In Vitro Differentiation of Human Skin-Derived Cells into Functional Sensory Neurons-Like
Abstract
:1. Introduction
2. Materials and Methods
2.1. Isolation and Cultivation of Human SKPs
2.2. Induction of SN Differentiation from Human SKPs
2.3. Immunocytochemistry
2.4. Reverse Transcription—Quantitative Polymerase Chain Reaction (RT-qPCR)
2.5. Intracellular Ca2+ Measurement
2.6. Patch-Clamp
2.7. Statistical Analysis
3. Results
3.1. Characterization of Human SKPs
3.2. Induction of Neurogenesis
3.3. Analysis of the SKPs During Differentiation
3.4. Functional Analysis of SNs-Like Issued From SKPs
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
- Lee, G.; Chambers, S.M.; Tomishima, M.J.; Studer, L. Derivation of neural crest cells from human pluripotent stem cells. Nat. Protoc. 2010, 5, 688–701. [Google Scholar] [CrossRef]
- Guo, X.; Spradling, S.; Stancescu, M.; Lambert, S.; Hickman, J.J. Derivation of sensory neurons and neural crest stem cells from human neural progenitor hNP1. Biomaterials 2013, 34, 4418–4427. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Chambers, S.M.; Qi, Y.; Mica, Y.; Lee, G.; Zhang, X.-J.; Niu, L.; Bilsland, J.; Cao, L.; Stevens, E.; Whiting, P.; et al. Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nat. Biotechnol. 2012, 30, 715–720. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Denham, M.; Hasegawa, K.; Menheniott, T.; Rollo, B.; Zhang, D.; Hough, S.; Alshawaf, A.; Febbraro, F.; Ighaniyan, S.; Leung, J.; et al. Multipotent caudal neural progenitors derived from human pluripotent stem cells that give rise to lineages of the central and peripheral nervous system. Stem Cells Dayt. Ohio 2015, 33, 1759–1770. [Google Scholar] [CrossRef] [PubMed]
- Reinhardt, P.; Glatza, M.; Hemmer, K.; Tsytsyura, Y.; Thiel, C.S.; Höing, S.; Moritz, S.; Parga, J.A.; Wagner, L.; Bruder, J.M.; et al. Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling. PLoS ONE 2013, 8, e59252. [Google Scholar] [CrossRef]
- Compagnucci, C.; Barresi, S.; Petrini, S.; Billuart, P.; Piccini, G.; Chiurazzi, P.; Alfieri, P.; Bertini, E.; Zanni, G. Rho Kinase Inhibition Is Essential During In vitro Neurogenesis and Promotes Phenotypic Rescue of Human Induced Pluripotent Stem Cell-Derived Neurons With Oligophrenin-1 Loss of Function. Stem Cells Transl. Med. 2016, 5, 860–869. [Google Scholar] [CrossRef] [Green Version]
- Lee, S.H.; Jeong, S.K.; Ahn, S.K. An update of the defensive barrier function of skin. Yonsei Med. J. 2006, 47, 293–306. [Google Scholar] [CrossRef] [Green Version]
- Blanpain, C.; Fuchs, E. Epidermal Stem Cells of the Skin. Annu. Rev. Cell Dev. Biol. 2006, 22, 339–373. [Google Scholar] [CrossRef] [Green Version]
- Riekstina, U.; Muceniece, R.; Cakstina, I.; Muiznieks, I.; Ancans, J. Characterization of human skin-derived mesenchymal stem cell proliferation rate in different growth conditions. Cytotechnology 2008, 58, 153–162. [Google Scholar] [CrossRef] [Green Version]
- Woo, W.-M.; Oro, A.E. SnapShot: Hair Follicle Stem Cells. Cell 2011, 146, 334–334.e2. [Google Scholar] [CrossRef] [Green Version]
- Dupin, E.; Calloni, G.; Real, C.; Gonçalves-Trentin, A.; Le Douarin, N.M. Neural crest progenitors and stem cells. C. R. Biol. 2007, 330, 521–529. [Google Scholar] [CrossRef] [PubMed]
- Toma, J.G.; Akhavan, M.; Fernandes, K.J.; Barnabé-Heider, F.; Sadikot, A.; Kaplan, D.R.; Miller, F.D. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat. Cell Biol. 2001, 3, 778–784. [Google Scholar] [CrossRef] [PubMed]
- Toma, J.G.; McKenzie, I.A.; Bagli, D.; Miller, F.D. Isolation and characterization of multipotent skin-derived precursors from human skin. Stem Cells Dayt. Ohio 2005, 23, 727–737. [Google Scholar] [CrossRef]
- Fernandes, K.J.L.; McKenzie, I.A.; Mill, P.; Smith, K.M.; Akhavan, M.; Barnabé-Heider, F.; Biernaskie, J.; Junek, A.; Kobayashi, N.R.; Toma, J.G.; et al. A dermal niche for multipotent adult skin-derived precursor cells. Nat. Cell Biol. 2004, 6, 1082–1093. [Google Scholar] [CrossRef]
- Wong, C.E.; Paratore, C.; Dours-Zimmermann, M.T.; Rochat, A.; Pietri, T.; Suter, U.; Zimmermann, D.R.; Dufour, S.; Thiery, J.P.; Meijer, D.; et al. Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin. J. Cell Biol. 2006, 175, 1005–1015. [Google Scholar] [CrossRef] [PubMed]
- Biernaskie, J.; Sparling, J.S.; Liu, J.; Shannon, C.P.; Plemel, J.R.; Xie, Y.; Miller, F.D.; Tetzlaff, W. Skin-derived precursors generate myelinating Schwann cells that promote remyelination and functional recovery after contusion spinal cord injury. J. Neurosci. Off J. Soc. Neurosci. 2007, 27, 9545–9559. [Google Scholar] [CrossRef] [Green Version]
- Fernandes, K.J.L.; Kobayashi, N.R.; Gallagher, C.J.; Barnabé-Heider, F.; Aumont, A.; Kaplan, D.R.; Miller, F.D. Analysis of the neurogenic potential of multipotent skin-derived precursors. Exp. Neurol. 2006, 201, 32–48. [Google Scholar] [CrossRef]
- Kléber, M.; Lee, H.-Y.; Wurdak, H.; Buchstaller, J.; Riccomagno, M.M.; Ittner, L.M.; Suter, U.; Epstein, D.J.; Sommer, L. Neural crest stem cell maintenance by combinatorial Wnt and BMP signaling. J. Cell Biol. 2005, 169, 309–320. [Google Scholar] [CrossRef] [Green Version]
- Sieber-Blum, M.; Grim, M.; Hu, Y.F.; Szeder, V. Pluripotent neural crest stem cells in the adult hair follicle. Dev. Dyn. Off. Publ. Am. Assoc. Anat. 2004, 231, 258–269. [Google Scholar]
- Le Douarin, N.M.; Dupin, E. Multipotentiality of the neural crest. Curr. Opin. Genet. Dev. 2003, 13, 529–536. [Google Scholar] [CrossRef]
- Fuccillo, M.; Joyner, A.L.; Fishell, G. Morphogen to mitogen: The multiple roles of hedgehog signalling in vertebrate neural development. Nat. Rev. Neurosci. 2006, 7, 772–783. [Google Scholar] [CrossRef] [PubMed]
- Lee, H.-Y.; Kléber, M.; Hari, L.; Brault, V.; Suter, U.; Taketo, M.M.; Kemler, R.; Sommer, L. Instructive role of Wnt/beta-catenin in sensory fate specification in neural crest stem cells. Science 2004, 303, 1020–1023. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bhatt, S.; Diaz, R.; Trainor, P.A. Signals and switches in Mammalian neural crest cell differentiation. Cold Spring Harb. Perspect. Biol. 2013, 5, a008326. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lee, J.E. NeuroD and neurogenesis. Dev. Neurosci. 1997, 19, 27–32. [Google Scholar] [CrossRef]
- Mizuseki, K.; Sakamoto, T.; Watanabe, K.; Muguruma, K.; Ikeya, M.; Nishiyama, A.; Arakawa, A.; Suemori, H.; Nakatsuji, N.; Kawasaki, H.; et al. Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells. Proc. Natl. Acad. Sci. USA 2003, 100, 5828–5833. [Google Scholar] [CrossRef] [Green Version]
- Guha, U.; Gomes, W.A.; Samanta, J.; Gupta, M.; Rice, F.L.; Kessler, J.A. Target-derived BMP signaling limits sensory neuron number and the extent of peripheral innervation in vivo. Dev. Camb. Engl. 2004, 131, 1175–1186. [Google Scholar] [CrossRef] [Green Version]
- Lebonvallet, N.; Boulais, N.; Le Gall, C.; Chéret, J.; Pereira, U.; Mignen, O.; Bardey, V.; Jeanmaire, C.; Danoux, L.; Pauly, G.; et al. Characterization of neurons from adult human skin-derived precursors in serum-free medium: A PCR array and immunocytological analysis. Exp. Dermatol. 2012, 21, 195–200. [Google Scholar] [CrossRef]
- Sakka, M.; Leschiera, R.; Le Gall-Ianotto, C.; Gouin, O.; L’herondelle, K.; Buscaglia, P.; Mignen, O.; Philbé, J.-L.; Saguet, T.; Carré, J.-L.; et al. A new tool to test active ingredient using lactic acid in vitro, a help to understand cellular mechanism involved in stinging test: An example using a bacterial polysaccharide (Fucogel®). Exp. Dermatol. 2018, 27, 238–244. [Google Scholar] [CrossRef]
- Boisvert, E.M.; Engle, S.J.; Hallowell, S.E.; Liu, P.; Wang, Z.-W.; Li, X.-J. The Specification and Maturation of Nociceptive Neurons from Human Embryonic Stem Cells. Sci. Rep. 2015, 5, 16821. [Google Scholar] [CrossRef] [Green Version]
- Alshawaf, A.J.; Viventi, S.; Qiu, W.; D’Abaco, G.; Nayagam, B.; Erlichster, M.; Chana, G.; Everall, I.; Ivanusic, J.; Skafidas, E.; et al. Phenotypic and Functional Characterization of Peripheral Sensory Neurons derived from Human Embryonic Stem Cells. Sci. Rep. 2018, 8, 603. [Google Scholar] [CrossRef] [Green Version]
- Fernandes, K.J.L.; Toma, J.G.; Miller, F.D. Multipotent skin-derived precursors: Adult neural crest-related precursors with therapeutic potential. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2008, 363, 185–198. [Google Scholar] [CrossRef] [PubMed]
- Fernandes, K.J.L.; Miller, F.D. Isolation, expansion, and differentiation of mouse skin-derived precursors. Methods Mol. Biol. Clifton NJ 2009, 482, 159–170. [Google Scholar]
- Ernst, N.; Tiede, S.; Tronnier, V.; Kruse, C.; Zechel, C.; Paus, R. An improved, standardised protocol for the isolation, enrichment and targeted neural differentiation of Nestin+ progenitors from adult human dermis. Exp. Dermatol. 2010, 19, 549–555. [Google Scholar] [CrossRef] [PubMed]
- Zhu, Y.; Liu, Q.; Zhou, Z.; Ikeda, Y. PDX1, Neurogenin-3, and MAFA: Critical transcription regulators for beta cell development and regeneration. Stem. Cell Res. Ther. 2017, 8, 240. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Gene | Forward 5′->3′ | Reverse 5′->3′ |
---|---|---|
HNK-1 | GCT GAC GAC GAC AAC ACC TA | CGG TGT ACC AGC CAA CAA C |
p75NTR | GTC CCC CGC AGA GCC GTT GAG AAG | TGA ACC ACA CGC CCC CAC CAG AG |
NESTIN | CTC CAG AAA CTC AAG CAC C | TGA TTC CTG ATT CTC CTC TTC C |
BRN3A | CGT ACC ACA CGA TGA ACA GC | AGG AGA TGT GGT CCA GCA GA |
Pax6 | AGT GAA TCA GCT CGG TGG TGT CTT | TGC AGA ATT CGG GAA ATG TCG CAC |
Pax3 | TAC CAG CCC ACG TCT ATT CCA CAA | TTT GGT GTA CAG TGC TCG GAG GAA |
Sox1 | GGC TTT TGT ACA GAC GTT CCC | AAC CCA AGT CTG GTG TCA GC |
Sox9 | ACG GCT CCA GCA AGA ACA AG | TTG TGC AGA TGC GGG TAC TG |
Zic1 | AAA CTG GTT AAC CAA ATC CGC | CTC AAA CTC GCA CTT GAA GG |
Sox2 | GCA CAT GAA CGG CTG GAG CAA CG | TGC TGC GAG TAG GAC ATG CTG TAG G |
AP2 | TCT TGT CAC TTG CTC ATT GGG | GTT ACC CTG CTC ACA TCA CTA G |
Ngn1 | CAA-CCG-CAT-GCA-CAA-CTT-GA | GCG-TCT-CGA-TTT-TGG-TGA-GC |
Ngn2 | TGG-GTC-TGG-TAC-ACG-ATT-GC | GTC-TTC-TTG-ATG-CGC-TGC-AC |
Ngn3 | CAA-ACA-CCA-CAG-GAG-TCT-ATC-C | GGT-CTG-GGA-TCC-TTG-ATT-CTT-C |
PRDM12 | CAG-GTT-CTG-CTC-CTG-TTC-GT-3’ | TGT-GGG-AGG-TGT-TCA-ATG-AGG |
β-actin | GAG ACC TTC AAC ACC CCA GC | ATG TCA CGC ACG ATT TCC CT |
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Bataille, A.; Leschiera, R.; L’Hérondelle, K.; Pennec, J.-P.; Le Goux, N.; Mignen, O.; Sakka, M.; Plée-Gautier, E.; Brun, C.; Oddos, T.; et al. In Vitro Differentiation of Human Skin-Derived Cells into Functional Sensory Neurons-Like. Cells 2020, 9, 1000. https://doi.org/10.3390/cells9041000
Bataille A, Leschiera R, L’Hérondelle K, Pennec J-P, Le Goux N, Mignen O, Sakka M, Plée-Gautier E, Brun C, Oddos T, et al. In Vitro Differentiation of Human Skin-Derived Cells into Functional Sensory Neurons-Like. Cells. 2020; 9(4):1000. https://doi.org/10.3390/cells9041000
Chicago/Turabian StyleBataille, Adeline, Raphael Leschiera, Killian L’Hérondelle, Jean-Pierre Pennec, Nelig Le Goux, Olivier Mignen, Mehdi Sakka, Emmanuelle Plée-Gautier, Cecilia Brun, Thierry Oddos, and et al. 2020. "In Vitro Differentiation of Human Skin-Derived Cells into Functional Sensory Neurons-Like" Cells 9, no. 4: 1000. https://doi.org/10.3390/cells9041000
APA StyleBataille, A., Leschiera, R., L’Hérondelle, K., Pennec, J.-P., Le Goux, N., Mignen, O., Sakka, M., Plée-Gautier, E., Brun, C., Oddos, T., Carré, J.-L., Misery, L., & Lebonvallet, N. (2020). In Vitro Differentiation of Human Skin-Derived Cells into Functional Sensory Neurons-Like. Cells, 9(4), 1000. https://doi.org/10.3390/cells9041000