A.Bashirullah, S.R.Halsell and R.L.Cooperstock contributed equally to this workMaternally synthesized RNAs program early embryonic development in many animals. These RNAs are degraded rapidly by the midblastula transition (MBT), allowing genetic control of development to pass to zygotically synthesized transcripts. Here we show that in the early embryo of Drosophila melanogaster, there are two independent RNA degradation pathways, either of which is sufficient for transcript elimination. However, only the concerted action of both pathways leads to elimination of transcripts with the correct timing, at the MBT. The first pathway is maternally encoded, is targeted to specific classes of mRNAs through cis-acting elements in the 3Ј-untranslated region and is conserved in Xenopus laevis. The second pathway is activated 2 h after fertilization and functions together with the maternal pathway to ensure that transcripts are degraded by the MBT.

Vascularization of engineered bone tissue is critical for ensuring its survival after implantation and it is the primary factor limiting its clinical use. A promising approach is to prevascularize bone grafts in vitro using endothelial progenitor cells (EPC) derived from peripheral blood. Typically, EPC are added together with mesenchymal stem cells (MSC) that differentiate into osteoblasts. One problem with this approach is how to promote traditional tissue engineering bone survival with a minimally invasive method. In this study, we examined the effectiveness of administering to stimulate the release of peripheral blood stem cells and their co-culturing system for generating prevascularized engineered bone. Cells were isolated by Ficoll density gradient centrifugation and identified as EPC and MSC based on morphology, surface markers, and functional analysis. EPC and MSC were cocultured in several different ratios, and cell morphology and tube formation were assessed by microscopy. Expression of osteogenesis and vascularization markers was quantified by enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction, and histochemical and immunofluorescence staining. Increasing the proportion of EPC in the coculture system led to greater tube formation and greater expression of the endothelial cell marker CD31. An EPC:MSC ratio of 75:25 gave the highest expression of osteogenesis and angiogenesis markers. Cocultures adhered to a three-dimensional scaffold of strontium-doped calcium polyphosphate and proliferated well. Our findings show that coculturing peripheral blood-derived EPC and MSC may prove useful for generating prevascularized bone tissue for clinical use.

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These results suggest that PB MSCs are a new source of seed cells used in articular cartilage repair.