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The generation of three-dimensional cerebral organoids from human-induced pluripotent stem cells (hPSC) has facilitated the investigation of mechanisms underlying several neuropsychiatric disorders, including stress-related disorders, namely major depressive disorder and post-traumatic stress disorder. Generating hPSC-derived neurons, cerebral organoids, and even assembloids (or multi-organoid complexes) can facilitate research into biomarkers for stress susceptibility or resilience and may even bring about advances in personalized medicine and biomarker research for stress-related psychiatric disorders. Nevertheless, cerebral organoid research does not come without its own set of ethical considerations. With increased complexity and resemblance to in vivo conditions, discussions of increased moral status for these models are ongoing, including questions about sentience, consciousness, moral status, donor protection, and chimeras. There are, however, unique ethical considerations that arise and are worth looking into in the context of research into stress and stress-related disorders using cerebral organoids. This paper provides stress research-specific ethical considerations in the context of cerebral organoid generation and use for research purposes. The use of stress research as a case study here can help inform other practices of in vitro studies using brain models with high ethical considerations.
Non-human animal chimeras, containing human neurological cells, have been created in the laboratory. Despite a great deal of debate, the status of such beings has not been resolved. Under normal definitions, such a being could either be unconventionally human or abnormally animal. Practical investigations in animal sentience, artificial intelligence, and now chimera research, suggest that such beings may be assumed to have no legal rights, so philosophy could provide a different answer. In this vein, therefore, we can ask: What would a chimera, if it could think, think about? Thinking is used to capture the phenomena of a novel, chimeric being perceiving its terrible predicament as no more than a laboratory experiment. The creation of a thinking chimera therefore forces us to reconsider our assumptions about what makes human beings (potentially) unique (and other sentient animals different), because, as such, a chimera’s existence bridges our social and legal expectations about definitions of human and animal. Society has often evolved new social norms based on different kinds of (ir)rational contrivances; the imperative of non-contradiction, which is defended here, therefore requires a specific philosophical response to the rights of a thinking chimeric being.
Developmental biology is seemingly well understood, with development widely accepted as being a series of programmed changes through which an egg turns into an adult organism, or a seed matures into a plant. However, the picture is much more complex than that: is it all genetically controlled or does environment have an influence? Is the final adult stage the target of development and everything else just a build-up to that point? Are developmental strategies the same in plants as in animals? How do we consider development in single-celled organisms? In this concise, engaging volume, Alessandro Minelli, a leading developmental biologist, addresses these key questions. Using familiar examples and easy-to-follow arguments, he offers fresh alternatives to a number of preconceptions and stereotypes, awakening the reader to the disparity of developmental phenomena across all main branches of the tree of life.
The abnormal animal featured here is the calico cat, whose mottled fur is due to a phenomenon which affects all female mammals. Females inactivate one of their X chromosomes so they become mosaics. We discuss how some male/female mosaics have been used by researchers to probe genetic mechanisms.
The cause of hybrid sterility and inviability has not been analyzed in the fin-fish hybrid, although large numbers of hybridizations have been carried out. In this study, we produced allo-diploid hybrids by cross-fertilization between female goldfish (Carassius auratus) and male golden venus chub (Hemigrammocypris rasborella). Inviability of these hybrids was due to breakage of the enveloping layer during epiboly or due to malformation with serious cardiac oedema around the hatching stage. Spontaneous allo-triploid hybrids with two sets of the goldfish genome and one set of the golden venus chub genome developed normally and survived beyond the feeding stage. This improved survival was confirmed by generating heat-shock-induced allo-triploid hybrids that possessed an extra goldfish genome. When inviable allo-diploid hybrid cells were transplanted into goldfish host embryos at the blastula stage, these embryos hatched normally, incorporating the allo-diploid cells. These allo-diploid hybrid cells persisted, and were genetically detected in a 6-month-old fish. In contrast, primordial germ cells taken from allo-diploid hybrids and transplanted into goldfish hosts at the blastula stage had disappeared by 10 days post-fertilization, even under chimeric conditions. In allo-triploid hybrid embryos, germ cells proliferated in the gonad, but had disappeared by 10 weeks post-fertilization. These results showed that while hybrid germ cells are inviable even in chimeric conditions, hybrid somatic cells remain viable.
This introductory chapter begins with background. First it describes the shape of Florentine intellectual culture in the early sixteenth century. It then summarizes the political changes in the city beginning with the 1490s through the naming of Cosimo as leader, and summarizes Cosimo’s early years. The years of instability that preceding Cosimo's reign had harmed the city’s intellectual and cultural life. Cosimo worked both to maximize the city’s independence from foreign control and to reestablish the city’s importance as a cultural center. The Florentine Bernardo Segni later summarized these early successes, beginning with the reopening of the university at Pisa and the founding of the Accademia Fiorentina. As a resut, a new generation of scholars built their intellectual careers in the city. They took a particular interest in the study of the Florentine language, its letters, and Florentine culture. Segni also singled out some of the era’s principal scholars: Piero Vettori, Benedetto Varchi, and Giovan Battista Gelli. They, along with Pierfrancesco Giambullari, Vincenzio Borghini, and several others, are the principal focus of this study.
The use of chimeric molecules fusing several antigenic determinants is a promising strategy for the development of low-cost, standardized and reliable kits to determine specific antibodies. In this study, we designed and assessed a novel recombinant chimera that complements the performance of our previously developed chimera, CP1 [FRA and SAPA antigens (Ags)], to diagnose chronic Chagas disease. The new chimeric protein, named CP3, is composed of MAP, TcD and TSSAII/V/VI antigenic determinants. We compared the performance of both chimeric Ags using a panel of 67 Trypanosoma cruzi-reactive sera and 67 non-reactive ones. The sensitivity of CP3 vs CP1 was 100 and 90.2%, and specificity was 92.5 and 100%, respectively. The mixture of CP1 + CP3 achieved 100% of sensitivity and specificity. More importantly, an additional subset of 17 sera from patients with discordant results of conventional serological methods was analysed; the CP1 + CP3 mixture allowed us to accurately classify 14 of them with respect to IIF, the usual technique used in most of the reference centres. These results show an improved performance of the CP1 + CP3 mixture in comparison with enzyme-linked immunosorbent assay and indirect haemagglutination commercial assays.
Suppose that a colleague proposed a fantastic experiment: to introduce human stem cells into a neonatal mouse so that its entire brain developed into “human-like” neuronal structures. The colleague claimed it would still be a mouse, and that its chimeric brain would be nothing like a “human” one. It would not, as a result, have a moral status beyond its nonhuman animal origins. Thus, the “human neuron mouse” would allow scientists to tinker with human-like neurology in ways that would be precluded if it were a human being, and that would promise to lead to substantial understanding of the destructive and incurable brain diseases that befall humanity. The colleague does admit, however, that for reasons of comparative fidelity, experiments in human patients would be scientifically preferable, although in this case, neither ethically justified nor legally permitted. For that reason, it might be desirable to create a human brain in a nonhuman primate, where it would be more likely that significant human-like neuronal development would occur, but still could not become a person. This article explores the significance of a “human neuron chimpanzee,” and suggests that contradictions in the design of the experiment make it unethical to proceed in either murine or primate models.
Primordial germ cell (PGC) transplant is a promising tool in aquaculture; however, successful use of this technique requires in depth knowledge of the early stages of embryo and larval development. The aim of this study was to analyse the effect of different temperatures (22, 26, and 30°C) on the early development of B. amazonicus. The newly fertilized eggs were distributed into tanks with controlled temperature and oxygenation. Samples were collected at pre-established times and analysed under light and fluorescence microscopy. Temperature influenced the speed and duration of each stage of early development, including hatching time. The highest pronuclei fusion rate was observed 8 min post-fertilization (mpf) at 22 and 26°C, and 6 mpf at 30°C. The duration of the 512–1000 blastomeres phase during in the blastocyst stage was 1 h 30 min at 22°C, and 25 min at 26 and 30°C. Hatching occurred at 24 h 30 mpf at 22°C, 16 h post-fertilization (hpf) at 26°C, and 11 h 30 mpf at 30°C. The rate of morphologically normal larvae was 88.34% at 22°C, 90.49% at 26°C, and 73% at 30°C. Malformations of the head, yolk sac, heart, and tail were observed in all temperatures. Nevertheless, B. amazonicus embryos were able to develop satisfactory in all three temperatures tested. These results enable embryo manipulation at different temperatures to optimize the micromanipulation time of embryos and larvae for biotechnological studies.
The objective of this study was to compare developmental capacity of rabbit chimeric embryos and the allocation of the EGFP gene expression to the embryoblast (ICM) or embryonic shield. We produced chimeric embryos (TR<>N) by synchronous transfer of two or three blastomeres at the 16-cell stage from transgenic (TR) into normal host embryos (N) at the same stage. In the control group, two to three non-transgenic blastomeres were used to produce chimeric embryos. The TR embryos were produced by microinjection of EGFP into both pronuclei of fertilized rabbit eggs. The developmental rate and allocation of EGFP-positive cells of the reconstructed chimeric embryos was controlled at blastocyst (96 h PC) and embryonic shield (day 6) stage.
All chimeric embryos (120/120, 100%) developed up to blastocyst stage. Using fluorescent microscope, we detected green signal (EGFP expression). In 90 chimeric (TR<>N) embryos (75%). Average total number of cells in chimeric embryos at blastocyst stage was 175 ± 13.10, of which 58 ± 2.76 cells were found in the ICM area. The number of EGFP-positive cells in the ICM area was 24 ± 5.02 (35%). After the transfer of 50 chimeric rabbit embryos at the 16-cell stage, 20 embryos (40%) were flushed from five recipients on day 6 of pregnancy, of which five embryos (25%) were EGFP positive at the embryonic shield stage.
Our results demonstrate that transgenic blastomeres in synchronous chimeric embryos reconstructed from TR embryos have an ability to develop and colonize ICM and embryonic shield area.
The objective of this study was to compare in vitro developmental capacity of zona-free aggregated rabbit chimeric embryos and the allocation of EGFP (enhanced green fluorescence protein) gene expression to the inner cell mass (ICM). We produced chimeric embryos by synchronous aggregation of zona-free blastomeres from embryonic cell nuclear transfer (EMB-NT) or somatic cell nuclear transfer (SC-NT) and blastomeres from normal zona-free embryos (N) at the 16-cell stage. In the control group, transgenic (TR) and normal zona-free embryos were used to produce chimeric embryos (TR<>N). EMB-NT embryos were produced by fusion of enucleated oocytes with embryonic cells, which were derived from 32-cell stage transgenic embryos bearing the EGFP gene. The SC-NT embryos were produced by fusing enucleated oocytes with cumulus cells, which were derived from homozygotes transgenic for the EGFP gene female oocytes at 16 h post-coitum. Nuclei of transgenic blastomeres emitted a green signal under fluorescence microscopy. Zona-free EMB-NT or zona-free SC-NT rabbit embryos, both with EGFP fluorescence, as well as TR and zona-free rabbit embryos with no fluorescence (EMB-NT<>N, SC-NT<>N, TR<>N) were aggregated on day 2.5 and evaluated on day 5. The proportion of EMB-NT<>N embryos that developed to the blastocyst stage was significantly higher compared with SC-NT derived cells (p<0.05), but significantly lower than in TR<>N chimeric blastocysts (p<0.001). Similarly, a higher proportion (p<0.001) of EGFP-positive cells allocated to ICM of chimeric blastocysts was revealed in TR<>N chimeras (55%), compared with EMB-NT<>N (35%) and SC-NT<>N (21%). Our results indicate that synchronous chimeric embryos reconstructed from TR embryos were better able to develop and colonize the ICM area than EMB-NT and SC-NT embryos. In this study we have demonstrated for the first time that rabbit NT-derived embryos are able to develop into chimeric blastocysts and participate in the ICM area.
Studies with intact preimplantation mouse embryos and some types of chimaeric aggregates have shown that the most advanced cells are preferentially allocated to the inner cell mass (ICM) rather than the trophectoderm. Thus, differences between 4-cell and 8-cell stage embryos could contribute to the tendency for tetraploid cells to colonise the trophectoderm more readily than the ICM in 4-cell tetraploid[harr ]8 cell diploid chimaeras. The aim of the present study was to test whether 4-cell stage embryos in 4-cell diploid[harr ]8-cell diploid aggregates contributed equally to all lineages present in the E12.5 conceptus. These chimaeras were compared with those produced from standard aggregates of two whole 8-cell embryos and aggregates of half an 8-cell embryo with a whole 8-cell embryo. As expected, the overall contribution of 4-cell embryos was lower than that of 8-cell embryos and similar to that of half 8-cell stage embryos. In the 4-cell[harr ]8-cell chimaeras the 4-cell stage embryos did not contribute more to the trophectoderm than the ICM derivatives. Thus, differences between 4-cell and 8-cell embryos cannot explain the restricted tissue distribution of tetraploid cells previously reported for 4-cell tetraploid[harr ]8-cell diploid chimaeras. It is suggested that cells from the more advanced embryo are more likely to contribute to the ICM but, for technical reasons, are prevented from doing so in simple aggregates of equal numbers of whole 4-cell and whole 8-cell stage embryos.
Studies of tetraploid[harr ]diploid (4n[harr ]2n) mouse chimaeras have demonstrated unequal contributions of 4n
cells to different tissues of the midgestation conceptus. Such a pattern has also been reported in chimaeras as
early as E3.5d, which show an enhanced contribution of 4n cells to the mural trophectoderm (Everett &
West, 1996). In this study, sectioned 4n[harr ]2n and 2n[harr ]2n control chimaeric blastocysts were digitised and
reconstructed in 3 dimensions (3-D). The 3-D images revealed only limited mixing of cells from the 2
contributing embryos of individual blastocysts in both chimaera groups. Consequently, the distribution
pattern of the 2 cell types was dependent on the spatial relationship between the orientation of the
blastocyst and the boundary between the 2 clusters of cells. The distribution patterns observed were not
strikingly different for 4n[harr ]2n and 2n[harr ]2n chimaeras, each showing some transgenic positive cell
contribution in all 3 identifiable developmental lineages. It was notable, however, that in all 4n[harr ]2n
blastocysts at least some 4n cells were located adjacent to the blastocyst cavity. Such a consistent pattern
was not evident in 2n[harr ]2n chimaeras. This study has demonstrated the value of 3-D reconstructions for the
analysis of spatial relationships of 2 cell populations in chimaeric mouse blastocysts.
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