Generation of IPSC-derived neurons from an Alzheimer’s Disease patient carrying a novel PSEN1 T119I mutation

ISAJA, LUCIANA; MARAZITA, MARIELA; RODRÍGUEZ VARELA, MARÍA SOLEDAD; MUCCI, SOFÍA; ITZCOVICH, TATIANA; CHREM-MENDEZ, PATRICIO; NIIKADO, MATIAS; FERRIOL LAFFOUILLERE, SOFÍA LUJÁN; ALLEGRI, RICARDO; MARTINETTO, HORACIO; SEVLEVER, GUSTAVO EMILIO; SCASSA, MARÍA ELIDA; SURACE, EZEQUIEL IGNACIO; ROMORINI, LEONARDO

San Francisco

Congreso; International Society for Stem Cell Research (ISSCR) annual meeting.; 2021

ISSR

Alzheimer’s disease (AD) is the main cause of dementia in adults. It is estimated that 5% ofcases are caused by inherited mutations in genes such as Presenilin-1 (PSEN1). Previously, ourgroup reported a novel heterozygous variant in PSEN1 (c.356C>T; p.T119I) in an Argentinefamily with early- and late-onset AD. In order to functionally validate this variant, we aimed togenerate a human induced pluripotent stem cell (hiPSC) line from dermic fibroblasts (DF) of amale mutation carrier. To this purpose, we first infected patient DF with a STEMCCA lentiviralvector encoding the Yamanaka reprogramming factors (OCT-4, KLF4, SOX2 y c-MYC) andobtained a line termed FFAD1.2. The cells exhibited typical hiPSCs morphologicalcharacteristics (formation of compact multicellular colonies with a high nucleus/cytoplasm ratioand distinct colony borders) and high Alkaline Phosphatase (AP) activity. Moreover, we alsoobserved robust expression of different stemness-associated markers (OCT-4, NANOG,SSEA4, TRA1-80, and TRA1-60) analyzed by immunofluorescence and RT-qPCR. Also, usinga non-directed method of differentiation (embryoid bodies assay) we demonstrated that this linehad the pluripotent potential to be differentiated into cells from the three germinal layers(mesoderm, endoderm and ectoderm) as judged by immunofluorescence analysis of Smoothmuscle actin (SMA), Alpha-fetoprotein (AFP) and βIII-tubulin (TUJ1) differentiation markersexpression, respectively. Finally, the FFAD1.2c4 presented the same normal karyotype (46,XY) and PSEN1 T119I genotype as the parental DF cells and silenced ectopic expression ofYamanaka gene. Following the generation and characterization of the hiPSC line, we performeda 2D differentiation protocol to generate hiPSC-derived neurons carrying this mutation. Afterthree weeks of differentiation, we observed cells morphologically similar to neurons whichexpressed high levels of TUJ1 and MAP2, two neuron-specific cytoskeletal protein, analyzed byimmunofluorescence. Overall, we successfully obtained a hiPSCs line (FFAD1.2) carrying theAD PSEN1 T119I genotype and achieved to directedly differentiate it to neuronal lineage.