Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs

Abstract

The isolation of human induced pluripotent stem cells (iPSCs) offers a new strategy for modelling human disease. Recent studies have reported the derivation and differentiation of disease-specific human iPSCs. However, a key challenge in the field is the demonstration of disease-related phenotypes and the ability to model pathogenesis and treatment of disease in iPSCs. Familial dysautonomia (FD) is a rare but fatal peripheral neuropathy, caused by a point mutation in the IKBKAP gene involved in transcriptional elongation. The disease is characterized by the depletion of autonomic and sensory neurons. The specificity to the peripheral nervous system and the mechanism of neuron loss in FD are poorly understood owing to the lack of an appropriate model system. Here we report the derivation of patient-specific FD-iPSCs and the directed differentiation into cells of all three germ layers including peripheral neurons. Gene expression analysis in purified FD-iPSC-derived lineages demonstrates tissue-specific mis-splicing of IKBKAP in vitro. Patient-specific neural crest precursors express particularly low levels of normal IKBKAP transcript, suggesting a mechanism for disease specificity. FD pathogenesis is further characterized by transcriptome analysis and cell-based assays revealing marked defects in neurogenic differentiation and migration behaviour. Furthermore, we use FD-iPSCs for validating the potency of candidate drugs in reversing aberrant splicing and ameliorating neuronal differentiation and migration. Our study illustrates the promise of iPSC technology for gaining new insights into human disease pathogenesis and treatment.

Figure 1. Establishment of FD-iPSCs from patient fibroblasts

(a-b) FD patient fibroblasts (a) were converted into FD-iPSCs (b, c) following lentiviral transduction with OCT4, SOX2, KLF4, and cMYC. d, Nanog protein expression in FD-iPSC line. e, Flow-cytometry analysis of FD-iPSCs for pluripotent surface markers. f, Karyotype analysis of FD-iPSCs. g, Bisulfite sequencing analysis of NANOG promoter in FD-fibroblast and FD-iPSC clones. h, Global gene expression patterns were compared among FD-fibroblast, FD-iPSCs and human ESCs. i, Teratoma from FD-iPSCs showed three germ-layer differentiation as illustrated by the presence of endodermal epithelia expressing β-catenin, Pax6+ neuroectodermal precursors and mesodermal collagen+ cells. j, Sequencing result showed the 2507+6T>C mutation of IKBKAP in FD-iPSC. k, Analysis of IKBKAP RT-PCR products in mRNA derived from normal and FD-specific fibroblasts and pluripotent stem cells. All scale bars correspond to 50μm.

Figure 2. FD-iPSC derived cell lineages model the tissue specificity of FD IKBKAP splicing defect

a, Undifferentiated iPSCs were defined by NANOG expression and quantitative analysis of TRA1-81. b-f, FD-iPSCs were directed towards specific lineages and purified by flow-cytometric sorting for appropriate surface markers. The cell types analyzed included AP2+ neural crest precursors purified based on HNK1+ expression (b), FACS purified Forse1+ neural rosette cells (Pax6+) (c), hematopoietic cells that were harvested from colony forming unit culture and sorted with CD45 marker (d), FACS isolated CD144+ (VE-cadherin) endothelial cells (CD105+) from blast colony culture (e), FACS isolated CXCR4+ endodermal precursors (Sox17+) from ActivinA-induced differentiation culture (f). IKBKAP RT-PCR products of FACS purified lineages (bottom panel). The ratio of normal:mutant splicing (g) and expression of normal IKBKAP transcript (h) are shown. n = 3 – 6; **, P < 0.01; ***, P < 0.001. All values are mean ± s.d. All scale bars correspond to 50μm.

Figure 3. Molecular and functional characterization of FD-iPSC derived neural crest precursor cells

a, List of the unselected top increased (blue) and top decreased (red) genes comparing FD-iPSC versus [C14 and H9] derived neural crest cells as assessed by microarray analysis. b-c, Representative images and quantifications of MASH1 (b) and Tuj1 (c) expression in spontaneously differentiated neural crest cells derived from FD-iPSC and C14-iPSC. n = 4; **, P < 0.01; ***, P < 0.001. c, inset, Representative image of putative sensory neuron progeny (Brn3a+/Periperin+) from FD-iPSC derived neural crest cells. d-e, Representative images of wound healing assay (fixed at 48 hrs after scratching) and paxillin staining (fixed at 1.5 hrs after plating) of FD-iPSC and C14-iPSC derived neural crest cells. n = 4; *, p < 0.05. Quantification of paxillin staining was performed by counting the number of Paxillin puncta marking focal adhesion complex. All values are mean ± s. d. All scale bars correspond to 50μm.

Figure 4. Validating kinetin as a candidate compound for treating FD-iPSC derived neural crest cells

a, Gel image of RT-PCR products for IKBKAP splicing rescued by kinetin treatment in control and FD-iPSC derived neural crest. b, Quantification of band intensity of FD-iPSC derived neural crest cells normalized by GAPDH and the ratio of normal and mutant spliced IKBKAP transcripts. n = 5; *, p < 0.05; ***, P < 0.001. c-d, MASH1 (c) and Tuj1 (d) expression in neuronal differentiation with kinetin treated neural crest cells derived from FD-iPSC . n = 4; ***, P < 0.001. (E-F) Results of wound healing assay (e) and paxillin staining (f) of kinetin treated FD-iPSC derived neural crest cells. n = 4 - 6; ***, P < 0.001. All values are mean ± s. d.