The current idea is that cells that comprise the ICM have lost the ability to generate TE cells and vice versa and are considered pluripotent instead of totipotent. As development progresses through peri- and post-implantation stages, the cells of the ICM continue to differentiate into the embryonic part ectoderm, mesoderm, endoderm and germline , and contribute as well to the extra-embryonic part of the embryo placenta, amnion, chorion and umbilical cord.
Interestingly, despite the shared traits of self-renewal and pluripotency, stem cells can differ vastly in transcriptional profile, epigenetic profile, morphology and culture requirements. It is becoming clear that the differences observed between ESC types is the result of differing developmental stages of the ICM precursor cells that give rise to the stem cells. In mice, different types of self-renewing and pluripotent stem cell populations can be generated in culture from post-implantation embryonic day E 5.
Despite the shared traits of self-renewal and pluripotency between mouse ESCs mESCs and mEpiSCs, including transcriptional profile, epigenetic profile, morphology and culture conditions, there are vast differences between these two different stem cell types. It has thus become clear that pluripotency is not a fixed state, but exists in at least two distinct forms Nichols and Smith , Although human embryonic stem cells hESCs are derived from preimplantation embryos Thomson et al.
Therefore, speculation arose that the human ICM progresses in vitro through an epiblast-like state before hESCs emerge in culture. Detailed analysis of the morphological changes of the human ICM during the hESC derivation process indeed revealed the formation of a transient structure that emerged 2—3 days after blastocyst plating Fig.
Derivation of human embryonic stem cells hESCs. During this period, a transient structure emerges in culture, the post-inner cell mass intermediate PICMI. The newly derived hESC line can undergo unlimited propagation. Further investigations on the characteristics of the PICMI, an obligatory hESC-precursor, will help to get a more clear understanding of the human pluripotent states. Optimizing culture conditions, by inhibition or activation of key signalling pathways important for pluripotency, could help in increasing this efficiency.
Small molecules, both agonists and antagonists of specific signalling pathways, have been shown to influence lineage fate decisions during early embryo development in mice and humans Nichols and Smith, ; Yamanaka et al. In humans, the culture of 8-cell stage Day 3 embryos up to Day 6, to the blastocyst stage, in 2i medium significantly increased the number of NANOG-positive cells in the ICMs of good quality blastocysts, however, it did not affect the number of GATA6- Gata-binding protein 6 positive hypoblast cells Van der Jeught et al.
B After 3 days of culture in medium containing 2i, the good quality blastocyst embryos showed a significantly increased number of NANOG-positive epiblast cells green in the inner call mass ICM , while the number of GATA6-positive hypoblast cells red was unaffected. Treatment of human 8-cell stage Day 3 embryos with SB for 3 more days, to the blastocyst stage, significantly increased the number of NANOG-positive epiblast cells in the human ICMs in both good and poor quality blastocysts, while the number of GATA6-positive hypoblast cells remained unchanged Van der Jeught et al.
This suggests that other signalling pathways may regulate hypoblast formation during human preimplantation development. It was tested whether the increase in the number of epiblast cells in the blastocyst embryo increased the derivation efficiency of hESCs.
Both culture with 2i and SB of 8-cell stage Day 3 human embryos until blastocyst stage Day 6 allowed PICMI formation and subsequent hESC derivation, however, at same efficiency as embryos that did not receive such treatment Van der Jeught et al.
Even though an enhanced number of epiblast cells did not lead to a higher derivation efficiency of novel hESC lines using a limited number of available embryos, an upgraded combination of small molecules should be capable of enhancing PICMI formation rates as well as subsequent hESC derivation. Fan et al. This combination increased the derivation efficiency of hESCs from poor quality embryos Fan et al. Similarly, it has been shown that treatment with insulin increases the epiblast cell number in mice, leading to a higher mESC derivation efficiency Campbell et al.
The PICMI is an epithelialized structure that can grow either flat or ball like and is surrounded by a layer of endoderm separated by a basement membrane. The PICMI displays a unique transcriptional profile of a mix of early and late epiblast markers as well as early germ cell markers O'Leary et al.
Different states of pluripotency in hESCs. It is not known yet whether individual PICMI cells fluctuate between early and late epiblast stages, are co-expressing both types of marker genes, or if the PICMI cells contain a mixed population of cells at both early and late epiblast stages. O'Leary et al. However, the analysis of X chromosome inactivation status after subsequent hESC derivation revealed that some lines showed reactivation of the inactivated X chromosome XaXa , which was attributed to the hypoxic conditions the hESCs were derived in, whereas other lines showed a mixed phenotype with and without H3K27me3 Xi-indicative nuclear spots O'Leary et al.
During assisted reproductive technology ART , the patient undergoes controlled hormonal ovarian hyperstimulation, aimed towards retrieving a number of oocytes that is high enough to create a cohort of embryos, from which the ones with highest implantation potential are selected to be transferred to the patient directly or for cryopreservation to be transferred at a later stage Alpha Scientists in Reproductive and Embryology, ; Willis et al.
Moreover, it was reported that blastocysts originating from PQEs are as efficient in generating hESCs as the ones originating from good quality embryos Strom et al. This could be explained by the presence of good quality ICMs in blastocysts with compromised implantation potential due to an inferior TE quality della Ragione et al. In addition to the correlation between embryo morphology and blastocyst implantation competency and hESC derivation potential O'Leary et al.
Moreover, the capacity of a good quality ICM blastocyst to successfully generate novel hESCs was also dependent on the age of the mother O'Leary et al. In this respect, Boroviak et al. Any precursors of EpiSCs present in those cultures would probably differentiate and disappear naturally as EpiSCs do not survive well after single cell passaging. Avoiding complete single cell dissociation and using manual dissociation instead of trypsinization, it was shown indeed that mouse ICMs could be cultured to give rise to both mESCs and EpiSCs Najm et al.
During the preimplantation period of mouse development, two important lineage segregation steps lead to the formation of the epiblast reviewed in Kuijk et al. At this stage, the TE cells form the surface of the embryo that contacts directly the zona pellucida and express the marker Cdx2 ; whereas ICM cells are localized inside the embryo and are Oct4 Octamer-binding transcription factor 4 positive.
The second segregation step involves the segregation of Gata6 -positive primitive endoderm PrE cells and of Nanog -positive epiblast EPI cells that emerge from the Oct4 positive ICM cells of the expanding blastocyst.
Interestingly, the polarization of the EPI cells in the implanting embryo is accompanied by a process of cavitation that leads to the formation of the proamniotic cavity. This cellular rearrangement in the embryo, during early implantation, does not seem to involve apoptosis, but instead involves the activation of signalling pathways by the PrE and its derivative visceral endoderm, including the bone morphogenetic protein BMP signalling pathway Coucouvanis and Martin, ; Bedzhov and Zernicka-Goetz, This model proposes that cells of the extra-embryonic lineages secrete extracellular matrix proteins that form a basal membrane enveloping the EPI, and thereby creates a niche for its maturation during implantation Bedzhov and Zernicka-Goetz, PICMI: the mother of all stem cells in mouse and human.
With the discovery and derivation of mouse EpiSCs Brons et al. Interestingly, this metastable heterogeneity, at least in mice, seems to exist also at the level of DNA methylation. A novel field of investigating the transcriptional profile and DNA methylation of cells to a single-cell resolution will hopefully shed more light on the significance of heterogeneity Grun et al. The conversion from mEpiSCs to mESCs has been achieved by several research groups by adapting the culture conditions and overexpressing several transcription factors reviewed in Festuccia et al.
The appearance of the silent X is easily recognizable by the characteristic perinuclear spot, corresponding to the inactive X chromosome Nichols and Smith , One of the best-studied examples is the differential usage regulated by DNA methylation of the distal and proximal enhancer to regulate the expression of Oct4 in mESCs as well as in the ICM and primordial germ cells and mEpiSCs and in the egg cylinder or epiblast, respectively Yeom et al.
Different culture media used to generate human naive pluripotent stem cells. Moreover in that study, Forskolin, a protein kinase A pathway stimulator that induces expression of KLF4 and KLF2, was shown to transiently substitute for the need for ectopic transgene expression.
However, mhESCs could be maintained only for several passages. Valamehr et al. Ware et al. In a different study Chan et al. Theunissen et al. Remarkably, the resulting reset hESCs could be cultured in defined medium without serum products or growth factors.
Moreover, the metabolism of these reset hESCs was altered, resulting in an increased mitochondrial respiration. These aforementioned findings open the debate about which pluripotency state is preferred for future clinical applications. As derivatives from primed hESCs are already being used in clinical trials Schwartz et al. Should different somatic cells be tested for reprogramming to specific cell types providing a tailored approach?
Considerable differences have been shown in the differentiation propensity of different primed hESC lines Osafune et al. In fact, the majority of the in vitro differentiation protocols are plagued by a relatively low yield of differentiated cells and contamination with undesired cell types.
Moreover, defined and predictable protocols for directed differentiation and methods of cell delivery have yet to be optimized. Based on germ cell differentiation experiments, the answer to what may be the ultimate pluripotent state to use in regenerative medicine remains uncertain.
This was proven by differentiating transgenic lines with either a Blimp1 or Stella reporter construct in the presence of BMP4 Hayashi and Surani, This transient stage resembled the early pluripotent epiblast that gives rise to PGCs in vivo Hayashi et al. The in vitro differentiation of these mEpiLCs to PGC-like cells was more efficient, and in two landmark studies in mice functional oocytes and sperm could be obtained Hayashi et al.
This remarkable achievement was thus based on the careful choice of the starting population of stem cells. Not much is known about the best state of pluripotency for germ cell formation from human pluripotent stem cells. Primed state hESCs have been extensively used to generate precursor gametes, however, derivatives have not reached beyond the premeiotic stage without the need for genetic manipulation Handel et al.
Moreover, it was shown that the endodermal lineage marker SOX17 could contribute to specify the germ cell fate in humans Irie et al. Interestingly, similar to the role of ActA in facilitating human germ cell development in vivo Coutts et al. The generation and cryopreservation of PICMIs from embryos cryopreserved but no longer needed for reproductive purposes, as well as from the fresh PQEs in cycle cohorts, could be beneficial to ART patients. Similar to cord blood banking for the potential treatment of diseases, cryopreserved PICMIs could be an important resource for the regenerative treatment of born siblings.
Routinely extending embryo culture beyond the blastocyst stage for PQEs and thawed blastocysts would require optimized culture conditions and increased efficiency, similar to the early trials of ART.
In any event, IVF centres could play an increasingly important role in the future application of stem cells in regenerative medicine. Additional studies may prove that the PICMI represents the human peri-implantation embryo, a unique stage during human development that is under studied. Therefore, by understanding the PICMI's metabolism and genome-wide transcriptional profile, we could gain precious insight into the mechanism of implantation in humans.
Could the mechanisms responsible for the blastocyst's transition into a PICMI be comparable or related to its potential to implant? By studying the interaction between cultured human endometrial cells with both normal and aneuploid PICMI structures, our understanding of the events responsible for human embryo implantation would increase substantially.
Implantation failure is thought to be a major cause for the low efficiency of human fecundity. The development of new experimental systems to better understand implantation in humans would undoubtedly benefit a large number of ART patients.
We would like to thank Ferring Company Aalst, Belgium for financial support. Development ; : — Google Scholar. Allegrucci C , Young LE. Differences between human embryonic stem cell lines. Hum Reprod Update ; 13 : — The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod ; 26 : — Epigenetic reversion of post-implantation epiblast to pluripotent embryonic stem cells.
Nature ; : — Bedzhov I , Zernicka-Goetz M. Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation. Haploid sex cells gametes are produced so that at fertilization a diploid zygote forms. This tutorial is an in-depth study guide regarding male and female reproductive physiology Read More.
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Embryonic Index: ICM. The inner cell mass ICM forms within the blastocyst, prior to its implantation within the uterus. The ICM is a cellular mass on one side of the hollow interior of the round embryo, the outer layer of which is called the trophoblast.
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