Activin A-derived human embryonic stem cells show increased competence to differentiate into primordial germ cell-like cells

Protocols for specifying human primordial germ cell-like cells (hPGCLCs) from human embryonic stem cells (hESCs) remain hindered by differences between hESC lines, their derivation methods, and maintenance culture conditions. This poses significant challenges for establishing reproducible in vitro models of human gametogenesis. Here, we investigated the influence of activin A (ActA) during derivation and maintenance on the propensity of hESCs to differentiate into PGCLCs. We show that continuous ActA supplementation during hESC derivation (from blastocyst until the formation of the post-inner cell mass intermediate [PICMI]) and supplementation (from the first passage of the PICMI onwards) is beneficial to differentiate hESCs to PGCLCs subsequently. Moreover, comparing isogenic primed and naive states prior to differentiation, we showed that conversion of hESCs to the 4i-state improves differentiation to (TNAP [tissue nonspecific alkaline phosphatase]+/PDPN [podoplanin]+) PGCLCs. Those PGCLCs expressed several germ cell markers, including TFAP2C (transcription factor AP-2 gamma), SOX17 (SRY-box transcription factor 17), and NANOS3 (nanos C2HC-type zinc finger 3), and markers associated with germ cell migration, CXCR4 (C-X-C motif chemokine receptor 4), LAMA4 (laminin subunit alpha 4), ITGA6 (integrin subunit alpha 6), and CDH4 (cadherin 4), suggesting that the large numbers of PGCLCs obtained may be suitable to differentiate further into more mature germ cells. Finally, hESCs derived in the presence of ActA showed higher competence to differentiate to hPGCLC, in particular if transiently converted to the 4i-state. Our work provides insights into the differences in differentiation propensity of hESCs and delivers an optimized protocol to support efficient human germ cell derivation

An updated view into the cell cycle kinetics of human T lymphocytes and the impact of irradiation

Even though a detailed understanding of the proliferative characteristics of T lymphocytes is imperative in many research fields, prior studies have never reached a consensus on these characteristics, and on the corresponding cell cycle kinetics specifically. In this study, the general proliferative response of human T lymphocytes to phytohaemagglutinin (PHA) stimulation was characterized using a carboxyfluorescein succinimidyl ester-based flow cytometric assay. We were able to determine when PHA-stimulated T lymphocytes complete their first division, the proportion of cells that initiate proliferation, the subsequent division rate of the cells, and the impact of irradiation on these proliferative properties. Next, we accurately visualized the cell cycle progression of dividing T lymphocytes cultured in whole blood using an adapted 5-ethynyl-2’-deoxyuridine pulse-chase method. Furthermore, through multiple downstream analysis methods, we were able to make an estimation of the corresponding cell cycle kinetics. We also visualized the impact of X-rays on the progression of the cells through the cell cycle. Our results showed dose-dependent G2 arrest after exposure to irradiation, and a corresponding delay in G1 phase-entry of the cells. In conclusion, utilizing various flow cytometric assays, we provided valuable information on T lymphocyte proliferation characteristics starting from first division to fully dividing cells

An updated view into the proliferative characteristics of human T lymphocytes and the effects of irradiation

Introduction Human T lymphocytes are widely used in radiobiology research and in these studies, detailed knowledge about T lymphocyte proliferation characteristics and corresponding cell cycle kinetics is often key. To present day, however, the available information on these characteristics remains quite conflicting. Many prior reports are based on outdated techniques and never reached a consensus on the proliferative properties of these cells. Materials and Methods In this study, the general proliferative response of human T lymphocytes to phytohaemagglutinin (PHA) stimulation was characterized using a carboxyfluorescein succinimidyl ester (CFSE)-based flowcytometric assay. We were able to determine when PHA-stimulated T lymphocytes complete their first division, the proportion of cells that initiate proliferation, the subsequent division rate of the cells, and the impact of irradiation on these proliferative properties. Using an adapted 5- ethynyl-2’-deoxyuridine (EdU) pulse-chase method, the cell cycle progression of both non-irradiated and irradiated PHAresponsive T lymphocytes cultured in whole blood was visualized and assessed. Through multiple downstream analysis methods, an estimation of the corresponding cell cycle kinetics was made. Results T lymphocytes complete a first cell cycle between 48 and 72 hours after PHA stimulation. After irradiation of resting T lymphocytes with doses of 1 and 2 Gy of X-rays, no significant impact was observed on the proportion of cells that initiate proliferation and on the subsequent division rate of these cells. The cell cycle phase (G1, S, and G2/M) durations was estimated to be 7, 6.1, and 3.5 hours, respectively. The total cell cycle time was predicted to be 16.6 hours. Furthermore, our results showed dose-dependent G2 arrest after exposure to 2 and 4 Gy of X-rays in the S phase, and a corresponding delay in G1 phase-entry of the cells. When exposed to irradiation in the G1 phase, no pronounced G1 arrest could be observed. This study provides valuable information on T lymphocyte proliferative characteristics starting from first division to fully dividing cells, based on various flow-cytometric assays. Furthermore, we propose a feasible EdU pulse-chase method to chase T lymphocytes cultured in whole blood, which provided us with a model that better represents the in vivo situation

A novel S-micronucleus assay for radiosensitivity detection

Background and aim The assessment of chromosomal radiosensitivity (RS), a characteristic of patients with genetic disorders such as ATM, NBS, is often done by the G0- micronucleus (MN) assay. This standard G0-MN assay is performed on lymphocytes irradiated in the G0-phase of the cell cycle, thus focusing on defects in the non-homologous end joining repair pathway. In this study, we propose a novel S-MN assay that irradiates the cells during the S phase of the cell cycle, and thus focusses on chromosomal radiosensitivity due to defects in the homologous recombination repair pathway. Methods Peripheral blood samples were collected from 20 healthy individuals and 7 patients with a confirmed or suspected defect in a DNA repair gene. Furthermore, blood samples from 2 donors were collected repeatably over a period of 12 months. Whole blood was cultured with phytohaemagglutinin-M as mitogen. After 96 hours, the cells were pulse-labeled with the S phase marker 5-ethynyl-2’-deoxyuridine (EdU) and irradiated in vitro with 0.5, 1, and 2 Gy of 220 kV X-rays. After an additional 24 hours of culture with cytochalasin B, the cells were fixed and MNi were scored in EdU-positive binucleated cells. Results and conclusion We demonstrate that the marker EdU can be successfully used for the identification of lymphocytes irradiated during the S phase, thus allowing the assessment of HR-mediated repair. Furthermore, we show the suitability of EdU as a novel staining method to manually score MNi. Evaluation of the inter-individual and intra-individual variability across healthy donor samples demonstrate the robustness and reproducibility of this novel assay. Finally, our results show an enhanced RS profile in two patients with a known mutation in ATM and ATRIP. A minor increase in MNi yield was observed for a patient carrying a heterozygous NBS mutation. Altogether, we show the value of the novel S-MN assay as an additional cytogenetic assay, and we demonstrate the applicability of this assay in evaluating chromosomal radiosensitivity in patients with a DNA repair defect

Radiosensitivity analysis in patients with a primary immunodeficiency disease

Primary Immune Deficiency Diseases (PIDs) are life-threatening genetic disorders of the immune system. A subset of PIDs is caused by mutations in genes involved in the repair of DNA double-strand breaks, by which affected patients may also be radiosensitive. For many reasons PID patients can be exposed to radiation (bone marrow transplantation, radiotherapy, diagnostic imaging), but this may pose serious risks to radiosensitive subjects. Surprisingly, radiosensitivity testing is currently not included in the workup of PIDs in most European countries. The main goal of our study is to translate radiosensitivity analysis in the diagnostic workup for PIDs in Belgium. To this aim, two cell-cycle specific in vitro radiosensitivity assays will be included in the standard diagnostic procedures in patients with suspected PID at the Ghent University Hospital (reference center for Belgium): (1) the G0 cytokinesis-block micronucleus assay (CBMN) which is about to be translated into the clinical practice, (2) the S/G2 CBMN which has been developed in our labs with positive proof-of-concept but which must be further optimized before translation. Both assays will be performed on peripheral blood lymphocytes of the patients. Furthermore, radiosensitivity assays will also be optimized for fibroblasts, obtained from skin biopsies of patients. In our study design, radiosensitivity analysis will be the central core of two innovative diagnostic and therapeutic algorithms, which will include immunophentyping, direct subsequent genetic analysis and guide optimal patient care. Micronucleus analysis in lymphocytes of patients is currently ongoing and first results will be presented. We believe that inclusion of radiosensitivity testing will represent a major improvement in the timely diagnosis and management of patients affected by these life-threatening, heterogeneous and difficult-to-diagnose diseases

Assessment of radiation sensitivity in patients with a primary immunodeficiency disease

Primary Immune Deficiency Diseases (PIDs) are life-threatening genetic disorders of the immune system. A subset of PIDs is caused by mutations in genes involved in the repair of DNA double-strand breaks, by which affected patients may also be radiosensitive. For many reasons PID patients can be exposed to radiation (bone marrow transplantation, radiotherapy, diagnostic imaging), but this may pose serious risks to radiosensitive subjects. Surprisingly, radiosensitivity testing is currently not included in the workup of PIDs in most European countries. The main goal of our study is to translate radiosensitivity analysis in the diagnostic workup for PIDs in Belgium. To this aim, two cell-cycle specific in vitro radiosensitivity assays will be included in the standard diagnostic procedures in patients with suspected PID at the Ghent University Hospital (reference center for Belgium): (1) the G0 cytokinesis-block micronucleus assay (CBMN) which is about to be translated into the clinical practice, (2) the S/G2 CBMN which has been developed in our labs with positive proof-of-concept but which must be further optimized before translation. Both assays will be performed on peripheral blood lymphocytes of the patients. Furthermore, radiosensitivity assays will also be optimized for fibroblasts, obtained from skin biopsies of patients. In our study design, radiosensitivity analysis will be the central core of two innovative diagnostic and therapeutic algorithms, which will include immunophentyping, direct subsequent genetic analysis and guide optimal patient care. Micronucleus analysis in lymphocytes of patients is currently ongoing and first results will be presented. We believe that inclusion of radiosensitivity testing will represent a major improvement in the timely diagnosis and management of patients affected by these life-threatening, heterogeneous and difficult-to-diagnose diseases