Uncovering low-level mosaicism in human embryonic stem cells using high throughput single cell shallow sequencing

Abstract

Human pluripotent stem cells (hPSCs) have significant levels of low-grade genetic mosaicism, which commonly used techniques fail to detect in bulk DNA. These copy number variations remain a hurdle for the clinical translation of hPSC, as their effect in vivo ranges from unknown to dangerous, and the ability to detect them will be necessary as the field advances. As such there is need for techniques which can efficiently analyse genetic content in single cells with higher throughput and lower costs. We report here on the use of the Fluidigm C1 single cell WGA platform in combination with shallow whole genome sequencing to analyse the genetic content of single hPSCs. From a hPSC line carrying an isochromosome 20, 56 single cells were analysed and found to carry a total of 50 aberrations, across 23% of cells, which could not be detected by bulk analysis. Aberrations were predominantly segmental gains, with a fewer number of segmental losses and aneuploidies. Interestingly, 40% of the breakpoints seen here correspond to known DNA fragile sites. Our results therefore demonstrate the feasibility of single cell shallow sequencing of hPSC and further expand upon the biological importance and frequency of single cell mosaicism in hPSC.

Gains of 12p13.31 delay WNT-mediated initiation of hPSC differentiation and promote residual pluripotency in a cell cycle dependent manner

“…The smallest known gain of 20q.11.21 is less than 1Mb in size, and we show here gains of 12p13.31 as small as 2Mb, both falling below the detection threshold of typically used banding techniques. Furthermore, both gains have also been identified as low level mosaics in hPSC (Jacobs et al, 2014;Keller et al, 2019). Given the impact of gains of 12p13.31 on hPSC differentiation, this raises the possibility of unidentified mosaic populations interfering with differentiation outcomes by creating heterogenous cell populations, potentially impacting research outcomes.…”

Advanced Materials to Enhance Central Nervous System Tissue Modeling and Cell Therapy

“…Another challenge associated with stem cell manufacturing is the need for reliable and cost-effective high-throughput analytics to determine identity, purity, and potency of the cell products in vitro, amenable to high-scale production. [166][167][168] Detailed characterization of lineage commitment dynamics of differentiating hESCs that has reached unprecedented resolution with the use of single-cell RNAseq [169][170][171][172] will aid in the development of improved analytics as well as enhanced purification methods to obtain high yields of highly pure cell populations. [169][170][171][172] Remaining challenges associated with implantation of stem cell-derived products into the CNS can also be tackled by engineering advanced materials.…”

Evaluation of tools for identifying large copy number variations from ultra-low-coverage whole-genome sequencing data

“…Low-coverage high-throughput single cell sequencing has also emerged in recent years and has been applied to study e.g. low-level mosaicism introduced by differing CNVs in cell subpopulations in cultured hESC samples [ 13 ]. In addition to the versatility of applications of low-coverage sequencing, the advantages of this approach also include lower costs and less computational resources and storage capacity compared to high-coverage sequencing.…”