Partial reprogramming strategy for intervertebral disc rejuvenation by activating energy switch

Abstract Rejuvenation of nucleus pulposus cells (NPCs) in degenerative discs can reverse intervertebral disc degeneration (IDD). Partial reprogramming is used to rejuvenate aging cells and ameliorate progression of aging tissue to avoiding formation of tumors by classical reprogramming. Understanding the effects and potential mechanisms of partial reprogramming in degenerative discs provides insights for development of new therapies for IDD treatment. The findings of the present study show that partial reprogramming through short‐term cyclic expression of Oct‐3/4, Sox2, Klf4, and c‐Myc (OSKM) inhibits progression of IDD, and significantly reduces senescence related phenotypes in aging NPCs. Mechanistically, short‐term induction of OSKM in aging NPCs activates energy metabolism as a “energy switch” by upregulating expression of Hexokinase 2 (HK2) ultimately promoting redistribution of cytoskeleton and restoring the aging state in aging NPCs. These findings indicate that partial reprogramming through short‐term induction of OSKM has high therapeutic potential in the treatment of IDD.

Partial reprogramming ameliorates cellular aging and mice aging state. Moreover, Hutchinson-Gilford progeria syndrome (HGPS) induced aging cell hallmarks such as nuclear envelope abnormalities, shorter telomere size, and increased oxidative stress are reversed by partial programming (Ocampo et al., 2016). In addition, transient reprogramming of nuclear factors resets epigenetic clock, reduces inflammation of chondrocytes, and restores the youthful regenerative response in human muscle stem cells (Sarkar et al., 2020).
Furthermore, the short-term induction of Oct4,Sox2,and Klf4 (OSK) promotes axon regeneration after injury, and reverses vision loss in an aged mouse model of glaucoma (Lu et al., 2020). Transient cyclic reprogramming in the dentate gyrus (DG) increases the survival of newborn DG neurons during their maturation and increases synaptic plasticity in mature neurons (Rodríguez-Matellán et al., 2020). These findings indicate that partial reprogramming is a potential strategy for slowing aging.
Low back pain (LBP) is strongly associated with intervertebral disc degeneration (IDD) and a major cause of disability worldwide.
LBP imposes an enormous clinical and socioeconomic burden on society ("Global, regional, and national mortality among young people aged 10-24 years, 1950-2019: a systematic analysis for the Global Burden of Disease Study 2019," 2021). Intervertebral disc (IVD) combines with a gel-like nucleus pulposus (NP) and circumferentially annulus fibrosus (AF) (Roberts et al., 2006). NP plays essential roles in maintaining homeostasis by secreting type II collagen and proteoglycans to form extracellular matrix (ECM) (Wang et al., 2013). Although, the precise pathogenesis of IDD remains elusive, recuperating NP aging state can arrest progression of IDD. IVD exhibits unique features such as avascularity, hypoxia, acidic environment, low nutrition, and low cellularity (Sakai & Grad, 2015). Therefore, glycolysis is the main energy metabolism pathway in NP cells (Madhu et al., 2020). Aging NP cells have low glycolysis levels resulting in severe energy stress and high levels of reactive oxygen species (ROS). Yamanaka factors have high potential in enhancing the process of glycolysis (Sone et al., 2017). Therefore, the present study sought to explore whether the upregulation of expression of Yamanaka factors in aging NP cells can ameliorate the energy stress and restore the young state. Moreover, that the role of partial reprogramming by Yamanaka factors in treatment of IDD was evaluated.
In the current study, surgically induced IDD mouse model was established to explore the effect of short-term cyclic induction of OSKM in vivo. In addition, NP cells (NPCs) were isolated and cultured to P6 to establish a replicative senescence (RS) model of NPCs (aging NPCs). This model was used to explore the potential mechanism of amelioration of IDD in vitro. The findings showed that short-term cyclic OSKM induction ameliorates progression of IDD. Partial reprogramming inhibits phenotypes associated with aging in aging NPCs by upregulating the expression of hexokinase 2 (HK2) to activate energy metabolism and promote redistribution of the cytoskeleton (Scheme 1). These changes induce a "younger" S C H E M E 1 Schematic Diagram of partial reprogramming in aging NPCs state in aging NPCs. These results indicate that partial reprogramming induced by OSKM improves aging state of NPCs and delays progression of IDD.

| In vivo short-term cyclic reprogramming ameliorates intervertebral disc degeneration
Metabolic disease and muscle injury in aging mice can be ameliorated by transient expression of Yamanaka factors in vivo (Ocampo et al., 2016); however, studies have not elucidated whether this kind of partial reprogramming in nucleus pulposus cells (NPCs) can promote degenerative disc regeneration. Therefore, the effect of cyclic OSKM induction in 4F mice from 8 weeks of age was explored in the present study and the genotype for each mouse was  Figure 1e). Further, three groups including 4F mice at 2 weeks of age (2 week group), 12 months of age (12 month group), and 12 months of age received dox cyclic treatment for 2 weeks (12 month@cyclic doxycycline) to explore the effect of age on IVD.
Histological analysis showed that dox-induced 2-week-old 4F mice presented less degenerative changes compared with the 12 month group (Supplementary Figure S1g-h). 5-mC% is an indicator of the level of DNA methylation. Notably, DNA hypomethylation is directly proportional to cell aging (Murgatroyd et al., 2010). The findings showed that 5-mC% significantly increased after dox cyclic treatment of IVD in aged 4F mice (Supplementary Figure S1i).
Moreover, the expression of aging relative gene was improved at varying degrees (Supplementary Figure S1k-q). In summary, these findings indicate that short-term cyclic reprogramming in 4F mice alleviates surgically induced IDD and age-related IVD can be also reversed through cyclic OSKM induction.
Notably, NPCs reach the state of RS faster compared with HDFs (Ji et al., 2018). In the present study, the RS model of NPCs (P6) was used to explore whether short-term induction of OSKM promotes regeneration of aging NPCs. NPCs were isolated from IVD of 4F mice, then cultured to P6 (aging). The short-term induction of OSKM was performed by doxycycline administration for 2 days (dox2d). The ex-

F I G U R E 1
In vivo short-term cyclic reprogramming ameliorates intervertebral disc degeneration. (a) Overview of the experimental set-up on induction of doxycycline after surgery and representation of cyclic doxycycline administration protocol. (b) 4 factor mice were administered with doxycycline (1 mg/ml) through drinking water or not for 2 days and then the levels of Oct4, Sox2, Klf4, and c-Myc from NP tissue were determined by Western blotting. (c,d) HE (c) and Safranin O (d) staining of disc sections in control groups and treatment groups at Week 4 and Week 14 after surgery and blank groups (water groups, n = 5; doxycycline groups, n = 5; blank groups, n = 5). Scale bar = 50 μm. (e) Histological score of mice in control groups and treatment groups mice, as determined by Safranin O staining. ***p < 0.001, ****p < 0.0001 unpaired two-sample Student's t-test

| Maintenance of rejuvenated aging-associated phenotypes after cyclic induction of OSKM
Doxycycline treatment was stopped at the fourth day for a duration of 4 days (dox4d-4d) to evaluate the short-term effect or for a duration of 8 days (dox4d-8d) for a long-term effect after induction of OSKM in aging NPCs to explore if the effect was reproducible. In addition, induction was performed for extra 4 days These results demonstrate that maintenance of the ameliorated F I G U R E 3 Maintenance of the rejuvenated aging-associated phenotypes after induction of OSKM. (a) Cultured primary 4F aging NPCs were treated with doxycycline (1 μg/ml) for 4 days (dox4d); 4 days and stopped for 4 days (dox4d-4d); 4 days and stopped for 8 days (dox4d-8d); 4 days and stopped for 4 days and then treated for 4 days (dox4d-4d+4d). Levels of sox9 were determined by Western blotting. (b-e) qPCR analysis of stress response genes in the p53 pathway in 4F aging NPCs after doxycycline treatment. (f,h) Immunofluorescence and quantification of γH2AX expression in 4F aging NPCs after doxycycline treatment. Scale bar, 25 μm. (g,i) Immunofluorescence and quantification of γH2AX and H3K9me3 expression in 4F aging NPCs after doxycycline treatment. Scale bar, 25 μm. (j) Levels of ROS in 4F aging NPCs after doxycycline treatment. (k) Proliferation of aging NPCs cultured in inducing OSKM lasting 2 or 4 days as determined by EdU incorporation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, according to one-way ANOVA with Bonferroni correction. Data are presented as mean ± SEM aging-associated phenotypes can be achieved when induction is stopped for short periods (like 4 days). However, the effect is lost when induction treatment is terminated for a long period (like 8 days). Notably, short-term cyclic induction again restores rejuvenation of short-term induction of OSKM.

| Partial reprogramming promotes redistribution of cytoskeleton organization
The previous findings showed younger morphological change in aging NPCs modulated by short-term induction of OSKM (Figure 2d).
Further analysis was performed to explore whether cytoskeleton was redistributed after the short-term induction of OSKM. Fluorescent staining of F-actin and γ-H2AX revealed higher organized network of fibers in short-term induction aging NPCs consistent with low level of nuclear DNA double-strand breaks. Notably, disorganization of F-actin fibers was observed in aging NPCs which accumulated in the sub-membrane region (Supplementary Figure S4a). In addition, highly organized cytoskeleton consistent with the significant increase in level of H3K9me3 was observed through immunofluo- Notably, induction in the dox4d-4d+4d group restored distribution of fibers. The analysis of mean influorescence intensity of F-actin showed similar results (Figure 4c). Expression of CDC42 and p70 S6 kinase, which are the regulators of cytoskeleton (Ip et al., 2011;Nobes & Hall, 1995), was significantly upregulated at dox4d and at dox4d-4d. Notably, the expression was restored to the aging state at dox4d-8d, and was activated at dox4d-4d+4d (Supplementary Figure S4e, Figure 4f). The cell surface showed a similar trend in Factin fibers after induction with dox ( Figure 4d). These findings imply that induction of OSKM promoted redistribution of cytoskeleton organization. Glycolysis was promoted by short-term induction and reversed to aging level at dox4d-4d and dox4d-8d. However, dox4d-4d+4d activated glycolysis (Figure 5a,c). Notably, OxPhos was activated and showed a significant increase, which is contrary to classical reprogramming. The results showed that dox4d promoted OxPhos process in aging NPCs, and the activation was abrogated at dox4d-4d and dox4d-8d, mainly on the level of basic respiration. Maximal respiration gradually decreased at dox4d-4d and dox4d-8d. However, dox4d-4d+4d activated OxPhos (Figure 5b,d,e). Further analysis was conducted to determine the mechanism underlying the enriched energy metabolism. RNA-seq genes associated with glycolysis and tricarboxylic acid cycle (TAC) were selected for analysis. The findings showed that several respiratory enzymes involved in glycolysis and TAC were significantly upregulated (Figure 5f,g). Analysis showed that hexokinase 2 (HK2) played a crucial role in activating energy metabolism in aging NPCs under the induction of OSKM. The expression of HK2 was significantly upregulated compared with respiratory enzymes (Figure 5h). 3-Bromopyruvic acid (3-Br PA), a specific inhibitor of HK2 was used to verify the function of HK2 in redistribution of cytoskeleton (Huang et al., 2020). Immunofluorescence of oct3/4 and F-actin was performed to determine the level of organization of the cytoskeleton. The results showed more disorganized F-actin fibers after treatment of NPC cells with 3-Br PA under induction of dox ( Figure 5i). Moreover, a significant decrease in number of fibers was observed (Figure 5j). The cell surface was enlarged after addition of 3-Br PA (Figure 5k). These results indicate that the activation of HK2 promoted energy metabolism resulting in redistribution of cytoskeleton in aging NPCs which were induced by OSKM.

| Enhancement of energy metabolism promotes rejuvenated phenotypes in aging NPCs induced by OSKM
Further analysis was conducted to explore whether rejuvenated phe-

| DISCUSS ION
Partial reprogramming is a potential strategy to delay senescent process (Ocampo et al., 2016). The findings of the present study showed that partial reprogramming significantly relieved IDD in a needle puncture model and age-related model. In the current study, 32-G needle was used to induce changes in neutral zone mechanics of IVD without histology changes, ultimately causing a gradual degeneration process. Notably, 4F mice under dox therapy showed less disc damage, which provides a basis for the development of novel therapies for IDD treatment.
NPCs are the key functional cells that maintain homeostasis of the vertebral disc (Bian et al., 2021). Therefore, analysis was performed to explore the changes in aging NPCs to further understand partial reprogramming in vertebral disc. Physiological aging has similar features as RS, including high levels of DNA damage, changes in chromatin conformation, increased ROS production, decreased proliferation, activated apoptosis and initiation of senescence-associated secretory phenotype (SASP) (Boulestreau et al., 2021;Guarente, 2008;Haigis & Sinclair, 2010;Kennedy & Lamming, 2016;Soultoukis & Partridge, 2016;Steffen & Dillin, 2016). The phenotypes associated with aging achieve recovery at different degree, through short-term expression of Yamanaka factors. Moreover, the rejuvenation was achieved through short-term cyclic induction, indicating that partial reprogramming can rejuvenate aging NPCs. Notably, the identity of aging NPCs was not changed at any time point similar to the findings on partial reprogramming in vitro. SOX9 is a marker for determining the fate and differentiation of chondrocyte lineage (Lefebvre & Dvir-Ginzberg, 2017). A previous study reported a positive relationship between SOX9 expression, cellularity, and extracellular matrix in disc, implying that SOX9 plays a crucial role in the function of NPCs (Tsingas et al., 2020). SOX9 level showed parallel increase F I G U R E 5 Enhancement of energy metabolism boosts redistribution of cytoskeleton organization in aging NPCs induced by OSKM. (a,b) ECAR determined by glycolysis stress test (Injection 1: Glucose, Injection 2: Oligomycin, Injection 3: 2-DG), OCR level as determined by MitoStress test (Injection 1: Oligomycin, Injection 2: FCCP, Injection 3: Antimycin A/Rotenone) using Seahorse Instrument. Measurements were performed every 5 min per timepoint for each condition. Data shown in c, d represent mean ± SD for 6 wells, representative of one of the three independent experiments. (c) Quantification of glycolysis from one timepoint in Glycolysis Stress Test (mean ± SD for n = 6 wells). (d,e) Quantification of basal respiration and maximal respiration for each timepoint using MitoStress test (mean ± SD for n = 6 wells). (f,g) RNA sequencing analysis of 4F aging NPCs treated with doxycycline (1 μg/ml) for 4 days compared with untreated 4F aging NPCs. Heatmap and volcano plots showing expression levels of respiratory enzymes associated genes. (h) qPCR analysis of respiratory enzymes associated genes in glycolysis and tricarboxylic acid cycle (TAC) in 4F aging NPCs. (i,j) Immunofluorescence and quantification of F-actin in 4F aging NPCs subjected to short-term expression of OSKM in the presence of 3-Br PA (HK2 specific inhibitor). Scale bar, 25 μm. (k) Quantification of cell area surface in 4F aging NPCs subjected to short-term expression of OSKM in the presence of 3-Br PA (HK2 specific inhibitor). Scale bar, 25 μm. **p < 0.01, ****p < 0.0001 according to one-way ANOVA with Bonferroni correction and decrease with the use and withdrawal of OSKM in the present study. This indicates that OSKM significantly upregulates expression of SOX9. Therefore, short-term induction of OSKM can restore the function of aging NPCs by upregulating SOX9 expression. A significant decrease in levels of catabolism factors such as mmp13 and adamts5 was reported previously (Sarkar et al., 2020). Anabolism F I G U R E 6 Enhancement of energy metabolism promotes rejuvenated phenotypes in aging NPCs induced by OSKM. (a-d) Expression levels of stress response genes in the p53 pathway in 4F aging NPCs subjected to short-term induction of OSKM in the presence of 3-Br PA as determined by qPCR analysis. (e-g) Expression levels of senescence-associated catabolism index MMP13, ADAMTS5, and anabolism index COL2 in 4F aging NPCs subjected to short-term expression of OSKM in the presence of 3-Br PA as determined by qPCR analysis. (h,i) Immunofluorescence and quantification of γH2AX level in 4F aging NPCs subjected to short-term expression of OSKM in the presence of 3-Br PA. Scale bar, 25 μm. (j,k) Immunofluorescence and quantification of H3K9me3 level in 4F aging NPCs subjected to short-term expression of OSKM in the presence of 3-Br PA. Scale bar, 25 μm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 according to unpaired two-sample Student's t-test factors such as collagen II, which directly reflects the function of NPCs had higher expression at dox2d time point than that at dox4d. Moreover, the higher expression of collagen II was observed shortterm after induction (dox4d-4d) than the level at dox4d indicating that lasting induction affects expression of collagen II. Induction of OSKM enhanced aging NPCs proliferation. However, long-term induction was unfavorable for function recovery. Induction-activated proliferation may impede collagen II synthesis; thus, short-term cyclic induction should be used to balance proliferation and function recovery in aging NPCs.
Cytoskeleton plays a key role in maintaining cell shape and elasticity as well as cell division and proliferation (Ikeda et al., 2003).
Short-term cyclic induction of Yamanaka factors promotes reorganization of disordered cytoskeleton, and restores cell proliferation (Jeganathan et al., 2016). A well-organized cytoskeleton in NPCs ensures that discs endure compression stress and prevent IVDD progression (Ke et al., 2021). Partial reprogramming is thus a potential novel method for reorganizing disordered cytoskeleton, and provides new insights into development of effective therapy for inhibiting IVD degeneration. Furthermore, energy metabolism pathway showed significant changes during partial reprogramming in aging NPCs, compared with classical reprogramming (Zhang et al., 2012). Glycolysis and OxPhos pathways were significantly enriched. Cyclic induction results demonstrated that OSKM acted like a "energy switch" to initiate energy metabolism. These changes can be attributed to the aging NPCs state because glycolysis and OxPhos levels are significantly low in aging cells (Azzu & Valencak, 2017). Aging NPCs have no enough energy to achieve rejuvenation unlike young cells in classical reprogramming. Partial reprogramming provides enough energy for the proliferation of aging NPCs.
Short-term induction of OSKM can be used to improve energy metabolism in aging NPCs and rescue its state. RNA sequence analysis between aging NPCs and induced aging NPCs showed that HK2 expression was significantly high in partial reprogramming process, implying that HK2 plays a key role in initiating energy metabolism during partial reprogramming. Therefore, HK2 is a potential target for activating energy metabolism in aging NPCs and for treatment of IDD.
In summary, the findings of the present study show that partial reprogramming through short-term cyclic induced OSKM in vivo abrogates progression of IDD. Mechanistically, partial reprogramming in vitro promotes energy metabolism as a "energy switch" by upregulating expression of hexokinase 2 (HK2) ultimately promoting redistribution of cytoskeleton and rejuvenating aging state in aging NPCs. Further studies should explore the relationship between proliferation and function recovery, and other effect factors in partial reprogramming to achieve rejuvenation of aging cells and provide a basis for senescence treatment.

| E XPERIMENTAL PROCEDURE S
Experimental procedures are described in the Supplementary S2.

ACK N OWLED G EM ENTS
This study was supported by grants from the National Natural Science Foundation of China (NO. 82072481), the health foundation (WKJ-ZJ-1903) of Zhejiang Province; and National Natural Science Foundation of China (NO. 82002327).

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.