Selective Elimination of Culture-Adapted Human Embryonic Stem Cells with BH3 Mimetics

Abstract

SummaryThe selective survival advantage of culture-adapted human embryonic stem cells (hESCs) is a serious safety concern for their clinical application. With a set of hESCs with various passage numbers, we observed that a subpopulation of hESCs at late passage numbers was highly resistant to various cell death stimuli, such as YM155, a survivin inhibitor. Transcriptome analysis from YM155-sensitive (YM155S) and YM155-resistant (YM155R) hESCs demonstrated that BCL2L1 was highly expressed in YM155R hESCs. By matching the gene signature of YM155R hESCs with the Cancer Therapeutics Response Portal dataset, BH3 mimetics were predicted to selectively ablate these cells. Indeed, short-course treatment with a sub-optimal dose of BH3 mimetics induced the spontaneous death of YM155R, but not YM155S hESCs by disrupting the mitochondrial membrane potential. YM155S hESCs remained pluripotent following BH3 mimetics treatment. Therefore, the use of BH3 mimetics is a promising strategy to specifically eliminate hESCs with a selective survival advantage.

TPX2 Amplification-Driven Aberrant Mitosis in Long-Term Cultured Human Embryonic Stem Cells

“…Through massive array comparative genomic hybridization (aCGH), the sub-chromosomal amplification of the 20q11.21 locus has been reported to be the most frequent copy number variant (CNV) with normal a karyotype (4,7–9) derived from in vitro culture because the gain of 20q11.21 does not occur in normal embryos (10). In particular, BCL2L1 in 20q11.21 has been identified as a driver mutation for survival advantages in cultures (11, 12), which also results from an independent loss-of-function mutation in p53 (13). Other than the survival advantage conferred by the amplification of BCL2L1 or the TP53 mutation, the other genetic alterations have not been closely characterized, with a few notable exceptions (14), although various abnormal behaviors, such as tumor formation (15, 16) and impaired differentiation (17), in their progenies or hPSCs themselves were reported in animal models.…”

“…Trisomy of chromosomes 12 or 17, which is frequent in embryonal carcinomas (21), is commonly found in hPSCs due to the survival benefit of these abnormalities in culture (20). Recent studies demonstrated that aneuploid hPSCs (with trisomy in chromosome 12) showed increased proliferation and impaired differentiation in vivo (12, 17) and also developed tumors when differentiated cells were transplanted (15,16,22). However, to date, further molecular mechanisms underlying chromosomal instability (CIN) upon in vitro propagation have remained largely unknown, with the exception of a few studies that have revealed that reduced serum response factor (SRF) expression (23) or escape from mitotic cell death upon mitotic errors is responsible for ongoing CIN in aneuploid hPSCs (24).…”

Salicylic diamines selectively eliminate residual undifferentiated cells from pluripotent stem cell-derived cardiomyocyte preparations

“…Residual PSCs in a PSC-derived cell population represent a major obstacle for the clinical translation of stem cell research. Various strategies have been reported for the removal of contaminating PSCs ranging from genetic 18 , 27 – 34 and immunologic 9 – 14 selection methods, metabolic 23 – 26 or phototoxic ablation strategies 18 , 51 to PSC-selective cytotoxic compounds 35 – 42 . Among them, cytotoxic SMs represent a very attractive approach because they offer simplicity, accessibility, scalability and flexibility in combinatorial application at specific times, durations and doses in various platforms without the need for additional cell processing or genetic modification.…”

“…Among them, small synthetic molecules (SMs) represent powerful tools because they offer simplicity, accessibility, scalability and flexibility in combinatorial application at specific times, durations and doses, which represents an advantage compared to genetic and biologic approaches. Although several SMs have already demonstrated their utility for selective PSC removal 35 – 42 , rigorous assessment of their effects on differentiated cells has not been performed in most of these studies and not all available SMs might be equally suitable for purging of PSCs from different PSC-derivatives.…”

Structure-Activity Relationship Analysis of YM155 for Inducing Selective Cell Death of Human Pluripotent Stem Cells

“…Human ESCs (H9, Wicell Research Institute) and hiPSCs (SES8) (Lee et al, 2010) were maintained in ESC medium (Stem Cell Technology, # 29106). For treatment of YM155 and YM155 analogs, hESCs or hiPSCs were cultured in mTeSR1 medium (with 50× supplement, 0.1% gentamycin) on Matrigel (Corning, # 354277)-coated 60mm tissue culture dishes (Corning, # 430166) or tissue culture 6, 12, 24-well plate (Falcon, # 353046, # 353043, # 353047), as described previously (Cho et al, 2015, 2016, 2018; Jeong et al, 2017b; Kim S. Y. et al, 2017). Smooth muscle cells (hASMC and SMC3) (Lee et al, 2010) were cultured in SMCM medium (ScienCell Research Laboratories, # 1101).…”