PIBF1 regulates trophoblast syncytialization and promotes cardiovascular development

Lee, Jong Geol; Yon, Jung-Min; Kim, Globinna; Lee, Seul-Gi; Kim, C-Yoon; Cheong, Seung-A; Kim, Hyun-Yi; Yu, Jiyoung; Kim, Kyunggon; Sung, Young Hoon; Yoo, Hyun Ju; Woo, Dong-Cheol; Rho, Jin Kyung; Ha, Chang Hoon; Pack, Chan-Gi; Oh, Seak Hee; Lim, Joon Seo; Han, Yu Mi; Hong, Eui-Ju; Seong, Je Kyung; Lee, Han-Woong; Lee, Sang-Wook; Lee, Ki-Up; Kim, Chong Jai; Nam, Sang-Yoon; Cho, You Sook; Baek, In-Jeoung

doi:10.1038/s41467-024-45647-8

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Published: 19 February 2024

PIBF1 regulates trophoblast syncytialization and promotes cardiovascular development

Nature Communications volume 15, Article number: 1487 (2024) Cite this article

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Abstract

Proper placental development in early pregnancy ensures a positive outcome later on. The developmental relationship between the placenta and embryonic organs, such as the heart, is crucial for a normal pregnancy. However, the mechanism through which the placenta influences the development of embryonic organs remains unclear. Trophoblasts fuse to form multinucleated syncytiotrophoblasts (SynT), which primarily make up the placental materno-fetal interface. We discovered that endogenous progesterone immunomodulatory binding factor 1 (PIBF1) is vital for trophoblast differentiation and fusion into SynT in humans and mice. PIBF1 facilitates communication between SynT and adjacent vascular cells, promoting vascular network development in the primary placenta. This process affected the early development of the embryonic cardiovascular system in mice. Moreover, in vitro experiments showed that PIBF1 promotes the development of cardiovascular characteristics in heart organoids. Our findings show how SynTs organize the barrier and imply their possible roles in supporting embryogenesis, including cardiovascular development. SynT-derived factors and SynT within the placenta may play critical roles in ensuring proper organogenesis of other organs in the embryo.

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Introduction

Successful placentation at the first trimester is essential for the placental function of the following gestational periods and subsequent outcome of pregnancy: inadequate early placentation is associated with a higher risk of miscarriage¹, which is the most common form of pregnancy loss and occurs mainly in the first trimester² and can manifest in later pregnancy complications. In particular, proper development of the early placenta is crucial for the normal development of embryonic organs, such as the heart: the heart-cardiovascular system is the first organ to develop during organogenesis³, and the placenta and the embryonic heart arise in the first trimester and develop concurrently in the gestational timeline. Their defects frequently co-exist, as seen in vivo⁴ and clinical studies^5,6,7, and the phenomenon of inter-related influence between the two organs has been referred to as the placenta-heart axis⁸. Over the past 20 years, numerous in vivo studies have suggested that proper placentation is a prerequisite for normal cardiovascular development^{4,9,10,11,12,13}. However, the cellular and molecular mechanisms of how the placenta supports the development of the embryo proper, including the cardiovascular system, have yet to be well-defined.

Syncytiotrophoblast (SynT), a multinucleated layer formed by the continuous fusion of mononucleated cytotrophoblasts (CTB), serves as the primary barrier for exchange between maternal and fetal circulations, and its adequate development is a hallmark of successful placentation: defective syncytialization and altered expression of its modulators underpin numerous pregnancy-related complications¹⁴. Moreover, various placental cell types, including vascular and perivascular cells, also participate in barrier formation¹⁵. Regarding the placenta-heart axis, as a part of efforts to elucidate its cause-effect relationship, one step has recently been achieved through a massive in vivo phenotyping study: among trophoblast cells, SynT is proven to be a leading contributor to developmental heart disease¹⁶. However, the factors that organize the materno-fetal interface and the mechanisms by which the primary placenta influences the developing embryo have not been elucidated.

Progesterone immunomodulatory binding factor 1 (PIBF1) carries an essential role in maternal immune tolerance during pregnancy, as pregnant lymphocyte-produced PIBF1 inhibits natural killer (NK) cell activity¹⁷ and suppresses premature parturition under uterine stress¹⁸. The maternal level of PIBF1 increases continuously throughout pregnancy until term, and a low level of PIBF1 is associated with adverse pregnancy outcomes^{19,20,21,22,23,24}. Concurrently, PIBF1 is highly expressed in SynT in the human placenta during the first trimester and its expression declines at term²⁵, indicating the spatiotemporally different role of PIBF1 depending on the compartments of the mother and conceptus.

In this study, we found that SynT, a product of trophoblast fusion, interacts with adjacent vascular cells to initiate the formation of the exchange barrier in the primary placenta, and this process is orchestrated by trophoblast-expressed and SynT-derived PIBF1. Our findings suggest that a healthy placenta with proper SynT is required for normal early embryonic organogenesis, including cardiovascular development. The enhanced manifestation of cardiovascular features seen in PIBF1-treated heart organoids further implies the possible role of PIBF1 in cardiovascular development.

Results

Essential roles of PIBF1 in trophoblast syncytialization

We first investigated whether PIBF1 mediates syncytialization using a BeWo cell line. CRISPR/Cpf1-mediated gene targeting against exon 2 or 4 of human PIBF1 (Supplementary Fig. 1) generated PIBF1 knockout (KO) BeWo cell lines (Supplementary Fig. 2a). While cell fusion was induced in wild-type (WT) cells by the addition of adenylate cyclase activator forskolin (Fig. 1a), syncytia failed to form in PIBF1 KO BeWo cells (BeWo KO line #1 in Supplementary Fig. 2a) (Fig. 1a). This was accompanied by decreased expression of fusogenic gene ERVFRD-1 (Syncytin 2) and endocrine hormone gene chorionic gonadotropin beta (CGB) (Fig. 1b). Human CG (hCG), which is mainly produced by SynT and secreted into the maternal compartment at high levels during the first trimester²⁶, was detected at significantly lower levels in terms of secretion and protein in PIBF1-deficient cells, regardless of forskolin stimulation (Fig. 1c, d). These results were also replicated in PIBF1 exon 4-targeted KO cells (BeWo KO line #2) (Supplementary Fig. 2b–e), indicating the role of PIBF1 in BeWo trophoblast fusion.

**Fig. 1: PIBF1 is required for trophoblast syncytialization.**

Next, to determine whether PIBF1 mediates the differentiation of human trophoblast stem cells (hTSC) to SynT, we generated PIBF1 KO hTSC (Supplementary Fig. 1 and TS KO line #1 in Supplementary Fig. 3a, b). As a result, PIBF1 KO hTSC showed no changes in cell proliferation (Supplementary Fig. 3c), with TS markers slightly increased in stem cell conditions (Supplementary Fig. 3d, e). However, PIBF1 KO hTSC displayed a defective differentiation capacity into β-hCG-expressing SynT in both 2D and 3D cultures (Fig. 1e). This was consistent with the observation that PIBF1 KO hTSC at a basal state exhibited a declined expression of GCM1 (Supplementary Fig. 3f), a classical master regulator of SynT formation²⁷. The resultant SynT derived from PIBF1 KO hTSC showed decreased levels of SynT markers (Fig. 1f) and β-hCG secretion (Fig. 1g), while the levels of TS markers remained abnormally high (Fig. 1h). In addition, PIBF1 KO hTSCs also demonstrated a defective differentiation capacity into extravillous trophoblasts (EVTs) (Supplementary Fig. 3g, h). Considering that GCM1 was expressed at low levels in PIBF1 KO hTSC and has recently been refocused for its contribution to EVT differentiation²⁷, the possible role of PIBF1 in EVT development needs further investigation.

To examine the in vivo differentiation potency, PIBF1 KO hTSCs were engrafted subcutaneously into severe immunodeficient male mice (Fig. 1i). Hosts injected with hTSCs contained a substantial and detectable amount of hCG in serum and urine, while those with PIBF1 KO did not (Fig. 1j, k). The engrafted lesions of PIBF1 KO were indistinguishable from those of WT in terms of gross size (Fig. 1l), and histologically, both genotypes exhibited similar expression of the pan-trophoblast marker cytokeratin 7 (KRT7) (Fig. 1m). However, differentiated trophoblast populations marked with β-hCG or HLA-G were largely absent in lesions developed from PIBF1 KO hTSCs (Fig. 1m). Additionally, PIBF1 KO hTSC lines other than KO line #1 were obtained using CRISPR/Cpf1-mediated gene targeting (Supplementary Fig. 1 and Supplementary Fig. 4a, b), among which most of the results regarding TSC stemness and SynT/EVT differentiation capacities were also replicated in PIBF1 KO hTSC line #2 (Supplementary Fig. 4c–f). These findings with hTSCs suggest that PIBF1 mediates the cell-to-cell fusion of trophoblasts and the differentiation of TSC into SynT.

Full-length PIBF1 (fPIBF) is a nucleus- or centrosome-associated 90 kDa protein, while the smaller 35 kDa secretory form (sPIBF) is localized cytoplasmically²⁸. To further validate our findings, we overexpressed fPIBF and/or sPIBF in PIBF1-deficient BeWo cells (Fig. 1n). Consequently, the restoration of fPIBF or sPIBF induced cell-to-cell fusion, which was further enhanced by the simultaneous restoration of both variants (Fig. 1o). Notably, exogenous PIBF treatment (Supplementary Fig. 5a) in PIBF1 KO BeWo cells did not result in any significant improvement in syncytial formation (Fig. 1p and Supplementary Fig. 5b, c), indicating that PIBF1 mediates the cell-to-cell fusion event only when expressed and produced endogenously.

In summary, our data demonstrate that endogenous PIBF1 plays indispensable roles in trophoblast differentiation and syncytialization.

Angiogenic role of SynT-derived PIBF1

In the developing human placenta, a small fraction of mononuclear cytotrophoblasts (CTBs) retain the properties of trophoblast stem cells (TSCs) and differentiate into multinucleated syncytiotrophoblasts (SynT), which, along with villous endothelial cells (ECs), establish a materno-fetal exchange interface as well as a physical and immunological barrier^29,30. However, the mechanisms by which these cell populations communicate to establish the multi-layered structure are yet unclear. In this context, we hypothesized that syncytialized trophoblasts could secrete PIBF1 to build the complex interface, as sPIBF can be secreted by immune cells and act as a cytokine^18,28. To address this, we first measured cellular and secreted levels of PIBF1 in TSC, SynT, and EVT using multiple reaction monitoring, a highly selective and sensitive technique for quantifying targeted proteins³¹. As a result, the intracellular level of PIBF1 was comparable among TSC, SynT, and EVT (Fig. 2a), while secreted PIBF1 was significantly more abundant in SynT or EVT than in TSC (Fig. 2a).

**Fig. 2: Trophoblast-derived PIBF1 mediates vasculature formation in vitro.**

Next, to gain more detailed insights into the interaction between SynT and EC, we designed a SynT-EC transwell co-culture to verify whether syncytialized trophoblasts were capable of recruiting vascular endothelial cells (Fig. 2b). We found that hTSC-derived SynT or BeWo cells fused by forskolin recruited a remarkably higher number of HUVECs compared to non-differentiated hTSC or non-fused cells (Supplementary Fig. 6a, b), implying SynT-derived paracrine or endocrine signaling to endothelial cells. When PIBF1 was deficient in hTSC, as expected, this endothelial recruiting ability was lacking (Fig. 2c). PIBF1 KO BeWo cells also displayed defects in endothelial recruitment, which was rescued by the restoration of fPIBF and/or sPIBF (Fig. 2d). Notably, reintroducing sPIBF resulted in a higher level of endothelial recruitment compared to that of fPIBF (Fig. 2d), suggesting that fused trophoblast-derived signals encourage endothelial cell homing to the site of the materno-fetal interface, with sPIBF being the primary mediating factor.

As the placental blood vessels consist of several cell populations, including ECs and pericytes, we tested which vascular cell types were affected by PIBF1 in angiogenesis. Endothelial cells derived from the umbilical vein and artery (HUVEC and HUAEC, respectively) were adopted due to their different profiles of endothelial identity^32,33. As a result, recombinant sPIBF enhanced indices of endothelial migration and tube formation in a dose-dependent manner in HUVEC (Fig. 2e–g) via activation of angiogenesis-related signaling cascades (Fig. 2h and Supplementary Fig. 6c), and similarly in HUAEC (Fig. 2i, j and Supplementary Fig. 6d). It also dose-dependently increased the migratory property of placental pericytes (Fig. 2k and Supplementary Fig. 6e).

The N-terminal region of PIBF1, ranging from amino acids 1-184, is a biologically active site³⁴ and is equivalent to the recombinant active domain containing a fragment of PIBF1 (aPIBF) used in our study (Supplementary Fig. 5a). The angiogenic effects of recombinant fPIBF and aPIBF proteins were validated in HUVEC (Supplementary Fig. 6f-h), HUAEC (Supplementary Fig. 6i–k), and placental pericytes (Supplementary Fig. 6l). In contrast, the C-terminal fragment of PIBF1 (cPIBF) showed no changes in endothelial migration, tube formation, or pericyte migration (Supplementary Fig. 6f–l). This indicates that the angiogenic property of PIBF1 is located in the N-terminal region encoded by exons 2-4, which are known to exert its immunological activity³⁴.

Collectively, these results show that SynT-derived PIBF1 recruits vascular and perivascular cells to form vessel-like structures and promotes their angiogenesis, suggesting that PIBF1 may play a role in the development of the placenta’s circulatory system and the feto-maternal interface.

Defective syncytialization and formation of the materno-fetal interface in placenta lacking Pibf1

We next investigated whether the loss of Pibf1 affects placentation and subsequent embryogenesis by evaluating Pibf1-null placentas and embryos. Pibf1^+/- intercrosses produced no Pibf1^−/− offspring (Supplementary Fig. 7a), which was not associated with the implantation potential of blastocysts (Supplementary Fig. 7b). Instead, Pibf1-null embryos experienced developmental retardation from E9.5-10.5 (Supplementary Fig. 7c, d). At this stage, when the primary placenta begins to form³⁵ and Pibf1 is deleted in all cell lineages of the labyrinthine interhaemal membrane (Fig. 3a), the loss of Pibf1 led to defective placentation and embryogenesis (growth restriction in size, failed tail turning, pericardial effusion) (Fig. 3b). Pibf1-null placentas were morphologically and functionally disrupted, as evidenced by an underdeveloped labyrinth with decreased fetal-derived blood vessels (Fig. 3c, d) and reduced fatty acid transfer capacity (Fig. 3e, Supplementary Data 1).

**Fig. 3: Trophoblast syncytialization and vascular labyrinth integrity of the placenta are impaired in E10.5 *Pibf1*-null mice.**

Possible mechanisms of Pibf1 regarding the failed development of Pibf1-null placenta and embryos can be explored in light of previous literature. Pregnancy state and outcome are associated with maternal PIBF1 levels²⁴. However, Pibf1-null lethality did not result from maternal Pibf1 haploinsufficiency, as the abnormal appearances of Pibf1-null embryos were also replicated in WT foster mothers (Supplementary Fig. 8a, b). Meanwhile, Pibf1 was first described as a suppressor of conventional cytolytic natural killer (NK) cells¹⁷, while uterine NK (uNK) cells dampen IL17-producing helper T (Th17)-mediated inflammation, which occurs locally in the maternal decidua, to promote immune tolerance³⁶ (Supplementary Fig. 8c). We therefore examined whether embryonic loss of Pibf1 impacted the uNK population. However, no significant change was observed in their proportion among immune cells or absolute number in Pibf1^+/- decidua harboring Pibf1^−/− embryos (Supplementary Fig. 8d, e). In addition, functional markers of uNK cells such as granzyme B (cytotoxic granule contents³⁷), TNF-α and IFN-γ (pro-inflammatory cytokines³⁸), and CD107a (a proxy for degranulation³⁹) also did not show significant changes in Pibf1^+/- decidua with Pibf1^−/− embryos (Supplementary Fig. 8f), suggesting that embryonic loss of Pibf1 did not have a substantial impact on the population and function of uNK cells. Furthermore, in a Th17-deficient maternal immune background (Supplementary Fig. 8g), Pibf1^−/− embryos still exhibited morphological abnormalities (Supplementary Fig. 8h), similar to those detected in littermates from Pibf1 WT or heterozygous mothers. These data suggest, at least in part, that the maternal compartments of the conceptus were genetically or immunologically non-pathogenic to the early defects of the Pibf1^−/− embryo.

As PIBF1 plays essential roles in TSC differentiation and trophoblast fusion in vitro, we investigated whether Pibf1-null placentas have defects in SynT formation. In Pibf1^−/− placentas, defective labyrinth formation was accompanied by decreased trophoblast proliferation (Fig. 3f). Among TSCs and their lineages, the labyrinthine trophoblast within the interhaemal membrane affected by Pibf1 deletion was SynT-II: while s-TGCs developed typically (Ctsq in Fig. 3g, alkaline phosphatase positive in Fig. 3h), SynT-II markers (Gcm1, Synb, Mct4) were significantly decreased, with those of SynT-I (Syna, Mct1) not showing statistically significant changes (P = 0.232 and 0.495 in Syna and Mct1 in Student’s t-test, respectively) (Fig. 3g). This was further confirmed by MCT1/4 co-staining (Fig. 3i). Moreover, at an ultrastructural level in Pibf1^−/− labyrinth, the interhaemal membranes were few and abnormally increased in thickness, and most importantly, SynT-II failed to undergo fusion (Fig. 3j). SynT-I also exhibited visible abnormalities (excessively increased thickness, vacuoles, and electron-dense areas) (Fig. 3j). In terms of vascular development within the placenta, Pibf1^−/− embryo-derived blood vessel formation (Laminin-positive) declined, along with an increased degree of apoptosis (TUNEL-positive) (Fig. 3k); moreover, the reduced placental vasculature was site-specific (only in the labyrinth) (Fig. 3l), which is consistent with our histological data.

Collectively, these data indicate that the loss of Pibf1 leads to impaired cell-cell fusion in trophoblasts and their interaction with the endothelium, and consequently a failure to form a materno-fetal interface. This suggests that defective primary placentation is a major cause of the observed lethality.

Syncytialization and trophoblast-endothelial interaction recovered in a functional placenta harboring Pibf1-intact trophoblast

Pibf1 displayed a ubiquitous expression pattern throughout the placenta and embryo (Supplementary Fig. 9a, b), making it difficult to determine which compartments are involved in the embryonic-lethal phenotype of Pibf1^−/− embryos. To distinguish between trophoblast-intrinsic and embryo lineage-induced effects within the placenta, we generated conditional Pibf1 KO (cKO) conceptuses using Meox2-cre (Supplementary Fig. 10), a transgenic line for epiblast-specific cre-recombinase expression (endothelium, fetal blood cell), while leaving PIBF1 expression intact in trophectoderm-derived cells (S-TGC, SynT-I, SynT-II) within the placental labyrinth⁴⁰ (Fig. 4a and Supplementary Fig. 11a). Pibf1-null embryos associated with a Pibf1-retained placenta (∆/+) were normally detected at an expected Mendelian ratio (Fig. 4b), and both embryos and placentas were fully recovered at E10.5 (Fig. 4c–f), a stage when complete knockout counterparts were morphologically or histologically disrupted. Importantly, this rescue was accompanied by normal states of TSC and its differentiated lineages, especially SynT-II (Fig. 4g, h), and the labyrinthine vasculature (Fig. 4i).

**Fig. 4: *Pibf1* is required in trophoblast development, but not endothelial/hematopoietic lineages, for normal syncytialization, placental vascularization, and subsequent embryo survival in mice.**

The placenta also serves as a hematopoietic organ during this period^41,42: briefly, primitive hematopoiesis first occurs in the yolk sac blood islands (<E8.5), then expands to the endothelial cells of major vessels in the conceptus, including the placenta (E9.5-10.5). Hematopoietic progenitors subsequently colonize the fetal liver during gestation and bone marrow after birth⁴³. We observed that the hematopoietic potential of Pibf1^−/− yolk sac and placenta were normal until E9.5 (Supplementary Fig. 12a–c), but aberrantly decreased at E10.5 (Fig. 4j and Supplementary Fig. 12a, d). This defect was recovered in the Pibf1-intact yolk sac and placenta, and even in the Pibf1 cKO embryo at E10.5 (Fig. 4j). Moreover, the rescued placenta displayed normal morphometry and nutrient transport function at E13.5, as identified in histological assessment and measurement of fatty acid transfer capacity (Supplementary Fig. 13a, b, Supplementary Data 2). At this stage, the distribution of SynT-I and -II was also comparable to that of the control placenta (Supplementary Fig. 13c).

In addition, regarding its subcellular characteristics, the full-length 90 kDa PIBF1 is localized in the nucleus or centrosome²⁸. Elevated apoptosis and expression of the apoptosis regulator Trp53 (Supplementary Fig. 14a) in Pibf1-null embryos prompted us to investigate the role of Trp53 in the viability of embryos lacking Pibf1. However, the embryonic function of Pibf1 was not related to the Trp53 pathway, as Pibf1;Trp53 double KO embryos phenocopied Pibf1 KO embryos (Supplementary Fig. 14b, c). The centrosome-associated protein PIBF1 is also reported to be essential for the formation of primary cilia^44,45,46, and its mutations were reported in some patients with ciliopathy^47,48,49. We found that while Pibf1^∆/∆;Meox2^cre/+ fetuses had an expected Mendelian ratio until E18.5, no cKO newborns were detected after birth (Supplementary Fig. 14d). Also, Pibf1^∆/∆;Meox2^cre/+ fetuses displayed phenotypic features of ciliopathy (Supplementary Fig. 14e–h), which could potentially contribute to postnatal fetal demise, irrespective of a functionally normal placenta.

To provide further insights into the roles of trophoblast syncytialization and its interaction with vascular cells, we dissected endothelial lineages of the labyrinthine interhaemal membrane (Fig. 4k) using two endothelial-specific cre lines—Cdh5-cre and Tie2-cre—that express each promoter-driven cre recombinase in endothelial lineages (Supplementary Fig. 11a)^50,51. Cdh5- or Tie2-cre-mediated Pibf1 cKO embryos were detected at E10.5 with expected normal Mendelian inheritance (Fig. 4l) and no apparent morphological anomalies (Fig. 4m, o). The histological index of their placentas was not significantly different from the control (Fig. 4m, n). As Tie2- or Cdh5-cre-expressing cells share the identity of both endothelial and hematopoietic lineages^50,51, the endothelial-specific Pibf1 cKO conceptuses were explored for functional hematopoietic activity in their hemogenic compartments, which displayed comparable levels among genotypes (Fig. 4p). Additionally, when Pibf1 was ablated in hematopoietic lineages using Vav1-, Cdh5-, or Tie2-cre lines, the hematopoietic functions of different Pibf1 cKO organs were normal at E13.5 (cKO^Vav1 and cKO^Tie2 in Supplementary Fig. 15a–d) and postnatally in adult (cKO^Cdh5 and cKO^Vav1 in Supplementary Fig. 15e, f). These hematopoiesis data suggest that proper syncytialization of trophoblasts may ensure normal hematopoietic development in both extra-embryonic and embryonic organs.

Collectively, these results indicate that normally syncytialized trophoblasts and their interaction with endothelium, orchestrated by Pibf1, are prerequisites for establishing a materno-fetal interface, a functional placenta, and, ultimately, embryo survival.

Impact of trophoblast syncytialization upon cardiovascular development of the embryo

As observed in the placenta with its global expression pattern, Pibf1 is also expressed in the developing heart of the early embryo (Supplementary Fig. 9b). Under our Pibf1 cKO strategies, the Cre lines are active in the whole heart (Pibf1^∆/∆;Meox2^cre/+) or part of the heart (endocardial lineages in Pibf1^∆/∆;Cdh5^cre/+, Pibf1^∆/∆;Tie2^cre/+) (Fig. 5a and Supplementary Fig. 11b). In line with the notion that the placenta and embryonic heart develop in parallel during early gestation and that their defects commonly co-exist^4,5,6,7,8, the abnormalities of Pibf1-null embryos rescued by the SynT-intact functional placenta also included a visibly underdeveloped heart (Fig. 3b). Moreover, syncytialized trophoblasts produce angiogenic factors such as PIBF1 (Fig. 2a), which might be able to potentially affect endothelial lineages or cardiac vasculature in the developing heart. In this context, we examined the impact of Pibf1-mediated trophoblast syncytialization on cardiovascular development in Pibf1 mutants. As seen in the whole mount CD31 stain, the vasculature of the yolk sac, intersomitic region, and heart was severely disrupted in Pibf1^−/− embryos; however, it was largely recovered in Pibf1 cKO embryos (Fig. 5b). Particularly, in the cardiac region, CD31-stained areas were significantly decreased in Pibf1^−/− hearts but were comparable among control and Pibf1 cKO hearts (Fig. 5c, d). In gross and histological appearances, the Pibf1^−/− hearts showed severe developmental delays, displaying failed right and left chamber specification, abnormally enlarged pericardial cavity, reduced chamber length, and decreased trabecular density. These cardiac defects were fully rescued in all three Pibf1 cKO mutant hearts (Fig. 5e, f), which was further confirmed histologically by the myocardial marker cTnT (Fig. 5g, h). The early cardiogenesis-related markers (myocardium development and cardiac jelly regulation)⁵² and coronary vascular development markers (pan-endothelial lineage and arterial/venous specification)⁵³ exhibited a similar pattern, as most of them were decreased in Pibf1^−/− hearts and recovered in those of all Pibf1 cKO mutants (Fig. 5i, j), under the influence of a functional SynT-bearing placenta. These data indicate that the cardiovascular development of the embryo depends on Pibf1-mediated trophoblast syncytialization and placentation, not the genetic state of Pibf1 in the heart or vascular system itself.

**Fig. 5: Placenta with properly syncytialized trophoblasts maintains embryonic development of vasculature and heart in mice.**

Additionally, concerning its immunotolerant functions under certain pregnancy environments^17,18 and the relationship between its maternal level and pregnancy outcome^{19,20,21,22,23,24,54}, we addressed whether loss of Pibf1 in the maternal immune system affects pregnancy and the offspring (Supplementary Fig. 16a). Pibf1^∆/∆;Vav1^cre/+ females (Pibf1-deficient in hematopoietic/immune cells⁵⁵) displayed normal reproductive functions (Supplementary Fig. 16b), and their pups showed normal growth rate (Supplementary Fig. 16c), indicating that the genetic deletion of Pibf1 in immune cells does not directly affect the maintenance of pregnancy in mice. Meanwhile, the function of immune cells might also be affected by PIBF1, which can be produced by other cells or tissues. For example, PIBF1 is highly expressed in endometrial stromal cells during the mid-secretory phase of the endometrium and is required for their decidualization⁵⁶. Therefore, further investigation is needed to understand the role of PIBF1 in maintaining pregnancy in maternal environments.

Manifestation of cardiovascular features facilitated by PIBF1 in heart organoids

To further investigate the effects of PIBF1 on in vitro cardiovascular features, we employed an induced pluripotent stem cell (iPSC)-derived human heart organoid (hHO) model, which can mimic the three-dimensional characteristics of the heart⁵⁷. After differentiating hHOs from iPSC-derived embryonic bodies under the influence of sPIBF (Fig. 6a), we confirmed that cTnT-positive cardiomyocytes were significantly more abundant in sPIBF-treated hHOs than in controls (Fig. 6b). Additionally, co-staining of CD31 and PDGFRβ, markers for endothelial cells and pericytes, respectively, revealed an increased distribution of CD31-positive cells in sPIBF1-treated hHOs compared to controls, while that of PDGFRβ remained unchanged (Fig. 6b). A wide range of rsPIBF doses (50–1000 ng/ml) also showed increases in endothelial and cardiomyocyte distributions within hHO (Supplementary Fig. 17a).

**Fig. 6: PIBF1 facilitates the manifestation of cardiovascular features in human heart organoids.**

Considering that hHO is a mixed population of several cells, such as endothelial cells and cardiomyocytes⁵⁷, it remained uncertain whether the impact of sPIBF on hHO is due to its direct effect on cardiomyocyte differentiation or indirect effect resulting from enhanced endothelial angiogenesis. To address this issue, we conducted a two-dimensional culture of human iPSC-derived cardiomyocytes⁵⁸, which revealed the direct impact of PIBF1 on cardiomyocyte differentiation (Fig. 6c).

Since human iPSC-derived hHOs exhibit electrophysiological signals that can mimic depolarization/repolarization coupling as functional features⁵⁷, we measured changes in the leading indicators of field potential (FP), an electrophysiological signal, using a multi-electrode array (MEA). There were no significant differences in beat period and field potential duration (FPD) (Fig. 6d); however, the spike amplitude was significantly higher in sPIBF-treated samples than in controls (Fig. 6d). Moreover, the angiogenic effect of sPIBF observed in endothelial cells and pericytes was further confirmed in hHOs using a Matrigel-based sprouting assay: Most of them were PDGFRβ positive (Fig. 6e). Notably, sPIBF-treated hHOs displayed a more vascularized appearance with longer sprouting lengths than vehicle-treated hHOs (Fig. 6e), which was increased in a dose-dependent manner with sPIBF (Supplementary Fig. 17b).

To better understand how sPIBF affects developing hearts, we analyzed the transcriptomes of vehicle- or sPIBF-treated hHOs and found that 240 genes were differentially regulated by sPIBF, with 105 differentially expressed genes (DEGs) increased and 135 decreased (Supplementary Fig. 18). Gene Ontology enrichment analysis of the DEGs (Supplementary Data 3 and 4) revealed that in hHO, compared to the undifferentiated embryonic body (EB), there was increased expression of markers not only for cardiac muscle but also for other mesenchymal tissues such as bone and fat (Fig. 6f, column 1 vs. columns 2-4 in heatmap). However, when treated with rsPIBF, the expression of other mesenchymal cell fate-related genes was significantly suppressed. Notably, the expression of cardiomyocyte-related genes was significantly increased (Fig. 6f, columns 2-4 vs. columns 5-7 in heatmap), strengthening the fate decision in muscle and heart development by sPIBF (Fig. 6g).

Taken together, PIBF1 encourages differentiation/maturation of cardiomyocytes and endothelial angiogenesis in the in vitro heart development. Along with our findings on the essential roles of SynT-derived PIBF1 in angiogenesis and primary placentation, this may contribute to the development of the cardiovascular system in the embryo.

Discussion

PIBF1-intact trophoblast cells led to their syncytialization and played a role in the formation of multiple layers within the interhaemal membrane, which is essential for the proper development of the labyrinthine structure through PIBF1 derived from SynT. Under the influence of the placenta harboring properly syncytialized trophoblasts, the abnormalities observed in Pibf1-null early embryos were restored in vivo, and severe cardiovascular defects were also recovered by the functional placenta. Further in vitro evidence using human heart organoids proposes a possible role of PIBF1 in cardiovascular development. These data collectively indicate the essential roles of SynT in primary placentation and embryo survival, and imply the possible existence of placenta-derived factors that affect embryogenesis, including cardiovascular development.

We sought to determine what makes the placenta a functional unit at such an early period, represented as the proper development of trophoblasts and lipid transport-associated signatures of the placenta in mice³⁵. We demonstrated that SynT is a key trophoblast subtype that contributes to primary placentation and early cardiovascular development. Disrupted cell-cell fusion in SynT-II is a significant finding of Pibf1^−/− interhaemal membrane and a defective SynT-I, where ultrastructural anomalies are also detected. This phenomenon is similarly seen in Ly6e KO⁵⁹ or Atp11a KO⁶⁰ placentas, where unfused SynTs affect the morphologies of adjacent cells within the interhaemal membrane. In line with our findings, a recent study reported that defective SynT-I is a shared phenotype among mutants with mid-gestational congenital heart disease (CHD)¹⁶.

Interestingly, mice deficient with Synb, a fusogenic gene required for SynT-II fusion, showed an altered syncytialization in SynT-II but resulted in viable offspring, albeit with a slightly reduced number⁶¹. This suggests that the defective formation of SynT itself may not be the sole event contributing to the early pathogenesis of CHD. In this regard, our data further delineate the roles of SynT in terms of communicating with adjacent placental vascular cells via secreted PIBF1 to promote the formation of the materno-fetal interface in the primary placenta. SynT-derived PIBF1 mediates the angiogenic properties of vascular ECs and perivascular pericytes, which form the cellular structure of capillary wall in developing organs, including the heart⁶², and also play essential roles in placental vasculature development^63,64,65,66. Given that SynT recruits ECs and activates their angiogenesis signaling, it is highly likely that other SynT-derived angiogenic or vasculogenic factors also serve as organizers of the placental barrier.

In the genetic approach using a Meox2-cre driver line, the vascular interaction between placenta and embryo (chorioallantoic connection and umbilical cord vessels) was evident in both Pibf1^−/− and Pibf1^Meox2 cKO conceptuses. However, the two conceptuses differed in terms of the existence of functional trophoblasts, especially SynT. Notably, we have demonstrated that SynT is essential for primary placentation and plays a crucial role in the process, which can have significant implications for embryo organogenesis, including the development of the cardiovascular system. Rescued abnormalities of Pibf1-null embryos under a normal SynT-bearing placenta might be a result of restored placental nutritional supply. However, considering the unique molecular and physiological aspects of PIBF1 as proven in this study, it is important to take into account the presence of placenta-derived factors beyond the classical placenta-heart concept which has been described as a parallel development of the two organs, and PIBF1 may be one of the factors that mediate this axis. Considering that the blood level of PIBF1 is detectable in both non-pregnant and pregnant women throughout gestational periods²⁴, it would be possible to detect PIBF1 in placental or embryonic circulation as well. This would allow for the elucidation of the range of PIBF1 levels in normal and pathological conditions of pregnancy. Likewise, SynT may be able to produce other factors essential for heart development in an endocrine manner. Identifying the placenta-derived factor critical for embryonic cardiovascular development is an important finding, considering its potential for clinical application in early pregnancy complications associated with fetal CHD.

However, several limitations could not be addressed in this study. As evidenced in our study, the state of Pibf1 on the maternal side did not impact Pibf1-null embryonic lethality, indicating that maternally derived PIBF1 is unable to cross the placental barrier and reach embryonic circulation. The presence of placenta-resident cells or tissues that can act as a source of PIBF1, delivering it directly into the embryonic bloodstream and ultimately to developing organs, is crucial. SynT is considered one of the potential candidates for this role, as it is in direct contact with maternal blood space in humans and interacts with the endothelium of embryonic vasculature in mice. In this context, we have demonstrated that PIBF1 is a factor derived from SynT that is essential for primary placentation and plays a crucial role in in-vitro cardiogenesis. However, in this study, we did not prove that placentally produced PIBF1 enters the fetal blood circulation to directly exert effects on the developing fetal heart. Thus, it is possible that the dysfunctional placenta of Pibf1 mutants affects heart development in ways unrelated to secreted PIBF1. Another limitation of this study is the temporal discrepancy between the expression of Pibf1 in early embryos before E5.5⁶⁷ and the periods when cre recombinase becomes active, which occurs since E5.5 in the Meox2-cre driver line⁴⁰. Although Pibf1-null embryos displayed a normal ICM/TE ratio in vitro (Supplementary Fig. 7b) and a normal gross phenotype until E8.5 in vivo (Supplementary Fig. 7c), the intracellular events in the blastocysts that can affect fate decisions of embryonic stem cells and subsequent lineages cannot be thoroughly determined solely by such gross phenotype detection. Thus, the role of Pibf1 in the peri-implantation embryonic cells before E5.5, a period outside of the Meox2-cre activity window, needs to be further investigated.

In addition, the ciliopathy-related phenotypes and the prenatal lethality of Pibf1^Meox2 cKO fetus are most likely embryonic proper in origin because extraembryonic lineages in mice lack primary cilium whereas epiblast-derived ones are ciliated⁶⁸. Unlike that in mice, human trophoblasts have abundant primary cilia⁶⁹. The primary cilia in the placenta are recently re-examined with their potential involvement in the pathogenesis of preeclampsia⁷⁰, and PIBF1 KO TSC fails to differentiate into EVT; therefore, further work is needed to understand the relationship between PIBF1, ciliogenesis, and EVT development and to develop animal models that can recapitulate the findings of human trophoblasts and placental development with or without cilia.

Also, it is noteworthy that the expression level of GCM1 was reduced following PIBF1 KO in hTSC and its differentiated SynT. GCM1 is exclusively expressed in the placenta of humans and mice^71,72,73, and is classically considered a master regulator of SynT formation^74,75. It has recently been shown that GCM1 also plays a role in EVT differentiation: when GCM1 was knocked down in hTSC, there was a decrease in their ability to differentiate into both SynT and EVT²⁷, which is highly similar to that observed in PIBF1 KO hTSC, suggesting that GCM1 may be an intrinsic factor in PIBF1-mediated trophoblast syncytialization. The relationship between PIBF1 and GCM1 needs to be further investigated.

In terms of early pregnancy management and complications, placenta-derived factors that can reach the embryo through feto-placental circulation have garnered significant interest⁷⁶, as they can impact prenatal organ development and postnatal health as seen in the cases of serotonin⁷⁷ and ELABELA⁷⁸. Our study uncovers the previously unknown role of PIBF1 as a placenta-derived factor and offers insights into the essential role of SynT in the placenta-heart axis (Fig. 7). Furthermore, considering the emerging field of neuroplacentology⁷⁹, the placenta-heart axis concept we have rediscovered here may be applied more broadly to other organs that develop simultaneously with the primary placenta.

Fig. 7: PIBF1 is crucial for trophoblast syncytialization, and syncytiotrophoblast-derived PIBF1 mediates the formation of a multi-layered feto-maternal interface in the placenta, contributing to the proper embryo development, including the cardiovascular system.

Methods

Cell culture

BeWo cells (CCL-98, ATCC) were cultured in Dulbecco’s modified Eagle’s medium–Ham’s F12 (DMEM/F12) medium (11320-033, Gibco) supplemented with 10% FBS (16000-044, Gibco). HUVECs were isolated from the human umbilical cord of donated pregnant women and were cultured in Medium 200 (M200500, Gibco) supplemented with large vessel endothelial supplement (LVES; A1460801, Gibco). HUAECs (8010, ScienCell Research Laboratories) were cultured in Endothelial Cell Growth Medium (1001, ScienCell Research Laboratories). Human pericytes from the placenta (Pl-PC; C-12980, PromoCell) were cultured in Pericyte Growth Medium 2 (C-28041, PromoCell). The culture medium for BeWo, HUVEC, HUEAC, and Pl-PCs contained 1% antibiotic-antimycotic solution (penicillin 100 U/ml, streptomycin 100 μg/ml, amphotericin B 25 μg/ml; 15240-096, Gibco).

Human trophoblast stem (hTS) cells (RCB4936) were provided by Riken BRC. The cells were cultured and maintained as previously described⁸⁰. Briefly, hTS cells were seeded in 5 μg/ml collagen IV (354233, Corning)-coated dish and cultured in TS medium (DMEM/F12 (Gibco)) supplemented with 0.1 mM 2-mercaptoethanol (2-ME; 16141-079, Gibco), 0.2% FBS (Gibco), 0.5% antibiotic-antimycotic, 0.3% BSA (017-22231, Wako), 1% ITS-X supplement (094-06761, Wako), 1.5 μg/ml L-ascorbic acid (013-12061, Wako), 50 ng/ml EGF (053-07871, Wako), 2 μM CHIR99021 (038-23101, Wako), 0.5 μM A83-01 (035-24113, Wako), 1 μM SB431542 (031-24291, Wako), 0.8 mM valproic acid (227-01071, Wako) and 5 μM Y27632 (036-24023, Wako)], and the culture medium was replaced every two days.

All cells were cultured in a humidified atmosphere with 5% CO₂ at 37 °C.

Establishment of PIBF1 KO cell lines

To target the PIBF1 gene in human cell lines, we used a lentiviral vector expressing CRISPR/AsCpf1 (Acidaminococcus sp. BV3L6 Cpf1), pY108 (Addgene plasmid #84739; http://n2t.net/addgene:84739; RRID:Addgene_84739). CRISPR RNAs specific for the human PIBF1 gene were designed using Benchling (https://www.benchling.com), and the following oligomers were purchased from Macrogen, Inc.: 5′−agatGAAACAACAGTTCCTACGGA−3′ and 5′−aaaaTCCGTAGGAACTGTTGTTTC−3′ for PIBF1 exon 2-specific crRNA; 5′−agatTTGAGACAGAAACAACTAGA−3′ and 5′− aaaaTCTAGTTGTTTCTGTCTCAA−3′ for PIBF1 exon 4-specific crRNA. Each pair of oligomers was annealed and cloned into pY108 using BsmBI restriction enzymes (New England BioLabs).

BeWo and hTS cells were transduced with a 1:1 mixture of culture medium and lentiviral supernatant containing hPIBF1 exon 2 or exon 4 targeting Cpf1-expressing vectors in the presence of polybrene (2.5 μg/ml; 107689, Sigma). After selection with puromycin (2 μg/ml; A1113803, Gibco) for 48 h, the transduced and selected cells were dissociated and seeded in 100-mm dishes at the lowest possible cell concentration that allows for single-cell growth. The cell was then allowed to grow until it formed a visible colony, and the single-cell colonies (n = 60 for each targeted exon) were selected after being seeded in a 96-well plate.

After examining the PIBF1 expression of BeWo and hTSC colonies using Western blotting, the colonies were expanded to larger culture plates. For subsequent sequence analysis in PIBF1 KO suspect colonies, the mutant PIBF1 locus was amplified by PCR reaction using the following primers: hPIBF1 exon 2, 5′-cctccccgcaggaactg-3′ and 5′-tgccgtttgaagacaataaagca-3′ (580 bp), hPIBF1 exon 4, 5′-tactgtcaggtttctgatttcctaa-3′ and 5′- aacaccattcaccatccatagc-3′ (443 bp). The PCR products from the colonies were cloned using a T-Blunt PCR Cloning kit (SOT02-K020, SolGent). The mutations were identified by Sanger sequencing analysis (Bionics, Seoul, Republic of Korea) of 20-25 clones from each colony. PIBF1 KO TS cells were used at passages 6–8.

Recombinant PIBF1

Recombinant proteins of full length (rfPIBF; H00010464-P01) and C-terminal fragments of PIBF1 (rcPIBF; H00010464-Q01) were purchased from Abnova. Human monomeric Fc of immunoglobulin fused form of PIBF1 (1 ~ 298 a.a, rsPIBF), which was expressed and purified from CHO cell, was purchased from Ybiologics (Daejeon, Republic of Korea).

Construction of the raPIBF1 expression plasmid

For the production of raPIBF, cDNA for the N-terminal of PIBF1 (1~184 a.a) was synthesized at Cosmogenetech (Seoul, Republic of Korea). The cDNA was used as the template for polymerase chain reaction (PCR) analysis. The N-terminal PIBF1 (1–184 amino acids) was amplified from the cDNA template using the upstream primer 5′-CCA GAA TTC ATG TCT CGA AAA ATT TCA AAG GAG TC introduced an EcoRI restriction site, while the downstream primer 5′-AAG GTC GAC AAG GTC TTC ATC TTT TGT TTC TTA GAC C contained a SalI site. PCR was performed as follows: 95 °C for 4 min, followed by 30 cycles at 95 °C for 25 s, 65 °C for 25 s, and 70 °C for 60 s; the reaction was then held at 70 °C for 20 min and finally cooled to 4 °C for standby. Subsequently, the PCR products of PIBF1 were double-digested with restriction enzymes, cloned into the pET-28a vector, and transformed into E. coli DH5α competent cells.

Induction and purification of raPIBF1

The pET-28a-raPIBF1 recombinant plasmid was transformed into E. coli BL21(DE3) competent cells using a heat-shock method at 42 °C for 100 sec and then transferred to ice for 5 min and cultured in LB medium containing kanamycin (50 µg/ml) at 37 °C for 12 h. The transformed E. coli suspension was inoculated into culture tubes containing 5 mL LB medium (50 µg/ml kanamycin). Following incubation at 37 °C, the tubes were centrifuged at 13 x g until the optical density (OD₆₀₀) reached 0.7, isopropyl β-D-1 thiogalactopyranoside (IPTG) was added at a concentration of 0.1 mM. The LB media was cultured at 20 °C for 12 h to express the recombinant protein. The bacterial cells were harvested by centrifugation at 15,000 x g for 30 min and resuspended in 10 mL of 50 mM Tris-HCl (pH 8.5) buffer and separated on 10% SDS-PAGE gels to determine the solubility of raPIBF1. The raPIBF1 protein was purified using a 6xHis Tag and Ni-NTA affinity chromatography. Bacterial pellets were lysed using a lysis buffer (50 mM Tris-HCl (pH 8.5), 150 mM NaCl buffer) with sonication for 10 min at 4 °C. After centrifugation at 15,000 x g for 30 min, the resulting supernatant was loaded onto a Ni-NTA column that had been equilibrated with lysis buffer. After washing with lysis buffer, elution buffer (200 mM NaCl and 300 mM imidazole (pH 8.5)) was used for elution. The purified raPIBF1 proteins were dialyzed using a 10 kDa cutoff centricon with a PBS solution.

BeWo experiments

Cell-to-cell fusion and fusion index quantification

BeWo cells were incubated with 20 μM forskolin (3828, Cell Signaling Technology) to induce cell fusion. After 48 h, the cultured cells were stained with Hoechst 33342 (H1399, Invitrogen) and 2 μM Di-8-ANEPPS (D3167, Invitrogen) for 30 min, and subsequently imaged by Zeiss LSM 880 Fast Airyscan Confocal system (Carl Zeiss, Jena, Germany).

In fluorescent images of six randomly selected fields from three or more independent experiments, the number of nuclei within non-fused or fused cells was counted. Cells with more than two nuclei were identified as fused cells, and the fusion index in each group was calculated as follows: (the number of nuclei in fused cells) / (total number of nuclei). The fusion index data were expressed relative to the forskolin-untreated WT control.

PIBF1 restoration in PIBF1 KO BeWo cells

The fragment of the cDNA region encoding the amino acids 1-757 (fPIBF) or 1-224 + 684–757 (sPIBF) of the human PIBF1 (NP_006337.2) was synthesized and inserted into pCAGGS expression vector⁸¹ by Cosmogenetech (Seoul, Republic of Korea). The resulting construct DNA (1 μg/ml) expressing fPIBF or sPIBF or empty vector control was transfected into PIBF1 KO BeWo cells using Lipofectamine LTX with PLUS reagent (15338100, Invitrogen) following the manufacturer’s instruction.

Treatment with recombinant PIBF1 on PIBF1 KO BeWo cells

PIBF1 KO BeWo cells were incubated with rsPIBF (400 ng/ml) or rfPIBF (400 ng/ml) simultaneously with 20 μM forskolin. After 48 h, the fusion index was obtained as described above.

In vitro angiogenesis assays

For migration assay, three scratches were made using a 200-μl pipette tip across the plates. After washing, cells were incubated without or with rsPIBF, rfPIBF, raPIBF, and rcPIBF at a dose of 20, 50, 100, and 200 ng/ml for 9 h (HUVEC), 14 h (HUAEC), or 8 h (Pl-PC). VEGF (20 ng/ml; 293-VE, R&D Systems) and PDGF-BB (50 ng/ml; PeproTech, 100-14B) were used as a positive control for ECs and Pl-PCs, respectively. The images of cell migration at 0 h and endpoint were analyzed with their migrated area using ImageJ, and the cell migration rate was calculated as follows: [(area at 0 h)–(area at endpoint)]/(area at 0 h) × 100 (%).

For tube forming assay, ECs were labeled with 10 μM CellTracker Green CMFDA Dye (C7025, Invitrogen) for 30 min, and then were seeded onto a Matrigel-coated plate and incubated without or with the recombinant PIBF1 isoforms at a dose of 50, 100, 200, and 400 ng/ml for 9 h. VEGF (50 ng/ml) was used as a positive control. The images were captured using EVOS M5000 Imaging System (Invitrogen), and the capillary-like structures formed by HUVECs and HUAECs were analyzed using ImageJ software (NIH).

To investigate rsPIBF-induced changes of angiogenesis-related signaling cascades in HUVECs, HUVECs were incubated with VEGF (20 ng/ml), rsPIBF (200 ng/ml), or vehicle for 30 min, and the cells were processed for Western blot analysis.

hTS cell experiments

Differentiation of hTS cells

Differentiation of TS cells into SynT or EVT was induced as previously described⁸⁰. Briefly, for SynT differentiation in a 2D [SynT(2D)] culture, TS cells were seeded in a collagen IV-coated dish and cultured in SynT(2D) medium [DMEM/F12 supplemented with 0.1 mM 2-ME, 0.5% antibiotic-antimycotic, 0.3% BSA, 1% ITS-X, 2.5 μM Y27632, 2 μM forskolin, and 4% KnockOut™ Serum Replacement (KSR; 10828028, Gibco)]. The cells were replaced with a fresh medium on day 3 and analyzed on day 6. In the case of SynT differentiation in a 3D [SynT(3D)] culture, TS cells were cultured in SynT(3D) medium [SynT(2D) medium supplemented with 50 ng/ml EGF]. The cells were treated with fresh medium on day 3 and analyzed on day 6.

For EVT differentiation, TS cells were seeded in a collagen IV-coated dish and cultured in EVT medium [DMEM/F12 (Gibco) supplemented with 0.1 mM 2-ME, 0.5% antibiotic-antimycotic, 0.3% BSA, 1% ITS-X, 7.5 μM A83-01, 2.5 μM Y27632, 4% KSR, and 100 ng/ml NRG1 (26941, Cell Signaling Technology)] and Matrigel was added to the medium at a final concentration of 2%. At day 3, the cells were replaced with EVT medium lacking NRG1 and supplemented with Matrigel at a final concentration of 0.5%. At day 6, when the cells reached ~80% confluence, the cells were passaged and cultured in the EVT medium lacking NRG1 and KSR with 0.5% Matrigel for an additional two days and analyzed on day 8.

In addition, the viability of TSCs was measured using Ez-cytox cell viability assay (EZ-1000, DoGenBio) according to the manufacturer’s instructions.

Engraftment of TS cells

10⁷ TSCs were suspended in 200 μl of a 1:2 mixture of Matrigel and DMEM/F12 containing 0.3% BSA and 1% ITS-X, and then subcutaneously injected into 6- to 8-week-old severe immune-deficient (SID) male mice. Lesions, urine, and serum were collected seven days after injection.

Trophoblast-endothelial transwell co-culture

A trophoblast-endothelial co-culture system was adapted with a slight modification of the previous method⁸². Briefly, hTS cells were plated in the bottom of 24-well chambers coated with 2.5 μg/ml collagen IV and were cultured in TS or SynT(2D) medium for 5 days, with a fresh medium change on day 3. BeWo cells in the bottom of the same 24-well chambers were transfected with fPIBF-expressing and/or sPIBF-expressing constructs or control vector for 24 h, and then stimulated with forskolin for 48 h.

After 5 days of differentiation or 48 h of forskolin stimulation, the culture media were replaced with a 1:1 mixed medium of Medium200/LVES and modified SynT(2D) (SynT(2D) medium lacking EGF, Y27632, and forskolin, which can affect endothelial invasion capacity^{83,84,85,86,87}) in HUVEC-SynT, or Medium200/LVES and DMEM/F12/10% FBS in HUVEC-BeWo.

Concurrently, HUVECs were placed for 24 h in an 8-μm pore size transwell insert (CLS3422, Corning) coated with Matrigel (354234, Corning), and then the endothelial inserts were transferred onto the differentiated SynT or forskolin-stimulated BeWo cultures. After 24 h of co-culture, HUVECs that invaded the lower surface of the membrane were stained with crystal violet. The number of invaded cells was counted in six fields randomly selected from each sample using ImageJ software (NIH).

MRM analysis

MRM method establishment for PIBF1 quantification

Representative tryptic peptide for PIBF1 was determined to be ELLHNIQLLK (molecular weight: 1219.72 Da, N-terminus of PIBF1 (55-64 aa)) using PeptideaAltas (http://www.peptideatlas.org/) archive. Stable isotope-labeled internal standard (SIS) peptide for ELLHNIQLLK (molecular weight: 1227.72 Da) was synthesized by incorporating a heavy isotope labeled lysine at the C-terminal (L-Lysine-13C6,15N2) with 98% purity (Peptron, South Korea) to perform multiple reaction monitoring (MRM) analysis. A list of possible b- and y-series product ions for a doubly charged peptide of ELLHNIQLLK was generated theoretically using Skyline software (64 bits, version 19.1.0.193, University of Washington, MacCoss Lab) with the m/z range from 300 to 1,400. Those theoretical transitions were optimized with default instrument parameters of HPLC system (1290 Infinity, Agilent Technologies) and triple quadrupole mass spectrometry (MS) with a jet stream ESI source (6495 Agilent, Agilent Technologies). The MS source settings were set as below: gas temperature was 250 °C, gas flow was 15 L/min, nebulizer was 30 psi, sheath gas temperature was 350 °C, sheath gas flow was 10 L/min, capillary was 3500 V for positive ion funneling, nozzle voltage was 300 V, iFunnel parameters for high-pressure RF was 90 V and low-pressure RF was 60 V. Mixture of light peptide and SIS peptide was injected directly into a reversed-phase analytical column (Zorbax Eclipse Plus C18 rapid resolution HD, 2.1 × 100 mm, 1.8-micron column, Agilent Technologies) with 40 °C oven temperature. The peptide sample was separated and eluted at 0.3 ml/min on a linear gradient of mobile phase B from 5% to 30% B in 19 min (Mobile phase A: water/0.1% FA, mobile phase B: acetonitrile/0.1% FA). The gradient was ramped to 70% B for 3 min and ramped to 90% for 3 min. And 5% B for 10 min to equilibrate the column for the next run. Resulting in the top three most intense transitions for the light peptide (Q1: 407.2550 m/z (triple charged), Q3: 501.3395 (y4, singly charged), 433.2663 (y7, doubly charged) and 489.8084 (y8, doubly charged)) and SIS peptide (Q1: 410.2550 m/z (triple charged), Q3: 509.3537 (y4, singly charged), 437.2734 (y7, doubly charged) and 493.8155 (y8, doubly charged)) were selected for collision energy optimization and retention time determination, respectively. Targeted MS acquisitions were performed using 3-min detection windows, a 100-msec cycle time. MRM was performed using 6 points of concentration samples ranging from 0 to 50 fmol with a blank matrix to determine the response curve. The limit of detection (LoD) using 3 times of standard deviation plus the average peak area of three blank runs and the limit of quantitation (LoQ) using 10 times of standard deviation plus the average peak area of three blank runs were determined using the reverse response curve.

Preparation of cell lysates and conditioned media for MRM analysis

hTSC, SynT, and EVT were incubated in the media without BSA for 24 h. Cell pellets were suspended in lysis buffer [5% SDS, 50 mM triethylammonium bicarbonate (TEAB), 1× Halt™ protease inhibitor cocktail (78429, Thermo Fisher Scientific), pH 8.5] and were boiled at 80 °C for 10 min. Supernatants were harvested by centrifugation at 18,000 × g for 20 min. Conditioned media (CM) of 1 ml from each cell were lyophilized to reduce the volume. Each dried pellet from the lyophilized CM was suspended in 300 μl of lysis buffer. The protein concentration of each supernatant from cell lysate and CM was measured with a BCA protein quantification kit (Pierce™ BCA Protein Assay Kit; 23225, Thermo Fisher Scientific). A 100 µg aliquot of proteins was digested using suspension-trapping (S-Trap) mini columns (CO2-mini-80, ProtiFi) as per the manufacturer’s protocol with Lys-C/trypsin mixture (V5071, Promega) and incubation for 16 h at 37 °C⁸⁸. The eluted peptide mixture was lyophilized using a cold trap and stored at −80 °C until use.

Quantification of PIBF1 using MRM

The peptide mixture from the cell lysate and the CM was dried and reconstituted using 0.1% FA to be 1 μg/ul. SIS peptide was spiked to be 50 fmol and targeted MRM analysis was performed using established MRM methods. Quantity information for each transition was extracted using Skyline, and the concentration of PIBF1 was calculated using a peak area of 50 fmol SIS peptide.

Measurement of hCG

The presence of hCG in the mouse urine sample was qualitatively detected using a commercial over-the-counter hCG pregnancy test kit. The quantitative level of hCG in the samples of mouse serum or conditioned media obtained from BeWo, SynT(2D), or SynT(3D) culture was measured using an ELISA kit (KA4005, Abnova) following the manufacturer’s instruction.

Human heart organoid (hHO) experiments

Generation of hHO and rsPIBF1 treatment

iPSC-derived hHOs were generated as previously described⁵⁷. Briefly, human iPSCs were cultured in mTeSR-TM1 medium (StemCell Technologies) containing 10 μM Y-27632 (1254, Tocris Bioscience) and 10% Matrigel (345277, Corning) for 3 days on a shaking incubator to form embryonic bodies (EBs). EBs were then cultured in RPMI1640 containing B27 supplement without insulin and 6 μM CHIR99021 (4423, Tocris Bioscience) for 2 days, and then in RPMI1640 containing B27 supplement without insulin and 2 μM C-59 (S7037, Selleckchem) for 2 days. On day 7, cells were cultured in Advanced MEM (AD-MEM) containing 1% GlutaMAX and 1% antibiotic-antimycotic (hHO medium).

To investigate the effect of rsPIBF on the development of hHO, cells were cultured in hHO medium (control group) or hHO medium supplemented with 50, 200, and 1000 ng/ml sPIBF (sPIBF group) on day 8 (one day after hHO induction). The medium was replaced every two days until days 19-21.

Sprouting assay

For sprouting assay, a 20-μl cold drop of Matrigel containing control or rsPIBF-treated hHOs was solidified at room temperature for 5 min, added with AD-MEM, and cultured for 4-6 days on a shaking incubator with the medium replaced every two days.

Multi-electrodes array

For multi-electrodes array (MEA), after control or sPIBF-treated hHOs were loaded in the MEA device (Maestro Edge version; Axion BioSystems, GA, USA), raw waveforms representing the field potential (FP) of each group were recorded for 3 minutes in AxIS software. Three leading indicators of recorded FP (spike amplitude, beat period, and field potential duration) were analyzed using the Cardiac Analysis Tool and AxIS Metric Plotting Tool (Axion BioSystems)^57,58.

Histology

For immunostaining of hHOs, control or rsPIBF-treated hHOs were fixed in 4% PFA overnight at 4 °C, dehydrated in a sucrose gradient (15-30%), embedded in OCT compound, cryosectioned in 9 μm thickness, and stored at –80 °C until following procedures.

RNA seq and Gene ontology (GO) analysis

Total RNA was isolated from EBs (day 3), control, or sPIBF-treated hHOs (day 20) using a Trizol Reagent kit (15596026, Invitrogen). Next-generation sequencing (NGS) was conducted by LifeGenomics (Suwon, Republic of Korea). Differentially expressed gene analysis was performed using DESeq2 version 1.36.0⁸⁹. The genes up- or down-regulated over 1.5 times with adjusted p-value (Benjamini-Hochberg correction) were regarded as significant. GO analysis was performed using clusterProfiler version 4.4.4⁹⁰. The statistical analysis and visualization were performed under the R version 4.2.2 and R studio Build 353 environments.

hiPSC-derived cardiomyocyte experiments

A two-dimensional culture of hiPSC-derived cardiomyocytes was performed as previously described⁵⁸ with slight modifications. Briefly, hiPSCs were seeded on Matrigel-coated dishes with mTeSRTM1 (StemCell Technologies) media containing 10 µM Y27632. The mTeSRTM1 media was changed every day until the confluency of hiPSCs reached 90%. First, to differentiate into the mesodermal lineage, hiPSCs were cultured for 1 day in RPMI1640 media containing B27 supplement without insulin (B27 media) supplemented with 6 μM CHIR99021 (Day 0). The next day (Day 1), it was replaced with B27 media containing 3 μM CHIR99021 and cultured for 1 day. Next, for induction into cardiac progenitors, B27 media containing 2 μM C59 (Sellckem), a Wnt signal inhibitor, was replaced on day 2. The next day (Day 3), it was replaced with B27 media containing 2 μM C59 and cultured for 1 day. From day 4 to day 10, the Advanced MEM (Gibco) medium (AD-MEM) containing 1% penicillin/streptomycin and 1% GlutaMAX (Gibco) was replaced every 2 days until the differentiated cells began beating.

To investigate the effect of rsPIBF on the differentiation of cardiomyocytes, sPIBF (200 ng/ml) was treated for 6 days (Day 4-10). After confirming beating, cells were detached using TrypLE (Gibco) and reseeded on Matrigel-coated dishes. On day 12, the hiPSC-CMs were fixed in 4% PFA for 20 min and stained with antibodies against cTnT and α-actinin, as listed in Supplementary Data 6.

Mice

All animal experiments were performed per the Korean Ministry of Food and Drug Safety guidelines. Experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committees (IACUC) of Asan Institute for Life Sciences (approval number: 2019-12-217). All mice were maintained in the specific pathogen-free facility of the Laboratory of Animal Research at Asan Medical Center (AMCLAR) at an ambient temperature of 23 ± 1 °C and 50 ± 5% humidity, with ad libitum access to a commercial CHOW diet (Purina Laboratory Rodent Diet, 38057) and standard laboratory water and a 12-h dark/light cycle, which was applied throughout all mouse studies. The protocol for conventional knockout of Pibf1 (Pibf1^em1Hwl/J)⁹¹ and Trp53 (Trp53^em1Baek/J)⁹² in mice was described previously. ICR and SID (NOD/J-Prkdc^em1BaekIl2rg^em1Baek/J) mice were purchased from OrientBio (Gyeonggi-do, Republic of Korea) and GEM Biosciences (Chenogju, Republic of Korea), respectively. Meox2-cre (B6.129S4-Meox2^tm1(cre)Sor/J, 003755), Vav1-iCre (B6N.Cg-Commd10^{Tg(Vav1-icre)A2Kio}/J, 018968), Cdh5-cre (B6;129-Tg(Cdh5-cre)1Spe/J, 017968), and R26R reporter (B6.129S4-Gt(ROSA)26Sor^tm1Sor/J, 003474) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Tie2-cre (B6.Cg-Tg(Tek-cre)1Ywa/J, JAX 008863) and Il17a knockout (B6.129P2-Il17a^tm1Yiw) mice were generously provided from Dr. Chang Hoon Ha and Dr. Kyung Jin Lee, respectively, from Asan Medical Center (Seoul, Republic of Korea).

To obtain tissue samples of conceptus at different gestational ages, 7-week-old female mice were mated at a 1:1 ratio with male mice in the evening. The day of the vaginal plug formation was considered embryonic day (E) 0.5. Pregnant mice were then randomly assigned to groups with different experiments, euthanized at the designated time point, and the tissue samples from the different parts of the conceptus were snap-frozen and stored at −80 °C or fixed in 4% PFA for further analysis.

Generation of Pibf1 cKO mice

To generate single guide RNAs (sgRNAs) used for floxed PIBF1 mice, the following oligomers were purchased from Macrogen, Inc.: 5′−taggGCTATTTCAGTTTCTCTAAG−3′ and 5′−aaacCTTAGAGAAACTGAAATAGC−3′ for Pibf1 intron 1-specific sgRNA; 5′−taggACTCTATTTCTGCTTCCCGA−3′ and 5′−aaacTCGGGAAGCAGAAATAGAGT−3′ for Pibf1 intron 17-specific sgRNA. Each pair of oligomers was annealed and cloned into pUC57-sgRNA vector, a gift from Xingxu Huang (Addgene plasmid # 51132; http://n2t.net/addgene:51132;RRID:Addgene_51132), using BsaI restriction enzymes (New England BioLabs), and the cloned vectors were used as a template for sgRNA in vitro transcription. The following single-stranded deoxynucleotide donor DNAs were purchased from Integrated DNA Technologies, Inc.: 5′−tgtaactcccttgtttaaatacgacacgatttagtaaaatagttatagaactagctatttcagtttctctATAACTTCGTATAATGTATGCTATACGAAGTTATaagaggaacgctaaagaaggaggtctgtgtggtgggggggaacaggaagagcttcagtacaatccgcctc−3′ for inserting a loxP into Pibf1 intron 1; 5′−caattctcctccttcaatatattgtgaatttctgttgaactgtgaaacagataactctatttctgcttccATAACTTCGTATAATGTATGCTATACGAAGTTATcgagggctgaaattacaaccatgaaccaccatacccaggtgaaactatttttttttagagatataacata−3′ for inserting a loxP into Pibf1 intron 17. Microinjection of fertilized eggs and their transfer to foster mothers were performed as previously described⁹¹. Genomic DNA samples obtained from the tail of embryos or 3-week-old pups were genotyped by using the primers listed in Supplementary Data 5.

Embryo outgrowth culture

Blastocysts were individually cultured on 0.1% gelatin-coated multi-well dishes in KnockOut DMEM (10829018) supplemented with 20% KnockOut Serum Replacement (10828010), 2 mM GlutaMAX (35050061), 0.1 mM non-essential amino acids (11140050), 50 U/ml penicillin-50 μg/ml streptomycin (15070063), 55 μM 2-mercaptoethanol (21985023), and 5 ng/ml leukemia inhibitory factor (A35933) (all reagents were purchased from Gibco) at 37 °C and 5% CO₂. On the 7th day of culture, outgrowths were photographed, collected, and subsequently genotyped by PCR.

Morphometric analysis of embryo and heart

The images of embryo outgrowths and dissected parts of the conceptus, including the yolk sac, embryo, and heart, were photographed using a ProgRes C5 camera and Capture Pro software (Jenoptik Optical Systems, Jena, Germany). Quantitative measurements of the areas of inner cell mass and trophectoderm in the outgrowths, the crown-rump length in whole-mount embryos, and the width and height of heart chambers in whole-mount hearts were carried out using ImageJ software (NIH).

Histology

Morphometric analysis of placenta

H&E-stained placental sections were examined for the areas of three main layers of the placenta (decidua basalis, junctional zone, labyrinth) and those of labyrinthine vascular spaces under a light microscope. Fetal blood sinuses were distinguished from maternal ones by their primitive nucleated erythrocytes. The blood sinuses in the labyrinth were examined and compartmentalized in three random non-overlapping ×40 fields of each sample. The extent of each area was quantified using ImageJ software (NIH).

Immunostaining

Heat-induced antigen-retrieved tissue sections were subjected to Vectastain Elite ABC-HRP system (PK6101, Vector Labs) for immunohistochemistry according to the manufacturer’s instructions. Primary antibodies were as follows: anti-Ki67 (1/10000; ab15580, Abcam), anti-Laminin (1/750; L9393, Sigma-Aldrich), anti-Perforin (1/200; ab16074, Abcam), and anti-PIBF1 (1/500; PMID:14634107³⁴). In the case of staining Perforin, peroxidase-conjugated donkey anti-rat IgG (712-035-150, Jackson ImmunoResearch Laboratories) was applied as a secondary antibody. Labyrinthine Ki67-positive trophoblast cells or decidual Perforin-positive cells were counted in three randomly selected ×40 fields of each sample.

For immunofluorescence staining, antigen-retrieved tissue sections were subjected to primary antibodies, subsequently captured by Alexa Fluor-conjugated secondary antibodies. In vitro samples, including fixed cells and hHO cryosections, were permeabilized with 0.1% Triton X-100 and subjected to antibodies. The sections were mounted with DAPI (D1306, Molecular Probes), and examined under Zeiss LSM 880 confocal microscope system (Carl Zeiss, Jena, Germany). CD31, cTnT, or PDGFRβ-stained areas were quantified using ImageJ software (NIH). The antibodies used for immunofluorescence staining are listed in Supplementary Data 6.

Alkaline phosphatase activity

OCT-embedded placentas were 6 μm-thick cryosectioned, incubated in alkaline phosphatase buffer (0.1 M NaCl, 0.1 M Tris-HCl, 0.05 M MgCl₂, 0.1% Tween 20, pH 9.5) containing 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT) color development substrate (S3731, Promega) according to the manufacturer’s instructions, and then counterstained with nuclear fast red.

Transmission electron microscopy

Isolated placentas were fixed in 2.5% glutaraldehyde at 4 °C for 4 h, fixed in 1% OsO₄ for 1 h at room temperature, and then embedded in Epon. Semithin sections (500 nm) were stained with toluidine blue, and the thickness of interhaemal membranes was measured in five randomly selected ×100 fields of each sample. Ultrathin sections (60 nm) were contrasted with uranyl acetate and lead citrate and observed using a Hitachi H-7100 transmission electron microscope (Hitachi, Nackashi, Japan) at 80 kV.

In situ hybridization

For in situ hybridization (ISH) of mouse Pibf1, samples were fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned into 5-μm-thick slices on SuperFrost Plus adhesion slides (J1800AMNZ, ThermoFisher Scientific). Following the manufacturer’s instructions, a chromogenic RNA ISH assay was performed using the RNAscope 2.5 HD detection kit (Brown) for FFPE tissues (322371, Advanced Cell Diagnostics). RNAscope probes (all from Advanced Cell Diagnostics) were used for mouse Ppib (313911), bacterial dapB (310043), and mouse Pibf1 (408871) was used as the positive control, negative control, and target probe, respectively.

Measurement of fatty acids

Gas chromatography coupled with mass spectrometry (GC/MS)-based measurement of placental and embryonic fatty acids (FAs) was previously described³⁵. Quantified data were expressed as a relative amount (%) to the wild-type group.

Laser micro-dissection

Dissecting the placenta into three main layers (decidua basalis, junctional zone, labyrinth) using laser micro-dissection of frozen sections was previously described³⁵.

Quantitative real-time PCR

cDNA synthesized with the SuperScript III First-Strand synthesis system (18080-051, Invitrogen) was employed as a template for quantitative real-time PCR reactions using the iQ SYBR Green Supermix (170-8882AP, Bio-Rad) and the primer sets (Supplementary Data 5) in the QuantStudio3 Real-Time PCR system (Applied Biosystems, CA, USA). Data were normalized by an internal control gene, such as RPS18, GAPDH, and mGapdh, and expressed relative to the expression level of the wild-type or control group.

In vitro fertilization and embryo transfer

Oocytes were collected from females superovulated with 5 IU pregnant mare serum gonadotropin (G4877, Sigma) and 5 IU human chorionic gonadotropin (CG10, Sigma) at a 48 h interval. The in vitro fertilized 2-cell stage embryos from Pibf1 heterozygous were transferred to pseudopregnant wild-type ICR or Il17a knockout female mice. Embryos at E10.5 to E11.0 were collected, photographed, and genotyped by PCR.

Fluorescence-activated cell sorting (FACS)

The mesometrial regions of conceptus covering decidua basalis were dissected and sampled at E8.5 or E10.5, chopped, and digested using 1 mg/ml collagenase type II (C6885, Sigma) and 1 U/ml DNase I (04536282001, Roche) at 37 °C for 30 min. The prepared single cells were Fc-blocked with anti-CD16/CD32 (553142, BD Biosciences) and then stained with antibodies listed in Supplementary Data 6. All incubation steps were carried out in D-PBS containing 2% FBS. Stained samples were assessed using the FACSCanto II system (BD Biosciences, CA, USA) and further analyzed using FlowJo software (Tree Star, OR, USA).

Whole-mount X-gal staining

4% PFA-fixed isolated parts of conceptus were incubated in detergent buffer (2 mM MgCl₂, 0.02% IGEPAL, and 0.01% sodium deoxycholate in PBS, pH 7.4) at 4 °C overnight. Samples were then incubated in detergent buffer containing 4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, and 1 mg/ml X-gal (B4252, Sigma) at 37 °C for overnight in the dark, post-fixed in 4% PFA overnight, and visualized under a stereo microscope. For histological analysis, X-gal-stained tissues were paraffin-embedded, 4 μm-thick cuts, and counterstained with nuclear fast red.

Hematopoiesis experiments

Colony-forming unit assay

Dissected parts of conceptus at different gestational ages were mechanically minced and enzymatically digested by incubating them in D-PBS containing 2% FBS and 0.1% collagenase type I (C0130, Sigma) at 37 °C for 30 min. In the case of preparing adult bone marrow samples, the femur and tibia of 4-month-old male mice were dissected, and their proximal and distal epiphysis were cut. Bone marrow was collected by flushing them with 2% FBS-containing D-PBS and dissociated by passing through a 70 μm cell strainer. Cells were cultured in a Methocult methylcellulose medium (M3434, Stem Cell Technologies) according to the manufacturer’s instructions. Colonies were morphologically distinguished and scored after 10-12 days of culture.

Complete blood count with white blood cell differential

Whole blood samples of 4-month-old male mice collected from the inferior vena cava were analyzed for hematologic parameters using ADVIA 2120i automatic blood analyzer (Siemens, Munich, Germany).

Whole-mount CD31 staining

4% PFA-fixed embryos or yolk sacs were blocked in 5% goat serum, incubated with anti-CD31 (1:200; 553370, BD Biosciences), and subsequently with AF594-conjugated secondary antibody (1:200; A21209, Invitrogen) in the dark. The immuno-stained whole mounts were visualized under Zeiss LSM 880 laser scanning confocal microscope system (Carl Zeiss, Jena, Germany). All incubation steps were performed in PBTx (PBS with 0.1% Tween 20 and 0.1% Triton X-100) at 4 °C overnight.

Western blotting

Protein lysates from cell or tissue samples were prepared using cell extraction buffer (FNN0011, Invitrogen) supplemented with protease inhibitor cocktail (Sigma, P2714) and phenylmethylsulfonyl fluoride (0754, Amresco), loaded in SDS-PAGE gels, transferred onto polyvinylidene difluoride membrane (IPVH00010, Millipore), and immunoblotted with primary and secondary antibodies as listed in Supplementary Data 6. The signals were detected using a SuperSignal West Pico chemiluminescent system (34080, Thermo Fisher Scientific).

Magnetic resonance imaging

MR imaging was performed at a 7.0 T/160 mm Parmascan preclinical scanner on Paravision 6.0 and equipped with a 72 mm diameter volume transmission coil, and a surface receive coil (Bruker Inc., Germany). The embryos are then placed into sealed plastic containers in preparation and immersed in 4% formaldehyde solution in PBS during MR scanning. The high-resolution 2D rapid acquisition with relaxation enhancement (2D RARE) sequence, fast spin-echo based T2 weighted (T2w) imaging was performed with axial (Ax) and sagittal (Sag) views – TR/TE = 4500/ 50 ms; average = 40; field of view (FOV) = 12 × 12 mm (Ax), 14 × 14 (Sag); RARE factor = 8; matrix = 256 × 256; slice thickness = 0.4 mm; slice number = 20 (no gap); and total scan time = 3 h 12 m. AsanJ-Morphometry™ is a dedicated software for ImageJ (NIH) used to indicate abnormalities in an embryo. The AsanJ-Morphometry software is open to the public for research purposes and downloadable at http://datasharing.aim-aicro.com/morphometry.

Statistical analysis

Experiments were replicated at least two times, and no data were excluded from the analyses. The χ² test was applied to test whether the genotype distribution of the mice from various breeding schemes obeyed the expected Mendelian inheritance patterns. Other data were analyzed by Student’s two-tailed unpaired t-test or one-way ANOVA followed by Tukey’s multiple comparison test. Statistical significance was determined using GraphPad Prism software (GraphPad Software, CA, USA). All data points in diagrams represent biological replicates. P values less than 0.05 were considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The RNA-seq data for rsPIBF-treated hHO generated in this study have been deposited in the GEO database under accession code GSE229265. All other data are available within the main text and the Supplementary Information files. Source data are provided with this paper.

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Acknowledgements

This study was supported by grants from the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2018M3A9D5A01087977 and 2020R1A2C2012912). This work was also supported by grants (2020-028, 2022-013, and 2023-062) from the Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea. We thank the core facilities of the GEAR Core, Confocal Microscope Core, Clinical Proteomics Core, Comparative Pathology Core, Electron Microscopy Core, Flowcytometry Core, Magnetic Resonance Core, Mammalian Genetics Core, Metabolomics Core, and Dep. of Laboratory Animal Research at the ConveRgence mEDIcine research center (CREDIT) at Asan Medical Center for the use of their shared equipment, services, and expertise. We are also grateful to Dr. B. Polgar and Dr. J. Szekeres-Bartho (University of Pecs, Hungary) for generously providing anti-PIBF1 antibodies.

Author information

Jong Geol Lee
Present address: Biological Resources Research Group, Bioenvironmental Science & Toxicology Division, Gyeongnam Branch Institute, Korea Institute of Toxicology (KIT), Jinju, 52834, Korea
These authors contributed equally: Jong Geol Lee, Jung-Min Yon.

Authors and Affiliations

Asan Institute for Life Sciences, Asan Medical Center, Seoul, 05505, Korea
Jong Geol Lee, Jung-Min Yon, Globinna Kim, Seung-A Cheong, Jiyoung Yu, Kyunggon Kim, Young Hoon Sung, Hyun Ju Yoo, Dong-Cheol Woo, Jin Kyung Rho, Chang Hoon Ha, Chan-Gi Pack, Joon Seo Lim, Ki-Up Lee, Chong Jai Kim, You Sook Cho & In-Jeoung Baek
Korea Mouse Phenotyping Center (KMPC), Seoul, 08826, Korea
Jong Geol Lee, Je Kyung Seong, Sang-Wook Lee & In-Jeoung Baek
Department of Cell and Genetic Engineering, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
Jung-Min Yon, Globinna Kim, Young Hoon Sung & In-Jeoung Baek
Department of Stem Cell Biology, School of Medicine, Konkuk University, Seoul, 05029, Korea
Seul-Gi Lee
College of Veterinary Medicine, Konkuk University, Seoul, 05029, Korea
C-Yoon Kim
NGeneS Inc, Ansan, 15495, Korea
Hyun-Yi Kim
Department of Digital Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
Kyunggon Kim & Hyun Ju Yoo
Department of Biomedical Engineering, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
Dong-Cheol Woo & Chan-Gi Pack
Department of Biochemistry and Molecular Biology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
Jin Kyung Rho, Chang Hoon Ha & Ki-Up Lee
Department of Pediatrics, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
Seak Hee Oh
Research Institute of Medical Science, Sungkyunkwan University School of Medicine, Seoul, 06351, Korea
Yu Mi Han
College of Veterinary Medicine, Chungnam National University, Daejeon, 34134, Korea
Eui-Ju Hong
College of Veterinary Medicine, Seoul National University, Seoul, 08826, Korea
Je Kyung Seong
Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, 03722, Korea
Han-Woong Lee
Department of Radiation Oncology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
Sang-Wook Lee
Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
Chong Jai Kim
College of Veterinary Medicine, Chungbuk National University, Cheongju, 28644, Korea
Sang-Yoon Nam
Division of Allergy and Clinical Immunology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, 05505, Korea
You Sook Cho

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Jong Geol Lee
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Jung-Min Yon
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Globinna Kim
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Seul-Gi Lee
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C-Yoon Kim
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Yu Mi Han
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Eui-Ju Hong
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Ki-Up Lee
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Chong Jai Kim
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You Sook Cho
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Contributions

I.-J.B. designed research. J.G.L., J.-M.Y., S.-G.L., C.-Y.K., J.Y., K.K., J.K.R., and C.H.H. carried out in vitro assays. J.G.L., J.-M.Y., G.K., S.-A.C., Y.H.S., and H.J.Y. carried out in vivo experiments. S.-G.L. and C.-Y.K. performed heart organoid experiments. D.-C.W. and C.-G.P. performed imaging experiments. K.K. and Y.S.C. produced recombinant proteins. J.G.L., J.-M.Y., H.-Y.K., S.-G.L., C.-Y.K., K.K., Y.S.C. and I.-J.B. analyzed data. I.-J.B. supervised the project and secured funding. J.G.L., J.-M.Y., C.-Y.K., J.S.L., Y.S.C. and I.-J.B. wrote the original paper. S.H.O., Y.M.H., E.-J.H., J.K.S., H.-W.L., S.-W.L., K.-U.L., C.J.K., S.-Y.N., and all other authors revised and edited the paper.

Corresponding authors

Correspondence to You Sook Cho or In-Jeoung Baek.

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Competing interests

J.G.L., J.-M.Y., K.K., Y.S.C., and I.-J.B. have filed a provisional patent application in the Korea patent office (composition for preventing and treating pregnant complications related to placenta and/or fetal cardiovascular abnormalities by targeting PIBF1). All the other authors declare no competing interests.

Ethics

HUVEC preparation was approved by the Institutional Review Board of the Asan Medical Center (approval ID: 2022-0664). Written informed consent was obtained from all participants or their families in accordance with the Declaration of Helsinki.

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Nature Communications thanks Julia Szekeres-Bartho and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Lee, J.G., Yon, JM., Kim, G. et al. PIBF1 regulates trophoblast syncytialization and promotes cardiovascular development. Nat Commun 15, 1487 (2024). https://doi.org/10.1038/s41467-024-45647-8

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Received: 09 June 2023
Accepted: 30 January 2024
Published: 19 February 2024
DOI: https://doi.org/10.1038/s41467-024-45647-8

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Subjects

Abstract

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Introduction

Results

Essential roles of PIBF1 in trophoblast syncytialization

Angiogenic role of SynT-derived PIBF1

Defective syncytialization and formation of the materno-fetal interface in placenta lacking Pibf1

Syncytialization and trophoblast-endothelial interaction recovered in a functional placenta harboring Pibf1-intact trophoblast

Impact of trophoblast syncytialization upon cardiovascular development of the embryo

Manifestation of cardiovascular features facilitated by PIBF1 in heart organoids

Discussion

Methods

Cell culture

Establishment of PIBF1 KO cell lines

Recombinant PIBF1

Construction of the raPIBF1 expression plasmid

Induction and purification of raPIBF1

BeWo experiments

Cell-to-cell fusion and fusion index quantification

PIBF1 restoration in PIBF1 KO BeWo cells

Treatment with recombinant PIBF1 on PIBF1 KO BeWo cells

In vitro angiogenesis assays

hTS cell experiments

Differentiation of hTS cells

Engraftment of TS cells

Trophoblast-endothelial transwell co-culture

MRM analysis

MRM method establishment for PIBF1 quantification

Preparation of cell lysates and conditioned media for MRM analysis

Quantification of PIBF1 using MRM

Measurement of hCG

Human heart organoid (hHO) experiments

Generation of hHO and rsPIBF1 treatment

Sprouting assay

Multi-electrodes array

Histology

RNA seq and Gene ontology (GO) analysis

hiPSC-derived cardiomyocyte experiments

Mice

Generation of Pibf1 cKO mice

Embryo outgrowth culture

Morphometric analysis of embryo and heart

Histology

Morphometric analysis of placenta

Immunostaining

Alkaline phosphatase activity

Transmission electron microscopy

In situ hybridization

Measurement of fatty acids

Laser micro-dissection

Quantitative real-time PCR

In vitro fertilization and embryo transfer

Fluorescence-activated cell sorting (FACS)

Whole-mount X-gal staining

Hematopoiesis experiments

Colony-forming unit assay

Complete blood count with white blood cell differential

Whole-mount CD31 staining

Western blotting

Magnetic resonance imaging

Statistical analysis

Reporting summary

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding authors

Ethics declarations

Competing interests

Ethics

Peer review

Peer review information

Additional information

Supplementary information

Source data

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