Direct cell reprogramming to study Alzheimer's disease

Abstract

Alzheimer’s disease (AD) is the most common neurodegenerative disease worldwide, affecting approximately 35 million people. AD is characterised by progressive deficits in memory, cognitive decline, and the eventual deterioration of bodily functions. Despite its high prevalence, the knowledge of causative disease mechanisms is limited, resulting in a current lack of effective treatments for AD. The limited availability of live human neurons for AD research has resulted in a reliance on inferences obtained from post-mortem tissue, animal models, and immortalised cell lines that hold inherent limitations of their accuracy to in vivo disease mechanisms. Ground-breaking research into cell reprogramming technologies, including the generation of induced pluripotent stem cells (iPSCs) and direct reprogramming techniques, has been a major focus for AD research due to the ability to generate live human neuron disease models. Research within our lab has previously detailed the direct reprogramming of adult human fibroblasts to induced neural progenitors (iNPs) which are capable of differentiating into mature neurons. Utilising this protocol to develop a cell based model of AD would provide an attractive option for further research into the mechanisms underlying AD. This study details the first attempt to generate a cholinergic neural subtype from SOX2/PAX6 SNIM® RNA reprogrammed iNPs. Following previously established basal forebrain cholinergic neuron differentiation protocols, we were successful in the generation of iNPs and neurons. However, these neurons lacked the expression of key cholinergic genes and proteins. The limited expression of key cholinergic genes identifies the requirement for protocol optimisation as the next step for future research, and the subsequent extension of SOX2/PAX6 SNIM® RNA reprogramming to generate iNPs and neurons from cells donated by patients with AD. In addition, we investigated the translation of our previously developed direct cell reprogramming strategy to the use of an alternative source of donor cells. Cell reprogramming research has increasingly seen the use of alternative cell sources to the traditional use of fibroblasts for cell reprogramming. Previous research has identified that donor somatic cell type affects the derived cell phenotypes and reprogramming efficiencies. Blood-derived donor cells have been shown to be capable of reprogramming into iPSCs and iNPs which, through the minimum invasiveness of collection, offers a potentially improved donor cell source for AD research. This study outlines the initial stages of developing a protocol for blood cell reprogramming to iNPs based on our current protocol for the generation of iNPs from fibroblasts via SOX2/PAX6 SNIM® RNA transfection. This study demonstrated that amenable hematopoietic progenitor cell populations can be isolated and successfully transfected with SNIM® RNA, supporting the extension of our previously developed SOX2/PAX6 SNIM® RNA reprogramming protocols. Variability in current trials identifies the need for further protocol optimisation prior to increasing the scale of research. However, this study identifies the potential extension of SNIM® RNA reprogramming protocols to use blood cells as an attractive alternative donor source for modelling AD.