Panther, L. | Search bioRxiv

bioRxiv2021

Generation of iPSC lines with high cytogenetic stability from peripheral blood mononuclear cells (PBMCs)

The utility of human induced pluripotent stem cells (hiPSCs) is contingent upon genomic integrity and stability. Recurrent genomic aberrations have been observed in human iPSC lines upon long-term culture, [~]10-25% demonstrate karyotype abnormalities. We describe a new and reliable non-integrating episomal plasmid reprogramming method for fresh (unexpanded) peripheral blood mononuclear cells (PBMC) into iPSCs (PBMC-iPSCs). PBMC-iPSCs produced using this method have a superior chromosome-level karyotype stability rate ([~]5% abnormality rate for all chromosomes; 2.8% for autosomes). After extended culture PBMC-iPSCs maintain a low rate of abnormalities (2% for autosomes). Deep coverage whole genome sequencing in a subset of PBMC-iPSC lines showed no shared single nucleotide polymorphisms (SNPs) or structural variants are introduced during reprogramming and maintenance of PBMC-iPSCs. iPSCs reprogrammed from unexpanded PBMCs have consistently high cytogenetic stability and minimal genomic aberrations, suggesting this method is highly suited for iPSCs in research and therapeutic clinical applications.

cell biology↗

bioRxiv2020

An integrated multi-omic analysis of iPSC-derived motor neurons from C9ORF72 ALS patients

Neurodegenerative diseases present a challenge for systems biology, due to the lack of reliable animal models and the difficulties in obtaining samples from patients at early stages of disease, when interventions might be most effective. Studying induced pluripotent stem cell (iPSC)-derived neurons could overcome these challenges and dramatically accelerate and broaden therapeutic strategies. Here we undertook a network-based multi-omic characterization of iPSC-derived motor neurons from ALS patients carrying genetically dominant hexanucleotide expansions in C9orf72 to gain a deeper understanding of the relationship between DNA, RNA, epigenetics and protein in the same pool of tissue. ALS motor neurons showed the expected C9orf72-related alterations to specific nucleoporins and production of dipeptide repeats. RNA-seq, ATAC-seq and data-independent acquisition mass-spectrometry (DIA-MS) proteomics were then performed on the same motor neuron cultures. Using integrative computational methods that combined all of the omics, we discovered a number of novel dysregulated pathways including biological adhesion and extracellular matrix organization and disruption in other expected pathways such as RNA splicing and nuclear transport. We tested the relevance of these pathways in vivo in a C9orf72 Drosophila model, analyzing the data to determine which pathways were causing disease phenotypes and which were compensatory. We also confirmed that some pathways are altered in late-stage neurodegeneration by analyzing human postmortem C9 cervical spine data. To validate that these key pathways were integral to the C9 signature, we prepared a separate set of C9orf72 and control motor neuron cultures using a different differentiation protocol and applied the same methods. As expected, there were major overall differences between the differentiation protocols, especially at the level of in individual omics data. However, a number of the core dysregulated pathways remained significant using the integrated multiomic analysis. This new method of analyzing patient specific neural cultures allows the generation of disease-related hypotheses with a small number of patient lines which can be tested in larger cohorts of patients.

neuroscience↗

bioRxiv2020

Generation of twenty four induced pluripotent stem cell lines from twenty four members of the Lothian Birth Cohort 1936

Cognitive decline is among the most feared aspects of ageing. We have generated induced pluripotent stem cells (iPSCs) from 24 people from the Lothian Birth Cohort 1936, whose cognitive ability was tested in childhood and in older age. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using non-integrating oriP/EBNA1 backbone plasmids expressing six iPSC reprogramming factors (OCT3/4 (POU5F1), SOX2, KLF4, L-Myc, shp53, Lin28, SV40LT). All lines demonstrated STR matched karyotype and pluripotency was validated by multiple methods. These iPSC lines are a valuable resource to study molecular mechanisms underlying individual differences in cognitive ageing and resilience to age-related neurodegenerative diseases.

cell biology↗