Footprint-free human induced pluripotent stem cells from articular cartilage with redifferentiation capacity: a first step toward a clinical-grade cell source.

Human induced pluripotent stem cells (iPSCs) are potential cell sources for regenerative medicine; however, clinical applications of iPSCs are restricted because of undesired genomic modifications associated with most reprogramming protocols. We show, for the first time, that chondrocytes from autologous chondrocyte implantation (ACI) donors can be efficiently reprogrammed into iPSCs using a nonintegrating method based on mRNA delivery, resulting in footprint-free iPSCs (no genome-sequence modifications), devoid of viral factors or remaining reprogramming molecules. The search for universal allogeneic cell sources for the ACI regenerative treatment has been difficult because making chondrocytes with high matrix-forming capacity from pluripotent human embryonic stem cells has proven challenging and human mesenchymal stem cells have a predisposition to form hypertrophic cartilage and bone. We show that chondrocyte-derived iPSCs can be redifferentiated in vitro into cartilage matrix-producing cells better than fibroblast-derived iPSCs and on par with the donor chondrocytes, suggesting the existence of a differentiation bias toward the somatic cell origin and making chondrocyte-derived iPSCs a promising candidate universal cell source for ACI. Whole-genome single nucleotide polymorphism array and karyotyping were used to verify the genomic integrity and stability of the established iPSC lines. Our results suggest that RNA-based technology eliminates the risk of genomic integrations or aberrations, an important step toward a clinical-grade cell source for regenerative medicine such as treatment of cartilage defects and osteoarthritis.

Nanosized fibers' effect on adult human articular chondrocytes behavior.

Tissue engineering with chondrogenic cell based therapies is an expanding field with the intention of treating cartilage defects. It has been suggested that scaffolds used in cartilage tissue engineering influence cellular behavior and thus the long-term clinical outcome. The objective of this study was to assess whether chondrocyte attachment, proliferation and post-expansion re-differentiation could be influenced by the size of the fibers presented to the cells in a scaffold. Polylactic acid (PLA) scaffolds with different fiber morphologies were produced, i.e. microfiber (MS) scaffolds as well as nanofiber-coated microfiber scaffold (NMS). Adult human articular chondrocytes were cultured in the scaffolds in vitro up to 28 days, and the resulting constructs were assessed histologically, immunohistochemically, and biochemically. Attachment of cells and serum proteins to the scaffolds was affected by the architecture. The results point toward nano-patterning onto the microfibers influencing proliferation of the chondrocytes, and the overall 3D environment having a greater influence on the re-differentiation. In the efforts of finding the optimal scaffold for cartilage tissue engineering, studies as the current contribute to the knowledge of how to affect and control chondrocytes behavior.

Bipolar radiofrequency plasma ablation induces proliferation and alters cytokine expression in human articular cartilage chondrocytes.

PURPOSE: The aim of the study was to determine the in vitro effects of plasma-mediated bipolar radiofrequency ablation on human chondrocyte compensatory proliferation and inflammatory mediator expression. METHODS: Human articular cartilage biopsy specimens, from total knee replacement, and human chondrocytes in alginate culture, from patients undergoing autologous chondrocyte implantation, were exposed to plasma ablation with a Paragon T2 probe (ArthroCare, Austin, TX). Instantaneous chondrocyte death was investigated with live/dead assays of biopsy specimens and cell cultures. Chondrocyte proliferation was determined by Hoechst staining of DNA on days 3 and 6. Messenger RNA expression of IL-1ß, IL-6, IL-8, tumor necrosis factor α, high-mobility group protein B1, matrix metalloproteinase 13, type IIA collagen, and versican was determined on days 3 and 6. RESULTS: Live/dead imaging showed a well-defined local margin of cell death ranging from 150 to 200 µm deep, both in the alginate gel and in the biopsy specimens exposed to plasma ablation. The ablation-exposed group showed a significant proliferation increase compared with control on day 3 (P < .043). There were significant increases compared with control in IL-6 expression on day 3 (P < .020) and day 6 (P < .045) and in IL-8 expression on day 3 (P < .048). No differences were seen for IL-1ß, tumor necrosis factor α, high-mobility group protein B1, matrix metalloproteinase 13, type II collagen, or versican. CONCLUSIONS: This study has shown that exposure to plasma-mediated ablation induces a well-defined area of immediate cell death and a short-term increase in proliferation with human articular chondrocytes in vitro. The exposure also alters cytokine expression for the same period, causing upregulation of IL-6 and IL-8. CLINICAL RELEVANCE: The results show the potential of plasma-mediated ablation to cause the onset of a tissue regeneration response with human articular cartilage.

Triphasic and quadriphasic waveforms are superior to biphasic waveforms for synchronized beating of cardiomyocytes.

BACKGROUND AND PURPOSE: Within pacemaker research few attempts have been made to find an optimal waveform phase sequence that synchronizes beating of cardiomyocytes at an electrode. Multielectrode arrays (MEAs) offer electrophysiological screening of cardiomyocytes serving as a system for preliminary screening of pacing waveform design. MATERIALS AND METHODS: The HL-1 cell line was cultured in MEAs until confluence and stimulated with biphasic, triphasic, and quadriphasic waveforms. The amplitudes required for synchronized beating of the cells were determined. RESULTS: Triphasic and quadriphasic waveforms were more efficient in eliciting synchronized beating of the HL-1 cells compared with the biphasic waveform because it allows significant reductions in synchronizing voltage amplitudes and reductions in supplied stimulus. CONCLUSION: The MEA system allows for a straightforward manner to investigate effects of waveform design on synchronized beating in cardiomyocytes in vitro. Increased number of phase changes in a pacing waveform seems to be the major reason for the reduction in synchronizing amplitudes.

Optimization of a chondrogenic medium through the use of factorial design of experiments.

The standard culture system for in vitro cartilage research is based on cells in a three-dimensional micromass culture and a defined medium containing the chondrogenic key growth factor, transforming growth factor (TGF)-ß1. The aim of this study was to optimize the medium for chondrocyte micromass culture. Human chondrocytes were cultured in different media formulations, designed with a factorial design of experiments (DoE) approach and based on the standard medium for redifferentiation. The significant factors for the redifferentiation of the chondrocytes were determined and optimized in a two-step process through the use of response surface methodology. TGF-ß1, dexamethasone, and glucose were significant factors for differentiating the chondrocytes. Compared to the standard medium, TGF-ß1 was increased 30%, dexamethasone reduced 50%, and glucose increased 22%. The potency of the optimized medium was validated in a comparative study against the standard medium. The optimized medium resulted in micromass cultures with increased expression of genes important for the articular chondrocyte phenotype and in cultures with increased glycosaminoglycan/DNA content. Optimizing the standard medium with the efficient DoE method, a new medium that gave better redifferentiation for articular chondrocytes was determined.