Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders

Abstract

A major challenge to the study and treatment of neurogenetic syndromes is accessing live neurons for study from affected individuals. Although several sources of stem cells are currently available, acquiring these involve invasive procedures, may be difficult or expensive to generate and are limited in number. Dental pulp stem cells (DPSC) are multipotent stem cells that reside deep the pulp of shed teeth. To investigate the characteristics of DPSC that make them a valuable resource for translational research, we performed a set of viability, senescence, immortalization and gene expression studies on control DPSC and derived neurons. We investigated the basic transport conditions and maximum passage number for primary DPSC. We immortalized control DPSC using human telomerase reverse transcriptase (hTERT) and evaluated neuronal differentiation potential and global gene expression changes by RNA-seq. We show that neurons from immortalized DPSC share morphological and electrophysiological properties with non-immortalized DPSC. We also show that differentiation of DPSC into neurons significantly alters gene expression for 1305 transcripts. Here we show that these changes in gene expression are concurrent with changes in protein levels of the transcriptional repressor REST/NSRF, which is known to be involved in neuronal differentiation. Immortalization significantly altered the expression of 183 genes after neuronal differentiation, 94 of which also changed during differentiation. Our studies indicate that viable DPSC can be obtained from teeth stored for ≥72hrs, these can then be immortalized and still produce functional neurons for in vitro studies, but that constitutive hTERT immortalization is not be the best approach for long term use of patient derived DPSC for the study of disease.

Stem Cells of Dental Origin: Current Research Trends and Key Milestones towards Clinical Application

“…Methods most applied to confirm functional neural transformation include the patch-clamp analysis of the voltage-dependent Na + or K + channels [139–141, 147] and the fluorescent detection of intracellular Ca 2+ flux upon stimulation with neurotransmitters [135]. …”

Culturing and Neuronal Differentiation of Human Dental Pulp Stem Cells

“…to be returned with the tooth. The families are instructed to store the tube of media at 4°C (in the refrigerator) but they often do not and yet results are still adequate (Urraca et al, 2015). …”

“…(Kiraly et al, 2009) for the induction of neurons from DPSC. The neurons produced by this protocol are positive for neuronal markers such as TUJ1 (Figure 2b) and NeuN as well as glial markers like GFAP and undergo expression changes indicative of neuronal differentiation including the expression of synapse associated and ion channel transcripts (Urraca et al, 2015). Cells from morphologically homogenous culture of dental pulp stem cells (passage 1-4) are seeded on poly-D-lysine coated 6-well plates for imaging and electrophysiological studies or T-25 flasks for molecular studies.…”

“…The neurons are maintained in this media for 3 to 6 weeks, until they reach maturity. At this stage the DPSC derived neurons are positive for neuronal marker MAP2 and most of the cultures show only residual levels of glial marker GFAP (Urraca et al, 2015). …”

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“…Their applications in the regenerative arena include dental pulp regeneration, tooth reconstruction, bone tissue engineering, and angiogenic, vasculogenic, endocrine, and neurologic cell therapy. With regard to bone tissue engineering, the strong osteogenic properties of DPSCs have encouraged their application in bone regeneration/repair, while their neurogenic potential has prompted their use as a means to understand neurogenetic disorders without resorting to the generation of induced pluripotent stem cells (iPSCs) . However, we still require novel strategies to derive the full regenerative potential from cells as plentiful and potentially utile as DPSCs.…”