Two sites of uterine insemination were compared with natural service for the recovery and fertili... more Two sites of uterine insemination were compared with natural service for the recovery and fertilization of sheep ova following multiple ovulation induced with an equine anterior pituitary extract (HAP) given after a period of progesterone treatment. Freshly ejaculated semen was placed directly into either the lumen of the body of the uterus or extremities of the uterine horns 24-30 h after HAP treatment. There was little difference between the two sites of uterine insemination in recovery and fertilization rates of ova. Recovery rates were slightly lower (6-9 %) following uterine insemination, but fertilization rates were considerably higher than in naturally mated ewes (96' 5 v. 59• 2 %). Overall, uterine insemination resulted in the recovery of 20 % more fertilized ova than after natural service. Progesterone-HAP treatment gave a marked degree of control over the time of ovulation and it is suggested that uterine inseminations 24 h after the conclusion of treatment be used when large numbers of ova of known age are required at predetermined times.
... Philip S Choi, Lisa Zakhary, Wen-Yee Choi, Sophie Caron, Ezequiel Alvarez-Saavedra, Eric A Mi... more ... Philip S Choi, Lisa Zakhary, Wen-Yee Choi, Sophie Caron, Ezequiel Alvarez-Saavedra, Eric A Miska, et al. ... These cells are totipotent (toti total) in nature, meaning that they can become any kind of cell in the body (9). Blastocyst ES cells are the cells in the ICM of blasto-cyst. ...
The numbers of primordial follicles in ovaries of ewe lambs, from two groups of Peppin Merinos th... more The numbers of primordial follicles in ovaries of ewe lambs, from two groups of Peppin Merinos that had been selected for (T group) and against (0 group) mUltiple births, were estimated at birth, 7 days and 5 months of age. Peripheral plasma and anterior pituitary levels of LH and FSH were determined by radioimmunoassay in the lambs at 5 months of age. These lambs were either entire or unilaterally ovariectomized (hemispayed) at 7 days of age. There was no difference in primordial follicle numbers between lambs at birth and 7 days of age, but when paired ovaries of hemispayed lambs at 7 days and 5 months of age were compared there was a significant increase in follicle numbers with age (34903 v. 48047). O-group lambs had more primordial follicles than T-group lambs at birth and 7 days of age (65501 v. 37797) and there was a similar but not significant difference at 5 months of age (53934 v. 44057). Peripheral plasma LH was higher in T-group than in O-group lambs and plasma LH levels were correlated to pituitary LH content (r = o• 75). Vesicular follicles larger than 2 mm in diameter were present only in ovaries of lambs with plasma LH levels above 3• 0 ng/m!. Pituitary FSH levels were considerably higher in hemispayed lambs than in entire lambs and there was a trend to higher levels in T-group lambs when compared with O-group lambs. It is concluded that genetic selection for the incidence of multiple births has resulted in changes in the number of ovarian primordial follicles of the post-natal lamb and it is suggested that these changes may be related to the levels of pituitary gonadotrophins.
Directed differentiation of human embryonic stem cells (hESCs) may yield models to study organoge... more Directed differentiation of human embryonic stem cells (hESCs) may yield models to study organogenesis, produce cells and tissues for therapies, and identify clinically relevant compounds for disease treatment. Optimal conditions for specific differentiation of hESCs are still being determined. Incorporation of fluorescent reporter genes will enable high-throughput screening to identify fate-specifying molecules. Ectopic expression, or silencing, of key developmental genes can also direct differentiation toward specific lineages. Here, we briefly overview various genetic modifications used to generate useful hESC lines. We identify strengths and limitations to each method and propose the most suitable approaches for different applications.
Two experiments were carried out to investigate the importance of progesterone and oestrogen for ... more Two experiments were carried out to investigate the importance of progesterone and oestrogen for the survival and development of fertilized ova in ovariectomized ewes. In experiment 1 ewes were treated with progesterone (P) alone or progesterone and 17 p-oestradiol (P + E2) following ovariectomy on the second (day 2) or sixth day (day 6) after mating to vasectomized rams. Fertilized ova were transferred to the ewes either immediately after ovariectomy or on day 6 after ovariectomy on day 2. In experiment 2 ewes that had been given an equine anterior pituitary extract to induce multiple ovulation were ovariectomized on day 2 after mating to fertile rams, and then treated with P or P+E2. On day 18 the uteri of all ewes were flushed to recover embryos. Treatment with P commenced immediately after ovariectomy and E2 was given from day 21-to day 5, to ewes ovariectomized on day 2. Ovarian vein blood was collected just prior to ovariectomy and was assayed for its progesterone and oestrogen contents. In both experiments oestradiol had no effect upon the proportion of ewes with normal embryos. In experiment 1 delay of transfer until day 6 after ovariectomy on day 2 increased the proportion of ewes with normal embryos [28 of 43 (65 %) v. 16 of 46 (35 %)]. Transfers on day 6 after ovariectomy on the same day tended to be less successful than transfers on day 6 after ovariectomy on day 2 [11 of 23 (48 %) v. 28 of 43 (65 %)]. In all ewes in experiment 2, and in experiment 1 where ovariectomies and transfers were carried out on the same day, survival and normal development of ova occurred only in ewes in which the ovarian vein progesterone levels at ovariectomy, expressed as progesterone per corpus luteum, fell within precise limits. There was no such relationship in ewes ovariectomized on day 2 and given progesterone prior to transfer on day 6. It would appear that the secretion of oestradiol during the luteal phase of the oestrous cycle in the ewe has little effect on the survival and development of ova. However, progesterone appears to have an important role. Further, it would appear that as early as the second day after oestrus the activity of the corpus luteum, as assessed by its progesterone secretion, may well influence the success or failure of pregnancy.
In Vitro Cellular & Developmental Biology – Animal, Feb 23, 2010
Human embryonic stem (hES) cells were originally isolated and maintained on mouse embryonic fibro... more Human embryonic stem (hES) cells were originally isolated and maintained on mouse embryonic fibroblast (MEF) feeder layers in the presence of fetal bovine serum (FBS). However, if the hES cells are to be used for therapeutic applications, it is preferable to regulatory authorities that they be derived and cultured in animal-free conditions to prevent mouse antigen contamination that would exacerbate an immune response to foreign proteins, and the potential risk of transmission of retroviral and other zoonotic pathogens to humans. As a step towards this goal, we derived a new hES cell line (MISCES-01) on human adult skin fibroblasts as feeder cells using serum replacement (SR) medium. The MISCES-01 cells have a normal diploid karyotype (46XX), express markers of pluripotency (OCT4, GCTM-2, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, and alkaline phosphatase) and following in vitro and in vivo differentiation, give rise to derivatives of the three primary germ layers. This cell line can be obtained for research purposes from the Australian Stem Cell Centre (http://www.stemcellcentre.edu.au).
The clinical translation of promising products, technologies and interventions from the disciplin... more The clinical translation of promising products, technologies and interventions from the disciplines of nanomedicine and cell therapy has been slow and inefficient. In part, translation has been hampered by suboptimal research practices that propagate biases and hinder reproducibility. These include the publication of small and underpowered preclinical studies, suboptimal study design (in particular, biased allocation of experimental groups, experimenter bias and lack of necessary controls), the use of uncharacterized or poorly characterized materials, poor understanding of the relevant biology and mechanisms, poor use of statistics, large between-model heterogeneity, absence of replication, lack of interdisciplinarity, poor scientific training in study design and methods, a culture that does not incentivize transparency and sharing, poor or selective reporting, misaligned incentives and rewards, high costs of materials and protocols, and complexity of the developed products, technologies and interventions. In this Perspective, we discuss special manifestations of these problems in nanomedicine and in cell therapy, and describe mitigating strategies. Progress on reducing bias and enhancing reproducibility early on ought to enhance the translational potential of biomedical findings and technologies. For a product, technology or intervention to warrant clinical trials, there must be sufficient preclinical evidence of safety and efficacy. However, the clinical translation of promising fundamental discoveries and preclinical approaches in nanomedicine and cell therapy, which hold great promise for the design of future medical interventions and for the improvement of Reprints and permissions information is available at www.nature.com/reprints.
The problems of sustaining placenta formation in embryos produced by nuclear transfer have emphas... more The problems of sustaining placenta formation in embryos produced by nuclear transfer have emphasized the need for basic knowledge about epiblast formation and gastrulation in bovine embryos. The aims of this study were to define stages of bovine post-hatching embryonic development and to analyse functional mechanisms of germ-layer formation. Embryos developed in vivo were collected after slaughter from superovulated cows on days 9, 11, 14 and 21 after insemination and processed for transmission electron microscopy (n = 26) or immunohistochemistry (n = 27) for potential germ-layer characterization (cytokeratin 8 for potential ectoderm; alpha-1-fetoprotein for potential endoderm; and vimentin for potential mesoderm). On day 9, the embryos were devoid of zona pellucida and presented a well-defined inner cell mass (ICM), which was covered by a thin layer of trophoblast cells (the Rauber's layer). Formation of the hypoblast from the inside of the ICM was ongoing. On day 11, the Rauber's layer was focally interrupted and adjacent underlying ICM cells formed tight junctions. The hypoblast, which formed a thin confluent cell layer, was separated from the ICM and the trophoblast by intercellular matrix. The embryos were ovoid to tubular and displayed a confluent hypoblast on day 14. The epiblast was inserted into the trophoblast epithelium and tight junctions and desmosomes were present between adjacent epiblast cells as well as between peripheral epiblast and trophoblast cells. In some embryos, the epiblast was more or less covered by foldings of trophoblast in the process of forming the amniotic cavity. Cytokeratin 8 was localized to the trophoblast and the hypoblast underlying the epiblast; alpha-1-fetoprotein was localized to most hypoblast cells underlying the trophoblast; and vimentin was localized to most epiblast cells. On day 21, the smallest embryos displayed a primitive streak and formation of the neural groove, whereas the largest embryos presented a neural tube, up to 14 somites and allantois development. These embryos depicted the gradual formation of the endoderm, mesoderm and ectoderm as well as differentiation of paraxial, intermediate and lateral plate mesoderm. Cytokeratin 8 was localized to the trophoblast, the hypoblast and the surface and neural ectoderm; and alpha-1-fetoprotein was localized to the hypoblast, but not the definitive endoderm, the intensity increasing with development. Vimentin was initially localized to some, but not all, cells positioned particularly in the ventral region of the primitive streak, to presumptive definitive endoderm cells inserted into the hypoblast, and to mesoderm. In conclusion, within 2 weeks of hatching, bovine embryos complete formation of the hypoblast and the epiblast, establishment of the amniotic cavity, ingression of epiblast cells for primitive streak formation, involution of cells through the node and the streak for endoderm and mesoderm fomation, neurulation and differentiation of the mesoderm. The recruitment of cells from the epiblast to form the primitive streak as well as the endoderm and mesoderm is associated with expression of the intermediate filament vimentin.
Chimeric antigen receptor (CAR)-T therapy has demonstrated remarkable outcomes for B cell maligna... more Chimeric antigen receptor (CAR)-T therapy has demonstrated remarkable outcomes for B cell malignancies, however, its application for T cell lymphoma, particularly cutaneous T cell lymphoma (CTCL), has been limited. Barriers to effective CAR-T cell therapy in treating CTCL include T cell aplasia in autologous transplants, CAR-T product contamination with leukemic T cells, CAR-T fratricide (when the target antigen is present on normal T cells), and tumor heterogeneity. To address these critical challenges, innovative CAR engineering by targeting multiple antigens to strike a balance between efficacy and safety of the therapy is necessary. In this review, we discuss the current obstacles to CAR-T cell therapy and highlight potential targets in treating CTCL. Looking forward, we propose strategies to develop more powerful dual CARs that are advancing towards the clinic in CTCL therapy.
Iranian Journal of Veterinary Research, Sep 20, 2010
Embryonic stem cells (ESCs) are originally derived from the ICM of blastocysts and are characteri... more Embryonic stem cells (ESCs) are originally derived from the ICM of blastocysts and are characterized by their ability to self-renew and their pluripotencies. Only a few reports have been published on ESC isolations and line establishment in animals, even fewer in horses. However, it is still important to isolate equine ESCs for animal biotechnology and therapeutic applications. In the present study, we tried to derive horse ESC lines from the ICM of blastocysts fertilized in vivo and maintain their pluripotencies in different conditions. The primary horse ESCs were able to self-renew when they were cultured in basic medium on γ-irradiated MEFs. After 15 passages, immunohistochemistry of the putative horse ESCs showed that some cells in the colonies were positive for Oct-4, SSEA-1, GCTM-2, TRA-1-60 and TRA-1-81. Moreover, to optimize the culture conditions, these putative horse ESCs were cultured in basic medium supplemented with human leukemia inhibitory factor (hLIF) only, human basic fibroblastic growth factor (hbFGF) only, or hbFGF plus hLIF with or without heterologous (MEF) feeder cells. Based on our results, the heterologous feeder (MEF) cells are necessary to maintain the undifferentiated state for horse ESCs, and ESC-like cell morphology of horse ESCs were well maintained in the basic medium supplemented with or without hLIF. This result suggested that hLIF was neither prerequisite nor negative for maintenance of horse ESCs; bFGF seemed to be negative for maintenance of horse ECSs and the combination of hLIF and bFGF was unable to improve the culture condition.
... Brunet. S., Maria, AS, Guillard, P. et al.(1999). Kinetochore fibers are not involved in the ... more ... Brunet. S., Maria, AS, Guillard, P. et al.(1999). Kinetochore fibers are not involved in the formation of (he first meiotic spindle in mouse oocytes, but control the exit from the first meiotic M phase./. Cell. ... Bioessays, 22, 351-63. Coonen, E., Harper, IC, Ramaekers, E C. et al.(1994). ...
The susceptibility of fertilized sheep ova and oocytes, with and without zonae pellucidae, to lys... more The susceptibility of fertilized sheep ova and oocytes, with and without zonae pellucidae, to lysis by complement plus antibodies in antisera raised against sheep ovaries, oocytes and sperm was examined in vitro. The presence of the zona reduced the proportion of ova lysed (8 % v. 57 %) and a large proportion of ova with zonae continued development in culture following exposure to antisera. Antisera to ovary and to oocytes lysed a higher proportion of ova than did antisera to sperm (51 and 56% v. 29%). There was no effect of age of fertilized ova (day 2, 4, 6; day 0 = day of oestrus) on susceptibility to lysis. Fluorescein-labelled antibodies from high-titre antisera to ovary and oocytes traversed the zona and attached to blastomeres. Following absorption of the antibody conjugates with sheep serum, fluorescein staining of ova with intact zonae was confined to the zona, whereas with zona-free ova the blastomeres were stained.
but surprising increase in the count of epididymo-deferential spermatozoa (P == 0.09) which could... more but surprising increase in the count of epididymo-deferential spermatozoa (P == 0.09) which could be related to a secretion or absorption defect of the luminal fluid. (Australia). 14.30-14.45 0-157. The gonadotrophin releasing hormone mRNA and protein expression in Vero cells
The survival, fertilization, development, and viability in vitro and in vivo of~nfertilized mouse... more The survival, fertilization, development, and viability in vitro and in vivo of~nfertilized mouse eggs frozen by slow cooling to -36oC or -80oC in 1.5M dimethyl sulphoxide (DMSO) was examined in a series of experiments which explored some of the problems in freezing the egg. DMSO was added to the eggs at either room temperature or at ooc. Maximum success rate (42% of frozen eggs developing to-two cells) was obtained when DMSO was added at OOC and the eggs slow cooled to -80°C. Removal of cumulus failed to improve freezing success rates. Addition of DMSO at temperatures above ooc significantly reduced the fertilizing capacity of eggs. Excessive exposure of eggs to temperatures. around l5°C also caused a significant reduction in fertilization rates. The effects of DMSO and cooling on fertilization are likely to be due to zona hardening by cortical granule release and to disorganization of the egg cytoskeleton and plasma membrane. These problems will be difficult to overcome if cryopreservation of the unfertilized human egg is preferred to the fertilized egg or early cleavage stage embryo in clinical in vitro fertilization. Fertil Steril 52:778, 1989
While optimizing the ultrarapid embryo freezing procedure we noted that embryo survival was lowes... more While optimizing the ultrarapid embryo freezing procedure we noted that embryo survival was lowest when gas bubbles formed in the straws. Here we report the influences of gassing the freezing solutions with 5% CO2 in air, degassing the solution, the pH of the medium and straw irradiation on the survival and development in vitro of 2-cell mouse embryos. Embryos were ultrarapidly frozen in medium M2 containing 3 M dimethyl sulphoxide and 0.25 M sucrose. Significantly fewer embryos survived freezing and thawing in gassed solutions. The subsequent development of intact embryos to blastocysts was similar in gassed and non-gassed solutions. Survival and development of embryos frozen in solutions of pH 7.0, 7.6 or 8.0 was similar, but fewer embryos developed to blastocysts after freezing in media at pH 6.0. In straws sterilized by gamma irradiation before use, embryo survival after freezing and thawing was dramatically reduced compared with that seen when non-irradiated straws were used. With non-gassed solutions significantly more gas bubbles formed in irradiated straws than in non-irradiated straws. Although bubble formation was significantly reduced in all straws by using degassed freezing solutions, this did not improve embryo survival. We conclude that under conditions normally used for ultrarapid freezing, dissolved gas and pH have less effect on embryo viability than the use of irradiated straws.
Two sites of uterine insemination were compared with natural service for the recovery and fertili... more Two sites of uterine insemination were compared with natural service for the recovery and fertilization of sheep ova following multiple ovulation induced with an equine anterior pituitary extract (HAP) given after a period of progesterone treatment. Freshly ejaculated semen was placed directly into either the lumen of the body of the uterus or extremities of the uterine horns 24-30 h after HAP treatment. There was little difference between the two sites of uterine insemination in recovery and fertilization rates of ova. Recovery rates were slightly lower (6-9 %) following uterine insemination, but fertilization rates were considerably higher than in naturally mated ewes (96' 5 v. 59• 2 %). Overall, uterine insemination resulted in the recovery of 20 % more fertilized ova than after natural service. Progesterone-HAP treatment gave a marked degree of control over the time of ovulation and it is suggested that uterine inseminations 24 h after the conclusion of treatment be used when large numbers of ova of known age are required at predetermined times.
... Philip S Choi, Lisa Zakhary, Wen-Yee Choi, Sophie Caron, Ezequiel Alvarez-Saavedra, Eric A Mi... more ... Philip S Choi, Lisa Zakhary, Wen-Yee Choi, Sophie Caron, Ezequiel Alvarez-Saavedra, Eric A Miska, et al. ... These cells are totipotent (toti total) in nature, meaning that they can become any kind of cell in the body (9). Blastocyst ES cells are the cells in the ICM of blasto-cyst. ...
The numbers of primordial follicles in ovaries of ewe lambs, from two groups of Peppin Merinos th... more The numbers of primordial follicles in ovaries of ewe lambs, from two groups of Peppin Merinos that had been selected for (T group) and against (0 group) mUltiple births, were estimated at birth, 7 days and 5 months of age. Peripheral plasma and anterior pituitary levels of LH and FSH were determined by radioimmunoassay in the lambs at 5 months of age. These lambs were either entire or unilaterally ovariectomized (hemispayed) at 7 days of age. There was no difference in primordial follicle numbers between lambs at birth and 7 days of age, but when paired ovaries of hemispayed lambs at 7 days and 5 months of age were compared there was a significant increase in follicle numbers with age (34903 v. 48047). O-group lambs had more primordial follicles than T-group lambs at birth and 7 days of age (65501 v. 37797) and there was a similar but not significant difference at 5 months of age (53934 v. 44057). Peripheral plasma LH was higher in T-group than in O-group lambs and plasma LH levels were correlated to pituitary LH content (r = o• 75). Vesicular follicles larger than 2 mm in diameter were present only in ovaries of lambs with plasma LH levels above 3• 0 ng/m!. Pituitary FSH levels were considerably higher in hemispayed lambs than in entire lambs and there was a trend to higher levels in T-group lambs when compared with O-group lambs. It is concluded that genetic selection for the incidence of multiple births has resulted in changes in the number of ovarian primordial follicles of the post-natal lamb and it is suggested that these changes may be related to the levels of pituitary gonadotrophins.
Directed differentiation of human embryonic stem cells (hESCs) may yield models to study organoge... more Directed differentiation of human embryonic stem cells (hESCs) may yield models to study organogenesis, produce cells and tissues for therapies, and identify clinically relevant compounds for disease treatment. Optimal conditions for specific differentiation of hESCs are still being determined. Incorporation of fluorescent reporter genes will enable high-throughput screening to identify fate-specifying molecules. Ectopic expression, or silencing, of key developmental genes can also direct differentiation toward specific lineages. Here, we briefly overview various genetic modifications used to generate useful hESC lines. We identify strengths and limitations to each method and propose the most suitable approaches for different applications.
Two experiments were carried out to investigate the importance of progesterone and oestrogen for ... more Two experiments were carried out to investigate the importance of progesterone and oestrogen for the survival and development of fertilized ova in ovariectomized ewes. In experiment 1 ewes were treated with progesterone (P) alone or progesterone and 17 p-oestradiol (P + E2) following ovariectomy on the second (day 2) or sixth day (day 6) after mating to vasectomized rams. Fertilized ova were transferred to the ewes either immediately after ovariectomy or on day 6 after ovariectomy on day 2. In experiment 2 ewes that had been given an equine anterior pituitary extract to induce multiple ovulation were ovariectomized on day 2 after mating to fertile rams, and then treated with P or P+E2. On day 18 the uteri of all ewes were flushed to recover embryos. Treatment with P commenced immediately after ovariectomy and E2 was given from day 21-to day 5, to ewes ovariectomized on day 2. Ovarian vein blood was collected just prior to ovariectomy and was assayed for its progesterone and oestrogen contents. In both experiments oestradiol had no effect upon the proportion of ewes with normal embryos. In experiment 1 delay of transfer until day 6 after ovariectomy on day 2 increased the proportion of ewes with normal embryos [28 of 43 (65 %) v. 16 of 46 (35 %)]. Transfers on day 6 after ovariectomy on the same day tended to be less successful than transfers on day 6 after ovariectomy on day 2 [11 of 23 (48 %) v. 28 of 43 (65 %)]. In all ewes in experiment 2, and in experiment 1 where ovariectomies and transfers were carried out on the same day, survival and normal development of ova occurred only in ewes in which the ovarian vein progesterone levels at ovariectomy, expressed as progesterone per corpus luteum, fell within precise limits. There was no such relationship in ewes ovariectomized on day 2 and given progesterone prior to transfer on day 6. It would appear that the secretion of oestradiol during the luteal phase of the oestrous cycle in the ewe has little effect on the survival and development of ova. However, progesterone appears to have an important role. Further, it would appear that as early as the second day after oestrus the activity of the corpus luteum, as assessed by its progesterone secretion, may well influence the success or failure of pregnancy.
In Vitro Cellular & Developmental Biology – Animal, Feb 23, 2010
Human embryonic stem (hES) cells were originally isolated and maintained on mouse embryonic fibro... more Human embryonic stem (hES) cells were originally isolated and maintained on mouse embryonic fibroblast (MEF) feeder layers in the presence of fetal bovine serum (FBS). However, if the hES cells are to be used for therapeutic applications, it is preferable to regulatory authorities that they be derived and cultured in animal-free conditions to prevent mouse antigen contamination that would exacerbate an immune response to foreign proteins, and the potential risk of transmission of retroviral and other zoonotic pathogens to humans. As a step towards this goal, we derived a new hES cell line (MISCES-01) on human adult skin fibroblasts as feeder cells using serum replacement (SR) medium. The MISCES-01 cells have a normal diploid karyotype (46XX), express markers of pluripotency (OCT4, GCTM-2, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, and alkaline phosphatase) and following in vitro and in vivo differentiation, give rise to derivatives of the three primary germ layers. This cell line can be obtained for research purposes from the Australian Stem Cell Centre (http://www.stemcellcentre.edu.au).
The clinical translation of promising products, technologies and interventions from the disciplin... more The clinical translation of promising products, technologies and interventions from the disciplines of nanomedicine and cell therapy has been slow and inefficient. In part, translation has been hampered by suboptimal research practices that propagate biases and hinder reproducibility. These include the publication of small and underpowered preclinical studies, suboptimal study design (in particular, biased allocation of experimental groups, experimenter bias and lack of necessary controls), the use of uncharacterized or poorly characterized materials, poor understanding of the relevant biology and mechanisms, poor use of statistics, large between-model heterogeneity, absence of replication, lack of interdisciplinarity, poor scientific training in study design and methods, a culture that does not incentivize transparency and sharing, poor or selective reporting, misaligned incentives and rewards, high costs of materials and protocols, and complexity of the developed products, technologies and interventions. In this Perspective, we discuss special manifestations of these problems in nanomedicine and in cell therapy, and describe mitigating strategies. Progress on reducing bias and enhancing reproducibility early on ought to enhance the translational potential of biomedical findings and technologies. For a product, technology or intervention to warrant clinical trials, there must be sufficient preclinical evidence of safety and efficacy. However, the clinical translation of promising fundamental discoveries and preclinical approaches in nanomedicine and cell therapy, which hold great promise for the design of future medical interventions and for the improvement of Reprints and permissions information is available at www.nature.com/reprints.
The problems of sustaining placenta formation in embryos produced by nuclear transfer have emphas... more The problems of sustaining placenta formation in embryos produced by nuclear transfer have emphasized the need for basic knowledge about epiblast formation and gastrulation in bovine embryos. The aims of this study were to define stages of bovine post-hatching embryonic development and to analyse functional mechanisms of germ-layer formation. Embryos developed in vivo were collected after slaughter from superovulated cows on days 9, 11, 14 and 21 after insemination and processed for transmission electron microscopy (n = 26) or immunohistochemistry (n = 27) for potential germ-layer characterization (cytokeratin 8 for potential ectoderm; alpha-1-fetoprotein for potential endoderm; and vimentin for potential mesoderm). On day 9, the embryos were devoid of zona pellucida and presented a well-defined inner cell mass (ICM), which was covered by a thin layer of trophoblast cells (the Rauber's layer). Formation of the hypoblast from the inside of the ICM was ongoing. On day 11, the Rauber's layer was focally interrupted and adjacent underlying ICM cells formed tight junctions. The hypoblast, which formed a thin confluent cell layer, was separated from the ICM and the trophoblast by intercellular matrix. The embryos were ovoid to tubular and displayed a confluent hypoblast on day 14. The epiblast was inserted into the trophoblast epithelium and tight junctions and desmosomes were present between adjacent epiblast cells as well as between peripheral epiblast and trophoblast cells. In some embryos, the epiblast was more or less covered by foldings of trophoblast in the process of forming the amniotic cavity. Cytokeratin 8 was localized to the trophoblast and the hypoblast underlying the epiblast; alpha-1-fetoprotein was localized to most hypoblast cells underlying the trophoblast; and vimentin was localized to most epiblast cells. On day 21, the smallest embryos displayed a primitive streak and formation of the neural groove, whereas the largest embryos presented a neural tube, up to 14 somites and allantois development. These embryos depicted the gradual formation of the endoderm, mesoderm and ectoderm as well as differentiation of paraxial, intermediate and lateral plate mesoderm. Cytokeratin 8 was localized to the trophoblast, the hypoblast and the surface and neural ectoderm; and alpha-1-fetoprotein was localized to the hypoblast, but not the definitive endoderm, the intensity increasing with development. Vimentin was initially localized to some, but not all, cells positioned particularly in the ventral region of the primitive streak, to presumptive definitive endoderm cells inserted into the hypoblast, and to mesoderm. In conclusion, within 2 weeks of hatching, bovine embryos complete formation of the hypoblast and the epiblast, establishment of the amniotic cavity, ingression of epiblast cells for primitive streak formation, involution of cells through the node and the streak for endoderm and mesoderm fomation, neurulation and differentiation of the mesoderm. The recruitment of cells from the epiblast to form the primitive streak as well as the endoderm and mesoderm is associated with expression of the intermediate filament vimentin.
Chimeric antigen receptor (CAR)-T therapy has demonstrated remarkable outcomes for B cell maligna... more Chimeric antigen receptor (CAR)-T therapy has demonstrated remarkable outcomes for B cell malignancies, however, its application for T cell lymphoma, particularly cutaneous T cell lymphoma (CTCL), has been limited. Barriers to effective CAR-T cell therapy in treating CTCL include T cell aplasia in autologous transplants, CAR-T product contamination with leukemic T cells, CAR-T fratricide (when the target antigen is present on normal T cells), and tumor heterogeneity. To address these critical challenges, innovative CAR engineering by targeting multiple antigens to strike a balance between efficacy and safety of the therapy is necessary. In this review, we discuss the current obstacles to CAR-T cell therapy and highlight potential targets in treating CTCL. Looking forward, we propose strategies to develop more powerful dual CARs that are advancing towards the clinic in CTCL therapy.
Iranian Journal of Veterinary Research, Sep 20, 2010
Embryonic stem cells (ESCs) are originally derived from the ICM of blastocysts and are characteri... more Embryonic stem cells (ESCs) are originally derived from the ICM of blastocysts and are characterized by their ability to self-renew and their pluripotencies. Only a few reports have been published on ESC isolations and line establishment in animals, even fewer in horses. However, it is still important to isolate equine ESCs for animal biotechnology and therapeutic applications. In the present study, we tried to derive horse ESC lines from the ICM of blastocysts fertilized in vivo and maintain their pluripotencies in different conditions. The primary horse ESCs were able to self-renew when they were cultured in basic medium on γ-irradiated MEFs. After 15 passages, immunohistochemistry of the putative horse ESCs showed that some cells in the colonies were positive for Oct-4, SSEA-1, GCTM-2, TRA-1-60 and TRA-1-81. Moreover, to optimize the culture conditions, these putative horse ESCs were cultured in basic medium supplemented with human leukemia inhibitory factor (hLIF) only, human basic fibroblastic growth factor (hbFGF) only, or hbFGF plus hLIF with or without heterologous (MEF) feeder cells. Based on our results, the heterologous feeder (MEF) cells are necessary to maintain the undifferentiated state for horse ESCs, and ESC-like cell morphology of horse ESCs were well maintained in the basic medium supplemented with or without hLIF. This result suggested that hLIF was neither prerequisite nor negative for maintenance of horse ESCs; bFGF seemed to be negative for maintenance of horse ECSs and the combination of hLIF and bFGF was unable to improve the culture condition.
... Brunet. S., Maria, AS, Guillard, P. et al.(1999). Kinetochore fibers are not involved in the ... more ... Brunet. S., Maria, AS, Guillard, P. et al.(1999). Kinetochore fibers are not involved in the formation of (he first meiotic spindle in mouse oocytes, but control the exit from the first meiotic M phase./. Cell. ... Bioessays, 22, 351-63. Coonen, E., Harper, IC, Ramaekers, E C. et al.(1994). ...
The susceptibility of fertilized sheep ova and oocytes, with and without zonae pellucidae, to lys... more The susceptibility of fertilized sheep ova and oocytes, with and without zonae pellucidae, to lysis by complement plus antibodies in antisera raised against sheep ovaries, oocytes and sperm was examined in vitro. The presence of the zona reduced the proportion of ova lysed (8 % v. 57 %) and a large proportion of ova with zonae continued development in culture following exposure to antisera. Antisera to ovary and to oocytes lysed a higher proportion of ova than did antisera to sperm (51 and 56% v. 29%). There was no effect of age of fertilized ova (day 2, 4, 6; day 0 = day of oestrus) on susceptibility to lysis. Fluorescein-labelled antibodies from high-titre antisera to ovary and oocytes traversed the zona and attached to blastomeres. Following absorption of the antibody conjugates with sheep serum, fluorescein staining of ova with intact zonae was confined to the zona, whereas with zona-free ova the blastomeres were stained.
but surprising increase in the count of epididymo-deferential spermatozoa (P == 0.09) which could... more but surprising increase in the count of epididymo-deferential spermatozoa (P == 0.09) which could be related to a secretion or absorption defect of the luminal fluid. (Australia). 14.30-14.45 0-157. The gonadotrophin releasing hormone mRNA and protein expression in Vero cells
The survival, fertilization, development, and viability in vitro and in vivo of~nfertilized mouse... more The survival, fertilization, development, and viability in vitro and in vivo of~nfertilized mouse eggs frozen by slow cooling to -36oC or -80oC in 1.5M dimethyl sulphoxide (DMSO) was examined in a series of experiments which explored some of the problems in freezing the egg. DMSO was added to the eggs at either room temperature or at ooc. Maximum success rate (42% of frozen eggs developing to-two cells) was obtained when DMSO was added at OOC and the eggs slow cooled to -80°C. Removal of cumulus failed to improve freezing success rates. Addition of DMSO at temperatures above ooc significantly reduced the fertilizing capacity of eggs. Excessive exposure of eggs to temperatures. around l5°C also caused a significant reduction in fertilization rates. The effects of DMSO and cooling on fertilization are likely to be due to zona hardening by cortical granule release and to disorganization of the egg cytoskeleton and plasma membrane. These problems will be difficult to overcome if cryopreservation of the unfertilized human egg is preferred to the fertilized egg or early cleavage stage embryo in clinical in vitro fertilization. Fertil Steril 52:778, 1989
While optimizing the ultrarapid embryo freezing procedure we noted that embryo survival was lowes... more While optimizing the ultrarapid embryo freezing procedure we noted that embryo survival was lowest when gas bubbles formed in the straws. Here we report the influences of gassing the freezing solutions with 5% CO2 in air, degassing the solution, the pH of the medium and straw irradiation on the survival and development in vitro of 2-cell mouse embryos. Embryos were ultrarapidly frozen in medium M2 containing 3 M dimethyl sulphoxide and 0.25 M sucrose. Significantly fewer embryos survived freezing and thawing in gassed solutions. The subsequent development of intact embryos to blastocysts was similar in gassed and non-gassed solutions. Survival and development of embryos frozen in solutions of pH 7.0, 7.6 or 8.0 was similar, but fewer embryos developed to blastocysts after freezing in media at pH 6.0. In straws sterilized by gamma irradiation before use, embryo survival after freezing and thawing was dramatically reduced compared with that seen when non-irradiated straws were used. With non-gassed solutions significantly more gas bubbles formed in irradiated straws than in non-irradiated straws. Although bubble formation was significantly reduced in all straws by using degassed freezing solutions, this did not improve embryo survival. We conclude that under conditions normally used for ultrarapid freezing, dissolved gas and pH have less effect on embryo viability than the use of irradiated straws.
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Papers by Alan Trounson