Hiromasa Hara

Interspecies organogenesis generates autologous functional islets

Islet transplantation is an established therapy for diabetes. We have previously shown that rat pancreata can be created from rat pluripotent stem cells (PSCs) in mice through interspecies blastocyst complementation. Although they were functional and composed of rat-derived cells, the resulting pancreata were of mouse size, rendering them insufficient for isolating the numbers of islets required to treat diabetes in a rat model. Here, by performing the reverse experiment, injecting mouse PSCs into Pdx-1-deficient rat blastocysts, we generated rat-sized pancreata composed of mouse-PSC-derived cells. Islets subsequently prepared from these mouse–rat chimaeric pancreata were transplanted into mice with streptozotocin-induced diabetes. The transplanted islets successfully normalized and maintained host blood glucose levels for over 370 days in the absence of immunosuppression (excluding the first 5 days after transplant). These data provide proof-of-principle evidence for the therapeutic potential of PSC-derived islets generated by blastocyst complementation in a xenogeneic host.

High incidence of multiple aster formation in vitrified-warmed bovine oocytes after in vitro fertilization

In vitro-matured bovine oocytes do not tolerate vitrification as well as mature murine or human oocytes. Delayed first cleavage in vitrified and in vitro-fertilized bovine oocytes may be responsible for the decreased yield of blastocysts in vitro. Because formation of sperm-aster and the subsequent assembly of microtubule network play an important role for migration and fusion of both pronuclei, aster formation in vitrified-warmed oocytes was analyzed by confocal laser-scanning microscopy. At 10 h post-insemination (hpi), proportions of oocytes fertilized normally were comparable between the vitrified and fresh control groups (67 and 70%, respectively). Proportions of oocytes that exhibited microtubule assembly were similar between the two groups (95% each), but the proportion of oocytes with multiple asters was higher in the vitrified group when compared with the fresh control group (68 vs 29%, P < 0.05). Both migration and development of two pronuclei were adversely affected by multiple aster formation. In the next experiment, multiple asters observed in 5.5 vs 8 hpi pronuclear zygotes were located near the male pronucleus, suggesting that those multiple asters were not the cytoplasmic asters of maternal origin. In conclusion, multiple aster formation frequently observed in vitrified-warmed bovine oocytes may be related to loss of ooplasmic function responsible for normal microtubule assembly from the sperm-aster.

Rescue of Vitrified-Warmed Bovine Oocytes with Rho-Associated Coiled-Coil Kinase Inhibitor

ABSTRACT Cryotolerance of matured bovine oocytes is not fully practical even though a promising vitrification procedure with a ultrarapid cooling rate was applied. The present study was conducted to investigate whether recovery culture of vitrified-warmed bovine oocytes with an inhibitor (Y-27632) of Rho-associated coiled-coil kinase (ROCK) can improve the developmental potential after in vitro fertilization (IVF) and in vitro culture. Immediately after warming, almost all oocytes appeared to be morphological normal. Treatment of the postwarming oocytes with 10 μM Y-27632 for 2 h resulted in the significantly higher oocyte survival rate before IVF as well as higher cleavage rate and blastocyst formation rate. Quality analysis of the resultant blastocysts in terms of total cell number and apoptotic cell ratio also showed the positive effect of the Y-27632 treatment. Time-dependent change in mitochondrial activity of the vitrified-warmed oocytes was not influenced by ROCK inhibition during the period of recovery culture. However, the ability of ooplasm to support single-aster formation was improved by the ROCK inhibition. Thus, inhibition of ROCK activity in vitrified-warmed bovine oocytes during a short-term recovery culture can lead to higher developmental competence, probably due to decreased apoptosis and normalized function of the microtubule-organizing center.

Vitrification of ICSI- and IVF-derived bovine blastocysts by minimum volume cooling procedure: effect of developmental stage and age

The objective was to investigate the effects of developmental stage (fully-expanded or expanding blastocysts) and/or age (harvested on Days 7 or 8) on post-vitrification in vitro survival of bovine blastocysts derived from intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Post-warming survival (re-expansion of blastocoele within 24 h) of ICSI-derived fully-expanded blastocysts (80%) was similar to that of their IVF-derived counterparts (88%). However, the ability of ICSI-derived expanding blastocysts to survive vitrification procedures (61%) was lower than that of IVF-derived blastocysts (85%; P < 0.05), although the ICSI- and IVF-derived fresh blastocysts were of similar quality. The age of the blastocysts before vitrification did not affect cryotolerance for either ICSI-derived (73 and 59% for Days 7 and 8 embryos, respectively) or IVF-derived blastocysts (86% for both Days 7 and 8 embryos). At 24 h of post-warming culture, ICSI-derived blastocysts surviving vitrification contained a higher proportion of dead cells than their IVF-derived counterparts (5-13% vs. 2-4%; P < 0.05), but these proportions were not different from those of fresh control embryos. There was an adverse effect of vitrification on the ability of blastocysts to hatch within 72 h of culture only in IVF-derived Day 8 blastocysts (41 and 70% in vitrified and fresh control groups, respectively). In conclusion, the proportion of blastocysts that survived vitrification procedures was similar for ICSI- and IVF-derived bovine blastocysts if the former were cultured to the fully-expanded stage prior to vitrification, with no significant difference between embryos harvested on Day 7 versus Day 8.

Derivation of Embryonic Stem Cell Lines from Parthenogenetically Developing Rat Blastocysts

This study was undertaken to establish rat embryonic stem (ES) cells from parthenogenetically developing blastocysts. Ten blastocysts were prepared by treatment of ovulated rat oocytes with ionomycin and cycloheximide, and three alkaline phosphatase-positive ES cell lines were established using the N2B27 medium supplemented with mitogen activated protein kinase kinase inhibitor PD0325901, glycogen synthase kinase 3 inhibitor CHIR99021, rat leukemia inhibitory factor, and forskolin. Expression of stem cell marker genes (Oct-4, rNanog, Fgf-4, and Rex-1) was confirmed in all three ES cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR). Combined bisulfite restriction analysis showed that the differentially methylated region locus of five imprinted genes (H19, Meg3IG, Igf2r, Peg5, and Peg10) in these ES cells remained to be demethylated or was hypomethylated, which was similar to that in control ES cells established from normal blastocysts. Characteristics of the parthenogenetic blastocyst-derived ES cells were successfully transmitted to the next generation through a chimeric rat for one of the three ES cell lines. This is the first report on germline-competent (genuine) ES cells derived from parthenogenetically developing rat blastocysts.

Stimulatory effect of Rho-associated coiled-coil kinase (ROCK) inhibitor on revivability of in vitro-produced bovine blastocysts after vitrification

Inhibition of Rho-associated coiled-coil kinase (ROCK) activity promoted recovery and growth of frozen-thawed human embryonic stem cells. The primary objective was to determine if a ROCK inhibitor (Y-27632) in post-thaw culture medium improved revivability of vitrified IVP bovine blastocysts. Expanding or expanded blastocysts (7 d after IVF) were vitrified (minimum volume cooling procedure, using a Cryotop) in 15% ethylene glycol, 15% DMSO and 0.5M sucrose. When post-warm blastocysts were cultured in mSOF medium, survival rate (re-expansion of blastocoel at 24h of culture) was improved (P<0.05) by the addition of 10 microM Y-27632 (94.9+/-2.4%, mean+/-SEM) compared to a control (78.0+/-6.0%). Conversely, after 48 h of culture, there were no significant differences in hatching rate (62.8+/-11.1 vs. 59.6+/-9.4%) and mean total cell number (135.2+/-13.1 vs. 146.7+/-13.3). In non-vitrified IVP bovine blastocysts, the hatching rate on Day 9 was improved by Y-27632 (91.7+/-3.8 vs. 54.7+/-8.9%, P<0.05), with no difference in mean total cell number of blastocysts (230.0+/-23.0 vs. 191.2+/-22.2, P=0.23). In an additional experiment, Y-27632 was added to culture medium on either Day 0, Day 2, or Day 4 (and remained present until Day 8), resulting in no improvement in blastocyst yield compared to a control group (7.5+/-2.1, 31.4+/-2.3, 36.2+/-3.2, and 28.6+/-6.9%, respectively). In conclusion, adding a ROCK inhibitor to post-thaw culture medium improved revivability of IVP bovine blastocysts after vitrification and warming.

Microtubule assembly and in vitro development of bovine oocytes with increased intracellular glutathione level prior to vitrification and in vitro fertilization

Although vitrification is a useful technique for preservation of bovine oocytes, the yield of blastocysts derived from the vitrified oocytes is still low. We have recently reported a new type of cryoinjury, multiple aster formation, by which pronuclear migration and development of vitrified-warmed and in vitro-fertilized bovine oocytes are impaired. The aim of the present study was to investigate the effect of glutathione (GSH) content of vitrified bovine oocytes on multiple aster formation and subsequent in vitro development. Treatment of bovine cumulus-oocyte complexes with β-mercaptoethanol (βME) and L-cysteine (Cys) during in vitro maturation resulted in 2.5-fold higher GSH content not only in fresh control but also in vitrified-warmed oocytes. The percentage of normally fertilized zygotes exhibiting sperm aster(s) was >95% in all four groups (with or without βME/Cys × fresh control or vitrified). The frequency of multiple aster formation in vitrified oocytes (three-fold higher than that in fresh control oocytes) was not affected by the increased level of intracellular GSH with βME/Cys. Consequently, the migration and development of pronuclei as well as the yield of blastocysts from vitrified-warmed oocytes (17 versus 41%) were not improved. In addition, there was no effect of increased GSH level on the yield of blastocysts in fresh control groups.

High revivability of vitrified–warmed bovine mature oocytes after recovery culture with α-tocopherol

The objective of this study was to investigate whether developmental competence of vitrified-warmed bovine oocytes can be improved by antioxidant treatment during recovery culture. In experiment 1, one of the two antioxidants (either l-ascorbic acid or α-tocopherol) was added as a supplement to the recovery culture medium to which postwarming oocytes were exposed for 2 h before IVF. The exposure to α-tocopherol had a positive effect on rescuing the oocytes as assessed by the blastocyst yield 8 days after the IVF (35.1-36.3% vs 19.2-25.8% in untreated postwarming oocytes). Quality of expanding blastocysts harvested on Day 8 was comparable between α-tocopherol-treated vitrification group and fresh control group in terms of total cell number and chromosomal ploidy. In experiment 2, level of reactive oxygen species, mitochondrial activity, and distribution of cortical granules in α-tocopherol-treated postwarming oocytes were assessed. No obvious differences from the control data were found in these parameters. However, the treatment with α-tocopherol increased the percentage of zygotes exhibiting normal single aster formation (90.3% vs 48.0% in untreated postwarming oocytes; 10 h post-IVF). It was concluded that α-tocopherol treatment of vitrified-warmed bovine mature oocytes during recovery culture can improve their revivability, as shown by the high blastocyst yield and the higher mean total cell number in the blastocysts.

Knock-in of a histone H2B-tdTomato reporter into the Rosa26 locus allows visualization of cell nuclei in rats

Fusion of histoneH2Bwith green fluorescent protein has made it possible to observe chromosomal dynamics in living mouse cells under a laser-scanning confocal microscope (Kanda et al., 1998), while knocking the tdTomato gene into the rat Rosa26 locus allows for ubiquitous transgene expression (Kobayashi et al., 2012). The present study merged these two approaches inserting a gene fusion of histone H2B and tdTomato into the rat Rosa26 locus via embryonic-stem-cell-mediated transgenesis so that cell nuclei could be visualized during rat preimplantation development. PCR products of the human H2B, tdTomato, splice acceptor sequence, and IRES-Neo-SV40pAwere inserted into theNheI site of prRosa26-1with an in-fusion cloning kit (Kobayashi et al., 2012). The finalH2B-tdTomato targeting vector was linearized by SalI digestion (Fig. S1A). The rBLK2i-1 embryonic stem cells (RGD ID: 10054010, http://rgd.mcw.edu/wg/home) were cultured on mitomycin C-treated mouse embryonic fibroblasts in N2B27 medium containing 1mM mitogen-activated kinase kinase (MEK) inhibitor PD0325901, 3mM glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021, 1,000U/mL ESGRO 1 , and 10mM forskolin (Hirabayashi et al., 2014). Targeting vector (25mg) was introduced into 5 10 embryonic stem cells by electroporation at 800 V, 10mF in 500mL of N2B27 medium.Theelectroporated cellswere treatedwith 200mg/ mL G418 for 48 hr, resulting in 13 tdTomato-positive colonies seven days after the electroporation. Four out of the 13 embryonic stemcell linesweredetermined to carry a knockin at the Rosa26 locus (clones #1, 4, 5, 11; Fig. S1A) by PCR for tdTomato (primers: 50-GCGAGGAGGTCATCAAAGAG and 50-GATGACGGCCATGTTGTTGT) with AmpliTaq 1 DNA polymerase (Applied Biosystems, Foster City,CA), and for homologous recombination in theRosa26 locus (primers: 50-CAGAAAAGGCGGAGCGAGCCCAAG and 50-GGGCCCTCACATTGCCAAAAGACGG) with PrimeSTAR 1 GXL DNA polymerase (Takara Bio, Shiga, Japan). A total of 18 chimeric rats (female 7, male 11) were generated by injection of targeted embryonic stem cells (clone #1) into 64 Crlj:WI (RGD ID: 2312504) blastocysts, followed by transfer of the blastocysts to three pseudopregnant Crlj:WI recipients. Eighty-three F1 offspring were delivered from Crlj:WI females mated with six male chimeras (> 70% black-colored coat), and two out of eight black F1 rats were identified as H2B-tdTomato knock-in-positive by PCR for tdTomato. Normal viability and fertility of the homozygous knock-in rats demonstrated that this reporter is non-toxic and does not interfere with mitosis or meiosis. Parthenogenetic development of the established transgenic strain was observed from metaphase-II oocytes to blastocysts under an in vitro culture system (Fig. 1). Oocytes retrieved from superovulated, heterozygous knock-in females were denuded and cultured in mR1ECM medium after activation with ionomycin (5mM, 5min) followedbycycloheximideplus cytochalasinB (5mg/mLeach, 4 hr) treatment. Cell nuclei, including the meiotic plate and pronucleus, were successfully visualized by H2B-tdTomato expression during in vitro development. Thus, these H2B-tdTomato knock-in rats allow for the monitoring of chromosomal dynamics during embryonic development and may facilitate nuclear handling during rat cloning by somatic-cell nuclear transplantation.

Multiple aster formation is frequently observed in bovine oocytes retrieved from 1-day stored ovaries

We have recently reported that multiple aster formation after in vitro fertilization (IVF) was one of the factors that negatively affected the developmental competence of vitrified-warmed bovine matured oocytes, and that short-term culture of the post-warm oocytes with an inhibitor of Rho-associated coiled-coil kinase (ROCK) suppressed the multiple aster formation and improved the blastocyst yield. The present study was conducted to investigate whether increased multiple aster formation following IVF was involved in impaired developmental competence of stored ovary-derived bovine oocytes. Oocytes retrieved from 1-day stored ovaries had lower developmental potential to day 8 blastocysts when compared with those from fresh ovaries (37 versus 63%). Immunostaining of α-tubulin 10 h post-IVF revealed that a higher incidence of multiple aster formation occurred in oocytes retrieved from stored ovaries than from fresh ovaries (31 versus 15%). Treatment of post-in vitro maturated (post-IVM) oocytes with ROCK inhibitor for 2 h significantly suppressed the incidence of multiple aster formation (10 versus 32% in the control group). However, the suppression effect of ROCK inhibitor on multiple aster formation in IVM/IVF oocytes did not improve blastocyst yield from stored ovary-derived oocytes (41 versus 37% in the control group). These results suggested that the higher incidence of multiple aster formation by bovine ovary storage was not responsible for the decreased developmental competence of IVF oocytes.