Endochondral ossification is required for haematopoietic stem-cell niche formation.

Little is known about the formation of niches, local micro-environments required for stem-cell maintenance. Here we develop an in vivo assay for adult haematopoietic stem-cell (HSC) niche formation. With this assay, we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation.

Visualizing in vivo liposomal drug delivery in real-time.

Liposomes have tremendous potential for efficient small molecule delivery. Previous studies, however, have been hampered by an inability to monitor their distribution and release of contents. Here, the authors demonstrate the real time monitoring of small molecule delivery using luciferin as a model. To monitor the release of luciferin in vivo, luciferin was packaged in thermosensitive liposomes and delivered into transgenic mice that constitutively express luciferase. Their experiments show the thermally induced release of the liposomal content in real time. In addition, the model provides evidence that the thermosensitive liposomes are stable over a long period of time ( approximately 3 weeks), and still release their content upon heating. These data present a strategy to monitor liposomal drug delivery in vivo with luciferin.

Beta-catenin-dependent Wnt signaling in mandibular bone regeneration.

Osteoblasts are derived from two distinct embryonic lineages: cranial neural crest, and mesoderm. Both populations of cells are capable of forming bone and cartilage during fetal development and during adult bone repair, but whether they use equivalent molecular pathways to achieve osteoblast differentiation is unknown. We addressed this question in the context of cranial repair and focused on the role of Wnt signaling in mandibular skeletal healing. Transgenic Wnt reporter mice were used to pinpoint Wnt-responsive cells in the injury callus, and in situ hybridization was used to identify some of the Wnt ligands expressed by cells during the repair process. A gene transfer technique was employed to abrogate Wnt signaling during mandibular healing, and we found that reparative intramembranous ossification requires a functional Wnt pathway. Finally, we evaluated how constitutive activation of the Wnt pathway, caused by mutation of the LRP5 receptor, affected bone repair in the mandible. Taken together, these data underscore the functional requirement for Wnt signaling in cranial skeletal healing.

Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration.

The fetal skeleton arises from neural crest and from mesoderm. Here, we provide evidence that each lineage contributes a unique stem cell population to the regeneration of injured adult bones. Using Wnt1Cre::Z/EG mice we found that the neural crest-derived mandible heals with neural crest-derived skeletal stem cells, whereas the mesoderm-derived tibia heals with mesoderm-derived stem cells. We tested whether skeletal stem cells from each lineage were functionally interchangeable by grafting mesoderm-derived cells into mandibular defects, and vice versa. All of the grafting scenarios, except one, healed through the direct differentiation of skeletal stem cells into osteoblasts; when mesoderm-derived cells were transplanted into tibial defects they differentiated into osteoblasts but when transplanted into mandibular defects they differentiated into chondrocytes. A mismatch between the Hox gene expression status of the host and donor cells might be responsible for this aberration in bone repair. We found that initially, mandibular skeletal progenitor cells are Hox-negative but that they adopt a Hoxa11-positive profile when transplanted into a tibial defect. Conversely, tibial skeletal progenitor cells are Hox-positive and maintain this Hox status even when transplanted into a Hox-negative mandibular defect. Skeletal progenitor cells from the two lineages also show differences in osteogenic potential and proliferation, which translate into more robust in vivo bone regeneration by neural crest-derived cells. Thus, embryonic origin and Hox gene expression status distinguish neural crest-derived from mesoderm-derived skeletal progenitor cells, and both characteristics influence the process of adult bone regeneration.

Liposomal packaging generates Wnt protein with in vivo biological activity.

Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context.