Enhanced conversion of induced neuronal cells (iN cells) from human fibroblasts: Utility in uncovering cellular deficits in mental illness-associated chromosomal abnormalities

Abstract

The novel technology of induced neuronal cells (iN cells) is promising for translational neuroscience, as it allows the conversion of human fibroblasts into cells with postmitotic neuronal traits. However, a major technical barrier is the low conversion rate. To overcome this problem, we optimized the conversion media. Using our improved formulation, we studied how major mental illness-associated chromosomal abnormalities may impact the characteristics of iN cells. We demonstrated that our new iN cell culture protocol enabled us to obtain more precise measurement of neuronal cellular phenotypes than previous iN cell methods. Thus, this iN cell culture provides a platform to efficiently obtain possible cellular phenotypes caused by genetic differences, which can be more thoroughly studied in research using other human cell models such as induced pluripotent stem cells.

Keywords: Childhood-onset schizophrenia; Conversion rate; Copy number variations (CNVs); Fibroblasts; Process length; Schizophrenia; Translational research; iN cells.

Fig. 1

Media containing small molecule cocktails (SMC), valproic acid (VPA), and unoprostone (UNO) increases the iN cells conversion rate. (A) The SMC in combination with a single epigenetic modifier (VPA, 2 mM or TSA, 200 nM) in neuronal media promotes a general increase in the production of iN cells derived from fibroblasts of normal control subjects following 3 weeks in culture. These iN cells are functional and mostly glutamatergic in subtype (Kano et al., 2015). White bars indicate iN cells produced in neuronal media only or with the addition of one of the epigenetic modifiers. The combination of SMC with VPA induced a 4-fold increase in iN cells compared to the neuronal media plus VPA alone. (B) Cells from normal control subjects, cultured for 3 weeks in the SMC plus VPA (2 mM) and UNO (10 nM) (striped bar) media showed a further 1.4-fold increase in the iN cell conversion rate compared to cells in the SMC plus VPA media (black bar). The conversion rate is expressed as a fold change of MAP2 positive cells over DAPI positive cells in a 200x field. Means ± s.e.m. (*p<0.05, ***p<0.001). TSA, Trichostatin A; 5AZA, 5-azacytidine. The number of analyzed fields are shown in Supplementary Methods.

Fig. 2

iN cells from a patient with 16p11.2 duplication showed significantly higher conversion rate than other subjects. All cells were grown in the neuronal media containing small molecule cocktails (SMC), valproic acid (VPA) and unoprostone (UNO). Means ± s.e.m. (***p<0.001, ****p<0.0001). The number of analyzed fields are shown in Supplementary Methods.

Fig. 3

Similar morphological phenotypes were observed in iN calls from controls and patients with childhood-onset schizophrenia carrying different copy number variations. All cells were grown in the neuronal media containing SMC, VPA, and UNO. The soma size, process length, and process number were assessed in a 200x field. Means ± s.e.m. The numbers of analyzed cells are shown in Supplementary Methods. (A) Cell soma size (μm²). (B) The number of process. (C) Length of the longest process (μm). (D) Ratio of longest process length to cell soma size.