Genetic and Morphological Features of Human iPSC-Derived Neurons with Chromosome 15q11.2 (BP1-BP2) Deletions

Abstract

Background: Copy number variation on chromosome 15q11.2 (BP1-BP2) causes a deletion of CYFIP1, NIPA1, NIPA2 and TUBGCP5. Furthermore, it also affects brain structure and elevates the risk for several neurodevelopmental disorders that are associated with dendritic spine abnormalities. In rodents, altered cyfip1 expression changes dendritic spine morphology, motivating analyses of human neuronal cells derived from induced pluripotent stem cells (iPSCs; iPSC-neurons). Methods: iPSCs were generated from a mother and her offspring, both carrying the 15q11.2 (BP1-BP2) deletion, and a non-deletion control. Gene expression in the deletion region was estimated using quantitative real-time PCR assays. Neural progenitor cells (NPCs) and iPSC-neurons were characterized using immunocytochemistry. Results:CYFIP1, NIPA1, NIPA2 and TUBGCP5 gene expression was lower in iPSCs, NPCs and iPSC-neurons from the mother and her offspring in relation to control cells. CYFIP1 and PSD-95 protein levels were lower in iPSC-neurons derived from the copy number variant-bearing individuals using Western blot analysis. Ten weeks after differentiation, iPSC-neurons appeared to show dendritic spines, and qualitative analysis suggested that dendritic morphology was altered in 15q11.2-deletion subjects compared with control cells. Conclusions: The 15q11.2 (BP1-BP2) deletion is associated with a reduced expression of four genes in iPSC-derived neuronal cells; it may also be associated with altered iPSC-neuron dendritic morphology.

Low-Density Neuronal Cultures from Human Induced Pluripotent Stem Cells

“…We have previously reported on iPSC-derived neurons from 2 individuals with a microdeletion in the chromosome 15q11.2 region that leads to haploinsufficiency of TUBGCP1 , NIPA1 , NIPA2 , and CYFIP1 [13]. Altered differentiation patterns in neuronal and glial lineages of human iPSCs bearing the 15q11.2 (BP1-BP2) deletion have been reported by other groups [19].…”

“…Low-density areas in our culture system enabled analysis of dendritic spines without the need to express a fluorescent reporter construct in cultures, which is required for neurons in high-density neuronal cultures [13]. Dendritic filopodia and dendritic spines were identified by staining with FITC-phalloidin (Fig.…”

“…This deletion is associated with several neuropsychiatric disorders, including autism, schizophrenia, and intellectual disability [20]. Cells that carry the deletion or that are haploinsufficient for CYFIP1 exhibit an increased number of dendritic filopodia, indicating their immaturity [13,21]. To investigate whether such neuronal differentiation defects could be detected in our low-density neuronal culture system, we employed an iPSC line (71-500-9001) harboring the 15q11.2 (BP1-BP2) deletion (Fig.…”

“…In the present study, we describe a differentiation protocol for the generation of low-density neuronal cultures that enables rapid characterization of isolated neurons without the need for transfection with a fluorescent reporter construct [13]. The low-density cultures also enable facile electrophysiological recordings and unbiased neuronal morphological analysis.…”

“…Human iPSCs were maintained in supplemented mTeSR™1 medium (Stem Cell Technologies) under standard conditions (5% CO 2 , 37°C, and 100% humidity). Karyotyping and array comparative genomic hybridization were used to check for chromosomal abnormalities and to confirm deletions in the chromosome 15q11.2 region as described [13]. …”