Specification of chondrocytes and cartilage tissues from embryonic stem cells.

Osteoarthritis primarily affects the articular cartilage of synovial joints. Cell and/or cartilage replacement is a promising therapy, provided there is access to appropriate tissue and sufficient numbers of articular chondrocytes. Embryonic stem cells (ESCs) represent a potentially unlimited source of chondrocytes and tissues as they can generate a broad spectrum of cell types under appropriate conditions in vitro. Here, we demonstrate that mouse ESC-derived chondrogenic mesoderm arises from a Flk-1(-)/Pdgfrα(+) (F(-)P(+)) population that emerges in a defined temporal pattern following the development of an early cardiogenic F(-)P(+) population. Specification of the late-arising F(-)P(+) population with BMP4 generated a highly enriched population of chondrocytes expressing genes associated with growth plate hypertrophic chondrocytes. By contrast, specification with Gdf5, together with inhibition of hedgehog and BMP signaling pathways, generated a population of non-hypertrophic chondrocytes that displayed properties of articular chondrocytes. The two chondrocyte populations retained their hypertrophic and non-hypertrophic properties when induced to generate spatially organized proteoglycan-rich cartilage-like tissue in vitro. Transplantation of either type of chondrocyte, or tissue generated from them, into immunodeficient recipients resulted in the development of cartilage tissue and bone within an 8-week period. Significant ossification was not observed when the tissue was transplanted into osteoblast-depleted mice or into diffusion chambers that prevent vascularization. Thus, through stage-specific manipulation of appropriate signaling pathways it is possible to efficiently and reproducibly derive hypertrophic and non-hypertrophic chondrocyte populations from mouse ESCs that are able to generate distinct cartilage-like tissue in vitro and maintain a cartilage tissue phenotype within an avascular and/or osteoblast-free niche in vivo.

Gene and protein expression profile of naive and osteo-chondrogenically differentiated rat bone marrow-derived mesenchymal progenitor cells.

Adult mesenchymal progenitor cells (MPCs) are adherent stromal cells of non-haematopoietic origin derived from bone marrow and other tissues. Upon limited in vitro expansion, they retain their self-renewal capacity as well as their potential to differentiate into tissues of mesenchymal lineage, such as bone, cartilage, muscle, tendon and connective tissues. Amongst these tissues, cartilage is the only one with insufficient self-renewal capacity, thus MPCs would qualify as an excellent tool for therapeutic regeneration of focal cartilage lesions. However, optimal in vitro manipulation of MPCs is a prerequisite; identification and a better understanding of the molecular mechanisms regulating their differentiation pathways are needed. Despite wide usage of rats as a mammalian experimental model for preclinical fracture healing and orthopaedic tissue regeneration studies, basal gene and protein expression profiles of the osteo-chondrogenic differentiation lineages of adult rat MPCs have rarely been investigated. Therefore, this study was carried out for a quantitative RT-PCR based time-course profiling of osteo- and chondrogenesis related gene expression in undifferentiated and differentiated rat adult MPCs. In addition, with an antibody array analysis TIMP-1, MCP-1 and VEGFalpha-164 were detected in the culture supernatant and CINC-2 and beta-NGF in the cell lysate of MPCs according to their differentiation commitment. Identification of differentially expressed genes and proteins along the osteo-chondrogenic lineage provides a foundation for a more reproducible and reliable quality and differentiation control of rat bone marrow-derived MPCs used for osteochondrogenic differentiation studies.

Soluble signalling factors derived from differentiated cartilage tissue affect chondrogenic differentiation of rat adult marrow stromal cells.

BACKGROUND: Chondral defects show lack of proper regeneration whereas osteochondral lesions display limited regeneration capacity. Latter is probably due to immigration of chondroprogenitor cells from the subchondral bone. Known chondroprogenitor cells for cartilage tissues are multi-potent adult marrow stromal or mesenchymal stem cells (MSCs). In vitro chondrogenic differentiation of these precursor cells usually require cues from growth and signalling factors provided in vivo by surrounding tissues and cells. We hypothesise that signalling factors secreted by differentiated cartilage tissue can initiate and maintain chondrogenic differentiation status of MSCs. METHODS: To study such paracrine communication between allogenic rat articular cartilage and rat MSCs embedded in alginate beads a novel coculture system without addition of external growth factors has been established. RESULTS: Impact of cartilage on differentiating MSCs was observed at two different time points. Firstly, sustained expression of Sox9 was observed at an early stage which indicated induction of chondrogenic differentiation. Secondly, late stage repression of collagen X indicated pre-hypertrophic arrest of differentiation. In the culture supernatant we have identified vascular endothelial growth factor alpha (VEGF-164 alpha), matrix metalloproteinase (MMP) -13 and tissue inhibitors of MMPs (TIMP-1 and TIMP-2) which could be traced back either to the cartilage explant or to the MSCs under the influence of cartilage. CONCLUSION: The identified factors might be involved in regulation of collagen X gene and protein expression and therefore, may have an impact on the control and regulation of MSCs differentiation.

Influence of cellular microenvironment and paracrine signals on chondrogenic differentiation.

Articular cartilage disorders and injuries often result in life long chronic pain and compromised quality of life, thus regeneration of articular cartilage is a persistent challenge to medical science. One of the most promising therapeutic approaches is cell based tissue engineering which provides a healthy population of cells to the injured site and requires differentiated chondrocytes from the uninjured site as base material. Use of healthy chondrocytes has several limitations and an excellent alternative cell population could be adult marrow stromal cells/mesenchymal stem cells (MSCs) which are known to possess extensive proliferation potential and proven capability to differentiate into chondrocytes. Both, in vivo and in vitro pliability of MSCs and chondrocytes greatly depends on their microenvironment. Gene and protein expression profiles of both the cell types can be altered by soluble factors from surrounding tissue/ cells or by direct cellular contact. For MSC or chondrocyte-based cartilage repair, inhibition of hypertrophy and stabilization of the cartilaginous phenotype in the implant is a prerequisite for success and long lasting vitality of the repaired tissue.

CD45-positive cells of haematopoietic origin enhance chondrogenic marker gene expression in rat marrow stromal cells.

Adult mesenchymal stem cells (MSCs) can be readily isolated from bone marrow, expanded in culture and subsequently subjected to differentiation into various connective tissue lineages. In general, for animal studies separation of MSCs from other bone marrow-derived cells is achieved by sole adherence to plastic surface of tissue culture flasks; however, this procedure produces a heterogeneous cell population containing CD45-positive haematopoietic cells (HCs) and haematopoietic stem cells (HSCs). It is known, that mixed cell cultures consisting of cocultures of differentiated somatic cells with adult stem cells promote differentiation towards specific cell lineages. For determining the effect of the CD45-positive cell population on the differentiation potential of MSCs, we sorted out the bone marrow-derived adherent cells by immunomagnetic technique (MACS) to attain a subpopulation of CD45-depleted cells. Herein, we show that the presence of adherent CD45-positive HCs not only promote expression of the chondrogenic marker genes Col2a1, COMP and Sox9, but also of Col1a1, Col10a1 and to a certain degree Cbfa1 in MSCs when cultured in an appropriate three-dimensional environment. These observations constitute a step towards unravelling the influence of haematopoietic cells on chondrogenic differentiation of MSCs.