Generation of induced Pluripotent Stem Cells as disease modelling of NLSDM

Abstract

Neutral Lipid Storage Disease with Myopathy (NLSDM) is a rare defect of triacylglycerol metabolism, characterized by the abnormal storage of neutral lipid in organelles known as lipid droplets (LDs). The main clinical features are progressive myopathy and cardiomyopathy. The onset of NLSDM is caused by autosomal recessive mutations in the PNPLA2 gene, which encodes adipose triglyceride lipase (ATGL). Despite its name, this enzyme is present in a wide variety of cell types and catalyzes the first step in triacylglycerol lipolysis and the release of fatty acids. Here, we report the derivation of NLSDM-induced pluripotent stem cells (NLSDM-iPSCs) from fibroblasts of two patients carrying different PNPLA2 mutations. The first patient was homozygous for the c.541delAC, while the second was homozygous for the c.662G>C mutation in the PNPLA2 gene. We verified that the two types of NLSDM-iPSCs possessed properties of embryonic-like stem cells and could differentiate into the three germ layers in vitro. Immunofluorescence analysis revealed that iPSCs had an abnormal accumulation of triglycerides in LDs, the hallmark of NLSDM. Furthermore, NLSDM-iPSCs were deficient in long chain fatty acid lipolysis, when subjected to a pulse chase experiment with oleic acid. Collectively, these results demonstrate that NLSDM-iPSCs are a promising in vitro model to investigate disease mechanisms and screen drug compounds for NLSDM, a rare disease with few therapeutic options.

Keywords: Lipid droplets; Lipid metabolism; Myopathy; NLSDM; PNPLA2; iPSCs.

Fig. 1

Lipid droplets abnormal storage in different tissues of NLSDM patient 1, compared to a control subject. (A) Buffy coats stained with May-Grünwald-Giemsa (arrow indicates Jordans' anomaly in the granulocyte of patient 1). Magnification: 100 ×. (B) Phase contrast images of primary dermal fibroblasts obtained from a control subject and NLSDM patient (patient 1) whose fibroblasts were used to produce iPS cells. Arrows indicate abnormal TAGs accumulation in the NLSDM fibroblasts. Magnification: 40 ×. (C) Direct sequencing of PNPLA2 gene confirmed the presence of mutations in the two disease-specific iPSC lines (c.541delAC and c.662G > C respectively).

Fig. 2

Characterization and differentiation of patient-derived NLSDM-iPSC. (A) Immunostaining with primary antibodies anti-OCT4, anti-SSEA-4 and anti-TRA-1-81 revealed that NLSDM-iPSCs express markers of pluripotency, including OCT4 (red), SSEA4 (green), and TRA-1-81 (green). Nuclei were stained with DAPI. Magnification: 10 ×. (B) Real-time quantitative RT-PCR assays for eight pluripotency-associate genes in two patient-derived iPSCs compared with fibroblast cell lines (basal level reported as 1). Data represent the mean of three independent experiments, performed in triplicate. PCR reactions were normalized against an internal control (ACTB). (C) Quantification of TG content in iPSCs derived from 2 controls and 2 NLSDM fibroblast cell lines. The graph represents TG content obtained from three independent experiments. The analysis was performed using Student t-test. Thick bar: mean value; error bar: SD. P-values of < 0.05 and of < 0.01 were considered to be significant and indicated with “*”and “**”, respectively.

Fig. 3

NLSDM-iPSCs differentiation into all three embryonic germ layers in vitro. Immunostaining using primary antibodies anti-FOXA2, anti-α-SMA and anti-TUJ1 showed that markers for the three germ layers, endoderm-FOXA2, mesoderm-α-SMA and ectoderm-TUJ1, were expressed in differentiated NLSDM-iPSCs. Nuclei were stained with DAPI. Magnification: 20 ×.

Fig. 4

Evaluation of NLSDM pathophysiological characteristic in the disease-specific iPSCs. Nile Red stained microphotographs of control and NLSDM-iPSCs (A; magnification: 20 ×) revealed more LDs (yellow) in the NLSDM cells. (B) Analysis of LD number and dimensions observed in NLSDM-iPSCs, compared to control iPSCs (n = 3; 100 cells were analyzed for each experiment) was carried out by Student's t-test. Thick bar: mean value; error bar: SD. P-values of < 0.05 and of < 0.01 were considered to be significant and indicated with “*” and “**”, respectively. (C) Oleic acid pulse-chase experiments on control and NLSDM-iPSCs. Culture medium (CM: DMEM-F12 containing 20% KOSR, 100 μM non-essential amino acids, 10 ng/ul bFGF, 1% penicillin/streptomycin, 1% l-glutamine, 1% sodium pyruvate and 0.2% β-mercaptoethanol) was supplemented with OA (200 μM) and, after 18 h, iPSCs were washed and chased with fresh medium containing 5% KOSR and fatty acid free BSA (2% w/v) for 24 and 72 h. Cells were stained for neutral lipids with NR. Lipid droplets are in yellow. Magnification: 40 ×. (D) Analysis of LD number and dimension observed in NLSDM-iPSCs, compared to control iPSCs, during the oleic acid pulse-chase experiments. In two independent experiments 100 cells in each of 3 replicates were analyzed. Thick bar: mean value; error bar: SD. P-values of < 0.05 and of < 0.01 were considered to be significant and indicated with “*”and “**”, respectively.