CDK5 deficiency does not impair the differentiation of human induced pluripotent stem cells into dopaminergic neurons

CDK5, a kinase essential for neuronal development and function, becomes hyperactivated under neurotoxic and stress conditions, contributing to neurodegeneration. However, whether CDK5 is required for acquisition of a dopaminergic phenotype remains unclear. This work aimed to evaluate if CDK5 loss affects the differentiation of human induced pluripotent stem cells (hiPSCs) into dopaminergic neurons. We applied a 60-day differentiation protocol, based on the sequential addition of specific induction and maturation factors, to CDK5 wild-type (WT) and knockout (KO) hiPSCs (FN2.1 cell line). Samples were collected at days 15, 24 and 59 to monitor differentiation and characterize the resulting neuronal phenotype. Dopaminergic differentiation was assessed by RT-qPCR analysis of tyrosine hydroxylase (TH) and dopamine transporter (DAT), together with the ventral midbrain markers PITX3 and FOXA2. We also performed immunofluorescence detection of TH and PITX3. Both WT and CDK5 KO lines showed progressive increases in TH and PITX3 transcript expression throughout differentiation, accompanied by the generation of TH- and PITX3-positive neurons, indicating that CDK5 deficiency does not prevent the acquisition of a dopaminergic phenotype. These findings establish that FN2.1 CDK5 KO hiPSCs can be differentiated into dopaminergic neurons and provide a cellular model for further investigation of CDK5-dependent mechanisms in PD, including mitochondrial function and calcium homeostasis.