Identification of Spectral Modifications Occurring during Reprogramming of Somatic Cells

Recent technological advances in cell reprogramming by generation of induced pluripotent stem cells (iPSC) offer major perspectives in disease modelling and future hopes for providing novel stem cells sources in regenerative medicine. However, research on iPSC still requires refining the criteria of the pluripotency stage of these cells and exploration of their equivalent functionality to human embryonic stem cells (ESC). We report here on the use of infrared microspectroscopy to follow the spectral modification of somatic cells during the reprogramming process. We show that induced pluripotent stem cells (iPSC) adopt a chemical composition leading to a spectral signature indistinguishable from that of embryonic stem cells (ESC) and entirely different from that of the original somatic cells. Similarly, this technique allows a distinction to be made between partially and fully reprogrammed cells. We conclude that infrared microspectroscopy signature is a novel methodology to evaluate induced pluripotency and can be added to the tests currently used for this purpose

The Transition between Telomerase and ALT Mechanisms in Hodgkin Lymphoma and Its Predictive Value in Clinical Outcomes

International audienceBackground: We analyzed telomere maintenance mechanisms (TMMs) in lymph node samples from HL patients treated with standard therapy. The TMMs correlated with clinical outcomes of patients. Materials and Methods: Lymph node biopsies obtained from 38 HL patients and 24 patients with lymphadenitis were included in this study. Seven HL cell lines were used as in vitro models. Telomerase activity (TA) was assessed by TRAP assay and verified through hTERT immunofluorescence expression; alternative telomere lengthening (ALT) was also assessed, along with EBV status. Results: Both TA and ALT mechanisms were present in HL lymph nodes. Our findings were reproduced in HL cell lines. The highest levels of TA were expressed in CD30−/CD15− cells. Small cells were identified with ALT and TA. Hodgkin and Reed Sternberg cells contained high levels of PML bodies, but had very low hTERT expression. There was a significant correlation between overall survival (p < 10−3), event-free survival (p < 10−4), and freedom from progression (p < 10−3) and the presence of an ALT profile in lymph nodes of EBV+ patients. Conclusion: The presence of both types of TMMs in HL lymph nodes and in HL cell lines has not previously been reported. TMMs correlate with the treatment outcome of EBV+ HL patients

Patient-Derived iPSCs Reveal Evidence of Telomere Instability and DNA Repair Deficiency in Coats Plus Syndrome

Coats plus (CP) syndrome is an inherited autosomal recessive condition that results from mutations in the conserved telomere maintenance component 1 gene (CTC1). The CTC1 protein functions as a part of the CST protein complex, a protein heterotrimer consisting of CTC1&ndash;STN1&ndash;TEN1 which promotes telomere DNA synthesis and inhibits telomerase-mediated telomere elongation. However, it is unclear how CTC1 mutations may have an effect on telomere structure and function. For that purpose, we established the very first induced pluripotent stem cell lines (iPSCs) from a compound heterozygous patient with CP carrying deleterious mutations in both alleles of CTC1. Telomere dysfunction and chromosomal instability were assessed in both circulating lymphocytes and iPSCs from the patient and from healthy controls of similar age. The circulating lymphocytes and iPSCs from the CP patient were characterized by their higher telomere length heterogeneity and telomere aberrations compared to those in control cells from healthy donors. Moreover, in contrast to iPSCs from healthy controls, the high levels of telomerase were associated with activation of the alternative lengthening of telomere (ALT) pathway in CP-iPSCs. This was accompanied by inappropriate activation of the DNA repair proteins &gamma;H2AX, 53BP1, and ATM, as well as with accumulation of DNA damage, micronuclei, and anaphase bridges. CP-iPSCs presented features of cellular senescence and increased radiation sensitivity. Clonal dicentric chromosomes were identified only in CP-iPSCs after exposure to radiation, thus mirroring the role of telomere dysfunction in their formation. These data demonstrate that iPSCs derived from CP patients can be used as a model system for molecular studies of the CP syndrome and underscores the complexity of telomere dysfunction associated with the defect of DNA repair machinery in the CP syndrome

Contribution a l'etude des signaux regulant l'activation et la proliferation des lymphocytes T chez l'homme. Etude en culture semi-solide

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 78645 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Generation of induced pluripotent stem cell (iPSC) line from a patient with triple negative breast cancer with hereditary exon 17 deletion of BRCA1 gene

BRCA1 germline mutation confers hereditary predisposition for breast and ovarian cancer. To understand the physiopathology of mammary and ovarian epithelial cancer transformation, and to identify early driver molecular events, we have generated an iPSC line from a patient carrying a germline exon 17 deletion in BRCA1 gene (BRAC1Ex17 iPSC) in a high-risk family context. Blood cells were reprogrammed used non-integrative virus of Sendaï. The BRCA1-deleted iPSC had normal karyotype, harboured a deletion in the exon 17 of the BRCA1 gene, expressed pluripotent hallmarks and had the differentiation capacity into the three germ layers

Toxicity and phototoxicity of Hypocrellin A on malignant human cell lines, evidence of a synergistic action of photodynamic therapy with Imatinib mesylate

International audiencePhotodynamic therapy combines a photosensitizer, localised preferentially in malignant cells with light activation. Hypocrellin A (HA), a lipid-soluble peryloquinone, is considered as a high potential photosensitizer.We report dose and light irradiation effects of HA on HeLa, Calu and K562 cell lines, the latter including a subclone resistant to Imatinib mesylate (IM, Gleevec). All cell lines and subclones tested are sensitive to HA PDT.In the epithelial tumour cell lines, we observe a significant photosensitizing effect in the presence of HA. In the leukemic K562 cells, HA exposure led to an inhibitory effect, which was not seen in the K562 cells resistant to Imatinib mesylate. However, experiments using IM and HA led to a reversal of IM-resistant phenotype in this cell line, with evidence of a major sensitizing effect of photodynamic therapy.Overall our results suggest a phototoxicity of HA in epithelial cell lines and demonstrate for the first time, a synergy between IM and photodynamic therapy to circumvent IM-resistance

iPSC-Derived Hereditary Breast Cancer Model Reveals the BRCA1-Deleted Tumor Niche as a New Culprit in Disease Progression

International audienceTumor progression begins when cancer cells recruit tumor-associated stromal cells to produce a vascular niche, ultimately resulting in uncontrolled growth, invasion, and metastasis. It is poorly understood, though, how this process might be affected by deletions or mutations in the breast cancer type 1 susceptibility (BRCA1) gene in patients with a lifetime risk of developing breast and/or ovarian cancer. To model the BRCA1-deleted stroma, we first generated induced pluripotent stem cells (iPSCs) from patients carrying a germline deletion of exon 17 of the BRCA1 gene (BRCA1+/− who, based on their family histories, were at a high risk for cancer. Using peripheral blood mononuclear cells (PBMCs) of these two affected family members and two normal (BRCA1+/+) individuals, we established a number of iPSC clones via non-integrating Sendai virus-based delivery of the four OCT4, SOX2, KLF4, and c-MYC factors. Induced mesenchymal stem cells (iMSCs) were generated and used as normal and pathological stromal cells. In transcriptome analyses, BRCA1+/− iMSCs exhibited a unique pro-angiogenic signature: compared to non-mutated iMSCs, they expressed high levels of HIF-1α, angiogenic factors belonging to the VEGF, PDGF, and ANGPT subfamilies showing high angiogenic potential. This was confirmed in vitro through the increased capacity to generate tube-like structures compared to BRCA1+/+ iMSCs and in vivo by a matrigel plug angiogenesis assay where the BRCA1+/− iMSCs promoted the development of an extended and organized vessel network. We also reported a highly increased migration capacity of BRCA1+/− iMSCs through an in vitro wound healing assay that correlated with the upregulation of the periostin (POSTN). Finally, we assessed the ability of both iMSCs to facilitate the engraftment of murine breast cancer cells using a xenogenic 4T1 transplant model. The co-injection of BRCA1+/− iMSCs and 4T1 breast cancer cells into mouse mammary fat pads gave rise to highly aggressive tumor growth (2-fold increase in tumor volume compared to 4T1 alone, p = 0.01283) and a higher prevalence of spontaneous metastatic spread to the lungs. Here, we report for the first time a major effect of BRCA1 haploinsufficiency on tumor-associated stroma in the context of BRCA1-associated cancers. The unique iMSC model used here was generated using patient-specific iPSCs, which opens new therapeutic avenues for the prevention and personalized treatment of BRCA1-associated hereditary breast cancer

Generation of an induced pluripotent stem cell (iPSC) line from a patient with maturity-onset diabetes of the young type 3 (MODY3) carrying a hepatocyte nuclear factor 1-alpha (HNF1A) mutation

Heterozygous non-synonymous (p.S142F) mutation in HNF1A leads to maturity-onset diabetes of the young (MODY) type 3, which is a subtype of dominant inherited young-onset non-autoimmune diabetes due to the defect of insulin secretion from pancreatic beta cells. We generated induced pluripotent stem cells (iPSCs) from a patient with HNF1A p.S142F mutation. Cells from this patient, which were reprogrammed by non-integrative viral transduction had normal karyotype, harboured the HNF1A p.S142F mutation, expressed pluripotency hallmarks

Patient-Derived iPSCs Reveal Evidence of Telomere Instability and DNA Repair Deficiency in Coats Plus Syndrome

Coats plus (CP) syndrome is an inherited autosomal recessive condition that results from mutations in the conserved telomere maintenance component 1 gene (CTC1). The CTC1 protein functions as a part of the CST protein complex, a protein heterotrimer consisting of CTC1–STN1–TEN1 which promotes telomere DNA synthesis and inhibits telomerase-mediated telomere elongation. However, it is unclear how CTC1 mutations may have an effect on telomere structure and function. For that purpose, we established the very first induced pluripotent stem cell lines (iPSCs) from a compound heterozygous patient with CP carrying deleterious mutations in both alleles of CTC1. Telomere dysfunction and chromosomal instability were assessed in both circulating lymphocytes and iPSCs from the patient and from healthy controls of similar age. The circulating lymphocytes and iPSCs from the CP patient were characterized by their higher telomere length heterogeneity and telomere aberrations compared to those in control cells from healthy donors. Moreover, in contrast to iPSCs from healthy controls, the high levels of telomerase were associated with activation of the alternative lengthening of telomere (ALT) pathway in CP-iPSCs. This was accompanied by inappropriate activation of the DNA repair proteins γH2AX, 53BP1, and ATM, as well as with accumulation of DNA damage, micronuclei, and anaphase bridges. CP-iPSCs presented features of cellular senescence and increased radiation sensitivity. Clonal dicentric chromosomes were identified only in CP-iPSCs after exposure to radiation, thus mirroring the role of telomere dysfunction in their formation. These data demonstrate that iPSCs derived from CP patients can be used as a model system for molecular studies of the CP syndrome and underscores the complexity of telomere dysfunction associated with the defect of DNA repair machinery in the CP syndrome