Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease

Significance

Human pluripotent stem cells (hPSCs) continue to be underappreciated as a model for studying trophoblast differentiation. In this study, we provide a reproducible, two-step protocol by which hPSCs can be differentiated into bipotential cytotrophoblast (CTB) stem-like cells and subsequently into functional, terminally differentiated trophoblasts. In addition, we provide evidence that the response of hPSC-derived CTBs to low oxygen is similar to that of primary CTBs. Finally, using trisomy 21-affected hPSCs, we show, for the first time to our knowledge, that hPSCs can model a trophoblast differentiation defect. We propose that hPSCs are superior to other currently available models for studying human trophoblast differentiation.

Abstract

Trophoblast is the primary epithelial cell type in the placenta, a transient organ required for proper fetal growth and development. Different trophoblast subtypes are responsible for gas/nutrient exchange (syncytiotrophoblasts, STBs) and invasion and maternal vascular remodeling (extravillous trophoblasts, EVTs). Studies of early human placental development are severely hampered by the lack of a representative trophoblast stem cell (TSC) model with the capacity for self-renewal and the ability to differentiate into both STBs and EVTs. Primary cytotrophoblasts (CTBs) isolated from early-gestation (6–8 wk) human placentas are bipotential, a phenotype that is lost with increasing gestational age. We have identified a CDX2⁺/p63⁺ CTB subpopulation in the early postimplantation human placenta that is significantly reduced later in gestation. We describe a reproducible protocol, using defined medium containing bone morphogenetic protein 4 by which human pluripotent stem cells (hPSCs) can be differentiated into CDX2⁺/p63⁺ CTB stem-like cells. These cells can be replated and further differentiated into STB- and EVT-like cells, based on marker expression, hormone secretion, and invasive ability. As in primary CTBs, differentiation of hPSC-derived CTBs in low oxygen leads to reduced human chorionic gonadotropin secretion and STB-associated gene expression, instead promoting differentiation into HLA-G⁺ EVTs in an hypoxia-inducible, factor-dependent manner. To validate further the utility of hPSC-derived CTBs, we demonstrated that differentiation of trisomy 21 (T21) hPSCs recapitulates the delayed CTB maturation and blunted STB differentiation seen in T21 placentae. Collectively, our data suggest that hPSCs are a valuable model of human placental development, enabling us to recapitulate processes that result in both normal and diseased pregnancies.

Trophoblast is the first lineage specified in the developing embryo. Following blastocoel formation, cells segregate into the inner cell mass (ICM), which gives rise to the epiblast and embryo proper; the outer trophectoderm (TE) cells give rise to extraembryonic ectoderm, the epithelial portion of the placenta (1). Much of what we know about this first lineage specification comes from studies in mouse (2–4). In fact, the combination of abilities to derive trophoblast stem cells (TSCs) from mouse blastocysts and to evaluate the compartment-specific contribution of specific genes during embryogenesis has resulted in the identification of numerous transcription factors and signaling pathways involved in TE specification and placental development (2, 3, 5). Results from such studies have led to identification of Cdx2 (caudal type homeobox 2), a member of the caudal-related homeobox transcription factor gene family, the expression of which has been shown to be required for maintenance of TE and TSCs in the mouse embryo (6, 7). Other transcription factors, including Elf5 (Ets domain transcription factor) and Eomes (Eomesodermin), also have been shown to be required for maintenance of the TSC fate in the mouse (8, 9).

Significantly less is known about TE specification and the TSC niche in the human embryo (10, 11). Recent studies have pointed to major differences in gene expression between mouse and human preimplantation embryos (12, 13). One major distinction is the coexpression of CDX2 and POU5F1 (POU domain, class 5, homeobox 1)/OCT4 (octamer-binding protein 4) in the early TE of human embryos, whereas in the mouse embryo these two genes are expressed exclusively in the TE and ICM, respectively, each reciprocally regulating the expression of the other (6, 12, 13). Other differences include the lack of EOMES and ELF5 expression in human TE (13). Based on these findings, the absence of a proliferating trophoblast compartment in the early human embryo (1, 10), and the inability to derive human TSCs from such preimplantation embryos (14), it has been proposed that the human TSC niche may in fact reside in the early postimplantation placenta.

In the absence of a human TSC model, researchers have turned to human pluripotent stem cells (hPSCs). Since 2002, when Xu et al. (15) first published the finding that bone morphogenetic protein 4 (BMP4) induces the expression of trophoblast-related genes in hPSCs, multiple groups have used these cells as a model for studying trophoblast lineage specification (16–22). The majority of these studies, including our own (21), have used BMP4 in the presence of feeder-conditioned medium (FCM), resulting in the expression of some mesoderm markers and therefore generating doubt about the true identity of these hPSC-derived cells (23). Nevertheless, follow-up studies using more defined culture conditions have confirmed the identity of these cells as trophoblasts (20).

Most recently, Lee et al. (24) have proposed criteria for defining trophoblasts based on expression of a set of markers, including ELF5. Although a laudable attempt at standardization, this study fails to account for differences in gene expression across gestational age and falls short of defining syncytiotrophoblasts (STBs) (24). To confirm the utility of hPSCs for modeling trophoblast differentiation, we instead asked whether these cells can recapitulate functional phenotypes of primary trophoblasts during both normal development and disease. We previously have identified p63, a member of the p53 family of nuclear proteins, as a marker specific to proliferative cytotrophoblasts (CTBs) in the human placenta (21, 25, 26). We now have identified a subpopulation of CTBs in the early human placenta that are double-positive for p63 and CDX2; this CTB subpopulation is greatly reduced in the second trimester and is temporally associated with the loss of bipotential differentiation of CTBs (27). In addition, we describe a completely defined culture condition, containing BMP4, by which CDX2⁺/p63⁺ CTB stem-like cells can be efficiently and reproducibly derived from hPSCs. Furthermore, we show that hPSC-derived CTBs respond to low oxygen in a manner similar to primary CTBs. Finally, we provide the first, to our knowledge, proof-of-concept data for the ability of hPSCs to model a trophoblast differentiation defect, using trisomy 21 (T21)-affected hPSCs.

Results

Identification of a CDX2-p63 Double-Positive CTB Population in the Early Human Placenta.

The CTB, the trophoblast layer adjacent to the villous stroma, is the proliferative trophoblast compartment in the human placenta. The CTB layer is continuous in the first trimester and becomes discontinuous starting in the second trimester (10, 11). We previously identified p63 as a pan-CTB marker (25). We now have stained human placenta samples using an antibody to CDX2 and found that in early gestation (6 wk), CDX2, along with p63, was found in the majority of CTBs (Fig. 1A). However, unlike p63, which is maintained in the CTB until term (25), the number of CDX2⁺ CTBs diminished markedly as the first trimester progressed (Fig. 1 B and C). By 9-wk gestation only ∼50% of CTBs were positive for CDX2, and by 20-wk gestation no CDX2⁺ CTBs remained (Fig. 1 B and C).

Fig. 1.

CDX2 and p63 staining in early-gestation placenta tissues. (A, Upper) Immunofluorescence staining of CDX2 and p63. p63 (green) and CDX2 (red) overlap significantly in the CTB of early-gestation human placenta (6 wk). Nuclei were counterstained with DAPI (blue). (Magnification: 10×.) (Lower) Higher magnification (60×) images of the areas indicated by the white boxes in the upper panels. (B) Immunohistochemical staining of CDX2 and p63. p63 is localized in the nuclei of all CTBs, cells adjacent to the villous stroma, across gestation. CDX2, on the other hand, is present in only a subset (∼70%) of CTBs early (5–6 wk) in gestation, decreases significantly by 12-wk gestation, and is virtually absent by 20 wk. (Magnification: 40×.) (C) Quantification of the percent of CDX2 positivity in CTBs. The calculation is based on counting CTBs in five random areas under the 10× objective in each slide.

Generation of CTB Stem-Like Cells from hPSCs.

The majority of hPSC trophoblast differentiation protocols expose the cells to high concentrations of BMP4 (50 ng/mL or higher) in the presence of FCM (Fig. 2A). One previous study developed a minimal medium for trophoblast induction but also used high BMP4 concentrations and focused on the evaluation of CDX2 as the primary TE marker (17). Because high-dose BMP4 also can induce mesoderm (23), we asked whether, in that minimal medium (17), low-dose BMP4 (10 ng/mL) by itself is able to initiate trophoblast differentiation of hPSCs into CTBs, as evaluated by a comprehensive panel of markers, including p63 and CDX2. We initially used WA09/H9 ES cells (ESCs) but also tested this protocol on WA01/H1 and SIVF21 ESCs as well as on induced pluripotent stem cells (iPSCs) derived in the L.C.L. and Y. Liu laboratories, all of which were adapted to StemPro according to the manufacturer’s instructions, with similar results in each case (Fig. S1). We refer to this method as the “two-step protocol” (Fig. 2B).

Fig. 2.

Representations of trophoblast differentiation protocols. (A) Traditional (one-step) trophoblast differentiation protocol using BMP4 in the presence of FCM. This protocol was used by the majority of previous studies for trophoblast differentiation of hPSCs. (B) Our modified (two-step) trophoblast differentiation protocol. Following a 2-d “rest” in StemPro-based minimal medium (EMIM), hPSCs are cultured in EMIM plus 10 ng/mL BMP4 for up to 4 d to form uniformly flattened CTB stem-like colonies. These cells then are trypsinized and replated to be terminally differentiated into STBs and EVTs in FCM plus 10 ng/mL BMP4. H9, H9/WA09 cells; U, undifferentiated hPSCs in StemPro; D0, hPSCs after 2-d rest in EMIM; D1–D4, days after treatment with EMIM+BMP4; CTB+1 to CTB+6, days after replating in FCM+BMP4.

Fig. S1.

Generation of CTB stem-like cells and terminally differentiated trophoblasts from the H1/WA01 (H1) hESC line. (A) Following a 2-d rest in minimal medium, the colonies appear to tighten, and after only 2 d of EMIM-BMP4 treatment cells appear uniformly flattened. (B) qRT-PCR analysis showed that the pluripotency marker OCT4 is decreased and trophoblast-associated genes p63 and CDX2 are significantly increased following EMIM+BMP4 treatment, but the mesoderm-associated marker, Brachyury/T is not. Data were normalized to 18S and are expressed as fold change relative to undifferentiated H1 hESCs. (C) Changes in cell morphology after replating of H1-derived CTB stem-like cells and further differentiation in FCM+BMP4. Cells continued to show a flattened phenotype with numerous multinucleated STB-like cells appearing 4–6 d after replating in this medium. (D) qRT-PCR analysis of replated cells showed CTB markers such as CDX2 and p63 were reduced and differentiation markers such as CGB and HLA-G were induced. Data were normalized to 18S and are expressed as fold change relative to H1-derived CTB stem-like cells. (E) Secretion of total hCG, normalized to DNA content after 2–6 d of terminal differentiation of CTB stem-like cells in FCM+BMP4. All data are expressed as mean ± SD of biological triplicates; *P < 0.05.

In the first step, StemPro-adapted hPSCs were treated with a DMEM/F12-based minimal medium (EMIM) (17) for 2 d and then were cultured in EMIM containing 10 ng/mL BMP4 for up to 4 d (Fig. 2B). After 2 d in EMIM alone, the colonies appeared to tighten and become better delineated; following BMP4 treatment, cells uniformly adopted a more flattened appearance but continued to grow to near confluence by day 4 (Fig. 3A). We then proceeded to evaluate markers of undifferentiated CTBs by immunostaining: Compared with undifferentiated hPSCs, BMP4-treated cells showed loss of the pluripotency marker POU5F1/OCT4; conversely, the CTB-associated genes p63, CDX2, TEAD4 (TEA domain family member 4), KRT7 (keratin, type II cytoskeletal 7), and EGFR (EGF receptor) were all highly expressed in the hPSC-derived CTBs (Fig. 3B). In addition, YAP (Yes-associated protein) was localized to the nuclei of these hPSC-derived CTBs (Fig. 3B). After 4 d of BMP4 treatment, more than 95% of the cells were positive for both p63 and EGFR as measured by flow cytometry (Fig. 3C). Next, we investigated gene expression during CTB induction of hPSCs. Analysis by quantitative RT-PCR (qRT-PCR) showed that the expression of POU5F1/OCT4 decreased and the expression of p63, CDX2, and KRT7 increased significantly following BMP4 treatment (Fig. 3D). In addition, the mouse TSC-associated marker ELF5 was induced twofold, whereas the mesoderm-associated marker T/BRACHYURY was reduced from the basal levels present in StemPro-adapted cells and was not induced by low-dose BMP4 treatment (Fig. 3D). These data indicate that, with only 4 d of low-dose BMP4 treatment in this defined medium, hPSCs uniformly assume a CTB stem-like phenotype. Interestingly, as in isolated primary CTBs but unlike p63⁺ CTBs in vivo (26), hPSC-derived CTBs were not highly proliferative and could not be propagated under these culture conditions (Fig. S2).

Fig. 3.

Morphology and marker expression of hPSC-derived CTB stem-like cells. (A) Following a 2-d rest period in StemPro-based basal medium (EMIM), the colonies appear to tighten, and after 4 d of EMIM-BMP4 treatment cells appear uniformly flattened. (B) StemPro-adapted H9/WA09 (H9) cells show high expression of the pluripotency marker POU5F1/OCT4, but after 4 d of BMP4 treatment in EMIM the cells lose POU5F1/OCT4 and uniformly express CTB markers, including p63, CDX2, TEAD4, KRT7, and EGFR. YAP was localized to the nuclei of EMIM-BMP4–treated cells as well. Nuclei were counterstained with DAPI (blue). (C) Flow cytometric analysis of StemPro-adapted undifferentiated H9 cells and H9-derived CTB stem-like cells. Almost 95% of cells were positive for both p63 and EGFR following 4 d of EMIM-BMP4 treatment. (D) Gene-expression changes shown by qRT-PCR, during step 1 of the differentiation protocol, induction of the CTB stem-like cells. The pluripotency marker POU5F1/OCT4 is decreased, and the trophoblast-associated genes p63, CDX2, KRT7, and ELF5 are increased following EMIM-BMP4 treatment. The mesoderm-associated marker Brachyury/T was not induced during EMIM-BMP4 treatment; in fact, basal levels in StemPro-adapted H9 cells were markedly reduced following this differentiation. Data were normalized to 18S and are expressed as fold change relative to undifferentiated H9 cells. Data are expressed as means ± SD of biological triplicates; *P < 0.05 in comparison with values in StemPro-adapted undifferentiated H9 cells.

Fig. S2.

Proliferative capacity of hESC-derived CTB stem-like cells. H9/WA09 hESCs were differentiated into CTB stem-like cells following a 2-d rest in minimal medium and 4 d of EMIM+BMP4 treatment. Cells were fixed and stained with Ki67 and p63 and counterstained with DAPI.

Differentiation Capacity of hPSC-Derived CTBs.

To determine the multipotency of hPSC-derived CTBs, we tested their ability to differentiate terminally into both STBs and extravillous trophoblasts (EVTs). Because the cells reach confluency at day 4 after treatment with EMIM-BMP4, we attempted to replate the cells at a lower density (∼2–4 × 10⁴ cells/cm²). To replate hPSC-derived CTBs, we tested different cell-detachment methods, including trypsin, StemDS, and EDTA; we obtained the most consistent results with trypsin. We also tested replating on Geltrex both in the minimal medium EMIM and in FCM and found that cells plated significantly better in the presence of FCM (Fig. S3A).

Fig. S3.

FCM is required for optimal terminal trophoblast differentiation. H9/WA09 hESCs were differentiated into CTB stem-like cells and then were replated in EMIM+BMP4 or FCM+BMP4. (A) Cells were imaged at day 1 after plating, showing significantly fewer cells plated in EMIM. (B–D) qPCR (B), HLA-G FACS (C), and hCG ELISA (D) were performed on cells at day 4 after replating. hCG secretion was normalized to DNA content. Data are expressed as mean ± SD of biological triplicates; *P < 0.05.

After replating and culture in FCM supplemented with 10 ng/mL human BMP4 (FCM+BMP4), the cells continued to differentiate (Fig. 4). Morphologically, 4–6 d after FCM+BMP4 treatment, cells continued to show an epithelial (flattened) phenotype with numerous multinucleated cells (Fig. 4A). Based on qRT-PCR analysis, CTB markers such as CDX2 and p63 were reduced, and differentiation markers, including CGB (chorionic gonadotropin subunit beta; an STB marker) and HLA-G (an EVT marker), were induced (Fig. 4B). Loss of CTB markers was further evident by immunostaining, showing loss of expression of CDX2, TEAD4, and YAP following culture in FCM-BMP4 (Fig. 4C). Functional trophoblast differentiation was confirmed by the secretion of hCG (human CG, the pregnancy hormone and a marker of STBs) and MMP2 (matrix metalloproteinase-2, an EVT marker) (Fig. 4D). In addition, we confirmed the presence of invasive HLA-G⁺ EVTs using a Matrigel invasion assay followed by immunostaining (Fig. 4E). The presence of FCM appeared to be optimal for differentiation, because differentiation in minimal medium with BMP4 resulted in significantly lower levels of differentiation markers, including hCG secretion and surface HLA-G⁺ cells (Fig. S3 B–D). Together, based on both marker expression and function, these data confirm the bipotential differentiation ability of hPSC-derived CTBs in FCM+BMP4.

Fig. 4.

Step 2: differentiation of H9/WA09 (H9)-derived CTB stem-like cells into STBs and EVTs. (A) Cell morphologic changes after replating of H9-derived CTB stem-like cells and further differentiation in FCM+BMP4. Cells continued to show a flattened phenotype with numerous multinucleated STB-like cells appearing around 4 d after replating in this medium. (B) qRT-PCR analysis showed CTB markers such as CDX2 and p63 were reduced and differentiation markers such as CGB (in STBs) and HLA-G (in EVTs) were induced. Data were normalized to 18S and are expressed as fold change relative to H9-derived CTB stem-like cells. Data are expressed as means ± SD of biological triplicates; *P < 0.05 in comparison with the values of CTB stem-like cells. (C) Immunostaining of replated H9-derived CTBs shows the loss of CTB markers such as CDX2, TEAD4, and YAP following treatment with FCM+BMP4. Nuclei were counterstained with DAPI (blue). (D) Secretion of total hCG and MMP2 after 2–6 d of terminal differentiation of CTB stem-like cells in FCM+BMP4. Data were normalized to total DNA content and are expressed as means ± SD of biological triplicates; *P < 0.05 in comparison with the values of CTB stem-like cells. (E) Matrigel invasion assay. StemPro-adapted H9 cells and H9-derived CTBs were plated on Matrigel-coated membranes with 8-μm pores. After 6 d of further culture in StemPro (H9 cells) or FCM+BMP4 (H9-derived CTBs), cells that had invaded the underside of the membranes in the invasion chamber were immunostained for KRT7 (red) and HLA-G (green). Nuclei were counterstained with DAPI (blue).

Behavior of hPSC-Derived CTBs in Low Oxygen.

The effect of low oxygen on the differentiation of STBs has been well established. Specifically, the production of hCG- and STB-associated transcripts is known to be inhibited by low oxygen tension (28, 29). We replated hPSC-derived CTBs in either 20% or 2% oxygen and continued to differentiate them in FCM+BMP4 (step 2). Under 2% oxygen, the cells produced significantly less hCG (Fig. 5A and Fig. S4A); they also showed a significant decrease in the expression of STB-associated transcripts, including CGA (chorionic gonadotropin subunit alpha), CGB, and PSG4 (pregnancy-specific β-1-glycoprotein 4) (Fig. 5B).

Fig. 5.

Low oxygen tension inhibits the differentiation of hESC-derived CTBs into STBs. (A) Decreased hCG secretion at 2, 3, and 4 d after replating of CTBs in 2% oxygen compared with CTBs replated in 20% oxygen, as measured by ELISA and normalized to DNA content. (B) qRT-PCR analysis of the STB markers CGA, CGB, and PSG4. Data were normalized to 18S and are expressed as fold change relative to hESC-derived CTB stem-like cells. All data are expressed as means ± SD of biological triplicates; *P < 0.05 in comparison of cells in 20% oxygen and cells in 2% oxygen on the same day.

Fig. S4.

Response of SIVF-disomy hESC-derived CTBs to hypoxia and ARNT knockdown. (A) Decreased hCG secretion 4 d after CTBs were replated in 2% oxygen as compared with cells replated in 20% oxygen, measured by ELISA and normalized to DNA content. (B) qRT-PCR analysis of HIF complex targets in scramble or shARNT-infected SIVF-disomy hESC-derived CTBs 2 d after replating in 20% vs. 2% oxygen. shARNT-infected cells show blunted induction in 2% oxygen. Data were normalized to 18S and are expressed as fold change relative to scramble/20% oxygen. (C) Surface expression of EGFR (a CTB marker) and HLA-G (an EVT marker) was analyzed by flow cytometry of scramble and shARNT-infected SIVF-disomy hESC-derived CTBs replated in 20% or 2% oxygen for 2 d. All samples are compared with isotype. (D) Two days after CTB stem-like cells were replated in 2% oxygen, the EVT marker HLA-G is increased in scramble control cells but not in shARNT-infected cells. Data were normalized to 18S and are expressed as fold change relative to scramble control samples in 20% oxygen. Data are expressed as mean ± SD of biological triplicates. *P < 0.05.

Culture under low oxygen tension is also known to promote the initial differentiation of CTBs into HLA-G⁺ EVTs (30), although further differentiation into fully mature, invasive EVTs is inhibited (31); in addition, an intact hypoxia-inducible factor (HIF), a transcription factor complex mediating a significant portion of the cellular response to low oxygen (32), has been shown to be required for the differentiation of rodent TSCs into trophoblast giant cells, which are analogous to human EVTs (33, 34). To test the effect of different oxygen tensions on EVT differentiation, we generated hESCs stably expressing either a scrambled shRNA or shRNA specific to ARNT (aryl hydrocarbon receptor nuclear translocator; also called “HIF1β”); which is required for the formation of an intact HIF complex (32). We used two different hESC lines, H9/WA09 and SIVF-disomy hESCs (see below). ARNT-null hESCs showed significantly reduced expression of ARNT at the protein level (Fig. 6A), although they still stabilized HIF1α under low oxygen conditions (Fig. 6B). They showed a blunted response to culture in low oxygen, as demonstrated by decreased induction of HIF-dependent genes (Fig. 6C and Fig. S4B). Also, although CTBs derived from scrambled shRNA-expressing hESC showed a doubling of HLA-G⁺ cells under 2% oxygen as measured by flow cytometry, ARNT-null hESC-derived CTBs maintained the same proportion of HLA-G⁺ cells in both 2% and 20% oxygen (Fig. 6D and Fig. S4C). This finding was not the result of improved plating, because hESC-derived CTBs showed similar replating efficiency in both 20% and 2% oxygen (Fig. S5). In addition, under 2% oxygen, ARNT-null hESC-derived CTBs also had decreased secretion og MMP2 (Fig. 6E), a marker specific to HLA-G⁺ EVTs in the human placenta (35, 36). Finally, under 2% oxygen, ARNT-null hESC-derived CTBs showed a significantly lower induction of the EVT-associated genes HLA-G and HTRA4 (high-temperature requirement protein A4) (Fig. 6F and Fig. S4D) (37).

Fig. 6.

Low oxygen tension induces EVT differentiation in an HIF complex-dependent manner. (A) Western blot for ARNT in undifferentiated hESCs stably infected with lentivirus expressing either scrambled shRNA or ARNT-specific shRNA (shARNT). (B) Western blot for HIF1α in hESCs stably infected with lentivirus expressing either scrambled shRNA or ARNT-specific shRNA, differentiated into CTBs, and then replated and cultured in 2% oxygen for the indicated number of hours. (C) qRT-PCR analysis of HIF target genes in CTBs, derived from either scrambled- or ARNT-specific shRNA-infected hESC, at day 4 postdifferentiation in FCM+BMP4 (day CTB+4) in 20% or 2% oxygen. Data were normalized to 18S and are expressed as fold change relative to scrambled/20% oxygen. (D) Surface expression of HLA-G (an EVT marker) was analyzed by flow cytometry of scrambled and shARNT-infected hESC-derived CTBs cultured in 20% or 2% oxygen for 2 d after replating. All samples are compared with isotype. (E) Increased MMP2 secretion at 2 (CTB+2) and 3 (CTB+3) d after replating of scrambled shRNA- and shARNT-infected hESC-derived CTBs in 2% oxygen as measured by ELISA and normalized to DNA content. (F) qRT-PCR analysis of EVT-associated genes HLA-G and HTRA4 in scrambled and shARNT-infected hESC-derived CTBs 2 d after replating in 20% or 2% oxygen. Data were normalized to 18S and are expressed as fold change relative to scrambled/20% oxygen. All data are expressed as means ± SD of biological triplicates; *P < 0.05.

Fig. S5.

Effect of oxygen tension on replating of hESC-derived CTBs. WA09/H9 hESCs were differentiated into CTB stem-like cells, replated in FCM+BMP4 in either 20% or 2% oxygen, and counted. Data are expressed as mean ± SD of biological triplicates. The difference between the two conditions was not statistically significant.

T21: Comparison of Primary and hPSC-Derived CTBs in the Context of Disease.

T21 is a common aneuploidy known mostly for associated fetal malformations, including structural cardiac defects and neurodevelopmental delay. However, T21 placentas also show developmental abnormalities, including prolonged maintenance of a continuous CTB layer well into the second trimester and abnormalities of STB formation (38–41). We evaluated six T21 placentas and five gestational age-matched euploid placentas by CDX2 immunohistochemistry and found that T21 chorionic villi contained a higher percentage of CDX2⁺ CTBs (Fig. 7 A and B). We next asked whether T21-affected hPSCs showed any defects in trophoblast differentiation. We used two T21 hPSC lines: one hESC line (SIVF21) and one iPSC line (C1); as control in each case we used a subclone of the same cell line that had lost its extra copy of chromosome 21 (the SIVF21-disomy hESC line and the C5 iPSC line, respectively). T21-hPSCs showed a delay in the induction of the trophoblast lineage, as measured by surface EGFR expression (Fig. 7C and Fig. S6). In addition, T21-hPSCs also showed a slower decrease in the expression of the pluripotency factor POU5F1/OCT4 and an exaggerated induction of CDX2 (Fig. 7D).

Fig. 7.

Both T21 placenta and T21-hPSC–derived CTBs showed delay in differentiation to CTBs. (A) Six T21 and five normal (euploid) placentas, all between 14- and 15-wk gestational age, were immunostained for CDX2. Representative micrographs show a continuous layer of CDX2⁺ CTBs in T21 villi and fewer such cells in normal villi. (B) For each of the areas of the placenta shown in A, five random areas were selected. CDX2-positive and -negative CTBs were quantified. Data are expressed as means ± SD; *P < 0.05. (C) Flow cytometry showed maximum expression of EGFR (a surface CTB marker) on day 6 in T21-hPSCs and on day 4 in the disomy control hPSCs, showing the delay in CTB induction in the T2-hPSCs. All results are shown in comparison with the isotype control. (D) Based on qRT-PCR, T21 cells showed a delay both in the reduction of the pluripotency marker POU5F1/OCT4 and in the induction of the CTB marker CDX2. CDX2 levels also were higher and were maintained longer in T21-hPSCs. Data were normalized to 18S and are expressed as fold change relative to disomy day 0. Data are expressed as means ± SD of biological triplicates; *P < 0.05.

Fig. S6.

BMP4-induced trophoblast differentiation of disomy (C5) and T21 (C1) isogenic iPSC lines. (A) Flow cytometry showed maximum expression of EGFR (a CTB marker) on day 6 in C1 (T21) and on day 4 in the C5 (disomy control) iPSCs, showing a delay in CTB induction in C1 cells. All samples are compared with their respective isotype. (B) qRT-PCR of cells during the first step of trophoblast differentiation shows a reduction of the pluripotency marker POU5F1/OCT4 in both iPSC lines but a higher induction of CDX2 in T21-iPSCs. Data were normalized to 18S and are expressed as fold change relative to disomy day 0. (C) qRT-PCR of cells during the second step of trophoblast differentiation showed higher levels of CGA and CGB in T21-iPSCs. (D) hCG secretion was slightly higher in the T21-iPSCs, but the difference was not statistically significant. Data were normalized to the total DNA content. (E) Both T21- and disomic iPSCs were differentiated into CTBs (4 and 6 d of EMIM+BMP4 treatment for disomy and T21-iPSCs, respectively). Four days after replating of these iPSC-derived CTBs, cells were fixed and stained with ZO-1 (red) and DAPI (blue), and the fusion index was calculated. T21-iPSCs showed a reduction in fusion index. All data are expressed as mean ± SD of biological triplicates; *P < 0.05.

During the second step of trophoblast differentiation, T21-hPSC–derived CTBs showed maintenance of the CTB markers CDX2 and ELF5 (Fig. 8A). We determined the fusion index of the differentiating cells by staining for ZO-1 (zona occludens protein 1) and DAPI and counting both the number of nuclei and the number of multinucleated STBs (Fig. 8 B and C). CTBs derived T21-hPSCs from showed a lower fusion index (Fig. 8 B and C and Fig. S6); they also showed increased hCG secretion and altered expression of the transcripts of the hCG components CGA and CGB (Fig. 8 D and E and Fig. S6). Finally, Activin-A has been shown to rescue the fusion defect in primary T21 CTBs (40); we therefore treated CTBs derived from T21-hPSCs with Activin-A during the second step of trophoblast differentiation and found that this treatment restored the fusion index of these cells to the level in the disomy cells (Fig. 8F).

Fig. 8.

T21-hPSC–derived CTBs showed blunted fusion, which can be rescued morphologically by Activin A treatment. (A) For the second step, T21 CTBs showed delay in the loss of the CTB markers CDX2 and ELF5. Data were normalized to 18S and are expressed as fold change relative to disomy day CTB+2. (B) Both T21 and disomy hESCs were differentiated into CTBs (with 4 and 6 d of EMIM+BMP4 treatment for normal and T21-hPSCs, respectively). Four days after the hESC-derived CTBs were replated, cells were fixed and stained with ZO-1 (red) and DAPI (blue). (C) Cells and nuclei were counted in five random areas of the slide, and the fusion index was calculated by the indicated formula. (D) By qRT-PCR, CGA was higher in T21 hESCs, but CGB expression was similar in both lines. (E) hCG secretion was higher in T21-hESC–derived trophoblasts. Data were normalized to the total DNA content. (F) Both T21 and disomy hESCs were differentiated into CTBs (with 4 and 6 d of EMIM+BMP4 treatment for normal and T21-hESCs, respectively). The CTBs then were replated and cultured in FCM+BMP4 with or without Activin A for 4 d. Cells were fixed again and stained with ZO-1 and DAPI, and the fusion index was calculated as stated above. All data are expressed as mean ± SD; *P < 0.05.

Discussion

Previous studies of trophoblast differentiation of hPSCs have focused on the induction of CDX2, a transcription factor known to be required for the maintenance of TSCs in the mouse (6, 7) and that also is expressed in the early human TE (12, 13). However, because CDX2 is also a marker of early mesendoderm precursors (23), and because short-term, high-dose BMP4 treatment is known to induce this lineage (42), assessment of cells using this marker alone is insufficient to establish TE identity. We have identified another marker, p63, which identifies proliferative CTBs in the early human placenta (21, 25, 26). p63 (specifically, the N-terminal–truncated form of this protein known as “∆Np63”) has been identified previously as a marker of stem cells in stratified epithelia (43) and also is known to be induced by BMP4 during epidermal differentiation of pluripotent stem cells of both mouse and human origin (44). In this study we confirm that the early human placenta is composed of a population of CDX2⁺-p63⁺ double-positive CTBs, which we propose as the CTB “stem” cell compartment. p63 has been shown to be enriched, at least at the RNA level, in even earlier stages of human trophoblast, specifically, in the TE of human blastocysts (45). Therefore it is possible that this CDX2⁺-p63⁺ double-positive population may emerge in the human blastocyst and persist in a subset of trophoblasts in the early postimplantation placenta.

This finding is significant, particularly because of the controversy surrounding the BMP4-based model of trophoblast induction of hPSCs. These models have been questioned, based mostly on observations in the mouse system, in which signaling through BMP receptors appears to be required only for mesoderm induction and not for trophoblast lineage specification (23, 46). The identification of BMP4-inducible p63, which is dispensable for mouse placentation (47), as an early marker of human proliferative CTB, may explain why BMP4 induces the TE lineage in hPSCs but appears not to be required for the same process in mice (46).

Here we outline a simple two-step protocol by which we can reproducibly generate first a pure population of CDX2⁺/p63⁺ CTB stem-like cells and subsequently a combination of both hCG-producing STBs and MMP2-producing, invasive EVTs. This protocol is superior to previously described protocols in several ways. First, it is a robust protocol that uses low-dose BMP4 to generate a uniform population of CTB stem-like cells, consistently, in a short period from StemPro-adapted hPSCs, without induction of the mesoderm marker T/BRACHYURY. We routinely performed flow cytometric analysis, using EGFR as a marker of CTBs, to monitor the day on which surface expression of this marker peaks and to assign those cells as hPSC-derived CTBs before replating for the second step. Second, the ability to replate the hPSC-derived CTBs allows large numbers of differentiated trophoblasts to be obtained starting with a relatively small number of undifferentiated hPSCs. Finally, and perhaps most importantly, unlike previous one-step protocols (including both FCM+BMP4 and BAP), our two-step protocol gives us the ability to separate the study of trophoblast lineage specification (hPSC → CTB) from terminal trophoblast differentiation (CTB → STB and EVT). This ability is important, particularly because it allows the characterization of different stages of trophoblast differentiation and will improve comparative studies with primary cells.

One of the major reasons hPSCs have not been widely used for studying early implantation events has been the paucity of data regarding their ability to model human trophoblast differentiation during both normal development and disease. One recent study showed blunted trophoblast differentiation of hESCs carrying an unbalanced chromosomal translocation t (11, 22) after BMP4 treatment (48). Because this translocation is commonly associated with miscarriage, the authors argued that the hESCs carrying this chromosomal aberration are a good model for studying implantation failure (48). However, because the mechanism of implantation failure in these cases is not known, the model could not be validated further. We decided to evaluate two well-established phenomena in trophoblast differentiation: the response of CTBs to low oxygen tension and the differentiation defects of T21-affected CTBs.

The culture of primary CTBs in low oxygen is known to inhibit hCG production and reduce the expression of STB-associated genes (28, 29), a phenotype that was replicated with hPSC-derived CTBs in our study. The effect of low oxygen on EVT differentiation is more complex. Low oxygen tension appears to induce the first step of EVT differentiation, seen as the expansion of the HLA-G⁺ cell column EVTs, in both first-trimester human placental explants (31, 49) and isolated primary trophoblasts (30). However, differentiation into mature, invasive EVT appears to be thwarted in low oxygen (29, 49, 50). Using our two-step protocol, we evaluated the first step of EVT differentiation, which involves the induction of HLA-G expression, and found that in low (2%) oxygen the surface HLA-G⁺ cells were expanded in a HIF complex-dependent manner. We could not evaluate the second step of EVT differentiation (i.e., invasion) independently, because we could not replate the HLA-G⁺ cells.

We next turned to another well-studied model of a human trophoblast differentiation defect: T21. As mentioned previously, it is known that T21 placentas maintain a continuous layer of CTBs well into the second trimester (38). We evaluated these placentas further by immunohistochemistry and noted that CDX2 is maintained longer in the CTBs of T21 placentas than in euploid placentas. Interestingly, with BMP4 treatment, T21-hPSCs induced CDX2 at much higher levels and also maintained this marker longer after the induction of terminal trophoblast differentiation (step 2). More importantly, and again similar to previous studies using primary T21 CTBs (39–41), T21-hPSC–derived CTBs showed a defect in the formation of multinucleated STBs, which was alleviated with Activin-A treatment. These data show, for the first time to our knowledge, a well-established trophoblast differentiation defect that can be recapitulated using hPSCs.

There remain some limitations to the use of hPSCs for modeling trophoblast differentiation. In particular, to date we have been unable to maintain the p63⁺/CDX2⁺ CTBs in an undifferentiated state. Several factors may be at play here, including suboptimal culture conditions and/or insufficient expression of factors promoting self-renewal. With respect to the former, we are in the process of performing small-molecule screens and have identified factors that maintain p63 and CDX2; nevertheless, these factors do not appear to be sufficient for maintaining self-renewal in this cell population. This result has led us to conclude that other factors are likely required for CTB stem cell maintenance. Candidates for such factors, insufficiently induced following BMP4 treatment of hPSCs, include ELF5, an imprinted gene and a member of the Ets family of transcription factors, which is required for maintenance of the TSC compartment in mice (51). In mice, the Elf5 promoter is hypomethylated specifically in TSCs, in which this gene is expressed, and is hypermethylated in ESCs, in which the gene is silent (9). ELF5 is expressed in human trophoblasts of the early placenta, where its promoter has been shown to be hypomethylated (52). In contrast, ELF5 is not expressed in the TE of the human preimplantation blastocyst (13). As in previous studies (20), we do see induction of ELF5 following BMP4 treatment; however, this induction is weak (1.5- to twofold in all lines tested) and does not reach the levels of expression seen in postimplantation CTBs (24). Therefore, it is possible that the hPSC-derived CTBs are more similar to preimplantation TE than to early postimplantation CTBs. In addition, the variability of expression of imprinted genes in different hPSC lines has been described in both the undifferentiated and differentiated states and has been attributed mostly to epigenetic instability during derivation and culture (53, 54). For this reason, it is likely that the culture conditions for both the propagation of hPSCs and their subsequent differentiation into trophoblasts must be optimized in a way that promotes the expression of the trophoblast-associated imprinted genes, such as ELF5. Finally, high expression of ELF5 has been noted recently in new hESC lines derived from earlier-stage human embryos. These lines have an enhanced ability to differentiate into trophoblast and reportedly maintain the TSC state (55); thus, it is also possible that only early-stage embryos have the correct epigenetic landscape for the derivation and maintenance of true human TSCs.

We also are still in the process of defining the culture conditions required for inducing pure subpopulations of either STBs or EVTs. Under the current culture conditions, HLA-G⁺ cells appear to peak 2 d after the replating of hPSC-derived CTBs in FCM+BMP4, whereas the number of multinucleated hCG-secreting cells peaks 4 d after replating. That these cell subpopulations peak at different times and diminish soon thereafter may indicate that they are competing with each other and/or that the culture conditions are suboptimal for their maintenance and maturation. Additional optimization of culture conditions, including changes in extracellular matrix and 3D culture, may be required to enhance further the utility of hPSC-derived trophoblasts as a model. Overall, however, given the proper expression of many trophoblast-associated genes, in proper progression from CTB induction to STB/EVT differentiation, the proper response of the CTB stem-like cells to low oxygen, and the ability to recapitulate an STB differentiation defect with T21-affected iPSCs, we maintain that the BMP4-based protocol, particularly the two-step protocol described herein, provides a bona fide model for the study of early human trophoblast differentiation. Currently, the only other human model that provides a bipotential trophoblast progenitor cell population is primary CTBs isolated from first-trimester placental tissue, to which access is highly limited. Other human trophoblast cell lines, whether derived from choriocarcinoma (trophoblastic tumor) tissue or isolated primary trophoblasts, represent previously committed trophoblast lineages (either villous or extravillous) and thus do not provide a platform for evaluating lineage segregation and TSC fate decisions. Our two-step protocol presents a system for studying both the initial specification of the human trophoblast lineage and subsequent differentiation into both villous and extravillous lineages. We therefore propose that, except in primary cells, our two-step protocol is superior to other currently available models for studying early human trophoblast differentiation.

Materials and Methods

hPSC Culture and Differentiation.

The hPSC aspect of the research was performed under a protocol approved by the University of California, San Diego Institutional Review Board and Embryonic Stem Cell Research Oversight Committee. Before differentiation, ESCs (WA09/H9 and WA01/H1 lines from WiCell and SIVF21 trisomy and disomy lines) and iPSCs [C1 (T21) and C5 (disomy) lines] (56) were cultured under feeder-free conditions in StemPro (Invitrogen) medium with 12 ng/mL recombinant basic FGF (bFGF) (Invitrogen) on plates coated with Geltrex (1:200; BD Biosciences). In the first step, cells were plated relatively sparsely (8 × 10³/cm² for WA09/H9 and 10⁵/cm² for SIVF, C1, and C5 cell lines). Cells were passaged with StemDS (ScienCell Research Laboratories) or Accutase (Life Technology) every 3–5 d. Only passages 10–20 post-StemPro adaptation were used for all experiments described here. For the first phase of TE differentiation, hPSCs were first treated with a StemPro (Thermo Fisher)-based basal medium [KnockOut DMEM (Thermo Fisher)/F12 containing 1% insulin-transferrin-selenium A, 1× nonessential amino acid, 2% (wt/vol) BSA, 2 mM l-glutamine, 100 ng/mL heparin sulfate] designated as “EMIM” (17) for 2 d. The cells then were switched to EMIM medium containing 10 ng/mL human BMP4 (R&D Systems) for 2–4 d, at which point they were designated hPSC-derived CTBs. For terminal differentiation, the hPSC-derived CTBs were passaged using 0.05% trypsin and were replated on Geltrex-coated plates in FCM [KnockOut DMEM/F12 containing 20% (vol/vol) KnockOut serum replacement (Knockout SR; Thermo Fisher), 1× GlutaMAX (Thermo Fisher), and 1× nonessential amino acid, 0.1 mM 2-mercaptoethanol; conditioned on irradiated mouse embryonic fibroblasts for 24 h], supplemented with 10 ng/mL human BMP4 for the indicated number of days. For Activin-A rescue experiments, in addition to FCM+BMP4, the cells were treated with 5 ng/mL Activin A (Stemgent). For culture under low oxygen, cells were incubated in an Xvivo system under 2% oxygen (BioSpherix). For all experiments in hypoxia, the medium was changed, and cells were lysed (for RNA or protein analysis) or fixed (for flow cytometry) in the Xvivo work chamber under 2% oxygen.

Generation of ARNT-Knockdown hESC Lines.

pLKO.1-based scrambled shRNA was obtained from Addgene. A scrambled shRNA and a set of five Mission shRNA Lentiviral constructs targeting the human ARNT gene were purchased and packaged for transduction according to the manufacturer’s instructions (Sigm-Aldrich a). HEK293FT cells were transfected with the shRNA constructs Delta 8.2 and VSV-G using Lipofectamine 2000 (Sigma-Aldrich) according to the manufacturer’s instructions. Lentiviral supernatants were concentrated with PEG-it virus precipitation solution (System Biosciences). The concentrated viral particles then were incubated with target cells in the presence of 10 mg/mL Polybrene (Sigma-Aldrich). Packaging and infection efficiency were tested using a GFP-expressing lentivirus (in the same pCMV-lentiviral construct used for ARNT). Of the five ARNT-specific shRNA constructs, three worked best, based on protein knockdown in HEK293T cells; therefore, subsequent experiments were performed with this subset of three shRNAs pooled together. hESCs were stably infected; after 3 d of infection, cells were treated with 5 μg/mL puromycin (Sigma-Aldrich) to select cells stably transduced over at least three passages. Infection efficiency was determined by GFP expression. Knockdown efficiency was determined by Western blot.

Human Placental Tissue Collection and Immunohistochemistry.

Human placental tissues were collected under a protocol approved by the Human Research Protections Program Committee of the University of California, San Diego Institutional Review Board; all patients gave informed consent for the collection and use of these tissues. Serial sections of formalin-fixed, paraffin-embedded placental tissues were stained with mouse anti-p40 (∆Np63-specific antibody, clone BC28; Biocare Medical) and rabbit anti-CDX2 (clone EPR2764Y; Abcam) antibodies, using a Ventana Discovery Ultra automated immunostainer with standard antigen retrieval and reagents per the manufacturer’s protocol.

Flow Cytometric Analysis.

For flow cytometry, cells were fixed and permeabilized with either 4% (wt/vol) paraformaldehyde for 10–15 min or with 100% ice-cold methanol for 30 min and then were washed three times in PBS supplemented with 10% (vol/vol) FBS. Cells then were incubated at room temperature for 1 h in flow cytometry (FC) buffer (0.5% BSA and 1% FBS in PBS) with an allophycocyanin (APC)-conjugated mouse anti-human EGFR antibody (clone AY13; BioLegend), phycoerythrin (PE)-conjugated mouse anti–HLA-G (MEM-G/9; EXBIO), or FITC-conjugated mouse anti-human p63 antibody (clone 4A4; Santa Cruz). APC- or PE-conjugated mouse IgG (clone MPOC-21; BioLegend), or FITC-conjugated mouse IgG (Santa Cruz) was used as isotype IgG control. Cells were washed three times with FC buffer, and analysis was carried out using a BD FACS-Canto Flow cytometer.

Immunostaining of Cells.

For immunofluorescence staining, cells grown on Geltrex-coated coverslips were fixed with 4% (wt/vol) paraformaldehyde in PBS at room temperature for 10 min. Cells then were permeabilized with 0.3% Triton X-100 for 10 min and incubated with primary antibodies, including mouse anti-p63 (4A4 clone; Sigma-Aldrich), rabbit anti-Ki67 (ab15580; Abcam), mouse anti-KRT7 (clone OV-TL 12/30; Invitrogen), mouse anti-YAP (sc-15407; Santa Cruz), rabbit anti-EGFR (sc-03; Santa Cruz), rabbit anti-TEAD4 (HPA056896; Abcam), rabbit anti-CDX2 (clone EPR2764Y; Abcam), rabbit anti-OCT4 (ab19857; Abcam), rabbit anti–ZO-1 (ab59720; Abcam), or mouse anti–HLA-G (clone 4H84; Abcam) and were visualized by Alexa Fluor 488- or 595-conjugated goat secondary antibodies (Invitrogen). For nuclear staining, cells were incubated with DAPI (Invitrogen) for 5 min.

RNA Isolation and Quantitative Real-Time PCR.

Total RNA was extracted using either the mirVana RNA Isolation Kit (Ambion) or the NucleoSpin RNA II Kit (Macherey-Nagel) according to the manufacturer’s protocol. The purity and concentration of the samples were assessed with a NanoDrop 2000/2000c Spectrophotometer (Thermo Fisher Scientific). cDNA was prepared from 500 ng RNA using iScript (Bio-Rad) in a 20-μL reaction and was diluted 1:5 with nuclease-free water. qRT-PCR was performed using 4 μL of the diluted cDNA, along with 500 nM of each primer and POWER SYBR Green PCR master mix (Applied Biosystems) in a total reaction volume of 20 μL. qRT-PCR was performed using a System 7300 instrument (Applied Biosystems) or Step One Plus (Applied Biosystems) and a one-step program: 95 °C for 10 min; 95 °C for 30 s; 60 °C for 1 min, for 40 cycles. Unless otherwise stated, each experiment was performed in triplicate, and all results were normalized against 18S rRNA. Relative mRNA expression levels, compared with 18S rRNA, were determined by the comparative cycle threshold (ΔΔC_T) method. All primer pairs (Table S1) were checked for specificity using BLAST analysis and were checked by both agarose gel electrophoresis and thermal dissociation curves to ensure amplification of a single product with the appropriate size and melting temperature.

Table S1.

qPCR primers

Primer name	Primer sequence
18S	F 5′-CGC CGC TAG AGG TGA AAT TCT-3′
	R 5′-CGA ACC TCC GAC TTT CGT TCT-3′
POU5F1 (OCT4)	F 5′-TGG GCT CGA GAA GGA TGT G-3′
	R 5′-GCA TAG TCG CTG CTT GAT CG-3′
TP63 (ΔNp63 isoform)	F 5′-CTG GAA AAC AAT GCC CAG A-3′
	R 5′-AGA GAG CAT CGA AGG TGG AG-3′
KRT7	F 5′-AGG ATG TGG ATG CTG CCT AC-3′
	F 5′-CAC CAC AGA TGT GTC GGA GA-3′
CDX2	F 5′-TTC ACT ACA GTC GCT ACA TCA CC-3′
	R 5′-TTG ATT TTC CTC TCC TTT GCT C-3′
ELF5	F 5′-AGT CTG CAC TGA CAT TTT CTC ATC-3′
	R 5′-CAG AAG TCC TAG GGG CAG TC-3′
T/BRACHYURY	F 5′-AGT TGA GGA CAG CAG GTT TTA GTT-3′
	R 5′-GCT GAT TGT CTT TGG CTA CT-3′
CGA	F 5′-CAA CCG CCC TGA ACA CAT CC-3′
	R 5′-CAG CAA GTG GAC TCT GAG GTG-3′
CGB	F 5′-ACC CTG GCT GTG GAG AAG G-3′
	R 5′-ATG GAC TCG AAG CGC ACA-3′
PSG4	F 5′-CCA GGG TAA AGC GAC CCA TT-3′
	R 5′-AGA ATA TTG TGC CCG TGG GT-3′
HLA-G	F 5′-CAG ATA CCT GGA GAA CGG GA-3′
	R 5′-CAG TAT GAT CTC CGC AGG GT-3′
HTRA4	F 5′-AAA GAA CTG GGG ATG AAG GAT TC-3′
	R 5′-TGA CGC CAA TCA CAT CAC CAT-3′
VEGFA	F 5′-AGG CCA GCA CAT AGG AGA GA-3′
	F 5′-TTT CTT GCG CTT TCG TTT TT-3′
ANKRD37	F 5′-GTC GCC TGT CCA CTT AGC C-3′
	F 5′-GCT GTT TGC CCG TTC TTA TTA CA-3′
SLC2A1	F 5′-ATA CTC ATG ACC ATC GCG CTA G-3′
	F 5′-AAA GAA GGC CAC AAA GCC AAA TG-3′

Cell Invasion Assay.

Matrigel-coated Transwell membranes (8.0-μm pore inserts in 24-well BioCoat chambers; BD Biosciences) were used to perform in vitro invasion assays. hPSCs and hPSC-derived CTBs were collected with StemDS or 0.05% trypsin, respectively. Cells were washed in cold 1× PBS and then were resuspended in StemPro medium (hPSCs) or FCM plus 10 ng/mL BMP4 (hPSC-derived CTBs). Cells (1 × 10⁴ per well) were placed in the upper chamber, and StemPro plus bFGF (for hPSCs) or FCM+BMP4 for (hPSC-derived CTBs) was placed in the lower chamber. After 4 d, cells on the upper surface were removed by scrubbing with a cotton swab; filters then were fixed with 4% paraformaldehyde and stained with mouse anti-KRT7 and rabbit anti-HLA-G antibodies (as detailed above). Cells were visualized by goat anti-rabbit Alexa Fluor 488- and goat anti-mouse Alexa Fluor 595-conjugated secondary antibodies. For nuclear staining, cells were incubated with DAPI for 5 min.

hCG Hormone and MMP2 Secretion Assays.

Cell-culture supernatants were collected and stored at −80 °C until use. Levels of total hCG were quantified using the hCG ELISA Kit (HC251F; Calbiotech Inc.), and levels of total MMP2 were quantified using the human MMP2 ELISA Kit (ab100606; Abcam) according to the manufacturers’ protocols. The results were normalized to total DNA content, quantified by DNeasy (Qiagen).

Statistical Analysis.

Unless otherwise stated, the data presented are the mean ± SD of biological triplicates; the results shown are representative of three to five independent experiments, each performed using a different preparation of cells. Student’s t test was performed, and P values below 0.05 were taken to indicate a statistically significant difference between the populations sampled.