Induced pluripotent stem cell model recapitulates pathologic hallmarks of Gaucher disease

Abstract

Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid β-glucocerebrosidase gene. To model GD, we generated human induced pluripotent stem cells (hiPSC), by reprogramming skin fibroblasts from patients with type 1 (N370S/N370S), type 2 (L444P/RecNciI), and type 3 (L444P/L444P) GD. Pluripotency was demonstrated by the ability of GD hiPSC to differentiate to all three germ layers and to form teratomas in vivo. GD hiPSC differentiated efficiently to the cell types most affected in GD, i.e., macrophages and neuronal cells. GD hiPSC-macrophages expressed macrophage-specific markers, were phagocytic, and were capable of releasing inflammatory mediators in response to LPS. Moreover, GD hiPSC-macrophages recapitulated the phenotypic hallmarks of the disease. They exhibited low glucocerebrosidase (GC) enzymatic activity and accumulated sphingolipids, and their lysosomal functions were severely compromised. GD hiPSC-macrophages had a defect in their ability to clear phagocytosed RBC, a phenotype of tissue-infiltrating GD macrophages. The kinetics of RBC clearance by types 1, 2, and 3 GD hiPSC-macrophages correlated with the severity of the mutations. Incubation with recombinant GC completely reversed the delay in RBC clearance from all three types of GD hiPSC-macrophages, indicating that their functional defects were indeed caused by GC deficiency. However, treatment of induced macrophages with the chaperone isofagomine restored phagocytosed RBC clearance only partially, regardless of genotype. These findings are consistent with the known clinical efficacies of recombinant GC and isofagomine. We conclude that cell types derived from GD hiPSC can effectively recapitulate pathologic hallmarks of the disease.

Fig. 1.

Generation and characterization of L444P/RecNciI (type 2) GD hiPSC. (A) Staining of GD hiPSC with antibodies to the stem cell and pluripotency markers SOX2, SSEA-3, SSEA-4, NANOG, TRA-1–60 TRA-1–81, and OCT4. (Scale bar, 100 μm.) (B) (a) GD hiPSC cells gave rise to benign cystic teratomas in NOG-SCID mice. (b–f) H&E staining of teratoma cells from the three germ layers. (b) Glandular structure. (c) Pigmented neural epithelium and rosettes. (d) Intestinal epithelium. (e) Cartilage. (f) Bone. (Magnification: 20×.)

Fig. 2.

Directed differentiation of type 2 GD hiPSC to monocyte/macrophages. (A and B) FACS analysis of L444P/RecNciI hiPSC-monocyte/macrophages. Histograms show the percentage of cells stained with antibodies to specific markers (Lower) and isotype controls (Upper). (A) CD14 expression in GD hiPSC-monocytes. (B) Expression of CD14 (a), CD163 (b), and CD68 (c) in GD hiPSC-macrophages. (C) May–Grünwald–Giemsa staining showing the appearance of GD hiPSC-macrophages with different morphologies (a–c). (Scale bar, 20 μm.) (D) (a, b, d, and e) Phagocytosis of opsonized RBC by GD hiPSC-macrophages. (c and f) Non-opsonized RBC control. (a–c) Live-cell images of GD hiPSC-macrophages. (d–f ), May–Grünwald–Giemsa staining of GD hiPSC-macrophages. (Scale bars, 20 μm.) (E) RT-PCR analysis showing the induction of TNF-α, IL-10, IL-12p35, and IL-12p40 mRNA in response to LPS treatment. Numbers in the ordinates represent the fold-activation of corresponding cytokines compared with the nontreated condition. P values for fold-cytokine induction in GD hiPSC-macrophages compared with control hiPSC-macrophages are P < 0.0138 (TNF-α), P < 0.1 (IL-10), P < 0.0698 (IL-12p35), and P < 0.009 (IL-12p40).

Fig. 3.

Phenotype of GD hiPSC-macrophages. (A) Low levels of GC enzymatic activity (Upper) and GC protein (Lower) in N370S/N370S, L444P/L444P, and L444P/RecNciI hiPSC- vs. control hiPSC-macrophages (iMϕ). (B) Staining of L444P/RecNciI (a and b) and control (c and d) hiPSC-macrophages with rabbit antibodies to GlcCer (G-Cer, red) and nuclear staining with DAPI (blue). (C) HPLC-MS/MS analysis showing the level of glucosylsphingosine in H9/hESC-, control hiPSC-, N370S/N370S, L444P/L444P, and L444P/RecNciI-hiPSC-macrophages. (D) Kinetics of phagocytosed RBC clearance in different lines of L444P/RecNciI mutant macrophages. H9/hESC- (black), control hiPSC- (red), and three lines of L444P/RecNciI GD hiPSC-macrophages [#3 (blue), #4 (purple), and #16 (green)] were incubated with opsonized RBC, and the time course of RBC clearance was followed. The ordinate represents the percent of GD hiPSC-macrophages containing visible RBC. On day 2, P < 0.0001. (E) Significant numbers of phagocytosed RBC are still visible 24 h after ingestion by L444P/RecNciI hiPSC-macrophages. L444P/RecNciI hiPSC-macrophages (a and b) and control hiPSC-macrophages (c and d) were incubated with opsonized RBC as described above, and cells were stained with May–Grünwald–Giemsa 24 h later. Representative microscopic images are shown. Arrows indicate RBC remnants. (F) Comparison of the time courses of RBC clearance in type 1 N370S/N370S (blue), type 2 L444P/RecNciI (purple), and type 3 L444P/L444P (green) macrophages, in control hiPSC- (red), and in H9/hESC-macrophages (black). On day 2, P values corresponding to the type 1, 2, and 3 genotypes compared with the controls were P < 0.008, P < 0.0001, and P < 0.001, respectively. (G) Phenotypic correction of mutant phenotype by recombinant GC. Untreated control hiPSC-macrophages (purple), untreated L444P/RecNciI hiPSC-macrophages (blue), and L444P/RecNciI hiPSC-macrophages treated with 0.02 (green), 0.04 (red), 0.08 (light blue), and 0.24 (pink) U/mL recombinant GC, as described in SI Materials and Methods, were assayed for RBC clearance. On day 2, P values for 0.02, 0.04, 0.08, and 0.24 U/mL GC were P < 0.0015, P < 0.005, P < 0.0005, and P < 0.0001, respectively. (H) Isofagomine treatment increases GC enzyme activity in mutant macrophages. L444P/RecNciI, L444P/L444P, N370S/N370S, and control hiPSC-macrophages were incubated in the presence or absence of 60 μM isofagomine for 5 d; then GC activity was determined. Numbers represent fold-increase of GC activity in treated vs. untreated cells. (I) Time course of phagocytosed RBC clearance by GD hiPSC-macrophages in the absence or presence of isofagomine. L444P/RecNciI hiPSC-macrophages were incubated in the absence (blue) or presence of 30 μM (green), 60 μM (red), or 100 μM (black) isofagomine for 5 d and then were assayed for RBC clearance as described in D. Untreated H9/hESC macrophages were used as a control (pink). Isofagomine treatment was continued for the duration of the RBC clearance assay. On day 2, P values for 30 μM and 60 μM isofagomine were P < 0.0002 and P < 0.0006, respectively. (Scale bars, 20 μm in B and E.)

Fig. 4.

Differentiation of GD hiPSC to neuronal cells. L444P/RecNciI hiPSC were differentiated to neuronal cells as described in SI Materials and Methods, and were stained with antibodies to the indicated markers. (A–E) Characterization of neuronal rosette progenitors. (A) DAPI (blue). (B) MAP2 (red) overlaid with DAPI. (C) Tuj1 (red). (D) SOX2 (red). (E) GFAP (green) overlaid with Tuj1 (red). (F–K) Characterization of GD hiPSC neuronal cells extending from rosettes. (F) Tuj1 (green). (G) MAP2 (green). (H) GABA (green). (I) DβH (green). (J) TH (green). (K) O4 (red) overlaid with DAPI. (L) Low levels of GC enzymatic activity in N370S/N370S, L444P/L444P, and L444P/RecNciI hiPSC- vs. control hiPSC-neurons. (Scale bars, 100 μm in A–G, 20 μm in H–K.)