Culturing and Neuronal Differentiation of Human Dental Pulp Stem Cells

Sarita Goorha1, Lawrence T. Reiter2

1 Department of Neurology, University of Tennessee Health Science Center, Memphis, Tennessee, 2 Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, Tennessee
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 21.6
DOI:  10.1002/cphg.28
Online Posting Date:  January, 2017
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Abstract

A major issue in studying human neurogenetic disorders, especially rare syndromes affecting the nervous system, is the ability to grow neuronal cultures that accurately represent these disorders for analysis. Although there has been some success in generating induced pluripotent stem (iPS) cells from both skin and blood, there are still limitations to the collection and production of iPS cells from these biospecimens. We have had significant success in collecting and growing human dental pulp stem (DPS) cells from exfoliated teeth sent to our laboratory by the parents of children with a variety of rare neurogenetic syndromes. This protocol outlines our current methods for the growth and expansion of DPS cells from exfoliated (baby) teeth. These DPS cells can be differentiated into a variety of cell types including osteoblasts, chondrocytes, and mixed neuron and glial cultures. Here we provide our protocol for the differentiation of early passage DPS cell cultures into neurons for molecular studies. © 2017 by John Wiley & Sons, Inc.

Keywords: dental pulp stem cells; SHED teeth; deciduous teeth; stem cells; neurogenetics; rare disorders

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Collection and Transportation of Exfoliated Teeth
  • Basic Protocol 2: Dental Pulp Extraction
  • Basic Protocol 3: Passage, Freezing, and Thawing of DPS Cell Cultures
  • Basic Protocol 4: Differentiation of DPS Cells into Mixed Neuronal Cultures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Collection and Transportation of Exfoliated Teeth

  Materials
  • Transport medium (see recipe)
  • 15‐ml conical tube
  • Parafilm
  • Liquid biohazard shipping bag and box (e.g., Saf‐T‐Pak, cat. no. STP‐210EXMT)

Basic Protocol 2: Dental Pulp Extraction

  Materials
  • Washing medium (see recipe)
  • DPS cell culture medium (see recipe)
  • 10 mg/ml collagenase, type I (e.g., Thermo Fisher Scientific, cat no. 17100017)
  • Dispase II (e.g., Sigma‐Aldrich, cat. no. 4942078001)
  • Pasteur pipet
  • Dental tool to crack teeth or handheld grinding tool (e.g., Dremel)
  • Forceps
  • Petri dish
  • Dissection microscope
  • Razor blade
  • 15‐ml centrifuge tube
  • Centrifuge
  • 37°C incubator with and without 5% CO 2
  • 12‐well culture plate, poly‐D‐lysine coated (e.g., Fisher Scientific, cat. no. 08‐774‐269)
NOTE: All solutions and equipment coming in contact with living cells must be sterile and aseptic technique should be used.

Basic Protocol 3: Passage, Freezing, and Thawing of DPS Cell Cultures

  Materials
  • Cultured DPS cells on a 12‐well plate (see protocol 2)
  • Washing medium (see recipe)
  • HyClone HyQTase (e.g., GE Life Sciences, cat. no. SV30030.01)
  • DPS cell culture medium (see recipe)
  • Freezing medium: DPS cell culture medium supplemented with 15% (v/v) DMSO
  • 37°C, 5% CO 2 humidified incubator
  • 15‐ml centrifuge tube
  • Centrifuge
  • Cryotubes
  • Nalgene Cryo 1°C freezing container
  • Cryopreservation storage unit (–196°C)
NOTE: All solutions and equipment coming in contact with DPS cells must be sterile and aseptic technique should be used.NOTE: All culture incubations are carried out in a humidified 37°C, 5% CO2 incubator.

Basic Protocol 4: Differentiation of DPS Cells into Mixed Neuronal Cultures

  Materials
  • Cultured DPS cells in plates or flasks (see protocol 3)
  • Washing medium (see recipe)
  • Epigenetic reprogramming medium (see recipe)
  • Neural differentiation medium (see recipe)
  • Phosphate‐buffered saline (PBS; see appendix 2D)
  • Neural maturation medium (see recipe)
  • 37°C, 5% CO 2 incubator
NOTE: All media for the induction of DPS cells to neurons must be prepared fresh before addition to the cells.
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Figures

Videos

Literature Cited

Literature Cited
  Arthur, A., Rychkov, G., Shi, S., Koblar, S.A., and Gronthos, S. 2008. Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells 26:1787‐1795. doi: 10.1634/stemcells.2007‐0979.
  Granader, Y.E., Bender, H.A., Zemon, V., Rathi, S., Nass, R., and Macallister, W.S. 2010. The clinical utility of the Social Responsiveness Scale and Social Communication Questionnaire in tuberous sclerosis complex. Epilepsy Behav. 18:262‐266. doi: 10.1016/j.yebeh.2010.04.010.
  Gronthos, S., Arthur, A., Bartold, P.M., and Shi, S. 2011. A method to isolate and culture expand human dental pulp stem cells. Methods Mol. Biol. 698:107‐121. doi: 10.1007/978‐1‐60761‐999‐4_9.
  Gronthos, S., Brahim, J., Li, W., Fisher, L.W., Cherman, N., Boyde, A., DenBesten, P., Robey, P.G., and Shi, S. 2002. Stem cell properties of human dental pulp stem cells. J. Dent. Res. 81:531‐535. doi: 10.1177/154405910208100806.
  Kiraly, M., Kadar, K., Horvathy, D.B., Nardai, P., Racz, G.Z., Lacza, Z., Varga, G., and Gerber, G. 2011. Integration of neuronally predifferentiated human dental pulp stem cells into rat brain in vivo. Neurochem. Int. 59:371‐381. doi: 10.1016/j.neuint.2011.01.006.
  Kiraly, M., Porcsalmy, B., Pataki, A., Kadar, K., Jelitai, M., Molnar, B., Hermann, P., Gera, I., Grimm, W.D., Ganss, B., Zsembery, A., and Varga, G. 2009. Simultaneous PKC and cAMP activation induces differentiation of human dental pulp stem cells into functionally active neurons. Neurochem. Int. 55:323‐332. doi: 10.1016/j.neuint.2009.03.017.
  Marchetto, M.C., Winner, B., and Gage, F.H. 2010. Pluripotent stem cells in neurodegenerative and neurodevelopmental diseases. Hum. Mol. Genet. 19:R71‐76. doi: 10.1093/hmg/ddq159.
  Nosrat, I.V., Smith, C.A., Mullally, P., Olson, L., and Nosrat, C.A. 2004. Dental pulp cells provide neurotrophic support for dopaminergic neurons and differentiate into neurons in vitro; implications for tissue engineering and repair in the nervous system. Eur. J. Neurosci. 19:2388‐2398. doi: 10.1111/j.0953‐816X.2004.03314.x.
  Pick, M., Stelzer, Y., Bar‐Nur, O., Mayshar, Y., Eden, A., and Benvenisty, N. 2009. Clone‐ and gene‐specific aberrations of parental imprinting in human induced pluripotent stem cells. Stem Cells 27:2686‐2690. doi: 10.1002/stem.205.
  Urraca, N., Memon, R., El‐Iyachi, I., Goorha, S., Valdez, C., Tran, Q.T., Scroggs, R., Miranda‐Carboni, G.A., Donaldson, M., Bridges, D., and Reiter, L.T. 2015. Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders. Stem Cell Res. 15:722‐730. doi: 10.1016/j.scr.2015.11.004.
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