Background
The study of early embryonic development in mammals has provided fundamental information on how the fertilized egg transitions through successive developmental stages to form a complex organism such as ourselves. It is a remarkable journey that has fascinated me since my days as a graduate student in Cambridge. The mouse embryo has been my main study system of choice but this has always been in the service of understanding human development and improving human pregnancy outcomes. The Marshall Lab at Cambridge was also the research base for Bob Edwards when I was a student. So I was keenly aware of the direct translational implications of work from mouse embryo culture to human IVF. Working with Richard Gardner, I helped establish the mouse blastocyst as a model system to study lineage decision-making in development and identified that the epiblast cells of the inner cell mass were the original pluripotent cells (Gardner and Rossant, 1979).
The blastocyst in both the mouse and the human contains three distinct lineages by the time of implantation in the uterus. There is an outer polarized epithelium, the trophectoderm (TE), which gives rise to the trophoblast cells in the placenta. The trophectoderm encloses a blastocoelic cavity, at one end of which is a group of cells called the inner cell mass (ICM). The ICM consists of the pluripotent epiblast which contributes to all cells and tissues of the embryo proper as well as some extraembryonic membranes such as the amnion. The layer of primitive endoderm (PrE) on the blastocoelic surface of the ICM gives rise primarily to extraembryonic endoderm of the yolk sac. Thus, of the three lineages of the blastocyst, only the epiblast can be considered to be truly pluripotent. However, it is not totipotent as it has lost the capacity to generate TE and PrE of the conceptus. The fate and potential of the different lineages has been determined by ever-increasingly sophisticated chimera and lineage tracing experiments and the signaling pathways and transcription factors involved in specifying cell fate are fairly well understood (reviewed (Rossant, 2018)).
The beginnings of pluripotent stem cell research also date back to the 1970s. Embryonal carcinoma cell lines with some properties similar to the ICM had been isolated from mouse teratocarcinomas (Cronmiller and Mintz, 1978; Martin and Evans, 1974). They could contribute to some normal tissues in chimeras, just like epiblast cells, but the resulting chimeras succumbed to growth of tumors derived from the cell lines (Papaioannou et al., 1978). The concept that it might be possible to capture the pluripotent state of the epiblast by directly culturing blastocysts in vitro was clearly in the wind. Many groups tried to derive such cell lines and two groups, Martin Evans and Matt Kaufman (Evans and Kaufman, 1981), and Gail Martin (Martin, 1981), succeeded in 1981. The derivation of mouse embryonic stem (ES) cells marked the beginning of a revolution in mouse genetics and the foundation of all future pluripotent stem cell research and applications. It was not until 1998, however, that Jamie Thomson first reported the derivation of human embryonic stem cells from excess IVF blastocysts (Thomson et al., 1998). The ethical concerns raised by the use of human embryos for this research was a definite concern for many inside and outside the field. It was the discovery of induced pluripotent stem cells (iPSC) by Yamanaka and colleagues in 2006/7 (Takahashi et al., 2007; Takahashi and Yamanaka, 2006) that really led to an exponential surge of interest in pluripotent stem cell research and its potential applications to understanding and treating human disease.
The correlation between the properties of the embryo itself and its derived stem cells is of ongoing interest and certainly generates some controversy.
Different mouse stem cell states and their relationship to the early embryo
Mouse ES and iPSC grown under so-called ‘naïve’ conditions (Ying et al., 2008) show gene expression, epigenetic profiles and X-inactivation status that are highly similar to the epiblast of the blastocyst itself. Importantly they also behave like epiblast cells when reintroduced into the early embryo, contributing to all lineages of the fetus itself, including the germ line. It is actually possible to produce entirely ES-derived mice by aggregating ES cells with tetraploid embryos – the so-called ‘tetraploid complementation assay” (Nagy et al., 1993). In fact, carefully maintained mouse ES cells can routinely generate complete mice when introduced into diploid precompaction 8-cell embryos (Poueymirou et al., 2007), a process that has allowed rapid phenotypic screening of genetic alterations (Cox et al., 2010).
What about the other lineages of the blastocyst? Can they produce stem cell lines in vitro? My lab derived permanent cell lines from both the TE and the PrE of the mouse blastocyst and showed that they could self-renew, differentiate appropriately in culture and contribute to the expected lineages in vivo in chimeras.
ES cells, trophoblast stem (TS) cells (Tanaka et al., 1998) and Extraembryonic endoderm (XEN) cells (Kunath et al., 2005) are derived under conditions that reflect the known growth factor requirements within the embryo itself. Most notably, different levels of FGF/ERK signaling act to specify cell fate (EPI vs PrE) or maintain proliferation (postimplantation TE) in the embryo itself. Other pathways, in particular the Hippo signaling pathway (Cockburn et al., 2013), also play key roles in establishing cell fate in the embryo itself. There are still many open questions on the details of the process of lineage commitment in the mouse embryo but the overall correlation between lineage behavior in the embryo and in its derived stem cells holds firm.
Recent work has improved the derivation of TS cells (Lee et al., 2019) and XEN cells (Ohinata et al., 2022) to resemble even more closely their progenitors at the blastocyst stage.
Capturing different cell states in stem cell lines in vitro has been extended to other stages of development. It is obviously of interest to ask whether it is actually possible to capture the earlier totipotent state in culture. A stem cell state that reflects some of the properties of the 2-cell stage of development, the 2C-like cell, has been reported to arise spontaneously in standard ES cell cultures and has been proposed to represent the totipotent state of the 2-cell embryo (Macfarlan et al., 2012). In the mouse, the two-cell stage is the time of the major activation of the zygotic genome (ZGA), which is marked by transient activation of retroposons and a set of genes only found at this stage of development. 2C-like cells share many of these gene profiles and may well represent a useful model to study the mechanisms of ZGA. However, they cannot be maintained in a stable state indefinitely in culture (Genet and Torres-Padilla, 2020) and their ability to contribute to all lineages in later development is not well documented (Macfarlan et al., 2012).
There have been a number of studies claiming to derive pluripotent cell lines with extended potential to generate both ICM and trophectoderm derivatives, usually beginning with ES cells. Although many of these do show altered properties from standard ES cells and some gene expression typical of earlier stages, most of them do not show highly convincing capacity to generate TE either in vitro or in vivo. We reassessed the capacity of two of the best documented cell lines from the Liu (Yang et al., 2017b) and Deng (Yang et al., 2017b) labs for their chimeric potential and showed that, although the cells could occasionally be found in the TE lineages, they were not fully transformed into trophoblast and continued to express ES markers (Posfai et al., 2021). Two more recent papers have used spliceosome inhibition (Shen et al., 2021) and chemical-induced chromatin remodeling (Yang et al., 2022) respectively to shift the cellular state of ES cells towards a more stable totipotent blastomere-like cell. While closer to the blastomere state, the complex chemical interference needed to cause this shift in potential still requires to be understood in terms of its relationship to the progress of totipotency to pluripotency in the embryo itself.
The epiblast cells of the blastocyst undergo further morphological and gene expression changes as the embryo implants in the uterus, leading up to the major germ layer specification events of gastrulation. They do retain full pluripotency during these transition stages but undergo various epithelial reorganizations to form the egg cylinder stage. Epiblast stem cells (EpiSC) can be derived from early postimplantation embryos in the presence of FGF (Brons et al., 2007; Tesar et al., 2007) (see Fig 3) and represent the pre-gastrulation stage epiblast, the so-called primed state. They do not respond to induction of germ cell fate and they cannot contribute to normal development after injection into the blastocyst. More recently the Smith group has proposed that there is also a formative stage of the epiblast in the early post-implantation period during which the epiblast cells exit from the naïve pluripotent state, gain responsiveness to germ cell induction and are prepared for later lineage responsiveness at the gastrulation stage (Kalkan and Smith, 2014; Smith, 2017). Several groups have isolated stem cell lines with some properties of the formative state (reviewed (Pera and Rossant, 2021)). The relationship between these various cell lines and the stages of epiblast development in the embryo is by no means entirely clear. Pluripotency seems to be a somewhat flexible state of being!
Human development and stem cell states
When human ES cells and iPSC were first derived, they were grown under conditions that included FGF and ERK activation, leading to the general conclusion that they were closer to mouse EpiSC than the naïve pluripotent state. It took time and considerable effort to develop culture conditions that could transform human ES cells to a stable naïve state, but this has now been achieved in a number of labs (Takashima et al., 2014; Theunissen et al., 2014). Expression profiling confirms that these cells are closer to the early ICM of the human blastocyst, including in their X-inactivation status, although this state seems to be relatively transient in the embryo itself. As the embryo implants there is a fairly extended period of period of 4-5 days where the post-implantation epiblast persists in a relatively stable state of gene expression (Nakamura et al., 2016) whilst undergoing expansion in numbers. Both formative and primed ES cells in humans can be considered to represent different phases of this transition. At this stage, as in the mouse, the exact identities of the different cell states and their comparison to the embryo itself are unclear (reviewed (Pera and Rossant, 2021)).
It is becoming clear, however, that human naïve stem cells do have a broader lineage potential than their mouse equivalents. Several groups have shown that naïve hES cells retain some potential to differentiate down the TE pathway, depending on appropriate culture conditions (Cinkornpumin et al., 2020; Dong et al., 2020; Guo et al., 2021; Io et al., 2021). This parallels experimental data showing that isolated ICMs from the mature human blastocyst can still generate TE in outgrowth culture ((Guo et al., 2021)). This is in contrast to results from the mouse where ICM cells clearly lose TE potential after initiation of blastocyst formation (Posfai et al., 2017).
Recently two groups have reported that it is possible to identify a subset of cells in naïve hES cultures that express many of the properties of the 8-cell blastomere stage of development, which marks the time of major ZGA in the human embryo (Mazid et al., 2022; Taubenschmid-Stowers et al., 2022). Similar to the mouse 2C-like cells, these 8C-like cells express features of zygotic genome activation and share common markers of ZGA like HERV-L and Trpx1. These cells may be useful for studying human ZGA but, like the mouse, this is not a stable state that can be used to promote totipotent development.
What about stable stem cell lines from the TE and hypoblast in human? Again, such lines have been derived but not with as much ease as from the mouse blastocyst. Human TS cells have been derived from both blastocysts and early villus biopsies (Okae et al., 2018). They seem to have the lineage potential of the early postimplantation cytotrophoblast, rather than the TE of the blastocyst. There have been two reports of isolating extraembryonic endoderm-like cells (Linneberg-Agerholm et al., 2019) or yolk sac-like cells (YSLC) (Mackinlay et al., 2021) from human ES cells, which may have some properties of the hypoblast.
More details on current understanding of human embryo development and its relationship to stem cell states is found in Rossant and Tam (Rossant and Tam, 2022).
Stem-cell derived blastocyst models from mouse to human
There are still many gaps in our knowledge of the molecular processes of blastocyst formation in humans and there are several groups working to fill those gaps with direct data from human embryos in culture. However, use of human embryos for research is limited by regulation or legislation in many jurisdictions and, even where permissible, the supply is limited. As with other approaches to studying human development using stem cell-derived organoids, there is considerable interest in using stem cells to model early development. Here I focus on the production of so called blastoids as models of the blastocyst itself. The Rivron lab generated structures resembling blastocysts by controlled aggregation of mouse ES cells and TS cells (Rivron et al., 2018). The primitive endoderm lineage was not well represented in these original blastoids. They could cause a decidual response in the uterus but did not develop further.
Blastoids with more primitive endoderm cells were produced when mouse extended potential ES cells (Yang et al., 2017a; Yang et al., 2017b), were either combined with TS cells (Sozen et al., 2019) or cultured in suspension culture alone (Li et al., 2019). Although these blastoids were closer in morphology and expression profiles to the blastocyst, they still failed to show embryo development after implantation. In the case of blastoids derived entirely from extended potential ES cells (Li et al., 2019), our re-analysis of the gene expression profiles did not support a bona fide TE identity for all of the putative TE cells in the blastoids (Posfai et al., 2021).
Clearly even in the mouse, the blastoid is not yet really equivalent to the blastocyst. In the mouse one always has the gold standard of the embryo to fall back on when trying to validate the system. When generating human stem cell-based embryo models, it is still very important to be able to compare with the embryo itself, even though there is a limitation on the temporal extent of study. Thus, the potential to be able to generate large numbers of human embryo models from stem cells is very attractive. The logical place to start with generation of human blastoids might be expected to be combinations of human blastocyst-derived stem cells, in a similar manner to the mouse. However, there have been no published reports on successful generation of human blastoids from combining human ES and TS cells. Instead, there have been a series of reports claiming to generate blastoids directly from human ES cells by various culture manipulations without addition of any specific extraembryonic cell types (Fan et al., 2021; Kagawa et al., 2022; Liu et al., 2021; Sozen et al., 2021; Yanagida et al., 2021; Yu et al., 2021). One study claimed to produce iblastoids as an intermediate during the process of reprogramming adult cells to iPSC (Liu et al., 2021), while most other studies began with human ES cells in a putative naïve state. Given the reports that ES cells in the naïve state retain some TE potential (Cinkornpumin et al., 2020; Dong et al., 2020; Guo et al., 2021; Io et al., 2021), these groups claim to have revealed this potential in a relatively controlled manner so as to produce blastoids at reasonable efficiency. Some of the reported human blastoids do show quite striking resemblance to the blastocyst itself in both morphology and gene expression, but in-depth comparisons of the gene expression profile of the cell types in the blastoid with the embryo itself always need to be made. Formation of an epithelial cyst with enclosed pluripotent cells is not sufficient to confirm a functional TE phenotype. Our reanalysis of the published data shows that all human blastoids contain undefined cell types at various proportions (Zhao et al, BioRxiv). Further, the claimed TE lineage in the blastoids derived during reprogramming is closer in expression profile to amnion than TE. Clearly it is early days for generating reproducible, homogeneous human blastoids that can mimic early developmental stages in vitro. More refinement of culture conditions and careful comparison with normal blastocysts and early post-implantation stages will be needed to validate these potentially powerful experimental models.
It has been suggested that broader use of stem cell-derived blastoids could avoid some of the regulatory and ethical issues of human embryo research. A blastoid may resemble the products of conception but is not derived by the union of egg and sperm. Its genotype reflects the diploid genotype of its founding cell line. However, blastoids do come with their own legal and ethical concerns. Although currently blastoids, even in the mouse, are clearly not functional embryo equivalents, would further improvements bring them closer to such capacity? When would a stem cell model be considered to have crossed the line and become an embryo? In some jurisdictions such as Australia, blastoids are already considered as requiring the same regulatory oversight as embryos themselves (Matthews and Morali, 2020) and the US NIH is not currently funding such research. The recently revised ISSCR Stem Cell Guidelines took these issues into consideration and proposed that integrated stem cell models like blastoids (with cell types potentially able to generate a functional placenta and embryo) should be subject to special oversight and restricted to short term culture (Lovell-Badge et al., 2021), while still recognizing that they are not embryo equivalents. The guidelines also specifically prohibit transfer of any stem cell-based embryo model (including blastoids, gastruloids and other models) to the uterus of a human or animal host.
Conclusions
Many years of research on mouse embryo development and differentiation have provided the tools, the fundamental knowledge and the practical applications that underlie the current excitement about exploring human development more directly. The advent of single cell genomics and transcriptomics, in vivo live imaging, CRISPR gene editing has provided new insights into understanding lineage development in both mouse and human embryos. The development of stem-cell based integrated and non-integrated embryo models (Rossant and Tam, 2021; Weatherbee et al., 2021) provides new avenues for human embryology research but with a clear need to be validated against normal embryonic processes. Overall, increased scientific knowledge of the mechanisms of embryogenesis will help us understand the basis of both genetic developmental anomalies and non-genetic congenital diseases, as well as shedding light on the causes of early embryo loss and pregnancy disorders. Embryo and stem cell research have always been closely intertwined and there is no doubt that will continue in the future to great effect.
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