Enhanced derivation of osteogenic cells from murine embryonic stem cells after treatment with HepG2-conditioned medium and modulation of the embryoid body formation period: application to skeletal tissue engineering.

Despite the considerable progress made in directing embryonic stem cell (ESC) differentiation to therapeutically useful lineages, several issues remain to be resolved before ESCs can be used for cell therapy: 1) increasing the efficiency of specific lineage generation, and 2) developing time- and cost-effective culture systems for controlling ESC differentiation. Our study aimed to develop efficient methods to enhance mesodermal differentiation and thereby upregulate osteogenic differentiation of ESCs. Specifically, murine ESCs (mESCs) were cultured in the presence of 50% conditioned medium (CM) from the human hepatocarcinoma cell line HepG2, which resulted in enhanced mesoderm formation during embryoid body (EB) formation in the CM-treated mESCs (CM-mESCs). By varying the length of EB culture time, we achieved the selective control and stimulation of osteogenic differentiation and suppression of cardiogenic differentiation. Hence, reducing the EB culture of the CM-mESCs to 1 day resulted in 5-10-fold enhancement of osteogenic differentiation, as determined by bone nodule formation, higher alkaline phosphatase activity, the presence of well-organized osteoblast-cadherin in the bone nodules, and increased cbfa-1/runx2 gene expression. In contrast, increasing the EB culture of the CM-mESCs to 5 days resulted in three- to four-fold enhanced cardiogenic differentiation. These findings for development of highly efficient culture systems and protocols for mESC differentiation into osteogenic lineage that are time- and cost-effective can be used in skeletal tissue engineering applications.

Chondrogenic differentiation of human embryonic stem cells: the effect of the micro-environment.

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.

Derivation of distal airway epithelium from human embryonic stem cells.

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.

Enhanced derivation of osteogenic cells from murine embryonic stem cells after treatment with ionic dissolution products of 58S bioactive sol-gel glass.

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.

In vitro differentiation and in vivo mineralization of osteogenic cells derived from human embryonic stem cells.

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.

Time- and concentration-dependent effects of dissolution products of 58S sol-gel bioactive glass on proliferation and differentiation of murine and human osteoblasts.

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.

Cells from different regions of the intervertebral disc: effect of culture system on matrix expression and cell phenotype.

STUDY DESIGN: This study examined how the culture system and region of cellular origin affect disc cell morphology and extracellular matrix production. OBJECTIVE: To determine the role of the cell populations in the different regions of the adult intervertebral disc in maintaining gradients in composition across the disc. SUMMARY OF BACKGROUND DATA: It is not known whether the steep profiles in composition across the intervertebral disc are maintained by distinct cell populations or whether differences in cell metabolism are determined by changes in the physical environment across the disc. Very little information exists on the matrix produced by cells from the mature, non-notochordal nucleus pulposus. METHODS: Cells were extracted from articular cartilage, nucleus pulposus, and the inner and outer anulus fibrosus of caudal discs from 18- to 24-month-old steers cultured in alginate or collagen gels or in monolayer. The effect of culture system and cell origin on cell morphology and matrix synthesis was measured using 35S-sulphate labeling and indirect immunolocalization. RESULTS: Distinct morphologic differences between cells from different regions cultured in monolayer were retained through two passages. The rate of sulfate incorporation varied with cell type. Immediately after isolation, it was two- to threefold greater for nucleus cells than for cells from the disc inner anulus or articular cartilage. The rate was lowest for outer anulus cells. It also varied with culture system. For all cell types, the incorporation rate was highest in alginate and lowest in monolayer. Immunolocalization showed that nucleus cells stained strongly for all proteoglycan epitopes, whereas outer anulus cells stained least and in monolayer produced little proteoglycan. CONCLUSIONS: The disc has at least three distinct cell populations, which differ in morphology and in amount and type of matrix they produce. Cells from mature nucleus pulposus produced sulfated glycosaminoglycans at a high rate in contrast to reported results for notochordal nucleus cells. Alginate, although an appropriate culture system for inner anulus and nucleus cells, may not be a suitable medium for outer anulus cells.