
==== Front
Mol Ther
Mol Ther
Molecular Therapy
1525-0016
1525-0024
American Society of Gene & Cell Therapy

S1525-0016(24)00400-3
10.1016/j.ymthe.2024.06.018
Original Article
Dedifferentiation-like reprogramming of degenerative nucleus pulposus cells into notochordal-like cells by defined factors
Zhang Yuang 125
Liang Chengzhen 1235
Xu Haibin 125
Li Yi 12
Xia Kaishun 12
Wang Liyin 4
Huang Xianpeng 12
Chen Jiangjie 12
Shu Jiawei 12
Cheng Feng 12
Shi Kesi 12
Wang Jingkai 123
Tao Yiqing 123
Wang Shaoke 12
Zhang Yongxiang 12
Li Hao 12
Feng Shoumin 12
Li Fangcai lifangcai@zju.edu.cn
123∗
Zhou Xiaopeng doctorzxp@zju.edu.cn
123∗∗
Chen Qixin zrcqx@zju.edu.cn
123∗∗∗
1 Department of Orthopedic Surgery, the Second Affiliated Hospital, Zhejiang University School of Medicine; Hangzhou City, Zhejiang Province 310009, China
2 Orthopedics Research Institute of Zhejiang University; Hangzhou City, Zhejiang Province 310009, China
3 Key Laboratory of Motor System Disease Research and Precision Therapy of Zhejiang Province; Hangzhou City, Zhejiang Province 310009, China
4 Department of Ophthalmology and Vision Science, Eye and ENT Hospital, Fudan University; Shanghai 200031, China
∗ Corresponding author: Fangcai Li, Department of Orthopedic Surgery, the Second Affiliated Hospital, Zhejiang University School of Medicine; Hangzhou City, Zhejiang Province 310009, China. lifangcai@zju.edu.cn
∗∗ Corresponding author: Xiaopeng Zhou, Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou City, Zhejiang Province 310009, China. doctorzxp@zju.edu.cn
∗∗∗ Corresponding author: Qixin Chen, Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou City, Zhejiang Province 310009, China. zrcqx@zju.edu.cn
5 These authors contributed equally

15 6 2024
07 8 2024
15 6 2024
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© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The extensive degeneration of functional somatic cells and the depletion of endogenous stem/progenitor populations present significant challenges to tissue regeneration in degenerative diseases. Currently, a cellular reprogramming approach enabling directly generating corresponding progenitor populations from degenerative somatic cells remains elusive. The present study focused on intervertebral disc degeneration (IVDD) and identified a three-factor combination (OCT4, FOXA2, TBXT [OFT]) that could induce the dedifferentiation-like reprogramming of degenerative nucleus pulposus cells (dNPCs) toward induced notochordal-like cells (iNCs). Single-cell transcriptomics dissected the transitions of cell identity during reprogramming. Further, OCT4 was found to directly interact with bromodomain PHD-finger transcription factor to remodel the chromatin during the early phases, which was crucial for initiating this dedifferentiation-like reprogramming. In rat models, intradiscal injection of adeno-associated virus carrying OFT generated iNCs from in situ dNPCs and reversed IVDD. These results collectively present a proof-of-concept for dedifferentiation-like reprogramming of degenerated somatic cells into corresponding progenitors through the development of a factor-based strategy, providing a promising approach for regeneration in degenerative disc diseases.

Graphical abstract

Chen and colleagues present a novel reprogramming strategy that directly reversed dNPCs into corresponding progenitor cells, successfully alleviating IVDD in a rat model. Their dissection of critical chromatin dynamics underlying the reprogramming process provides new insights into degenerative tissue repair.

Keywords

degenerative disease
tissue regeneration
reprogramming
intervertebral disc degeneration
dedifferentiation
transcriptional factor
chromatin remodeling
nucleus pulposus cells
notochordal-like cells
==== Body
pmcIntroduction

Degenerative diseases can indeed pose significant challenges to tissue renewal and regeneration due to the absence of stem and progenitor cell pool coupled with somatic cell dysfunction.1,2 Restoring in situ stem and progenitor populations could enable robust cell repopulation in degenerated tissues. Recent advancements in direct reprogramming strategies have enabled the transformation of adult cells into tissue-specific stem cells with remarkable regenerative capabilities.3 However, these strategies predominantly require initial cells with relatively high plasticity.4,5 In contrast, numerous degenerative tissues lack suitable initial cell sources, where originally functional somatic cells continue to exist, but undergo extensive degeneration.2,6 While several reprogramming approaches are feasible in normal somatic cells, they exhibit limited efficacy in degenerative somatic cells.7,8 Thus, there is a pressing need to pioneer an approach that can directly induce corresponding progenitor cells from these degenerative somatic cells.

Based on the natural dedifferentiation process, several reprogramming strategies have primarily reversed normal terminally committed cells into progenitor populations by reactivating progenitor-specific gene networks.4,9,10 However, unlike the dedifferentiation of normal terminally committed cells, recent research has identified multiple adverse modulators in degenerative somatic cells that may impede progenitor-specific gene activation and fate conversion.11,12 Overcoming the reprogramming barriers underlying degenerative cellular status might be crucial for dedifferentiation-like reprogramming toward progenitor cells. Recently, the transient expression of the Yamanaka factors, namely OCT4, SOX2, KLF4, and c-MYC (OSKM), effectively reversed adult cells from degenerative states into normal states, highlighting the pivotal role of factor-mediated disruption of epigenetic imprints in resetting the degenerative status.13,14,15 Nevertheless, further dedifferentiation into stem/progenitor states by extending the periods of OSKM induction has proven uncontrollable and conducive to teratoma formation.16 Hence, there is a need to develop a novel factor-based strategy that can overcome the epigenetic restrictions underlying degenerative states and concomitantly reactivate progenitor-specific gene networks. This strategy could potentially directly achieve tissue-specific dedifferentiation from degenerative somatic cells into their corresponding progenitor cells.

Intervertebral disc degeneration (IVDD), standing as a well-characterized example of degenerative diseases, is elicited by nucleus pulposus (NP) dysfunction. During IVD development, notochordal cells (NCs) play a dominant role in forming NP tissue and maintaining local homeoestasis. Recent fate-mapping studies have unveiled distinct cellular alterations within the NP region during IVDD, wherein NCs, the precursor cells of NP cells (NPCs), irreversibly degenerate into degenerative NPCs (dNPCs) and lose their self-renewal capacity.17 Terminally differentiated dNPCs exhibit extensive degenerative features, posing challenges in reprogramming, such as reduced proliferation, inflammatory phenotypes, and epigenetic alterations.7,18,19,20,21 Additionally, the lack of suitable substitute cells for in vivo reprogramming necessitates current reprogramming therapies to rely on exogenous transplantation.22,23 While transplantation-independent studies targeting dNPCs aim to ameliorate their degenerative phenotypes, achieving robust cell repopulation remains elusive.13 Thus, this study aimed to develop a novel factor-based strategy to overcome these challenges by reprogramming dNPCs into induced notochordal-like cells (iNCs), providing a promising therapeutic strategy for IVDD treatment.

Herein, a unique factor combination composed of notochord-specific factors and the Yamanaka factor was identified. Notably, this combination achieved the dedifferentiation-like reprogramming of dNPCs into iNCs, thereby restoring typical notochordal-like molecular features and functional phenotypes. Moreover, transcriptomic changes during this reprogramming process were detected through comprehensive transcriptome analyses. Further analysis revealed that OCT4 directly interacted with the nucleosome remodeling factor, bromodomain PHD-finger transcription factor (BPTF), to remodel the chromatin during the early phases, which was critical for initiating this dedifferentiation-like reprogramming. Furthermore, overexpressing selected reprogramming factors successfully generated iNCs from in situ dNPCs in puncture-induced IVDD models. Our findings illustrate the feasibility of dedifferentiation-like reprogramming of degenerative somatic cells by adopting an appropriate factor-based strategy, providing new insights into degenerative disc repair.

Results

Identifying notochord development-specific factors regulating human NC degeneration as basic reprogramming factors

Initially, we sequenced the mRNA of embryonic NCs from embryonic notochord, nNPCs from normal NP samples, and dNPCs from degenerated NP samples to dissect cellular differences (Figure 1A). Compared with NCs and nNPCs, the gene sets associated with notochordal development, cell proliferation and transcription activation were progressively downregulated, while extracellular matrix (ECM) disassembly related genes were upregulated in dNPCs (Figure 1A). Further Gene Ontology (GO) enrichment analyses also revealed that dNPCs exhibited inflammatory phenotypes under multiple stimuli, while NCs high expressed genes for cell growth and tissue homeostasis (Figure 1B). These transcriptome signatures suggested the pathological cellular status of dNPCs. Furthermore, we validated the differences of cellular phenotypes through histological analysis. We collected both normal NP specimens (Pfirrmann grades of disc degeneration I and II) and degenerated NP specimens (Pfirrmann grades of disc degeneration IV and V) (Figures S1A and S1B; Table S1). Consistent with sequencing results, vacuolar NC residues highly expressed notochordal markers KRT8, SHH, and gelatinous ECM component COL2 in normal specimens, while fibroblastic dNPCs highly expressed COL1 and MMP3 in degenerated NP specimens from both young and old patients (Figures 1C–1E). These NC residues secreted abundant gel-like matrix, significantly more than the fibroblastic dNPCs (Figure S1C). The replacement of NCs by dNPCs in human NP results in a notable dysregulation of local ECM composition, which was associated with IVDD.Figure 1 Identifying basic reprogramming factors from human NC to dNPC degeneration

(A) The expressions of gene sets including NC development, ECM disassembly, cell proliferation, and transcription-activation using heatmap among NCs, nNPCs, and dNPCs. (B) The differential analysis (left) and GO analysis (middle and right) of NCs and dNPCs. (C) The comparison of KRT8, SHH, COL2, COL1, and MMP3 expressions in NCs and dNPCs, n = 3. (D and E) Immunofluorescence staining (D) and box plot of the normalized values (E) for KRT8, SHH, COL2, COL1, and MMP3 in three distinct groups. Scale bar, 250 μm. n = 4 (F) GSEA analysis of human NCs and dNPCs. (G) The PPI network shows hub genes for primitive streak formation and notochord development (bottom), and the top 10 genes identified using maximum neighborhood component method (top). (H) The expression of FT in NCs (n = 6) and dNPCs (n = 7). (I) Protein expression levels (left) and the quantitative result (right) of FT in NCs and dNPCs. (J) Immunofluorescence in NCs after knockdown of FT for dNPCs marker MMP3 and COL1, notochordal marker KRT8 and SHH, and NP functional marker COL2 and ADAMTS5. Scale bar, 100 μm. n = 3. Data are expressed as mean ± SD. Statistics were compared using Student’s unpaired t test (C, H, and I) and one-way ANOVA (E).

Previous studies have achieved the dedifferentiation of terminally committed cells into progenitor populations by overexpressing factors for tissue pattern formation and reactivating gene regulatory networks, without undergoing a pluripotent state.9,10 We performed Gene Set Enrichment Analysis (GSEA) and found large downregulated genes in dNPCs were associated with primitive streak formation and notochord development (Figure 1F). To identify hub genes for notochordal tissue formation, a protein-protein interaction network analysis was conducted on genes enriched in these processes using the CytoScape software (Figure 1G). Among the top 10 genes identified in the network using the MNC method, the well-known transcription factors FOXA2 and TBXT (FT) showed significant downregulations in dNPCs (Table S2), which have been reported as pivotal regulators of the notochord gene regulatory network that promote notochord development and formation. Their misexpression in other cell types has been independently shown to activate notochordal-like transcriptional profiles and induce notochordal fate. Recent studies have exposed that FOXA224,25 and TBXT24,26,27 act synergistically to govern a regulatory network, driving notochord fate specification in Ciona and mouse compared with individual action or combined use with other factors, raising the possibility that their ectopic co-expression in human somatic cells might induce NCs conversion. Therefore, notochordal genes FT were identified as potential hub genes to restate notochordal expression patterns in dNPCs. We sequenced more samples to validate their differential expressions, and also detected their expressions at the protein level (Figures 1H and 1I). To further verify their function, we knocked them down in primary NCs. A significant degeneration toward dNPCs was observed, especially after the knockdown of both FT (Figures 1J, S1D, and S1E). Therefore, the notochord-specific factors FT might be essential for maintaining human NC phenotypes and were considered the potential reprogramming factors.

Adding OCT4 to reprogramming factor combinations is essential for dNPCs to iNCs conversion

To avoid the disturbance of resident stem/progenitor cells in obtained dNPCs, fluorescence-activated cell sorting (FACS) was performed to purify dNPCs as CD90−TIE2− cells to eliminate mesenchymal stem cells (MSCs) and NP progenitor cells (NPPCs) (Figures S2A and 2A). Purified dNPCs exhibited typical fibroblastic phenotypes while no NCs markers were positive, confirming their homogeneous identity as initial cells (Figures S2B and S2C). Then, to determine whether FT could convert dNPCs into iNCs, dNPCs were introduced with FT using lentiviral vectors and cultured in NP precursor-conditioned medium for dedifferentiation-like phenotypic maintenance.28 The conversion was preliminarily assessed by the expression of early notochordal markers KRT8 and NOTO and degenerative phenotypic indicators COL1A1 and COL2A1. While FT co-overexpression limitedly elicited KRT8, NOTO, and COL2A1 expressions and slightly downregulated COL1A1 expression compared with single-factor or vehicle transfection, the improvements were still modest compared with primary NCs (Figures 2B and 2C). Hence, FT overexpression moderately improved the phenotype of human dNPCs, but was insufficient to induce dedifferentiation-like reprogramming.Figure 2 A combination of OFT promotes dNPC to iNC conversion

(A) Schematic diagram of transcriptional factor screening and reprogramming process. (B) All potential factor combinations for dNPC to iNC conversion. (C) The KRT8, NOTO, COL1A1, and COL2A1 expressions of dNPCs treated with FT. n = 3. (D) The KRT8, NOTO, COL1A1, and COL2A1 expressions of dNPCs treated with different combinations of OCT4, c-MYC, KLF4, and basic reprogramming factors FT. n = 3. (E) The OCT4, FOXA2, and TBXT expression of OFT+ dNPCs after GFP/mCherry FACS sorting. Scale bar, 100 μm. (F and G) Morphological differences between iNCs (OFT treatment) and dNPCs (empty-vector treatment) were visualized under a bright field (F) and quantified using CellProfiler (G). Scale bar, 80 μm. (H) Immunofluorescence staining (left) and the quantitative result (right) of KI67+ cells in vehicle-transducted dNPCs and OFT-transducted dNPCs. Scale bar, 250 μm. n = 6. (I) Immunofluorescence staining of GFP-positive iNCs for the notochordal markers. Scale bar, 100 μm. Data are expressed as mean ± SD. Statistics were compared using one-way ANOVA (C and D) and Student’s unpaired t test (H).

Given the refractoriness of the dNPCs and the widespread decrease of chromatin accessibility during IVDD, we introduced Yamanaka factors OCT4 (O), c-MYC (M), and KLF4 (K), which have been documented to disrupt the epigenetic imprint to reverse degenerative status.13 These factors were also found to be involved in the conversion of somatic cells into progenitor cells in other cell types.29,30,31 We then introduced various combinations of these factors alongside the basic factors into human dNPCs (Figure 2B). Among all the three-factor combinations (OFT, MFT, KFT), OFT showed the most significant improvement in COL1A1, COL2A1, and notochordal gene expressions (Figure 2D). The four- or five-factor combinations were generally less effective than OFT (Figure 2D), which might be attributed to the dilution of each transfected construct and a decreased ratio of OFT+ cells with multiple-factor transfections. We also assessed the ratio of Ki67+ cells and NOTO+ cells among all factor combinations using immunofluorescence, and OFT also exhibited the most significant enhancement in these evaluations (Table S3). Consequently, the OFT was identified as the optimal combination to induce the dNPC to iNC conversion in subsequent experiments.

To further identify the dedifferentiation-like reprogramming, we transfected dNPCs with OCT4-GFP and FOXA2-TBXT-mCherry and isolated OFT+ cells using FACS (Figures 2E and S2D–S2F). Morphologically, large vesicles were observed in the cytoplasm of OFT+ cells, consistent with typical notochordal characteristics (Figures 2F and S3A). To accurately distinguish morphological changes, we performed a cellular morphological feature analysis, which showed that OFT+ cells transformed into a short shuttle or circular-like shape from irregular and elongated shuttle-like morphology, making them more closely resemble natural NCs (Figure 2G). Meanwhile, OFT transduction also significantly increased the KI67-positive cell populations compared with vehicle transduction, indicating that many OFT+ cells re-entered into proliferating states (Figure 2H). We examined more notochordal properties at 14 days. Immunofluorescence staining demonstrated that notochordal secretory markers SHH, CHRD, and NOG were positively expressed in OFT+ cells (Figure 2I). Importantly, OFT overexpression did not induce a significant increase in pluripotent marker NANOG, indicating that the dedifferentiation-like reprogramming would not induce induced pluripotent stem cell (iPSC) generation (Figure S3B). To assess the risk of tumorigenesis after OFT transduction, we cultured iNCs for 30 days and analyzed their karyotypes, revealing normal karyotypes for OFT+ cells (Figure S3C).

Overall, based on evaluations of morphological features and molecular characteristics, the exogenous overexpression of OCT4, in combination with FT, effectively dedifferentiated dNPCs into iNCs.

OFT partially improves dNPCs dysfunction and endows them with progenitor properties

To identify the reversal of degenerative status, we mainly evaluated the improvements in dysregulated ECM secretion. Compared with dNPCs, iNCs exhibited higher expressions of COL2 and ACAN but lower expressions of COL1, MMP3, and ADAMTS5 (Figures 3A, 3B, S4A, and S4B). Additionally, iNCs secreted abundant GAGs (Figure 3C). Although the expression of matrix-degrading enzymes MMP13 and ADAMTS4 was not reduced, the improvements in other markers indicated that iNCs significantly restored the anabolism for ECM generation and partially ameliorated the catabolism for ECM degradation.Figure 3 Characterization of iNCs as the NP progenitor

(A) The COL1, COL2, and MMP3 expressions of dNPCs and iNCs were assessed by immunocytochemical staining. Scale bar, 80 μm. (B) The expressions of ECM-related proteins among dNPCs, iNCs, and NCs. (C) The Alcian blue staining for three groups (dNPCs, iNCs, and NCs) was measured. Scale bar, 100 μm. (D) The nNPCs, dNPCs, and iNCs were stained with KRT19, GD2, TIE2, ACAN, and COL2 on D14 under three-dimensional pellet culture in NPC-differentiation medium. Scale bar, 200 μm. (E) Relative protein expressions of nNPCs, dNPCs, and iNCs in 14-day differentiation and normal culture measured by wester blot results. (F) OCT4, FOXA2, and TBXT expression of dNPCs after OFT overexpression and 14-day differentiation culture. (G) Kyoto Encyclopedia of Genes and Genomes pathway enrichment of genes in different comparation groups (iNCs vs dNPCs, iNCs vs nNPCs). (H) Immunofluorescence staining (left) and the quantitative result (right) for dNPCs (top chamber) co-cultured with iNCs/nNPCs/dNPCs/NCs (bottom chamber) for COL2, ACAN, MMP3, and F-actin. Scale bar, 100 μm. n = 3. Data are expressed as mean ± SD. Statistics were compared using one-way ANOVA (H).

Furthermore, we characterized the properties of iNCs as NPC progenitors. As NPC progenitors, NCs can differentiate into NPCs and maintain the NPC population.32 To assess the potential for NPC-like differentiation, we cultured iNCs in a three-dimensional differentiation culture system. NPC differentiation-related markers significantly elevated in iNCs. In contrast, these elevations were limited in dNPCs after 14 days (Figures 3D, 3E, and S4C). Meanwhile, early NC gene expressions declined during 14-day differentiation (Figure S4D). Interestingly, OFT expression was also significantly decreased (Figures 3F and S4E). The downregulation of OFT expression during differentiation might contribute to the NPC-like differentiation of iNCs.

Moreover, NCs play a crucial role in intercellular communication, influencing the surrounding cells to restore their anabolic phenotypes.33,34 Functional enrichment analysis of iNCs compared with dNPCs and nNPCs showed differentially expressed genes were actively involved in intercellular signal interactions and cell-cell communications (Figures 3G and S5A). These iNCs highly expressed secretory proteins (Figure S5B). We constructed a co-culture system to confirm whether iNCs could positively affect coexisting dNPCs (Figure S5C). After 7 days of co-culture, NCs and iNCs significantly improved the degenerative phenotypes of dNPCs, especially with respect to anabolic phenotypes (Figures 3H and S5D). Altogether, iNCs exhibited typical NC molecular characteristics and properties as NP progenitors, including gelatinous-ECM secretion, NPC-like differentiation, and active paracrine secretion.

Single-cell RNA sequencing analyses characterize cellular alterations during reprogramming

To further decipher the cellular alterations during dedifferentiation-like reprogramming, we captured initial dNPCs (D0, 3 071 cells) and reprogrammed cells (D14, 6 635 cells) after quality control and performed single-cell RNA sequencing (scRNA-seq) using the 10× Genomics platform. In dNPC samples, the mean reads and median genes per cell were 121,802 and 6,560 respectively. In iNC samples, the mean reads and median genes per cell were 50,175 and 4,392 respectively. Using t-distributed stochastic neighbor embedding analysis, we identified five distinctive clusters based on their characteristic gene signatures (C1 and C2 for dNPC samples and C3–C5 for reprogrammed samples) (Figures 4A and 4B).Figure 4 Single-cell transcriptome analysis comprehensively reveals the transition of cell fate underlying reprogramming

(A) t-Distributed stochastic neighbor embedding visualization of individual cells before (left) and after (right) reprogramming. (B) Stacked bar chart illustrating the distribution of cell sub-clusters in dNPCs and iNCs samples. (C) Visualization of corresponding marker genes for fcNPCs, imNPCs, hsNPCs, and NCs across all cells. (D) Dot plot displaying the expression of respective marker genes within all five sub-clusters. (E) The enrichment scores of different gene sets including ECM disassembly, notochord development, and dedifferentiation in cell subpopulations. (F) Pseudotime trajectory axis showed the cell conversion throughout direct reprogramming. (G–I) Gene expression dynamics of the specified genes across the pseudotime trajectory. (J) The differentially upregulated genes in hsNPCs (left) and NCs (right) were evaluated by GO enrichment analysis. (K) CFU-fibroblastic (S) (top) and CFU-fibroblastic (F) (bottom) derived from Noggin+ NCs in methylcellulose medium at 14 days. Immunocytochemical staining of them showed CFU-S expressed higher COL2 and ACAN than CFU-F. Scale bar, 200 μm. (L) Frequency of CFU-S and CFU-F derived from iNCs and dNPCs. n = 5. Data are expressed as mean ± SD. Statistics were compared using Student’s unpaired t test (L).

Using established gene sets,32,35,36,37,38 we identified the main cell subtypes. In dNPC samples, SOD2, COL1A1, and MMP3 marked fibrocartilaginous NPCs (fcNPC, C1), while PFN1, NEK10, and CFLAR indicated inflammatory NPCs (imNPC, C2) (Figure 4C). The former exhibited a fibrocartilaginous gene expression pattern (Figures 4D and S5E), while the latter were associated with inflammatory reactions (Figure 4D). Expectedly, there were barely any signs of NC, NPPC, or MSC types in the dNPC samples, suggesting that iNCs did not originate from initial stem or progenitor cells.39 iNCs samples were divided into three subclusters: COL2A1 and SOX9 for homeostasis NPCs (hsNPC, C3) and NOG, FZD3, and STMN2 for NCs (C4) (Figure 4C). Specifically, C3 was enriched in CXCL14, TEK, and GUCY1A1 expressions (Figure 4D). The high expressions of COL2A1 and SOX9 suggest a restored gelatinous ECM synthesis.36 C4 highly expressed early notochordal markers KRT8, ENKD1, PAX6, SALL3, and RTN1 (Figure 4D). C5 lacked expressions of dNPC, nNPC, and NC markers, considered as transitional/intermediate reprogramming cells (tRC).

We analyzed the expression differences of specific gene sets in different subpopulations. Notably, although both NC and hsNPC significantly improved ECM disassembly and dedifferentiation-related gene sets, only NC subcluster upregulated notochord development-related genes (Figure 4E). Furthermore, we reconstructed the cellular transition trajectories using Monocle 2 to explain the cellular hierarchy before and after reprogramming. The pseudotime trajectory showed a one-way linear transformation, where fcNPCs gradually converted into imNPCs, tRCs, and hsNPCs, and finally transformed into NCs (Figure 4F). We tracked the dynamic changes of specific gene sets along the transition axis. Fibrocartilaginous genes were downregulated companied with an overall upward of notochordal genes along trajectories (Figures 4G and 4H). Additionally, markers for early NPC stages initially increased and then decreased to a low level, with the highest peak occurring in the middle stage of the trajectory (Figure 4I). Hence, the identification of different stages within these subpopulations robustly supported a dedifferentiation-like process during dNPCs to iNCs conversion.

To determine the potential functions of iNCs subtypes, we performed a GO enrichment analysis for hsNPCs and NCs. While genes upregulated in hsNPC were mainly related to terms associated with ECM organization, genes upregulated in the NC subpopulation were enriched in cell regeneration and somatic stem cell population maintenance, indicating that the NC subpopulation played a role in cell repopulation as primary NCs (Figure 4J). To further validate the function of the NC subpopulation for cell repopulation, we assessed their colony-forming ability. Noggin was selected to be used as the sorting indicator for its high expression in the NC subpopulation. The FACS-sorted Noggin+ NC subpopulation generated more colony-forming units (CFU)-spherical, while CFU-fibroblastic formation was similar to dNPCs (Figures 4K, 4L, and S5F). These results unveiled the distinct roles of different subpopulations within reprogrammed cells, highlighting the emergence of a specific NC subpopulation with a robust capacity for cellular repopulation during the dedifferentiation-like reprogramming.

OCT4 promotes BPTF-mediated chromatin remodeling during the early stages to initiate dedifferentiation-like reprogramming

To explore the molecular dynamics underlying the OFT-induced reprogramming process, we sequenced mRNA isolated from reprogrammed cells at multiple time points (days 3 [D3], D7, D14), along with dNPCs, nNPCs, and natural NCs. Principal component analysis showed that reprogrammed cells gradually acquired transcriptomic signatures of natural NCs (Figure 5A). When assessing gene sets related to important functions, we observed that dNPCs were enriched in degeneration of the ECM and activation of MMPs. d3iNCs preferentially performed DNA replication and oxidative phosphorylation, indicating high requirements for extensive material synthesis and energy metabolism at the early stages.40,41 d7iNCs exhibited the most enrichment in DNA replication with decreased oxidative phosphorylation. Notably, d14iNCs showed the most significant enrichment in notochord development and cell-cell communications. These distinctive cell properties at different time points suggested a dynamic transition during the 14-day OFT induction (Figure 5B).Figure 5 OCT4 promotes BPTF-mediated chromatin loosening during the early stages to drive dNPC to iNC conversion

(A) Principal component analysis of human dNPCs, nNPC, NCs, and iNCs for different time points. (B) single-sample GSEA for the mRNA-seq result of iNCs for different time points. (C) Heatmap (left) and individual expression patterns (middle and right) showing gene expression of reprogrammed cells for different time points. (D) GO enrichment of four gene modules from k-means analysis. (E) GSEA analysis for differentially expressed genes between human d3iNCs and dNPCs samples. (F) Heatmap and Metagene plot of ATAC-seq signal of human dNPCs, and iNCs for different time points. (G) Immunofluorescence staining (right) and dot plot of the normalized values (left) of H3K27me3 and H3K4me3 in reprogrammed cells for different time points (dNPCs, d3iNCs, d7iNCs, d14iNCs). Scale bar, 100 μm. n = 3 (H3K27me3); n = 4 (H3K4me3). (H) overexpressed OFT interacted with chromatin-remodeling proteins BPTF in d3iNCs and dNPCs by co-IP. (I) Heatmap and Metagene plot of ATAC-seq signal of FT group, OFT group, and OFT group treated with BPTF inhibitor BZ-1. (J) The ATAC-seq data presents genomic views for the KRT8 and COL2A1 gene loci. (K) Immunofluorescence staining (left and middle) and dot plot of the normalized values (right) of H3K27me3 and H3K4me3 in FT group, OFT group, and OFT group treated with BPTF inhibitor BZ-1. Scale bar, 100 μm. n = 5(H3K27me3); n = 4 (H3K4me3). (L) The quantitative results of immunofluorescence staining of iNCs treated with BPTF inhibition. n = 3. Data are expressed as mean ± SD. Statistics were compared using one-way ANOVA (G, K, and L).

To observe the critical molecular events initiating this process, all genes were divided into four modules based on their expression patterns. Specifically, fibroblastic ECM-related genes were rapidly downregulated during early stages, while notochordal genes dynamically increased at different time points, indicating that the elimination of degenerative phenotypes occurred earlier than the acquisition of notochordal identity (Figures 5C and S5G). Notably, GO analysis showed that module III was enriched in chromatin-related changes, which increased rapidly from an early time and was highly expressed during the entire process (Figures 5C and 5D). GSEA based on gene expressions of D0 and D3 also showed similar results (Figure 5E). To better observe the changes of cellular chromatin states, we conducted transposase-accessible chromatin sequencing (ATAC-seq) on iNCs at multiple time points (D3, D7, and D14), along with dNPCs. The data revealed that the normalized signal intensity in cells was obviously increased from D3, indicating an early stage increase in chromatin accessibility during this reprogramming process (Figure 5F). Histone markers H3K27me3 and H3K4me3 are nuclear markers representing transcriptional chromatin silencing and activation, respectively, which significantly changed from D3 (Figure 5G). Additionally, we analyzed the expression differences of chromatin regulation-related genes in different iNCs subpopulations. However, these genes showed a similar elevation in all iNCs subtypes, indicating that chromatin status alterations might be more important for initiating dedifferentiation-like reprogramming than notochordal phenotype formation (Figure S5H).

Given the significant influence of OCT4 on the dedifferentiation-like reprogramming of dNPCs and the reported interactions between OCT4 and chromatin remodeling proteins,42,43 we further determined whether OCT4 played a pivotal role in regulating genes related to chromatin remodeling. We conducted co-immunoprecipitation (co-IP) and mass spectrometry (MS) on iNC samples to screen for proteins that interacted with OCT4 and identified interacting proteins (Table S4). Among these, BPTF, the largest member of the human nucleosome remodeling factor NURF, stood out, as it has been increasingly proved to increase chromatin accessibility.44 However, the direct interaction between OCT4 and BPTF during reprogramming remained unclarified. In d3iNCs, we confirmed the interaction between OCT4 and BPTF by co-IP combined with immunoblotting (Figure 5H). In contrast, there were no significant interactions between BPTF and FOXA2 or TBXT (Figure 5H). Furthermore, to verify whether BPTF directly affects cell conversions, we downregulated BPTF expression using the inhibitor BZ-1 in dNPCs before introducing OFT. ATAC-seq results and immunofluorescence staining showed a restored chromatin repression after BPTF inhibition (Figures 5I and 5K). Specifically, we examined the chromatin accessibility associated with the representative genes, including KRT8 and COL2A1, and found a decrease after BPTF inhibition (Figure 5J). Compared with normal iNCs, BPTF inhibition also decreased the reprogramming efficiency (Figure 5L). These findings collectively indicated that OCT4 directly activated the chromatin remodeling protein BPTF during the early stages, which is a critical step in dNPC to iNC conversion.

AAV2-mediated OFT overexpression converts in situ dNPCs into iNCs in degenerated NP

To assess the potential of in situ dNPCs dedifferentiation-like reprogramming, we induced IVDD in a rat-tail puncture model as previously published.45 It has been established that NCs are abundant in healthy rat discs until degeneration sets in, and they are known to play a crucial role in maintaining NP homeostasis.46 Injury-induced IVDD accelerates the degeneration of resident NCs.47 To minimize interference from preexisting NCs, we harvested IVD samples at two months after puncture.48,49,50 Immunofluorescence analysis revealed typical degenerative signs in punctured NP tissue (Figures 6A and 6B). Simultaneously, notochordal genes were almost absent, indicating that normal NCs occupied a very low proportion and had been replaced by dNPCs (Figures S6A–S6G). Consequently, the delivery of OFT was performed at the two-month post-puncture time point.Figure 6 Regenerating iNCs in degenerated rat NP via AAV2-mediated OFT delivery

(A) Experimental outline for assessing in situ reprogramming in rat IVDD model. (B) The expressions of COL2, COL1, and MMP3 were assessed using immunofluorescence in rat disc region after puncture for two months. Scale bar, 200 μm. (C) Illustration shows the transfection and converting process for in situ dNPCs (D) The FLAG and MMP3 expression levels in situ were assessed by immunofluorescence staining after delivery for 3 weeks. Scale bar, 800 μm. (E) Expression of OFT in dNPCs in situ measured by immunofluorescence staining after AAV2-mediated delivery for 3 weeks. Scale bar, 250 μm. (F) OFT expression in dNPCs in situ is measured by RT-qPCR after AAV2-mediated delivery for 3 weeks. n = 5. (G) Immunofluorescence staining (left) and the quantitative result (right) of KI67 in situ after delivery for 4 weeks. Scale bar, 800 μm. n = 4. (H) Immunofluorescence staining of transfected dNPCs in situ (left) and box plot of the normalized values (right) for notochordal markers SHH, NOG, SALL3, TIE2, and overexpressed OCT4 after AAV2-mediated delivery for 4 weeks. Scale bar, 100 μm. n = 4. (I) Cell apoptosis of GAL3+ cells and KRT8+ cells was measured by TUNEL staining. Scale bar, 300 μm. Data are expressed as mean ± SD. Statistics were compared using Student’s unpaired t test (G and H) and one-way ANOVA (F and I).

Subsequently, we designed an adeno-associated virus (serotype 2) (AAV2) system encoding OFT and introduced it into IVDD rats through intradiscal delivery (2 × 1010 viral genomes per disc) (Table S5). At specific time points, we harvested rat IVDs to assess OFT overexpression, iNCs generation, and degenerative tissue changes (Figures 6A and 6C). To confirm the successful transfection of OFT in the NP region, we detected FLAG tag expression after 3 weeks of AAV2 injection, which was used to label the exogenous TBXT protein. Positive staining in the OFT group confirmed effective transfection in dNPCs (Figure 6D). RT-qPCR results demonstrated high expression levels of OFT in the OFT group (Figure 6F). Tissue immunofluorescence further revealed their colocalization in the nucleus (Figure 6E). Subsequently, we assessed whether OFT successfully induced the dedifferentiation-like reprogramming at 4 weeks after injection. Compared with the vehicle group, a significant increase in KI67+ cells was observed in the OFT group (Figure 6G). Moreover, the OFT group highly expressed notochordal genes NOG, SALL3, SHH, and NP progenitor marker TIE2, suggesting iNCs generation (Figures 6H and S7A). TUNEL staining, co-stained with KRT8 and GAL3, revealed that OFT treatment resulted in reduced cellular apoptosis compared with the vehicle group, particularly in KRT8 and GAL3-positive regions, indicating improved cell viability in discs after OFT reprogramming (Figure 6I).

dNPCs to iNCs reprogramming effectively reverses IVDD

Subsequently, we evaluated the overall recovery of rat IVDD by measuring disc height using X-ray imaging every month after treatment. Compared with the degeneration and vehicle groups, the decrease in disc height was less pronounced in OFT groups (Figures 7A and S7B). Micro-computed tomography (CT) evaluation revealed that the OFT group exhibited milder cartilage endplate wear with a more intact NP, while the discs from the degeneration and vehicle groups had almost indistinguishable NP boundaries and worn endplates (Figure 7B). Hematoxylin and eosin (H&E) staining and safranin O-fast green (S-O) staining at different time points provided details of histological degeneration. NPC morphology, a rich ECM, and clear NP/annulus fibrosus compartmentalization were better preserved in the OFT group (Figures 7C, 7D, S7C, and S7D). Specifically, the evaluation of histological score revealed that the OFT group had a lesser degree of histological degeneration (Figure S7E).45 Biomechanical functions including compressive and stretching mechanical properties of rat IVD, were assessed at week 16. The mechanical properties of the OFT group were closer to naive levels, indicating better preservation of disc function (Figure 7E).Figure 7 OFT effectively ameliorates rat IVDD in vivo

(A) Representative radiographs (middle) and quantitative statistics (bottom) at different time points of rat caudal vertebrae (Co7/Co8, Co8/Co9) after AAV2-mediated OFT delivery. DHI was used to quantitatively evaluate the disc height changes (top). DHI%, post-punctured DHI/pre-punctured DHI × 100. n = 3. (B) Micro-CT imaging of rat caudal vertebrae at 16 weeks. The picture illustrates the coronal position (top) and horizontal position (bottom). Scale bar, 600 μm. (C and D) H&E staining (C) and S-O staining (D) of rat discs for different groups at 4 and 16 weeks. Scale bar, 600 μm. (E) The representative compression curve (top) and tensile curve (bottom) of different groups at 16 weeks were assessed by a universal material testing machine. (F and G) Immunofluorescence staining of different groups at 16 weeks for catabolic marker MMP3 (F), as well as ECM component COL2, ACAN, COMP, and CS (G). Scale bar, 600 μm. (H) The collagen constituent and organization of different groups at 16 weeks were visualized using picrosirius red staining. Scale bar, 600 μm. (I) RT-qPCR result of control, vehicle, and OFT group at 16 weeks for NP marker COL2, ACAN, SOX9, and fibrocartilaginous marker COL1, COL3, and fibronectin expression. n = 3. Data are expressed as mean ± SD. Statistics were compared using one-way ANOVA (I).

Furthermore, to assess the safety of OFT transduction in vivo, we initially conducted subcutaneous transplantation of in vitro-converted OFT+ iNCs into highly immunodeficient mice. A 9-week follow-up showed no notable tumor formation (Figure S7F). Moreover, we directly examined whether tumorigenicity was present in OFT-AAV2-infected rat discs. There was no expression of pluripotent cell markers or cellular nuclear atypia in both the OFT and vehicle groups (Figures S7G and 7C). These results confirmed the in vivo safety of OFT-mediated dNPC dedifferentiation-like reprogramming.

Furthermore, we investigated whether OFT treatment restored the abundance of ECM in discs. Compared with the vehicle group, typical ECM components were more abundant in the OFT group, while MMP3 was less prevalent, indicating improved ECM preservation (Figure 7G). RT-qPCR results also revealed a similar result (Figure 7I). Additionally, picrosirius red staining identified milder fibrosis in the OFT group (Figures 7H and S7H). Overall, dNPC dedifferentiation-like reprogramming preserved a relatively healthy NP and mitigated IVDD in the rat model.

Discussion

Through the screening of various factor combinations, a novel combination capable of reprogramming dNPCs into iNCs was identified. These iNCs effectively attenuated degenerative phenotypes and restored typical molecular signatures similar to those of natural NCs. Furthermore, OCT4 was found to play a vital role in activating BPTF-dependent chromatin remodeling to initiate the dedifferentiation-like reprogramming of dNPCs. Our reprogramming strategy offers a promising approach for in situ progenitor cell regeneration in degenerative diseases, including IVDD, effectively overcoming the limitations of traditional cell transplantation.

This study explored the fate plasticity of dNPCs to identify an effective strategy for their dedifferentiation-like reprogramming. Unlike previous dedifferentiation-like reprogramming whose initial cells were typically contingent upon somatic cells with a certain degree of plasticity, terminally differentiated host cells in some degenerative diseases such as IVDD and osteoarthritis generally display degenerative status, signifying a loss of regenerative potential.10,22,51 Achieving dedifferentiation-like reprogramming in such degenerated tissues remains challenging. Recently, various adverse factors underlying pathological cellular status have been identified, including inflammatory phenotypes, low proliferation, and epigenetic imprints, which have been shown to significantly impede the fate conversion.7,20,21 Nevertheless, whether certain critical barriers associated with the degenerative cellular status may also restrict dedifferentiation-like reprogramming remains elusive. Through the screening of various factor combinations, this study identified a novel factor combination comprising notochord-specific factors and Yamanaka factors for dNPC to iNC conversion. Earlier studies have recognized tissue-specific developmental factors as potential reprogramming factors, enabling dedifferentiation-like reprogramming.9,14 Given the similarity of the reprogramming route, a two-factor combination, FT, was initially screened owing to their pivotal roles in notochord formation and phenotype maintenance.24,52,53 However, while FT knockdown significantly promoted NC degeneration, co-overexpressing FT only marginally enhanced dNPC phenotypes. Therefore, unlike previous dedifferentiation strategies, solely applying notochordal-specific factors, FT exhibited limited capacities for the dedifferentiation-like reprogramming of dNPCs, probably ascribed to the refractoriness of the degenerative status in initial cells.7 Hence, additional factors were incorporated to enhance dNPCs plasticity. Canonical Yamanaka factors have been shown to remodel the genetic imprints of degenerated somatic cells.14 Considering their key roles in neural development and lineage reprogramming, SOX2 is first excluded.54,55 Among various combinations, OFT induced the expression of numerous notochordal markers and ECM anabolic markers. Therefore, besides lineage-specific factors FT, the inclusion of OCT4 was essential for dedifferentiation-like reprogramming of dNPCs.56,57 Our results emphasize the impact of degenerative status of initial cells on dedifferentiation-like reprogramming and successfully achieved the fate conversion of degenerative cells by developing a factor-based strategy.

Distinctive molecular dynamics in intermediate phases have been reported to play an instrumental role in cell conversions.40 Our results demonstrated that OCT4 activated BPTF-mediated chromatin remodeling during the early stages to initiate dNPC to iNC conversion. Of note, OFT induced extensive alterations in chromatin remodeling-related genes and a rapid increase in chromatin accessibility from D3, occurring earlier than the acquisition of notochordal identity. Comparable transient changes have also been observed in partial reprogramming-induced rejuvenation.13,30 Therefore, relaxation of pre-existing chromatin repression underlying degeneration seems to be crucial for initiating reprogramming from dNPCs. OCT4, rather than FT, was identified via MS and co-IP analysis as the pioneer factor that directly activated BPTF to increase chromatin accessibility. To our knowledge, this is the first study to document the direct interaction between OCT4 and the BPTF protein. Similar roles, where OCT4 recruits and cooperates with other chromatin-remodeling proteins like BRG1 and Baf155, have been reported to establish an accessible chromatin state that facilitates the binding of other factors, thereby driving iPSC reprogramming.42,43 The NC-specific transcription factors FT in our combinations have been proven to bind to specific chromatin regions to activate the notochordal gene regulatory network.24,25,58 However, the repressive chromatin state of dNPCs could inhibit FT’s transcriptional activation. Herein, the inhibition of OCT4-activated BPTF restored the repressive chromatin state, hindering iNC reprogramming. Therefore, OCT4-activated BPTF-mediated chromatin remodeling was critical for initiating this dedifferentiation-like reprogramming, which accounted for the enhanced reprogramming effects after the addition of OCT4 to FT. Importantly, a repressive chromatin state depositing during cell differentiation and degeneration has been noted in various cell types and acted as a barrier to reprogramming.19,21 In some degenerative diseases, such as age-related macular degeneration and osteoarthritic degeneration, the decline of cellular status is closely associated with an increased repressive chromatin state.59,60,61 This degeneration-related epigenetic deposition suppresses cellular plasticity and potential fate conversion. Moreover, a cellular reprogramming originating from degenerative cells showed was relatively inefficient and yielded incomplete remodeling compared with reprogramming under physiological conditions.7,62 The incomplete transcriptional activation of reprogramming factors due to transcriptional repression in repressive chromatin regions might directly mediate this incomplete remodeling of reprogrammed cells. Thus, the supplementation of OCT4 or alternative factors to remodel the repressive chromatin associated with degeneration might facilitate reprogramming and ameliorate the phenotypes of these reprogrammed cells. Hence, from a broader perspective, the role of OCT4 in the relaxation of pre-existing chromatin repression could also be essential for dedifferentiation-like reprogramming of other degenerated somatic cells into progenitor state and should be considered during the design of future reprogramming strategies.

Nevertheless, our study has some unsolved limitations. First, while our study demonstrates the anabolic superiority of iNCs, the recovery of catabolic phenotypes, such as the expressions of ECM degrading enzymes, and cellular viability remain limited and falls short of that of natural NCs (Figure S8A). In contrast with iPSC reprogramming, which resets cells to a pluripotent state, our direct conversion approach does not involve transitioning through an induced pluripotent stage. Consequently, this reprogramming strategy is unable to completely erase all signatures associated with the degenerated phenotype of initial cells, particularly in terminally differentiated human cells.63 The introduction of small molecules with anti-catabolic or anti-apoptotic effects to the current factor combination might assist in erasing the degenerative phenotypes of reprogrammed cells. Second, despite demonstrating that OCT4 activates BPTF-mediated chromatin loosening in dNPC to iNC conversion, the downstream gene loci of BPTF and their exact relationships with FT remains enigmatic. Further investigations, such as integrative chromatin immunoprecipitation sequencing and ATAC-seq analysis, are anticipated to expand our understanding of the cooperative interactions among multiple factors and their roles in dNPC to iNC conversion. Third, we did not track the fate of iNCs in vivo. Considering the harsh local microenvironment, such as the high inflammatory response and dysregulated ECM, these cells may not exhibit sustained viability. Further evaluation of in-situ iNCs phenotypes at multiple time points could elucidate this issue, providing more insights into in vivo reprogramming for IVDD therapy.

In summary, our findings demonstrated the successful dedifferentiation-like reprogramming of dNPCs into iNCs using a novel factor-based strategy. The OCT4-activated BPTF-dependent chromatin remodeling during the early stages was critical for initiating this conversion process. On a broader level, this exploration of the cellular plasticity of degenerated somatic cells for dedifferentiation-like reprogramming provides a fresh perspective on treating degenerative diseases.

Materials and methods

Human subjects

Human NP specimens were obtained from orthopedics department of the Second Affiliated Hospital of Zhejiang University School of Medicine. Aborted human embryo samples were obtained from the Obstetrics and Gynecology department of the Second Affiliated Hospital of Zhejiang University. All donors uniformly signed informed consent forms, allowing the use of their discarded tissues for this research. All experimental procedures were performed in adherence to protocols approved by the institutional research ethics committee of Zhejiang University (Approval No.2022-0967).

Animals

Sprague Dawley rats and BALB/c-nu mice were purchased from Zhejiang Chinese Medical University Laboratory Animal Research Center. The experimental procedures in this study were all approved by the Animal Care and Use Committee of Zhejiang University and performed under proper protocols as previously described.45

IVDD model

Three-month-old (male, n = 120; 250–300 g) Sprague Dawley rats were first anesthetized by intraperitoneal injection of 1% pentobarbital sodium (0.4 mL/100 g). After anesthesia, 20G sterile needles (Hamilton Company) were used to puncture their discs (coccygeal vertebrae [Co] Co7/8, Co8/9, and Co9/10 per animal). The needle penetrated the annulus fibrosus on one side of the disc into the NP region and was rotated 360° and held for 30 s to induce degeneration. After surgery, the animals recovered in a warm and ventilated cage. After 2 months, we randomly divided the rats into four groups: OFT-AAV2 group (puncture treatment, received 2 μL solutions, 2 × 1010 vg per disc), vehicle-AAV2 group (puncture treatment, received 2 μL solutions, 2 × 1010 vg per disc), degeneration group (only puncture treatment), and control group (no treatment). We used 2-μL Hamilton microsyringes to perform AAV2 or PBS delivery.

Histological analyses

Caudal vertebrae were harvested after 3, 4, 8, 12, and 16 weeks of treatment. After being washed in PBS, samples were fixed in 4% paraformaldehyde (Biosharp, 143174) for 48 h, decalcified in 10% EDTA for 1 month, dehydrated, embedded, and sliced into 5-μm paraffin sections using a microtome (Leica, MultiCut). H&E staining was performed to evaluate the whole structure of discs and cell number of each part (annulus fibrosus, NP, and cartilage endplate). S-O staining was performed to evaluate the distributions of proteoglycan and cartilaginous content. Picrosirius red staining was performed for collagen fiber characterization within the NP region. These stainings enable us to comprehensively assess the tissue degeneration of IVDs under different interventions. H&E and S-O staining were visualized using an optical ortho microscope (Leica, dm3000). The heterogeneity of collagen organization was visualized under polarized light. Histological scores of every group were rated based on five indexes, from 5 points (no degeneration) to 15 points (severe degeneration).45

Immunochemistry

To assess the expressions of specific proteins in situ, deparaffinized sections from human and rat samples were heated in sodium citrate buffer solution (pH 6.5) for 3 h at 65°C for antigen retrieval and incubated in the sealing solution (5% BSA in PBS) for 90 min at 37°C. Blocked sections were then incubated with primary antibodies overnight at 4°C. Then, sections were washed three times in PBS with 0.5‰ Tween and incubated for 1 h at 37°C with secondary antibodies (1:500, Alexa Fluor 488, 647, Abcam). When co-labeling specific proteins and apoptotic cells on sections, TUNEL staining is performed after incubation with secondary antibodies. After being mounted with a histology mounting medium containing DAPI (F6057, Sigma), all sections were imaged using a fluorescence microscope (Leica, DM6B). The proportion of staining positive areas and cells was quantified using ImageJ software.

Disc height measurement

The disc height of every group was evaluated using a molybdenum target radiographic image unit (GE mammography DMR Bucky 18 × 24, GE Healthcare). After 4, 8, 12, and 16 weeks of treatment, anesthetized rats were placed supine. Radiographs of rat tails were taken using a computed radiographic plate system (AGFA Diagnostic Center Compact Plus, AGFA HealthCare) and quantitatively analyzed using ImageJ software. The disc height index (DHI) was used to evaluate the degree of disc height narrowing (DHI% = post-punctured DHI/pre-punctured DHI × 100).

Mechanical strengths analyses

Stretching and compression tests were performed on the IVDs of rats' tails after 4 months of treatment using the universal testing machine (Zwick/Roell Z020). After muscles and tendons adhered to IVDs and vertebral bodies were fully removed, the tails were prepared into segments of the vertebrae-disc-vertebrae. Disc diameters and heights were measured and recorded using the vernier caliper. Then, tension and compression testing were separately performed on the samples at 1 mm/min and 1 mm/min until the rat disc was ruptured. The ratio of the load to total cross-sectional area and the ratio of the displacement to disc height were calculated to evaluate the stress and strain, respectively.

Micro-CT evaluation

Fresh rat tail samples were harvested after 16 weeks of treatment and processed into individual IVDs with one of three adjacent vertebral bodies. Ordinary and discography imaging of rat discs were respectively scanned using a micro-CT imaging system (SKYSCAN 1276, BRUCKER) before and after soaking in iodixanol (Visipaque, GE) at 4°C for 48 h. Iodixanol is a low cytotoxic contrast agent used in discography for clear visualization of the IVD structure.64 Coronal images were analyzed to evaluate the wear of cartilaginous endplates adjacent to the IVD. Three-dimensional reconstruction was performed using CTvox software to evaluate the compartment demarcation of the IVD.

Bulk and scRNA-seq and analyses

Bulk RNA-seq

For identifying notochord development-specific factors, total RNA of natural NCs (passages 3–4) from embryonic notochord, nNPCs from normal NP samples (passages 3–4), and dNPCs (passages 3–4) from degenerated NP samples was isolated. For dissecting the transcriptional changes during reprogramming, total RNA of reprogrammed cells of D3, D7, and D14, as well as from dNPCs was isolated. Each sample consisted of 500,000 cells, and we conducted at least three independent experimental replicates for each group. The samples were then submitted to Wuhan Metware Biotechnology Company for mRNA-seq analysis.

scRNA-seq

dNPCs without OFT transduction and 14 day-OFT+dNPCs were prepared into living cell suspensions (>10,000 cells per sample). Two independent samples from two donors were initially pooled before sequencing (patient 1: Pfirrmann grade IV, 51 years old; patient 2: Pfirrmann grade V, 55 years old). Fresh living cell suspensions were then submitted to Hangzhou Lianchuan Biotechnology Company for 10× Genomics Chromium scRNA-seq analysis.

ATAC-seq data analysis

Cell samples processed akin to RNA-seq, were used for ATAC-seq library construction following established protocols (two independent replicates for each group). Briefly, cells from each pooled sample were dissociated to yield single-cell suspensions. Subsequently, these cells were suspended in nuclear isolation buffer and subjected to repeated washing using nuclear wash buffer as per the standard nuclear isolation procedure. Following this, 50,000 nuclei were pelleted, cultured with transposase at 37°C for 30 min. The Library Prep Kit (Vazyme) were used to purify the transposed DNA, which was used for the library generation via PCR amplification. The library was further purified with a Hyperactive ATAC-Seq Library Prep Kit according to the manufacturer’s instructions and sequenced using the Illumina Novaseq 6000 platform by Repugene Technology Company.

Cell isolation and culture from human specimens

Degenerative NP specimens were obtained from patients with spinal stenosis or IVD herniation (Pfirrmann grades IV and V; n = 13). Healthy NP specimens were obtained from patients with scoliosis or spinal fracture (Pfirrmann grades I and II; n = 6). Detailed characteristics of patient, including gender, age, disc degeneration grading, and surgical segment, are listed in Table S1. Part of the patient samples was fixed in 4% para-formaldehyde for 48 h, dehydrated, embedded, and sliced into 5-μm paraffin sections using a microtome (Leica, MultiCut) for further histological analyses. The other part of the patient samples was used to obtain primary cells. After being washed in sterile PBS (pH 7.4) three times, human NP samples were extracted and digested (0.2% type II collagenase) in serum-free DMEM (Gibco) for 30 min at 37°C. Then, digested samples were centrifuged to remove collagenase. Human NPCs were then cultured in DMEM supplemented with 1% antibiotics (100 U/mL penicillin/streptomycin) and 15% fetal bovine serum (FBS) (Gibco) under 5% CO2 at 37°C.

Human embryos (3–6 weeks) were obtained from patients who obtained medical abortions and washed in sterile PBS (pH 7.4) three times. The notochordal tissue was isolated in the super-clean bench under the 20× anatomic microscope. Then, the obtained tissue was minced by a sterile knife blade and digested using TrypLE Express Enzyme (Gibco) for 30 min at 37°C. After centrifugation to remove TrypLE Express Enzyme, human NCs were cultured in the NC expand medium28 and used for subsequent experiments within five passages.

Retrovirus vectors and cell transduction

To overexpress target genes in dNPCs in vitro, six types of lentivirus (LV) carrying specific genes (LVOCT4, LVc-MYC, LVKLF4, LVFOXA2, LVTBXT, and LVFOXA2-TBXT) and empty vectors were constructed by Genechem Technology (Table S5). DNA sequencing was used to verify the sequences of plasmids. dNPCs (passage 2) were seeded in 12-well plates (1.0 × 105 per well) and cultured under 37°C with 5% CO2. When purified dNPCs grew to 80% confluence, LV (LVOCT4, LVc-MYC, LVKLF4, LVFOXA2, LVTBXT: multiplicity of infection [MOI] = 1:30; LVFOXA2-TBXT: MOI = 1: 50) and Poly P agent (1:50, 500 ng/mL, Cat. No. REVG005, Genechem Technology) were added into the DMEM with 15% FBS and replaced the original medium of dNPCs. After 18 h, the fresh medium was replaced to continuously culture transduced dNPCs. After 2 days, the original medium was replaced by the NP precursor-conditioned medium.28 The conditional medium was changed every 2 days for continuous cultivation.

Production of AAV2

The AAV2-OFT was sourced from Genechem Technology. Transfer plasmid of AAV-OFT were constructed by inserting rat OCT4, FOXA2, and TBXT cDNA into an AAV vector containing CAG promoter. Then, the resulting pAAVs (refer to Table S5), rep-cap plasmid, and helper plasmid were subsequently co-transfected into 293 cells to produce AAV2 vector. After 3 days, the virus was harvested from the cell lysates and culture media. Subsequently, the virus underwent purification via iodixanol gradient ultracentrifugation. Virus titers were determined using quantitative real-time PCR (titers = 1.0 × 1013 vector genomes per mL).

Purification of LV-transducted dNPCs

dNPCs transducted with OFT for 3 days were digested into single cells suspension in the dark (LVOCT4-GFP + LVFOXA2-TBXT-mCherry). Samples were passed through 35-μm filters. Cell sorting was then performed to collect fluorescent protein-positive cells using Beckman MoFlo fractional flow cytometry with accompanying software. Purified positive cells were then cultured in NP precursor-conditioned medium.

Small interfering RNA transfection

To knock down the expression of FT, NCs were seeded in a twelve-well plate with round coverslips (for immunofluorescence) or without round coverslips (for RT-qPCR). Upon reaching 50% confluence, the original medium was replaced with a fresh NC expand medium (1,000 μL). Subsequently, a transfection mixture composed of Opti-MEM medium (200 μL, Gibco), small interfering RNA (siRNA) (50 pM, Genepharma), and siRNA-Mate (10 μL, G04002, Genepharma) was added into the culture medium. The cells were then continuously transfected for 48–72 h. The siRNA sequences were as follows: FOXA2 siRNA (5′-CCAUUAUGAACUCCUCUUATT-3′); TBXT siRNA (5′-CCUAUGCUCAUCGGAACAATT-3′).

Cell morphology analyses

To better visualize cellular morphology, dNPCs and iNCs were fixed with 4% PFA (Biosharp), permeabilized with 0.3% Triton X-100, and stained with anti-α-tubulin-antibody (Abcam). After nuclei staining using DAPI (Cell Signaling Technology, 4083s), samples were imaged on a laser confocal fluorescence microscopy (Leica, STELL ARI S5, Germany). Cell shape features were analyzed using CellProfiler Analyst.

Quantitative real-time PCR

Total RNA from tissues or cultured cells was isolated using the Trizol reagent (Takara Bio) and then quantified by microvolume spectrophotometers (701-058112, Thermo Fisher Scientific). The subsequent reverse transcription of RNA samples (500 ng/10 μL for each group) was performed using the Prime Script RT Master Mix kit (RR037A, Takara) in a StepOnePlus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific) under standard conditions. Primers were designed to evaluate the expressions of marker genes for NCs (KRT8, SALL3, NOTO, SHH, and GAL3), normal NPCs (SOX9 and KRT19), ECM components (COL2A1, ACAN, COL3A1, FN1, and COL1A1), ECM-degrading enzymes (MMP3, MMP10, MMP13, and ADAMTS5), cellular pluripotency (NANOG), and reprogramming factors (OCT4, FT) (Table S6). They were synthesized by Sangon Biotech. Gene-specific primers (forward primer: 1 μL, 0. 5 μM; reversed primer: 1 μL, 0.5 μM), cDNA (1 μL, template amount = 5 ng total RNA), and TB green Premix Ex Taq II (10 μL, 0.4 μM, RR820 A/B, Takara) were mixed (20 μL, final reaction volume), and the real-time PCR reactions were conducted in the Fast Real-Time PCR System (ABI 7500, Thermo Fisher Scientific) under standard conditions.

Western blot

Total proteins were isolated from cells using RIPA lysis buffer (Beyotime) with 1% phosphatase and protease inhibitors (Beyotime). The protein concentrations were quantified by BCA Kit (Beyotime). We electrophoresed 30 μg loading amount of every sample in the pre-cast 10% Tris gel using a BioRad Criterion system. Then, we transferred the separated proteins to Polyvinylidene fluoride (PVDF) membranes (BioRad). After being sealed using 5% skim milk/1× TBST at room temperature for 2 h, the separated proteins with PVDF membranes were incubated with specific primary antibodies overnight at 4°C. Primary antibodies were selected to evaluate the protein expressions for reprogramming factors (OCT4, FOXA2, and TBXT), ECM components (COL1, ACAN, and COL2), ECM degrading enzymes (MMP3, MMP13, ADAMTS4, and ADAMTS5), normal NPCs (SOX9), and chromatin remodeling factor (BPTF) (Table S7). Membranes were subsequently incubated with the horseradish peroxidase-conjugated secondary antibody (1:1,000, Beyotime) in 1% skim milk/1× TBST for 1 h at room temperature. After being washed three times with PBS, we detected protein signals with the enhanced chemiluminescence substrate (FD8020, Fude Biology). Relative protein expression was quantified and analyzed using ImageJ software.

MS

Protein lysates of dNPCs and d3iNCs were immunoprecipitated using anti-Flag-tag agarose beads, followed by separation on PAGE gels. The resulting gel lanes were then submitted to Shanghai Zhongke New Life Biotechnology Co., Ltd, for MS analysis. Subsequently, proteins identified in control and OFT (overexpression) lanes were selected for further filtering analysis. Proteins with unique peptides of less than one in OFT samples were excluded. The remaining proteins were ranked based on the ratio of their abundance in overexpression and control lanes. The proteins associated with chromatin remodeling in high-ranking positions are considered the target proteins of interest.

Immunofluorescence analyses

All groups of cells were washed three times in PBS and fixed with 4% PFA (Biosharp) at room temperature for 10 min. After being washed three times in PBS, cells were permeabilized using 0.3% Triton X-100 in PBS for 8–10 min. After being blocked in the sealing solution (5% BSA in PBS) at 37°C for 1 h, cells were incubated with specific primary antibodies overnight at 4°C. The content was washed three times in PBS again and underwent subsequent incubation with corresponding secondary antibodies (1:500, Alexa Fluor 488, 555, 647, Abcam) was performed at 37°C for 1 h. After nuclei staining using DAPI (Cell Signaling Technology, 4083s), cells were imaged by laser confocal fluorescence microscopy (Leica, STELLARIS5). The quantification of cell number and cellular fluorescence intensity were analyzed using ImageJ software. All primary antibodies used are listed in Table S7.

Colony formation assay

We made 1.0 × 103 dNPCs and NOG+ NCs into single-cell suspensions, seeded into the 6-well plate, and cultured in a methyl-cellulose-based medium (04230, STEMCELL Technologies). Colonies containing more than 10 cells were observed and counted after 14 days under phase-contrast microscopy.

Evaluation of NPC-like differentiation of iNCs

dNPCs and iNCs (2 × 105) in 15 mL centrifuge tubes (Corning) were centrifuged (300×g, 5 min) and cultured in a normal medium for 24 h to form spheres. Over the next 14 days, the culture medium of the differentiation group was replaced by an induction medium (DMEM supplemented with 1% FBS, 10 ng/mL TGF-b3, 5 mg/mL insulin, 0.17 mM ascorbic acid-2-phosphate, and 5 ng/mL GDF5) while the undifferentiation group remained unaltered. After 14 days, spheres were fixed and dehydrated, with the optimal cutting temperature compound (4583) embedded, and fast froze with liquid nitrogen. Last, they were sectioned into 6-μm-thick slices for immunofluorescence analyses.

dNPCs and iNCs (3 × 105) were seeded in a T25 culture flask. Grouping and differentiation cultures were consistent with the above protocol. On days 0, 3, 7, 10, and 14, cells were harvested for PCR analysis. Cellular protein was isolated from different groups at D0 and D14 for western blot analysis.

Alcian blue staining

The adherent cells seeded in 96-well plates were fixed with 4% PFA (Biosharp) for 10 min, and stained with Alcian Blue dyes (C0153S-1, Beyotime) at room temperature for 30 min. After that, they were washed in PBS. The results of the staining process were observed and imaged using the inverted microscope (CKX53, Olympus).

Co-culture system

The transwell co-culture system was used to evaluate the positive regulatory effect of iNCs. The transwell inserts with 0.4 μm translucent membranes were used to allow the penetrations of the bioactive proteins and ionic molecules without cellular migration. The lower chamber was cultured with iNCs, dNPCs, NCs or nNPCs, and the dNPCs were seeded in the upper chamber. After culture for 7 days, RT-qPCR and immunofluorescence were conducted to evaluate the phenotype of dNPCs in the upper chamber.

Karyotype analysis

dNPCs and dNPCs transfected with OFT for 30 days were obtained, and a karyotype analysis was performed by Hangzhou Kayotapu Biological Technology Co., Ltd.

Assessment of transplanted iNCs viability in vivo

dNPCs and iNCs were washed with PBS and incubated with 5 μg/mL DiR (Thermo Fisher Scientific) at 37°C for 30 min with 5% CO2. After centrifuging and washing with PBS three times, labeled cells were suspended in the serum-free medium at a density of 1 × 106 cells/100 μL. Then, DiR-labeled cells (1 × 106) were injected subcutaneously into BALB/c-nu mice (6-week-old, female). The fluorescence intensities of transplanted cells were assessed using the live fluorescence imaging system (IVIS Spectrum, PerkinElmer) every week until fluorescence disappearance. All detected fluorescence intensities were normalized based on the initial fluorescence intensity.

Statistical analysis

All experiments were independently performed and repeated at least three times. The results are expressed as means and standard deviations (SD). Statistical data were analyzed using GraphPad Prism version 8.3 software. The two-tailed Student’s unpaired t test was applied for comparisons of two groups. The one-way ANOVA tests with Turkey’s post hoc were used for comparisons of multiple groups. Statistical significance: ns, not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Data and code availability

All experimental data and materials for this article can be obtained from the corresponding author upon reasonable request.

Supplemental information

Document S1. Figures S1–S8 and Tables S1–S7

Document S2. Article plus supplemental information

Acknowledgments

We thank Jing Xue for assisting the acquisition of clinic samples. We thank Junying Li from Bio-ultrastructure analysis Lab of Analysis center of Agrobiology and environmental sciences, Zhejiang University. This study was supported by the Nature Science Foundation of Zhejiang Province (LY24H060001 ), the 10.13039/501100001809 National Natural Science Foundation of China (No .82002327 , No. 82172457, No. 82202681 ), the 10.13039/501100012166 National Key Research and Development Program of China (No. 2022YFC2407202 ), and the 10.13039/501100019540 Science Fund for Distinguished Young Scholars of Zhejiang Province (LR23H060001 ), the State Key Program of National Natural Science of Zhejiang Province (LZ22H090003).

Author contributions

F.-C.L., X.-P.Z., and Q.-X.C. designed and directed this study. Y.-A.Z., C.-Z.L., and H.-B.X. performed the experiments. Y.L., K.-S.X., and J.-J.C. performed western blot experiments and data analysis. X.-P.H., J.-W.S., and F.C. worked on the virus transfection. K.-S.S. and J.-K.W. performed RT-qPCR experiments. S.-M.F. and Y.-Q.T. conducted the immunostaining experiments. L.-Y.W. and Y.-A.Z. wrote the manuscript. X.-P.Z. and H.L. edited the manuscript. S.-K.W. and Y.-X.Z. conducted the in vivo experiments.

Declaration of interests

All authors in this work declare that they have no competing interests.

Supplemental information can be found online at https://doi.org/10.1016/j.ymthe.2024.06.018.
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