A Sendai virus-based expression system directs efficient induction of chondrocytes by transcription factor-mediated reprogramming

In this study, we developed an SeV vector that directly induces chondrocytes from MEFs by simultaneously expressing three reprogramming factors (SOX9_H131A/K398A, KLF4, and c-MYC) and showed that the newly developed SeV vector was superior to our previous RV vector in inducing chondrocytes. First, the SeV-based vector induced chondrocytes more efficiently than the RV vectors (Figs. 1 and 2), which is consistent with the high efficiency of gene delivery and robust expression of reprogramming factors by SeV-based vectors reported for reprogramming somatic cells into iPSCs¹¹ and fibroblasts directly into cardiomyocytes^11,12. The high expression levels of reprogramming factors from SeV vectors are possibly due to their high copy number (10⁴–10⁵ copies per cell)¹³ compared to only ~ 10 copies per cell of RVs¹⁵. Second, the SeV vector achieved more immediate expression of reprogramming factors than our previous RV vectors. This is presumably because SeVs possess an RNA-dependent RNA polymerase that instantaneously initiates transcription and replication in the cytoplasm¹⁶. In contrast, RVs undergo reverse transcription and integration into the host genome⁷, where viral gene expression typically occurs after host cell division¹⁷, which is entirely dependent on the cellular transcriptional machinery. Third, the SeV vector showed a relatively uniform expression of transgenes¹¹, which ensured the production of a more homogenous population of induced chondrocytes. SeV vectors incorporate multiple exogenous genes into a single vector, from which the genes are expressed in a relatively constant stoichiometry. In addition, the levels of transgene expression among the infected cells varied to a lesser extent than those expressed by the RV vectors, for which the expression varied widely with the integrated genomic sites. Overall, the newly developed SeV vector enables the efficient induction of a more homogenous population of chondrocytes in a shorter period of cell culture and is therefore better suited for the scalable production of chondrocytes.

The presence of integrated transgenes in the genome is an obvious cause of concern when transcription factor-induced chondrocytes are used in regenerative medicine, largely because of their potential to form tumors after transplantation. This is particularly true when the reprogramming system requires c-Myc and Klf4, which act as oncogenes^18,19. Indeed, chondrocytes induced by RV vector-directed reprogramming have been reported to generate tumors after transplantation³. Considering that SeV is a negative-sense single-stranded RNA virus (NSRV), it is generally believed that SeV does not integrate into the host genome. However, some reports have suggested that RNA viruses that do not generate DNA intermediates for replication can accidentally integrate into the host genome^20,21,22. As shown in Fig. 4c, we did not observe integration of the current SeV vector into the genome of induced chondrocytes, at least during the period required for cell culture, indicating that the current SeV vector poses little concern for integration into the host genome.

Despite these substantial advantages of SeV vectors over RV vectors, certain limitations must be overcome to further improve the transcription factor-mediated induction of chondrocytes. As shown in Fig. 5b, some chondrocytes appeared enlarged, possibly indicating hypertrophic changes due to differentiation via the endochondral ossification pathway. Articular chondrocytes in osteoarthritic lesions often display a hypertrophic phenotype that is believed to play a causative role in OA progression. Although gene expression analyses indicated that SeV-induced chondrocytes did not display any evident gene expression signature of hypertrophic chondrocytes, they may easily respond to external cues to become hypertrophic. If these chondrocytes are implanted into an osteoarthritic lesion, they can exacerbate rather than ameliorate OA. To prevent this untoward hypertrophic differentiation, an SeV vector carrying a dominant negative form of a transcription factor (such as Runx or Osterix) that plays crucial roles in chondrocyte hypertrophy and subsequent differentiation into osteocytes may be helpful^23,24.

Another limitation of current SeV vectors is their immunogenicity. Although the SeV vector used in this study lacks envelope-related genes and largely escapes the innate immune response, it still elicits an acquired immune response²⁵ that could eliminate transplanted cells in vivo. For instance, in immunodeficient mice, transplanted SeV-infected cells remain intact for at least 60 days; however, they disappear after only a few days in immunocompetent mice, even when immunosuppressants are administered²⁶. Therefore, removal of the SeV vector from induced chondrocytes before implantation may be beneficial for the long-term preservation of implanted cells. Possible methods for removing SeV vectors from reprogrammed cells include the use of a vector backbone derived from temperature-sensitive mutations²⁷, treatment of reprogrammed cells with an siRNA targeting the SeV L gene¹¹, inclusion of a target sequence of cell-type-specific miRNAs,²⁸ or installation of a Csy4 endoribonuclease-based system in the vector^29,30. The incorporation of these technologies into the current vector may lead to the development of an SeV vector that generates chondrocytes free of the SeV vector initially used to induce them.

In summary, the SeV vector-based direct reprogramming system reported in this study not only represents a considerable advancement in generating induced chondrocytes for cell therapy but also provides a solid foundation for further improvements in the scalable production of vector-free chondrocytes.

Methods

Preparation of viral vectors

The recombinant DNA experiments performed in this study were approved by the Recombinant DNA Experiment Committee of the University of Tsukuba (approval numbers: 190121, 210260, and 220144). Complementary DNAs encoding human SOX9_H131A/K398A, KLF4, c-MYC, and blasticidin resistance genes were inserted into the SeVdp vector¹¹ (Fig. 1a). Using the SeVdp vector and expression vectors for SeV proteins, a virus-containing culture medium was prepared as described previously¹¹, except that NIH3T3 cells were used to determine the titer. The retroviral vectors were prepared as described previously⁵.

Induction of chondrocytes using the SeV-based direct reprogramming system

MEFs were prepared from C57BL/6J mouse embryos as described previously³¹. MEFs were cultured at 37℃ in an atmosphere of 5% O₂ and 5% CO₂ in DMEM containing 10% (v/v) fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin (hereafter referred to as standard medium). The MEFs were seeded in a 24-well plate at a density of 4 × 10⁴ cells/well and were infected with SeV on the next day at a multiplicity of infection (MOI) of approximately 1.0 for 24 h at 32℃ in an atmosphere of 5% CO₂. The virus-infected cells were then cultured at 37℃ in an atmosphere of 5% O₂ and 5% CO₂ for 24 h. The uninfected cells were removed by culturing in standard medium containing 8 µg/mL blasticidin for 3 d. The surviving cells were further cultured at 37℃ in an atmosphere of 5% O₂ and 5% CO₂ in standard medium without blasticidin, and the medium was changed every 2 d. The chondrogenic medium was DMEM containing 1% (v/v) fetal bovine serum, 10 ng/mL GDF5 (Biolegend, San Diego, CA, USA; 779506), 10 ng/mL TGF-β1 (Biolegend, 580702), 50 µg/mL L-ascorbic acid 2-phosphate (Sigma, St Louis, MO, USA; A8960), 0.1 µM dexamethasone (Nacalai Tesque, Inc., Tokyo, Japan; 11107-64), 1 mM sodium pyruvate (Nacalai Tesque, Inc.; 06977-34), and ITS (Thermo Fisher Scientific Inc., Waltham, MA, USA; 41400045). Phase-contrast images of the cells were acquired using a Nikon ECLIPS TS100 microscope.

Preparation of stromal vascular fraction (SVF) cells derived from inguinal white adipose tissue (iWAT) and mouse dermal fibroblasts (MDFs)

iWAT was isolated from a 5-month-old female mouse and was minced. The minced iWAT was dispersed using collagenase (Roche, ~ 1.5 units/g tissue) and dispase II (Roche, ~ 2.4 units/g tissue) in phosphate buffered saline (PBS) containing 10 mM CaCl₂ (~ 1 ml/g tissue) at 37℃ for 1 h. The dispersed cells were centrifuged at 700 × g for 10 min, and the pellet was isolated as SVF cells. The SVF cells suspended in standard medium were filtered through a 70 μm strainer and cultured at 37℃ in an atmosphere of 20% O₂ and 5% CO₂. To isolate MDFs, the hair around the chest of the same female mouse was shaved, and the exposed skin was removed and minced. The skin tissue was dispersed using collagenase and dispase II, and the dispersed cells were filtered and cultured as described for the SVF cells. Chondrocytes were induced using the same method as used for MEFs, except that the cells were cultured in the presence of 20% O₂ instead of 5% O₂.

Alcian blue staining

The induced chondrocytes were washed with PBS and fixed in methanol at room temperature for 2 min. After removing methanol, the cells were washed using 0.1 M HCl and stained with Alcian blue staining solution (pH 2.5, Nacalai Tesque, Inc.) at room temperature for approximately 60 min. The cells were washed with 0.1 M HCl.

Immunofluorescence staining

The virus-infected cells were washed with PBS and fixed using 10% formalin (v/v) at room temperature for 10 min. Then, the cells were washed with PBS, treated with 50 mM NH₄Cl at room temperature for 10 min, treated with 0.1% (w/v) Triton X-100 at room temperature for 5 min, and washed with PBS. The cells were then incubated with the primary antibody in 0.1% (w/v) saponin/PBS at room temperature for 60 min. The cells were washed with PBS and incubated with fluorescent-labeled secondary antibodies in 0.1% (w/v) saponin/PBS at room temperature for 60 min. After washing with 0.1% (w/v) saponin/PBS, the cells were treated with one drop of VECTASHIELD containing DAPI (Nacalai Tesque, Inc.). Fluorescent images were acquired using an All-in-One Fluorescence Microscope BZ-710 (Keyence corp.). The antibodies and dilutions used for immunofluorescence staining are listed in Supplementary Table S1.

Immunoblotting

MEFs were seeded in 60 mm plate at a density of 4 × 10⁵ cells/plate and infected with RVs or SeV. The virus-infected cells were collected 2, 5, 10, and 15 days after virus infection and suspended in radioimmunoprecipitation assay buffer containing protease inhibitor cocktail, 1 µM MG132, and 0.5 mM phenylmethylsulfonyl fluoride (approximately 5 × 10⁵ cells/50 µL). After brief sonication using Bioruptor II (30 s ON-30 s OFF, two cycles, power: high; BM Equipment Co., Ltd., Tokyo, Japan), the insoluble fraction was removed via centrifugation at 20,000 × g at 4℃ for 5 min. The cell extracts (approximately 28 µg protein each) were used for immunoblotting with an anti-DYKDDDDK (Flag-SOX9) antibody (1:1,500 dilution). Chemiluminescent images were acquired using a Fusion FX7. EDGE (M&S Instruments Inc.) and quantification were performed using the Evolution Capt software, and the original images of the blots are shown in Supplementary Fig. S1.

Reverse transcription and quantitative PCR (RT-qPCR)

Total RNA was extracted from the induced chondrocytes and RT-qPCR was performed as described previously⁵. The primer sets used for qPCR are listed in Supplementary Table S2. P-values were calculated using the Student’s t-test. To quantify the SeV genomic RNA, total RNA was purified from virus-infected cells and used for RT-qPCR. The amount of SeV genomic RNA was calculated based on a standard curve constructed using the plasmid DNA vector used for the SeV preparation.

Genomic PCR

Chondrocytes were induced by RV or SeV in a 12-well plate (approximately 10⁵ cells/well) for 10 d and lysed using 100 µL of lysis buffer (50 mM Tris-HCl, pH 7.5, 1% SDS, 20 mM EDTA, 0.1 M NaCl) containing 50 µg/mL RNase A at 37℃ for 15 min. Proteinase K (2 µg) was added and the cells were incubated at 55℃ for 5 h. Genomic DNA was extracted using 100 µL of phenol/chloroform and precipitated by adding 100 µL of 2-propanol. After centrifugation at 15,500 × g at 4℃ for 7 min, the pellet was rinsed with 70% ethanol. The pellet was dissolved in 10 µL of TE (pH 8.0). For genomic PCR, 10 ng of genomic DNA or 1 ng of the plasmid vector was used as the template. To detect the virus vector-derived sequence (Flag-tagged Sox9m-coding gene), PCR was performed using KOD-plus-Neo (TOYOBO) with a two-step cycle (94℃, 2 min > [98℃, 10 s; 68℃, 30 s] × 35 cycles > 4℃). To detect Gapdh, PCR was performed using a three-step cycle (94℃, 2 min > [98℃, 10 s; 58℃, 30 s; 68℃, 20 s] × 35 cycles > 4℃). The primers used for PCR are listed in Supplementary Table S3. The PCR products were analyzed by electrophoresing on a 1% agarose gel. Original images of the gels are shown in Supplementary Fig. S2.

Transplantation of SeV-induced chondrocytes in mice

The animal experiments performed in this study were approved by the Animal Experimental Committee of the University of Tsukuba (approval numbers: 22–488 and 23–312). All experiments were performed in accordance with the relevant guidelines, regulations, and ARRIVE guidelines. MEFs were infected with SeV at an MOI approximately 1.0 in 100 mm plates, and uninfected cells were removed by treating with blasticidin (8 µg/mL) for 3 d. The virus-infected cells were cultured without blasticidin for 1 d and transplanted subcutaneously into the backs of immunodeficient mice (BALB/cAJc1-nu/nu, 6 weeks old, female). The mice were euthanized via cervical dislocation under isoflurane anesthesia 3 weeks after transplantation and the grafts were isolated. Paraffin sections were prepared from the grafts and stained using Alcian Blue. Immunofluorescence staining of paraffin-embedded sections was performed according to standard protocols using anti-COL2A1 or anti-COL1A1 antibody (Santa Cruz Biotechnology). Total RNA was extracted from the grafts and gene expression was analyzed using RT-qPCR.

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• Source: https://www.nature.com/articles/s41598-024-77508-1