Low-Density Neuronal Cultures from Human Induced Pluripotent Stem Cells

Abstract

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented possibility to investigate defects occurring during neuronal differentiation in neuropsychiatric and neurodevelopmental disorders, but the density and intricacy of intercellular connections in neuronal cultures challenge currently available analytic methods. Low-density neuronal cultures facilitate the morphometric and functional analysis of neurons. We describe a differentiation protocol to generate low-density neuronal cultures (∼2,500 neurons/cm²) from human iPSC-derived neural stem cells/early neural progenitor cells. We generated low-density cultures using cells from 3 individuals. We also evaluated the morphometric features of neurons derived from 2 of these individuals, one harboring a microdeletion on chromosome 15q11.2 and the other without the microdeletion. An approximately 7.5-fold increase in the density of dendritic filopodia was observed in the neurons with the microdeletion, consistent with previous reports. Low-density neuronal cultures enable facile and unbiased comparisons of iPSC-derived neurons from different individuals or clones.

R430: A potent inhibitor of DNA and RNA viruses

“…hiPSC line 73-56010-02 was employed in this study 23 , 31 . It was generated from fibroblasts derived from skin biopsy samples which were collected from healthy individuals via 4 mm full thickness punch biopsies under local anesthesia.…”

Childhood-Onset Schizophrenia: Insights from Induced Pluripotent Stem Cells

“…Moreover, at 10 weeks of differentiation qualitative analysis of iPSC-derived neurons provided tentative evidence that dendritic morphology was altered in 15q11.2-deletion carriers relative to control. In support of this view, Dimitrion et al [109] observed in a follow up study that in low-density neuronal cultures the density of dendritic filopodia was strongly increased in neurons with the microdeletion (i.e., the maternally-derived iPSC line) relative to the control.…”

Dysregulation of protein synthesis and dendritic spine morphogenesis in ASD: studies in human pluripotent stem cells

“…Disruption of CYFIP expression is associated with intellectual disability and ASD. Human iPSCs and iPSC-derived neurons with autism-risk copy number variation on chromosome 15q11.2 show reduction in CYFIP1 gene expression [90], and these neurons display 7.5-fold increase in filopodia density compared to neurons from control individual [91]. It should be noted that the micro-deletion on chromosome does not only affect the CYFIP1 gene but also the neighboring genes NIPA1, NIPA2, and TUBGCP5, which encode proteins for Mg 2+ transport and tubulin-interacting protein, respectively.…”

“…Towards this end, it would be desirable to confirm findings of monogenetic ASD patient-derived iPSC by generating isogenic iPSCs that harbor the particular gene mutations, which can now be readily achieved by genome editing such as CRISPR-Cas9. Furthermore, the maturity of dendritic spines formed in cultured human stem cell-derived neurons may also be a concern for detailed study of dendritic spine morphology defects in ASD [90,91], although the presence of mature spines in iPSC-derived neurons has been recently demonstrated [76]. Synapse maturation may be enhanced by co-culture between human iPSCderived neurons and mouse hippocampal neurons in slices, in which the human neurons can be integrated into the preexisting neural circuits in a region-specific manner.…”