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    • Although Sca is another conserved marker present

    2018-11-06

    Although Sca-1 is another conserved marker present in both stromal and epithelial SCs of many tissues, including hematopoietic AP1903 manufacturer (Spangrude et al., 1988), mouse incisor pulp progenitor cells (Balic et al., 2010), lung (Kim et al., 2005), prostate (Lawson et al., 2007), and pancreas (Rovira et al., 2010) SCs, Sca-1 based sorting did not enrich the sphere-forming DESCs. Moreover, immunostaining demonstrated that Sca-1 was mainly expressed in the middle and center of DESC spheres where TA cells resided (Fig. 6B). This suggests that Sca-1 is expressed more in TA cells instead of DESCs in the dental epithelium, although it cannot be excluded that the DESC sphere culture described herein did not provide a suitable environment for propagating Sca-1+ DESCs. The sphere-forming efficiency varied considerably between different populations. CD49fBright cells consistently had a 2.8 fold higher sphere-forming activity than unsorted cells (0.05% unsorted, 0.14% CD49fBright). Although Lgr5-GFP+ cells were scarce (0.4%) in the CL, we were able to isolate Lgr5-GFP+ cells for sphere-forming assays and the sphere-forming efficiency was 0.6%, which was a 12-fold increase compared to unsorted cells. No K5-H2BGFP retaining cells could form spheres in culture, suggesting that the sphere culture system did not contained factors needed to activate slow-cycling DESCs. FACS analyses showed that only around 2% of cells in the CL region are CD44+. CD44 is intensively expressed in presecretory ameloblasts, the SI, odontoblasts, and the wall of blood vessels, but not in the OEE or at the IEE-OEE junction in developing human tooth germ (Felszeghy et al., 2001). The enzyme-based cell dissociation process may remove surface markers and it takes longer than 3h to replenish, which may affect the quantity and quality of surface marker based cell sorting. However, this influence is enzyme- and surface marker-specific (Mulder et al., 1994; Ford et al., 1996). It appears that dispase and collagenase dissociation does not affect the cell sorting based on surface marker in SCs of other organs, such as prostate, breast, and skin (Stingl et al., 2006; Lukacs et al., 2010; Szabo et al., 2012). Our data here also showed that dispase and collagenase treatments did not affect CD49fBright-based cell sorting for DESCs. However, it cannot be ruled out that the negative results for CD44 and Sca-1 based sorting were due to the removal of these two surface markers by dispase and collagenase. In summary, this is the first report on both in vivo and in vitro characterization of slow-cycling and Lgr5+ DESCs in the mouse incisor CL. As the majority of both slow-cycling and Lgr5+ DESCs highly expressed CD49f, CD49fBright can serve as a surface marker for identifying and isolating DESCs. Furthermore, the in vitro DESC sphere culture system provides a new venue to expand and maintain DESCs for exploring the potential of DESCs in tooth regeneration.
    Acknowledgments We thank Allan Prejusa and Jonathan Lei for the help in FACS cell sorting and analysis; Drs. Elaine Fuchs, Adam Glick, Hans Clevers, Mingyao Liu, Michael M. Shen, and Frank Costantini for providing H2B-GFP, K5rtTA, Lgr5, Lgr5, Lgr4, Nkx3., and ROSA26EYFP mice; Dr. Jan C.C. Hu for amelogenin antibody; Dr. Hidemitsu Harada for sharing the CL dissection experience; Drs. Nick Barker and Hans Clevers for mLgr5, hLgr5, and mOlfm4 cDNAs; and Dr. Stefan Siwko for critical reading of the manuscript. This work was supported by the National Institutes of HealthK08DE020883 (to JYC), T32DE018380 (RD), CA96824 (FW), CA140388 (WLM, FW), CPRIT110555 (FW, WLM), Komen Breast Cancer Foundation (WLM), and the John S. Dunn Research Foundation (WLM).
    Introduction The hematopoietic system is the most sensitive tissue in the body to the effects of ionizing radiation. Radiation-induced damage to hematopoietic stem cells (HSCs) results in bone marrow failure, which can cause anemia, infection and hemorrhage in irradiated individuals (Mauch et al., 1995; Chao, 2007). In addition to its acute effects, the induction of oxidative stress and DNA damage in HSCs are thought to underlie the increased risks that irradiated individuals have for developing long-term complications, including myelofibrosis, myelodysplasia and acute leukemia (Wang et al., 2010; Yahata et al., 2011; Ivanov et al., 2012). Currently, hematopoietic failure following exposure to ionizing radiation is treated with the cytokine granulocyte colony-stimulating factor (G-CSF) (MacVittie et al., 2005; Dainiak, 2010); however, in the absence of endogenous hematopoietic recovery bone marrow transplantation is the only definitive cure. Thus, discovering the mechanisms responsible for regenerating HSCs and restoring functional hematopoiesis may improve future therapies for hematopoietic radiation injury.