Investigating the Impact of Adhesome Modulation on Induced Pluripotent Stem Cell Genome Stability

Introduction: : Induced pluripotent stem cells (iPSCs) have revolutionized fields such as regenerative medicine, disease modeling, and drug discovery. However, they still present challenges for large-scale adoption including high costs, low reprogramming efficiencies, and genomic instability during reprogramming [1,2]. These genomic abnormalities, including copy number variations (CNVs) and point mutations, can negatively impact downstream applications of iPSCs [1-3].

Reprogramming-induced genomic instability has been linked to the activation of oncogenic pathways, silencing of tumor suppressors, and disruption of developmental gene regulatory networks [1-3]. While advances in genomic sequencing have improved detection of these abnormalities, effective strategies to prevent their occurrence remain underdeveloped [3].

The optimization of iPSC technologies has previously leveraged biophysical cues, mechanotransduction, and mechanosignaling to improve reprogramming efficiency. Investigations in this domain typically involve modifications of culture substrates or the targeted regulation of genes and proteins involved in cellular responses to the microenvironment [4]. In the context of cancer, these interactions have been found to contribute to genomic instability [5,6]. However, the effects of these manipulations on chromosomal stability during reprogramming have not been explored.

In this investigation, we focused on genes that participate in cell-cell and cell-extracellular matrix interactions (collectively termed the “adhesome”) during iPSC generation. Preliminary results from an shRNA screen targeting 103 genes revealed a top candidate, SHROOM3, whose knockdown increased reprogramming efficiency in primary and secondary reprogramming models. We further investigate the effects of SHROOM3 knockdown on genomic stability in the search to provide a more efficient and higher genetic fidelity iPSC generation method.

Materials and

Methods: : Reprogramming was performed using an immortalized BJ fibroblast-derived cell line, known as hiF-Ts, which contain a doxycycline-inducible OKSM (stem cell transcription factor) cassette. Prior to the induction of reprogramming, the hiF-Ts were seeded and transduced with SHROOM3 shRNA or control (lacZ, a non-mammalian gene) shRNA expressing lentiviral vectors. To initiate reprogramming, growth media was supplemented with doxycycline.

On days 6, 9, 12, and 15 of reprogramming, samples were singularized, MEF depleted, and processed for RNA extraction, library preparation, and sequencing. Libraries were prepared using the 10x Genomics Chromium according to the manufacturer’s protocol and sequencing was performed on a NovoGene NovaSeq 6000.

The generated sequencing read files were aligned to the human genome and filtered to remove ambient RNA signatures prior to further analysis. R and Seurat were used to perform quality control, normalization, sample integration, clustering, cell type identification, and visualization.

Day 6 fibroblasts (earliest timepoint) and day 15 iPSCs (latest timepoint) in each knockdown condition were identified using Seurat and their raw sequencing files identified and isolated. These files were inputs for scAllele [7] and SComatic [8] to infer the number of mutations present at the beginning and end of reprogramming; these values were subtracted to calculate the number of mutations introduced during reprogramming.

CNVs were calculated using the hPSC Genetic Analysis Kit from STEMCELL Technologies according to the manufacturer’s protocol.

Results, Conclusions, and Discussions:: Leveraging our existing reprogramming time course single-cell RNA sequencing data, we sought to understand the impacts of SHROOM3 knockdown on genomic stability. To assess the accumulation of point mutations and short insertions and deletions over the course of reprogramming, we used and compared two computational mutation calling pipelines [7,8]. According to these inferences, control knockdown cells acquired 5,390 (scAllele) and 6,995 (SComatic) new variants during reprogramming, whereas SHROOM3 knockdown cells acquired only 898 and 1,251 new variants, respectively (Fig. 1a). This reduction in variant burden suggests that SHROOM3 knockdown may protect cells from reprogramming-induced mutagenesis.

To further investigate this potential protective effect, we employed an orthogonal, qPCR-based method to assess CNVs in our iPSCs generated in each condition at the eight most common sites for karyotypic abnormalities in PSCs. Our results revealed copy number gains in the control knockdown condition, with 6 of 8 loci showing significant deviations from diploid levels (average copy number: 2.84). In contrast, the SHROOM3 knockdown condition exhibited minimal CNVs with only 4 of 8 loci altered and an average copy number of 1.93, closely matching the diploid control DNA (Fig. 1b,c). Importantly, the autosomal CNV distribution in SHROOM3 knockdown iPSCs was not statistically different from the control DNA, suggesting near-native chromosomal integrity at these sites.

Our findings present SHROOM3 as a potential regulator of genome integrity during reprogramming in addition to a factor for improving reprogramming efficiency. Preliminary pathway analysis using our single-cell RNA sequencing data suggests this shift may be linked to increased expression of native antioxidants and upregulation of DNA repair pathways. These results highlight the potential of adhesome interactions to modulate genomic stability and overcome this negative byproduct of reprogramming.

Acknowledgements and/or References (Optional):: This work was supported, in large part, by a National Institutes of Health (NIH) New Innovator Award Number DP2CA250382-01 to T.L.D. N.O. was partially supported by the NIH under Award Number T32NS082174. T.A.Q.P. was partially supported by the NIH under Award Number T32HL116270. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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