A deficitary model of CDK5 does not impairs neuronal differentiation of human pluripotent stem cells

Abstract

CDK5/P35 is a complex involved in neuronal homeostasis and de- velopment that was described as a critical player for neuronal surviv- al. Besides, its deregulation is linked with neurodegenerative pathol- ogies such as Alzheimer Disease and Parkinson Disease. For that reason, we generated a deficitary CDK5 genetic model in neurons derived from human pluripotent stem cells. For this purpose, we used CRISPR/Cas9 technology to generate human embryonic and induced pluripotent stem cells (hESCs and hiPSCs, respectively) KO-CDK5 lines. CDK5 protein expression levels were analyzed by western blot in samples obtained from clones where indels caused by CRISPR/Cas9 editing were detected by DNA sequencing. We obtained CDK5-/- clones for H9 hESCs and FN2.1 hiPSCs lines and a CDK5+/- clone for H9 hESCs line. Then, neural stem cells (NSC) were derived from the CDK5 KO clones using a commercial neural induction medium and their phenotype was validated by im- munofluorescence staining using antibodies that recognize specific lineage markers (SOX-1, SOX-2, NESTIN and PAX-6). Finally, NSC obtained from the heterozygous CDK5+/- KO H9 hESCs clone were differentiated into neurons using a 2D-based protocol and their phe- notype was validated by immunofluorescence staining of neuronal specific markers (TUJ-1 and MAP2). In conclusion, we managed to obtain NSC-neurons from CDK5-/- and CDK5+/- clones, determin- ing that CDK5 is not essential for NSC generation. Besides, neuro- nal differentiation was achieved for H9 CDK5+/- clone, indicating that the CDK5 deficiency does not impair the generation of NSC-derived neurons. This result allows us to account with a CDK5-defi- cient model to further study its participation in neuronal homeostasis dysfunctions.