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    • The gene expression changes that occurred

    2018-11-06

    The gene expression changes that occurred during the differentiation process were consistent with our expectations of changes that must occur during neurogenesis. For example, there was a positive enrichment for genes involved in synaptogenesis and a negative enrichment for genes involved in progression of the cell cycle (see Supplemental Table 8 — DAVID Analysis). This is completely expected of any cell that is transitioning from a proliferative state to a quiescent neuronal state. We must be careful, however, not to assume that hTERT immortalization produces DPSC derived neurons that are functionally indistinguishable to neurons from primary DPSCs. In fact, our GSEA analysis indicates that many transcripts with NRSF/REST transcription factor order Seliciclib were significantly up-regulated during the process of differentiation of primary DPSCs to neurons (Fig. 5A and Supplemental Table 5), despite the fact that REST transcript levels did not increase significantly. We can now show that REST protein is indeed down-regulated in both immortalized and non-immortalized DPSCs during the process of differentiation, which supports our finding of an enrichment in REST regulated transcripts during the differentiation process (Fig. 6). However, some of the same transcripts that have REST binding site motifs and were up-regulated during differentiation reverted back to the previous expression levels in immortalized DPSC neurons (Fig. 6B). Furthermore, in a parallel set of experiments, we demonstrate that both osteogenesis and adipogenesis are significantly less efficient in constitutively hTERT immortalized DPSCs versus non-immortalized DPSCs (El-Iyachi et al., 2015). These results lead us to conclude that constitutive hTERT immortalization may not be useful for most experimental investigations of genetic disorders. Recently, Page et al. showed that a doxycycline inducible hTERT constructs could be used to maintain mesenchymal stem cells (MSCs) in culture long term up to p50 with no ill effects on subsequent differentiation if the hTERT gene is turned back off (Piper et al., 2012). One viable option for maintaining DPSCs from subjects with rare neurogenetic disorders long term may be to use an inducible promoter system for hTERT expression in DPSCs which can also be turned off prior to differentiation into neurons. It should also be noted that other methods, perhaps those recently used to make iPSCs from DPSCs (Griesi-Oliveira et al., 2014), may also be appropriate for studies involving particular syndromes. In fact, it appears that making iPSCs from DPSCs may be more efficient than making iPSCs from fibroblasts (Yan et al., 2010). Nevertheless, we will always have the option of using primary DPSCs for our experiments, despite the fact that this is a limited resource and especially scarce when studying rare syndromes. However, the utility of DPSCs is not just limited to disorders involving neuronal function. DPSCs are able to differentiate into a variety of different cell lineages (Morad et al., 2013; Verma et al., 2014; Young et al., 2013). In the near future, DPSCs may be used for tissue engineering, possible treatment of neural tissue injury, degenerative diseases, and as well as regenerative dentistry. DPSCs even have the ability to integrate into host brain tissue in the mammalian brain, migrate to injured areas and express neuronal specific markers as well as voltage dependent sodium and potassium channels (Marchetto et al., 2010). Understanding the culture conditions, characteristics, and potential of DPSCs may prove beneficial to all fields of health care. By establishing the technical boundaries of this renewable resource, we have provided a baseline for future studies of neurogenetic syndromes at the molecular and cellular biological levels.
    Acknowledgments We would like to thank the families who contributed teeth to the study. We also thank Dr. Michael A. Dyer for his critical review of the manuscript and Dr. Amanda Preston for edits to the revised manuscript. RNAseq was performed with the assistance of the UTHSC Molecular Resource Center. These experiments were funded in part by NIHR21NS075709-02 to L.T.R. and a Dental School Alumni Fund scholarship to R.M.