[Show abstract][Hide abstract] ABSTRACT: Pluripotent mouse embryonic stem (ES) cells differentiate in vitro spontaneously into cell types of all three primary germ layers when cultivated as cell aggregates, so-called 'embryoid bodies'. Many reports have shown that this system recapitulates cellular developmental processes and gene expression patterns of early embryogenesis. During ES cell differentiation, efficient and directed differentiation into a specific cell type is influenced by many parameters, for example, the batch of the serum used or the application of growth factors and signalling molecules. Because all ES cell lines are considered to be pluripotent, one should not expect remarkable differences regarding their spontaneous differentiation efficiencies. However, here we show that different ES cell lines exhibit a variable degree of spontaneous chondrogenic differentiation indicating that lines with a specific differentiation capacity could be selected. This is an important aspect if ES cells are applied for tissue regeneration.
Preview · Article · Mar 2005 · Cell Biology International
[Show abstract][Hide abstract] ABSTRACT: Our knowledge of cellular differentiation processes during chondro- and osteogenesis, in particular the complex interaction of differentiation factors, is still limited. We used the model system of embryonic stem (ES) cell differentiation in vitro via cellular aggregates, so called embryoid bodies (EBs), to analyze chondrogenic and osteogenic differentiation. ES cells differentiated into chondrocytes and osteocytes throughout a series of developmental stages resembling cellular differentiation events during skeletal development in vivo. A lineage from pluripotent ES cells via mesenchymal, prechondrogenic cells, chondrocytes and hypertrophicchondrocytes up to osteogenic cells was characterized. Furthermore, we found evidence for another osteogenic lineage, bypassing the chondrogenic stage. Together our results suggest that this in vitro system will be helpful to answer so far unacknowledged questions regarding chondrogenic and osteogenic differentiation. For example, we isolated an as yet unknown cDNA fragment from ES cell-derived chondrocytes, which showed a developmentally regulated expression pattern during EB differentiation. Considering ES cell differentiation as an alternative approach for cellular therapy, we used two different methods to obtain pure chondrocyte cultures from the heterogenous EBs. First, members of the transforming growth factor (TGF)-beta family were applied and found to modulate chondrogenic differentiation but were not effective enough to produce sufficient amounts of chondrocytes. Second, chondrocytes were isolated from EBs by micro-manipulation. These cells initially showed dedifferentiation into fiboblastoid cells in culture, but later redifferentiated into mature chondrocytes. However, a small amount of chondrocytes isolated from EBs transdifferentiated into other mesenchymal cell types, indicating that chondrocytes derived from ES cells posses a distinct differentiation plasticity.
[Show abstract][Hide abstract] ABSTRACT: The in vitro differentiation of ES cells, which closely recapitulates embryonic cell differentiation processes, has been used as a model system to analyze cell differentiation. This is an alternative way to investigate the consequences of loss of function mutations after gene targeting if knock-out mice cannot be generated. Because of their broad differentiation capacity ES cells are also discussed as an experimental approach to generate cells for transplantation. A common method to differentiate ES cells in vitro is their cultivation as cell aggregates, so-called “embryoid bodies (EBs).” The efficiency of ES cell-derived chondrocyte differentiation is found to be influenced by several parameters, such as the batch of fetal calf serum used for cultivation or the size of EBs. For a reproducible pattern of chondrogenic and osteogenic differentiation it is an important prerequisite to generate EBs of the same size. This is achieved by differentiation of ES cells via hanging drop-cultivation. This chapter describes some basic techniques of ES cell cultivation, the methods used for chondrogenic and osteogenic differentiation of ES cells, for characterization of the specific cell types and for isolation of chondrocytes from EBs. Furthermore, the chapter briefly summarizes approaches to enhance the differentiation efficiency.
No preview · Article · Feb 2003 · Methods in Enzymology
[Show abstract][Hide abstract] ABSTRACT: Evidence exists that cells of mesenchymal origin show a differentiation plasticity that depends on their differentiation state. We used in vitro differentiation of embryonic stem cells through embryoid bodies as a model to analyze chondrogenic and osteogenic differentiation because embryonic stem cells recapitulate early embryonic developmental phases during in vitro differentiation. Here, we show that embryonic stem cells differentiate into chondrocytes, which progressively develop into hypertrophic and calcifying cells. At a terminal differentiation stage, cells expressing an osteoblast-like phenotype appeared either by transdifferentiation from hypertrophic chondrocytes or directly from osteoblast precursor cells. Chondrocytes isolated from embryoid bodies initially dedifferentiated in culture but later re-expressed characteristics of mature chondrocytes. The process of redifferentiation was completely inhibited by transforming growth factor beta3. In clonal cultures of chondrocytes isolated from embryoid bodies, additional mesenchymal cell types expressing adipogenic properties were observed, which suggests that the subcultured chondrocytes indeed exhibit a certain differentiation plasticity. The clonal analysis confirmed that the chondrogenic cells change their developmental fate at least into the adipogenic lineage. In conclusion, we show that chondrocytic cells are able to transdifferentiate into other mesenchymal cells such as osteogenic and adipogenic cell types. These findings further strengthen the view that standardized selection strategies will be necessary to obtain defined cell populations for therapeutic applications.
No preview · Article · Jan 2003 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: Primary cultures or established cell lines of vertebrates are commonly used to analyse the mutagenic, embryotoxic or teratogenic potential of environmental factors, drugs and xenobiotics in vitro. However, these cellular systems do not include developmental processes from early embryonic stages up to terminally differentiated cell types. An alternative approach has been offered by permanent lines of pluripotent stem cells of embryonic origin, such as embryonic carcinoma (EC), embryonic stem (ES) and embryonic germ (EG) cells. The undifferentiated stem cell lines are characterized by nearly unlimited self-renewal capacity and have been shown to differentiate in vitro into cells of all three primary germ layers. Pluripotent embryonic stem cell lines recapitulate cellular developmental processes and gene expression patterns of early embryogenesis during in vitro differentiation, data which are summarized in this review. In addition, recent studies are presented which investigated mutagenic, cytotoxic and embryotoxic effects of chemical substances using in vitro systems of pluripotent embryonic stem cells. Furthermore, an outlook is given on future molecular technologies using embryonic stem cells in developmental toxicology and embryotoxicology.
Full-text · Article · Jan 2002 · Toxicology in Vitro
[Show abstract][Hide abstract] ABSTRACT: Differentiation of mouse embryonic stem (ES) cells via embryoid bodies was established as a suitable model to study development in vitro. Here, we show that differentiation of ES cells in vitro into chondrocytes can be modulated by members of the transforming growth factor-beta family (TGF-beta(1), BMP-2 and -4). ES cell differentiation into chondrocytes was characterized by the appearance of Alcian blue-stained areas and the expression of cartilage-associated genes and proteins. Different stages of cartilage differentiation could be distinguished according to the expression pattern of the transcription factor scleraxis, and the cartilage matrix protein collagen II. The number of Alcian-blue-stained areas decreased slightly after application of TGF-beta(1), whereas BMP-2 or -4 induced chondrogenic differentiation. The inducing effect of BMP-2 was found to be dependent on the time of application, consistent with its role to recruit precursor cells to the chondrogenic fate.
Full-text · Article · May 2000 · Mechanisms of Development