Gain of 20q11.21 in Human Pluripotent Stem Cells Impairs TGF-β-Dependent Neuroectodermal Commitment

Abstract

Summary Gain of 20q11.21 is one of the most common recurrent genomic aberrations in human pluripotent stem cells. Although it is known that overexpression of the antiapoptotic gene Bcl-xL confers a survival advantage to the abnormal cells, their differentiation capacity has not been fully investigated. RNA sequencing of mutant and control hESC lines, and a line transgenically overexpressing Bcl-xL , shows that overexpression of Bcl-xL is sufficient to cause most transcriptional changes induced by the gain of 20q11.21. Moreover, the differentially expressed genes in mutant and Bcl-xL overexpressing lines are enriched for genes involved in TGF-β- and SMAD-mediated signaling, and neuron differentiation. Finally, we show that this altered signaling has a dramatic negative effect on neuroectodermal differentiation, while the cells maintain their ability to differentiate to mesendoderm derivatives. These findings stress the importance of thorough genetic testing of the lines before their use in research or the clinic.

Functional in vivo and in vitro effects of 20q11.21 genetic aberrations on hPSC differentiation

“…Both results suggest that there are hurdles in ectodermal lineage commitment in hPSCs due to the CNV gain at 20q11.21. This result is consistent with a recent study that reported that CNV gain at 20q11.21 in hPSCs leads to impaired neuroectodermal differentiation 40 . Markouli et al 40 .…”

Genetic barcoding reveals clonal dominance in iPSC-derived mesenchymal stromal cells

“…This region includes the gene BCL2L1, an antiapoptotic factor that favors clonal outgrowth [40,41]. Notably, this genetic aberration may also impair the differentiation of pluripotent cells [41], and this might explain why iPSCs of donor 1 repeatedly failed to differentiate towards iMSCs.…”

Genomic Stability Testing of Pluripotent Stem Cells

“…Recurrent genetic aberrations in PSCs are not only genotypically similar to those seen in cancer, but the consequential cellular phenotypes are comparable as well. Effects include escaping apoptosis (Avery et al., 2013; Merkle et al., 2017), decreased differentiation capacity (Markouli et al., 2019), increased tumorigenicity (Ben‐David et al., 2014), and faster cell cycling (Barbaric et al., 2014). Therefore, detecting an abnormality can help explain difficulty with a directed differentiation protocol or indicate a newly adopted culture practice as suboptimal.…”

“…Current Protocols in Stem Cell Biology capacity (Markouli et al, 2019), increased tumorigenicity (Ben-David et al, 2014), and faster cell cycling (Barbaric et al, 2014). Therefore, detecting an abnormality can help explain difficulty with a directed differentiation protocol or indicate a newly adopted culture practice as suboptimal.…”

“…PSCs can acquire genetic changes during routine cell culture with certain changes reported as recurrent (Baker et al., 2007; Draper et al., 2004) and pervasive across cell lines and culture platforms (Taapken et al., 2011). Causes of these recurrent changes are still being determined, but the functional consequences enhance variant cells with a competitive advantage over wild‐type cells via pro‐growth, anti‐apoptotic, and anti‐differentiation phenotypes (Avery et al., 2013; Barbaric et al., 2014; Markouli et al., 2019; Zhang et al., 2019). Variant cells may overtake the culture and supplant wild‐type cells entirely.…”