Mesenchymal stem cells undergoing apoptosis recover cyclophosphamide-induced cleft palate through modulation of NLR signaling mediated pyroptosis

Abstract

Background

Cleft palate is a common developmental disorder in craniofacial region, which cause the severe postnatal oral dysfunction. Mesenchymal stem cell (MSC) therapy has been revealed as a promising therapeutic approach in fetal developmental defects. However, since placental barrier blocks the transfer of cells from maternal circulation, how the MSC exert therapeutic effects and biological function to repair developmental injuries and maintain tissue homeostasis remains elusive.

Methods

Cyclophosphamide (CP)-induced cleft palate mice were used with replenishment of adipose-derived MSC (ADSC) or apoptotic vesicles (apoVs) derived from MSC, which of cleft palate were characterized by morphological analysis. PKH26 labeling and TUNEL assay were used in tracing of MSC or MSC-derived apoVs. Mechanistical studies were assessed by combinations of RNA-seq analysis, proteomic re-analysis and immunoassay.

Results

Exogenous MSC recovered the CP-induced cleft palate in E14.5 and E16.5 fetuses. Intriguingly, we unexpectedly found that MSC decreased rapidly and underwent apoptosis in maternal placenta. Accordingly, we assumed MSC-derived apoVs were the mediator of MSC exerting therapeutic effects. MSC-derived apoVs were injected into the pregnant mice and remarkedly improved CP-induced cleft palate, which were traced in fetal multiple organs, particularly in palatine shelves. Mechanistically, transcriptomic and proteomic analysis revealed that MSC-derived apoVs suppressed NLR signaling associated gene expression. Subsequent experimental validation confirmed that MSC-derived apoVs inhibited NLRP3-Caspase-1 mediated pyroptosis in palatal tissues, thereby contributing to the rescue of cleft palate phenotype.

Conclusions

Our studies revealed exogenous MSC underwent apoptosis and released apoVs to repair fetal cleft palate defects, which mechanistically reduced the NLR signaling mediated pyroptosis in palatal shelves.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-025-04652-4.

Introduction

Cleft palate is a common congenital disorder associated with significant morbidity, including difficulties in feeding, speech and hearing, which affects approximately 1 in 700 live births worldwide [1]. However, the therapeutics of cleft palate has been limited on multiple surgeries at postnatal stage, and an effective therapeutic approach directly intervening palatal defects during development remains to be investigated [2, 3]. Mechanistically, the pathogenesis of cleft palate is arisen from a failure in the strictly regulated palate developmental step, including processes of palatal shelves (PS) elevation to horizontal position and bilateral PS meeting and fusing at midline [4, 5].

Cyclophosphamide (CP), a widely used alkylating chemotherapeutic agent, is known to induce developmental abnormalities including cleft palate [6–8]. In vivo, CP is metabolized into active derivatives such as phosphoramide mustard and acrolein. These metabolites cause DNA cross-linking and strand breaks, interfering with cell division and inducing pyroptosis in female reproductive system and developing embryos, particularly within the PS [9–11]. Disrupted cellular proliferation and differentiation at this critical stage impairs the elevation and fusion of the PS, resulting in cleft palate formation. CP has also been reported to induce oxidative stress by increasing reactive oxygen species (ROS) production, overwhelming antioxidant defenses, and damaging cellular components [12–14]. Furthermore, CP may alter the expression of developmental genes and inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), which are implicated in craniofacial patterning [15, 16]. Collectively, these factors contribute to the teratogenic effects of CP and underscore the need for targeted interventions to mitigate these outcomes.

Mesenchymal stem cells (MSCs) are multipotent stromal cells that reside in various tissues such as adipose, umbilical cord, and bone marrow [17–19]. Based on their powerful proliferative/multilineage differentiation properties and potent immunomodulatory capacities, MSCs have been widely applied in a variety of disease therapy, such as in systemic lupus erythematosus (SLE), osteoporosis and ischemic heart failure [20–22]. Importantly, in the context of pregnancy, MSC-based therapies have shown promise in alleviating preeclampsia and promoting fetal-maternal immune tolerance [23, 24]. Beyond direct cell engraftment, increasing evidence supports that MSCs exert paracrine effects through releasing and transferring extracellular vesicles (EVs), including exosomes, microvesicles and apoptotic vesicles (apoVs), with carrying miRnomes and proteomes to modulate fetal-maternal microenvironmental homeostasis [25, 26].

Recent studies have revealed that MSC-derived apoVs possess potent regenerative and immunomodulatory properties. In the field of bone regeneration, apoVs have been shown to enhance osteogenic activity by delivering specific proteins and miRNAs [27]. They have also been successfully employed in clinical studies using lyophilized formulations for bone repair and hemostasis [28]. In addition to bone healing, MSC-apoVs also promote wound repair by enhancing angiogenesis and tissue remodeling, and they modulate immune responses by influencing macrophage polarization and attenuating inflammatory signaling [29, 30]. Due to their low immunogenicity, structural stability, and modifiability for targeted delivery, apoVs are considered promising candidates for clinical translation [31, 32]. However, whether transplanted MSCs or MSC-derived ApoVs directly contribute to the recovery developmental defects of fetuses, particular of cleft palate, remains elusive.

The placental barrier is composed of the trophoblastic epithelium covering the villi, the chorionic connective tissue, and fetal capillary endothelium, which collectively limit the bidirectional transfer of biological substances between the maternal and fetal circulations [33]. Since biological or chemical molecules with molecular weights exceeding 1000 Da are typically restricted by the placental barrier, there are limited therapeutic approaches available for addressing pregnant-related disorders [34]. As lipid bilayer-bound nanoparticles generated from cells, EVs are capable of inheriting and encapsulating a number of cellular contents, including DNA, RNA, proteins and lipids [35, 36], extensively contributing organismal homeostasis maintenance and pathogenesis [37–39]. Accumulating studies have documented the potential regulatory roles of EVs in maternal-fetal communication [40, 41]. Notably, placenta-derived EVs have been identified based on placenta-specific miRNAs and proteins, and they mediate signaling crosstalk between maternal and fetal compartments under both physiological and pathological conditions [42, 43]. Furthermore, fetal-derived exosomes have been revealed to travel to maternal circulation and participate in intercellular signaling [44]. However, whether the maternal circulating EVs transfer to fetuses to exert biological function remains unclear. Moreover, whether and how the MSC-derived EVs can cross this barrier to deliver therapeutic benefits to the developing fetus requires further investigation.

In this study, we firstly show that exogenous MSCs administration can recover developmental cleft palate at embryonic days 14.5 (E14.5) and E16.5. To dissect how MSCs might cross the placental barrier and exert therapeutic effects on the fetus, PKH26 labeled MSCs were traced them to uterus of female mice. Unexpectedly, we found that the transplanted MSCs underwent apoptosis and disappeared within the uterine environment. Subsequent immunofluorescence analyses showed that these apoptotic MSCs released apoptotic products, termed as apoVs, which were detected in the fetal circulation. Accordingly, we proved that MSC-derived apoVs were capable of effectively rescuing cleft palate, indicating their critical mechanistical role in mediating the therapeutic benefits of MSC therapy. Furthermore, transcriptomic and bioinformatic analyses of fetal palate tissue and MSC-derived apoVs revealed that apoVs attenuated the aberrant upregulation of gene expression in cleft palate fetuses, particularly within the NOD-like receptor (NLR) signaling pathway associated with pyroptosis.

Materials and methods

The work has been reported in line with the ARRIVE guidelines 2.0.

Animals

All animal experiments were conducted in accordance with the protocol approved by the Fujian Normal University Institutional Animal Care and Use Committee. Male and female C57BL/6J mice, aged 6 to 8 weeks, were purchased from Shanghai Laboratory Animal Center (SLAC, Shanghai, China) and housed in the specific pathogen-free (SPF) facility at the Fujian Normal University Animal Center. The mice were maintained under controlled environment conditions (23 ± 2 °C, 50% ± 5% humidity) with a 12-hour light/dark cycle and were provided with ad libitum access to food and water.

Treatments

After 1- to 2- week acclimatization period, mice were bred naturally at a ratio of one male to up to three females. Pregnant mice were then randomly assigned to one of the four treatment groups: (1) Vehicle control group (n = 12), received 0.1 mL/kg body weight (b.w.) PBS via intraperitoneal injection (i.p.) and tail vein injection; (2) CP group (n = 12), received 20 mg/kg b.w. CP i.p.; (3) CP + MSCs group (n = 6), received 20 mg/kg b.w. CP i.p. and 1 × 10⁶ MSCs via tail vein injection; (4) CP + ApoVs group (n = 6), received 20 mg/kg b.w. CP i.p. and 6 × 10⁹ ApoVs via tail vein injection. Pregnant mice were administered the specified treatment or control dose of CP, MSCs, or ApoVs on gestational day E11.5 (with day E0.5 corresponding to the day of copulatory plug detection). Pregnant mice were euthanized by cervical dislocation following isoflurane anesthesia and sacrificed on E14.5 or E16.5.

Histological analysis

Staged embryos were collected from timed pregnant females in cold PBS and imaged using an Axio Zoom.V16 stereo zoom microscope equipped with an Axiocam 506 color camera (Carl Zeiss, Germany). Embryonic heads were carefully dissected and fixed in 4% paraformaldehyde (PFA) at 4 °C overnight. After fixation, the samples were dehydrated through a graded ethanol series and embedded in paraffin. Serial sections were cut at a thickness of 7 μm and subjected to standard hematoxylin and eosin (H&E) staining, as previously described [45].

Isolation, culture and apoptosis induction of adipose-derived mesenchymal stem cells (ADSCs)

Adipose tissue was harvested from the inguinal fat pads of 6- to 8-week-old C57BL/6J mice under sterile conditions. The tissue was finely minced and digested in PBS with 4 mg/ml collagenase type I (C0103, Sigma-Aldrich, USA) and 8 mg/ml dispase II (D4693, Sigma-Aldrich, USA) at 37 °C for 60 min with gentle agitation. The digested mixture was filtered through a 70 μm cell strainer (352350, Corning, USA) to remove debris and centrifuged at 1500 rpm for 5 min. The resulting cell pellet was resuspended in DMEM (11965092, Gibco, USA) supplemented with 10% FBS (10099141, Gibco, USA) and 1% penicillin-streptomycin (15140122, Gibco, USA), and plated in T25 flasks. Non-adherent cells were removed after 24 h by replacing the medium. Adherent cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂, with medium changes every 2–3 days, until cells 70–80% confluency was achieved. For apoptosis induction, the ADSCs were washed twice with 0.1 μm-filtered PBS, and α-MEM (12561056, Gibco, USA) containing 500 nM staurosporine (STS; ALX-380-014-M005, Enzo life sciences, USA) was added to the culture dish. Cells were incubated for 6 h to induce apoptosis. Apoptotic ADSCs were observed and captured using an Axio Observer 5 microscope (Carl Zeiss, Germany).

Isolation, quantification, and characterization of ApoVs

ApoVs were isolated following a previously reported sequential centrifugation protocol [46]. Briefly, STS-treated apoptotic MSCs were collected, and cell debris was removed by centrifugation at 800 × g for 10 min at 4 °C, followed by 2,000 × g for 10 min at 4 °C. The resulting supernatant was further centrifuged at 16,000 × g for 30 min at 4 °C to isolate apoVs. ApoVs were resuspended in PBS and stored on ice. To prevent coagulation effects, apoVs were infused with 10U/mL heparin (H3393, Sigma-Aldrich, USA) and administered via tail vein injection. Quantification of apoVs was performed using nanoparticle tracking analysis to determine particle size and electric potential. Measurements were analyzed with ZetaView PMX120 (Particle Metrix, Germany). For characterization, apoVs were stained with antibodies targeting CD9 (1:100; sc-13118, Santa Cruz Biotechnology, USA), and CD81 (1:100; ab125011, Abcam, USA) and analyzed using flow cytometry (NovoCyte, ACEA Biosciences, USA). Annexin V-FITC (640906, BioLegend, USA) was used to confirm their apoptotic origin.

Transmission electron microscope

ApoVs were diluted in acetone and dispersed via ultrasonic oscillation for 15 min. The resulting dispersion was carefully deposited onto carbon-coated copper grids and allowed to dry at room temperature. TEM imaging was performed using a Tecnai G2 Spirit 120 kV transmission electron microscope (FEI, USA).

Labeling of ApoVs

ApoVs were resuspended in Diluent C (Sigma-Aldrich, USA) to achieve a final concentration suitable for labeling. An equal volume of 4 µM PKH26 (PKH26PCL, Sigma-Aldrich, USA) dye solution, prepared in Diluent C, was added to the ApoVs suspension and mixed gently for 5 min at room temperature. The reaction was terminated by adding an equal volume of complete culture medium, followed by centrifugation at 16,000 × g for 30 min at 4 °C to remove unbound dye. The labeled ApoVs were washed twice with sterile PBS to ensure thorough removal of excess dye and then resuspended in culture medium.

Total RNA isolation and quantitative real-time PCR

Total RNA was isolated from palatal tissues of E14.5 fetal mice using RNAiso reagent (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. First-strand cDNA synthesis was performed using the PrimeScript™ RT reagent kit (Takara Bio Inc.). Quantitative real-time PCR (qRT-PCR) was carried out using the SYBR Premix Ex Taq system (Takara Bio Inc.) on a Step One Plus Real-Time PCR System (Applied Bio-Systems, USA). Gene-specific primers were used to determine the relative mRNA expression levels of target genes. Primer sequences are listed in Supplementary Table 1. Relative expression levels were calculated using the 2^−ΔΔCt method, with Gapdh as the internal control.

Immunofluorescence (IF) staining

Paraffin sections were deparaffinized, rehydrated, and subjected to antigen retrieval using 10 mM sodium citrate buffer. After blocking with 5% normal serum at room temperature for 45 min, sections were incubated overnight at 4 °C with primary antibodies against Caspase-1 (1:100; 24232, Cell Signaling Technology, USA). The sections were then washed in PBST (PBS + 0.1% Triton X-100) and incubated with Alexa Fluor-conjugated secondary antibodies (1:500; Invitrogen, USA) for 1 h at room temperature. Nuclei were counterstained with DAPI (1:1000; D1306, Thermo Fisher, USA). After final washes, the sections were mounted with antifade mounting medium and imaged using a laser scanning confocal microscope (LSM780, Carl Zeiss, Garmany). Confocal images were captured and analyzed under identical settings to ensure consistency.

Western blot assay

Palates from E14.5 mouse embryos were carefully dissected and lysed on ice in RIPA buffer supplemented with 1% protease inhibitor cocktail (11836170001, Roche, Switzerland). Total protein concentrations were determined using a BCA protein assay kit (23225, Invitrogen, USA) according to the manufacturer’s protocol. Equal amounts of protein were resolved on 12% SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF) membranes (ISEQ00010, Millipore, USA). The membranes were blocked with 5% BSA for 1 h at room temperature and then incubated with primary antibodies against Caspase-1(1:1000; 24232, Cell Signaling Technology, USA) and NLRP3 (1:1000; F0335, SelleckChem, USA) following the manufacturer’s instructions. After washing in Tris-buffered saline (TBS) containing 0.1% Tween-20, the membranes were incubated with fluorescently labeled secondary antibodies (LI-COR, USA). Protein bands were visualized using the Odyssey CLx imaging system (LI-COR, USA).

TUNEL assay

Apoptotic MSCs were fixed in 4% PFA and permeabilized according to standard protocols. Following the manufacturer’s instructions, cells were incubated with TUNEL reagent (G3250, Promega, USA) for 60 min at 37 °C. After counterstaining with DAPI, TUNEL-positive cells were visualized and quantified under a fluorescence microscope (Carl Zeiss, Garmany).

RNA sequencing (RNA-seq) analysis

Palates from E14.5 mouse embryos were harvest, and total RNA was extracted using the RNeasy Mini Kit (74106, QIAGEN, Germany) following the manufacturer’s instruction. The concentration and purity of RNA samples were assessed using a NanoDrop™ OneC spectrophotometer (Thermo Fisher, USA). RNA sequencing was performed on the MGISEQ-2000 platform. Sequenced read quality was evaluated using Fastp (version 0.23.0), and raw reads were filtered with Trimmomatic (version 0.36) to remove low-quality and adapters. Clean reads were aligned to the mouse reference genome (mm10) using the STAR aligner (version 2.5.3a) with default parameters. Aligned reads were quantified using FeatureCounts, and expression levels were calculated as Reads Per Kilobase of transcript per Million mapped reads (RPKM). Differentially expressed genes (DEGs) between groups were identified using the edgeR package (version 3.12.1), with normalization and filtering criteria set to fold change (FC) > 2 and adjusted p-value < 0.05. Gene ontogeny (GO) and KEGG pathway enrichment analyses were performed using DAVID Bioinformatics Resources (http://david.ncifcrf.gov) based on the identified DEGs. Visualization of data was conducted using the GOplot package in R.

Proteomic Re-analysis

Proteins significantly upregulated in ApoVs (FC > 2, adjusted p-value < 0.05), as identified by Zhang et al., were selected for further functional annotation and pathway enrichment analysis using the GO and KEGG databases.

Statistical analysis

Data are presented as mean ± SD of at least triplicate measurements. Statistical significance was analyzed using one-way ANOVA. P values less than 0.05 were considered statistically significant. Graph analysis was performed using GraphPad Prism 8.00 (GraphPad Software, USA).

Results

Isolation and characterization of MSC and MSC-derived ApoVs

As previously described [47], murine MSCs were isolated from mouse adipose tissue and exhibited a typical spindle-shaped fibroblast-like morphology (Fig. 1A). These MSCs demonstrated proliferative capacity (Fig. 1B) and multilineage differentiation potential, including osteogenic and adipogenic differentiation (Fig. 1C and D). Flow cytometry analysis showed that the MSCs expressed positive surface markers CD73, CD90, and CD105, and lacked expression of the hematopoietic and endothelial markers of CD31 and CD45 (Fig. 1E). To harvest apoVs, cultured MSCs were treated with staurosporine (STS) to induce apoptosis, which was evident by cellular shrinkage after 6–8 h of treatment (Fig. 1F). TUNEL staining further confirmed the execution phase of apoptosis in MSCs (Fig. 1G). Transmission electron microscopy (TEM) and nanoparticle track analysis (NTA) verified the size of MSC-derived apoVs (Fig. 1H and I), consistent with previous reports [48]. ApoVs surface marker profiles were estimated by flow cytometry analysis, which positively expressed apoptotic marker Annexin V, and EV-associated markers CD9 and CD63 (Fig. 1J) [48, 49].

Fig. 1.

Characterization of MSCs and MSC-derived apoVs. A MSCs morphology. Scale bar, 50 μm. B Crystal violet staining of MSCs showing the proliferation capacity. Scale bar, 50 μm. C Alizarin red staining of MSCs showing the osteogenic differentiation capacity. Scale bar, 50 μm. D Oil red O staining of MSCs showing the adipogenic differentiation capacity. Scale bar, 50 μm. E Flow cytometry analysis of apoVs negative markers CD31 and CD 45, and positive markers CD73, CD90 and CD105. F MSC-derived apoVs morphology. Scale bar, 50 μm. G Detection of cell apoptosis using TUNEL staining. Scale bar, 50 μm. H Representative TEM images of apoVs. Scale bar, 100 nm. I The NTA results showing the size of apoVs in 1 × PBS buffer (pH 7.4). J Flow cytometry analysis of apoVs markers Annexin V, CD9 and CD63

MSCs transplantation ameliorates CP-induced cleft palate in fetuses

As a commonly used chemotherapeutic drugs, CP is known to induce multiple tissue impairments and dysfunctions, which was proved to cause fetal loss or malformations [50]. To establish a fetal injury model, we intraperitoneally injected CP at a dose of 20 mg/kg into pregnant mice at E14.5, and harvested fetuses at E14.5 and E16.5, the key developmental stages of palatal morphogenesis. Compared to control fetuses, CP-exposed fetuses exhibited significantly reduced body size, shortened tails and limb atrophy (Fig. 2A and B, left panels). Histological examination using hematoxylin and eosin (H&E) staining revealed that the PS in CP-treated fetuses failed to elevate to a horizontal position and displayed incomplete fusion at the midline, leading to visible clefting (Fig. 2A and B, right panels). These defects were more pronounced at E16.5, where PS development and midline continuity were completely absent.

Fig. 2.

MSCs ameliorates cleft palate in CP-induced fetal mice. A Gross morphological appearance and representative H&E staining of craniofacial regions in E14.5 fetuses (n = 3 per group). Right panels show enlarged views of boxed areas. CP-treated fetuses display reduced body size and palatal shelf elevation defects; MSC-treated fetuses exhibit partial restoration of craniofacial development and PS elevation. B Corresponding data in E16.5 fetuses (n = 3 per group). MSC treatment restores PS morphology and promotes successful midline fusion. Scale bars, 500 μm for lower magnification, 100 μm for higher magnification

To assess the therapeutic potential of MSCs, we injected 1 × 10⁶ MSCs intravenously into CP-treated pregnant mice at E11.5. Gross morphological evaluation showed that MSCs injection effectively improved fetal body size and limb formation at both E14.5 and E16.5 (Fig. 2A and B, left panels). Notably, histological analyses demonstrated improved PS elevation and restored midline fusion, indicating a significant reversal of the cleft palate phenotype (Fig. 2A and B, right panels).

MSCs cross the placental barrier by undergoing apoptosis in the maternal uterus and releasing ApoVs into the fetal circulation

To decipher how MSCs contribute to the rescue of fetal cleft palate, we investigated the biodistribution of transplanted MSCs. MSCs were labeled with PKH26 dye and intravenously injected into pregnant female mice. We found that systemically injected PKH26-MSCs preferentially accumulated in the uterus, particularly within the endometrium (EM), but not in the perimetrium (PM) or myometrium (MM) (Fig. 3A and E). Importantly, the number of MSCs markedly declined within 1 day post-injection and had almost disappeared by day 5 (Fig. 3A and E). Based on previous studies [46, 51], we hypothesized that these infused MSCs underwent apoptosis. TUNEL assays combined with PKH26-labeling confirmed that the transplanted MSCs underwent apoptosis after infusion (Fig. 3B). Recent evidence suggests that apoptosis plays an important role in MSC therapy, and that MSC-derived apoptotic products, termed as apoVs, exerted extensive therapeutic effects in multiple diseases [52]. In line with these findings and our results, we assumed that transplanted MSCs undergo apoptosis and release apoVs, which can cross the placental barrier and function as “communication bridges” linking maternal circulation to the fetal organismal microenvironment.

Fig. 3.

MSCs cross the placental barrier through undergoing apoptosis in maternal uterus and releasing apoVs to fetuses. A Tracing of PKH26-labeled MSCs (red) in the maternal uterus. Right panel (a) are higher magnification views of the left boxed regions, indicating region of endometrium (EM), perimetrium (PM) and myometrium (MM). Right panel (b) are higher magnification views of the left boxed regions, indicating region of EM and lumen. Scale bars, 50 μm for lower magnification, 10 μm for higher magnification. B Detection of PKH26-labeled MSCs (red) using TUNEL staining (Green) in the maternal uterus. Right panel (a) and (b) are higher magnification views of the left boxed regions, indicating region of EM. Scale bars, 50 μm for lower magnification, 10 μm for higher magnification. C Tracing of PKH26-labeled MSC-derived apoVs (red) in fetal craniofacial regions. Right panel (a) and (b) are higher magnification views of the left boxed regions, indicating the PS regions. Scale bars, 50 μm for lower magnification, 10 μm for higher magnification. D Tracing of PKH26-labeled MSC-derived apoVs (red) in the fetal multiple organs, including liver, lung, heart and kidney. Right panels are higher magnification views of the left boxed regions. Scale bars, 50 μm for lower magnification, 10 μm for higher magnification. E Quantification of fluorescence intensity of PKH26-labeled MSCs (red) after tracing 1 days, 3 days and 5 days. F Quantification of fluorescence intensity of PKH26-labeled MSC-derived apoVs (red) in fetal multiple organs, including liver, lung, heart and kidney. Data are presented as mean ± SD. *p < 0.05 by one-way ANOVA

To trace fetal biodistribution, PKH26-labeled apoVs were injected into pregnant mice at E11.5, and fetal tissues were examined at E14.5. Immunofluorescence analysis revealed that PKH26-apoVs accumulated in the fetal craniofacial region, especially enriched in the PS (Fig. 3C). Interestingly, PKH26-apoVs were also detected in multiple fetal organs, including the liver, heart and lung, but not the kidney (Fig. 3D and F). Collectively, these results demonstrate that MSC-derived apoVs can traverse the placental barrier and exert biological function in fetal recipient tissues.

MSC-derived apoptotic vesicles rescued CP-induced cleft palate in fetuses

To determine whether apoVs are the functional mediators of MSC therapeutic effects, we intravenously injected 6 × 10⁹ apoVs into CP-treated pregnant mice on E11.5 and examined fetal morphology at E14.5 and E16.5. ApoVs treatment partially reversed CP-induced developmental defects, including improved fetal body size, limb morphology, and craniofacial features (Fig. 4A and B, left panels). H&E-stained coronal sections of the palatal region revealed that apoVs significantly restored PS elevation and enhanced palatal fusion at the midline at both developmental stages (Fig. 4A and B, right panels). These above results showed the apoptosis/apoVs play an important role in mediating MSCs function and therapy.

Fig. 4.

MSC-derived apoVs ameliorates cleft palate in CP-induced fetal mice. A Gross morphological appearance and representative H&E staining of craniofacial regions in E14.5 fetuses (n = 3 per group). Right panels show enlarged views of boxed areas. ApoVs partially rescue body morphology and PS development. B Similar assessments in E16.5 fetuses (n = 3 per group), showing improved PS fusion and palatal continuity following apoVs treatment. Scale bars, 500 μm for lower magnification, 100 μm for higher magnification

Systemically infused MSC-derived ApoVs restore altered NLR signaling in the fetal palate

To explore the underlying mechanism by which MSC-derived apoVs rescue cleft palate in CP-induced fetal impairment, we performed RNA-seq analysis on fetal palate tissues collected at E14.5. Compared to the control (Ctrl) group, CP-treated fetuses exhibited 427 differentially expressed genes (DEGs), consisting of 348 up-regulated and 79 down-regulated genes (Fig. 5A). Importantly, we observed substantial transcriptomic changes following apoVs treatment in CP-exposed fetuses, with 171 genes upregulated and 686 downregulated compared to the CP group (Fig. 5B). Heatmap analysis revealed that the majority of aberrantly upregulated genes in the CP group were attenuated by apoVs treatment (Fig. 5C). Further intersection analysis identified 292 DEGs that were upregulated in the Ctrl vs. CP comparison and subsequently downregulated in the CP vs. apoVs comparison (Fig. 5D). Gene ontology (GO) enrichment analysis of these 292 genes indicated that apoVs primarily modulate gene expression associated with immune system and inflammatory response (Fig. 5E).

Fig. 5.

Systemically infused MSC-derived apoVs rescue altered NLR signaling pathway of fetal palate. A Volcano plot of DEGs in Ctrl and CP fetal mice (n = 3 per group). B Volcano plot of DEGs in CP and CP + apoVs fetal mice (n = 3 per group). C Clustered heatmap detailing of DEGs in Ctrl, CP and CP + apoVs fetal mice (Log₂(fold change) > 1.0 and < −1.0, P value < 0.05). D Venn diagram showing the number of up-regulated gene (Ctrl vs. CP) and down regulated gene (CP vs. CP + apoVs) (Log₂(fold change) > 1.0 and < −1.0, P value < 0.05). E GO analysis of the intersection of up-regulated gene (Ctrl vs. CP) and down regulated gene (CP vs. CP + apoVs) (Log₂(fold change) > 1.0 or < −1.0). F KEGG pathway enrichment analysis of the intersection of up-regulated gene (Ctrl vs. CP) and down regulated gene (CP vs. CP + apoVs) (Log₂(fold change) > 1.0 and < −1.0)

To further investigate the signaling pathways involved, kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed that the NOD-like receptor (NLR) signaling pathway is involved in the regulatory mechanism through which apoVs exert therapeutic effects in CP-induced cleft palatal fetuses (Fig. 5F). Notably, we re-analysed proteomic data of MSC-derived apoVs from a previously published study [49], which demonstrated that “immune system”-related proteins were abundant enriched within the “organismal systems” category (Supplementary Fig. 1 A). KEGG analysis of these “immune system” proteins further indicated that apoVs carry NLR signaling-related proteins derived from MSCs (Supplementary Fig. 1B). Together, these findings suggest that MSC-derived apoVs may reach the fetal palate and modulate NLR signaling-mediated pyroptosis, thereby contributing to the phenotypic rescue of cleft palate.

MSC-derived ApoVs ameliorate CP-induced cleft palate by suppressing NLR signaling-mediated pyroptosis

Building on transcriptomic and proteomic findings that implicated the NLR signaling in the pathogenesis of CP-induced cleft palate, we next aimed to investigate whether pyroptosis mediated by this pathway contributes to this developmental defect and whether it can be suppressed by MSC-derived apoVs treatment. Quantitative PCR analysis revealed significantly elevated mRNA expression of pyroptosis-related genes, including Caspase-1, Nlrp3, Gsdmd, Il-18, and Il-1β in fetal palatal tissues from the CP group. Notably, these increases were markedly attenuated following administration of MSC-derived apoVs (Fig. 6A). Immunofluorescence staining further revealed substantial accumulation of Caspase-1 positive cells within the PS of CP-treated fetuses, whereas this pyroptotic response was significantly diminished in the apoVs-treated group (Fig. 6B and C). Consistent with these findings, western blot analysis confirmed upregulation of NLRP3 and Caspase-1 protein levels in the CP group, both of which were reduced upon treatment with MSC-derived apoVs (Fig. 6D and E). Together, these results support the involvement of NLR signaling-mediated pyroptosis in the etiology of CP-induced cleft palate and demonstrate that MSC-derived apoVs can suppress this inflammatory pathway to promote palatal tissue recovery.

Fig. 6.

MSC-derived apoVs suppress NLR signaling mediated pyroptosis in CP-induced fetal palatal shelves. A Quantitative RT-PCR analysis of Caspase-1, Nlrp3, Gsdmd, Il-18, and Il-1β mRNA levels in palatal tissues from Ctrl, CP and CP + ApoVs groups at E14.5 (n = 3 per group). B Representative immunofluorescence staining for Caspase-1 in fetal palatal shelves (n = 3 per group). Scale bars, 50 μm for lower magnification, 25 μm for higher magnification. C Quantification of fluorescence intensity of Caspase-1 positive cells per field. D Representative western blot bands of NLRP3 and Caspase-1 proteins in fetal palatal tissues. GAPDH served as loading control. Full-length blots are presented in Supplementary Fig. 2. E Quantification of NLRP3 and Caspase-1 protein levels normalized to GAPDH (n = 3 per group). Data are presented as mean ± SD. *p < 0.05, **p < 0.01 by one-way ANOVA

Discussion

Palatal development stands as a finely coordinated process with multiple gene interactions and specific environmental triggers, contributing to the complex pathogenesis of cleft palate [1, 53]. As demonstrated in the previous researches, cleft palate can be recovered by surgery, dental correction, speech therapy and psychosocial intervention [54, 55]. Nevertheless, current therapeutics of cleft palate still lack of effective cure approaches with functionality and reproducibility to regenerate the palatal defects [33, 56]. Recently, MSCs as regenerative potential cells were considered to treat developmental defects during pregnancy, especially when combined with engineering strategies [57–59]. However, whether systemically transplanted MSCs can directly recover fetal defects remains to be unveiled. In this study, we demonstrate for the first time that systemic administration of MSCs in CP-induced pregnant mice effectively restored fetal developmental abnormalities, particularly cleft palate. Histological analysis revealed that MSC transplantation corrected impaired palatal development, including elevation of the PS to a horizontal orientation and their fusion at the midline. These findings suggest that systemic MSC infusion may have future applications in regenerative medicine for maternal and fetal developmental disorders.

Given that the placenta barrier limits the transfer of most biological substances from maternal to fetal circulation, we deciphered mechanisms by which MSC therapy exerts effects on fetal tissues. In vivo tracing experiments revealed that transplanted MSCs underwent apoptosis within the maternal uterus and subsequently released apoVs into the fetal compartment. While earlier studies have emphasized the role of MSC engraftment and paracrine signaling in therapeutic efficacy [60, 61], increasing evidence over the past decade has revealed that apoptosis also plays a critical role in MSC therapy. Rather than being a passive process of cellular clearance, apoptosis enables MSCs to release intracellular contents, membrane fragments, and vesicles with biological activity [52]. Recent studies have shown transplanted MSCs undergo apoptosis to exert specific therapeutic effects [51], of which apoVs have been documented that are extensively involved in a variety of disease therapy, such as immune disorders, osteoporosis, skin injuries, ovarian impairments, and more [29, 45, 46, 62]. Specifically, it is indicated that MSC-derived apoVs ameliorate impaired ovary, further recover female fertility and fetal abnormalities [45]. However, whether MSC-derived apoVs influence fetal development during pregnancy is still unknown. In this study, we found that MSC-derived apoVs effectively rescue fetal cleft palate, which revealed a previously unrecognized regulatory link between maternal apoVs and fetal development. Although traditionally viewed as a restrictive barrier, the placenta is in fact a lipid membrane, which may permit the passage of lipid bilayer vesicles such as apoVs (~ 100 nm in diameter) [49]. These characteristics make apoVs well suited to traverse the placental barrier and exert biological effects in the fetus, suggesting a novel mechanism of MSC therapy via maternal-to-fetal communication.

Understanding how apoVs participate in the mechanistic regulation of pregnant biological behavior and therapeutic outcomes will inform future applications of apoVs in developmental disease therapy and regenerative medicine. In transcriptomic analysis, we observed that MSC-derived apoVs restored the aberrant gene expression in palatal tissues of CP-induced cleft palatal models, particularly by modulating NLR signaling. Combining with proteomic re-analysis, we further confirmed that apoVs carry NLR signaling related proteins to interplay with fetal recipient organ. In this regard, NLR signaling are specific pathogen-associated molecular patterns, triggering a number of inflammatory and immune reaction. As to the toxin such as CP, NLRs are activated to initiate anti-inflammatory signaling cascades, which induce Caspase-1 activation to drive inflammatory lytic cell death, pyroptosis [63–65]. Furthermore, recent researches have demonstrated that MSC-derived EVs can target NLR signaling to ordinate recipient immune microenvironment and regulate pyroptosis [66–68]. Nevertheless, whether systemic apoptotic signals coordinate with NLR/Caspase-1-mediated pyroptosis is not elucidated enough. Based on our findings, we propose that MSC-derived apoVs directly regulate fetal NLR signaling, offering novel insight into the molecular link between maternal apoptotic signaling and fetal inflammatory impairments. Nonetheless, the specific mechanisms by which apoVs regulate the NLR pathway remain to be elucidated. In particular, it is not yet clear whether proteins or microRNAs within apoVs directly interact with key components of the NLR signaling cascade. Future studies should incorporate genetic approaches such as CRISPR/Cas9-mediated knockout or overexpression of key target genes (e.g., Nlrp3, Caspase-1, Il1b) in fetal or organoid models to define causal relationships.

From a translational perspective, our findings offer promising implications for prenatal therapy. ApoVs represent a cell-free, immunologically inert alternative to cell transplantation, which may overcome several safety and ethical concerns associated with MSC therapies. Notably, apoVs exhibited the capacity to traverse the placental barrier and accumulate in fetal organs, including the palate, heart, and liver. This indicates their potential utility for targeted fetal interventions. Nevertheless, several critical challenges remain to be addressed, including optimal dosage, timing of administration, long-term safety, and standardization of apoVs production. Moreover, although rodent placentae differ anatomically from human placentae, similar vesicle trafficking mechanisms have been reported in primates and human tissues [69, 70]. Further studies in large animal models and human placental systems are needed before clinical application.

Conclusions

In summary, our study demonstrates that systemically administered MSCs can effectively rescue CP-induced fetal cleft palate, primarily through the release of apoVs that cross the placental barrier and act on fetal tissues. These apoVs accumulate in the fetal craniofacial region, including the palatal shelves, and restore impaired palatal development. Through transcriptomic and proteomic analyses, we identify the NLR signaling–mediated pyroptosis pathway as a critical target of apoV activity in the fetal palate. These findings not only establish a novel mechanistic link between maternal MSC apoptosis and fetal therapeutic response but also suggest a previously unrecognized mode of intercellular communication between maternal and fetal compartments. Our work provides strong support for the application of MSC-derived apoVs as a non-cellular therapeutic strategy for congenital defects, offering a potential new avenue for prenatal regenerative interventions. Future studies are warranted to further dissect the molecular cargo of apoVs and clarify their downstream targets and mechanisms in fetal development.

Abbreviations

ADSCs

Adipose-derived mesenchymal stem cells

apoVs

Apoptotic vesicles

CP

Cleft palate

DEGs

Differentially expressed genes

DMEM

Dulbecco’s modified eagle medium

EM

Endometrium

EVs

Extracellular vesicles

FBS

Fetal bovine serum

FC

Fold change

GO

Gene ontogeny

H&E

Hematoxylin and eosin

IF

Immunofluorescence

KEGG

Kyoto encyclopedia of genes and genomes

LSM

Laser scanning confocal microscope

MEM

Minimum essential medium

MM

Myometrium

MSCs

Mesenchymal stem cells

NLR

NOD-like receptor

NTA

Nanoparticle track analysis

PM

Perimetrium

PS

Palatal shelves

qRT-PCR

Quantitative real-time polymerase chain reaction

RNA-seq

Ribonucleic acid sequencing

RPKM

Reads Per Kilobase of transcript per Million mapped reads

SLE

Systemic lupus erythematosus

SPF

Specific pathogen-free

STS

Staurosporine

TEM

Transmission electron microscope

TUNEL

Terminal-deoxynucleotidyl Transferase Mediated dUTP Nick-End Labeling

Author contributions

Xi Zheng and Yu Fu designed the study, performed transcriptomic, proteomic analysis, and drafted the manuscript. Hongbin Lu, Lizhen Lan, Xiaohan Geng, and Peiyuan Zhang performed histological experiments and immunoassay. Bingdong Sui performed MSCs and apoVs identification experiments. Xuefeng Hu conceived the project and supervised the experiments. All authors reviewed and approved the final manuscript.

Funding

This work was supported by National Natural Science Foundation of China (No. 81771034).

Data availability

The RNA-seq data generated during this study have been deposited in the NCBI Sequence Read Archive (SRA) under accession number PRJNA1271243. All other data supporting the findings are included in this article and its supplementary information files.

Declarations

Ethics approval and consent to participate

This research does not contain clinical experiments. The animal studies were conducted in compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal experiments were approved by Fujian Normal University Institutional Animal Care and Use Committee (Title: The regulatory mechanism of Shox2/Alx1 signaling axis in the osteogenesis of hard palate; Approval Number: IACUC-20230013; Date of approval: 10/03/2023).

Consent for publication

All authors confirm their consent for publication.

Competing interests

The authors declare that they have no competing interests.