Precision assembly of complex cellular microenvironments using holographic optical tweezers.

The accurate study of cellular microenvironments is limited by the lack of technologies that can manipulate cells in 3D at a sufficiently small length scale. The ability to build and manipulate multicellular microscopic structures will facilitate a more detailed understanding of cellular function in fields such as developmental and stem cell biology. We present a holographic optical tweezers based technology to accurately generate bespoke cellular micro-architectures. Using embryonic stem cells, 3D structures of varying geometries were created and stabilized using hydrogels and cell-cell adhesion methods. Control of chemical microenvironments was achieved by the temporal release of specific factors from polymer microparticles positioned within these constructs. Complex co-culture micro-environmental analogues were also generated to reproduce structures found within adult stem cell niches. The application of holographic optical tweezers-based micromanipulation will enable novel insights into biological microenvironments by allowing researchers to form complex architectures with sub-micron precision of cells, matrices and molecules.

Revealing cytokine-induced changes in the extracellular matrix with secondary ion mass spectrometry.

Cell-secreted matrices (CSMs), where extracellular matrix (ECM) deposited by monolayer cell cultures is decellularized, have been increasingly used to produce surfaces that may be reseeded with cells. Such surfaces are useful to help us understand cell-ECM interactions in a microenvironment closer to the in vivo situation than synthetic substrates with adsorbed proteins. We describe the production of CSMs from mouse primary osteoblasts (mPObs) exposed to cytokine challenge during matrix secretion, mimicking in vivo inflammatory environments. Time-of-flight secondary ion mass spectrometry data revealed that CSMs with cytokine challenge at day 7 or 12 of culture can be chemically distinguished from one another and from untreated CSM using multivariate analysis. Comparison of the differences with reference spectra from adsorbed protein mixtures points towards cytokine challenge resulting in a decrease in collagen content. This is supported by immunocytochemical and histological staining, demonstrating a 44% loss of collagen mass and a 32% loss in collagen I coverage. CSM surfaces demonstrate greater cell adhesion than adsorbed ECM proteins. When mPObs were reseeded onto cytokine-challenged CSMs they exhibited reduced adhesion and elongated morphology compared to untreated CSMs. Such changes may direct subsequent cell fate and function, and provide insights into pathological responses at sites of inflammation.

Investigation of localized delivery of diclofenac sodium from poly(D,L-lactic acid-co-glycolic acid)/poly(ethylene glycol) scaffolds using an in vitro osteoblast inflammation model.

Nonunion fractures and large bone defects are significant targets for osteochondral tissue engineering strategies. A major hurdle in the use of these therapies is the foreign body response of the host. Herein, we report the development of a bone tissue engineering scaffold with the ability to release anti-inflammatory drugs, in the hope of evading this response. Porous, sintered scaffolds composed of poly(D,L-lactic acid-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) were prepared with and without the anti-inflammatory drug diclofenac sodium. Analysis of drug release over time demonstrated a profile suitable for the treatment of acute inflammation with ∼80% of drug released over the first 4 days and a subsequent release of around 0.2% per day. Effect of drug release was monitored using an in vitro osteoblast inflammation model, comprised of mouse primary calvarial osteoblasts stimulated with proinflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). Levels of inflammation were monitored by cell viability and cellular production of nitric oxide (NO) and prostaglandin E2 (PGE2). The osteoblast inflammation model revealed that proinflammatory cytokine addition to the medium reduced cell viability to 33%, but the release of diclofenac sodium from scaffolds inhibited this effect with a final cell viability of ∼70%. However, releasing diclofenac sodium at high concentrations had a toxic effect on the cells. Proinflammatory cytokine addition led to increased NO and PGE2 production; diclofenac-sodium-releasing scaffolds inhibited NO release by ∼64% and PGE2 production by ∼52%, when the scaffold was loaded with the optimal concentration of drug. These observations demonstrate the potential use of PLGA/PEG scaffolds for localized delivery of anti-inflammatory drugs in bone tissue engineering applications.

Comparison of osteogenic differentiation of embryonic stem cells and primary osteoblasts revealed by responses to IL-1ß, TNF-α, and IFN-γ.

There are well-established approaches for osteogenic differentiation of embryonic stem cells (ESCs), but few show direct comparison with primary osteoblasts or demonstrate differences in response to external factors. Here, we show comparative analysis of in vitro osteogenic differentiation of mouse ESC (osteo-mESC) and mouse primary osteoblasts. Both cell types formed mineralized bone nodules and produced osteogenic extracellular matrix, based on immunostaining for osteopontin and osteocalcin. However, there were marked differences in the morphology of osteo-mESCs and levels of mRNA expression for osteogenic genes. In response to the addition of proinflammatory cytokines interleukin-1ß, tumor necrosis factor-α, and interferon-γ to the culture medium, primary osteoblasts showed increased production of nitric oxide (NO) and prostaglandin E2 (PGE2) at early time points and decreases in cell viability. In contrast, osteo-mESCs maintained viability and did not produce NO and PGE2 until day 21. The formation of bone nodules by primary osteoblasts was reduced markedly after cytokine stimulation but was unaffected in osteo-mESCs. Cell sorting of osteo-mESCs by cadherin-11 (cad-11) showed clear osteogenesis of cad-11(+) cells compared to unsorted osteo-mESCs and cad-11(-) cells. Moreover, the cad-11(+) cells showed a significant response to cytokines, similar to primary osteoblasts. Overall, these results show that while osteo-mESC cultures, without specific cell sorting, show characteristics of osteoblasts, there are also marked differences, notably in their responses to cytokine stimuli. These findings are relevant to understanding the differentiation of stem cells and especially developing in vitro models of disease, testing new drugs, and developing cell therapies.

Delivery of definable number of drug or growth factor loaded poly(DL-lactic acid-co-glycolic acid) microparticles within human embryonic stem cell derived aggregates.

Embryoid bodies (EBs) generated from embryonic stem cells are used to study processes of differentiation within a three dimensional (3D) cell environment. In many instances however, EBs are dispersed to single cell suspensions with a subsequent monolayer culture. Moreover, where the 3D integrity of an EB is maintained, cytokines or drugs of interest to stimulate differentiation are often added directly to the culture medium at fixed concentrations and effects are usually limited to the outer layers of the EB. The aim of this study was to create an EB model with localised drug and or growth factor delivery directly within the EB. Using poly(DL-lactic acid-co-glycolic acid) microparticles (MPs) with an average diameter of 13µm, we have demonstrated controllable incorporation of defined numbers of MPs within human ES cell derived EBs, down to 1 MP per EB. This was achieved by coating MPs with human ES cell lysate and centrifugation of specific ratios of ES cells and MPs to form 3D aggregates. Using MPs loaded with simvastatin (pro or active drug) or BMP-2, we have demonstrated osteogenic differentiation within the 3D aggregates, maintained in culture for up to 21days, and quantified by real time QPCR for osteocalcin. Immunostaining for RUNX2 and osteocalcin, and also histochemical staining with picrosirius red to demonstrate collage type 1 and Alizarin red to demonstrate calcium/mineralisation further demonstrated osteogenic differentiation and revealed regional staining associated with the locations of MPs within the aggregates. We also demonstrated endothelial differentiation within human ES cell-derived aggregates using VEGF loaded MPs. In conclusion, we demonstrate an effective and reliable approach for engineering stem aggregates with definable number of MPs within the 3D cellular structure. We also achieved localised osteogenic and endothelial differentiation associated with MPs releasing encapsulated drug molecules or cytokines directly within the cell aggregate. This provides a powerful tool for controlling and investigating differentiation within 3D cell cultures and has applications to drug delivery, drug discovery, stem cell biology, tissue engineering and regenerative medicine.

Early gene regulation of osteogenesis in embryonic stem cells.

The early gene regulatory networks (GRNs) that mediate stem cell differentiation are complex, and the underlying regulatory associations can be difficult to map accurately. In this study, the expression profiles of the genes Dlx5, Msx2 and Runx2 in mouse embryonic stem cells were monitored over a 48 hour period after exposure to the growth factors BMP2 and TGFß1. Candidate GRNs of early osteogenesis were constructed based on published experimental findings and simulation results of Boolean and ordinary differential equation models were compared with our experimental data in order to test the validity of these models. Three gene regulatory networks were found to be consistent with the data, one of these networks exhibited sustained oscillation, a behaviour which is consistent with the general view of embryonic stem cell plasticity. The work cycle presented in this paper illustrates how mathematical modelling can be used to elucidate from gene expression profiles GRNs that are consistent with experimental data.

Engineering an in-vitro model of rodent cartilage.

OBJECTIVES: The purpose of this study was to identify a cell source, scaffold substrate and culture environment suitable for use in engineering an in-vitro model of rodent cartilage. METHODS: The chondrogenic activity and stability of cells isolated at Day 18 of gestation was assessed under normoxia and hypoxia using a cytokine stimulation assay and gene expression analysis. The ability of the selected cells seeded in fibrous electrospun scaffolds to form cartilaginous tissue during longterm static and dynamic culture was assessed using immunocytochemistry and biochemical analysis. KEY FINDINGS: Rodent fetal chondrocytes appear to have enhanced phenotypic stability compared with other cell sources. Following 16 weeks under static culture, the engineered constructs were found to have greater cellularity and collagen content that native rodent cartilage. CONCLUSIONS: A cell source, scaffold and culture environment have been identified that support the generation of in-vitro rodent cartilage. In future work, cytokine treatment of the engineered tissues will take place to generate in-vitro osteoarthritis models.

Noninvasive detection and imaging of molecular markers in live cardiomyocytes derived from human embryonic stem cells.

Raman microspectroscopy (RMS) was used to detect and image molecular markers specific to cardiomyocytes (CMs) derived from human embryonic stem cells (hESCs). This technique is noninvasive and thus can be used to discriminate individual live CMs within highly heterogeneous cell populations. Principal component analysis (PCA) of the Raman spectra was used to build a classification model for identification of individual CMs. Retrospective immunostaining imaging was used as the gold standard for phenotypic identification of each cell. We were able to discriminate CMs from other phenotypes with >97% specificity and >96% sensitivity, as calculated with the use of cross-validation algorithms (target 100% specificity). A comparison between Raman spectral images corresponding to selected Raman bands identified by the PCA model and immunostaining of the same cells allowed assignment of the Raman spectral markers. We conclude that glycogen is responsible for the discrimination of CMs, whereas myofibril proteins have a lesser contribution. This study demonstrates the potential of RMS for allowing the noninvasive phenotypic identification of hESC progeny. With further development, such label-free optical techniques may enable the separation of high-purity cell populations with mature phenotypes, and provide repeated measurements to monitor time-dependent molecular changes in live hESCs during differentiation in vitro.

Maintenance of pluripotency in human embryonic stem cells cultured on a synthetic substrate in conditioned medium.

Realizing the potential clinical and industrial applications of human embryonic stem cells (hESCs) is limited by the need for costly, labile, or undefined growth substrates. Here we demonstrate that trypsin passaging of the hESC lines, HUES7 and NOTT1, on oxygen plasma etched tissue culture polystyrene (PE-TCPS) in conditioned medium is compatible with pluripotency. This synthetic culture surface is stable at room temperature for at least a year and is readily prepared by placing polystyrene substrates in a radio frequency oxygen plasma generator for 5 min. Modification of the polystyrene surface chemistry by plasma etching was confirmed by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), which identified elemental and molecular changes as a result of the treatment. Pluripotency of hESCs cultured on PE-TCPS was gauged by consistent proliferation during serial passage, expression of stem cell markers (OCT4, TRA1-60, and SSEA-4), stable karyotype and multi-germlayer differentiation in vitro, including to pharmacologically responsive cardiomyocytes. Generation of cost-effective, easy-to-handle synthetic, defined, stable surfaces for hESC culture will expedite stem cell use in biomedical applications.

Engineering embryonic stem-cell aggregation allows an enhanced osteogenic differentiation in vitro.

Pluripotent embryonic stem (ES) cells hold great promise for the field of tissue engineering, with numerous studies investigating differentiation into various cell types including cardiomyocytes, chondrocytes, and osteoblasts. Previous studies have detailed osteogenic differentiation via dissociated embryoid body (EB) culture in osteoinductive media comprising of ascorbic acid, beta-glycerophosphate, and dexamethasone. It is hoped that these osteogenic cultures will have clinical application in bone tissue repair and regeneration and pharmacological testing. However, differentiation remains highly inefficient and generates heterogeneous populations. We have previously reported an engineered three-dimensional culture system for controlled ES cell-ES cell interaction via the avidin-biotin binding complex. Here we investigate the effect of such engineering on ES cell differentiation. Engineered EBs exhibit enhanced osteogenic differentiation assessed by cadherin-11, Runx2, and osteopontin expression, alkaline phosphatase activity, and bone nodule formation. Results show that cultures produced from intact EBs aggregated for 3 days generated the greatest levels of osteogenic differentiation when cultured in osteoinductive media. However, when cultured in control media, only engineered samples appeared to exhibit bone nodule formation. In addition, polymerase chain reaction analysis revealed a decrease in endoderm and ectoderm expression within engineered samples. This suggests that engineered ES cell aggregation has increased mesoderm homogeneity, contributing to enhanced osteogenic differentiation.

Mathematical modelling of tissue-engineered angiogenesis.

We present a mathematical model for the vascularisation of a porous scaffold following implantation in vivo. The model is given as a set of coupled non-linear ordinary differential equations (ODEs) which describe the evolution in time of the amounts of the different tissue constituents inside the scaffold. Bifurcation analyses reveal how the extent of scaffold vascularisation changes as a function of the parameter values. For example, it is shown how the loss of seeded cells arising from slow infiltration of vascular tissue can be overcome using a prevascularisation strategy consisting of seeding the scaffold with vascular cells. Using certain assumptions it is shown how the system can be simplified to one which is partially tractable and for which some analysis is given. Limited comparison is also given of the model solutions with experimental data from the chick chorioallantoic membrane (CAM) assay.

Controlled embryoid body formation via surface modification and avidin-biotin cross-linking.

Cell-cell interaction is an integral part of embryoid body (EB) formation controlling 3D aggregation. Manipulation of embryonic stem (ES) cell interactions could provide control over EB formation. Studies have shown a direct relationship between EB formation and ES cell differentiation. We have previously described a cell surface modification and cross-linking method for influencing cell-cell interaction and formation of multicellular constructs. Here we show further characterisation of this engineered aggregation. We demonstrate that engineering accelerates ES cell aggregation, forming larger, denser and more stable EBs than control samples, with no significant decrease in constituent ES cell viability. However, extended culture >/=5 days reveals significant core necrosis creating a layered EB structure. Accelerated aggregation through engineering circumvents this problem as EB formation time is reduced. We conclude that the proposed engineering method influences initial ES cell-ES cell interactions and EB formation. This methodology could be employed to further our understanding of intrinsic EB properties and their effect on ES cell differentiation.

Tissue engineering: strategies, stem cells and scaffolds.

Tissue engineering scaffolds are designed to influence the physical, chemical and biological environment surrounding a cell population. In this review we focus on our own work and introduce a range of strategies and materials used for tissue engineering, including the sources of cells suitable for tissue engineering: embryonic stem cells, bone marrow-derived mesenchymal stem cells and cord-derived mesenchymal stem cells. Furthermore, we emphasize the developments in custom scaffold design and manufacture, highlighting laser sintering, supercritical carbon dioxide processing, growth factor incorporation and zoning, plasma modification of scaffold surfaces, and novel multi-use temperature-sensitive injectable materials.

Clinical applications of musculoskeletal tissue engineering.

BACKGROUND: Current surgical techniques for the repair of the musculoskeletal system can be often limited by the availability, quality and quantity of materials, such as grafts to effect repair. This has led to the exploration and development of novel methods of intervention based on tissue engineering and regenerative medicine. SOURCE OF DATA: This review summarizes the successes and investigations which are happening to date in the field of musculoskeletal tissue engineering. This is based on an extensive literature search and through basic research being performed by the authors. AREAS OF AGREEMENT: Due to the constraints surrounding certain surgical techniques and restrictions on their use, novel procedures are required for the repair and regeneration of damaged tissues. AREAS OF CONTROVERSY: The choice of cell type has caused much debate within the tissue-engineering field. However it is widely accepted that currently only autologous primary/adult stem cells are fit for transplantation, until such times that optimized differentiation and selection protocols exist for embryonic stem cells. GROWING POINTS: The current results of the clinical cases utilizing tissue engineered constructs for bone and cartilage repair provide insights for improvement of these techniques thus allowing treatments to become increasingly viable. AREAS TIMELY FOR DEVELOPING RESEARCH: There is a need to better understand the integration of scaffolds and cell populations into the target tissue. This should provide vital information influencing scaffold manufacturing procedures and cell selection.

Autonomic nerve trauma at radical hysterectomy: the nerve content and subtypes within the superficial and deep uterosacral ligaments.

The authors previously demonstrated nerve trunks and autonomic ganglia of the hypogastric plexus within the uterosacral ligament (USL) and the cardinal ligaments. The nerve content of these ligaments is greatest closer to the pelvic sidewalls and diminishes toward the insertion of the ligaments into the uterus, with the greater nerve content in the USL. Here the authors determine whether the nerve content of the superficial and deep portion of the USLs, where they are divided at a radical hysterectomy, differ. Biopsies were taken from the right and left superficial and deep USL in 6 patients during radical hysterectomy for early-stage cervical cancer. Indirect immunofluorescence was performed using primary antibodies to (1) the panneuronal marker PGP 9.5, (2) the parasympathetic marker vasoactive intestinal peptide, (3) the sympathetic markers tyrosine hydroxylase and neuropeptide-Y, (4) the sensory and nociceptive nerve marker substance P, and (5) the sensory and sensory-motor nerve marker calcitonin gene-related peptide. The percentage area of immunoreactivity (PAI) was determined using a computer-assisted image analyzer as an objective measure of nerve content. There was a lower nerve content in the superficial USL compared with the deep USL. The PAI of the deep USL was greater than that of the superficial USL for all the nerve markers (P < .05). The PAI was greatest for sympathetic and sensory/nociceptive nerve markers. There were relatively more sympathetic nerve fibers than parasympathetic nerve fibers in the deep USL. These data provide further indirect evidence that pelvic dysfunction following radical hysterectomy is associated with division of the deep portion of the USL.

Accelerated formation of multicellular 3-D structures by cell-to-cell cross-linking.

The three-dimensional (3-D) arrangement of cells within tissues is integral to their development and function. Advances in stem cell science and regenerative medicine have stimulated interest in the replication of this architecture in vitro. We have developed a versatile method for controlling short-term cell-cell and cell-matrix interactions via a facile cell surface engineering process that enables the rapid formation of specific 3-D interactions for a range of cell types. We demonstrate that chemical modification of cell surfaces and matrix proteins can artificially accelerate the cell adhesion process and confirm the ability to control the formation of multicellular aggregates with defined architectures and heterotypic cell types. Direct comparison with a natural aggregation process seen during differentiation of embryonic stem (ES) cells revealed increased expression of developmental regulatory proteins and a concomitant enhancement of ES cell differentiation. Furthermore, this new methodology has numerous applications in generating layered structures. For example, we demonstrate improved transfer of therapeutic human keratinocytes onto a dermal layer in a skin repair model.

Characterization of human fetal osteoblasts by microarray analysis following stimulation with 58S bioactive gel-glass ionic dissolution products.

Bioactive glasses dissolve upon immersion in culture medium, releasing their constitutive ions in solution. There is evidence suggesting that these ionic dissolution products influence osteoblast-specific processes. Here, we investigated the effect of 58S sol-gel-derived bioactive glass (60 mol % SiO2, 36 mol % CaO, 4 mol % P2O5) dissolution products on primary osteoblasts derived from human fetal long bone explant cultures (hFOBs). We used U133A human genome GeneChip oligonucleotide arrays to examine 22,283 transcripts and variants, which represent over 18,000 well-substantiated human genes. Hybridization of samples (biotinylated cRNA) derived from monolayer cultures of hFOBs on the arrays revealed that 10,571 transcripts were expressed by these cells, with high confidence. These included transcripts representing osteoblast-related genes coding for growth factors and their associated molecules or receptors, protein components of the extracellular matrix (ECM), enzymes involved in degradation of the ECM, transcription factors, and other important osteoblast-associated markers. A 24-h treatment with a single dosage of ionic products of sol-gel 58S dissolution induced the differential expression of a number of genes, including IL-6 signal transducer/gp130, ISGF-3/STAT1, HIF-1 responsive RTP801, ERK1 p44 MAPK (MAPK3), MAPKAPK2, IGF-I and IGFBP-5. The over 2-fold up-regulation of gp130 and MAPK3 and down-regulation of IGF-I were confirmed by real-time RT-PCR analysis. These data suggest that 58S ionic dissolution products possibly mediate the bioactive effect of 58S through components of the IGF system and MAPK signaling pathways.

Dose- and time-dependent effect of bioactive gel-glass ionic-dissolution products on human fetal osteoblast-specific gene expression.

Bioactive glasses dissolve upon immersion in culture medium, and release their constitutive ions into solution. There has been some evidence suggesting that these ionic-dissolution products influence osteoblast-specific processes. Here, the effect of 58S sol-gel-derived bioactive glass (60% SiO(2), 36% CaO, 4% P(2)O(5), in molar percentage) on primary osteoblasts derived from human fetal long bone explant cultures is investigated, and it is hypothesized that critical concentrations of sol-gel-dissolution products (consisting of a combination of simple inorganic ions) can enhance osteoblast phenotype in vitro by affecting the expression of a number of genes associated with the differentiation and extracellular matrix deposition processes. Cells were exposed to a range of 58S dosages continuously for a period of 4-14 days in monolayer cultures. Quantitative real-time RT-PCR analysis of a panel of osteoblast-specific markers showed a varied gene expression pattern in response to the material. The highest concentration of Ca and Si tested (96 and 50 ppm, respectively) promoted upregulation of gene expression for most markers (including alkaline phosphatase, osteocalcin, and osteopontin) at the latest time point, compared to non-58S-treated control, although this observation was not statistically significant. The same 58S concentration produced higher ALP activity levels and increased proliferation throughout the culture period, compared to lower dosages tested; however, the results generated were again not statistically significant. The data overall suggest that no significant effect can be ascribed to the ionic products of 58S bioactive gel-glass dissolution tested here and their ability to stimulate osteoblastic marker gene expression.

Novel surface entrapment process for the incorporation of bioactive molecules within preformed alginate fibers.

A physical entrapment technique has been developed for the surface engineering of preformed alginate fibers. Surface engineering was carried out at room temperature in aqueous solutions without additional solvent, a catalyst/initiator, a chemical cross-linking agent, or a temperature increase. Entrapment of surface-modifying molecules was achieved by exposing the alginate fibers to a Na(+)-rich NaCl/CaCl2 mixture solution, which caused the formation of a moderate dissociation layer into which the modifier could diffuse within a few seconds. The surface dissociation was then reversed by the addition of a large excess of multivalent cations, which resulted in collapse of the interface and immobilization of the modifying species. Rhodamine-tagged poly(ethylene glycol)s of different molecular weights were used as model molecules to investigate the effect of process parameters on the entrapment efficiency. It was found that the entrapment efficiency as well as the distribution of the modifier within the alginate fibers was determined by several factors, including the NaCl/CaCl2 ratio in the preswelling solution, exposure time, and concentration and molecular weight of the modifiers. The morphology of the fibers was not significantly changed in terms of shape and size after the entrapment process. By this technique, poly(L-lysine) (PLL) coupled with cell adhesion peptide sequence GRGDS (PLL-GRGDS) was entrapped within alginate fibers, and it was demonstrated that the modification promoted the attachment of mouse 3T3 fibroblasts.

Differentiation of osteoblasts from murine embryonic stem cells by overexpression of the transcriptional factor osterix.

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.