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.

   Keywords: Mesenchymal stem cells, Apoptosis, Apoptotic vesicles, Cleft
   palate, NLR signaling, Pyroptosis

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
   [[38]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 [[39]2, [40]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 [[41]4, [42]5].

   Cyclophosphamide (CP), a widely used alkylating chemotherapeutic agent,
   is known to induce developmental abnormalities including cleft palate
   [[43]6–[44]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 [[45]9–[46]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 [[47]12–[48]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 [[49]15, [50]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
   [[51]17–[52]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 [[53]20–[54]22]. Importantly, in the context of pregnancy,
   MSC-based therapies have shown promise in alleviating preeclampsia and
   promoting fetal-maternal immune tolerance [[55]23, [56]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 [[57]25, [58]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 [[59]27]. They have also been
   successfully employed in clinical studies using lyophilized
   formulations for bone repair and hemostasis [[60]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 [[61]29, [62]30]. Due to their low immunogenicity, structural
   stability, and modifiability for targeted delivery, apoVs are
   considered promising candidates for clinical translation [[63]31,
   [64]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 [[65]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 [[66]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 [[67]35, [68]36], extensively
   contributing organismal homeostasis maintenance and pathogenesis
   [[69]37–[70]39]. Accumulating studies have documented the potential
   regulatory roles of EVs in maternal-fetal communication [[71]40,
   [72]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 [[73]42, [74]43].
   Furthermore, fetal-derived exosomes have been revealed to travel to
   maternal circulation and participate in intercellular signaling
   [[75]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 × 10^6 MSCs via tail vein
   injection; (4) CP + ApoVs group (n = 6), received 20 mg/kg b.w. CP i.p.
   and 6 × 10^9 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 [[76]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% CO[2], 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 [[77]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 ([78]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 [[79]47], murine MSCs were isolated from mouse
   adipose tissue and exhibited a typical spindle-shaped fibroblast-like
   morphology (Fig. [80]1A). These MSCs demonstrated proliferative
   capacity (Fig. [81]1B) and multilineage differentiation potential,
   including osteogenic and adipogenic differentiation (Fig. [82]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. [83]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. [84]1F). TUNEL staining further confirmed the
   execution phase of apoptosis in MSCs (Fig. [85]1G). Transmission
   electron microscopy (TEM) and nanoparticle track analysis (NTA)
   verified the size of MSC-derived apoVs (Fig. [86]1H and I), consistent
   with previous reports [[87]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. [88]1J) [[89]48, [90]49].

Fig. 1.

   [91]Fig. 1
   [92]Open in a new tab

   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 [[93]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. [94]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. [95]2A and B, right panels). These
   defects were more pronounced at E16.5, where PS development and midline
   continuity were completely absent.

Fig. 2.

   [96]Fig. 2
   [97]Open in a new tab

   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 × 10^6 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. [98]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. [99]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. [100]3A and
   E). Importantly, the number of MSCs markedly declined within 1 day
   post-injection and had almost disappeared by day 5 (Fig. [101]3A and
   E). Based on previous studies [[102]46, [103]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. [104]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 [[105]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.

   [106]Fig. 3
   [107]Open in a new tab

   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. [108]3C). Interestingly, PKH26-apoVs were also detected in
   multiple fetal organs, including the liver, heart and lung, but not the
   kidney (Fig. [109]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 × 10^9 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. [110]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. [111]4A and B,
   right panels). These above results showed the apoptosis/apoVs play an
   important role in mediating MSCs function and therapy.

Fig. 4.

   [112]Fig. 4
   [113]Open in a new tab

   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. [114]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. [115]5B). Heatmap analysis revealed that
   the majority of aberrantly upregulated genes in the CP group were
   attenuated by apoVs treatment (Fig. [116]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. [117]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. [118]5E).

Fig. 5.

   [119]Fig. 5
   [120]Open in a new tab

   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 (Log[2](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) (Log[2](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) (Log[2](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) (Log[2](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. [121]5F). Notably, we
   re-analysed proteomic data of MSC-derived apoVs from a previously
   published study [[122]49], which demonstrated that “immune
   system”-related proteins were abundant enriched within the “organismal
   systems” category (Supplementary Fig. [123]1 A). KEGG analysis of these
   “immune system” proteins further indicated that apoVs carry NLR
   signaling-related proteins derived from MSCs (Supplementary
   Fig. [124]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. [125]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. [126]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. [127]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.

   [128]Fig. 6
   [129]Open in a new tab

   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. [130]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 [[131]1,
   [132]53]. As demonstrated in the previous researches, cleft palate can
   be recovered by surgery, dental correction, speech therapy and
   psychosocial intervention [[133]54, [134]55]. Nevertheless, current
   therapeutics of cleft palate still lack of effective cure approaches
   with functionality and reproducibility to regenerate the palatal
   defects [[135]33, [136]56]. Recently, MSCs as regenerative potential
   cells were considered to treat developmental defects during pregnancy,
   especially when combined with engineering strategies [[137]57–[138]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 [[139]60,
   [140]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 [[141]52]. Recent studies have shown transplanted
   MSCs undergo apoptosis to exert specific therapeutic effects [[142]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 [[143]29, [144]45,
   [145]46, [146]62]. Specifically, it is indicated that MSC-derived apoVs
   ameliorate impaired ovary, further recover female fertility and fetal
   abnormalities [[147]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) [[148]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 [[149]63–[150]65]. Furthermore, recent researches have
   demonstrated that MSC-derived EVs can target NLR signaling to ordinate
   recipient immune microenvironment and regulate pyroptosis
   [[151]66–[152]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 [[153]69, [154]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.

Supplementary Information

   [155]Supplementary material 1.^ (14.4KB, docx)
   [156]Supplementary material 2.^ (306.9KB, pdf)
   [157]Supplementary material 3.^ (370.9KB, pdf)

Acknowledgements