Enhanced disc regeneration through CRISPR/Cas9-mediated SOX9 and TGFβ1 coexpression in tonsil-derived mesenchymal stromal cells

Abstract

Background

Intervertebral disc (IVD) degeneration, a primary cause of chronic low back pain, currently lacks treatments that target its underlying pathological mechanisms. Tonsil-derived mesenchymal stromal cells (ToMSCs) have shown promise for IVD regeneration; however, their therapeutic potential is limited by the harsh microenvironment of degenerated discs. This study investigated whether ToMSCs engineered to co‐overexpress SOX9 and TGFβ1 using a tetracycline‐off (Tet‐off) regulatory system could enhance extracellular matrix (ECM) restoration and reduce inflammation in degenerative IVDs.

Methods

We used CRISPR/Cas9 technology to generate ToMSCs that express SOX9, TGFβ1, or both factors under Tet-off regulation. Gene expression was confirmed by Western blot and qRT-PCR analyses. In vitro studies assessed chondrogenic differentiation capacity, while in vivo assessments were performed using a rat tail needle puncture model of IVD degeneration. After administering the CRISPR-engineered ToMSCs, we monitored mechanical allodynia with the von Frey test over six weeks. Therapeutic outcomes were evaluated through T2‐weighted MRI and histological analysis.

Results

In vitro experiments showed that ToMSCs co-expressing SOX9 and TGFβ1 exhibited superior chondrogenic differentiation compared to cells expressing a single factor. In vivo studies demonstrated that dual-factor expressing ToMSCs significantly improved disc hydration (as confirmed by MRI), enhanced ECM synthesis—particularly aggrecan and type II collagen—and reduced inflammation compared to single-factor treatments. These improvements were accompanied by reduced mechanical allodynia, indicating functional recovery.

Conclusion

Our study demonstrates that ToMSCs engineered to co-express SOX9 and TGFβ1 effectively promote IVD regeneration by enhancing ECM production and reducing inflammation. This dual-factor approach represents a promising therapeutic strategy for treating degenerative disc disease and warrants further investigation for clinical application.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-025-04600-2.

Introduction

Intervertebral disc (IVD) degeneration is a primary cause of chronic discogenic low back pain (LBP) and imposes a significant global health burden by affecting pain, mobility, and quality of life [1–6]. Current treatment modalities, including surgical and pharmacological interventions, primarily provide symptom relief without addressing the underlying pathophysiological mechanisms [7–9]. This unmet clinical need underscores the urgency for innovative regenerative strategies that restore both the structural and functional integrity of degenerated discs.

Mesenchymal stromal cells (MSCs) have emerged as promising candidates for IVD regeneration due to their tissue repair and immunomodulatory properties [10, 11]. However, the harsh microenvironment of degenerated IVDs—characterized by hypoxia, acidic pH, and inflammatory mediators—often limits their regenerative efficacy [12]. Standalone MSC therapies frequently fail to fully restore IVD structure and function, highlighting the need for enhanced therapeutic approaches.

Among various MSC sources, tonsil-derived MSCs (ToMSCs) have attracted attention because of their high proliferation rates and lower immunogenicity compared to adipose-derived or bone marrow-derived MSCs [13, 14]. However, isolating autologous ToMSCs from adult patients is clinically challenging, as tonsillectomy is rarely performed in adults [15, 16]. In this study, we obtained ToMSCs from discarded tissue during pediatric tonsillectomy, which is a common surgical procedure. This method is considered a clinically advantageous approach because it allows cells to be obtained without invasive procedures.

According to recent studies, ToMSCs exhibit elevated levels of stanniocalcin-1, a protein that is crucial for maintaining their proliferative capacity and regulating oxidative stress by reducing mitochondrial reactive oxygen species, which are key contributors to IVD degeneration [17]. In our previous study, a genetically engineered ToMSC-based tetracycline-off (Tet-off) regulatory system expressing transforming growth factor beta 1 (TGFβ1), insulin-like growth factor 1 (IGF-1), and bone morphogenetic protein 7 (BMP-7) enhanced ECM regeneration and reduced inflammation in an IVD degeneration model [18]. Although this approach improved disc repair, further refinement was necessary to enhance chondrogenic differentiation and ECM synthesis [19].

Building on these findings, the present study focuses on SOX9 and TGFβ1, two critical regulators of IVD homeostasis and cartilage formation [20, 21]. SOX9, a member of the Sry-type HMG box (SOX) family, regulates type II procollagen expression, which is essential for chondrocyte function and IVD development [22, 23]. TGFβ1, a member of the TGF-β superfamily, promotes ECM synthesis and is vital for IVD development and maintenance [20]. Recent research has shown that human umbilical cord-derived MSCs (hUC-MSCs) transfected with SOX9 and TGFβ1 exhibit enhanced chondrogenesis and promote IVD regeneration [24].

While these results are promising, we fully acknowledge the potential oncogenic risks associated with the overexpression of TGFβ1 and SOX9. TGFβ1 has a context-dependent dual role in tumorigenesis, and SOX9 dysregulation has been implicated in various cancers [25]. To mitigate these risks, we genetically engineered ToMSCs by integrating SOX9 and TGFβ1 transgenes into the adeno-associated virus integration site 1 (AAVS1) “safe harbor” locus using the RNA-guided CRISPR/Cas9 system. Furthermore, transgene expression was regulated by the Tet-off inducible system, allowing temporal control of transgene expression and minimizing the risk caused by continuous overexpression. We evaluated the engineered ToMSCs in an in vivo rat model of IVD degeneration, focusing on ECM regeneration and inflammation modulation. This study provides foundational evidence for multi-factorial genetic engineering approaches in developing next-generation MSC-based regenerative therapies that overcome conventional treatment limitations.

Materials and methods

Purification and culture of tonsil-derived mesenchymal stromal cells

ToMSCs were isolated using a modified version of a previously published procedure. Discarded tonsil fragments were obtained from a child undergoing tonsillectomy at the Department of Otolaryngology, Bundang Cha Hospital (Sungnam, Korea), with written informed consent provided by the child’s parents. The research protocol was approved by the Institutional Review Board of Bundang Cha Hospital (approval number: 2021-03-016-006). The tonsil tissue was washed twice with 1 × phosphate-buffered saline (PBS) (Welgene, Gyeongsan, Korea), minced into small pieces, and digested for 30 min at 37 °C in RPMI 1640 medium (Invitrogen, Waltham, MA, USA) containing 10 µg/mL DNase I (Sigma-Aldrich, St. Louis, MO, USA) and 210 U/mL collagenase type I (Invitrogen). The digested tissue was filtered through a wire mesh and then washed twice with RPMI 1640 supplemented with 20% fetal bovine serum (FBS) (Gibco, Waltham, USA), followed by an additional wash with RPMI 1640 containing 10% FBS. Mononuclear cells were isolated using Ficoll-Paque (GE Healthcare, Little Chalfont, UK) density gradient centrifugation. Cells (1 × 10⁸) were seeded into T-125 culture flasks containing Dulbecco’s modified Eagle’s medium F12 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS, 100 µg/mL streptomycin (Invitrogen), and 100 U/mL penicillin (Invitrogen). The medium was replaced after 48 h to remove non-adherent cells.

Flow cytometry analysis

The phenotypic profile of ToMSCs was characterized using flow cytometry. At least 20,000 cells were suspended in 100 µL of 1× PBS containing 2% FBS and incubated with fluorescently labeled antibodies: FITC-HLA-ABC (Thermo Fisher Scientific), PE-HLA-DR (BD Biosciences, Franklin Lakes, NJ, USA), APC-CD44, PE-CD73, PE-CD90, and APC-CD105 (all from Miltenyi Biotec, Teterow, Germany). PE-CD31 (BD Pharmigen), FITC-CD45 (MiltenyiBiotec), PE-CD34, APC-CD43, and FITC-CD3 (all from BioLegend, San Diego, CA, USA).

Trilineage differentiation of ToMSC

To evaluate the differentiation capacity of ToMSCs into chondrocytes, osteocytes and adipocytes, the cells were seeded onto tissue culture dishes at a density of 1 × 10⁴ cells/cm² in DMEM/F12 supplemented with 10% FBS. At 70–80% confluency, the cells were cultured in chondrogenic, osteogenic or adipogenic differentiation media (The StemPro^® Chondrogenesis Differentiation Kit, The StemPro^® Osteogenesis Differentiation Kit, The StemPro^® Adipogenesis Differentiation Kit, all from Invitrogen Gibco), with the media replaced every 3–4 days. After 21 days of differentiation, the cells were fixed with 4% paraformaldehyde (PFA) for 30 min, stained with Alcian blue (for chondrogenesis), Alizarin red S (for osteogenesis), and Oil red O (for adipogenesis), and subsequently visualized under a light microscope.

Plasmid construction

pAAVS1-puro-Tetoff-SOX9-TGFβ1-CAG-tTA-Advanced

The Tet-off promoter and tTA gene were obtained from pTRE-TIGHT (Takara Bio, Shiga, Japan) and pAAV-Tetoffbidir-Alb-luc (Addgene, Watertown, MA, USA), respectively. SOX9 and TGFβ1 cDNAs were sourced from FUW-tetO-SOX9 and TGFB1_pLX307 (both from Addgene). The genes were combined using P2A sequences to create a single cistronic gene cassette under the Tet-off promoter. A 6His tag was added to the C-terminal of TGFβ1 for antibody detection. The final construct was generated by subcloning all components into the pAAVS1-puro-CAG plasmid.

pAAVS1-puro-Tetoff-SOX9-CAG-tTA-Advanced

The Tet-off promoter and tTA gene were sourced from pTRE-TIGHT and pAAV-Tetoffbidir-Alb-luc (Addgene), respectively. SOX9 cDNA was obtained from FUW-tetO-SOX9 (Addgene) and expressed as a single cistronic gene cassette under the Tet-off promoter. A 6His tag was added to the C-terminus of SOX9 for detection. The final construct was created by subcloning all components into the pAAVS1-puro-CAG plasmid.

pAAVS1-puro-Tetoff-TGFβ1-CAG-tTA-Advanced

The Tet-off promoter and tTA gene were sourced from pTRE-TIGHT and pAAV-Tetoffbidir-Alb-luc (Addgene), respectively. TGFβ1 cDNA was obtained from TGFβ1_pLX307 (Addgene) and expressed as a single cistronic gene cassette under the Tet-off promoter. A 6His tag was added to the C-terminus of TGFβ1 for detection. The final construct was generated by subcloning all DNA components into the pAAVS1-puro-CAG plasmid.

Knock-in gene editing using CRISPR/Cas9

The AAVS1 locus, a well-established safe harbor site [26], was chosen as the integration site for the knock-in gene cassette. ToMSCs (passage 2 or 3) were transfected using a Neon transfection system (Invitrogen) with the following parameters: 990 V, 40 ms, 2 pulses, 6.25 µg of Cas9 protein, 15 µg of sgRNA, and 1 µg of donor DNA. Three donor DNA constructs were used: pAAVS1-puro-Tetoff-SOX9-TGFβ1-CAG-tTA-Advanced, pAAVS1-puro-Tetoff-SOX9-CAG-tTA-Advanced, and pAAVS1-puro-Tetoff-TGFβ1-CAG-tTA-Advanced. Following electroporation, cells were selected using 0.8 µg/mL puromycin (Sigma-Aldrich) for 48 h, followed by 0.16 µg/mL puromycin to isolate knock-in colonies. The resulting stable cell lines were designated as ToMSC-Tetoff: SOX9-TGFβ1-6His, ToMSC-Tetoff: SOX9-6His, and ToMSC-Tetoff: TGFβ1-6His.

Western blots

Cells were lysed in RIPA buffer (Thermo Fisher Scientific) to extract total protein. Protein samples (20 µg per lane) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Sigma-Aldrich). Membranes were blocked with 5% skim milk for 1 h at room temperature with continuous shaking. Primary antibodies were applied overnight at 4 °C: anti-β-actin (1:100,000, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-His tag antibody (1:2,000, abm, Thermo Fisher Scientific). After three washes with PBST (1 × PBS, 0.2% Tween-20), membranes were incubated with appropriate secondary antibodies (goat anti-rabbit or goat anti-mouse IgG H&L [HRP], LI-COR, Lincoln, NE, USA) for 2 h at room temperature. Following three additional PBST washes, membranes were analyzed using an Odyssey CLx Imager (LI-COR).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from ToMSC-Tetoff: SOX9-TGFβ1-6His, ToMSC-Tetoff: SOX9-6His, ToMSC-Tetoff: TGFβ1-6His, and naïve ToMSC samples using an RNA purification kit (MACHEREY-NAGEL GmbH & Co., Düren, Germany). cDNA was synthesized from 100 ng of RNA using a cDNA synthesis kit (TOYOBO, Osaka, Japan). qRT-PCR was performed using Power SYBR Green Master Mix (Thermo Fisher Scientific).

Alcian blue staining

For chondrogenesis assays, ToMSC-Tetoff: SOX9-TGFβ1-6His, ToMSC-Tetoff: SOX9-6His, ToMSC-Tetoff: TGFβ1-6His, and naïve ToMSCs were seeded at 1 × 10⁴ cells/cm² in 24-well plates containing DMEM with 10% FBS. When cells reached 80% confluence, the medium was replaced with chondrogenic differentiation medium, which was changed every 3 days. As a control, naïve ToMSCs were cultured in DMEM/F12 supplemented with 10% FBS without chondrogenic induction for 21 days. After 21 days of culture, all cells were fixed with 4% formaldehyde and stained with 1% Alcian blue (Sigma-Aldrich) in 0.1 N HCl for 30 min. The staining was visualized under a light microscope and subsequently eluted with 0.1 N HCl. Optical density (OD) was measured at 620 nm using a spectrophotometer (Tecan, Infinite 200 Pro).

Animal surgical procedures

Thirty-six female Sprague-Dawley rats (9 weeks old, 200–220 g) were obtained from Orient Bio Inc. (Seongnam-si, Gyeonggi-do, Republic of Korea). Animals were housed at 22 ± 1 °C with 50 ± 1% relative humidity and a 12-hour light/dark cycle. Standard rodent chow and water were provided ad libitum during a one-week acclimatization period. All procedures followed the guidelines of the Institutional Animal Care and Use Committee (IACUC) of CHA Bundang Medical Center (approval number: IACUC 230180). The animal studies were performed in compliance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments).

Prior to surgery, the peritoneal area was sterilized with 70% alcohol. Anesthesia was administered intraperitoneally using Zoletil (50 mg/kg; Virbac Laboratories, Carros, France) combined with xylazine (Rompun; 10 mg/kg; Bayer, Seoul, Republic of Korea). The tail skin was sterilized with 70% alcohol followed by povidone-iodine. A dorsal longitudinal skin incision exposed the coccygeal intervertebral discs. For the sham group, only a skin incision was made. In the remaining 30 rats, IVD degeneration was induced by puncturing the coccygeal discs (Co6/7 and Co7/8) using a 21-gauge sterile spinal needle bent at 5 mm to limit penetration. The needle was inserted dorsoventrally, rotated 360° twice, and held in place for 30 s [18, 27, 28]. Postoperative care included administration of analgesics (Ketoprofen, SCD Pharm. Co., Ltd., Seoul, Republic of Korea) and antibiotics (cefazolin, Chong Kun Dang, Seoul, Republic of Korea). During recovery, rats were maintained at 37 °C on heating pads.

Experimental design

Two weeks after IVD degeneration, rats were randomized into five experimental groups (n = 6 per group): 1) Injury group: 2 µL PBS injected into punctured coccygeal discs (Co6/7 and Co7/8); 2) ToMSC group: 2 µL PBS containing 2 × 10⁴ ToMSCs; 3) ToMSC-Tetoff: SOX9-6His group: 2 µL PBS containing 2 × 10⁴ ToMSCs engineered to overexpress SOX9; 4) ToMSC-Tetoff: TGFβ1-6His group: 2 µL PBS containing 2 × 10⁴ ToMSCs engineered to overexpress TGFβ1; 5) ToMSC-Tetoff: SOX9-TGFβ1-6His group: 2 µL PBS containing 2 × 10⁴ ToMSCs co-overexpressing SOX9 and TGFβ1. All injections were performed using a Hamilton syringe with a 31-gauge needle, targeting the center of the degenerated coccygeal discs (Co6/7 and Co7/8). To prevent immune rejection, cyclosporine (100 µg/mL; Cipol-N, Chong Kun Dang, Seoul, Republic of Korea) was administered in the drinking water from two days before cell injection until euthanasia. Six weeks post-implantation, rats were euthanized via CO₂ asphyxiation, and coccygeal discs were harvested for radiologic and histologic analyses.

Mechanical allodynia

Behavioral nociception was evaluated using the von Frey test to assess mechanical allodynia and pain-related behavior [29]. Testing was conducted one day before cell implantation (baseline) and on days 1, 7, 14, 21, 28, 35, and 42 post-implantation. Each rat was placed individually in a plastic observation chamber with a mesh floor and acclimated for 20 min before testing to minimize interference from exploratory behaviors. Mechanical stimuli were applied to the tail using an electronic von Frey device (e-VF; Ugo Basile Biological Instruments, model 37000-007). The withdrawal threshold was defined as the average force (g) required to elicit a paw withdrawal response, determined over three trials.

Histological analysis

Six weeks post-implantation, rats were euthanized using CO₂ inhalation. Coccygeal disc tissues with adjacent vertebral bodies were harvested and fixed in 10% neutral buffered formalin for one week. Tissues were decalcified using Rapid Cal Immuno (BBC Biochemical, Mount Vernon, WA, USA) for two weeks, embedded in paraffin, and sectioned into 6-µm slices using a microtome (Leica, Wetzlar, Germany). Sections were dewaxed, rehydrated, and stained with Safranin O (Sigma-Aldrich) to assess proteoglycan distribution and quantity. Stained sections were mounted and scanned using a C-mount camera adapter (U-TVO.63XC; Olympus, Tokyo, Japan). IVD degeneration was evaluated using a comprehensive 16-point scoring system [30, 31] with five subcategories: nucleus pulposus (NP) morphology, NP cellularity, annulus fibrosus (AF) morphology, endplate morphology, and the boundary integrity between the NP and AF.

Higher weights were assigned to degenerative features including alterations in NP shape and area, NP cellular morphology, AF lamellar organization, and endplate disruptions. Each subcategory was scored from 0 (normal) to 2 (severe degenerative changes), with total scores ranging from 0 (normal) to 16 (most severe degeneration).

Additional sections were stained with hematoxylin and eosin (H&E) for morphological evaluation. Tissue morphology and NP area were quantified using FIJI ImageJ software (version 1.54f, National Institutes of Health, Bethesda, MD, USA). All analyses were performed by two independent, blinded observers to ensure objectivity and reliability.

Magnetic resonance imaging (MRI)

Disc structural changes and degeneration were assessed six weeks post-implantation using a 9.4-T MRI spectrometer (Bruker BioSpec, Billerica, MA, USA). T2-weighted imaging was performed in both axial and coronal planes with the following parameters: repetition time (TR) 5,000 ms; echo time (TE) 30 ms; matrix size 150 × 150; field of view 15 × 15 mm; and slice thickness 0.5 mm with 0-mm interslice spacing. The MRI index (product of NP area and average signal intensity) was used to quantify disc degeneration [18, 32]. The high-signal intensity region in the mid-coronal T2-weighted image, corresponding to the NP, was designated as the region of interest. Signal intensity was measured using FIJI ImageJ software and compared to normal rat coccygeal discs. Two blinded observers performed independent assessments.

Immunofluorescence and immunohistochemistry staining

Immunohistochemical analysis assessed disc matrix components (aggrecan and type II collagen), while immunofluorescence analysis evaluated the pain marker calcitonin gene-related peptide (CGRP), the catabolic enzyme matrix metalloproteinase-13 (MMP-13), and pro-inflammatory cytokines (tumor necrosis factor-alpha [TNF-α] and interleukin-1β [IL-1β]). For immunostaining, tissue sections were dewaxed, rehydrated, and incubated with primary antibodies at the following dilutions: aggrecan (1:200; Abcam, Cambridge, UK), type II collagen (1:200; Abcam, Cambridge, UK), CGRP (5 µg/mL; Abcam, Cambridge, UK), Tie2 (1:200; R&D Systems, Minneapolis, MN, USA), Brachyury (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), human nuclei antibody (1:200; Merck KGaA, Darmstadt, Germany), TNF-α (1:50; Invitrogen, Carlsbad, CA, USA), MMP-13 (1:200; Proteintech, Rosemont, IL, USA), and IL-1β (1:200; R&D Systems, Minneapolis, MN, USA). Detailed specifications of primary and secondary antibodies, including their dilution ratios, are provided in Table S1. This analysis enabled evaluation of disc matrix components, inflammatory markers, and catabolic enzymes, providing insights into the pathophysiology of disc degeneration and repair.

Immunohistochemistry staining

For immunohistochemistry, antigen retrieval was conducted by heating sections in pH 9.0 Tris-EDTA buffer (Biosesang, Yongin-si, Republic of Korea) at 95 °C for 10 min, followed by a 30 min cooling period in the same buffer. To inactivate endogenous peroxidase and alkaline phosphatase, sections were treated with BLOXALL blocking solution for 10 min. After a single wash with 1 × PBS, sections were incubated with blocking serum (Vectastain Elite ABC HRP kit, Vector Laboratories, Newark, CA, USA) for 20 min to prevent nonspecific binding. Primary antibodies diluted in blocking serum were applied to the sections and incubated for 24 h at 4 °C. After washing with PBS, sections were incubated with a biotinylated secondary antibody (Vectastain Elite ABC HRP kit) for 30 min at room temperature. Following another PBS wash, an Avidin–Biotin Complex (ABC) solution (Vectastain Elite ABC HRP kit) was applied for 30 min. Staining was developed using DAB substrate (Vectastain Elite ABC HRP kit) for 10 min.

The stained sections were then washed with tap water, dehydrated through graded ethanol solutions (70%, 80%, 90%, and 99%), cleared in xylene, and mounted with coverslips using Canada balsam.

Immunofluorescence staining

Antigen retrieval for immunofluorescence staining followed the same protocol as for immunohistochemistry. After retrieval, sections were blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature to prevent nonspecific binding. Primary antibodies diluted in 5% BSA were applied and incubated for 2 h at room temperature. After three washes with 1 × PBS, sections were incubated with Alexa Fluor 488 and 568 secondary antibodies (1:200, Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Nuclear staining was performed with DAPI (1:10,000; Thermo Fisher Scientific, Waltham, MA, USA) diluted in Dulbecco’s PBS (DPBS, Cytiva, Marlborough, MA, USA) for 10 min. Sections were washed with PBS and mounted using Dako Fluorescent Mounting Medium (Agilent, Santa Clara, CA, USA).

Image capture and quantitative analysis

Digitally scanned images of stained sections were obtained using a digital slide scanner (Axio Scan.Z1, Carl Zeiss, Oberkochen, Germany). Quantitative analysis of immunostaining was conducted using FIJI ImageJ software (version 1.54f, National Institutes of Health, Bethesda, MD, USA). Analysis included calculating the percentage of the area positive for aggrecan and type II collagen, and determining the number of positive cells relative to DAPI for CGRP, Tie2, Brachyury, MMP-13, TNF-α, IL-1β, and human nuclei [18, 32, 33].

Statistical analysis

Data were analyzed using GraphPad Prism (version 8.0.2, GraphPad Software, La Jolla, CA, USA) and are presented as mean ± standard deviation (SD). Statistical significance was determined using one-way or two-way analysis of variance (ANOVA) with Tukey’s post-test. A p-value of less than 0.05 was considered statistically significant.

Results

Characterization of ToMSCs

ToMSCs isolated from tonsil tissue displayed the typical spindle-shaped morphology of MSCs (Fig. 1A). Flow cytometry analysis using appropriate isotype controls confirmed that not only ToMSCs were positive for MSC surface markers CD44, CD73, CD90, and CD105 but also negative for the T cell marker, CD3, the endothelial marker, CD31, and hematopoietic lineage markers, CD34, CD43, and CD45 (Fig. 1B). In addition, ToMSCs expressed HLA class I (HLA-ABC) antigens but not HLA class II (DRB1) (Fig. 1B), consistent with standard MSC characteristics. Moreover, we confirmed that ToMSCs could differentiate into chondrocytes, osteoblasts and adipocytes, demonstrating their multipotency (Fig. 1C).

Fig. 1.

Characteristics and flow cytometric analysis of tonsil-derived mesenchymal stromal cells (ToMSCs). A Morphology of ToMSCs observed by light microscopy (×40). Scale bar, 100 μm. B Flow cytometric analysis of ToMSCs using antibodies specific to common MSC surface positive markers (CD44, CD73, CD90, CD105), negative surface markers (CD3, CD31, CD34, CD43 and CD45) and HLA antigens (class I and II). C Chondrogenic, osteogenic and adipogenic differentiation of ToMSCs was assessed by staining with Alcian blue, Alizarin red S and Oil red O, respectively. Scale bar, 100 μm

Precise targeting of transgenes to the AAVS1 safe harbor locus via CRISPR/Cas9

Building on previous research demonstrating ToMSCs’ chondrogenic potential [34, 35], we aimed to enhance this capability through genetic modification by overexpressing SOX9, TGFβ1, and their combination under Tet-off promoter control. Using CRISPR/Cas9-mediated homology-directed repair, transgenes were inserted into the AAVS1 locus—a well-established safe harbor site that tolerates disruption without compromising cellular function [26] (Fig. 2A). Successful integration was confirmed by PCR analyses, which showed the expected bands of 1.2 kb and 1.1 kb using primer pairs F1/R1 and F2/R2, respectively (Fig. 2B, Sequences of PCR primers and sgRNA are in Supplementary Table S2).

Fig. 2.

CRISPR/Cas9-mediated knock-in of a transgene into the AAVS1 safe harbor site in ToMSC chromosomes. A Schematic diagram of the homology-directed repair-mediated knock-in process. B In-target knock-in confirmation by PCR (cropped image, uncropped gel image of Fig. 2B is in Supplementary Figure S1)

Evaluations of transgene expression by Western blot and qRT-PCR

Western blot analysis was conducted to assess transgene expression in three cell lines: ToMSC-Tetoff: SOX9-TGFβ1-6His, ToMSC-Tetoff: SOX9-6His, and ToMSC-Tetoff: TGFβ1-6His. In accordance with the Tet-off system design, transgene expression was observed only in the absence of doxycycline (Fig. 3A). qRT-PCR confirmed significant transgene expression in doxycycline-free conditions, with knock-in cell lines showing substantially higher SOX9 or TGFβ1 expression compared to naïve ToMSCs (Fig. 3B).

Fig. 3.

Analysis of transgenic expression by western blots and qRT-PCR. A Western blot analysis of transgene expression in Tetoff ToMSC cell lines (cropped image, uncropped blots image of Fig. 3A is in Supplementary Figure S2). B Quantitative RT-PCR analysis of transgene expression levels

In vitro chondrogenic differentiation of engineered ToMSCs

Chondrogenic potential was evaluated by culturing ToMSC-Tetoff: SOX9-TGFβ1-6His, ToMSC-Tetoff: SOX9-6His, ToMSC-Tetoff: TGFβ1-6His, and naïve ToMSCs in chondrogenic differentiation medium. After 21 days, Alcian blue staining revealed enhanced chondrocyte differentiation in engineered ToMSCs relative to naïve ToMSCs (Fig. 4). The ToMSC-Tetoff: SOX9-TGFβ1-6His group showed the highest staining intensity, indicating that co-expression of SOX9 and TGFβ1 promoted stronger chondrogenesis than either factor alone (Fig. 4). As a control, naïve ToMSCs were cultured in DMEM/F12 supplemented with 10% FBS without chondrogenic induction for 21 days and subsequently stained with Alcian blue; naïve ToMSCs were not stained as shown in Supplementary Figure S3.

Fig. 4.

Comparison of chondrogenic differentiation potential between genetically engineered ToMSCs and wild-type ToMSCs. Alcian blue staining (left panel) and quantification (right panel) of cells cultured in chondrogenic differentiation medium for 21 days. ***p < 0.001.**p < 0.01, *p < 0.05

Anti-allodynic effect of ToMSC-Tetoff: SOX9-TGFβ1-6His

Disc degeneration was induced in rat tail discs (Co6/7 and Co7/8) using a 21-gauge spinal needle, followed by treatment administration two weeks later (Fig. 5A). Treatment groups received either ToMSC, ToMSC-Tetoff: SOX9-6His, ToMSC-Tetoff: TGFβ1-6His, or ToMSC-Tetoff: SOX9-TGFβ1-6His, while the injury group received PBS.

Fig. 5.

Schematic diagram of the surgical procedure and assessment of mechanical allodynia following ToMSC treatment in a rat tail needle-puncture model. A Surgical procedure using a 21-gauge spinal needle followed by injection of PBS, ToMSC, ToMSC-Tetoff: SOX9-6His, ToMSC-Tetoff: TGFβ1-6His, or ToMSC-Tetoff: SOX9-TGFβ1-6His. B Assessment of mechanical allodynia using the von Frey test was performed after injection (PBS or ToMSC treatment) on days − 1, 1, 7, 14, 21, 28, 35, and 42. Statistical analysis was performed using two-way ANOVA followed by the Tukey’s post-hoc test. ##p < 0.01, ###p < 0.001, injury group vs. ToMSC group; $$p < 0.01, $$$p < 0.001, injury group vs. ToMSC-Tetoff: SOX9-6His group; &p < 0.05, &&p < 0.01, &&&&p < 0.0001, injury group vs. ToMSC-Tetoff: TGFβ1-6His group; **p < 0.01, **p < 0.0001, injury group vs. ToMSC-Tetoff: SOX9-TGFβ1-6His group

Mechanical allodynia—a characteristic of LBP associated with peripheral and central sensitization—was evaluated using the von Frey test on the ventral tail surface [36, 37]. Measurements were taken one day before injection and weekly for six weeks post-treatment.

The injury (control) group maintained significantly lower withdrawal thresholds compared to the sham (normal) group, indicating persistent hypersensitivity due to disc damage (Fig. 5B).

Although all treatment groups showed gradual improvement in withdrawal thresholds, the ToMSC-Tetoff: SOX9-TGFβ1-6His group demonstrated the most pronounced and sustained therapeutic effect, with significant pain relief beginning on Day 28 and continuing until withdrawal thresholds approached normal levels by Day 42.

Enhanced disc regeneration with ToMSC-Tetoff: SOX9-TGFβ1-6His in the rat tail injury model

MRI analysis

T2-weighted MRI evaluated disc regeneration six weeks post-treatment (Fig. 6A). This imaging technique visualizes tissue hydration levels via signal intensity [38, 39]. The sham group exhibited bright, homogeneous NP signals indicative of high water content and structural integrity, whereas the injury group displayed diminished, blurred NP regions reflecting severe dehydration and disc degeneration.

Fig. 6.

Improved structural and functional recovery of IVD following ToMSC treatments. A, B T2-weighted MRI scans at 6 weeks post-IVD needle-puncture injury (A) and quantitative analysis of MRI indices (B) across six experimental groups. C-E Representative images of IVD sections stained with H&E and safranin O (C), quantitative analysis of histological score (D), and measurement of NP area (E). Statistical analysis was performed using one-way ANOVA followed by the Tukey’s post-hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Quantitative assessment using the MRI index (NP area × mean signal intensity) revealed superior outcomes in the ToMSC-Tetoff: SOX9-TGFβ1-6His group (Fig. 6B). This group achieved significantly higher scores (66.02 ± 20.01%) compared to the injury group (15.11 ± 3.71%), demonstrating enhanced NP regeneration and structural recovery [40].

Histological evaluation

H&E and safranin O staining were used to assess morphological structure and proteoglycan distribution within the NP region. In the sham group, the NP and AF regions displayed normal cellular organization with a clearly defined NP-AF boundary (Fig. 6C). In contrast, the injury group exhibited substantial degeneration characterized by reduced NP cell density, loss of the NP-AF boundary, and structural collapse, including annular ruptures and clustered NP cells. Safranin O staining further illustrated proteoglycan content: the sham group showed strong staining intensity, whereas the injury group displayed significantly reduced staining, reflecting severe proteoglycan loss.

Among the treatment groups, the ToMSC-Tetoff: SOX9-TGFβ1-6His group demonstrated the most pronounced regenerative outcomes. This group maintained a relatively well-defined NP-AF boundary, exhibited increased NP cell density, and showed improved tissue organization compared to both the injury group and other treated groups. Histological scoring based on H&E and safranin O staining showed that the ToMSC-Tetoff: SOX9-TGFβ1-6His group achieved significantly lower degeneration scores (3.25 ± 1.5%) than the ToMSC group (12 ± 2%), ToMSC-Tetoff: SOX9-6His group (8.75 ± 3.38%), and ToMSC-Tetoff: TGFβ1-6His group (8.25 ± 0.96%) (Fig. 6D). Additionally, this group exhibited a significantly larger NP area than both the injury and ToMSC groups, indicating improved structural restoration (Fig. 6E). Together, these findings validate the superior regenerative potential of co-expressing SOX9 and TGFβ1 in promoting NP recovery and overall disc integrity.

ToMSC-Tetoff: SOX9-TGFβ1-6His demonstrated the best restoration of ECM integrity

IVD degeneration is primarily driven by the degradation of ECM components, including aggrecan and type II collagen, which are essential for maintaining disc structure and function. This degradation is mediated by inflammatory cytokines that activate MMPs [41]. Immunohistochemical staining revealed a statistically significant decrease in the expression of aggrecan and type II collagen in the injury group (7.81 ± 4.36% and 8.56 ± 1.31%, respectively) compared to healthy disc tissues in the sham group (66.45 ± 2.84% and 65.70 ± 3.03%, respectively) (Fig. 7A). Conversely, all treatment groups showed increased expression of these critical ECM components relative to the injury group. Among these, the ToMSC-Tetoff: SOX9-TGFβ1-6His group (48.36 ± 4.25% and 50.65 ± 7.083%, respectively) demonstrated the highest levels of aggrecan and type II collagen expression (Fig. 7B and C). These findings highlight the superior efficacy of SOX9 and TGFβ1 co-expression in restoring ECM composition in degenerated discs compared to the individual expression of either factor, demonstrating its potential as an advanced regenerative therapy.

Fig. 7.

Matrix protein preservation in the disc nucleus pulposus (NP) following ToMSC treatment. A Immunohistochemical staining of aggrecan (i) and type II collagen (ii) at low magnification (top) and high magnification (bottom). Black rectangles indicate the NP regions shown at higher magnification. B Quantification of aggrecan-positive area percentage in the NP. C Quantification of type II collagen-positive area percentage in the NP. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. All data are presented as mean ± standard deviation (SD)

ToMSC-Tetoff: SOX9-TGFβ1-6His exhibited the best preservation of endogenous disc NP progenitor cell

Immunofluorescence staining for Brachyury and Tie2, markers associated with NP progenitor cells, was conducted to evaluate the effects of treatments on NP cell phenotype. Previous in vitro studies have shown that Brachyury promotes ECM synthesis in NP cells through the transcriptional regulation of Smad3 [42, 43].

In our study, immunofluorescence staining revealed that the expression levels of brachyury and Tie2 were significantly higher in the healthy disc tissue of the sham group (92.51 ± 4.90% and 93.69 ± 3.53%, respectively) compared to the injury group (18.12 ± 1.995% and 21.67 ± 2.96%, respectively) (Fig. 8A). All treatment groups demonstrated increased expression of these markers relative to the injury group; notably, the ToMSC-Tetoff: SOX9-TGFβ1-6His group (79.91 ± 2.794% and 75.91 ± 2.06%, respectively) exhibited the highest levels (Fig. 8B and C). These findings suggest that promoting brachyury and Tie2 expression in degenerated discs may support the repair process by upregulating ECM synthesis and preserving the NP progenitor cell phenotype.

Fig. 8.

Maintenance of endogenous NP cell phenotype following ToMSC treatment. A Immunofluorescence staining for brachyury (red), Tie2 (green), and nuclei (DAPI, blue) with merged signals. White rectangles indicate NP regions shown at higher magnification. B Quantification of brachyury-positive cells in the NP. C Quantification of Tie2-positive cells in the NP. The percentage of immunopositive cells was calculated relative to total DAPI-positive cells in low-power fields of the NP. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. **p < 0.01; ***p < 0.001; ****p < 0.0001. Data are presented as mean ± SD

ToMSC-Tetoff: SOX9-TGFβ1-6His demonstrates the highest sell retention and proliferation in IVD tissues

Immunofluorescence staining for human nuclear antigen (HNA) was conducted to assess the retention and proliferation of transplanted ToMSCs in IVD tissues. HNA expression—a marker for human-derived cells—was absent in the sham and injury groups, which did not receive cell injections. In contrast, HNA expression was observed exclusively in the treatment groups, confirming successful retention of transplanted cells (Fig. 9A). Among these, the ToMSC-Tetoff: SOX9-TGFβ1-6His group (15.82 ± 3.31%) exhibited the highest HNA expression, indicating superior cell survival and retention at six weeks post-transplantation (Fig. 9B).

Fig. 9.

Assessment of ToMSC retention in the NP following ToMSC treatment. A Immunofluorescence staining for human nuclear antigen (red) and nuclei (DAPI, blue), with merged signals at 6 weeks post-treatment. White rectangles indicate NP regions shown at higher magnification. B Quantification of human nuclear antigen-positive cells relative to total DAPI-positive cells in low-power fields of the NP. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05; ***p < 0.001; ****p < 0.0001; ns, not significant. Data are presented as mean ± SD

The statistical analysis further demonstrated that the HNA expression was significantly higher in the ToMSC-Tetoff: SOX9-TGFβ1-6His group than in the other treatment groups. These results suggest that SOX9 and TGFβ1 co-expression promotes the survival and proliferation of transplanted cells within degenerated IVDs, while the control groups showed no evidence of exogenous cell presence, as expected.

ToMSC-Tetoff: SOX9-TGFβ1-6His demonstrated the greatest decrease in catabolic enzymes and pro-inflammatory cytokines in the disc NP

MMP-13 plays a critical role in the degradation of type II collagen, a major structural component of the IVD, and its expression is upregulated by pro-inflammatory cytokines, thereby accelerating degeneration [44]. MMP-13 expression is known to be upregulated by pro-inflammatory cytokines, exacerbating the degenerative process.

Immunohistochemical staining for MMP-13 revealed a significant increase in its expression in the injury group (81.71 ± 2.04%) compared to the sham group (11.95 ± 2.136%) (Fig. 10A, B). In contrast, treatment groups showed reduced MMP-13 expression, with the ToMSC-Tetoff: SOX9-TGFβ1-6His group (26.62 ± 3.92%) exhibiting the most pronounced decrease.

Fig. 10.

Effects of ToMSC treatment on pro-inflammatory markers in the NP. A Immunofluorescence staining for matrix metalloproteinase-13 (MMP-13) (green) and nuclei (DAPI, blue), with merged images at 6 weeks post-treatment. White rectangles indicate NP regions shown at higher- magnification. B Quantification of MMP-13-positive cells in the NP. C Immunofluorescence staining for tumor necrosis factor-alpha (TNF-α, red) and interleukin-1β (IL-1β, green), and nuceli (DAPI, blue), with merged images. White rectangles indicate NP regions shown at higher magnification. D Quantification of TNF-α-positive cells in the NP. E Quantification of IL-1β-positive cells in the NP. The percentage of immunopositive cells was calculated relative to total DAPI-positive cells. in low-power fields of the NP. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. **p < 0.05; ***p < 0.001; ****p < 0.0001. Data are presented as mean ± SD

To further assess the inflammatory environment, co-immunofluorescence staining for IL-1β and TNF-α was performed (Fig. 10C). Results showed significantly increased expression of these pro-inflammatory cytokines in the injury group (95.51 ± 1.40% and 93.61 ± 3.22%, respectively) (Fig. 10D). Treatment groups demonstrated reduced cytokine expression, with the ToMSC-Tetoff: SOX9-TGFβ1-6His group exhibiting the lowest levels of IL-1β (57.41 ± 6.78%) and TNF-α (49.54 ± 3.84%). These findings indicate a strong interaction between ECM degradation and inflammatory responses, where elevated MMP-13, IL-1β, and TNF-α contribute to the progression of IVD degeneration [45]. ToMSC-Tetoff: SOX9-TGFβ1-6His therapy effectively suppressed the expression of both MMP-13 and pro-inflammatory cytokines, highlighting its potential to inhibit ECM degradation and reduce inflammation. To summarize, ToMSC-Tetoff: SOX9-TGFβ1-6His therapy demonstrates a dual mechanism of action—namely, both mitigating inflammatory responses and preventing ECM breakdown. As such, it offers a promising strategy to promote recovery and structural integrity in degenerated IVDs.

ToMSC-Tetoff: SOX9-TGFβ1-6His shows the greatest reduction of injury-induced pain

Immunofluorescence staining for CGRP, a marker associated with pain, was performed to evaluate chronic discogenic LBP in degenerated disc [46]. The sham group showed the lowest CGRP expression (13.85 ± 2.56%), while the injury group exhibited significantly increased expression (90.90 ± 2.27%), consistent with pain and inflammation following IVD injury (Fig. 11A). Among the treatment groups, the ToMSC-Tetoff: SOX9-TGFβ1-6His group (43.61 ± 4.94%) demonstrated the most significant reduction in CGRP expression, to a level lower than that observed in the other treatment groups and the injury group (Fig. 11B). These findings suggest that ToMSC-Tetoff: SOX9-TGFβ1-6His therapy effectively reduces CGRP expression, potentially mitigating chronic discogenic LBP and associated inflammation resulting from IVD injury. This underscores its potential as a targeted therapeutic approach for pain relief in degenerated discs.

Fig. 11.

Pain reduction in the NP following ToMSC-Tetoff-SOX9-TGF β1-6His treatment. A Immunofluorescence staining for calcitonin gene receptor protein (CGRP, red) and nuclei (DAPI, blue), with merged images at 6 weeks post-treatment. White rectangles indicate NP regions shown at higher magnification. B Quantification of CGRP-positive cells in the NP. The percentage of immunopositive cells was calculated relative to total DAPI-positive cells in low-power fields of the NP. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05; ****p < 0.0001. Data are presented as mean ± SD

Discussion

This study demonstrates the therapeutic potential of ToMSC-Tetoff: SOX9-TGFβ1-6His for regenerating degenerated IVDs by enhancing ECM synthesis, modulating inflammation, and alleviating pain in a rat tail needle puncture model. Our findings highlight significant improvements in disc hydration and structural integrity, as validated by T2-weighted MRI and detailed histological examination. Notably, increased proteoglycan content and preserved NP morphology were key indicators of successful disc regeneration, supporting the conclusion that co-expression of SOX9 and TGFβ1 in ToMSCs effectively enhances the regenerative process in degenerated IVDs.

Our previous study on ToMSC-Tetoff-TGFβ1-IGF-1-BMP-7 [18] identified several limitations that were addressed in the current research. First, we incorporated Alcian blue staining to confirm chondrogenic differentiation and sulfated glycosaminoglycan production, addressing the previous lack of direct evidence for chondrocyte-like cell differentiation. Second, we streamlined our approach by focusing exclusively on SOX9 and TGFβ1, thereby eliminating potential complications associated with IGF-1 and BMP-7—whose roles in the avascular, hypoxic disc environment remain unclear. Notably, BMP-7 has been linked to osteogenic differentiation, posing challenges for its use in IVD repair. Finally, the simultaneous overexpression of multiple growth factors in our previous study hindered the ability to discern their individual contributions, underscoring the need for a more targeted strategy.

Our in vitro analysis provided important insights into the differential effects of SOX9 and TGFβ1. TGFβ1 alone demonstrated superior efficacy in promoting ECM synthesis compared to SOX9, as evidenced by Alcian blue staining intensity (Fig. 4). This observation aligns with TGFβ1’s established role in mediating ECM deposition through Smad signaling pathways [47], while SOX9 primarily functions as a master transcriptional regulator of chondrogenic genes [23]. However, our in vivo results revealed that the combination of SOX9 and TGFβ1 produced markedly superior regenerative outcomes compared to either factor alone. This synergistic effect is consistent with previous studies [21, 24] and suggests complex molecular interactions between these factors in the context of IVD regeneration.

The enhanced therapeutic efficacy of ToMSC-Tetoff: SOX9-TGFβ1-6His was validated through multiple analytical approaches. T2-weighted MRI and histological assessments confirmed enhanced proteoglycan retention and improved NP matrix integrity. Particularly noteworthy was the preservation of NP progenitor cell markers (Brachyury and Tie2), suggesting maintained cellular homeostasis within the degenerative environment—a significant finding given the adverse effects of the degenerated disc microenvironment on cell survival and function. Furthermore, the treatment effectively modulated inflammatory responses by downregulating key mediators including TNF-α, IL-1β, and MMP-13, while also reducing CGRP expression, indicating its potential for comprehensive pain management.

Our findings are consistent with previous research highlighting the synergistic benefits of combined SOX9 and TGFβ1 treatment. Lee et al. demonstrated that TGFβ1 enhances SOX9 transcriptional activity, thereby maximizing proteoglycan synthesis [21]. This mechanistic insight was further supported by Khalid et al., who reported that hUC-MSCs transfected with SOX9 and TGFβ1 exhibited enhanced ECM synthesis, characterized by increased levels of both aggrecan and type II collagen [22]. Coricor et al. further elucidated the molecular mechanism underlying this synergy, demonstrating that TGFβ1 stabilizes the SOX9 protein through serine 211 phosphorylation, involving both Smad-dependent and p38 MAPK pathways [47]. Our study builds upon these findings by demonstrating the therapeutic relevance of this synergy in IVD regeneration.

Immunological factors remain an important consideration when applying human cells in vivo. Considering the low immunogenicity of ToMSCs and the lack of general need for immunosuppressive therapy, we used immunocompetent Sprague-Dawley (SD) rats and administered immunosuppressive agents (oral cyclosporine) [48–53]. Additionally, the NP, a key target of our study among IVDs, forms a blood-nucleus pulposus barrier (BNB) due to its avascular and anatomically isolated characteristics. This structural feature makes it relatively immune privileged [54]. A recent study supports this concept, showing that when human iPSC-derived MSCs were xenotransplanted into the IVD of immunocompetent SD rats and immunodeficient nude rats, the immune response and cell survival rates were similar [55].

However, in our experimental model, we induced needle puncture to mimic IVD degeneration prior to cell transplantation. This damage is likely to provoke immune responses when human cells are transplanted into the compromised BNB. In this context, we applied the immunosuppressant cyclosporine A (starting oral administration two days prior to transplantation and maintaining it throughout the experimental period) to minimize immune responses. Cyclosporine A is known to effectively inhibit T-cell activation [56] and the same dose (100 mg/L in drinking water) was used in previous xenotransplantation studies [57]. Another study demonstrated that short- to medium-term (4–12 weeks) xenotransplantation survival is possible in immunocompetent rodent models without immunosuppressive therapy [58].

Immunodeficient models (e.g., nude or SCID mice) can eliminate xenogeneic immune responses and are useful for studying intrinsic regenerative mechanisms, but in this study, we used a normal immune function model with a controlled immunosuppressed state to better mimic the host immune environment. While this approach is not entirely identical to the clinical setting, it allowed for the evaluation of short-term graft survival and therapeutic efficacy in a more immunologically relevant context than a complete immunodeficiency system.

Beyond the immunological aspects, this study presents notable limitations that must be taken into consideration when interpreting the results.

First, anatomical and physiological differences between rat and human IVDs limit the direct applicability of this study. To compensate for this, validation using human IVD tissue culture or large animal models that better mimic human physiological structures is necessary.

Second, although no immediate adverse effects were observed during the study period, the long-term safety of sustained TGFβ1 and SOX9 expression remains a concern. To address this, future studies will incorporate additional safety strategies. One approach involves the use of suicide gene systems, such as inducible caspase-9 (iCASP9), which can selectively eliminate potential tumors resulting from transgene overexpression. Additionally, the integration of reporter genes will enable noninvasive tracking of transplanted cells, allowing real-time monitoring of their location and migration. This will facilitate the evaluation of off-target effects and help minimize the risk of unintended tumor formation or spread to non-target tissues [59, 60].

Third, the survival of transplanted cells could be confirmed at the 6-week time point using HNA immunofluorescence staining, but this method was limited to a single-point analysis and could not track the dynamic behavior or distribution changes of cells in real time. These limitations can be addressed in the future through a non-invasive cell tracking system based on reporter genes, which could play an important role in understanding the mechanisms of cell therapy effects and evaluating the movement of cells to non-target tissues [61].

Meanwhile, despite the meaningful therapeutic effects demonstrated in this study, several unresolved questions remain. For example, the appropriateness of the intervention timing during disease progression, age-related changes in cellular responsiveness, and the impact of mechanical load conditions on therapeutic effects have not yet been sufficiently elucidated. In the future, considering these variables, the development of a more sophisticated delivery system that precisely targets therapeutic effects, ensures sustainable therapeutic effects, and minimizes off-target effects will be essential for clinical applications.

Looking forward, our findings provide a robust foundation for developing comprehensive therapeutic strategies that combine ECM synthesis, inflammation modulation, and pain relief. The demonstrated synergy between SOX9 and TGFβ1 opens new avenues for therapeutic intervention in IVD degeneration. Further research should address long-term efficacy, delivery optimization, and clinical translation to establish ToMSC-Tetoff: SOX9-TGFβ1-6His as a viable treatment option for degenerative disc disease-related chronic low back pain. Additionally, combining this approach with other therapeutic modalities, such as biomaterial scaffolds or mechanical stimulation, may further enhance treatment outcomes. Integrating these findings with emerging technologies in tissue engineering and regenerative medicine may ultimately lead to more effective treatments for this challenging condition.

Conclusions

In this study, we demonstrated that ToMSC-Tetoff: SOX9-TGFβ1-6His therapy represents a promising therapeutic approach for IVD regeneration through its orchestrated mechanisms: robust ECM synthesis and preservation, potent inflammation suppression, and significant pain alleviation. The synergistic effects of SOX9 and TGFβ1 co-expression in ToMSCs were conclusively demonstrated by enhanced structural integrity, marked reductions in inflammatory mediators, and substantial improvements in mechanical sensitivity within treated IVDs.

Our findings establish that ToMSC-Tetoff: SOX9-TGFβ1-6His therapy strategically targets fundamental aspects of IVD degeneration by preserving NP progenitor cell populations, suppressing catabolic enzyme activity, and reducing inflammatory markers. This innovative therapeutic strategy represents a major advancement in cell-based treatments for chronic LBP and degenerative disc diseases, establishing a solid foundation for clinical translation. Further investigation is essential to evaluate long-term outcomes, safety profiles, and scalability in clinical settings. The demonstrated efficacy of ToMSC-Tetoff: SOX9-TGFβ1-6His therapy opens promising new avenues for treating degenerative disc diseases, potentially transforming treatment outcomes for patients suffering from chronic LBP.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

IVD

Intervertebral disc

LBP

Low back pain

MSCs

Mesenchymal stromal cell

ToMSCs

Tonsil-derived mesenchymal stromal cell

Tet-off

Tetracycline-off system

CRISPR/Cas9

Clustered regularly interspaced short palindromic repeats /CRISPR-associated protein 9

SOX9

SRY-box transcription factor 9

TGFβ1

transforming growth factor beta 1

ECM

Extracellular matrix

MRI

Magnetic resonance imaging

TNF-α

Tumor necrosis factor-α

IL-1β

Interleukin-1β

NP

Nucleus pulposus

AF

Annulus fibrosus

Co

Coccygeal disc

MMP-13

Matrix metalloproteinase-13

HNA

Human nuclei antigen

CGRP

Calcitonin gene-related peptide

Author contributions

I.H., D.Y.H. designed the study; D.H.K. performed the material development; S.L., Y.Y., M.S.B., Y.K., H.C. and H.E.S. performed the in-vivo experiments. S.L., Y.Y., D.H.K., S.B.A., I.H. and D.Y.H. wrote the paper with input from all authors. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT: Ministry of Science and ICT) (RS-2024-00347403) and a grant of Korean Cell-Based Artificial Blood Project funded by the Korean government (The Ministry of Science and ICT, The Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of Food and Drug Safety) (grant number: RS-2023-KH141187).

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Consent of Committee on the Bioethics and consent to participate: (1) Title of the approved project: Characterization and functional enhancement of tonsil-derived stromal cells (2). Name of the institutional approval committee: Bundang CHA Hospital, CHA University, Republic of Korea (3). Approval number: CHAMC 2021-03-016-006 (4). Date of approval: April 1st, 2022. Written informed consent for tonsil tissue was obtained from the parents of the children.

Consent of the Committee on the Ethics of Animal Experiments of the Local Ethics Commission: (1) Title of the approved project: Bone and cartilage regeneration experiments for spine and disc diseases (2). Name of the institutional approval committee: the Institutional Animal Care and Use Committee (IACUC) of CHA Bundang Medical Center, CHA University, Republic of Korea (3). Approval Number: IACUC-230180 (4). Date of approval: November 1st, 2023. The animal studies were conducted according to the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments).

Consent for publication

Not applicable.

Competing interests

The authors declare no potential conflict of interest.