Partial reprogramming strategy for intervertebral disc rejuvenation by activating energy switch

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.     

Senescence promotes in vivo reprogramming through p16INK4a and IL‐6

Cellular senescence is a damage response aimed to orchestrate tissue repair. We have recently reported that cellular senescence, through the paracrine release of interleukin‐6 (IL6) and other soluble factors, strongly favors cellular reprogramming by Oct4, Sox2, Klf4, and c‐Myc (OSKM) in nonsenescent cells. Indeed, activation of OSKM in mouse tissues triggers senescence in some cells and reprogramming in other cells, both processes occurring concomitantly and in close proximity. In this system, Ink4a/Arf‐null tissues cannot undergo senescence, fail to produce IL6, and cannot reprogram efficiently; whereas p53‐null tissues undergo extensive damage and senescence, produce high levels of IL6, and reprogram efficiently. Here, we have further explored the genetic determinants of in vivo reprogramming. We report that Ink4a, but not Arf, is necessary for OSKM‐induced senescence and, thereby, for the paracrine stimulation of reprogramming. However, in the absence of p53, IL6 production and reprogramming become independent of Ink4a, as revealed by the analysis of Ink4a/Arf/p53 deficient mice. In the case of the cell cycle inhibitor p21, its protein levels are highly elevated upon OSKM activation in a p53‐independent manner, and we show that p21‐null tissues present increased levels of senescence, IL6, and reprogramming. We also report that Il6‐mutant tissues are impaired in undergoing reprogramming, thus reinforcing the critical role of IL6 in reprogramming. Finally, young female mice present lower efficiency of in vivo reprogramming compared to male mice, and this gender difference disappears with aging, both observations being consistent with the known anti‐inflammatory effect of estrogens. The current findings regarding the interplay between senescence and reprogramming may conceivably apply to other contexts of tissue damage.     

Proliferation Rate of Somatic Cells Affects Reprogramming Efficiency

The discovery of induced pluripotent stem (iPS) cells provides not only new approaches for cell replacement therapy, but also new ways for drug screening. However, the undefined mechanism and relatively low efficiency of reprogramming have limited the application of iPS cells. In an attempt to further optimize the reprogramming condition, we unexpectedly observed that removing c-Myc from the Oct-4, Sox-2, Klf-4, and c-Myc (OSKM) combination greatly enhanced the generation of iPS cells. The iPS cells generated without c-Myc attained salient pluripotent characteristics and were capable of producing full-term mice through tetraploid complementation. We observed that forced expression of c-Myc induced the expression of many genes involved in cell cycle control and a hyperproliferation state of the mouse embryonic fibroblasts during the early stage of reprogramming. This enhanced proliferation of mouse embryonic fibroblasts correlated negatively to the overall reprogramming efficiency. By applying small molecule inhibitors of cell proliferation at the early stage of reprogramming, we were able to improve the efficiency of iPS cell generation mediated by OSKM. Our data demonstrated that the proliferation rate of the somatic cell plays critical roles in reprogramming. Slowing down the proliferation of the original cells might be beneficial to the induction of iPS cells.     

c-Myc对诱导性多能干细胞的诱导效率及全能性影响的研究

该研究探讨转录因子c—Myc对多能诱导干细胞（inducedpluripotentstemcells,iPS）诱导效率及形成的iPS克隆全能性的影响。将Yam,dnaka四因子（Oct4、Sox2、Klf4和C—Myc,OSKM）和不舍C。Myc的三因子（OSK）病毒分别感染OG2小鼠成纤维细胞（mouseembryofibroblast,MEF）,诱导成为iPSN胞,通过计数iPS克隆形成数目和流式细胞仪分析iPS克隆的绿色荧光蛋白（GFP）阳性比例。比较OSKM和OSK诱导iPS的效率。分别挑取三株OSK和OSKM诱导的iPS克隆,采用荧光定量PCR法、碱性磷酸酶fAP）染色法和免疫荧光法检测iPS克隆的干性相关蛋白的表达;采用畸胎瘤实验、嵌合体形成实验和四倍体补偿实验检测iPS克隆的全能性。结果显示：在该实验室诱导体系里,OSK病毒感染MEF后形成的iPS克隆数目明显增多,GFP阳性克隆的比例增加。OSK和OSKM诱导iPS克隆的干细胞基本特征,包括形态、AP染色、干细胞特异性基因表达、三胚层形成均没有明显差异。然而,OSK诱导iPS克隆在形成嵌合体小鼠的全能性评估实验中的阳性比例高于OSKM克隆,且只有OSK克隆株能够获得生殖系传递小鼠和四倍体补偿小鼠。该研究表明,在实验诱导体系里去除C—Myc的三因子OSK诱导iPS的效率显著高于OSKM四因子,且三因子诱导iPS克隆具有更好的全能性。     

Activation of DNA demethylases attenuates aging‐associated arterial stiffening and hypertension

DNA methylation increases with age. The objective of this study was to investigate whether compound H, a potential activator of DNA demethylases, attenuates aging‐related arterial stiffness and hypertension. Aged mice (24–27 months) and adult mice (12 months) were used. Pulse wave velocity (PWV), a direct measure of arterial stiffness, and blood pressure (BP) were increased significantly in aged mice. Notably, daily treatments with compound H (15 mg/kg, IP) for 2 weeks significantly attenuated the aging‐related increases in PWV and BP. Compound H abolished aging‐associated downregulation of secreted Klotho (SKL) levels in both kidneys and serum likely by enhancing DNA demethylase activity and decreasing DNA methylation. Aging‐related arterial stiffness was associated with accumulation of stiffer collagen and degradation of compliant elastin which are accompanied by increased expression of MMP2, MMP9, TGF‐β1, and TGF‐β3. These changes were effectively attenuated by compound H, suggesting rejuvenation of aged arteries. Compound H also rescued downregulation of Sirt1 deacetylase, AMPKα, and eNOS activities in aortas of aged mice. In cultured smooth muscle cells (SMCc) Klotho‐deficient serum upregulated expression of MMPs and TGFβ which, however, was not affected by compound H. In conclusion, compound H attenuates aging‐associated arterial stiffness and hypertension by activation of DNA demethylase which increases renal SKL expression and consequently circulating SKL levels leading to activation of the Sirt1‐AMPK‐eNOS pathway in aortas of aged mice.     

mTOR-regulated senescence and autophagy during reprogramming of somatic cells to pluripotency: A roadmap from energy metabolism to stem cell renewal and aging

Molecular controllers of the number and function of tissue stem cells may share common regulatory pathways for the nuclear reprogramming of somatic cells to become induced Pluripotent Stem Cells (iPSCs). If this hypothesis is true, testing the ability of longevity-promoting chemicals to improve reprogramming efficiency may provide a proof-of-concept validation tool for pivotal housekeeping pathways that limit the numerical and/or functional decline of adult stem cells. Reprogramming is a slow, stochastic process due to the complex and apparently unrelated cellular processes that are involved. First, forced expression of the Yamanaka cocktail of stemness factors, OSKM, is a stressful process that activates apoptosis and cellular senescence, which are the two primary barriers to cancer development and somatic reprogramming. Second, the a priori energetic infrastructure of somatic cells appears to be a crucial stochastic feature for optimal successful routing to pluripotency. If longevity-promoting compounds can ablate the drivers and effectors of cellular senescence while concurrently enhancing a bioenergetic shift from somatic oxidative mitochondria toward an alternative ATP-generating glycolytic metabotype, they could maximize the efficiency of somatic reprogramming to pluripotency. Support for this hypothesis is evidenced by recent findings that well-characterized mTOR inhibitors and autophagy activators (e.g., PP242, rapamycin and resveratrol) notably improve the speed and efficiency of iPSC generation. This article reviews the existing research evidence that the most established mTOR inhibitors can notably decelerate the cellular senescence that is imposed by DNA damage-like responses, which are somewhat equivalent to the responses caused by reprogramming factors. These data suggest that fine-tuning mTOR signaling can impact mitochondrial dynamics to segregate mitochondria that are destined for clearance through autophagy, which results in the loss of mitochondrial function and in the accelerated onset of the glycolytic metabolism that is required to fuel reprogramming. By critically exploring how mTOR-regulated senescence, bioenergetic infrastructure and autophagy can actively drive the reprogramming of somatic cells to pluripotency, we define a metabolic roadmap that may be helpful for designing pharmacological and behavioral interventions to prevent or retard the dysfunction/exhaustion of aging stem cell populations.     

Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells

The discovery of direct cell reprogramming and induced pluripotent stem (iPS) cell technology opened up new avenues for the application of non-viral, transposon-based gene delivery systems. The Sleeping Beauty (SB) transposon is highly advanced for versatile genetic manipulations in mammalian cells. We established iPS cell reprogramming of mouse embryonic fibroblasts and human foreskin fibroblasts by transposition of OSKM (Oct4, Sox2, Klf4 and c-Myc) and OSKML (OSKM + Lin28) expression cassettes mobilized by the SB100X hyperactive transposase. The efficiency of iPS cell derivation with SB transposon system was in the range of that obtained with retroviral vectors. Co-expression of the miRNA302/367 cluster together with OSKM significantly improved reprogramming efficiency and accelerated the temporal kinetics of reprogramming. The iPS cells displayed a stable karyotype, and hallmarks of pluripotency including expression of stem cell markers and the ability to differentiate into embryoid bodies in vitro. We demonstrate Cre recombinase-mediated exchange allowing simultaneous removal of the reprogramming cassette and targeted knock-in of an expression cassette of interest into the transposon-tagged locus in mouse iPS cells. This strategy would allow correction of a genetic defect by site-specific insertion of a therapeutic gene construct into ‘safe harbor’ sites in the genomes of autologous, patient-derived iPS cells.     

Reprogramming of HeLa cells upon DNMT3L overexpression mimics carcinogenesis

Previously we had discovered loss of DNA methylation at the DNMT3L promoter, an enzymatically-inactive DNA methyltransferase, in squamous cell carcinoma of cervix indicating association between cancer and DNMT3L. This study extends this correlation further by identifying the role of DNMT3L in nuclear reprogramming, an event central to the process of carcinogenesis. We show that in cervical cancer cell lines, overexpression of DNMT3L, which functions by regulating the activity of DNMT3A and DNMT3B, increased cellular proliferation and anchorage-independent growth. Importantly, increased DNMT3L expression resulted in changed morphology of cells but this change was gradual and observed only after several passages. Interestingly, confluent cultures of DNMT3L-overexpressing HeLa cell colonies had characteristics of iPS cells. Concomitant with the morphological changes, expression pattern of genes important in nuclear reprogramming, development and cell cycle were observed to have significantly changed. Many imprinted genes, the known targets of DNMT3L, were downregulated. The slow nature of morphological changes and genome-wide nuclear reprogramming observed upon DNMT3L overexpression reinforces its role in carcinogenesis.     

Cuttine edge: T cells from aged mice are resistant to depletion early during virus infection

Aging is associated with decreased expansion of T cells upon stimulation. In young mice, infection induces a transient T cell depletion followed by the development of an Ag-specific T cell response that controls the infection. We found that T cells were depleted early after infection with E55 + murine leukemia retrovirus in young, but not aged, mice. Adoptive transfer experiments showed donor T cells of young, but not aged, mice were depleted due to apoptosis in various tissues of young recipients. However, T cells of neither young nor aged donors were depleted in aged recipients. These results indicate that both environmental and intrinsic cellular properties limit depletion of T cells of aged mice and suggest a novel explanation for the decreased T cell response associated with aging.