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Cartilage injuries and osteoarthritis are leading causes of disability in developed countries. The regeneration of damaged articular cartilage using cell transplantation or tissue engineering holds much promise but requires the identification of an appropriate cell source with a high proliferative propensity and consistent chondrogenic capacity. Human fetal mesenchymal stem cells (MSCs) have been isolated from a range of perinatal tissues, including first-trimester bone marrow, and have demonstrated enhanced expansion and differentiation potential. However, their ability to form mature chondrocytes for use in cartilage tissue engineering has not been clearly established. Here, we compare the chondrogenic potential of human MSCs isolated from fetal and adult bone marrow and show distinct differences in their responsiveness to specific growth factors. Transforming growth factor beta 3 (TGFβ3) induced chondrogenesis in adult but not fetal MSCs. In contrast, bone morphogenetic protein 2 (BMP2) induced chondrogenesis in fetal but not adult MSCs. When fetal MSCs co-stimulated with BMP2 and TGFβ3 were used for cartilage tissue engineering, they generated tissue with type II collagen and proteoglycan content comparable to adult MSCs treated with TGFβ3 alone. Investigation of the TGFβ/BMP signaling pathway showed that TGFβ3 induced phosphorylation of SMAD3 in adult but not fetal MSCs. These findings demonstrate that the initiation of chondrogenesis is modulated by distinct signaling mechanisms in fetal and adult MSCs. This study establishes the feasibility of using fetal MSCs in cartilage repair applications and proposes their potential as an in vitro system for modeling chondrogenic differentiation and skeletal development studies.

Embryonic stem cells (ESCs) are being investigated increasingly for their potential as a cell source for tissue engineering. Antibiotics are regularly used in ESC culture media to control contamination, although they can be cytotoxic and interfere with protein synthesis. Our aim was to examine the effects of the frequently used antibiotics gentamicin and combined penicillin and streptomycin on ESC culture using differentiation of murine ESC into type II pneumocytes as a model. Antibiotics reduced the expression of the specific marker for type II pneumocytes, SPC mRNA, by up to 60%. We also identified an adverse effect on the growth rate of differentiating embryoid bodies, causing a significant ( p < 0.05) reduction of up to 40%, and an increase in population doubling time of up to 48%. No contamination was seen in any of the cultures. Our findings suggest that the routine use of antibiotics in ESC culture should be avoided as it may reduce the efficiency of the culture system.

Coculture of stem/progenitor cells with mature cells or tissues can drive their differentiation toward required lineages. Thus, we hypothesized that coculture of murine embryonic stem (ES) cells with embryonic mesenchyme from distal lung promotes the differentiation of pneumocytes. Murine ES cells were differentiated to embryoid bodies (EBs) and cultured for 5 or 12 days with pulmonary mesenchyme from embryonic day 11.5 or 13.5 murine embryos, in direct contact or separated by a membrane. Controls included EBs cultured alone or with embryonic gut mesenchyme. Histology revealed epithelium-lined channels in directly cocultured EBs, whereas EBs grown alone showed little structural organization. The lining cells expressed cytokeratin and thyroid transcription factor 1, an early developmental marker in pulmonary epithelium. Differentiation of type II pneumocytes specifically was demonstrated by the presence of surfactant protein C (SP-C) in some of the epithelial cells. None of these markers was seen in EBs cultured alone or with embryonic gut mesenchyme. Indirect coculture of EBs with lung mesenchyme resulted in a 14-fold increase in SP-C gene expression. Thus, provision of an appropriate microenvironment, in the form of pulmonary mesenchyme, appears to promote the differentiation of ES cells toward lung epithelium. Our findings may have applications in regenerative medicine strategies and the engineering of lung tissue.

Osterix is a transcription factor crucial for the normal development of the osteoblast. Here we have investigated whether the osteogenic differentiation of murine embryonic stem (ES) cells can be induced by overexpression of osterix. Differentiation was initiated by formation of embryoid bodies (EB) which were then dispersed and cultured in alpha-minimum essential medium supplemented with L-ascorbate phosphate and alpha-glycerophosphate for up to 21 days. osterix was found to induce expression of several osteoblast-specific markers, as confirmed by immunostaining and real-time RT-PCR. The expression of genes encoding osteocalcin and Cbfa1 was upregulated and the formation of mineralized bone nodules was significantly increased by osterix transfection. In combination with dexamethasone, bone nodule formation was further increased in osterix-transfected cells. Expression of both Sox-9 and PPAR-gamma, genes that are associated with chondrocyte and adipocyte differentiation, was initially increased in the osterix-transfected cells but was downregulated after day 7. This suggests that the process of osterix-induced differentiation of ES cells involves transition through an intermediate bi- or tripotential progenitor cell population. In conclusion, this cell differentiation strategy is useful not only for generating osteoblastic cells from ES cells, but also for investigating factors that influence this process and potentially delineating the ontogeny of the osteoblast.

Bone loss is a significant clinical problem, and treatments utilizing donated graft material are limited. To meet future demands in the healthcare industry, there has been a shift of outlook toward the use of bioactive materials for tissue regeneration. A number of in vivo and in vitro studies have highlighted the potential of the bioactive glass ceramic 45S5 Bioglass as a synthetic regenerative scaffold. The application of sol-gel processing techniques has led to the synthesis of mesoporous bioactive glasses with greater textural and compositional variety. In this study, we evaluated the effects of supplemented tissue culture medium containing up to 203 ppm silica prepared by static soaking of particles of 58S sol-gel bioactive glass (58% SiO(2), 33% CaO, 9% P(2)O(5)) on the in vitro proliferation and differentiation of murine and human primary osteoblasts. These extracts had a higher silica content than those used previously in studies of 45S5 Bioglass, because of the faster rates of ion exchange permitted by the higher surface area-to-volume ratio of mesoporous glass. We found that osteoblasts from both species increased their proliferation in response to the glass-conditioned medium. In addition, the extent to which supplemented medium could alter cell differentiation varied with time in culture. Proliferation induced by supplemented medium paralleled effects induced by treatment with basic fibroblast growth factor, a known mitogenic growth factor for osteoblasts. Bone nodule formation was also increased by exposure to the glass-conditioned medium and this effect was positively correlated with the dose of glass used to prepare the medium. Apoptosis was stimulated by glass-conditioned medium in murine osteoblasts, but inhibited in human osteoblasts. These data demonstrate the bioactive effects of dissolution products derived from sol-gel materials on primary osteoblasts and complements in vivo studies that indicate the suitability of this material as a bone graft substitute.

The pluripotency and high proliferative index of embryonic stem (ES) cells make them a good potential source of cells for tissue engineering purposes. We have shown that ES cells can be induced to differentiate in vitro into pulmonary epithelial cells (type II pneumocytes) using a serum-free medium designed for the maintenance of mature distal lung epithelial cells in culture (SAGM). However, the resulting cell cultures were heterogeneous. Our aim in this study was to attempt to increase pneumocyte yield and differentiation state by determining which medium components enhance the differentiation of pneumocytes and modifying the medium accordingly. Quantitative RT-PCR was used to measure changes in the expression of a type II pneumocyte-specific gene, surfactant protein C (SPC), in response to alterations in the cell culture medium. Results suggested that most individual SAGM growth factors were inhibitory for type II pneumocyte differentiation, with the largest increases in SPC expression (approximately threefold) being observed upon removal of retinoic acid and triiodothryonine. However, large standard deviations occurred between replicates, illustrating the highly variable nature of ES cell differentiation. Nevertheless, these observations represent an initial step towards achieving directed differentiation of pneumocytes from stem cells that could lead to their purification for tissue engineering purposes.

We have previously shown osteogenic differentiation of mouse embryonic stem (ES) cells and temporal enrichment with osteoblastic cells, by stimulation with serum-containing culture medium supplemented with beta-glycerophosphate, ascorbate, and dexamethasone. In our present study we have used similar culture conditions to further investigate osteogenic differentiation of mouse ES cells. Using reverse transcription-polymerase chain reaction (RT-PCR) we demonstrated the expression of genes associated with osteoblast differentiation including the bone matrix protein osteocalcin and the transcription factor Cbfa-1/runx2. Furthermore, results of cDNA microarray analysis, and subsequent RT-PCR analysis of differentiating ES cells after exposure to osteogenic stimuli, revealed a combination of upregulation of genes involved in osteoblast differentiation including osteopontin, HSP-47, and IGF-II coupled with downregulation of genes involved in differentiation of other phenotypes such as the neuroectoderm factor Stra-13. Finally, we have applied magnetically activated cell-sorting methods to ES cell cultures treated with osteogenic stimuli and, using an antibody to cadherin-11, have purified a subpopulation of cells with osteoblastic characteristics.

Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after, but not before, 14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair.

We have previously induced differentiation of embryonic stem cells (ESC) to specific phenotypes by manipulating the culture conditions, including the use of indirect co-culture. In this study, we hypothesized that co-culture with primary chondrocytes can induce human embryonic stem cells (hESC) to differentiate towards the chondrocyte lineage. Co-cultures of hESC and chondrocytes were established using well inserts, with control comprising hESC grown alone or with fibroblasts. After 28 days, after removal of the chondrocyte inserts, hESC differentiation was assessed, by morphology, immunocytochemistry, and reverse transcription polymerase chain reaction. hESC, co-cultured or grown alone, were also implanted into SCID mice on a poly-D, L-lactide scaffold, harvested 35 days later and assessed in the same way. hESC co-cultured with chondrocytes formed colonies and secreted extracellular matrix containing glycosaminoglycans (GAG). Quantitative assay showed increased synthesis of sulfated GAG in co-culture as compared with control hESC grown alone for the same period (p < 0.0001). In addition, co-cultured hESC expressed Sox 9 and collagen type II, unlike control hESC. Co-culture with fibroblasts did not induce chondrogenic differentiation. The implanted constructs with co-cultured hESC contained significantly more type II collagen (p < 0.01), type I collagen (p < 0.05), total collagen (p < 0.01), and GAG (p < 0.01) than those with hESC grown alone. Thus, we show for the first time differentiation of hESC to chondrocytes. Our results confirm the potential of the culture micro-environment to influence ESC differentiation and could provide the basis for future generation of chondrogenic cells for use in tissue repair and increase our understanding of the mechanisms that direct differentiation.

Calcitonin gene-related polypeptide (CGRP) has been localised immunochemically within the rat and guinea pig anterior uvea to nerve fibres of trigeminal origin. As with substance P (1-3) the level of CGRP in the iris-ciliary body is depleted after thermal damage to the Gasserian ganglion and elevated in chronically sympathectically denervated eyes. Unlike substance P, a potent pupillary constrictor (4,5), CGRP has no notable miotic action, but does, however, cause an elevation of the intraocular pressure (IOP) accompanied by disruption of the blood-aqueous barrier. It is proposed that the diverse actions of these two sensory neuropeptides conjointly mediate the antidromic ocular injury response.