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.

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.

Depleting Macrophages In Vivo with Clodronate-Liposomes.

In order to decipher the role of macrophages in vivo, it could be necessary to establish a model of macrophage depletion in the whole animal. One method to obtain animal models efficiently depleted in macrophages in different tissues (bone marrow, spleen, liver, lungs, brain, gut, peritoneal cavity, lymph nodes/vessels) and blood is the use of a clodronate-liposome solution.Here, we describe the protocol used to deplete efficiently macrophages in mouse bone marrow.

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.

The role of p63 in germ cell apoptosis in the developing testis.

The fetal and neonatal development of male germ cells (gonocytes) is a poorly understood but crucial process for establishing fertility. In rodents, gonocytes go through two phases of proliferation accompanied by apoptosis and separated by a quiescent period during the end of fetal development. P63 is a member of the P53 gene family that yields six isoforms. We detected only the p63 protein and no p53 and p73 in the nucleus of the gonocytes of mouse testes. We report for the first time the ontogeny of each p63 mRNA isoform during testis development. We observed a strong expression of p63gamma mRNA and protein when gonocytes are in the quiescent period. In vitro treatment with retinoic acid prevented gonocytes from entering the quiescent period and was correlated with a reduced production of p63gamma isoform mRNA. We investigated the function of p63 by studying the testicular phenotype of P63-null mice. P63 invalidation slightly, but significantly increased the number of gonocytes counted during the quiescent period. As P63-null animals die at birth we used an original organ culture that mimicked neonatal in vivo development to study further the testicular development. P63 invalidation resulted in a sharply increased number of gonocytes during the culture period due to a decrease in spontaneous apoptosis with no change in proliferation. P63 invalidation also caused abnormal morphologies in the germ cells that were also found in P63(+/-) adult male mice. Thus, p63 appears as an important regulator of germ cell development.

Essential role for mitochondrial thioredoxin reductase in hematopoiesis, heart development, and heart function.

Oxygen radicals regulate many physiological processes, such as signaling, proliferation, and apoptosis, and thus play a pivotal role in pathophysiology and disease development. There are at least two thioredoxin reductase/thioredoxin/peroxiredoxin systems participating in the cellular defense against oxygen radicals. At present, relatively little is known about the contribution of individual enzymes to the redox metabolism in different cell types. To begin to address this question, we generated and characterized mice lacking functional mitochondrial thioredoxin reductase (TrxR2). Ubiquitous Cre-mediated inactivation of TrxR2 is associated with embryonic death at embryonic day 13. TrxR2(TrxR2(-/-)minus;/TrxR2(-/-)minus;) embryos are smaller and severely anemic and show increased apoptosis in the liver. The size of hematopoietic colonies cultured ex vivo is dramatically reduced. TrxR2-deficient embryonic fibroblasts are highly sensitive to endogenous oxygen radicals when glutathione synthesis is inhibited. Besides the defect in hematopoiesis, the ventricular heart wall of TrxR2(TrxR2(-/-)minus;/TrxR2(-/-)minus;) embryos is thinned and proliferation of cardiomyocytes is decreased. Cardiac tissue-restricted ablation of TrxR2 results in fatal dilated cardiomyopathy, a condition reminiscent of that in Keshan disease and Friedreich's ataxia. We conclude that TrxR2 plays a pivotal role in both hematopoiesis and heart function.

Testis-specific expression of the nuclear form of phospholipid hydroperoxide glutathione peroxidase (PHGPx).

The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) is present in at least three different isoforms in testis: as a cytosolic, as a mitochondrial, and as a nuclear protein. We have recently shown that a sperm nucleus-specific glutathione peroxidase (snGPx) is identical to the mitochondrial and cytosolic forms of PHGPx apart from its N-terminus. This arginine-rich N-terminus of snGPx, reminiscent of protamines, is encoded by an alternative exon located in the first intron of the PHGPx gene and is responsible for nuclear localisation and chromatin binding of snGPx [Pfeifer et al., FASEB J. 15 (2001), pp. 1236-1238]. By using a combination of techniques including selective cloning of mRNA 5'-ends, RT-PCR, and S1 analyses, we provide evidence that the transcript encoding the nuclear form is generated by transcription initiation at an alternative promoter and not by alternative splicing. We show that the major transcription start region is located at -12 to -14 upstream of the AUG translation initiation site of the sperm nucleus-specific exon and lacks a TATA box. Two minor TATA-less transcription initiation sites are located at around -30 and -45. We have shown by in situ hybridisation that snGPx expression in testis, like protamine expression, is restricted to late stages of spermatogenesis whereas PHGPx expression is only found in spermatocytes and early spermatids. These findings have to be taken into account when studying either the differential regulation of PHGPx and snGPx expression in testis or the impact of putative mutations in snGPx on male fertility in man.