Abstract
Pathophysiology of osteoarthritis (OA) is characterized by progressive loss of articular cartilage in the knee-joints. To impart regenerative ability in lowly metabolizing chondrocytes, the bone marrow stem cells (BMSCs) has recently been recognized as a superior alternative treatment for OA. However, study of primary BMSCs-mediated chondrogenesis is difficult due to progressive cellular aging and replicative senescence. To obtain a therapeutic cell population for OA, BMSCs were immortalized by human papilloma virus (HPV)-16 E6/E7 along with mCherry luciferase (mCL), a gene marker for non-invasive imaging, and designated as iBMSCs-mCL. Next, their cell morphology, population doubling time (PDT) and colony forming ability (CFU) were evaluated. Furthermore, pluripotency and immunophenotypic markers were investigated. To deduce therapeutic ability, iBMSCs-mCL were intra-articularly injected into right knee of anterior cruciate ligament transaction (ACLT)-OA mice model and tracked through non-invasive bioluminescence imaging. Cell morphology of iBMSCs-mCL was similar to parental BMSCs. PDT and CFU ability of iBMSCs-mCLs were significantly increased. Pluripotency and immunophenotypic markers were highly expressed in iBMSC-mCL. Long-term survival and tri-lineage differentiation particularly chondrogenic potential of iBMSCs-mCL were also demonstrated in vitro and then in vivo which was monitored through non-invasive imaging. Intensive bioluminescent signals in iBMSCs-mCL administered knee-joint indicated a marked in vivo survival and proliferation of iBMSCs-mCL. Immunohistochemical staining for type II collagen (IHC of Col II) and alcian blue & safranin o staining of proteoglycans also corroborated cartilage regeneration by iBMSCs-mCL. Conclusively, iBMSCs-mCL maintains stemness and in vivo cartilage regeneration potential suggesting a promising avenue for development of OA therapeutics.
Highlights
Osteoarthritis (OA) is characterized by the senescence of articular chondrocytes with aging and deterioration of cartilaginous matrix contributing to structural aberrations [1]
The immortalized Bone marrow-derived Mesenchymal stem cells (MSCs) (BMSCs) were designated as immortalized bone marrow stem cells (iBMSCs) and further transduced with imaging selection markers including luciferase and mCherry
The iBMSCs and iBMSCs-mCherry luciferase (mCL) both displayed a spindleshaped and fibroblast-like morphology at passage 25 resembling the parental BMSCs at passage 1, and showed bioluminescence signal (Figure 1A)
Summary
Osteoarthritis (OA) is characterized by the senescence of articular chondrocytes with aging and deterioration of cartilaginous matrix contributing to structural aberrations [1]. Due to lack of vasculature, the articular cartilage possessing sparse population of chondrocytes is unable to mount an adequate healing response to cartilaginous injury. Bone marrow-derived MSCs (BMSCs) can be induced to chondrogenic lineage under various cultural conditions [3,4,5]. BMSCs could lose its phenotype, differentiation potential and the termination of proliferation during long-term in vitro cultures [6]. The limited life span of stem cells represents a hurdle in pre-clinical investigation and therapeutic development. To overcome such limitations, attempts have been made to generate cell lines displaying stable stem cell phenotypes and unlimited proliferation
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