iPSC-derived forebrain neurons from FXS individuals show defects in initial neurite outgrowth

Abstract

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and is closely linked with autism. The genetic basis of FXS is an expansion of CGG repeats in the 5'-untranslated region of the FMR1 gene on the X chromosome leading to the loss of expression of the fragile X mental retardation protein (FMRP). The cause of FXS has been known for over 20 years, yet the full molecular and cellular consequences of this mutation remain unclear. Although mouse and fly models have provided significant understanding of this disorder and its effects on the central nervous system, insight from human studies is limited. We have created human induced pluripotent stem cell (iPSC) lines from fibroblasts obtained from individuals with FXS to enable in vitro modeling of the human disease. Three young boys with FXS who came from a well-characterized cohort representative of the range of affectedness typical for the syndrome were recruited to aid in linking cellular and behavioral phenotypes. The FMR1 mutation is preserved during the reprogramming of patient fibroblasts to iPSCs. Mosaicism of the CGG repeat length in one of the patient's fibroblasts allowed for the generation of isogenic lines with differing CGG repeat lengths from the same patient. FXS forebrain neurons were differentiated from these iPSCs and display defective neurite initiation and extension. These cells provide a well-characterized resource to examine potential neuronal deficits caused by FXS as well as the function of FMRP in human neurons.

FIG. 1.

Fibroblasts from fragile X syndrome (FXS) individuals. (A) Fibroblasts were isolated from skin biopsies from four individuals: three diagnosed with FXS and one unaffected control. (B) FMR1 CGG repeat length analysis of fibroblasts from the four individuals shows that the FXS individuals' fibroblasts have >435 repeats, while those from unaffected control have 31, within the normal range. Repeat length analysis of FX13 suggests either mosaicism for the full mutation or PCR artifact due to the high CG content. We cannot distinguish between these possibilities because the limitations of the repeat length assay preclude a more precise definition of the repeat length of the full mutation. Fibroblasts from the FXS mosaic individual (FX11) had two different repeat lengths for FMR1: one that fell in the full-mutation range and one in the premutation range. (C) Average level of methylation at each CpG site in the FMR1 promoter region as determined by bisulfate sequencing. The FXS lines have significant methylation of the FMR1 promoter, while control fibroblasts do not. (D) Quantitative reverse transcription (RT)–polymerase chain reaction (PCR) for the FMR1 gene shows the affected FXS fibroblasts express reduced FMR1 compared with control cells. Expression is normalized to control. Error bars=SE, n=3. (E) Immunoblot for FMRP in the fibroblasts shows that control cells express FMRP while the FXS cells do not.

FIG. 2.

FXS induced pluripotent stem cells (iPSCs) preserve characteristic genetic and epigenetic marks of FMR1 silencing. iPSCs were generated from individual fibroblast lines. (A) Phase-contrast images and immunofluorescence of selected iPSC lines show expression of pluripotency transcription factors OCT4 and SOX2 and cell surface markers Tra1-81 and SSEA4, but not the neuronal transcription factor PAX6. Scale bars=100 μm. (B) Gel electrophoresis for CGG repeat length confirms that repeat length in iPSCs from FXS individuals is preserved after reprogramming. The FX11-7 fibroblasts had two populations of cells with different CGG repeat lengths (mosaic) and isogenic full-mutation and premutation iPSCs were generated from these cells. (C) Average level of methylation at each CpG site in the FMR1 promoter region as determined by bisulfate sequencing. The FXS lines have significant methylation of the FMR1 promoter, while control iPSCs do not. (D) Quantitative RT-PCR for the FMR1 gene shows that the full-mutation FXS iPSCs (FX08-1, FX11-7, and FX13-2) express almost no FMR1 compared with control. The FX11-9U premutation clone expresses normal to high levels of FMR1, whereas its isogenic full-mutation counterpart, FX11-7, expresses no detectable FMR1. Expression is normalized to control C603-4 line. Error bars=SE, n=3. (E) Immunoblot for FMRP in the fibroblasts shows that control cells express FMRP while the FXS cells do not. Color images available online at www.liebertpub.com/scd

FIG. 3.

FXS and control iPSCs generate forebrain neurons in vitro. (A) Representative immunofluorescence of 6-week-old iPSC-derived neurons shows that control neurons express FMRP while FXS neurons do not. (B) Immunofluorescence for the neuronal marker βIII-tubulin and the forebrain transcription factor FOXG1 indicates both FXS and control neurons express FOXG1, confirming their forebrain identity. Scale bars=20 μm. Color images available online at www.liebertpub.com/scd

FIG. 4.

Human FXS forebrain neurons have reduced neurite initiation and outgrowth. Low-magnification images of human forebrain neurospheres from control (A) and FXS (B) iPSCs cultured on laminin and labeled by immunocytochemistry for neural-specific βIII-tubulin (purple) and F-actin using fluorescent phalloidin (green). Note the decreased number and length of projections in FXS neurospheres as compared with control neurospheres after 2 DIV. Also note that many βIII-tubulin-positive neurons migrate away from the FXS neurosphere (arrowheads). High-magnification differential interference contrast images of live growth cones from WT (C) and FXS neurons (D) at indicated times. Note rapid process extension by control forebrain neuron compared with FXS neuron. (E) Comparison of neurite lengths and number of neurites/neurosphere for control and FXS neurospheres in fixed cultures (***P<0.05), as well as the average rate of neurite outgrowth from control and FXS spheres by live cell imaging (***P<0.05). Color images available online at www.liebertpub.com/scd