Cardiac differentiation is modulated by anti-apoptotic signals in murine embryonic stem cells.

BACKGROUND: Embryonic stem cells (ESCs) serve as a crucial ex vivo model, representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos. ESCs exhibit a unique combination of self-renewal potency, unlimited proliferation, and pluripotency. The latter is evident by the ability of the isolated cells to differentiate spontaneously into multiple cell lineages, representing the three primary embryonic germ layers. Multiple regulatory networks guide ESCs, directing their self-renewal and lineage-specific differentiation. Apoptosis, or programmed cell death, emerges as a key event involved in sculpting and forming various organs and structures ensuring proper embryonic development. However, the molecular mechanisms underlying the dynamic interplay between differentiation and apoptosis remain poorly understood. AIM: To investigate the regulatory impact of apoptosis on the early differentiation of ESCs into cardiac cells, using mouse ESC (mESC) models - mESC-B-cell lymphoma 2 (BCL-2), mESC-PIM-2, and mESC-metallothionein-1 (MET-1) - which overexpress the anti-apoptotic genes Bcl-2, Pim-2, and Met-1, respectively. METHODS: mESC-T2 (wild-type), mESC-BCL-2, mESC-PIM-2, and mESC-MET-1 have been used to assess the effect of potentiated apoptotic signals on cardiac differentiation. The hanging drop method was adopted to generate embryoid bodies (EBs) and induce terminal differentiation of mESCs. The size of the generated EBs was measured in each condition compared to the wild type. At the functional level, the percentage of cardiac differentiation was measured by calculating the number of beating cardiomyocytes in the manipulated mESCs compared to the control. At the molecular level, quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA expression of three cardiac markers: Troponin T, GATA4, and NKX2.5. Additionally, troponin T protein expression was evaluated through immunofluorescence and western blot assays. RESULTS: Our findings showed that the upregulation of Bcl-2, Pim-2, and Met-1 genes led to a reduction in the size of the EBs derived from the manipulated mESCs, in comparison with their wild-type counterpart. Additionally, a decrease in the count of beating cardiomyocytes among differentiated cells was observed. Furthermore, the mRNA expression of three cardiac markers - troponin T, GATA4, and NKX2.5 - was diminished in mESCs overexpressing the three anti-apoptotic genes compared to the control cell line. Moreover, the overexpression of the anti-apoptotic genes resulted in a reduction in troponin T protein expression. CONCLUSION: Our findings revealed that the upregulation of Bcl-2, Pim-2, and Met-1 genes altered cardiac differentiation, providing insight into the intricate interplay between apoptosis and ESC fate determination.

Establishment and characterization of prostate organoids from treatment-naïve patients with prostate cancer.

Three-dimensional (3D) organoid culture systems are emerging as potential reliable tools to investigate basic developmental processes of human disease, especially cancer. The present study used established and modified culture conditions to report successful generation and characterization of patient-derived organoids from fresh primary tissue specimens of patients with treatment-naïve prostate cancer (PCa). Fresh tissue specimens were collected, digested enzymatically and the resulting cell suspensions were plated in a 3D environment using Matrigel as an extracellular matrix. Previously established 12-factor medium for organoid culturing was modified to create a minimal 5-factor medium. Organoids and corresponding tissue specimens were characterized using transcriptomic analysis, immunofluorescent analysis, and immunohistochemistry. Furthermore, patient-derived organoids were used to assess the drug response. Treatment-naïve patient-derived PCa organoids were obtained from fresh radical prostatectomy specimens. These PCa organoids mimicked the heterogeneity of corresponding parental tumor tissue. Histopathological analysis demonstrated similar tissue architecture and cellular morphology, as well as consistent immunohistochemical marker expression. Also, the results confirmed the potential of organoids as an in vitro model to assess potential personalized treatment responses as there was a differential drug response between different patient samples. In conclusion, the present study investigated patient-derived organoids from a cohort of treatment-naïve patients. Derived organoids mimicked the histological features and prostate lineage profiles of their corresponding parental tissue and may present a potential model to predict patient-specific treatment response in a pre-clinical setting.

The Use of Stem Cell-Derived Organoids in Disease Modeling: An Update.

Organoids represent one of the most important advancements in the field of stem cells during the past decade. They are three-dimensional in vitro culturing models that originate from self-organizing stem cells and can mimic the in vivo structural and functional specificities of body organs. Organoids have been established from multiple adult tissues as well as pluripotent stem cells and have recently become a powerful tool for studying development and diseases in vitro, drug screening, and host-microbe interaction. The use of stem cells-that have self-renewal capacity to proliferate and differentiate into specialized cell types-for organoids culturing represents a major advancement in biomedical research. Indeed, this new technology has a great potential to be used in a multitude of fields, including cancer research, hereditary and infectious diseases. Nevertheless, organoid culturing is still rife with many challenges, not limited to being costly and time consuming, having variable rates of efficiency in generation and maintenance, genetic stability, and clinical applications. In this review, we aim to provide a synopsis of pluripotent stem cell-derived organoids and their use for disease modeling and other clinical applications.

Epidermal Growth Factor Is Essential for the Maintenance of Novel Prostate Epithelial Cells Isolated From Patient-Derived Organoids.

Prostate cancer (PCa) is the second leading cause of cancer-related mortality and morbidity among males worldwide. Deciphering the biological mechanisms and molecular pathways involved in PCa pathogenesis and progression has been hindered by numerous technical limitations mainly attributed to the limited number of cell lines available, which do not recapitulate the diverse phenotypes of clinical disease. Indeed, PCa has proven problematic to establish as cell lines in culture due to its heterogeneity which remains a challenge, despite the various in vitro and in vivo model systems available. Growth factors have been shown to play a central role in the complex regulation of cell proliferation among hormone sensitive tumors, such as PCa. Here, we report the isolation and characterization of novel patient-derived prostate epithelial (which we named as AUB-PrC) cells from organoids culture system. We also assessed the role of epidermal growth factor (EGF) in culturing those cells. We profiled the AUB-PrC cells isolated from unaffected and tumor patient samples via depicting their molecular and epithelial lineage features through immunofluorescence staining and quantitative real-time PCR (qRT-PCR), as well as through functional assays and transcriptomic profiling through RNA sequencing. In addition, by optimizing a previously established prostate organoids culture system, we were able to grow human prostate epithelial cells using growth medium and EGF only. With these data collected, we were able to gain insight at the molecular architecture of novel human AUB-PrC cells, which might pave the way for deciphering the mechanisms that lead to PCa development and progression, and ultimately improving prognostic abilities and treatments.

CYR61/CCN1 expression in resected pancreatic ductal adenocarcinoma: A retrospective pilot study of the interaction between the tumors and their surrounding microenvironment.

BACKGROUND: CCN1 is an extracellular matrix-associated protein thought to be implicated in tumor-stromal interaction in several solid tumors. The aim of our pilot study was to evaluate the correlation between CCN1 expression in stromal cells, pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma cells in resected pancreatic ductal adenocarcinoma (PDAC) specimens, and correlate that clinically. METHODS: A total of 42 paraffin-embedded PDAC tumor specimens were stained for CCN1 and evaluated via immunohistochemical (IHC) analysis. Statistical analysis was performed to correlate between CCN1 expression profiles in tumor tissues and clinicopathological parameters of patients. RESULTS: Our results showed CCN1 (CYR61) gene was highly expressed in PDAC tissues relative to other organ specific tumor tissues. Also, moderate and overexpression of CCN1 in PanIN was associated with PanIN grade 3 tissues. A statistically significant association was found between PanIN CCN1 scores on one hand and cancer stage, cancer grade, and CCN1 expression among ductal tumor cells and adjacent stromal cells on the other hand. DISCUSSION: The associations demonstrated suggest that CCN1 might be contributing to a substantial role in the interaction between the pancreatic tumors on one hand and their surrounding microenvironment and their precursors on the other hand; hence, it might serve as a potential therapeutic target for PDAC.

EMT Markers in Locally-Advanced Prostate Cancer: Predicting Recurrence?

Background: Prostate cancer (PCa) is the second most frequent cause of cancer-related death in men worldwide. It is a heterogeneous disease at molecular and clinical levels which makes its prognosis and treatment outcome hard to predict. The epithelial-to-mesenchymal transition (EMT) marks a key step in the invasion and malignant progression of PCa. We sought to assess the co-expression of epithelial cytokeratin 8 (CK8) and mesenchymal vimentin (Vim) in locally-advanced PCa as indicators of EMT and consequently predictors of the progression status of the disease. Methods: Co-expression of CK8 and Vim was evaluated by immunofluorescence (IF) on paraffin-embedded tissue sections of 122 patients with PCa who underwent radical prostatectomies between 1998 and 2016 at the American University of Beirut Medical Center (AUBMC). EMT score was calculated accordingly and then correlated with the patients' clinicopathological parameters and PSA failure. Results: The co-expression of CK8/Vim (EMT score), was associated with increasing Gleason group. A highly significant linear association was detected wherein higher Gleason group was associated with higher mean EMT score. In addition, the median estimated biochemical recurrence-free survival for patients with < 25% EMT score was almost double that of patients with more than 25%. The validity of this score for prediction of prognosis was further demonstrated using cox regression model. Our data also confirmed that the EMT score can predict PSA failure irrespective of Gleason group, pathological stage, or surgical margins. Conclusion: This study suggests that assessment of molecular markers of EMT, particularly CK8 and Vim, in radical prostatectomy specimens, in addition to conventional clinicopathological prognostic parameters, can aid in the development of a novel system for predicting the prognosis of locally-advanced PCa.

The synthetic retinoid ST1926 attenuates prostate cancer growth and potentially targets prostate cancer stem-like cells.

Retinoids are vitamin A derivatives that regulate crucial biological processes such as cellular proliferation, apoptosis, and differentiation. The use of natural retinoids in cancer therapy is limited due to their toxicity and the acquired resistance by cancer cells. Therefore, synthetic retinoids were developed, such as the atypical adamantyl retinoid ST1926 that provides enhanced bioavailability and reduced toxicity. We have assessed the in vitro and in vivo antitumor properties and mechanism of action of ST1926 in targeting cancer stem-like cells population of human prostate cancer (PCa) cell lines, DU145 and PC3, and mouse PCa cell lines, PLum-AD and PLum-AI. We demonstrated that ST1926 substantially reduced proliferation of PCa cells and induced cell cycle arrest, p53-independent apoptosis, and early DNA damage. It also decreased migration and invasion of PCa cells and significantly reduced prostate spheres formation ability in vitro denoting sufficient eradication of the self-renewal ability of the highly androgen-resistant cancer stem cells. Importantly, ST1926 potently inhibited PCa tumor growth and progression in vivo. Our results highlight the potential of ST1926 in PCa therapy and warrant its clinical development.

Sphere-Formation Assay: Three-Dimensional in vitro Culturing of Prostate Cancer Stem/Progenitor Sphere-Forming Cells.

Cancer Stem Cells (CSCs) are a sub-population of cells, identified in most tumors, responsible for the initiation, recurrence, metastatic potential, and resistance of different malignancies. In prostate cancer (PCa), CSCs were identified and thought to be responsible for the generation of the lethal subtype, commonly known as Castration-Resistant Prostate Cancer (CRPC). In vitro models to investigate the properties of CSCs in PCa are highly required. Sphere-formation assay is an in vitro method commonly used to identify CSCs and study their properties. Here, we report the detailed methodology on how to generate and propagate spheres from PCa cell lines and from murine prostate tissue. This model is based on the ability of stem cells to grow in non-adherent serum-free gel matrix. We also describe how to use these spheres in histological and immuno-fluorescent staining assays to assess the differentiation potential of the CSCs. Our results show the sphere-formation Assay (SFA) as a reliable in vitro assay to assess the presence and self-renewal ability of CSCs in different PCa models. This platform presents a useful tool to evaluate the effect of conventional or novel agents on the initiation and self-renewing properties of different tumors. The effects can be directly evaluated through assessment of the sphere-forming efficiency (SFE) over five generations or other downstream assays such as immuno-histochemical analysis of the generated spheres.

Modeling Human Neurological and Neurodegenerative Diseases: From Induced Pluripotent Stem Cells to Neuronal Differentiation and Its Applications in Neurotrauma.

With the help of several inducing factors, somatic cells can be reprogrammed to become induced pluripotent stem cell (iPSCs) lines. The success is in obtaining iPSCs almost identical to embryonic stem cells (ESCs), therefore various approaches have been tested and ultimately several ones have succeeded. The importance of these cells is in how they serve as models to unveil the molecular pathways and mechanisms underlying several human diseases, and also in its potential roles in the development of regenerative medicine. They further aid in the development of regenerative medicine, autologous cell therapy and drug or toxicity screening. Here, we provide a comprehensive overview of the recent development in the field of iPSCs research, specifically for modeling human neurological and neurodegenerative diseases, and its applications in neurotrauma. These are mainly characterized by progressive functional or structural neuronal loss rendering them extremely challenging to manage. Many of these diseases, including Parkinson's disease (PD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) have been explored in vitro. The main purpose is to generate patient-specific iPS cell lines from the somatic cells that carry mutations or genetic instabilities for the aim of studying their differentiation potential and behavior. This new technology will pave the way for future development in the field of stem cell research anticipating its use in clinical settings and in regenerative medicine in order to treat various human diseases, including neurological and neurodegenerative diseases.

HTR-8/SVneo cell line contains a mixed population of cells.

INTRODUCTION: The placenta, a transient organ in humans, is essential for pregnancy maintenance and fetal development. Trophoblast and stromal cells are the main cell types present in human placenta. Trophoblast cells are derivatives of the trophectoderm layer and fulfill the endocrine, exchange, invasion and implantation processes of the placenta, whereas stromal cells are of extraembryonic mesenchymal origin and are important for villous formation and maintenance. Different cell lines were developed to study trophoblast functions including BeWo, JEG-3 and JAR from chorioncarcinoma while HTR-8/SVneo was developed using first trimester extravillous trophoblast infected with simian virus 40 large T antigen (SV40). These cell lines are largely used to study trophoblast functions including cell fusion, migration and invasion. Therefore, the purity of each cell lines is crucial in order to be able to use them as a model recapitulating trophoblast cells. METHODS: HTR-8/SVneo, BeWo, JEG-3 and JAR were analyzed for epithelial and mesenchymal markers using immunofluorescence, real time PCR and Western blot. RESULTS: Our results showed that HTR-8/SVneo cell line contains two populations of cells suggesting the presence of trophoblast and stromal/mesenchymal cells. While all cells in BeWo, JEG-3 and Jar are positive for the trophoblast/epithelial marker CK7, HTR-8/SVneo cells contained few clusters of CK7 positive cells. Interestingly, vimentin expression was detected in a subset of HTR-8/SVneo cells and was completely absent from all other tested placental cell lines. DISCUSSION: Our results unveil the presence of a heterogeneous population of trophoblast and stromal cells within HTR-8/SVneo cell line. This mixed population of cells should be taken into consideration when using this cell line to study trophoblast functions.

MicroRNA-34a regulates WNT/TCF7 signaling and inhibits bone metastasis in Ras-activated prostate cancer.

Aberrant activation of Ras and WNT signaling are key events that have been shown to be up-regulated in prostate cancer that has metastasized to the bone. However, the regulatory mechanism of combinatorial Ras and WNT signaling in advanced prostate cancer is still unclear. TCF7, a WNT signaling-related gene, has been implicated as a critical factor in bone metastasis, and here we show that TCF7 is a direct target of miR-34a. In samples of prostate cancer patients, miR-34a levels are inversely correlated with TCF7 expression and a WNT dependent gene signature. Ectopic miR-34a expression inhibited bone metastasis and reduced cancer cell proliferation in a Ras-dependent xenograft model. We demonstrate that miR-34a can directly interfere with the gene expression of the anti-proliferative BIRC5, by targeting BIRC5 3'UTR. Importantly, BIRC5 overexpression was sufficient to reconstitute anti-apoptotic signaling in cells expressing high levels of miR-34a. In prostate cancer patients, we found that BIRC5 levels were positively correlated with a Ras signaling signature expression. Our data show that the bone metastasis and anti-apoptotic effects found in Ras signaling-activated prostate cancer cells require miR-34a deficiency, which in turn aids in cell survival by activating the WNT and anti-apoptotic signaling pathways thereby inducing TCF7 and BIRC5 expressions.

The plasminogen activation system modulates differently adipogenesis and myogenesis of embryonic stem cells.

Regulation of the extracellular matrix (ECM) plays an important functional role either in physiological or pathological conditions. The plasminogen activation (PA) system, comprising the uPA and tPA proteases and their inhibitor PAI-1, is one of the main suppliers of extracellular proteolytic activity contributing to tissue remodeling. Although its function in development is well documented, its precise role in mouse embryonic stem cell (ESC) differentiation in vitro is unknown. We found that the PA system components are expressed at very low levels in undifferentiated ESCs and that upon differentiation uPA activity is detected mainly transiently, whereas tPA activity and PAI-1 protein are maximum in well differentiated cells. Adipocyte formation by ESCs is inhibited by amiloride treatment, a specific uPA inhibitor. Likewise, ESCs expressing ectopic PAI-1 under the control of an inducible expression system display reduced adipogenic capacities after induction of the gene. Furthermore, the adipogenic differentiation capacities of PAI-1(-/-) induced pluripotent stem cells (iPSCs) are augmented as compared to wt iPSCs. Our results demonstrate that the control of ESC adipogenesis by the PA system correspond to different successive steps from undifferentiated to well differentiated ESCs. Similarly, skeletal myogenesis is decreased by uPA inhibition or PAI-1 overexpression during the terminal step of differentiation. However, interfering with uPA during days 0 to 3 of the differentiation process augments ESC myotube formation. Neither neurogenesis, cardiomyogenesis, endothelial cell nor smooth muscle formation are affected by amiloride or PAI-1 induction. Our results show that the PA system is capable to specifically modulate adipogenesis and skeletal myogenesis of ESCs by successive different molecular mechanisms.

p38 mitogen activated protein kinase controls two successive-steps during the early mesodermal commitment of embryonic stem cells.

Embryonic stem (ES) cells differentiate in vitro into all cell lineages. We previously found that the p38 mitogen activated kinase (p38MAPK) pathway controls the commitment of ES cells toward either cardiomyogenesis (p38 on) or neurogenesis (p38 off ). In this study, we show that p38α knock-out ES cells do not differentiate into cardiac, endothelial, smooth muscle, and skeletal muscle lineages. Reexpression of p38MAPK in these cells partially rescues their mesodermal differentiation defects and corrects the high level of spontaneous neurogenesis of knock-out cells. Wild-type ES cells were treated with a p38MAPK-specific inhibitor during the differentiation process. These experiments allowed us to identify 2 early independent successive p38MAPK functions in the formation of mesodermal lineages. Further, the first one correlates with the regulation of the expression of Brachyury, an essential mesodermal-specific transcription factor, by p38MAPK. In conclusion, by genetic and biochemical approaches, we demonstrate that p38MAPK activity is essential for the commitment of ES cell into cardiac, endothelial, smooth muscle, and skeletal muscle mesodermal lineages.