon reprogramming of somatic cells into iPSCs [34][35][36][37]. This effect was seen regardless of somatic cell type used for reprogramming.…”

Transdifferentiation of human endothelial progenitors into smooth muscle cells

“…Finally, we performed a principal component analysis on the entire gene expression signature of our control and experimental samples (EPC, UASMC, iSMC) as well as control samples from previously published studies: Skeletal Muscle 24,25 , Brain 26 , Liver 26 , Umbilical Vein and Coronary Artery Endothelial Cells 27 , Aortic Smooth Muscle Cells 28,29 , Dermal Fibroblasts 30 . This analysis confirmed that the transdifferentiated iSMC closely resemble the control cells including adult aorta SMC and umbilical artery SMC while at the same time being significantly different from the starting EPC population (Figure 3G).…”

“…Gray circles mark genes predicted to belong in the particular genetic network but found not to be significantly upregulated in iSMC as compared to EPC control cells. (G) Principal component analysis performed on the normalized signal values for each of the chip probes as well as probes from previously published studies: Skeletal Muscle 24,25 , Brain 26 , Liver 26 , Umbilical Vein and Coronary Artery Endothelial Cells 27 , Aortic Smooth Muscle Cells 28,29 , Dermal Fibroblasts 30 .…”

X-linked Adrenoleukodystrophy

“…Head trauma may be considered as such an environment factor capable to initiate the cerebral form of X-ALD [285]. The recent development of X-ALDinduced pluripotent stem cells differentiated into oligodendrocytes or neurons would provide very informative tools [286,287]. The Abcd1 null mouse model does not develop cerebral demyelination and does not permit us to understand the relationships between VLCFA accumulation, demyelination, inflammation, oxidative stress, and cell death in brain.…”