PROCESSES LENGTHENING AND NEURONAL ADHERENCE ARE DEREGULATED IN CDK5-DEFICIENT HUMAN EMBRYONIC STEM CELL-DERIVED NEURONS

MUCCI, SOFÍA; RODRÍGUEZ VARELA, MARÍA SOLEDAD; ALLIO, CAMILA PAOLA; ISAJA, LUCIANA; FERRIOL LAFFOUILLERE, SOFÍA LUJÁN; SEVLEVER, GUSTAVO EMILIO; SCASSA, MARÍA ELIDA; ROMORINI, LEONARDO

Mar del Plata

Congreso; REUNIÓN CONJUNTA SAIC SAI&FAIC SAFIS 2022; 2022

SAIC SAI&FAIC SAFIS

CDK5 is an essential molecule involved in neuronal survival and homeostasis, thus making the study of the consequences of CDK5 deficiency in a human neuronal model relevant. For this reason, we aim to analyze the differentially expressed genes in WT and KO-CDK5 (CRISPR/Cas9 edited) H9 human embryonic stem cells (hESCs)-derived neurons. Initially, we differentiated WT and KO- CDK5 H9 cell lines into neurons using a defined medium and vali- dated their phenotype by immunostaining with neuronal lineage-de- fining markers (TUJ-1, MAP2, MAP5). Next, an RNA-seq analysis (n=3) was performed with RNA isolated from WT and KO-CDK5 hESCs-derived neurons. We found 920 differentially expressed genes between WT and KO-CDK5 hESCs-derived neurons by us- ing the DESeq2 package in R Studio (alpha = 0.01). Through a GO analysis of biological processes, we found that among the differ- entially expressed genes 137 participate in neurogenesis and neu- ronal differentiation. Next, we proceed to the validation of 5 genes found differentially expressed by RNA-seq analysis by RT-qPCR. Then, we studied processes lengthening in WT and KO-CDK5 H9 hESCs-derived neurons. We found that KO-CDK5 neurons showed a marked increase in the length of their processes with respect to the WT counterparts. In addition, the adherence of KO-CDK5 deficient neurospheres to Geltrex pre-coated plates was remarkably reduced compared to WT neurospheres (10.40%±2.95 vs. 78.12%±7.37, re- spectively). In conclusion, although CDK5 deficiency did not impair neural differentiation of hESCs, its disruption altered the transcrip- tome of hESCs-derived neurons, reduced process lengthening and decreased cell adhesion.