In the current study, we investigated the functional significance of NRXN1 deletions in neurodevelopment, using hiPSCs and hESCs as in vitro models. During differentiation of neural stem cells into mature neurons, we found that knockdown of a single neuronal gene NRXN1 leads to systematic perturbations of expression levels of several neurodevelopment-related pathways. Additionally, we also observed reduced astrocytes differentiation potential, and a strong positive correlation between expression of α-NRXN1 and the astrocyte marker GFAP. Altogether, our results suggested that NRXN1 deletion most likely influenced the synapse function and neuronal connectivity. The relationship between NRXN1 knockdown and inhibition of astrocytes differentiation is intriguing and is worthy of follow-up studies. Both the disturbed neuronal pathways and the inhibition of astrocytes differentiation could play a role in the molecular pathophysiology of several neurodevelopmental diseases, such as autism and schizophrenia.
One of the interesting findings in our study is the reduced astrocytes generation from neural stem cells after NRXN1
knockdown. Astrocytes are star shaped glial cells present in central nerve system and spinal cord, and they have several important functions 
. They are the most abundant cell types in mammalian brains and are involved in the physical structure and buffer of the neural system. They could regulate the electrical pulse between neurons, control the neural transmitters uptake and release, modulate synapse transmission and help repair the neural system. Astrocytes could also form synapse between neural synapse and release gliotransmitter, forming tripartite synapse and dynamically regulate the synapse transmission 
. The delicate tree structure of astrocytic cell and the fact that each astrocyte can potentially contact 300,000 neurons, provide the topology that ensures multiple levels of interaction between neurons and astrocytes. Although astrocytes are not capable to generate electrical signals like neurons, they respond with elevated intracellular calcium concentration to mechanical or chemical stimulus 
. As the astrocytes actively participate in neuron synapse formation and signing transduction, the defect of astrocytes function may have severe consequence for neural system. Previous studies already suggested that the abnormal glial-neuronal communication may be involved in pathogenesis of autism 
and schizophrenia 
. Given the strong association between astrocytes and the synapse, it is reasonable to speculate that the deletion of NRXN1
may influence astrocytes differentiation, thus contributing to pathogenesis of diseases with impaired synaptic adhesion and transmission.
Recent advances in hESCs and hiPSCs research have made it possible to establish in vitro
model systems to study complex neurodevelopmental disorders, for which animal models are generally not available or not ideal to use in specific scientific contexts. Neuronal system is generally difficult to study, as live neurons are not readily available from the patients to understand the molecular pathophysiology of the diseases. Although hESCs are more widely used for studies of molecular mechanisms, we expect that patient-specific hiPSCs will find more use in future genetic studies. These iPS cells can be generated from skin fibroblasts or peripheral blood monocytes 
, which share the same genetic background as the patients themselves, thus enabling more refined studies in vitro
. By analyzing morphological, electrophysiological, transcriptional and functional differences of neurons derived from specific patients and control subjects, we may better understand the molecular mechanism of diseases pathogenesis, and ultimately help develop better individualized diagnosis and therapeutic tools.
We believe that one of the previously unrecognized advantages of using stem cells model to study complex neuropsychiatric diseases is to understand the functional impacts of CNVs by modulating gene expression. CNVs may account for a significant proportion of human phenotypic variation, including disease susceptibility 
. Deletion or duplications of one or more genes may lead to dosage-dependent gene expression changes, disrupt regulatory elements, generate novel fusion products, or act by way of position effect, with various possible positive and negative consequences, including imprinting and differential allelic expression 
. Given that several single-gene deletions and recurrent genomic deletions serve as highly penetrant disease susceptibility factors in neuropsychiatric diseases, it is expected that stem cell models can be readily used to screen the functional significance of CNVs in large scale, using a combination of gene knockin and knockdown techniques, coupled with cell biology studies (morphological analysis, electrophysiology analysis, etc) and molecular biology studies (gene expression, methylation, histone modification, etc).
In conclusion, the combination of stem cells models with targeted gene knockdown and high-throughput transcriptome sequencing clearly provided a novel approach for studying the functional significance of CNVs in complex neuropsychiatric diseases. The models we established here would help confirm the roles of candidate CNVs or other genetic alternations identified from previous genetic studies (CNV association, candidate gene association and genome-wide association studies), and help discover additional diseases susceptibility genes and pathways. Finally, these in vitro models may facilitate drug discovery by serving as therapeutic models for drug screening, and may facilitate pharmacogenomic studies to understand how differences in genetic background impact treatment responses.