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]
- 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]
- Blanpain, C.; Fuchs, E. Epidermal Stem Cells of the Skin. Annu. Rev. Cell Dev. Biol. 2006, 22, 339–373. [Google Scholar] [CrossRef]
- 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]
- Woo, W.-M.; Oro, A.E. SnapShot: Hair Follicle Stem Cells. Cell 2011, 146, 334–334.e2. [Google Scholar] [CrossRef]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
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 |
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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