Human pluripotent stem cells as a model of trophoblast 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 Stem Cell 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 placenta, 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 (syncytiotrophoblasts, STBs) and invasion and maternal Significantly less is known about TE specification and the TSC vascular remodeling (extravillous trophoblasts, EVTs). Studies of niche in the human embryo (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 embryos (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 cytotrophoblasts (CTBs) isolated from meobox 1)/OCT4 (octamer-binding protein 4) in the early TE of early-gestation (6–8 wk) human placentas 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 pregnancies. Human pluripotent stem cells (hPSCs) continue to be underap- preciated as a model for studying trophoblast differentiation. In human pluripotent stem cells | cytotrophoblast | extravillous trophoblast | this study, we provide a reproducible, two-step protocol by which syncytiotrophoblast | 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 blastocoel formation, cells segregate into the response of hPSC-derived CTBs to low oxygen is similar to that the inner cell mass (ICM), which gives rise to the epiblast 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 ectoderm, 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 blastocysts 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 mesoderm 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.