Functional Characterization of IPSC-Derived Brain Cells as a Model for X-Linked Adrenoleukodystrophy

Abstract

X-ALD is an inherited neurodegenerative disorder where mutations in the ABCD1 gene result in clinically diverse phenotypes: the fatal disorder of cerebral childhood ALD (cALD) or a milder disorder of adrenomyeloneuropathy (AMN). The various models used to study the pathobiology of X-ALD disease lack the appropriate presentation for different phenotypes of cALD vs AMN. This study demonstrates that induced pluripotent stem cells (IPSC) derived brain cells astrocytes (Ast), neurons and oligodendrocytes (OLs) express morphological and functional activities of the respective brain cell types. The excessive accumulation of saturated VLCFA, a "hallmark" of X-ALD, was observed in both AMN OLs and cALD OLs with higher levels observed in cALD OLs than AMN OLs. The levels of ELOVL1 (ELOVL Fatty Acid Elongase 1) mRNA parallel the VLCFA load in AMN and cALD OLs. Furthermore, cALD Ast expressed higher levels of proinflammatory cytokines than AMN Ast and control Ast with or without stimulation with lipopolysaccharide. These results document that IPSC-derived Ast and OLs from cALD and AMN fibroblasts mimic the respective biochemical disease phenotypes and thus provide an ideal platform to investigate the mechanism of VLCFA load in cALD OLs and VLCFA-induced inflammatory disease mechanisms of cALD Ast and thus for testing of new therapeutics for AMN and cALD disease of X-ALD.

Fig 1. Morphological and specific marker characterization of fibroblast-derived IPSC.

(A) Fibroblasts from a male healthy or patient with AMN or cALD disease were transduced with retroviral vectors expressing reprogramming factors OCT4, SOX2, NANOG, LIN28, KLF4, and c-MYC as described under methods. IPSC colony before isolation. (B) A putative control IPSC line was isolated and expanded under feeder-free maintenance medium for human IPSC, colonies growth was observed for 5 days by phase contrast image. (C-D) Control, AMN or cALD IPSC expressed the SOX2 and SSEA4 markers of pluripotency. (E) Summary chart depicts the markers for IPSC lines that were characterized. Scale bars represent 200 μm.

Fig 2. Characterization of pluripotency in the Control, AMN and cALD IPSC.

Representative images from different cell types (Control, AMN and cALD) for the 3 embryonic germ layers in vitro. Control, AMN and cALD IPSC lines generated cell types of all three embryonic germ layers (endoderm, AFP; mesoderm, α-SMA; ectoderm, Nestin), as embryoid bodies as decribed under method section (scale bars, 100 μm).

Fig 3. IPSC-derived neural precursor cells differentiation and characterization.

(A) Protocol for direct differentiation of human stem cell lines (IPSC) into neural precursor cells. After EBs formation from day 4–9, cells were differentiated as embryoid bodies from day 9–16 in neural induction media where neural rosette structure was selected and plated and second passage cells were analyzed. (B-D) Representative immunostaining results for NPC cultures from different IPSC (Control, AMN and cALD) shows SOX9⁺, PAX6⁺ and Nestin⁺ NPC cells (scale bars, 200 μm). (E) Summary chart of observed positive and negative markers for NPC characterization.

Fig 4. IPSC-derived brain cells differentiation and characterization.

(A) Protocols for differentiation of IPSC-derived NPC into different brain cell types (neurons, Ast and OLs). (B) Morphology of different brain cell types as phase contrast images at the end of differentiation protocol, (scale bars, 100 μm). (C) Representative positive (as differentiation efficiency markers) and negative (as cell culture purity markers) immunostaining for respective markers for Neurons (NeuN and β-Tubulin as positive staining), OLs (MBP as positive staining) and Ast (GFAP as positive staining) from control cells. (D-E) Representative positive immunostaining for AMN and cALD cells for neuron markers (β-Tubulin), for Ast (GFAP) and for OLs markers (MBP).

Fig 5. Biochemical characterization of IPSC-derived Ast, neurons and OLs.

(A) Quantification of the OLs marker: galactocerebrosides purified from OLs, neurons and Ast by densitometric scanning in Control, AMN and cALD cells. (B) Quantification of the fluorescence (n = 2) related to calcium influx stimulation by glutamate in control cells (neurons, OLs and Ast). (C-D) quantification of mRNA levels of IL-1β, TNFα and IL-6 in Control, AMN and cALD Ast by RT-qPCR (n = 3; n is the number of independent measurements from independent preparation of cells). mRNA levels were standardized with mRNA level of the GAPDH. Data are represented as mean±SD. *P<0.05; **P<0.01

Fig 6. Functional characterization of different IPSC-derived brain cells.

(A-B) quantification of mRNA levels of IL-1β and IL-6 respectively in neurons, OLs and Ast of different cell lines (Control, AMN and cALD) by RT-qPCR (n = 2; n is the number of independent preparation of cells). mRNA levels were quantified in stimulated (cytokines or LPS with cytokines) or unstimulated cells as described in the method section. (C) Quantification of mRNA levels of GFAP in Control, AMN and cALD Ast treated with cytokines or LPS with cytokines by RT-qPCR (n = 3; n is the number of independent preparation of cells). mRNA levels were standardized with mRNA level of the GAPDH. Data are represented as % of Control mean±SD. *P<0.05.

Fig 7. Characterization of IPSC-derived brain cells from AMN and cALD for metabolic defect.

(A-B) Quantification of VLCFA (C26:0 μg/mg of protein or ratio C26:0/C22:0) in neurons, OLs and Ast from different cell lines (Control, AMN and cALD) by GC. In brief, fatty acids methyl ester was prepared directly from different cell types as described in Material and Methods. (A) VLCFA (C26:0 and C22:0) were measured as area percent of total FAs and expressed as ratio of C26:0/22:0 or (B) normalized to protein and presented as absolute amount per mg of protein in Control, AMN and cALD OLs and Ast. Results represent the means±SD from three different cell differentiation experiments; *P<0.05. (C) Quantification of mRNA levels of ELOVL1 in different IPSC-derived brain cells from Control, AMN and cALD by RT-qPCR (n = 3); n is the number of independent preparation of cells. mRNA levels were standardized with mRNA level of the GAPDH or RPLP0. Data are represented as mean±SD. *P<0.05.