Individual Cell Lines Research Articles

Abstract A57: Drug sensitivity and resistance testing (DSRT) of clinically important compounds on primary ovarian cancer cell lines

Abstract The heterogeneity of chemoresponses in high-grade serous ovarian cancer (HGS-OvCa) presents a major clinical challenge. The inherited or acquired nonresponsiveness to current therapies is likely to be one reason for relapse and treatment failure. Better models for predicting effective treatment options and drug combinations are needed. Although most tumors initially respond to standard platinum-taxane combination therapy, treatment resistance eventually evolves in 80-90% of the patients. Drug sensitivity and resistance testing (DSRT) was performed by using high-throughput screening (HTS) of 306 small-drug molecules on six primary cell lines from patients with disseminated HGS-OvCa. Commonly available ovarian cancer cell line OVCAR8 was used as a control. The drug effect was investigated with help of a quantitative scoring approach, drug sensitivity score (DSS). Sixteen drugs of clinical interest were investigated in more detail. Drug sensitivity varied greatly within primary cell lines. Out of 306 compounds, 29 were effective on the most resistant cell line, whereas 102 compounds showed effect on the most sensitive line. We found that 9 out of the 16 clinically important compounds gave no response on the tested cell lines. Seven compounds provided response on the screened cell lines: four HSP90 inhibitors, an HSP70 inhibitor, a Wee1 inhibitor, and a proteasome inhibitor. Of these seven compounds, the most effective drug compounds (the Wee1 inhibitor and two HSP90 inhibitors) were chosen for validation on primary HGS-OvCa cell lines to verify the HTS result. The effectiveness of currently used clinical treatment-line drugs, cisplatin and paclitaxel, was compared to the putative novel drug compounds on the primary cell lines and the control cell line. One primary cell line was completely resistant to the validated drug compounds as well as to cisplatin and paclitaxel combination treatment. Five cell lines were sensitive to cisplatin-paclitaxel treatment, but the sensitivity to the three most effective compounds varied greatly. The commonly available cell line OVCAR8 was the most sensitive of all tested cell lines. In conclusion, we found three potential drug compounds of clinical interest effective for disseminated HGS-OvCa. The analyses demonstrate that patient-derived primary HGS-OvCa cell lines have unique heterogeneous characteristics that are likely to have an effect on the choice of best drug candidates for each individual cell line, and furthermore for each patient. Consequently, more combinatorial drug treatment strategies should be validated to find new specific personalized drug treatment strategies. Citation Format: Pia Roering, Piia Mikkonen, Swapnil Potdar, Krister Wennerberg, Johanna Hynninen, Seija Grénman, Annika Auranen, Olli Carpén, Katja Kaipio. Drug sensitivity and resistance testing (DSRT) of clinically important compounds on primary ovarian cancer cell lines. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A57.

The oncomir face of microRNA-206: A permanent miR-206 transfection study

MiR-206 is a remarkable miRNA because it functions as a suppressor miRNA in rhabdomyosarcoma while at the same time, as previously showed, it can act as an oncomiRNA in SMARCB1 immunonegative soft tissue sarcomas. The aim of this study was to investigate the effect of miR-206 on its several target genes in various human tumorous and normal cell lines. In the current work, we created miR-206-overexpressing cell lines (HT-1080, Caco2, iASC, and SS-iASC) using permanent transfection. mRNA expression of the target genes of miR-206 (SMARCB1, ACTL6A, CCND1, POLA1, NOTCH3, MET, and G6PD) and SMARCB1 protein expression were examined with quantitative real-time polymerase chain reaction, immunoblotting, immunocytochemistry, and flow cytometry. MiRNA inhibition was used to validate our results. We found a diverse silencing effect of miR-206 on its target genes. While an overall tendency of downregulation was noted, expression profiles of individual cell lines showed large variability. Only CCND1 and MET were consistently downregulated. MiR-206 had an antiproliferative effect on a normal human fibroblast cell line. A strong silencing effect of SMARCB1 in miR-206 transfected SS-iASC was most likely caused by the synergic influence of the SS18-SSX1 fusion protein and miR-206. In the same cell line, a moderate decrease of SMARCB1 protein expression could be observed with immunocytochemistry and flow cytometry. In the most comprehensive analysis of miR-206 effects so far, a modest but significant downregulation of miR-206 targets on the mRNA level was confirmed across all cell lines. However, the variability of the effect shows that the action of this miRNA is largely cell context-dependent. Our results also support the conception that the oncomiR effect of miR-206 on SMARCB1 plays an important but not exclusive role in SMARCB1 immunonegative soft tissue sarcomas so it can be considered important in planning the targeted therapy of these tumors in the future. Impact statement Mir-206 is a very unique microRNA because it can act as a suppressor miRNA or as an oncomiRNA depending on the tumor tissue. In SMARCB1 negative soft tissue sarcomas miR-206 is overexpressed, so thus in epithelioid and synovial sarcomas it functions as an oncomiRNA. MiR-206 has diverse silencing effects on its target genes. We found that the action of miR-206 is largely cell context dependent. The oncomiR role of miR-206 is crucial but not exclusive in SMARCB1 negative soft tissue sarcomas and miR-206 has an antiproliferative effect on a normal human fibroblast cell line. Expressions of miR-206 targets observed in tumors can only be reproduced in the corresponding tumorous cell lines. This is the first study which examined the permanent effect of miR-206 on its target genes in normal, tumor, and genetically engineered cell lines.

Integrative Modeling Identifies Key Determinants of Inhibitor Sensitivity in Breast Cancer Cell Lines.

Cancer cell lines differ greatly in their sensitivity to anticancer drugs as a result of different oncogenic drivers and drug resistance mechanisms operating in each cell line. Although many of these mechanisms have been discovered, it remains a challenge to understand how they interact to render an individual cell line sensitive or resistant to a particular drug. To better understand this variability, we profiled a panel of 30 breast cancer cell lines in the absence of drugs for their mutations, copy number aberrations, mRNA, protein expression and protein phosphorylation, and for response to seven different kinase inhibitors. We then constructed a knowledge-based, Bayesian computational model that integrates these data types and estimates the relative contribution of various drug sensitivity mechanisms. The resulting model of regulatory signaling explained the majority of the variability observed in drug response. The model also identified cell lines with an unexplained response, and for these we searched for novel explanatory factors. Among others, we found that 4E-BP1 protein expression, and not just the extent of phosphorylation, was a determinant of mTOR inhibitor sensitivity. We validated this finding experimentally and found that overexpression of 4E-BP1 in cell lines that normally possess low levels of this protein is sufficient to increase mTOR inhibitor sensitivity. Taken together, our work demonstrates that combining experimental characterization with integrative modeling can be used to systematically test and extend our understanding of the variability in anticancer drug response.Significance: By estimating how different oncogenic mutations and drug resistance mechanisms affect the response of cancer cells to kinase inhibitors, we can better understand and ultimately predict response to these anticancer drugs.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/15/4396/F1.large.jpg Cancer Res; 78(15); 4396-410. ©2018 AACR.

DONOR CHIMERISM IN PATIENTS WITH PRIMARY IMMUNODEFICIENCY AFTER ALLOGENEIC HEMATOPOIETIC STEM CELLS TRANSPLANTATION

Allogeneic stem cell transplantation (alloHSCT) is effective curative option for a broad range of primary immunodeficiencies (PIDs). Hematopoietic chimerism monitoring in patients with various PIDs and its connection with the outcome of alloHSCT is of great interest. In this study 16 alloHSCT in patients with PIDs were included. Three-year overall survival was 72.2 ± 12.0 %. Full donor chimerism (FDC) was achieved in 13 (81.25 %) patients. Prolonged persistence of mixed chimerism (MC) was observed in 3 (18.75 %) patients. In patients with MC in the peripheral blood, circulating T-cells are completely or predominantly of donor origin, whereas granulocytes are predominantly or completely recipient cells, and chimerism in B-cells differs significantly from 0 % chimerism to FDC. In patients with PIDs, engraftment of individual cell lines (split chimerism) could be observed. In some patients chimerism decreased during the first year after alloHSCT with its subsequent stabilization. Increasing MC is not associated with transplant rejection in PIDs. FDC in patients with PIDs provides restoration of all cell lines participating in the immune response regardless of the diagnosis, but it is associated with more frequent development of «graft-versus-host» disease (GVHD), which is a serious complication of alloHSCT and can lead to treatment-related mortality (TRM). MC/split chimerism, in which the frequency of development of GVHD is less, can also provide the formation of a full immune response and correction of other disease manifestations, but only when replacing defective cell lines according to the diagnosis.

Abstract 4631: Expression of ganglioside GD2 in Ewing sarcoma cells is not associated with specific functional characteristics or stem cell features

Abstract Safe and effective targeting of cancer with chimeric antigen receptor (CAR) engineered T cells relies on the presence of adequate tumor-associated surface antigens. We have found that Ewing sarcomas (EwS) express ganglioside GD2 and are recognized by T cells engineered with GD2-specific CARs. In an extended series of 97 EwS patient samples, 52 were GD2pos by immunohistochemistry. Based on reports that GD2 in breast cancer defines a malignant population with stem cell characteristics, we hypothesized that GD2 expression in EwS is associated with high capacity to self-renew and reinitiate tumor growth. Among 15 individual EwS cell lines with variable surface expression of GD2 by flow cytometry, GD2 expression levels were not associated with the capacities to proliferate and expand in vitro, form colonies in semi-solid medium, nor with their chemosensitivity, assessed by cell viabilities in the presence of increasing concentrations of the cytotoxic drug doxorubicin. Subpopulations with GD2hi phenotype selected from GD2pos and from GD2low EwS cell lines by cell sorting maintained their GD2hi and GD2low expression status during subsequent cultures over several weeks. The two subpopulations did not have different in vitro growth, colony-formation capacity, or chemosensitivity. In xenografting experiments, GD2hi and GD2low subpopulations of two EwS cell lines initiated tumors with comparable efficacies. To obtain direct evidence that GD2 surface expression is irrelevant for the biology of EwS cells, we performed genetic knockdown of the GD3S gene which drives biosynthesis of GD2 by CRISPR/Cas9 gene editing. GD3S gene editing resulted in effective elimination of GD2 surface expression in the GD2hi EwS cell lines TC-71, VH-64 and A4573. The knockdown did not affect the capacity of the cells to proliferate, form colonies in soft agar in vitro, nor their chemosensitivity compared to wild-type EwS cells from the individual cell lines. We conclude that GD2 expression in EwS cells, other than reported in breast cancer, is not associated with distinct functional features. Specifically, GD2 does not affect the growth characteristics, clono- and tumorigenicity and chemosensitivity of the tumor cells. Elimination of GD2pos subpopulations from heterogeneous tumors by CAR T cell targeting is therefore unlikely to eradicate the disease and will have to be combined with additional targeting strategies. Citation Format: Sareetha Kailayangiri, Bianca Altvater, Silke Jamitzky, Stefanie Lesch, Jan-Henrik Mikesch, Sonja Schelhaas, Schaefers Michael, Jutta Meltzer, Nicole Farwick, Wolfgang Hartmann, Eva Wardelmann, Petra Fischer, Jendrik Hardes, Claudia Rossig. Expression of ganglioside GD2 in Ewing sarcoma cells is not associated with specific functional characteristics or stem cell features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4631.

Abstract 3356: The EWS/FLI1 transcriptome is characterized by marked heterogeneity across Ewing sarcoma cell lines

Abstract BACKGROUND: Ewing sarcoma (ES) is the second most common pediatric bone cancer. ES cells are dependent on the activity of the EWS/FLI1 transcription factor for cell survival. EWS/FLI1 drives proliferation, dysregulates the cell cycle and establishes a block in differentiation. Despite the known dependence of Ewing sarcoma on EWS/FLI1, the exact identity of all the downstream targets of EWS/FLI1 is not known. While gene signatures have been reported for EWS/FLI1, these fail to capture the marked heterogeneity in the EWS/FLI1 transcriptome or correlate these expression changes with cellular phenotype. METHODS: Next generation RNA sequencing with deep coverage was used to generate a comprehensive analysis of the EWS/FLI1 transcriptome by silencing EWS/FLI1 in 5 different ES cell lines at two different time points. These results were organized into gene expression networks and signaling nodes were identified by weighted correlation network analysis (WGCNA). The networks were validated by a 500-gene signature of EWS/FLI1 targets using a next generation sequencing capture assay and the Illumina platform. The cellular endpoint of EWS/FLI1 silencing was evaluated by caspase 3,7 cleavage to measure apoptosis, BGAL staining to measure senescence and scratch assays to capture migration. RESULTS: Next generation RNA sequencing with siRNA silencing of EWS/FLI1 revealed marked heterogeneity in the EWS/FLI1 transcriptome among cell lines. Targets of EWS/FLI1 that were common across cell lines exhibited small magnitude gene expression changes (log fold <2), while large magnitude gene expression changes were unique to individual cell lines (log fold>2). When the log fold change in expression was restricted to greater than 2, only 13 common repressed targets and 4 common induced targets were identified in all 5 cell lines. In addition, silencing of EWS/FLI1 induced dramatically different phenotypes across cell lines, where p53 wild-type cell lines underwent senescence and the p53 mutant cell lines did not. Most cell lines did not continue to proliferate with EWS/FLI1 silencing with the exception being the widely used A673 cell line. Cellular migration did not change with EWS/FLI1 silencing in many of the cell lines in contrast to A673 cells. CONCLUSIONS: EWS/FLI1 drives different gene expression signatures and different phenotypes in unique cell lines. This diversity needs to be considered as EWS/FLI1 directed therapies are developed. Citation Format: Susan M. Kitchen-Goosen, Megan J. Bowman, Marie Adams, Penny Berger, Patrick J. Grohar. The EWS/FLI1 transcriptome is characterized by marked heterogeneity across Ewing sarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3356.

A combined tissue-engineered/insilico signature tool patient stratification in lung cancer.

Patient‐tailored therapy based on tumor drivers is promising for lung cancer treatment. For this, we combined in vitro tissue models with in silico analyses. Using individual cell lines with specific mutations, we demonstrate a generic and rapid stratification pipeline for targeted tumor therapy. We improve in vitro models of tissue conditions by a biological matrix‐based three‐dimensional (3D) tissue culture that allows in vitro drug testing: It correctly shows a strong drug response upon gefitinib (Gef) treatment in a cell line harboring an EGFR‐activating mutation (HCC827), but no clear drug response upon treatment with the HSP90 inhibitor 17AAG in two cell lines with KRAS mutations (H441, A549). In contrast, 2D testing implies wrongly KRAS as a biomarker for HSP90 inhibitor treatment, although this fails in clinical studies. Signaling analysis by phospho‐arrays showed similar effects of EGFR inhibition by Gef in HCC827 cells, under both 2D and 3D conditions. Western blot analysis confirmed that for 3D conditions, HSP90 inhibitor treatment implies different p53 regulation and decreased MET inhibition in HCC827 and H441 cells. Using in vitro data (western, phospho‐kinase array, proliferation, and apoptosis), we generated cell line‐specific in silico topologies and condition‐specific (2D, 3D) simulations of signaling correctly mirroring in vitro treatment responses. Networks predict drug targets considering key interactions and individual cell line mutations using the Human Protein Reference Database and the COSMIC database. A signature of potential biomarkers and matching drugs improve stratification and treatment in KRAS‐mutated tumors. In silico screening and dynamic simulation of drug actions resulted in individual therapeutic suggestions, that is, targeting HIF1A in H441 and LKB1 in A549 cells. In conclusion, our in vitro tumor tissue model combined with an in silico tool improves drug effect prediction and patient stratification. Our tool is used in our comprehensive cancer center and is made now publicly available for targeted therapy decisions.

Bioprocessing of Recombinant CHO-K1, CHO-DG44, and CHO-S: CHO Expression Hosts Favor Either mAb Production or Biomass Synthesis.

Chinese hamster ovary (CHO) cells comprise a variety of lineages including CHO-DXB11, CHO-K1, CHO-DG44, and CHO-S. Despite all CHO cell lines sharing a common ancestor, extensive mutagenesis, and clonal selection has resulted in substantial genetic heterogeneity among them. Data from sequencing show that different genes are missing in individual CHO cell lines and each cell line harbors a unique set of mutations with relevance to the bioprocess. However, not much literature is available about the influence of genetic differences of CHO on the performance of bioprocess operations. In this study, the host cell-specific differences among three widely used CHO cell lines (CHO-K1, CHO-S, and CHO-DG44) and recombinantly expressed the same monoclonal antibody (mAb) in an isogenic format by using bacterial artificial chromosomes (BACs) as transfer vector in all cell lines is examined. Cell-specific growth and product formation are studied in batch, fed-batch, and semi-continuous perfusion cultures. Further, two different cell culture media are used to investigate their effects. The authors find CHO cell line-specific preferences for mAb production or biomass synthesis that are determined by the host cell line. Additionally, quality attributes of the expressed mAb are influenced by the host cell line and media.

The deregulated microRNAome contributes to the cellular response to aneuploidy

BackgroundAneuploidy, or abnormal chromosome numbers, severely alters cell physiology and is widespread in cancers and other pathologies. Using model cell lines engineered to carry one or more extra chromosomes, it has been demonstrated that aneuploidy per se impairs proliferation, leads to proteotoxic as well as replication stress and triggers conserved transcriptome and proteome changes.ResultsIn this study, we analysed for the first time miRNAs and demonstrate that their expression is altered in response to chromosome gain. The miRNA deregulation is independent of the identity of the extra chromosome and specific to individual cell lines. By cross-omics analysis we demonstrate that although the deregulated miRNAs differ among individual aneuploid cell lines, their known targets are predominantly associated with cell development, growth and proliferation, pathways known to be inhibited in response to chromosome gain. Indeed, we show that up to 72% of these targets are downregulated and the associated miRNAs are overexpressed in aneuploid cells, suggesting that the miRNA changes contribute to the global transcription changes triggered by aneuploidy. We identified hsa-miR-10a-5p to be overexpressed in majority of aneuploid cells. Hsa-miR-10a-5p enhances translation of a subset of mRNAs that contain so called 5’TOP motif and we show that its upregulation in aneuploids provides resistance to starvation-induced shut down of ribosomal protein translation.ConclusionsOur work suggests that the changes of the microRNAome contribute on one hand to the adverse effects of aneuploidy on cell physiology, and on the other hand to the adaptation to aneuploidy by supporting translation under adverse conditions.

Investigation of model stacking for drug sensitivity prediction

BackgroundA significant problem in precision medicine is the prediction of drug sensitivity for individual cancer cell lines. Predictive models such as Random Forests have shown promising performance while predicting from individual genomic features such as gene expressions. However, accessibility of various other forms of data types including information on multiple tested drugs necessitates the examination of designing predictive models incorporating the various data types.ResultsWe explore the predictive performance of model stacking and the effect of stacking on the predictive bias and squared error. In addition we discuss the analytical underpinnings supporting the advantages of stacking in reducing squared error and inherent bias of random forests in prediction of outliers. The framework is tested on a setup including gene expression, drug target, physical properties and drug response information for a set of drugs and cell lines.ConclusionThe performance of individual and stacked models are compared. We note that stacking models built on two heterogeneous datasets provide superior performance to stacking different models built on the same dataset. It is also noted that stacking provides a noticeable reduction in the bias of our predictors when the dominant eigenvalue of the principle axis of variation in the residuals is significantly higher than the remaining eigenvalues.

Autophagy‑mediated adaptation of hepatocellular carcinoma cells to hypoxia‑mimicking conditions constitutes an attractive therapeutic target.

Hepatocellular carcinoma has extremely poor prognosis. In cancerous liver tissues, aberrant proliferation of cancer cells leads to the creation of an area where an immature vascular network is formed. Since oxygen is supplied to cancer tissues through the bloodstream, a part of the tumor is exposed to hypoxic conditions. As hypoxia is known to severely reduce the effectiveness of existing anticancer agents, novel valid therapeutic targets must be identified for the treatment of hepatocellular carcinoma. Generally, autophagy has been reported to play an important role in the adaptation of cancer cells to hypoxia. However, the exact role and significance of this process vary depending on the cancer type, requiring detailed analysis in individual primary tumors and cell lines. In the present study, we examined autophagy induced by cobalt chloride, a hypoxia‑mimicking agent, in hepatocellular carcinoma cells with the aim to evaluate the validity of this process as a potential therapeutic target. We observed that treatment with cobalt chloride induced autophagy, including the intracellular quality control mechanism, in an AMPK‑dependent manner. Furthermore, treatment with autophagy inhibitors (bafilomycin and LY294002) resulted in significant, highly‑selective cytotoxicity and apoptosis activation under hypoxia‑mimicking conditions. The knockdown of AMPK also revealed significant cytotoxicity in hypoxia‑mimicking conditions. These results clearly demonstrated that autophagy, especially mitophagy, was induced by the AMPK pathway when hepatocellular carcinoma cells were subjected to hypoxic conditions and played an important role in the adaptation of these cells to such conditions. Thus, autophagy may constitute an attractive therapeutic target for the treatment of hepatocellular carcinoma.

Synthesis and biological evaluation of estrone 3-O-ether derivatives containing the piperazine moiety

A series of new estrone derivatives were designed and synthesized, and their structures were confirmed by spectroscopic methods. All new estrone derivatives were investigated for their in vitro cytotoxic efficacies against a panel of three human prostate cancer cell lines (PC-3, LNCaP, and DU145). The derivatives 6, 7, 10, 15, 16, 20, 21, 22, 24 and 26 showed important cytotoxic actions against individual carcinoma cell line collections. Moreover, antagonistic activities of compounds (7, 15, 16 and 21) towards a1-ARs (α1A, α1B, and α1D) were further evaluated using dual-luciferase reporter assays, and the compounds 16 and 21 exhibited better a1-ARs subtype selectivity. The structure–activity relationship (SAR) suggested that the substitute’s type and position on the phenyl group leads to the interesting variations within pharmacological effects of resultant molecular systems.

A desirability-based multi objective approach for the virtual screening discovery of broad-spectrum anti-gastric cancer agents.

Gastric cancer is the third leading cause of cancer-related mortality worldwide and despite advances in prevention, diagnosis and therapy, it is still regarded as a global health concern. The efficacy of the therapies for gastric cancer is limited by a poor response to currently available therapeutic regimens. One of the reasons that may explain these poor clinical outcomes is the highly heterogeneous nature of this disease. In this sense, it is essential to discover new molecular agents capable of targeting various gastric cancer subtypes simultaneously. Here, we present a multi-objective approach for the ligand-based virtual screening discovery of chemical compounds simultaneously active against the gastric cancer cell lines AGS, NCI-N87 and SNU-1. The proposed approach relays in a novel methodology based on the development of ensemble models for the bioactivity prediction against each individual gastric cancer cell line. The methodology includes the aggregation of one ensemble per cell line using a desirability-based algorithm into virtual screening protocols. Our research leads to the proposal of a multi-targeted virtual screening protocol able to achieve high enrichment of known chemicals with anti-gastric cancer activity. Specifically, our results indicate that, using the proposed protocol, it is possible to retrieve almost 20 more times multi-targeted compounds in the first 1% of the ranked list than what is expected from a uniform distribution of the active ones in the virtual screening database. More importantly, the proposed protocol attains an outstanding initial enrichment of known multi-targeted anti-gastric cancer agents.

Genetic and epigenetic factors which modulate differentiation propensity in human pluripotent stem cells.

Human pluripotent stem cell (hPSC) lines are known to have a bias in their differentiation. This gives individual cell lines a propensity to preferentially differentiate towards one germ layer or cell type over others. Chromosomal aberrations, mitochondrial mutations, genetic diversity and epigenetic variance are the main drivers of this phenomenon, and can lead to a wide range of phenotypes. Our aim is to provide a comprehensive overview of the different factors which influence differentiation propensity. Specifically, we sought to highlight known genetic variances and their mechanisms, in addition to more general observations from larger abnormalities. Furthermore, we wanted to provide an up-to-date list of a growing number of predictive indicators which are able to identify differentiation propensity before the initiation of differentiation. As differentiation propensity can lead to difficulties in both research as well as clinical translation, our thorough overview could be a useful tool. Combinations of the following key words were applied as search criteria in the PubMed database: embryonic stem cells, induced pluripotent stem cells, differentiation propensity (also: potential, efficiency, capacity, bias, variability), epigenetics, chromosomal abnormalities, genetic aberrations, X chromosome inactivation, mitochondrial function, mitochondrial metabolism, genetic diversity, reprogramming, predictive marker, residual stem cell, clinic. Only studies in English were included, ranging from 2000 to 2017, with a majority ranging from 2010 to 1017. Further manuscripts were added from cross-references. Differentiation propensity is affected by a wide variety of (epi)genetic factors. These factors clearly lead to a loss of differentiation capacity, preference towards certain cell types and oftentimes, phenotypes which begin to resemble cancer. Broad changes in (epi)genetics, such as aneuploidies or wide-ranging modifications to the epigenetic landscape tend to lead to extensive, less definite changes in differentiation capacity, whereas more specific abnormalities often have precise ramifications in which certain cell types become more preferential. Furthermore, there appears to be a greater, though often less considered, contribution to differentiation propensity by factors such as mitochondria and inherent genetic diversity. Varied differentiation capacity can also lead to potential consequences in the clinical translation of hPSC, including the occurrence of residual undifferentiated stem cells, and the transplantation of potentially transformed cells. As hPSC continue to advance towards the clinic, our understanding of them progresses as well. As a result, the challenges faced become more numerous, but also more clear. If the transition to the clinic is to be achieved with a minimum number of potential setbacks, thorough evaluation of the cells will be an absolute necessity. Altered differentiation propensity represents at least one such hurdle, for which researchers and eventually clinicians will need to find solutions. Already, steps are being taken to tackle the issue, though further research will be required to evaluate any long-term risks it poses.

Using intracellular markers to identify a novel set of surface markers for live cell purification from a heterogeneous hIPSC culture

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can provide sources for midbrain dopaminergic (mDA) neural progenitors (NPCs) for cell therapy to treat Parkinson’s disease (PD) patients. However, the well-known line-to-cell line variability in the differentiation capacity of individual cell lines needs to be improved for the success of this therapy. To address this issue, we sought to identify mDA NPC specific cell surface markers for fluorescence activated cell sorting (FACS). Through RNA isolation after sorting for NPCs based on staining for cell-specific transcription factors followed by microarray, we identified two positive cell surface markers (CORIN and CD166) and one negative cell surface marker (CXCR4) for mDA NPC sorting. These three markers can enrich floor plate NPCs to 90% purity, and the sorted NPCs more efficiently differentiate to mature dopaminergic neurons compared to unsorted or CORIN+ alone mDA NPCs. This surface marker identification strategy can be used broadly to facilitate isolation of cell subtypes of interest from heterogeneous cultures.

Unique Responses of Hepatocellular Carcinoma and Cholangiocarcinoma Cell Lines toward Cantharidin and Norcantharidin.

The present study aimed to investigate whether cell lines from human gastric and liver cancers respond differently toward cantharidin (CTD) and norcantharidin (NCTD) than other types of cancer cells. We first established the half maximal inhibitory concentrations (IC50s) of CTD for a large panel of cancer cell lines representing the 12 major types of human cancers and the mode of cell death induced by the two compounds. We next compared the growth inhibitory effects as well as the corresponding modes of action of CTD and NCTD. The IncuCyte ZOOM system was used as a semi-high throughput means to define IC50s and 90% inhibitory doses (IC90s) as a reference for the maximal tolerable doses (MTDs) for the two compounds in 72 cancer cell lines. Classical clonogenic survival assay was used to assess the anti-proliferative effect of CTD on selected cell lines of interest. In addition, DNA content-based flow was used to interrogate the modes of cell death following CTD or NCTD exposure. The results of these experiments led to several findings. 1). Cell lines representing hepatocellular carcinomas (HCCs) and cholangiocarcinomas (CCs) were among the most sensitive toward CTD, consistent with the previous clinical study of this compound and its source of origin, Mylabris. 2). Among the individual cell lines of a given cancer types, the sensitivity trends for CTD and NCTD did not exhibit a good correlation. 3) CTD and NCTD caused distinctive cytotoxic effects on HepG2 cells. Specifically, while a cytostatic effect is the primary cause of growth inhibition of CTD, cytotoxic effect is the main contributing factor for the growth inhibition of NTCD. These results indicate that liver cancer cell lines are among the most sensitive to CTD and that CTD and NCTD exhibit their effects through distinct mechanisms.

Tailoring Mathematical Models to Stem-Cell Derived Cardiomyocyte Lines Can Improve Predictions of Drug-Induced Changes to Their Electrophysiology.

Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) have applications in disease modeling, cell therapy, drug screening and personalized medicine. Computational models can be used to interpret experimental findings in iPSC-CMs, provide mechanistic insights, and translate these findings to adult cardiomyocyte (CM) electrophysiology. However, different cell lines display different expression of ion channels, pumps and receptors, and show differences in electrophysiology. In this exploratory study, we use a mathematical model based on iPSC-CMs from Cellular Dynamic International (CDI, iCell), and compare its predictions to novel experimental recordings made with the Axiogenesis Cor.4U line. We show that tailoring this model to the specific cell line, even using limited data and a relatively simple approach, leads to improved predictions of baseline behavior and response to drugs. This demonstrates the need and the feasibility to tailor models to individual cell lines, although a more refined approach will be needed to characterize individual currents, address differences in ion current kinetics, and further improve these results.

CHO-Omics Review: The Impact of Current and Emerging Technologies on Chinese Hamster Ovary Based Bioproduction.

CHO cells are the most prevalent platform for modern bio-therapeutic production. Currently, there are several CHO cell lines used in bioproduction with distinct characteristics and unique genotypes and phenotypes. These differences limit advances in productivity and quality that can be achieved by the most common approaches to bioprocess optimization and cell line engineering. Incorporating omics-based approaches into current bioproduction processes will complement traditional methodologies to maximize gains from CHO engineering and bioprocess improvements. In order to highlight the utility of omics technologies in CHO bioproduction, the authors discuss current applications as well as limitations of genomics, transcriptomics, proteomics, metabolomics, lipidomics, fluxomics, glycomics, and multi-omics approaches and the potential they hold for the future of bioproduction. Multiple omics approaches are currently being used to improve CHO bioprocesses; however, the application of these technologies is still limited. As more CHO-omic datasets become available and integrated into systems models, the authors expect significant gains in product yield and quality. While individual omics technologies provide incremental improvements in bioproduction, the authors will likely see the most significant gains by applying multi-omics and systems biology approaches to individual CHO cell lines.

Impaired oxidative stress response characterizes HUWE1-promoted X-linked intellectual disability

Mutations in the HECT, UBA and WWE domain-containing 1 (HUWE1) E3 ubiquitin ligase cause neurodevelopmental disorder X-linked intellectual disability (XLID). HUWE1 regulates essential processes such as genome integrity maintenance. Alterations in the genome integrity and accumulation of mutations have been tightly associated with the onset of neurodevelopmental disorders. Though HUWE1 mutations are clearly implicated in XLID and HUWE1 regulatory functions well explored, currently much is unknown about the molecular basis of HUWE1-promoted XLID. Here we showed that the HUWE1 expression is altered and mutation frequency increased in three different XLID individual (HUWE1 p.R2981H, p.R4187C and HUWE1 duplication) cell lines. The effect was most prominent in HUWE1 p.R4187C XLID cells and was accompanied with decreased DNA repair capacity and hypersensitivity to oxidative stress. Analysis of HUWE1 substrates revealed XLID-specific down-regulation of oxidative stress response DNA polymerase (Pol) λ caused by hyperactive HUWE1 p.R4187C. The subsequent restoration of Polλ levels counteracted the oxidative hypersensitivity. The observed alterations in the genome integrity maintenance may be particularly relevant in the cortical progenitor zones of human brain, as suggested by HUWE1 immunofluorescence analysis of cerebral organoids. These results provide evidence that impairments of the fundamental cellular processes, like genome integrity maintenance, characterize HUWE1-promoted XLID.

HLA and Histo-Blood Group Antigen Expression in Human Pluripotent Stem Cells and their Derivatives

One prerequisite for a successful clinical outcome of human pluripotent stem cell (hPSC) based therapies is immune compatibility between grafted cells/tissue and recipient. This study explores immune determinants of human embryonic stem cell lines (hESC) and induced human pluripotent stem cell (hiPSC) lines and hepatocyte- and cardiomyocyte-like cells derived from these cells. HLA class I was expressed on all pluripotent hPSC lines which upon differentiation into hepatocyte-like cells was considerably reduced in contrast to cardiomyocyte-like cells which retained class I antigens. No HLA class II antigens were found in the pluripotent or differentiated cells. Histo-blood group carbohydrate antigens SSEA-3/SSEA-4/SSEA-5, Globo H, A, Lex/Ley and sialyl-lactotetra were expressed on all hPSC lines. Blood group AB(O)H antigen expression was in accordance with ABO genotype. Interestingly, only a subpopulation of A1O1 cells expressed A. During differentiation of hPSC, some histo-blood group antigens showed congruent alteration patterns while expression of other antigens differed between the cell lines. No systematic difference in the hPSC cell surface tissue antigen expression was detected. In conclusion, hPSC and their derivatives express cell surface antigens that may cause an immune rejection. Furthermore, tissue antigen expression must be established for each individual stem cell line prior to clinical application.