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

Mariko Horiia,b,1, Yingchun Lia,b,1, Anna K. Wakelanda,b,1, Donald P. Pizzoa, Katharine K. Nelsona,b, Karen Sabatinib,c, Louise Chang Laurentb,c, Ying Liud,e,f, and Mana M. Parasta,b,2

aDepartment of Pathology, University of California, San Diego, La Jolla, CA 92093; bSanford Consortium for Regenerative Medicine, University of California, San Diego, La Jolla, CA 92093; cDepartment of Reproductive Medicine, University of California, San Diego, La Jolla, CA 92093; dDepartment of Neurosurgery, Center for and Regenerative Medicine, University of Texas Health Sciences Center, Houston, TX 77030; eThe Senator Lloyd and B. A. Bentsen Center for Stroke Research, University of Texas Health Sciences Center, Houston, TX 77030; and fThe Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Sciences Center, Houston, TX 77030

Edited by R. Michael Roberts, University of Missouri–Columbia, Columbia, MO, and approved May 25, 2016 (received for review March 24, 2016) Trophoblast is the primary epithelial cell type in the , a Elf5 (Ets domain transcription factor) and Eomes (Eomeso- transient organ required for proper fetal growth and develop- dermin), also have been shown to be required for maintenance of ment. Different trophoblast subtypes are responsible for gas/nutrient the TSC fate in the mouse (8, 9). exchange (, STBs) and invasion and maternal Significantly less is known about TE specification and the TSC vascular remodeling (extravillous , EVTs). Studies of niche in the human (10, 11). Recent studies have pointed to early human placental development are severely hampered by the major differences in gene expression between mouse and human lack of a representative trophoblast stem cell (TSC) model with the preimplantation (12, 13). One major distinction is the capacity for self-renewal and the ability to differentiate into both coexpression of CDX2 and POU5F1 (POU domain, class 5, ho- STBs and EVTs. Primary (CTBs) isolated from meobox 1)/OCT4 (octamer-binding protein 4) in the early TE of early-gestation (6–8 wk) human are bipotential, a phe- human embryos, whereas in the mouse embryo these two genes are notype that is lost with increasing gestational age. We have iden- expressed exclusively in the TE and ICM, respectively, each recip- tified a CDX2+/p63+ CTB subpopulation in the early postimplantation rocally regulating the expression of the other (6, 12, 13). Other human placenta that is significantly reduced later in gestation. We differences include the lack of EOMES and ELF5 expression in describe a reproducible protocol, using defined medium containing human TE (13). Based on these findings, the absence of a prolif- bone morphogenetic protein 4 by which human pluripotent stem erating trophoblast compartment in the early human embryo (1, 10), cells (hPSCs) can be differentiated into CDX2+/p63+ CTB stem-like and the inability to derive human TSCs from such preimplantation cells. These cells can be replated and further differentiated into embryos (14), it has been proposed that the human TSC niche may STB- and EVT-like cells, based on marker expression, hormone secre- in fact reside in the early postimplantation placenta. tion, and invasive ability. As in primary CTBs, differentiation of In the absence of a human TSC model, researchers have turned hPSC-derived CTBs in low oxygen leads to reduced human chorionic to human pluripotent stem cells (hPSCs). Since 2002, when Xu gonadotropin secretion and STB-associated gene expression, in- et al. (15) first published the finding that bone morphogenetic + stead promoting differentiation into HLA-G EVTs in an hypoxia- protein 4 (BMP4) induces the expression of trophoblast-related inducible, factor-dependent manner. To validate further the utility genes in hPSCs, multiple groups have used these cells as a model of hPSC-derived CTBs, we demonstrated that differentiation of tri- for studying trophoblast lineage specification (16–22). The ma- somy 21 (T21) hPSCs recapitulates the delayed CTB maturation and jority of these studies, including our own (21), have used BMP4 blunted STB differentiation seen in T21 placentae. Collectively, our data suggest that hPSCs are a valuable model of human placental Significance development, enabling us to recapitulate processes that result in both normal and diseased . Human pluripotent stem cells (hPSCs) continue to be underap- preciated as a model for studying trophoblast differentiation. In human pluripotent stem cells | | | this study, we provide a reproducible, two-step protocol by which | placenta hPSCs can be differentiated into bipotential cytotrophoblast (CTB) stem-like cells and subsequently into functional, terminally dif- rophoblast is the first lineage specified in the developing ferentiated trophoblasts. In addition, we provide evidence that Tembryo. Following formation, cells segregate into the response of hPSC-derived CTBs to low oxygen is similar to that the (ICM), which gives rise to the and of primary CTBs. Finally, using trisomy 21-affected hPSCs, we embryo proper; the outer trophectoderm (TE) cells give rise to show, for the first time to our knowledge, that hPSCs can model a extraembryonic , the epithelial portion of the placenta trophoblast differentiation defect. We propose that hPSCs are (1). Much of what we know about this first lineage specification superior to other currently available models for studying human comes from studies in mouse (2–4). In fact, the combination of trophoblast differentiation. abilities to derive trophoblast stem cells (TSCs) from mouse and to evaluate the compartment-specific contribution Author contributions: M.M.P. designed research; M.H., Y. Li, A.K.W., D.P.P., K.K.N., and K.S. performed research; M.H., Y. Li, A.K.W., L.C.L., and Y. Liu contributed new reagents/ of specific genes during embryogenesis has resulted in the iden- analytic tools; M.H., Y. Li, A.K.W., K.S., L.C.L., and M.M.P. analyzed data; and M.H., Y. Li, tification of numerous transcription factors and signaling pathways A.K.W., and M.M.P. wrote the paper. involved in TE specification and placental development (2, 3, 5). The authors declare no conflict of interest. Results from such studies have led to identification of Cdx2 This article is a PNAS Direct Submission. (caudal type homeobox 2), a member of the caudal-related ho- 1M.H., Y. Li, and A.K.W. contributed equally to this work. meobox transcription factor gene family, the expression of which 2To whom correspondence should be addressed. Email: [email protected]. has been shown to be required for maintenance of TE and TSCs This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in the mouse embryo (6, 7). Other transcription factors, including 1073/pnas.1604747113/-/DCSupplemental.

E3882–E3891 | PNAS | Published online June 20, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1604747113 Downloaded by guest on September 29, 2021 in the presence of feeder-conditioned medium (FCM), resulting proof-of-concept data for the ability of hPSCs to model a tro- PNAS PLUS in the expression of some markers and therefore phoblast differentiation defect, using trisomy 21 (T21)-affected generating doubt about the true identity of these hPSC-derived hPSCs. cells (23). Nevertheless, follow-up studies using more defined culture conditions have confirmed the identity of these cells as Results trophoblasts (20). Identification of a CDX2-p63 Double-Positive CTB Population in the Most recently, Lee et al. (24) have proposed criteria for defining Early Human Placenta. The CTB, the trophoblast layer adjacent to trophoblasts based on expression of a set of markers, including the villous stroma, is the proliferative trophoblast compartment ELF5. Although a laudable attempt at standardization, this study in the human placenta. The CTB layer is continuous in the first fails to account for differences in gene expression across gestational trimester and becomes discontinuous starting in the second tri- age and falls short of defining syncytiotrophoblasts (STBs) (24). mester (10, 11). We previously identified p63 as a pan-CTB To confirm the utility of hPSCs for modeling trophoblast dif- marker (25). We now have stained human placenta samples using ferentiation, we instead asked whether these cells can recapitu- an antibody to CDX2 and found that in early gestation (6 wk), late functional phenotypes of primary trophoblasts during both CDX2, along with p63, was found in the majority of CTBs (Fig. normal development and disease. We previously have identified 1A). However, unlike p63, which is maintained in the CTB until + p63, a member of the p53 family of nuclear proteins, as a marker term (25), the number of CDX2 CTBs diminished markedly as specific to proliferative cytotrophoblasts (CTBs) in the human the first trimester progressed (Fig. 1 B and C). By 9-wk gestation placenta (21, 25, 26). We now have identified a subpopulation of only ∼50% of CTBs were positive for CDX2, and by 20-wk ges- + CTBs in the early human placenta that are double-positive for tation no CDX2 CTBs remained (Fig. 1 B and C). p63 and CDX2; this CTB subpopulation is greatly reduced in the second trimester and is temporally associated with the loss of Generation of CTB Stem-Like Cells from hPSCs. The majority of bipotential differentiation of CTBs (27). In addition, we describe hPSC trophoblast differentiation protocols expose the cells to a completely defined culture condition, containing BMP4, by high concentrations of BMP4 (50 ng/mL or higher) in the pres- + + which CDX2 /p63 CTB stem-like cells can be efficiently and ence of FCM (Fig. 2A). One previous study developed a minimal reproducibly derived from hPSCs. Furthermore, we show that medium for trophoblast induction but also used high BMP4 hPSC-derived CTBs respond to low oxygen in a manner similar concentrations and focused on the evaluation of CDX2 as the to primary CTBs. Finally, we provide the first, to our knowledge, primary TE marker (17). Because high-dose BMP4 also can induce

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-gesta- tion human placenta (6 wk). Nuclei were counter- stained with DAPI (blue). (Magnification: 10×.) (Lower) ×

Higher magnification (60 ) images of the areas indi- BIOLOGY cated by the white boxes in the upper panels. (B)Im- munohistochemical staining of CDX2 and p63. p63 is DEVELOPMENTAL 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. (Magnifi- cation: 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.

Horii et al. PNAS | Published online June 20, 2016 | E3883 Downloaded by guest on September 29, 2021 low-dose BMP4 treatment in this defined medium, hPSCs uni- formly 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).

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 tropho- blasts (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. 2. Representations of trophoblast differentiation protocols. (A) Tradi- After replating and culture in FCM supplemented with 10 ng/mL tional (one-step) trophoblast differentiation protocol using BMP4 in the human BMP4 (FCM+BMP4), the cells continued to differentiate presence of FCM. This protocol was used by the majority of previous studies (Fig. 4). Morphologically, 4–6dafterFCM+BMP4 treatment, cells for trophoblast differentiation of hPSCs. (B) Our modified (two-step) tro- continued to show an epithelial (flattened) phenotype with nu- phoblast differentiation protocol. Following a 2-d “rest” in StemPro-based merous multinucleated cells (Fig. 4A).BasedonqRT-PCRanalysis, minimal medium (EMIM), hPSCs are cultured in EMIM plus 10 ng/mL BMP4 CTB markers such as CDX2 and p63 were reduced, and differen- for up to 4 d to form uniformly flattened CTB stem-like colonies. These cells tiation markers, including CGB (chorionic gonadotropin subunit then are trypsinized and replated to be terminally differentiated into STBs beta; an STB marker) and HLA-G (an EVT marker), were in- and EVTs in FCM plus 10 ng/mL BMP4. H9, H9/WA09 cells; U, undifferenti- B ated hPSCs in StemPro; D0, hPSCs after 2-d rest in EMIM; D1–D4, days duced (Fig. 4 ). Loss of CTB markers was further evident by after treatment with EMIM+BMP4; CTB+1toCTB+6, days after replating immunostaining, showing loss of expression of CDX2, TEAD4, and in FCM+BMP4. YAP following culture in FCM-BMP4 (Fig. 4C). Functional tro- phoblast differentiation was confirmed by the secretion of hCG (human CG, the hormone and a marker of STBs) and mesoderm (23), we asked whether, in that minimal medium (17), MMP2 (matrix metalloproteinase-2, an EVT marker) (Fig. 4D). In + low-dose BMP4 (10 ng/mL) by itself is able to initiate trophoblast addition, we confirmed the presence of invasive HLA-G EVTs differentiation of hPSCs into CTBs, as evaluated by a com- using a Matrigel invasion assay followed by immunostaining (Fig. prehensive panel of markers, including p63 and CDX2. We ini- 4E). The presence of FCM appeared to be optimal for differen- tially used WA09/H9 ES cells (ESCs) but also tested this protocol tiation, because differentiation in minimal medium with BMP4 on WA01/H1 and SIVF21 ESCs as well as on induced pluripotent resulted in significantly lower levels of differentiation markers, in- + stem cells (iPSCs) derived in the L.C.L. and Y. Liu laboratories, cluding hCG secretion and surface HLA-G cells (Fig. S3 B–D). all of which were adapted to StemPro according to the manu- Together, based on both marker expression and function, these data facturer’s instructions, with similar results in each case (Fig. S1). confirm the bipotential differentiation ability of hPSC-derived CTBs We refer to this method as the “two-step protocol” (Fig. 2B). in FCM+BMP4. In the first step, StemPro-adapted hPSCs were treated with a DMEM/F12-based minimal medium (EMIM) (17) for 2 d and Behavior of hPSC-Derived CTBs in Low Oxygen. The effect of low then were cultured in EMIM containing 10 ng/mL BMP4 for up oxygen on the differentiation of STBs has been well established. to 4 d (Fig. 2B). After 2 d in EMIM alone, the colonies appeared Specifically, the production of hCG- and STB-associated tran- to tighten and become better delineated; following BMP4 scripts is known to be inhibited by low oxygen tension (28, 29). treatment, cells uniformly adopted a more flattened appearance We replated hPSC-derived CTBs in either 20% or 2% oxygen but continued to grow to near confluence by day 4 (Fig. 3A). We and continued to differentiate them in FCM+BMP4 (step 2). then proceeded to evaluate markers of undifferentiated CTBs Under 2% oxygen, the cells produced significantly less hCG (Fig. by immunostaining: Compared with undifferentiated hPSCs, 5A and Fig. S4A); they also showed a significant decrease in the BMP4-treated cells showed loss of the pluripotency marker expression of STB-associated transcripts, including CGA (chorionic POU5F1/OCT4; conversely, the CTB-associated genes p63, gonadotropin subunit alpha), CGB, and PSG4 (pregnancy-specific CDX2, TEAD4 (TEA domain family member 4), KRT7 (keratin, β-1-glycoprotein 4) (Fig. 5B). type II cytoskeletal 7), and EGFR (EGF receptor) were all highly Culture under low oxygen tension is also known to promote + expressed in the hPSC-derived CTBs (Fig. 3B). In addition, YAP the initial differentiation of CTBs into HLA-G EVTs (30), al- (Yes-associated protein) was localizedtothenucleiofthese though further differentiation into fully mature, invasive EVTs is hPSC-derived CTBs (Fig. 3B). After 4 d of BMP4 treatment, inhibited (31); in addition, an intact hypoxia-inducible factor more than 95% of the cells were positive for both p63 and EGFR (HIF), a transcription factor complex mediating a significant as measured by flow cytometry (Fig. 3C). Next, we investigated portion of the cellular response to low oxygen (32), has been gene expression during CTB induction of hPSCs. Analysis by shown to be required for the differentiation of rodent TSCs into quantitative RT-PCR (qRT-PCR) showed that the expression of trophoblast giant cells, which are analogous to human EVTs POU5F1/OCT4 decreased and the expression of p63, CDX2, (33, 34). To test the effect of different oxygen tensions on EVT and KRT7 increased significantly following BMP4 treatment differentiation, we generated hESCs stably expressing either a (Fig. 3D). In addition, the mouse TSC-associated marker ELF5 scrambled shRNA or shRNA specific to ARNT (aryl hydrocar- was induced twofold, whereas the mesoderm-associated marker bon receptor nuclear translocator; also called “HIF1β”); which is T/BRACHYURY was reduced from the basal levels present in required for the formation of an intact HIF complex (32). We StemPro-adapted cells and was not induced by low-dose BMP4 used two different hESC lines, H9/WA09 and SIVF-disomy hESCs treatment (Fig. 3D). These data indicate that, with only 4 d of (see below). ARNT-null hESCs showed significantly reduced

E3884 | www.pnas.org/cgi/doi/10.1073/pnas.1604747113 Horii et al. Downloaded by guest on September 29, 2021 PNAS PLUS

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, dur- ing 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 ex- pressed as means ± SD of biological triplicates; *P < 0.05 in comparison with values in StemPro-adapted undifferentiated H9 cells.

expression of ARNT at the protein level (Fig. 6A), although T21: Comparison of Primary and hPSC-Derived CTBs in the Context of they still stabilized HIF1α under low oxygen conditions (Fig. Disease. T21 is a common aneuploidy known mostly for associ- 6B). They showed a blunted response to culture in low oxygen, ated fetal malformations, including structural cardiac defects and as demonstrated by decreased induction of HIF-dependent neurodevelopmental delay. However, T21 placentas also show genes (Fig. 6C and Fig. S4B). Also, although CTBs derived developmental abnormalities, including prolonged maintenance from scrambled shRNA-expressing hESC showed a doubling of of a continuous CTB layer well into the second trimester and HLA-G+ cells under 2% oxygen as measured by flow cytom- abnormalities of STB formation (38–41). We evaluated six T21 etry, ARNT-null hESC-derived CTBs maintained the same placentas and five gestational age-matched euploid placentas by proportion of HLA-G+ cellsinboth2%and20%oxygen(Fig. CDX2 immunohistochemistry and found that T21

+ BIOLOGY 6D and Fig. S4C). This finding was not the result of improved contained a higher percentage of CDX2 CTBs (Fig. 7 A and B).

plating, because hESC-derived CTBs showed similar replating We next asked whether T21-affected hPSCs showed any defects DEVELOPMENTAL efficiency in both 20% and 2% oxygen (Fig. S5). In addition, in trophoblast differentiation. We used two T21 hPSC lines: one under 2% oxygen, ARNT-null hESC-derived CTBs also had hESC line (SIVF21) and one iPSC line (C1); as control in each decreased secretion og MMP2 (Fig. 6E), a marker specific to case we used a subclone of the same cell line that had lost its extra + HLA-G EVTs in the human placenta (35, 36). Finally, under copy of chromosome 21 (the SIVF21-disomy hESC line and the 2% oxygen, ARNT-null hESC-derived CTBs showed a signif- C5 iPSC line, respectively). T21-hPSCs showed a delay in the in- icantly lower induction of the EVT-associated genes HLA-G duction of the trophoblast lineage, as measured by surface EGFR and HTRA4 (high-temperature requirement protein A4) (Fig. expression (Fig. 7C and Fig. S6). In addition, T21-hPSCs also 6F and Fig. S4D)(37). showed a slower decrease in the expression of the pluripotency

Horii et al. PNAS | Published online June 20, 2016 | E3885 Downloaded by guest on September 29, 2021 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 differenti- ation 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. Nu- clei were counterstained with DAPI (blue). (D) Secre- tion 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 Matri- gel-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).

factor POU5F1/OCT4 and an exaggerated induction of CDX2 and C and Fig. S6); they also showed increased hCG secretion (Fig. 7D). and altered expression of the transcripts of the hCG compo- During the second step of trophoblast differentiation, T21- nents CGA and CGB (Fig. 8 D and E and Fig. S6). Finally, hPSC–derived CTBs showed maintenance of the CTB markers Activin-A has been shown to rescue the fusion defect in pri- CDX2 and ELF5 (Fig. 8A). We determined the fusion index of mary T21 CTBs (40); we therefore treated CTBs derived from the differentiating cells by staining for ZO-1 (zona occludens T21-hPSCs with Activin-A during the second step of tropho- protein 1) and DAPI and counting both the number of nuclei blast differentiation and found that this treatment restored and the number of multinucleated STBs (Fig. 8 B and C). CTBs the fusion index of these cells to the level in the disomy cells derived T21-hPSCs from showed a lower fusion index (Fig. 8 B (Fig. 8F).

E3886 | www.pnas.org/cgi/doi/10.1073/pnas.1604747113 Horii et al. Downloaded by guest on September 29, 2021 BMP4 to generate a uniform population of CTB stem-like cells, PNAS PLUS 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 ex- pression 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

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 mea- sured 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.

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 “ΔNp63”) 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 tropho- blast, specifically, in the TE of human blastocysts (45). Therefore + + it is possible that this CDX2 -p63 double-positive population may emerge in the human and persist in a subset of trophoblasts in the early postimplantation placenta. Fig. 6. Low oxygen tension induces EVT differentiation in an HIF complex- This finding is significant, particularly because of the contro- dependent manner. (A) Western blot for ARNT in undifferentiated hESCs stably versy surrounding the BMP4-based model of trophoblast in- infected with lentivirus expressing either scrambled shRNA or ARNT-specific duction of hPSCs. These models have been questioned, based shRNA (shARNT). (B) Western blot for HIF1α in hESCs stably infected with len- mostly on observations in the mouse system, in which signaling tivirus expressing either scrambled shRNA or ARNT-specific shRNA, differenti- through BMP receptors appears to be required only for meso- ated into CTBs, and then replated and cultured in 2% oxygen for the indicated derm induction and not for trophoblast lineage specification (23, 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 post- 46). The identification of BMP4-inducible p63, which is dis- + +

differentiation in FCM BMP4 (day CTB 4) in 20% or 2% oxygen. Data were BIOLOGY pensable for mouse placentation (47), as an early marker of normalized to 18S and are expressed as fold change relative to scrambled/20% human proliferative CTB, may explain why BMP4 induces the oxygen. (D) Surface expression of HLA-G (an EVT marker) was analyzed by flow DEVELOPMENTAL TE lineage in hPSCs but appears not to be required for the same cytometry of scrambled and shARNT-infected hESC-derived CTBs cultured in 20% or process in mice (46). 2% oxygen for 2 d after replating. All samples are compared with isotype. (E) Here we outline a simple two-step protocol by which we can Increased MMP2 secretion at 2 (CTB+2) and 3 (CTB+3) d after replating of + + reproducibly generate first a pure population of CDX2 /p63 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- CTB stem-like cells and subsequently a combination of both associated genes HLA-G and HTRA4 in scrambled and shARNT-infected hESC- hCG-producing STBs and MMP2-producing, invasive EVTs. derived CTBs 2 d after replating in 20% or 2% oxygen. Data were normalized to This protocol is superior to previously described protocols in 18S and are expressed as fold change relative to scrambled/20% oxygen. All data several ways. First, it is a robust protocol that uses low-dose are expressed as means ± SD of biological triplicates; *P < 0.05.

Horii et al. PNAS | Published online June 20, 2016 | E3887 Downloaded by guest on September 29, 2021 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.

specification (hPSC → CTB) from terminal trophoblast differ- thwarted in low oxygen (29, 49, 50). Using our two-step protocol, entiation (CTB → STB and EVT). This ability is important, we evaluated the first step of EVT differentiation, which involves particularly because it allows the characterization of different the induction of HLA-G expression, and found that in low (2%) + stages of trophoblast differentiation and will improve compara- oxygen the surface HLA-G cells were expanded in a HIF complex- tive studies with primary cells. dependent manner. We could not evaluate the second step One of the major reasons hPSCs have not been widely used for of EVT differentiation (i.e., invasion) independently, because we + studying early implantation events has been the paucity of data could not replate the HLA-G cells. regarding their ability to model human trophoblast differentiation We next turned to another well-studied model of a human during both normal development and disease. One recent study trophoblast differentiation defect: T21. As mentioned previously, showed blunted trophoblast differentiation of hESCs carrying an it is known that T21 placentas maintain a continuous layer of unbalanced chromosomal translocation t (11, 22) after BMP4 CTBs well into the second trimester (38). We evaluated these treatment (48). Because this translocation is commonly associated placentas further by immunohistochemistry and noted that CDX2 with , the authors argued that the hESCs carrying this is maintained longer in the CTBs of T21 placentas than in euploid chromosomal aberration are a good model for studying implan- placentas. Interestingly, with BMP4 treatment, T21-hPSCs in- tation failure (48). However, because the mechanism of implan- duced CDX2 at much higher levels and also maintained this tation failure in these cases is not known, the model could not be marker longer after the induction of terminal trophoblast differ- validated further. We decided to evaluate two well-established entiation (step 2). More importantly, and again similar to previous phenomena in trophoblast differentiation: the response of CTBs studies using primary T21 CTBs (39–41), T21-hPSC–derived to low oxygen tension and the differentiation defects of T21- CTBs showed a defect in the formation of multinucleated STBs, affected CTBs. which was alleviated with Activin-A treatment. These data show, The culture of primary CTBs in low oxygen is known to inhibit for the first time to our knowledge, a well-established trophoblast hCG production and reduce the expression of STB-associated differentiation defect that can be recapitulated using hPSCs. genes (28, 29), a phenotype that was replicated with hPSC-derived There remain some limitations to the use of hPSCs for mod- CTBs in our study. The effect of low oxygen on EVT differenti- eling trophoblast differentiation. In particular, to date we have + + ation is more complex. Low oxygen tension appears to induce the been unable to maintain the p63 /CDX2 CTBs in an undif- first step of EVT differentiation, seen as the expansion of the ferentiated state. Several factors may be at play here, including + HLA-G cell column EVTs, in both first-trimester human pla- suboptimal culture conditions and/or insufficient expression of cental explants (31, 49) and isolated primary trophoblasts (30). factors promoting self-renewal. With respect to the former, we However, differentiation into mature, invasive EVT appears to be are in the process of performing small-molecule screens and

E3888 | www.pnas.org/cgi/doi/10.1073/pnas.1604747113 Horii et al. Downloaded by guest on September 29, 2021 PNAS PLUS

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.

have identified factors that maintain p63 and CDX2; neverthe- stability during derivation and culture (53, 54). For this reason, it less, these factors do not appear to be sufficient for maintaining is likely that the culture conditions for both the propagation of self-renewal in this cell population. This result has led us to hPSCs and their subsequent differentiation into trophoblasts must conclude that other factors are likely required for CTB stem cell be optimized in a way that promotes the expression of the trophoblast- maintenance. Candidates for such factors, insufficiently induced associated imprinted genes, such as ELF5. Finally, high expres- following BMP4 treatment of hPSCs, include ELF5, an imprinted sion of ELF5 has been noted recently in new hESC lines derived gene and a member of the Ets family of transcription factors, from earlier-stage human embryos. These lines have an en- which is required for maintenance of the TSC compartment in hanced ability to differentiate into trophoblast and reportedly mice (51). In mice, the Elf5 promoter is hypomethylated specifi- maintain the TSC state (55); thus, it is also possible that only cally in TSCs, in which this gene is expressed, and is hyper- early-stage embryos have the correct epigenetic landscape for methylated in ESCs, in which the gene is silent (9). ELF5 is the derivation and maintenance of true human TSCs. expressed in human trophoblasts of the early placenta, where its We also are still in the process of defining the culture condi- promoter has been shown to be hypomethylated (52). In contrast, tions required for inducing pure subpopulations of either STBs

+ BIOLOGY ELF5 is not expressed in the TE of the human preimplantation or EVTs. Under the current culture conditions, HLA-G blastocyst (13). As in previous studies (20), we do see induction of cells appear to peak 2 d after the replating of hPSC-derived DEVELOPMENTAL ELF5 following BMP4 treatment; however, this induction is weak CTBs in FCM+BMP4, whereas the number of multinucleated hCG- (1.5- to twofold in all lines tested) and does not reach the levels of secreting cells peaks 4 d after replating. That these cell subpopu- expression seen in postimplantation CTBs (24). Therefore, it is lations peak at different times and diminish soon thereafter may possible that the hPSC-derived CTBs are more similar to preim- indicate that they are competing with each other and/or that the plantation TE than to early postimplantation CTBs. In addition, culture conditions are suboptimal for their maintenance and the variability of expression of imprinted genes in different hPSC maturation. Additional optimization of culture conditions, in- lines has been described in both the undifferentiated and differ- cluding changes in extracellular matrix and 3D culture, may be entiated states and has been attributed mostly to epigenetic in- required to enhance further the utility of hPSC-derived trophoblasts

Horii et al. PNAS | Published online June 20, 2016 | E3889 Downloaded by guest on September 29, 2021 as a model. Overall, however, given the proper expression of many determined by GFP expression. Knockdown efficiency was determined by trophoblast-associated genes, in proper progression from CTB Western blot. induction to STB/EVT differentiation, the proper response of the CTB stem-like cells to low oxygen, and the ability to recapitulate Human Placental Tissue Collection and Immunohistochemistry. Human pla- an STB differentiation defect with T21-affected iPSCs, we main- cental tissues were collected under a protocol approved by the Human Research Protections Program Committee of the University of California, San Diego tain that the BMP4-based protocol, particularly the two-step Institutional Review Board; all patients gave informed consent for the collection protocol described herein, provides a bona fide model for the and use of these tissues. Serial sections of formalin-fixed, paraffin-embedded study of early human trophoblast differentiation. Currently, the placental tissues were stained with mouse anti-p40 (ΔNp63-specific antibody, only other human model that provides a bipotential trophoblast clone BC28; Biocare Medical) and rabbit anti-CDX2 (clone EPR2764Y; Abcam) progenitor cell population is primary CTBs isolated from first- antibodies, using a Ventana Discovery Ultra automated immunostainer with trimester placental tissue, to which access is highly limited. Other standard antigen retrieval and reagents per the manufacturer’s protocol. human trophoblast cell lines, whether derived from choriocarci- noma (trophoblastic tumor) tissue or isolated primary trophoblasts, Flow Cytometric Analysis. For flow cytometry, cells were fixed and permeabilized – represent previously committed trophoblast lineages (either villous with either 4% (wt/vol) paraformaldehyde for 10 15 min or with 100% ice- or extravillous) and thus do not provide a platform for evaluating cold methanol for 30 min and then were washed three times in PBS sup- plemented with 10% (vol/vol) FBS. Cells then were incubated at room lineage segregation and TSC fate decisions. Our two-step protocol temperature for 1 h in flow cytometry (FC) buffer (0.5% BSA and 1% FBS in presents a system for studying both the initial specification of the PBS) with an allophycocyanin (APC)-conjugated mouse anti-human EGFR human trophoblast lineage and subsequent differentiation into antibody (clone AY13; BioLegend), phycoerythrin (PE)-conjugated mouse both villous and extravillous lineages. We therefore propose anti–HLA-G (MEM-G/9; EXBIO), or FITC-conjugated mouse anti-human p63 that, except in primary cells, our two-step protocol is superior antibody (clone 4A4; Santa Cruz). APC- or PE-conjugated mouse IgG (clone to other currently available models for studying early human MPOC-21; BioLegend), or FITC-conjugated mouse IgG (Santa Cruz) was used trophoblast differentiation. as isotype IgG control. Cells were washed three times with FC buffer, and analysis was carried out using a BD FACS-Canto Flow cytometer. Materials and Methods hPSC Culture and Differentiation. The hPSC aspect of the research was Immunostaining of Cells. For immunofluorescence staining, cells grown on performed under a protocol approved by the University of California, San Geltrex-coated coverslips were fixed with 4% (wt/vol) paraformaldehyde in Diego Institutional Review Board and Research Over- PBS at room temperature for 10 min. Cells then were permeabilized with sight Committee. Before differentiation, ESCs (WA09/H9 and WA01/H1 lines 0.3% Triton X-100 for 10 min and incubated with primary antibodies, from WiCell and SIVF21 trisomy and disomy lines) and iPSCs [C1 (T21) and C5 including mouse anti-p63 (4A4 clone; Sigma-Aldrich), rabbit anti-Ki67 (disomy) lines] (56) were cultured under feeder-free conditions in StemPro (ab15580; Abcam), mouse anti-KRT7 (clone OV-TL 12/30; Invitrogen), mouse (Invitrogen) medium with 12 ng/mL recombinant basic FGF (bFGF) (Invitrogen) anti-YAP (sc-15407; Santa Cruz), rabbit anti-EGFR (sc-03; Santa Cruz), rabbit on plates coated with Geltrex (1:200; BD Biosciences). In the first step, cells anti-TEAD4 (HPA056896; Abcam), rabbit anti-CDX2 (clone EPR2764Y; – were plated relatively sparsely (8 × 103/cm2 for WA09/H9 and 105/cm2 for SIVF, Abcam), rabbit anti-OCT4 (ab19857; Abcam), rabbit anti ZO-1 (ab59720; – C1, and C5 cell lines). Cells were passaged with StemDS (ScienCell Research Abcam), or mouse anti HLA-G (clone 4H84; Abcam) and were visualized by Laboratories) or Accutase (Life Technology) every 3–5 d. Only passages 10–20 Alexa Fluor 488- or 595-conjugated goat secondary antibodies (Invitrogen). post-StemPro adaptation were used for all experiments described here. For the For nuclear staining, cells were incubated with DAPI (Invitrogen) for 5 min. first phase of TE differentiation, hPSCs were first treated with a StemPro (Thermo Fisher)-based basal medium [KnockOut DMEM (Thermo Fisher)/F12 RNA Isolation and Quantitative Real-Time PCR. Total RNA was extracted using containing 1% insulin-transferrin-selenium A, 1× nonessential amino acid, 2% either the mirVana RNA Isolation Kit (Ambion) or the NucleoSpin RNA II Kit ’ (wt/vol) BSA, 2 mM L-glutamine, 100 ng/mL heparin sulfate] designated as (Macherey-Nagel) according to the manufacturer s protocol. The purity and “EMIM” (17) for 2 d. The cells then were switched to EMIM medium containing concentration of the samples were assessed with a NanoDrop 2000/2000c 10 ng/mL human BMP4 (R&D Systems) for 2–4 d, at which point they were Spectrophotometer (Thermo Fisher Scientific). cDNA was prepared from designated hPSC-derived CTBs. For terminal differentiation, the hPSC-derived 500 ng RNA using iScript (Bio-Rad) in a 20-μL reaction and was diluted 1:5 CTBs were passaged using 0.05% trypsin and were replated on Geltrex-coated with nuclease-free water. qRT-PCR was performed using 4 μL of the diluted plates in FCM [KnockOut DMEM/F12 containing 20% (vol/vol) KnockOut serum cDNA, along with 500 nM of each primer and POWER SYBR Green PCR master replacement (Knockout SR; Thermo Fisher), 1× GlutaMAX (Thermo Fisher), and mix (Applied Biosystems) in a total reaction volume of 20 μL. qRT-PCR was 1× nonessential amino acid, 0.1 mM 2-mercaptoethanol; conditioned on irra- performed using a System 7300 instrument (Applied Biosystems) or Step One diated mouse embryonic fibroblasts for 24 h], supplemented with 10 ng/mL Plus (Applied Biosystems) and a one-step program: 95 °C for 10 min; 95 °C for human BMP4 for the indicated number of days. For Activin-A rescue experi- 30 s; 60 °C for 1 min, for 40 cycles. Unless otherwise stated, each experiment ments, in addition to FCM+BMP4, the cells were treated with 5 ng/mL Activin was performed in triplicate, and all results were normalized against 18S rRNA. A (Stemgent). For culture under low oxygen, cells were incubated in an Xvivo Relative mRNA expression levels, compared with 18S rRNA, were determined by ΔΔ system under 2% oxygen (BioSpherix). For all experiments in hypoxia, the the comparative cycle threshold ( CT) method. All primer pairs (Table S1)were medium was changed, and cells were lysed (for RNA or protein analysis) or checked for specificity using BLAST analysis and were checked by both agarose fixed (for flow cytometry) in the Xvivo work chamber under 2% oxygen. gel electrophoresis and thermal dissociation curves to ensure amplification of a single product with the appropriate size and melting temperature. Generation of ARNT-Knockdown hESC Lines. pLKO.1-based scrambled shRNA was obtained from Addgene. A scrambled shRNA and a set of five Mission Cell Invasion Assay. Matrigel-coated Transwell membranes (8.0-μm pore in- shRNA Lentiviral constructs targeting the human ARNT gene were purchased serts in 24-well BioCoat chambers; BD Biosciences) were used to perform in and packaged for transduction according to the manufacturer’s instructions vitro invasion assays. hPSCs and hPSC-derived CTBs were collected with (Sigm-Aldrich a). HEK293FT cells were transfected with the shRNA constructs StemDS or 0.05% trypsin, respectively. Cells were washed in cold 1× PBS and Delta 8.2 and VSV-G using Lipofectamine 2000 (Sigma-Aldrich) according to then were resuspended in StemPro medium (hPSCs) or FCM plus 10 ng/mL 4 the manufacturer’s instructions. Lentiviral supernatants were concentrated BMP4 (hPSC-derived CTBs). Cells (1 × 10 per well) were placed in the upper with PEG-it virus precipitation solution (System Biosciences). The concen- chamber, and StemPro plus bFGF (for hPSCs) or FCM+BMP4 for (hPSC-derived trated viral particles then were incubated with target cells in the presence of CTBs) was placed in the lower chamber. After 4 d, cells on the upper surface 10 mg/mL Polybrene (Sigma-Aldrich). Packaging and infection efficiency were removed by scrubbing with a cotton swab; filters then were fixed with were tested using a GFP-expressing lentivirus (in the same pCMV-lentiviral 4% paraformaldehyde and stained with mouse anti-KRT7 and rabbit anti-HLA-G construct used for ARNT). Of the five ARNT-specific shRNA constructs, three antibodies (as detailed above). Cells were visualized by goat anti-rabbit Alexa worked best, based on protein knockdown in HEK293T cells; therefore, Fluor 488- and goat anti-mouse Alexa Fluor 595-conjugated secondary anti- subsequent experiments were performed with this subset of three shRNAs bodies. For nuclear staining, cells were incubated with DAPI for 5 min. 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 hCG Hormone and MMP2 Secretion Assays. Cell-culture supernatants were transduced over at least three passages. Infection efficiency was collected and stored at −80 °C until use. Levels of total hCG were quantified

E3890 | www.pnas.org/cgi/doi/10.1073/pnas.1604747113 Horii et al. Downloaded by guest on September 29, 2021 using the hCG ELISA Kit (HC251F; Calbiotech Inc.), and levels of total MMP2 ACKNOWLEDGMENTS. Uli Schmidt (Stem Cell Laboratory) derived the original PNAS PLUS were quantified using the human MMP2 ELISA Kit (ab100606; Abcam) SIVF21 ESC line from which the subsequent SIVF21-disomy ESC line was derived according to the manufacturers’ protocols. The results were normalized to through subculture. This work was supported by California Institute for Regen- total DNA content, quantified by DNeasy (Qiagen). erative Medicine (CIRM) New Faculty Award RN2-00931, CIRM Physician-Scientist Award RN3-06396, and NIH/National Institute of Child Health and Development Grant R01-HD071100. M.H. was supported through CIRM Research and Training ± Statistical Analysis. Unless otherwise stated, the data presented are the mean Grant TG2-01154 to the University of California, San Diego (UCSD). A.K.W. was SD of biological triplicates; the results shown are representative of three to supported by UCSD Respiratory Biology Training Grant T32-HL098062. Y. Liu was five independent experiments, each performed using a different preparation supported by CIRM Grant RT1-01107-1, the Memorial Hermann Foundation- of cells. Student’s t test was performed, and P values below 0.05 were taken to Staman Ogilvie Fund, the Bentsen Stroke Center Fund, and Mission Connect– indicate a statistically significant difference between the populations sampled. Texas Institute for Rehabilitation and Research Foundation.

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