ARHGDIAConfers Selective Advantage to Dissociated Human Pluripotent Stem Cells

Abstract

Human pluripotent stem cells (hPSCs) have generated significant interest in the scientific community based on their potential applications in regenerative medicine. However, numerous research groups have reported a propensity for genomic alterations during hPSC culture that poses concerns for basic research and clinical applications. Work from our laboratory and others has demonstrated that amplification of chromosomal regions is correlated with increased gene expression. To date, the phenotypic association of common genomic alterations remains unclear and is a cause for concern during clinical use. In this study, we focus on trisomy 17 and a list of candidate genes with increased gene expression to hypothesize that overexpressing 17q25 located ARHGDIA will confer selective advantage to hPSCs. HPSC lines overexpressing ARHGDIA exhibited culture dominance in cocultures of overexpression lines with nonoverexpression lines. Furthermore, during low-density seeding, we demonstrate increased clonality of our ARHGDIA lines against matched controls. A striking observation is that we could reduce this selective advantage by varying the hPSC culture conditions with the addition of ROCK inhibitor (ROCKi). This work is unique in (1) demonstrating a novel gene that confers selective advantage to hPSCs when overexpressed and may help explain a common trisomy dominance, (2) providing a selection model for studying culture conditions that reduce the appearance of genomically altered hPSCs, and (3) aiding in elucidation of a mechanism that may act as a molecular switch during culture adaptation.

Standardization of Cell Culture Conditions and Routine Genomic Screening under a Quality Management System Leads to Reduced Genomic Instability in hPSCs

“…We found that the most abundant de novo pathogenic alterations acquired by hPSCs under ISO9001:2015 were an insertion in 14q23.3 (which has been associated to a cancerous phenotype) [66], followed by an insertion in 14q32 (linked to predisposition to early clonal hematopoiesis leading to myeloid neoplasms) [70], the 20q11.21 amplification and trisomies in chromosomes 8 and 12. Given that all of these alterations confer a selective advantage to variants which could lead to malignant phenotypes, it is clear that despite the improved genomic stability of hPSCs, genetic alterations which may potentially jeopardize the safety or impair the function of hPSCs are not completely removed from cultured cells.…”

“…Remarkably, most of these alterations have been associated with various cancers, notably the association of trisomy of chromosome 12 with embryonal carcinoma cells [60] or testicular germ cell tumors [61], or 20q11.21 amplification with colorectal cancer [62], cervical cancer [63] and tumorigenic transformation [64,65]. Regarding subchromosomal alterations besides gains in chromosome 20 and a small insertion in 14q23.3 [66], none of the detected CNAs is, to our knowledge, associated with malignant transformation or impaired functional properties of cultured hPSCs.…”

Distinct gene programs underpinning 'disease tolerance' and 'resistance' in influenza virus infection

“…Arhgdia was co-expressed with program T in all cell types and under various conditions. Arhgdia is known as a regulator of Rho proteins and its activity improves survival of stem cells 47 and kidney functions 48 . However, its more general role in control of disease tolerance has not been reported.…”