Simplified drug efficacy evaluation system for vasopressin neurodegenerative disease using mouse disease-specific induced pluripotent stem cells.

Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disorder in which vasopressin-secreting neurons degenerate over time due to the production of mutant proteins. We have demonstrated therapeutic effects of chemical chaperones in an FNDI mouse model, but the complexity and length of this evaluation were problematic. In this study, we established disease-specific mouse induced pluripotent stem cells (iPSCs) from FNDI-model mice and differentiated vasopressin neurons that produced mutant proteins. Fluorescence immunostaining showed that chemical chaperones appeared to protect vasopressin neurons generated from iPSCs derived from FNDI-model mice. Although KCL stimulation released vasopressin hormone from vasopressin neurons generated from FNDI-derived iPSCs, vasopressin hormone levels did not differ significantly between baseline and chaperone-added culture. Semi-quantification of vasopressin carrier protein and mutant protein volumes in vasopressin neurons confirmed that chaperones exerted a therapeutic effect. This research provides fundamental technology for creating in vitro disease models using human iPSCs and can be applied to therapeutic evaluation of various degenerative diseases that produce abnormal proteins.

Generation of hypothalamic neural stem cell-like cells in vitro from human pluripotent stem cells.

When damaged, restoring the function of the hypothalamus is currently impossible. It is unclear whether neural stem cells exist in the hypothalamus. Studies have reported that adult rodent tanycytes around the third ventricle function as hypothalamic neural stem cell-like cells. However, it is currently impossible to collect periventricular cells from humans. We attempted to generate hypothalamic neural stem cell-like cells from human embryonic stem cells (ESCs). We focused on retina and anterior neural fold homeobox (RAX) because its expression is gradually restricted to tanycytes during the late embryonic stage. We differentiated RAX::VENUS knockin human ESCs (hESCs) into hypothalamic organoids and sorted RAX+ cells from mature organoids. The isolated RAX+ cells formed neurospheres and exhibited self-renewal and multipotency. Neurogenesis was observed when neurospheres were transplanted into the mouse hypothalamus. We isolated RAX+ hypothalamic neural stem cell-like cells from wild-type human ES organoids. This is the first study to differentiate human hypothalamic neural stem cell-like cells from pluripotent stem cells.

Subcutaneous transplantation of human embryonic stem cells-derived pituitary organoids.

Introduction: The pituitary gland, regulating various hormones, is central in the endocrine system. As spontaneous recovery from hypopituitarism is rare, and exogenous-hormone substitution is clumsy, pituitary replacement via regenerative medicine, using pluripotent stem cells, is desirable. We have developed a differentiation method that in mice yields pituitary organoids (POs) derived from human embryonic stem cells (hESC). Efficacy of these POs, transplanted subcutaneously into hypopituitary mice, in reversing hypopituitarism was studied. Methods: hESC-derived POs were transplanted into inguinal subcutaneous white adipose tissue (ISWAT) and beneath dorsal skin, a relatively avascular region (AR), of hypophysectomized severe combined immunodeficient (SCID) mice. Pituitary function was evaluated thereafter for ¾ 6mo, assaying basal plasma ACTH and ACTH response to corticotropin-releasing hormone (CRH) stimulation. Histopathologic examination of organoids 150d after transplantation assessed engraftment. Some mice received an inhibitor of vascular endothelial growth factor (VEGF) to permit assessment of how angiogenesis contributed to subcutaneous engraftment. Results: During follow-up, both basal and CRH-stimulated plasma ACTH levels were significantly higher in the ISWAT group (p < 0.001 - 0.05 and 0.001 - 0.005, respectively) than in a sham-operated group. ACTH secretion also was higher in the ISWAT group than in the AR group. Histopathologic study found ACTH-producing human pituitary-cell clusters in both groups of allografts, which had acquired a microvasculature. POs qPCR showed expression of angiogenetic factors. Plasma ACTH levels decreased with VEGF-inhibitor administration. Conclusions: Subcutaneous transplantation of hESC-derived POs into hypopituitary SCID mice efficaciously renders recipients ACTH-sufficient.

Functional Lactotrophs in Induced Adenohypophysis Differentiated From Human iPS Cells.

Prolactin (PRL), a hormone involved in lactation, is mainly produced and secreted by the lactotrophs of the anterior pituitary (AP) gland. We previously reported a method to generate functional adrenocorticotropic hormone-producing cells by differentiating the AP and hypothalamus simultaneously from human induced pluripotent stem cells (iPSCs). However, PRL-producing cells in the induced AP have not been investigated. Here, we confirmed the presence of PRL-producing cells and evaluated their endocrine functions. We differentiated pituitary cells from human iPSCs using serum-free floating culture of embryoid-like aggregates with quick reaggregation (SFEB-q) method and evaluated the appearance and function of PRL-producing cells. Secretion of PRL from the differentiated aggregates was confirmed, which increased with further culture. Fluorescence immunostaining and immunoelectron microscopy revealed PRL-producing cells and PRL-positive secretory granules, respectively. PRL secretion was promoted by various prolactin secretagogues such as thyrotropin-releasing hormone, vasoactive intestinal peptide, and prolactin-releasing peptide, and inhibited by bromocriptine. Moreover, the presence of tyrosine hydroxylase-positive dopaminergic nerves in the hypothalamic tissue area around the center of the aggregates connecting to PRL-producing cells indicated the possibility of recapitulating PRL regulatory mechanisms through the hypothalamus. In conclusion, we generated pituitary lactotrophs from human iPSCs; these displayed similar secretory responsiveness as human pituitary cells in vivo. In the future, this is expected to be used as a model of human PRL-producing cells for various studies, such as drug discovery, prediction of side effects, and elucidation of tumorigenic mechanisms using disease-specific iPSCs. Furthermore, it may help to develop regenerative medicine for the pituitary gland.

Cryopreservation of canine spermatozoa using a skim milk-based extender and a short equilibration time.

Although useful spermatozoa cryopreservation techniques have been established, long-term equilibration seems to be required before freezing the spermatozoa of many species, including dogs. The fertility of cryopreserved dog spermatozoa from five males for a reduced equilibration period (0, 30, 60, 120 and 180 min) in a skim milk (SM)-based extender containing raffinose was evaluated in the present study. When the sperm was diluted with the extender at room temperature (RT) and cryopreserved without equilibration, the proportion of total motile spermatozoa (TMS) after thawing was lower (27%) than when the sperm was equilibrated for 30 min (33%), 60 min (32%), 120 min (44%; p < .05) or 180 min (29%). The proportion of TMS increased as the equilibration time increased and peaked at 120 min. Acrosome integrity was significantly lower in the cryopreserved spermatozoa that had not undergone the initial equilibration than in the equilibrated spermatozoa (p < .05). The normal rate of acrosomes increased with the extension of the first equilibration and peaked at 120 min. When frozen-thawed spermatozoa that had been diluted at RT and subjected to an initial equilibration lasting 60 or 180 min were transcervically inseminated into recipients, there were no differences in the delivery rate, litter size or breeding efficiency. In the cryopreservation of canine spermatozoa using a SM-based extender, even if the initial equilibration time was shortened to 60 min, the results were comparable to those obtained when the conventional method (with an initial equilibration time of 180 min) was used.

In vitro maturation of canine oocytes co-cultured with bovine and canine granulosa cell monolayers.

The present study investigated the effects of bovine granulosa cell monolayers (BGML) and canine granulosa cell monolayers (CGML) on nuclear maturation of canine oocytes with and without cumulus cells. Cumulus-oocyte complexes (COCs) or cumulus-free oocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM, control group), DMEM with BGML (BGML group), or DMEM with CGML (CGML group) for 72 h at 38.5 °C in 5% CO(2), 5% O(2,) and 90% N(2). All media were supplemented with 10% of FCS, 50 ng/mL of EGF, 2 µg/mL of estradiol-17ß, 0.1 IU/mL of hCG, 0.1 IU/mL of FSH, 0.25 mM of pyruvic acid, 100 µM of ß-mercaptoethanol, 100 IU/mL of penicillin, and 100 µg/mL of streptomycin. In cumulus-enclosed oocytes retrieved from ovaries at estrus and/or diestrus, the highest percentage of M-II oocytes (P < 0.05) was present in the BGML group (27.0%) compared with the CGML group (7.9%) and the control group (3.5%). In cumulus-free oocytes collected from ovaries at estrus and/or diestrus, the proportions of M-II oocytes co-cultured with the CGML were low (3.0%) and similar (P > 0.05) to proportions achieved with control (3.0%). However, the presence of BGML improved (P < 0.05) the ability of denuded oocytes to develop into M-II (10.2%). The BGML group had the highest overall meiotic resumption (P < 0.05), and least oocyte degeneration (P < 0.05) among experimental groups. In conclusion, BGML had a positive impact on the in vitro maturation system, as well as meiotic resumption of canine oocytes.

Beneficial effect of hyperbaric oxygen therapy on the follicular survival in the mouse ovary after transplantation.

A large proportion of follicles are lost during the initial ischemia that occurs after transplantation of ovarian tissues. Thus, the effect of hyperbaric oxygen therapy (HBO) on the follicular loss of ovarian tissues after transplantation was examined in mice. Ovarian slices from ICR mice were transplanted under the kidney capsule in ovariectomized ICR. Hyperbaric oxygen with 100% oxygen was initiated for 30 min at 2.5 atmospheres absolute immediately after transplantation, and this treatment was repeated at 48-h intervals for 2 weeks. The number of follicles was dramatically reduced at 2 weeks post transplantation. However, HBO was significantly effective in enhancing the survival of transplanted ovarian follicles. The survival rates of primordial and primary follicles in ovarian tissues of mice with HBO were significantly higher than those without HBO. These results indicate HBO can be effectively used for the enhancement of survival of transplanted ovarian tissues.

Cryopreservation of canine embryos.

The assisted reproductive techniques (ARTs) such as in vitro fertilization, embryo transfer, and cryopreservation of gametes have contributed considerably to the development of biomedical sciences in addition to improving infertility treatments in humans as well as the breeding of domestic animals. However, ARTs used in canine species have strictly limited utility when compared with other mammalian species, including humans. Although successful somatic cell cloning has been reported, artificial insemination by frozen semen to date is only available for the improved breeding and reproduction for companion and working dogs as well as guide dogs for the blind. We describe here the successful cryopreservation of embryos and subsequent embryo transfer in dogs. Canine embryos were collected from excised reproductive organs after artificial insemination and subsequently cryopreserved by a vitrification method. When the 4-cell to morula stage of cryopreserved embryos were nonsurgically transferred into the uteri of nine recipient bitches using a cystoscope, five recipients became pregnant and four of them delivered a total of seven pups. The cryopreservation of embryos in canine species will facilitate the transportation and storage of genetic materials and will aid in the elimination of vertically transmitted diseases in dogs. In addition, this technique will contribute to the improved breeding of companion and working dogs such as guide dogs, drug-detecting dogs, and quarantine dogs.

Effect of bovine cumulus-oocyte complexes-conditioned medium on in-vitro maturation of canine oocytes.

PURPOSE: To investigate the ability of medium conditioned with bovine cumulus-oocyte complexes (COCs) to support nuclear maturation of canine oocytes recovered from domestic dog ovaries. METHODS: Cumulus-oocyte complexes were obtained from ovaries of domestic bitches (8 months old to 7 years old), and in-vitro maturation was evaluated in TCM-199 supplemented with different concentrations (0, 20, 30 or 50%) of bovine COCs-conditioned medium (BCM). The canine COCs were cultured for 72 or 96 h at 38.5°C in 5% CO2, 5% O2 and 90% N2. The bovine COCs-conditioned medium was obtained from culture of bovine COCs with TCM-199 supplemented with 5% FCS for 22 h at 38.5°C in 2% CO2, 98% air. RESULTS: The proportion of germinal vesicle breakdown (GVBD) after 72 h was significantly higher (P < 0.05) in medium supplemented with 30% BCM (20.7%) compared with the control group (13.4%). The rates of GVBD-MII stage were significantly higher (P < 0.05) when oocytes were matured with BCM at concentration of 30% (41.5%) compared with control (26.6%) after 72 h in-vitro culture. After 96 h in-vitro culture, the oocytes matured in medium supplemented with 30% BCM (5.5%) showed a significant increase (P < 0.05) in the proportion of MII compared with control (0.7%). However, increasing the cultivation time from 72 to 96 h resulted in an increase in oocyte degeneration rate. CONCLUSIONS: The results suggested that bovine COCs-conditioned medium supplementation significantly increased nuclear maturation of canine oocytes.

Effect of graft site and gonadotrophin treatment on follicular development of canine ovarian grafts transplanted to NOD-SCID mice.

PURPOSE: To investigate the effect of graft site and gonadotrophins administration on the number and survival rate of follicles of canine ovarian grafts transplanted to NOD-severe combined immune deficiency (SCID) mice. METHODS: Fresh ovarian cortex slices obtained from immature bitches were grafted subcutaneously (SC), under kidney capsule (KC) or into ovarian bursa (OB) in NOD-SCID mice. Two months after surgery, the mice allocated into non-treated and treated gonadotrophins groups that injected with porcine follicle stimulating hormone during 7 days and human chorionic gonadotrophin 48 h later. Ovarian grafts were collected after 10 h of last injection and processed for histology. RESULTS: The number of transitional and preantral follicles under KC and into OB was significantly higher in gonadotrophins-treated mice than those who received saline. Furthermore, the survival rates of primary, transitional and preantral follicles under KC and into OB grafts were significantly higher than those placed SC in the treated gonadotrophins group, and in the non-treated gonadotrophins group; the proportion of primary and preantral follicle survival was significantly higher under KC and into OB than SC grafts. CONCLUSIONS: In canine ovarian xenografting, administration of gonadotrophin could be effective for improvement of survival of transplanted ovary. Furthermore, the grafting into OB appeared to be better than grafting under KC, which in turn is better than SC.

Vitrification of canine cumulus-oocyte complexes in DAP213 with a cryotop holder.

Purpose: The effects of the cryoprotectant and the container (holder) used for the vitrification of canine germinal vesicle stage oocytes were examined to improve the cryopreservation method for canine oocytes and embryos. Methods: Canine cumulus-oocyte complexes (COCs) were collected from ovaries, and were vitrified with E30S (30% ethylene glycol and 0.5 M sucrose) or DAP213 (2 M dimethyl sulfoxide, 1 M acetamide, and 3 M propylene glycol) solution held by a cryotube or cryotop sheets. After warming, the oocytes were stained with propidium iodide for the assessment of their plasma membrane integrity. Results: In all the vitrification groups, more than 65% of the vitrified oocytes displayed a normal morphology (E30S-top, 65.6%; DAP-tube, 67.3%; DAP-top, 80.0%). However, when assessed by propidium iodide staining, the viability of oocytes in the DAP-top group (43.6%) was higher than that in the E30S-top group (21.3%, P < 0.05). Furthermore, the viability of the oocytes in the DAP-top group (43.6%) was higher than that in the DAP-tube group (4.1%, P < 0.05). Conclusions: These results suggest that a combination of DAP213 as the cryoprotectant and a cryotop sheet as the holder improved viability after the vitrification of canine oocytes at the germinal vesicle stage.

Pronuclear formation of freeze-dried canine spermatozoa microinjected into mouse oocytes.

PURPOSE: The aim of the present study was to investigate the fertilizing capacity of fresh, frozen-thawed and freeze-dried canine spermatozoa. METHODS: After canine spermatozoa were injected into mouse oocytes, the rates of oocyte activation, male pronuclear formation and chromosomal aberrations were investigated. RESULTS: The rates of oocyte activation were comparable (90.6-100%), no matter the sperm type injected. The percentage of male pronuclear formation was higher (P < 0.001) in the freeze-dried spermatozoa (92.3%) than the fresh (61.5%) and frozen-thawed (69.2%) spermatozoa. However, the chromosomal damage in the oocytes injected with freeze-dried spermatozoa was higher (72.9%: P < 0.001) than with fresh (26.9%) and frozen-thawed (21.4%) spermatozoa. CONCLUSIONS: These data indicate using mouse oocytes that freeze-dried canine spermatozoa may potentially fertilize canine oocytes although chromosomal damage is frequently generated.

Artificial insemination with canine spermatozoa frozen in a skim milk/glucose-based extender.

Due to the recent outbreak of avian influenza, transportation of frozen canine semen with egg yolk has been sharply restricted. Thus, there is urgent need to develop a novel egg yolk-free extender for freezing canine spermatozoa. In the present study, the effect of using skim milk/glucose (SG)-based extender without egg yolk on the motility and fertilizing capacity of canine spermatozoa frozen-thawed in the presence of glycerol was examined. There was a tendency for the proportion of motile spermatozoa exposed to SG-based extender for 3 h to be higher than that exposed for 1 h, but the difference was not significant. The motility and other viability parameters of canine spermatozoa after thawing were similar to those obtained with an egg yolk-based extender. When spermatozoa frozen with SG-based extender containing glycerol after 3 h exposure were transcervically inseminated into 2 recipient bitches, a total of 6 pups were obtained. These results suggest that a simple extender composed of skim milk, glucose and glycerol is useful for cryopreservation of canine spermatozoa, which may contribute to improved exchange of genetic material and efficient production of companion and working dogs, such as guide dogs for the blind.