Amniotic fluid-derived stem cells: potential factories of natural and mimetic strategies for congenital malformations

Abstract

Background:

Mesenchymal stem cells (MSCs) derived from gestational tissues offer a promising avenue for prenatal intervention in congenital malformations although their application is hampered by concerns related to cellular plasticity and the need for invasive, high-risk surgical procedures. Here, we present naturally occurring exosomes (EXOs) isolated from amniotic fluid-derived MSCs (AF-MSCs) and their mimetic analogs (MIMs) as viable, reproducible, and stable alternatives. These nanovesicles present a minimally invasive therapeutic option, addressing the limitations of MSC-based treatments while retaining therapeutic efficacy.

Methods:

MIMs were generated from AF-MSCs by combining sequential filtration steps through filter membranes with different porosity and size exclusion chromatography columns. A physicochemical, structural, and molecular comparison was conducted with exosomes (EXOs) released from the same batch of cells. Additionally, their distribution patterns in female mice were evaluated following in vivo administration, along with an assessment of their safety profile throughout gestation in a mouse strain predisposed to neural tube defects (NTDs). The possibility to exploit both formulations as mRNA-therapeutics was explored by evaluating cell uptake in two different cell types(fibroblasts, and macrophages) and mRNA functionality overtime in an in vitro experimental setting as well as in an ex vivo, whole embryo culture using pregnant C57BL6 dams.

Results:

Molecular and physiochemical characterization showed no differences between EXOs and MIMs, with MIMs determining a threefold greater yield. Biodistribution patterns following intraperitoneal administration were comparable between the two particle types, with the uterus being among targeted organs. No toxic effects were observed in the dams during gestation, nor were there any malformations or significant differences in the number of viable versus dead fetuses detected. MIMs delivered a more intense and prolonged expression of mRNA encoding for green fluorescent protein in macrophages and fibroblasts. An ex-vivo whole embryo culture demonstrated that MIMs mainly accumulate at the level of the yolk sac, while EXOs reach the embryo.

Conclusions:

The present data confirms the potential application of EXOs and MIMs as suitable tools for prevention and treatment of NTDs and proposes MIMs as prospective vehicles to prevent congenital malformations caused by in utero exposure to drugs.

Introduction

Congenital malformations, which encompass a wide range of structural and functional abnormalities present at birth, remain a leading cause of infant morbidity and mortality worldwide, necessitating innovative therapeutic approaches to improve outcomes and prevent lifelong disability. Prenatal surgical repair using regenerative strategies, such as biomaterials, stem cells, or a combination of both, has been proposed to reduce their severity. However, these procedures often carry significant risks for both the mother and the infant [1,2,3]. As an example, despite evidence that prenatal surgery significantly improves clinical outcome for infants affected by spina bifida by reducing the need for ventriculoperitoneal shunt placement, motor function and mental development improvements, preterm labor, uterine dehiscence, neonatal death, and preterm birth still remain highly prevalent upon treatment [4, 5]. Mesenchymal stem cell (MSC)-based approaches, especially those derived from gestational tissues (i.e., placental tissues, umbilical cord), have been widely studied as potential strategies to create an in utero pro-regenerative environment, due to the role they play in mediating embryo-maternal communication [6]. Advantages in the use of these tissues over adult counterparts include the possibility to establish a cell-banking system as they can yield a great number of cells noninvasively and without posing unnecessarily complex ethical issues [7]. Transamniotic therapy mediated by placental and amniotic fluid derived MSCs (AF-MSCs) has demonstrated a protective effect for the treatment of fetal and neonatal congenital disorders [8]. It is now widely established that MSCs act as trophic mediators, modulating the function of surrounding endogenous cells by releasing paracrine signals (growth factors, cytokines, chemokines, and extracellular vesicles (EVs) [9, 10]. MSC-derived EVs, including exosomes (EXOs; 50-130 nm in size), are formed through plasma membrane invagination and multivesicular body formation [11]. By maintaining parental physicochemical and molecular properties [12, 13], displaying inherent targeting capabilities and endogenous homing markers (which makes them able to cross biological barriers), EXOs are currently considered as promising diagnostic and therapeutic tools [14]. In addition to exerting similar effects to those associated with the cells they are released by, EXOs have been proposed as natural delivery systems able to increase the efficiency and targeted specificity of therapeutics [15]. Our laboratory has recently developed an efficient approach to utilize EXOs as reconfigurable systems for the delivery of a chemotherapeutic agent, doxorubicin (DOXO), for the treatment of advanced ovarian cancer [16]. We also established a platform based on a cell extrusion approach as a versatile system to produce therapeutics (called IDEM, immune-derived exosome mimetics) that retain the molecular features of EXOs with an increased structural stability. When loaded with DOXO, these particles IDEM showed an incremental encapsulation efficiency (EE) compared to values reported in literature for naturally released EXOs [17], a marked release that guarantees an increased uptake by target cancer cells, in 2D and 3D culture systems, as well as a more effective cytotoxic and apoptotic effect of DOXO-loaded particles compared to the free drug.

In this study, we propose generating EXOs and mimetics (MIMs) from AF-MSCs as reconfigurable tools with potential future applications in the treatment (or prevention) of congenital malformations. A comprehensive physicochemical and molecular characterization was performed along with the assessment of the distribution patterns of fluorescently labelled MIMs and EXOs when in vivo administered in female mice as well as their safety profile throughout gestation in a mouse strain predisposed to neural tube defects (NTDs), which are among the most severe and prevalent human congenital malformations, affecting on average 1.9 per 1000 live births worldwide [18]. The possibility to exploit both formulations as reconfigurable mRNA-therapeutics was explored by evaluating cell uptake (using two different cell types, fibroblasts, and macrophages) and mRNA functionality overtime in an in vitro experimental setting as well as in an ex vivo whole embryo culture. The latter was performed as a proof-of-concept system to determine differences in the biodistribution potential between natural and mimetic strategies during pregnancy.

Materials and methods

Cell culture

Amniotic fluid MSCs (AF-MSCs) were purchased from Celprogen and maintained using a Mesenchymal Stem Cell Growth kit (ATCC). Murine macrophages (J774 cell line) were purchased from ATCC and cultured in High Glucose-Dulbecco's Modified Eagle Medium (HG-DMEM) (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher Scientific), 1% L-glutamine and 100 U/ml Penicillin–Streptomycin (PS) solution (Sigma-Aldrich). Fibroblast (MRC-5 cell line) cultures were maintained in F12-DMEM (Gibco) supplemented with 15% heat-inactivated FBS, 1% L-glutamine and 100 U/ml PS solution (Sigma-Aldrich). Culture conditions were established at 37 °C and 5% CO₂.

Exosome and mimetics production

AF-MSCs (10 × 10⁶) at passage 3 (P3) were grown in standard media supplemented with Exo-free FBS for 24 h. Media and cells were collected and processed following previously established protocols to isolate naturally released EXOs and to produce MIMs, respectively [16]. EXOs were isolated by subjecting media to a series of centrifugations required to remove the cellular component (500 × g for 5 min) and any debris (2000 × g for 30 min). The remaining supernatant was passed through 0.22 mm PES membrane filter (CellTreat) and then concentrated using 10KDa Amicon ultra centrifugal filters (Millipore). Total exosome isolation reagent (Invitrogen) was then added in a 1:1 ratio to the volume obtained after the Amicon-based concentration process. The solution was mixed by vortexing for 30 s and incubated overnight at 4 °C. The next day, the sample was centrifuged at 10,000 X g for 1 h at 4 °C. The concentrated solution was centrifuged at 10,000 X g for 1 h at 4 °C, and the pellet was resuspended in 0.22 mm filtered PBS. Mimetics (MIM) were produced by deconstructing and reconstructing cells through porous membranes of decreasing size. Briefly, AF-MSCs were harvested and washed twice in PBS. The PBS-resuspended pellet was then filtered through 10 mm-filter Pierce™ spin cups (ThermoFisher) and centrifuged at 14,000 X g for 10 min at 4 °C. The pelleted flow-through was resuspended in PBS and the same process repeated. Consequently, the pellet was passed through 8 mm filters (Merck-Millipore) with the same centrifuge settings as before. The pellet was finally resuspended in 150 µL of 0.22 µm-filtered PBS and run through G-50 Sephadex high-capacity spin columns (Sigma Aldrich) for further purification of the solution. Figure 1a shows the steps required for MIM production. MIM were also generated utilizing Frozen AF-MSCs (F-MIMs) to evaluate the feasibility of this approach without the need to manipulating fresh cells. Exosomes and mimetics were stored at -80 °C or immediately used for downstream applications.

Fig. 1

AF-MSC-derived mimetics production and characterization. a Schematics of mimetics (MIMs) production compared to naturally released exosomes (EXOs): MIM production occurs through filtered-membrane centrifugation steps while EXO extraction from culture media by using the Total Exosome Isolation Reagent (TEIR). Concentration (particle/ml) b and size c values for mimetics (MIMs, in red) and exosomes (EXOs, in blue) obtained by NTA (n = 10). Statistically significant differences (p*** < 0.001) were observed between the two particle type formulations in terms of yield starting from the same number of AF-MSCs (10 millions/batch). d Total protein content (expressed in mg) shows a reduction in MIMs compared to EXOs (n = 3, p*** < 0.001). e Protein array displays comparable qualitative molecular profiles between MIMs and EXOs. f Biodistribution of fluorescently labelled EXOs and MIMs in various organs of female mice after intraperitoneal injection. The bar graphs represent the particle accumulation (signal reported as photons total flux, p/s) in each organ for EXOs and MIMs. Significant differences in LNP accumulation were observed between the groups in the lungs (p < 0.05) and liver (p < 0.01), where EXOs exhibit greater uptake compared to MIMs. Both groups show comparable levels of accumulation in the ovaries, brain, spleen, and kidneys, with no significant differences observed. (n = 3 mice/group; mean ± SD). g Schematic illustrating organ-specific biodistribution of EXOs and MIMs in female mice, organized based on decreasing total photon flux signal. The organs are ranked in order of particle accumulation

AF-MSC derived exosomes and mimetics characterization

Nanoparticles Tracking Analysis (NTA). EXO and MIM samples were analyzed according to the MISEV2018 Minimal information for studies of EVs [19]. The NS300 Nanosight System (Malvern) was used to determine size and concentration. A 100X dilution in 0.1 µm filtered PBS was prepared for each sample. For each batch, particle counts were normalized to the cell source by standardizing the yield to the number of cells seeded. Specifically, the yield was normalized against 10 million cells to ensure consistency across batches. This approach allowed to account for any variation as MIMs and EXOs were obtained from the same cell batch. The instrument was calibrated using 100 nm polystyrene beads prior to each run, and measurements were conducted in triplicate to ensure reproducibility. Particle size distribution, as well as the total particle number, were assessed to confirm the successful isolation of EXOs and MIMs (30–150 nm range). Yield consistency between samples was verified by comparing particle concentrations across independent isolations, with less than 10% variation observed between replicates. Five videos of 60 s each were recorded for each sample, and the threshold was kept constant at 5. Measurements for both formulations were repeated n = 10 times to obtain statistically robust data.

Evaluation of exosomal markers. Total protein concentration in EXOs and MIMs was determined by using Pierce BCA Protein Assay (Pierce) and the presence of 8 specific exosomal markers (CD63, EpCAM, ANXA5, TSG101, GM130, FLOT1, ICAM, ALIX and CD81) was assessed using Exo-Check™ Exosome Antibody Array (System Biosciences) and following manufacturer’s instructions.

mRNA encapsulation and encapsulation efficiency (EE%) assessment

The N1-methylpseudouridine-substituted mRNA used in this study was sourced from the RNA Therapeutic Core at Houston Methodist Research Institute (Houston, USA). As a proof-of-concept, mRNA encoding for nuclear green fluorescent protein (GFP) was encapsulated within EXOs and MIMs. Encapsulation was achieved using the Exo-Fect™ Exosome Transfection Kit (System Biosciences), which was employed according to the manufacturer’s protocol. To ensure that only encapsulated mRNA was present in the final preparations, unencapsulated mRNA was removed using an Exosome Spin Column (Invitrogen). The EE (%) was evaluated through the Quant-iT™ RiboGreen RNA Assay Kit (Invitrogen), which offers high sensitivity, detecting RNA within the range of 1–200 ng. This assay is widely regarded for its ability to selectively bind RNA, providing an accurate measure of the amount encapsulated. To distinguish encapsulated mRNA from free mRNA, 0.1% Triton-X-100 was added to the samples and incubated for 10 min at room temperature (RT). This detergent disrupts the lipid bilayer of EXOs and MIMs, releasing encapsulated contents for quantification. A comparison of RNA concentrations before and after treatment allowed for precise EE determination. The mRNA content in both EXO and MIM samples was measured by exciting the samples at 485 nm and reading emission at 530 nm using a fluorescence microplate reader (Synergy H4 Hybrid Plate Reader, Biotek). This method enabled the accurate quantification of GFP-mRNA, providing critical insight into the encapsulation capacity of each vesicle type.

EXO- and MIM-mediated cellular uptake and GFP-mRNA expression

To assess EXO- and MIM-mediated uptake by human fetal fibroblasts (MRC-5) and mouse macrophages (J774), both cell types were seeded into 6-well plates at a density of 1 × 10⁴ cells/cm² and allowed to adhere overnight. The following day, particles were labeled using 5 µM Vybrant DiD dye (ThermoFisher), a lipophilic tracer that integrates into the lipid membranes of vesicles. The staining was conducted in a final volume of 500 µl at 37 °C for 10 min to ensure sufficient dye incorporation. After staining, unbound dye was removed by ultracentrifugation at 40,000 × g for 1 h at 4 °C, followed by resuspension of the pellet in 1 ml of 0.22 μm filtered PBS (Gibco). To further purify the samples, exosome spin columns (Invitrogen) were utilized. DiD-labeled EXOs and MIMs, standardized to a concentration of 1 × 10⁸ particles, were then added to each well containing MRC-5 and J774 cells. Quantitative evaluation of cellular uptake was conducted at multiple time points (4, 8, and 12 h post-treatment) using fluorescence microscopy. Fluorescent signals from the DiD dye were visualized to track particle internalization and provide a direct measure of cellular uptake dynamics over time. In parallel, to assess the functionality of the encapsulated mRNA and its stability within the vesicles, both MRC-5 and J774 cells were exposed to GFP-mRNA-loaded EXOs and MIMs for extended periods (24, 48, and 72 h). After each incubation period, cells were processed for flow cytometry to determine GFP expression levels, thus evaluating mRNA translation efficiency within the recipient cells. This approach enabled the assessment of not only uptake, but also the ability of the two formulations to protect mRNA from degradation and support functional protein production over time. A similar protocol was employed to evaluate the efficacy of frozen AF-MSCs (F-MIMs) as delivery systems, ensuring a robust comparison of the various vesicle types in terms of both uptake and mRNA functionality in different cell types.

Fluorescence microscopy. At 3 different time points (4, 8, and 12 h), DiD-labelled cells were washed twice in pre-warmed PBS at pH 7.4, fixed in 4% paraformaldehyde (PFA) for 10 min at RT, and washed three times in PBS for 5 min/wash. Imaging was achieved by using a Nikon fluorescence microscope. The DiD dye, which emits in the far-red spectrum, was detected using appropriate filters for excitation at 644 nm and emission at 665 nm. The fluorescent signal was analyzed to track vesicle internalization, providing quantitative data on the extent of cellular uptake over time.

Flow cytometry. Flow cytometry was employed to quantify the percentage of GFP-expressing cells. At each designated time point (24, 48, 72 h), J774 and MRC-5 cells were harvested and analyzed for the presence of GFP expression. GFP expressing cells were identified using the 488 nm excitation laser. Mean fluorescence intensity (MFI) was accounted for to evaluate changes in the levels of GFP expression overtime. Ten thousand events per sample were acquired with a BD LSR Fortessa™ flow cytometer, and the FCS/SSC parameters were used to gate cells. Flow Cytometry Standard (FCS) files were analyzed using Flowjo software.

Ex vivo whole embryo culture

Ex vivo studies were conducted following the approved protocol AN-7618 established by Baylor College of Medicine’s Institutional Animal Care and Use Committee (IACUC) in accordance with the guidelines of the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals, as well as adhering to the ARRIVE guidelines 2.0. Animal protocol title “Intervention strategies for non-folate responsive neural tube defects”, approval date 11/17/2023. Three pregnant C57BL6 dams (2–4 months old) were euthanized on gestational day 8.5 according to the IACUC-approved “Euthanasia in rodents policy” and the CCM policy of “Euthanasia of adults and neonatal rodents in Smartbox units” by using automated CO₂ euthanasia chambers. The uterus was resected and placed in warm HEPES-buffered Tyrode’s Solution (Thermo Scientific) for dissection. Using forceps, the uterus was peeled away from the conceptus, and the decidual capsule and Reichardt’s membrane were carefully removed to leave the embryo and yolk sac intact. Embryos (n = 7/experimental group) were randomly assigned to two groups (EXOs and MIMs) and were cultured in 100% immediately centrifuged rat serum (Envigo) containing 10⁸ EXOs or MIMs for 24 h while rotating in roller bottles at 37.5 °C. Prior to culture, the rat serum was equilibrated with a 5% O₂/5% CO₂ gas mixture (AirGas) by gently blowing the gas mixture over the surface of the serum within the roller bottle for approximately 60 s. Each roller bottle contained 4 mL of serum and no more than 4 embryos were cultured per bottle. After 24 h, embryos were removed from the culture bottles, washed briefly in PBS, and the embryo was then separated from the yolk sac. Localization of exosomes or mimetics was assessed qualitatively by confocal microscopy as reported below.

Confocal microscopy

Embryos and yolk sacs exposed to exosomes or mimetics were fixed on ice for 30 min in 4% PFA. They were then washed twice in PBS before being placed in 1 mL of blocking buffer (1% BSA in PBS) in a microcentrifuge tube. The microfuge tube was pre-incubated with blocking buffer 1 h prior to prevent the embryos and yolk sacs from sticking to the walls of the tube. The embryos and yolk sacs were incubated in blocking buffer for 1 h while rotating at RT. Hoechst (1 μg/mL) and Phalloidin-iFlour 488 or Phalloidin-iFlour 594 (1:1000) (Abcam, ab176753/ab176757) were added to the blocking buffer and the embryos and yolk sacs were incubated overnight while rotating at 4 °C. They were then washed in 3, 1 mL volumes of PBS (1 h per wash while rotating at RT). Whole embryos and yolk sacs were then imaged using a CSU-W1 Spinning Disk Confocal system (Nikon Center of Excellence, CPEH, Baylor College of Medicine).

Animal studies

Animal studies were conducted following approved protocol (AN-9175) established by Baylor College of Medicine’s Institutional Animal Care and Use Committee (IACUC) in accordance with the guidelines of the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals, as well as adhering to the ARRIVE guidelines 2.0. Animal protocol title “Biomimetic therapeutics for congenital malformations”, approval date 10/04/2023. Animals were euthanized according to the IACUC-approved “Euthanasia in rodents policy” and the CCM policy of “Euthanasia of adults and neonatal rodents in Smartbox units” by using automated CO₂ euthanasia chambers.

Biodistribution studies. Female C57BL/6 J mice (8 weeks old) were purchased from The Jackson Laboratory (USA) and kept in a specified pathogen-free environment with a 12-h light/dark cycle. MIMs were stained using Vybrant™ DiD Cell-Labeling Solution (Invitrogen) according to manufacturer’s guidelines. Mice were prepared using 2.5% inhaled isoflurane. Intraperitoneal (i.p.) injection was performed with DiD-stained EXOs or MIMs (1 × 10⁹) in 300 µL PBS, or PBS only (Sham). Particle biodistribution was assessed through ex vivo fluorescent imaging of explanted organs after euthanasia through isoflurane inhalation on days 1 after injection. Fluorescent signal was detected using IVIS® SpectrumCT imaging system (Perkin Elmer, USA) and analyzed with Living Image® 4.0 software. Regions of interest (ROIs) were drawn at the injection site and for each organ using the software’s ROI tool, and the fluorescent signals were expressed as radiance photons. Signal normalization in EXOs and MIMs was performed by subtracting the total flux [p/s] of the epi-fluorescent signal from the sham group (PBS).

Assessment of EXO and MIM toxicology profile. Pregnant dams (n = 5 per experimental group) of the Swiss-Vancouver/Fnn (SWV) strain were administered i.p. injections of equal amounts of EXOs or MIMs at a concentration of 10⁹ particles in 300 µl of PBS, every other day throughout gestation, beginning on the day of plug detection and continuing until embryonic day 15.5 (E15.5, autopsy day). The body weight of the dams was monitored and recorded throughout the treatment period. On E15.5, the dams were euthanized, and blood samples were collected via cardiac puncture for the analysis of blood cell types and compared across groups. Maternal organs were harvested and weighed. Fetuses were collected and evaluated for number (to determine litter size), viability (against deaths/resorptions), and the presence of malformations induced by the treatment. A direct comparison was conducted with untreated mice to assess potential toxic effects associated with continuous treatment using EXOs and MIMs.

Statistical analysis

Data was initially reported as mean, standard deviation, standard error, median, minimum and maximum considering two different categories or groups of exosomes produced by MIMs versus standard extraction EXOs. In a sequence, normality was tested. Number (yield) and diameter (size in nm) were compared between groups using Independent-Samples Mann–Whitney U Test, and differences were considered significant when p < 0.05. For protein quantification, mRNA EE, MFI, a two-tailed Student’s t-test was performed. All graphs show average values and standard deviation. Differences among multiple groups in toxicology studies were assessed using one-way analysis of variance (ANOVA).

Results

Mimetics display exosomal size and molecular moieties

Nanoparticle tracking analysis (NTA) was used to determine size and concentration of the two formulations. Starting from the same number of AF-MSCs (1 × 10⁶), the optimized procedure allowed to produce 2.74 × 10¹⁰ MIMs compared to 1.15 × 10⁹ obtained following standard protocols for the isolation of natural EXOs from culture media (Fig. 1b), showing a 2.38-fold increase compared to natural counterparts (p < 0.001). The same yield was not obtained when MIMs were produced from frozen cells (Supplementary Fig. 1a). MIMs presented an average size of 113 ± 28 nm while EXOs 130 ± 54 nm, respectively (Fig. 1c). No differences in terms of size were found between MIMs and F-MIMs (113 ± 28 vs 105 ± 9.09, respectively) (Supplementary Fig. 1b). Evaluation of total proteins showed a reduction in MIMs compared to EXOs (Fig. 1d), although qualitative analysis confirmed the presence of specific exosomal markers (Cd63, EpCAM, ANXA5, TSG101, CD81, ALIX, ICAM, FLOT1, GM130) with no differences between the two particle types (Fig. 1e).

Mimetics and exosomes show a similar biodistribution pattern in vivo 

The biodistribution profile of fluorescently labelled (DiD)-EXOs and MIMs was assessed upon i.p. administration (Fig. 1f). Twenty-four hrs after injection, data were acquired and normalized against the sham group (PBS only). The biodistribution analysis revealed that both groups exhibit preferential accumulation in the lungs, liver, and ovaries. Notably, EXOs demonstrated significantly higher accumulation in the lungs (p < 0.05) and liver (p < 0.01) compared to MIMs. In contrast, the ovaries displayed similar levels of particle accumulation in both groups, with no significant differences observed. A more modest accumulation was detected in the kidneys, brain and spleen. In these organs, the biodistribution profile was similar between groups, with no statistically significant differences in nanoparticle levels. A schematic displaying levels of intensity across organs is shown in Fig. 1g.

Mimetic- and exosome-based treatments are safe in a sensitive mouse model 

Pregnant dams treated with either EXOs or MIMs were compared to controls to assess potential toxic effects due to the continuous treatment. A schematic representation of the experimental design is reported in Fig. 2a. Throughout gestation, dams treated with EXOs and MIMs exhibited normal weight gain with no significant differences compared to the sham group, suggesting no systemic toxicity from either treatment (Fig. 2b). At E15.5, blood analysis from treated dams revealed a significant reduction in monocytes caused by EXO and MIM. In addition, MIMs significantly reduced red blood cells and increased mean corpuscular volume (Table 1), during this period. Examination of maternal organs showed no differences in relative organ weight between EXOs, MIMs and Sham (Fig. 2c). Fetal assessment revealed that the number (Fig. 2d), viability (vs deaths/resorptions, Fig. 2e), and development of fetuses (Fig. 2f) from dams treated with EXOs or MIMs were comparable to those found in Sham. No significant malformations or developmental abnormalities were observed in any of the groups.

Fig. 2

Evaluation of the toxicological profile of EXOs and MIMs throughout gestation. a A schematic representation of the experimental design. Particles (dosage 10^9) were administered intraperitoneally every other day during gestation, starting from the detection of the plug to gestational day 15.5. Dam weight was recorded throughout pregnancy, and at E15.5, fetuses, maternal organs, and blood were collected for analysis. b A graph showing the change in dam weight (in grams) over time in response to treatment with EXOs and MIMs, compared to control (PBS only, Sham). Data represent the average of 5 animals per group. c Comparison of relative weights of maternal explanted organs across groups (EXOs, MIMs, and Sham), expressed as the ratio between organ weight and body weight (in mg). Data represent the average of 5 animals per group. d Litter size and e the percentage of normal fetuses in dams treated with EXOs and MIMs, compared to the control group (Sham). Data represent the average of 5 animals per group. f Fetal weight (in mg) collected from dams treated with EXOs and MIMs, compared to Sham

Table 1 Hematological values for EXO- and MIM-treated dams in comparison to untreated SWV mice. The results represent the mean ± standard deviation (n = 5)

mRNA-GFP delivered through mimetics maintain expression overtime in different cell types

The cell uptake of DiD-labelled MIMs was tested on human fetal fibroblasts (MCF-5) and murine macrophages (J774). There was a differential cell uptake of MIMs, with J774 cells incorporating more particles at early time points than their fibroblastic counterparts (Fig. 3a). EE for mRNA was found comparable between MIMs and EXOs, being assessed around 49.79 ± 2.61 and 50.87 ± 6.11, respectively (Fig. 3b). After assessing mRNA EE, the expression of the mRNA encoding for GFP delivered through MIMs was quantitatively evaluated on macrophages (Fig. 3c) and fibroblasts (Fig. 3d) at 24, 48, and 72 h. Flow cytometry data show GFP-mRNA loaded MIMs and EXOs display a different trend as mRNA mediators when administered to J774 cells. In particular, the percentage of GFP-positive cells increases overtime when delivered by MIMs, with the highest expressions levels being reached at 72 h (up to 90%). However, in the EXO group, a slight reduction in the number of positive cells is observed overtime, with 92% of GFP-positive cells being found at 24 h. A similar trend between MIMs and EXOs is observed when administered to fibroblasts. In this case, the percentage of GFP-positive cells is assessed around 68 ± 2.58 and 63.22 ± 3.33 for EXOs and MIMs at 24 h and increases for both treatments up to 86.32 ± 1.81 and 84.42 ± 3.14 at 72 h, respectively. Accordingly, the MFI associated with GFP expression was found to increase overtime in J774 cells treated with MIMs, with values recorded at 48 and 72 h (602.75 ± 10.91 and 832.25 ± 12, respectively, being statistically highly significant (P < 0.01) compared to their EXO counterparts where decreasing values were found (391.5 ± 7.5 and 356.25 ± 15.68, respectively (Fig. 3e). On the other hand, fibroblasts uptake of mRNA mediated by MIMs showed a statistically significant increase in the MFI only at 72 h, compared to EXOs (952.25 ± 8.01 vs 568.75 ± 11.44) (Fig. 3f). When F-MIMs were administered to cells, differential uptake patterns were observed depending on the cell type as well as on the preparation method. F-MIMs are easily taken up by J774 cells although the number of GFP-positive cells and the MFI, fade overtime compared to freshly prepared MIMs (Supplementary Fig. 1c). Comparable trends between MIMs from fresh and frozen MSCs, were found upon administration to fibroblasts at 24 and 72 h, although it was found doubled at 48 h for F-MIMs and only at 72 h for fresh MIMs (Supplementary Fig. 1d).

Fig. 3

Differential cellular uptake of fluorescently labelled and mRNA-loaded mimetics. a Fluorescence microscope images showing DiD-labelled mimetics taken up by two cell lines, murine macrophages (J774) and human fibroblasts (MRC-5) at early time points (4, 8 and 12 h). (Magnification: 10X, Scale bar:100 µm). b Graph showing mRNA-GFP encapsulation efficiency (EE) in MIMs and EXOs as revealed by Quant-it™ RiboGreen RNA Assay Kit. (n = 3). Time-dependent appearance of GFP fluorescence expressed as percentage of GFP-positive cells in c macrophages and d fibroblasts observed by flow cytometry after 24 h, 48 h and 72 h post transfection with MIMs. EXOs are used for comparison. (Data represented as mean ± SD, n = 4). Quantification of changes in the mean fluorescent intensity detected in macrophages e and fibroblasts f at each time point. Untreated cells used as baseline. (n = 4, p*** < 0.001)

Mimetic and exosome biodistribution differs in ex vivo whole embryo culture

The potential biodistribution of MIMs as a prospective therapeutic strategy for the treatment of congenital malformations was determined by using standard embryo cultures. Murine embryos at E8.5 were cultured for 24 h in the presence of DiD-labelled, GFP-mRNA-loaded MIMs or EXOs (Fig. 4a). At the end of the incubation period the yolk sac was dissected from the embryos and both components were observed using confocal microscopy, revealing the presence of DiD signal within the yolk sac upon exposure to both formulations (Fig. 4b). Signal associated to the expression of mRNA encoding for GFP was colocalized with the presence of MIMs and EXOs. However, while EXOs were found to be able to reach the embryos, as demonstrated by the expression of the DiD signal and GFP expression (although to limited extents), no signal was found upon exposure to MIMs (Fig. 4c). EXOs were localized for the most part in the cranial and ventral regions.

Fig. 4

Mimetics and exosome biodistribution in whole embryo culture. a Schematic representation of whole embryo culture established to define mimetics (MIMs) and exosomes (EXOs) distribution ex vivo. Embryos were isolated from pregnant dams and cultured in presence of MIMs or EXOs (1 × 10⁸) administered in culture media for 24 h. Confocal microscopy images showing explanted yolk sac, b and embryos, c upon exposure to DiD-labelled (red), mRNA-GFP-loaded (green) MIMs or EXOs. Phalloidin (gray) and Hoechst (blue) were used to counterstain actin filaments and DNA, respectively. Scale bars: 500 µm

Discussion

Tissue engineering approaches (including their combination with bioactive cells) represent promising treatment options to repair structural birth defects [20,21,22,23]. Yet, the application of these bioengineering technologies are invasive and only limited advancements have been made in these clinical scenarios [24, 25]. The stem cell therapy alternative, mainly involving cells isolated from gestational tissues, have been reported to improve animal survival and facilitate in utero tissue repair in genetically and mechanically induced spina bifida [8, 26,27,28]. Cell therapy poses several inherent risks and challenges, including  issues with scalability, cell differentiation abilities and aging, bystander effect which reduces cell potency, number of cells reaching target sites, and therapeutic outcome [29].

With this work, we present evidence suggesting that nanotherapeutics derived from AF-MSCs could serve as promising minimally invasive strategies for the prenatal repair of congenital anomalies. While EXOs from AF-MSCs have already been reported to retain parental cell molecular moieties and exert protective and regenerative potential [30, 31], we compared them to those associated to their mimetic counterparts produced from the same cells. MIMs were obtained through a recently established process [16], with the aim to potentially exploit them as alternative, therapeutic delivery systems. Our data suggest that both, EXOs and MIMs, could be used as scalable approaches to be tailored for individual clinical applications. Production of MIMs yields a 2.38-fold greater concentration than natural EXOs isolated from the same number of source cells (fresh AF-MSCs, 1 × 10⁶). This trend is in line with previously acquired data, showing a 2–48 fold increase in the production of MIMs obtained from immune cells (IDEM) through the same process compared to natural EXOs [16]. To reduce variability and improve consistency across experiments, EXOs and MIMs were derived from the same batch, simultaneously exploiting the supernatant for isolation of EXOs and cells for MIMs. By utilizing a unified source for both particles, we minimized biological variability and ensured that any differences observed between the EXO and MIM populations were intrinsic to their biophysical properties, rather than being confounded by differences in cell batches or culture conditions. In terms of size, MIMs closely resemble their natural counterparts, with a diameter (113 ± 28 nm) which falls within the expected range for natural EXOs (130 ± 54 nm) [19, 32], although more heterogeneous when compared to IDEMs, and display a similar cohort of exosomal proteins. Recently, Sayyed et al. produced cell-derived nanovesicles from human adipose-derived-MSCs by cell extrusion with a mean diameter of 177.3 ± 2 nm and a yield of 1 × 10¹⁰ from 1 × 10⁶ cells, surpassing the size produced in the current data, but showing a lower yield than was obtained in the present work [33]. On the other hand, Zhang et al. reported a mean diameter of mimetic nanovesicles of 126.9 ± 3.0 nm, with a yield of 16 × 10⁹ particles from 1 × 10⁶ cells, and a total protein concentration of 122.8 µg per batch [34]. Their yield and protein content were 20-fold higher than what they observed for natural EXOs. In our study, the yield of EXOs isolated from the same number of cells as MIMs was greater than the ones reported by these authors, although an overall similar trend in protein concentration was noted. MIM production from MSCs by Lu et al. generated EVs with a peak diameter mostly between 100–200 nm and a yield of approximately 1.6 × 10⁶ [35]. It should be borne in mind that the diameter and composition of naturally secreted EXOs vary according to cell type and physiologic status, and environmental conditions, such as extracellular stimuli they are exposed to (including culture media and oxygen levels)[36]. Also, techniques used for EXO extraction, including variations in centrifugation protocols, type of rotor and g-force are aspects that play an important role in magnifying the yield, purity, protein content, and size of these EVs [37, 38]. Such variables should also be pondered in the production of MIMs and may explain differences encountered between the present results (in terms of yield, size and protein content) and data reported by others [33,34,35]. The feasibility of using frozen AF-MSCs to generate MIMs (F-MIMs) was evaluated to simplify the procedure by avoiding manipulation of fresh cells was also evaluated. Frozen cells produced a lower yield compared to fresh ones, but their diameter did not differ significantly, although the range of variation in the diameter of F-MIMs (min, 97.91, max: 119.01) was smaller than what was observed for MIMs (min: 85, max: 141). Cellular uptake evaluation demonstrated a reduction in the expression of GFP-positive macrophages over time, while a similar pattern of increased fibroblasts’ GFP expression was observed for MIMs and F-MIMs at 24 and 72 h, suggesting cryopreservation does not fully prevent the onset of apoptosis, impacting on cell recovery, which may explain the lower yield of F-MIMs and the differences observed when frozen cells were used [39].

The assessment of EV biodistribution is a pivotal stepping‐stone in the evaluation of their physiological significance, especially when it comes to the development of therapeutics [40], as it can determine the level of on‐target or off-target effects. Biodistribution is influenced by a variety of factors, including their cell origin, isolation techniques, and surface marker profiles [41]. To explore their biodistribution patterns and allow for a comparative analysis between naturally derived EXOs and engineered MIMs in terms of their organ-specific accumulation, fluorescently labeled particles were administered i.p. into female mice. While previous studies have reported the preferential accumulation of EVs in the lungs and liver following systemic and i.p. administration, although to different extents [42, 43], by looking into the female reproductive organs, our study reveals that EXOs and MIMs preferentially accumulate also in the uterus and ovaries. This unanticipated localization suggests AF-derived EXOs and MIMs have an inherent affinity or organotropism for reproductive tissues and may hold significant potential for targeted therapeutic interventions in the female reproductive system. The ability of naturally occurring EXOs to evade immune surveillance likely contributes to this biodistribution pattern [44]. Their small size, biocompatibility, and immune-modulatory surface proteins enable them to avoid rapid clearance by macrophages and other components of the innate immune system [14, 45]. This immune evasion mechanism not only enables EXOs to persist longer in circulation but also enhances their ability to accumulate in specific tissues where immune activity is naturally modulated. Based on this, our study suggests that MIMs may exhibit properties similar to those of EXOs; however, a more in-depth evaluation of their circulating levels at earlier time points is necessary to provide further insights.

Given the potential of EXOs and MIMs to specifically target uterine and ovarian tissues, we conducted a toxicology study using a well-established mouse model highly susceptible to drug-induced toxicity and NTDs, the SWV/Fnn strain [46]. EXOs or MIMs were administered i.p. every other day during a 15-day gestation period. Throughout the study, no significant changes were observed in the dams, as indicated by consistent maternal body weight and stable organ weights. Furthermore, the treatment did not impact the number of fetuses collected or result in a significant increase in fetal mortality or embryo resorption, suggesting that repeated administration of EXOs or MIMs during pregnancy is well tolerated in NTD-susceptible mouse model and supporting the safety of these vesicles for future therapeutic applications targeting reproductive tissues. The alterations produced by EXOs and MIMs in the hematological pattern of the SWV/Fnn females must be interpreted with caution, since these parameters may be influenced by variables such as age, gestation, liver and spleen functions, nutritional factors, among others [47, 48]. The MIMs reduced RBC and hemoglobin, increasing mean corpuscular volume, but these alterations did not lead to values outside the normal range for SWV/Fnn [49]. In addition, monocytes were significantly reduced by both EXOs and MIMs, but the greatest reduction was produced by EXOs when compared to MIMs. Most importantly, the reduction produced by MIMs (0.6 ± 0.2) kept monocyte counts above the norms for these animals (0.26 ± 0.17), whereas EXOs (0.17 ± 0.0) consistently reduced monocytes below the normal values, demonstrating that MIMs did impact innate immunity. To the best of our knowledge, this study is the first to comprehensively document the biodistribution of EVs in female mice, alongside the toxicological profile throughout gestation. In particular, the observed organotropism associated to EXOs and MIMs could be exploited for drug delivery specifically targeting uterine and ovarian tissues.

RNA encapsulation into EXOs represents a promising therapeutic strategy to various conditions, allowing for a more precise and ample control of protein expression than gene replacement therapy [50, 51]. In an effort to utilize both formulations for RNA therapeutic development, EXOs and MIMs were transfected with mRNA encoding GFP. Comparable values in terms of encapsulation rate were found between EXOs and MIMs (approximately 50%). This result contrasts with some reports in the literature, where mRNA loading into EXOs has been achieved with efficiencies as high as 90% [52] and highlights the influence of different loading strategies. Various methods, such as electroporation, passive diffusion, and specific transfection reagents, can significantly impact the loading outcome, leading to variability across studies [53]. Nonetheless, GFP-mRNA loaded within DiD-labeled MIMs and EXOs was efficiently delivered and expressed by two different cell lines, demonstrating the marked ability of MIMs to preserve mRNA functionality. Over time, we found that MIMs resulted in a greater percentage of GFP-positive macrophages and fibroblasts. In contrast, EXOs demonstrated a different temporal behavior within these cell populations. Specifically, the percentage of GFP-positive macrophages was initially higher during the first 24 h post-treatment with EXOs but decreased over time. However, in fibroblasts, the pattern of GFP expression induced by EXOs was similar to that observed with MIMs containing GFP mRNA. These findings suggest that the RNA cargo loading and delivery efficiency varies depending on the target cell type and function (i.e., varying capabilities for endocytosis and other uptake mechanisms) as well as on the type of nanoparticle used (i.e., composition, surface chemistry, size and shape [54]). Notably, MIMs were associated with a more prolonged expression of the delivered mRNA. The sustained and increasing expression of GFP mRNA delivered by MIMs indicates that this formulation may offer significant advantages over natural EXOs for mRNA encapsulation and delivery in therapeutic applications [16]. This differential uptake and sustained expression highlight the potential of MIMs as a more effective vehicle for mRNA-based therapies.

Next, we tested the plausibility of applying the technology of MIMs and EXOs as a potential therapeutic strategy for congenital malformations by using ex-vivo whole embryo culture. Our data showed that embryos explanted at E9.5 and cultured in a “soup” of DiD-labelled, mRNA-loaded EXOs or MIMs for 24 h, display the presence of a colocalization of signals at the level of the yolk sac where they expressed encapsulated GFP-mRNA. Importantly, no differences were found between the two formulations in support of embryo growth, yet only EXOs reached the embryo. As a semi-permeable barrier that allows for the exchange of nutrients, gases, and signaling molecules between the mother and fetus, this finding is not surprising. EVs have been reported as paracrine mediators of labor and delivery [55]. The ability of nanoparticles to cross the placenta is influenced by several recognized factors. Among these, lipid charge plays a significant role, with cationic nanoparticles demonstrating a higher likelihood of placental crossing compared to their anionic counterparts [56]. Additionally, the size of the nanoparticles (smaller particles exhibit greater placental uptake) and the gestational age at the time of exposure are crucial determinants [57]. Furthermore, the utilization of sensitive detection and quantitation methods is essential for understanding the extent and impact of nanoparticle accumulation in these regions. This includes assessing how exposure duration, clearance, and dosage affect placental transfer and accumulation.

While the accumulation of MIMs at the level of the yolk sac deserves a more detailed evaluation on the molecular mechanisms detaining them from crossing the placenta, data obtained here suggest their potential role as reconfigurable drug delivery tools to prevent the teratogenicity caused by maternal intake of drugs known to be toxic for the fetus, such as various anti-seizure medications which remain a hurdle in the treatment of pregnant women with seizure disorders [58,59,60], and other non-epileptic conditions [61]. In these instances, the chronic use of such substances is usually warranted to obtain adequate seizure control during pregnancy, raising serious concerns for pregnant women and those in childbearing age [62]. Malformations caused by these drugs are frequently severe and include NTDs, congenital cardiac and craniofacial defects [63]. On the other hand, since EXOs reach embryonic tissues and are primarily expressed in its cranial and ventral portions, they may be best suited for the prenatal repair of NTDs and other birth defects by loading these nanovesicles with cell-specific cargoes such as proteins, lipids, and nucleic acids [64]. This is not to say that despite the lack of direct contact with the embryo, MIMs may still play a role in the delivery of such molecules and be used in prenatal regenerative medicine through targeted delivery of genetic material to cells at the yolk sac by crosstalk and intercellular communication, due to its role in embryonic development [65, 66]. Further studies are warranted to best understand these mechanisms.

Conclusions

The heterogenous nature of naturally secreted EXOs requires a complex, time-consuming extraction processes resulting in significantly lower yield compared to MIMs and limiting their use for clinical application. The lack of standardized protocols limits the ability to compare results reported by others, and affects the consistency of RNA transfection system used to encapsulate mRNA into MIMs and/or EXOs. Although RNA transfection  is considered a convenient method [50], EE remained within a 50% margin. The loading of EXOs and mimetic counterparts with mRNA remains a challenge to be overcome in future studies [67]. The present data suggests that  MIMs represent as a promising strategy for high-throughput applications representing a better prospect for future clinical use as vehicles to reduce the incidence of congenital malformations secondary to in utero exposure to antiseizure medications and confirms the potential of EXOs as minimally invasive strategies to reduce the severity of NTD-associated abnormalities during prenatal repair. This finding opens new avenues for research into the mechanisms that drive this selective biodistribution. It also highlights the potential to tailor AF-MSC-derived EXO- or MIM-based delivery systems for more effective targeting ofreproductive organs. Translational research utilizing these strategies is warranted to better comprehend the impact and extent of the present evidence for clinical applications.