Manipulation of gene function in embryonic stem cells by either over expression or downregulation is critical for understanding their subsequent cell fate. We have developed a tetracycline-inducible short hairpin RNA interference (shRNAi) for human embryonic stem cells (hESCs) and demonstrated doxycycline dose-dependent knockdown of the transcription factor OCT4 and the cell surface antigen β2-microglobulin. The induced knockdown of OCT4 resulted in rapid differentiation of hESCs with a significant increase in transcription of genes associated with trophoblast and endoderm lineages, the extent of which was controlled by the degree of induction. Transgene toxicity, which may occur in conditional over-expression strategies with hESCs, was not observed with wild-type Tet repressor protein. The system allows efficient, reversible, and long-term downregulation of target genes in hESCs and enables the generation of stable transfectants for the knockdown of genes essential for cell survival and self-renewal, not necessarily possible by nonconditional shRNAi methods.

Human embryonic stem cells (hESCs) replicate in vitro by the process of self-renewal, whilst maintaining their pluripotency. Understanding the pathways involved in the regulation of this process will assist in developing fully-defined conditions for the robust proliferation of hESCs necessary for therapeutic applications. We previously demonstrated that sphingosine-1-phosphate (S1P) plays an important role in survival and proliferation of hESCs. and here the key signaling pathways and downstream targets of S1P were investigated in a representative cell line (Shef 4). A significant rise in ERK1/2 activation with S1P treatment was witnessed in hESCs maintained on murine embryonic fibroblasts (MEFs) exhibiting significantly higher levels of active ERK1/2 than those grown on Matrigel. RT-PCR and microarray analysis of micro-dissected, non-differentiated hESC revealed 1049 differentially expressed genes in S1P treated preparations compared with controls (n = 3). S1P regulated apoptosis through several BCL-2 family members, including BAX and BID, with increased expression of cell cycle progression genes associated with proliferation of hESC cultures. S1P treatment also increased expression of cell adhesion genes specifically cadherins and integrins. However, gene expression associated with pluripotency was decreased with S1P treatment indicating that an increased rate of hESC turnover (higher proliferation and lower apoptosis) may be balanced by an increased susceptibility to differentiate.

The antigenic profile of human embryonic stem (ES) and embryonal carcinoma (EC) cells has served as a key element of their characterization, with a common panel of surface and intracellular markers now widely used. Such markers have been used to identify cells within the 'undifferentiated state', yet it appears that this categorization may be an oversimplification, because a number of sub-states appear to exist within this state. To increase the resolution of the undifferentiated state, we have generated eight novel monoclonal antibodies, all capable of recognizing undifferentiated human ES and EC cells, and herein describe their characterization. The reactivity of these antibodies against a range of cell lines is reported, as well as their developmental regulation, basic biochemistry and reactivity in immunohistochemistry of testicular germ cell tumours. Our data reveal a range of reactivity for all antibodies against both ES and EC cells, suggesting that these markers will afford recognition of unique sub-states within the undifferentiated stem cell compartment.

Given the growing body of research seeking to examine adverse childhood experiences (ACEs) and intimate partner violence (IPV) among sexual and gender minority (SGM) individuals, Institutional Review Boards must consider whether participating in violence research is emotionally distressing for SGM people. Yet, little research has studied SGM participants’ reactions to participating in research on ACEs, IPV, and minority stress. Thus, the current study examined reactions, including negative emotional reactions, to participating in violence research among SGM young adults. In total, 230 participants who self-identified as a sexual minority (30.1% also identified as a gender minority) in a dating relationship completed a cross-sectional assessment on ACEs, IPV (including identity abuse victimization and perpetration), minority stress (i.e., internalized homo/bi/transphobia), and reactions to research participation. Results indicated that participants identifying as a gender minority had significantly higher negative emotional reactions to study participation compared to cisgender participants, but this increase among gender minority individuals was small. In addition, gender minority participants and those with higher minority stress (i.e., internalized trans/bi/homo-negativity) and ACEs reported significantly higher negative emotional reactions to participation. Furthermore, gender minority participants scored worse on a scale indicating appreciation for contributing to research. Finally, reporting IPV victimization and perpetration was not associated with negative emotional reactions. Findings suggest that questions assessing minority stress and negative childhood experiences may be more emotionally salient or stressful for gender minority participants compared to questions measuring IPV.

Background: Coexisting subgroups of cells within a tumor are always evolving. Understanding the molecular events that drive this evolution remains a high priority for understanding dynamic response to treatment. However, models that accurately map individual clones’ activity and sensitivity to treatment remain a challenge. To fill this gap, we established monoclonal cell populations (MCP) from a cell line and used this model system to dissect coexisting cellular and molecular events within an individual tumor and modulated response to anti-EGFR treatment in Non-Small Cell Lung Cancers (NSCLC). Materials/Methods: MCPs were established from the commercially available H1975 EGFR-mutant NSCLC cell line. Parental cells were resuspended at a density of 10 cells per mL and 100 μL of cell suspension was added to each well of a 96-well plate. Eight plates were used, and each well was inspected 24 hours post-seeding by two or more independent scientists. Wells with individual cells were monitored every 3-4 days and followed throughout the expansion process. Once MCPs were established, morphological comparison was performed using the opensource QPath software and clones were classified as circular, branched, or mixed. Selected clones were then treated with 0.7 μM of Osimertinib for 72 hours, in line with the IC50 value of the parental line and treatment responses were compared. Results: Of the 120 single cells detected, 25 expanded successfully and 16 were treated with Osimertinib. Rates of clonal expansion varied from 19 to 49 days and the most prominent morphology was branched (68.75%), followed by mixed (18.75%) and circular (12.5%). No direct correlation was observed between rate of expansion and cell morphology. Response to Osimertinib varied significantly across the clones compared to the parental line from which they were established. Cell viability after 72 hours of treatment with Osimertinib was similar between the parental line and 5 of the 25 clones. For the remaining clones, cell viability was significantly lower in the monoclonal cell subpopulations compared to the parental line. Specifically, cell viability decreased by 40% compared to the parental cell in 5 clones. In the most sensitive clones, cell viability was reduced by 63% and 75% compared to the parental. Conclusion: 25 monoclonal cell subpopulations were successfully established and expanded from the H1975 cell line. MCPs showed significant variations in their growth rate and sensitivity to Osimertinib suggesting that this approach may help identify coexisting mechanisms of response to treatment within an individual tumor. Defining molecular profiles of coexisting MCPs may lead to the identification of biomarkers that may assist physicians in predicting response to treatment or design combination regimens that concomitantly target subclones within the tumor microecology. Citation Format: Claire Blanchard, Kyle Avery, Chelsea Ward, Abduljalil M. Alsubaie, Elisa Baldelli, Mariaelena Pierobon. Establishment of monoclonal cell subpopulations as model systems for functionally exploring resistance mechanisms in EGFR mutant non-small cell cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 138.