Reprogramming fibroblasts and peripheral blood cells from a C9ORF72 patient: A proof‐of‐principle study

Abstract

As for the majority of neurodegenerative diseases, pathological mechanisms of amyotrophic lateral sclerosis (ALS) have been challenging to study due to the difficult access to alive patients' cells. Induced pluripotent stem cells (iPSCs) offer a useful in vitro system for modelling human diseases. iPSCs can be theoretically obtained by reprogramming any somatic tissue although fibroblasts (FB) remain the most used cells. However, reprogramming peripheral blood cells (PB) may offer significant advantages. In order to investigate whether the choice of starting cells may affect reprogramming and motor neuron (MNs) differentiation potential, we used both FB and PB from a same C9ORF72‐mutated ALS patient to obtain iPSCs and compared several hallmarks of the pathology. We found that both iPSCs and MNs derived from the two tissues showed identical properties and features and can therefore be used interchangeably, giving the opportunity to easily obtain iPSCs from a more manageable source of cells, such as PB.

Modelling frontotemporal dementia using patient-derived induced pluripotent stem cells

“…Nonetheless, it is also challenging as similar to other repeat expansions, the C9ORF72 HRE is prone to genomic instability (196) leading to HRE mosaicism which is an additional source of genetic heterogeneity in iPSC cultures. Examples of HRE genomic instability and subsequent mosaicism in cell cultures are evident across published C9ORF72 iPSC studies (197)(198)(199)(200)(201). Moreover, all three potential disease mechanisms leading to neurodegenerative phenotypes co-exist in patientderived iPSC models, and although this adds to their physiological relevance, the contribution of each to cellular phenotypes cannot be easily dissected.…”

NEK1haploinsufficiency worsens DNA damage but not defective ciliogenesis inC9ORF72patient-derived iPSC-motoneurons

“…Primary fibroblasts of the double mutant C9ORF72 / NEK1 patient were isolated from skin biopsy as previously described (Onesto et al, 2016) after written informed consent and approval by the ethics committee (2015-03-31-07). iPSC reprogramming was performed using the CytoTune®-iPS 2.0 Sendai Reprogramming Kit ( Thermo Fisher Scientific ) as previously described (Bardelli et al, 2020). The obtained iPSC clones were cultured in E8 medium ( Thermo Fisher Scientific ) and passaged twice a week using 0.5 mM PBS/EDTA.…”

“…Expression of stemness markers was assessed by reverse-transcription PCR (RT-PCR) using the primers already described (Bardelli et al, 2020). iPSC lines derived from a wild-type healthy individual (Bossolasco et al, 2018) and a mutant C9ORF72 patient were previously generated and characterized as described (Bardelli et al, 2020).…”

“…Briefly, iPSCs were grown in suspension for 7 days to generate embryoid bodies (EBs) and they were then plated on Matrigel-coated coverslips and grown in E8 medium for additional 10 days. Expression of stemness markers was assessed by reverse-transcription PCR (RT-PCR) using the primers already described (Bardelli et al, 2020). iPSC lines derived from a wild-type healthy individual (Bossolasco et al, 2018) and a mutant C9ORF72 patient were previously generated and characterized as described (Bardelli et al, 2020).…”

Balancing serendipity and reproducibility: Pluripotent stem cells as experimental systems for intellectual and developmental disorders

“…Heat-stabilized bFGF can reduce fluctuations in cell signaling and spontaneous differentiation ( Daniszewski et al., 2018 ; Lotz et al., 2013 ). Studies have assessed specific aspects of iPSC culture, including effects of different maintenance media on gene expression ( Daniszewski et al., 2018 ) and of different somatic cells of origin on motor neuron differentiation ( Bardelli et al., 2020 ). Systematic, comprehensive review and meta-analysis will be needed to understand how iPSC derivation methods and/or culture conditions affect neural differentiation.…”