Establishment of macaque trophoblast stem cell lines derived from cynomolgus monkey blastocysts.

The placenta forms a maternal-fetal junction that supports many physiological functions such as the supply of nutrition and exchange of gases and wastes. Establishing an in vitro culture model of human and non-human primate trophoblast stem/progenitor cells is important for investigating the process of early placental development and trophoblast differentiation. In this study, we have established five trophoblast stem cell (TSC) lines from cynomolgus monkey blastocysts, named macTSC #1-5. Fibroblast growth factor 4 (FGF4) enhanced proliferation of macTSCs, while other exogenous factors were not required to maintain their undifferentiated state. macTSCs showed a trophoblastic gene expression profile and trophoblast-like DNA methylation status and also exhibited differentiation capacity towards invasive trophoblast cells and multinucleated syncytia. In a xenogeneic chimera assay, these stem cells contributed to trophectoderm (TE) development in the chimeric blastocysts. macTSC are the first primate trophoblast cell lines whose proliferation is promoted by FGF4. These cell lines provide a valuable in vitro culture model to analyze the similarities and differences in placental development between human and non-human primates.

strictly restricted to the inner cell mass (ICM) 6 , hence questioning the suitability of HTR8/SVneo cells. The BeWo cell line derived from human choriocarcinoma has also been used as a human trophoblast in vitro model 7 . The BeWo has syncytialization and invasion abilities 8,9 ; however, choriocarcinoma cells might represent unnatural features compared to endogenous trophoblasts. Trans-differentiation of human ESC to trophoblast-like cells by BMP4 treatment has also been adopted 2 . However, the adequacy of trans-differentiation system remains somewhat controversial, since the gene expression profile of the resulting trophoblast-like cells do not resemble that of primary trophoblast cells, moreover they express other cell lineage markers 2,10 . Recently, the trans-differentiation protocol from human ESC to trophoblast-like cells was improved in so-called BAP treatment 11 . This newer treatment succeeded to suppress the upregulation of mesoderm markers including T (Brachyury) 11 , although the difference in global gene expression profile between trans-differentiated human ESC and primary trophoblast remains 12 .
In mice, the trophoblast stem cells, which have the potential to differentiate both in vivo and in vitro into all trophoblast subtypes, were established from the preimplantation blastocysts and extraembryonic ectoderm of post-implantation embryos in the presence of fibroblast growth factor 4 (FGF4) 13 . This useful in vitro model has revealed the underlying mechanisms of trophoblast differentiation and placental development. Attempts to establish human TSCs by employing the same strategy used for mouse TSCs has been unsuccessful 14 , suggesting that establishment of in vitro human TSCs may depend on certain exogenous factors, which remains different from mouse TSCs. Okae et al., recently succeeded in establishing human TSC lines from blastocysts and early gestation placentae, highlighting the essential roles of epidermal growth factor (EGF) and a WNT activator in the establishment and maintenance of human TSCs 15 . This study clearly showed that an exogenous FGF4 is not required for the proliferation of human TSCs, though the direct effect of FGF4 on human TSCs was not examined.
Although both human and mouse have the discoidal hemochorial placenta, there are substantial differences between these two species in placental formation processes after implantation 16,17 . Successful establishment of human TSCs employing a group of exogenous factors, completely different from mouse TSCs, raised questions on whether this dependency on different factors reflect the species-specific differences between mice and humans, or if it could be generalized as cellular differences between rodents and primates 15 . Establishment of TSCs from non-human primates will offer valuable insights to address this important question. Previous reports show the establishment of TSC lines from rhesus monkey blastocysts 18 . Although these cell lines were capable of differentiating into both invasive cells and multinucleated cells; they also expressed the ICM/ESC marker, OCT4, and the marker for STBs, chorionic gonadotropin (CG) 18,19 , without induction of differentiation, making it uncertain if these cells are genuine TSCs. Accordingly, the establishment of trophoblastic stem/progenitor cell lines from a non-human primate, whose placental structure is similar to that of humans, remains an important issue. To address this, we aimed to derive cell lines from the cynomolgus monkey blastocysts, and successfully obtained stable proliferating trophoblastic cell lines exhibiting differentiation potential, by adopting the modified culture conditions for mTSCs without using EGF and WNT activator.
Results establishment of cynomolgus monkey trophoblastic cell lines from blastocysts. We attempted to establish trophoblastic cell lines from cynomolgus monkey blastocysts by adopting the conditions used for the establishment and maintenance of mouse TSC, with slight modifications. In the original protocol, mouse TSC lines are established in the presence of FGF4, heparin, and mouse embryonic fibroblast cells (MEF) 13,20 . For the maintenance of mouse TSCs, MEF can be replaced by the MEF-conditioned medium (MEF-CM) or by activin A 20,21 . We, therefore, co-cultured a single cynomolgus monkey blastocyst (#2) and MEF in the TS medium supplemented with 50% MEF-CM, activin A, FGF4, and heparin (abbreviated as 50CM/AFH in Fig. 1A). In addition to AFH; BMS493, a pan-RAR inverse agonist; and Y-27632, a ROCK inhibitor (abbreviated as BY in Fig. 1A) were also added because these reagents appeared to facilitate maintenance of mouse TSCs in a serum-free, knockout serum replacement (KSR)-supplemented condition (manuscript in preparation). Another blastocyst (#1) was also co-cultured with MEF, but without AFHBY as a control. Both blastocysts attached to the surface of tissue culture dish displaying cellular outgrowth by the 5 th day of culture, although the embryos did not hatch completely (Fig. S1A). The blastocyst outgrowths were passaged to expand on the 12 th day of culture, and 70% MEF-CM was added (70CM) instead of co-culturing with MEF. As a result, a stable growing cell line forming a flat monolayer of colonies with epithelial cell-like appearance, named macTSC#2, was obtained from blastocyst #2 after another passage in AFHBY-containing media (Fig. 1A,B). The macTSC#2 cells steadily grew for at least 132 days (4 days × 33 passages) without an apparent reduction in their growth potential (Fig. 1C). A cell line, indistinguishable from macTSC#2, albeit with slower cell growth, was obtained unexpectedly from the blastocyst without AFHBY and named macTSC#1 (Figs. 1A and S1B).
To elucidate the factors required for the growth of macTSC#2, we cultured the cells with several combinations of factors used in the AFHBY condition, but in the absence of MEF. The combination of FGF4 and its co-factor heparin (FH) promoted cell growth, whereas activin A was dispensable. Removal of FH or all the factors slowed the growth rate of cells, but cells grew stably unlike mouse TSCs (Figs. 1D and S1C). We also observed a positive effect of BMS493 and Y-27632 (BY) on cell growth and decided to use the FHBY condition for routine maintenance of macTSC lines. Under this condition, growth of macTSC#1 was also stimulated (Fig. S1E), and 49.0% and 40.7% of macTSC#1 and #2 cells, respectively, remained euploid after 21 -30 (#1) and 20 -35 (#2) passages (Fig. S1D,F).
The adequacy of the FHBY condition was verified by culturing another set of blastocysts (#3-#5, Fig. 1A). While the establishment of macTSC#3 cell line was done using the same initial condition as was used for macTSC#2, thereby demonstrating the reproducibility of the procedure, the two other macTSC lines; namely, macTSC#4 and #5, were obtained using the FHBY condition, showing the dispensability of MEF. Cells of macTSC#3-#5 lines also had an indistinguishable appearance from macTSC#1 and #2 (Fig. S1B).  to both ESC 24 and embryonic fibroblast cells of cynomolgus monkey (Fig. 2B). The FF36 region was methylated moderately in macTSC#2; however, this region was methylated similarly in ESCs, unlike in mouse and human ESC. The EG01 region was hypermethylated in ESC, again unlike mouse and human ESC. Analysis of macTSC#1 gave similar results (Fig. S2A,B). These epigenetic features in T-E T-DMRs also supported that macTSCs were of trophoblastic lineage. Thus, the bisulfite sequencing analysis revealed a DNA methylation profile of macTSCs consistent with their trophoblastic origin.
Expression of miRNAs of primate-specific chromosome 19 miRNA cluster (C19MC). Another criterion by Lee et al. for defining early human trophoblast was the high expression of C19MC miRNAs 22 . These miRNAs are expressed almost exclusively in the human placenta, but also in ESC and some other tissues [25][26][27] . C19MC miRNAs are highly conserved in the macaque genome, and some of them were expressed in first trimester and term placenta 28 . We detected the expression of all C19MC miRNAs examined in macTSC#2. Four of them (miR-1323, miR-525, miR-520h, and miR-519d) showed significantly higher expression in macTSC#2 than in somatic tissues (Fig. 3), in which these three miRNAs were almost undetectable. Even though the expression of miR-517s (the primer set detected all subtypes of miR-517) was detected in somatic tissues, it was also detected in macTSC#2 at a comparable level to that in the placenta. Expression pattern of those C19MC miRNAs was detected at a similar level in macTSC#2 as compared to the other macTSC lines (Fig. S3). These results suggested that macTSCs, derived from cynomolgus monkey blastocysts, express members of the primate trophoblast specific C19MC miRNAs. expression of genes related to placental development. To explore similarities among macTSC lines, and their transcriptomic characteristics, we performed RNA-seq analyses. First, we compared the correlation of gene expression profiles of all macTSC lines with ESC, fibroblast, and placenta. As shown in Fig. 4A, all macTSCs formed a close cluster apart from the ESC, fibroblast, and placenta, suggesting that all macTSC lines have similar gene expression profiles even though they were established under different culture conditions. Gene ontology (GO) term enrichment analysis of the genes with >2-fold higher expression in all macTSC lines than in ESC revealed the terms associated with angiogenesis, positive regulation of epithelial cell migration, and placenta development (Fig. 4B). Importantly, some genes such as KRT7, GATA3, TFAP2C, VGLL1, and HAND1, which are known to be highly expressed in trophoblasts, were also highly expressed in macTSCs compared to ESCs (Table S1). In contrast, genes with >2-fold lower expression in macTSC lines than in ESC were associated with somatic cell differentiation (Fig. S4). These results collectively indicated that macTSCs are cells of trophoblast origin.
Recent reports showed human VGLL1 gene expression in proliferative, undifferentiated CTBs residing in the early human placenta 29 . The robust expression of VGLL1 in macTSCs thus implied that the macTSCs might have the potential to differentiate. To test this, we treated the cells with dbcAMP in the absence of FHBY, and analyzed gene expression of each trophoblast subtype markers, since an increase in cellular cAMP is known to induce differentiation of human trophoblast cells 8,30 . First, we analyzed the expression of CTB markers by qRT-PCR (Fig. 4C). The expression levels of TEAD4, which is widely used as an undifferentiated trophoblast marker 31 , were downregulated by the FHBY removal regardless of the presence of dbcAMP. GATA3, known as a trophoblast marker 22 , was also downregulated. On the other hand, EpCAM expression was not reduced significantly by the removal of FHBY, but significantly downregulated by the dbcAMP treatment. In contrast, STB markers, GCM1, CGA, and CGB were upregulated significantly by dbcAMP treatment (Fig. 4D). In addition, expression of the putative fusogenic endogenous retroviral genes, ERVV-1, and ERVV-2 32 , was also increased (Fig. 4D). As shown in Fig. 4E, HTRA4, ITGA5, and CDH5, which are used as EVT markers, were also upregulated significantly by dbcAMP treatment. One of the invasiveness-related matrix metalloproteinase genes, MMP2, was also induced by dbcAMP treatment. Although RNA-seq indicated upregulation of another member of MMP gene family, MMP9, in all the five macTSCs (Table S2), the expression of MMP9 did not change by culture conditions based on the primer set used. www.nature.com/scientificreports www.nature.com/scientificreports/ Differentiation of multinucleated STBs from macTSC. Human and non-human primate trophoblast cells differentiate to form multinucleated STBs, which secrete CG via cell fusion. Since results from qRT-PCR suggested that dbcAMP induced macTSC differentiation, we investigated whether macTSC#2 differentiates into Enriched GO terms associated with the genes with >2-fold expression in all macTSCs compare to that in ESC. PANTHER (www.pantherdb.org) was used for GO analyses. Gene ratio, the ratio of the analyzed genes in the gene set of the GO term; Count, the number of analyzed genes in the GO term. P-values were calculated using Fisher's exact test with Bonferroni correction. (C,D,E) qRT-PCR for analyzing the expression of CTB (C), STB (D) and EVT (E) markers. The mean values (±SD) of technical replicates for biological duplicates, normalized by the expression of ACTB, were shown relative to those of cells maintained under the FHBY condition (arbitrarily set as 1). −dbcAMP, basal TS medium without FHBY and dbcAMP; +dbcAMP, basal TS medium with 1 mM dbcAMP. *p < 0.05 vs. FHBY (Two-tailed Student's t-test).
STBs. The CG protein in dbcAMP-treated macTSC#2 was easily detectable by immunofluorescence (Fig. 5A). Furthermore, to detect the formation of multinucleated trophoblasts, cells were stained for E-Cadherin to visualize the cell border, and the frequency of appearance of multinucleated (≥ three nuclei per cell) cells was analyzed. While 5% and 3% of nuclei were fused to form multinucleated cells under FHBY and -dbcAMP conditions, respectively, dbcAMP treatment increased appearance frequency of such nuclei to 15% (Fig. 5B,C and Table S3). Macaque placenta secretes progesterone and estradiol during the gestation 33 . To see if differentiated macTSCs produce and secrete these steroid hormones, we measured the amount of progesterone and estradiol secreted in the culture media by ELISA (Fig. 5D,E). While there was no difference in secreted estradiol between FHBY and +dbcAMP conditions (Fig. 5E), the amount of progesterone was significantly increased in +dbcAMP condition (Fig. 5D). It is not known if there is no estrogen secretion, or the level was too low to detect over the background. These analyses suggested that at least some of the cells differentiated to the CG-positive, progesterone-producing STBs upon dbcAMP treatment of macTSC#2, but the differentiated STBs may not be fully functional.
Differentiation of invasive EVT from macTSC. Human and non-human primate CTBs, especially those located in cell column, differentiate into another cell type including the invasive EVTs. To address whether macTSCs also differentiate towards EVT, we investigated the expression of EVT marker protein, and the invasiveness of macTSC. MMP2 and MMP9 reportedly degrade gelatin. We first performed gelatin zymography to detect MMP2 and MMP9 activity in the macTSC#2 cell extract, and the results show augmented level of MMP2 protein by the dbcAMP treatment (Fig. 6A). Similarly, the dbcAMP-treated macTSC#2 showed increased expression of Mafa-AG protein by immunofluorescence compared to cells in the FHBY condition (Fig. 6B).
The expression pattern of HLAs can be used to evaluate the subtype of human trophoblast cells 2 . While a lack of HLA-C ortholog, the only HLA type I molecule expressed in the human EVT, in the macaque genome hampers a straight application of this evaluation, cynomolgus STB and EVT were intensely stained by anti-human HLA type I antibodies 34,35 . However, undifferentiated CTB-like cells are detectable by the expression of HLA-A and -B ortholog. The flow cytometric analysis by using a pan anti-human HLA class I antibody, W6/32, and the 25D3 antibody for detecting Mafa-AG 35 , showed an increase in the ratio of HLA class I + /Mafa-AG + putative EVT-like population from 23.6% to 32.4% by dbcAMP treatment at the expense of HLA class I − /Mafa-AG − population (Fig. 6C, S5, and S6). This result suggested that some cells with EVT-like MHC expression profile existed even under the FHBY condition and that dbcAMP shifted the distribution of cell populations towards EVT-like cells.
Finally, we analyzed the invasiveness of macTSC#2 cells by employing the Matrigel invasion assay 36 . Cells were cultured for four days on the cell culture inserts, with or without Matrigel-coating, followed by counting the number of nuclei of invaded or migrated cells to calculate invasion-to-migration ratio (% invasion index). Although these analyses showed the presence of invasive EVT-like cells even in the FHBY condition in agreement with the flow cytometry results mentioned above, the ratio of invasive to migratory cells was increased significantly by the dbcAMP treatment (Fig. 6D,E). These results demonstrated that a part of macTSC#2 also acquired the invasive EVT-like phenotype upon dbcAMP treatment.
Gene expression profile of macTSCs under the dbcAMP condition. The dbcAMP treatment induce human trophoblast differentiation toward multinucleated STB 8,30 . In this study, however, invasive EVT-like cells www.nature.com/scientificreports www.nature.com/scientificreports/ also appeared upon dbcAMP treatment of macTSC#2 (Figs. 5 and 6), suggesting that dbcAMP treatment also trigger EVT cell fate in macTSCs. To further resolve this question, we analyzed comprehensive gene expression profiles of macTSCs with dbcAMP by RNA-seq. From the result of PCA, the gene expression profiles of all five macTSC lines were slightly changed (Fig. 7A). The GO analysis of >2-fold upregulated genes after dbcAMP treatment in all macTSC lines showed enrichment of terms related to positive regulation of cell migration, vasculature development, and angiogenesis (Fig. 7B). Remarkably, these upregulated genes commonly included GCM1, MMP2, CDH5, and HTRA4, which are expressed exclusively or preferentially in the human placenta (Table S4) [37][38][39] . GO terms related to cell proliferation were enriched in >2-fold downregulated genes, (Figs. 7C and S7) reflecting the inhibition of cell growth by dbcAMP treatment. In addition, gene set enrichment analyses showed the upregulation of the genes associated with not only cell-cell fusion, but also epithelial to mesenchymal transition, the terms linked to multinucleated STB and invasive EVT formation, respectively (Fig. 7C). These results also suggested that all macTSC lines had the potential to differentiate towards both STB and EVT. www.nature.com/scientificreports www.nature.com/scientificreports/ contribution of mactSc to the te region of mouse blastocysts. Given that macTSCs were derived from blastocysts and showed the potential to differentiate toward both STB and EVT, we hypothesized that macTSCs will retain a TE cell-like feature at least partially. We addressed this question by performing a xenogeneic chimera assay by aggregating macTSC cells with mouse eight-cell stage embryos. For this purpose, several Venus-overexpressing macTSC lines from macTSC#2 and macTSC#4 were established to locate macTSC-derived cells in the xenogeneic chimeric blastocysts, and two of them, macTSC#2-Venus and macTSC#4-Venus, were used in the assay (Figs. 8A and S8). Notably, macTSCs were incorporated successfully into 31% (macTSC#2) and 46% (macTSC#4) of aggregates (Figs. 8B,C and S9). All macTSC cells were located at TE region, but not at ICM. These results demonstrated that macTSCs could participate in blastocyst formation by being a part of TE.

Discussion
To date, human TSCs were not derived using similar or a variation of the conditions used for the derivation of mouse TSCs 14 . Moreover, no study investigated the effect of FGF4 on macaque trophoblast cells, underestimating the role of FGF4 in the proliferation of primate trophoblast stem/progenitor cells. In this study, we obtained a new type of cell lines, macTSC#1~#5, from cynomolgus monkey blastocysts in TS medium with FGF4, heparin, BMS493, and Y-27632. Although the exact origin of the cells is unknown, macTSCs met the recently proposed criteria for the definition of early human trophoblast cells 22 , such as expression of trophoblast/placenta-specific genes including the primate-specific C19MC miRNAs, and the hypomethylation of ELF5 promoter (Figs. 2A, 3 and 6C). Previously identified T-E T-DMRs also showed DNA methylation status characteristic of trophoblast cell lineage (Fig. 2B). In addition, dbcAMP treatment not only enhanced the expression of STB and EVT marker genes/proteins, but also increased the formation of multinucleated cells and their invasiveness (Figs. 4, 5, and 6). Taken together, we conclude that the cell lines derived from cynomolgus monkey blastocysts, in this study, are essentially the trophoblastic cells with differentiation potential. The FGF4 signaling is essential for www.nature.com/scientificreports www.nature.com/scientificreports/ the maintenance of the undifferentiated state, and for promoting the proliferation of mouse TSCs 13,40 . FGF4 also enhanced proliferation of macTSCs in this study, although it appeared dispensable for the maintenance of macTSCs' undifferentiated state (Fig. 1C,D). Thus, the macTSCs are the first primate trophoblast stem cell lines whose proliferation is promoted by FGF4.
It should be noted that we cannot rule out the possibility that the macTSC line is a heterogeneous cell population, including progenitors of both STB and EVT, and that the dbcAMP treatment may have induced differentiation of both these progenitor cells. In the flow cytometric analysis, around 30% of cells were pan HLA class I + /Mafa-AG − in any conditions (Fig. 6C). Although the identity of this population is ambiguous, freshly isolated cynomolgus monkey trophoblast showed higher intensity in W6/32 staining compared with isotype control 35 . Besides, W6/32 and 25D3 antibodies recognized STB in chorionic villi of cynomolgus monkey placenta 35 . Thus, pan HLA class I + /Mafa-AG − population may be composed of CTB-and STB-like cells, and pan HLA class I + / Mafa-AG + population might include not only EVT-but also a few STB-like cells. Another concern regarding the heterogeneity is a possible contamination of ICM-derived cells. We would like to point out that the cell lines were established in three different initial culture conditions. One of the three conditions did not even include any exogenous factors (Fig. 1A). If there were any ICM-derived cells survived, they likely differentiated into the cells with different phenotypes depending on the culture conditions. However, the five cell lines showed highly similar gene expression profiles to each other (Fig. 4A), implying that there is no or minimal contamination of ICM-derived cells in the established cell lines. A single-cell RNA-sequencing or a single-cell cloning and subsequent differentiation anlyses can conclude if macTSCs are indeed multipotential stem cells. This caveat will be addressed in a future study.
RNA-seq analyses of macTSCs revealed dbcAMP-dependent upregulation of CD9 (Table S2), which is proposed as a marker for putative human EVT progenitor cells 41 . This might suggest that macTSCs are in a developmentally immature, and an undifferentiated state compared to EVT progenitor cells, which are known as cell column trophoblast located in the tip of villi, and that dbcAMP induced differentiation of a part of macTSCs towards EVT through a temporal EVT progenitor state. RNA-seq and subsequent GO term enrichment analyses showed that genes associated with the terms related to the cardiovascular system, such as "ventricular septum morphogenesis, " and "artery morphogenesis, " were upregulated upon dbcAMP treatment (Fig. 7B). A recent finding showing placental defects are highly prevalent in mutant mice with embryonic abnormalities in heart, brain, and vascular system may partly explain our GO term enrichment results 42 . In other words, genes with relevant www.nature.com/scientificreports www.nature.com/scientificreports/ functions in trophoblast or placenta are likely associated with the terms related to the cardiovascular system. We, therefore, consider that the GO analysis result is not conflicting with the feature of trophoblast cells.
Vandevoort et al. have previously reported the derivation of TSC lines from TE of rhesus monkey blastocysts, plated on collagen-coated dishes after initial expansion with rhesus embryonic feeder cells, but without any exogenous factors 18 . While these cells expressed trophoblast marker genes such as KRT7 and CD9, CDX2 expression was undetectable by both immunohistochemistry and RT-PCR. In addition, the expression of CGB, one of the STB markers, was observed without induction of differentiation, suggesting that the rhesus TE-derived cells are actually in a more differentiated state than our macTSCs. It was noted that the expression of CDX2 increased in the rhesus TE-derived cells when cultured in the presence of Activin A 19 . In contrast, there was no expression of CDX2 in macTSCs (Fig. S10), even in the presence of Activin A. Furthermore, the effect of FGF4 on the rhesus monkey TSCs was not investigated.
Recently, human TSCs (hTSCs) were established from blastocysts and early gestation placentae 15 . Both hTSCs and macTSCs expressed CTB marker genes, such as TEAD4, TP63, and FZD5 in the undifferentiated state, while upregulation of STB and EVT markers, including CGA, CGB, MMP2, ITGA5, and CD9 was induced by the differentiation condition (Figs. 5, 6 and Table S2). Thus, hTSCs and macTSCs show similarities in the expression pattern of each of the trophoblast marker. In addition, both macTSCs and hTSCs have differentiation capacity toward STBs and EVTs. However, the requirement of exogenous factors is significantly different. While the EGF and Wnt signaling activator were essential for the maintenance of hTSCs and CTBs 15,43 , PD153035, an EGF inhibitor, caused slight reduction without complete inhibition of the proliferation of macTSCs in the presence of FGF4 (Fig. S11). Thereby, demonstrating that the effect of EGF-like activities in the FBS, if present, on the proliferation of macTSCs is negligibly weak. FGF4 was not essential to maintain human TSCs, whereas it enhanced the proliferation of macTSCs. Both human and macaque have the discoidal hemochorial placenta that comprised mostly of CTB, STB, EVT, and extraembryonic mesodermal cells 16,44 . In humans, after the attachment of the blastocyst on the uterine wall, trophoblasts invade decidua to lead the whole embryo to embed within endometrium 16 . In the macaque, the blastocyst does not invade decidua and stays at the surface of the uterine wall resulting in the formation of a secondary placenta on the opposite side of the primary implantation site 45 . The observed difference in the requirement of growth factors between hTSCs and macTSCs could be due to a difference in the placentation process described above. Induction of macTSCs' differentiation toward STB by dbcAMP treatment was seemingly inefficient compared to that of hTSCs 15 , regardless of statistically significant increase in the marker gene expression and the fusion index. Considering this argument along with the fact that macTSCs have a relatively unstable karyotype, the FHBY condition may be suboptimal. It would be worthwhile to investigate similarities in transcriptome and epigenome of the hTSCs and macTSCs, furthermore, how macTSCs may perform when grown in hTSCs culture conditions should be investigated in future studies.
In mice, TSC lines demonstrated relevant roles of certain transcription factors (TFs), such as Cdx2, Elf5, and Eomes 46 , in the specification, maintenance, and differentiation of TSCs. However, specific roles of these TFs in the primate trophoblast lineage remain obscure, mostly due to lack of appropriate model systems, as recently discussed by Knöfler et al. 47 . As these authors suggested, the availability of the recently developed hTSCs 15 , and 3D placental organoids 43,48 will accelerate the functional analysis of such TFs. Thereby, deepening our knowledge about TSCs and key TFs involved in trophoblast lineage. The establishment of macTSC lines, as in the present study, can facilitate implementation of such research experiments. Knowing more molecularly-specific similarities and differences between the trophoblast of human and non-human primates is a prerequisite for pre-clinical studies. The macTSCs provide a useful tool for revealing key TF networks that are common to the primate trophoblast, and for delineating human-specific and macaque-specific functions of underlying TFs. In addition, these non-human primate TSCs will provide cellular proxy for in vivo analyses, such as the chimeric analysis, which are prohibited in humans for ethical reasons. Such attempt may reveal whether macTSCs show the TE-like feature and contribute to the placenta in a future study.

Methods
Reagents. All reagents were purchased from Wako (Osaka, Japan) unless otherwise noted. All PCR primers were purchased from Sigma-Aldrich (Tokyo, Japan) or Eurofins Genomics (Tokyo, Japan). establishment of trophoblastic cell lines from cynomolgus monkey blastocysts. Cynomolgus monkey blastocysts (five embryos in total) were transferred to the University of Tokyo, and cultured for 2-8 days with or without mitomycin C-treated mouse embryonic fibroblasts (MEF) in basal culture medium (TS medium) consisting of RPMI-1640 medium supplemented with 20% fetal bovine serum (FBS, Biowest, Nuaillé, France), 2 mM L-glutamine, 1 mM sodium pyruvate, and 0.1 mM 2-mercaptoethanol, and exogenous reagents as indicated in Fig. 1A. The reagents used were 10 ng/ml Activin A (R&D Systems, Minneapolis, US), 25 ng/ml FGF4, 1 µg/ml Heparin (Sigma-Aldrich), 1 µM BMS493 (TOCRIS, Bristol, UK), and 10 µM Y-27632. The cells were incubated at 37 °C in a humidified atmosphere of 5% CO 2 , and the culture medium changed once every two days after the blastocysts firmly attached to the dish. When blastocyst outgrowth expanded, it was trypsinized and re-plated on a larger multi-well plate or dish. qRt-pcR. cDNA was synthesized using SuperScript ® III Reverse Transcription (Invitrogen). qRT-PCR was performed using THUNDERBIRD ™ SYBR ® qPCR Mix (TOYOBO, Osaka, Japan). Amplification and fluorescence detection were carried out using Light Cycler ® 96 (Roche, Basel, Switzerland). Primers used are shown in Table S5. fluorescence immunocytochemistry. Cells grown on coverslips were fixed with 4% paraformaldehyde in PBS(-). Fixed cells were then permeabilized with 0.1% Triton-X in PBS(-) and incubated with 5% BSA, 0.1% Tween-20 in PBS(-) overnight. Subsequently, the cells were incubated with primary antibodies at 4 °C overnight.
Primary antibodies used in this study are listed in Table S6. The signals were visualized by Alexa Fluor ® 488 goat anti-mouse IgG (A11029, Invitrogen) or Alexa Fluor ® 594 goat anti-rabbit IgG (A11037, Invitrogen). After nuclear counter-staining with DAPI (DOJINDO), the cells were mounted on glass slides with VECTASHIELD ® H-1000 (Vector Laboratories, Burlingame, US). The cells containing three or more nuclei were defined as the multinucleated cells in this study. The fusion index (%) was calculated by the following formula: [(the number of nuclei in the multinucleated cells in a field of microscopic view) -(the number of multinucleated cells in a field of microscopic view)]/(total number of nuclei in a field of microscopic view)] × 100 (Table S3). 25D3 antibody, which has been shown to detect Mafa-AG, a cynomolgus monkey homolog of human HLA-G 35,49 , was kindly provided by Dr. Thaddeus G. Golos, University of Wisconsin-Madison.
Measurement of steroid hormones in culture medium by ELISA. After the 6-days differentiation of macTSC#2, culture medium was replaced to a fresh TS medium containing 0.1% FBS and further cultured for 9 hours. The culture medium was then collected, centrifuged to remove the dead cells and debris, and the supernatant was used for ELISA. One ml of culture medium was extracted twice with 2 ml of diethyl ether and incubated at −80 °C for 20-30 minutes. The supernatant was transferred to a new glass tube and dried up at 55 °C. The extract was diluted with 200 µl of ELISA Buffer in Progesterone ELISA Kit (Cayman Chemical, Ann Arbor, US) or Assay Buffer in DetectX ® SERUM 17β-ESTRADIOL Enzyme Immunoassay Kit (Arbor Assays, Ann Arbor, US). The measurement of progesterone and estradiol was performed by Progesterone ELISA Kit and DetectX ® SERUM 17β-ESTRADIOL Enzyme Immunoassay Kit following manufacturers' instructions.
protein extraction and zymography. Total protein was extracted with RIPA buffer containing 1 mM PMSF. Total proteins (5 µg) were then separated by SDS-PAGE using 10% acrylamide gel containing 1.0 mg/ml gelatin. After the electrophoresis, the gel was incubated twice in the renaturation solution (2.5% Triton X-100) for 40 minutes each followed by overnight incubation in developing buffer (50 mM Tris-HCl pH 7.4, 200 mM NaCl, 5 mM CaCl 2 , and 1% Triton X-100) at 37 °C. After incubation, the gel was stained with Coomassie blue stain and de-stained using 30% methanol/10% acetic acid solution until the bands became visible.

Establishment of Mafa-AG/Venus-overexpressing macTSC lines. A macTSC#2 line overexpress-
ing Mafa-AG was established to set the threshold in flow cytometric analysis. A full-length ORF of Mafa-AG was amplified by using a primer set that added a 3xFlag tag at the N-terminus (Table S5) and cloned into pEN-TR ™ /D-TOPO ® (Invitrogen). The 3xFlag-Mafa-AG cDNA insert was transferred into a destination vector, including Venus and puromycin-resistant genes 50 using Gateway ® LR Clonase ™ II (Invitrogen). The resulting Mafa-AG/Venus-expression vector was transfected into macTSC by using jetPRIME ® in vitro DNA & siRNA Transfection Reagent (Polyplus Transfection, Illkirch, France) for 6 hours in the FHBY condition. Transfected cells were selected with 10 µg/ml puromycin for 48 hours after the transfection. Single Venus-positive colonies were picked and expanded separately in the FHBY condition. A cell line with a uniform and strong Venus fluorescence was used in the present study. flow cytometry. Cells were harvested with trypsin, suspended in 1% BSA/PBS(-) and incubated at room temperature for 30 min. Then, 10 µg of the primary antibodies were added, followed by another incubation for 30 min at room temperature. Refer to Table S6 for the primary and secondary antibodies used in this assay. The dead cells were stained with propidium iodide (Sigma-Aldrich). The expression of Mafa-AG and HLA class I was analyzed by FACSVerse ™ Flow Cytometer. Isotype controls of anti-Mafa-AG and anti-HLA class I antibody was used to draw a threshold line (Fig. S5).
invasion assay. macTSC#2 cells were pre-cultured in the proliferative (FHBY) or differentiation (+dbcAMP) conditions. macTSC#2 cells in the proliferative or differentiated state were cultured for four days in FluoroBlok ™ cell culture inserts with 8.0 µm pore size (Corning, New York, US) covered with Matrigel ® matrix (Corning) in FHBY or +dbcAMP conditions. Then, 1.5 × 10 3 (FHBY) or 1.0 × 10 4 (+dbcAMP) cells were placed on the insert. The inserts were filled with the TS medium supplemented with 0.1% FBS so that the cells get attracted to the TS medium supplemented with 20% FBS in the bottom wells. Cell nuclei on the lower surface of the inserts were stained with Hoechst 33342 (Lonza, Basel, Switzerland) and photographed. Migration assay, without Matrigel coating, was also performed in parallel as a control. The invasion indices, [(number of invaded cells/number of migrated cells) × 100] (%), were calculated for each condition from 6 (FHBY) and 21 (+dbcAMP) randomly chosen microscopic images each of biological duplicate (835 and 364 total nuclei in FHBY and +dbcAMP conditions, respectively). Since, dbcAMP treatment severely block cell proliferation, more images were required to achieve enough number of nuclei.

Establishment of Venus-overexpressing macTSC lines. macTSC#2 and #4 lines overexpressing
Venus was established for xenogeneic chimera assay. The 3x Flag Venus-expression vector 50 was transfected into macTSC using jetPRIME ® in vitro DNA & siRNA transfection reagent (Polyplus Transfection, Illkirch, France) for six hours in the FHBY condition. Transfected cells were selected with 10 µg/ml puromycin for 48 hours after the transfection. Single Venus-positive colonies were selected and expanded separately in the FHBY condition.
Western blotting. Total protein was extracted with RIPA buffer containing 1 mM PMSF. Total proteins (5 µg) were separated by SDS-PAGE and transferred onto Immobilon PVDF membrane (Millipore, Darmstadt, Germany). The membrane was incubated in blocking buffer (5% BSA, 0.1% Tween-20/TBS) for 1 hour, followed by overnight incubation with anti-GFP antibody or IgG (Table S6) at 4 °C. After rinsing with wash buffer (0.05% Tween-20/TBS), the membrane was incubated with the secondary antibody diluted in blocking buffer at room temperature for 1 hour. The protein of interest was detected using ImmunoStar Basic.