scholarly journals Lineage segregation, pluripotency and X-chromosome inactivation in the pig pre-gastrulation embryo

2018 ◽  
Author(s):  
Priscila Ramos-Ibeas ◽  
Fei Sang ◽  
Qifan Zhu ◽  
Walfred W.C. Tang ◽  
Sarah Withey ◽  
...  
Keyword(s):  
X Chromosome ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Stem Cell Differentiation ◽  
Human Embryos ◽  
Early Human Development ◽  
Mammalian Embryogenesis ◽  
Primed State ◽  
Post Implantation ◽  
Early Human

AbstractHigh-resolution molecular programs delineating the cellular foundations of mammalian embryogenesis have emerged recently. Similar analysis of human embryos is limited to pre-implantation stages, since early post-implantation embryos are inaccessible. Notwithstanding, we previously suggested conserved principles of pig and human early development. For further insight on pluripotent states and lineage delineation, we analysed pig embryos at single cell resolution. Here we show progressive segregation of inner cell mass and trophectoderm in early blastocysts, and then of epiblast and hypoblast in late blastocysts. We detected distinct pluripotent states, first as a short ‘naïve’ state followed by a protracted primed state. Dosage compensation with respect to the X-chromosome in females is attained via X-inactivation in late epiblasts. Detailed human-pig comparison is a basis towards comprehending early human development and a foundation for further studies of human pluripotent stem cell differentiation in pig interspecies chimeras.

scholarly journals BMP-treated human embryonic stem cells transcriptionally resemble amnion cells in the monkey embryo

Biology Open ◽  
2021 ◽  
Author(s):  
Sapna Chhabra ◽  
Aryeh Warmflash
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Single Cell ◽  
Human Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Types ◽  
Human Embryos ◽  
Early Human

Human embryonic stem cells (hESCs) possess an immense potential to generate clinically relevant cell types and unveil mechanisms underlying early human development. However, using hESCs for discovery or translation requires accurately identifying differentiated cell types through comparison with their in vivo counterparts. Here, we set out to determine the identity of much debated BMP-treated hESCs by comparing their transcriptome to recently published single cell transcriptomic data from early human embryos (Xiang et al., 2019). Our analyses reveal several discrepancies in the published human embryo dataset, including misclassification of putative amnion, intermediate and inner cell mass cells. These misclassifications primarily resulted from similarities in pseudogene expression, highlighting the need to carefully consider gene lists when making comparisons between cell types. In the absence of a relevant human dataset, we utilized the recently published single cell transcriptome of the early post implantation monkey embryo to discern the identity of BMP-treated hESCs. Our results suggest that BMP-treated hESCs are transcriptionally more similar to amnion cells than trophectoderm cells in the monkey embryo. Together with prior studies, this result indicates that hESCs possess a unique ability to form mature trophectoderm subtypes via an amnion-like transcriptional state.

scholarly journals BMP-treated human embryonic stem cells transcriptionally resemble amnion cells in the monkey embryo

2021 ◽  
Author(s):  
Sapna Chhabra ◽  
Aryeh Warmflash
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Single Cell ◽  
Human Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Types ◽  
Human Embryos ◽  
Early Human

AbstractHuman embryonic stem cells (hESCs) possess an immense potential to generate clinically relevant cell types and unveil mechanisms underlying early human development. However, using hESCs for discovery or translation requires accurately identifying differentiated cell types through comparison with their in vivo counterparts. Here, we set out to determine the identity of much debated BMP-treated hESCs by comparing their transcriptome to the recently published single cell transcriptomes of early human embryos in the study Xiang et al 2019. Our analyses reveal several discrepancies in the published human embryo dataset, including misclassification of putative amnion, intermediate and inner cell mass cells. These misclassifications primarily resulted from similarities in pseudogene expression, highlighting the need to carefully consider gene lists when making comparisons between cell types. In the absence of a relevant human dataset, we utilized the recently published single cell transcriptome of the early post implantation monkey embryo to discern the identity of BMP-treated hESCs. Our results suggest that BMP-treated hESCs are transcriptionally more similar to amnion cells than trophectoderm cells in the monkey embryo. Together with prior studies, this result indicates that hESCs possess a unique ability to form mature trophectoderm subtypes via an amnion-like transcriptional state.

scholarly journals The selection arena in early human blastocysts resolves the pluripotent inner cell mass

2018 ◽  
Author(s):  
Manvendra Singh ◽  
Thomas J Widmann ◽  
Vikas Bansal ◽  
Jose L Cortes ◽  
Gerald G Schumann ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Major Gene ◽  
Cell Mass ◽  
Endogenous Retrovirus ◽  
Early Embryo ◽  
Human Embryos ◽  
Pluripotent Cells ◽  
Early Human

Is the human early embryo unique in lacking an inner cell mass (ICM) and having parallel rather than step-wise development? Here we reanalyse single-cell transcriptomic data and stain human embryos in situ to reveal both classical step-wise development and the missing ICM, a transcriptomic homolog of macaque ICM, that differentiates to epiblast and primitive endoderm. This apparent classicism obscures numerous features that render our blastocyst phylogenetically distinct: unlike mice, human epiblast has hallmarks of self-renewal and we have abundant, previously unrecognized, blastocyst non-committed cells (NCCs), part of an apoptosis-mediated quality control/purging process. Comparative transcriptomics further reveals the transcriptomes of the pluripotent cells to be especially fast evolving, rendering all primate embryos unique. Rapid transcriptome turnover is in large part owing to endogenous retrovirus H (ERVH) activity, ERVH being associated with recent major gene expression gain and loss events of pluripotency-associated genes. Each species is characterised by the ERVHs that are active and the neighbour genes whose expression are in turn modulated. The current portfolio of naive cultures, putative in vitro mimics of pluripotent cells, are both developmentally and phylogenetically "confused" in part owing to a lack of HERVH expression.

scholarly journals Opening the black box: Stem cell–based modeling of human post-implantation development

2018 ◽  
Vol 218 (2) ◽  
pp. 410-421 ◽  
Author(s):  
Kenichiro Taniguchi ◽  
Idse Heemskerk ◽  
Deborah L. Gumucio
Keyword(s):  
Stem Cell ◽  
Human Development ◽  
Experimental Models ◽  
Uterine Wall ◽  
Black Box ◽  
Cellular Mechanisms ◽  
Early Human Development ◽  
Post Implantation ◽  
Early Human

Proper development of the human embryo following its implantation into the uterine wall is critical for the successful continuation of pregnancy. However, the complex cellular and molecular changes that occur during this post-implantation period of human development are not amenable to study in vivo. Recently, several new embryo-like human pluripotent stem cell (hPSC)–based platforms have emerged, which are beginning to illuminate the current black box state of early human post-implantation biology. In this review, we will discuss how these experimental models are carving a way for understanding novel molecular and cellular mechanisms during early human development.

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

2019 ◽  
Author(s):  
Yi Zheng ◽  
Jianping Fu
Keyword(s):  
Stem Cells ◽  
Primordial Germ Cells ◽  
Primitive Streak ◽  
Human Embryos ◽  
Vitro Fertilization ◽  
Human Embryology ◽  
Post Implantation ◽  
Advance Knowledge ◽  
Early Human

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.

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

Development ◽  
1989 ◽  
Vol 107 (3) ◽  
pp. 597-604 ◽  
Author(s):  
K. Hardy ◽  
A.H. Handyside ◽  
R.M. Winston
Keyword(s):  
Cell Death ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Cell Numbers ◽  
Human Blastocyst ◽  
Development In Vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/− 8.1 cells, which increased to 84.4 +/− 5.7 and 125.5 +/− 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/− 6.0 and 40.3 +/− 5.0, respectively) and then doubled on day 7 (80.6 +/− 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/− 4.0 and 41.9 +/− 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/− 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.

2. Embryonic stem cells

2021 ◽  
pp. 21-37
Author(s):  
Jonathan Slack
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Practical Importance ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Human Embryos ◽  
Mouse Es Cells ◽  
Stage Embryo

‘Embryonic stem cells’ focuses on embryonic stem (ES) cells, which are grown in tissue culture from the inner cell mass of a mammalian blastocyst-stage embryo. Human ES cells offer a potential route to making the kinds of cells needed for cell therapy. ES cells were originally prepared from mouse embryos. Although somewhat different, cells grown from inner cell masses of human embryos share many properties with mouse ES cells, such as being able to grow without limit and to generate differentiated cell types. Mouse ES cells have so far been of greater practical importance than those of humans because they have enabled a substantial research industry based on the creation of genetically modified mice.

From fibroblasts and stem cells: implications for cell therapies and somatic cloning

2005 ◽  
Vol 17 (2) ◽  
pp. 125 ◽  
Author(s):  
Wilfried A. Kues ◽  
Joseph W. Carnwath ◽  
Heiner Niemann
Keyword(s):  
Stem Cells ◽  
Adult Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Human Embryos ◽  
Somatic Stem Cells ◽  
Primary Fibroblast ◽  
Cell Therapies

Pluripotent embryonic stem cells (ESCs) from the inner cell mass of early murine and human embryos exhibit extensive self-renewal in culture and maintain their ability to differentiate into all cell lineages. These features make ESCs a suitable candidate for cell-replacement therapy. However, the use of early embryos has provoked considerable public debate based on ethical considerations. From this standpoint, stem cells derived from adult tissues are a more easily accepted alternative. Recent results suggest that adult stem cells have a broader range of potency than imagined initially. Although some claims have been called into question by the discovery that fusion between the stem cells and differentiated cells can occur spontaneously, in other cases somatic stem cells have been induced to commit to various lineages by the extra- or intracellular environment. Recent data from our laboratory suggest that changes in culture conditions can expand a subpopulation of cells with a pluripotent phenotype from primary fibroblast cultures. The present paper critically reviews recent data on the potency of somatic stem cells, methods to modify the potency of somatic cells and implications for cell-based therapies.

6 ANEUPLOIDY TOLERANCE IN RHESUS MACAQUE PRE-IMPLANTATION EMBRYOS VIA MICRONUCLEI FORMATION, CELLULAR FRAGMENTATION, AND BLASTOMERE EXCLUSION

2017 ◽  
Vol 29 (1) ◽  
pp. 110 ◽  
Author(s):  
B. L. Daughtry ◽  
J. L. Rosenkrantz ◽  
N. Lazar ◽  
N. Redmayne ◽  
K. A. Nevonen ◽  
...  
Keyword(s):  
Rhesus Macaque ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Chromosomal Mosaicism ◽  
Human Embryos ◽  
Cleavage Stage ◽  
Chromosomal Breakage

A primary contributor to in vitro fertilization (IVF) failure is the presence of unbalanced chromosomes in pre-implantation embryos. Previous array-based and next-generation sequencing (NGS) studies determined that ~50 to 80% of human embryos are aneuploid at the cleavage stage. During early mitotic divisions, many human embryos also sequester mis-segregated chromosomes into micronuclei and concurrently undergo cellular fragmentation. We hypothesised that cellular fragmentation represents a response to mis-segregated chromosomes that are encapsulated into micronuclei. Here, we utilised the rhesus macaque pre-implantation embryo as a model to study human embryonic aneuploidy using a combination of EevaTM time-lapse imaging for evaluating cell divisions, single-cell/-fragment DNA-Sequencing (DNA-Seq), and confocal microscopy of nuclear structures. Results from our time-lapse image analysis demonstrated that there are considerable differences in the timing of the first and third mitotic divisions between rhesus blastocysts and those that arrested before this stage in development (P < 0.01; ANOVA). By examining the chromosome content of each blastomere from cleavage stage embryos via DNA-Seq, we determined that rhesus embryos have an aneuploidy frequency up to ~62% (N = 26) with several embryos exhibiting chromosomal mosaicism between blastomeres (N = 6). Certain blastomeres also exhibited reciprocal whole chromosomal gains or losses, indicating that these embryos had undergone mitotic non-disjunction early in development. In addition, findings of reciprocal sub-chromosomal deletions/duplications among blastomeres suggest that chromosomal breakage had occurred in some embryos as well. Embryo immunostaining for the nuclear envelope protein, LAMIN-B1, demonstrated that fragmented cleavage-stage rhesus embryos often contain micronuclei and that cellular fragments can enclose DNA. Our DNA-Seq analysis confirmed that cellular fragments might encapsulate whole and/or partial chromosomes lost from blastomeres. When embryos were immunostained with gamma-H2AX, a marker of chromatin fragility, we observed distinct foci solely in micronuclei and DNA-containing cellular fragments. This suggests that micronuclei may be ejected from blastomeres through the process of cellular fragmentation and, once sequestered, these mis-segregated chromosomes become highly unstable and undergo DNA degradation. Finally, we also observed that ~10% of embryos prevented cellular fragments or large blastomeres from incorporating into the inner cell mass or trophectoderm at the blastocyst stage (n = 5). Upon confocal imaging, multiple nuclei and intense gamma-H2AX foci were found in a large unincorporated blastomere in one of the blastocysts. Altogether, our findings demonstrate that the rhesus embryo responds to segregation errors by eliminating chromosome-containing micronuclei via cellular fragmentation and/or selecting against aneuploid blastomeres that fail to divide during pre-implantation development with significant implications for human IVF.

102. THE EFFECT OF INSULIN ON EMBRYONIC STEM CELL PROGENITOR CELLS IN THE MOUSE BLASTOCYST

2009 ◽  
Vol 21 (9) ◽  
pp. 21
Author(s):  
J. M. Campbell ◽  
I. Vassiliev ◽  
M. B. Nottle ◽  
M. Lane
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Number ◽  
Human Embryos ◽  
Cell Stage ◽  
Stage Of Development

Human ESCs are produced from embryos donated at the mid-stage of pre-implantation development. This cryostorage reduced viability. However, it has been shown that this can be improved by the addition of growth factors to culture medium. The aim of the present study was to examine whether the addition of insulin to embryo culture medium from the 8-cell stage of development increases the number of ES cell progenitor cells in the epiblast in a mouse model. In vivo produced mouse zygotes (C57Bl6 strain) were cultured in G1 medium for 48h to the 8-cell stage, followed by culture in G2 supplemented with insulin (0, 0.17, 1.7 and 1700pM) for 68h, at 37 o C , in 5% O2, 6%CO2, 89% N2 . The number of cells in the inner cell mass (ICM) and epiblast was determined by immunohistochemical staining for Oct4 and Nanog. ICM cells express Oct4, epiblast cells express both Oct4 and Nanog. The addition of insulin at the concentrations examined did not increase the ICM. However, at 1.7pM insulin increased the number of epiblast cells (6.6±0.5 cells vs 4.1±0.5, P=0.001) in the ICM, which increased the proportion of the ICM that was epiblast (38.9±3.7% compared to 25.8±3.4% in the control P=0.01). This indicates that the increase in the epiblast is brought about by a shift in cell fate as opposed to an increase in cell division. The effect of insulin on the proportion of cells in the epiblast was investigated using inhibitors of phosphoinositide3-kinase (PI3K) (LY294002, 50µM); one of insulin's main second messengers, and p53 (pifithrin-α, 30µg/ml); a pro-apoptotic protein inactivated by PI3K. Inhibition of PI3K eliminated the increase caused by insulin (4.5±0.3 cells versus 2.2±0.3 cells, P<0.001), while inhibition of p53 increased the epiblast cell number compared to the control (7.1±0.8 and 4.1±0.7 respectively P=0.001). This study shows that insulin increases epiblast cell number through the activation of PI3K and the inhibition of p53, and may be a strategy for improving ESC isolation from human embryos.

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