We have previously established that the integrity of the induced blood–brain barrier (iBBB) formed by brain microvascular endothelial cells derived from the iPSC of 22q11.2 DS (22q11.2 Deletion Syndrome, also called DiGeorge Syndrome) patients is compromised. We tested the possibility that the haploinsufficiency of CRKL, a gene within the 22q11.2 DS deletion region, contributes to the deficit. The CRKL is a major substrate of the Abl tyrosine kinase, and the Abl/CRKL signaling pathway is critical for endothelial barrier functions. Imatinib, an FDA-approved drug, inhibits Abl kinase and has been used to treat various disorders involving vascular leakages. To test if imatinib can restore the compromised iBBB, we treated the patient’s iBBB with imatinib. After treatment, both trans-endothelial electrical resistance and solute permeability returned to comparable levels of the control iBBB. Correspondingly, changes in tight junctions and endothelial glycocalyx of the iBBB were also restored. Western blotting showed that imatinib increased the level of active forms of the CRKL protein. A transcriptome study revealed that imatinib up-regulated genes in the signaling pathways responsible for the protein modification process and down-regulated those for cell cycling. The KEGG pathway analysis further suggested that imatinib improved the gene expression of the CRKL signaling pathway and tight junctions, which agrees with our expectations and the observations at protein levels. Our results indicate that the 22q11.2DS iBBB is at least partially caused by the haploinsufficiency of CRKL, which can be rescued by imatinib via its effects on the Abl/CRKL signaling pathway. Our findings uncover a novel disease mechanism associated with 22q11.2DS.

Calcium plays an important role in regulating various neuronal activities in human brains. Investigating the dynamics of the calcium level in neurons is essential not just for understanding the pathophysiology of neuropsychiatric disorders but also as a quantitative gauge to evaluate the influence of drugs on neuron activities. Accessing human brain tissue to study neuron activities has historically been challenging due to ethical concerns. However, a significant breakthrough in the field has emerged with the advent of utilizing patient-derived human induced pluripotent stem cells (iPSCs) to culture neurons and develop brain organoids. This innovative approach provides a promising modeling system to overcome these critical obstacles. Many robust calcium imaging analysis tools have been developed for calcium activity analysis. However, most of the tools are designed for calcium signal detection only. There are limited choices for in-depth downstream applications, particularly in discerning differences between patient and normal calcium dynamics and their responses to drug treatment obtained from human iPSC-based models.Moreover, end-user researchers usually face a considerable challenge in mastering the entire analysis procedure and obtaining critical outputs due to the steep learning curve associated with these available tools. Therefore, we developed CalciumZero, a user-friendly toolbox to satisfy the unmet needs in calcium activity studies in human iPSC-based 3D-organoid/neurosphere models. CalciumZero includes a graphical user interface (GUI), which provides end-user iconic visualization and smooth adjustments on parameter tuning. It streamlines the entire analysis process, offering full automation with just one click after parameter optimization. In addition, it includes supplementary features to statistically evaluate the impact on disease etiology and the detection of drug candidate effects on calcium activities. These evaluations will enhance the analysis of imaging data obtained from patient iPSC-derived brain organoid/neurosphere models, providing a more comprehensive understanding of the results.