Identification and Specification of the Mouse Skeletal Stem Cell

SummaryHow are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues

Adipose-Derived Stem Cells: A Review of Signaling Networks Governing Cell Fate and Regenerative Potential in the Context of Craniofacial and Long Bone Skeletal Repair

Improvements in medical care, nutrition and social care are resulting in a commendable change in world population demographics with an ever increasing skew towards an aging population. As the proportion of the world’s population that is considered elderly increases, so does the incidence of osteodegenerative disease and the resultant burden on healthcare. The increasing demand coupled with the limitations of contemporary approaches, have provided the impetus to develop novel tissue regeneration therapies. The use of stem cells, with their potential for self-renewal and differentiation, is one potential solution. Adipose-derived stem cells (ASCs), which are relatively easy to harvest and readily available have emerged as an ideal candidate. In this review, we explore the potential for ASCs to provide tangible therapies for craniofacial and long bone skeletal defects, outline key signaling pathways that direct these cells and describe how the developmental signaling program may provide clues on how to guide these cells in vivo. This review also provides an overview of the importance of establishing an osteogenic microniche using appropriately customized scaffolds and delineates some of the key challenges that still need to be overcome for adult stem cell skeletal regenerative therapy to become a clinical reality

Integration of Multiple Signaling Pathways Determines Differences in the Osteogenic Potential and Tissue Regeneration of Neural Crest-Derived and Mesoderm-Derived Calvarial Bones

The mammalian skull vault, a product of a unique and tightly regulated evolutionary process, in which components of disparate embryonic origin are integrated, is an elegant model with which to study osteoblast biology. Our laboratory has demonstrated that this distinct embryonic origin of frontal and parietal bones confer differences in embryonic and postnatal osteogenic potential and skeletal regenerative capacity, with frontal neural crest derived osteoblasts benefitting from greater osteogenic potential. We outline how this model has been used to elucidate some of the molecular mechanisms which underlie these differences and place these findings into the context of our current understanding of the key, highly conserved, pathways which govern the osteoblast lineage including FGF, BMP, Wnt and TGFβ signaling. Furthermore, we explore recent studies which have provided a tantalizing insight into way these pathways interact, with evidence accumulating for certain transcription factors, such as Runx2, acting as a nexus for cross-talk

Enhanced Activation of Canonical Wnt Signaling Confers Mesoderm-Derived Parietal Bone with Similar Osteogenic and Skeletal Healing Capacity to Neural Crest-Derived Frontal Bone

<div><p>Bone formation and skeletal repair are dynamic processes involving a fine-tuned balance between osteoblast proliferation and differentiation orchestrated by multiple signaling pathways. Canonical Wnt (cWnt) signaling is known to playing a key role in these processes. In the current study, using a transgenic mouse model with targeted disruption of <i>axin2</i>, a negative regulator of cWnt signaling, we investigated the impact of enhanced activation of cWnt signaling on the osteogenic capacity and skeletal repair. Specifically, we looked at two calvarial bones of different embryonic tissue origin: the neural crest-derived frontal bone and the mesoderm-derived parietal bone, and we investigated the proliferation and apoptotic activity of frontal and parietal bones and derived osteoblasts. We found dramatic differences in cell proliferation and apoptotic activity between <i>Axin2</i>^<i>-/-</i> and wild type calvarial bones, with <i>Axin2</i>^<i>-/-</i> showing increased proliferative activity and reduced levels of apoptosis. Furthermore, we compared osteoblast differentiation and bone regeneration in <i>Axin2</i>^<i>-/-</i> and wild type neural crest-derived frontal and mesoderm-derived parietal bones, respectively. Our results demonstrate a significant increase either in osteoblast differentiation or bone regeneration in <i>Axin2</i>^<i>-/-</i> mice as compared to wild type, with <i>Axin2</i>^<i>-/-</i> parietal bone and derived osteoblasts displaying a “neural crest-derived frontal bone-like” profile, which is typically characterized by higher osteogenic capacity and skeletal repair than parietal bone. Taken together, our results strongly suggest that enhanced activation of cWnt signaling increases the skeletal potential of a calvarial bone of mesoderm origin, such as the parietial bone to a degree similar to that of a neural crest origin bone, like the frontal bone. Thus, providing further evidence for the central role played by the cWnt signaling in osteogenesis and skeletal-bone regeneration.</p></div

<i>In vivo</i> calvarial healing of <i>Axin2</i>^<i>-/-</i> and wild type frontal and parietal bones.

<p><b>(A</b>) Two-millimeter (2mm) defects were created in the frontal and parietal bones of 7 month-old <i>Axin2</i>^<i>-/-</i> and wild type mice (n = 3). Quantification of defect repair according to microCT-scan results. Statistical analysis was conducted utilizing the Mann-Whitney Test. P-values: *P≤ 0.05. (<b>B</b>) Pentachrome staining of coronal sections of skull at post-operative week 8 showing the repair of calvarial bone defects as determined by yellow color. Bone regeneration was higher in <i>Axin2</i>^<i>-/-</i> frontal and parietal bones as compared to wild type bones. <b>(C)</b> Histogram showing the distance between the osteogenic fronts (dashed) and marked by arrows (Objective magnification 5x).</p

Enhanced proliferative activity of <i>Axin2</i>^<i>-/-</i> FOb and POb.

<p>(<b>A</b>) <i>In vitro</i> BrdU assay performed on <i>Axin2</i>^<i>-/-</i> and wild type FOb and POb cells undergoing differentiation reveals increased proliferative activity in <i>Axin2</i>^<i>-/-</i> cells than corresponding wild type. (<b>B</b>) <i>in vivo</i> PCNA immunostaining performed on coronal sections derived from frontal and parietal bones of pN21 <i>Axin2</i>^<i>-/-</i> and wild type mice, also indicates an increase in proliferation of <i>Axin2</i>^<i>-/-</i> frontal and parietal bones compared wild type. (<b>C</b>) Quantification of PCNA staining obtained by calculating the percentage of PCNA positive cells over the total cell number counted at least in five equivalent areas of each bone, indicates the lowest cell proliferation activity in wild type parietal bone, whereas <i>Axin2</i>^<i>-/-</i> parietal bone displays activity similar to that of wild type frontal bone. Scale bar = 150 μm.</p

<i>In vitro</i> differentiation and osteogenic-related gene expression analysis of <i>Axin2</i>^<i>-/-</i> and wild type FOb and POb cells.

<p>(<b>A)</b> quantitative PCR analysis performed on wt and <i>Axin2</i>^<i>-/-</i>FOb and POb showing a significant upregulation of cWnt signaling target genes in both, <i>Axin2</i>^<i>-/-</i>FOb and POb as compared to wt osteoblasts. (<b>B</b>) enhanced activation of cWnt signaling in <i>Axin2</i>^<i>-/-</i>FOb and POb is further confirmed by indirect immunofluorescence analysis showing larger number of cells with positive nuclear staining for active β-catenin. Positive nuclear staining is more dramatic in <i>Axin2</i>^<i>-/-</i>POb. Nuclear counterstaining was performed with DAPI (objective magnification 10x). Scale bars = 50μm. (<b>C</b>) Cells were cultured with differentiation medium for 18 days. Mineralization of extracellular matrix as assessed by alizarin red staining indicates a more robust mineralization in <i>Axin2</i>^<i>-/-</i> FOb and POb as compared to corresponding wild type FOb and POb. (<b>D</b>) Magnification of alizarin red stained bone nodules. (<b>E</b>) Quantification of alizarin red staining as showed above (panel A) confirms enhanced osteogenic capacity of FOb and POb cells derived from <i>Axin2</i>^<i>-/-</i> mice. (<b>F</b>) RT-PCR analysis of osteogenic markers showing significant higher up-regulation of the <i>Runx2</i>, <i>Alpl</i> and <i>Bglap</i> in <i>Axin2</i>^<i>-/-</i> FOb and POb. (<b>G</b>) Histograms representing quantification of each electrophoretic band obtained by Image J program. Each band was normalized to its <i>Gapdh</i> content. *P≤ 0.05.</p

Small Molecule Inhibition of Transforming Growth Factor Beta Signaling Enables the Endogenous Regenerative Potential of the Mammalian Calvarium

Current approaches for the treatment of skeletal defects are suboptimal, principally because the ability of bone to repair and regenerate is poor. Although the promise of effective cellular therapies for skeletal repair is encouraging, these approaches are limited by the risks of infection, cellular contamination, and tumorigenicity. Development of a pharmacological approach would therefore help avoid some of these potential risks. This study identifies transforming growth factor beta (TGFβ) signaling as a potential pathway for pharmacological modulation in vivo. We demonstrate that inhibition of TGFβ signaling by the small molecule SB431542 potentiates calvarial skeletal repair through activation of bone morphogenetic protein (BMP) signaling on osteoblasts and dura mater cells participating in healing of calvarial defects. Cells respond to inhibition of TGFβ signaling by producing higher levels of BMP2 that upregulates inhibitory Smad6 expression, thus providing a negative feedback loop to contain excessive BMP signaling. Importantly, study on human osteoblasts indicates that molecular mechanism(s) triggered by SB431542 are conserved. Collectively, these data provide insights into the use of small molecules to modulate key signaling pathways for repairing skeletal defects