Epigenetic Enzymes, Age, and Ancestry Regulate the Efficiency of Human iPSC Reprogramming.

Epigenetic enzymes regulate higher-order chromatin architecture and cell-type specific gene expression. The ATPase BRG1 and the SWI/SNF chromatin remodeling complex are epigenetic enzymes that regulate chromatin accessibility during steady and transitional cell states. Experiments in mice show that the loss of BRG1 inhibits cellular reprogramming, while studies using human cells demonstrate that the overexpression of BRG1 enhances reprogramming. We hypothesized that the variation of SWI/SNF subunit expression in the human population would contribute to variability in the efficiency of induced pluripotent stem cells (iPSC) reprogramming. To examine the impact of an individual's sex, ancestry, and age on iPSC reprogramming, we created a novel sex and ancestry balanced cohort of 240 iPSC lines derived from human dermal fibroblasts (DF) from 80 heathy donors. We methodically assessed the reprogramming efficiency of each DF line and then quantified the individual and demographic-specific variations in SWI/SNF chromatin remodeling proteins and mRNA expression. We identified BRG1, BAF155, and BAF60a expression as strongly correlating with iPSC reprogramming efficiency. Additionally, we discovered that high efficiency iPSC reprograming is negatively correlated with donor age, positively correlated with African American descent, and uncorrelated with donor sex. These results show the variations in chromatin remodeling protein expression have a strong impact on iPSC reprogramming. Additionally, our cohort is unique in its large size, diversity, and focus on healthy donors. Consequently, this cohort can be a vital tool for researchers seeking to validate observational results from human population studies and perform detailed mechanistic studies in a controlled cell culture environment. Stem Cells 2018;36:1697-1708.

A co-culture genome-wide RNAi screen with mammary epithelial cells reveals transmembrane signals required for growth and differentiation.

INTRODUCTION: The extracellular signals regulating mammary epithelial cell growth are of relevance to understanding the pathophysiology of mammary epithelia, yet they remain poorly characterized. In this study, we applied an unbiased approach to understanding the functional role of signalling molecules in several models of normal physiological growth and translated these results to the biological understanding of breast cancer subtypes. METHODS: We developed and utilized a cytogenetically normal clonal line of hTERT immortalized human mammary epithelial cells in a fibroblast-enhanced co-culture assay to conduct a genome-wide small interfering RNA (siRNA) screen for evaluation of the functional effect of silencing each gene. Our selected endpoint was inhibition of growth. In rigorous postscreen validation processes, including quantitative RT-PCR, to ensure on-target silencing, deconvolution of pooled siRNAs and independent confirmation of effects with lentiviral short-hairpin RNA constructs, we identified a subset of genes required for mammary epithelial cell growth. Using three-dimensional Matrigel growth and differentiation assays and primary human mammary epithelial cell colony assays, we confirmed that these growth effects were not limited to the 184-hTERT cell line. We utilized the METABRIC dataset of 1,998 breast cancer patients to evaluate both the differential expression of these genes across breast cancer subtypes and their prognostic significance. RESULTS: We identified 47 genes that are critically important for fibroblast-enhanced mammary epithelial cell growth. This group was enriched for several axonal guidance molecules and G protein-coupled receptors, as well as for the endothelin receptor PROCR. The majority of genes (43 of 47) identified in two dimensions were also required for three-dimensional growth, with HSD17B2, SNN and PROCR showing greater than tenfold reductions in acinar formation. Several genes, including PROCR and the neuronal pathfinding molecules EFNA4 and NTN1, were also required for proper differentiation and polarization in three-dimensional cultures. The 47 genes identified showed a significant nonrandom enrichment for differential expression among 10 molecular subtypes of breast cancer sampled from 1,998 patients. CD79A, SERPINH1, KCNJ5 and TMEM14C exhibited breast cancer subtype-independent overall survival differences. CONCLUSION: Diverse transmembrane signals are required for mammary epithelial cell growth in two-dimensional and three-dimensional conditions. Strikingly, we define novel roles for axonal pathfinding receptors and ligands and the endothelin receptor in both growth and differentiation.

Differential responses to retinoic acid and endocrine disruptor compounds of subpopulations within human embryonic stem cell lines.

The heterogeneous nature of stem cells is an important issue in both research and therapeutic use in terms of directing cell lineage differentiation pathways, as well as self-renewal properties. Using flow cytometry we have identified two distinct subpopulations by size, large and small, within cultures of human embryonic stem (hES) cell lines. These two cell populations respond differentially to retinoic acid (RA) differentiation and several endocrine disruptor compounds (EDC). The large cell population responds to retinoic acid differentiation with greater than a 50% reduction in cell number and loss of Oct-4 expression, whereas the number of the small cell population does not change and Oct-4 protein expression is maintained. In addition, four estrogenic compounds altered SSEA-3 expression differentially between the two cell subpopulations changing their ratios relative to each other. Both populations express stem cell markers Oct-4, Nanog, Tra-1-60, Tra-1-80 and SSEA-4, but express low levels of differentiation markers common to the three germ layers. Cloning studies indicate that both populations can revive the parental population. Furthermore, whole genome microarray identified approximately 400 genes with significantly different expression between the two populations (p<0.01). We propose the differential response to RA in these populations is due to differential gene expression of Notch signaling members, CoupTF1 and CoupTF2, chromatin remodeling and histone modifying genes that render the small population resistant to RA differentiation. The findings that hES cells exist as heterogeneous populations with distinct responses to differentiation signals and environmental stimuli will be relevant for their use for drug discovery and disease therapy.

Ancestry-dependent gene expression correlates with reprogramming to pluripotency and multiple dynamic biological processes.

Induced pluripotent stem cells (iPSCs) can be derived from differentiated cells, enabling the generation of personalized disease models by differentiating patient-derived iPSCs into disease-relevant cell lines. While genetic variability between different iPSC lines affects differentiation potential, how this variability in somatic cells affects pluripotent potential is less understood. We generated and compared transcriptomic data from 72 dermal fibroblast-iPSC pairs with consistent variation in reprogramming efficiency. By considering equal numbers of samples from self-reported African Americans and White Americans, we identified both ancestry-dependent and ancestry-independent transcripts associated with reprogramming efficiency, suggesting that transcriptomic heterogeneity can substantially affect reprogramming. Moreover, reprogramming efficiency-associated genes are involved in diverse dynamic biological processes, including cancer and wound healing, and are predictive of 5-year breast cancer survival in an independent cohort. Candidate genes may provide insight into mechanisms of ancestry-dependent regulation of cell fate transitions and motivate additional studies for improvement of reprogramming.

Mitochondrial hTERT exacerbates free-radical-mediated mtDNA damage.

Telomerase is often re-activated in human cancers and is widely used to immortalize cells in culture. In addition to the maintenance of telomeres, telomerase has been implicated in cell proliferation, genomic instability and apoptosis. Here we show that human telomerase reverse transcriptase (hTERT) is targeted to the mitochondria by an N-terminal leader sequence, and that mitochondrial extracts contain telomerase activity. In seven different human cell lines, mitochondrial telomerase increases hydrogen-peroxide-mediated mitochondrial DNA damage. hTERT expression did not alter the rate of hydrogen peroxide breakdown or endogenous cellular levels. Because the damaging effects of hydrogen peroxide are mediated by divalent metal ions (Fenton chemistry), we examined the levels of bioavailable metals. In all cases, higher levels of chelatable metals were found in hTERT-expressing cells. These results suggest that mitochondrial telomerase sensitizes cells to oxidative stress, which can lead to apoptotic cell death, and imply a novel function of telomerase in mitochondrial DNA transactions.

Antisense blocking of BRCA1 enhances sensitivity to plumbagin but not tamoxifen in BG-1 ovarian cancer cells.

Previous studies have shown that reduction in BRCA1 mRNA and protein can result in increased proliferation of BG-1 ovarian cancer cells in both in vitro and in vivo conditions, suggesting that BRCA1 may normally act as a growth inhibitor in these cells. Also, there are other reports that suggest that wild-type BRCA1 protein may repress estrogen receptor (ER) function either directly or indirectly. However, response to antiestrogen drugs in BRCA1-blocked ER-positive ovarian cancer cells has not been reported, and this served as the rationale for this study. We analyzed the effect of tamoxifen, emodin, and plumbagin in BRCA1-blocked ER-positive BG-1 ovarian cancer cells. For all three drugs, BRCA1-blocked cells were more sensitive than the corresponding control cells as assessed by MTT assay; however, only plumbagin showed a statistically significant difference in mean viability (P < 0.05). All three drugs induced loss of mitochondrial membrane potential (DeltaPsi(m)), nuclear condensation, DNA fragmentation, and morphological changes, as observed after 6 h of drug treatment, suggesting apoptosis induction in both BRCA1-blocked and control cells. However, apoptosis induction was greater in BRCA1-blocked cells, the efficacy being in the order of plumbagin > tamoxifen > emodin. The dose of plumbagin needed to kill 50% was 5 microM in the control cells and 2.68 microM for the BRCA1-blocked cells, indicating that the latter was about twofold more sensitive to plumbagin than the wild-type cells. This throws light on the fact that plumbagin may have chemotherapeutic potential as an anticancer agent in BRCA1-mutated ovarian cancer patients.