25122
Establishment of a Human Induced Pluripotent Stem Cell-Based Model of Kleefstra Syndrome
Objectives: The aim of the present study was to establish a patient-derived in vitro disease model of KS accompanied by ASD (KS+ASD) using the iPSC technology.
Methods: Blood samples from a patient with KS+ASD carrying a premature termination codon mutation in the euchromatic histone lysine methyltransferase 1 (EHMT1) gene and two healthy subjects were taken after ethical approval and obtaining written informed consent. Diagnosis of the subjects was confirmed with ADOS and ADI-R. Mononuclear cells were isolated from the blood and genetically reprogrammed by a non-integrating gene delivery system. Differentiation of iPSCs into neuronal precursor cells and glutamatergic neurons was induced by a dual-SMAD inhibition protocol. Neurite morphology was measured by using an Operetta® High Content Imaging System (PerkinElmer). EHMT1 gene expression was investigated by using reverse transcription-quantitative PCR and Western blotting.
Results: The iPSCs showed embryonic stem cell morphology, normal karyotype, expressed pluripotency markers, and were able to spontaneously differentiate into cells of the three germ layers. iPSCs were successfully differentiated into neurons; this was demonstrated by neuron specific immunolabeling for MAP2 and NF200, electronmicroscopic visualization of synapses and electrophysiological detection of ionic (sodium, potassium) currents and action potentials. Determination of EHMT1 mRNA and protein expression demonstrated functional haploinsufficiency of the gene in the patient-derived cell cultures. iPSC-derived neuronal cell cultures were investigated in order to detect any substantial phenotypical differences between neurons originated from the KS+ASD case and neurotypical subjects. Neurite morphology was significantly compromised on multiple endpoints, including full and maximal length of neurites, number of neurite roots and endings in the KS+ASD condition in comparison to controls. The number of dendritic protrusions (filopodia or immature dendritic spines) was also reduced in the KS+ASD cultures compared to controls. Calcium currents evoked by glutamate did not differ between KS+ASD and controls however, administration of acetyl-choline induced larger calcium currents in the KS+ASD cell cultures. Gene expression patterns of 180 ASD-associated candidate genes were investigated by qRT-PCR, showing significantly altered expression of ARX, SIX3, and HCN1 genes relative to both controls.
Conclusions: The present iPSC-derived neuronal cultures represent an excellent in vitro model system for KS which may serve to obtain a better understanding of the underlying pathophysiology of KS and potentially that of ASD.