It is well known that the formation of a fully functional placenta is essential for a successful pregnancy and healthy fetus.1 As an intricate organ, the placenta consists of different types of trophoblast cells, and they cooperate delicately to ensure correct and harmonious placenta throughout the gestation.2 The placenta is the sole site for waste and nutrient exchange between mother and fetus. Meanwhile, the placenta also secretes hormones into the maternal bloodstream and fetal circulation, which promote the maintenance of pregnancy and development of the fetus. Dysfunction of the placenta would result in a series of clinical symptoms, such as pre-eclampsia, recurrent spontaneous abortion, fetal growth restriction.3–7 Therefore, figuring out the details of the cellular organization and regulatory mechanisms in the development of trophoblasts might be beneficial to prevent placenta-derived pregnancy complications.
The stem cell line is a type of cell that retains the potential ability of self-renewal. Under the certain differentiated medium condition, they can be induced into specific cells with special functions. Stem cells like induced pluripotent stem cells, embryonic stem cells, and adult stem cells have largely broadened our horizon of the mystery of the origin of life. Meanwhile, considering their extraordinary regenerative abilities, stem cell therapy offers the potential for treating diseases such as diabetes mellitus, and heart disease.8–10 Janet Rossant's lab derived the mouse trophoblast stem cell (mTSC) from the extraembryonic ectoderm and the blastocysts with the supplement of fibroblast growth factor 4 (FGF4).11 Following a similar strategy, TSC from rabbit and monkey blastocysts were derived.12,13 However, although such efforts have been made, the establishment of human trophoblast stem cell (hTSC) encounter many difficulties. Some analogous cell types were derived and showed some characteristics similar to TSCs; however, they are not real TSCs.14 Surprisingly, Takahiro Arima's lab successfully generated the hTSC from blastocysts and early placenta with optimized culture conditions.15 Shortly afterward, self-renewing trophoblast organoids were established.16,17 Importantly, these cytotrophoblast cells (CTBs) maintain the capacity to give rise to the downstream trophoblast lineages, which provide a powerful tool to understand the pathogenesis of pregnancy disorders originating from trophoblast defects.
The fetal-derived placenta and maternal decidua compose the maternal-fetal interface. The accurate communication between trophoblasts and decidual immune cells at the interface facilitates the delicate balance between fetal and maternal immune systems.18,19 The trophectoderm of the blastocyst gives rise to the different types of trophoblast cells which constitute the full functional placenta.20 Besides, the stromal cells, together with immune cells like natural killer (NK) cell, T cells, macrophage, consist of the maternal side.21–24 Although cells at the interface have been traditionally classified as above, whether new cell types exist, and what are their specific functions remain an ongoing focus. Here, we mainly focus on the progress of the establishment of in-vitro stem cell culture systems and elucidate the cellular organization and components of cell types at the maternal-fetal interface in the first trimester.
Human trophoblast stem cells isolation and culture
Over the past decade, many researchers have focused on the isolation and establishment of hTSC. Attempts to derive hTSC from human blastocyst-stage embryos based on culture conditions used for mTSC derivation failed, which might due to the fact that fibroblast growth factor (FGF) signaling is unlikely necessary for the maintenance of hTSC.25 Different from the expression of fibroblast growth factor receptor-2 (FGFR2) in mTSC, FGFR2 is not easily detected in the expanded human blastocysts, while specifically observed in the cytotrophoblast layer of placenta by 5 weeks of gestation. Suggesting that FGF signaling is initiated later in human development, and a potential source of self-renewing FGF-dependent hTSC niche may exist in the chorionic villi of post-implantation placenta.25
A traditional method employing percoll gradient was used to separate hTSC from term villous tissue. The isolated cells were morphologically larger, bi-nucleated and cytokeratin 14 positive, a marker of a subset of CTBs.26 James et al. have attempted to isolate hTSC from the first-trimester placenta using the Hoechst side population technique in combination with two sequential trypsin digests, and 98.5% of the obtained mononuclear cells expressed trophoblast marker, cytokeratin 7, but do not express β4 integrin.27 Examining the expression of murine TSC markers in primary human first trimester placental tissue showed that a subset of villous cytotrophoblasts (vCTBs) express caudal type homeobox 2 (CDX2), nanog homeobox (NANOG), POU class 5 homeobox 1 (POU5F1 or OCT4), stage-specific embryonic antigen-4 (SSEA4), and SRY-box 2 (SOX2), indicating that they could be the less differentiated CTBs.28,29 And a CDX2/tumor protein p63 (TP63) double-positive subpopulation isolated from early-gestation (6–8 weeks) human placentas reduced significantly in late gestation. They further differentiated into syncytiotrophoblast (STB)-like and extravillous cytotrophoblasts (EVTs)-like cells based on marker expression and hormone secretion, implying that the hTSC may exist in the early placenta villous.30
A lot of work studying the hTSC-like cells were conducted on HTR8/SVneo cell line. HTR8/SVneo cells were generated by transformation of primary EVTs and characterized with a gene expression profile more similar to vCTBs than EVTs.31 HTR8/SVneo cells express CDX2, notch receptor 1 (NOTCH1), NANOG, and SOX2, but not cytokeratin-7 and OCT4.32 Similar to human embryonic stem cell, HTR8/SVneo cells represent a self-replicating capability and reform spheroid structures and could repopulate the surface of the chorionic villi.32 Evidence indicates that the HTR8/SVneo cell population possibly contain a TSC population. The vCTBs positive for both interleukin 7 receptor (IL7R) and interleukin 1 receptor type 2 (ILIR2) failed to express differentiated trophoblast markers, while the Hoechst side-population of HTR8/SVneo cells express IL7R and ILIR2.33 Further, the Hoechst side-population of HTR8/SVneo cells express a high level of CDX2 and bone morphogenetic protein 4 (BMP4) but a lower level of beta-human chorionic gonadotropin (β-HCG), human leucocyte antigen-G (HLA-G), and α5/β3-integrin and could form colonies that up-regulate markers associated with STB and EVTs differentiation.33,34
The RNA sequencing (RNA-seq) of isolated CTBs, EVTs, and STB from first-trimester placentas were performed, and the functional annotation of the gene lists conducted with ConsensusPathDB showed that genes related to Wnt and epidermal growth factor (EGF) signal transduction pathways were overrepresented in the CTBs and may be required for proliferation of various epithelial stem cells. Based on the results above, proliferative human CTBs cell lines from both human blastocysts and first-trimester placental samples were established in a medium composed of CHIR99021 (a Wnt activator), EGF, Y27632 (a Rho-associated protein kinase inhibitor), A83-01 (a transforming growth factor-beta [TGF-β] inhibitor), SB431542 (a TGF-β inhibitor), and valproic acid (a histone deacetylase inhibitor).15 These proliferative human CTBs preferentially differentiate into EVT-like cells upon CHIR99021 withdrawal and differentiate into STB-like cells in a basal medium supplemented only with Y27632. Furthermore, the transcriptome profiling of hTSC and the TS-derived EVT-like and STB-like cells were similar to those of primary cultured corresponding compartments. More importantly, these cells can sustain the proliferative ability for over 5 months (up to 150 population doublings).15
Bioartificial organoids and organs architected with TSCs or placental tissues
Cells interact with each other and orient polarity to initiate the origin of tissue morphogenesis,35 which could develop into 3D organoids architected from multipotent stem cells. The culture medium based on a defined cocktail of factors applied for the establishment of several human epithelial progenitor cell lines facilitated establishing hTSC. By embedding the villous vCTBs from first-trimester placental tissues (6–7 weeks) in Matrigel containing A83-01, Noggin (a BMP signaling inhibitor), EGF, R-spondin (a Wnt signaling activator), CHIR99021 and prostaglandin E2, sixteen different organoids were established from early human placenta tissues with 100% derivation efficiency.16 These cells could further develop into CTB organoids after 2–3 weeks. These organoids could be sustained for more than 5 months before passage 13 and patterned with apical and basal layers. However, the multiple rows of mononuclear, KRT7+ cells in the outer layers of the organoids appeared like epithelial progenitors with E-cadherin positive signals and CTBs marker HAl1, while the multinucleated inner layers with micro-villous membrane secreting chorionic gonadotrophin beta (CGβ) and deprived of vimentin frankly mimicked STB.16 Markers used to identify villous trophoblast cells, such as transcription factor AP-2 alpha (AP-2α), AP-2γ, and GATA binding protein 3 (GATA3) expressed properly in the CTB organoids and markers of stemness like CDX2, TP63, and TEA domain transcription factor 4 (TEAD4) appeared in the outer layers. E74 like ETS transcription factor 5 (ELF5) is expressed in vCTBs but not in post-mitotic STB cells and invasive EVTs, and the proximal ELF5 promoter was hypo-methylated in the organoids, indicative of vCTBs origins. ELF5 interacts with transcription factors, CDX2 and eomesodermin (EOMES), and the high proliferation ability of ELF5 and CDX2 double-positive cells demarcates a putative TS cell compartment in the early human placenta.36 Withdrawal of R-Spondin and CHIR99021 from organoid cultures decreased the expression of Wnt stem cell receptor (LGR5), the CTBs self-renewal markers ELF5, CDX2, and TP63, also the STB marker ENDOU and CGβ, but increased the EVTs markers HLA-G, manifesting the EVTs differentiation,16 indicating that the hypo-methylation of ELF5 promoter is a vital marker suggestive of hTSC self-renewal ability. Apart from the identification of regular villous trophoblast cell markers, data from RNA-seq also proffered a high similarity of gene expression profiles between CTB organoids and primary human trophoblasts. Not only CTB organoids characterized early vCTB and the overlying layer of STB, but the withdrawal of Wnt also promoted EVT-like outgrowth with an expression of HLA-G at the edge of the organoids.16
In the mouse model, ESC and TSC stem cell-embryos (ETS-embryos) construct within a three-dimensional scaffold of extracellular matrix in a medium that sustains single ESCs and small clumps of TSCs to self-assemble.37 Most of the structures with both ESCs and TSCs formed with a cylindrical architecture composed of single adjoining ESC and TSC compartments. ETS-embryos mimicked formation of embryonic structure and spatiotemporally morphogenetic events in natural embryogenesis. Staining of cell adhesion marker (E-cadherin) indicated the cavitation of ETS-embryos at sequential time points. After 96 hours, both cavities in ESC and TSC compartments united into a whole lumen resembling pro-amniotic cavity. The ESC compartment provided TGF signal for a renewal of TSC compartment. By adding 10 μM Activin/TGF-β receptor inhibitor (SB431542), the TSC compartment failed to cavitate in a vast number of ETS-embryos. Although a small number of ESCs cultured in ECM could form self-assembling rosettes and induce mesoderm commitment, ESC-derived rosettes could foster robust mesoderm induction by interactions with extraembryonic stem cells.
Recently, besides ESC and TSC compartments, extra-embryonic endoderm stem cells were aggregated to form a more embryo-like structure (ETX-embryoids). Not only did this ETX-embryoids exhibit lumenogenesis, asymmetric patterns of gene expression for markers of mesoderm and primordial germ cell precursors, and formation of anterior visceral endoderm-like tissues, they could efficiently initiate implantation with elevated markers from TSC compartment, such as Cb1, Cb2, Igf2, and trigger decidualization with expression of Cox2 and Pl1 in pseudo-pregnant mouse uterus.38 These assembled in vitro models of mammalian embryogenesis might facilitate to understand better how trophectoderm “talks” with other parts of the embryo during post-implantation morphogenesis. Based on this strategy, human EST-embryos or ETX-embryoids in prospect could be assembled under specific in vitro systems and applied to researches retarded by ethical issues in vivo.
Apart from organoids constructed with TSC, placenta endowed with vascular networks offers outstanding opportunities for tissue engineering. Bioartificial organs generated by tissue engineering proffers accessible alternatives for orthotopic transplantation to overcome donor organ shortages and, compared with organoids, macro-scale systems for basic studies. Decellularized human placenta and cotyledons with ample arterial and venous networks were naturally significant for tissue engineering. The vascular structures of stroma were remained intact without gross bulging or weakening and nucleated cells so that it was suitable to be served as scaffolds harboring empty vascular and luminal spaces with ECM protrusions, which could be adopted to generate “hepatized placenta.” More importantly, the integrity of this scaffolds could be stored in saline at 4°C or cryopreserved after lyophilization for months at −80°C, which may facilitate batched production and transportation. After ensuring of the suitability of this scaffolds, including storage at −80°C, trimmed donor sheep hepatic tissues were injected through the capsule and confirmed well perfusion and integrity. To avoid sealing of vascular, before grafted to host, excess placental tissue was trimmed again. After transplantation (up to 45 days), the latex perfused hepatized placenta was verified by casts, and the grafts caused no morbidity or mortality. Subsequently, blood flow perfused into sinusoids of hepatic sheets, and other cell types populated the placental capsule. Moreover, hepatocytes in graft expressed connexon-32, albumin, and contained glycogen which patterned position-specifically to fulfill a hepatic function. Hepatized banked placental grafts making up to 20% by volume of the liver could replace the deficient functions of acute liver failure (ALF) with 85% partial hepatectomy. Seventy-one percent of animals with ALF plus hepatized placenta survived compared to 0% survival rate within a group of ALF without a hepatized placenta.39 This study illustrated that human placenta offers superior organizational scaffold for tissue engineering.
Now the successful establishment of hTSC lines from the human blastocyst and the first-trimester placenta was achieved, and the CTB organoids could mimic the primary placenta villous for the most part, indicating that the hTSC niche could be specified before the blastocyst stage and may be located in the placental villous later. However, the concrete molecular mechanism and detailed development process of human placenta and its different trophoblast subtypes remain poorly understood. How hTSC maintain its self-renewal ability and which signaling is pivotal for the proliferation and directional differentiation of hTSC need further investigation.
The potential signaling pathways in maintenance of the TSCs
Various signaling pathways have been reported to maintain the proliferation of first trimester CTBs. First-trimester CTBs could produce Wnt molecules and express Fzd receptors, while human endometrial cells express various Wnt ligands, suggesting that autocrine and paracrine activation of the Wnt pathway may regulate trophoblast development and function.40–42 Among the different ligands, Wnt5a, secreted by villous and decidual stromal cells, promotes primary CTBs proliferation and survival by activating the MAPK pathway and suppressing camptothecin-induced apoptosis.43 Besides, Notch receptors and ligands expressed in diverse cytotrophoblast populations of the first and second-trimester placenta.44 The Notch receptors (Notch2 and Notch3) and membrane-anchored ligands (delta-like ligand-1 and -4 and Jagged-1 and -2) expressed in vCTBs, cell column trophoblasts (CCTs), and EVTs. Notch1 and Notch4 exclusively expressed in CCTs and vCTBs, respectively.45 While Notch1 acts as a key factor promoting the development of progenitors of the extravillous trophoblast lineage, the function of Notch4 in the hTSC-contained vCTBs remains unclear.46 TGF-β superfamily members are thought to play vital roles in placentation. Investigation of expression and localization of the canonical TGF-b targets Smad2/3 and their regulators (Smad4 and Smad7) in the first-trimester placenta showed that activated pSmad2L was mainly detected in the cytoplasm of vCTBs, while pSmad2C located in the cytoplasm of CCTs and the cytoplasm/nuclei of EVTs.47 All those data demonstrated that complicated signaling networks might play roles in the vCTBs during human placentation. And the successful hTSC model will be very helpful for exploring the concrete roles of signaling pathways in the stemness maintenance of human trophoblasts.
New cell subtypes identified at the maternal-fetal interface
In the first trimester, decidual natural killer (dNK) cells are the main type of lymphocytes fraction at the maternal-fetal interface with over 70% occupation.48,49 dNK cells produce a vast array of growth factors, angiogenic factors, and cytokines, which helps to remodel the decidua and spiral arteries, promote trophoblast invasion and immune tolerance, increase the availability of maternal blood at the implantation site.50–52 Dysregulation of the dNK cells was implicated in pregnancy disorders. Klentzeris et al. showed that endometrial biopsies from the unexplained infertility patients had substantially fewer NK cells than the normal counterparts.53 Research from Fu et al. demonstrated that the reduction of CD56bright CD27+ NK cells would fail to maintain inflammation suppression caused by Th17 cells, thus caused recurrent spontaneous abortions.54 Since the dNK cells are quite different from the peripheral NK cells,48 a better understating of the immunological nature of dNK subsets is an urgent task. Different cell surface makers could be further applied to identify more detailed subgroups of the NK cells. CD49A (also known as ITGA1) and CD9 are well-recognized markers of tissue-resident dNK cells. Combining with additional markers identified by single-cell transcriptome, Vento-Tormo et al. showed that three subtypes of dNK existed in the decidua.55 For dNK1, they contain more cytoplasmic granules and express a panel of specific markers, including CD39, B4GALNT1, and CYP26A1. Meanwhile, dNK1 subset express high-affinity receptor for the dimeric form of HLA-G molecules, LILRB1, indicating the potential regulatory functions of EVTs and making them functionally analogous to the previously found pregnancy trained decidual NK cells, which were characterized by the open chromatin of interferon-gamma and vascular endothelial growth factor (VEGF)-alpha, and higher potential secretion of interferon-gamma and VEGF-alpha.56 However, the rest of the two types like dNK2, with the expression of markers of ANXA1 and ITGB2; and KLRB1, CD160, and CD103 for the dNK3 are less known to us presently. Taken together, the mapping of the molecular patterns helps to illuminate how dNK cells participate in a tolerant microenvironment at the maternal-fetal interface.
Macrophages make up the second-largest cell population and constitute about 20%–30% of leukocytes at the maternal-fetal interface.57 Macrophages could differentiate into given phenotypes with diverse biological functions. By simplified classification, macrophages have been divided into M1 and M2 subtypes based on their activation.22,58 M1 macrophages are characterized by antimicrobial and pro-inflammatory properties, while M2 macrophages are anti-inflammatory.18 M1 macrophages express toll-like receptor-2, toll-like receptor-4, CD80, CD86, and major histocompatibility complex-II surface phenotypes, and release various cytokines and chemokines, such as tumor necrosis factor-alpha, interleukin 6 (IL-6), IL-12, chemokine (C-X-C motif) ligand 9, and chemokine (C-X-C motif) ligand 10.59–61 The proportion and number of M1/M2 macrophages are variable during gestation periods to establish fetal-maternal tolerance, thus protect the fetus from the maternal immune microenvironment. Macrophages are polarized into an alternatively activated state to sustain fetal-maternal tolerance, indicating that the immunosuppressive M2 macrophages are essential for a successful pregnancy.22 Like dNK cells, decidual macrophages cultured in-vitro secreted high level of matrix metalloproteinase 9 and VEGF-alpha,62 indicating that macrophages are important participators in spiral arteries remodeling and trophoblast invasion.63 Also, macrophages act as phagocytes to phagocytose apoptotic cell debris, which is important to promote trophoblast invasion and protect the adjacent cells from overwhelming inflammatory damages.64,65
In the first trimester, 10%–20% of T cells comprise the decidual immune cells.66 They could be further divided into two groups: 40%–75% of cells are CD8+ cytotoxic T lymphocytes, and 30%–45% are CD4+ helper T cells.67–69 The importance of T cells in maintaining pregnancy has become increasingly apparent. Impaired expression of PD-1 and Tim-3 in CD8+ T cells are associated with miscarriage,70 suggesting the important role of CD8+ T cells in normal pregnancy. Simultaneously, CD4+ regulatory T cells (Tregs) could facilitate an invasion of trophoblast and access to the maternal blood supply at early pregnancy.71 Observational studies of human samples have demonstrated the presence of Tregs cells in the human decidua, and the disturbance of Tregs might be a potential cause of human infertility, recurrent spontaneous abortions, and other pregnancy complications.72–76 Meanwhile, the secretion of IL-17 by TH17 was reported to be a mediator of preeclampsia.77–80 During pregnancy, paternally derived alloantigen by the embryo would threaten pregnancy success, while high populations of Tregs in the decidual tissues showed the suppressive effects of Tregs on fetus-specific and nonspecific responses.19,81
The human placenta is composed of a variety of different trophoblastic cell types and vascular networks, which endow the placenta with the functional roles of promoting fetal viability and growth. Floating and anchoring villi consist of the structure of the placenta, constituted of several types of trophoblast cells.82,83 The mononucleated CTBs are progenitor trophoblast cells. CTBs can fuse to form the multinucleated STB cell layer, which covers floating chorionic villi, providing the interface between mother and fetus for gas exchange and nutrient transport.82,84 Alternatively, the proliferative CTBs could also differentiate into the invasive EVTs, anchoring the placenta to the decidua and facilitating the nutrient transfer.85,86 The traditional classification of trophoblast types mainly based on the locations and basic growth features. While how many more subtypes might exist, what are their destinies, and how they perfectly cooperate to form a fabulous placenta, remain a focus ongoing. The RNA-seq technique on the single-cell level has been proved to be a powerful tool to identify new cell types in different tissues.87,88 Based on the transcriptome characteristics of placental cells from first-trimester, three sub-groups of CTBs were dissected out.89 The first subtype exhibited potential of proliferative activity with the high expression of cell cycle-associated genes (such as CDK1, CCNB1, RRM2), indicating they might be the pool of proliferative stem cells.89 While the second subtype showed the least proliferate capacity but were fusion-competent trophoblast cells, with a high expression of Syncytin-2, a sufficient factor to induce cell fusion with a cell that don not express Syncytin-2. Functional prediction indicated that this cell category was associated with pregnancy steroid hormones.89 Previous studies have demonstrated that impairment of the syncytin-mediated fusion of STBs leads to preterm birth, intrauterine growth retardation,90,91 indicating that a dysregulated fusion of the second subtype of CTBs might be origins of some clinical complications. Moreover, the third group showed cell cycle potential while without Syncytin-2 expression, which needs to be further determined. Meanwhile, Liu's study also discovered that different EVT subtypes exist in the 8-week human villi. Based on their transcriptome, EVTs from the 8-week villi could also be divided into three subgroups.89 With a subgroup highly express DNA replication-associated factor RRM2, suggesting that this group have proliferative potential. The receptor activity regulation and immune response characterized other unfamiliar subtypes, which needs further investigation. Accordingly, the elaborated elucidation of the cellular organization of the maternal-fetal interface provides us new insights into the complexity of placentation.
The current knowledge about the human trophoblast differentiation to STB and EVTs are most originate from the evidence from the trophoblast cell lines or primary cultured cells, or the comparative expression patterns between the human and mouse models. The recently established methods for derived hTSC both in 2D or 3D culture will undoubtedly promote the research about hTSC self-renew and differentiation greatly, providing us with a clearer roadmap for the genetic and epigenetic pathway underlying the specification of different trophoblast subtypes. Combining with the in vivo expression pattern, it may pave the way for our understanding of the micro-environment for the trophoblast development. Meanwhile, it has been reported that the stem-cell-based reconstructive embryo-like structure can be used for in vitro culture to post-implantation stage or even can implant into the endometrium. So, it will be a great help if the combination of human ESC and TSC will recapitulate the early human embryo-like structure or post-implantation development observed in mouse since this developmental stage is still a black box for human mainly due to the ethical restrictions. The single-cell sequencing is well developed in recent years and has proven its great help for the study in the fetal-maternal interface. Many different cell types have been identified, and the potential interactions between these cell types were also discovered. For the further study, utilizing the hTSC model and other cells in the fetal-maternal interface, it may be critical to dissect out the function of different interactions between the trophoblast and other maternal or villi core cell types. And this technology can also be applied to the pathological pregnancy, such as the preeclampsia, to explore the mechanism of the disease from the aspect of different cell types. In short, the recently developed hTSC and the single-cell sequencing approach are two main breakthroughs for the placenta research, and the appropriate employment of these approaches will greatly promote our understanding of the blueprint of trophoblast development and interaction of different trophoblast subtypes with the other cell types in the fetal-maternal interface both in physiological and pathological pregnancy.
This work was supported in parts by the National Key R&D Program of China (2017YFC1001402 to H.W., 2018YFC1004102 to J.L.), National Natural Science Foundation of China (81490744 to H.W., 31600945 to J.L. and 31701016 to J.W.), Fujian Natural Science Foundation (2017J01071 to J.L.), the Fundamental Research Funds for the Central Universities (20720180041 J.L.) and Foundation from Key Laboratory of Reproduction Regulation of NPFPC (2017KF01 to J.L.). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.
Conflicts of Interest
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