Direct Reprogramming of the Marmoset Dermal Fibroblasts into Neural Stem Cells and Neurons

Abstract

AbstractBackgroundThe neuropathological hallmarks of Alzheimer’s Disease (AD) are characterized by the accumulation of beta‐amyloid (Ab) plaques and neurofibrillary tangles (NFTs) of a hyperphosphorylated form of the microtubule‐associated protein tau (MAPT) in the brain. However, Ab and tau accumulate in the brain decades before the clinical symptoms emerge and the biological basis for the relationship between abnormal aggregation of AD‐related proteins and neuronal deterioration remains unknown. Cell models play an imperative role in understanding the pathogenesis of the disorder. For this, the present studies aimed to establish a marmoset induced neurons directly converted from fibroblasts (induced neurons; iNs) as well as induced neural stem cells (iNSCs) that are further differentiated into neurons to quantify the spontaneous presence of AD‐related pathology in marmoset cell cultures in vitro.MethodMarmoset fibroblasts were cultured from skin biopsies and reprogrammed into iNSCs by transfection with episomal reprogramming vectors carrying the Oct4, Sox2, Nanog, Lin28, Klf4, and L‐Myc and culture in the presence of small molecule inhibitors CHIR99021 and SB431542. The iNSCs were subsequently differentiated into neurons. Direct neuronal conversion from the marmoset fibroblast was performed by co‐overexpression of Ngn2 and Ascl1 under the control of the doxycycline‐inducible tetOn promoter and supplementation of medium containing doxycycline in combination with a defined cocktail of small molecule‐based pathway modulators.ResultHighly proliferative marmoset iNSCs were established and expressed neural stem cell markers, including Sox2 and Pax6 when detected by immunofluorescence. iNSCs were differentiated into neurons and stained the neuron‐specific markers such as Tuj1, Map2, and NeuN. The Ngn2 and Ascl1 overexpressed marmoset fibroblasts underwent a morphological change into a neuron‐like structure within a few hours of neuronal induction and the resultant iNs were expressed the pan‐neuronal markers in the neurite‐like projection and cell bodies.ConclusionThe directly reprogrammed iNSC and iNs have been shown to have neuronal characteristics and offer a cellular model system to quantify the natural incidence of AD‐related pathology in marmosets. The marmoset cellular system of AD will provide mechanistic insight into the etiology of the disease and may serve as a beneficial tool for drug discovery to treat the disorder.