Article

Prolonged Expansion of Human Nucleus Pulposus Cells Expressing Human Telomerase Reverse Transcriptase Mediated by Lentiviral Vector

Department of Orthopaedic Surgery, Navy General Hospital, No.6 Fu-cheng Road, Beijing, 100048, PR China.
Journal of Orthopaedic Research (Impact Factor: 2.99). 01/2014; 32(1). DOI: 10.1002/jor.22474
Source: PubMed

ABSTRACT

Human degenerative disc disease (DDD) is characterized by progressive loss of human nucleus pulposus (HNP) cells and extracellular matrix, in which the massive deposition are secreted by HNP cells. Cell therapy to supplement HNP cells to degenerated discs has been thought to be a promising strategy to treat DDD. However, obtaining a large quality of fully functional HNP cells has been severely hampered by limited proliferation capacity of HNP cells in vitro. Previous studies have used lipofectamine or recombinant adeno-associated viral (rAAV) vectors to deliver human telomerase reverse transcriptase (hTERT) into ovine or HNP cells to prolong the activity of nucleus pulposus cells with limited success. Here we developed a lentiviral vector bearing both hTERT and a gene encoding green fluorescence protein (L-hTERT/EGFP). This vector efficiently mediated both hTERT and EGFP into freshly isolated HNP cells. The expressions of both transgenes in L-hTERT/EGFP transduced HNP cells were detected up to day 210 post viral infection, which was twice as long as rAAV vector did. Furthermore, we observed restored telomerase activity, maintained telomere length, delayed cell senescence, and increased cell proliferation rate in those L-hTERT/EGFP transduced HNP cells. Our study suggests that lentiviral vector might be a useful gene delivery vehicle for HNP cell therapy to treat DDD. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res XX:XXX-XXX, 2013.

Full-text preview

Available from: onlinelibrary.wiley.com
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cell-based therapies may hold significant promise for the treatment of early stage degeneration of the intervertebral disc (IVD). Given their propensity to proliferate and ability to form multiple tissue types, mesenchymal stem cells (MSCs) have been proposed as a potential cell source to promote repair of the nucleus pulposus (NP). However, for any successful cell-based therapy, a carrier biomaterial may be essential for targeted delivery providing key biophysical and biochemical cues to facilitate differentiation of MSCs. Two widely used biomaterials for NP regeneration are chitosan and alginate. The primary objective of this study was to assess the influence of alginate and chitosan hydrogels on bone marrow stem cells (BM) and NP cells in isolation or in coculture. A secondary objective of this study was to investigate coculture seeding density effects of BM and NP cells and simultaneously explore which cell type is responsible for matrix formation in a cocultured environment. Porcine NP and BM cells were encapsulated in alginate and chitosan hydrogels separately at two seeding densities (4x10(6) and 8x10(6) cells/mL) or in coculture (1:1, 8x10(6) cells/mL). Constructs (diameter=5 mm, height=3 mm) were maintained under IVD-like conditions [low-glucose, low (5%) oxygen] with or without transforming growth factor-beta 3 (TGF-beta 3) supplementation for 21 days. Results demonstrated differential viability depending on hydrogel type. NP cells remained viable in both biomaterial types whereas BM viability was diminished in chitosan. Further, hydrogel type was found to regulate sulfated glycosaminoglycan (sGAG) and collagen accumulation. Specifically, alginate better supports sGAG accumulation and collagen type II deposition for both NP and BM cell types compared with chitosan. Having identified that alginate more readily supports cell viability and matrix accumulation, we further explored additional effects of seeding density ratios (NP:BM-1:1, 1:2) for coculture studies. Interestingly, in coculture conditions, the BM cell population declined in number while NP cells increased, indicating that MSCs may in fact be signaling NP cells to proliferate rather than contributing to matrix formation. These findings provide exciting new insights on the potential of MSCs for NP tissue regeneration strategies.
    Full-text · Article · Sep 2014 · Tissue Engineering Part A
  • [Show abstract] [Hide abstract]
    ABSTRACT: As a main contributing factor to low back pain, intervertebral disc degeneration (IDD) is the fundamental basis for various debilitating spinal diseases. The pros and cons of current treatment modalities necessitate biological treatment strategies targeting for reversing or altering the degeneration process in terms of molecules or genes. The advances in stem cell research facilitate the studies aiming for possible clinical application of stem cell therapies for IDD. Human NP cells are versatile with cell morphology full of variety, capable of synthesizing extracellular matrix components, engulfing substances by autophagy and phagocytosis, mitochondrial vacuolization indicating dysfunction, expressing Fas and FasL as significant omens of immune privileged sites. Human discs belong to immune privilege organs with functional FasL expression, which can interact with invasive immune cells by Fas-FasL regulatory machinery. IDD is characterized by decreased expression level of FasL with dysfunctional FasL, which in turn unbalances the interaction between NP cells and immune cells. Certain modulation factors might play a role in the process, such as miR-155. Accumulating evidence indicates that Fas-FasL network expresses in a variety of stem cells. Given the expression of functional FasL and insensitive Fas in stem cells (we term as FasL privilege), transplantation of stem cells into the disc may regenerate the degenerative disc by not only differentiating into NP-like cells, increasing extracellular matrix, but also reinforce immune privilege via interaction with immune cells by Fas-FasL network.
    No preview · Article · Nov 2014 · Current Stem Cell Research & Therapy
  • [Show abstract] [Hide abstract]
    ABSTRACT: Intervertebral disc (IVD) degeneration is a complicated process that involves both age-related change and tissue damage caused by multiple stresses. In a degenerative IVD, cellular senescence accumulates and is associated with reduced proliferation, compromised self-repair, increased inflammatory response, and enhanced catabolic metabolism. In this review, we decipher the senescence mechanism of IVD degeneration by interpreting how aging coordinates with age-related, microenvironment-derived stresses in promoting disc cell senescence and accelerating IVD degeneration. After chronic and prolonged replication, cell senescence may occur as a natural part of the disc aging process, but can potentially be accelerated by growth factor deficiency, oxidative accumulation, and inflammatory irritation. While acute disc injury, excessive mechanical overloading, diabetes, and chronic tobacco smoking contribute to the amplification of senescence-inducing stresses, the avascular nature of IVD impairs the immune-clearance of the senescent disc cells, which accumulate in cell clusters, demonstrate inflammatory and catabolic phenotypes, deteriorate disc microenvironment, and accelerate IVD degeneration. Anti-senescence strategies, including telomerase transduction, supply of growth factors, and blocking cell cycle inhibitors, have been shown to be feasible in rescuing disc cells from early senescence, but their efficiency for disc regeneration requires more in vivo validations. Guidelines dedicated to avoiding or alleviating senescence-inducing stresses might decelerate cellular senescence and benefit patients with IVD degenerative diseases.
    No preview · Article · Oct 2015 · Osteoarthritis and Cartilage
Show more