Engineering placenta-like organoids containing endogenous vascular cells from human-induced pluripotent stem cells

Abstract

The placenta is an essential organ that maintains the health of both the fetus and its mother. Understanding the development of human placenta has been hindered by the limitations of existing animal models and monolayer cell cultures. Models that can recapitulate the essential aspects of human placental multicellular components and vasculature are still lacking. Herein, we presented a new strategy to establish placenta-like organoids with vascular-like structures from human-induced pluripotent stem cells in a defined three-dimensional (3D) culture system. The resulting placenta-like tissue resembles first-trimester human placental development in terms of complex placental components and secretory function. The multicellular tissue was characterized by the inclusion of trophoblasts (cytotrophoblasts, syncytiotrophoblasts, extravillous trophoblasts, and other endogenous vascular cells), which were identified by immunofluorescence, flow cytometry analyses, real-time quantitative reverse transcription polymerase chain reaction and single-cell RNA-seq. Moreover, the 3D tissue was able to secrete the placenta-specific hormone human chorionic gonadotropin β (hCG-β) and vascular endothelial growth factor A (VEGFA). The tissue responded to the inflammatory factor tumor necrosis factor-α (TNF-α) and VEGF receptor inhibitors. This new model system can represent the major features of placental cellular components, and function, which have not been realized in 2D monolayer cultures. The developed tissue system might open new avenues for studying normal early human placental development and its disease states.

Keywords: 3D culture; human‐induced pluripotent stem cells; in vitro model; placenta; trophoblasts; vasculature.

FIGURE 1

Schematic of early human placental development in vivo and in vitro. (a) Early human placental development in vivo. During pregnancy, the oocyte combines with sperm to form the zygote, thereby triggering embryogenesis. After fertilization, the blastocyst segregates into two lineages, the trophectoderm (TE), and the inner cell mass (ICM). The TE gives rise to the epithelial portion of the human placenta. As the main component of human placenta, the trophoblast is composed of three subtypes: CTBs, STBs, and EVTs. The multinucleated STBs line the outermost surface of the human placenta and subsequently form the major cellular barrier between the feus and mother. The EVTs invade into the decidua and remodel the maternal blood supply. (b) Illustration of human placental model generation in vitro. hiPSCs were seeded onto micropillar chips and treated with BMP4 to generate 3D clusters with trophoblast and mesodermal lineages under 3D culture conditions. The 3D clusters gradually grew into millimeter‐sized tissues when treated with a cohort of factors (e.g., VEGFA, bFGF, and R‐spondin 1). The formed 3D tissue contained trophoblast subtypes (CTBs, STBs, and EVTs) and vascular cells. (c) Characterization of hiPSC‐derived placenta‐like 3D tissue through immunofluorescence staining, qRT‐PCR, single‐cell RNA‐seq, and flow cytometry.

FIGURE 2

Differentiation of hiPSCs into placenta‐like tissue containing endogenous vascular cells under 3D culture conditions. (a) Schematic of the protocol used for differentiating hiPSCs into placenta‐like organoids. KSRM (KSR medium), SB (SB‐451342), CHIR (CHIR99021). The hiPSCs were exposed to a low‐oxygen environment from Days 0 to 12. At Day 12, the formed tissue was transferred to a 21% oxygen environment. (b) Representative phase contrast images of the formed 3D tissue from Day 1 to Day 20. Scale bar = 100 μm. (c and d) Confocal micrographs of the CTB marker CDX2 and the endothelial cell marker CD31 in 3D cultures at 15 days of differentiation (c) and in primary placenta (n = 3; 6–8 weeks gestation in first‐trimester) (d). (e–g) 3D tissue immunofluorescence analysis for the markers of EVTs (HLA‐G), STBs (ENDOU), and endothelial cells (CD31), respectively. (h and i) Confocal micrographs depicting the expression of the pericyte marker PDGFβ and the endothelial marker CD31 in the primary placenta (n = 3; 6–8 weeks gestation in first‐trimester) and in 3D tissue at Day 15. (j) 3D construction of the endothelial marker CD31 and pericyte marker PDGFβ in placental organoid at 19 days taken across several z‐stacks and combined into a single image by the extended focus module of the confocal microscope software. (k) Confocal micrographs showing the expression of the endothelial marker VE‐cad and the trophoblast marker E‐cad in Day 16 placenta‐like organoids. (l) 3D construction of the endothelial marker VE‐cad and the trophoblast marker E‐cad in 3D tissue. (m) Small endothelial tube marker CD31 in 3D cultures at Day 19 covered by collagen type IV (Col IV).

FIGURE 3

Single‐cell transcriptome atlas of the 3D tissue formed from hiPSCs. (a) UMAP plot displaying 16,311 cells at Days 9 and 24. Unsupervised clustering identified 12 clusters, which were labeled by different colors. These clusters were clustered into five cell types based on the expression of different markers. (b–f) Violin plots demonstrated the expression plot of cell‐specific genes among different clusters in the placental tissues, including proliferating cells, STBs, vascular cells, CTBs, and EVTs.

FIGURE 4

Expression of CTB‐, endothelial cell‐, and pericyte‐related markers in the formed placenta‐like 3D tissue. (a) qRT‐PCR demonstrating the relative mRNA expression of CDX2, P63, and KRT17 in the 3D tissue at 0, 10, and 20 days of differentiation (n = 3). Student's t‐test were used for data analysis (*p < 0.05; **p < 0.01; ***p < 0.001). (b) GATA3 staining of the 3D tissue at Day 16, which revealed the expression of a trophoblast marker in the 3D culture. (c and d) Immunofluorescence analysis for the trophoblast marker KRT7, proliferative cell marker Ki67, and trophoblast stem cell markers CDX2 and P63 in Day 16 3D tissue. (e–g) Flow cytometry analysis of Days 10 and 24 3D tissue stained for CD140 and CD49b to identify proliferating trophoblast progenitor cells and pericytes, respectively. (h) qRT‐PCR to identify mesodermal markers at 0, 10, and 20 days of differentiation. Three independent experiments were conducted. The data were analyzed using Student's t‐test (*p < 0.05; **p < 0.0; ***p < 0.001). (i and j) Flow cytometry analysis of the endothelial cell marker CD31 (i) and the vascular progenitor marker KDR (j) in the tissue at 10 days. Primary HUVECs served as a positive control.

FIGURE 5

Characterization of STB and EVT in the placenta‐like tissue. (a) The relative mRNA expression of STB markers CYP19A1 and CGA in Day 0, 10, and 20 placenta‐like organoids. Data were analyzed using Student's t‐test (*p < 0.05; **p < 0.01). (b and c) Confocal micrographs showing the expression of the endothelial cell marker CD31 and STB markers ENDOU and CGA in the 3D tissue at 16–20 days. (d) Immunofluorescence images showing CD31 and ENDOU expression in primary placenta (n = 3; 6–8 weeks gestation in first‐trimester). (e) Electron transmission microscopy images of multinucleated STBs. The right picture showed the enlarged square areas in the left picture. The microvilli were indicated by red arrowheads. nu, nucleus. (f) Expression of the endothelial cell marker vWF and the EVT marker HLA‐G in day 19 3D cultures detected by immunofluorescence analysis. (g) Staining of Day 19 3D tissue for vWF and αSMA, markers of endothelial cells and pericytes, respectively. (h and i) CD31 staining to detect small endothelial tubes in Day 19 tissue (h) and primary first‐trimester tissue (i). The endothelial cells appeared to be covered by extracellular matrix collagen type IV (Col IV). Scale bars are indicated in the images.

FIGURE 6

Functional characterization of placenta‐like tissue containing endogenous vascular cells. (a) Secretion of hCG‐β by the 3D tissue (n = 11). (b) Secretion of VEGFA from the formed 3D tissues (n = 5). (c) Electron transmission microscopy images of endothelial cells. Red arrowheads indicate Weibel–Palade bodies in the endothelial cells. (d) TNF‐α‐mediated activation of placental tissues detected by the induction of ICAM‐1 expression in endothelial cells (CD31). ICAM‐1 expression was determined after 24 h of TNF‐α treatment (100 ng/ml). Experiments were independently repeated three times with similar results. DAPI was used to counterstain the nuclei. (e) Quantitative analysis of the ICAM‐1 fluorescence intensity in the formed tissue after exposure to TNF‐α stimulation. Data are presented as the mean ± SEM. The data were analyzed using Student's t‐test (*p < 0.05). (f) Total immunofluorescence analysis of the placenta‐like tissue (Day 17) treated with VEGFR inhibitors for 3 days. Scale bar = 50 μm. (g and h) Quantitative analysis of PDGFβ and CD31 fluorescence intensity in the 3D tissue under different conditions. Data are presented as the mean ± SEM. The data were analyzed using Student's t‐test (*p < 0.05)