*4.7. Human*

X-ALD patient skin fibroblasts have, for several years, constituted one of the rare in vitro models of the disease. In 1980, Moser et al. demonstrated for the first time that the accumulation of VLCFAs observed in the brain and adrenals of patients is also present in primary fibroblasts, thus validating this model for X-ALD studies, at least at the biochemical level [138]. Since then, this cellular model has become a platform for a broad variety of analyses concerning lipid metabolism, X-ALD diagnosis, functional characterization of peroxisomal ABC transporters, cellular consequences of *ABCD1* deficiency, and screening of therapeutic compounds. Great scientific advances have emerged from this handy model, but its skin origin is a limitation in pathogenesis studies. Indeed, the gene regulation and function in skin fibroblasts are very far from those of neural, glial and microglial cells.

The involvement of peripheral blood mononuclear cells (PBMCs) in the inflammation feature of X-ALD was early suspected, since PBMCs from X-ALD patients produce higher levels of inflammatory cytokines than control ones [139,140]. Used in gene therapy, the CD34+ PBMCs (lymphoid and myeloid progenitors) transduced with normal *ABCD1* can efficiently correct the clinical phenotype of the X-ALD patients [82]. Moreover, AMN monocytes have a pro-inflammatory expression pattern and, after differentiation into macrophages, are not able to switch to an anti-inflammatory regenerative state [141]. *Abcd2*, whose expression level is extremely low in these cells, could be a therapeutic target [142]. Therefore, human monocytes can be used to study the inflammatory process and identify compounds capable of inducing *ABCD2* expression, correcting VLCFA level, β-oxidation, and inflammatory features [44,58].

The development of the iPSC (induced pluripotent stem cell) technology offers the opportunity to study disease-involved cells with a chosen mutation and a phenotype matching physiology. Several iPSC models have successfully been obtained from skin fibroblasts of cALD and AMN patients [143–147]. Gene expression profiling shows that X-ALD iPSCs have differentially expressed genes compared to control iPSCs, among which some are positively correlated to the severity of the disease (cALD versus AMN) [148]. When iPSCs are differentiated into oligodendrocytes or astrocytes, the VLCFA level is increased and is higher in cALD differentiated cells than in AMN cells, whereas no VLCFA accumulation is observed in neurons [144]. iPSC-derived astrocytes show pro-inflammatory features that also correlate with the severity of the phenotype. The differentiation of microglia from iPSC also seems to be a promising model, as differentiated microglia show the main phenotype of primary fetal and adult human microglia including phagocytic and inflammatory capacity [146]. In addition, cALD iPSCs differentiated in brain microvascular endothelial cells show impaired BBB function as well as lipid metabolism modifications and interferon activation [149], and could lead to the study of an important factor of brain pathogenesis in X-ALD. Altogether, these works show that iPSC-derived brain cells should allow the study of the pathogenesis of X-ALD in detail, permit the identification of biomarkers, and screen new therapeutic molecules. Co-culture experiments are expected to provide new insight into intercellular communication in the brain.

In conclusion, for forty years, enormous progress has been made in the knowledge of peroxisomal ABC transporters thanks to the development and the use of cell and animal models. If no model exactly mimics the human X-ALD, there is no doubt that the new technological developments will offer opportunities to progress in the study of the role of peroxisomal ABC transporters in the neuronal, glial, and microglial intercellular communications.
