Three-dimensional human placenta-like bud synthesized from induced pluripotent stem cells

Placental dysfunction is related to the pathogenesis of preeclampsia and fetal growth restriction, but there is no effective treatment for it. Recently, various functional three-dimensional organs have been generated from human induced-pluripotent cells (iPSCs), and the transplantation of these iPSCs-derived organs has alleviated liver failure or diabetes mellitus in mouse models. Here we successfully generated a three-dimensional placental organ bud from human iPSCs. The iPSCs differentiated into various lineages of trophoblasts such as cytotrophoblast-like, syncytiotrophoblast-like, and extravillous trophoblast-like cells, forming organized layers in the bud. Placental buds were transplanted to the murine uterus, where 22% of the buds were successfully engrafted. These iPSC-derived placental organ buds could serve as a new model for the study of placental function and pathology.

The placenta is a multifunctional organ that supports the developing fetus.Improper placental development in the early stages of pregnancy can result in fetal growth restriction, preeclampsia, and miscarriage 1,2 .Surprisingly, there are few in-vitro experimental models of the early stages of human placental development, although these would improve our understanding of the physiological aspects of placentation and our ability to develop novel treatments for placental dysfunctions.
Trophoblasts are specialized cells of the placenta that originate from the trophectoderm, the outer layer of the blastocyst 3 .The placenta is composed of three types of trophoblasts: cytotrophoblasts (CTBs), syncytiotrophoblasts (STBs) and extravillous trophoblasts (EVTs).Proliferative CTBs can differentiate into either STBs or EVTs.STBs form a multinuclear layer in the outer surfaces of the placental villi and are responsible for gas/nutrient exchange and hormone production.EVTs invade the lumina of the spiral arteries as well as the decidua and the myometrium, where they contribute to anchoring the placenta and directing a sufficient supply of maternal blood into it.The differentiation of trophoblasts can be observed through changes in the expression patterns of specific gene markers.Trophoblast ectoderm (TE) is represented by caudal type homeobox 2 (CDX-2)/(CDX2).Tumor protein p63 (TP63)/TP63 is a proliferative CTB marker and cytokeratin 7 (CK7)/keratin 7 (KRT7) is a pan-trophoblast or CTB marker.The STB markers are human chorionic gonadotropin (hCG)/chorionic gonadotropin subunit beta 3 (CGB3) and syncytin-1/endogenous retrovirus group W member 1 (ERVW1).The EVT marker is human leukocyte antigen G (HLA-G)/(HLAG), and the cell column trophoblast marker is peroxisome proliferator-activated receptor gamma (PPAR-γ)/(PPARG ).
Three-dimensional organ buds, including liver, kidney, and pancreas buds, have been generated in vitro by culturing tissue-specific progenitors from induced pluripotent stem cells (iPSCs) with human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (MSCs) [4][5][6] .Elsewhere, trophoblast-like cells have been created from human embryonic stem cells by adding bone morphogenetic protein 4 (BMP4) 7 .Here, we attempted to generate three-dimensional human placenta-like organ buds from iPSCs with the aid of endothelial and mesenchymal cells.

Results
Differentiation of iPS cells into trophoblast lineage by BMP4 treatment.First, iPSCs were treated with 100 ng/mL of BMP4 in a three-dimensional (3D) gel matrix and cultured for eight days (Fig. 1A).To observe the subsequent differentiation of two cell lines of iPSCs (201B7 and 409B2) into trophoblast-like cells, we analyzed representative markers of the trophoblast lineage as a function of time (Fig. 1B-H).mRNA expression levels of the pluripotent cell markers, sex determining region Y-box2 (SOX2)/(SOX2), Nanog homeobox (NANOG), and octamer-binding transcription factor 4 (OCT4)/POU class 5 homeobox 1 (POU5F1) were high in iPSCs and day 0 cells (Fig. 1B-D), but gradually decreased from day 2, indicating differentiation into trophoblasts.In contrast, mRNA expression levels of caudal type homeobox 2 (CDX2), a TE marker, was low in iPSCs and day 0 cells but abruptly increased after two days of BMP4 treatment, suggesting that iPSCs had started to differentiate into TE-like cells by this time (Fig. 1E).Starting on day 4, however, this increase was reversed: CDX2 expression decreased to and then remained at a modest level, suggesting the further differentiation of TElike cells into CTB-and STB-like cells (Fig. 1E).
The mRNA levels of PPARG , KRT7, and TP63 were low in iPSCs and day 0 cells, but increased from day 2, reaching a peak at day 4 in 201B7 cells (Fig. 1F-H).On the other hand, in 409B2 cells, the mRNA levels of those genes peaked on day 6 (Fig. 1F-H).This suggests that on days 4-6 of BMP4 treatment, iPSCs differentiate into cell column trophoblast-and CTB-like cells, and that the rate of differentiation into CTBs is relatively slow in 409B2 compared to 201B7.Subsequently, the mRNA expression levels of PPARG , KRT7, and TP63 decreased slightly (Fig. 1F-H), suggesting that these cells further differentiated into STB-like cells after day 4 of BMP4 treatment, although they retained some degree of CTB character.
The mRNA expression levels of the STB markers human chorionic gonadotropin (hCG)/ chorionic gonadotropin subunit beta 3 (CGB3) and syncytin-1/endogenous retrovirus group W member 1 (ERVW1) remained low from the iPSC stage through day 2-4, suggesting that differentiation into the STB phenotype did not occur until day 2-4 (Fig. 1I,J).From day 4 in 201B7 and day 6 in 409B2, however, CGB3 and ERVW1 mRNA increased (Fig. 1I,J), indicating that BMP-4-treated iPSCs were differentiating into STB-like cells.Similarly, human leucocyte antigen-G (HLAG) mRNA increased from day 4-6 of BMP4 treatment (Fig. 1K), implying that some portion of iPSCs were differentiating into EVT-like cells.Since CTB-like differentiation was relatively slower in 409B2 than in 201B7 (approximately 2 days behind), the rate of differentiation into STB-and EVT-like cells were concomitantly slower in 409B2 compared to 201B7.
Next, we compared the expression levels of cytokeratin-7 (CK7) and HLA-G between day 0 and 4 of BMP4 treatment by immunofluorescence in 201B7 iPSCs.CK7 was not detected before BMP4 treatment (day 0) but was highly expressed in the cytoplasm on day 4 (Fig. 1L).Similarly, HLA-G was not expressed on day 0 but was observed around the nucleus on day 6 (Fig. 1M).These changes in CK7 and HLA-G protein expression were compatible with those in their mRNA expression (Fig. 1G,K).Collectively, these results demonstrate that iPSCs were successfully differentiated into trophoblast-like cells by BMP4 treatment, and this trophoblast differentiation was universally observed in different iPS cell lines.
Generation of three-dimensional placental organ buds from iPSCs.Next, we attempted to generate a placental organ bud from iPSCs.We began this experiment with iPSCs that had been stimulated with BMP4 for four days.Trophoblast-like cells treated with BMP4 were mixed with human mesenchymal stem cells (MSCs) from the amnion and human umbilical vein endothelial cells (HUVECs) from the umbilical cord in a 3D-gel matrix (Fig. 2A).One day after mixture, they started to self-organize into three-dimensional cells in both 201B7 and 409B2 iPSCs (Fig. 2B,C).Self-organization did not occur in the absence of HUVEC or MSCs (Supplementary Fig. 1).We were also able to generate placental buds from trophoblast-like cells that had been treated with BMP4 for either 2 or 6 days, but not for 8 days (Supplementary Fig. 2).To characterize the STB-like differentiation and viability in these placental organ buds, we assayed hCG levels in the conditioned medium as a function of time.The hCG levels in the culture medium of placental buds derived from 201B7 iPSCs gradually increased from day 2 to 8 after seeding (Fig. 2D).The culture period of organ buds created using 409B2 iPSCs was extended to 21 days (Fig. 2C).The size of the buds remained apparently unchanged until day 7-8, but gradually shrank from day 11-12.Similar to the placental buds derived from 201B7 iPSCs, the concentration of hCG in the culture medium also gradually increased and reached a peak on days 6-8.However, it then began to decrease, reaching almost zero on days 16 and 21 (Fig. 2E).In addition, the number of GFP-labeled 409B2 iPSCs gradually decreased in the placental buds, becoming almost nonexistent on days 17 and 21.These data suggest that the placental organ buds remain functional for at least one week, but begin to weaken beyond about two weeks and may not be viable by three weeks.
Immunohistochemical analysis of a placental bud obtained from 201B7 iPSCs revealed that CK7 was ubiquitously expressed in the organ bud (Fig. 3A).hCG was expressed inside the organ bud, where it formed islandlike aggregates (Fig. 3A).In contrast to hCG, HLA-G staining, although less intense than CK7 and hCG, was observed at the periphery compared to the interior of the bud (Fig. 3A), implying that these trophoblast-like cells were forming layers dependent on their character.CD31 (the marker for HUVECs)-and CD90 (the marker for MSCs)-positive cells were located in different places in the bud (Fig. 3B).The localization of differentiated iPSCs-derived trophoblast-like cells, HUVECs, and MSCs was similar in the placental bud generated using 409B2 iPSCs (Fig. 3C,D).This indicates that the added HUVECs and MSCs maintain their original properties, but during organ bud differentiation, both cells seem to migrate to different locations.Collectively, our results demonstrated that induced-trophoblast cells from iPSCs were able to form three-dimensional structures with the assistance of MSCs and HUVECs.In addition, differentiated trophoblast-like cells formed layers within the bud.Syncytium-like cells as identified by hCG were localized inside the bud, while EVT-like cells as identified by HLA-G formed the outer layer of the bud.
Transplantation of placental bud to the NOD/SCID mouse uterus.Finally, we tested the orthotopic transplantation of these placental organ buds into immunodeficient mice.To induce a pseudo-pregnant condition, a total of nine NOD/SCID mice were pretreated with progesterone starting four days before transplantation (Fig. 4A).On the day of transplantation, 100 ng of estradiol was subcutaneously injected.After lapa-Vol:.(1234567890 rotomy, placental buds were transplanted into the uterine lumen using a syringe with an 18 gauge needle.Hormonal supplementation was administered for seven days and engraftment was judged on the seventh day after transplantation (Fig. 4A).We observed that placental buds had successfully engrafted and survived in two of the nine injected mice (22%).These two engrafted organ buds were then histologically examined.
In case 2, the placental organ bud (Fig. 5A) was engrafted inside the uterus (Fig. 5B).Interestingly, a blood vessel-like structure was observed in the center of the bud (Fig. 5B).HLA-G-positive EVT-like trophoblast cells were observed beneath the attachment site, forming an outer layer of the bud (Fig. 5C).In addition, HLA-G was also expressed in cells that construct blood vessel-like tissues in the buds of organs (Fig. 5D).Next to this EVT-like layer, hCG-positive cells formed islets inside the bud (Fig. 5E,F).These inner hCG-positive islets were clearly separated from the outer HLA-G-positive EVT-like layer (Fig. 5G).The EVT layer and the hCG islets surrounded a blood perfusion area that was rich in red blood cells (Fig. 5G).Again, the attached placental organ bud was able to differentiate properly in vivo such that EVT-like cells resided outside the bud and hCG-positive STB-like cells were localized next to the EVT-like cells.This suggests that a transplanted organ bud can sense its surrounding environment, and that signals from the environment might direct bud migration and differentiation in the proper direction.

Discussion
Using human iPSCs and immunodeficient mice, we successfully demonstrated that (1) trophoblast-like cells can be induced with BMP4 treatment, (2) three-dimensional placenta-like organ buds can be induced from iPSCs with the aid of MSCs and HUVECs in a 3D-gel matrix, and (3) synthesized placental organ buds can be transplanted to the uteruses of immunodeficient mice, whereupon trophoblast-like cells can migrate and differentiate in the proper direction within the organ bud.
(1) BMP4 induces trophoblast differentiation from iPSCs.In 2002, Xu et al. succeeded in differentiating human embryonic stem cells (ES cells) into trophoblasts by adding BMP4 7 .In their experiment, trophoblast-like morphological changes were observed on day 2 of treatment with 100 ng/mL BMP4.We followed their methods in our iPSC experiment and observed that our iPSCs lost their pluripotency (as evidenced by the decrease in SOX2, NANOG, and POU5F1 mRNA) and gained trophoblast-like features (as evidenced by the increases in CDX2, PPARG , KRT7, TP63, and CGB3 mRNA) from day 2. The trophoblast-like differentiation pattern of iPSCs induced by BMP4 is very similar to that of ES cells by Xu's.The methodological difference between the two is the cell source: Xu used ES cells, whereas we used iPSCs.ES cells are derived from human blastocysts, which poses ethical concerns.The advantage of using iPSCs is that iPSCs can be produced from somatic cells derived from skin and blood, and those somatic cells can be harvested relatively easily and with fewer ethical issues.Moreover, since iPSCs can be made from the patient's own somatic cells, there is less risk of rejection in the clinical application of cell and organ transplantation.In 2016, Horii et al. showed that human pluripotent stem cells can be differentiated into CTB-like cells by treatment with low-dose BMP4 (10 ng/mL) 8 .Further, these cells can be differentiated into STBs and EVT-like cells in the presence of feeder-conditioned medium containing 10 ng/mL of BMP4 8 .Thus, BMP4 seems to be a master regulator in the early stages of human trophoblast differentiation 9 .
In human embryos, the pluripotency-associated transcription factor OCT4/POU5F1 is initially expressed in the eight-cell stage at three days post-fertilization (dpf) 10 .OCT4/POU5F1 expression was high in both the inner cell mass and the TE cells at 5 dpf, but was downregulated in the TE cells by 6 dpf 10 .In our iPSCs, downregulation of POU5F1 mRNA started around day 2 of BMP4 treatment, which suggests that our iPSCs started to differentiate into TE cells around this stage.It is worth noting that, in mouse ES cells, forced repression of Oct3/4 induces differentiation toward the TE lineage, whereas overexpression of Oct3/4 induces differentiation into extraembryonic endoderm 11 .This suggests that the suppression of OCT4/POU5F1 is necessarily for stem cells to differentiate toward the TE lineage.
The transcription factor CDX2 also appears to regulate cell differentiation toward TE in early embryos.In human embryos, CDX2 is not expressed at the eight-cell or morula stage at 3-4 dpf; rather, it is first detectable in TE at 5 dpf 10 .Interestingly, CDX2 expression overlaps with that of OCT4 in TE cells at 5 dpf 10 .In mouse embryos, CDX2 is required for the maintenance of TE and trophoblast stem cells 12 .Both downregulation of Oct3/4 and upregulation of Cdx2 can trigger the differentiation of mouse ES cells toward TE 12 .Similarly, Cdx2 was required for the development of a functional TE lineage in a mouse Cdx2 -/-blastocyst experiment 13 .Cdx2 is essential for the segregation of the ICM and TE lineages at the blastocyst stage by ensuring the repression of Oct4 and Nanog in the TE lineage 13 .Thus, a well-coordinated transition from OCT4 to CDX2 seems to be critical for the differentiation of iPSCs into TE cells.
TP63, which is expressed in proliferative CTBs, is known to play an important role in maintaining of the stem cell sate of CTBs 14 .BMP4-treated human ES cells show decreased expression of TP63 during differentiation from CTBs to STBs, and forced down-regulation of TP63 inhibits differentiation of human ES cells into functional trophoblasts 15 .In the present experiments, it was confirmed that TP63 was transiently increased in BMP4-treated iPSCs, and the expression changes of other trophoblast-related markers showed that the addition of BMP4 induced iPSCs to differentiate into CTB-like cells and then into STB-like and EVT-like cells.
(2) Placental organ bud.Takebe et al. succeeded in producing a liver organ bud from iPSCs 6 , with MSCs and HUVECs helping to induce a 3D structure.These stromal cell populations are necessary not only for forming a 3D structure but also for inducing self-condensation 5 .Takebe et al. also used their self-condensation method to produce diverse organ buds such as a pancreatic islet 5 .In our study, likewise, we were not able to produce a placental organ bud in the absence of MSCs.The presence of HUVECs and MSCs is also necessary for generating vascularized tissues; the addition of HUVECS, in particular, enables the rapid formation of a functional vasculature 5 .Takebe et al. also reported that HUVECs and MSCs release various growth factors and cytokines that enhance the survival and functionality of organ buds 5 .MSCs can differentiate into osteocytes, adipocytes, chondrocytes, and cardiomyocytes, and were initially expected to serve as "cell replacements 16 .Recently, however, the role demanded of MSCs has been changing to that of "paracrine providers" 16 .MSCs produce a variety of growth factors, including vascular endothelial growth factor, fibroblast growth factor-2, and M-, G-colony stimulating factors.MSCs also secrete cytokines such as IL-10 and TGF-β in a paracrine manner and have antiinflammatory effects.Therefore, 3D placental organs with MSCs may be advantageous for the implantation of placental organ buds.Isolation of human mesenchymal stem cells from amnion.Separation and isolation of MSCs were performed as previously described 21 .Human fetal membranes were obtained during caesarean deliveries, and the amnion was manually separated from the chorion beneath.Amnion tissues were then minced with scissors and digested with brightase-C/dispase 1 solution (brightase-C 40 μg/ml, #892431, Nippi, and dispase 1 200PU/ ml, #386-02271, Wako) for 90 min at 37 °C with shaking.Digested tissues were filtered through a mesh strainer (Falcon #352360, Thermo Fisher Scientific) and centrifuged at 400 × g for 5 min at room temperature.The dissociated amnion cells were suspended in minimal essential medium (MEM) α (#12571063, Gibco) supplemented with 10% fetal bovine serum, 100 U/mL of penicillin and 100 ng/mL of streptomycin.Isolated MSCs were seeded in uncoated plastic dishes with same medium.The culture was maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 .After three to four days in culture, the non-adherent cells were removed, and the adherent cells were maintained in culture until they reached 80% confluence.The passage was performed using 0.5% trypsin-EDTA (#15400054, Gibco).Written informed consent was obtained from each patient.All procedures were approved by the Ethics Committee of the Kyoto University Graduate School of Medicine (G0325) and carried out in accordance with the Ethics Guidelines form Medical and Health Research Involvingt Human Subjects of the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Ministry of Health, Labour and Walfare of Japan.

Isolation of HUVECs.
Separation and isolation of HUVECs were performed as previously described 22 .
Briefly, the vein in the umbilical cord was flushed with PBS and one end of the vein was clamped.Collagenase was added into the other end of the vein until moderate distention of the vein was observed, and the cord was then incubated at 37 °C for 40 min.After incubation, the cord was massaged gently; the clamped end was then cut above the clamp and cells were collected into 50 mL tubes.Tubes were centrifuged at 1500 rpm for 5 min and the supernatant was aspirated.The pellet was resuspended in 6 ml of HuMedia-EG2 (#KE-2150S, Kurabo) and plated in 6 cm plates.The culture was maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 .The passage was performed using 0.5% trypsin-EDTA.

Generation of three-dimensional placental bud in vitro.
To generate the placental organ bud, we used the methods reported by Takebe et al. 23 .iPSCs were differentiated with BMP4 to a stage equivalent to day 4. 1.0 × 10 6 human iPSC-derived trophoblast-like cells, 0.8 × 10 6 to 1.0 × 10 6 HUVECs and 2.0 × 10 5 human MSCs were combined and centrifuged at 1500 rpm for 5 min at room temperature.The supernatant was discarded and the pellet was resuspended in 1 mL of a 1:1 mixture of HuMedia-EG2 (0.5 mL) and conditioning medium from HUVECs (0.5 mL).The final concentration of iPSC-derived trophoblast-like cells was 1.0 × 10 6 cells/mL.1 mL of mixed cells was then plated in each well of a pre-solidified Geltrex-coated 24-well plate (iPSCs: 1.0 × 10 6 cells/ well).Fluorescent images of organ buds from 409B2 iPSCs were captured by Olympus IX71 Inverted System Microscope.
Quantitative real-time PCR.Quantitative RT-PCR was used to determine the relative levels of gene expression (LightCycler 480 System, Roche Life Sciences).Primer sequences used for amplifications are shown in Table 1.Unless otherwise stated, each experiment was performed in triplicate, and all results were normalized against GAPDH.Relative mRNA expression levels were determined according to the comparative cycle threshold (ΔΔCT) method.SYBR Green was used to detect amplification.

Analysis of hCG.
The media from induced trophoblast cells were collected for ELISA analysis and stored at − 20 °C.The secreted hCG was assayed using a Human hCG ELISA kit (abcam, #ab100533) according to the manufacturer's instructions.

Figure 2 .
Figure 2. Placental organ buds synthesized from iPSCs.(A) Our scheme for synthesizing placental organ buds from iPSCs.(B) Representative images of three-dimensional placental buds after co-culture with HUVECs and MSCs for 0-3 days.201B7 iPSCs treated with BMP4 for 4 days (D4) were utilized.Bars, 5 mm.(C) Representative images of placental buds after co-culture with HUVECs and MSCs for 0-21 days.409B2 iPSCs treated with BMP4 for 4 days (D4) were utilized.Macroscopic views (upper panels) and the views under fluorescence microscope (lower panels).Note that 409B2 iPSCs contain GFP, so the iPSCs were identified by green fluorescence.Bars, 5 mm.(D) hCG levels in the medium of placental organ buds from 0 to 8 days.201B7 iPSCs treated with BMP4 for 4 days (D4) were utilized.iPS represents the cells before BMP4 treatment.hCG levels were statistically analyzed by ANOVA, and the hCG level on each day was compared to that of iPSCs in post-hoc test.n = 3 in each group.(E) hCG levels in the medium of placental organ buds from 0 to 21 days.409B2 iPSCs treated with BMP4 for 4 days (D4) were utilized.iPS represents the cells before BMP4 treatment.hCG levels were statistically analyzed by ANOVA, and the hCG level on each day was compared to that of iPSCs in post-hoc test.n = 3 in each group.**, P < 0.01.

Figure 5 .
Figure 5. Trophoblast layers forming in the transplanted placental organ bud.Histological examination of case 2. (A) Macroscopic image of the transplanted organ bud in the NOD/SCID mouse uterus (case 2).A black arrow head indicates the bud.Bar, 1 mm.(B) H&E staining of case 2. Lower (left) and higher magnification (right).Black arrow head indicates the vascular-like structure inside the organ bud.(C, D) Immunohistochemistry of HLA-G.(C) Lower magnification (middle), and (D) higher magnification of the vasculature-like structure.(E, F) Immunohistochemistry of hCG.(E) Lower and (F) higher magnification.(G) Scheme of the trophoblast layers and blood-perfused area in the organ bud.All bars, 300 µm.

Table 1 .
Primer sequences used for qPCR.