Electrical stimulation induces direct reprogramming of human dermal fibroblasts into hyaline chondrogenic cells
Introduction
Articular cartilage is a hyaline cartilage which consists of chondrocytes and extracellular matrix composed of types II collagens, proteoglycans, and other matrix proteins. Because articular cartilage cannot spontaneously regenerate in vivo, the loss of cartilage due to trauma or aging-induced degeneration generally results in osteoarthritis [[1], [2], [3]]. Although many studies have developed various techniques to repair cartilage tissues, most current cartilage repair techniques eventually lead to the formation of fibrocartilage that expresses types I and II collagen [4,5]. However, because the presence of type I collagen impairs the development of cartilage-specific matrix architecture and mechanical function, fibrocartilage expressing high levels of type I collagen results in morbidity and functional impairment of articular cartilage. Therefore, the regeneration of hyaline cartilage which does not express type I collagen is essential for the cartilage repair [5,6].
A large number of hyaline chondrogenic cells can be produced by chondrogenesis of stem cells. However, embryonic stem cells and induced pluripotent stem (iPS) cells have potential for tumorigenicity due to the possible presence of residual undifferentiated cells and thus significantly restrict their use in clinical application [7]. To overcome the clinical risks, direct reprogramming methods in which somatic cells, such as fibroblasts, are reprogrammed directly into induced desired functional cell types without the pluripotency stage have been developed [8]. Moreover, the direct reprogramming has the advantages such as rapid production of customized cell types through direct conversion, easiness to obtain sufficient cell amounts and avoidability of immune response because of ability to use the somatic cells such as skin derived fibroblasts, which and can expand easily and generate autologous target cells, as cell sources for direct reprogramming [9]. For these advantages, many studies revealed that fibroblasts could be converted to several lineages including neurons, cardiomyocytes, and hepatocytes by ectopic expression of target specific transcription factors, application of mRNAs, and exposure to small molecules [[10], [11], [12]]. Recently, a previous study also showed that gene transduction of two reprogramming factors (c-Myc, Klf4) and SOX9 directly induced dermal fibroblast to transdifferentiate into chondrogenic cells without transition through a pluripotent state and form histologically homogenous hyaline cartilage [13]. However, although the direct reprogramming avoids the pluripotent state, the gene integration induced teratoma formation probably due to gene transduction-derived genomic instability [14]. Moreover, the direct reprogramming via gene transduction show low efficiency of induction and thus take a lot of time and high cost to obtain enough amount of cell sources for cell therapy [9]. Therefore, the new strategy is required for developing direct reprogramming technology with high safety, low cost and high efficiency for future clinical application.
Recently, there are increasing evidences that physical factors have an influence in determining the fates of stem cells. It was reported that electrical stimulation (ES) and electromagnetic field (EMF) inducers improve healing of cartilage defects by increasing cell proliferation, glycosaminoglycan synthesis, and the expression of extracellular matrix genes, and by reducing the production of inflammatory mediators [[15], [16], [17]]. Moreover, our recent studies demonstrated that ES can drive chondrogenesis of mesenchymal stem cells with higher efficiency than chondrogenic growth factors [18]. These data implicate that ES may be the potential inducer for direct reprogramming into hyaline chondrogenic cells. In this study, we found that ES with optimized condition induces human dermal fibroblasts (HDFs) to be reprogrammed directly into hyaline chondrogenic cells without exogenous growth factors or gene transduction.
Section snippets
Cell culture and light microscopy observations
The HDF cells were isolated from adult skin (GIBCO). The cells were cultured in high glucose DMEM (Sigma) supplemented with 10% fetal bovine serum (maintenance medium) in polystyrene dishes at 37 °C under 5% CO2. HDF were cultured in a high-density micromass (2 × 107 cells/ml) and then the growth medium was refilled at 35 mm dishes. After 3 day of culture in the maintenance medium either with or without electrical stimulation (ES). Microscopic observation was performed with a phase contrast
Electrical stimulation induces aggregation of human dermal fibroblasts
It was known that cellular condensation precedes chondrogenesis and represents an important step in the process of cartilage formation during limb skeletogenesis [19,20]. In addition, in vitro studies reported that mesenchymal stem cells are aggregated into large condensations and then differentiated into chondrocytes [21]. Our previous study also showed that the optimized ES induced chondrogenic differentiation through condensation of mesenchymal stem cells [18]. Thus, we speculated that
Discussion
Although the effects of ES has been poorly understood in the context of regenerative medicine, our previous study showed that ES induces mesenchymal stem cells to differentiate into chondrogenic cells. Herein, we have demonstrated that ES drives direct reprogramming of HDFs into hyaline chondrogenic cells by significantly increasing the expression of chondrogenic markers and decreasing the expression of the fibroblastic marker in HDFs (Fig. 2). Since autologous chondrocytes transplantation
Acknowledgements
This work was supported by Eulji University in 2015 and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science (NRF-2016R1D1A1B03935005).
References (34)
Treatment of a full-thickness articular cartilage defect in the femoral condyle of an athlete with autologous bone-marrow stromal cells
Osteoarthritis Cartilage
(2007)Cartilage healing after microfracture in osteoarthritic knees
Arthroscopy
(2006)Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors
Cell
(2010)Induction of chondrogenic cells from dermal fibroblast culture by defined factors does not involve a pluripotent state
Biochem. Biophys. Res. Commun.
(2011)- et al.
Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components
Exp. Cell Res.
(2001) Gene-induced chondrogenesis of primary mesenchymal stem cells in vitro
Mol. Ther.
(2005)Cartilage repair: generations of autologous chondrocyte transplantation
Eur. J. Radiol.
(2006)Platelet-derived growth factor A modulates limb chondrogenesis both in vivo and in vitro
Mech. Dev.
(2000)Molecular interactions of the IGF system
Cytokine Growth Factor Rev.
(2005)The healing and regeneration of articular cartilage
J. Bone Joint Surg. Am
(1988)
Articular cartilage lesions of the knee
Am. J. Sports Med.
Knee osteoarthritis prevalence, risk factors, pathogenesis and features: part I
Caspian J. Intern. Med.
Arthroscopic subchondral bone plate microfracture technique augments healing of large chondral defects in the radial carpal bone and medial femoral condyle of horses
Vet. Surg.
Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies
Nat. Med.
The emergence of direct programming and the loss of cell fate hierarchies
Nat. Rev. Mol. Cell Biol.
Direct reprogramming of adult cells: avoiding the pluripotent state
Stem Cells Cloning
Direct conversion of fibroblasts to functional neurons by defined factors
Nature
Cited by (18)
Polypyrrole-based structures for activation of cellular functions under electrical stimulation
2024, BioelectrochemistryNovel method to repair articular cartilage by direct reprograming of prechondrogenic mesenchymal stem cells
2021, European Journal of PharmacologyCitation Excerpt :Thus, it is essential to develop effective and safe methods to differentiate MSCs. Increasing evidence suggests that ES that influences the fate of stem cells and is more efficient than chondrogenic growth factors (Kwon et al., 2016; Lee et al., 2019). However, the clinical application of this direct reprogramming warrants further work.
From wearables to implantables-clinical drive and technical challenges
2020, Wearable Sensors: Fundamentals, Implementation and ApplicationsTargeting cell plasticity for regeneration: From in vitro to in vivo reprogramming
2020, Advanced Drug Delivery ReviewsCitation Excerpt :Fibroblasts have been reported to more efficiently convert to functional cardiomyocytes in response to nanotopographical cues in 2D [188], and to an increased expression of matrix metalloproteinases (MMPs) when confined in the fibrin based 3D hydrogel environment [189]. Exposure of dermal fibroblasts to electrical stimulation resulted in increased secretion of several growth factors including TGF-β1, PDGF-AA, and IGFBP-2, 3, and facilitated its reprogramming to hyaline cartilaginous tissue [190] and their stimulation using radio electric conveyed fields (RECF) resulted in their conversion to several cell types including neural, cardiac and skeletal muscle like cells [191]. Direct conversion of somatic cells into target somatic tissues helps scrutinize the basis of cellular plasticity in a physiological context.