Use of RUNX2 expression to identify osteogenic progenitor cells derived from human embryonic stem cells.

We generated a RUNX2-yellow fluorescent protein (YFP) reporter system to study osteogenic development from human embryonic stem cells (hESCs). Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP(+) cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP(+) cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development.

Functional assessment of hematopoietic niche cells derived from human embryonic stem cells.

To evaluate hematopoietic niche cell populations isolated from human embryonic stem cells (hESCs), we tested the ability of hESC-derived stromal lines to support CD34(+) umbilical cord blood (UCB)- and hESC-derived CD34(+)45(+) cells in long-term culture initiating cell (LTC-IC) assays. Specifically, these hematopoietic populations were cocultured with hESC-derived mesenchymal stromal cells (hESC-MSCs) and hESC-derived endothelial cells (hESC-ECs), and then assessed for their LTC-IC potential in comparison to coculture with bone marrow (BM)-derived MSCs and the mouse stromal line M2-10B4. We found that the hESC-derived stromal lines supported LTC-ICs from UCB similar to M2-10B4 cells and better than BM-MSCs. However, none of the stromal populations supported LTC-IC from hESC-derived CD34(+)45(+) cells. Engraftment data using the output from LTC-IC assays showed long-term repopulation (12 weeks) of NSG mice to correlate with LTC-IC support on a given stromal layer. Therefore, hESC-derived stromal lines can be used to efficiently evaluate putative hematopoietic stem/progenitor cells derived from hESCs or other cell sources.

Induction of fracture repair by mesenchymal cells derived from human embryonic stem cells or bone marrow.

Development of novel therapeutic approaches to repair fracture non-unions remains a critical clinical necessity. We evaluated the capacity of human embryonic stem cell (hESC)-derived mesenchymal stem/stromal cells (MSCs) to induce healing in a fracture non-union model in rats. In addition, we placed these findings in the context of parallel studies using human bone marrow MSCs (hBM-MSCs) or a no cell control group (n = 10-12 per group). Preliminary studies demonstrated that both for hESC-derived MSCs and hBM-MSCs, optimal induction of fracture healing required in vitro osteogenic differentiation of these cells. Based on biomechanical testing of fractured femurs, maximum torque, and stiffness were significantly greater in the hBM-MSC as compared to the control group that received no cells; values for these parameters in the hESC-derived MSC group were intermediate between the hBM-MSC and control groups, and not significantly different from the control group. However, some evidence of fracture healing was evident by X-ray in the hESC-derived MSC group. Our results thus indicate that while hESC-derived MSCs may have potential to induce fracture healing in non-unions, hBM-MSCs function more efficiently in this process. Additional studies are needed to further modify hESCs to achieve optimal fracture healing by these cells.

Human embryonic stem cell-derived CD34+ cells function as MSC progenitor cells.

Mesenchymal stem/stromal cells (MSCs) have been isolated from various tissues and utilized for an expanding number of therapies. The developmental pathways involved in producing MSCs and the phenotypic precursor/progenitor cells that give rise to human MSCs remain poorly defined. Human embryonic stem cells (hESCs) have the capability to generate functional hemato-endothelial cells and other mesoderm lineage cells. hESC-derived CD73(+) cells have been isolated and found to have similar phenotypic and functional characteristics as adult MSCs. Here we demonstrate hESC-derived CD34(+)CD73(-) cells can serve as MSC progenitor cells with the ability to differentiate into adipocytes, osteoblasts and chondrocytes. Additionally, gene array analysis of hESC-derived MSCs show substantially different gene expression compared to bone marrow (BM)-derived MSCs, especially with increased expression of pluripotent and multipotent stem cell and endothelial cell-associated genes. The isolation of functional MSCs from hESC-derived CD34(+)CD73(-) cells provides improved understanding of MSC development and utilization of pluripotent stem cells to produce MSCs suited for novel regenerative therapies.

Responses of intramembranous bone and sutures upon in vivo cyclic tensile and compressive loading.

Cranial vault and facial sutures interpose between mineralized bones of the skull, and may function analogously to appendicular and cranial base growth plates. However, unlike growth plates that are composed of chondrocyte lineage, cranial and facial sutures possess heterogeneous cell lineages such as mesenchymal cells, fibroblasts, and osteoblasts, in addition to vascular-derived cells. Despite recently intensified effort, the biological responses of intramembranous bone and sutures to mechanical loading are not well understood. This study was designed to investigate whether brief doses of tensile or compressive forces induce modeling and growth responses of intramembranous bone and sutures. In different groups of growing rabbits in vivo, cyclic tensile or compressive forces at 1 N and 8 Hz were applied to the maxilla for 20 min/day over 12 consecutive days. Computerized histomorphometric analyses revealed that the average sutural widths of both the premaxillomaxillary suture (PMS) and nasofrontal suture (NFS) loaded in either tension or compression were significantly higher than age- and sex-matched sham controls (P<0.01). The average cell densities of tension- or compression-loaded PMS and NFS were significantly higher than sham controls (P<0.01). The average osteoblast occupied sutural bone surface loaded under tension was significantly higher than that of sham control (P<0.05). Interestingly, tensile loading significantly reduced the average osteoclast surface, in comparison to sham control (P<0.05). For the NFS, tensile loading significantly increased the average osteoblast occupied sutural bone surface, in comparison with that of sham control (P<0.05). Also for the NFS suture, compression significantly reduced the average sutural osteoclast surface in comparison with sham control (P<0.05). Taken together, the present data suggest that high-frequency cyclic forces in either tension or compression induce modeling and growth changes in cranial sutures. Due to the structural complexity of cranial vault and facial sutures, either tensile or compressive forces likely are transmitted as shear stresses and upregulate genes and gene products responsible for sutural growth.

Adult stem cell driven genesis of human-shaped articular condyle.

Uniform design of synovial articulations across mammalian species is challenged by their common susceptibility to joint degeneration. The present study was designed to investigate the possibility of creating human-shaped articular condyles by rat bone marrow-derived mesenchymal stem cells (MSCs) encapsulated in a biocompatible poly(ethylene glycol)-based hydrogel. Rat MSCs were harvested, expanded in culture, and treated with either chondrogenic or osteogenic supplements. Rat MSC-derived chondrogenic and osteogenic cells were loaded in hydrogel suspensions in two stratified and yet integrated hydrogel layers that were sequentially photopolymerized in a human condylar mold. Harvested articular condyles from 4-week in vivo implantation demonstrated stratified layers of chondrogenesis and osteogenesis. Parallel in vitro experiments using goat and rat MSCs corroborated in vivo data by demonstrating the expression of chondrogenic and osteogenic markers by biochemical and mRNA analyses. Ex vivo incubated goat MSC-derived chondral constructs contained cartilage-related glycosaminoglycans and collagen. By contrast, goat MSC-derived osteogenic constructs expressed alkaline phosphatase and osteonectin genes, and showed escalating calcium content over time. Rat MSC-derived osteogenic constructs were stiffer than rat MSC-derived chondrogenic constructs upon nanoindentation with atomic force microscopy. These findings may serve as a primitive proof of concept for ultimate tissue-engineered replacement of degenerated articular condyles via a single population of adult mesenchymal stem cells.

Expression of in vivo mechanical strain upon different wave forms of exogenous forces in rabbit craniofacial sutures.

Sutures are fibrous joints between craniofacial bones, providing an interesting model for studying the biomechanics of the interface between soft and mineralized tissues. To explore whether different wave forms of exogenous forces induce corresponding sutural strain wave forms, sutural strain of the premaxillomaxillary suture (PMS) and nasofrontal suture (NFS) of New Zealand White rabbits (N = 8) was recorded upon application of static, sine- and square-wave forces against the maxilla from 1 N to 5 N in 1 N increments. The PMS demonstrated compressive strain, whereas the NFS tensile strain. Despite a tenfold difference in peak PMS strain (- 1451 +/- 512 micro(epsilon)) and NFS strain (141 +/- 39 micro(epsilon)) in response to 5 N cyclic forces, wave forms of exogenous forces were expressed as corresponding wave forms of sutural strain in both the PMS and NFS. Peak sutural strain was similar upon static and sine-wave cyclic loading. Thus, cells and matrix components of fibrous sutural tissue experience different wave forms of exogenous forces as corresponding wave forms of tissue-borne mechanical strain. Current craniofacial orthopedic therapies exclusively utilize static forces to change the shape of craniofacial bones via mechanically induced bone apposition and resorption. The present data provide room for exploring whether cyclic forces capable of inducing different sutural strain wave forms may accelerate sutural anabolic or catabolic responses.

Biomechanics of craniofacial sutures: orthopedic implications.

Sutures are soft connective tissue articulations between craniofacial bones. Suture mechanics deals with patterns of mechanical stress experienced in sutures resulting from natural activities such as mastication and exogenous forces such as orthopedic loading. Patterns of sutural mechanical stress can be delineated readily as sutural strain using strain gages attached over the suture. In mastication, complex sutural strain patterns have been elucidated in a few species. Mechanical stresses are not transmitted in the skull as a continuing gradient, for different sutures are capable of redefining a propagating mechanical force as predominately tensile or compressive strain. Exogenous mechanical forces with engineering waveforms such as static and sine wave at different frequencies induce corresponding waveforms and rates of sutural strain, providing the basis for applying novel mechanical stimuli to engineer sutural growth. The available data on suture mechanics converge to a hypothetical theme that mechanical forces regulate sutural growth by inducing sutural mechanical strain. Various orthopedic therapies, including headgear, facemask, and functional appliances may induce sutural strain, leading to modification of otherwise natural suture growth.

Suture growth modulated by the oscillatory component of micromechanical strain.

Sutures are fibrous connective tissue articulations between intramembranous craniofacial bones. Sutures are composed of fibroblastic cells with their matrices in the center and osteogenic cells in the periphery producing a matrix that is mineralized during skeletal growth. Whether oscillatory forces stimulate sutural growth is unknown. In the present work, we applied static and cyclic forces with the same peak magnitude of 5N to the maxilla in growing rabbits and quantified (1) acute in vivo sutural bone strain responses and (2) chronic growth responses in the premaxillomaxillary suture (PMS) and nasofrontal suture (NFS). Bone strain recordings showed that the waveforms of static force and 1-Hz cyclic force were expressed as corresponding static and cyclic sutural strain patterns in both the PMS and NFS, with the mean peak PMS strain (-1451 +/- 137 microepsilon for the cyclic and -1572 +/- 138 microepsilon for the static) approximately 10-fold higher than the mean peak NFS strain (124 +/- 9 microepsilon for the cyclic and 134 +/- 9 microepsilon for the static). Strain polarity was the opposite: compressive for the PMS but tensile for the NFS. However, on application of repetitive 5N cyclic and static forces in vivo for 10 minutes/day over 12 days, cyclic loading induced significantly greater sutural widths for the compressed PMS (95.1 +/- 8.3 microm) than sham control (69.8 +/- 8.2 microm) and static loading (58.9 +/- 2.8 microm; p < 0.01). Interestingly, the same trend was true for the NFS under tensile strain: significantly greater sutural width for cyclic loading (267.4 +/- 64.2 microm) than sham control (196.0 +/- 10.1 microm) and static loading (169.9 +/- 11.4 microm). Cell counting in 110 x 110 microm grids laid over sutures disclosed significantly more sutural cells on repetitive cyclic loading than sham control and static loading (p < 0.05) for both the PMS and NFS. Fluorescent labeling of newly formed sutural bone demonstrated more osteogenesis on cyclic loading in comparison with sham control and static loading. Thus, the oscillatory component of cyclic force or more precisely the resulting cyclic strain experienced in sutures is a potent stimulus for sutural growth. The increased sutural growth by cyclic mechanical strain in the tensed NFS and compressed PMS suggests that both microscale tension and compression induce anabolic sutural growth response.

Strain induced osteogenesis of the craniofacial suture upon controlled delivery of low-frequency cyclic forces.

Static forces have been used for more than a century to modulate osteogenesis of craniofacial sutures in not only laboratory research, but also clinical practice. Whether cyclic forces more effectively stimulate sutural osteogenesis than static forces is unknown. Here, the premaxillomaxillary sutures of growing rabbits received in vivo exogenous static forces with peak magnitude of 2 Newtons, or cyclic forces also at 2 Newtons but with frequencies of 0.2 Hz and 1 Hz. The static force and two cyclic forces did not evoke significant differences in the peak magnitude of static bone strain (506 microstrain 182; mean S.D.), 0.2-Hz cyclic strain (436 microstrain 191) or 1-Hz cyclic strain (461 microstrain 229). However, cyclic forces at 0.2 Hz delivered to the premaxillomaxillary suture for 10 min/d over 12 days (120 cyclic per day) induced significantly more craniofacial growth (p< 0.01), marked sutural separation, and islands of newly formed bone, in comparison with both sham controls and static force of matching peak magnitude. The bone strain threshold of approximately 500 microstrain for inducing sutural osteogenesis is lower than the minimum effective strain capable of inducing bone apposition in long bones. These data demonstrate, for the first time, that application of brief doses of cyclic forces induces sutural osteogenesis more effectively than static forces with matching peak magnitude.

Characterization of PC12 cell proliferation and differentiation-stimulated by ECM adhesion proteins and neurotrophic factors.

Among the various elements which influence axonal outgrowth in vivo is the physicochemical interaction of actively outgrowing nerve fibers with the various substrata they encounter during differentiation. Several experiments have explored the role of the extracellular matrix (ECM) in the control of neuronal differentiation. The nature, however, of the interactions between neurons and components of the ECM during regeneration and development are largely a matter of speculation. Although previous studies have already explored the influence of a number of ECM adhesion proteins and neurotrophic factors on neurite outgrowth, none have been carried in a systematic approach that allows for the simultaneous comparison of different surface conditions in relation to different neurotrophic factors. Motivated by the necessity of establishing controlled environments that allow for the rational design of stable neuronal/biomaterial interfaces, the long-term effects of NGF and FGF-2 on the behavior of PC12 cells plated on collagen and laminin modified surfaces were evaluated. A pheochromocytoma cell line derived from transplantable rat adrenal medulla, PC12 cells have been commonly employed as an instructive model for studying the underlying mechanisms of neuronal differentiation. Long-term characterization of PC12 proliferation and neuronal differentiation for an experimental duration of 7-22 days was achieved by both qualitatively and quantitatively assaying for cell count, neurite number, neurite mean length, and neurite stability. Neurite stability was determined in terms of resistance to loss after neurotrophic factor (NGF/FGF-2) withdrawal. The present findings demonstrate that ECM adhesion proteins collagen and laminin are equally effective in promoting PC12 proliferation. It was noted as well that NGF supplemented collagen cultures are significantly more efficient in providing long-term support to PC12 differentiation in terms of neurite number, mean length, and stability.