CTRP3缺失促进腱向诱导的犬脂肪源性干细胞呈现腱样基质特征。 机翻

Abstract

Introduction

Tendon injuries heal through disorganized extracellular matrix (ECM) deposition rather than the restoration of native collagen architecture, thus resulting in suboptimal functional recovery. Although adipose-derived stem cells (ADSCs) are attractive for tendon regeneration because of their accessibility and expandability, their tenogenic differentiation efficiency is lower than that of tendon-derived stem cells (TDSCs). C1q/TNF-related protein 3 (CTRP3) is an adipokine implicated in ECM remodeling and fibrosis; however, its role in tendon lineage regulation is unknown.

Objective

CTRP3 (C1q/TNF-related protein 3) contributes to extracellular-matrix remodeling and, in tendon, is associated with a pathological fibrotic matrix signature; we therefore reasoned that reducing CTRP3 would favor a more mature tendon-like matrix. Using a loss-of-function approach based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), we investigated whether CTRP3 disruption alters tenogenic marker expression, at both the transcript and protein levels, and the tendon-associated ECM marker profile of canine ADSCs relative to wild-type ADSCs and native TDSCs.

Methods

CRISPR/Cas9-mediated CTRP3–disrupted ADSCs were generated and validated using sequencing, quantitative polymerase chain reaction, and Western blotting. Wild-type ADSCs, CTRP3-disrupted ADSCs, and TDSCs were subjected to tenogenic induction for 0–3 weeks. Tenogenic transcriptional and ECM markers were analyzed at mRNA and protein levels.

Results

CTRP3-disrupted ADSCs showed increased collagen type I and tenomodulin (TNMD) expression during tenogenic induction compared with wild-type ADSCs. COL III expression showed a transient increase followed by a decline, with lower expression in CTRP3-disrupted cADSCs than in WT cADSCs at week 2. CTRP3 disruption increased early Mkx and Scx expression at the earliest time points but did not consistently alter the early transcriptional program, which rose comparably across all groups. CTRP3-disrupted cADSCs showed partial, marker- and time-point-dependent overlap with native cTDSCs, mainly for selected late ECM markers rather than across the overall transcript and protein profiles.

Conclusion

CTRP3 disruption shifted the tendon-associated ECM marker profile during the tenogenic differentiation of ADSCs, with reduced COL III accumulation, rather than uniformly enhancing tenogenic differentiation. Although early transcriptional changes were observed, the more consistent differences were found in late tendon-associated ECM markers. The modulation of CTRP3 may represent a candidate strategy worth further investigation for stem cell–based tendon regeneration in veterinary medicine.

Keywords: CTRP3, Adipose-derived stem cells, Tendon differentiation, Tenogenesis, Extracellular matrix, Canine

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Introduction

Tendons are highly specialized connective tissues composed of hierarchically organized collagen type I (COL I) fibrils that enable efficient force transmission and musculoskeletal function [1], [2]. However, following an injury, the restoration of this native hierarchical structure is limited. Healing often proceeds through disorganized extracellular matrix (ECM) deposition and increased collagen type III (COL III) accumulation, resulting in scar-like remodeling and compromised biomechanical properties [3], [4]. These limitations underscore the need to identify the molecular regulators that coordinate ECM maturation during stem cell–mediated tendon repair.

Mesenchymal stem cells (MSCs) have been widely investigated for tendon regeneration owing to their multilineage capacity and paracrine effects [5]. Among the available sources, adipose-derived stem cells (ADSCs) are particularly attractive because they can be obtained with minimal morbidity and can be readily expanded efficiently in vitro. However, ADSCs often exhibit incomplete tenogenic differentiation and a limited capacity to reproduce the organized ECM structure of native tendon tissue [6], [7]. Tendon-derived stem cells (TDSCs) possess lineage priming toward tendon-specific gene networks and are often used as a reference for tenogenic differentiation; however, their application is limited by donor site availability and harvesting constraints. Collectively, these observations suggest that factors beyond early lineage commitment contribute to the inferior regenerative performance of ADSCs.

Tenogenic differentiation involves a coordinated cascade of transcriptional activation and ECM remodeling. Early specification is driven by transcription factors such as Scx and Mkx, whereas mature tendon formation is characterized by a COL I–rich matrix and tenomodulin (TNMD) expression [8], [9], [10], [11]. Established protocols using growth factors such as transforming growth factor beta-3 (TGF-β3) and growth differentiation factor-7 (GDF-7) can induce tenogenic gene expression [12], [13], [14]; however, ADSCs often exhibit incomplete differentiation [6], [7]. Although ECM organization is widely recognized as a key determinant of tendon structure and function [3], [4], [11], it remains unclear whether incomplete matrix maturation is a primary limitation of ADSC-based tenogenic differentiation. In particular, effective tendon regeneration requires the transition from a provisional COL III–rich matrix to a mature COL I–dominant architecture [15], [16], [17], [18]. These observations highlight the need to identify the intrinsic regulators of ECM remodeling during tenogenic differentiation.

C1q/TNF-related protein 3 (CTRP3) is a secreted adipokine implicated in metabolic regulation, inflammation, and ECM remodeling [19], [20], [21]. CTRP3 has been reported to modulate phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinase (MAPK) signaling pathways [22], [23], [24]. Although CTRP3 exerts protective, anti-fibrotic effects in several metabolic and cardiovascular tissues [25], it plays a distinct pathological role in tendon, where it is up-regulated in tendinopathy and promotes degenerative extracellular-matrix remodeling, including proteoglycan accumulation, collagen disorganization, and an increased type III collagen signature, whereas its blockade ameliorates tendinopathy [26]. However, whether CTRP3 functions as a regulator of ECM maturation during stem cell–mediated tenogenic differentiation has not yet been directly investigated. Given the matrix-modulatory properties of CTRP3, its suppression may influence progression from an immature reparative matrix to a more organized tendon-like extracellular architecture.

Tendon injuries such as common calcaneal (Achilles) tendon disruption are clinically important in dogs, are frequently associated with chronic degeneration and prolonged recovery, and carry high complication rates after surgical repair [27]; effective regenerative options therefore remain limited. Because adipose tissue provides an accessible and abundant autologous cell source, canine ADSCs represent a clinically relevant candidate for cell-based tendon regeneration. In this study, we determined whether CTRP3 disruption alters tenogenic marker expression and the tendon-associated ECM marker profile of canine ADSCs (cADSC). We employed a loss-of-function approach based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to directly evaluate the role of CTRP3 in ADSC tenogenesis. We hypothesized that CTRP3 acts as a regulatory constraint on ECM remodeling, and its disruption promotes the transition toward a more mature tendon-like matrix characterized by increased COL I and reduced COL III. These findings provide insight into the intrinsic mechanisms governing stem cell–based tendon regeneration and suggest potential strategies for improving the therapeutic application of ADSCs.

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Materials and methods

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Cell isolation and culture

Tendon tissues were aseptically collected from four adult male Beagle dogs (approximately 1 year of age) that had been euthanized as part of an unrelated experimental protocol (IACUC approval no. SNU-200709-4-5). Tendon-derived cells were obtained from the calcaneal (common calcaneal) tendon and the biceps tendon, and adipose-derived cells were obtained from subcutaneous adipose tissue. The use of the archived tendon and adipose tissues and all subsequent experimental procedures in the present study were approved by the Institutional Animal Care and Use Committee of Seoul National University (IACUC approval no. SNU-220428-3). The samples were repeatedly washed with Dulbecco's phosphate-buffered saline (dPBS; Welgene Inc., Gyeongsan-si, Gyeongsangbuk-do, Republic of Korea) to remove blood and debris, and any remaining non-tendinous tissue was carefully trimmed away. The cleaned tendon pieces were finely minced and digested with 0.3% collagenase type I (3 mg/mL; Gibco BRL, Grand Island, NY, USA) at 37 °C for 1 h under gentle agitation. The resulting cell suspension was passed through a 70-μm strainer and cultured in low-glucose Dulbecco's Modified Eagle Medium (DMEM; Welgene Inc.) supplemented with 10% fetal bovine serum (FBS; 35-015-CV, Corning Inc., Corning, NY, USA) and 1% penicillin–streptomycin (Gibco BRL) at 37 °C in a humidified 5% CO2 atmosphere. cADSCs were isolated from subcutaneous adipose tissue by using an identical isolation procedure. These enzymatic (collagenase) digestion procedures for cTDSCs and cADSCs were based on previously described methods with minor modifications [28], [29]. After 24 h, the cells were rinsed with dPBS to remove nonadherent debris, and the culture medium was refreshed every 48 h. Cells were seeded 2.3 × 104 cells/cm2 and subcultured at 80%–90% confluence. Cells were used for subsequent experiments at passages P3–P5. The tenogenic and stem-cell identity of the isolated cTDSCs was supported by tri-lineage (adipogenic, chondrogenic, and osteogenic) differentiation assessed by RT-PCR for LPL, COL2A1, and RUNX2, respectively, and by immunocytochemistry for Scx, Mkx, and SOX2 (Supplementary Fig. S1).

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Pre-electroporation preparation

Prior to electroporation, cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C in a humidified 5% CO2 incubator and passaged at a 1:5 ratio upon reaching approximately 80% confluence. Complete medium and trypsin–ethylenediaminetetraacetic acid were prewarmed for 30 min before use, whereas single guide RNA (sgRNA) aliquots and Cas9 protein were stored on ice until ribonucleoprotein (RNP) assembly.

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Canine complement CTRP3 gene (C1QTNF3) target selection and sgRNA design

C1QTNF3 was targeted using CanFam3.1, with a focus on exon 1. sgRNA candidates were designed using CHOPCHOP and selected on the basis of predicted on-target activity and specificity. The sgRNA target sequences, protospacer-adjacent motif (PAM) sites, and genomic coordinates are provided in Supplementary Table S1.

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In vitro sgRNA synthesis

The sgRNAs were synthesized using the GeneArt Precision gRNA Synthesis Kit (Invitrogen, Thermo Fisher Scientific, A29377) following the manufacturer's protocol. Briefly, sgRNA DNA templates containing a T7 promoter were generated. In vitro transcription was performed, and sgRNAs were purified to remove enzymes and unincorporated nucleotides. sgRNA concentration was measured using NanoDrop (Thermo Scientific), and aliquots were stored at −80 °C until use.

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Cas9–sgRNA RNP assembly

Genome editing was performed using Cas9–sgRNA RNP complexes. For each reaction, recombinant Cas9 protein (Invitrogen, A36499) and sgRNA were mixed at a 1:1 mass ratio (1 μg Cas9 + 1 μg sgRNA) in a nuclease-free microtube, briefly centrifuged, and incubated for 10 min at room temperature to allow RNP formation.

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Electroporation platform and workstation setup

Electroporation was performed using the Neon Transfection System (Thermo Fisher Scientific) with the 10-μL tip format. The Neon pipette station was prepared according to the manufacturer's instructions by filling the Neon tube with 3 mL of E buffer prior to pulsing. Electroporation was performed using freshly prepared cell suspensions and R buffer mixtures, and the cells were immediately recovered in a pre-warmed complete medium after pulsing.

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Electroporation parameter optimization using piggyBac green fluorescent protein (GFP) reporter delivery

To optimize the electroporation conditions for cADSCs, delivery efficiency was screened using a GFP reporter plasmid. A piggyBac GFP plasmid and piggyBac transposase plasmid were mixed at a 1:1 ratio (500 ng each), and Neon Resuspension Buffer R was added to a final volume of 10 μL. cADSCs were harvested by trypsinization, washed once with dPBS, counted, and aliquoted at 5 × 105 cells per condition. Cells were pelleted, the supernatant was removed completely, and the pellet was resuspended directly in the 10-μL plasmid/R-buffer mixture immediately before electroporation. Electroporation was performed using the preloaded optimization program of the Neon system (#2–#16; varying voltage, pulse width, and pulse number). After pulsing, the cells were immediately expelled into a pre-warmed complete medium, gently triturated to disperse the clumps, and seeded into a six-well plate. GFP expression was assessed by fluorescence microscopy on day 1 post-electroporation, and the relative GFP + cell abundance was quantified using ImageJ (version 1.54p, National Institutes of Health, Bethesda, MD, USA) to compare delivery efficiency across programs.

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Co-electroporation of CTRP3 RNP and GFP reporter plasmid

To enrich successfully electroporated cells, CTRP3 Cas9 RNP complexes were co-electroporated with a separate GFP expression plasmid that was used solely as a transient reporter. Cas9 protein and sgRNA (1 μg each) were incubated for 10 min at room temperature to assemble the RNPs. A GFP expression plasmid (500 ng) was added, Neon Resuspension Buffer R was used to adjust the mixture to 11 μL, and 10 μL was electroporated using Neon program #9 (1400 V, 30 ms, 1 pulse). The cells were immediately transferred to a pre-warmed complete medium and plated for recovery. GFP expression was monitored on days 1 and 3 post-electroporation, and GFP-positive cells were isolated by fluorescence-activated cell sorting (FACS) on day 3 for downstream genotyping.

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Flow cytometry and GFP + cell sorting

To enrich electroporated cells, GFP + populations were isolated by FACS at the National Center for Inter-University Research Facilities at Seoul National University. Data acquisition and sorting were performed using BD FACSDiva software (v9.0.1). Cells were harvested on day 3 post-electroporation, resuspended in phosphate-buffered saline (PBS) + 2% FBS, filtered through a 40-μm cell strainer, and kept on ice before sorting. Gating was established using an untransfected control to define background fluorescence, followed by forward scatter (FSC)/side scatter gating to exclude debris, and doublet discrimination using FSC height/FSC area. The sorted GFP-positive cells were expanded for 5–8 days as a pooled edited population without single-cell clonal isolation, and subsequent analyses were performed using this edited cell population. This population is hereafter referred to as CTRP3-disrupted cADSCs. During this expansion, the edited (pooled) cADSC population initially appeared to proliferate more slowly after electroporation but subsequently expanded sufficiently for the downstream tenogenic differentiation experiments; however, cell viability and proliferation rates were not quantitatively assessed.

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Genomic DNA (gDNA) extraction

gDNA was extracted from the pooled cell populations by using a QIAamp DNA kit (51306, Qiagen, Hilden, Germany) according to the manufacturer's protocol. DNA concentration was measured using a NanoDrop spectrophotometer and normalized for polymerase chain reaction (PCR) where applicable.

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PCR amplification of the CTRP3 target locus

The PCR amplification of the CTRP3 target region was performed using i-StarTaq DNA Polymerase (25162; iNtRON Biotechnology). Each reaction contained 10 pmol of forward primer, 10 pmol of reverse primer, and approximately 150 ng of gDNA per sample (unless otherwise specified). Samples were amplified for 35 cycles by using the following cycling conditions recorded in this workflow: 30 s at 94 °C (denaturation), 40 s at 58 °C (annealing), and 44 s at 72 °C (extension) per cycle. For assays employing two primer sets, annealing was performed at 58 °C for primer_1 and 64 °C for primer_2 (primer sequences are provided in Supplementary Table S2). The PCR products were resolved on 1.5% agarose gels and visualized using the iBright Imaging System (Thermo Fisher Scientific).

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T7 endonuclease I (T7E1) mismatch cleavage assay

Genome editing was assessed using a T7E1 mismatch cleavage assay (New England Biolabs). The PCR amplicons were subjected to heteroduplex formation according to standard procedures and then digested with T7E1. For each digestion reaction (20 μL total), 16 μL of PCR product was mixed with 1.8-μL nuclease-free water, 2-μL T7E1 reaction buffer, and 0.2-μL T7 endonuclease I, followed by incubation at 37 °C for 15 min. The digested products were resolved by agarose gel electrophoresis and imaged using the iBright Imaging System.

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Gel excision and purification of PCR products

For the sequence-level quantification of editing outcomes, PCR products were gel excised and purified using a QIAquick Gel Extraction Kit (Qiagen), according to the manufacturer's instructions. Purified DNA was eluted in a final volume of 20 μL, and DNA concentration was measured prior to cloning.

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Western blot

CTRP3-disrupted cADSCs were washed twice with ice-cold PBS and lysed in ice-cold RIPA buffer (Gen Depot, Grand Island, NY, USA) supplemented with 1% protease inhibitor cocktail (Gen Depot). Lysates were incubated for 30 min on ice with intermittent vortexing and clarified by centrifugation at 12,000 × g for 10 min at 4 °C. The supernatants were collected, and the total protein concentrations were determined using the Bradford assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein (20 μg per lane) were mixed with 5× reducing Laemmli sample buffer and denatured at 100 °C for 5 min. Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis with 10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature and incubated overnight at 4 °C with a primary antibody against CTRP3 (1:500, MBS151520, MyBioSource, San Diego, CA, USA). After washing with TBST, the membranes were incubated with a horseradish peroxidase–conjugated secondary antibody (1:10,000, AB_228341, Thermo Fisher Scientific) for 1 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence substrate (Bio-Rad) and imaged using an LAS 4000 mini system (GE Healthcare, Lafayette, CO, USA). Band intensities were quantified using ImageJ and normalized to β-actin (ab8227, 1:1,000, Abcam, Cambridge, UK) as a loading control. All Western blotting experiments were performed independently four times by using separate cell preparations.

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Thymine–adenine (TA) cloning and Sanger sequencing

Given that the bulk Sanger sequencing of mixed amplicons yields overlapping chromatograms, PCR products were TA-cloned to isolate individual alleles for indel identification and editing frequency estimation. PCR amplicons spanning the CTRP3 target site were gel purified using the QIAquick Gel Extraction Kit (Qiagen) and eluted in 20 μL of elution buffer. Purified amplicons were cloned using the All-in-One PCR Cloning Kit (BIOFACT) according to the manufacturer's instructions, with minor empirical adjustments to optimize colony recovery. For the initial cloning attempt, approximately 150 ng of the gel-purified PCR product was used per reaction and incubated at room temperature for 55 min. The cloning reaction mixture was mixed with chemically competent E. coli and incubated on ice for 55 min before plating on lysogeny broth agar supplemented with ampicillin. Plates were incubated at 37 °C for ∼17 h, after which colonies were picked for sequencing. For subsequent cloning attempts, the insert input was increased to ∼500 to 550 ng and the room temperature incubation was extended to 2 h, whereas the 55-min on-ice incubation with competent cells were maintained. Under these conditions, colonies were recovered from both the unsorted transfected and GFP-sorted populations. Colonies were selected from multiple plates after approximately 15 h of incubation and subjected to Sanger sequencing (Cosmo Genetech, Seoul, Republic of Korea). Sequence traces were aligned to CanFam3.1 reference amplicon, and clones were scored as edited when insertions or deletions were detected at or near the predicted cut site. Editing efficiency was calculated as the ratio of edited clones to the total number of sequenced clones for each population.

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Tenogenic induction

Cells were maintained in DMEM supplemented with 10% FBS, 50 μg/mL L-ascorbic acid (A5960, Sigma-Aldrich, St. Louis, MO, USA), 10 ng/mL TGF-β3 (100-36E, PeproTech Inc., Rocky Hill, NJ, USA), and 50 ng/mL GDF-7 (120-37, PeproTech Inc.) [30], [31]. The medium was replaced every 4 days with fresh supplements/growth factors. No antibiotics were used to maintain optimal viability during long-term culture [32], [33], [34]. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2 for up to 3 weeks.

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Real-Time quantitative PCR (RT-qPCR)

Total RNA was isolated at weekly intervals using QIAzol reagent (Qiagen). RNA purity and concentration were determined using a microplate reader (Epoch, Gen 5.2 software; BioTek Instruments Inc., Winooski, VT, USA). First-strand complementary DNA (cDNA) was generated using PrimeScript II First-Strand cDNA Synthesis Kit (Takara Bio Inc., Otsu, Japan). RT-qPCR was performed on an ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) by using SYBR Premix Ex Taq (Takara Bio, Inc.) and gene-specific primers (Table 1). Relative messenger RNA (mRNA) expression levels were calculated using the 2–ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as the internal control for normalization. Quantitative PCR was performed on an Applied Biosystems StepOnePlus Real-Time PCR System using a two-step (shuttle) protocol: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. A melt (dissociation) curve analysis was performed after amplification to confirm a single product (single melt peak). Each sample was analyzed in technical triplicate. All quantitative PCR experiments were independently performed using four separate cell preparations (n = 4).

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Immunocytochemistry and fluorescence quantification

Cells were plated in 48-well culture dishes at a density of 2.7 × 103 cells per well. For fixation, the cells were washed three times with PBS and treated with 4% paraformaldehyde for 5 min at room temperature, followed by washing with PBS. Permeabilization was achieved using 0.2% Triton X-100 for 5 min. After rinsing, nonspecific binding was blocked with 10% FBS in PBS for 30–60 min. Cells were then incubated overnight at 4 °C with primary antibodies, including Scx (1:300; NBP3-07598, Novus Biologicals), Mkx (1:400; NBPI-89912, Novus Biologicals, Centennial, CO, USA), TnC (1:300; sc-25328, Santa Cruz Biotechnology, Dallas, TX, USA), TNMD (1:500; BS-7525R, Bioss Inc.), Collagen type I (1:400, MA1-26771, Invitrogen, Carlsbad, CA, USA) and collagen type III (1:400, CSI 007-01-02, Invitrogen). After washing with PBS, the cells were incubated for 1 h at room temperature with Alexa Fluor–conjugated anti-mouse and anti-rabbit secondary antibodies (Santa Cruz Biotechnology; catalog nos. sc-516248 and sc-533656; both diluted 1:500). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; R37606, Invitrogen) for 5 min, followed by final PBS washes. Fluorescent images were obtained using an EVOS XL Core Imaging System (Thermo Fisher Scientific). Immunocytochemistry was performed on tenogenically induced cTDSCs, WT cADSCs, and CTRP3-disrupted cADSCs at 0, 1, 2, and 3 weeks after the onset of induction, with all groups processed in parallel under identical staining conditions. To confirm the specificity of the immunofluorescence signal, a negative control in which the primary antibody was omitted (secondary antibody only) was included and showed no specific staining. Four independent biological replicates (n = 4 donor animals) were used for immunocytochemical analyses.

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Fluorescence quantification

The fluorescence intensity was quantified using ImageJ [35]. For each cell, a region of interest (ROI) encompassing the whole cell was drawn along the cell boundary identified from the corresponding phase-contrast image, so that both nuclear and cytoplasmic/extracellular marker signals were captured within the ROI. The DAPI channel was used to identify and count individual nuclei. Within each whole-cell ROI, the integrated density (IntDen) of the marker signal and that of the DAPI signal were measured, and the marker signal was normalized to the nuclear signal on a per-cell basis (normalized intensity = marker IntDen/DAPI IntDen). Identical acquisition settings (including exposure time and gain) and a common threshold were applied across all compared images. Because DAPI fluorescence itself can vary with staining and chromatin condensation, this normalization has inherent limitations, and the normalized intensities were interpreted as relative, complementary measures rather than absolute quantities. Five microscopic fields were analyzed per sample under identical image-acquisition settings, and the resulting values were averaged to a single value per biological replicate (n = 4 donor animals). Because fluorescence intensity is sensitive to image-acquisition settings and does not capture the full-population distribution, these protein-level measurements were interpreted alongside, and as complementary to, the transcript-level RT-qPCR data rather than as a stand-alone endpoint.

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Statistical analysis

For each biological replicate (independent cell preparation), values from multiple microscopic fields (technical replicates) were averaged to a single value, and all analyses were performed on biological replicates (n = 4 for RNA; n = 4 for protein). Because independent cultures were seeded simultaneously and harvested separately at weeks 0, 1, 2 and 3, samples at different time points represent independent observations; accordingly, an ordinary (between-subjects) two-way ANOVA was used, with cell group and induction time as factors and their interaction. For each group, n = 4 corresponds to four independent donor-derived cell preparations (biological replicates). Although tendon- and adipose-derived cells originated from four donor animals, the cell preparations were cryobanked and retrieved as independent stocks, and donor-level identity linking individual preparations back to specific animals was not retained. Per-animal pairing across preparations therefore could not be reconstructed, so donor was not modeled as a blocking factor and all groups were analyzed as independent (between-subjects) samples. Marker expression showed marked mean–variance dependence: homogeneity of variance (Levene's test) was satisfied after logarithmic transformation but not on the linear scale, so ANOVA was performed on log-transformed values, with normality assessed by the Shapiro–Wilk test. When the group × time interaction was significant, between-group differences within each time point and within-group differences across time points were evaluated by Tukey's post-hoc multiple-comparison test using the pooled error term from the two-way model; the Games–Howell test was used where variances remained heterogeneous. All analyses were performed using GraphPad Prism (version 10.4; GraphPad Software, San Diego, CA, USA). Data are presented as mean ± SD, and significance was set at p < 0.05.

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Results

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Generation and validation of CTRP3-disrupted cADSCs

CRISPR/Cas9-mediated genome editing successfully targeted the exon 1 of CTRP3 to generate CTRP3-disrupted cADSCs (Fig. 1A). Strong GFP fluorescence was detected in CTRP3-disrupted cADSCs after electroporation (Fig. 1B), whereas wild type (WT) cADSCs exhibited minimal GFP signals under identical imaging conditions. RT-qPCR demonstrated a significant reduction in CTRP3 mRNA expression in CTRP3-disrupted cADSCs compared with that in WT cADSCs (p < 0.001; Fig. 1C). Western blot analysis further showed markedly reduced CTRP3 protein expression in CTRP3-disrupted cADSCs, with β-actin used as a loading control (Fig. 1D). Densitometric quantification confirmed a significant reduction in CTRP3-disrupted cADSCs relative to WT cADSCs (0.23 ± 0.01 vs 0.63 ± 0.03, normalized to β-actin; mean ± SD; n = 4; p < 0.001). Clone sequencing analysis identified indel mutations in 24 of the 32 clones analyzed, corresponding to an editing efficiency of 75% (Fig. 1E). These data confirm the efficient CRISPR/Cas9-mediated disruption of CTRP3 in cADSCs.

[图：Generation and validation of CTRP3-disrupted cADSCs Schematic illustration of the CRISPR/Cas9-mediated genome editing st]

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Morphological changes during tenogenic induction

Representative phase-contrast images show progressive morphological changes during the 3-week tenogenic induction period in canine tendon-derived stem cells (cTDSCs), WT cADSCs, and CTRP3-disrupted cADSCs (Fig. 2). In all groups, the cells gradually adopted elongated spindle-shaped morphologies with increased alignment and cellular density.

[图：Morphological changes during tenogenic induction Representative phase-contrast images showing morphological changes in c]

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CTRP3 disruption does not consistently alter early tenogenic transcriptional activation

RT-qPCR revealed temporal changes in the expression of tenogenic markers during differentiation (Fig. 3). The expressions of early transcription factors Mkx and Scx increased during the initial phase of induction in all groups (Fig. 3A). Late tendon-associated genes, such as COL I and TNMD, gradually increased throughout the induction period (Fig. 3B), whereas TnC, a matricellular glycoprotein of the provisional tendon extracellular matrix that is up-regulated by mechanical loading and during tendon remodeling [36], displayed a transient increase during the early-to-intermediate phase of induction before declining at later time points (Fig. 3C). However, no consistent differences were observed between CTRP3-disrupted and WT cADSCs across the time points.

[图：Time-dependent expression of tenogenic markers during differentiation. Relative mRNA expression of early transcription f]

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Comparison of tenogenic gene expression among cell types

At baseline (week 0), cTDSCs generally showed higher expression of tenogenic markers than the ADSC-derived groups, whereas CTRP3-disrupted cADSCs displayed intermediate levels relative to WT cADSCs (Fig. 4). During early induction, Mkx and Scx expressions were elevated in both CTRP3-disrupted cADSCs and cTDSCs compared with WT cADSCs (Fig. 4A).

[图：Between-group comparison of tenogenic gene expression during differentiation. The relative mRNA expression levels of ear]

By contrast, at later stages of differentiation, cTDSCs showed the highest mRNA expression of the late tendon-associated genes COL I and TNMD, whereas, relative to WT cADSCs, CTRP3-disrupted cADSCs showed significantly higher COL I and TNMD mRNA expression at early time points (weeks 0–1) that diminished thereafter (Fig. 4). The corresponding increases in COL I and TNMD in CTRP3-disrupted cADSCs were evident at the protein level (Fig. 7B). TnC mRNA peaked at week 1 in all groups and then declined; relative to WT cADSCs, CTRP3-disrupted cADSCs showed higher TnC expression at weeks 0 and lower expression at week 2 and 3 (Fig. 4C).

[图：Between-group comparison of protein expression during differentiation. Protein expressions of early transcription factor]

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Protein expression of tenogenic markers during induction

Representative immunocytochemical staining confirmed the expression of COL I, Mkx, Scx, TnC, and TNMD in all groups during tenogenic induction; COL I is shown across the induction period, whereas the remaining markers are shown at their peak expression (Fig. 5). Quantitative comparisons of these markers are presented in Fig. 6, Fig. 7.

[图：Representative immunocytochemical staining of tenogenic markers in cTDSCs, WT cADSCs, and CTRP3-disrupted cADSCs Represe]

[图：Protein-level expression of tenogenic markers during tenogenic induction. Protein expression of early transcription fact]

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Quantitative analysis of tenogenic protein expression

Quantitative immunofluorescence intensity (integrated density [IntDen]) analysis revealed significant time- and group-dependent differences in the expression of tenogenic markers (Fig. 6). The early transcription factors Mkx and Scx increased significantly during the first two weeks of induction in all groups. Mkx rose to a peak at week 2 and then decreased at week 3, with this reduction reaching statistical significance only in cTDSCs. Scx peaked sharply at week 1 and then declined at later time points rather than reaching a plateau (Fig. 6A). COL I and TNMD levels increased steadily over the 3-week induction period, with significantly higher expression in CTRP3-disrupted cADSCs and cTDSCs than in WT cADSCs (Fig. 6B). TnC showed a transient increase followed by a reduction at later stages, and COL III displayed a transient peak at week 2 in all groups before declining by week 3, with CTRP3-disrupted cADSCs showing lower COL III than WT cADSCs at week 2 (Fig. 6C). Two-way ANOVA identified significant main effects of group and time, as well as a group × time interaction, for all markers (COL I, Mkx, Scx, TNMD, COL III, and TnC) (p < 0.001).

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Comparison of protein expression among cell types

A cross-group comparison of protein expression at matched time points showed differences in tenogenic marker profiles among the cell sources (Fig. 7). Among the early transcription factors (Fig. 7A), Mkx differed between CTRP3-disrupted and WT cADSCs mainly at baseline, whereas Scx was significantly higher in CTRP3-disrupted than in WT cADSCs at several time points (weeks 0, 2 and 3); overall, the differences in these early factors were less consistent than those observed for the late ECM markers. For the late tendon-associated markers (Fig. 7B), COL I and TNMD were significantly higher in CTRP3-disrupted cADSCs than in WT cADSCs across the later induction period. At baseline, cTDSCs showed higher expression than the cADSC-derived groups for some, but not all, markers (Fig. 7A–C). Overall, CTRP3-disrupted cADSCs approached the protein expression profile of cTDSCs for several late ECM markers, although this resemblance was marker- and time-point-dependent (Fig. 7).

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Discussion

This study showed that the genetic disruption of CTRP3 shifts cADSC tenogenic differentiation toward a more tendon-like extracellular matrix (ECM) marker profile [37], rather than uniformly increasing early transcriptional commitment. The differences associated with CTRP3 disruption were more evident at the level of late ECM markers than at early transcriptional activation, suggesting that matrix-associated changes are a prominent feature of this phenotype. Although CTRP3-disrupted cADSCs showed stage-dependent changes in early tenogenic regulators, the most consistent differences were observed in late tendon-associated markers, including increased COL I and TNMD expression at the protein level and reduced COL III accumulation at week 2. These findings are consistent with CTRP3 disruption favouring a more tendon-like ECM marker profile, although the downstream mechanisms remain to be defined.

Notably, for the late tendon-associated markers COL I and TNMD, the effect of CTRP3 disruption differed between the transcript and protein levels in a temporally coherent manner. At the mRNA level, CTRP3-disrupted cADSCs showed significantly higher COL I and TNMD expression than WT cADSCs at early time points (weeks 0–1), and these differences diminished to non-significance by weeks 2–3 (Fig. 4). At the protein level, by contrast, the corresponding differences were significant and sustained throughout the 3-week induction period (Fig. 7). This temporal offset—an earlier, transient transcriptional difference followed by a sustained protein-level difference—is consistent with the post-transcriptional regulation and progressive extracellular accumulation that characterize matrix proteins such as collagen, in which transcript abundance may plateau or decline while the encoded protein continues to accumulate. We therefore interpret the CTRP3-disruption phenotype for these late ECM markers primarily from the protein-level and time-course data, and regard the earlier mRNA changes as an initial transcriptional phase rather than a discrepancy between datasets.

Early transcription factors Scx and Mkx are established regulators of tendon lineage commitment and are robustly induced in both WT and CTRP3-disrupted cADSCs during early differentiation [8], [10]. The induction of these genes confirmed that the culture conditions effectively activated the tenogenic program [38]. Mkx expression tended to be higher in CTRP3-disrupted cADSCs at baseline (week 0), whereas Scx was higher in CTRP3-disrupted than in WT cADSCs at several time points rather than specifically at the later stages, thus suggesting a stage-dependent pattern of early tenogenic marker expression [8], [9], [10], [39]. However, these differences were not consistently sustained across the time points, indicating that CTRP3 disruption did not primarily enhance early transcriptional commitment.

CTRP3 disruption was associated with a marked reduction in COL III expression, which is typically enriched in early repair tissue and scar-like tendon and is linked to inferior mechanical performance compared with COL I–dominant tendon tissue [16], [17], [18], [40], [41], [42]. Together with the progressive increase in COL I and TNMD, these findings indicate a shift in marker expression toward a more tendon-like, COL I–dominant ECM profile; however, the structural organization and mechanical maturation of the matrix were not directly assessed.

CTRP3 has been implicated in fibrotic matrix remodeling in other tissues through the activation of PI3K/Akt and MAPK pathways [22], [23], [24], [25]. These findings raise the hypothesis that CTRP3 may influence tendon-associated ECM marker expression; however, the relevant signaling mechanisms and structural consequences remain to be determined. Although the underlying signaling mechanisms were not directly investigated in the present study, the observed expression patterns were consistent with previous reports linking the modulation of signaling activity to reduced fibrotic remodeling and improved matrix organization [16], [17], [18], [23], [43]. Collectively, these findings suggest that CTRP3 may influence tendon-associated ECM marker expression, although its precise regulatory role remains to be established. Its suppression in vitro was associated with features of a COL I–dominant ECM marker profile and a shift away from fibroticlike remodeling; however, whether CTRP3 exerts distinct effects under pathological versus regenerative conditions remains to be determined.

An important consideration is the nature of the CTRP3 disruption achieved in this study. The CRISPR/Cas9-mediated loss-of-function approach enabled the functional evaluation of CTRP3 in cADSC tenogenesis. Rather than generating single cell–derived knockout clones, CTRP3-disrupted cADSCs were analyzed as a pooled edited population with high but incomplete editing efficiency. Accordingly, the observed phenotype likely reflects the collective behavior of cells with varying degrees of CTRP3 reduction rather than a uniform knockout state. Nevertheless, the consistent decrease in CTRP3 expression at both mRNA and protein levels, together with reproducible changes in ECM markers, is consistent with a possible role for CTRP3 in modulating tendon-associated ECM marker expression.

This interpretation is particularly relevant because CTRP3 is a secreted adipokine rather than a lineage-defining transcription factor. Therefore, its disruption may influence tenogenic differentiation less through the direct initiation of tendon lineage programs and more through the alteration of the extracellular signaling milieu that governs matrix remodeling and maturation. These findings suggest that CTRP3 may act as an intrinsic factor associated with tendon-associated ECM marker expression during ADSC tenogenesis, although its mechanistic role was not directly examined. Studies have shown that secreted factors regulate stem cell differentiation by modulating the extracellular microenvironment rather than through direct transcriptional control [20], [21]. Notably, the fact that partial genetic disruption was sufficient to induce measurable changes in COL I, TNMD, and COL III expression raises the possibility that a reduction in CTRP3 level, rather than its complete loss, is sufficient to influence matrix remodeling; however, because editing efficiency and dose–response were not directly quantified, this dosage-sensitivity remains to be tested. Similar dose-dependent effects have been reported in stem cell systems, where a partial reduction in signaling components can lead to measurable changes in differentiation outcomes without complete loss of function [23].

However, CRISPR/Cas9-mediated editing may introduce off-target effects or trigger compensatory cellular responses. Rescue experiments to restore CTRP3 expression have not yet been performed. Although the convergence of CTRP3-disrupted cADSCs toward a TDSC-like profile supports the role of CTRP3 loss-of-function, the precise causal and pathway-level mechanisms remain to be established.

This study has a few limitations that should be considered. First, the findings were derived from an in vitro differentiation model, and the effects of CTRP3 disruption on tendon repair were not evaluated in vivo. Future studies using tendon injury models will be required to determine whether CTRP3-modified cADSCs improve matrix organization, biomechanical properties, and functional recovery. Second, the downstream signaling pathways have not been directly investigated and warrant further study. Third, the matrix ultrastructure and biomechanical characteristics were not assessed, and these will be important for determining whether the observed molecular changes translate into functionally meaningful tendon regeneration [44], [45], [46].

Despite these limitations, this study provides significant new insights into the context-dependent function of CTRP3 in tendon lineage regulation [8], [19], [21]. Consistent with the pathological role of CTRP3 reported in tendon [26], [47], and in contrast to its protective, anti-fibrotic actions in metabolic and cardiovascular tissues [25], our CRISPR/Cas9-based loss-of-function analysis of ADSCs suggests that CTRP3 suppression is associated with a shift of the ECM marker profile toward a more tendon-like, COL I–dominant composition and with a reduction of the fibrotic COL III signature [15], [16]. Importantly, these findings indicate that CTRP3 disruption was more consistently associated with changes in late ECM markers than with changes in early transcriptional markers during tenogenic differentiation. This may represent an intrinsic regulatory process distinct from traditional extrinsic induction strategies that rely on growth factor supplementation (e.g., TGF-β3, GDF-7) [12], [13], [48]. Because CTRP3 is an endogenous, secreted regulator, its modulation may offer a complementary intrinsic strategy to influence the tendon-associated ECM marker profile of ADSCs, potentially reducing reliance on exogenous growth factors in stem cell–based tendon regeneration for veterinary applications.

Modulating such internal molecular restraints is an alternative approach to modifying the tendon-associated ECM marker profile of ADSCs for veterinary regenerative applications. From a translational perspective, cTDSCs are often used as a reference cell type for tenogenic potential [49] but are limited by donor-site morbidity and tissue availability [6], [7]. By contrast, cADSCs offer practical advantages in terms of accessibility and expandability but often fail to achieve coordinated tenogenic maturation [50], [51]. The present findings indicate that modulating intrinsic regulators such as CTRP3 can shift the tendon-associated ECM marker profile during cADSC tenogenic induction without requiring tendon harvesting. The partial overlap of CTRP3-disrupted cADSCs with cTDSCs in selected late ECM markers provides a rationale for future studies aimed at improving stem cell–based tendon regeneration strategies.

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Conclusion

The genetic disruption of CTRP3 shifted cADSC tenogenic differentiation toward a more tendon-like ECM marker profile, rather than uniformly increasing early transcriptional activation. CTRP3-disrupted cADSCs showed increased COL I and TNMD expression at the protein level, reduced COL III accumulation, and partial, marker- and time-point-dependent overlap with native cTDSCs for selected late ECM markers. These findings support further investigation of CTRP3 as a potential modulator of tendon-associated ECM marker expression during cADSC tenogenesis.

Ethics approval and consent to participate

All animal procedures were reviewed and approved by the IACUC of Seoul National University (approval no. SNU-220428-3). The tissue samples used in this study were obtained from dogs euthanized as part of a separate IACUC-approved project (IACUC approval no. SNU-200709-4-5), and no animals were sacrificed for the purpose of this research. No additional institutional permission or owner consent was required for sample collection, in accordance with institutional policies and national regulations. All experimental procedures adhered to the institutional and national ethical standards for animal use in scientific studies.

Consent for publication

This manuscript contains no data related to identifiable individuals.

Authors’ contributions

SHK and JHL contributed equally to this study SHK conceived and designed the study and performed the tenogenic differentiation experiments. JHL designed and conducted the CRISPR/Cas9-mediated gene editing experiments and performed editing validation analyses. SHK and JHL analyzed the data and drafted and revised the manuscript. GJ supervised the study and critically revised the manuscript. BJK provided oversight for the study, contributed to the interpretation of the results, and critically revised the manuscript. All authors reviewed and approved the final version of the manuscript.

Ethics declaration

This study was conducted in accordance with the following guidelines for animal welfare and/or reporting: Seoul National University Institutional Animal Care and Use Committee (IACUC) guidelines and applicable national regulations of the Republic of Korea. This study was approved by the Institutional Animal Care and Use Committee of Seoul National University. (Approval No. SNU-220428-3; SNU-200709-4-5)

Declaration of generative AI and AI-assisted technologies in the manuscript preparation process

During the preparation of this work, the authors used ChatGPT (OpenAI) to assist with language editing and improving the clarity of the manuscript. The authors reviewed and edited the output as needed and take full responsibility for the content of the published article.

Funding

This research was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (grant no. 2023R1A2C1003001). The funding organization was not involved in the design of the study; the acquisition, analysis, and interpretation of data; or the writing of the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

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Contributor Information

Goo Jang, Email: snujang@snu.ac.kr.

Byung-Jae Kang, Email: bjkang81@snu.ac.kr.

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Supplementary data

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References

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