Intratracheal administration of clinical-grade mesenchymal stem cell-derived extracellular vesicles reduces lung injury in a rat model of bronchopulmonary dysplasia.

Mesenchymal stem cells (MSCs) prevent the onset of bronchopulmonary dysplasia (BPD) in animal models, an effect that seems to be mediated by their secreted extracellular vesicles (EVs). The aim of this study was to compare the protective effects of intratracheally (IT) administered MSCs versus MSC-EVs in a hyperoxia-induced rat model of BPD. At birth, rats were distributed as follows: animals raised in ambient air for 2 wk ( n = 10), and animals exposed to 60% oxygen for 2 wk and treated with IT-administered physiological solution ( n = 10), MSCs ( n = 10), or MSC-EVs ( n = 10) on postnatal days 3, 7, and 10. The sham-treated hyperoxia-exposed animals showed reductions in total surface area of alveolar air spaces, and total number of alveoli ( Nalv), and an increased mean alveolar volume (Valv). EVs prompted a significant increase in Nalv ( P < 0.01) and a significant decrease in Valv ( P < 0.05) compared with sham-treated animals, whereas MSCs only significantly improved Nalv ( P < 0.05). Small pulmonary vessels of the sham-treated hyperoxia-exposed rats also showed an increase in medial thickness, which only EVs succeeded in preventing significantly ( P < 0.05). In conclusion, both EVs and MSCs reduce hyperoxia-induced damage, with EVs obtaining better results in terms of alveolarization and lung vascularization parameters. This suggests that IT-administered EVs could be an effective approach to BPD treatment.

Cryopreserved Subcutaneous Adipose Tissue for Fat Graft.

UNLABELLED: Cryopreservation of subcutaneous white adipose tissue (sWAT) avoids multiple surgeries in patients subjected to reconstructive procedure. Fat grafts were performed subcutaneously on 26 mice treated with fresh (13 mice) or cryopreserved (13 mice) human sWAT. Cytofluorometry for CD marker expression of stem cells, differentiation capability, and in vivo survival of fat grafts were evaluated. In vitro analysis evidenced that cryopreservation did not affect the stem potential of samples. In vivo MRI showed that grafts were well preserved in 13 mice treated with fresh sWAT, whereas in 13 animals treated with thawed fat, graft volumes were strongly reduced after 1 week. Ultrastructural studies performed both on fresh and thawed specimens demonstrated that grafts performed with thawed sWAT are able to store lipids more slowly with respect to grafts performed with fresh sWAT and adipocytes maintained a multilocular appearance. Collected data demonstrated that the protocol of cryopreservation could maintain the regenerative capability of the sWAT, but the rate of reabsorption after fat grafting is higher using cryopreserved sWAT. Maintaining the stem potential of sWAT after cryopreservation is a very important aspect for reconstructive and regenerative medicine. The employment of cryopreserved sWAT represents an interesting goal for surgeons. Surely there is the necessity to improve the protocol of cryopreservation. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266 .

Trichlorfon-induced polyploidy and nondisjunction in mouse oocytes from preantral follicle culture.

Trichlorfon (TCF), an organophosphate insecticide and potent inhibitor of choline esterases, was previously shown to induce first meiotic nondisjunction and spindle aberrations in isolated, follicle cell-denuded mouse oocytes maturing in vitro. To explore dose-response and direct and indirect, potentially synergistic effects of TCF on the somatic cells and the oocyte within a follicle, we presently employed preantral follicle culture. 100 microg/ml TCF added at the time of hormonally stimulated resumption of meiosis of follicle cell-enclosed mouse oocytes, 16 h before in vitro ovulation, induced significant rises in first meiotic nondisjunction in oocytes from preantral follicle culture. Lower concentrations (6 microg/ml TCF) disturbed polar body formation. Nuclear maturation to meiosis II in absence of cytokinesis resulted in significant increases in polyploidy. Oocytes maturing in follicles in the presence of TCF had aberrant second meiotic spindles. Influences of TCF on somatic cell function were evident by reduced follicular mucification in vitro and deceased progesterone production. In contrast to TCF, acetylcholine (0.1-100 microM) increased progesterone production. The observations therefore suggest that TCF influences oocyte maturation and folliculogenesis directly and indirectly. High TCF is aneugenic at first meiotic division in oocytes, irrespective of the presence or absence of follicle cells. At lower concentrations TCF interferes with spindle formation, chromosome congression at meiosis II, and coordination of nuclear and cytoplasmic maturation, posing risks for second meiotic errors. The observations suggest that chronic TCF exposure during maturation in the follicle may predispose oocytes to the formation of chromosomally unbalanced preimplantation embryos after fertilization.

In vitro effects of dexamethasone on mouse ovarian function and pre-implantation embryo development.

The effect of dexamethasone (5-80 microg/ml) on ovarian function and embryo development was studied in mice. The follicle bio-assay revealed no effects of DEX up to 40 microg/ml on folliculogenesis and oogenesis, whereas 80 microg/ml hampered follicle differentiation and oocyte maturation. Androgen, estrogen and progestin secretion patterns were strongly impaired at all doses levels. However, the ovulation-induced progesterone increase indicating that the steroid pathway was activated in presence of DEX. Applying the oil-free mouse embryo assay no alteration of DEX on the first cleavage stages were observed whereas blastocyst rate decreased from 20 microg/ml DEX onwards, and hatching capacity was already impaired in presence of 10 microg/ml DEX. In conclusion, steroidogenesis was affected from 5 microg/ml onwards and the minimum effective inhibitory dose was set at 10 microg/ml for early embryo development. Based on these in vitro findings, physiological or therapeutic levels of glucocorticosteroids are unlikely to affect female fertility.

Ovarian follicle bioassay reveals adverse effects of diazepam exposure upon follicle development and oocyte quality.

A mouse ovarian follicle bioassay was used to study folliculogenesis and oocyte quality in vitro. Diazepam (DZ) was chosen as test compound to evaluate the system for its ability to detect possible effects of chemicals on reproduction. The bioassay is suitable to analyze the influence of DZ on each of the follicular components at any stage of development. A dose finding study revealed that follicle growth, differentiation and steroidogenesis were significantly disturbed by > or =5 microg/ml DZ. A transient exposure procedure was used to examine stage-specific sensitivities of oogenesis to DZ. The oocyte appeared to be most vulnerable during its growth process within the follicle. Resumption of meiosis was disturbed dose-dependently with reduced oocyte quality after chronic exposure to > or =2.5 microg/ml DZ. The bioassay is a highly efficient and informative tool to assess the hazards of chemical compounds for female fertility and to elucidate their mechanisms of action.