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. Author manuscript; available in PMC: 2025 Aug 25.
Published in final edited form as: Placenta. 2025 Jun 6;168:35–45. doi: 10.1016/j.placenta.2025.05.029

A human cytotrophoblast-villous endothelium-fetal organ multi-cell model and the impact on gene and protein expression in placenta cytotrophoblast, fetal hepatocytes and fetal kidney epithelial cells

Helen N Jones a,b, Alyssa Williams a,b, Rebecca L Wilson a,b,*
PMCID: PMC12376020  NIHMSID: NIHMS2098320  PMID: 40505174

Abstract

Introduction:

Appropriate fetal growth requires multi-directional, coordinated communication between maternal, placental, and fetal systems. Disruptions in these signaling arms can have deleterious consequences for fetal growth and lead to fetal developmental adaptations associated with short- and long-term morbidities. This proof-of-concept human cell model study aimed to identify the effects of altered trophoblast culture conditions and human insulin-like 1 growth factor (hIGF1) nanoparticle gene therapy on fetal liver hepatocytes and kidney epithelial cells.

Methods:

We utilized human cell lines: BeWo choriocarcinoma cells (trophoblast), Human Placental Micro-Vascular Endothelial Cells, and WRL68 (hepatocytes) or HEK293T/17 (kidney epithelium), in a co-culture model designed to mimic cytotrophoblast-villous endothelium-fetal organ communication.

Results:

Trophoblast stress response mechanisms were increased by culturing BeWo cells in growth media without fetal bovine serum (FBS). BeWo cells were also cultured without FBS and treated with a hIGF1 nanoparticle gene therapy which is known to mitigate cellular stress mechanisms. BeWo cells without FBS support had increased expression of cellular stress mechanisms but not when IGF1 was over-expressed with a transient hIGF1 nanoparticle gene therapy. BeWo cells without FBS and without FBS + hIGF1 nanoparticle gene therapy had increased expression of gluconeogenesis and glycolysis rate-limiting enzymes. Gene and protein expression in fetal liver and kidney cells was not impacted by increased trophoblast stress or hIGF1 nanoparticle gene therapy.

Discussion:

Our data demonstrated that cytotrophoblast, cultured without FBS support, turn on mechanisms involved in glucose production. Whether this is reflected in vivo remains uninvestigated but may represent a placental compensation mechanism in complicated pregnancies.

Keywords: Placenta, Fetal liver, Fetal kidney, Developmental programming, Cell culture

1. Introduction

Advances in epidemiological research and animal studies have illuminated a significant association between the in utero environment and the likelihood of developing diseases, such as diabetes and cardiovascular disease, later in life [1,2]. This association fundamentally alters the idea that a person’s vulnerability to diseases solely arises from genetic and postnatal environmental interactions. The Developmental Origins of Health and Disease (DOHaD) hypothesis predominantly links fetal growth restriction (FGR), as a result of placental insufficiency and inadequate nutrient and oxygen delivery to the fetus, with increased risk of developing physiological deficits including glucose intolerance, insulin resistance and hypertension, as early as adolescence [3]. However, there is emerging evidence that fetal overgrowth, as a consequence of excessive nutrient supply to the fetus, can also have a profound impact on lifelong health and disease [4]. These scenarios ultimately underscore the critical role of placental function and fetal nutrient status in fetal developmental programming and shaping long-term health outcomes.

Appropriate in utero fetal growth requires the placenta to delicately balance fetal demands with maternal supply [5]. The placenta functions as an endocrine organ that coordinates the transfer of nutrients and waste between mother and fetus and facilitates gas exchange [6]. Nutrient transfer specifically, occurs via facilitated diffusion (glucose and lactate), active transport with the aid of carrier proteins, endocytosis and/or exocytosis and increases as the fetal growth rate increases [7]. The functional area for nutrient transfer in the placenta is the chorionic villous tissue, which contains the placental blood vessels that connect directly to fetal circulation via the umbilical cord [8]. Chorionic villi are covered in a multinucleated syncytium which is in direct contact with the maternal blood within the intervillous space. Underneath this syncytial layer lies the cytotrophoblast cells, placental macrophages, fibroblasts and capillary endothelium containing the fetal blood [9]. Hence, nutrients must cross several layers of cells and basement membranes in order to move between maternal and fetal circulations.

It has long been understood that maternal-placental-fetal communication is multi-directional, particularly when it comes to fetal growth [5]. The fetus communicates with the mother via the placenta which in turn produces the hormones and factors necessary to promote appropriate maternal metabolism and behaviors but may be constrained depending on the maternal state. Hence, dysfunction in any of these signaling arms can lead to alterations in placental function and fetal growth, ultimately initiating a cascade of developmental adaptations with adverse consequences both in the short and long term. Placental secretion of proteins including hormones [10], cytokines and growth factors [11], as well as glycoproteins, steroid hormones and more recently extracellular vesicles [12], have been heavily studied to better understand their implications to maternal pregnancy adaptation as well as fetal developmental programming of adverse long-term health. For example, insulin-like 1 growth factor (IGF1) is a growth factor produced by the placenta from the first trimester of pregnancy [13]. IGF1 is needed for appropriate placental growth and development and influences transport of nutrients including glucose and amino acids across the placenta [14]. In humans, both maternal and fetal circulating levels of IGF1 are lower in cases of FGR compared to appropriate for gestational age cases [15,16]. Transgenic mouse models have also confirmed the necessity for placental Igf1 in the promotion of fetal growth [17,18].

Whilst there are many extrinsic and intrinsic causes of perturbed fetal growth, the commonality between the causes is placental dysfunction [19–21]. Hence, therapeutic interventions which target the placenta offer unparalleled opportunities to not only prevent adverse maternal and fetal outcomes in the short-term perinatal period but also mitigate the risk of major diseases throughout an individual’s lifespan. Whilst prior investigations into the use of IGF1 as a potential therapeutic intervention have had mixed results [22–24], we have been successful in showing that specifically increasing placental gene expression of human IGF1 (hIGF1) maintains or mitigates against reduced fetal weight in various animal models [25–27]. Placental gene expression of hIGF1 is increased with a plasmid that contains the hIGF1 gene under the control of a placenta-specific promotor (PLAC1 or CYP19a1), with cellular uptake aided by the use of a non-viral polymer nanoparticle [26,28,29]. Our animal studies are supported by in vitro investigations in human placenta models which confirm the ability to manipulate cytotrophoblast nutrient transporter expression and prevent increased cell death under oxidative stress condition by treatment with the hIGF1 nanoparticle [29,30].

In addition to comprehensively characterizing the placental response to hIGF1 gene therapy, we also understand that manipulating the placenta indirectly impacts fetal liver and kidney development and function [31]. More specifically, improving development and function with hIGF1 gene therapy mitigates FGR-associated changes in gene and protein expression of factors relating to glucose metabolism in the fetal liver [31] and blood pressure regulation in the fetal kidneys [32]. Overall, suggesting the potential to prevent or reverse developmental programming of metabolic health deficits with an in utero intervention. In this proof-of-concept translational, human cell model study we aimed to identify the impacts of culturing trophoblast in conditions without support and with hIGF1 nanoparticle treatment on gene expression of glucose metabolism-related factors in fetal liver hepatocytes and regulators of kidney epithelial cell function important for blood pressure regulation. In light of data from our in vivo studies in a guinea pig model of placental insufficiency, we hypothesized that increased trophoblast stress (through culturing without FBS) would result in changes to placental-fetal signaling that would influence gene and protein expression in fetal liver and kidney cells. To test this hypothesis, we utilized human cell lines: BeWo choriocarcinoma cells (trophoblast), Human Placental Micro-Vascular Endothelial Cells (HPMVEC; placenta capillary endothelium), and WRL68 (fetal liver hepatocytes) or HEK293T/17 (fetal kidney epithelium), in a multi-layer model designed to mimic cytotrophoblast-villous endothelium-fetal organ communication (Schematic 1).

Schematic 1.

Schematic 1.

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Visual representation of the multi-cell model designed to mimic cytotrophoblast-villous endothelium-fetal organ communication. To alter trophoblast response mechanisms prior to co-culture, BeWo cells were treated 1 of 3 ways: growth media + dialyzed FBS and − human insulin-like 1 growth factor (hIGF1) nanoparticle (sham nanoparticle); growth media − FBS and − hIGF1 nanoparticle; growth media − FBS and + hIGF1 nanoparticle. After 24 h in treatments (designated 0 h), BeWo cells were placed into the apical transwell chamber of a porous transwell insert. + FBS − hIGF1 nanoparticle BeWo cells were provided media with dialyzed FBS and without nanoparticle. − FBS − hIGF1 nanoparticle and − FBS + hIGF1 nanoparticle BeWo cells were provided media without FBS and without nanoparticle. Human placenta microvascular endothelial cells (HPMVECs) had been grown onto the under-side of the transwell insert for 24 h prior to co-culture. Human fetal liver hepatocytes (WRL68) or human fetal kidney epithelial cells (HEK293T/17) had been placed into the basal chamber of the culture plate for 24 h prior to co-culture. Media in the basal chamber was WRL68 or HEK293T/17 culture media with dialyzed FBS. Cells were co-cultured together for 24 h and 48 h. Culture media from both chambers was collected at 0 h, 24 h and 48 h. BeWo and WRL68 or HEK293T/17 cells were collected at 0 h, 24 h and 48 h to analyzed gene and protein expression.

2. Materials and methods

2.1. Cell culture reagents

Gibco’s Ham’s F-12K (Kaighn’s) Medium, Gibco’s DMEM (Dulbecco’s Modified Eagle Medium), Gibco’s heat-inactivated Fetal Bovine Serum (FBS), Gibco’s Trypsin-EDTA (0.05 %) and Gibco’s Phosphate Buffered Saline (PBS) were purchased from Thermo Fisher Scientific. Corning’s 0.4 μm, 12 mm, 12-well Transwell Permeable Polycarbonate Membrane Inserts and Plates, Corning’s Penicillin-Streptomycin solution and Cell Application’s Attachment Factor solution were purchased from Fisher Scientific. HyClone™ Dialyzed FBS was purchased from Cytiva. 20 mm sterile, round cover slips were obtained from Quality Biological.

2.2. Nanoparticle formation

PHPMA115-b-PDMEAMA115 co-polymer was synthesized and char-acterized by Dr. Mukesh Gupta (Vanderbilt University) [26]. Plasmids were cloned from a pEGFP-C1 plasmid (Clonetech Laboratories). For the sham nanoparticle, the CMV promotor was replaced by a CYP19A1 promotor and the GFP gene was replaced by a non-coding Antisense GFP gene. For the hIGF1 nanoparticle, the CMV promotor was replaced by a CYP19A1 promotor and the GFP gene was replaced by a human IGF1 gene. Lyophilized PHPMA115-b-PDMEAMA115 co-polymer was reconstituted (10 mg/mL) in sterile saline. Nanoparticles were formed by combining 10 μg of plasmid with 20 μL reconstituted polymer and made to a total volume of 200 μL with sterile saline under aseptic conditions at room temperature.

2.3. Cell culture

BeWo (CCL98), WRL68 (CL48) and HEK293T/17 (CRL11268) cell lines were purchased from ATCC. Human Placenta Micro-Vascular Endothelial Cells (HPMVEC) were isolated from a normal term placenta as previously reported [33]. BeWo (passages 3–8) cells were maintained in Ham’s F-12 media with 10 % FBS and 1 % penicillin-streptomycin. WRL68 and HEK293T/17 cells (both passages 3–8) were maintained in DMEM media with 10 % FBS and 1 % penicillin-streptomycin. HPMVECs (passages 3–8) were maintained on flasks pre-coated with Attachment Factor in DMEM media with 20 % FBS and 1 % penicillin-streptomycin. All cells were incubated under humidity at 37 °C and 5 % CO2.

2.4. Experimental procedure

The experimental procedure is outlined in Schematic 1. All experiments were performed under aseptic conditions. On day 1, 1×106 BeWo cells were seeded onto T25 flasks under the following conditions: + FBS − hIGF1 = Ham’s F-12 media +10 % dialyzed FBS (depleted from low-molecular weight components of <10,000 MW) and 1 % penicillin-streptomycin, − FBS − hIGF1 and − FBS + hIGF1 = Ham’s F-12 media with 1 % penicillin-streptomycin only. Cells were allowed to adhere for 4 h before sham nanoparticle (+ FBS − hIGF1 and − FBS − hIGF1) or hIGF1 nanoparticle (− FBS + hIGF1) was added. 5×105 HPMVECs were seeded onto the under-side of the transwell insert that had been pre-coated with Attachment Factor in 200 μL of normal growth media. Transwell inserts with the HPMVECs were transferred to the incubator with the transwell inserts inverted for 24 h to allow for attachment of the cells. 1×105 WRL68 or HEK293T/17 cells were seeded into the bottom of a 12-well plate and culture in DMEM media with 10 % dialyzed FBS and 1 % penicillin-streptomycin. On day 2, the culture media covering the HPMVECs was removed and discarded. Culture media from the WRL68 or HEK293T/17 cells was removed, collected and designated timepoint 0 (T0). The HPMVEC transwell inserts were returned to standard culture position within the 12-well plates containing the WRL68 or HEK293T/17 cells. Fresh DMEM media with 10 % dialyzed FBS and 1 % penicillin-streptomycin was placed into the basolateral chamber of the plate. For the BeWo cells, culture media was removed, collected and designated T0. Cells were washed briefly with PBS to remove any remaining nanoparticles not endocytosed by the cells. BeWo cells were then trypsinized and 1×105 cells were seeded into the inner chamber of the HPMVEC transwell insert. For + FBS − hIGF1, fresh Ham’s F-12 media with 10 % dialyzed FBS and 1 % penicillin-streptomycin was added to the apical transwell insert chamber. For − FBS − hIGF1 and − FBS + hIGF1, fresh Ham’s F-12 media with 1 % penicillin-streptomycin only was added to the apical transwell insert chamber. Remaining BeWo cells not seeded into the transwell insert were collected for RNA extraction and confirmation of increased IGF1 gene expression. On day 3 (T24) and day 4 (T48), BeWo and WRL68 or HEK293T/17 cells were collected for RNA extraction and protein extraction. WRL68 or HEK293T/17 cells cultured on cover-slips placed in the bottom of the 12-well plate prior to seeding on day 1 were fixed for morphological analysis. Media from both the apical transwell insert chamber (BeWo) and basolateral transwell chamber (HPMVEC with WRL68 or HEK293T/17) was also collected at T24 and T48.

2.5. RNA extractions and quantitative PCR (qPCR)

After media was collected from the apical and basolateral transwell insert chambers, HPMVEC cells were removed from the underside of the transwell insert using a clean Kimwipe to prevent potential contamination. BeWo and WRL68 or HEK293T/17 cells were washed with PBS and lysed with 350 μL of RLT buffer (Qiagen). RNA was extracted using the RNeasy Mini Plus kit (Qiagen) following standard manufacturers protocol. A total of 1 μg of RNA was converted to complementary DNA (cDNA) using the High-capacity cDNA Reverse Transcription kit (Applied Biosystems) and diluted to 1:100. For qPCR, 2.5 μl of cDNA was mixed with 10 μl of PowerUp SYBR green (Applied Biosystems), 1.2 μl of primers at a concentration of 10 nM, and water to make up a total reaction volume of 20 μl. Genes assessed are provided in Supplemental Table S1. Stability of reference genes ACTB, TBP, HPRT and B2M in the cells was determined using Normfinder [34], and gene expression was normalized to the geometric mean of all four. Reactions were performed using the Quant3 Real-Time PCR System (Applied Biosystems), and relative mRNA expression calculated using the comparative CT method with the Design and Analysis Software v2.6.0 (Applied Biosystems).

2.6. Western blot

BeWo and WRL68 or HEK293T/17 cells were prepared for protein extractions as described above for RNA extractions. Cells were lysed in 300 μL of RIPA buffer containing protease and phosphatase inhibitors. Lysates were centrifuged, supernatant collected and protein concentrations determined using Pierce™ Coomassie Plus Assay Kit (Thermo Fisher Scientific) following manufacturer’s protocol. 15–20 μg of protein was mixed with Bolt® SDS Loading Buffer (Invitrogen) and Reducing Agent (Invitrogen) and denatured by heating at 95 °C for 10 min. The lysates and a pre-stain protein ladder (PageRuler, Thermo Fisher Scientific) were then run on a 4–12 % Tris-Bis precast gel (Invitrogen) following manufacturers protocols and transferred onto nitrocellulose membranes using the Bolt Mini Electrophoresis unit (Invitrogen). Membranes were placed into 5 % skim-milk in Tris-buffered Saline containing Tween 20 (TBS-T) and incubated overnight at 4 °C. Primary antibodies are provided in Supplemental Table S1 and were applied for 2 h at room temperature or overnight at 4 °C. The membranes were then washed 3 times in fresh TBS-T, and then further incubated with a HRP conjugated secondary (Cell Signaling 7074 and 7076, 1:2000) for 2 h at room temperature. Protein bands were visualized by chemiluminescence using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) on the ChemiDoc Imager (Bio-Rad) and signal intensity of the protein bands calculated using Image Lab software (version 6.1, Bio-Rad), normalized to bActin.

2.7. Immunofluorescence

WRL68 or HEK293T/17 cells co-cultured with BeWo and HPMVEC and grown on coverslips were fixed with ice-cold 50 % ethanol:50 % methanol for 10 min and dried at room temperature. Coverslips were then washed with PBS and antigen retrieval performed by incubating in 3 % BSA and 0.1 % Triton-X in PBS for 10 min at room temperature. After washing again in PBS, a protein block (Animal-free Block, Thermo Fisher Scientific) was applied to the coverslips for 15 min at room temperature before incubating in primary antibodies (WRL68: Vimentin and Ki67. HEK293T/17: Vimentin and ZO1. Supplemental Table S1) for 1 h at 37 °C. Primary antibodies were washed off with PBS and coverslips were further incubated with Alexa-488 and Alexa-647 secondary antibodies (Invitrogen) and 4′,6-diamidino-2-phenylindole (DAPI) for 1 h at room temperature. Coverslips were then mounted onto microscope slides with Prolong Diamond Antifade mountant (Thermo Fisher Scientific). Staining was visualized using the AxioScan Scanning Microscope (Zeiss) and Zen Imaging software, v3.9 (Zeiss).

2.8. Media analysis

Glucose and lactate in BeWo and WRL68 or HEK293T/17 culture media was measured using the YSI model 2700 Glucose/Lactate Analyzer following standard protocols and normalized to total protein.

2.9. Statistics

All experiments were performed in triplicate on 6 independent passages (n = 6 passages). All statistical analyses were performed using SPSS Statistics 29 software. Distribution assumptions were checked with a Q-Q-Plot. Statistical significance was determined using Generalized Linear Modelling with treatment of the BeWo cells (+FBS − hIGF1, − FBS − hIGF1 or − FBS + hIGF1) the main effect. Statistical significance was considered at P ≤ 0.05. For statistically significant results, a Bonferroni post hoc analysis was performed. Results are reported as estimated marginal means ± standard error of the mean (SEM).

3. Results

3.1. Expression of cell stress markers were elevated in BeWo cells after 24h culture in media without FBS and prevented with hIGF1 over-expression

We analyzed expression of cell stress, DNA damage markers and glucose metabolism-related factors after 24 h in BeWo cells cultured without FBS and treated with hIGF1 nanoparticle. IGF1 gene expression increased after 20 h of hIGF1 nanoparticle treatment in − FBS + hIGF1 BeWo cells when compared to + FBS − hIGF1 and − FBS − hIGF1 BeWo cells and remained increased throughout the co-culture experiments (Supplemental Fig. S1A and S1B). IGF2 expression was similar between + FBS − hIGF1, − FBS − hIGF1 and − FBS + hIGF1 and did not change across the co-culture period (Supplemental Fig. S1C and S1D). FBS deprivation (− FBS − hIGF1 BeWo cells) increased gene expression of NFE2L2 and TP53 (cell stress markers), SOD1 and SOD2 (antioxidants) and protein expression of H2A.X(S139) (DNA damage marker) and when compared to +FBS − hIGF1 (Fig. 1A–E, respectively). Protein expression of Catalase trended towards an increase when compared to + FBS − hIGF1 (Fig. 1F).

Fig. 1. Expression of cellular stress markers in BeWo cells following 24 h culture in no FBS media and 20 h of sham or insulin-like 1 growth factor (hIGF1) nanoparticle treatment.

Fig. 1.

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A. Gene expression of cellular defense transcription factor NFE2L2 was increased in − FBS − hIGF1 BeWo cells when compared + FBS − hIGF1 and − FBS + hIGF1. B. Gene expression of cell stress response transcription factor TP53 was increased in − FBS − hIGF1 BeWo cells when compared + FBS − hIGF1 and trended towards and increase when compared to − FBS + hIGF1. C. Gene expression of antioxidant SOD1 was increased in − FBS − hIGF1 and − FBS + hIGF1 BeWo cells when compared to + FBS − hIGF1. D. Gene expression of antioxidant SOD2 was increased in − FBS − hIGF1 BeWo cells when compared + FBS − hIGF1 and − FBS + hIGF1. E. Protein expression of DNA damage marker H2A.X(S139) was increased in − FBS − hIGF1 and − FBS + hIGF1 BeWo cells when compared to + FBS − hIGF1. Representative Western blot is provided. F. Protein expression of antioxidant Catalase trended towards an increase in − FBS − hIGF1 BeWo cells when compared to + FBS − hIGF1 and − FBS + hIGF1. Data are estimated marginal mean ± SEM calculated using generalized linear modelling. n = 12 independent passages. *P < 0.05; **P < 0.01; ***P < 0.001. NFE2L2: nuclear factor, erythroid 2-like 2. TP53: tumor protein 53. SOD1/2: superoxide dismutase 1/2. H2A.X(S139): phospho- Histone H2A.X. Representative Western blot images: +− =+ FBS − hIGF1, − = − FBS − hIGF1, −+= − FBS + hIGF1.

hIGF1 nanoparticle gene therapy in FBS-deprived BeWo cells (− FBS + hIGF1) normalized NFE2L2 and TP53 to + FBS − hIGF1 levels (Fig. 1A and B, respectively). SOD1 expression remained elevated when compared to + FBS − hIGF1 (Fig. 1C) while SOD2 expression was not increased and similar to + FBS − hIGF1 (Fig. 1D). H2A.X(S139) expression was elevated compared to + FBS − hIGF1 (Fig. 1E) whilst Catalase protein expression trended toward a decrease compared to − FBS − hIGF1 (Fig. 1F). There was no difference in protein expression of antioxidant Thioredoxin with either Starvation or hIGF1 nanoparticle treatment and protein expression of cleaved-PARP was not detected (data not shown).

3.2. Stressed BeWo cells utilized less glucose from the media and produced more lactate after 24 h in culture, which was not prevented by IGF1 over-expression

Glucose concentration in BeWo media with FBS and without FBS prior to culturing with cells was similar (60.8 mg/dL or 3.4 mM vs. 63.7 mg/dL or 3.5 mM). Bewo media with FBS contained trace amounts of lactate (0.14 mg/dL) whilst BeWo media without FBS contained no lactate. After 24 h, FBS-deprived BeWo cells (both with and without hIGF1 nanoparticle gene therapy) showed 25 % less glucose utilization compared to + FBS − hIGF1 BeWo cells (42 % decreased glucose concentration in the media)(Table 1). Lactate production was higher in FBS-deprived cells (− FBS − hIGF1: 19.8 mg/dL; − FBS + hIGF1: 20.28 mg/dL) compared to + FBS − hIGF1 (15.36 mg/dL)(Table 1).

Table 1.

Concentration of glucose and lactate in media.

BeWo-WRL68 Co-Culture
Glucose (mg/dL normalized to total protein) Lactate (mg/dL normalized to total protein)
+ FBS − hIGF1 − FBS − hIGF1 − FBS + hIGF1 + FBS − hIGF1 − FBS − hIGF1 − FBS + hIGF1
EMM SEM EMM SEM EMM SEM EMM SEM EMM SEM EMM SEM
Media Only Ham’s F12 60.80 63.70 63.70 0.14 0.00 0.00
DMEM 213.00 213.00 213.00 0.19 0.19 0.19
T0 Apical 35.55 2.47 47.70a 3.32 48.91a 3.40 15.36 1.47 19.80a 1.80 20.28a 1.94
Basal 193.45 11.83 193.45 11.83 193.45 11.83 4.22 0.22 4.22 0.22 4.22 0.22
T24 Apical 354.62 36.73 411.79a 33.00 400.77a 31.86 45.81 5.33 55.38a 6.44 50.58 5.88
Basal 276.49 19.21 302.72 21.03 271.98 18.89 27.60 2.23 26.49 2.14 25.30 2.04
T48 Apical 281.06 13.66 322.78a 15.68 298.51 14.51 85.11 4.33 84.14 4.28 78.65 4.00
Basal 154.83 7.26 163.45 7.66 148.88 6.98 46.60 2.05 45.11 1.98 41.76 1.83
BeWo-HEK293T/17 Co-Culture
Glucose (mg/dL normalized to total protein) Lactate (mg/dL normalized to total protein)
+ FBS − hIGF1 − FBS − hIGF1 − FBS + hIGF1 + FBS − hIGF1 − FBS − hIGF1 − FBS + hIGF1
EMM SEM EMM SEM EMM SEM EMM SEM EMM SEM EMM SEM
Media Only Ham' s F12 60.80 63.70 63.70 0.14 0.00 0.00
DMEM 213.00 213.00 213.00 0.19 0.19 0.19
T0 Apical 35.55 2.47 47.70a 3.32 48.91a 3.40 15.36 1.47 19.80a 1.80 20.28a 1.94
Basal 161.97 17.87 161.97 17.87 161.97 17.87 2.66 0.33 2.66 0.33 2.66 0.33
T24 Apical 338.35 22.33 294.13a 19.41 382.16a,b 25.22 32.19 2.83 25.52a 2.24 31.48b 2.77
Basal 203.75 18.16 214.21 19.09 225.60 20.10 16.07 1.63 15.30 1.55 16.43 1.66
T48 Apical 298.09 36.34 277.75 33.86 287.32 35.02 61.89 5.46 51.42a 4.71 58.50b 5.16
Basal 117.70 9.87 119.89 10.89 115.12 9.66 25.68 1.38 25.95 1.39 24.93 1.34
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EMM = estimated marginal means; SEM = standard error of mean.

a

significantly different (P < 0.05) to + FBS − hIGF1 of the same row.

b

significantly different (P < 0.05) to − FBS − hIGF1 of the same row.

In FBS-deprived BeWo cells, glucose transporter SLC2A1 expression was similar to those not deprived of FBS but trended for an increase when compared hIGF1 nanoparticle gene therapy treated BeWo cells (Fig. 2A). Gluconeogenesis enzyme G6PC expression increased in both FBS-deprived groups (with/without hIGF1) compared to + FBS − hIGF1 (Fig. 2B). hIGF1 nanoparticle gene therapy increased FBP1 and PCK2 compared to sham nanoparticle gene therapy treated groups (Fig. 2C and D, respectively). PCK1 mRNA was not detected (no amplification after 40 qPCR cycles) in BeWo cells (data not shown). Glycolysis enzymes expression (HK2, PFKP and LDHA) was similar between sham nanoparticle gene therapy treated BeWo cells but increased with hIGF1 nanoparticle gene therapy (Fig. 2E–G, respectively). LDHB and LDHC expression increased in both FBS-deprived groups compared to + FBS − hIGF1 (Fig. 2H and I, respectively).

Fig. 2. Gene expression of glucose metabolism-related factors in BeWo cells following 24 h culture in no FBS media and 20 h of sham or insulin-like 1 growth factor (hIGF1) nanoparticle treatment.

Fig. 2.

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A. Gene expression of glucose transporter SLC2A1 in − FBS − hIGF1 BeWo cells was similar to + FBS − hIGF1 BeWo cells but trended for an increase when compared to − FBS + hIGF1 BeWo cells. B. Gene expression of gluconeogenesis enzyme G6PC was increased in − FBS − hIGF1 and − FBS + hIGF1 BeWo cells compared to + FBS − hIGF1. C. PCK2 expression was increased in − FBS + hIGF1 BeWo cells compared to + FBS − hIGF1 and − FBS − hIGF1. D. FBP1 expression was increased in − FBS + hIGF1 BeWo cells compared to + FBS − hIGF1 and − FBS − hIGF1. E. Gene expression of glycolysis enzymes HK2 was similar between + FBS − hIGF1 and − FBS − hIGF1 BeWo cells but increased in − FBS + hIGF1. F. Gene expression of PFKP was similar between + FBS − hIGF1 and − FBS − hIGF1 BeWo cells but increased in − FBS + hIGF1. G. Gene expression of LDHA was similar between + FBS − hIGF1 and − FBS − hIGF1 BeWo cells but increased in − FBS + hIGF1. H. Expression of LDHB was increased in − FBS − hIGF1 and − FBS + hIGF1 BeWo cells compared to + FBS − hIGF1. I. Expression of LDHC was increased in − FBS − hIGF1 and − FBS + hIGF1 BeWo cells compared to + FBS − hIGF1. Data are estimated marginal mean ± SEM calculated using generalized linear modelling. n = 12 independent passages. *P < 0.05; **P < 0.01; ***P < 0.001. SLC2A1: solute carrier family 2 member 1. G6PC: glucose-6-phosphatase. FBP1: Fructose-1,6-bisphosphatase 1. PCK2: Phosphoenolpyruvate carboxykinase 2. HK2: hexokinase 2. PFKP: Phosphofructokinase, platelet. LDHA/B/C: lactate dehydrogenase A/B/C.

3.3. Increasing cell stress mechanisms or over-expressing IGF1 in BeWo cells had no impact on WRL68 gene express of glucose metabolism-related factors

We assessed expression of glucose metabolism-related factors in the WRL68 cell line after 24 h and 48 h in co-culture with BeWo cells and HPMVECs to determine whether altering trophoblast stress responses impacted fetal hepatocytes. There was no difference in the gene expression of SLC2A1, gluconeogenesis enzymes (G6PC, FBP1, FBP2, PCK1, PCK2), glycolysis enzymes (HK2, PFKP, LDHA, LDHB, LDHC) nor cell stress markers (NFE2L2, TP53) in WRL68 cells co-culture with + FBS − hIGF1, − FBS − hIGF1 or − FBS + hIGF1 BeWo cells (Supplemental Fig. S2). Irrespective of BeWo treatment, WRL68 gene expression of SLC2A1, FBP1, FBP2 and LDHA was reduced after 24 h in co-culture compared to 0 h but increased back to similar levels as 0 h after 48 h in co-culture (Supplemental Fig. S2). Gene expression of G6PC, FBP2, PCK1, PCK2, HK2, PFKP, LDHB, LDHC, NFE2L2 and TP53 was reduced in co-cultured WRL68 cells at 24 h compared to 0 h and remained lower at 48 h compared to 0 h (Supplemental Fig. S2). Morphologically WRL68 cells expanded and were indistinguishable when comparing co-cultured with + FBS − hIGF1, − FBS − hIGF1 and − FBS + hIGF1 BeWo cells (Supplemental Fig. S3).

3.4. Co-culturing BeWo cells with WRL68 cells was associated with reversal of cell stress responses in BeWo cells and reduced gene expression of gluconeogenesis/glycolysis enzymes

To further understand the implications of co-culturing BeWo, HPMVEC and WRL68 cells, we analyzed expression of cellular stress markers and glucose metabolism-related factors in BeWo cells. In + FBS − hIGF1 BeWo cells, co-culture with HPMVECs and WRL68 cells resulted in: reduced expression of NFE2L2 at 24 h and 48 h (Fig. 3A), decreased TP53 expression at 48 h (Fig. 3B), increased SOD1 and SOD2 gene expression at 24 h and 48 h (Fig. 3C & D), stable H2A.X(S139) levels (Fig. 3E) and decreased Catalase protein at 24 h and 48 h (Fig. 3F).

Fig. 3. Gene expression of cellular stress markers in + FBS/− human insulin-like 1 growth factor (hIGF1) nanoparticle BeWo Cells, − FBS/− hIGF1 nanoparticle BeWo cells or − FBS/+ hIGF1 nanoparticle BeWo cells co-cultured with WRL68 cells and HPMVECs.

Fig. 3.

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A. Irrespective of BeWo treatment, gene expression of NFE2L2 was reduced at 24 h in co-culture and remained lower at 48 h compared to 0 h. B. For all BeWo treatments, TP53 expression did not change when comparing 0 h–24 h but was decreased at 48 h compared to 0 h. C. SOD1 in + FBS − hIGF1 BeWo cells was higher at 24 h and 48 h when compared to 0 h. In − FBS − hIGF1 and − FBS + hIGF1 BeWo cells, SOD1 expression was lower at 48 h compared to 0 h and 24 h. D. SOD2 in + FBS − hIGF1 BeWo cells was higher at 24 h and 48 h when compared to 0 h. In − FBS − hIGF1 and − FBS + hIGF1 BeWo cells, SOD2 expression was lower at 48 h compared to 0 h and 24 h. E. In + FBS − hIGF1 BeWo cells, protein expression of H2A.X (S139) remained unchanged across the co-culture with WRL68 cells. H2A.X(S139) expression in − FBS − hIGF1 and − FBS + hIGF1 BeWo cells was lower at 24 h and 48 h compared to 0 h. Representative Western blot image to the right of graph. F. Catalase protein expression decreased at 24 h and 48 h in co-culture when compared to 0 h, irrespective of BeWo treatment. Representative Western blot image to the right of graph. Data are estimated marginal mean ± SEM calculated using generalized linear modelling. n = 6 independent passages. *P < 0.05; **P < 0.01; ***P < 0.001. HPMVEC: human placenta microvascular endothelial cells. NFE2L2: nuclear factor, erythroid 2-like 2. TP53: tumor protein 53. SOD1/2: superoxide dismutase 1/2. H2A.X(S139): phospho- Histone H2A.X. Representative Western blot images: +− =+ FBS − hIGF1, − = − FBS − hIGF1, −+= − FBS + hIGF1.

In FBS-deprived BeWo cells (− FBS − hIGF1), co-culture with HPMVECs and WRL68 cells led to: decreased NFE2L2 expression at 24 h and 48 h that matched + FBS − hIGF1 BeWo cells (Fig. 3A), decreased TP53 and SOD1 expression by 48 h and comparable to levels in + FBS − hIGF1 BeWo cells at 48 h (Fig. 3B & C), an inital SOD2 increase at 24 h followed by a reduction at 48 h (Fig. 3D) and reduced H2A.X(S139) and Catalase protein expression in by 24 h that was comparable to levels in + FBS − hIGF1 BeWo cells at 48 h (Fig. 3E & F).

In co-cultured − FBS + hIGF1 BeWo cells, gene expression of NFE2L2, TP53 and SOD2 and protein expression of H2A.X(S139) and Catalase at 24 h and 48 h changed in a similar manner to − FBS − hIGF1 BeWo cells (Fig. 3). SOD1 gene expression was lower at 48 h and lower than + FBS − hIGF1 BeWo cells at the 48 h timepoint (Fig. 3C).

Gene expression of SLC2A1, G6PC, FBP1, and PCK2 in + FBS − hIGF1 BeWo cells did not change when co-cultured with WRL68 cells (Supplemental Fig. S4). Gene expression of HK2 and PFKP was increased at 24 h and 48 h while LDHA and LDHC were lower at 48 h (Supplemental Fig. S4). In FBS-deprived BeWo cells (with/without hIGF1), gene expression of gluconeogenesis enzymes that were higher at 0 h were reduced by 48 h, and comparable to + FBS − hIGF1 BeWo cells at 48 h (Supplemental Fig. S4). Changes in gene expression of glycolysis enzymes occurred in a similar manner across the co-culture period with HPMVECs and WRL68 cells and were no different from + FBS − hIGF1 BeWo cells at 48 h (Supplemental Fig. S4).

3.5. Co-culture of BeWo and WRL68 cells resulted in increased media glucose concentrations in both the apical and basal transwell insert chambers

In + FBS − hIGF1 BeWo cells co-cultured with HPMVECs and WRL68 cells, there was a ~5X increase in glucose in the apical transwell chamber (BeWo side) and 30 % increase in the basal chamber (WRL68 side) at 24 h (Table 1). Glucose concentration decreased 21 % and 44 % in apical and basal transwell chambers, respectively between 24 h and 48 h (Table 1). Lactate concentrations increased ~32,600 % in the apical transwell chamber and ~14,400 % in the basal chamber at 24 h (Table 1) and continued to increase in both transwell chambers between 24 h and 48 h (Table 1).

In co-cultured FBS-deprived BeWo cells (with/without hIGF1), glucose concentrations were ~5X higher in the apical transwell chamber at 24 h and higher than the apical transwell chamber of + FBS − hIGF1 group. Glucose in the basal chamber was ~42 % higher at 24 h in the − FBS − hIGF1 group but only ~28 % higher in the − FBS + hIGF1 group at 24 h (Table 1). A similar decreasing pattern for glucose between 24 h and 48 h occurred, irrespective of hIGF1 nanoparticle gene therapy. Lactate concentrations increased in both the apical and basal transwell chambers at 24 h and 48 h and in a similar manner to + FBS − hIGF1 group (Table 1).

3.6. Increasing cell stress mechanisms or over-expressing IGF1 in BeWo cells had no impact on gene expression of angiogenic factors in fetal kidney epithelial HEK293T/17 cells

Similar to further understanding the implications of manipulating trophoblast function on fetal hepatocytes, we assessed the expression of important kidney epithelial cell function factors in the HEK293T/17 cell line after 24 h and 48 h in co-culture with BeWo cells. There was no difference in the gene expression of NFE2L2, TP53, SOD1, SOD2, TGFb, VEGF, IGF1R and ACE in HEK293T/17 cells co-culture with + FBS − hIGF1, − FBS − hIGF1 or − FBS + hIGF1 BeWo cells (Supplemental Fig. S5). Irrespective of BeWo manipulation, HEK293T/17 gene expression of NFE2L2, TP53, SOD1, SOD2, TGFb, VEGF and IGF1R was reduced after 24 h in co-culture when compared to 0 h (Supplemental Fig. S5). Gene expression of NFE2L2, TP53, SOD1, SOD2, TGFb was increased at 48 h in co-culture when compared to 24 h. VEGF, IGF1R and ACE gene expression was higher at 48 h in co-culture with BeWo cells when compared to 0 h and 24 h. Morphologically HEK293T/17 cells expanded and were indistinguishable when comparing those co-cultured with + FBS − hIGF1, − FBS − hIGF1 and − FBS + hIGF1 BeWo cells (Supplemental Fig. S6).

3.7. Co-culturing BeWo cells with HEK293T/17 cells increased cell stress responses in BeWo cells and gene expression of gluconeogenesis/glycolysis enzymes

To further elucidate whether signals from fetal cells or media glucose concentrations were more influential on changes to trophoblast gene and protein expression, we assessed expression of cellular stress markers and glucose metabolism-related factors in BeWo cells co-cultured with HPMVECs and HEK293T/17 cells. Co-cultured + FBS − hIGF1 BeWo cells showed: increased NFE2L2 and TP53 expression at 24 h and 48 h (Fig. 4A & B), stable SOD1 and SOD2 gene expression and Catalase protein expression (Fig. 4C, D & 4F) and increased protein expression of H2A.X(S139) from 24 h onwards (Fig. 4D).

Fig. 4. Gene expression of cellular stress markers in + FBS/− human insulin-like 1 growth factor (hIGF1) nanoparticle BeWo Cells, − FBS/− hIGF1 nanoparticle BeWo cells or − FBS/+ hIGF1 nanoparticle BeWo cells co-cultured with HEK293T/17 cells and HPMVECs.

Fig. 4.

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A. In + FBS − hIGF1 and − FBS + hIGF1 BeWo cells, gene expression of NFE2L2 was increased at 24 h and remained higher at 48 h when compared to 0 h NFE2L2 expression did not change in − FBS − hIGF1 BeWo cells across the co-culture. B. In + FBS − hIGF1 and − FBS + hIGF1 BeWo cells, gene expression of TP53 was increased at 24 h and remained higher at 48 h when compared to 0 h. In − FBS − hIGF1 BeWo cells, expression of TP53 was higher at 48 h compared to 0 h and 24 h. C. SOD1 gene expression did not change in + FBS − hIGF1 BeWo cells across the co-culture period. SOD1 expression was decreased at 24 h and 48 h compared to 0 h in − FBS − hIGF1 and − FBS + hIGF1 co-cultured BeWo cells. D. SOD2 gene expression did not change across the co-culture period for all BeWo treatments. E. Protein expression of H2A.X(S139) increased in at 48 h when compared to 0 h for all BeWo treatments. Representative Western blot image to the right of graph. F. Catalase protein expression did not change across the co-culture period in + FBS − hIGF1 and − FBS + hIGF1 BeWo cells. Catalase expression was reduced in the − FBS − hIGF1 BeWo cells at 24 h and 48 h compared to 0 h. Representative Western blot image to the right of graph. Data are estimated marginal mean ± SEM calculated using generalized linear modelling. n = 6 independent passages. *P < 0.05; **P < 0.01; ***P < 0.001. HPMVEC: human placenta microvascular endothelial cells. NFE2L2: nuclear factor, erythroid 2-like 2. TP53: tumor protein 53. SOD1/2: superoxide dismutase 1/2. H2A.X(S139): phospho- Histone H2A.X. Representative Western blot images: +− =+ FBS − hIGF1, – = − FBS − hIGF1, −+= − FBS + hIGF1.

In FBS-deprived (− FBS − hIGF1) BeWo cells there was: sustained elevated NFE2L2 expression comparable to elevated levels in + FBS − hIGF1 BeWo cells (Fig. 4A), further increased TP53 by 48 h that was higher than + FBS − hIGF1 BeWo cells at 48 h (Fig. 4B), reduced SOD1 expression at 24 h and 48 (Fig. 4C), no change in SOD2 expression (Fig. 4D) and increased H2A.X(S139) protein expression and decreased Catalase protein expression by 48 h (Fig. 4E & F).

Gene expression changed with co-culture in FBS-deprived hIGF1 nanoparticle gene therapy (− FBS + hIGF1) BeWo cells matched + FBS − hIGF1 BeWo cells except for reduced SOD1 expression at 24 h and 48 h (Fig. 4C).

SLC2A1 increased progressively in BeWo cells co-cultured with HPMVECs and HEK293T/17 cells from 24 h, irrespective of BeWo treatment (Fig. 5A). + FBS − hIGF1 BeWo cells showed increased expression of G6PC, FBP1, PCK2, HK2, PFKP, LDHA, LDHB, and LDHC by 48 h (Fig. 5B–I). FBS-deprived cells (with/without hIGF1) maintained elevated G6PC expression (Fig. 5B). Gene expression of FBP1 and PCK2 were higher in − FBS − hIGF1 BeWo cells by 48 h, but unchanged − FBS + hIGF1 at 24 h and 48 h (Fig. 5C & D).

Fig. 5. Gene expression of glucose metabolism-related factors in + FBS/− human insulin-like 1 growth factor (hIGF1) nanoparticle BeWo Cells, − FBS/− hIGF1 nanoparticle BeWo cells or − FBS/+ hIGF1 nanoparticle BeWo cells co-cultured with HEK293T/17 cells and HPMVECs.

Fig. 5.

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A. Gene expression of SLC2A1 was increased in BeWo cells co-cultured with HEK293T/17 cells at 24 h, and further again at 48 h when compared to 0 h, irrespective of BeWo treatment. B. In + FBS − hIGF1 BeWo cells, gene expression of G6PC increased at 24 h when compared to 0 h and remained higher at 48 h compared to 0 h. In − FBS − hIGF1 and − FBS + hIGF1 BeWo cells, G6CP gene expression did not change at 24 h and 48 h compared to 0 h. C. In + FBS − hIGF1 and − FBS − hIGF1 BeWo cells, gene expression of FBP1 increased at 48 when compared to 0 h and 24 h. FBP1 expression was lower in − FBS + hIGF1 BeWo cells at 24 h compared to 0 h and 48 h. D. In + FBS − hIGF1 and − FBS − hIGF1 BeWo cells, PCK2 increased at 24 h compared to 0 h, and remained increased at 48 h compared to 0 h. PCK2 expression was higher in − FBS + hIGF1 BeWo cells at 24 h compared to 0 h and 48 h. E. In + FBS − hIGF1 and − FBS − hIGF1 BeWo cells, HK2 increased at 48 h when compared to 0 h and 24 h. F. In + FBS − hIGF1 BeWo cells, PFPK increased at 48 h when compared to 0 h and 24 h. In − FBS − hIGF1 BeWo cells, PFKP expression increased at 48 h compared to 0 h and 24 h. Expression of PFKP in − FBS + hIGF1 BeWo cells remained unchanged across the co-culture period. G. In + FBS − hIGF1 BeWo cells, LDHA increased at 24 h when compared to 0 h, and remained increased at 48 h compared to 0 h. In − FBS − hIGF1 BeWo cells, LDHA increased at 48 h compared to 0 h. In − FBS + hIGF1 BeWo cells, LDHA decreased at 48 h compared to 24 h. H. In + FBS − hIGF1 BeWo cells, LDHB increased at 24 h when compared to 0 h, and remained increased at 48 h compared to 0 h. In − FBS − hIGF1 BeWo cells, LDHB decreased at 24 h compared to 0 h. In − FBS + hIGF1 BeWo cells, LDHB decreased at 24 h and 48 h compared to 0 h. I. In + FBS − hIGF1 BeWo cells, LDHC increased at 24 h when compared to 0 h, and remained increased at 48 h compared to 0 h. In − FBS − hIGF1 BeWo cells, LDHC increased at 48 h compared to 0 h. In − FBS + hIGF1 BeWo cells, LDHC increased at 24 h compared to 0 h. Data are estimated marginal mean ± SEM calculated using generalized linear modelling. n = 6 independent passages. *P < 0.05; **P < 0.01; ***P < 0.001. HPMVEC: human placenta microvascular endothelial cells. SLC2A1: solute carrier family 2 member 1. G6PC: glucose-6-phosphatase. FBP1: Fructose-1,6-bisphosphatase 1. PCK2: Phosphoenolpyruvate carboxykinase 2. HK2: hexokinase 2. PFKP: Phosphofructokinase, platelet. LDHA/B/C: lactate dehydrogenase A/B/C.

3.8. Co-culture of BeWo and HEK293T/17 cells resulted in increased media glucose concentrations in the apical insert chamber but not the basal chamber

In + FBS − hIGF1 BeWo cells co-cultured with HPMVECs and HEK293T/17, glucose concentrations in the apical transwell chamber (BeWo side) increased ~4.5X at 24 h and decreased 12 % by 48 h (Table 1). Glucose concentrations in the basal chamber (HEK293T/17) increased only 4 % at 24 h and decreased 42 % by 48 h (Table 1). Lactate concentrations increased ~22,900 % (apical) and ~8300 % (basal) by 24 h and further increased 92 % (apical) and 60 % (basal) from 24 h to 48 h (Table 1).

In FBS-deprived (− FBS − hIGF1) BeWo cells, glucose concentrations increased ~3.6X in the apical chamber at 24 h and were lower compared to + FBS − hIGF1; there was no change in basal chamber (Table 1). Glucose concentrations decreased 6 % (apical) and 44 % (basal) between 24 h and 48 h (Table 1). There was lower initial lactate production (25 mg/dL in apical transwell chamber) compared to + FBS − hIGF1 at 24 h and by 48 h, lactate in the apical transwell chamber remained lower (Table 1) Lactate in the basal chamber matched + FBS − hIGF1 (Table 1).

With hIGF1 nanoparticle gene therapy, glucose in the apical transwell chamber at 24 h was higher (~5X) compared to other groups (Table 1). Basal chamber glucose concentration changed similar to − FBS − hIGF1 (Table 1). By 48 h glucose and lactate concentrations in both transwell chambers were comparable to other groups (Table 1).

4. Discussion

Fetal growth requires multi-directional maternal-placental-fetal communication, and dysfunction in any aspect of this communication can result in perturbation of fetal development with life-long consequences. In this proof-of-concept study, we aimed to assess the impacts of altered trophoblast stress response mechanisms and trophoblast-specific hIGF1 nanoparticle treatment on fetal liver hepatocytes and fetal kidney epithelial cells. We confirmed that after 24 h culture in media without FBS, expression levels of trophoblast cellular stress mechanisms were elevated as well as expression of gluconeogenesis rate-limiting enzymes (Fig. 6A). When trophoblast cells were stressed but IGF1 over-expressed with a transient hIGF1 nanoparticle gene therapy, cellular stress mechanisms were not increased, however upregulation of enzymes in the gluconeogenesis and glycolysis pathways remained (Fig. 6B). Our original hypothesis was that altering trophoblast stress mechanisms would result in changes to placental-fetal signaling that would influence gene and protein expression in fetal liver and kidney cells; this was not observed. Unexpectedly, we found the potential for trophoblast to not only transport glucose but also produce glucose, which goes beyond the scope of the current manuscript but worth further investigation. Overall, this study furthers our understanding of the mechanisms linking placental function and fetal glucose status in fetal developmental programming and shaping long-term health outcomes.

Fig. 6. Summary of gene changes in the gluconeogenesis/glycolysis pathway in BeWo cells.

Fig. 6.

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A. Summary of gene expression changes (purple arrows/equals) in gluconeogenesis/glycolysis pathway genes in BeWo cells cultured for 24 h in media without FBS compared to BeWo cells cultured in media with FBS. B. Summary of gene expression changes in gluconeogenesis/glycolysis pathway genes in BeWo cells cultured for 24 h in media without FBS and with human insulin-like 1 growth factor (hIGF1) nanoparticle gene therapy compared to BeWo cells cultured in media with FBS (purple arrows/equals) and compared to BeWo cells cultured in media without FBS (orange arrows/equals). SLC2A1: solute carrier family 2 member 1. G6PC: glucose-6-phosphatase. FBP1: Fructose-1,6-bisphosphatase 1. PCK2: Phosphoenolpyruvate carboxykinase 2. HK2: hexokinase 2. PFKP: Phosphofructokinase, platelet. LDHA/B/C: lactate dehydrogenase A/B/C. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Placental insufficiency is often associated with dysfunctional trophoblast phenotypes [35]. Increased oxidative stress and defects in trophoblast proliferation and differentiation pathways are pathogenic hallmarks. In the current study, we confirmed that culturing BeWo cells for 24 h in media without the support of FBS resulted in increased expression of cell stress and DNA damage markers as well as reduced utilization of glucose from the media. Increased expression of gluconeogenesis and glycolysis enzyme genes in the trophoblast was also observed which has also been shown in term placentas and associated with mitochondrial dysfunction [36,37]. We speculate that, upregulation in the gene expression of these gluconeogenesis/glycolysis enzymes is a cell response to redirect resources from the TCA cycle to other processes, however further investigations beyond the scope of the current manuscript are required. Increased expression of IGF1 with the hIGF1 gene therapy increased expression of additional enzymes within the gluconeogenesis and glycolysis pathways whilst also preventing increased expression of cell stress markers. We have previously shown that hIGF1 gene therapy protected against both increased cell death and decreased mitochondrial activity in BeWo cells stressed with hydrogen peroxide [29]. These current data confirm the positive effect of increasing IGF1 expression on molecular mechanisms in stressed trophoblast cells.

The original aim of this study was to determine how increased trophoblast stress would result in changes to placental-fetal signaling that would influence gene and protein expression in fetal liver and kidney cells. In our BeWo-HPMVEC-fetal liver WRL68 co-culture model we identified a significant increase in glucose within the apical transwell BeWo culture media within 24 h of co-culture which was higher when − FBS − hIGF1 BeWo cells were present. This was likely due to the transport of glucose, facilitated by specific glucose transporters like SLC2A1/GLUT1 in the BeWo down the concentration gradient from the high glucose media in the basal compartment [38]. In addition, liver hepatocytes can produce glucose via gluconeogenesis [39]. Whilst we can only speculate that the increased glucose within the co-culture media at 24 h was due to WRL68 production, we hypothesize that there is signaling from the BeWo cells that rapidly increases glucose production within the WRL68 cells resulting in alleviation of cell stress mechanisms within the BeWo cells. We chose not to reduce glucose in the WRL68 culture media as our aim was to specifically look at manipulation of the trophoblast. However, whether similar outcomes would be observed if glucose was also lower in the basal chamber of the co-culture system is an interesting future direction.

Co-culture of BeWo, HPMVECs and fetal kidney epithelial HEK293T/17 cells showed no difference in HEK293T/17 morphology or expression of key genes relating to angiogenesis and blood pressure regulation with BeWo manipulation. However, whilst co-cultured BeWo and WRL68 cells showed decreased cellular stress mechanisms in − FBS − hIGF1 BeWo cells, cellular stress mechanisms were increased in + FBS − hIGF1 BeWo cells when co-cultured with HEK293T/17. Additionally, there was a significant increase in glucose the apical transwell media within 24 h of co-culture that was associated with increased gene expression of glycolysis and gluconeogenesis enzymes in BeWo cells by 48 h. Upregulation of glycolysis and gluconeogenesis enzymes expression was not observed when BeWo cells were co-cultured with WRL68 cells and thus provides evidence for the possibility that trophoblast cells, in addition to transporting glucose, can produce glucose. The idea that trophoblast can produce glucose in addition to transporting it is not novel but often debated [40]. Efforts to determine whether the placenta has the capacity to produce glucose have focused on G6PC, the key enzyme which catalyzes the terminal reaction of glucose from glucose-6-phosphate [41]. Whilst most highly expressed and active in the liver, G6PC activity is not liver-exclusive and has been found in other tissues including the placenta [42]. It is also possible that in this study, increased glucose in the apical transwell media is from BeWo transport of glucose from stores within the HPMVECs and/or HEK293T/17 cells and requires further investigation beyond the current scope.

One of the strengths of this study was the utilization of a culture system which mimics the cytotrophoblast-endothelial environment. The structure of the chorionic villous tissue is complex, requiring multiple cell types with highly specialized functions to work cooperatively to coordinate nutrient, oxygen and waste exchange between maternal and fetal circulations. Nutrients from the maternal blood and trophoblast-derived endocrine factors must cross a layer of endothelial cells prior to entering the placental-fetal circulation, hence we chose to include HPMVECs within the culture system. However, our hypothesis centered on the impact of ‘stressing’ (culturing in media without FBS) or ‘stressing’ and treating trophoblast with a hIGF1 gene therapy on fetal liver and kidney cells. The HPMVECs only presented as an intermediary cell type to better recapitulate the in vivo situation. As we did not collect or analyze the HPMVECs, we cannot be certain of their influence on the outcomes observed in both the BeWo and WRL68 or HEK293T/17 cells. As this was a proof-of-concept study, further investigations into the absolute role of the HPMVECs or whether outcomes would be different if syncytialized BeWo cells or isolated primary trophoblast cells were incorporated are beyond the scope but present interesting future directions.

In conclusion, we hypothesized that increased trophoblast stress would result in changes to placental-fetal signaling that would influence gene and protein expression in fetal liver and kidney cells. Our data did not support this hypothesis but instead demonstrated that cytotrophoblast, under culture conditions without FBS, activate mechanisms involved in glucose production. Whether this is reflected in vivo remains uninvestigated but may represent a placental compensation mechanism in complicated pregnancies. Most importantly, this study highlights the complexities of the placenta and the often-overlooked challenges in studying such a dynamic and multifaceted system.

Supplementary Material

Supplemental Material

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.placenta.2025.05.029.

Funding

This study was funded by Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) awards K99HD109458 (RLW) and R01HD090657 (HNJ).

Footnotes

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

CRediT authorship contribution statement

Helen N. Jones: Writing – review & editing, Resources, Funding acquisition. Alyssa Williams: Methodology, Investigation. Rebecca L. Wilson: Writing – original draft, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

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