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    • Given the importance of the master regulatory

    2018-11-09

    Given the importance of the master regulatory HOX genes for patterning during embryogenesis, we expect that the ectopic expression of a specific HOX code could allow for enhanced development of desired tissues in vitro. One example is human HOXB4, which is highly expressed in the hematopoietic stem and progenitor cell compartment in the adult bone marrow. Its ectopic expression in differentiating mouse embryonic stem LY 2109761 promotes the formation of long-term repopulating hematopoietic stem cells in vitro (Pilat et al., 2005, 2013). Several studies have already successfully reported the generation of MSCs from human iPSCs using special media formulations (Chen et al., 2012; Frobel et al., 2014; Sheyn et al., 2016). However, in this work we present the successful direct programming of iPSCs toward VW-typical MSCs by ectopic expression of a small set of HOX genes, namely HOXB7, HOXC6, and HOXC8. As the activity of homeotic selector proteins are highly conserved throughout evolution, it is very likely that our results will also hold true for human iPSCs. The possibility and feasibility of obtaining patient-specific VW-MSCs from iPSCs in large amounts by forward programming could potentially open avenues toward novel, MSC-based therapies. Finally, it remains to be shown whether this approach could also be used for enforcing transdifferentiation of other somatic cell types toward MSCs, thus avoiding the need for prior reprogramming of those cells back to the pluripotent stage.
    Experimental Procedures
    Author Contributions
    Acknowledgments We thank V. Jendrossek and P. Horn for hosting our groups and their support in this project. We also thank I. Spratte and S. Skibbe for excellent technical assistance and K. Lennartz for the help with FACS. The work was supported by the UK Essen/IFORES (D/107-81040, D.K.) and BMBFFKZ 01GN0815 (M.Z.).
    Introduction The cranial vault undergoes intramembranous bone formation and provides mechanical support and protection for the brain. Important elements of the calvarial structure are the sutures that narrow during appositional bone growth and remain as an active site of bone formation during calvarial development. In 2007, Lana-Elola et al. showed that during calvarial development, mesenchymal cells of the sutures differentiate to form calvarial bone and their differential fate depends on positioning, with cells occupying the midline of the suture mesenchyme less prone to differentiation. In 2014, Ouyang et al. published a thorough observation about PRX1-expressing cells of the postnatal calvaria, showing that these cells have qualities of skeletal stem cells (SSCs), reside in the calvarial sutures, and are distinct from postnatal cells of the sutures expressing Collagen 1 (COL1). Zhao et al. (2015) and, more recently, Maruyama et al. (2016) showed that various populations of SSCs reside in postnatal calvarial sutures, supporting the logic that the stem cell niche of the calvaria is contained in the suture mesenchyme. Additional research about SSCs of the calvaria and their niche is required to devise therapeutic strategies which, by targeting a specific population of SSCs, can promote calvarial bone regeneration or perturb the development of craniofacial malformations, such as craniosynostosis. The pair-related homeobox gene Prx1 (also known as Prrx1) is a transcription factor that is highly expressed during limb bud formation and craniofacial development (Martin and Olson, 2000). PRX1-expressing cells and their postnatal progeny also have a central role in bone homeostasis and fracture repair since inactivation of RANKL (receptor activator of nuclear factor κB ligand) expression in these cells causes osteopetrosis (Xiong et al., 2011) and inactivation of BMP2 (bone morphogenetic protein 2) expression generates a low-bone-mass phenotype, with bones prone to factures and unable to repair (Tsuji et al., 2006). In culture, PRX1-expressing cells isolated from the periosteum of postnatal long bones (Kawanami et al., 2009) or postnatal calvaria (Ouyang et al., 2014) can differentiate into chondroblasts and osteoblasts upon induction. Additional studies show that PRX1 averts differentiation of cells into committed osteoblasts (Lu et al., 2011) or adipocytes (Du et al., 2013). Together, the evidence suggests that expression of Prx1 is a unique marker to identify and characterize SSCs of the calvarial bones. We therefore hypothesize that postnatal cells expressing PRX1 (pnPRX1+) of the calvaria represent an SSC population that resides exclusively in the sutures and is required for calvarial bone regeneration. Using intravital microscopy (IVM) (Lo Celso et al., 2009), we performed lineage-tracing analyses and lineage-ablation analyses to show that pnPRX1+ cells reside exclusively in the sutures, are required for regeneration of calvarial bone defects, and are dispensable for postnatal calvarial development.