In vivo MR imaging of magnetically labeled human embryonic stem cells.

INTRODUCTION: Human embryonic stem cells (hES) have emerged as a potentially new therapeutic approach for treatment of heart and other diseases applying the concept of regenerative medicine. A method for in vivo visualization and tracking of transplanted hES would increase our understanding of in vivo hES behavior in both experimental and clinical settings. The aim of this study was to evaluate the feasibility of magnetic labeling and visualization of hES with magnetic resonance imaging (MRI). METHODS: hES were established and expanded according to standard procedures. After expansion, the cells were cultured under feeder free conditions and magnetically labeled by addition of dextran-coated Ferrum-oxide particles (Endorem) to the medium. Accumulation of small particles of iron-oxide (SPIO) in hES was assessed by Prussian blue staining and electron microscopy. For in vitro MRI, the labeled and unlabeled hES were examined in cell solution and after transplantation into explanted mouse heart ( approximately 100,000 cells) on a Bruker Avance DMX 500 vertical magnet at 11.75 T. A multi-slice, multi spin-echo T(2)-weighted images were obtained. For in vivo imaging, the experiments were performed on male Sprague-Dawley using Bruker Biospec 2.35 T magnet. The hES were directly injected ( approximately 500,000 cells) after surgical procedure (thoracotomy) into anterior left ventricular (LV) wall. Multi-slice T(2)-weighted gradient echo images were obtained using cardiac gating. RESULTS: hES appeared to be unaffected by magnetic labeling and maintained their ability to proliferate and differentiate. No additive agent for membrane permeabilisation was needed for facilitation of intracellular SPIO accumulation. Prussian blue and electron microscopy have revealed numerous iron particles in the cytoplasm of hES. On T(2)-weighted images, the labeled cells have shown well-defined hyopintense areas at the site of injection in anterior LV wall both in vitro and in vivo. CONCLUSIONS: It is feasible to magnetically label and visualize hES both in vitro and in vivo. MR visualization of magnetically labeled hES may be a valuable tool for in vivo tracking of hES.

Clonal populations of chondrocytes with progenitor properties identified within human articular cartilage.

The aim of the present study was to identify and characterize progenitor properties of human articular chondrocytes selected by using agarose suspension culture. In this chondrogenic selective culture condition, about 3.6% of seeded surplus chondrocytes from patients undergoing articular chondrocyte transplantation proliferated and formed cell clusters after 6 weeks. Phase-contrast microscopy and transmission electron microscopy revealed four different types of cell clusters differing in cellular content and matrix production. Based on their morphological features, they were named the homogenous (H), the homogenous matrix (HM), the differentiated matrix (DM) and the differentiated (D) cell clusters. All cell clusters showed positive safranin O staining, and matrix was positive for antibodies detecting type II collagen and aggrecan. The clusters were further demonstrated to express the genes for fibroblast growth factor receptor 3, type IIA collagen and type IIB collagen, while type X collagen was not expressed. After subcloning, the H and HM clusters demonstrated the best proliferative capacity. Chondrocytes from these two cell clusters also showed phenotypic plasticity in chondrogenic, adipogenic as well as osteogenic assays. This study demonstrates that existing subpopulations of cells with chondroprogenitor properties can be isolated from human adult articular cartilage using agarose suspension cultures.

Phenotypic plasticity of human articular chondrocytes.

BACKGROUND: Progenitor cells in mesenchymal tissues are important in the maintenance of tissue homeostasis and regeneration capacity. Articular cartilage is a tissue with a very low capacity for repair. One explanation could be the lack of chondrogenic progenitor cells within the adult tissue. As a test of chondrogenic differentiation potential, we examined the ability of isolated chondrocytes to take on several phenotypic identities within the mesenchymal lineage by applying culture techniques and markers used in the study of the phenotypic plasticity of marrow-derived mesenchymal stem cells (MSCs). METHODS: Culture-expanded human articular chondrocytes were analyzed for chondrogenic, adipogenic, and osteogenic capacity in defined in vitro culture systems. The osteochondrogenic potential of cells loaded into porous calcium-phosphate ceramic cubes implanted into mice was also determined. RESULTS: The different assays demonstrated that culture-expanded chondrocytes have the potential to form cartilage in pellet mass cultures, to form adipose cells in dense monolayer cultures, and to form a calcium-rich matrix in an osteogenic assay. In the in vitro assays, a variability of phenotypic plasticity was demonstrated among the donors. In contrast with MSCs, chondrocytes formed cartilage only (and not bone) in the in vivo osteochondrogenic assay. CONCLUSIONS: These results suggest that, within articular cartilage, there are chondrogenic cells that exhibit a level of phenotypic plasticity that is comparable with that of MSCs. However, there was a difference in the expression of bone in the in vivo assay.

Autologous chondrocytes used for articular cartilage repair: an update.

Articular cartilage in adults has a poor ability to self-repair after a substantial injury; however, it is not known whether there is a cartilage resurfacing technique superior to the existing techniques. It is not satisfactory that at the beginning of the new millennium, there still is a lack of randomized studies comparing different cartilage repair techniques and there still is little knowledge of the natural course of a cartilaginous lesion. To date, various articular cartilage resurfacing techniques have the potential to improve the repair of cartilage defects and reduce the patient's disability. One such cartilage repair technique is autologous chondrocyte transplantation combined with a periosteal graft. Since the first patient was operated on in 1987, much interest in cartilage repair and cell engineering has emerged. The experience with autologous chondrocyte transplantation during the past 13 years with in vitro chondrocyte expansion, cartilage harvest, and postoperative biopsy technique is discussed, and the latest followup of 213 consecutive patients in different subgroups with 2 to 10 years followup is presented. The technique gives stable long-term results with a high percentage of good to excellent results (84%-90%) in patients with different types of single femoral condyle lesions, whereas patients with other types of lesions have a lower degree of success (mean, 74%).