Use of PIBF1 or vector comprising gene that encodes same for preventing and treating pregnancy complications associated with placenta or fetal cardiovascular abnormalities

Abstract

The present invention relates to use of PIBF1 or a vector comprising a gene that encodes same for preventing and treating pregnancy complications associated with placenta or fetal cardiovascular abnormalities, and, more specifically, to: a pharmaceutical composition comprising PIBF1 or a vector comprising a gene encoding same for preventing and treating pregnancy complications associated with placenta or fetal cardiovascular abnormalities; and a method for preventing and treating pregnancy complications associated with placenta or fetal cardiovascular abnormalities by using the composition.

Description

Use of a vector comprising PIBF1 or a gene encoding it for the prevention or treatment of pregnancy complications associated with placental or fetal cardiovascular abnormalities

[0001] The present invention relates to the use of a vector comprising PIBF1 or a gene encoding it for the prevention or treatment of pregnancy complications associated with placental or fetal cardiovascular abnormalities, and more particularly, to a pharmaceutical composition comprising a vector comprising PIBF1 or a gene encoding it for the prevention or treatment of pregnancy complications associated with placental or fetal cardiovascular abnormalities, and a method for preventing or treating pregnancy complications associated with placental or fetal cardiovascular abnormalities using the same.

[0002] A proper placenta ensures normal development of embryonic organs. Several lines of evidence suggest that the placenta controls cardiovascular development (non-patent references 1 to 5), which is described as the placenta-heart axis (non-patent references 6 and 7). However, the cellular and molecular mechanisms underlying this paradigm remain poorly understood. Progesterone immunomodulatory binding factor 1 (PIBF1) is well known to play a crucial role in immune tolerance during pregnancy (non-patent references 8 and 9). Its levels continue to rise throughout pregnancy, eventually reaching maternal levels (non-patent references 10 and 11). Simultaneously, in the human placenta, its expression is highly expressed in syncytiotrophoblasts (SynT) in early pregnancy and declines at term. SynT, a multinucleated layer formed by the continuous fusion of mononuclear trophoblasts (CTBs), provides the primary barrier for exchange between the maternal and fetal circulations. However, the factors that constitute the maternal-fetal interface and the mechanisms by which the placenta influences the developing cardiovascular system of the embryo have not yet been fully elucidated.

[0003] Against this backdrop, the present inventors have confirmed that SynT, as a result of trophoblast cell fusion, interacts with adjacent vascular cells to initiate the formation of an exchange barrier in the placenta and maintain embryonic cardiovascular development, which is regulated by trophoblast-expressed PIBF1 and SynT-derived PIBF1, and in particular, when a specific form of PIBF1 protein, such as a secretory form, is processed, it activates the signaling mechanism for migration and angiogenesis of embryonic-derived vascular endothelial cells and pericytes, which are major cells of the placenta, thereby promoting angiogenesis in the placenta and promoting cardiovascular development of the fetus along the formed placental circulatory system, thereby demonstrating that it is effective in preventing, improving, or treating pregnancy-related diseases related to placental or fetal cardiovascular abnormalities, thereby completing the present invention.

[0004] [Prior Art Literature]

[0005] [Non-patent literature]

[0006] (Non-patent Document 1) V. Perez-Garcia et al., Placentation defects are highly prevalent in embryonic lethal mouse mutants. Nature 555, 463-468 (2018)

[0007] (Non-patent Document 2) Y. Barak et al., PPAR gamma is required for placental, cardiac, and adipose tissue development. Mol Cell 4, 585-595 (1999)

[0008] (Non-patent Document 3) RH Adams et al., Essential role of p38alpha MAP kinase in placental but not embryonic cardiovascular development. Mol Cell 6, 109-116 (2000)

[0009] (비특허문헌 4) G. D. Raffel et al., Ott1 (Rbm15) is essential for placental vascular branching morphogenesis and embryonic development of the heart and spleen. Mol Cell Biol 29, 333-341 (2009)

[0010] (비특허문헌 5) E. O. Maruyama et al., Extraembryonic but not embryonic SUMO-specific protease 2 is required for heart 10 development. Sci Rep 6, 20999 (2016)

[0011] (비특허문헌 6) M. Hemberger, J. C. Cross, Genes governing placental development. Trends Endocrinol Metab 12, 162-168 (2001)

[0012] (비특허문헌 7) E. J. Camm, K. J. Botting, A. N. Sferruzzi-Perri, Near to One's Heart: The Intimate Relationship Between the Placenta and Fetal Heart. Front Physiol 9, 629 (2018)

[0013] (비특허문헌 8) J. Szekeres-Bartho, B. Polgar, PIBF: the double edged sword. Pregnancy and tumor. Am J Reprod Immunol 64, 77-86 (2010)

[0014] (Non-patent Document 9) B. Huang et al., Interleukin-33-induced expression of PIBF1 by decidual B cells protects against preterm labor. Nat Med 23, 128-135 (2017)

[0015] (Non-patent Document 10) MK Lim et al., Characterization of serum progesterone and progesterone-induced blocking factor (PIBF) 20 levels across trimesters in healthy pregnant women. Sci Rep 10, 3840 (2020)

[0016] (Non-patent Document 11) B. Polgar, E. Nagy, E. Miko, P. Varga, J. Szekeres-Bartho, Urinary progesterone-induced blocking factor concentration is related to pregnancy outcome. Biol Reprod 71, 1699-1705 (2004)

[0017] Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing, improving or treating pregnancy-related diseases associated with placental or fetal cardiovascular abnormalities using a specific form of PIBF1 protein.

[0018] Another object of the present invention is to provide a method for preventing, improving or treating pregnancy-related diseases associated with placental or fetal cardiovascular abnormalities using a specific form of PIBF1 protein.

[0019] Another object of the present invention is to provide the use of a specific form of PIBF1 protein for the manufacture of a medicament for the prevention, improvement or treatment of pregnancy-related diseases associated with placental or fetal cardiovascular abnormalities.

[0020] To solve the above-described problem, the present invention provides a pharmaceutical composition for preventing, improving or treating pregnancy complications related to placental or fetal cardiovascular abnormalities, comprising (i) a PIBF1 (Progesterone immunomodulatory binding factor 1) protein or a fragment thereof; or (ii) a vector comprising a gene encoding the PIBF1 protein or a fragment thereof.

[0021] The present invention also provides a composition comprising (i) a PIBF1 protein or a fragment thereof; or (ii) a vector comprising a gene encoding a PIBF1 protein or a fragment thereof, for use in preventing, improving, or treating pregnancy complications associated with placental or fetal cardiovascular abnormalities.

[0022] Additionally, the present invention provides a method for preventing, improving or treating pregnancy complications associated with placental or fetal cardiovascular abnormalities, comprising administering to a subject in need thereof a composition comprising (i) a PIBF1 (Progesterone immunomodulatory binding factor 1) protein or a fragment thereof; or (ii) a vector comprising a gene encoding a PIBF1 protein or a fragment thereof.

[0023] Furthermore, the present invention provides the use of the aforementioned composition for the manufacture of a medicament for preventing, improving or treating pregnancy-related diseases associated with placental or fetal cardiovascular abnormalities.

[0024] In the present invention, the PIBF1 protein may include the amino acid sequence of sequence number 1.

[0025] In the present invention, the fragment of the PIBF1 protein may include a part of the amino acid sequence of the N-terminus of the PIBF1 protein or a part of the amino acid sequence of the N-terminus and a part of the amino acid sequence of the C-terminus of the PIBF1 protein.

[0026] In the present invention, some amino acid sequences of the N-terminus may include at least amino acid residues from positions 1 to 184 in the amino acid sequence of SEQ ID NO: 1.

[0027] In the present invention, some amino acid sequences of the C-terminus may include at least amino acid residues from positions 684 to 757 in the amino acid sequence of SEQ ID NO: 1.

[0028] In the present invention, some amino acid sequences of the N-terminus may include the amino acid sequence of SEQ ID NO: 2, and some amino acid sequences of the N-terminus and some amino acid sequences of the C-terminus may include the amino acid sequence of SEQ ID NO: 3.

[0029] In the present invention, the pharmaceutical composition or the composition can increase PIBF1 (Progesterone immunomodulatory binding factor 1) protein having a molecular weight of 25 to 90 kDa in the mother.

[0030] In the present invention, the pharmaceutical composition or the composition may exhibit one or more effects selected from the group consisting of the following a) to d):

[0031] a) Formation of vascular-like structures in the embryo;

[0032] b) Angiogenesis in the embryo;

[0033] c) differentiation and maturation of embryonic cardiomyocytes; and

[0034] d) Formation of the maternal-fetal interface.

[0035] In the present invention, pregnancy complications related to placental or fetal cardiovascular abnormalities may include recurrent miscarriage, preeclampsia, or fetal heart defects.

[0036] The pharmaceutical composition according to the present invention promotes the formation of blood vessels in the placenta by activating the signaling mechanisms related to the migration and angiogenesis of embryonic-derived vascular endothelial cells and pericytes, which are the main cells of the placenta, and promotes the development of the cardiovascular system of the fetus along the formed circulatory system of the placenta, and is useful for preventing, improving or treating various pregnancy-related diseases related to placental or fetal cardiovascular abnormalities, such as recurrent miscarriage, preeclampsia or fetal heart defects.

[0037] Figure 1 shows a schematic of two crRNAs targeting exon 2 or 4 of human PIBF1.

[0038] Figures 2a to 2e show the results showing that trophoblast syncytium is disrupted in PIBF1KO BeWo. Figure 2a shows the mutant PIBF1 sequences observed in PIBF1KO BeWo cell lines #1 and #2 using CRISPR / Cpf1 targeting human PIBF1 exons 2 and 4, respectively. Figure 2b shows trophoblast fusion assays in WT and BeWo PIBF1 KO cell line #2, with BeWo borders and nuclei stained with Di-8-ANEPPS (Di-8; green) and Hoechst (blue). White and red dotted lines indicate the plasma membrane and nuclei within fused cells. Data are expressed relative to the WT control. Figure 2c shows the expression of the SynT marker in FSK-treated PIBF1 KO BeWo cell line #2. Data were normalized using RPS18 and expressed relative to the WT data. Figure 2d shows the level of hCG secreted by FSK-treated PIBF1 KO BeWo cell line #2. Figure 2e shows the expression of β-hCG and PIBF1 in FSK-treated PIBF1 KO BeWo cell line #2. β-Actin was used as an internal control. Data are expressed as mean ± SEM (Figures 2b and 2d) or mean ± minimum to maximum (Figure 2c). ****P < 0.0001 in a one-way ANOVA.

[0039] Figures 3a to 3p show the results showing that PIBF1 is required for trophoblast fusion. Figure 3a shows the results of trophoblast fusion assays in WT and PIBF1KO BeWo cells. After 48 h of incubation with forskolin (FSK, 20 μM), the BeWo border and nuclei were stained with Di-8-ANEPPS (Di-8; green) and Hoechst (blue), respectively. White and red dotted lines indicate the plasma membrane and nuclei within fused cells. Figure 3b shows the expression of SynT markers in FSK-treated PIBF1KO BeWo cells. Figure 3c shows the level of hCG secreted by FSK-treated PIBF1KO BeWo cells. Figure 3d shows the expression of β-hCG and PIBF1 in FSK-treated PIBF1KO BeWo cells. β-Actin was used as an internal control. Figure 3e shows phase contrast images of PIBF1KO hTSC-derived syncytiotrophoblast cells (TS-SynT) in 2D (top) and 3D cultures (bottom) and immunostaining for β-hCG (green) in 2D PIBF1KO TS-SynT (middle). White and red dotted lines indicate the plasma membrane and nucleus within the fused cells. Figure 3f shows the expression of SynT markers in PIBF1KO TS-SynT. Figure 3g shows the level of hCG secreted by PIBF1KO TS-SynT. Figure 3h shows the expression of TS markers in PIBF1KO TS-SynT. Figure 3i shows a schematic diagram of PIBF1KO hTSC engraftment in immunodeficient NSG mice. Figure 3j shows the serum hCG levels in NSG mice injected subcutaneously with PIBF1KO hTSCs. Figure 3k shows representative images of an over-the-counter hCG pregnancy test of urine samples collected from PIBF1KO hTSC-injected mice. Figure 3l shows macroscopic villous lesions developed from transplanted PIBF1KO hTSCs.Figure 3m shows H&E and immunofluorescence staining results for KRT7 (white), β-hCG (green), and HLA-G (red) in PIBF1KO hTSC-derived lesions. Figure 3n shows the expression of PIBF1 in FSK-treated PIBF1KO cells reintroduced with full-length PIBF1 (fPIBF) and / or secreted PIBF1 (sPIBF). Figure 3o shows the results of fusion assays in PIBF1KO BeWo cells after restoration of fPIBF and / or sPIBF. Figure 3p shows the results of fusion assays in PIBF1KO BeWo cells supplemented with recombinant fPIBF1 (rfPIBF) and / or sPIBF1 (rsPIBF). Fusion assay data [Figures 3a, 3o, and 3p] are expressed relative to the data of the WT control. Transcriptional expression data [3b, 3f, and 3h] are normalized to Gapdh and expressed relative to WT. Data are presented as mean ± SEM in Figures 3a, 3c, 3g, 3j, 3o, and 3p or as mean ± minimum to maximum in Figures 3b, 3f, and 3f. *,**,***,****P<0.05, 0.01, 0.001, and 0.0001, respectively, by one-way ANOVA (Figures 3a to 3c, 3o, and 3p) or Student's t-test (Figures 3f, 3h, and 3j).

[0040] Figures 4a to 4h show that PIBF1KO impairs the differentiation function of TSCs with little effect on self-renewal ability. Figure 4a shows the mutant PIBF1 sequence observed in PIBF1 KO TSCs using CRISPR / Cpf1 targeting human PIBF1 exon 2. Figure 4b shows the expression of PIBF1 in PIBF1 KO TSCs, with β-actin used as an internal control. Figure 4c shows the cell proliferation rate of PIBF1KO TSCs under stem cell conditions. Data are expressed relative to WT at 72 hours and are expressed as mean ± SEM. Figures 4d and 4e show the expression of TS marker transcripts (Figure 4d) and protein levels (Figure 4e) in PIBF1KO TSCs under stem cell conditions. Figure 4f shows the expression of GCM1 transcripts in undifferentiated PIBF1KO TSCs. Figure 4g shows the expression of HLA-G and MMP2 in PIBF1KO TSC-derived EVT (TS-EVT). Figure 4h shows phase contrast and HLA-G (green) immunostaining images in PIBF1KO TS-EVT. Transcript expression data (Figures 4d, 4f, and 4g) were normalized to GAPDH, expressed relative to WT, and presented as mean ± minimum to maximum. Student's t test, **P < 0.01; ****P < 0.0001.

[0041] Figures 5a to 5c show results demonstrating that exogenous treatment with recombinant PIBF1 (rPIBF) does not affect the reduced syncytium formation in PIBF1 KO BeWo cells. Figure 5a shows a schematic diagram of rPIBF used in the present invention, including the full-length (fPIBF), secreted form (sPIBF), active domain (aPIBF), and C-terminal fragment (cPIBF) of PIBF1. Figure 5b shows representative images of PIBF1 KO BeWo cell line #1 treated with recombinant sPIBF (rsPIBF) or rfPIBF. Figure 5c shows fusion assays in PIBF1 KO BeWo cell line #2 supplemented with rsPIBF or rfPIBF. Data are expressed relative to WT and are presented as mean ± SEM. **P<0.01 by one-way ANOVA.

[0042] Figures 6a to 6s show results demonstrating that trophoblast-derived PIBF1 mediates vasculature formation and the expression of cardiovascular characteristics in vitro. Figure 6a shows the results of multiple reaction monitoring (MRM) analysis of intracellular and secreted PIBF1 in hTSCs, TS-SynTs, and TS-EVTs. A P value less than 0.05 indicates statistical significance in a one-way ANOVA. ns, not significant. Figure 6b shows a schematic diagram of endothelial recruitment by hTSC-derived SynTs or fused BeWo cells. HUVECs that invaded the lower chamber containing hTSC-derived SynTs or FSK-treated BeWo cells for 24 hours were stained with crystal violet (CV) and counted. Figure 6c shows the recruitment assay of HUVECs by differentiated SynTs derived from WT and PIBF1KO hTSCs (top panel) and the recruitment assay of HUVECs by restoration of fPIBF and / or sPIBF to PIBF1KO BeWo cells (bottom panel). Figure 6d shows representative images of migration (top panel) and tube formation assays (bottom panel) in rsPIBF-treated HUVECs. Figures 6e and 6f show quantified data from migration (Fig. 6e) and tube formation assays (Fig. 6f) in rsPIBF-treated HUVECs. Figure 6g shows the expression of angiogenesis-related signaling cascades in rsPIBF-treated HUVECs. Data were normalized to β-actin. Figures 6h and 6i show migration (Figure 6h) and tube formation assays (Figure 6i) in rsPIBF-treated HUAECs. Figure 6j shows migration assays in rsPIBF-treated placental pericytes. Figure 6k shows a schematic diagram of iPSC differentiation into hHO and rsPIBF application. Figure 6l shows immunostaining for cTnT, CD31, and PDGFRβ in rsPIBF-treated hHO.Figure 6m shows immunostaining of cTnT and α-actinin in rsPIBF-treated hiPSC-derived 2D-cultured cardiomyocytes (n = 8 per group). Figure 6n shows multielectrode array (MEA) results of rsPIBF-treated hHO. FPD, field potential duration. Figure 6o shows sprouting assay results of rsPIBF-treated hHO. Sprouting was confirmed by phase contrast and PDGFRβ staining images. Figures 6p and 6q show the dose effect of sPIBF1 on hHO development. Figure 6p shows immunostaining of cTnT and CD31 in hHO treated with rsPIBF at doses of 50, 200, and 1000 ng / ml, and Figure 6q shows sprouting assays in hHO treated with rsPIBF at doses of 50, 200, and 1000 ng / ml. Figure 6r visualizes the expression patterns of genes significantly up- or down-regulated in sPIBF-treated hHO compared to the untreated group using a heatmap. Two clusters of genes were identified by hierarchical clustering based on the expression patterns, and representative GO terms of each cluster were visualized using bar graphs (the width and color of the bars represent gene proportions and p-values, respectively). Figure 6s visualizes the top five enriched GO terms (large dots) for genes significantly upregulated by sPIBF treatment, along with their associated genes (small dots). The size of the large dots represents the number of associated genes. The color of the small dots represents the binary logarithm of the gene fold change due to sPIBF treatment. The lines connecting the genes associated with the GO terms are colored uniquely according to the GO term. Recruitment (Figure 6c), migration (Figures 6e, 6h, and 6j), tube formation (Figures 6f and 6i), and protein band intensities (Figure 6g) are expressed relative to the WT control and are expressed as the mean ± SEM. One-way ANOVA (Figures 6a, 6c, and 6e to 6j) indicates *P<0.05; **P<0.05.01; ***P<0.001; ****P<0.0001. The intensity of fluorescently labeled cells (Figs. 6l and 6m) and sprouting length data (Fig. 6o) were expressed relative to the control group. Data were expressed as mean ± SEM (Figs. 6l to 6o). *P<0.05; ***P<0.001; ****P<0.0001 in a two-tailed Student's t-test (Figs. 6l to 6o).

[0043] Figures 7a to 7l show results demonstrating that PIBF1 regulates vasculature formation by regulating the recruitment and angiogenesis of ECs and placental pericytes (Pl-PCs) in vitro. Figure 7a shows HUVEC recruitment by hTSC-derived SynT in a transwell invasion assay, and Figure 7b shows HUVEC recruitment by confluent BeWo cells in a transwell invasion assay. Data are expressed relative to the control group and are presented as the mean ± SEM. ***P < 0.001, ****P < 0.0001 by Student's t test. Figure 7c is a representative blot image of the angiogenesis-related signaling cascade in rsPIBF-treated HUVECs. β-Actin was used as an internal control. Figures 7d and 7e are representative images of migration and tube formation assays in rsPIBF-treated HUAECs (Figure 7d) and a representative image of migration assays in rsPIBF-treated Pl-PCs (Figure 7e). Figures 7f to 7h are representative images of migration and tube formation assays in rfPIBF, raPIBF, or rcPIBF-treated HUVECs (Figure 7f), the quantified results of their migration (Figure 7g), and the quantified results of the tube formation assay (Figure 7h). Figures 7i to 7k are representative images of migration and tube formation assays in rfPIBF, raPIBF, or rcPIBF-treated HUAECs (Figure 7i), the quantified results of their migration (Figure 7j), and the quantified results of the tube formation assay (Figure 7k). Figure 7l shows migration assays in Pl-PCs treated with rfPIBF, raPIBF, or rcPIBF. Migration (Figures 7g, 7j, and 7l) and tube formation data (Figures 7h and 7k) are expressed relative to the control group and are presented as mean ± SEM. One-way ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0044] Figure 8 shows a Volcano plot of all genes expressed in rsPIBF-treated human cardiac organoids. All expressed genes are plotted on the X-axis on a log2 scale, and the calculated probability (p-value) on the Y-axis on a -log10 scale. Red dots represent differentially expressed genes (DEGs) that were significantly up- or down-regulated with a fold change >1.5 at an adjusted p-value <0.05. Vertical and horizontal dashed blue lines represent thresholds.

[0045] Figures 9a to 9d show that Pibf1-null mice exhibit intrauterine lethality, and the time window of impaired embryonic development is related to the first placental period. Figure 9a shows Pibf1 + / - Shows the expected and observed genotype distributions in newborns after cross-crossing. ****P < 0.0001 in the χ2 test. Figure 9b shows Pibf1 - / - The area quantification of inner cell mass (ICM), trophectoderm (TE), and their ratio (ICM / TE) determined by embryo growth culture is shown, and the data are expressed as mean ± SEM. Figure 9c shows Pibf1 at E8.5-13.5. + / - The genotype distribution of the crossover is shown. † Developmentally delayed and has several defects (reduced size, pericardial effusion, and failure to rotate the tail). ‡ Almost resorbed with little residue. Figure 9d shows Pibf1 at E8.5-13.5. - / - The results of the head-rump length measurements of embryos are presented, and the data are expressed as the mean ± SEM. **P < 0.05; ***P < 0.01 by Student's t test. Pibf1 - / - The head-rump length of the embryo was considered to be 0 at E13.5, at which stage Pibf1 - / - Embryos were largely resorbed with few remnants used for genotyping and were therefore not statistically compared with the WT group.

[0046] Figures 10a to 10l show the results showing that placental trophoblast fusion and vascular labyrinth integrity were impaired in Pibf1-null mice. Figure 10a shows Pibf1 - / - A schematic diagram of the lineage containing Pibf1 deletion in the conceptus and labyrinth interhaemal membrane is shown. S-TG: sinusoidal trophoblast giant cell. ST-I and ST-II: syncytiotrophoblast type I and II, respectively. EC: endothelial cell. Figure 10b shows placental histology and Pibf1 - / - Gross outline of the embryo. The black solid line indicates the maximum thickness of the labyrinth. dec: decidua. jz: junctional zone. lab: labyrinth. ch: chorion. al: allantois. Figures 10c and 10d show Pibf1 - / - The gross histological evaluation of placental morphology (Fig. 10c) and the morphological analysis of the labyrinthine vascular bed (Fig. 10d) are shown in the placenta. mbs and fbs: maternal and fetal blood spaces, respectively. Figure 10e shows Pibf1 measured by gas chromatography coupled with mass spectrometry (GC-MS). - / - Fatty acid (FA) contents of placentas and embryos at E9.5-10.5 are shown. Data are expressed as relative amounts (%) (colored bars) compared to wild type (WT; gray bars) (n=6 / genotype). The term "acid" is omitted except for docosahexaenoic acid (DHA). NEFA: nonessential FA; EFA: essential FA. Figure 10f shows Pibf1 expression as determined by the percentage of Ki67-positive cells in the labyrinth trophoblast. - / - This shows the proliferation of labyrinthine trophoblasts in the placenta. Figure 10g shows Pibf1 - / -Expression of markers for TSCs and their lineages in the placenta is shown. GlyT: glycogen trophoblast. SpT: spongiocyte trophoblast. Figure 10h shows Pibf1 labeled with endogenous alkaline phosphatase. - / - The distribution of maternal blood sinusoidal cover sinusoidal TGC (S-TGC) in the labyrinth is shown in Fig. 10i. Pibf1 - / - Immunofluorescence staining of MCT1 (red) and MCT4 (green) to detect SynT-I (arrowhead) and -II (arrow) layers, respectively, in the placenta. MCT4 signal is Pibf1 - / - Excessive reduction in the labyrinth. m and f: maternal and fetal blood sinuses, respectively. Figure 10j shows Pibf1 - / - Ultrastructure of the mesentery in the labyrinth is shown. The upper panel shows a semi-thin section stained with toluidine blue (TB) and the thickness of the mesentery, and the lower panel shows a pseudo-colored transmission electron microscopy (EM) image of an ultra-thin section (bottom). ii, ii', and ii" indicate lack of fusion within SynT-II. Hashtags indicate areas of excessive electron density in SynT-I. ec: endothelial cell, i: SynT-I, ii: SynT-II. Figure 10k shows Pibf1. - / - Immunostaining of laminin and TUNEL to mark the endothelial lining of fetal blood vessels and apoptotic cells in the placenta. TUNEL-positive signals indicate Pibf1 - / - In the labyrinth (arrow), only cells associated with fetal blood vessels or fetal red blood cells were detected. Figure 10l shows laser microdissection of Pibf1 - / -Expression of endothelial vasculature markers in placental sections is shown. Experimental samples were obtained at E10.5 unless otherwise specified. Transcript expression data (Figs. 10g and 10l) were normalized to Gapdh and expressed relative to WT data. Data are expressed as mean ± SEM in Figs. 10c to 10f, 10j, and 10l, or mean ± minimum to maximum in Fig. 10g. *, **, *** P<0.05, 0.01, and 0.001, respectively, by Student's t-test.

[0047] Figures 11a to 11g show that the viability of Pibf1-null embryos is not related to uterine immune tolerance to natural killer (uNK) cells / lymphocytes or maternal genetic capacity of Pibf1. Figure 11a shows Pibf1 + / - In vitro fertilization (IVF) and embryo transfer (ET) of outbred embryos into wild-type pregnant female mice are shown. Figure 11b shows Pibf1 + / - Genotype distribution of E10.5 embryos from wild-type mothers after crossbreeding (left) and their Pibf1 - / - A whole-body image of the embryo (right) is shown. † Developmentally delayed and has several defects (reduced size, pericardial effusion, and failure of tail rotation). Figure 11c shows immune tolerance and Pibf1 regulated by the balance between uterine NK (uNK) and helper T 17 (Th17) cells in the decidua. - / - Schematic diagram of a lineage containing Pibf1 deletion in the conceptus. Figure 11d shows Pibf1 deletion at E8.5 and E10.5. - / - CD3e in the mesometrial region of the conceptus - CD122 + DBA + Fluorescence-activated cell sorting (FACS) analysis of uNK cells is shown, and data are expressed as mean ± SEM. Figure 11e shows Pibf1 - / -Quantification of perforin-positive uNK cells in the decidual zone of the conceptus, data are expressed as mean ± SEM. Figure 11f shows Pibf1 + / - IVF and ET of Il17-deficient pseudopregnant female mice from crossbred embryos are shown. Figure 11g shows Pibf1 + / - Genotype distribution of E10.5 embryos from Il17-deficient mothers after crossbreeding (left) and their Pibf1 - / - A whole-body image of the embryo (right) is shown. †Development is delayed and has several defects (reduced size, pericardial effusion, and failure to rotate the tail).

[0048] Figures 12a and 12b illustrate epiblast-specific Pibf1 knockout using a Meox2-driven cre line. Figure 12a shows the generation of Pibf1 conditional knockout (cKO) mice using CRISPR / Cas9. Figure 12b shows Meox2 cre Genotyping analysis to confirm the presence of a Pibf1 deletion is shown. Genomic DNA samples prepared from the tails of E13.5 embryos were used as templates for PCR, and agarose gel electrophoresis assays were performed after PCR. Primer positions are indicated by black or colored arrows on the relevant alleles. Colored dotted boxes indicate the absence of the wild-type or Pibf1 floxed allele (red) and the presence of the recombinant allele (blue) in the Pibf1cKO mutant.

[0049] Figures 13a to 13p show results showing that Pibf1 is required for normal syncytialization, placental vascularization, and subsequent embryonic survival in mice, but not in the endothelial / hematopoietic lineage, in trophoblast development. Figure 13a shows Meox2 cre (cKO Meox2 ) is a schematic representation of a line containing Pibf1 deletion in the mesentery and a conceptus with a conditional knockout (cKO) driven by a cKO. Figure 13b is a schematic representation of a line containing Pibf1 deletion in the mesentery and a conceptus with a conditional knockout (cKO) driven by a cKO. Meox2The expected and observed genotype distribution in crossbreeding is shown in Figure 13c. Meox2 The histology and gross appearance of the placenta of the embryo are shown in Figures 13d and 13e. Meox2 The gross histological evaluation of placental morphology (Fig. 13d) and morphological analysis of the labyrinthine vascular layer (Fig. 13e) in the placenta are shown. Figure 13f shows cKO Meox2 The results of measuring the head-to-rump length in embryos are shown in Fig. 13g. cKO Meox2 The expression of markers for TSC and its lineage in the placenta is shown in Fig. 13h. cKO Meox2 Immunofluorescence staining of MCT1 (red) and MCT4 (green) to detect SynT-I (arrowhead) and -II (arrow) layers, respectively, in the placenta is shown in Fig. 13i. cKO Meox2 The expression of markers for endothelial vasculature in laser microdissection cross-sections of placentas is shown in Fig. 13j. Pibf1KO and cKO mice were determined by colony forming unit (CFU) assay. Meox2 The hematopoietic potential in the yolk sac (ys), embryo proper (em), and placenta (pl) of the embryo is shown. CFU-GEMM = CFU-granulocyte, erythrocyte, macrophage, megakaryocyte. CFU-GM = CFU-granulocyte, macrophage. BFU-E = erythroblast forming unit. Figure 13k shows Cdh5 cre (cKO Cdh5 ) or Tie2 cre (cKO Tie2 ) is a schematic representation of a lineage containing Pibf1 deletion in the mesentery and conceptus with an endothelium-specific cKO (ecKO) driven by the cKO. Figure 13l is a schematic representation of a lineage containing Pibf1 deletion in the mesentery and conceptus with an ecKO driven by the cKO. Cdh5 or cKO Tie2 This shows the expected and observed genotypic distribution in crossbreeding. Figure 13m shows cKO Cdh5 and cKO Tie2The histology and macroscopic appearance of the placenta of the ecKO embryos are shown. Figure 13n shows the gross histological evaluation of the ecKO placenta. Figure 13o shows the results of measurements of the head-to-rump length in ecKO embryos. Figure 13p shows the hematopoietic potential of the ecKO mutants as determined by the CFU assay. Unless otherwise specified, experimental samples were obtained at E10.5. Transcript expression data (Figures 13g and 13i) were normalized to Gapdh and expressed relative to the control data, and CFU (Figures 13j and 13p) were expressed relative to the control data. The number of each bar represents the sample size (n) in Figures 13j and 13p. Data are expressed as mean ± SEM in Figures 13d to 13f, 13i, 13j, and 13n to 13p, or as mean ± minimum to maximum in Figure 13g. ns: χ 2 Not significant in the test. **, *** P<0.01, 0.001 in one-way ANOVA.

[0050] Figures 14a and 14b show Meox2-, Cdh5-, or Tie2-driven cre recombinase activity in the placenta (Fig. 14a) and embryonic heart (Fig. 14b). Males carrying Meox2-, Cdh5-, or Tie2-cre alleles were mated with female R26R-lacZ reporters, and embryos, placentas, or hearts were obtained at E10.5 or E15.5, and whole-tissue X-gal staining was performed. Embryos carrying R26R-lacZ but negative for the cre allele were used as negative controls. Dec: decidua; jz, junctional zone; labyrinth: labyrinth. fbs: fetal blood space; LaT: labyrinth trophoblast; a: atrium; v: ventricle. ra and la: right and left atrium; rv and lv: right and left ventricle; oft: outflow tract; ecc: endocardial ridge.

[0051] Figures 15a to 15d provide supporting data on defective hematopoietic tissue development in Pibf1-null conceptuses. Figure 15a shows Pibf1 at E8.5-10.5. - / - Whole-body images of the yolk sac (YS) and Pibf1 at E10.5 - / - Histological findings of YS. Pibf1 at E10.5 - / - Whole-tissue images of YS show reduced vascularity and pale appearance. Asterisk indicates Pibf1 - / - At E10.5 YS, blood islands are distended, disorganized, and contain few red blood cells. Figure 15b shows Pibf1 at E8.5. - / - Expression of markers for hematopoiesis (left) and globin (right) of primitive erythroid cells in YS is shown. Figure 15c shows Pibf1 expression at E9.5 as determined by colony forming unit (CFU) assay. - / - The hematopoietic potential of the yolk sac (ys), embryo proper (em), and placenta (pl) is shown. Data are expressed relative to the wild type (WT). The number in each bar represents the sample size (n). CFU-GEMM = CFU-granulocytes, erythrocytes, macrophages, megakaryocytes. CFU-GM = CFU-granulocytes, macrophages. BFU-E = erythroblast forming unit. Figure 15d shows Pibf1 at E10.5. - / - Expression of markers for globin in hematopoietic (left) and primitive erythroid cells (right) in the placenta is shown. Transcript expression data (Figures 15b and 15d) were normalized using Gapdh and expressed relative to WT. Data are presented as mean ± SEM (Figures 15b and 15d). *P < 0.05; **P < 0.01 by Student's t test.

[0052] Figures 16a to 16c are Pibf1 / ;Meox2 cre / + Supporting data for the placental phenotype are shown. Figure 16a shows Meox2 at E13.5. cre -Pibf1cKO(cKO) Meox2) shows the gross histological evaluation of the placenta. Data are expressed as mean ± SEM. Figure 16b shows cKO at E13.5 as measured by gas chromatography coupled with mass spectrometry (GC-MS). Meox2 Fatty acid (FA) content of placentas and embryos is shown. Data are expressed as relative amounts (%) (colored bars) compared to WT (gray bars) (n=6 / genotype). The term “acid” is omitted except for DHA. NEFA: nonessential FA; EFA: essential FA. Figure 16c shows immunofluorescence staining of MCT1 (red) and MCT4 (green) to detect SynT-I and -II layers, respectively, in cKOMeox2 placentas at E13.5. m and f: maternal and fetal blood sinuses, respectively.

[0053] Figures 17a to 17f show results showing that loss of Pibf1 in the hematopoietic lineage does not interfere with normal hematopoiesis. Figure 17a shows the results of Vav1-cre (cKO) at E13.5 Vav1 ) or Tie2-cre (cKO Tie2 ) shows the gross images of the fetus and fetal liver of hematopoietic-specific Pibf1cKO using a line. The yellow dotted line outlines the fetal liver. Figure 17b shows the cKO Vav1 and cKO Tie2 The results of measuring the head-rump length of an E13.5 fetus are shown. Figure 17c shows cKO Vav1 and cKO Tie2 Cellularity analysis of the E13.5 fetal liver of cKO mice. Figure 17d shows the cellularity analysis of cKO mice at E13.5. Vav1 and cKO Tie2 The results of CFU assay of the yolk sac, fetal liver, and placenta of cKO are shown in Figures 17e and 17f. Cdh5 and cKO Vav1Results of CFU assays of total blood cell counts by bone marrow (Fig. 17e) and leukocyte differentiation (Fig. 17f) in adult mice (4 months old) are shown. Data are expressed relative to the control group and are presented as mean ± SEM in Figs. 17c to 17f. The numbers in each bar represent the sample size (n) in Figs. 17d and 17e.

[0054] Figures 18a to 18j show the results showing that placentas with appropriately fused trophoblasts maintain embryonic development of vascular structures and hearts in mice. Figure 18a shows the results of whole-body Pibf1KO (Pibf1 - / - ), and the outer layer-specific cKO of the embryo (cKO Meox2 ) or endothelial-specific cKO (cKO Cdh5 , cKO Tie2) shows a schematic diagram of the lineage containing Pibf1 deletion in the heart. Endo: endocardium, Myo: myocardium, Epi: epicardium. Figure 18b shows whole-tissue CD31 staining of the yolk sac, intersomitic vasculature (ISV), and heart in Pibf1 mutant embryos. The yellow dotted line in the yolk sac image indicates a continuous vessel with a diameter of ≥40 μm. Figures 18c and 18d show CD31 staining of histological sections from Pibf1 mutant hearts (Figure 18c) and quantification of the CD31-stained area (Figure 18d). a: atrium; la and ra: left and right atrium; v: ventricle; lv and rv: left and right ventricle. Figure 18e shows whole-tissue images and histological examination results of Pibf1KO and cKO hearts. White lines indicate the maximum length from edge to edge of the right and left ventricles. Neither the left nor the right ventricle was identified in the Pibf1KO heart. Figure 18f compares the ventricular dimensions of Pibf1KO and cKO hearts measured from whole-tissue images. Figures 18g and 18h ​​show immunostaining of cTnT in histological sections from Pibf1 mutant hearts (Figure 18g) and quantification of the cTnT staining area (Figure 18h). Figures 18i and 18j show the expression of markers for coronary vascular development (Figure 18i) and heart formation (Figure 18j) in Pibf1KO and cKO hearts. Unless otherwise specified, experimental samples were obtained at E10.5. Transcript expression data (Figures 18i and 18j) were normalized to Gapdh and expressed relative to control data. Data are presented as mean ± SEM in Figures 18d, 18f, and 18h, or mean ± minimum to maximum in Figures 18i and 18j. *, **, ***P<0.05, 0.01, 0.001 in one-way ANOVA.

[0055] Figures 19a and 19b show the results of measurements of the height and width of the atria and ventricles in Pibf1 mutant hearts. As shown in Figure 19a, in E10.5 whole-body hearts, the atrial and ventricular areas were captured from the ventral and dorsal views, respectively, for measurement and quantified as shown in Figure 19b. Data are expressed as the mean ± SEM. Pibf1 - / - The ventricular height and width of the heart were considered as 0, and Pibf - / - Because the hearts did not form distinct right and left ventricles, no statistical comparisons were made between groups.

[0056] Figures 20a to 20h show that Pibf1-null embryonic lethality is not Trp53-dependent, and ciliary disease-related abnormalities are induced by Pibf1 in late pregnancy. Δ / Δ ;Meox2 cre / + The results show that it appears in the fetus. Figure 20a shows E10.5Pibf1 - / - Expression of Trp53 in embryos is shown, and β-actin was used as an internal control. Figure 20b shows Pibf1 - / - ,Trp53 - / - A crossbreeding plan for obtaining embryos is shown. Figure 20c shows Trp53-deficient Pibf1 + / - Genotype distribution of outcross E10.5 embryos (top panel) and their Pibf1 - / - ;Trp53 - / - Whole-body images of embryos (bottom panel) are shown. †Developmental delay and several defects (reduced size, pericardial effusion, and failure of tail rotation). Figure 20d shows cKO at E18.5 and postnatally. Meox2 It shows the expected and observed genotype distribution of the cross. χ 2 ***P < 0.001 in the test. Figures 20e and 20f show cKO Meox2The overall morphology of an E15.5 fetus (Fig. 20e) and an enlarged view of the limbs (Fig. 20f) are shown. Arrows, arrowheads, and asterisks indicate polydactyly, microphthalmia, and subcutaneous edema. Figure 20g shows a cKO Meox2 This is a magnetic resonance image of an E15.5 fetus. Arrows, arrowheads, and asterisks indicate microphthalmia, subcutaneous edema, and hydrocephalus. Figure 20h shows a cKO fetus. Meox2 Immunofluorescence staining to detect Arl13b / acetylated tubulin double-positive primary cilia (arrowheads) in the spinal cord of an E15.5 fetus. cc: central canal; L: lumen.

[0057] Figures 21a to 21c show results demonstrating that Pibf1 deletion in maternal blood and immune cells does not interfere with normal fertility. Figure 21a shows a mating plan for monitoring pregnancy and outcome. As shown in Figure 21b, Pibf1 f / f or cKO Vav1 Females (8 weeks old) were mated with wild-type males for 4 months, and pups from three different litters per genotype were monitored for body weight for 8 weeks as shown in Fig. 21c.

[0058] Figure 22 is a schematic diagram showing that PIBF1 functions as an essential factor for trophoblast fusion and that syncytiotrophoblast (SynT)-derived PIBF1 functions as an embryonic cardiovascular growth factor to maintain the placental-heart axis. Trophoblast stem cells (TSCs) differentiate into syncytiotrophoblasts through cell-to-cell fusion via PIBF1, and PIBF1 secreted from differentiated syncytiotrophoblasts interacts with placental embryonic angiogenic cells, such as endothelial cells and pericytes, to promote angiogenesis, thereby establishing the interhaemal membrane that constitutes the maternal-fetal interface of the primary placenta [SynT-I and SynT-II: syncytiotrophoblast types I and II; S-TGC: sinusoidal trophoblast giant cell].

[0059] Hereinafter, the present invention will be described in more detail.

[0060] All technical terms used in this invention, unless otherwise defined, have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention.

[0061] In the present invention, we have elucidated a novel mechanism regulating placental, fetal vascular, and cardiac development by identifying a new secretory cell of PIBF1, which was known as a protein that regulates immune cell-derived immune tolerance during pregnancy. Specifically, the inventors have confirmed that PIBF1 protein or a fragment thereof is essential for trophoblast differentiation and fusion into SynT in human trophoblast and mouse placenta, and that SynT-derived PIBF1 enables communication between SynT and adjacent vascular cells, thereby developing a vascular network in the primary placenta, and further influences the early development of the embryonic cardiovascular system in an endocrine manner, thereby demonstrating the potential of PIBF1 protein or a fragment thereof to prevent or treat pregnancy complications associated with placental or fetal cardiovascular abnormalities.

[0062] Accordingly, the first aspect of the present invention relates to a pharmaceutical composition for preventing, improving or treating pregnancy complications associated with placental or fetal cardiovascular abnormalities, comprising (i) a PIBF1 (Progesterone immunomodulatory binding factor 1) protein or a fragment thereof; or (ii) a vector comprising a gene encoding a PIBF1 protein or a fragment thereof.

[0063] In this regard, the present invention provides a composition comprising (i) a PIBF1 (Progesterone immunomodulatory binding factor 1) protein or a fragment thereof; or (ii) a vector comprising a gene encoding a PIBF1 protein or a fragment thereof, for use in the prevention, improvement, or treatment of pregnancy complications associated with placental or fetal cardiovascular abnormalities.

[0064] The present invention also provides the use of a composition comprising (i) a PIBF1 protein or a fragment thereof; or (ii) a vector comprising a gene encoding a PIBF1 protein or a fragment thereof, for the manufacture of a medicament for preventing, improving, or treating pregnancy complications associated with placental or fetal cardiovascular abnormalities.

[0065] In the present invention, the PIBF1 protein may include, but is not limited to, its full-length form or a portion of the amino acid sequence at the N-terminus or a portion of the amino acid sequence at the N-terminus and a portion of the amino acid sequence at the C-terminus of the PIBF1 protein.

[0066] In humans, the PIBF1 protein may include an amino acid sequence disclosed in NCBI Reference Sequence: NP_006337.2 or a portion thereof, but may include, without limitation, an amino acid sequence that exhibits substantially the same or corresponding efficacy as the protein. The PIBF1 protein may also include an isoform or a precursor thereof.

[0067] In the present invention, the PIBF1 protein may comprise or consist of the amino acid sequence of the following SEQ ID NO: 1, but may be included without limitation as long as it comprises or consists of an amino acid sequence that exhibits substantially the same or corresponding efficacy as the PIBF1 protein. Specifically, it may comprise an amino acid sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more with the amino acid sequence of SEQ ID NO: 1. For example, it includes amino acid sequences having sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. The "% sequence identity" for an amino acid sequence is determined by comparing a comparison region with two optimally aligned sequences, wherein the portion of the amino acid sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence for the optimal alignment of the two sequences (which does not include additions or deletions).

[0068] MSRKISKESKKVNISSSLESEDISLETTVPTDDISSSEEREGKVRITRQLIERKELLHNIQLLKIELSQKTMMIDNLKVDYLTKIEELEEKLND ALHQKQLLTLRLDNQLAFQQKDASKYQELMKQEMETILLRQKQLEETNLQLREKAGDVRRNLRDFELTEEQYIKLKAFPEDQLSIPEYVSVRFYE LVNPLRKEICELQVKKNILAEELSTNKNQLKQLTETYEEDRKNYSEVQIRCQRLALELADTKQLIQQGDYRQENYDKVKSERDALEQEVIELRR KHEILEASHMIQTKERSELSKEVVTLEQTVTLLQKDKEYLNRQNMELSVRCAHEEDRLERLQAQLEESKKAREEMYEKYVASRDHYKTEYENKLH DELEQIRLKTNQEIDQLRNASREMYERENRNLREARDNAVAEKERAVMAEKDALEKHDQLLDRYRELQLSTESKVTEFLHQSKLKSFESERVQLLQEETARNLTQCQLECEKYQKKLEVLTKEFYSLQASSEKRITELQAQNSEHQARLDIYEKLEKELDEIIMQTAEIENEDEAERVLFSYGYGANVP TTAKRRLKQSVHLARRVLQLEKQNSLILKDLEHRKDQVTQLSQELDRANSLLNQTQQPYRYLIESVRQRDSKIDSLTESIAQLEKDVSNLNKEKS ALLQTKNQMALDLEQLLNHREELAAMKQILVKMHSKHSENSLLLTKTEPKHVTENQKSKTLNVPKEHEDNIFTPKPTLFTKKEAPEWSKKQKMKT (SEQ ID NO: 1)

[0069] In the present invention, the fragment of the PIBF1 protein may include or consist of a partial amino acid sequence of the N-terminus of the PIBF1 protein or a partial amino acid sequence of the N-terminus and a partial amino acid sequence of the C-terminus of the PIBF1 protein.

[0070] Specifically, the N-terminal portion of the amino acid sequence may include or consist of at least amino acid residues from positions 1 to 184 in the amino acid sequence of SEQ ID NO: 1. For example, the N-terminal portion of the amino acid sequence essentially consists of at least amino acid residues from amino acid positions 1 to 184 based on the amino acid sequence of SEQ ID NO: 1, and may include amino acid residues up to any amino acid position prior to amino acid position 757 without limitation.

[0071] Additionally, some amino acid sequences of the C-terminus may include or consist of at least amino acid residues from positions 684 to 757 in the amino acid sequence of SEQ ID NO: 1. For example, some amino acid sequences of the C-terminus essentially consist of at least amino acid residues from amino acid positions 684 to 757 based on the amino acid sequence of SEQ ID NO: 1, and may include amino acid residues from any amino acid position after amino acid position 1 to the end of the C-terminus without limitation.

[0072] In addition, when the fragment of the PIBF1 protein includes a part of the amino acid sequence of the N-terminus and a part of the amino acid sequence of the C-terminus, it may be a fused form of the part of the amino acid sequence of the N-terminus and the part of the amino acid sequence of the C-terminus described above. Preferably, it may include or consist of at least amino acid residues from positions 1 to 184 and at least amino acid residues from positions 684 to 757 based on the amino acid sequence of SEQ ID NO: 1, and more preferably, it may include or consist of at least amino acid residues from positions 1 to 224 and at least amino acid residues from positions 684 to 757 based on the amino acid sequence of SEQ ID NO: 1. At this time, the part of the amino acid sequence of the N-terminus may include any amino acid residue from positions 1 to 224 based on the amino acid sequence of SEQ ID NO: 1 without limitation.

[0073] In the present invention, the N-terminal part of the amino acid sequence may include or consist of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto, and may include without limitation any amino acid sequence that exhibits substantially the same or corresponding efficacy as the PIBF1 protein fragment, or consist of the amino acid sequence. Specifically, it may include an amino acid sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more with the amino acid sequence of SEQ ID NO: 2. For example, it includes amino acid sequences having sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. The "% sequence identity" for an amino acid sequence is determined by comparing a comparison region with two optimally aligned sequences, wherein the portion of the amino acid sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence for the optimal alignment of the two sequences (which does not include additions or deletions).

[0074] MSRKISKESKKVNISSSLESEDISLETTVPTDDISSSEEREGKVRITRQLIERKELLHNIQLLKIELSQKTMMIDNLKVDYLTKIEELEEKLNDALHQKQLLTLRLDNQLAFQQKDASKYQELMKQEMETILLRQKQLEETNLQLREKAGDVRRNLRDFELTEEQYIKLKAFPEDQLSIPEYVS (SEQ ID NO. 2)

[0075] In the present invention, the N-terminal part of the amino acid sequence and the C-terminal part of the amino acid sequence may include or consist of the amino acid sequence of SEQ ID NO: 3, but is not limited thereto, and may include without limitation any amino acid sequence that exhibits substantially the same or corresponding efficacy as the PIBF1 protein fragment, or consist of the amino acid sequence. Specifically, it may include an amino acid sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more with the amino acid sequence of SEQ ID NO: 3. For example, it includes amino acid sequences having sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. The "% sequence identity" for an amino acid sequence is determined by comparing a comparison region with two optimally aligned sequences, wherein the portion of the amino acid sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence for the optimal alignment of the two sequences (which does not include additions or deletions).

[0076] MSRKISKESKKVNISSSLESEDISLETTVPTDDISSSEEREGKVRITRQLIERKELLHNIQLLKIELSQKTMMIDNLKVDYLTKIEELEEKLNDALHQKQLLTLRLDNQLAFQQKDASKYQELMKQEMETILLRQKQLEETNLQLREKA GDVRRNLRDFELTEEQYIKLKAFPEDQLSIPEYVSVRFYELVNPLRKEICELQVKKNILAEELSTNKNQLKQLTEELAAMKQILVKMHSKHSENSLLLTKTEPKHVTENQKSKTLNVPKEHEDNIFTPKPTLFTKKEAPEWSKKQKMKT (SEQ ID NO: 3)

[0077] Any amino acid sequence that exhibits substantially the same or equivalent efficacy as the PIBF1 protein may be included without limitation. Specifically, an amino acid sequence having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% with the amino acid sequence of SEQ ID NO: 1 may be included. For example, it includes amino acid sequences having sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. The "% sequence identity" for an amino acid sequence is determined by comparing a comparison region with two optimally aligned sequences, wherein a portion of the amino acid sequence in the comparison region may include additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions) for the optimal alignment of the two sequences.

[0078] The present invention also encompasses polypeptides having an amino acid sequence substantially identical to the PIBF1 protein or fragments thereof, as well as fragments of the polypeptides. A polypeptide having a substantially identical amino acid sequence refers to a protein having the same function as that used in the present invention, regardless of amino acid sequence homology. A fragment of the polypeptide also refers to a protein having the same function as that used in the present invention, regardless of the length of the fragment.

[0079] The protein used in the present invention may have some of its amino acids substituted, and the amino acid substitutions of the present application may be non-conservative substitutions. The non-conservative substitutions may include altering amino acid residues of the target protein or polypeptide in a non-conservative manner, such as, for example, replacing an amino acid residue having a specific side chain size or specific property (e.g., hydrophilicity) with an amino acid residue having a different side chain size or different property (e.g., hydrophobicity).

[0080] The amino acid substitutions may also be conserved substitutions. Conserved substitutions may involve altering amino acid residues of a target protein or polypeptide in a conserved manner, such as replacing an amino acid residue having a particular side chain size or a particular characteristic (e.g., hydrophilicity) with an amino acid residue having the same or similar side chain size or the same or similar characteristic (e.g., still hydrophilicity). Such conserved substitutions generally do not significantly affect the structure or function of the resulting protein. In the present application, an amino acid sequence variant, a fragment thereof, or a variant thereof with one or more amino acid substitutions, which is a mutant of a recombinant protein, may comprise a conserved amino acid substitution that does not significantly alter the structure or function of the protein.

[0081] For example, mutual substitutions between amino acids in each of the following groups may be considered conservative substitutions in the present application:

[0082] A group of amino acids with nonpolar side chains: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine.

[0083] A group of uncharged amino acids with polar side chains: glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0084] A group of negatively charged amino acids with polar side chains: aspartic acid and glutamic acid.

[0085] A group of positively charged basic amino acids: lysine, arginine, and histidine.

[0086] A group of amino acids containing phenyl: phenylalanine, tryptophan, and tyrosine.

[0087] The recombinant proteins, polypeptides and / or amino acid sequences included in the present invention may also be understood to include at least the following scope: variants or homologues having the same or similar function as the recombinant proteins or polypeptides.

[0088] In the present invention, the variant may be a protein or polypeptide produced by substitution, deletion, or addition of one or more amino acids compared to the amino acid sequence of the recombinant protein and / or the polypeptide. For example, the functional variant may include a protein or polypeptide having an amino acid change by substitution, deletion, and / or insertion of at least one amino acid, for example, substitution, deletion, and / or insertion of 1-30, 1-20, or 1-10, or alternatively, for example, 1, 2, 3, 4, or 5 amino acids. The functional variant may substantially retain the biological properties of the protein or the polypeptide prior to the change (e.g., substitution, deletion, or addition). For example, the functional variant may retain 60%, 70%, 80%, 90%, or 100% or more of the biological activity of the protein or the polypeptide prior to the change.

[0089] In the present invention, the homologue may be a protein or polypeptide having at least about 80% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or the polypeptide.

[0090] In the present invention, the homology generally refers to the similarity, analogousness or association between two or more sequences. The "percentage of sequence homology" can be calculated by comparing two aligned sequences in a comparison window to determine the number of positions at which the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) exists, and dividing the number of matching positions by the total number of positions to provide the number of matching positions in the comparison window (i.e., window size), and multiplying the result by 100 to provide the percentage of sequence homology. Alignment to determine the percentage of sequence homology can be performed in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve maximal alignment within the full-length sequences being compared or within the target sequence region. The homology can also be determined by the following methods: FASTA and BLAST. The FASTA algorithm is described, for example, in W.R. Pearson and D.J. Lipman's "Improved Tool for Biological Sequence Comparison", Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D, J. Lipman and W.R.Pearson's "Fast and Sensitive Protein Similarity Search", Science, 227:1435-1441, 1989, and a description of the BLAST algorithm is given in S. Altschul, W. Gish, W. Miller, EW Myers and D. Lipman, "A Basic Local Alignment Search Tool", Journal of Molecular Biology, 215: 403-410, 1990.

[0091] The mRNA of a gene encoding the PIBF1 protein or a fragment thereof may include, but is not limited to, the base sequence disclosed in NCBI Reference Sequence: NM_006346.4 or a portion thereof.

[0092] Preferably, it may include or consist of a base sequence encoding the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, but may include without limitation a base sequence that exhibits substantially the same or corresponding efficacy as the base sequence or consists of the base sequence. Specifically, it may include a base sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more with the base sequence encoding the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. For example, it includes base sequences having sequence homology of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. The "% sequence homology" for a base sequence is determined by comparing a comparison region with two optimally aligned sequences, wherein a portion of the base sequence in the comparison region may include additions or deletions (i.e., gaps) compared to a reference sequence for the optimal alignment of the two sequences (which does not include additions or deletions).

[0093] The present invention also encompasses polynucleotides and fragments thereof having a base sequence substantially identical to the above base sequence. A polynucleotide having a substantially identical base sequence refers to a gene encoding a recombinant protein having the same function as that used in the present invention, regardless of sequence homology. A fragment of the polynucleotide also refers to a polynucleotide encoding a recombinant protein having the same function as that used in the present invention, regardless of the length of the fragment.

[0094] In the present invention, the pharmaceutical composition can increase PIBF1 protein having a molecular weight of 25 to 90 kDa in the mother.

[0095] At this time, if the PIBF1 protein is a secretory protein, the secretory PIBF1 protein may have various molecular weights, for example, 25 to 45 kDa, preferably 30 to 40 kDa, and more preferably 35 kDa, but is not limited thereto. Specifically, the secretory PIBF1 protein may include or consist of the amino acid sequence of SEQ ID NO: 3.

[0096] The composition of the present invention may comprise a mixture of the PIBF1 protein and a fragment thereof, or may comprise a vector comprising a gene encoding the PIBF1 protein and a vector comprising a gene encoding a fragment of the PIBF1 protein.

[0097] The PIBF1 protein of the present invention or a fragment thereof; or a vector comprising a gene encoding the PIBF1 protein or a fragment thereof, can promote blood vessel formation in the placenta by activating a signaling mechanism related to migration and angiogenesis of embryonic-derived vascular endothelial cells and pericytes, and can promote cardiovascular development of the fetus along the formed placental circulatory system.

[0098] Therefore, the pharmaceutical composition according to the present invention can be effectively used for the prevention or treatment of pregnancy complications related to placental or fetal cardiovascular abnormalities by exhibiting any one or more effects selected from the group consisting of the following a) to d): a) formation of blood vessel-like structures in the embryo; b) angiogenesis in the embryo; c) differentiation and maturation of embryonic cardiomyocytes; and d) formation of the maternal-fetal interface.

[0099] In the present invention, pregnancy complications related to placental or fetal cardiovascular abnormalities may include recurrent miscarriage, preeclampsia, or fetal heart defects.

[0100] In this specification, the term "habitual miscarriage" means two to three or more repeated miscarriages before the fifth month (20 weeks) of pregnancy, and preferably includes, but is not limited to, miscarriages caused by placental or fetal cardiovascular abnormalities.

[0101] In this specification, the above "preeclampsia (PE)" means that hypertension (140 / 90 mmHg or higher) and proteinuria (daily urine output 0.3 g or higher and / or dipstick testing 1+ or higher) occur after the 20th week of pregnancy. Depending on the time of onset and delivery, preeclampsia can be classified into early-onset preeclampsia (or early-onset preeclampsia, EP), which develops early and results in delivery before the 34th week, and late-onset preeclampsia (or late-onset preeclampsia, LP), which develops in the middle or late gestation and results in delivery after the 34th week, for example, between the 37th and 41st weeks.

[0102] As used herein, the term "prevention" refers to any action that inhibits or delays the onset of a disease or condition. In the present invention, it refers to delaying or inhibiting the onset of pregnancy complications related to placental or fetal cardiovascular abnormalities.

[0103] As used herein, the term "improvement" refers to any action that improves or beneficially alters the symptoms of a disease or condition. In the present invention, it refers to improving or beneficially altering the symptoms of pregnancy complications associated with placental or fetal cardiovascular abnormalities.

[0104] In this specification, the term "treatment" means any act of delaying, stopping or reversing the progression of a disease or condition, and in the present invention, means alleviating, alleviating or eliminating symptoms of pregnancy complications associated with placental or fetal cardiovascular abnormalities, or reversing them.

[0105] When the pharmaceutical composition of the present invention is used in the form of a vector, the vector may be of any type, for example, a plasmid vector or minicircle DNA.

[0106] However, typically, the vector of the present invention may be a viral vector. The viral vector may be based on, for example, herpes simplex virus, adenovirus or lentivirus. The viral vector may be an adeno-associated virus (AAV) vector or a derivative thereof. The viral vector derivative may be a chimeric, shuffled or capsid modified derivative. The viral vector may comprise an AAV genome derived from a naturally derived serotype, isolate or clade of AAV. The serotype may be, for example, AAV2, AAV5 or AAV8.

[0107] The efficacy of gene therapy generally relies on the appropriate and effective delivery of donated DNA. This process is typically mediated by viral vectors. Adeno-associated virus (AAV), a member of the parvovirus family, is commonly used in gene therapy. Wild-type AAV, containing viral genes, integrates its genomic material into chromosome 19 of the host cell. The AAV single-stranded DNA genome comprises two inverted terminal repeats (ITRs) containing structural (cap) and packaging (rep) genes, and two open reading frames.

[0108] The composition of the present invention can be administered to a patient in need thereof for the purpose of preventing or treating pregnancy complications associated with placental or fetal cardiovascular abnormalities.

[0109] In the present invention, the term "patient" means any single entity requiring treatment, including humans, cows, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, guinea pigs, rabbits, chickens, insects, etc. In addition, any subject participating in a clinical research trial that does not exhibit any clinical findings of a disease, a subject participating in an epidemiological study, or a subject used as a control group is included as a subject.

[0110] The composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration.

[0111] The pharmaceutical composition of the present invention may be in various oral or parenteral dosage forms. When formulating the pharmaceutical composition, it may be prepared using one or more buffers (e.g., saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrating agents, or surfactants, diluents, or excipients.

[0112] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing one or more compounds with at least one excipient, such as starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethyl-cellulose or gelatin. For example, tablets or sugar-coated tablets can be obtained by mixing an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and then processing the mixture into a granule mixture.

[0113] In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, or preservatives may be included. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as disintegrants in some cases, and anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives may be additionally included.

[0114] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, or suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.

[0115] The pharmaceutical composition of the present invention may be administered orally or parenterally, and may be formulated according to methods known in the art in the form of an external skin injection for parenteral administration; an injection for intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine, epidural, or intracerebrovascular injection; a transdermal injection; or a nasal inhalation agent.

[0116] In the case of the above injection, it must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for the injection include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal may be additionally included. In addition, the injection may in most cases additionally include isotonic agents such as sugars or sodium chloride.

[0117] Transdermal administration includes forms such as ointments, creams, lotions, gels, topical solutions, pastes, liniments, and aerosols. Transdermal administration, as described above, refers to topically administering a pharmaceutical composition to the skin, thereby delivering an effective amount of the active ingredient contained in the pharmaceutical composition into the skin.

[0118] For inhalation administration, the proteins used according to the present invention can conveniently be delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or another suitable gas. For pressurized aerosols, the dosage unit can be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in the well-known prescription book of pharmaceutical chemistry (Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour).

[0119] In addition, the pharmaceutical composition of the present invention can be prepared and administered in any formulation known to be suitable for administering protein drugs.

[0120] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. That is, the total effective amount of the composition of the present invention may be administered to a patient as a single dose, or may be administered as multiple doses in a fractionated treatment protocol for long-term administration. It is important to consider all of the above factors and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0121] The dosage of the pharmaceutical composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. The daily dosage may be administered in one to several divided doses, such that, when parenterally administered, the PIBF1 protein or its fragment is preferably administered in an amount of 0.001 to 50 mg, more preferably 0.01 to 30 mg per kg of body weight per day, and, when orally administered, the PIBF1 protein or its fragment is preferably administered in an amount of 0.001 to 100 mg, more preferably 0.01 to 10 mg per kg of body weight per day, based on the present invention. However, since the dosage may increase or decrease depending on the administration route, severity of obesity, sex, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0122] The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

[0123] A second aspect of the present invention relates to a method for preventing or treating pregnancy complications associated with placental or fetal cardiovascular abnormalities, comprising administering to a subject in need thereof a composition comprising (i) a PIBF1 (Progesterone immunomodulatory binding factor 1) protein or a fragment thereof; or (ii) a vector comprising a gene encoding a PIBF1 protein or a fragment thereof.

[0124] In the method of the present invention, the "subject" may be used with the same meaning as the "patient" described above. More specifically, it may be a mammal, including a human, at risk of developing pregnancy complications related to placental or fetal cardiovascular abnormalities, or a mammal, including a human, that has developed pregnancy complications related to placental or fetal cardiovascular abnormalities, wherein the mammal includes, but is not limited to, a human, a cow, a horse, a sheep, a pig, a goat, a camel, an antelope, a dog, a cat, a guinea pig, a rabbit, a mouse, a rat, etc.

[0125] In the method of the present invention, the description of the composition, effect, administration route, number of administrations, dosage, etc. of the composition administered to the subject is the same as described above, and therefore, description thereof is omitted.

[0126] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0127] [Example 1]

[0128] Cell culture and establishment of PIBF1 KO cell lines

[0129] 1-1. Cell culture

[0130] 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% fetal bovine serum (FBS) (16000-044, Gibco). Human ultraviolet endothelium-derived cells (HUVECs), isolated from donated human umbilical cords using collagen I, were cultured in Medium 200 (M200500, Gibco) supplemented with large vessel endothelial growth factor (LVES; A1460801, Gibco). Human ultraviolet endothelium-derived cells (HUAECs) (8010, ScienCell Research Laboratories) were cultured in Endothelial Cell Growth Medium (1001, ScienCell Research Laboratories). Human placental pericytes (Pl-PC; C-12980, PromoCell) were cultured in Pericyte Growth Medium 2 (C-28041, PromoCell). BeWo, HUVEC, HUEAC, and Pl-PC culture media contained 1% antibiotic-antimycotic (penicillin 100 U / ml, streptomycin 100 μg / ml, amphotericin B 25 μg / ml; 15240-096, Gibco).

[0131] Human trophoblast stem (hTS) cells (RCB4936) were provided by Riken BRC. The cells were cultured and maintained as described in the literature [H. Okae et al., Derivation of Human Trophoblast Stem Cells. Cell Stem Cell 22, 50-63 e56 (2018)]. Briefly, hTS cells were seeded on 5 μg / ml collagen IV (354233, Corning)-coated dishes and cultured in TS medium [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 Cells were cultured in DMEM / F12 (Gibco) supplemented with SB431542 (031-24291, Wako), 0.8 mM valproic acid (227-01071, Wako), and 5 μM Y27632 (036-24023, Wako), and the culture medium was changed every 2 days. All cells were cultured in a humidified atmosphere with 5% CO2 at 37°C.

[0132] 1-2. Establishment of PIBF1 KO cell line

[0133] To target the PIBF1 gene in human cell lines, a lentiviral vector expressing CRISPR / AsCpf1 (Acidaminococcus sp. BV3L6 Cpf1), pY108 (Addgene plasmid #84739; http: / n2t.net / addgene:84739; RRID:Addgene_84739) was used. CRISPR RNA specific for the human PIBF1 gene was designed using Benchling (https: / www.benchling.com), and the following oligomers were purchased from Macrogen, Inc.

[0134] Oligomer sequence SEQ ID NO. PIBF1 exon 2-specific crRNA 5′-agatGAAACAACAGTTCCTACGGA-3′45′-aaaaTCCGTAGGAACTGTTGTTTC-3′5 PIBF1 exon 4-specific crRNA 5′-agatTTGAGACAGAAACAACTAGA-3′65′-aaaaTCTAGTTGTTTCTGTCTCAA-3′7

[0135] Each oligomer pair was annealed and cloned into pY108 using BsmBI restriction enzyme from New England BioLabs, Inc. (NEB).

[0136] 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, transduced and selected cells were dissociated and seeded into 100 mm dishes at the lowest cell density that allowed single-cell growth. Cells were then grown until visible colonies were formed, and single-cell colonies (n = 60 for each target exon) were seeded into 96-well plates and then selected.

[0137] After examining PIBF1 expression in BeWo and hTSC colonies using Western blotting, the colonies were expanded to larger culture plates. For subsequent sequencing of suspected PIBF1 KO colonies, the mutant PIBF1 locus was amplified by PCR using the following primers.

[0138] Primer sequence size sequence number hPIBF1 exon 25′-cctccccgcaggaactg-3′580 bp 85′-tgccgtttgaagacaataaagca-3′9 hPIBF1 exon 45′-tactgtcaggtttctgatttcctaa -3′443b p 105′-aacaccattcaccatccatagc-3′11

[0139] PCR products from colonies were cloned using the T-Blunt PCR Cloning Kit (SOT02-K020, SolGent). Mutations were confirmed by Sanger sequencing of 20–25 clones from each colony (Bionics, Seoul, Korea). BeWo and hTS colonies harboring PIBF1 KO, in which the absence of PIBF1 expression was confirmed by Western blotting, were expanded to larger culture plates. PIBF1 KO TS cells were used at passages 6–8.

[0140] [Example 2]

[0141] recombinant PIBF1

[0142] The full-length recombinant protein of PIBF1 (rfPIBF; H00010464-P01) and the C-terminal fragment (rcPIBF; H00010464-Q01) were purchased from Abnova. In addition, the human Fc fusion form of PIBF1 (1–298 amino acids, rsPIBF), expressed and purified from CHO cells, was purchased from Ybiologics (Daejeon, South Korea). For production of raPIBF, the N-terminal cDNA of PIBF1 (1–184 amino acids) was synthesized by Bioneer (Daejeon, South Korea) and cloned into the Escherichia coli expression vector pET28.1a. This recombinant DNA vector was sequenced for deployment using the T7 promoter primer and transferred into the BL21(DE2) E. coli expression system. Two liters of LB medium were used to culture E. coli, and recombinant protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside at 0.D600 = 0.6 at 37°C and 150 rpm shaking conditions. The cell pellet was harvested after 12 h of incubation at 20°C using 3,000 rpm centrifugation. The pellet was lysed using 50 mM Tris-HCl buffer (pH 8.5) with 150 mM NaCl and 1X protease inhibitor cocktail, and purified using ion exchange chromatography using Q Sepharose Fast Flow (Cytiva, USA) and sequential size exclusion chromatography using Superdex 200 (Cytiva, USA) and 1X PBS buffer. The purity of the recombinant protein was confirmed using SDS-PAGE and Coomassie staining, and the protein solution was stored at -20°C until use.

[0143] [Example 3]

[0144] BeWo experiment

[0145] 3-1. Quantification of cell-to-cell fusion and fusion index

[0146] After 24 h of seeding in 20-mm confocal dishes, BeWo cells were cultured with 20 μM forskolin (3828, Cell Signaling Technology) to induce cell fusion, and the forskolin-containing medium was replaced every 24 h. After 48 h of treatment, the cultured cells were stained with Hoechst 33342 (H1399, Invitrogen) and 2 μM Di-8-ANEPPS (D3167, Invitrogen) for 30 min, washed twice in dye-free medium, and imaged with a Zeiss LSM 880 Fast Airyscan confocal system (Carl Zeiss, Jena, Germany). The number of nuclei in non-fused or fused cells was counted in fluorescence images of six randomly selected fields from at least three independent experiments using ImageJ (NIH). Cells with two or more nuclei were identified as fused cells, and the fusion index for each group was calculated as follows: (number of fused nuclei) / (total number of nuclei). Fusion index data were expressed relative to the forskolin-untreated WT control.

[0147] 3-2. Restoration of PIBF1 in PIBF1 KO BeWo cells

[0148] To reintroduce PIBF1, a fragment of the cDNA region encoding amino acids 1–757 (fPIBF) or 1–224 + 684–757 (sPIBF) of human PIBF1 (NP_006337.2) was synthesized and inserted into the pCAGGS expression vector (H. Niwa, K. Yamamura, J. Miyazaki, Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991).) by Cosmogenetech (Seoul, Korea). After confirming the correct insertion by restriction digestion analysis and sequencing, the resulting construct DNA (1 μg / ml) expressing fPIBF or sPIBF or an empty vector control was transfected into PIBF1 KO BeWo cells using Lipofectamine LTX with PLUS reagent (15338100, Invitrogen) according to the manufacturer's instructions. This was further confirmed by Western blotting of PIBF1 in lysates of transfected cells.

[0149] 3-3. Recombinant PIBF1 treatment of PIBF1 KO BeWo cells

[0150] PIBF1 KO BeWo cells were cultured with rsPIBF (400 ng / ml) or rfPIBF (400 ng / ml) simultaneously with 20 μM forskolin. After 48 h, fusion indices were obtained as described above.

[0151] 3-4. Cytotrophoblast-endothelial transwell co-culture

[0152] The cytotrophoblast-endothelial co-culture system was slightly modified from the method described in the literature [Y. Wang et al., Placental trophoblast-derived factors diminish endothelial barrier function. J Clin Endocrinol Metab 89, 2421-2428 (2004)]. Briefly, hTS cells were plated on 24-well chamber bottoms coated with 2.5 μg / ml collagen IV and cultured in TS or SynT (2D) medium for 5 days, with fresh medium replaced on day 3. BeWo cells in the same 24-well chamber bottoms were transfected with fPIBF expression and / or sPIBF expression constructs or a control vector for 24 h and then stimulated with forskolin for 48 h.

[0153] After 5 days of differentiation or 48 h of forskolin stimulation, the culture medium was replaced with a 1:1 mixture of Medium 200 / LVES and modified SynT(2D) (SynT(2D) medium without EGF, Y27632 and forskolin, which may affect endothelial invasion capacity) in HUVEC-SynT, or with Medium 200 / LVES and DMEM / F12 / 10% FBS in HUVEC-BeWo.

[0154] Simultaneously, HUVECs were placed on 8-μm pore-size Transwell inserts (CLS3422, Corning) coated with Matrigel (354234, Corning) for 24 h, and then the endothelial inserts were transferred to differentiated SynT or forskolin-stimulated BeWo cultures. After 24 h of co-culture, HUVECs that had invaded the lower surface of the membrane were stained with crystal violet. The number of invaded cells was counted in six randomly selected fields from each sample using ImageJ software (NIH).

[0155] [Example 4]

[0156] In vitro angiogenesis assay

[0157] For migration assays, when HUVECs, HUAECs, and Pl-PCs reached 95% confluence, three scratches were made across the plate using a 200-μl pipette tip. After washing, cells were incubated without, or with, rsPIBF, rfPIBF, raPIBF, and rcPIBF at doses 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 positive controls for ECs and Pl-PCs, respectively. Cell migration images at 0 h and endpoint were analyzed by the migrated area using ImageJ, and cell migration rate was calculated as follows: [(area at 0 h) - (area at endpoint)] / (area at 0 h) Υ 100(%).

[0158] For the tube formation assay, when HUVECs and HUAECs reached 80–90% confluence, ECs were labeled with 10 μM CellTracker Green CMFDA Dye (C7025, Invitrogen) for 30 min. Cells were then seeded onto Matrigel-coated plates and cultured for 9 h without, or with, recombinant PIBF1 isoforms at doses of 50, 100, 200, and 400 ng / ml. VEGF (50 ng / ml) was used as a positive control. Images were captured using an EVOS M5000 imaging system (Invitrogen), and capillary-like structures formed by HUVECs and HUAECs were analyzed using ImageJ software (NIH).

[0159] To investigate the changes in angiogenesis-related signaling cascades induced by rsPIBF in HUVECs, HUVECs were incubated with VEGF (20 ng / ml), rsPIBF (200 ng / ml), or vehicle for 30 min, and cells were processed for Western blot analysis.

[0160] [Example 5]

[0161] hTS cell experiments

[0162] 5-1. Differentiation of hTS cells

[0163] Differentiation of TS cells into SynT or EVT was induced as described previously [H. Okae et al., Derivation of Human Trophoblast Stem Cells. Cell Stem Cell 22, 50-63 e56 (2018).]. Briefly, for SynT differentiation in 2D [SynT(2D)] culture, TS cells were seeded on collagen IV-coated dishes 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)]. Cells were replaced with fresh medium on day 3 and analyzed on day 6. For SynT differentiation in 3D [SynT(3D)] culture, TS cells were cultured in SynT(3D) medium [SynT(2D) medium supplemented with 50 ng / ml EGF]. Fresh medium was added to the cells on day 3, and the cells were analyzed on day 6.

[0164] For EVT differentiation, TS cells were seeded onto collagen IV-coated dishes 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%. On day 3, cells were replaced with EVT medium lacking NRG1, and Matrigel was added at a final concentration of 0.5%. On day 6, when cells reached ~80% confluence, cells were subcultured and cultured for an additional 2 days in EVT medium lacking NRG1 and KSR containing 0.5% Matrigel, and analyzed on day 8.

[0165] 5-2. Cell viability assay

[0166] The viability of TSCs was measured using the Ez-cytox cell viability assay (EZ-1000, DoGenBio). Briefly, TSCs were grown in 96-well plates, and an aqueous tetrazolium solution was added at 24 and 72 h of culture, followed by incubation at 37°C for 1 h. The optical density at 450 nm was measured using a microplate reader (SpectraMax 190, Molecular Devices), and cell viability was calculated according to the manufacturer's instructions.

[0167] 5-3. Engraftment of TS cells

[0168] TSC grown to ~80% confluence was separated using TrypLE (12604-021, Gibco) and 10 7TSCs from dogs were resuspended in 200 μl of a 1:2 mixture of DMEM / F12 containing Matrigel, 0.3% BSA, and 1% ITS-X. The suspension was injected subcutaneously into 6- to 8-week-old SID male mice. Lesions, urine, and serum were collected 7 days after injection. Lesions were photographed, fixed overnight in 4% paraformaldehyde (PFA) at 4°C, embedded in paraffin, and stored until the next experiment. Urine and serum samples were stored at -80°C until use.

[0169] [Example 6]

[0170] MRM analysis

[0171] 6-1. Establishment of an MRM method for PIBF1 quantification

[0172] A representative tryptic peptide for PIBF1 was determined to be ELLHNIQLLK (molecular weight: 1219.72 Da, N-terminus of PIBF1 (55-64 aa)) using the PeptideAltas (http: / www.peptideatlas.org / ) archive. A stable isotope-labeled internal standard (SIS) peptide for ELLHNIQLLK (molecular weight: 1227.72 Da) was synthesized by incorporating heavy isotope-labeled lysine at the C-terminus (L-lysine-13C6,15N2) with 98% purity (Peptron, South Korea) for performing multiple reaction monitoring (MRM) analysis. A list of possible b- and y-series product ions for the doubly charged peptide ELLHNIQLLK was theoretically generated using Skyline software (64-bit, version 19.1.0.193, University of Washington, MacCoss Lab) with an m / z range of 300–1,400. These theoretical transitions were optimized for triple quadrupole mass spectrometry (MS) using the default instrument parameters of an HPLC system (1290 Infinity, Agilent Technologies) and a Jet Stream ESI Source (6495 Agilent, Agilent Technologies). MS source settings were as follows: gas temperature 250 °C, gas flow 15 L / min, nebulizer 30 psi, sheath gas temperature 350 °C, sheath gas flow 10 L / min, capillary 3500 V for cation funneling, nozzle voltage 300 V, iFunnel parameters for high pressure RF 90 V, and low pressure RF 60 V. The mixture of light peptides and SIS peptides was separated on a reversed-phase analytical column (Zorbax Eclipse Plus C18 rapid resolution HD, 2.1 Y 100 mm, 1.The peptide samples were directly injected into an 8-micron column (Agilent Technologies). The peptide samples were separated and eluted at 0.3 ml / min with a linear gradient of mobile phase B from 5% to 30% B over 19 min (mobile phase A: water / 0.1% FA, mobile phase B: acetonitrile / 0.1% FA). The gradient was 70% B over 3 min and then 90% B over 3 min. The column was equilibrated with 5% B for 10 min for the next run. The top three most intense transitions for the light peptide (Q1: 407.2550 m / z (triple charge), Q3: 501.3395 (y4, singly charged), 433.2663 (y7, doubly charged), and 489.8084 (y8, doubly charged)) and the SIS peptide (Q1: 410.2550 m / z (triple charge), 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 acquisition was performed using a 3-min detection window with a 100-msec cycle time. MRM was performed using six sample points with concentrations ranging from 0 to 50 fmol along with a blank matrix to determine the response curve. The limit of detection (LoD) using the average peak area of ​​three blank runs + three standard deviations and the limit of quantitation (LoQ) using the average peak area of ​​three blank runs + 10 standard deviations were determined using the inverse response curve.

[0173] 6-2. Preparation of cell lysate and conditioned medium for MRM analysis

[0174] hTSCs, SynTs, and EVTs were cultured in BSA-free medium 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 boiled at 80°C for 10 min. The supernatants were harvested by centrifugation at 18,000 × g for 20 min. 1 ml of conditioned medium (CM) from each cell was lyophilized to reduce the volume. Each dried pellet of lyophilized CM was suspended in 300 μl of lysis buffer. The protein concentrations of the cell lysates and each supernatant of CM were measured using a BCA protein quantification kit (Pierce™ BCA Protein Assay Kit; 23225, Thermo Fisher Scientific). A 100 μg aliquot of protein was digested using a suspension trapping (STrap) mini column (CO2-mini-80, ProtiFi) with a Lys-C / trypsin mixture (V5071, Promega) according to the manufacturer's protocol and incubated at 37 °C for 16 h [A. Zougman, PJ Selby, RE Banks, Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis. Proteomics 14, 1006-1000 (2014)]. The eluted peptide mixture was lyophilized using a cold trap and stored at -80 °C until use.

[0175] 6-3. Quantification of PIBF1 using MRM

[0176] The peptide mixture from cell lysate and CM was dried and reconstituted to 1 μg / μl with 0.1% FA. 50 fmol of SIS peptide was spiked in, and targeted MRM analysis was performed using an established MRM method. Quantitative information for each transition was extracted using Skyline, and the concentration of PIBF1 was calculated using the peak area of ​​50 fmol of SIS peptide.

[0177] [Example 7]

[0178] hCG measurement

[0179] The presence of hCG in mouse urine samples was qualitatively detected using a commercially available hCG pregnancy test kit. Additionally, hCG levels were quantitatively measured in mouse serum or conditioned medium samples from BeWo, SynT(2D), or SynT(3D) cultures using an ELISA kit (KA4005, Abnova) according to the manufacturer's instructions.

[0180] [Example 8]

[0181] Human heart organoid (hHO) experiment

[0182] 8-1. Production of hHO and processing of rsPIBF1

[0183] iPSC-derived hHO were generated as described previously [SG Lee et al., Generation of human iPSC-derived heart organoids structurally and functionally similar to the heart. Biomaterials 290, 121860 (2022)]. Briefly, human iPSCs were cultured in mTeSR-TM1 medium (StemCell Technologies) containing 10 μM Y-27632 (1254, Tocris Bioscience) and 10% Matrigel (345277, Corning) in a shaking incubator for 3 days to form embryoid bodies (EBs). EBs were then cultured in RPMI1640 containing B27 supplement and 6 μM CHIR99021 (4423, Tocris Bioscience) without insulin for 2 days, followed by culture in RPMI1640 containing B27 supplement and 2 μM C-59 (S7037, Selleckchem) without insulin for 2 days. On day 7, cells were cultured in Advanced MEM (AD-MEM) containing 1% GlutaMAX and 1% antibiotic-antimycotic (hHO medium).

[0184] To determine the effect of rsPIBF on the development of hHO, cells were cultured in hHO medium (control group) or hHO medium containing 200 ng / ml sPIBF (sPIBF group) on day 8 (1 day after hH2O induction), and the medium was changed every other day until days 19-21.

[0185] 8-2. Germination assay

[0186] For germination assays, 20-μl cold drops of Matrigel containing control or rsPIBF-treated hHOs were solidified at room temperature for 5 min, AD-MEM was added, and cultured for 4–6 days in a shaking incubator with media replacement every 2 days.

[0187] 8-3. Multi-electrode array

[0188] For the multielectrode array (MEA), control or sPIBF-treated hHO were loaded onto the MEA device (Maestro Edge version, Axion BioSystems, GA, USA), and then the raw waveforms representing the field potential (FP) of each group were recorded for 3 min in the AxIS software. The three main indices of the recorded FP (spike amplitude, beat duration, and field potential duration) were analyzed using the Cardiac Analysis Tool and the AxIS Metric Plotting Tool (Axion BioSystems) [SG Lee et al., Generation of human iPSCs-derived heart organoids structurally and functionally similar to the heart. Biomaterials 290, 121860 (2022); SG Lee et al., Development and validation of dual-cardiotoxicity evaluation method based on analysis of field potential and contractile force of human iPSC-derived cardiomyocytes / multielectrode assay platform. Biochem Biophys Res Commun 555, 67-73 (2021)].

[0189] 8-4. Histology

[0190] 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 at 9 μm thickness, and stored at −80 °C until further procedures.

[0191] 8-5. RNA seq and Gene Ontology (GO) Analysis

[0192] Total RNA was isolated from EB (day 3) and control or sPIBF-treated hHO (day 20) using the Trizol reagent kit (15596026, Invitrogen). Next-generation sequencing (NGS) was performed at LifeGenomics (Suwon, South Korea). Differentially expressed gene analysis was performed using DESeq2 version 1.36.0 [M.I. Love, W. Huber, S. Anders, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550 (2014)]. Genes up- or down-regulated by ≥1.5-fold with an adjusted p-value (Benjamini-Hochberg correction) were considered significant. GO analysis was performed using clusterProfiler version 4.4.4 [T. Wu et al., clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innovation (Camb)2, 100141 (2021)] was used. Statistical analysis and visualization were performed in R version 4.2.2 and R Studio Build 353 environment.

[0193] [Example 9]

[0194] hiPSC-derived cardiomyocyte experiments

[0195] Two-dimensional culture of hiPSC-derived cardiomyocytes was performed as described in the literature [Lee, SG et al. Development and validation of dual-cardiotoxicity evaluation method based on analysis of field potential and contractile force of human iPSC-derived cardiomyocytes / multielectrode assay platform. Biochem. Biophys. Res. Commun. 555, 67-73 (2021).]. Briefly, hiPSCs were seeded on Matrigel-coated dishes containing mTeSRTM1 (StemCell Technologies) medium containing 10 μM Y27632. The mTeSRTM1 medium was replaced daily until hiPSC confluency reached 90%. First, to differentiate into mesodermal lineage, hiPSCs were cultured for 1 day in RPMI1640 medium containing insulin-free B27 supplement (B27 medium) supplemented with 6 μM CHIR99021 (day 0). The following day (day 1), the medium was replaced with B27 medium containing 3 μM CHIR99021 and cultured for 1 day. Next, to induce cardiac progenitors, the B27 medium containing 2 μM C59 (Sellckem), a Wnt signaling inhibitor, was replaced on day 2. The following day (day 3), the medium was replaced with B27 medium containing 2 μM C59 and cultured for 1 day. From day 4 to day 10, Advanced MEM (Gibco) medium (AD-MEM) containing 1% penicillin / streptomycin and 1% GlutaMAX (Gibco) was replaced every 2 days until the differentiated cells started beating.

[0196] To investigate the effect of rsPIBF on cardiomyocyte differentiation, sPIBF (200 ng / ml) was treated for 6 days (days 4–10). After confirming beating, cells were detached using TrypLE (Gibco) and reseeded onto Matrigel-coated dishes. On day 12, hiPSC-CMs were fixed in 4% PFA for 20 min and stained with antibodies against cTnT (Abcam, ab45932, rabbit 1 / 200) and α-actinin (Sigma, A7811, mouse 1 / 200).

[0197] [Example 10]

[0198] Mouse preparation and generation of Pibf1 cKO mice

[0199] 10-1. Preparing the mouse

[0200] All animal experiments were conducted in accordance with the guidelines of the Ministry of Food and Drug Safety. Experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Asan Institute for Life Sciences (Approval No.: 2019-12-217). All mice were maintained in a specific pathogen-free facility at the Asan Medical Center Animal Research Institute (AMCLAR), with free access to standard laboratory food and water ad libitum, and a 12-h dark / light cycle.

[0201] The protocols for conventional knockout of Pibf1 (Pibf1em1Hwl / J) and Trp53 (Trp53em1Baek / J) in mice were described in [Y.H. Sung et al., Knockout mice created by TALEN-mediated gene targeting. Nat Biotechnol 31, 23-24 (2013)] and [Y. Kim et al., Generation of knockout mice by Cpf1-mediated gene targeting. Nat Biotechnol 34, 808-810 (2016)], respectively. ICR and severe immunodeficiency (SID, NOD / J-Prkdcem1BaekIl2rgem1Baek / J) mice were purchased from OrientBio (Gyeonggi-do, South Korea) and GEM Biosciences (Cheongju, South Korea), respectively. Meox2-cre (B6.129S4-Meox2tm1(cre)Sor / J, 003755), Vav1-iCre (B6N.Cg-Commd10Tg(Vav1-icre)A2Kio / J, 018968), Cdh5-cre (B6;129-Tg(Cdh5-cre)1Spe / J, 017968), and R26R reporter (B6.129S4-Gt(ROSA)26Sortm1Sor / J, 003474) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Tie2-cre (B6.Cg-Tg(Tek-cre)1Ywa / J, JAX 008863) and Il17a knockout (B6.129P2-Il17atm1Yiw) mice were provided by Dr. Chang-Hoon Ha and Dr. Kyung-Jin Lee, respectively, at Asan Medical Center (Seoul, Korea).

[0202] To obtain tissue samples from conceptuses of different gestational ages, 7-week-old female mice were mated with male mice in the evening at a 1:1 ratio. The day of vaginal plug formation was considered embryonic day (E) 0.5. Pregnant mice were then randomly assigned to different experimental groups and euthanized at designated time points. Tissue samples from different parts of the conceptus were dissected under a stereomicroscope (SMZ1000; Nikon, Tokyo, Japan). Tissues were either rapidly frozen in liquid nitrogen and stored at -80°C for further analysis or fixed in 4% paraformaldehyde (PFA).

[0203] 10-2. Generation of Pibf1 cKO mice

[0204] To generate single guide RNAs (sgRNAs) for use in floxed PIBF1 mice, the following oligomers were purchased from Macrogen, Inc.

[0205] Oligomer sequence Sequence number Pibf1 intron 1-specific sgRNA 5′-taggGCTATTTCAGTTTCTCTAAG-3′125′-aaacCTTAGAGAAACTGAAATAGC-3′13 Pibf1 intron 17-specific sgRNA 5′-taggACTCTATTTCTGCTTCCCGA-3′145′-aaacTCGGGAAGCAGAAATAGAGT-3′15

[0206] Each oligomer pair was annealed and cloned into the pUC57-sgRNA vector (Addgene plasmid # 51132; http: / n2t.net / addgene:51132; RRID:Addgene_51132) provided by Xingxu Huang using BsaI restriction enzyme from New England BioLabs, Inc. (NEB), and the cloned vector was used as a template for sgRNA in vitro transcription.

[0207] The following single-stranded deoxynucleotide donor DNA was purchased from Integrated DNA Technologies, Inc.

[0208] Single-stranded deoxynucleotide donor DNA sequence Sequence number Single-stranded deoxynucleotide donor DNA for insertion of loxP into Pibf1 intron 1 5′-tgtaactcccttgtttaaatacgacacgatttagtaaaatagttatagaactagctatttcagtttctctATAACTTCGTATAATGTATGCTATACGAAGTTATaagaggaacgctaaagaaggaggtctgtgtggtgggggggaacaggaagagcttcagtacaatccgcctc-3′ Single-stranded deoxynucleotide donor DNA for insertion of loxP into Pibf1 intron 17 DNA5′-caattctcctccttcaatatattgtgaatttctgttgaactgtgaaacagataactctatttctgcttccATAACTTCGTATAATGTATGCTATACGAAGTTATcgagggctgaaattacaaccatgaaccaccatacccaggtgaaactatttttttttagagatataacata-3′17

[0209] Microinjection of embryos and transfer to surrogate mothers were performed as described previously [YH Sung et al., Knockout mice created by TALEN-mediated gene targeting. Nat Biotechnol 31, 23-24 (2013).]. Briefly, after superovulation and mating, embryos were collected from the oviducts of 3-4-week-old female mice with a C57BL / 6J (B6J) background, and sgRNA, Cas9 mRNA, and ssODN were co-injected into the cytoplasm of pronuclear stage embryos.

[0210] After incubation at 37°C for 24 hours, two-cell embryos were transferred to pseudopregnant ICR surrogate mothers. Genomic DNA samples obtained from the tails of embryos or 3-week-old pups were genotyped using the primers listed in Table 5.

[0211] List of primers used for genotyping Primer sequence (5′→3′) Sequence number Forward Reverse PCR genotyping Pibf 13′ flox AGTGTAGTGTCTTCAAGATTCATTCCCTCCCAGCTCCCTGTAGT 18 (forward) / 19 (reverse) Meox2-cre 1' GGGACCACCTTCTTTTGGCTTC 2' AAGATGTGGAGAGTTCGGGGTAG 3' CCAGATCCTCCTCAGAAATCAGC 202122 Pibf 15′flox & excised 1' CTTGGTAACTCACGCAAGGAT 2' CCTTGGCAATTTTGCGAGACA 3' CCTCCCAGCTCCCTGTAGT 232425

[0212] [Example 11]

[0213] Outgrowth culture

[0214] After Pibf1 heterozygous intercrossing, the collected blastocysts were individually cultured in 0.1% gelatin-coated multiwell dishes at 37°C and 5% CO2 in KnockOut DMEM (10829018) supplemented with 20% KnockOut Serum Replacement (10828010), 2 mM GlutaMAX (35050061), 0.1 mM nonessential 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). On the 7th day of culture, the products were photographed and collected, and then genotyped by PCR.

[0215] [Example 12]

[0216] Morphological analysis of embryos and hearts

[0217] Images of embryonic products and dissected parts of the embryos, including the yolk sac, embryo, and heart, were taken using a ProgRes C5 camera and Capture Pro software (Jenoptik Optical Systems, Jena, Germany). Quantitative measurements of the inner cell mass area and ectodermal area of ​​the products, the crown-rump length (CRL) of whole-mount embryos, and the width and height of the ventricles of whole-mount hearts were performed using ImageJ software (NIH).

[0218] [Example 13]

[0219] histology

[0220] Tissues were fixed in 4% PFA, embedded in paraffin, and sectioned (4–5 μm thick). The sections were dewaxed, rehydrated, and stained with hematoxylin and eosin (H&E) or immunohistochemically stained.

[0221] 13-1. Morphological analysis of the placenta

[0222] H&E-stained placental sections were examined under a light microscope to examine the three major layers of the placenta (the basal layer of the decidua, the junctional layer, and the labyrinth) and the labyrinth vascular space. Fetal blood sinuses were distinguished from maternal blood sinuses by the presence of primitive nucleated red blood cells. The labyrinth sinuses were examined and segmented into three random, non-overlapping Y40 fields for each sample. The extent of each region was quantified using ImageJ software (NIH).

[0223] 13-2. Immunostaining

[0224] For immunohistochemistry, tissue sections were subjected to heat-induced antigen retrieval in citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0). The sections were then blocked with 5% normal goat serum, incubated with primary antibodies, washed, and then incubated with biotinylated goat anti-rabbit IgG (PK6101, Vector Labs) according to the manufacturer's instructions. The primary antibodies used for immunohistochemistry were as follows: anti-Ki67 (1 / 10,000; ab15580, Abcam), anti-laminin (1 / 750; L9393, Sigma-Aldrich), and anti-perforin (1 / 200; ab16074, Abcam). For perforin staining, peroxidase-conjugated donkey anti-rat IgG (712-035-150, Jackson ImmunoResearch Laboratories) was used as the secondary antibody. Signals were visualized using a liquid DAB+ substrate chromogen system (K3467, Dako). Labyrinthine Ki67-positive trophoblast cells or decidual perforin-positive cells were counted in three randomly selected Y40 fields from each sample.

[0225] For immunofluorescence staining, heat-induced antigen retrieval sections incubated with or without mouse Ig blocking reagent (PK2200, Vector Labs) were blocked with 5% normal goat serum, treated with primary antibodies, and then captured with Alexa Fluor (AF)-conjugated secondary antibodies. For in vitro samples, fixed cells or hHO cryosections were washed, blocked, and permeabilized with 0.1% Triton X-100 before treatment with primary and subsequent secondary antibodies. Sections were mounted with DAPI (D1306, Molecular Probes) and examined on a Zeiss LSM 880 confocal microscope system (Carl Zeiss, Jena, Germany). The areas stained with CD31, cTnT, or PDGFRβ were quantified using ImageJ software (NIH). Antibodies used for immunofluorescence staining are listed in Table 6.

[0226] Target Supplier Identification Host Dilution Immunofluorescence Staining β-hCGAbcamab53087Rabbit1 / 100HLA-GAbcamab7759Mouse1 / 100KRT7Abcamab181598Rabbit1 / 1000GATA3Cell Signaling Technology5852Rabbit1 / 200TEAD4Abcamab58310Mouse1 / 100TP63Cell Signaling Technology13109Rabbit1 / 100cTnTAbcamab45932Rabbit1 / 200CD31MilliporeCBL468Mouse1 / 200PDGFRβAbcamab32570Rabbit1 / 200MCT1SigmaAB1286-IChicken1 / 150MCT4Santa Cruzsc-376140Mouse1 / 150CD31Abcamab28364Rabbit1 / 200cTnTAbcamab209813Rabbit1 / 500ARL13BProteintech17711-1-APRabbit1 / 500Acetylated TubulinProteintech66200-1-IgMouse1 / 1000Anti-chicken IgY, AF488InvitrogenA11039Goat1 / 200Anti-mouse IgG, AF594InvitrogenA11005Goat1 / 200Anti-mouse IgG, AF488InvitrogenA11001Goat1 / 200Anti-rabbit IgG, AF488InvitrogenA11008Goat1 / 200Anti-rabbit IgG, AF555InvitrogenA21429Chlorine 1 / 200

[0227] 13-3. Alkaline phosphatase activity

[0228] Isolated placentas were embedded in Tissue-Tek OCT compound (25608-903, VWR), and 6-μm-thick cryosections were incubated in alkaline phosphatase (AP) 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) chromogenic substrate (S3731, Promega) according to the manufacturer's instructions. The sections were then counterstained with nuclear fast red.

[0229] 13-4. Transmission electron microscope

[0230] The isolated placentas were washed with fresh 0.1 M phosphate buffer (pH 7.4) and fixed with 2.5% glutaraldehyde in the same buffer at 4 °C for 4 h. The labyrinth area of ​​the placenta was measured at 1-mm 3 The tissues were cut into fragments and further fixed overnight at 4°C. After washing three times for 10 min each in fresh 0.1 M phosphate buffer (pH 7.4), the tissues were fixed in 1% OsO4 for 1 h at room temperature, washed three times, dehydrated with ethanol and propylene oxide, and embedded in Epon according to standard techniques. Subthin (500 nm) and ultrathin (60 nm) sections were cut from the blocks. The subthin sections were stained with toluidine blue and examined under a light microscope. The thickness of the blood septum was measured in five randomly selected Y100 fields of each sample. The ultrathin sections were contrasted with uranyl acetate and lead citrate and observed at 80 kV using a Hitachi H-7100 transmission electron microscope (Hitachi, Nackashi, Japan).

[0231] [Example 14]

[0232] Fatty acid measurement

[0233] Gas chromatography coupled with mass spectrometry (GC / MS)-based determination of placental and embryonic fatty acids (FAs) was performed as described previously [JG Lee et al., Lipid signatures reflect the function of the murine primary placentation dagger. Biol Reprod 106, 583-596 (2022)]. Briefly, embryonic or placental tissue samples were homogenized in chloroform / methanol (2 / 1 v / v) and subjected to liquid-liquid extraction. The lipid-containing organic phase was collected, dried in a vacuum centrifuge, and hydrolyzed. After the addition of hexane, the upper phase containing the extracted FAs was vacuum-dried and reacted with BCl3-MeOH (12% w / w) at 60 °C for 30 min. The hexane extraction was repeated, and after the addition of anhydrous sodium sulfate to the upper hexane phase, the supernatant samples were subjected to GC / MS analysis. Calibration curves were generated using commercially available FA methyl esters. All lipid standards were purchased from Avanti-Polar Lipids and Sigma.

[0234] FA methyl esters were analyzed using an Agilent 7890 / 5975 GCMSD system equipped with an HP-5 MS 30 m × 0.25 mm × 0.25 μm column (19091S-433, Agilent) using helium (99.99%) as the carrier gas. The GC-optimized temperature program was as follows: initial hold at 50 °C for 2 min; 10 °C / min to 120 °C; 10 °C / min to 250 °C; hold at 250 °C for 15 min; and a final hold at 300 °C for 5 min to purge the column. A 5-min solvent delay and scan mode were applied. Extracted ion chromatograms corresponding to specific FAs were used for quantification. The quantified data were expressed as relative amounts (%) to the wild-type group.

[0235] [Example 15]

[0236] Laser microdissection

[0237] Isolated placentas were embedded in OCT compound and cryosectioned onto polyethylene naphthalate (PEN) membrane-coated glass slides (LCM0522, Thermo Fisher Scientific). Placental compartments corresponding to the labyrinth, junction, and decidua were microdissected and collected using a Leica 6500 Laser Microdissection System (Leica, Germany).

[0238] [Example 16]

[0239] Quantitative real-time PCR

[0240] According to the manufacturer's instructions, total RNA was isolated from BeWo cells, TSCs, and whole tissues of the yolk sac or placenta using the Trizol reagent kit (15596026, Invitrogen), and from embryonic hearts or laser-microdissected placentas using the RNeasy Micro kit (74004, Qiagen). cDNA synthesized with the SuperScript III First-Strand Synthesis System (18080-051, Invitrogen) was used as a template for quantitative real-time PCR reactions using iQ SYBR Green Supermix (170-8882AP, Bio-Rad) and primer sets (Table 7) on a QuantStudio3 real-time PCR system (Applied Biosystems, CA, USA). Data were normalized to internal control genes such as RPS18, GAPDH, and mGapdh, and expressed relative to the expression levels of the wild-type or control.

[0241]

[0242] [Example 17]

[0243] In vitro fertilization and embryo transfer

[0244] Oocytes were collected from superovulated females treated with 5 IU pregnant mare serum gonadotropin (G4877, Sigma) and 5 IU human chorionic gonadotropin (CG10, Sigma) at 48-h intervals. In vitro fertilized two-cell embryos heterozygous for Pibf1 were transferred into pseudopregnant wild-type ICR or Il17a knockout female mice. Embryos from E10.5 to E11.0 were collected, photographed, and genotyped by PCR.

[0245] [Example 18]

[0246] Fluorescence-activated cell sorting (FACS)

[0247] For FACS analysis, the intermediate layer region covering the decidual basal region of embryos was dissected and sampled at E8.5 or E10.5, minced, and digested with 1 mg / ml collagenase type II (C6885, Sigma) and 1 U / ml DNase I (04536282001, Roche) at 37°C for 30 min. Red blood cells were removed with ACK lysis buffer (A1049201, Gibco) for 5 min at room temperature and washed twice in ice-cold D-PBS. Prepared single cells were Fc-blocked with anti-CD16 / CD32 (553142, BD Biosciences) and then stained with FITC-conjugated Dolichos Biflorus Agglutinin (DBA) (20 μg / ml; L32474, Invitrogen) and the following antibodies: anti-CD3e-PerCP-Cy5.5 (1 / 100; 551163, BD Biosciences) and anti-CD122-PE (1 / 100; 12-1221-82, eBioscience). All incubation steps for FACS analysis were performed in D-PBS containing 2% FBS. Stained samples were evaluated using a FACSCanto II system (BD Biosciences, CA, USA) and further analyzed using FlowJo software (Tree Star, OR, USA).

[0248] [Example 19]

[0249] Whole tissue X-gal staining

[0250] Isolated sections of the conceptus were fixed in 4% PFA at 4°C for 3 h, and whole embryos, whole hearts, or 1-mm-thick transverse sections of the placenta were incubated overnight at 4°C in a washing buffer (2 mM MgCl2, 0.02% IGEPAL, and 0.01% sodium deoxycholate in phosphate-buffered saline (PBS), pH 7.4). After brief washing three times with the washing buffer, the samples were incubated in the dark at 37°C overnight in a washing buffer containing 4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, and 1 mg / ml X-gal (B4252, Sigma), postfixed overnight in 4% PFA, and visualized under a stereomicroscope. For histological analysis, X-gal-stained tissues were paraffin-embedded, sectioned at 4 μm thickness, and counterstained with nuclear fast red.

[0251] [Example 20]

[0252] Hematopoietic experiments

[0253] 20-1. Colony forming unit assay

[0254] Dissected fetal sections of different gestational ages were mechanically minced and enzymatically digested by incubation in D-PBS containing 2% FBS and 0.1% collagenase type I (C0130, Sigma) at 37°C for 30 min. For the preparation of adult bone marrow samples, the femurs and tibias of 4-month-old male mice were dissected, and the proximal and distal epiphyses were cut off. The bone marrow was collected by washing with D-PBS containing 2% FBS and passed through a 70 μm cell strainer. The cells were washed, resuspended in Iscove's MDM containing 2% FBS (07700, Stem Cell Technologies), plated in Methocult methylcellulose medium (M3434, Stem Cell Technologies), and cultured at 37°C in an incubator with 5% CO2. Colonies of CFU-granulocytes, macrophages (CFU-GM), erythroid burst forming units (BFU-E), and CFU-granulocytes, erythrocytes, macrophages, megakaryocytes (CFU-GEMM) were scored after 10-12 days of culture.

[0255] 20-2. Complete blood count using white blood cell differentiation

[0256] Whole blood samples from 4-month-old male mice were collected from the inferior vena cava into EDTA-containing tubes, and hematological parameters were analyzed using an ADVIA 2120i automated hematology analyzer (Siemens, Munich, Germany).

[0257] [Example 21]

[0258] Whole tissue CD31 staining

[0259] Embryos or yolk sacs were fixed in 4% PFA for 2 h at 4 °C, briefly washed three times in PBTx (PBS containing 0.1% Tween 20 and 0.1% Triton X-100), and blocked in 5% goat serum. Samples were then incubated with anti-CD31 (1:200; 553370, BD Biosciences), washed, and then incubated with AF594-conjugated secondary antibody (1:200; A21209, Invitrogen) in the dark. After washing in the dark, immunostained whole tissues were visualized on a Zeiss LSM 880 laser scanning confocal microscope system (Carl Zeiss, Jena, Germany). All incubation steps of this whole tissue staining procedure were performed overnight at 4 °C in PBTx unless otherwise specified.

[0260] [Example 22]

[0261] Western blotting

[0262] 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 onto SDS-PAGE gels, transferred to polyvinylidene difluoride membranes (IPVH00010, Millipore), and immunoblotted with primary and secondary antibodies listed in Table 8. Signals were detected using a SuperSignal West Pico chemiluminescence system (34080, Thermo Fisher Scientific).

[0263] Target Supplier Identification Host Dilution Western Blot β-hCGAbcamab54410Rabbit 1 / 1000PIBF1Szekeres-Bartho, J.PMID: 14634107 *Rabbit 1 / 1000 Phospho-PI3K p85 Cell Signaling Technology 4228 Rabbit 1 / 1000 PI3K p85 Cell Signaling Technology 4257 Rabbit 1 / 1000 Phospho-AKTCell Signaling Technology 4060 Rabbit 1 / 1000 AKTCell Signaling Technology 4691 Rabbit 1 / 1000 Phospho-eNOSCell Signaling Technology 9571 Rabbit 1 / 1000 eNOSCell Signaling Technology 32027 Rabbit 1 / 1000 Phospho-p38 MAPK Cell Signaling Technology 4511 Rabbit 1 / 1000 p38 MAPK Santa Cruzsc7972 Mouse 1 / 1000 Phospho-p44 / 42 MAPK (ERK1 / 2) Cell Signaling Technology 4377 Rabbit 1 / 1000 p44 / 42 MAPK (ERK1 / 2)Cell Signaling Technology4695Rabbit1 / 1000Phospho-STAT3Cell Signaling Technology9145Rabbit1 / 1000STAT3Cell Signaling Technology9139Mouse1 / 1000TRP53Santa Cruzsc6243Rabbit1 / 1000β-ACTINSanta Cruzsc47778Mouse1 / 1000anti-mouse IgG, HRP-linkedCell Signaling Technology7076SHorse1 / 5000anti-rabbit IgG, HRP-linkedCell Signaling Technology7074SGoat1 / 5000

[0264] *Literature [B. Polgar et al., Molecular cloning and immunologic characterization of a novel cDNA coding for progesterone-induced blocking factor.J Immunol171, 5956-5963 (2003)]

[0265] [Example 23]

[0266] Magnetic resonance imaging

[0267] MR imaging was performed on a 7.0 T / 160 mm Parmascan preclinical scanner in Paravision 6.0, equipped with a 72 mm diameter volume transmit coil and a surface receive coil (Bruker Inc., Germany). Embryos were then placed in sealed plastic containers and immersed in a 4% formaldehyde solution in phosphate-buffered saline (PBS) during MR scanning. High-resolution 2D rapid acquisition, fast spin-echo-based T2-weighted (T2w) imaging using a relaxation-enhanced (2D RARE) sequence was performed in axial (Ax) and sagittal (Sag) views - TR / TE = 4500 / 50 ms; averages = 40; field of view (FOV) = 12 x 12 mm (Ax), 14 x 14 (Sag); RARE factor = 8; matrix = 256 x 256; slice thickness = 0.4 mm; slice number = 20 (no gap); The total scan time was 3 hours and 12 minutes. AsanJ-Morphometry is a proprietary software program from ImageJ (NIH) used to highlight embryonic abnormalities. AsanJ-Morphometry software is publicly available for research purposes and can be downloaded from http: / / datasharing.aim-aicro.com / morphometry.

[0268] [Example 24]

[0269] Infertility test

[0270] Female fertility is 2 months old Pibf1 f / f or Pibf1 Δ / Δ ;Vav1 cre / +Females were mated with 3-month-old wild-type males previously shown to exhibit normal mating behavior, and then measured. For 4 months, each female was monitored for the number of pups born and delivered. The female and male pups from each litter of three were then monitored for changes in weight gain for 8 weeks.

[0271] Data Analysis and Statistics

[0272] Experiments were repeated at least twice, and no data were excluded from the analysis. The χ2 test was applied to test whether the genotype distribution of mice obtained from different breeding schemes followed the expected Mendelian inheritance pattern. Other data were analyzed using 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 the diagrams represent biological replicates. The lines, boxes, and whiskers in the boxplots represent the median, first and third quartiles, and the minimum and maximum distribution of values, respectively. In other diagrams, data are expressed as the mean ± SEM. A P value less than 0.05 was considered statistically significant.

[0273] <Experimental Results>

[0274] [Experimental Example 1]

[0275] The essential role of PIBF1 in trophoblast syncytialization

[0276] We investigated whether PIBF1 mediates syncytia using the BeWo cell line. CRISPR / Cpf1-mediated gene targeting of exon 2 or 4 of human PIBF1 (Fig. 1) generated a PIBF1 knockout (KO) BeWo cell line (Fig. 2a). When the degree of cell fusion was induced in wild-type (WT) cells by adding the adenylate cyclase activator forskolin (Fig. 3a), syncytia were not formed in PIBF1 KO BeWo cells (KO line #1 in Fig. 2a) compared to WT (Fig. 3a), and the expression of the fusion gene ERVFRD-1 (Syncytin 2) and the endocrine hormone gene chorionic gonadotropin beta (CGB) was reduced (Fig. 3b).

[0277] Human CG (hCG) is primarily produced by SynT and secreted into the maternal compartment at high levels during the first trimester. Its secretion and protein levels were significantly lower in PIBF1-deficient cells, regardless of forskolin stimulation (Figs. 3c and 3d). These results were also reproduced in PIBF1 exon 4-targeted knockout (KO) cells (KO line #2) (Figs. 2b to 2e), indicating an essential role for PIBF1 in BeWo trophoblast fusion.

[0278] Next, to determine whether PIBF1 mediates the differentiation of human trophoblast stem cells (hTSCs) into SynTs, we generated PIBF1 KO hTSCs (Fig. 1, Fig. 4a and 4b). As a result, PIBF1 KO TSCs showed no changes in cell proliferation (Fig. 4c) and a slight increase in TS markers in the stem cell state (Fig. 4d and 4e). However, PIBF1 KO hTSCs differentiated into defective SynTs with suppressed β-hCG expression in both 2D and 3D cultures (Fig. 3e), consistent with the observation that basal PIBF1 KO hTSCs exhibited reduced expression of GCM1, a typical master regulator of SynT formation (Fig. 4f). SynT derived from PIBF1 KO hTSCs showed reduced levels of SynT markers (Fig. 3f) and β-hCG secretion (Fig. 3g), whereas TS markers remained abnormally high (Fig. 3h).

[0279] Additionally, PIBF1 KO hTSCs were also defective in their ability to differentiate into extravillous trophoblasts (EVTs) (Fig. 4g and 4h).

[0280] Recently, it has been reported that GCM1 contributes to EVT differentiation [MJ Jeyarajahet et al., The multifaceted role of GCM1 during trophoblast differentiation in the human placenta.Proc Natl Acad Sci US A119, e2203071119 (2022)], and the low expression of GCM1 in PIBF1 KO hTSCs suggests that further investigation into the role of PIBF1 in EVT development is needed in the future.

[0281] To investigate the in vivo differentiation potential of hTSCs following PIBF1 deletion, PIBF1 KO hTSCs were subcutaneously transplanted into immunodeficient male mice (Fig. 3i). Hosts transplanted with hTSCs of the wild-type genotype contained detectable amounts of hCG in their serum and urine, whereas PIBF1 KO hTSCs did not (Figs. 3j and 3k). The overall size of the cell population transplanted with PIBF1 KO hTSCs was not different from that of the WT (Fig. 3l), and histologically, the pan-trophoblast marker cytokeratin 7 (KRT7) was similarly expressed in both genotypes (Fig. 3m). However, differentiated trophoblast cells (SynT, EVT) labeled with β-hCG or HLA-G were largely absent from the cell population differentiated from PIBF1 KO hTSCs (Fig. 3m). These data using hTSCs indicate that PIBF1 mediates cell-to-cell fusion of the trophoblast itself and differentiation of TSCs into SynTs.

[0282] Full-length PIBF1 (fPIBF) is a 90-kDa protein associated with the nucleus or centrosome, whereas the smaller, 35-kDa secreted form (sPIBF) is localized in the cytoplasm [M. Lachman et al., PIBF (progesterone-induced blocking factor) is overexpressed in highly proliferating cells and associated with the centrosome. Int J Cancer 112, 51-60 (2004)]. To further validate our findings, we overexpressed fPIBF and / or sPIBF in PIBF1-deficient BeWo cells (Fig. 3n). Consequently, restoration of fPIBF- or sPIBF-induced cell-cell fusion was further enhanced by simultaneous reintroduction of both mutants (Fig. 3o). Notably, exogenous PIBF treatment in PIBF1 KO BeWo (Fig. 5a) did not significantly improve syncytium formation (Fig. 3p, Fig. 5b, and 5c), indicating that PIBF1 mediates cell-cell fusion events only when endogenously expressed and produced. Collectively, these data demonstrate that endogenous PIBF1 plays an essential role in trophoblast differentiation and syncytium formation.

[0283] [Experimental Example 2]

[0284] The angiogenic role of SynT-derived PIBF1

[0285] During human placental development, mononuclear cytotrophoblasts (CTBs) function as TSCs and differentiate into multinucleated SynTs, which, together with villous endothelial cells (ECs), play a role in establishing a physical and immunological barrier as well as the maternal-fetal exchange interface. However, how these cell populations communicate with each other to establish the multilayered structure remains unknown. In this context, we hypothesized that fused trophoblast cells might secrete PIBF1 to establish the complex interface, as sPIBF can be secreted by immune cells and act as a cytokine.

[0286] To address this issue, we first measured the cellular and secreted levels of PIBF1 in TSCs, SynTs, and EVTs using multiple reaction monitoring, a highly selective and sensitive technique for quantifying target proteins. While intracellular levels of PIBF1 were similar in TSCs, SynTs, and EVTs (Fig. 6a), secreted PIBF1 was significantly more abundant in SynTs and EVTs than in TSCs (Fig. 6a), suggesting a potential role for trophoblast-derived PIBF1 in placental development.

[0287] Next, to gain more insight into the interaction between SynT and endothelial cells (ECs), we designed SynT-EC transwell co-cultures to determine whether syncytial trophoblast cells could recruit vascular endothelial cells (Fig. 6b). Consequently, we found that hTSC-derived SynT or BeWo cells fused with forskolin recruited significantly more HUVECs compared to undifferentiated hTSCs or unfused cells (Figs. 7a and 7b), suggesting SynT-derived paracrine or endocrine signaling to endothelial cells.

[0288] When PIBF1 was depleted in hTSCs, this endothelial recruitment capacity was impaired, as expected (Fig. 6c). PIBF1KO BeWo cells also exhibited defects in endothelial recruitment, but this was restored by exogenous introduction of intracellular fPIBF and / or sPIBF (Fig. 6c). Notably, reintroduction of sPIBF resulted in a higher level of endothelial recruitment compared to fPIBF (Fig. 6c), suggesting that a fused trophoblast-derived signal promotes endothelial cell homing to the maternal-fetal interface, and that sPIBF may be the primary mediator.

[0289] Because placental blood vessels are composed of multiple cell populations, including ECs and pericytes, we tested the vascular cell types whose angiogenesis is influenced by PIBF. Endothelial cells derived from the umbilical vein and artery (HUVECs and HUAECs, respectively) were selected because of their different endothelial identity profiles. As a result, recombinant sPIBF dose-dependently enhanced endothelial migration and tube formation indices in HUVECs (Figures 6d-6f) and HUAECs (Figures 6h and 6i, Figure 7d) through activation of angiogenesis-related signaling cascades. Furthermore, it dose-dependently enhanced the migration properties of placental pericytes (Figures 6j and 7e). The N-terminal region, spanning amino acids 1-184, is the biologically active region of PIBF1 and is identical to the recombinant activation domain of PIBF1 (aPIBF) of the present invention (Figure 5a). Recombinant proteins of fPIBF and aPIBF were also validated for their angiogenic effects in HUVECs (Figures 7F-H), HUAECs (Figures 7I-K), and placental pericytes (Figure 7L). In contrast, the C-terminal fragment of PIBF1 (cPIBF) did not alter endothelial migration / tube formation or pericyte migration (Figures 7F-L), indicating that the angiogenic property of PIBF1 resides in the N-terminal portion encoded by exons 2-4, which was previously shown to have immunological activity.

[0290] Taken together, these results demonstrate that SynT-derived PIBF1 recruits blood vessels and perivascular cells to form vessel-like structures and promotes angiogenesis, suggesting that PIBF1 may play a role in the development of the placental circulation and the fetal-maternal interface.

[0291] [Experimental Example 3]

[0292] Expression of cardiovascular features promoted by PIBF1 in cardiac organoids

[0293] To further confirm the effects of sPIBF on cardiovascular characteristics in vitro, we utilized an induced pluripotent stem cell (iPSC)-derived human cardiac organoid (hHO) model that can mimic the three-dimensional function of the heart.

[0294] After differentiation of hHOs from iPSC-derived embryoid bodies under conditions influenced by sPIBF (Fig. 6k), we confirmed that the distribution of cTnT, a cardiomyocyte-specific marker, was significantly higher in the sPIBF-treated hHO group compared to the control group (Fig. 6l). Furthermore, co-staining of CD31 and PDGFRβ, markers for endothelial cells and pericytes, respectively, revealed an increased distribution of CD31 in sPIBF1-treated hHO compared to the control group, whereas PDGFRβ remained unchanged (Fig. 6l). A wide range of rsPIBF doses (50-1000 ng / ml) also showed an increase in the distribution of endothelial and cardiomyocytes in hHO (Fig. 6p).

[0295] Given that hHO is a mixed population of multiple cells, including endothelial cells and cardiomyocytes, it remains unclear whether the effect of sPIBF on hHO is due to a direct effect on cardiomyocyte differentiation or an indirect effect via increased endothelial angiogenesis. To address this issue, we performed two-dimensional cultures of human iPSC-derived cardiomyocytes and confirmed that PIBF1 can directly influence cardiomyocyte differentiation (Fig. 6m).

[0296] Because human iPSC-derived hHO exhibits functional characteristics that mimic depolarization / repolarization coupling and reflect electrophysiological signals, changes in the field potential (FP), a leading indicator of electrophysiological signals, were measured using a multielectrode array (MEA). Results showed no significant differences in beating period and field potential duration (FPD) (Fig. 6n). However, spike amplitude was significantly higher in sPIBF compared to the control group (Fig. 6n).

[0297] Furthermore, the angiogenic effects of sPIBF observed in endothelial cells and pericytes were further confirmed in hHO using a Matrigel-based sprouting assay: the majority were PDGFRβ positive (Fig. 6o). Notably, sPIBF-treated hHO exhibited a more vascularized appearance with longer sprouting lengths than vehicle-treated hHO (Fig. 6o), which increased in a sPIBF dose-dependent manner (Fig. 6q).

[0298] Next, to better understand the effects of sPIBF on the developing heart, we analyzed the transcriptomes of hHO treated with vehicle or sPIBF and found that 240 genes were differentially regulated by sPIBF, of which 105 differentially expressed genes (DEGs) were upregulated and 135 were downregulated (Fig. 8). Gene Ontology analysis of the DEGs revealed that compared to undifferentiated embryoid bodies (EBs), hHO exhibited increased expression of markers for not only cardiac muscle but also other mesenchymal tissues, such as bone and fat (Fig. 6r, column 1 vs. columns 2-4 of the heatmap). However, treatment with rsPIBF significantly suppressed the expression of other mesenchymal cell fate-related genes. In particular, the expression of cardiomyocyte-related genes was significantly increased (Fig. 6r, columns 2-4 vs. columns 5-7 of the heatmap). This enhances the fate determination of muscle and heart development by sPIBF (Fig. 6s).

[0299] In summary, PIBF1 promotes cardiomyocyte differentiation / maturation and endothelial angiogenesis in in vitro cardiac development. Together with our findings demonstrating the essential role of SynT-derived PIBF1 in angiogenesis and primary placental formation, this may contribute to embryonic cardiovascular development.

[0300] [Experimental Example 4]

[0301] Incomplete syncytialization and maternal-fetal interface formation in placentas lacking Pibf1

[0302] Next, we evaluated Pibf1-null placentas and embryos to determine whether loss of Pibf1 in trophoblast cells affects placental and subsequent embryonic development. + / - Crossbreeding is Pibf1 - / - did not produce offspring (Fig. 9a), which was not related to the implantation potential of the blastocyst (Fig. 9b). Instead, Pibf1 - / - The embryos were developmentally retarded after E9.5–10.5 (Figs. 9c and 9d). At this stage, the primary placenta begins to develop, and Pibf1 is deleted from all cell lineages of the labyrinthine interhaemal membrane (Fig. 10a). Loss of Pibf1 leads to defective placental and embryonic development (growth restriction, failure of tail rotation, and pericardial effusion) (Fig. 10b). Pibf1 - / - The placenta was morphologically and functionally disrupted, as evidenced by a reduced number of fetal-derived blood vessels (Figs. 10c and 10d) and an underdeveloped labyrinth with reduced fatty acid transport capacity (Fig. 10e).

[0303] The possible mechanism of Pibf1 in the developmental failure of the Pibf1-null placenta and embryo can be explored by considering the literature [MK Lim et al., Characterization of serum progesterone and progesterone-induced blocking factor (PIBF) levels across trimesters in healthy pregnant women. Sci Rep10, 3840 (2020)]. Pregnancy status and outcome are related to maternal PIBF1 levels. However, Pibf1 - / - Because the abnormal embryonic appearance was also reproduced in wild-type surrogate mothers, Pibf1-null lethality was not due to maternal Pibf1 haploinsufficiency (Figs. 11a and 11b).

[0304] Meanwhile, Pibf1 was first described as a suppressor of conventional cytolytic natural killer (NK) cells [J. Szekeres-Bartho, B. Polgar, PIBF: the double-edged sword. Pregnancy and tumor. Am J Reprod Immunol 64, 77-86 (2010)], and uterine NK (uNK) cells promote immune tolerance by attenuating IL-17-producing helper T (Th17)-mediated inflammation that occurs locally in the maternal decidua [B. Fu et al., Natural killer cells promote immune tolerance by regulating inflammatory Th17 cells at the human maternal-fetal interface. Proc Natl Acad Sci US A110, E231-240 (2013)] (Fig. 11c). Therefore, we examined whether embryonic loss of Pibf1 affects the uNK population. However, Pibf1 - / - Pibf1 containing embryo + / - No significant changes in the proportion or absolute number of immune cells were observed in the decidua (Figs. 11d and 11e). Furthermore, in a Th17-deficient maternal immune background (Fig. 11f), Pibf1 - / - Embryos were still morphologically abnormal, similar to those found in littermates from Pibf1 wild-type or heterozygous mothers (Fig. 11g). These data suggest that, at least in part, the maternal compartment of the embryo is genetically or immunologically Pibf1 - / - Explains that it is not the cause of early embryonic defects.

[0305] Because PIBF1 plays an essential role in in vitro TSC differentiation and trophoblast fusion, we investigated whether Pibf1-null placentas were defective in SynT formation. - / -In the placenta, incomplete labyrinth formation was accompanied by decreased proliferation of trophoblast cells (Fig. 10f). Among TSCs and their lineages, the labyrinth trophoblast cells within the mesangium affected by Pibf1 deletion were SynT-II: while s-TGCs developed normally (Ctsq in Fig. 10g, alkaline phosphatase positivity in Fig. 10h), SynT-II markers (Gcm1, Synb, Mct4) were significantly reduced, while SynT-I (Syna, Mct1) remained unchanged (Fig. 10g). This was further confirmed by MCT1 / 4 co-staining (Fig. 10i). Moreover, Pibf1 - / - At the ultrastructural level of the labyrinth, the blood-membrane barrier was found to be thin and abnormally thickened, and most importantly, SynT-II failed to fuse (Fig. 10j). SynT-I also had visible abnormalities (excessively thickened, vacuolated, and electron-dense areas) (Fig. 10j). In terms of vascular development within the placenta, Pibf1 - / - Embryonic-derived angiogenesis (laminin positivity) was reduced along with increased apoptosis detected (TUNEL positivity) (Fig. 10k), and the reduced placental vasculature was site-specific only in the labyrinth, in accordance with histological data (Fig. 10l).

[0306] Collectively, these data indicate that loss of Pibf1 leads to impaired cell-cell fusion and interaction of trophoblasts with the endothelium, resulting in failure to form the maternal-fetal interface, and suggest that defective primary placentation is a major cause of lethality.

[0307] [Experimental Example 5]

[0308] Pibf1 Restores Synapse and Trophoblast-Endothelial Interactions in Functional Placentas Containing Intact Trophoblasts

[0309] To distinguish between trophoblast-intrinsic and embryonic lineage-inducing effects within the placenta, conditional Pibf1 KO (cKO) concepti were generated using Meox2-cre (Figs. 12a and 12b), a transgenic line for epiblast-specific cre-recombinase expression (endothelium, fetal blood cells) while retaining expression in trophectoderm-derived cells (S-TGC, SynT-I, SynT-II) within the placental labyrinth (Figs. 13a and 14a). Pibf1-null embryos associated with Pibf1-bearing placentas ( / +) were detected normally at the expected Mendelian ratios (Fig. 13b), and embryos and placentas were fully recovered at E10.5, a stage at which the complete knockout counterparts were morphologically and histologically disrupted (Figs. 13c to 13f). Importantly, this recovery was accompanied by a differentiation of lineages from the normal state of TSCs, particularly SynT-II (Figs. 13g and 13h) and labyrinthine vasculature (Fig. 13i).

[0310] The placenta also acts as a hematopoietic organ during this period. In summary, primitive hematopoiesis initially arises from the yolk sac blood islands ( <E8.5), 그런 다음 태반(E9.5-10.5)을 포함한 태아의 주요 혈관의 내피세포로 확장되며, 그 후 조혈 전구체는 임신 중 태아 간과 출생 후 골수를 콜로니화한다. 본 발명에서, Pibf1 - / - The hematopoietic capacity of the yolk sac and placenta was normal until E9.5 (Figs. 15a–15c), but was abnormally reduced at E10.5 (Figs. 13j, 15a and 15d). However, this defect was restored in the yolk sac and placenta with intact Pibf1, and even in Pibf1 cKO embryos at E10.5 (Fig. 13j). Moreover, the restored placentas exhibited normal morphometric and nutrient transport functions at E13.5, as confirmed by histological evaluation and fatty acid transport capacity measurements, respectively (Figs. 16a and 16b). At this stage, the distribution of SynT-I and -II was similar to that of the control placenta (Fig. 16c).

[0311] Furthermore, with regard to subcellular properties, the full-length 90-kDa PIBF1 is localized to the nucleus or centrosomes. Increased apoptosis and expression of the apoptosis regulator Trp53 in Pibf1-null embryos (Fig. 20a) prompted us to investigate the role of Trp53 in the survival of embryos lacking Pibf1. However, the embryonic function of Pibf1 was not related to the Trp53 pathway, as Pibf1;Trp53 double KO embryos phenotypically replicated Pibf1 KO embryos (Figs. 20b and 20c). The centrosome-associated protein PIBF1 has also been reported to be essential for primary cilium formation, and mutations have been reported in some ciliopathies. Pibf1 Δ / Δ ;Meox2 cre / + Although the fetuses showed the expected Mendelian ratios until E18.5, no cKO neonates were found after birth (Fig. 20d). In addition, Pibf1 Δ / Δ ;Meox2 cre / + The fetus exhibited phenotypic features of ciliary disease (Figs. 20e to 20h), which may contribute to postnatal fetal death regardless of a functionally normal placenta.

[0312] To provide further insight into the role of trophoblast syncytia and their interactions with vascular cells seen in vitro, we dissected the endothelial cell lineage of the labyrinth mesentery using two endothelial-specific cre lines, Cdh5-cre and Tie2-cre (Fig. 14a), which each express promoter-driven cre recombinase in the endothelial lineage (Fig. 13k). As a result, Cdh5- or Tie2-cre-mediated Pibf1 cKO embryos were detected at E10.5 without obvious morphological abnormalities (Figs. 13m and 13o) and with the expected normal Mendelian inheritance ratio (Fig. 13l), and the histological indices of the placenta were not significantly different from those of the control (Figs. 13m and 13n). Because Tie2- or Cdh5-cre expressing cells share the identity of both endothelial and hematopoietic cell lineages, endothelial-specific Pibf1 cKO conceptuses were probed for functional hematopoietic activity in the hematopoietic compartment, which showed similar levels between genotypes (Fig. 13p). Furthermore, when Pibf1 was deleted from the hematopoietic lineage using Vav1-, Cdh5-, or Tie2-cre lines, the hematopoietic functions of other Pibf1 cKO organs were restored prenatally at E13.5 (cKO in Figs. 17a to 17d). Vav1 and cKO Tie2 ) after birth (cKO in Figs. 17e and 17f Cdh5 and cKO Vav1 ) were all normal. These hematopoietic data suggest that proper engulfment of trophoblast cells can ensure normal hematopoietic development in both extraembryonic and embryonic organs. These results indicate that normally engulfed trophoblast cells and their interaction with the endothelium, orchestrated by Pibf1, are prerequisites for establishing the maternal-fetal interface, a functional placenta, and ultimately embryonic survival.

[0313] [Experimental Example 6]

[0314] Effects of trophoblast syncytialization on embryonic cardiovascular development

[0315] According to the Pibf1 cKO strategy, the cre line was able to produce whole heart (Pibf1 Δ / Δ ;Meox2 cre / + ) or some (Pibf1 Δ / Δ ;Cdh5 cre / + , Pibf1 Δ / Δ ;Tie2 cre / + ) is activated in the developing heart (Fig. 18a and Fig. 14b). The placenta and embryonic heart develop in parallel during early pregnancy, and their defects commonly coexist. Embryonic lethality was rescued by a functional placenta with intact SynT, including the previously identified underdeveloped heart of Pibf1-null embryos (Fig. 10b). Moreover, syncytialized trophoblasts produce angiogenic factors such as PIBF1 (Fig. 6a), which could potentially affect the endothelial lineage or cardiovascular architecture of the developing heart. In this context, we investigated the impact of Pibf1-mediated trophoblast syncytialization on cardiovascular development in Pibf1 mutants. As shown by whole-mount CD31 staining, Pibf1 - / - In embryos, the yolk sac, intersomitic region, and vascular structures of the heart were severely destroyed, but most of them were recovered in Pibf1cKO mutant embryos (Fig. 18b). In particular, the CD31-stained area in the heart region was Pibf1 - / - Although significantly reduced in the heart, it was similar between the control and Pibf1cKO hearts (Figs. 18c and 18d). In macroscopic and histological appearance, Pibf1 - / -The heart showed severe developmental delay, including hypodevelopment of the right and left ventricles, abnormally dilated pericardial cavities, reduced ventricular length, and reduced trabecular density. However, these cardiac defects were completely rescued in all three Pibf1cKO mice with normal PIBF1 expression in placental trophoblasts (Figs. 18e and 18f). This was further confirmed histologically by the cardiac marker cTnT (Figs. 18g and 18h). Early cardiac markers (regulation of myocardial development and cardiac morphogenesis) and coronary vascular development markers (panendothelial lineage and arterial / venous specification) showed similar patterns. Most of them were Pibf1 - / - It was reduced in the heart and restored in all Pibf1cKO mutants affected by functional SynT-containing placentas (Figs. 18i and 18j).

[0316] These data indicate that embryonic cardiovascular development depends on Pibf1-mediated trophoblast syncytialization and placental formation, rather than on the genetic status of Pibf1 in the heart or vasculature itself.

[0317] Furthermore, we addressed the impact of loss of Pibf1 in maternal immune cells on pregnancy, outcome, and offspring, with respect to the relationship between immune tolerance function and maternal levels in specific pregnancy settings and pregnancy outcomes (Fig. 21a). As a result, Pibf1 Δ / Δ ;Vav1 cre / +Females (Pibf1-deficient in hematopoietic / immune cells [J. de Boer et al., Transgenic mice with hematopoietic and lymphoid specific expression of Cre. Eur J Immunol 33, 314-325 (2003)]) exhibited normal reproductive function (Fig. 21b), and their delivered pups showed normal growth rates (Fig. 21c), indicating that genetic deletion of Pibf1 in maternal immune cells does not directly affect the maintenance of pregnancy in mice. On the other hand, the function of immune cells may be influenced by PIBF1, which can be produced in 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 decidualization. Therefore, further investigation is needed to understand the role of PIBF1 in maintaining pregnancy in the maternal environment.

[0318] This patent application was conducted with the support of the National Research Foundation of Korea (① Project No.: 2018M3A9D5A01087977, Research Project Title: Research on the establishment and utilization of a multifaceted placental phenotypic analysis platform, Research period: 2018.09. 20 ~ 2023.11. 19; ② Project No.: 2020R1A2C2012912, Research Project Title: Functional analysis of novel lipid metabolism proteins that regulate spermatogenesis and research on male infertility treatment strategies using them, Research period: 2020.03. 01 ~ 2024.02. 29; ③ Project No.: RS-2024-00342508, Research Project Title: Verification of the hypothesis of the placenta-embryonic organ development axis during pregnancy, Research period: 2024.05. 01 ~ 2029.04. 30). It is the result.

Claims

1. (i) PIBF1 (Progesterone immunomodulatory binding factor 1) protein or a fragment thereof; or (ii) A pharmaceutical composition for preventing or treating pregnancy complications associated with placental or fetal cardiovascular abnormalities, comprising a vector comprising a gene encoding PIBF1 protein or a fragment thereof.

2. A pharmaceutical composition according to claim 1, wherein the PIBF1 protein comprises an amino acid sequence of sequence number 1.

3. A pharmaceutical composition according to claim 1, wherein the fragment of the PIBF1 protein comprises a part of the amino acid sequence of the N-terminus of the PIBF1 protein or a part of the amino acid sequence of the N-terminus and a part of the amino acid sequence of the C-terminus of the PIBF1 protein.

4. A pharmaceutical composition according to claim 3, wherein the N-terminal amino acid sequence comprises at least amino acid residues from positions 1 to 184 in the amino acid sequence of SEQ ID NO:

1.

5. A pharmaceutical composition according to claim 3, wherein the amino acid sequence of the C-terminus includes at least amino acid residues from positions 684 to 757 in the amino acid sequence of SEQ ID NO:

1.

6. A pharmaceutical composition according to claim 3, wherein a portion of the amino acid sequence at the N-terminus includes an amino acid sequence of SEQ ID NO: 2, and a portion of the amino acid sequence at the N-terminus and a portion of the amino acid sequence at the C-terminus include an amino acid sequence of SEQ ID NO:

3.

7. A pharmaceutical composition according to any one of claims 1 to 6, which increases PIBF1 (Progesterone immunomodulatory binding factor 1) protein having a molecular weight of 25 to 90 kDa in the mother.

8. A pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmaceutical composition exhibits at least one effect selected from the group consisting of the following a) to d): a) Formation of vascular-like structures in the embryo; b) Angiogenesis in the embryo; c) differentiation and maturation of embryonic cardiomyocytes; and d) Formation of the maternal-fetal interface.

9. A pharmaceutical composition according to claim 1, wherein the pregnancy complications associated with placental or fetal cardiovascular abnormalities include recurrent miscarriage, preeclampsia, or fetal heart defects.

10. A method for preventing or treating pregnancy complications associated with placental or fetal cardiovascular abnormalities, comprising administering to a subject in need thereof a composition comprising (i) a PIBF1 (Progesterone immunomodulatory binding factor 1) protein or a fragment thereof; or (ii) a vector comprising a gene encoding a PIBF1 protein or a fragment thereof.

11. A method for preventing or treating pregnancy complications related to placental or fetal cardiovascular abnormalities, wherein the PIBF1 protein comprises the amino acid sequence of sequence number 1 in claim 10.

12. A method for preventing or treating pregnancy complications related to placental or fetal cardiovascular abnormalities, wherein the fragment of the PIBF1 protein comprises a portion of the amino acid sequence at the N-terminus of the PIBF1 protein or a portion of the amino acid sequence at the N-terminus and a portion of the amino acid sequence at the C-terminus of the PIBF1 protein.

13. A method for preventing or treating pregnancy complications related to placental or fetal cardiovascular abnormalities, wherein the N-terminal amino acid sequence comprises at least amino acid residues from positions 1 to 184 in the amino acid sequence of SEQ ID NO:

1.

14. A method for preventing or treating pregnancy complications related to placental or fetal cardiovascular abnormalities, wherein the amino acid sequence of the C-terminus comprises at least amino acid residues from positions 684 to 757 in the amino acid sequence of SEQ ID NO:

1.

15. A method for preventing or treating pregnancy complications related to placental or fetal cardiovascular abnormalities, wherein, in paragraph 12, a part of the amino acid sequence at the N-terminus comprises an amino acid sequence of SEQ ID NO: 2, and a part of the amino acid sequence at the N-terminus and a part of the amino acid sequence at the C-terminus comprise an amino acid sequence of SEQ ID NO:

3.

16. A method for preventing or treating pregnancy complications related to placental or fetal cardiovascular abnormalities, wherein the composition increases PIBF1 (Progesterone immunomodulatory binding factor 1) protein having a molecular weight of 25 to 90 kDa in the mother, according to any one of claims 10 to 15.

17. A method for preventing or treating pregnancy complications related to placental or fetal cardiovascular abnormalities, wherein the composition exhibits at least one effect selected from the group consisting of the following a) to d): a) Formation of vascular-like structures in the embryo; b) Angiogenesis in the embryo; c) differentiation and maturation of embryonic cardiomyocytes; and d) Formation of the maternal-fetal interface.

18. A method for preventing or treating pregnancy complications related to placental or fetal cardiovascular abnormalities, wherein the pregnancy complications related to placental or fetal cardiovascular abnormalities in paragraph 10 include recurrent miscarriage, preeclampsia, or fetal heart defects.

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