The first report of the derivation of embryonic stem (ES) cell lines from human blastocysts had major implications for research into developmental biology and regenerative medicine. Finding efficient and reproducible methods to derive therapeutically useful cells from an ES cell source is a key feature of many regenerative medicine strategies. We have previously demonstrated that it is possible to induce osteogenic differentiation of murine ES cells by supplementing the culture medium with ascorbic acid, beta-glycerophosphate, and dexamethasone. This study investigated whether methods for driving osteogenic differentiation developed with murine ES cells could be applied successfully to human ES cells. The H1 line was propagated in vitro on murine feeder layers and shown to be pluripotent by expression of the markers Oct-4 and SSEA-4. Subsequently, differentiation was initiated via embryoid body (EB) formation and, after 5 days in suspension culture, cells harvested from EBs were replated in a medium containing osteogenic supplements. We found that the treatment regimen previously identified as optimal for murine ES cells, and in particular the addition of dexamethasone at specific time points, also induced the greatest osteogenic response from human ES cells. We identified mineralizing cells in vitro that immunostained positively for osteocalcin and found an increase in expression of an essential bone transcription factor, Runx2. When implanted into SCID mice on a poly-D, L-lactide (PDLLA) scaffold, the cells had the capacity to give rise to mineralized tissue in vivo. After 35 days of implantation, regions of mineralized tissue could be identified within the scaffold by von Kossa staining and immunoexpression of the human form of osteocalcin. We did not see any evidence of teratoma formation. These data therefore demonstrate the derivation of osteoblasts from pluripotent human ES cells with the capacity to form mineralized tissue both in vitro and in vivo. We have also shown that a culture methodology established for differentiation of murine ES cells was entirely transferable to human ES cells. Further development of this technology will result in the capacity to generate sufficient yields of osteogenic cells for use in skeletal tissue repair.

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The pluripotency of embryonic stem cells (ESC) is offering new opportunities in tissue engineering and cell therapy. We have shown previously that alveolar epithelial cells, specifically type II pneumocytes, can be derived from murine ESC and hypothesized that a similar protocol could be used successfully on human ESC. Undifferentiated human ESC were induced to form embryoid bodies that were transferred into adherent culture conditions and grown in a medium designed for the maintenance of mature small airway epithelium. On inverted microscopy, the generated cells showed the cobblestone-like morphology of epithelium. The presence of surfactant protein C, a specific marker of type II pneumocytes, and its corresponding RNA were demonstrated by immunostaining and reverse transcription polymerase chain reaction, respectively. Electron microscopy revealed frequent cells with the typical ultrastructure of type II pneumocytes. This study provides evidence for in vitro induction of the differentiation from human ESC of alveolar type II cells, which have the potential for therapeutic use or construction of an in vitro model of human lung.

Embryonic stem (ES) cells represent a potentially useful cell source for tissue regeneration. Previously, using factors known to enhance differentiation and mineralization of primary osteoblasts, we were able to generate cell populations enriched with osteoblasts from a murine ES cell source. Dexamethasone was a potent inducer of osteoblast differentiation and the timing of stimulation markedly increased the proportion of osteoblast lineage cells. This study examined whether inorganic stimuli derived from bioactive glasses could affect the differentiation of osteoblasts in an ES-cell based system. Previous work has demonstrated the ability of soluble ions released from bioactive glasses undergoing dissolution in vitro to stimulate gene expression characteristic of a mature phenotype in primary osteoblasts. We report here on the potential of soluble extracts prepared from 58S sol-gel bioactive glass to further enhance lineage-specific differentiation in murine ES cells. Differentiation of ES cells into osteogenic cells was characterized by the formation of multilayered, mineralized nodules. These nodules contained cells expressing the transcription factor runx2/cbfa-1, and deposition of osteocalcin in the extracellular matrix was detected by immunostaining. When differentiating cells were placed in an osteoblast maintenance medium supplemented with soluble extracts prepared from bioactive glass powders, we observed increased formation of mineralized nodules (98 +/- 6%, mean +/- SEM) and alkaline phosphatase activity (56 +/- 14%, mean +/- SEM) in a pattern characteristic of osteoblast differentiation. This effect of the glass extracts exhibited dose dependency, with alkaline phosphatase activity and nodule formation increasing with extract concentrations. Compared with medium supplemented with dexamethasone, which had previously been used to enhance osteoblast lineage derivation, the glass extracts were as effective at inducing formation of mineralized nodules by murine ES cells. When glass extracts were used in combination with dexamethasone, a further increase in the number of nodules was observed (110 +/- 16%; cf. 83 +/- 7% for dexamethasone alone). This study demonstrates the capacity of an entirely inorganic material to stimulate differentiation of ES cells toward a lineage with therapeutic potential in tissue-engineering applications.

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