In vivo reduction of RAD51-mediated homologous recombination triggers aging but impairs oncogenesis.

Homologous recombination (HR) is a prominent DNA repair pathway maintaining genome integrity. Mutations in many HR genes lead to cancer predisposition. Paradoxically, the implication of the pivotal HR factor RAD51 on cancer development remains puzzling. Particularly, no RAD51 mouse models are available to address the role of RAD51 in aging and carcinogenesis in vivo. We engineered a mouse model with an inducible dominant-negative form of RAD51 (SMRad51) that suppresses RAD51-mediated HR without stimulating alternative mutagenic repair pathways. We found that in vivo expression of SMRad51 led to replicative stress, systemic inflammation, progenitor exhaustion, premature aging and reduced lifespan, but did not trigger tumorigenesis. Expressing SMRAD51 in a breast cancer predisposition mouse model (PyMT) decreased the number and the size of tumors, revealing an anti-tumor activity of SMRAD51. We propose that these in vivo phenotypes result from chronic endogenous replication stress caused by HR decrease, which preferentially targets progenitors and tumor cells. Our work underlines the importance of RAD51 activity for progenitor cell homeostasis, preventing aging and more generally for the balance between cancer and aging.

Fetal Muse-based therapy prevents lethal radio-induced gastrointestinal syndrome by intestinal regeneration.

BACKGROUND: Human multilineage-differentiating stress enduring (Muse) cells are nontumorigenic endogenous pluripotent-like stem cells that can be easily obtained from various adult or fetal tissues. Regenerative effects of Muse cells have been shown in some disease models. Muse cells specifically home in damaged tissues where they exert pleiotropic effects. Exposition of the small intestine to high doses of irradiation (IR) delivered after radiotherapy or nuclear accident results in a lethal gastrointestinal syndrome (GIS) characterized by acute loss of intestinal stem cells, impaired epithelial regeneration and subsequent loss of the mucosal barrier resulting in sepsis and death. To date, there is no effective medical treatment for GIS. Here, we investigate whether Muse cells can prevent lethal GIS and study how they act on intestinal stem cell microenvironment to promote intestinal regeneration. METHODS: Human Muse cells from Wharton's jelly matrix of umbilical cord (WJ-Muse) were sorted by flow cytometry using the SSEA-3 marker, characterized and compared to bone-marrow derived Muse cells (BM-Muse). Under gas anesthesia, GIS mice were treated or not through an intravenous retro-orbital injection of 50,000 WJ-Muse, freshly isolated or cryopreserved, shortly after an 18 Gy-abdominal IR. No immunosuppressant was delivered to the mice. Mice were euthanized either 24 h post-IR to assess early small intestine tissue response, or 7 days post-IR to assess any regenerative response. Mouse survival, histological stainings, apoptosis and cell proliferation were studied and measurement of cytokines, recruitment of immune cells and barrier functional assay were performed. RESULTS: Injection of WJ-Muse shortly after abdominal IR highly improved mouse survival as a result of a rapid regeneration of intestinal epithelium with the rescue of the impaired epithelial barrier. In small intestine of Muse-treated mice, an early enhanced secretion of IL-6 and MCP-1 cytokines was observed associated with (1) recruitment of monocytes/M2-like macrophages and (2) proliferation of Paneth cells through activation of the IL-6/Stat3 pathway. CONCLUSION: Our findings indicate that a single injection of a small quantity of WJ-Muse may be a new and easy therapeutic strategy for treating lethal GIS.

In Vivo Monitoring of Polycythemia Vera Development Reveals Carbonic Anhydrase 1 as a Potent Therapeutic Target.

Current murine models of myeloproliferative neoplasms (MPNs) cannot examine how MPNs progress from a single bone marrow source to the entire hematopoietic system. Thus, using transplantation of knock-in JAK2V617F hematopoietic cells into a single irradiated leg, we show development of polycythemia vera (PV) from a single anatomic site in immunocompetent mice. Barcode experiments reveal that grafted JAK2V617F stem/progenitor cells migrate from the irradiated leg to nonirradiated organs such as the contralateral leg and spleen, which is strictly required for development of PV. Mutant cells colonizing the nonirradiated leg efficiently induce PV in nonconditioned recipient mice and contain JAK2V617F hematopoietic stem/progenitor cells that express high levels of carbonic anhydrase 1 (CA1), a peculiar feature also found in CD34+ cells from patients with PV. Finally, genetic and pharmacologic inhibition of CA1 efficiently suppresses PV development and progression in mice and decreases PV patients' erythroid progenitors, strengthening CA1 as a potent therapeutic target for PV. SIGNIFICANCE: Follow-up of hematopoietic malignancies from their initiating anatomic site is crucial for understanding their development and discovering new therapeutic avenues. We developed such an approach, used it to characterize PV progression, and identified CA1 as a promising therapeutic target of PV. This article is highlighted in the In This Issue feature, p. 265.

Deleterious effect of bone marrow-resident macrophages on hematopoietic stem cells in response to total body irradiation.

Bone marrow (BM) resident macrophages interact with a population of long-term hematopoietic stem cells (LT-HSCs) but their role on LT-HSC properties after stress is not well defined. Here, we show that a 2 Gy-total body irradiation (TBI)-mediated death of LT-HSCs is associated with increased percentages of LT-HSCs with reactive oxygen species (ROS) and of BM resident macrophages producing nitric oxide (NO), resulting in an increased percentage of LT-HSCs with endogenous cytotoxic peroxynitrites. Pharmacological or genetic depletion of BM resident macrophages impairs the radio-induced increases in the percentage of both ROS+ LT-HSCs and peroxynitrite+ LT-HSCs and results in a complete recovery of a functional pool of LT-HSCs. Finally, we show that after a 2 Gy-TBI, a specific decrease of NO production by BM resident macrophages improves the LT-HSC recovery, whereas an exogenous NO delivery decreases the LT-HSC compartment. Altogether, these results show that BM resident macrophages are involved in the response of LT-HSCs to a 2 Gy-TBI and suggest that regulation of NO production can be used to modulate some deleterious effects of a TBI on LT-HSCs.

Tumor resistance to radiotherapy is triggered by an ATM/TAK1-dependent-increased expression of the cellular prion protein.

In solid cancers, high expression of the cellular prion protein (PrPC) is associated with stemness, invasiveness, and resistance to chemotherapy, but the role of PrPC in tumor response to radiotherapy is unknown. Here, we show that, in neuroblastoma, breast, and colorectal cancer cell lines, PrPC expression is increased after ionizing radiation (IR) and that PrPC deficiency increases radiation sensitivity and decreases radiation-induced radioresistance in tumor cells. In neuroblastoma cells, IR activates ATM that triggers TAK1-dependent phosphorylation of JNK and subsequent activation of the AP-1 transcription factor that ultimately increases PRNP promoter transcriptional activity through an AP-1 binding site in the PRNP promoter. Importantly, we show that this ATM-TAK1-PrPC pathway mediated radioresistance is activated in all tumor cell lines studied and that pharmacological inhibition of TAK1 activity recapitulates the effects of PrPC deficiency. Altogether, these results unveil how tumor cells activate PRNP to acquire resistance to radiotherapy and might have implications for therapeutic targeting of solid tumors radioresistance.

Ultra-high-dose-rate FLASH and Conventional-Dose-Rate Irradiation Differentially Affect Human Acute Lymphoblastic Leukemia and Normal Hematopoiesis.

PURPOSE: Ultra-high-dose-rate FLASH radiation therapy has been shown to minimize side effects of irradiation in various organs while keeping antitumor efficacy. This property, called the FLASH effect, has caused enthusiasm in the radiation oncology community because it opens opportunities for safe dose escalation and improved radiation therapy outcome. Here, we investigated the impact of ultra-high-dose-rate FLASH versus conventional-dose-rate (CONV) total body irradiation (TBI) on humanized models of T-cell acute lymphoblastic leukemia (T-ALL) and normal human hematopoiesis. METHODS AND MATERIALS: We optimized the geometry of irradiation to ensure reproducible and homogeneous procedures using eRT6/Oriatron. Three T-ALL patient-derived xenografts and hematopoietic stem/progenitor cells (HSPCs) and CD34+ cells isolated from umbilical cord blood were transplanted into immunocompromised mice, together or separately. After reconstitution, mice received 4 Gy FLASH and CONV-TBI, and tumor growth and normal hematopoiesis were studied. A retrospective study of clinical and gene-profiling data previously obtained on the 3 T-ALL patient-derived xenografts was performed. RESULTS: FLASH-TBI was more efficient than CONV-TBI in controlling the propagation of 2 cases of T-ALL, whereas the third case of T-ALL was more responsive to CONV-TBI. The 2 FLASH-sensitive cases of T-ALL had similar genetic abnormalities, and a putative susceptibility imprint to FLASH-RT was found. In addition, FLASH-TBI was able to preserve some HSPC/CD34+ cell potential. Interestingly, when HSPC and T-ALL were present in the same animals, FLASH-TBI could control tumor development in most (3 of 4) of the secondary grafted animals, whereas among the mice receiving CONV-TBI, treated cells died with high leukemia infiltration. CONCLUSIONS: Compared with CONV-TBI, FLASH-TBI reduced functional damage to human blood stem cells and had a therapeutic effect on human T-ALL with a common genetic and genomic profile. The validity of the defined susceptibility imprint needs to be investigated further; however, to our knowledge, the present findings are the first to show benefits of FLASH-TBI on human hematopoiesis and leukemia treatment.

A genetic variant controls interferon-ß gene expression in human myeloid cells by preventing C/EBP-ß binding on a conserved enhancer.

Interferon ß (IFN-ß) is a cytokine that induces a global antiviral proteome, and regulates the adaptive immune response to infections and tumors. Its effects strongly depend on its level and timing of expression. Therefore, the transcription of its coding gene IFNB1 is strictly controlled. We have previously shown that in mice, the TRIM33 protein restrains Ifnb1 transcription in activated myeloid cells through an upstream inhibitory sequence called ICE. Here, we show that the deregulation of Ifnb1 expression observed in murine Trim33-/- macrophages correlates with abnormal looping of both ICE and the Ifnb1 gene to a 100 kb downstream region overlapping the Ptplad2/Hacd4 gene. This region is a predicted myeloid super-enhancer in which we could characterize 3 myeloid-specific active enhancers, one of which (E5) increases the response of the Ifnb1 promoter to activation. In humans, the orthologous region contains several single nucleotide polymorphisms (SNPs) known to be associated with decreased expression of IFNB1 in activated monocytes, and loops to the IFNB1 gene. The strongest association is found for the rs12553564 SNP, located in the E5 orthologous region. The minor allele of rs12553564 disrupts a conserved C/EBP-ß binding motif, prevents binding of C/EBP-ß, and abolishes the activation-induced enhancer activity of E5. Altogether, these results establish a link between a genetic variant preventing binding of a transcription factor and a higher order phenotype, and suggest that the frequent minor allele (around 30% worldwide) might be associated with phenotypes regulated by IFN-ß expression in myeloid cells.

A multimodal treatment of carbon ions irradiation, miRNA-34 and mTOR inhibitor specifically control high-grade chondrosarcoma cancer stem cells.

BACKGROUND AND PURPOSE: High-grade chondrosarcomas are chemo- and radio-resistant cartilage-forming tumors of bone that often relapse and metastase. Thus, new therapeutic strategies are urgently needed. MATERIAL AND METHODS: Chondrosarcoma cells (CH-2879) were exposed to carbon-ion irradiation, combined with miR-34 mimic and/or rapamycin administration. The effects of treatment on cancer stem cells, stemness-associated phenotype, radioresistance and tumor-initiating properties were evaluated. RESULTS: We show that high-grade chondrosarcoma cells contain a population of radioresistant cancer stem cells that can be targeted by a combination of carbon-ion therapy, miR-34 mimic administration and/or rapamycin treatment that triggers FOXO3 and miR-34 over-expression. mTOR inhibition by rapamycin triggered FOXO3 and miR-34, leading to KLF4 repression. CONCLUSION: Our results show that particle therapy combined with molecular treatments effectively controls cancer stem cells and may overcome treatment resistance of high-grade chondrosarcoma.

Phagocytosis of Wnt inhibitor SFRP4 by late wound macrophages drives chronic Wnt activity for fibrotic skin healing.

Human and murine skin wounding commonly results in fibrotic scarring, but the murine wounding model wound-induced hair neogenesis (WIHN) can frequently result in a regenerative repair response. Here, we show in single-cell RNA sequencing comparisons of semi-regenerative and fibrotic WIHN wounds, increased expression of phagocytic/lysosomal genes in macrophages associated with predominance of fibrotic myofibroblasts in fibrotic wounds. Investigation revealed that macrophages in the late wound drive fibrosis by phagocytizing dermal Wnt inhibitor SFRP4 to establish persistent Wnt activity. In accordance, phagocytosis abrogation resulted in transient Wnt activity and a more regenerative healing. Phagocytosis of SFRP4 was integrin-mediated and dependent on the interaction of SFRP4 with the EDA splice variant of fibronectin. In the human skin condition hidradenitis suppurativa, phagocytosis of SFRP4 by macrophages correlated with fibrotic wound repair. These results reveal that macrophages can modulate a key signaling pathway via phagocytosis to alter the skin wound healing fate.

Publisher Correction: Progenitors from the central nervous system drive neurogenesis in cancer.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Prion protein deficiency impairs hematopoietic stem cell determination and sensitizes myeloid progenitors to irradiation.

Highly conserved among species and expressed in various types of cells, numerous roles have been attributed to the cellular prion protein (PrPC). In hematopoiesis, PrPC regulates hematopoietic stem cell self-renewal but the mechanisms involved in this regulation are unknown. Here we show that PrPC regulates hematopoietic stem cell number during aging and their determination towards myeloid progenitors. Furthermore, PrPC protects myeloid progenitors against the cytotoxic effects of total body irradiation. This radioprotective effect was associated with increased cellular prion mRNA level and with stimulation of the DNA repair activity of the Apurinic/pyrimidinic endonuclease 1, a key enzyme of the base excision repair pathway. Altogether, these results show a previously unappreciated role of PrPC in adult hematopoiesis, and indicate that PrPC-mediated stimulation of BER activity might protect hematopoietic progenitors from the cytotoxic effects of total body irradiation.

Human CCR6+ Th17 Lymphocytes Are Highly Sensitive to Radiation-Induced Senescence and Are a Potential Target for Prevention of Radiation-Induced Toxicity.

PURPOSE: This study addresses the sensitivity of different peripheral CD4+ T-lymphocyte subsets to irradiation (IR) and identifies potential targets for the prevention or treatment of radiation-induced toxicity. METHODS: This study was performed on peripheral blood mononuclear cells or sorted peripheral memory lymphocytes of CCR6+ mucosa-homing Th17/CCR6negTh and regulatory T subtypes of healthy volunteers. Cells were irradiated with a 2 Gy with or without pharmacologic inhibitors of different signaling pathways. Senescence of irradiated cells was assessed by resistance to apoptosis and determination of various senescence-associated biomarkers (senescence associated b-galactosidase activity, p16Ink4a-, p21Cdkn1a-, gH2A.X-, H2A.J expression). Cytokine production was measured in supernatants of irradiated cells by Luminex technology. RESULTS: Not all CD4+ memory T lymphocyte subsets were equally radiosensitive. High sensitivity of CCR6+Th17 lymphocytes to IR-induced senescence was shown by expression of the histone variant H2A.J, higher SA-b-Gal activity, and upregulation of p16Ink4a and p21Cdkn1a expression. Lower Annexin V staining and cleaved caspase-3, and higher expression of antiapoptotic genes Bcl-2 and Bcl-xL LF, showed that CCR6+Th17 lymphocytes were more resistant to IR-induced apoptosis than CCR6neg memory Th and regulatory T lymphocytes. After a 2 Gy IR, both CCR6+Th17 and CCR6neg cells acquired a moderate senescence-associated secretory phenotype, but only CCR6+Th17 cells secreted interleukin 8 (IL-8) and vascular endothelial growth factor-A (VEGF-A). Pharmacologic targeting of reactive oxygen species (ROS), mitogen-activated protein kinases (MAPKs), and mammalian target of rapamycin (mTOR) signaling pathways prevented the expression of senescent markers and IL-8 and VEGF-A expression by CCR6+Th17 cells after IR. CONCLUSIONS: This study suggests that IR induces senescence of CCR6+Th17 lymphocytes associated with secretion of IL-8 and VEGF-A that may be detrimental to the irradiated tissue. ROS-MAPKs signaling pathways are candidate targets to prevent this CCR6+Th17-dependent radiation-induced potential toxicity. Finally, the ratio of circulating H2A.J+ senescent CCR6+ Th17/CD4+ T lymphocytes may be a candidate marker of individual intrinsic radiosensitivity.

Interplay between FACT subunit SPT16 and TRIM33 can remodel chromatin at macrophage distal regulatory elements.

BACKGROUND: Cell type-specific use of cis-acting regulatory elements is mediated by the combinatorial activity of transcription factors involved in lineage determination and maintenance of cell identity. In macrophages, specific transcriptional programs are dictated by the transcription factor PU.1 that primes distal regulatory elements for macrophage identities and makes chromatin competent for activity of stimuli-dependent transcription factors. Although the advances in genome-wide approaches have elucidated the functions of these macrophage-specific distal regulatory elements in transcriptional responses, chromatin structures associated with PU.1 priming and the underlying mechanisms of action of these cis-acting sequences are not characterized. RESULTS: Here, we show that, in macrophages, FACT subunit SPT16 can bind to positioned nucleosomes directly flanking PU.1-bound sites at previously uncharacterized distal regulatory elements located near genes essential for macrophage development and functions. SPT16 can interact with the transcriptional co-regulator TRIM33 and binds to half of these sites in a TRIM33-dependent manner. Using the Atp1b3 locus as a model, we show that FACT binds to two positioned nucleosomes surrounding a TRIM33/PU.1-bound site in a region, located 35 kb upstream the Atp1b3 TSS, that interact with the Atp1b3 promoter. At this - 35 kb region, TRIM33 deficiency leads to FACT release, loss of the two positioned nucleosomes, RNA Pol II recruitment and bidirectional transcription. These modifications are associated with higher levels of FACT binding at the Atp1b3 promoter, an increase of RNA Pol II recruitment and an increased expression of Atp1b3 in Trim33-/- macrophages. CONCLUSIONS: Thus, sequestering of SPT16/FACT by TRIM33 at PU.1-bound distal regions might represent a new regulatory mechanism for RNA Pol II recruitment and transcription output in macrophages.

TRIM33 deficiency in monocytes and macrophages impairs resolution of colonic inflammation.

BACKGROUND: Mature myeloid cells play a crucial role in Crohn's disease (CD) but the molecular players that regulate their functions in CD are not fully characterized. We and others have shown that TRIM33 is involved in the innate immune response and in the inflammatory response but TRIM33 role in intestinal inflammation is not known. In this study, we investigated the role of TRIM33 in myeloid cells during dextran sulfate sodium (DSS)-induced colitis. METHODS: We study the role of TRIM33 during DSS-induced colitis which mimics intestinal inflammation using mice deleted for Trim33 only in mature myeloid cells (Trim33-/- mice) FINDINGS: We first show that Trim33 mRNA level is decreased in CD patient's blood monocytes suggesting a role of TRIM33 in CD. Using Trim33-/- mice, we show that these mice display an impaired resolution of colonic inflammation with an increased number of blood and colon monocytes and a decreased number of colonic macrophages. Trim33-/- monocytes are less competent for recruitment and macrophage differentiation. Finally, during resolution of inflammation, Trim33-/- colonic macrophages display an impaired M1/M2 switch and express a low level of membrane-bound TNF that is associated with an increased number of colonic neutrophils. INTERPRETATION: Our study shows an important role of TRIM33 in monocytes/macrophages during DSS-induced colitis and suggests that the decreased expression of TRIM33 in CD patient's blood monocytes might not be a consequence but might be involved in CD progression. FUND: La Ligue contre le Cancer (équipe labelisée), INSERM, CEA, Université Paris-Diderot, Université Paris-Sud.

Progenitors from the central nervous system drive neurogenesis in cancer.

Autonomic nerve fibres in the tumour microenvironment regulate cancer initiation and dissemination, but how nerves emerge in tumours is currently unknown. Here we show that neural progenitors from the central nervous system that express doublecortin (DCX+) infiltrate prostate tumours and metastases, in which they initiate neurogenesis. In mouse models of prostate cancer, oscillations of DCX+ neural progenitors in the subventricular zone-a neurogenic area of the central nervous system-are associated with disruption of the blood-brain barrier, and with the egress of DCX+ cells into the circulation. These cells then infiltrate and reside in the tumour, and can generate new adrenergic neurons. Selective genetic depletion of DCX+ cells inhibits the early phases of tumour development in our mouse models of prostate cancer, whereas transplantation of DCX+ neural progenitors promotes tumour growth and metastasis. In humans, the density of DCX+ neural progenitors is strongly associated with the aggressiveness and recurrence of prostate adenocarcinoma. These results reveal a unique crosstalk between the central nervous system and prostate tumours, and indicate neural targets for the treatment of cancer.

Hematopoietic stem and progenitor cell responses to low radiation doses - implications for leukemia risk.

Studies of the responses of hematopoietic stem and progenitor cells (HSPCs) to low doses of ionizing radiation formed an important aspect of the RISK-IR project ( www.risk-ir.eu ). A brief overview of these studies is presented here. The findings confirm the sensitivity of HSPCs to radiation even at low doses, and illustrate the substantial impact that differentiation state has upon cell sensitivity. The work provides mechanistic support for epidemiological findings of leukemia risk at dose levels used in diagnostic CT imaging, and further suggests that low-dose irradiation may facilitate bone marrow transplantation, a finding that could lead to refinements in clinical practice.

Low-Dose Irradiation Promotes Persistent Oxidative Stress and Decreases Self-Renewal in Hematopoietic Stem Cells.

Despite numerous observations linking protracted exposure to low-dose (LD) radiation and leukemia occurrence, the effects of LD irradiation on hematopoietic stem cells (HSCs) remain poorly documented. Here, we show that adult HSCs are hypersensitive to LD irradiation. This hyper-radiosensitivity is dependent on an immediate increase in the levels of reactive oxygen species (ROS) that also promotes autophagy and activation of the Keap1/Nrf2 antioxidant pathway. Nrf2 activation initially protects HSCs from the detrimental effects of ROS, but protection is transient, and increased ROS levels return, promoting a long-term decrease in HSC self-renewal. In vivo, LD total body irradiation (TBI) does not decrease HSC numbers unless the HSC microenvironment is altered by an inflammatory insult. Paradoxically, such an insult, in the form of granulocyte colony-stimulating factor (G-CSF) preconditioning, followed by LD-TBI facilitates efficient bone marrow transplantation without myeloablation. Thus, LD irradiation has long-term detrimental effects on HSCs that may result in hematological malignancies, but LD-TBI may open avenues to facilitate autologous bone marrow transplantation.

NRF2 Activation Impairs Quiescence and Bone Marrow Reconstitution Capacity of Hematopoietic Stem Cells.

Tissue stem cells are maintained in quiescence under physiological conditions but proliferate and differentiate to replenish mature cells under stressed conditions. The KEAP1-NRF2 system plays an essential role in stress response and cytoprotection against redox disturbance. To clarify the role of the KEAP1-NRF2 system in tissue stem cells, we focused on hematopoiesis in this study and used Keap1-deficient mice to examine the effects of persistent activation of NRF2 on long-term hematopoietic stem cells (LT-HSCs). We found that persistent activation of NRF2 due to Keap1 deficiency did not change the number of LT-HSCs but reduced their quiescence in steady-state hematopoiesis. During hematopoietic regeneration after bone marrow (BM) transplantation, persistent activation of NRF2 reduced the BM reconstitution capacity of LT-HSCs, suggesting that NRF2 reduces the quiescence of LT-HSCs and promotes their differentiation, leading to eventual exhaustion. Transient activation of NRF2 by an electrophilic reagent also promotes the entry of LT-HSCs into the cell cycle. Taken together, our findings show that NRF2 drives the cell cycle entry and differentiation of LT-HSCs at the expense of their quiescence and maintenance, an effect that appears to be beneficial for prompt recovery from blood loss. We propose that the appropriate control of NRF2 activity by KEAP1 is essential for maintaining HSCs and guarantees their stress-induced regenerative response.

[Hopes of high dose-rate radiotherapy]. / Les promesses du haut débit de dose en radiothérapie.

In this review, we present the synthesis of the newly acquired knowledge concerning high dose-rate irradiations and the hopes that these new radiotherapy modalities give rise to. The results were presented at a recent symposium on the subject.

Spermatogonial stem cells and progenitors are refractory to reprogramming to pluripotency by the transcription factors Oct3/4, c-Myc, Sox2 and Klf4.

The male germinal lineage, which is defined as unipotent, produces sperm through spermatogenesis. However, embryonic primordial germ cells and postnatal spermatogonial stem cells (SSCs) can change their fate and convert to pluripotency in culture when they are not controlled by the testicular microenvironment. The mechanisms underlying these reprogramming processes are poorly understood. Testicular germ cell tumors, including teratoma, share some molecular characteristics with pluripotent cells, suggesting that cancer could result from an abnormal differentiation of primordial germ cells or from an abnormal conversion of SCCs to pluripotency in the testis. Here, we investigated whether the somatic reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc (OSKM) could play a role in SSCs reprogramming and induce pluripotency using a doxycycline-inducible transgenic Col1a1-4F2A-OSKM mouse model. We showed that, in contrast to somatic cells, SSCs from adult mice are resistant to this reprogramming strategy, even in combination with small molecules, hypoxia, or p53 deficiency, which were previously described to favour the conversion of somatic cells to pluripotency. This finding suggests that adult SSCs have developed specific mechanisms to repress reprogramming by OSKM factors, contributing to circumvent testicular cancer initiation events.