Phosphoproteomic profiling reveals a defined genetic program for osteoblastic lineage commitment of human bone marrow-derived stromal stem cells.

Bone marrow-derived mesenchymal stem cells (MSCs) differentiate into osteoblasts upon stimulation by signals present in their niche. Because the global signaling cascades involved in the early phases of MSCs osteoblast (OB) differentiation are not well-defined, we used quantitative mass spectrometry to delineate changes in human MSCs proteome and phosphoproteome during the first 24 h of their OB lineage commitment. The temporal profiles of 6252 proteins and 15,059 phosphorylation sites suggested at least two distinct signaling waves: one peaking within 30 to 60 min after stimulation and a second upsurge after 24 h. In addition to providing a comprehensive view of the proteome and phosphoproteome dynamics during early MSCs differentiation, our analyses identified a key role of serine/threonine protein kinase D1 (PRKD1) in OB commitment. At the onset of OB differentiation, PRKD1 initiates activation of the pro-osteogenic transcription factor RUNX2 by triggering phosphorylation and nuclear exclusion of the histone deacetylase HDAC7.

Concise Review: Patency of Small-Diameter Tissue-Engineered Vascular Grafts: A Meta-Analysis of Preclinical Trials.

Several patient groups undergoing small-diameter (<6 mm) vessel bypass surgery have limited autologous vessels for use as grafts. Tissue-engineered vascular grafts (TEVG) have been suggested as an alternative, but the ideal TEVG remains to be generated, and a systematic overview and meta-analysis of clinically relevant studies is lacking. We systematically searched PubMed and Embase databases for (pre)clinical trials and identified three clinical and 68 preclinical trials ([>rabbit]; 873 TEVGs) meeting the inclusion criteria. Preclinical trials represented low to medium risk of bias, and binary logistic regression revealed that patency was significantly affected by recellularization, TEVG length, TEVG diameter, surface modification, and preconditioning. In contrast, scaffold types were less important. The patency was 63.5%, 89%, and 100% for TEVGs with a median diameter of 3 mm, 4 mm, and 5 mm, respectively. In the group of recellularized TEVGs, patency was not improved by using smooth muscle cells in addition to endothelial cells nor affected by the endothelial origin, but seems to benefit from a long-term (46-240 hours) recellularization time. Finally, data showed that median TEVG length (5 cm) and median follow-up (56 days) used in preclinical settings are relatively inadequate for direct clinical translation. In conclusion, our data imply that future studies should consider a TEVG design that at least includes endothelial recellularization and bioreactor preconditioning, and we suggest that more standard guidelines for testing and reporting TEVGs in large animals should be considered to enable interstudy comparisons and favor a robust and reproducible outcome as well as clinical translation.

Neonatal epicardial-derived progenitors aquire myogenic traits in skeletal muscle, but not cardiac muscle.

BACKGROUND/OBJECTIVES: Epicardium-derived progenitor cells (EPDCs) differentiate into all heart cell types in the embryonic heart, yet their differentiation into cardiomyocytes in the adult heart is limited and poorly described. This may be due to EPDCs lacking myogenic potential or the inert adult heart missing regenerative signals essential for directed differentiation of EPDCs. Herein, we aimed to evaluate the myogenic potential of neonatal EPDCs in adult and neonatal mouse myocardium, as well as in skeletal muscle. The two latter tissues have an intrinsic capability to develop and regenerate, in contrast to the adult heart. METHODS: Highly purified mouse EPDCs were transplanted into damaged neonatal and adult myocardium as well as regenerating skeletal muscle. Co-cultures with skeletal myoblasts were used to distinguish fusion independent myogenic conversion. RESULTS: No donor EPDC-derived cardiomyocytes were observed in hearts. In contrast, a remarkable contribution of EPDCs to skeletal muscle myofiber formation was evident in vivo. Furthermore, co-cultures of EPDCs with myoblasts showed that EPDCs became part of multinucleated fibers and appeared to acquire myogenic traits independent of a fusion event. Fluorescence activated cell sorting of EPDCs co-cultured with and without myoblasts and subsequent qRT-PCR of 64 transcripts established that the myogenic phenotype conversion was accomplished through induction of a transcriptional myogenic program. CONCLUSION: These results suggest that EPDCs may be more myogenic than previously anticipated. But, the heart may lack factors for induction of myogenesis of EPDCs, a scenario that should be taken into consideration when aiming for repair of damaged myocardium by stem cell transplantation.

Stem cell survival is severely compromised by the thymidineanalog EdU (5-ethynyl-2'-deoxyuridine), an alternative to BrdU for proliferation assays and stem cell tracing.

Stem cell therapy has opened up the possibility of treating numerous degenerating diseases. However, we are still merely at the stage of identifying appropriate sources of stem cells and exploring their full differentiation potential. Thus, tracking the stem cells upon in vivo engraftment and during in vitro co-culture is very important and is an area of research embracing many pitfalls. 5-Ethynyl-2'-deoxyuridine (EdU), a rather new thymidine analog incorporated into DNA, has recently been suggested to be a novel highly valid alternative to other dyes for labeling of stem cells and subsequent tracing of their proliferation and differentiation ability. However, our results herein do not at any stage support this recommendation, since EdU severely reduces the viability of stem cells. Accordingly, we found that transplanted EdU-labeled stem cells hardly survive upon in vivo transplantation into regenerating muscle, whereas stem cells labeled in parallel with another dye survived very well and also participated in myofiber formation. Similar data were obtained upon in vitro myogenic culture, and further analysis showed that EdU reduced cell numbers by up to 88 % and increased the cell volume of remaining cells by as much as 91 %. Even at low EdU concentrations, cell survival and phenotype were substantially compromised, and the myogenic differentiation potential was inhibited. Since we examined both primary derived cells and cell lines from several species with the same result, this appears to be a common trait of EdU. We therefore suggest that EdU labeling should be avoided (or used with precaution) for stem cell tracing purposes.