DNA Damage Response and Replication Stress in Mouse Embryonic Stem Cells

DNA replication is a central cellular process that allows duplication of the genetic material before its proper segregation during cell division. The DNA damage response (DDR) protects cells from deleterious mutations during replication and helps maintain genome stability in face of exogenous genotoxic stress. Such pathways must be particularly robust in stem cells, since they are constantly self-renewing and capable of differentiating into all other specialized cells. The main function of embryonic stem cells (ESCs) is to proliferate and differentiate into multiple cell types spatiotemporally, without compromising on their self-renewal capacity. The high proliferative capacity of ESCs is often coupled to rapid G1-S transition and elevated levels of CDKs and other cell cycle regulators. In this study, we show that mouse ESCs surprisingly experience endogenous DNA replication stress (RS), which is characterized by high basal levels of the ATR-dependent DDR marker γH2AX, chromatin recruitment of the single stranded DNA (ssDNA) binding proteins RPA and Rad51, accumulation of ssDNA gaps/nicks, increased replication fork reversal and slow fork progression. Strikingly, all these hallmarks of RS are quickly lost upon induction of differentiation, before cells stop proliferating. Furthermore, PARP1 activity - previously shown to be involved in replication of damaged DNA in somatic cells - is required to protect replication fork integrity in unperturbed ESCs. Our working hypothesis, which will be directly addressed in the next weeks, is that origin firing factors are rate limiting in ESCs, leading to inheritance of partially replicated DNA during fast cell cycle progression. Indeed, overexpression of the firing factor Cdc45 and/or altering the cell cycle length by inhibiting CDK activity, reduces DDR signalling in ESCs. Haematopoietic stem cells (HSCs) possess the ability to give rise to all the cells of the haematopoietic system. HSCs are mainly quiescent and are activated upon injury or inflammation to bring about tissue homeostasis. Stimulation of mice with interferon alpha (IFN-α) specifically activates dormant HSCs. Preliminary observations in this study suggest that most "activated" HSCs exhibit elevated γH2AX staining. These results suggest that HSCs that exit from dormancy may experience RS, similarly to actively proliferating ESCs. Collectively, the main findings in this study suggest that the active DDR in proliferating stem cells signals incomplete replication inherited during fast cell cycle progression

The Dynamic Nature of Human Dermal Fibroblasts Is Defined by Marked Variation in the Gene Expression of Specific Cytoskeletal Markers

The evidence for fibroblast heterogeneity is continuously increasing, and recent work has shed some light on the existence of different sub-populations of fibroblasts in the human skin. Although we now have a more precise understanding of their distribution in the human body, we do not know whether their properties are predictive of where these cells derive from or whether these sub-types have functional consequences. In this study, we employed single-cell transcriptomics (10X Genomics) to study gene expression and segregate fibroblast sub-populations based on their genetic signature. We report the differential expression of a defined set of genes in fibroblasts from human skin, which may contribute to their dynamicity in vivo and in vitro. We show that the sub-population of fibroblasts expressing cytoskeletal markers, such as ANXA2, VIM, ACTB, are enriched in an adult skin sample. Interestingly, this sub-population of fibroblasts is not enriched in a neonatal skin sample but becomes predominant when neonatal fibroblasts are cultivated. On the other hand, the fibroblast sub-populations expressing COL1A1 and ELN are enriched in neonatal skin but are reduced in the adult skin and in fibroblasts from neonatal skin that are cultured in vitro. Our results indicate that fibroblasts are a dynamic cell type, and while their genetic make-up changes markedly, only a handful of genes belonging to the same functional pathway govern this alteration. The gene expression pattern of cytoskeletal markers may help in identifying whether the fibroblasts were isolated from an adult or an infant or whether they were cultivated, and this information could be useful for quality control in clinics and in cell banking. Furthermore, this study opens additional avenues to investigate the role of these markers in defining the complexity of human dermal fibroblasts

The Dynamic Nature of Human Dermal Fibroblasts Is Defined by Marked Variation in the Gene Expression of Specific Cytoskeletal Markers

The evidence for fibroblast heterogeneity is continuously increasing, and recent work has shed some light on the existence of different sub-populations of fibroblasts in the human skin. Although we now have a more precise understanding of their distribution in the human body, we do not know whether their properties are predictive of where these cells derive from or whether these sub-types have functional consequences. In this study, we employed single-cell transcriptomics (10X Genomics) to study gene expression and segregate fibroblast sub-populations based on their genetic signature. We report the differential expression of a defined set of genes in fibroblasts from human skin, which may contribute to their dynamicity in vivo and in vitro. We show that the sub-population of fibroblasts expressing cytoskeletal markers, such as ANXA2, VIM, ACTB, are enriched in an adult skin sample. Interestingly, this sub-population of fibroblasts is not enriched in a neonatal skin sample but becomes predominant when neonatal fibroblasts are cultivated. On the other hand, the fibroblast sub-populations expressing COL1A1 and ELN are enriched in neonatal skin but are reduced in the adult skin and in fibroblasts from neonatal skin that are cultured in vitro. Our results indicate that fibroblasts are a dynamic cell type, and while their genetic make-up changes markedly, only a handful of genes belonging to the same functional pathway govern this alteration. The gene expression pattern of cytoskeletal markers may help in identifying whether the fibroblasts were isolated from an adult or an infant or whether they were cultivated, and this information could be useful for quality control in clinics and in cell banking. Furthermore, this study opens additional avenues to investigate the role of these markers in defining the complexity of human dermal fibroblasts

Poly(ADP-ribosyl)gycohydrolase (PARG) prevents the accumulation of unusual replication structures during unperturbed S phase

Poly(ADP-ribosyl)ation (PAR) has been implicated in various aspects of the cellular response to DNA damage and genome stability. Although 17 human poly(ADP-ribose) polymerase (PARP) genes have been identified, a single poly(ADP-ribose) glycohydrolase (PARG) mediates PAR degradation. Here we investigated the role of PARG in the replication of human chromosomes. We show that PARG depletion affects cell proliferation and DNA synthesis, leading to replication-coupled H2AX phosphorylation. Furthermore, PARG depletion or inhibition per se slows down individual replication forks similarly to mild chemotherapeutic treatment. Electron microscopic analysis of replication intermediates reveals marked accumulation of reversed forks and ssDNA gaps in unperturbed PARG-defective cells. Intriguingly, while we found no physical evidence for chromosomal breakage, PARG defective cells displayed both ATM and ATR activation, as well as chromatin recruitment of standard double strand break repair factors, such as 53BP1 and RAD51. Overall, these data prove PAR degradation essential to promote resumption of replication at endogenous and exogenous lesions, preventing idle recruitment of repair factors to remodeled replication forks. Furthermore, they suggest that fork remodeling and restart are surprisingly frequent in unperturbed cells and provide a molecular rationale to explore PARG inhibition in cancer chemotherapy

Role of transabdominal ultrasound of lung bases and follow-up in premature neonates with respiratory distress soon after birth

Background: Chest radiography has been the traditional method of diagnostic evaluation of patients of hyaline membrane disease (HMD). Lung sonography (USG) has been lately explored as an alternative modality. Aims: To explore the application of transabdominal USG of lung bases (TASL) in the evaluation of HMD in premature neonates with respiratory distress soon after birth. Settings and Design: Tertiary care institutional setup. Study duration-18 months. Follow-up-variable, up to 1 month. Prospective descriptive study. Materials and Methods: Eighty-eight consecutive patients admitted in the neonatal intensive care unit (NICU) with gestational age <32 weeks having respiratory distress within 6 h of birth were enrolled. The diagnosis of HMD was made if the patient had negative gastric shake test and/or suggestive chest radiograph. TASL was performed in all patients within the first 24 h of life and biweekly subsequently. USG was interpreted as normal, HMD pattern, or broncho-pulmonary dysplasia (BPD) pattern. Biweekly follow-up was done for patients showing HMD till normalization of the sonographic HMD pattern, development of the sonographic BPD pattern, or death/discharge of the neonate from the hospital. Results and Conclusions: TASL showed 85.7% sensitivity, 75% specificity, 88.88% positive predictive value, and 69.2% negative predictive value for the diagnosis of HMD. The abnormal sonographic findings on day 14 had 94.1% accuracy for prediction of eventual occurrence of clinical BPD. TASL is complementary to chest radiograph in the diagnosis of HMD. It is also useful for the early prediction of BPD with the potential of reducing the cumulative radiation dose to these neonates

Human Basal and Suprabasal Keratinocytes Are Both Able to Generate and Maintain Dermo&ndash;Epidermal Skin Substitutes in Long-Term In Vivo Experiments

The basal layer of human interfollicular epidermis has been described to harbour both quiescent keratinocyte stem cells and a transit amplifying cell population that maintains the suprabasal epidermal layers. We performed immunofluorescence analyses and revealed that the main proliferative keratinocyte pool in vivo resides suprabasally. We isolated from the human epidermis two distinct cell populations, the basal and the suprabasal keratinocytes, according to the expression of integrin &beta;4 (i&beta;4). We compared basal i&beta;4+ or suprabasal i&beta;4&minus; keratinocytes with respect to their proliferation and colony-forming ability and their Raman spectral properties. In addition, we generated dermo&ndash;epidermal substitutes using freshly isolated and sorted basal i&beta;4+ or suprabasal i&beta;4&minus; keratinocytes and transplanted them on immuno-compromised rats. We show that suprabasal i&beta;4&minus; keratinocytes acquire a similar proliferative capacity as basal i&beta;4+ keratinocytes after two weeks of culture in vitro, with expression of high levels of i&beta;4 and downregulation of K10 expression. In addition, both basal i&beta;4+ and suprabasal i&beta;4&minus; keratinocytes acquire authentic self-renewing properties during the in vitro 3D-culture phase and are able to generate and maintain a fully stratified epidermis for 16 weeks in vivo. Therefore, against the leading dogma, we propose that human suprabasal keratinocytes can retro-differentiate into true basal stem cells in a wound situation and/or when in contact with the basement membrane

Human Basal and Suprabasal Keratinocytes Are Both Able to Generate and Maintain Dermo–Epidermal Skin Substitutes in Long-Term In Vivo Experiments

The basal layer of human interfollicular epidermis has been described to harbour both quiescent keratinocyte stem cells and a transit amplifying cell population that maintains the suprabasal epidermal layers. We performed immunofluorescence analyses and revealed that the main proliferative keratinocyte pool in vivo resides suprabasally. We isolated from the human epidermis two distinct cell populations, the basal and the suprabasal keratinocytes, according to the expression of integrin β4 (iβ4). We compared basal iβ4+ or suprabasal iβ4− keratinocytes with respect to their proliferation and colony-forming ability and their Raman spectral properties. In addition, we generated dermo–epidermal substitutes using freshly isolated and sorted basal iβ4+ or suprabasal iβ4− keratinocytes and transplanted them on immuno-compromised rats. We show that suprabasal iβ4− keratinocytes acquire a similar proliferative capacity as basal iβ4+ keratinocytes after two weeks of culture in vitro, with expression of high levels of iβ4 and downregulation of K10 expression. In addition, both basal iβ4+ and suprabasal iβ4− keratinocytes acquire authentic self-renewing properties during the in vitro 3D-culture phase and are able to generate and maintain a fully stratified epidermis for 16 weeks in vivo. Therefore, against the leading dogma, we propose that human suprabasal keratinocytes can retro-differentiate into true basal stem cells in a wound situation and/or when in contact with the basement membrane