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World J Stem Cells. Jun 26, 2026; 18(6): 116526
Published online Jun 26, 2026. doi: 10.4252/wjsc.116526
Therapeutic effects of human amniotic epithelial cell-derived and umbilical cord blood mesenchymal stem cell-derived exosomes on preeclamptic rats
Lu-Yao Han, Wen-Zhu Zhang, Yu Zhang, Wan Zhong, Jing-Li Sun, Jian Shen, Department of Obstetrics and Gynecology, General Hospital of Northern Theater Command (Heping Campus), Shenyang 110000, Liaoning Province, China
Lu-Yao Han, China Medical University, Shenyang 110000, Liaoning Province, China
ORCID number: Wen-Zhu Zhang (0000-0001-7040-3268); Jing-Li Sun (0000-0003-1285-7645); Jian Shen (0000-0002-3374-7958).
Co-first authors: Lu-Yao Han and Wen-Zhu Zhang.
Author contributions: Han LY and Zhang WZ contributed equally to this manuscript and are co-first authors. Han LY and Zhong W contributed to data curation; Han LY contributed to formal analysis, investigation, and writing-original draft; Zhang WZ contributed to project administration, writing-review & editing; Zhang Y contributed to writing-review & editing; Sun JL contributed to methodology; Shen J contributed to conceptualization, supervision, and funding acquisition. All authors have read and approved the final version to be published.
Supported by Shenyang Science and Technology Plan in 2022, No. 22-321-32-16.
Institutional animal care and use committee statement: All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Medical Research Ethics Committee of the General Hospital of Northern Theater Command (Approval No. 2024-19).
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Data sharing statement: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Corresponding author: Jian Shen, PhD, Department of Obstetrics and Gynecology, General Hospital of Northern Theater Command (Heping Campus), No. 5 Guangrong Street, Heping District, Shenyang 110000, Liaoning Province, China. shenjian99@sina.com
Received: November 28, 2025
Revised: December 31, 2025
Accepted: March 25, 2026
Published online: June 26, 2026
Processing time: 208 Days and 23.4 Hours

Abstract
BACKGROUND

Preeclampsia (PE) is a serious complication in pregnancy. It is one of the primary causes of maternal and perinatal mortality. Human amniotic epithelial cells (hAECs) and mesenchymal stem cells (MSCs) derived from human umbilical cord blood (hucbMSCs) are both perinatal stem cells, capable of expressing characteristic stem cell surface markers. MSC-derived exosomes (MSCs-exos) exhibit functional properties comparable to those of MSCs, while offering advantages such as greater biological stability and the ability to circumvent potential complications associated with MSC-based therapy. This study utilized hAEC-derived exosomes (hAECs-exos) and hucbMSC-derived exosomes (hucbMSCs-exos) to treat preeclamptic rats and investigate their therapeutic effects.

AIM

To investigate the therapeutic effects of hAECs-exos and hucbMSCs-exos on PE in rats.

METHODS

Thirty-two pregnant rats were divided into four groups (normal pregnancy, PE, and two exosome-treated groups; n = 8). The PE model was induced by L-arginine methyl ester. From gestation day 12, the treatment groups received hAECs-exos or hucbMSCs-exos for 7 days, while controls received normal saline. Blood pressure, urinary protein, fetal/placental weight, and tissue analyses were performed. Quantitative data are expressed as the mean ± SD. Differences among multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by the LSD-t test for pairwise comparisons. A P value < 0.05 was considered statistically significant.

RESULTS

Compared to the normal pregnancy group, rats in the PE group exhibited significantly elevated blood pressure and 24-hour urinary protein levels, indicating successful model establishment. Furthermore, the PE group showed significantly increased levels of interleukin-6, tumor necrosis factor-α, soluble fms-like tyrosine kinase-1, and malondialdehyde, along with decreased fetal/placental weight, levels of interleukin-10, placental growth factor, vascular endothelial growth factor, superoxide dismutase, and placental CD31 expression (P < 0.05). Treatment with both exosomes significantly reversed all these alterations compared to the PE group (P < 0.05). Histological analysis further confirmed that the treatments markedly alleviated renal and placental pathological damage induced by PE.

CONCLUSION

This study demonstrates that both hAECs-exos and hucbMSCs-exos have therapeutic effects in rats with PE, potentially through mechanisms involving the inhibition of oxidative stress and inflammatory responses.

Key Words: Human amniotic epithelial cells; Human umbilical cord blood mesenchymal stem cells; Preeclampsia; Exosomes; Vascular endothelial growth factor; Placental growth factor

Core Tip: Preeclampsia (PE) is a serious obstetric syndrome, and effective therapeutic options are required urgently. This study demonstrates that exosomes derived from human amniotic epithelial cells and umbilical cord mesenchymal stem cells can function as innovative cell-free agents. Treatment with these exosomes effectively ameliorated key pathological features, including hypertension, proteinuria, and placental damage, in a rat model of PE. The underlying mechanisms involve multi-target modulation of angiogenic and inflammatory pathways. These findings highlight the clinical translation potential of exosomes derived from human amniotic epithelial cells and umbilical cord mesenchymal stem cells as novel therapeutic strategies for PE.



INTRODUCTION

Preeclampsia (PE) is a prevalent and severe pregnancy complication characterized by hypertension and proteinuria occurring after 20 weeks of gestation. Left uncontrolled, it can advance to multiple organ dysfunction, contributing significantly to maternal and perinatal mortality rates[1]. PE affects an estimated 2%-8% of pregnant women globally[2]. The precise pathogenesis of PE remains incompletely understood. Current understanding implicates impaired trophoblast remodeling of uterine spiral arteries, resulting in placental ischemia, hypoxia, oxidative stress, heightened inflammatory immune responses, endothelial cell damage, disrupted angiogenesis, mitochondrial dysfunction, maternal cardiovascular impairment, and immune dysregulation at the maternal-fetal interface[3,4]. Therefore, comprehensive exploration of the etiology and molecular mechanisms of PE, and the quest for safe and efficacious treatment strategies are imperative for safeguarding maternal and infant health.

Human amniotic epithelial cells (hAECs) and mesenchymal stem cells (MSCs) derived from human umbilical cord blood (hucbMSCs) are perinatal stem cell types. hAECs express key stem cell surface markers, suggesting the retention of stem cell properties[5]. hAECs exhibit immunomodulatory features, anti-inflammatory effects, and paracrine functions by secreting diverse cytokines that facilitate tissue repair, cell proliferation, migration, angiogenesis, and antioxidant, antifibrotic and antiapoptotic activity[6-8]. hucbMSCs demonstrate robust self-renewal capacity and multipotent differentiation potential. These cells possess regenerative, pluripotent and immunomodulatory attributes, enabling differentiation into various tissue cell types and secretion of numerous growth factors and trophic factors[9,10].

Exosomes are small extracellular vesicles ranging from 30 nm to 150 nm in diameter, released by cells across diverse organs and tissues, ubiquitous in organisms[11]. Laden with bioactive molecules such as RNAs, proteins and lipids, exosomes mediate intercellular signaling through multiple pathways, eliciting effects such as promoting angiogenesis, inhibiting apoptosis and inflammation, and combating fibrosis. MSC-derived exosomes (MSCs-exos) mirror the functions of MSCs but offer advantages such as enhanced stability, reduced immunogenicity, decreased pulmonary retention, blood-brain barrier penetrance, and circumvention of potential MSC-related issues such as chromosomal abnormalities, tumorigenesis, thrombosis, and immune rejection[12,13]. Consequently, leveraging exosomes in clinical settings emerges as an optimal strategy for managing diverse ailments.

In this study, hAEC-derived exosomes (hAECs-exos) and hucbMSC-derived exosomes (hucbMSCs-exos) were administered to preeclamptic rats. The efficacy of these exosomes was evaluated through the assessment of pertinent biomarkers, and their underlying mechanisms of action were initially investigated. The ultimate goal was to establish a theoretical framework and empirical groundwork for the clinical management of hypertensive disorders during pregnancy.

MATERIALS AND METHODS
Sources of exosomes

Exosomes from hAECs and hubcMSCs were obtained from Liaoning Shengjing Stem Cell Technology Co. Ltd.

Experimental animals

Sixteen healthy male Sprague-Dawley rats, aged 8-10 weeks and weighing 250 ± 20 g, along with 32 healthy female rats weighing 220 ± 20 g, were obtained from Beijing Huafukang Bioscience Co. Ltd. The animals were provided with an animal quality certificate number No. 110322230100948264 and an animal use license number SCXK (Beijing) 2019-0008. They underwent a 1-week acclimatization period in an environment maintained at 18-22 °C, relative humidity of 50%-70%, and a 12-hour light-dark cycle, with ad libitum access to food and water. This experimental protocol was approved by the Animal Medical Research Ethics Committee branch of the hospital (Ethics number: No. 2024-19).

Main reagents and equipment

L-arginine methyl ester (L-NAME) (Sigma, WI, United States), total protein assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu Province, China), enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Keaibo Biotechnology, Shanghai, China), hematoxylin and eosin (HE) staining kit (Shanghai Beyotime Biotechnology, Shanghai, China), immunohistochemistry hypersensitive SP kit (Fuzhou Maixin Biotechnology Development, Fuzhou, Fujian Province, China), CD31 antibody (Abcam, United Kingdom), BP-201A noninvasive animal blood pressure monitor (Beijing Ruanlong Biotechnology, Beijing, China), metabolic cage (Suzhou Shiguxiu Laboratory Animal Equipment, Suzhou, Jiangsu Province), flow cytometer (BD, NJ, United States), and multifunctional microplate reader (Nanjing Detie Biotechnology, Nanjing, Jiangsu Province, China) were utilized in the study.

Isolation, culture, and identification of hAECs and hucbMSCs

Isolation, culture, and identification of hAECs: The cleaned amniotic membrane tissue was placed in phosphate buffered saline (PBS) supplemented with 100 U/mL gentamicin and 2.5 μg/mL amphotericin B. After a 20-minute incubation period, the tissue was transferred to a culture flask and it with trypsin-EDTA three to four times. The digested fluid was harvested, the digestion process was halted by adding KnockOut™ serum replacement, filtered through a 200-mesh cell sieve, centrifuged, and the supernatant discarded. The cell pellet was resuspended in Dulbecco’s modified Eagle’s medium/F12 containing 10% KnockOut™ serum replacement, 10 ng/mL epidermal growth factor, 1% GlutaMAX, and 1% nonessential amino acids. The cells were inoculated into a 25-cm2 culture flask at 2.5 × 106/L and cultured in a 37 °C incubator with 5% CO2 and saturated humidity. The medium was replaced after 24 hours and cell expansion proceeded to the first passage (P1).

hAECs were harvested and adjusted to a density of 106 cells/mL. Subsequently, 1 mL of the cell suspension was processed. Following a wash with cold PBS, the cells were resuspended in 100 μL PBS. A 5-μL aliquot of monoclonal antibody was introduced, with a control group devoid of antibodies serving as the negative control. The cells were incubated at 4 °C in darkness for 30 minutes, followed by three washes with cold PBS. Flow cytometry was used to assess expression of CD29, CD31, CD34, CD90, CD105, HLA-DR and SSEA-4. The differentiation potential was evaluated using human adipose-derived MSC-specific adipogenic, osteogenic and chondrogenic induction media.

Isolation, culture and identification of hucbMSCs: Umbilical cord blood samples were diluted with PBS at a 1:1 ratio, followed by isolation of mononuclear cells (MNCs) through gradient density centrifugation using Ficoll-Paque (density = 1.077 g/mL). Subsequently, 20 mL diluted blood was layered onto an equal volume of Ficoll-Paque in a 50-mL centrifuge tube and centrifuged at 600 × g for 20 minutes at room temperature. The resulting MNC layer was carefully transferred to a new sterile 50-mL centrifuge tube. After a wash with PBS, the isolated MNCs were resuspended in StemMACS™ MSC expansion medium supplemented with 10% autologous serum and seeded in a six-well plate. The plate was incubated at 37 °C in a humidified atmosphere with 5% CO2. The medium was replaced after 1 day to eliminate nonadherent cells. Upon observation of fibroblast-like cell colonies reaching 80%-90% confluence, adherent cells were detached using trypsin solution, and the cells were passaged continuously until reaching P3.

hucbMSCs were harvested and adjusted to a density of 106 cells/mL. One milliliter of the cell suspension was removed, washed with cold PBS, and resuspended in 100 μL PBS. Five μL monoclonal antibody was added, and a group without antibody was designated as the negative control. The cells were incubated at 4 °C in the dark for 30 minutes, followed by three washes with cold PBS. A flow cytometer was used to assess expression of CD11b, CD34, CD45, CD73, CD90 and CD105. The differentiation potential was evaluated by exposing the cells to hAECMSC-specific adipogenic, osteogenic and chondrogenic induction media.

Extraction and identification of hAECs-exos and hucbMSCs-exos

Extraction of exosomes: hAECs and hucbMSCs were cultured in T75 flasks until reaching 80% confluence. The cells were washed twice with PBS and incubated in Dulbecco’s modified Eagle’s medium/F12 in a CO2 incubator at 37 °C with 5% CO2 and saturated humidity. After 48 hours, the supernatant was harvested and subjected to sequential centrifugation steps at 300 × g for 5 minutes, 200 × g for 15 minutes, and 13000 × g for 35 minutes at 4 °C. The supernatant was filtered through a 0.22-μm sterile filter, and processed via ultrafiltration with discard of the lower liquid phase. Following resuspension in PBS, the sample underwent centrifugation at 150000 × g for 3 hours at 4 °C. The supernatant was discarded, and the precipitate was collected. The concentration of hAECs-exos and hucbMSCs-exos was determined using a BCA protein quantification kit, while the Exo count was assessed through nanoparticle tracking analysis (NTA). The samples were stored at -80 °C.

Transmission electron microscopy: Transmission electron microscopy (TEM) was set to the operational state. Ten microliters of purified hAECs-exos and hucbMSCs-exos were transferred onto copper grids, allowed to settle for 5 minutes, and filter paper was used to remove excess liquid at the grid edge. Phosphotungstic acid (10 μL) was applied to the grids, followed by drying and imaging at 80-120 kV.

NTA: The concentration of exosomes were determined by a nanoparticle tracking analyzer. The system was calibrated with 100 nm polystyrene microspheres prior to measurement. Purified exosomes were appropriately diluted in 1 × PBS buffer and introduced into the sample chamber for analysis.

Western blotting: Proteins (30 μg) were separated using sodium-dodecyl sulfate gel electrophoresis at 80 V followed by 120 V. The proteins were transferred from the separation gel to a polyvinylidene difluoride (PVDF) membrane at a constant current of 200 mA. The PVDF membrane was incubated in a blocking solution containing 5% nonfat milk powder at room temperature for 2 hours. The PVDF membrane was sectioned based on the molecular weights of the proteins and immersed in a diluted primary antibody hybridization solution (CD9 antibody 1:100, CD63 antibody 1:100, and CD81 antibody 1:100) for overnight incubation at 4 °C. The membrane underwent five washes with Tris-buffered saline-Tween for 5 minutes each. Horseradish-peroxidase-labeled secondary antibody (diluted 1:10000 with 5% bovine serum albumin) was applied, and the membrane was incubated at room temperature for 1 hour. The PVDF membrane was washed five times with Tris-buffered saline-Tween for 5 minutes each. A mixture of ECL luminescent solutions A and B at a 1:1 ratio was applied to the PVDF membrane, followed by imaging using a chemiluminescence imaging system.

Establishment and grouping of PE models: After 1 week of adaptive feeding, female and male rats were housed together at a 2:1 ratio at 17:00. The presence of a milky white gelatinous substance in the vaginal canal of female rats before 08:00 the following morning was indicative of a vaginal plug, confirming pregnancy and designating that day as gestational day (GD). The pregnant rats were randomly assigned to four groups of eight: Normal pregnancy (NP), PE, hAECs-exos treatment, and hucbMSCs-exos treatment. On GD5, the PE, hAECs-exos and hucbMSCs-exos groups received intraperitoneal injections of 1 mL L-NAME at 150 mg/kg for 7 consecutive days, while the NP group received 1 mL normal saline. On GD12, 6 hours post-L-NAME injection, the hAECs-exos and hucbMSCs-exos groups were intravenously administered 1 mg/kg hAECs-exos and hucbMSCs-exos, respectively, for 7 consecutive days. In parallel, the NP and PE groups were given 1 mL normal saline.

Measurement of blood pressure in rats: Rats’ blood pressure was noninvasively monitored on GD 4, 8, 11, 15 and 18, with three measurements obtained during each session in a calm state, followed by calculation of the mean value.

Determination of 24-hour urinary protein in rats: From 08:00 on GD4 to 08:00 the following day, metabolic cages were used to collect 24-hour urine samples from rats in each experimental group. The urine volume was quantified, and the urine protein concentration was determined using the Coomassie Brilliant Blue method.

Specimen collection and processing: Delivery was via cesarean section at GD19, and fetal mouse and placental weights were recorded. Subsequently, 5 mL blood was extracted from the inferior vena cava, and the upper-layer serum(1.5 mL) was isolated by centrifugation, which was then preserved at -80 °C. Portions of placental and kidney tissues were allocated into cryotubes and stored at -80 °C, with another portion being immersed in 4% paraformaldehyde and shielded from light.

HE staining of kidney and placental tissues: The kidney and placenta tissues from each group of rats were fixed, embedded in paraffin, sectioned, and subjected to HE staining to assess morphological alterations.

ELISA detection: ELISA kits were used to quantify interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and soluble fms-like tyrosine kinase (sFlt)-1 levels in rat serum. Placental tissue weighing 100 mg was homogenized in 0.9 mL normal saline using an ultrasonic cell crusher. Following centrifugation at 300 rpm for 10 minutes, the resulting supernatant was analyzed using ELISA kits to determine placental growth factor (PLGF), vascular endothelial growth factor (VEGF), superoxide dismutase (SOD) and malondialdehyde (MDA) levels in the placental tissue.

Immunohistochemical detection: Immunohistochemistry was used to assess CD31 expression in rat placental tissues across all experimental groups. Tissue samples were examined microscopically, processed with Image-J software, and the proportion of positively stained cells was quantified.

Statistical analysis

Statistical analysis was conducted utilizing SPSS 26 software. Measurement data were presented as mean ± SD. One-way analysis of variance was used to assess variances among multiple groups, with post hoc least significant difference t tests utilized for pairwise group comparisons. Statistical significance was defined as P < 0.05.

RESULTS
Identification of hAECs

Under microscopic examination, primary amniotic epithelial cells exhibited a consistent morphology and were organized in a characteristic cobblestone pattern. Subsequent cultivation in adipogenic, osteogenic and chondrogenic differentiation media demonstrated the capacity of hAECs to manifest lipid droplets, calcium nodules, and acid mucopolysaccharides, indicative of their potential for adipogenic, osteogenic and chondrogenic differentiation (Figure 1). Flow cytometry of P1 hAECs indicated positive expression of CD29, CD90, CD105 and SSEA-4, and negative expression of CD31, CD34 and HLA-DR (Figure 2).

Figure 1
Figure 1 Microscopic observation (40 × magnification) and trilineage differentiation outcomes of primary cultured human amniotic epithelial cells. A: Morphology of human amniotic epithelial cells (hAECs) under light microscopy, scale bar = 100 μm; B: Outcome of adipogenic differentiation of hAECs, scale bar = 50 μm; C: Osteogenic differentiation of hAECs, scale bar = 50 μm; D: Chondrogenic differentiation of hAECs, scale bar = 200 μm.
Figure 2
Figure 2  Detection of expression of surface markers on human amniotic epithelial cells by flow cytometry.
Identification of hucbMSCs

Under microscopic examination, hucbMSCs exhibited consistent morphology. Subsequent culture in adipogenic, osteogenic and chondrogenic differentiation media demonstrated their capacity to produce lipid droplets, calcium nodules and acid mucopolysaccharides, confirming their adipogenic, osteogenic and chondrogenic differentiation potential (Figure 3). Flow cytometry at P3 indicated positive expression of CD73, CD90 and CD105, and negative expression of CD11b, CD34 and CD45 (Figure 4).

Figure 3
Figure 3 Microscopic (40 ×) observations and trilineage differentiation outcomes of primary cultured mesenchymal stem cells derived from human umbilical cord blood. A: Morphology of mesenchymal stem cells derived from human umbilical cord blood (hucbMSCs) under light microscopy, scale bar = 100 μm; B: Outcomes of adipogenic differentiation of hucbMSCs, scale bar = 50 μm; C: Osteogenic differentiation of hucbMSCs, scale bar 50 μm; D: Chondrogenic differentiation of hucbMSCs, scale bar = 200 μm.
Figure 4
Figure 4  Flow cytometry detection of surface marker expression on mesenchymal stem cells derived from human umbilical cord blood.
Identification of hAECs-exos

TEM showed that hAECs-exos had round or oval vesicles with a saucer-like shape (Figure 5). NTA indicated that 97.3% of the cells had a diameter distribution of 121.3 ± 71.9 (full width at half maximum) nm, consistent with typical exosome size. Western blotting confirmed the presence of exosome-specific markers CD9, CD63 and CD81 on the surface.

Figure 5
Figure 5 Identification of human amniotic epithelial cell exosomes. A: Morphological examination of human amniotic epithelial cell exosomes (hAECs-exos) using transmission electron microscopy (scale bar = 100 nm); B: Analysis of hAECs-exo particle size distribution via nanoparticle tracking analysis; C: Assessment of CD9, CD63 and CD81 surface protein expression on hAECs-exos by western blotting. hAECs-exos: Human amniotic epithelial cell exosomes; hAECs: Human amniotic epithelial cells.
Identification of hucbMSCs-exos

TEM showed the saucer-like morphology of hucbMSCs-exos (Figure 6). NTA revealed that 97.3% of the cell diameters were around 126.4 ± 80 (full width at half maximum) nm, consistent with typical exosome size. Western blot analysis confirmed the presence of exosome surface markers CD9, CD63, and CD81 on the membrane of hucbMSCs-exos.

Figure 6
Figure 6 Identification of exosomes from mesenchymal stem cells derived from human umbilical cord blood. A: Morphological examination of exosomes from mesenchymal stem cells derived from human umbilical cord blood (hucbMSCs-exos) using transmission electron microscopy (scale bar = 100 nm); B: Analysis of hucbMSCs-exos particle size distribution via nanoparticle tracking analysis; C: Evaluation of CD9, CD63 and CD81 surface protein expression in hucbMSCs-exos by western blotting. hucbMSCs-exos: Exosomes from mesenchymal stem cells derived from human umbilical cord blood; hucbMSCs: Mesenchymal stem cells derived from human umbilical cord blood.
Blood pressure measurement in rats

On GD4, there were no significant differences in systolic blood pressure among the four rat groups (Table 1 and Figure 7A) (P > 0.05). Following L-NAME intraperitoneal injection on GD8 and GD11, systolic blood pressure in the PE, hAECs-exos and hucbMSCs-exos groups significantly exceeded that in the NP group (P < 0.05), confirming successful establishment of the PE rat model. Throughout gestation, blood pressure in the PE group exhibited a progressive rise. Subsequent to exosome intervention, on GD15 and GD18, systolic blood pressure in the hAECs-exos and hucbMSCs-exos groups significantly decreased compared to that in the PE group (P < 0.05). There was no significant disparity between the two treatment cohorts (P > 0.05).

Figure 7
Figure 7 Changes in systolic blood pressure and 24-hours urinary protein in rats. A: Changes in systolic blood pressure in rats; B: Changes in 24-hours urinary protein in rats. aP < 0.05, compared to the normal pregnancy group; bP < 0.05, compared to the preeclampsia group. NP: Normal pregnancy; PE: Preeclampsia; hAECs-exos: Human amniotic epithelial cell exosomes; hucbMSCs-exos: Exosomes from mesenchymal stem cells derived from human umbilical cord blood; GD: Gestational day.
Table 1 Measurement of systolic blood pressure in groups of rats at different gestational days, mean ± SD.
Group
GD4
GD8
GD11
GD15
GD18
NP111.75 ± 1.83112.50 ± 3.42114.50 ± 3.30112.25 ± 4.95113.00 ± 4.44
PE112.63 ± 1.30137.25 ± 2.44a143.00 ± 2.27a151.38 ± 3.74a155.38 ± 6.95a
hAECs-exos113.00 ± 3.96139.25 ± 3.15a149.13 ± 3.91a130.88 ± 3.40a,b121.50 ± 4.14a,b
hucbMSCs-exos110.88 ± 2.42138.25 ± 4.23a146.5 ± 4.66a128.38 ± 3.89a,b120.25 ± 4.98a,b
24-hour urinary protein measurement in rats

On GD4, there were no significant differences in 24-hour urinary protein levels among the four groups of rats (P > 0.05) (Table 2 and Figure 7B). Following intraperitoneal L-NAME injection, rats in the GD8, GD11, PE, hAECs-exos and hucbMSCs-exos groups exhibited significantly elevated 24-hour urinary protein levels compared to the NP group (P < 0.05). In the PE group, urinary protein levels continued to rise with advancing gestational age. Subsequent exosome administration led to a significant reduction in 24-hour urinary protein levels in the hAECs-exos and hucbMSCs-exos groups compared to the PE group on GD15 and GD18 (P < 0.05). Importantly, no significant difference was observed between the two treatment groups (P > 0.05).

Table 2 Protein determination of 24-hour urine in each group of rats, mean ± SD.
Group
GD4
GD8
GD11
GD15
GD18
NP2.67 ± 0.492.91 ± 0.332.68 ± 0.552.76 ± 0.582.84 ± 0.59
PE2.76 ± 0.294.46 ± 0.39a5.92 ± 0.68a7.07 ± 0.66a8.19 ± 1.07a
hAECs-exos2.77 ± 0.234.45 ± 0.73a6.18 ± 0.77a5.34 ± 0.76a,b4.70 ± 0.52a,b
hucbMSCs-exos2.67 ± 0.554.37 ± 0.46a6.24 ± 0.91a5.43 ± 0.71a,b4.94 ± 0.46a,b
Developmental status of fetal mice in each group

All fetal mice in the NP group exhibited normal development (Figure 8A). In contrast, within the PE group, one pregnant mouse experienced a miscarriage, leading to halted embryo development (Figure 8B), along with the observation of limb malformations, congestion, and edema in multiple fetal mice (Figure 8C). Fetal mice in the hAECs-exos and hucbMSCs-exos groups displayed larger sizes compared to those in the PE group, with significant improvements noted in limb malformations and congestion (Figure 8D and E). There was a significant reduction in the weights of fetal mice and placentas in the PE group compared to the NP group (P < 0.05) (Table 3). Conversely, the weight of fetal mice and placentas in the hAECs-exos and hucbMSCs-exos groups was increased compared to in the PE group (P < 0.05).

Figure 8
Figure 8 Morphological appearance of fetal mice. A: Fetal mice from the normal pregnancy group; B: Fetal mouse abortion images from the preeclampsia group; C: Fetal mice from the preeclampsia group; D: Fetal mice from the human amniotic epithelial cell exosomes group; E: Fetal mice from the exosomes from mesenchymal stem cells derived from human umbilical cord blood group.
Table 3 Development of fetal mice in each group, mean ± SD.
Group
Weight of fetal mice (g)
Placental weight (g)
NP3.08 ± 0.480.50 ± 0.07
PE1.77 ± 0.13a0.37 ± 0.05a
hAECs-exos2.36 ± 0.33a,b0.44 ± 0.05a,b
hucbMSCs-exos2.44 ± 0.43a,b0.45 ± 0.05a,b
HE staining of kidney and placenta tissues in rats

Histological examination of kidney sections revealed no pathological alterations in the NP group, demonstrating normal morphology with intact glomerular and tubular structures. Conversely, the PE group exhibited disrupted kidney tissue architecture characterized by enlarged glomeruli, epithelial cell exfoliation, narrowed tubular lumens, epithelial cell edema, and vacuolization. Remarkably, rats treated with hAECs-exos and hucbMSCs-exos showed significantly reduced renal damage compared to the PE group (Figure 9). Similarly, placental HE staining indicated that the NP group maintained an intact placental structure with normal morphology, abundant villous blood vessels, and no discernible pathological changes. In contrast, the PE group displayed disorganized placental tissue, reduced villous blood vessels, and abnormal cell proliferation. Treatment with hAECs-exos and hucbMSCs-exos effectively mitigated placental tissue damage (Figure 10).

Figure 9
Figure 9 Hematoxylin and eosin staining of rat kidneys (400 ×). A: The kidneys of rats in the normal pregnancy group; B: The kidneys of rats in the preeclampsia group; C: The kidneys of rats in the human amniotic epithelial cell exosomes group; D: The kidneys of rats in the exosomes from mesenchymal stem cells derived from human umbilical cord blood group. NP: Normal pregnancy; PE: Preeclampsia; hAECs-exos: Human amniotic epithelial cell exosomes; hucbMSCs-exos: Exosomes from mesenchymal stem cells derived from human umbilical cord blood.
Figure 10
Figure 10  Hematoxylin and eosin staining of placental tissues from rats (200 ×). A: The placental of rats in the normal pregnancy group; B: The placental of rats in the preeclampsia group; C: The placental of rats in the human amniotic epithelial cell exosomes group; D: The placental of rats in the exosomes from mesenchymal stem cells derived from human umbilical cord blood group. NP: Normal pregnancy; PE: Preeclampsia; hAECs-exos: Human amniotic epithelial cell exosomes; hucbMSCs-exos: Exosomes from mesenchymal stem cells derived from human umbilical cord blood.
Changes in IL-6, IL-10, TNF-α, and sFlt-1 in serum

Compared to the NP group, the serum levels of IL-6, TNF-α, and sFlt-1 in the PE group exhibited a significant increase, while IL-10 levels decreased significantly (P < 0.05) (Table 4 and Figure 11). Conversely, in comparison to the PE group, the hAECs-exos and hucbMSCs-exos groups demonstrated decreased levels of IL-6, TNF-α, and sFlt-1 in serum, along with an increase in IL-10 levels (P < 0.05). There was no significant disparity observed between the two treatment groups (P > 0.05).

Figure 11
Figure 11  Serum levels of interleukin-6, interleukin-10, tumor necrosis factor-α, and soluble fms-like tyrosine kinase-1. A: Interleukin-6; B: Interleukin-10; C: Tumor necrosis factor-α; D: Soluble fms-like tyrosine kinase-1. aP < 0.05, compared to the normal pregnancy group; bP < 0.05, compared to the preeclampsia group. IL: Interleukin; TNF: Tumor necrosis factor; sFLT-1: Soluble fms-like tyrosine kinase-1; NP: Normal pregnancy; PE: Preeclampsia; hAECs-exos: Human amniotic epithelial cell exosomes; hucbMSCs-exos: Exosomes from mesenchymal stem cells derived from human umbilical cord blood.
Table 4 Levels of cytokines in serum, mean ± SD.
Cytokine
NP
PE
hAECs-exos
hucbMSCs-exos
IL-6 (pg/mL)93.18 ± 9.75149.26 ± 13.71a120.15 ± 16.20a,b124.64 ± 9.73a,b
IL-10 (pg/mL)2.30 ± 0.580.87 ± 0.18a1.58 ± 0.39a,b1.41 ± 0.53a,b
TNF-α (pg/mL)7.30 ± 1.0111.20 ± 1.48a8.96 ± 1.23a,b9.43 ± 1.95a,b
sFlt-1 (ng/mL)0.22 ± 0.050.43 ± 0.10a0.33 ± 0.09a,b0.32 ± 0.12a,b
Changes in PLGF, VEGF, SOD, and MDA in the placenta

In comparison to the NP group, placental levels of PLGF, VEGF and SOD were notably reduced, while MDA levels were significantly elevated in the PE group (Table 5 and Figure 12) (P < 0.05). Conversely, the placental levels of PLGF, VEGF and SOD were elevated, and MDA levels were decreased in the hAECs-exos and hucbMSCs-exos groups compared to the PE group (P < 0.05). There was no significant difference observed between the two treatment groups (P > 0.05).

Figure 12
Figure 12  Comparison of placental growth factor, vascular endothelial growth factor, superoxide dismutase and malondialdehyde levels in the rat placenta. A: Placental growth factor; B: Vascular endothelial growth factor; C: Superoxide dismutase; D: Malondialdehyde. aP < 0.05, compared to the normal pregnancy group; bP < 0.05, compared to the preeclampsia group. PLGF: Placental growth factor; VEGF: Vascular endothelial growth factor; SOD: Superoxide dismutase; MDA: Malondialdehyde; NP: Normal pregnancy; PE: Preeclampsia; hAECs-exos: Human amniotic epithelial cell exosomes; hucbMSCs-exos: Exosomes from mesenchymal stem cells derived from human umbilical cord blood.
Table 5 Content of cytokines in placental tissues, mean ± SD.
Cytokine
NP
PE
hAECs-exos
hucbMSCs-exos
PLGF (pg/mL)50.46 ± 4.4536.47 ± 4.84a42.28 ± 5.89a,b42.17 ± 3.38a,b
VEGF (pg/mL)57.89 ± 9.3732.06 ± 5.40a44.60 ± 9.94a,b41.06 ± 9.39a,b
SOD (ng/mL)1.90 ± 0.201.34 ± 0.12a1.69 ± 0.25a,b1.58 ± 0.11a,b
MDA (nmol/mL)0.61 ± 0.070.99 ± 0.16a0.86 ± 0.11a,b0.86 ± 0.10a,b
Immunohistochemical staining

Immunohistochemical staining of placental tissue was used to assess CD31 endothelial cell expression. A notable reduction in the percentage of CD31-positive cells in the placental tissue of rats in the PE group compared to the NP group was observed (P < 0.05). Conversely, a significant increase in CD31-positive cells was evident in the placental tissue of rats in the hAECs-exos and hucbMSCs-exos groups compared to the PE group (P < 0.05) (Table 6 and Figure 13). No significant difference was observed between the two treatment cohorts (P > 0.05).

Figure 13
Figure 13  Immunohistochemical staining of placental tissues from rats (400 ×). A: Immunohistochemical staining results of CD31-positive cells in the placenta of rats in the normal pregnancy group; B: Immunohistochemical staining results of CD31-positive cells in the placenta of rats in the preeclampsia group; C: Immunohistochemical staining results of CD31-positive cells in the placenta of rats in the human amniotic epithelial cell exosomes group; D: Immunohistochemical staining results of CD31-positive cells in the placenta of rats in the exosomes from mesenchymal stem cells derived from human umbilical cord blood group. NP: Normal pregnancy; PE: Preeclampsia; hAECs-exos: Human amniotic epithelial cell exosomes; hucbMSCs-exos: Exosomes from mesenchymal stem cells derived from human umbilical cord blood.
Table 6 Proportion of CD31-positive cells in rat placental tissues, mean ± SD.
CD31 positive cell proportion by group (%)
NP41.26 ± 1.26
PE19.82 ± 1.57a
hAECs-exos31.13 ± 1.35a,b
hucbMSCs-exos32.33 ± 1.13a,b
DISCUSSION

PE is a prevalent complication in pregnancy, posing significant risks to both maternal and fetal health[14]. Abnormal placental development and dysfunction are currently recognized as primary factors contributing to PE. The existing therapeutic approaches for PE primarily aim to manage hypertension, extend gestation, and facilitate timely delivery; however, these strategies do not offer a definitive solution. Consequently, there is an urgent demand for the development of a safe and efficacious treatment modality to ameliorate clinical manifestations and adverse pregnancy outcomes associated with PE. Stem cells have garnered considerable attention in therapeutic investigations across various pathologies owing to their pluripotent nature, self-renewal capacity, and differentiation potential. Nonetheless, challenges such as cell sourcing, tumorigenic risks, and ethical considerations have impeded their progress. Recent studies have identified diverse stem cell populations within the placenta, offering a novel and ethically uncontroversial reservoir. Notably, exosomes derived from these stem cells exhibit properties akin to their parent cells while addressing concerns regarding tumorigenicity, thus presenting a promising avenue for therapeutic interventions.

In this study, L-NAME was utilized to induce a rat model of PE, and the therapeutic efficacy and underlying mechanisms of hAECs-exos and hucbMSCs-exos were investigated. There was a significant increase in systolic blood pressure and 24-hour urinary protein levels in PE rats, mirroring the clinical features of hypertension and proteinuria in patients. Additionally, fetal and placental weights were notably reduced in the PE group, consistent with fetal growth restriction commonly associated with PE. Following exosome administration, a marked decrease in blood pressure and urinary protein levels was observed, along with increased fetal and placental weights, indicating the beneficial effects of both exosome types on ameliorating clinical manifestations and pregnancy outcomes in PE rats. The study revealed that PE could induce acute kidney injury, endothelial cell impairment, and podocyte loss, potentially leading to kidney disorders[15]. Histological examination of rat kidneys using HE staining unveiled alterations in renal tissue structure in the PE group, characterized by enlarged glomerular volume, epithelial cell shedding, edematous renal tubular epithelial cells, and narrowed lumens. Subsequent exosome treatment significantly mitigated the pathological renal tissue damage, suggesting that hAECs-exos and hucbMSCs-exos effectively attenuate renal injury in PE rats.

VEGF regulates the invasive capacity of trophoblasts, promotes placental angiogenesis, enhances vascular permeability, and facilitates maternal-fetal material exchange[16]. PLGF exerts effects in promoting trophoblast proliferation, migration and differentiation, as well as facilitating placental angiogenesis and spiral artery remodeling[17]. Reduced PLGF expression hinders chorionic angiogenesis, impairs vascular endothelial function, and disrupts placental development[18]. sFlt-1 is an anti-angiogenic factor that can inhibit the biological activities of VEGF and PLGF, thereby suppressing angiogenesis, impairing trophoblasts, inducing spiral artery remodeling disorders, and reducing placental blood supply[19]. Normally, VEGF, PLGF and sFlt-1 are in equilibrium. In PE, heightened sFlt-1 levels disrupt this balance, inhibiting VEGF and PLGF functions, disturbing angiogenesis, impeding trophoblast invasion and differentiation, causing vascular endothelial dysfunction, and resulting in placental ischemia and hypoxia[20]. Experimental findings demonstrate increased VEGF and PLGF levels and decreased sFlt-1 levels in PE rats treated with exosomes. Rats treated with hAECs-exos and hucbMSCs-exos showed improved placental tissue pathology and enhanced chorionic angiogenesis, indicated by increased CD31-positive cell proportion in the placenta compared to the PE group. Therefore, it is hypothesized that hAECs-exos and hucbMSCs-exos may ameliorate endothelial cell damage, enhance placental angiogenesis, and boost placental blood flow by upregulating VEGF and PLGF expression and downregulating sFlt-1 expression, potentially offering a therapeutic approach for PE.

During a normal pregnancy, the differentiation balance between T helper 1 (Th1) and Th2 cells is tightly maintained. In the first trimester of pregnancy, T helper cells are predominantly differentiated into pro-inflammatory Th1 cells, which secrete TNF-α and interferon-γ. These cytokines facilitate the invasion of placental trophoblasts and vascular remodeling. Subsequently, the differentiation of T helper cells shifts toward an anti-inflammatory Th2 phenotype, with the secretion of IL-10 and IL-4. These anti-inflammatory cytokines help neutralize pro-inflammatory cytokines and inhibit the activation of Th17 and Th1 cells[21]. In the context of PE, the body exhibits immune imbalance, characterized by increased proportions of Th1 and Th17 cells, along with decreased numbers of regulatory T cells. This immune dysregulation leads to excessive release of inflammatory factors, which damage vascular endothelial cells and ultimately induce placental pathological changes[22]. Notably, insufficient IL-10 levels may impede T-cell differentiation, trophoblast invasion, and spiral artery remodeling, leading to placental ischemia and oxidative-inflammatory responses[23,24]. PE patients exhibit elevated proinflammatory markers such as TNF-α and IL-6 alongside decreased anti-inflammatory factors such as IL-4 and IL-10, with TNF-α and IL-6 levels serving as prognostic indicators for PE[25,26]. Analysis of rat serum revealed increased IL-6 and TNF-α levels in the PE group, while hAECs-exos and hucbMSCs-exos groups exhibited decreased IL-6 and TNF-α levels and increased IL-10 levels. Consequently, hAECs-exos and hucbMSCs-exos mitigate PE symptoms by suppressing inflammation, immune responses, and tissue damage.

During normal pregnancy, maternal oxidation and antioxidant systems maintain equilibrium, whereas the placental antioxidant defense is compromised in individuals with PE[27]. MDA is a byproduct of cell membrane lipid peroxidation due to increased free radicals during oxidative stress, disrupting cell membrane permeability and integrity, thereby affecting cellular functions. SOD is a crucial antioxidant enzyme that scavenges free radicals, protects cells from oxidative damage, and exhibits anti-inflammatory properties[28]. Studies indicate decreased SOD levels and increased MDA levels in PE patients[29]. Treatment of PE rats with exosomes resulted in decreased MDA expression and increased SOD expression in placental tissue, suggesting that hAECs-exos and hucbMSCs-exos can mitigate oxidative stress responses, thereby alleviating vascular and tissue damage and ameliorating PE symptoms.

This study compared the therapeutic effects of hAECs-exos with hucbMSCs-exos in a PE rat model, and indicated no significant difference in therapeutic efficacy. Both exosome types ameliorated clinical symptoms and pregnancy outcomes in PE rats, attenuated oxidative stress and inflammatory responses, and mitigated kidney and placental tissue damage. However, as compared with hucbMSCs, hAECs offer distinct advantages[30]: Easy procurement from postpartum medical waste without invasive procedures, ethical concerns; wide availability; simple isolation and culture procedures for rapid cell expansion; low major histocompatibility complex-1 expression; absence of major histocompatibility complex-2 expression reducing immunogenicity and immune rejection risks; non-tumorigenicity due to lack of telomerase; differentiation capacity into three germ layers akin to embryonic stem cells; rich lysosome content facilitating autophagy activation; and resilience to DNA damage from time, age, or environment as neonatal cells. These features position hAECs as an ideal candidate for regenerative medicine and cell therapy applications. We speculate that the absence of significant differences between the two types of exosomes in this study may be attributed to the small sample size and limitations of the detection indicators. Given that the cargoes of exosomes derived from different sources (e.g., microRNAs, proteomes) are inherently distinct, these differences may be manifested in terms of long-term safety, subtle patterns of immune regulation, or therapeutic efficacy against specific PE subtypes[31]. Further studies are required to elucidate these differences through omics-based component analysis and functional validation.

This study had the following limitations and shortcomings: (1) Existing studies have summarized established PE models[32], but it is generally acknowledged that a single model can hardly encompass the full spectrum of the pathogenic mechanisms underlying this disease. The PE model induced by L-NAME can stably elicit hypertension, proteinuria, placental/fetal growth restriction, abnormal placental morphology, as well as the vascular endothelial and inflammatory phenotypes of PE[33,34], which are highly consistent with the core characteristics of human PE. The advantage of the L-NAME model lies in its ability to induce the key features of PE in small animal models in a more cost-effective, convenient and easy-to-operate manner, thereby providing an effective approach for investigating candidate drugs targeting the key links in the pathogenesis of PE. However, this model lacks the “placenta-initiated” processes such as defective invasion of placental trophoblasts, which makes it unable to recapitulate the complete disease course of PE. Human PE is often accompanied by severe complications such as eclampsia and HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets), whereas such phenotypes rarely occur in the L-NAME model. In addition, this model fails to simulate the individual differences and genetic susceptibility of PE, thus exhibiting a gap compared with the multifactorial and polygenic complexity of human PE. Therefore, this model is more suitable for investigating the mechanisms related to blood pressure regulation and endothelial function, but it may be insufficient in simulating complex immune dysregulation; (2) The exosome identification results in this study only met the minimum criteria. Specific exosomal markers such as tumor susceptibility gene 101 and Alix were not detected, and the precise quantity of exosomes (particle count/body weight) used for each intervention was not determined; (3) This study did not track the biodistribution of exosomes in rats. Future studies will employ labeled exosomes (e.g., DiR fluorescent labeling) for in vivo imaging research to further identify the direct targets of exosomes, thereby providing a theoretical basis for determining clinical administration regimens; (4) This study lacked verification of the core signaling pathways, and the mechanistic interpretation was only at the level of phenotypic correlation without elaborating on the specific action pathways. Subsequent studies will improve gene sequencing and proteomic analyses, as well as relevant verification experiments such as western blot. Further detection will be performed on the expression changes of key signaling pathway proteins related to angiogenesis (e.g., VEGF receptor 2, endothelial nitric oxide synthase), inflammation-related proteins (e.g., phosphorylation level of nuclear factor kappa B p65) and oxidative stress-related proteins (e.g., nuclear factor erythroid 2-related factor 2, heme oxygenase-1), so as to explore the deeper molecular mechanisms; (5) Only a single dose (1 mg/kg) was used for intervention in this study, which precluded the determination of the dose-effect relationship. Future studies will adopt gradient dose research to identify the optimal therapeutic window; and (6) In addition, this study only observed up to GD19 and did not evaluate the long-term effects on maternal rats and their offspring after treatment. Subsequent research can extend the observation period to monitor the postpartum indicators of maternal rats and the growth and development of offspring.

CONCLUSION

In summary, both hAECs-exos and hucbMSCs-exos demonstrate notable therapeutic efficacy in PE rats, offering a novel avenue for investigating noncellular treatment modalities. Nonetheless, a comprehensive investigation into the precise molecular mechanisms and cellular signaling pathways underlying their therapeutic actions is warranted.

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Footnotes

Peer review: Externally peer reviewed.

Peer-review model: Single blind

Specialty type: Cell and tissue engineering

Country of origin: China

Peer-review report’s classification

Scientific quality: Grade B, Grade C

Novelty: Grade B, Grade C

Creativity or innovation: Grade B, Grade C

Scientific significance: Grade B, Grade C

P-Reviewer: Takegawa N, MD, PhD, Japan; Tougeron D, PhD, Assistant Professor, Researcher, France S-Editor: Wang JJ L-Editor: A P-Editor: Wang WB

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