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

<div><p>X-ALD is an inherited neurodegenerative disorder where mutations in the <i>ABCD1</i> 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 <i>ELOVL1</i> (<i>ELOVL</i> 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.</p></div

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

<p>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).</p

Functional characterization of different IPSC-derived brain cells.

<p>(A-B) quantification of mRNA levels of <i>IL-1β</i> and <i>IL-6</i> 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 <i>GFAP</i> 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 <i>GAPDH</i>. Data are represented as % of Control mean±SD. *P<0.05.</p

IPSC-derived brain cells differentiation and characterization.

<p>(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).</p

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

<p>(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 <i>ELOVL1</i> 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 <i>GAPDH</i> or <i>RPLP0</i>. Data are represented as mean±SD. *P<0.05.</p

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

<p>(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 <i>IL-1β</i>, <i>TNFα</i> and <i>IL-6</i> 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 <i>GAPDH</i>. Data are represented as mean±SD. *P<0.05; **P<0.01</p

15-deoxy-delta12,14-prostaglandin J2 attenuates endothelial-monocyte interaction: implication for inflammatory diseases-5

pIKKα using its specific antibody (Cell Signaling) (A). β actin was used as a control for equal content of protein loaded. bEND.3 cells were transfected with IKKα, NF-κB luciferase and pCMV-β-galactosidase constructs and treated with 15d-PGJ2 (5–20 μM) and TNFα (50 ng/ml). After 4 h of TNFα treatment, cells were processed for luciferase assay as described in 'Material and Methods' (B). Results were calculated as mean ± SD for 3 independent experiments. &&& p < 0.001 compared with control, !!! p < 0.001 compared with TNFα treatment and ### p > 0.001 compared with IKKα. bEND.3 cells were transfected with Gal-p65 or Gal-DBD in the presence or absence of flag-IKKα along with PTL-luciferase and PRL-TK reporter constructs as described in Material and Method. bEND.3 cells were pretreated with 15d-PGJ2 (10 μM) for 30 min followed by TNFα treatment. After 6 h of TNFα treatment (50 ng/ml), cells were processed for luciferase assay and results were normalized with PRL-TK luciferase activity in each sample (C). Total DNA content was normalized with pcDNA3. Results were calculated as mean ± SD for 3 independent experiments.<p><b>Copyright information:</b></p><p>Taken from "15-deoxy-delta12,14-prostaglandin J2 attenuates endothelial-monocyte interaction: implication for inflammatory diseases"</p><p>http://www.journal-inflammation.com/content/5/1/14</p><p>Journal of Inflammation (London, England) 2008;5():14-14.</p><p>Published online 8 Aug 2008</p><p>PMCID:PMC2531100.</p><p></p

15-deoxy-delta12,14-prostaglandin J2 attenuates endothelial-monocyte interaction: implication for inflammatory diseases-1

. Cells were harvested in Trizol reagent for RNA isolation and cDNA synthesis. RT-PCR analysis was done for ICAM-1 (A), VCAM-1 (B) and E-selectin (C). Results were calculated as mean ± SD for 3 independent experiments. Samples were examined in triplicates. &&& p < 0.001 compared with control (untreated and unstimulated cells) and !!! p < 0.001 as compared to TNFα treatment. For the quantitation of expression of surface CAMs, bEND.3 cells were treated with TNFα (50 ng/ml) in the presence or absence of 15d-PGJ2 (5–20 μM) for 6 h followed by flow cytometry analysis (D) (n = 2).<p><b>Copyright information:</b></p><p>Taken from "15-deoxy-delta12,14-prostaglandin J2 attenuates endothelial-monocyte interaction: implication for inflammatory diseases"</p><p>http://www.journal-inflammation.com/content/5/1/14</p><p>Journal of Inflammation (London, England) 2008;5():14-14.</p><p>Published online 8 Aug 2008</p><p>PMCID:PMC2531100.</p><p></p

IPSC-derived neural precursor cells differentiation and characterization.

<p>(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.</p

Morphological and specific marker characterization of fibroblast-derived IPSC.

<p>(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.</p