Development of an efficient single-cell cloning and expansion strategy for genome edited induced pluripotent stem cells

Abstract

Disease-specific human induced pluripotent stem cells (hiPSCs) can be generated directly from individuals with known disease characteristics or alternatively be modified using genome editing approaches to introduce disease causing genetic mutations to study the biological response of those mutations. The genome editing procedure in hiPSCs is still inefficient, particularly when it comes to homology directed repair (HDR) of genetic mutations or targeted transgene insertion in the genome and single cell cloning of edited cells. In addition, genome editing processes also involve additional cellular stresses such as trouble with cell viability and genetic stability of hiPSCs. Therefore, efficient workflows are desired to increase genome editing application to hiPSC disease models and therapeutic applications. Apart from genome editing efficiency, hiPSC survival following single-cell cloning has proved to be challenging and has thus restricted the capability to easily isolate homogeneous clones from edited hiPSCs. To this end, we demonstrate an efficient workflow for feeder-free single cell clone generation and expansion in both CRISPR-mediated knock-out (KO) and knock-in (KI) hiPSC lines. Using StemFlex medium and CloneR supplement in conjunction with Matrigel cell culture matrix, we show that cell viability and expansion during single-cell cloning in edited and unedited cells is significantly enhanced. Our reliable single-cell cloning and expansion workflow did not affect the biology of the hiPSCs as the cells retained their growth and morphology, expression of various pluripotency markers and normal karyotype. This simplified and efficient workflow will allow for a new level of sophistication in generating hiPSC-based disease models to promote rapid advancement in basic research and also the development of novel cellular therapeutics.

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

“…Unwanted editing outcomes are ignored when looking at pooled populations, and we recommend isolating and screening clonal cell lines to perform quantitative studies of desired cellular processes, as they provide a more reliable readout of protein expression, where variations in signal between cells within a population represent true cell-to-cell variability. Clonal isolation is notoriously inefficient in iPSCs, which are programmed to undergo dissociation-induced apoptosis upon loss of attachment or cell-cell contact ( Bhargava et al, 2022 ; Chen and Pruett-Miller, 2018 ; Singh, 2019 ; Tristan et al, 2023 ; Watanabe et al, 2007 ). We optimized a protocol for efficient single-cell recovery of 201B7 iPSCs after FACS, with up to 80% of wells in a 96-well plate showing clonal growth after 10 days ( Figure 3A and Figure 3—figure supplement 1A ).…”

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

“…Therefore, it is difficult to achieve the desirable knockdown in all the cultured cells. An alternative to generate knockout clones is the use of knock-out strategies in hiPSCs 54 and the use of these cells to generate airway epithelial cells. Another, albeit suboptimal, alternative is establishing an immortalized PBEC line in order to clonally expand gene-edited cells 55 .…”

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

“…Unwanted editing outcomes are ignored when looking at pooled populations, and we recommend isolating and screening clonal cell lines to perform quantitative studies of desired cellular processes, as they provide a more reliable readout of protein expression, where variations in signal between cells within a population represent true cell-to-cell variability. Clonal isolation is notoriously inefficient in iPSCs, which are programmed to undergo dissociation-induced apoptosis upon loss of attachment or cell-cell contact (Bhargava et al, 2022; Chen & Pruett-Miller, 2018; Singh, 2019; Tristan et al, 2023; Watanabe et al, 2007). We optimized a protocol for efficient single-cell recovery of 201B7 iPSCs after FACS, with up to 80% of wells in a 96-well plate showing clonal growth after 10 days (Fig.…”