scholarly journals Protocol for controlled modeling of human epiblast and amnion development using stem cells

Author(s):  
Yi Zheng ◽  
Jianping Fu

Abstract Due to the inaccessibility of post-implantation human embryos and the restriction on in-vitro fertilization (IVF) embryos cultured beyond 14 days, the knowledge of early post-implantation human embryogenesis remains extremely limited. Recently, we have developed a microfluidic in-vitro platform, based on human pluripotent stem cells (hPSCs), which is capable of recapitulating several key developmental landmarks of early human post-implantation embryonic development, including lumenogenesis of the epiblast (EPI), amniogenesis, and specification of primordial germ cells (PGCs) and of primitive streak (PS) cells. Given its controllability and reproducibility, the microfluidic platform provides a powerful experimental platform to advance knowledge of human embryology and reproduction. This protocol describes the preparation of the microfluidic device and its implementation for modeling human post-implantation epiblast and amnion development using hPSCs.

2006 ◽  
Vol 55 (6) ◽  
Author(s):  
Jacques Suaudeau

Un’ampia polemica si è sviluppata attorno alle cellule staminali: alcuni rivendicano una totale libertà di reperire le cellule staminali embrionali umane (hES) dagli embrioni provenienti dalla fecondazione in vitro o dal trasferimento nucleare (clonazione terapeutica), altri insistono sull’impiego di cellule staminali somatiche e di cellule del sangue del cordone ombelicale (UCB). Il fulcro di questa polemica è etica: infatti, il reperimento del primo tipo di cellule, in quanto richiede il sacrificio programmato di embrioni umani, solleva, a differenza del secondo tipo, questioni etiche. Molti tra coloro che reputano la ricerca sulle cellule hES eticamente accettabile ritengono che gli embrioni umani, prima dell’impianto uterino, non possono essere considerati ancora organismi individuali. Essi fondano la loro tesi su due considerazioni: l’elevata percentuale di perdita naturale di embrioni precoci e il verificarsi della gemellarità monozigotica. Recenti studi hanno, tuttavia, messo in crisi simile tesi, mostrando che l’embrione dei mammiferi funziona come unità biologica sia a livello citologico (gap junctions, tight junctions, compaction) sia a livello genetico (zigotic gene activation). Altri si dichiarano a favore della ricerca sulle cellule ES, giustificandola con la seguente argomentazione: un “essere” umano non può essere riconosciuto come tale dal punto di vista antropologico, finché non abbia raggiunto un elevato grado di “umanizzazione”. Tuttavia, l’errore di simile “prospettiva dello sviluppo” proviene dalla mancanza di un’attenta riflessione sul piano ontologico. Altri, pur riconoscendo che l’embrione umano, in quanto persona potenziale, merita grande rispetto, giustificano la distruzione di embrioni umani per reperire le cellule ES, ricorrendo all’argomento del “fine buono”. In questo caso, il principio morale intangibile che deve essere applicato è quello per il quale il fine non giustifica i mezzi. Ne deriva che la distruzione di embrioni umani per ottenere cellule ES è una eliminazione diretta e deliberata di un essere umano innocente, non giustificabile attraverso alcun argomento. Va, infine, posto il seguente quesito: è lecito usare linee di hES fornite da altri ricercatori o disponibili sul mercato? Tuttavia, una simile utilizzazione rientra nella categoria della cooperazione moralmente illecita ad atti ingiusti, sia in termini di cooperazione materiale immediata sia in termini di cooperazione formale. D’altra parte, la proposta di reperire linee di cellule ES da un singolo blastomero, ottenuto attraverso la biopsia di un embrione, sarebbe, senza dubbio, più rispettosa della vita umana nascente, ma comporterebbe altri problemi etici: essa, infatti, implicherebbe il ricorso alla fecondazione in vitro ed esporrebbe l’embrione a un rischio non indifferente. Quanto poi alla “riprogrammazione” di cellule somatiche a livello di cellule ES, pur essendo eticamente lecita, resta, allo stato corrente, un’ipotesi teorica. Il realismo pratico ed il rispetto della vita umana nascente ci spingono, dunque, a considerare come primaria la ricerca sulle cellule staminali adulte e sulle cellule del sangue del cordone ombelicale, che, nel campo della medicina rigenerativa, ha già dato risultati incoraggianti. ---------- A wide polemic has developed around stem cells: some claim a full freedom for deriving human embryonic stem cells (hES) from embryos coming from in vitro fertilization or from nuclear transfer (therapeutic cloning), others insist on the interest of somatic stem cells or stem cells from umbilical cord blood (UCB). The core of this polemic is ethical: in fact, getting the first type of cells, because of it needs the programmed sacrifice of human embryos, raise, unlike the second type, ethical questions. Many among those who think hES research as ethically acceptable consider that human embryos before implantation cannot be considered as individual organisms. They support their opinion on two considerations: the elevated percentage of natural loss of early embryos and the occurrence of monozygotic twinning. But, recent studies have removed a lot of their substance from these arguments, showing in particular that the mammalian embryo works as a biological unity at the cytological level (gap junctions, tight junctions, compaction) as well as at the genetic level (zigotic gene activation). Others pronounced themselves in favor of hES research, with the argument that a biological human “being” cannot be recognized as such from an anthropological standpoint until he has reached a consistent level of “humanization”. But, the error of this “developmental perspective” comes from its ignorance of a careful ontological reflection. Others, although they do recognize that the human embryo, as a possible person, deserves great respect, justify the destruction of human embryos human to get ES cells with the argument of the “good end”. In this case, the intangible moral principle that must be applied is that the goal doesn’t justify the means. It follows that the destruction of human embryos to get hES cells is a direct and deliberate elimination of an innocent human being that no argument can justify. Another question is: is it permissible to use hES cell lines from other researchers or available on the market? But, this use enters into the category of the illegitimate cooperation in evil, both in terms of immediate material cooperation, and in terms of formal cooperation. On the other hand, the proposal to derive hES cell lines from a single blastomere separated mechanically from an embryo while leaving alive this embryo would be more respectful of early human life, but brings in other ethical problems: it implicates the practice of in vitro fertilization in vitro, and exposes the embryo to a substantial risk. Regarding the “reprogramming” of somatic cells to the level of ES cells, although it is ethically permissible, is now more a theoretical hypothesis. Practical realism and respect of early human life invite therefore to give prime attention to research on adult stem cells and on stem cells from umbilical cord blood, that, in the field of the regenerative medicine, have given encouraging results.


Author(s):  
D. Gareth Jones

The advent of in vitro fertilization (IVF) marked a watershed in the scientific understanding of the human embryo. This, in turn, led to a renaissance of human embryology, accompanied by the ability to manipulate the human embryo in the laboratory. This ability has resulted in yet further developments: refinements of IVF itself, preimplantation genetic diagnosis, the derivation and extraction of embryonic stem cells, and even various forms of cloning. There are immense social and scientific pressures to utilize the artificial reproductive technologies in ways that have little or no connection with overcoming infertility. As the original clinical goals of IVF have undergone transformation ethical concerns have escalated, so much so that they are condemned by some as illustrations of ‘playing God’, while any babies born via some of these procedures are labelled as ‘designer babies’. Both terms reflect the fear and repugnance felt by some at the interference with the earliest stages of human life by the artificial reproductive technologies. It is at these points that bioethical analyses have an important contribution to make.


2020 ◽  
Vol 26 (6) ◽  
pp. 779-798
Author(s):  
Ana M Pereira Daoud ◽  
Mina Popovic ◽  
Wybo J Dondorp ◽  
Marc Trani Bustos ◽  
Annelien L Bredenoord ◽  
...  

Abstract BACKGROUND Studying the human peri-implantation period remains hindered by the limited accessibility of the in vivo environment and scarcity of research material. As such, continuing efforts have been directed towards developing embryo-like structures (ELS) from pluripotent stem cells (PSCs) that recapitulate aspects of embryogenesis in vitro. While the creation of such models offers immense potential for studying fundamental processes in both pre- and early post-implantation development, it also proves ethically contentious due to wide-ranging views on the moral and legal reverence due to human embryos. Lack of clarity on how to qualify and regulate research with ELS thus presents a challenge in that it may either limit this new field of research without valid grounds or allow it to develop without policies that reflect justified ethical concerns. OBJECTIVE AND RATIONALE The aim of this article is to provide a comprehensive overview of the existing scientific approaches to generate ELS from mouse and human PSCs, as well as discuss future strategies towards innovation in the context of human development. Concurrently, we aim to set the agenda for the ethical and policy issues surrounding research on human ELS. SEARCH METHODS The PubMed database was used to search peer-reviewed articles and reviews using the following terms: ‘stem cells’, ‘pluripotency’, ‘implantation’, ‘preimplantation’, ‘post-implantation’, ‘blastocyst’, ‘embryoid bodies’, ‘synthetic embryos’, ‘embryo models’, ‘self-assembly’, ‘human embryo-like structures’, ‘artificial embryos’ in combination with other keywords related to the subject area. The PubMed and Web of Science databases were also used to systematically search publications on the ethics of ELS and human embryo research by using the aforementioned keywords in combination with ‘ethics’, ‘law’, ‘regulation’ and equivalent terms. All relevant publications until December 2019 were critically evaluated and discussed. OUTCOMES In vitro systems provide a promising way forward for uncovering early human development. Current platforms utilize PSCs in both two- and three-dimensional settings to mimic various early developmental stages, including epiblast, trophoblast and amniotic cavity formation, in addition to axis development and gastrulation. Nevertheless, much hinges on the term ‘embryo-like’. Extension of traditional embryo frameworks to research with ELS reveals that (i) current embryo definitions require reconsideration, (ii) cellular convertibility challenges the attribution of moral standing on the basis of ‘active potentiality’ and (iii) meaningful application of embryo protective directives will require rethinking of the 14-day culture limit and moral weight attributed to (non-)viability. Many conceptual and normative (dis)similarities between ELS and embryos thus remain to be thoroughly elucidated. WIDER IMPLICATIONS Modelling embryogenesis holds vast potential for both human developmental biology and understanding various etiologies associated with infertility. To date, ELS have been shown to recapitulate several aspects of peri-implantation development, but critically, cannot develop into a fetus. Yet, concurrent to scientific innovation, considering the extent to which the use of ELS may raise moral concerns typical of human embryo research remains paramount. This will be crucial for harnessing the potential of ELS as a valuable research tool, whilst remaining within a robust moral and legal framework of professionally acceptable practices.


2010 ◽  
Vol 38 (2) ◽  
pp. 204-211 ◽  
Author(s):  
Lawrence Goldstein

Lost at times in the heat of debate about stem cell research, or any controversial advanced technology, is the need for precision in debate and discussion. The details matter a great deal, ranging from the need to use words that have precise definitions, to accurately quote colleagues and adversaries, and to cite scientific and medical results in a way that reflects the quality, rigor, and reliability of the work at issue. Regrettably, considerable inaccuracy has found its way into the debates about stem cells, on all sides, with consequent fogging of the issues.A key detail that is often overlooked in the debates about the uses of human embryonic stem cells in research comes from the nature of in vitro fertilization (IVF) treatment for infertility. Specifically, there are many frozen human embryos (more precisely called blastocysts) that are in excess of reproductive needs of the couple who generated them, and that must be either frozen indefinitely, donated to another couple, or destroyed.


2006 ◽  
Vol 68 (1) ◽  
Author(s):  
Larry J. Pittman

Many Americans die each day from diseases affecting the heart, liver, kidneys, brain and a whole host of other bodily organs. Scientific researchers are constantly trying to develop new treatments for such medical conditions. Presently, the research community is working hard to develop medical treatments using stem cells from human embryos. That process involves extracting stem cells from either excess embryos that are no longer needed for in vitro fertilization or from embryos that are created through therapeutic cloning. At the blastocyst stage, about five days after the beginning of an embryo, researchers extract stem cells from the embryo and place them in a petri dish where the cells divide to produce a line of millions of stem cells. These stem cells are undifferentiated, meaning that they are still capable of transforming themselves into many different types of cells that exist in the human body. The hope is that physicians and other medical personnel will one day be able to inject these stem cells into a patient’s diseased heart, kidney, brain, liver, spinal cord or other organ, and the stem cells willtransform themselves into the same type of cells that comprise the host organ. The expectation is that the stem cells will repair the patient’s heart or other organs by curing diseases and otherwise improving the patient’s medical condition and life expectancy.


2008 ◽  
Vol 20 (1) ◽  
pp. 97
Author(s):  
Y. H. Hao ◽  
D. Wax ◽  
Z. S. Zhong ◽  
C. N. Murphy ◽  
L. Spate ◽  
...  

Although transgenic animals have been successfully cloned, the process is still inefficient. One of the limitations is the use of somatic donor cells that have a limited lifespan. If a genetic modification is made, the selection process must be initiated and completed rapidly or the cells will undergo senescence. Identification of a stem cell that would proliferate rapidly and not undergo senescence would prove to be very valuable. Here we report attempts at cloning by using porcine skin-derived sphere stem cells to determine if they are a suitable donor cell type. Skin-derived stem cells were isolated from fetal skin and express the neural progenitor marker NES, as well as genes that may be critical for pluripotency such as POU5F1 and STAT3. The skin-derived stem cells proliferate rapidly in vitro and retain a normal karyotype after long-term culture. In the present study, skin-derived stem cells were cultured and frozen in liquid nitrogen from passage 1 to passage 8. To investigate the developmental potential of the skin-derived stem cells, we performed nuclear transfer (NT) and compared their preimplantation developmental efficiency to that of the embryos derived from in vitro fertilization (IVF). Cumulus–oocyte complexes (COCs) were aspirated from antral follicles of ovaries from prepubertal gilts. Approximately, groups of 50-70 COCs were matured in vitro in 500 µL TCM-199 per culture well for 40–44 h at 38.5�C, in a humidified atmosphere of 5% CO2 in air. The donor cells were thawed and cultured one day before NT; skin-derived stem cells were pipetted vigorously in PBS-EDTA to isolate individual cells. For IVF, cryopreserved ejaculated spermatozoa were thawed and washed and then resuspended with fertilization medium (mTBM). The MII oocytes were co-incubated with sperm for 6 h, and then transferred to PZM3 and cultured. For NT and IVF, respectively, the percent cleavage at 48 h in PZM3 was 64.9 � 8.2% (169/208) and 62.1 � 3.1% (94/184) (P > 0.05), the percent blastocysts after 6 days was 21.5 � 5.8% (53/208) and 25.2 � 3.4% (46/184) (P > 0.05), and the number of nuclei per blastocyst was 28.5 � 1.9 (NT, maximum was 58) and 16.8 � 4.0 (IVF, maximum was 31) (P < 0.05). To determine development post-implantation, some cloned embryos were cultured in PZM3 for 15.5 h and an average of 112 cloned embryos were transferred to the oviducts of four naturally cycling gilts on Day 0–1 of standing estrus. Three of the animals were pregnant: one of them farrowed two male piglets on August 14th, with the other two due on September 8th and 9th. Future studies will involve performing NT and ET on skin-derived stem cells from a higher passage number to determine if they would be suitable for genetic modification prior to NT.


2014 ◽  
Vol 8 (1) ◽  
pp. 43-52 ◽  
Author(s):  
Mathana Amaris Fiona Sivaraman ◽  
Siti Nurani Mohd Noor

Abstract Background: The use of embryos in embryonic stem cell research (ESCR) has elicited ethical controversies as it entails the destruction of 5-day old human embryos to harvest stem cells. Objective: To explore the ethical positions of Islam, Buddhism, Hinduism, and Catholicism concerning the use of (1) left-over embryos from in vitro fertilization (IVF) also known as ‘surplus’ embryos and (2) ‘research embryos’ which are created by scientists to conduct research using embryonic stem cells. Methods: The opinions of religious leaders of Buddhist, Hindu, and Catholic faiths in Malaysia pertaining to ESCR were examined via in-depth, semi-structured interviews while Islamic responses are collected from local writings related to the derivation of fatwa on this issue. Participants’ responses on the ethics of human stem cell research are presented as a reflection of various scriptural texts of these four religions. These are presented and supported with the help of international bioethics literature and focus on the use of ‘surplus’ embryos and ‘research’ embryos. Results: Islamic ethics deviate from Hindu and Buddhist teachings regarding saving of research embryos that have been created specifically for research and are considered as human lives only after 120 days fertilization. Hindu and Buddhists also underscore the sanctity of human life, but give priority to the alleviation of suffering in living adult humans. They generally encourage ESCR. Research is a knowledge-seeking endeavor considered noble by Islam. This is also a concept within Hindu and Buddhist philosophy; in particular, when potentially beneficial research goals are the basis. Catholicism also emphasizes sanctity of human life, but stresses also the inviolability of embryos from the moment of conception. Conclusion: Embryonic stem cell research is permissible and encouraged according to Hindu and Buddhist perspectives in view of the potential benefits of such research to society, with some reservations. This is similar to Islamic views on the ethics of ESCR. However, Catholicism differs from all the other three religions; it appears to discourage research in this field because of the likely violation of a sacred principle in Catholic teachings.


2013 ◽  
Vol 16 (3) ◽  
pp. 593-599 ◽  
Author(s):  
J. Opiela ◽  
M. Samiec

Abstract The efficiency of somatic cell cloning (somatic cell nuclear transfer; SCNT) as well as in vitro fertilization/in vitro embryo production (IVF/IVP) in mammals stay at relatively same level for over a decade. Despite plenty of different approaches none satisfactory break-through took place. In this article, we briefly summarize the implementation of mesenchymal stem cells (MSCs) for experimental embryology. The advantages of using MSCs as nuclear donors in somatic cell cloning and in vitro embryo culture are described. The description of results obtained with these cells in mammalian embryo genomic engineering is presented.


Cell Research ◽  
2021 ◽  
Author(s):  
Xiaoxiao Wang ◽  
Yunlong Xiang ◽  
Yang Yu ◽  
Ran Wang ◽  
Yu Zhang ◽  
...  

AbstractThe pluripotency of mammalian early and late epiblast could be recapitulated by naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. However, these two states of pluripotency may not be sufficient to reflect the full complexity and developmental potency of the epiblast during mammalian early development. Here we report the establishment of self-renewing formative pluripotent stem cells (fPSCs) which manifest features of epiblast cells poised for gastrulation. fPSCs can be established from different mouse ESCs, pre-/early-gastrula epiblasts and induced PSCs. Similar to pre-/early-gastrula epiblasts, fPSCs show the transcriptomic features of formative pluripotency, which are distinct from naïve ESCs and primed EpiSCs. fPSCs show the unique epigenetic states of E6.5 epiblast, including the super-bivalency of a large set of developmental genes. Just like epiblast cells immediately before gastrulation, fPSCs can efficiently differentiate into three germ layers and primordial germ cells (PGCs) in vitro. Thus, fPSCs highlight the feasibility of using PSCs to explore the development of mammalian epiblast.


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