Blastocyst-like embryo surrogates from stem cells in implantation models : Reproductive and Developmental Medicine

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Special Issue: Updates on Research in Endometrium

Blastocyst-like embryo surrogates from stem cells in implantation models

Tian, Siyu1; Ruan, Hanzhang1; Yeung, William Shu-Biu1,2,3,*; Lee, Yin Lau1,2,3,*

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Reproductive and Developmental Medicine 6(4):p 225-233, December 2022. | DOI: 10.1097/RD9.0000000000000042
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Abstract

Low fertility rate in humans

According to the World Health Organization, approximately 1 in every 7 couples suffer from reproductive disorders[1]. Infertility has become a major health care problem in many countries in recent decades. The low fertility rate is partly attributed to an increasing tendency toward delayed marriage, leading to childbearing at an advanced maternal age. Fertility naturally declines with age; therefore, the number of couples seeking in vitro fertilization treatments is progressively increasing.

Despite remarkable advancements in ovarian stimulation, culture media, and laboratory conditions, the success rate of in vitro fertilization treatment remains low, even after repeated transfers of good-quality embryos. Implantation failure is a major factor contributing to infertility. Successful pregnancy relies on synchronization between the developing human blastocyst and the receptive endometrium. The endometrium accounts for almost two-thirds of implantation failures[2]. The implantation success rate must be urgently improved; however, the physiological and molecular mechanisms underlying the implantation process in humans remains unclear[3].

Development of in vitro implantation models

A good in vitro model is required to study the dynamic fetal-maternal interaction during implantation. The first human in vitro implantation model was established in 1985[4]. Primary endometrial epithelial cells (EECs) were isolated from normal human endometrial biopsies at the time of ovulation. EECs were grown in vitro to form a monolayer in which in vitro fertilized human oocytes were placed. In this model, the process of adhesion was observed, but not invasion[4]. Apart from endometrial cells isolated from the fresh tissues, endometrial adenocarcinoma cell lines such as RL95-2 and HEC-1B are commonly used as receptive and non-receptive EECs respectively (reviewed by Hannan et al.[5]). In addition, three-dimensional (3D) endometrial models composed of primary EECs and stromal cells[6] have been also developed to better imitate the endometrial structure. These established in vitro implantation models using endometrial cell lines and primary endometrial cells have been reviewed elsewhere[3].

Numerous attempts have been made to culture endometrial tissues in vitro[7–10]. Mouse endometrial tissues cultured in vitro for 3 days were morphologically similar to those observed in pseudo-pregnant mice[10]. However, an in vitro culture system for human endometrial tissue that lasts >2 days is yet to be established. Attempts to develop an in vitro endometrial model are ongoing. New technologies, such as microfluidic culture[11,12] and organoid formation[13–17] have been developed for constructing endometrial models[18]. The ideal method for assessing human endometrial receptivity is to determine whether a human blastocyst can attach and implant onto the endometrium of interest. However, high-quality human embryos are not easily available for testing, and ethical concerns prevail regarding their use. In this review, we discuss the use of embryo surrogates for establishing in vitro models, with a special focus on stem cell–derived blastocyst-like embryo mimics. Fig. 1 summarizes the different human embryo surrogates used for in vitro implantation studies.

F1
Fig. 1.:
Comparison of different human embryo surrogates for in vitro implantation study. Comparison of the application, pros and cons of different human embryo surrogates, including human and mouse embryos, cancer cell–derived spheroid, primary trophoblast spheroid, hESC trophoblast spheroid (BAP-EB), and human blastoids for in vitro implantation study. BAP: BMP4, A83-01, PD173074.

Animal embryos for studying early implantation

To overcome the issues concerning the use of human embryos for research, mouse models have been widely utilized to study pre-implantation embryo development, implantation, and trophoblast development[19–23]. Mouse embryos are easy to obtain and relatively inexpensive compared with other mammalian embryos. Additionally, in vivo validation studies can be performed using a mouse model.

Multiple factors that influence the implantation process have been identified in studies using mouse implantation models. For example, the role of E-cadherin in implantation was confirmed by anti-body injection into mouse uterine horns[24]. A mouse in vivo implantation model was used to demonstrate the detrimental effect of peritoneal fluid taken from infertile women with endometriosis on implantation[25]. Using a mouse blastocyst and human endometrial epithelial cell line coculture model, it was found that CD98 was an important determinant of human endometrial receptivity[26].

In addition to mouse models, non-human primate models have also been used to study the implantation process[27–30]. For example, the effect of progesterone on endometrial receptivity was studied using macaque implantation models[28]. The baboon implantation model has been used to study the effects of chorionic gonadotropin (CG)[29]. However, the scarcity of research materials and ethical concerns have hindered the use of these models. Furthermore, the development of trophoblasts from the blastocyst to the placenta in mice is different from that in humans[31,32], the cellular interactions during implantation and placentation vary greatly even among primates[33], and the relevance of the information derived from animal models to humans is questionable.

Trophoblastic cell–derived spheroids for studying early implantation

Trophoblast cell lines established from malignant tissues (BeWo, JAR, and JEG) are commonly used to study trophoblast development[34,35]. 3D trophoblast spheroids can be generated from these cell lines in a gyratory shaker[36,37], low-attachment plates[38,39], or rotating glass tubes[40] to mimic human embryos during implantation. Among the 3 commonly used trophoblast cell lines, the JAr cells, which show similar expression patterns as the primary villous trophoblast cells[41] and secret β-hCG, estrone, and estradiol[5,42], have been used to study the mechanisms of trophoblast attachment[43]. Trophoblastic spheroids generated from BeWo cells, which mainly consist of cytotrophoblast (CTB)-like and syncytiotrophoblast (STB)-like cells, have been reported to be a good substitute for human embryos to explore the attachment and invasion processes onto the secretion phase of the human endometrium[44]. Alternatively, the extra-villous trophoblast signature of JEG-3[41] allowed its use in studying the invasion process of trophoblasts[45]. Indeed, significantly higher attachment and invasion rates were observed in spheroids derived from JEG-3 cells when compared with those derived from BeWo and JAr cells[46,47].

We and others have cocultured choriocarcinoma cell lines (eg, JEG-3, JAr)-derived spheroids with endometrial cells as an in vitro implantation model to study endometrial receptivity in the past decade[48–56]. Endocrine disruptors 2,3,7,8-tetrachlorodibenzo-p-dioxin[48] and perfluorooctanoic acid[49], and endometrial genes Dickoff-1[50] and olfactomedin-1[51] suppressed the attachment of JAr spheroids onto an EEC line (Ishikawa). In addition, forced expression of glycodelin suppressed endometrial cell migration and invasion but enhanced spheroid attachment[52].

Although trophoblastic spheroids have been robustly generated from cell lines, choriocarcinoma cell lines have abnormal karyotypes and higher invasive and adhesive abilities compared with normal human implanting embryos[57,58]. The manner of attachment may not fully represent the process in early embryo implantation. The well-differentiated status of these cell lines, which are already at a later developmental stage than the trophectoderm cells of blastocysts during implantation, also hinders the study of early human embryo implantation[59].

In addition to immortalized trophoblast cell lines derived from tumor cells, trophoblastic cell lines derived from normal first trimester placental tissues (HTR-8/SVneo) were used for the generation of trophoblastic spheroids[60] and to study trophoblast invasion[61]. However, HTR-8/SVneo cells contain 2 populations of cells, namely trophoblast cells and stromal/mesenchymal cells[62], which may not be appropriate for early implantation studies. Primary trophoblast cells isolated from human placental tissues can also be used as an alternative[59]. Holmberg et al. used low-attachment plates to generate blastocyst-like spheroids from human first trimester trophoblast cells (Sw.71)[40]. However, spheroid formation efficiency from the primary cells was very low, and no spheroid could be formed from the primary cells isolated from placenta at 10 to 12 weeks[63].

Stem cell-based models of embryo surrogates for implantation study

Pre-implantation embryo development

After fertilization, human embryos take approximately 5 days to develop into blastocysts. After compaction and cavitation, the first cell differentiation results in 2 cell lineages: inner cell mass (ICM) and trophectoderm (TE)[64,65]. The fluid-filled blastocoel, together with the adjacent ICM, is enclosed by the TE at the blastocyst stage[64,65]. Before embryo implantation, the ICM differentiates into the primitive endoderm (PE) and epiblast (EPI). After hatching from zona pellucida, the blastocyst is ready for implantation[64]. During implantation, the TE differentiates into CTB, the outermost layer of which later fuse to form STB, while the ICM further develops to form the bilaminar disc, which prepares for gastrulation[66].

The power of stem cell-based models

Embryonic development involves the precise control of numerous processes, including proliferation, differentiation, and morphogenesis, which determine the proper self-organization of the embryo. Although human or mammalian embryos can be used to study these processes in models, the limited number of available embryos impedes research on high-throughput pharmacological drug screening for toxicity, viral susceptibility, and disease treatments. Functional studies using genome editing in mammalian embryos are time-consuming and inefficient. Establishing an in vitro stem cell-based model of embryo development can help overcome this limitation.

Human pluripotent stem cells (hPSCs) have drawn much attention over the past 2 decades owing to their capability for indefinite proliferation, self-renewal, and differentiation into the 3 primary germ cell layers (ectoderm, endoderm, and mesoderm). hPSCs can be derived directly from the epiblast of blastocysts as embryonic stem cells (ESC)[67,68] or obtained by re-programming somatic cells as induced pluripotent stem cells (iPSC)[69]. Recently, naïve pluripotent stem cells[70] or totipotent-like expanded potential stem cells[71,72] with an earlier embryonic status have been reported. These in vitro models hold great promise for studying human embryonic development.

Differentiation of human embryonic stem cells into trophoblast-like cells

In 1998, hESCs were first reported to be capable of differentiating into trophoblasts, with secretion of β-hCG into the culture medium[68]. Because early human trophoblast cells were unavailable, trophoblast induction from hPSCs offered a new approach for studying early implantation and trophoblast development.

In 2002, it was first reported that bone morphogenetic protein 4 (BMP4) induced the differentiation of hESCs and iPSCs to trophoblast-like cells, which formed syncytial cells expressing trophoblast markers and secreting β-hCG[73]. However, hESCs treated with BMP4 alone also expressed high levels of mesoendoderm markers like Brachyury, WNT3, and Mix paired-like homeobox MIXLI, suggesting that BMP4 also induced hESCs to other extra-embryonic lineages[74,75]. It was later found that supplementation of fibroblast growth factor 2 (FGF2) inhibitor (PD173074, SU5402) with BMP4 reduced the proportion of mesoendodermal cells during trophoblast differentiation[76–78]. The effects of FGF2 inhibitors might be related to the actions of FGF2 in maintaining hESC pluripotency and inhibiting trophoblast differentiation[79,80]. Subsequent modification of the differentiation system identified inhibitors of TGF-β/Activin/Nodal signaling (SB431542 or A83-01) had additive effects in inducing trophoblast differentiation from hESC[81,82]. Indeed, TGF-β/Activin/Nodal signaling is one of the critical pathways maintaining the characteristics of hESC[83,84]. With the use of BMP4 and the 2 inhibitors A83-01, PD173074 (BAP), the derived trophoblast-like cells express trophoblastic markers (KRT7, HLA-G, CGB), secrete hormones (β-hCG, estrogen, and progesterone) and possess invasive ability[82,85]. The BAP-induced trophoblast from hESCs was reported to be a suitable model system for studying early human placentation[86].

Trophoblastic spheroid derived from hESC for implantation study

Over the years, extensive efforts have been made to generate blastocyst-like trophoblastic spheroids from PSC that mimic early implantation embryos. hESCs can differentiate into cystic embryoid bodies (EB) and form 3 embryonic germ layers[87]. Using a similar method, Gerami-Naini et al. established EB by culturing hESC clumps in suspension for 8 days. After explant into Matrigel, the trophoblastic cells in the EB secreted βhCG, progesterone, and estradiol-17β[88]. However, the EB in culture tended to fuse and form large aggregates, leading to impaired cell proliferation and differentiation[89]. Several improved protocols using stirred suspension system[90], rotating cell culture system[91], and rotary orbital culture system[92] were developed to solve these problems and allow large-scale EB formation. With the improved EB formation method, efficient trophoblast differentiation with β-hCG secretion and trophoblast marker (CK7, HLA-G and MMP2/9) expression from these EB have been reported[93].

Human blastocyst-like embryo surrogates derived from hESC for implantation study

In an attempt to develop a human blastocyst surrogate for studying early implantation, our group modified the BAP differentiation protocol[82] for generating trophoblastic spheroid (BAP-EB) from hESCs[94]. We first produced large numbers of EBs from hESCs by using AggreWell culture plates. The spheroids formed were induced to differentiate into the trophoblast lineage using BAP (BMP4, A83-01, PD173074). EB treated with BAP for 48 hours (BAP-EB-48h) formed a blastocoel-like cavity with a size similar to that of human blastocysts. During a 120-hour period of differentiation, the pluripotent marker (OCT4) was downregulated and markers representing early trophoblasts (CDX2, GATA3, CK7), STB (ERVW-1, CGB), and EVT (HLA-G, MMP2) were progressively induced.

BAP-EB differentiated for 72 hours (BAP-EB-72h) attached specifically onto receptive EEC lines but not non-receptive EEC nor non-endometrial cell lines[94]. Their attachment onto EEC lines is more specific than that of choriocarcinoma spheroids[95]. Critically, BAP-EB can only attach onto “receptive” primary EECs, but not onto “pre-receptive” primary EEC[94,95]. In addition, BAP-EB spread on the EEC and invaded through endometrial stromal cells, similar to the observations made in in vitro cocultures of human blastocysts with endometrial cells[96]. Using BAP-EB, we demonstrated the importance of Hippo signaling[95] and gap junction[97] in trophoblast development. Transcriptomic analysis revealed a trophectoderm-like signature in BAP-EB-48h and BAP-EB-72h, but a trophoblast-like signature in BAP-EB-96h[95]. In fact, the attachment competent BAP-EB-72h exhibited a gene signature similar to the polar TE (pTE) cells of Day 7 blastocysts, while the less competent BAP-EB-48h resembled the mural TE (mTE) of Day 7 blastocysts[95]. As human blastocysts adhered to the EECs with their pTE, but not the mTE during implantation[98], BAP-EB can be regarded as a physiological 3D model that mimics the early implantation of human embryos better than choriocarcinoma cell line-derived trophoblastic spheroids.

New models from stem cells for studying differentiation and early post-implantation

Apart from trophoblastic spheroids, other 3D peri-implantation models have been derived from PSCs for implantation and early developmental studies[99–101]. Models of gastrulating embryos (gastruloids) re-capitulating germ-layer specification and axial organization in post-implantation blastocysts have been established in mice[102–104] and humans[105–109]. Gastruloids have been used for studying post-implantation embryo development, such as cardiogenesis[110] and somitogenesis[111]. In addition, the development of the epiblast and amniotic ectoderm has been explored using a human post-implantation amniotic sac embryoid (PASE) model that resembles the human amnion and ectoderm[112,113]. However, these 3D peri-implantation models cannot fully re-capitulate human embryos during early implantation because they are not composed of all cell types within an embryo. Until recently, embryoids modeling the blastocyst stage of development, namely blastoids, have also been developed in mice[114] and humans[115,116].

Blastocyst-like blastoids from stem cells

Mouse blastoids

The first mouse blastocyst-like blastoid completely derived from stem cells was generated by aggregating mouse ESCs with trophoblast stem cell (TSC); mouse ESC were first aggregated for 24 hours before adding mouse TSCs[114]. Only 0.3% of the aggregates formed blastoids after 65 hours of culture[114]. Later, a more efficient protocol (5–50%) was developed by generating mouse blastoids solely from mouse pluripotent stem cells (mPSC)[117]. The blastoids formed were called induced self-organizing 3D blastocyst-like cysts (iBLCs), which resemble pre- or post- compacted embryos[117]. Simultaneously, mouse blastoids were successfully derived solely from a new type of stem cells called mouse extended pluripotent stem cell (mEPSC)[118]. Among the aggregates formed by mEPSC, approximately 15% assembled into blastocyst-like structures[118]. By modifying the protocol of Rivron et al.[114], Sozen et al.[119] used mEPSCs and TSCs to generate extended potential blastoids displaying signatures of mid- to late-blastocysts. Formation of cystic structures and PE-like layers were more efficient in blastoids derived from mEPSC when compared to mESC[119]. Switching from normoxia (20% O2) to hypoxia (5% O2) upon adding TSCs enhanced the blastoid formation efficiency from 2.5% to 15.17%[119]. However, without TSC, no blastocyst-like structure was observed[119]. This observation is contradictory to the findings that mouse blastoids could be formed solely from mEPSC[118], indicating that different blastoid culture systems might contribute to distinct results.

The developmental potential of blastoids was determined in vivo. Mouse blastoid-induced decidua formation after transfer to pseudo-pregnant mice[114,117–119]. In addition, permeable vessel construction[114,117,118], differentiation of trophoblast cell types[114,119], and extra-embryonic ectoderm-like and vesicle endoderm-like organization[117,119] were observed. Although the mouse blastoids reorganized to these initial post-implantation structures, they failed to develop fully as bonafide blastocysts[114,117–119]. The parietal endoderm was not fully formed[119] and tissues derived from blastoids were smaller than those from embryos[117,118]. More specifically, blood mononuclear cells were found around disfigured pycnotic tissues derived from blastoids[117]. Consequently, the blastoids were resorbed shortly after implantation[117,119]. It was proposed that the failure was due to the absence of Reichert’s basement membrane[119].

Human blastoids

In 2021, human blastocyst-like blastoids were successfully generated from iPSC re-programmed from human dermal fibroblasts[115], human EPSC[120], and naïve human PSC cultured in the 5i/L/A system[116] or PXGL (PD0325901, XAV939, Gö6983, and LIF) systems[121]. A comparison of the 4 protocols has been provided in Fig. 2.

F2
Fig. 2.:
Comparison of the 4 human blastoid formation protocols. Comparison of the protocol, efficiency, and components used for generating human blastoids.

Two different strategies were used to generate blastoids. While Liu et al. generated the “iBlastoid” using an all-in-one medium[115], the other 3 protocols chose 2-step methods[116,120,121]. In 2 of the protocols, the concentrations of the differentiation factors were gradually reduced to enable differentiation of the outer TE while preserving inner cell pluripotency[120,121]. In contrast, Yu et al. used separate sets of differentiation factors to drive cell differentiation toward TE or PE[116]. The culture duration also varied among the 4 approaches, ranging from 3[121], 6[115,120], and 9 days[116].

A83-01 (activin/NODAL/TGF-β pathway inhibitor) is a common factor used in all 4 protocols for TE differentiation and stem cell proliferation[115,116,120,121]. CHIR99201 (WNT signaling activator) was included for TE and PE differentiation in the 3 blastoid protocols[115,116,120]. While BMP4 was used to induce TE formation from iPSC[115] and hEPSC[120], PD0325901 (MEK/ERK signaling inhibitor) was commonly used in protocols starting from naïve human PSC for TE differentiation[116,121].

The blastoids formed from the 4 protocols were not only morphologically similar to blastocysts with proper allocations of ICM-, PE-, and TE-markers but also showed transcriptomic profiles resembling pre-implantation human blastocysts[115,116,120,121]. While the iBlastoid could give rise to naïve PSCs, primed PSCs, and TSCs[115], naïve ESC, TSC, and naïve extra-embryonic endoderm lines were derived from the human blastoid developed by Yu et al.[116].

Despite the huge potential of blastoids for studying early developmental processes, human blastoids have not been extensively used as a research tool for multiple reasons[122]. First, only a small portion of blastoids could undergo in vitro attachment and develop as human embryos[116]. Second, the developmental potential of blastoids beyond implantation is still doubtful[122]. Third, regular embryo development requires normal epigenetic and chromosomal features, which have not yet been revealed in blastoids[122]. Fourth, the protocols for generating blastoids are not standardized and their efficiencies vary widely. With different starting cell lines, starting cell numbers, cell status, and derivation methods, the reported efficiencies of blastoid generation were 5.8%–18%[115], 4%–28%[116], 7.2%[120], and 30%–80%[121]. Fifth, cell types that were not typically found in human blastocysts were frequently observed within the blastoids. Further optimization of the efficiency and specificity of blastoid formation is required. Developmental biologists should agree on a set of markers, guidelines, and ethics that define the best reproducible protocol for consistently generating blastoids[110].

Concluding remarks

Stem cell–derived trophoblastic spheroids or human blastoids that can re-capitulate the first wave of cell differentiation in human embryos are an important resource for understanding the spatiotemporal molecular changes occurring in pre- and early post-implantation blastocysts. Owing to ethical concerns, a 14-day rule is commonly used as a guideline for the in vitro culture of human embryos. Soon after the publication of studies reporting on blastoids, in May 2021, the International Society for Stem Cell Research (ISSCR) eliminated the prohibition on the cultivation and use of embryos beyond 14 days. With the unlimited production of blastoids for basic scientific research, the mechanisms of the earliest cell lineage differentiation and developmental failures, including early embryonic death and miscarriage, are expected to be deciphered. Additionally, blastoids can be used for large-scale pharmacological drug screening to identify potential drug candidates that improve early embryonal development and implantation. Currently, no tool can reliably assess endometrial receptivity. Using blastoids as embryo surrogates to evaluate endometrial receptivity and identify women with dysfunctional endometrium enables clinicians to better counsel couples who did not benefit from assisted reproductive treatment.

Acknowledgments

None

Author contributions

Y.L.L. and W.S.Y. did conceptualization. Y.L.L., W.S.Y., S.T., and H.R. did methodology. S.T., H.R., Y.L.L., and W.S.Y. did writing-original draft. Y.L.L and W.S.Y. did writing-review and editing.

Funding(s)

The work was supported in part by a General Research Fund (grant number: 17111414) from the Research Grants Council of Hong Kong; Health and Medical Research Fund (grant numbers: HMRF 04151546) from the Food and Health Bureau, Government of the Hong Kong Special Administrative Region; Shenzhen Science and Technology Program (KQTD20190929172749226); The University of Hong Kong-Shenzhen Hospital Fund for Shenzhen Key Medical Discipline (SZXK2020089).

Conflicts of interest

All authors declare no conflict of interest.

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Keywords:

In vitro implantation models; Stem cells; Embryo surrogates; Endometrium; Trophoblastic spheroid; Blastoids

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