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Cisplatin-encapsulated TRAIL-engineered exosomes from human chorion-derived MSCs for targeted cervical cancer therapy

Stem Cell Research & Therapy volume 15, Article number: 396 (2024) Cite this article

Abstract

Background

Cisplatin (DDP) is an efficacious and widely applied chemotherapeutic drug for cervical cancer patients who are diagnosed as metastatic and inoperable, or desiring fertility preservation. Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) selectively triggers cancer cells apoptosis by binding to cognate death receptors (DR4 and DR5). Mesenchymal stem cells-derived exosomes (MSCs-Exo) have been regarded as ideal drug carriers on account of their nanoscale, low toxicity, low immunogenicity, high stability, biodegradability, and abundant sources.

Methods

Human chorion-derived mesenchymal stem cells (hCD-MSCs) were isolated by adherent culture method. TRAIL-engineered hCD-MSCs (hCD-MSCsTRAIL) were constructed by lentivirus transfection, and its secreted Exo (hCD-MSCs-ExoTRAIL) were acquired by differential centrifugation and confirmed to overexpress TRAIL by western blotting. Next, nanoscale drug delivery systems (DDP & hCD-MSCs-ExoTRAIL) were fabricated by loading DDP into hCD-MSCs-ExoTRAIL via electroporation. The CCK-8 assay and flow cytometry were conducted to explore the proliferation and apoptosis of cervical cancer cells (SiHa and HeLa), respectively. Cervical cancer-bearing nude mice were constructed to examine the antitumor activity and biosafety of DDP & hCD-MSCs-ExoTRAIL in vivo.

Results

Compared with hCD-MSCs-Exo, hCD-MSCs-ExoTRAIL weakened proliferation and enhanced apoptosis of cervical cancer cells. DDP & hCD-MSCs-ExoTRAIL were proved to retard cervical cancer cell proliferation and propel cell apoptosis more effectively than DDP or hCD-MSCs-ExoTRAIL alone in vitro. In cervical cancer-bearing mice, DDP & hCD-MSCs-ExoTRAIL evidently hampered tumor growth, and its role in inducing apoptosis was mechanistically associated with JNK/p-c-Jun activation and survivin suppression. Moreover, DDP & hCD-MSCs-ExoTRAIL showed favorable biosafety in vivo.

Conclusions

DDP & hCD-MSCs-ExoTRAIL nanoparticles exhibited great promise for cervical cancer treatment as an Exo-based chemo-gene combinational therapy in clinical practice.

Graphical abstract

Introduction

Cervical cancer induces the fourth highest carcinoma-related mortality in females, presenting with about 661,021 newly diagnosed patients as well as 348,189 deaths in 2022 worldwide [1]. Current conventional treatments for cervical cancer consist of surgical resection, radiotherapy and chemotherapy [2]. Metastatic and inoperable recurrent cervical cancer holds serious threats to patients clinical prognosis [3], necessitating platinum-based chemotherapy as the preferentially recommended therapeutic regimen [4].

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) initiates programmed cell death in extrinsic pathway, and optionally mediates apoptosis of malignant cells while renders no toxicity on normal cells [5]. TRAIL triggers the apoptosis of cancer cells via specific binding to cognate death receptors (DR4 and DR5) [6]. However, clinical application of soluble TRAIL protein variant is restricted by its short plasma half-life, low biostability, and limited bioavailability [7]. Delivery of TRAIL by nanoparticle-based carriers is potentially considered as a promising approach [8].

Exosomes (Exo), are disc-shaped nanoparticles naturally released by cells, with a diameter of 30–150 nm [9]. Exo serve as promising nano-sized drug delivery carriers and their phospholipid bilayer membrane protect the loaded small-molecule drugs from degradation [10]. Cell-free therapy using mesenchymal stem cells-derived exosomes (MSCs-Exo) as drug transporters potentially avoids some adverse reactions of MSCs transplantation, such as immune rejection, teratogenicity, and tumorigenicity [11]. And MSCs-Exo exhibit stem cell-like tumor-homing capacity [12]. Therefore, MSCs-Exo have been regarded as ideal drug carriers with nanoscale, low immunogenicity, low toxicity, biodegradability, high stability, and rich sources [13]. Researches implicated that TRAIL delivery by MSCs-Exo was more effective in killing tumor cells than recombinant soluble TRAIL protein [14].

Our previous research outlined that human chorion-derived mesenchymal stem cells (hCD-MSCs) exhibited good performance on chemotaxis towards cervical cancer cells [15]. On the basis of this premise, hCD-MSCs were selected as the cell source to manufacture Exo (hCD-MSCs-Exo) for co-loading with TRAIL and Cisplatin (DDP) to treat cervical cancer in the present study. Firstly, lentivirus packaging technology was employed to construct TRAIL-engineered hCD-MSCs (hCD-MSCsTRAIL), and a novel pattern of Exo expressing TRAIL (hCD-MSCs-ExoTRAIL) was further isolated. Secondly, chemotherapeutic agent DDP was encapsulated into hCD-MSCs-ExoTRAIL by electroporation method, thus developing a drug delivery system of hCD-MSCs-Exo co-loaded with TRAIL and DDP (DDP & hCD-MSCs-ExoTRAIL). Subsequently, DDP & hCD-MSCs-ExoTRAIL were administered in cervical cancer cells and cervical cancer-bearing nude mice to observe its antitumor activity. Collectively, application of DDP & hCD-MSCs-ExoTRAIL drug delivery systems may be a feasible scheme for cervical cancer treatment.

Materials and methods

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

Cell lines and culture

The cervical squamous carcinoma cell line SiHa and cervical adenocarcinoma cell line HeLa were acquired from Shanghai Cell Biology Medical Research Institute, Chinese Academy of Sciences. The normal cervical epithelial cell line ECT1 was obtained from Shanghai BinSui Biological Technology Co., Ltd. High-glucose Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin solution (Pen-Strep, 100 mg/mL; Invitrogen, UK) was employed to cultivate SiHa and HeLa cells. And Eagle’s minimum essential medium (EMEM) was used to incubate ECT1 cells. Cells were cultured at 37 °C in a humidity-controlled atmosphere with 5% CO2.

Compounds

Cisplatin (DDP, #HY-17394; MCE, New Jersey, USA) was solubilized in distilled water to generate 2 mM solution and preserved at -20 °C, which was diluted to specific concentrations with complete DMEM medium before use.

Separation and incubation of hCD-MSCs

The separation and incubation of hCD-MSCs were performed based on our previously mentioned protocol [15]. Placental chorionic tissues from healthy and full-term pregnant women (25–30 years of age) were obtained following cesarean section, which received approval from ethics committee of the Second Affiliated Hospital of Wenzhou Medical University. The chorionic tissues were washed with phosphate-buffered saline solution (PBS) and chopped up completely, and subsequently dissociated with 1 mg/mL collagenase type II (Solarbio, China) for 1 h at 37 °C in a water-bath shaker. Thereafter, cell suspension underwent filtration utilizing a 100-µm nylon mesh cell filter to remove the tissue and cellular debris. Next, cell suspension was gathered and subjected to centrifugation at 1000 rpm for 10 min, and the supernatant was removed carefully. Afterwards, isolated hCD-MSCs were resuspended in low-glucose DMEM medium consisting of 10% FBS and 1% Pen-Strep. The cell culture medium was replaced at 2-day intervals and cells were passaged at a confluence density ranging from 80 to 90%.

Multipotent differentiation and immunophenotype characterizations of hCD-MSCs

The hCD-MSCs at passage 3 (P3) were selected to for multipotent differentiation and immunophenotype characterizations. An appraisement of the multipotency of hCD-MSCs towards adipogenic, osteogenic, and chondrogenic lineages was performed. Concisely, cells were inoculated in 24-well plates at a density of 5000 cells per well. When cell confluence reached 60-70%, cell culture medium was replaced with adipogenic differentiation medium (Gibco, USA), osteogenic differentiation medium (Gibco, USA), and chondrogenesis differentiation medium (Gibco, USA), respectively. 4% paraformaldehyde fixation for 30 min was administrated in cells 2 weeks later. Subsequently, cells were subjected to Oil red O staining (Solarbio, China), Alkaline phosphatase staining (Beyotime, China), and Alcian blue staining (Solarbio, China), respectively, for confirming the positive induction.

The immunophenotype of hCD-MSCs was characterized by flow cytometry with fluorescent-labeled monoclonal antibodies against CD90, CD73, CD44, CD45, CD34, HLA-ABC, and HLA-DR (ThermoFisher, USA). Cells were subjected to trypsinization and PBS washing, followed by incubation with respective antibodies for 20 min at room temperature in light-protecting condition. Afterwards, the samples were processed by CytoFLEX flow cytometry (Beckman Coulter, Fullerton, USA).

Immunohistochemistry (IHC)

The cervical cancer tissues and paired adjacent non-cancerous cervical tissues were incubated with primary antibody DR4 (1:200, Affinity Biosciences, #AF0304) following the guidelines of immunohistochemistry. Concisely, deparaffinized and rehydrated tissue sections were subjected to microwave oven for antigen retrieval. Next, the tissue sections were incubated with primary antibodies overnight at 4 ℃ followed by incubation with appropriate secondary antibodies. A freshly-prepared DAB solution was employed for chromogenic reaction and nuclei were counterstained with hematoxylin. Positive staining appeared in brown, and representative pictures were captured by microscope (Leica Microsystems, Wetzlar, Germany).

Western blotting assay

Total proteins were extracted from cell lysates and phenylmethanesulfonylfluoride (PMSF) was applied to prevent proteins from degradation. The protein quantification was done by bicinchoninic acid (BCA) protein assay kit (Beyotime, China). The prepared protein (40 µg/path) was resolved on polyacrylamide sodium dodecyl sulfate (SDS-PAGE) gels and subsequently electro-transferred onto polyvinylidene difluoride (PVDF) membranes. Following blocking with 5% milk, membranes were incubated with primary antibodies overnight at 4 ℃. Primary antibody of DR4 (1:2000, Affinity Biosciences, #AF0304) were employed and the anti-rabbit secondary antibodies conjugated with horseradish peroxidase (HRP) were employed to detect the protein bands. The protein bands were visualized with enzyme-linked chemiluminescence detection kit (ECL) under the Chemiluminescence Imaging System (ChemiScope 6000, CLiNX, China).

Lentivirus and transfection

The pL-CMV-GFP-TRAIL-LAMP2b lentiviral vector and the negative control vector pL-CMV-GFP-blank-LAMP2b were acquired from Shanghai Nuobai Biological Technology Co., Ltd. (China), which were co-cultured with HEK-293T cells by using Lipofectamine 2000 (Invitrogen, USA) transfection reagent with a lentivirus transfection system, respectively. Then, the resulting virus particles were applied to transfect hCD-MSCs. The cell model stably overexpressing TRAIL (hCD-MSCsTRAIL) was generated by selection with 10 µg/mL Blasticidin-S (Solarbio, China), and the protein expression of TRAIL (1:1000, CST, #3219) was examined by western blotting.

Exo extraction and characterization

To obtain hCD-MSCs and hCD-MSCsTRAIL secreted Exo, cells were first maintained in low-glucose DMEM medium consisting of 10% FBS and 1% Pen-Strep until cell density reached 60–70% confluence. Cells were subsequently cultured in low-glucose DMEM medium containing 10% exosome-free FBS and 1% Pen-Strep for an additional 48 h. Then, culture supernatant was collected followed by a differential centrifugation at 4℃, including 3,000 g for 10 min and 10,000 g for 30 min to eliminate cells and debris. Exo was extracted at 100,000 g for 90 min at 4℃ in a Type Ti32 rotor by ultracentrifugation (Beckman Coulter, USA). After meticulously eliminating the supernatant, Exo was washed in PBS followed by a 90 min spin at 100,000 g in a Type Ti41 rotor at 4℃, and ultimately resuspended in PBS for preservation at -80℃ for follow-up usage. Herein, Exo derived from hCD-MSCs and hCD-MSCsTRAIL were referred as hCD-MSCs-Exo and hCD-MSCs-ExoTRAIL, respectively.

The morphology of Exo was visualized and photographed with transmission electron microscopy (TEM; Tecnai G2 Spirit, Fei, USA). The size distribution of Exo was discerned by nanoparticle tracking analysis (NTA; ZetaView PMX 110, Particle Metrix, Germany). The protein content in Exo was determined by BCA protein assay according to manufacturer’s instructions. Moreover, representative markers (Alix, Annexin-V and Flotillin-1) and negative marker (GM130) of Exo were identified by Western blotting. Primary antibodies against Alix (1:1000, CST, #2171), Annexin-V (1:1000, CST, #8555), Flotillin-1 (1:1000, CST, #18634) and GM130 (1:1000, CST, #12480) were employed. The corresponding secondary antibodies conjugated with horseradish peroxidase (HRP) were visualized to inspect the protein bands.

Uptake of Exo in cervical cancer cells

The purified hCD-MSCs-Exo and hCD-MSCs-ExoTRAIL were labeled with PKH26 red fluorescent labeling kit (Umibio, China). Briefly, Exo suspension was mixed with 100 µM PKH26 staining working solution at room temperature for 30 min shielded from light, and the reaction was ceased by adding 100 µL low-glucose DMEM medium containing 10% exosome-free FBS. The PKH26-labeled Exo was resuspended with PBS and centrifuged at 100,000 g in a Type Ti41 rotor for 90 min at 4 ℃ to remove the excess dye. The collected Exo were named as PKH26-hCD-MSCs-Exo or PKH26-hCD-MSCs-ExoTRAIL.

The cervical cancer cells (SiHa and HeLa) were seeded onto coverslips in 6-well plates (2 × 105 cells/well) and cultured for 24 h at 37°C with 5% CO2. Afterwards, cells were grown with medium consisting of 4 µg/mL PKH26-hCD-MSCs-Exo or 4 µg/mL PKH26-hCD-MSCs-ExoTRAIL for 24 h shielded from light, at 37°C with 5% CO2. Subsequently, cells were treated with PBS rinsing, 4% paraformaldehyde fixing, and 4’, 6-diamidino-2-phenylindole (DAPI; Abcam, ab104139) dyeing. Eventually, cells were photographed with the laser scanning confocal microscope (Leica, Germany).

Cell viability assay

Cells were inoculated at a proper density in 96-well plates overnight and subsequently incubated with different vehicle or therapeutic regimens for 48 h. Thereafter, CCK-8 reagent (10 µL/well) was added for another incubation of 2 h, and the optical density (OD) at 450 nm was acquired with Micro-plate Reader (Bio-Rad, USA).

Cell apoptosis assay

Cells were inoculated at a proper density in 60-mm dish overnight and subsequently incubated with vehicle or therapeutic regimens for 48 h. Next, cells were gathered through trypsinization, resuspended in binding buffer, and double stained with Annexin V conjugated with phycoerythrin (PE) and 7-amino-actinomycin D (7-AAD) for 20 min avoiding light. Stained cells were evaluated with CytoFLEX flow cytometry (Beckman Coulter, Fullerton, USA).

Fabrication of DDP & hCD-MSCs-ExoTRAIL

To entrap drug DDP into hCD-MSCs-ExoTRAIL, 32 µg/mL of hCD-MSCs-ExoTRAIL and 6 µM of DDP were mingled with ice-cold PBS in 0.4 cm electroporation cuvette (Bio-Rad, USA). Afterwards, the mixture was electroporated by once pulse at 400 V, 150 µF capacitance using Gene Pulser Xcell (Bio-Rad, USA), and subsequently maintained at 37 ℃ for 30 min for the recovery of Exo membrane. Following the reaction termination, the mixture was ultracentrifugated at 100,000 g for 90 min to eliminate free DDP and obtain DDP & hCD-MSCs-ExoTRAIL nanoparticles. The platinum encapsulated in Exo was quantified using inductively coupled plasma mass spectrometry (ICP-MS) and DDP loading efficiency (%) in DDP & hCD-MSCs-ExoTRAIL nanoparticles was calculated. The traits of the fabricated DDP & hCD-MSCs-ExoTRAIL nanoparticles were described with TEM and NTA. For stability evaluation, nanoparticles were dispersed in pure PBS at -80 ℃ or 4 ℃. And the size distribution of nanoparticles was measured by dynamic light scattering (DLS) using a nanoparticle size analyzer at the days 0, 1, 2, 3, 5, 7.

Drug administration in tumor-bearing nude mice model

All animal experiments were in line with the regulations promulgated by the Institutional Animal Care and Use Committee of Wenzhou Medical University (No.wydw2021-0176). Female BALB/c nude mice (5 weeks of age) were ordered from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in standard rearing condition. To establish cervical cancer-bearing mice, 1.5 × 106 SiHa cells in 100 µL PBS were transplanted subcutaneously in the right buttock of mice aged 7 weeks. When tumors grow to around 30 mm3 (volume = ½ × length × width2, as examined with vernier caliper [16]), mice were randomized into 4 clusters with 4 mice per cluster and subsequently intratumorally administrated with PBS, DDP (2 mg/kg), hCD-MSCs-ExoTRAIL (2 µg/g), and DDP & hCD-MSCs-ExoTRAIL (as obtained from electroporation with 2 mg/kg DDP and 2 µg/g hCD-MSCs-EXOTRAIL), respectively. The administration was repeated at 4-day intervals for 4 cycles. The tumor size and mice body weight were monitored before administration every time, and the last measurement was done 4 days after the fourth treatment.

Four days post the fourth treatment, mice were anesthetized intraperitoneally with pentobarbital sodium (100 mg/kg, #P3761; Sigma, USA). Peripheral blood collection of mice via submandibular vein puncture was applied for blood routine and biochemistry inspection. Whereafter, mice were euthanized by cervical dislocation method, and major organs (heart, liver, spleen, lung, kidney) and tumors were harvested, 4% paraformaldehyde-fixed, dehydrated, paraffin-embedded and sectioned. Tissue sections of major organs were deparaffinized and subjected to hematoxylin-eosin (HE) staining for histopathological lesions assessment. Tumor slices were processed for fluorescent terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining to evaluate tumor cells apoptosis. Additionally, total proteins were extracted from frozen tumor tissues, and protein expressions of JNK (1:1000, abcam, #ab179461), c-Jun (1:1000, CST, #9165), p-c-Jun (1:1000, CST, #3270) and survivin (1:1000, CST, #2808) in tumors were detected by Western blotting.

Statistical analysis

Experimental data were analyzed and plotted with GraphPad Prism 8.0 software, and delineated as mean ± standard deviation (SD). Statistical criteria were validated by Student’s t-test for two groups and One-way analysis of variance (ANOVA) for three or more groups. The least significance method was employed for data exhibiting homogeneous variances, and data with nonhomogeneous variances were analyzed with Dunnett’s T3 method. Moreover, non-parametric data were compared by Mann-Whitney U tests for two groups and Kruskal-Wallis tests for three or more groups. Significant probability (P)-values are represented as****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05.

Results

Isolation and characterization of hCD-MSCs

Firstly, IHC staining presented that DR4 protein was higher expressed in cervical cancer tissues than adjacent non-cancerous cervical tissues (Fig. 1A). In comparison with ECT1 cells, Western blotting revealed that DR4 protein was highly expressed in cervical cancer cells SiHa and HeLa (Fig. 1B).

Fig. 1
figure 1

Characterizations of the cultured hCD-MSCs. (A) Immunohistochemistry (IHC) depicted the DR4 protein expression in cervical cancer tissues and adjacent normal tissues (original magnification, 200x). (B) Western blotting revealed DR4 protein expression in cervical cancer cells and normal cervical epithelial cells. (C) Flow cytometry analysis of specific surface markers of hCD-MSCs. (D-F) Morphological observation of the trilineage differentiation of hCD-MSCs (original magnification, 200x). Oil red staining reflected the adipogenic differentiation in (D). Alkaline phosphatase esterase staining reflected the osteogenic differentiation in (E). Alcian blue staining reflected the chondrogenic differentiation in (F). (G) Western blotting revealed the TRAIL protein expression in hCD-MSCsTRAIL. Full-length blots are presented in Supplementary Fig. 1E

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Microscopic observations displayed that the cultured hCD-MSCs were spindle-shaped and adherent growth as monolayer (Fig. s1). Moreover, hCD-MSCs were positive for mesenchymal surface markers CD90, CD73, CD44, and HLA-ABC (>95%), while negative for hematopoietic surface markers CD45, CD34, and myeloid marker HLA-DR (<2%) (Fig. 1C). After selective induction, Oil red O staining verified the intracellular lipid accumulation (Fig. 1D), and Alkaline phosphatase staining confirmed the cytoplasm calcium deposits (Fig. 1E), and Alcian blue staining validated the proteoglycan production (Fig. 1F), reflecting that hCD-MSCs presented multipotent differentiation properties comprising of adipogenesis, osteogenesis, and chondrogenesis, respectively. The features addressed above complied with the classical characteristics of MSCs, which revealed the successful extraction and propagation of hCD-MSCs from placental chorionic tissues.

Moreover, Western blotting (Fig. 1G) validated the successful transfection of pL-CMV-GFP-TRAIL-LAMP2b plasmid into hCD-MSCs, with a higher expression of TRAIL protein in transfected cells (hCD-MSCsTRAIL).

Identification and characterization of hCD-MSCs-Exo and hCD-MSCs-ExoTRAIL

Firstly, TEM exhibited that hCD-MSCs-Exo (Fig. 2A) and hCD-MSCs-ExoTRAIL (Fig. 2B) both manifested as a canonical saucer-shaped morphology. Secondly, NTA revealed that hCD-MSCs-Exo (Fig. 2C) and hCD-MSCs-ExoTRAIL (Fig. 2D) showed a similar size distribution and presented with a mean diameter of 136.3 ± 46.7 nm and 138.6 ± 47.4 nm, respectively. Thirdly, Western blotting confirmed that TRAIL protein was enriched in hCD-MSCs-ExoTRAIL in comparison with hCD-MSCs-Exo (Fig. 2E). Furthermore, both hCD-MSCs-Exo and hCD-MSCs-ExoTRAIL positively expressed exosome biomarker proteins Alix, Annexin-V and Flotillin-1, whereas the negative extracellular vesicles surface marker GM130 was not presented (Fig. 2E). Collectively, these findings validated the successful isolation and purification of hCD-MSCs-Exo and hCD-MSCs-ExoTRAIL.

Fig. 2
figure 2

Characterizations of the isolated hCD-MSCs-Exo and hCD-MSCs-ExoTRAIL. (A-B) Transmission. electron microscope (TEM) images of hCD-MSCs-Exo in (A) and hCD-MSCs-Exo TRAIL in (B). (C-D) Nanoparticle tracking analysis (NTA) of the size and distribution of hCD-MSCs-Exo in (C) and hCD-MSCs-Exo TRAILin (D). (E) Western blotting revealed TRAIL protein and specific molecular markers expressions in hCD-MSCs-Exo and hCD-MSCs-Exo TRAIL. Full-length blots are presented in Supplementary Fig. 2E

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Internalization of Exo by cervical cancer cells

After co-culturing of cervical cancer cells (SiHa and HeLa) with PKH26-hCD-MSCs-Exo or PKH26-hCD-MSCs-ExoTRAIL for 24 h, laser scanning confocal microscope (Fig. 3A for SiHa; Fig. 3B for HeLa) exhibited that red fluorescence (PKH26 labeled Exo) concentrated in cytoplasm surrounding the nuclei (stained as blue by DAPI). The results confirmed the uptake of hCD-MSCs-Exo and hCD-MSCs-ExoTRAIL by cervical cancer cells.

Fig. 3
figure 3

Impacts of hCD-MSCs-ExoTRAIL on proliferation and apoptosis of cervical cancer cells. (A-B) Laser scanning confocal microscope images of Exo internalization by SiHa cells (A) and HeLa cells (B) (original magnification, 1000x). (C-D) Effects of Exo (0, 0.5, 1, 2, 4, 8, 16, 32 µg/mL) on proliferative abilities of SiHa cells (C) and HeLa cells (D), n = 3. (E) Effects of Exo (8 µg/mL) on apoptosis of SiHa cells, n = 3. (F) Quantitative analysis of the results in (E) of SiHa cells. (G) Effects of Exo (8 µg/mL) on apoptosis of HeLa cells, n = 3. (H) Quantitative analysis of the results in (G) of HeLa cells. (****P < 0.0001)

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Impact of hCD-MSCs-ExoTRAIL on proliferation and apoptosis of cervical cancer in vitro

The proliferative ability of cervical cancer cells with the administration of various concentrations of hCD-MSCs-Exo and hCD-MSCs-ExoTRAIL (0, 0.5, 1, 2, 4, 8, 16, 32 µg/mL) for 48 h was assessed by CCK-8 assay. As exhibited in Fig. 3C, SiHa cells treated with hCD-MSCs-ExoTRAIL had a lower survival rate with respect to hCD-MSCs-Exo in a concentration-dependent pattern (P<0.05). Moreover, resembled results were detected in HeLa cells (P<0.05) (Fig. 3D).

The apoptosis of cervical cancer cells with the treatment of 8 µg/mL hCD-MSCs-Exo and hCD-MSCs-ExoTRAIL for 48 h was appraised by flow cytometry. As illustrated in Fig. 3E-F, hCD-MSCs-ExoTRAIL induced higher apoptosis of SiHa cells relative to hCD-MSCs-Exo (P<0.05). Additionally, similar results were obtained in HeLa cells (P<0.05) (Fig. 3G-H).

Above findings presented that the engineered hCD-MSCs-ExoTRAIL showed a significant advantage for attenuating proliferation and facilitating apoptosis of cervical cancer cells. Therefore, hCD-MSCs-ExoTRAIL was selected to pack with DDP.

Effects of DDP on proliferation of cervical cancer cells

CCK-8 assay was employed to evaluate the antiproliferative capacity of DDP with various concentrations (0, 2, 4, 6, 8, 10, 12, 14, 16, 18 µM) for 48 h against cervical cancer cells. DDP displayed concentration-dependent cytotoxicity to SiHa and HeLa cells (Fig. 4A-B). As shown in Fig. 4A, the IC50 value was 12 µM and the IC30 value was 6 µM for DDP in SiHa cells. As exhibited in Fig. 4B, the IC50 value was 8 µM and the IC30 value was 6 µM for DDP in HeLa cells. Therefore, 6 µM DDP at 48 h administration was applied in the following experiments.

Fig. 4
figure 4

Characterizations and impacts of DDP & hCD-MSCs-ExoTRAIL nanopaticles on proliferation and apoptosis of cervical cancer cells. (A-B) Effects of DDP (0, 2, 4, 6, 8, 10, 12, 14, 16, 18 µM) on proliferative abilities of SiHa cells (A) and HeLa cells (B), n = 3. (C) TEM images of DDP & hCD-MSCs-ExoTRAIL nanopaticles. (D) NTA of the size and distribution of DDP & hCD-MSCs-ExoTRAIL nanopaticles. (E) DLS analysis of DDP & hCD-MSCs-ExoTRAIL nanoparticles at the days 0, 1, 2, 3, 5, 7 stored at -80℃ or 4 ℃. (F-G) Proliferative abilities of SiHa cells (F) and HeLa cells (G) with the treatment of control, DDP, hCD-MSCs-ExoTRAIL and DDP & hCD-MSCs-ExoTRAIL, n = 3. (H-I) Apoptosis of SiHa cells (H) and HeLa cells (I) with the treatment of control, DDP, hCD-MSCs-ExoTRAIL and DDP & hCD-MSCs-ExoTRAIL, n = 3. (J) Quantitative analysis of the results in (H) of SiHa cells. (K) Quantitative analysis of the results in (I) of HeLa cells. (***P < 0.001, ****P < 0.0001)

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Characterization of DDP & hCD-MSCs-ExoTRAIL

Electroporation method was implemented to synthesize the drug delivery systems of hCD-MSCs-Exo co-loaded with TRAIL and DDP (DDP & hCD-MSCs-ExoTRAIL). As presented in Fig. 4C, TEM image exhibited that DDP & hCD-MSCs-ExoTRAIL remained the typical saucer-shaped appearance, demonstrating that Exo membrane structure showed no damage after DDP package by electroporation. Moreover, NTA described that the average diameter of DDP & hCD-MSCs-ExoTRAIL was 128.5 ± 36.3 nm (Fig. 4D). Next, DLS analysis exhibited that DDP & hCD-MSCs-ExoTRAIL appeared negligible size change at -80 ℃ or 4 ℃ for 7 days (Fig. 4E). In addition, ICP-MS analysis determined that the DDP loading efficiency of DDP & hCD-MSCs-ExoTRAIL was 32.12 ± 2.74%.

Effects of DDP & hCD-MSCs-ExoTRAIL on proliferation and apoptosis of cervical cancer in vitro

The CCK-8 and flow cytometry assay were performed by incubating cervical cancer cells with DMEM (control group), DDP (6 µM), hCD-MSCs-ExoTRAIL (32 µg/mL), and DDP & hCD-MSCs-ExoTRAIL (as obtained from electroporation with 6 µM DDP and 32 µg/mL hCD-MSCs-EXOTRAIL) for 48 h, to evaluate cell proliferation and apoptosis, respectively.

In comparison to control group, survival rates of SiHa cells in DDP, hCD-MSCs-ExoTRAIL, and DDP & hCD-MSCs-ExoTRAIL groups were lower, of which survival rate ranked the lowest in SiHa cells treated with DDP & hCD-MSCs-ExoTRAIL (P<0.05) (Fig. 4F). Moreover, similar results were revealed in HeLa cells (P<0.05) (Fig. 4G).

In contrast to control group, apoptosis of SiHa cells in DDP, hCD-MSCs-ExoTRAIL, and DDP & hCD-MSCs-ExoTRAIL groups were higher, of which apoptosis ranked the highest in SiHa cells treated with DDP & hCD-MSCs-ExoTRAIL (P<0.05) (Fig. 4H and J). Moreover, similar results were observed in HeLa cells (P<0.05) (Fig. 4I and K).

These results illustrated that DDP & hCD-MSCs-ExoTRAIL exhibited the greatest proliferation inhibiting and apoptosis promoting capacities of cervical cancer cells.

In vivo therapeutic antitumor activity and biosafety evaluation

The therapeutic antitumor activity was evaluated in cervical cancer-bearing nude mice with the treatment of PBS, DDP, hCD-MSCs-ExoTRAIL, and DDP & hCD-MSCs-ExoTRAIL, respectively. The tumors continuously grew in PBS group, which displayed as the most rapidly growth pattern among these four groups (P<0.05) (Fig. 5A). Moreover, tumors grew slowly with varying degrees in DDP, hCD-MSCs-ExoTRAIL, and DDP & hCD-MSCs-ExoTRAIL groups, of which tumors grew slowest in DDP & hCD-MSCs-ExoTRAIL group (P<0.05) (Fig. 5A). After treatment completion, the ex vivo tumor images and tumor weights from these four groups visually demonstrated the antitumor efficacy, of which tumor weights were the smallest in the DDP & hCD-MSCs-ExoTRAIL group (P<0.05) (Fig. 5B-C). These results described that DDP & hCD-MSCs-ExoTRAIL exerted the most potently inhibitory function on cervical cancer growth.

Fig. 5
figure 5

Therapeutic activity of DDP & hCD-MSCs-ExoTRAIL nanoparticles in cervical cancer-bearing nude mice. (A) Tumor growth curves of mice with the treatment of control, DDP, hCD-MSCs-ExoTRAIL and DDP & hCD-MSCs-ExoTRAIL, n = 4 mice per group. (B-C) Tumor tissue images (B) and tumor weights (C) of mice with the treatment of control, DDP, hCD-MSCs-ExoTRAIL and DDP & hCD-MSCs-ExoTRAIL, n = 4 mice per group. (D) TUNEL-DAPI fluorescence staining of tumor tissues reflecting the cell apoptosis (original magnification, 200x). (E) Western blotting revealed the expression of apoptotic-related proteins in tumor tissues. Full-length blots are presented in Supplementary Fig. 5E. (*P < 0.05, ***P < 0.001, ****P < 0.0001)

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Afterwards, TUNEL staining illustrated the apoptotic cells (stained as green fluorescence) in tumor tissues. As shown in Fig. 5D, the apoptotic cells were largely augmented in tumors treated with DDP & hCD-MSCs-ExoTRAIL versus to PBS, DDP, and hCD-MSCs-ExoTRAIL groups. Next, apoptosis-related proteins in tumor tissues were further investigated by Western blotting. In contrast with PBS group, the expression level of JNK and p-c-Jun proteins were elevated in DDP, hCD-MSCs-ExoTRAIL, and DDP & hCD-MSCs-ExoTRAIL groups, with the highest expression in DDP & hCD-MSCs-ExoTRAIL group (Fig. 5E). Survivin protein expression was decreased in DDP, hCD-MSCs-ExoTRAIL, and DDP & hCD-MSCs-ExoTRAIL groups in comparison with PBS group, with the lowest expression in DDP & hCD-MSCs-ExoTRAIL group (Fig. 5E). These results suggested that DDP & hCD-MSCs-ExoTRAIL exhibited the strongest induction of apoptosis in cervical cancer.

Additionally, body weight, histopathology of major organs, and primary blood parameters of mice were assessed to systematically monitor the biosafety profiles of DDP & hCD-MSCs-ExoTRAIL treatment in vivo. No notable differences were revealed regarding the body weights of mice following administration with different drug regimens (P>0.05) (Fig. 6A). Moreover, HE staining presented that no noticeable histopathological alterations or impairments were inspected in major organ tissues (heart, liver, spleen, lung, kidney) of mice treated with different drug regimens (Fig. 6B). Furthermore, blood routine (WBC, Lym, Neu, RBC, HB, MCV, HCT, PLT) (Fig. 6C-D) and plasma biochemistry inspection (Fig. 6E) indicating liver (ALT, AST, TP, ALB, GLO), kidney (BUN, CREA), and heart ( LDH) damages were implemented to perceive slight lesions that missed to be reflected by HE staining. More importantly, these biomarker levels among mice receiving different drug treatments displayed no obvious differences (P>0.05) and the CK-MB concentrations were less than 0.18 µg/L of all mice. These results documented that DDP & hCD-MSCs-ExoTRAIL showed favorable biosafety in vivo.

Fig. 6
figure 6

Biosafety evaluation of DDP & hCD-MSCs-ExoTRAIL nanoparticles in cervical cancer-bearing nude mice. (A) Body weight curves of mice with the treatment of control, DDP, hCD-MSCs-ExoTRAIL and DDP & hCD-MSCs-ExoTRAIL, n = 4 mice per group. (B) Hematoxylin-eosin (HE) staining reflected the pathological morphology of major organs (Heart, Liver, Spleen, Lung, and Kidney) of mice (original magnification, 200x). (C-D) Blood routine parameters of mice, n = 4 mice per group. (E) Plasma biochemistry parameters of mice, n = 4 mice per group

Full size image

Discussion

The most common pathological types of cervical cancer are cervical squamous cell carcinoma, which constitutes approximately 75% of cases, and cervical adenocarcinoma, accounting for roughly 20% of cases [17]. Lately, the demographic profile of cervical cancer patients has exhibited a noticeable shift towards a younger age group [18]. Neoadjuvant chemotherapy has emerged as a crucial and alternative therapeutic approach, particularly for reproductive-age cervical cancer patients aiming to preserve fertility [19]. DDP is an efficacious and widely applied chemotherapeutic drug for cervical cancer [20]. However, its non-selective cytotoxicity and dose-dependent toxic side effects necessitate the exploration of more efficient therapeutic regimens with lower-dose DDP [21]. Nanoscale drug delivery systems potentially benefit in ameliorating therapeutic performances of drugs because of the enhanced permeability and retention (EPR) effect [22]. Among these, MSCs-Exo have garnered significant attention as naturally occurring nano-sized drug delivery vehicles [23].

Shamili et al. [24] applied a non-viral polyplex, consisting of polyethylenimine 25 kDa and GFP-tagged TRAIL plasmid, to engineer TRAIL-expressing murine bone marrow-derived MSCs. The resultant Exo containing TRAIL (Exo-TRAIL) decreased cell viability and increased apoptosis of B16F0 mouse melanoma cells in contrast to Exo [24]. In the present study, DR4 protein was highly expressed in cervical cancer, indicating that DR4 could serve as target of nanoscale drug delivery systems for cervical cancer therapy. Then, we succeeded in isolating and culturing hCD-MSCs and subsequently obtained Exo from hCD-MSCs. Next, genetically engineered hCD-MSCsTRAIL were constructed by ligating TRAIL with exosomal transmembrane protein Lamp2b [25], and its secreted Exo were proved to highly express TRAIL, indicating that TRAIL loaded hCD-MSCs-Exo were successfully generated. In more detail, our study examined and depicted the shape, size, and specific biomarker proteins of the purified hCD-MSCs-Exo and hCD-MSCs-ExoTRAIL, and these characterizations fulfilled the classical criteria for authentication of Exosomes [26]. Next, hCD-MSCs-Exo and hCD-MSCs-ExoTRAIL were confirmed to be effectively taken up by cervical cancer cells. Moreover, hCD-MSCs-ExoTRAIL exerted tumor-suppressive functions by weakening proliferative capacity and enhancing apoptotic capacity of cervical cancer cells, suggesting that hCD-MSCs-ExoTRAIL were ideal drug transport vehicles.

As it stands, exogenous loading of therapeutic agents following Exo purification can be performed by co-cultivation, sonication, electroporation, extrusion, and freeze-thaw cycles [27]. Moreover, the drug loading efficacy is of fundamental importance for evaluating the effectiveness of the established nanoscale drug delivery systems [28]. Zhang et al. [29] documented that the loading efficiency of DDP in umbilical cord blood-derived M1 macrophage Exo was estimated to around 30% with mild sonication approach. Considering the dual antitumor functions of chemo-gene combinational therapy [30], we established the nanoscale drug delivery systems by means of co-loading of TRAIL and DDP with hCD-MSCs-Exo. Our study successfully developed DDP & hCD-MSCs-ExoTRAIL by encapsulating DDP into hCD-MSCs-ExoTRAIL, which maintained the saucer shape with a diameter averaging 128.5 ± 36.3 nm. Additionally, the DDP loading capacity was approximately 32.12 ± 2.74%, achieved by employing electroporation technique.

Jiang et al. [31] designed nanoscale drug delivery systems via entrapping triptolide in TRAIL-engineered exosomes (TRAIL-Exo) secreted by TRAIL-overexpressing macrophage Raw264.7 cells, which was confirmed to play a stronger impact on cytotoxicity and pro-apoptosis compared with triptolide in malignant melanoma. Our study further assessed the therapeutic functions of DDP & hCD-MSCs-ExoTRAIL in cervical cancer. The DDP & hCD-MSCs-ExoTRAIL provided evident advantages in retarding cell growth and propelling cell apoptosis with respect to single DDP or hCD-MSCs-ExoTRAIL in cervical cancer in vitro, which revealed that the fabricated DDP & hCD-MSCs-ExoTRAIL exhibited remarkable tumoricidal activity in both cervical squamous cell carcinoma and cervical adenocarcinoma.

The mitogen-activated protein kinase (MAPK) signaling pathway comprises ERK1/2, p38, c-Jun N-terminal kinase (JNK) and MEK cascades, which modulating multiple cellular activities like apoptosis, autophagy, proliferation and differentiation [32]. Although exceptions exist, p38 MAPK and JNK pathways are generally relative to apoptosis promotion [33]. Frensemeier et al. [34] revealed that DDP facilitated apoptosis of cervical cancer cells through activation of JNK and c-Jun phosphorylation. Moreover, Mahalingam et al. [35] illustrated that recombinant human TRAIL protein-mediated pro-apoptotic function on colon carcinoma cells was related to an increase of JNK and c-Jun phosphorylation. Additionally, Xu et al. [36] documented that DDP plus TRAIL protein exerted their function in promoting cell apoptosis via reducing the expression of antiapoptotic protein Survivin in triple-negative breast tumor cells. In our study, the results acquired from these cervical cancer-bearing nude mice revealed that nanoscale drug delivery systems (DDP & hCD-MSCs-ExoTRAIL) were able to lessen tumor burden and hamper tumor growth more efficiently than DDP or hCD-MSCs-ExoTRAIL alone by xenograft volumetric and weight analysis. Additionally, TUNEL assay observed that DDP & hCD-MSCs-ExoTRAIL exhibited a greater capacity to prompt apoptosis in cervical cancer tissues than DDP or hCD-MSCs-ExoTRAIL alone. Moreover, DDP & hCD-MSCs-ExoTRAIL displayed maximum upregulation of JNK and p-c-Jun protein and downregulation of Survivin protein in contrast to DDP or hCD-MSCs-ExoTRAIL alone in the cervical cancer of mice. Therefore, the activation of JNK/p-c-Jun and suppression of Survivin potentially was one mechanism by which DDP & hCD-MSCs-ExoTRAIL performed its role in inducing apoptosis of cervical cancer.

The biological safety of nanoscale drug delivery systems is a crucial precondition for application in vivo and future clinical translation [37]. Next, our current study inspected the biosafety profile of DDP & hCD-MSCs-ExoTRAIL in mice. Firstly, the body weight of mice received four different treatments revealed no apparent difference. Secondly, histopathological and blood paraments evaluation reflected that DDP & hCD-MSCs-ExoTRAIL showed no toxic side effect in major organs of mice. Therefore, our present findings provided evidence that the nanoscale drug delivery systems (DDP & hCD-MSCs-ExoTRAIL) displayed superior anti-tumor efficacy and favorable biosafety in cervical cancer.

As widely known, cisplatin is extensively employed as a chemotherapeutic agent for the treatment of cervical cancer, especially for advanced or metastatic or recurrent cervical cancer [38]. Unfortunately, cisplatin administration is generally restricted by insufficient tumor chemotherapy response [39]. Therefore, clinical treatment requires higher dosage of cisplatin to achieve the anticipated therapeutic effect, while generating dose-related toxicities as nephrotoxicity, neurotoxicity and other serious undesirable effects [40]. In our present study, a low dosage of cisplatin delivered by hCD-MSCs-ExoTRAIL exerted superior ability to inhibit tumor growth than free cisplatin. Moreover, the nanoscale drug delivery system DDP & hCD-MSCs-ExoTRAIL appeared no apparent systemic toxicity. Based on the above results, we will continue to explore the long-term effects of DDP & hCD-MSCs-ExoTRAIL on tumor recurrence and metastasis, and elaborate the potential immune responses and immunogenicity associated with DDP or hCD-MSCs-ExoTRAIL in mice in future studies.

Certainly, potential challenges in translating DDP & hCD-MSCs-ExoTRAIL from preclinical studies to clinical practice need to be considered and addressed in follow-up investigations. Firstly, the preparation of exosomes is a time-consuming and labor-intensive process. Therefore, the primary bottleneck for clinical trial is the large-scale and rapid production of exosomes. The method for isolating high purity exosomes efficiently and economically is urgently needed in the future. Secondly, although DDP & hCD-MSCs-ExoTRAIL delivery system exhibits great potential for cervical cancer treatment, there is still a long way to go for clinical application. In present study, only one dosage and treatment regimen was evaluated in cervical cancer in vitro and in vivo, and additional experiments are required to meliorate the drug delivery system. We need to improve DDP encapsulation efficiency, and explore the most appropriate drug concentration to ascertain the most effective combination of DDP and exosomes. Moreover, we need to optimize the administration mode and schedule of DDP & hCD-MSCs-ExoTRAIL. Thirdly, individualized characterization of cervical cancer patients influences the therapeutic efficacy of DDP & hCD-MSCs-ExoTRAIL. We need to conduct multi-center prospective clinical trials to validate applicability of DDP & hCD-MSCs-ExoTRAIL in cervical cancer patients. Moreover, we need to obtain the approval of regulatory agencies for clinical applications.

Conclusions

Collectively, the rich source of human chorion will guarantee an adequate amount of hCD-MSCs and hCD-MSCs-Exo. The TRAIL-engineered hCD-MSCs-Exo featured as a tumor-suppressor, and acted as an appropriate nano-carrier platform for DDP encapsulation. Compared with single DDP or hCD-MSCs-ExoTRAIL, the developed nanoscale drug delivery systems (DDP & hCD-MSCs-ExoTRAIL) empowered improved therapeutic efficacy in hampering cervical cancer progression both in vitro and in vivo. Moreover, the DDP & hCD-MSCs-ExoTRAIL nanoparticles possessed an acceptable biosafety profile, holding great promise for its employment in cervical cancer as chemo-gene combinational therapy in clinical practice. Fortunately, Exo-based drug delivery systems present bright prospects for cervical cancer treatment.

Data availability

The data and materials that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

WBC:

White blood cell

Lym:

Lymphocyte

Neu:

Neutrophil

RBC:

Red blood cell

HB:

Hemoglobin

MCV:

Mean corpuscular volume

HCT:

Hematocrit

PLT:

Platelet

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

TP:

Total protein

ALB:

Albumin

GLO:

Globulin

BUN:

Blood urea nitrogen

CREA:

Creatinine

CK-MB:

Creatine kinase-MB isoenzyme

LDH:

Lactate dehydrogenase

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Funding

This work was supported by National Natural Science Foundation of China (Grant No. 82172088) and Wenzhou Major Science and Technology Innovation Project (Grant No. ZY2022012). The sponsors of the study had no involvement in the data collection, analysis, or interpretation, or in the writing of the manuscript. The authors declare that they have not used Artificial Intelligence in this study.

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  1. Miaomiao Ye and Tingxian Liu contributed equally to this work.

Authors and Affiliations

  1. Zhejiang Provincial Clinical Research Center for Obstetrics and Gynecology, Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Wenzhou Medical University, No. 109 Xueyuan Xi Road, Wenzhou, Zhejiang, 325027, China

    Miaomiao Ye, Tingxian Liu, Liqing Miao, Huihui Ji, Zhihui Xu, Huihui Wang, Jian’an Zhang & Xueqiong Zhu

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Contributions

Conceptualization, X.Z., J.Z., and M.Y.; methodology, M.Y., T.L., L.M., and H.J.; formal analysis, M.Y., T.L., H.J. and L.M.; investigation, J.Z., M.Y., and Z.X.; writing-original draft, M.Y. and T.L.; writing-review and editing, M.Y., T.L., L.M., H.J., Z.X., H.W., J.Z. and X.Z.; visualization, T.L., L.M., and H.W. supervision, X.Z. and J.Z.; funding acquisition, X.Z. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Jian’an Zhang or Xueqiong Zhu.

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Ethics approval and consent to participate

This study has been approved by the ethics committee of the Second Affiliated Hospital of Wenzhou Medical University. And informed consent was obtained from all subjects involved in the study. The project was titled as “The establishment of a co-delivery Nano-granular system based on TRAIL-exosome, siRNA and cisplatin and its application in drug-resistant cervical cancer (No. 2016kykt31)”, and was approved on 03/10/2016. The animal experiments were in line with the regulations promulgated by the Institutional Animal Care and Use Committee of Wenzhou Medical University. The project was titled as “The mechanistic exploration of a novel biomimetic nanozyme based on TRAIL target for reversing radio-resistance of cervical cancer to synergistically improve immunotherapy (No. wydw2021-0176)”, and was approved on 03/10/2021. Shanghai Cell Biology Medical Research Institute and Shanghai BinSui Biological Technology Co. have confirmed that there was initial ethical approval for collection of human cells (SiHa, HeLa, ECT1), and that the donors had signed informed consent.

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Ye, M., Liu, T., Miao, L. et al. Cisplatin-encapsulated TRAIL-engineered exosomes from human chorion-derived MSCs for targeted cervical cancer therapy. Stem Cell Res Ther 15, 396 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-024-04006-6

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Stem Cell Research & Therapy

ISSN: 1757-6512