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.

Translational and oncologic significance of tertiary lymphoid structures in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma (PDAC) is an aggressive tumor with poor survival and limited treatment options. PDAC resistance to immunotherapeutic strategies is multifactorial, but partially owed to an immunosuppressive tumor immune microenvironment (TiME). However, the PDAC TiME is heterogeneous and harbors favorable tumor-infiltrating lymphocyte (TIL) populations. Tertiary lymphoid structures (TLS) are organized aggregates of immune cells that develop within non-lymphoid tissue under chronic inflammation in multiple contexts, including cancers. Our current understanding of their role within the PDAC TiME remains limited; TLS are complex structures with multiple anatomic features such as location, density, and maturity that may impact clinical outcomes such as survival and therapy response in PDAC. Similarly, our understanding of methods to manipulate TLS is an actively developing field of research. TLS may function as anti-tumoral immune niches that can be leveraged as a therapeutic strategy to potentiate both existing chemotherapeutic regimens and potentiate future immune-based therapeutic strategies to improve patient outcomes. This review seeks to cover anatomy, relevant features, immune effects, translational significance, and future directions of understanding TLS within the context of PDAC.