Model Of Cell Differentiation Research Articles

2D and 3D liver models

2D and 3D liver models

Abstract P3089: Etv2 Functions As A Pioneer Transcription Factor To Regulate Endothelial Lineage Development And Reprogramming

The vasculature is an essential organ for the delivery of blood and oxygen to all tissues of the body and thus relevant to the treatment of ischemic heart disease. The ETS-related transcription factor 2 (ETV2) is a transcription factor that is both necessary and sufficient for the development of endothelial and blood lineages. However, the molecular mechanism by which ETV2 promotes endothelial and blood development is not known and warrants further investigation. Here, we report that ETV2 functions as a novel pioneer transcription factor that relaxes closed chromatin and regulates endothelial development. Pioneer factors are a subset of transcription factors that bind closed chromatin and promote chromatin relaxation in order to drive the development and reprogramming of cell lineages. By comparing embryonic stem cell differentiation and fibroblast reprogramming models with single cell RNA-seq, ATAC-seq, ChIP-seq and in-vitro nucleosomal binding techniques, we demonstrated that ETV2 was able to bind nucleosomal DNA and function as a pioneer factor independent of the cellular context. We also determined that ETV2 executed a pioneering role by recruiting and directly interacting with the ATP-dependent chromatin remodeling enzyme BRG1 to remodel chromatin around endothelial genes to help maintain an open configuration, resulting in increased H3K27ac deposition. Collectively, these results that define a novel pioneer factor and epigenetic regulatory pathways that govern the specification and differentiation of endothelial progenitor cells, will serve as a platform for targeted therapies to promote cardiovascular regeneration.

Heat Shock Alters the Proteomic Profile of Equine Mesenchymal Stem Cells.

The aim of this research was to determine the impact of heat stress on cell differentiation in an equine mesenchymal stem cell model (EMSC) through the application of heat stress to primary EMSCs as they progressed through the cell specialization process. A proteomic analysis was performed using mass spectrometry to compare relative protein abundances among the proteomes of three cell types: progenitor EMSCs and differentiated osteoblasts and adipocytes, maintained at 37 °C and 42 °C during the process of cell differentiation. A cell-type and temperature-specific response to heat stress was observed, and many of the specific differentially expressed proteins were involved in cell-signaling pathways such as Notch and Wnt signaling, which are known to regulate cellular development. Furthermore, cytoskeletal proteins profilin, DSTN, SPECC1, and DAAM2 showed increased protein levels in osteoblasts differentiated at 42 °C as compared with 37 °C, and these cells, while they appeared to accumulate calcium, did not organize into a whorl agglomerate as is typically seen at physiological temperatures. This altered proteome composition observed suggests that heat stress could have long-term impacts on cellular development. We propose that this in vitro stem cell culture model of cell differentiation is useful for investigating molecular mechanisms that impact cell development in response to stressors.

Distinct strengths of mTORC1 control T-cell memory via transcriptional FOXO1 and metabolic AMPKα1 pathways in linear cell differentiation and asymmetric cell division models.

Distinct strengths of mTORC1 control T-cell memory via transcriptional FOXO1 and metabolic AMPKα1 pathways in linear cell differentiation and asymmetric cell division models.

Environmentally relevant exposure to TBBPA and its analogues may not drastically affect human early cardiac development

Tetrabromobisphenol A (TBBPA) and its substitutes and derivatives have been widely used as halogenated flame retardants (HFRs), in the past few decades. As a consequence, these compounds are frequently detected in the environment, as well as human bodily fluids, especially umbilical cord blood and breast milk. This has raised awareness of their potential risks to fetuses and infants. In this study, we employed human embryonic stem cell differentiation models to assess the potential developmental toxicity of six TBBPA-like compounds, at human relevant nanomolar concentrations. To mimic early embryonic development, we utilized embryoid body-based 3D differentiation in presence of the six HFRs. Transcriptomics data showed that HFR exposure over 16 days of differentiation only interfered with the expression of a few genes, indicating those six HFRs may not have specific tissue/organ targets during embryonic development. Nevertheless, further analyses revealed that some cardiac-related genes were dysregulated. Since the heart is also the first organ to develop, we employed a cardiac differentiation model to analyze the six HFRs’ potential developmental toxicity in more depth. Overall, HFRs of interest did not significantly disturb the canonical WNT pathway, which is an essential signal transduction pathway for cardiac development. In addition, the six HFRs showed only mild changes in gene expression levels for cardiomyocyte markers, such as NKX2.5, MYH7, and MYL4, as well as a significant down-regulation of some but not all the epicardial and smooth muscle cell markers selected. Taken together, our results show that the six studied HFRs, at human relevant concentrations, may impose negligible effects on embryogenesis and heart development. Nevertheless, higher exposure doses might affect the early stages of heart development.

ETV2 functions as a pioneer factor to regulate and reprogram the endothelial lineage.

The vasculature is an essential organ for the delivery of blood and oxygen to all tissues of the body and is thus relevant to the treatment of ischaemic diseases, injury-induced regeneration and solid tumour growth. Previously, we demonstrated that ETV2 is an essential transcription factor for the development of cardiac, endothelial and haematopoietic lineages. Here we report that ETV2 functions as a pioneer factor that relaxes closed chromatin and regulates endothelial development. By comparing engineered embryonic stem cell differentiation and reprogramming models with multi-omics techniques, we demonstrated that ETV2 was able to bind nucleosomal DNA and recruit BRG1. BRG1 recruitment remodelled chromatin around endothelial genes and helped to maintain an open configuration, resulting in increased H3K27ac deposition. Collectively, these results will serve as a platform for the development of therapeutic initiatives directed towards cardiovascular diseases and solid tumours.

Analysis of ionizing radiation induced DNA damage response in human adult stem cells and differentiated neurons

Findings of neurodegenerative features associated with human radiosensitive syndromes such as Ataxia telangiectasia suggest that DNA repair efficiency is crucial for maintaining the functional integrity of central nervous system. To gain a better understanding of ionizing radiation (IR) induced DNA damage response in undifferentiated and differentiated neural cell types and to evaluate the role of ATM in DNA double strand break (DSB) repair, an in vitro human neural cell differentiation model system was utilized in this study. As compared to adult stem cells, differentiated neurons displayed an attenuated DSB repair response (as judged by the persistence of 53BP1 foci) after IR exposure and the attenuation was even more pronounced in stem cells and neurons after suppression of ATM (Ataxia Telangiectasia Mutated) gene product suggesting the importance of ATM for an optimal DSB repair efficiency in human neural cell types. In corroboration with an attenuated DNA damage response, a sharp decline in the expression levels of several DSB repair genes was observed in neurons. Our results suggest that cellular differentiation modulates the expression of several genes thereby compromising the DSB repair fidelity in post mitotic neurons. Further studies are required to verify whether or not ATM mediated exacerbation of DNA repair deficiency in differentiated neurons leads to neurodegeneration.

Screening for modulators of the cellular composition of gut epithelia via organoid models of intestinal stem cell differentiation

The cellular composition of barrier epithelia is essential to organismal homoeostasis. In particular, within the small intestine, adult stem cells establish tissue cellularity, and may provide a means to control the abundance and quality of specialized epithelial cells. Yet, methods for the identification of biological targets regulating epithelial composition and function, and of small molecules modulating them, are lacking. Here we show that druggable biological targets and small-molecule regulators of intestinal stem cell differentiation can be identified via multiplexed phenotypic screening using thousands of miniaturized organoid models of intestinal stem cell differentiation into Paneth cells, and validated via longitudinal single-cell RNA-sequencing. We found that inhibitors of the nuclear exporter Exportin 1 modulate the fate of intestinal stem cells, independently of known differentiation cues, significantly increasing the abundance of Paneth cells in the organoids and in wild-type mice. Physiological organoid models of the differentiation of intestinal stem cells could find broader utility for the screening of biological targets and small molecules that can modulate the composition and function of other barrier epithelia.

3191 – DELINEATING HUMAN NATURAL KILLER CELL DEVELOPMENT THROUGH AN IN VITRO CELL- DIFFERENTIATION MAP AND BARCODE-CLONAL TRACING

Natural Killer (NK) cells represent a unique cytotoxic innate lymphoid lineage that holds great potential for anti-cancer immunotherapy. The process of human NK cell lineage specification and maturation from hematopoietic progenitors is poorly understood. While mouse and human NK cell developmental pathways converged overall in terms of molecular signatures and transcription factor requirements, inherent differences in surface marker repertoire and shared phenotypic features with other innate lymphoid subsets, highlight the need for a platform allowing direct modeling of human NK cell development. Employing an in-vitro differentiation system on OP9 stroma and multi-parameter FACS analysis, we established and characterized a NK cell developmental trajectory that recapitulates multiple diverse maturation stages proposed in a current model of human NK cell development ex-vivo in the bone marrow and tonsils. Differentiation kinetics, expression of transcription factors (Tbet, Eomes) and effector molecules (Perforin, Granzyme-B) confirmed that in-vitro NK cell maturation progressed through this developmental trajectory in a sequential manner. Functional assays including IFNg secretion, release of cytotoxic granules and direct killing of tumor cells supported the increased effector functions as differentiating NK cells progressed from the early to the final maturation stages. We also further characterized NK cell differentiation steps using transcriptomic profiles from single cell RNA-sequencing. Finally, we employ DNA-barcode-clonal tracing to study developmental relationship between different stages in our in vitro NK cell differentiation model. In summary, we established a framework that allows further understanding of human NK cell development and provides a platform for studies on identifying regulatory networks controlling this process. Natural Killer (NK) cells represent a unique cytotoxic innate lymphoid lineage that holds great potential for anti-cancer immunotherapy. The process of human NK cell lineage specification and maturation from hematopoietic progenitors is poorly understood. While mouse and human NK cell developmental pathways converged overall in terms of molecular signatures and transcription factor requirements, inherent differences in surface marker repertoire and shared phenotypic features with other innate lymphoid subsets, highlight the need for a platform allowing direct modeling of human NK cell development. Employing an in-vitro differentiation system on OP9 stroma and multi-parameter FACS analysis, we established and characterized a NK cell developmental trajectory that recapitulates multiple diverse maturation stages proposed in a current model of human NK cell development ex-vivo in the bone marrow and tonsils. Differentiation kinetics, expression of transcription factors (Tbet, Eomes) and effector molecules (Perforin, Granzyme-B) confirmed that in-vitro NK cell maturation progressed through this developmental trajectory in a sequential manner. Functional assays including IFNg secretion, release of cytotoxic granules and direct killing of tumor cells supported the increased effector functions as differentiating NK cells progressed from the early to the final maturation stages. We also further characterized NK cell differentiation steps using transcriptomic profiles from single cell RNA-sequencing. Finally, we employ DNA-barcode-clonal tracing to study developmental relationship between different stages in our in vitro NK cell differentiation model. In summary, we established a framework that allows further understanding of human NK cell development and provides a platform for studies on identifying regulatory networks controlling this process.

Endoderm and mesoderm derivatives in embryonic stem cell differentiation and their use in developmental toxicity testing

Embryonic stem cell differentiation models have increasingly been applied in non-animal test systems for developmental toxicity. After the initial focus on cardiac differentiation, attention has also included an array of neuro-ectodermal differentiation routes. Alternative differentiation routes in the mesodermal and endodermal germ lines have received less attention. This review provides an inventory of achievements in the latter areas of embryonic stem cell differentiation, with a view to possibilities for their use in non-animal test systems in developmental toxicology. This includes murine and human stem cell differentiation models, and also gains information from the field of stem cell use in regenerative medicine. Endodermal stem cell derivatives produced in vitro include hepatocytes, pancreatic cells, lung epithelium, and intestinal epithelium, and mesodermal derivatives include cardiac muscle, osteogenic, vascular and hemopoietic cells. This inventory provides an overview of studies on the different cell types together with biomarkers and culture conditions that stimulate these differentiation routes from embryonic stem cells. These models may be used to expand the spectrum of embryonic stem cell based new approach methodologies in non-animal developmental toxicity testing.

An integrative model of cancer cell differentiation with immunotherapy * *This work is dedicated to the memory of our colleague, director, and great friend Prof. Dr Alejandro José Fendrik (1953–2021).

In order to improve cancer treatments, cancer cell differentiation and immunotherapy are the subjects of several studies in different branches of interdisciplinary sciences. In this work, we develop a new population model that integrates other complementary ones, thus emphasizing the relationship between cancer cells at different differentiation stages and the main immune system cells. For this new system, specific ranges were found where transdifferentiation of differentiated cancer cells can occur. In addition, a specific therapy against cancer stem cells was analysed by simulating cytotoxic cell vaccines. In reference to the latter, the different combinations of parameters that optimize it were studied.

Novel generic models for differentiating stem cells reveal oscillatory mechanisms.

Understanding cell fate selection remains a central challenge in developmental biology. We present a class of simple yet biologically motivated mathematical models for cell differentiation that generically generate oscillations and hence suggest alternatives to the standard framework based on Waddington’s epigenetic landscape. The models allow us to suggest two generic dynamical scenarios that describe the differentiation process. In the first scenario, gradual variation of a single control parameter is responsible for both entering and exiting the oscillatory regime. In the second scenario, two control parameters vary: one responsible for entering, and the other for exiting the oscillatory regime. We analyse the standard repressilator and four variants of it and show the dynamical behaviours associated with each scenario. We present a thorough analysis of the associated bifurcations and argue that gene regulatory networks with these repressilator-like characteristics are promising candidates to describe cell fate selection through an oscillatory process.

Examining the Developmental Trajectory of an in Vitro Model of Mouse Primordial Germ Cells following Exposure to Environmentally Relevant Bisphenol A Levels.

Background:Animal-based studies indicate that bisphenol A (BPA) exposure is detrimental to reproductive health, but its impact on the earliest stages of germ cell development remains poorly defined.Objectives:Using a murine in vitro model of early germ cell specification and differentiation, we sought to assess whether exposure to low levels of BPA prior to formation of primordial germ cells (PGCs) alters their differentiation trajectory and unique molecular program.Methods:We used an established method of in vitro differentiation of mouse embryonic stem cells (ESCs) into epiblast-like cells (EpiLCs) followed by PGC-like cells (PGCLCs), which together recapitulate defined stages of early germ cell development. Cellular consequences were determined using hemocytometer-based cell counting, fixation, and intracellular staining, followed by flow cytometry/fluorescence-activated cell sorting (FACS) of cells exposed to increasing concentrations (range: ) of BPA. To interrogate and characterize gene expression differences resulting from BPA exposure, we also generated RNA-seq libraries from RNA extracted from FACS-purified PGCLCs and performed transcriptome analysis using bioinformatics-based approaches.Results:Exposure of EpiLCs to BPA resulted in higher numbers of cells that were associated with a higher proportion of cells in S-phase as well as a lower proportion undergoing apoptosis; this difference occurred in a concentration-dependent manner. Exposure also resulted in a greater fraction of EpiLCs showing signs of DNA damage. Remarkably, EpiLC exposure did not negatively affect PGC specification and resulted in a concentration-dependent effect on PGCLC proliferation in XX but not XY cells. PGCLC transcriptome analysis revealed an aberrant program with significant deregulation of X-linked genes and retrotransposon expression. Differential gene expression analysis also revealed the deregulation of genes associated with lipid metabolism as well as deregulated expression of genes associated with later stages of gametogenesis.Conclusions:To the best of our knowledge our findings represent the first characterization of the consequences of early BPA exposure on a model of mammalian PGC development, highlighting altered cell behavior, altered underlying pathways, and altered molecular processes. https://doi.org/10.1289/EHP8196

Investigating developmental effects of PFAS using a 3D human induced pluripotent stem cell differentiation model

Investigating developmental effects of PFAS using a 3D human induced pluripotent stem cell differentiation model

The Instructive and the Selective Models of Cell Differentiation

The Instructive and the Selective Models of Cell Differentiation

Transcriptome Analysis in a Primary Human Muscle Cell Differentiation Model for Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is caused by CTG-repeat expansions leading to a complex pathology with a multisystemic phenotype that primarily affects the muscles and brain. Despite a multitude of information, especially on the alternative splicing of several genes involved in the pathology, information about additional factors contributing to the disease development is still lacking. We performed RNAseq and gene expression analyses on proliferating primary human myoblasts and differentiated myotubes. GO-term analysis indicates that in myoblasts and myotubes, different molecular pathologies are involved in the development of the muscular phenotype. Gene set enrichment for splicing reveals the likelihood of whole, differentiation stage specific, splicing complexes that are misregulated in DM1. These data add complexity to the alternative splicing phenotype and we predict that it will be of high importance for therapeutic interventions to target not only mature muscle, but also satellite cells.

CREB3L2 Modulates Nerve Growth Factor-Induced Cell Differentiation.

Nerve growth factor (NGF) stimulates numerous cellular physiological processes, including growth, differentiation, and survival, and maintains the phenotype of several neuronal types. Most of these NGF-induced processes require adaptation of the secretory pathway since they involve extensive remodeling of membranes and protein redistribution along newly formed neuritic processes. CREB3 transcription factors have emerged as signaling hubs for the regulation of numerous genes involved in the secretory pathway and Golgi homeostasis, integrating stimuli from multiple sources to control secretion, posttranslational modifications and trafficking of proteins. Although recent studies have focused on their role in the central nervous system, little is known about their participation in cell differentiation. Therefore, we aimed to analyze the expression and signaling mechanism of CREB3 transcription factor family members, using the NGF-induced PC12 cell differentiation model. Results show that NGF treatment causes Golgi enlargement and a parallel increased expression of proteins and mRNAs encoding for proteins required for membrane transport (transport factors). Additionally, a significant increase in CREB3L2 protein and mRNA levels is detected in response to NGF. Both MAPK and cAMP signaling pathways are required for this response. Interestingly, CREB3L2 overexpression hampers the NGF-induced neurite outgrowth while its inhibition enhances the morphological changes driven by NGF. In agreement, CREB3L2 overexpressing cells display higher immunofluorescence intensity of Rab5 GTPase (a negative regulator of PC12 differentiation) than control cells. Also, Rab5 immunofluorescence levels decrease in CREB3L2-depleted cells. Taken together, our findings imply that CREB3L2 is an important downstream effector of NGF-activated pathways, leading to neuronal differentiation.

Abstract 185: Monitoring of stem cell differentiation to mature hepatocytes with a machine learning-based AI model

Abstract Human embryonic stem cells (hESCs) and pluripotent stem cells (iPSCs)-based disease modelings are potential platforms for cancer research and development of new cancer therapeutics. Differentiation of stem cells is an essential step for those disease models. For tissue-specific differentiation, hESCs or iPSCs are cultured in specific receipts of induction and differentiation media for developing different types of tissue cells such as muscle, skin, and liver providing further study or clinical applications. During lineage differentiation, researcher needs to closely monitor stem cell differentiation to be on track via checking cell morphological changes under microscope since this procedure has high probability of failed differentiation results (e.g., no differentiation or differentiating unwanted tissue types). However, monitoring via microscopy is labor-intensive and time-consuming, and also has high inter-observer variation issues. Therefore, it significantly impedes the progression of stem cell research and clinical applications nowadays. In recent years, machine learning has shown promising results in many applications of artificial intelligence (AI) in different fields, especially computer vision and image analysis. AI-based computational tool will bring benefits like high-throughput, high accuracy, and reproductivity in many medical applications. In stem cell culture and differentiation, we believe that applying this new technology will help researcher detect abnormal stem cell differentiation at the early stage via microscopy to save time, labor, and cost for the study and aggregate reproducible data along the process. To this end, we developed a machine learning-based AI model to assist in monitoring morphological changes of hESCs culture in bright-field microscopy images obtained from different differentiation stages to mature hepatocytes. We conducted a pilot study to train an AI model estimating efficiency of stem cell differentiation at Hepatic Progenitor Cell (HPC) stage, which is a critical checkpoint for hepatocyte differentiation. To prepare datasets for training, experienced researchers annotated the morphology of HPC in hundreds of microscope images and determined a differentiation result (success/fail) for every image. During the model training, the initial model was first trained by a training dataset consisting of 341 success and 366 fail HPC results. Subsequently, a smaller separate dataset comprising of 86 success and 51 fail HPC results was then used for cross-validation. Finally, the test set containing 64 success and 29 fail HPC results was used to evaluate the AI model performance. In result, the AI model presented an excellent performance (accuracy= 0.978 and F1 score= 0.975). Our study suggests a potential application of AI-assisted monitoring model for stem cell culture and differentiation in the future. Citation Format: Wei-Lei Yang, Zijun Huo, ShihYu Chen, Dandan Zhu, Tien-Jen Liu, Dung-Fang Lee. Monitoring of stem cell differentiation to mature hepatocytes with a machine learning-based AI model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 185.

Impact of Mutant Haemopoietic Transcription Factor GATA1s on Haemopoiesis

Neonates and children with Down Syndrome (DS), with constitutional Trisomy 21 (T21), have a 150-fold increased risk of developing Myeloid Leukaemia (ML-DS), with differentiation arrest of immature megakaryocyte-erythroid cells. Virtually all ML-DS patients have in utero-acquired somatic mutations in the gene encoding the megakaryocyte-erythroid transcription factor GATA1 leading to the production of an 84 amino acid N-terminal truncated form of the GATA1 protein (GATA1s). We, and others, have previously shown that this N-terminal domain is necessary to prevent excessive megakaryocytic proliferation. However, the mechanisms by which GATA1, but not GATA1s, restrains megakaryocyte proliferation are unclear.We generated knock-in murine ES cell models expressing biotinylated forms of either full length GATA1 or GATA1s protein. We established a large scale in vitro differentiation assay to study embryonic-fetal megakaryocyte differentiation to define the normal megakaryocytic differentiation pathway in GATA1-expressing cells. ES cells were differentiated into embryoid bodies (EBs), which were disaggregated after 6 days and CD41+c-kit+ cells were cultured on OP9 feeder layers with TPO, IL6 and IL11. Detailed examination of the differentiation kinetics including FACS-sorting of specific populations followed by re-culture or Biomark qRT-PCR analysis, showed complex differentiation pathways as wild type cells differentiated into both megakaryocyte and non-megakaryocyte (both erythroid and myeloid) fates. By contrast, GATA1s-expressing cells almost exclusively differentiated into megakaryocyte lineage. Furthermore, as immature CD41+ haemopoietic cells differentiate into the megakaryocyte lineage, they lose c-kit expression and CD41 expression increases. In the GATA1s-expressing cells compared to GATA1-expressing cells, there is a significant accumulation (5 to 10-fold) of a specific immature megakaryocyte CD41++c-kit+ population, partially blocked in differentiation. Cell cycle analysis showed an increase in cells in S-phase specifically in this population in GATA1s-expressing cells (44% compared to 27% in normal cells) together with a decrease in apoptosis (5% compared to 11% in normal cells). To determine the relevance of these findings in vivo , corresponding mouse models were generated and experiments on yolk sacs isolated at different stages of development are ongoing.Taken together, these data suggest that our in vitro murine ES cell differentiation model is a relevant tool to dissect the molecular mechanisms underlying GATA1s function in abnormal megakaryocytic differentiation and they are likely to provide insight into general mechanisms of how altered function of a critical lineage-affiliated transcription factor can lead to multiple changes in progenitor cell cycle and differentiation. DisclosuresVyas:Celgene Corporation: Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau.

SORBS2 is a genetic factor contributing to cardiac malformation of 4q deletion syndrome patients.

Chromosome 4q deletion is one of the most frequently detected genomic imbalance events in congenital heart disease (CHD) patients. However, a portion of CHD-associated 4q deletions without known CHD genes suggests unknown CHD genes within these intervals. Here, we have shown that knockdown of SORBS2, a 4q interval gene, disrupted sarcomeric integrity of cardiomyocytes and caused reduced cardiomyocyte number in human embryonic stem cell differentiation model. Molecular analyses revealed decreased expression of second heart field (SHF) marker genes and impaired NOTCH and SHH signaling in SORBS2-knockdown cells. Exogenous SHH rescued SORBS2 knockdown-induced cardiomyocyte differentiation defects. Sorbs2-/- mouse mutants had atrial septal hypoplasia/aplasia or double atrial septum (DAS) derived from impaired posterior SHF with a similar expression alteration. Rare SORBS2 variants were significantly enriched in a cohort of 300 CHD patients. Our findings indicate that SORBS2 is a regulator of SHF development and its variants contribute to CHD pathogenesis. The presence of DAS in Sorbs2-/- hearts reveals the first molecular etiology of this rare anomaly linked to paradoxical thromboembolism.