Abstract
This study comprehensively addresses the phenotype, function, and whole transcriptome of primary human and rodent Schwann cells (SCs) and highlights key species-specific features beyond the expected donor variability that account for the differential ability of human SCs to proliferate, differentiate, and interact with axons in vitro. Contrary to rat SCs, human SCs were insensitive to mitogenic factors other than neuregulin and presented phenotypic variants at various stages of differentiation, along with a mixture of proliferating and senescent cells, under optimal growth-promoting conditions. The responses of human SCs to cAMP-induced differentiation featured morphological changes and cell cycle exit without a concomitant increase in myelin-related proteins and lipids. Human SCs efficiently extended processes along those of other SCs (human or rat) but failed to do so when placed in co-culture with sensory neurons under conditions supportive of myelination. Indeed, axon contact-dependent human SC alignment, proliferation, and differentiation were not observed and could not be overcome by growth factor supplementation. Strikingly, RNA-seq data revealed that ~ 44 of the transcriptome contained differentially expressed genes in human and rat SCs. A bioinformatics approach further highlighted that representative SC-specific transcripts encoding myelin-related and axon growth-promoting proteins were significantly affected and that a deficient expression of key transducers of cAMP and adhesion signaling explained the fairly limited potential of human SCs to differentiate and respond to axonal cues. These results confirmed the significance of combining traditional bioassays and high-resolution genomics methods to characterize human SCs and identify genes predictive of cell function and therapeutic value.
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Andersen ND, Srinivas S, Pinero G, Monje PV (2016) A rapid and versatile method for the isolation, purification and cryogenic storage of Schwann cells from adult rodent nerves. Sci Rep 6(1):31781. https://doi.org/10.1038/srep31781
Anderson KD, Guest JD, Dietrich WD, Bunge MB, Curiel R, Dididze M, Green BA, Khan A et al (2017) Safety of autologous human Schwann cell transplantation in subacute thoracic spinal cord injury. J Neurotrauma 34(21):2950–2963. https://doi.org/10.1089/neu.2016.4895
Morrissey TK, Levi AD, Nuijens A, Sliwkowski MX, Bunge RP (1995) Axon-induced mitogenesis of human Schwann cells involves heregulin and p185erbB2. Proc Natl Acad Sci U S A 92(5):1431–1435. https://doi.org/10.1073/pnas.92.5.1431
Levi AD, Bunge RP, Lofgren JA, Meima L, Hefti F, Nikolics K, Sliwkowski MX (1995) The influence of heregulins on human Schwann cell proliferation. J Neurosci 15(2):1329–1340
Bunge MB, Monje PV, Khan A, Wood PM (2017) From transplanting Schwann cells in experimental rat spinal cord injury to their transplantation into human injured spinal cord in clinical trials. In: Dunnett SB, Björklund A (eds) Progress in brain research, vol. 231. Academic Press, Amsterdam, pp 107–133
Eldridge CF, Bunge MB, Bunge RP (1989) Differentiation of axon-related Schwann cells in vitro: II. Control of myelin formation by basal lamina. J Neurosci 9(2):625–638
Casella GT, Bunge RP, Wood PM (1996) Improved method for harvesting human Schwann cells from mature peripheral nerve and expansion in vitro. Glia 17(4):327–338. https://doi.org/10.1002/(SICI)1098-1136(199608)17:4<327::AID-GLIA7>3.0.CO;2-W
Levi AD (1996) Characterization of the technique involved in isolating Schwann cells from adult human peripheral nerve. J Neurosci Methods 68(1):21–26. https://doi.org/10.1016/0165-0270(96)00055-6
Morrissey TK, Bunge RP, Kleitman N (1995) Human Schwann cells in vitro. I. Failure to differentiate and support neuronal health under co-culture conditions that promote full function of rodent cells. J Neurobiol 28(2):171–189. https://doi.org/10.1002/neu.480280205
Morrissey TK, Kleitman N, Bunge RP (1995) Human Schwann cells in vitro. II. Myelination of sensory axons following extensive purification and heregulin-induced expansion. J Neurobiol 28(2):190–201. https://doi.org/10.1002/neu.480280206
Weiss T, Taschner-Mandl S, Bileck A, Slany A, Kromp F, Rifatbegovic F, Frech C, Windhager R et al (2016) Proteomics and transcriptomics of peripheral nerve tissue and cells unravel new aspects of the human Schwann cell repair phenotype. Glia 64(12):2133–2153. https://doi.org/10.1002/glia.23045
Ravelo KM, Andersen ND, Monje PV (2017) Magnetic-activated cell sorting for the fast and efficient separation of human and rodent Schwann cells from mixed cell populations. Chapter 6. In: Monje PV, Kim HA (eds) Schwann cells: methods and protocols, methods in molecular biology, vol. 1739. https://doi.org/10.1007/978-1-4939-7649-2_6
Monje PV, Bartlett Bunge M, Wood PM (2006) Cyclic AMP synergistically enhances neuregulin-dependent ERK and Akt activation and cell cycle progression in Schwann cells. Glia 53(6):649–659. https://doi.org/10.1002/glia.20330
Bastidas J, Athauda G, De La Cruz G, Chan WM, Golshani R, Berrocal Y, Henao M, Lalwani A et al (2017) Human Schwann cells exhibit long-term cell survival, are not tumorigenic and promote repair when transplanted into the contused spinal cord. Glia 65(8):1278–1301. https://doi.org/10.1002/glia.23161
Fregien NL, White LA, Bunge MB, Wood PM (2005) Forskolin increases neuregulin receptors in human Schwann cells without increasing receptor mRNA. Glia 49(1):24–35. https://doi.org/10.1002/glia.20091
Monje PV, Athauda G, Wood PM (2008) Protein kinase A-mediated gating of neuregulin-dependent ErbB2-ErbB3 activation underlies the synergistic action of cAMP on Schwann cell proliferation. J Biol Chem 283(49):34087–34100. https://doi.org/10.1074/jbc.M802318200
Pinero G, Berg R, Andersen ND, Setton-Avruj P, Monje PV (2016) Lithium reversibly inhibits Schwann cell proliferation and differentiation without inducing myelin loss. Mol Neurobiol 54(10):8287–8307. https://doi.org/10.1007/s12035-016-0262-z
Bacallao K, Monje PV (2015) Requirement of cAMP signaling for schwann cell differentiation restricts the onset of myelination. PLoS One 10(2):e0116948. https://doi.org/10.1371/journal.pone.0116948
Monje PV, Rendon S, Athauda G, Bates M, Wood PM, Bunge MB (2009) Non-antagonistic relationship between mitogenic factors and cAMP in adult Schwann cell re-differentiation. Glia 57(9):947–961. https://doi.org/10.1002/glia.20819
Monje PV, Soto J, Bacallao K, Wood PM (2010) Schwann cell dedifferentiation is independent of mitogenic signaling and uncoupled to proliferation: role of cAMP and JNK in the maintenance of the differentiated state. J Biol Chem 285(40):31024–31036. https://doi.org/10.1074/jbc.M110.116970
Wood PM (1976) Separation of functional Schwann cells and neurons from normal peripheral nerve tissue. Brain Res 115(3):361–375. https://doi.org/10.1016/0006-8993(76)90355-3
Wanner IB, Wood PM (2002) N-cadherin mediates axon-aligned process growth and cell-cell interaction in rat Schwann cells. J Neurosci 22(10):4066–4079
Soto J, Monje PV (2017) Axon contact-driven Schwann cell dedifferentiation. Glia 65(6):864–882. https://doi.org/10.1002/glia.23131
Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M et al (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29(1):15–21. https://doi.org/10.1093/bioinformatics/bts635
Anders S, Pyl PT, Huber W (2014) HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31(2):166–169. https://doi.org/10.1093/bioinformatics/btu638
Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26(1):139–140. https://doi.org/10.1093/bioinformatics/btp616
Hardcastle TJ, Kelly KA (2010) baySeq: empirical Bayesian methods for identifying differential expression in sequence count data. BMC Bioinformatics 11(1):422. https://doi.org/10.1186/1471-2105-11-422
Nikolsky Y, Kirillov E, Zuev R, Rakhmatulin E, Nikolskaya T (2009) Functional analysis of OMICs data and small molecule compounds in an integrated “knowledge-based” platform. Methods Mol Biol 563:177–196. https://doi.org/10.1007/978-1-60761-175-2_10
Porter S, Clark MB, Glaser L, Bunge RP (1986) Schwann cells stimulated to proliferate in the absence of neurons retain full functional capability. J Neurosci 6(10):3070–3078
Bacallao K, Monje PV (2013) Opposing roles of PKA and EPAC in the cAMP-dependent regulation of schwann cell proliferation and differentiation. PLoS One 8(12):e82354. https://doi.org/10.1371/journal.pone.0082354
Barbacci EG, Guarino BC, Stroh JG, Singleton DH, Rosnack KJ, Moyer JD, Andrews GC (1995) The structural basis for the specificity of epidermal growth factor and heregulin binding. J Biol Chem 270(16):9585–9589. https://doi.org/10.1074/jbc.270.16.9585
Ridley AJ, Davis JB, Stroobant P, Land H (1989) Transforming growth factors-beta 1 and beta 2 are mitogens for rat Schwann cells. J Cell Biol 109(6 Pt 2):3419–3424. https://doi.org/10.1083/jcb.109.6.3419
Jessen KR, Mirsky R (2005) The origin and development of glial cells in peripheral nerves. Nat Rev Neurosci 6(9):671–682. https://doi.org/10.1038/nrn1746
Bianchini D, De Martini I, Cadoni A, Zicca A, Tabaton M, Schenone A, Anfosso S, Akkad Wattar AS et al (1992) GFAP expression of human Schwann cells in tissue culture. Brain Res 570(1–2):209–217. https://doi.org/10.1016/0006-8993(92)90583-U
Bansal R, Warrington AE, Gard AL, Ranscht B, Pfeiffer SE (1989) Multiple and novel specificities of monoclonal antibodies O1, O4, and R-mAb used in the analysis of oligodendrocyte development. J Neurosci Res 24(4):548–557. https://doi.org/10.1002/jnr.490240413
Zorick TS, Lemke G (1996) Schwann cell differentiation. Curr Opin Cell Biol 8(6):870–876. https://doi.org/10.1016/S0955-0674(96)80090-1
Morgan L, Jessen KR, Mirsky R (1991) The effects of cAMP on differentiation of cultured Schwann cells: progression from an early phenotype (04+) to a myelin phenotype (P0+, GFAP-, N-CAM-, NGF-receptor-) depends on growth inhibition. J Cell Biol 112(3):457–467. https://doi.org/10.1083/jcb.112.3.457
Mirsky R, Dubois C, Morgan L, Jessen KR (1990) 04 and A007-sulfatide antibodies bind to embryonic Schwann cells prior to the appearance of galactocerebroside; regulation of the antigen by axon-Schwann cell signals and cyclic AMP. Development 109(1):105–116
Wood PM, Bunge RP (1975) Evidence that sensory axons are mitogenic for Schwann cells. Nature 256(5519):662–664. https://doi.org/10.1038/256662a0
Bunge RP, Bunge MB, Eldridge CF (1986) Linkage between axonal ensheathment and basal lamina production by Schwann cells. Annu Rev Neurosci 9(1):305–328. https://doi.org/10.1146/annurev.ne.09.030186.001513
Eldridge CF, Bunge MB, Bunge RP, Wood PM (1987) Differentiation of axon-related Schwann cells in vitro. I. Ascorbic acid regulates basal lamina assembly and myelin formation. J Cell Biol 105(2):1023–1034. https://doi.org/10.1083/jcb.105.2.1023
Bunge MB, Wood PM (2012) Realizing the maximum potential of Schwann cells to promote recovery from spinal cord injury. Handb Clin Neurol 109:523–540. https://doi.org/10.1016/B978-0-444-52137-8.00032-2
Mogha A, Benesh AE, Patra C, Engel FB, Schoneberg T, Liebscher I, Monk KR (2013) Gpr126 functions in Schwann cells to control differentiation and myelination via G-protein activation. J Neurosci 33(46):17976–17985. https://doi.org/10.1523/JNEUROSCI.1809-13.2013
Lee MM, Badache A, DeVries GH (1999) Phosphorylation of CREB in axon-induced Schwann cell proliferation. J Neurosci Res 55(6):702–712. https://doi.org/10.1002/(SICI)1097-4547(19990315)55:6<702::AID-JNR5>3.0.CO;2-N
Lin J, Luo J, Redies C (2010) Cadherin-19 expression is restricted to myelin-forming cells in the chicken embryo. Neuroscience 165(1):168–178. https://doi.org/10.1016/j.neuroscience.2009.10.032
Wanner IB, Guerra NK, Mahoney J, Kumar A, Wood PM, Mirsky R, Jessen KR (2006) Role of N-cadherin in Schwann cell precursors of growing nerves. Glia 54(5):439–459. https://doi.org/10.1002/glia.20390
Feltri ML, Graus Porta D, Previtali SC, Nodari A, Migliavacca B, Cassetti A, Littlewood-Evans A, Reichardt LF et al (2002) Conditional disruption of beta 1 integrin in Schwann cells impedes interactions with axons. J Cell Biol 156(1):199–209. https://doi.org/10.1083/jcb.200109021
Sparrow N, Manetti ME, Bott M, Fabianac T, Petrilli A, Bates ML, Bunge MB, Lambert S et al (2012) The actin-severing protein cofilin is downstream of neuregulin signaling and is essential for Schwann cell myelination. J Neurosci 32(15):5284–5297. https://doi.org/10.1523/JNEUROSCI.6207-11.2012
Walko G, Wogenstein KL, Winter L, Fischer I, Feltri ML, Wiche G (2013) Stabilization of the dystroglycan complex in Cajal bands of myelinating Schwann cells through plectin-mediated anchorage to vimentin filaments. Glia 61(8):1274–1287. https://doi.org/10.1002/glia.22514
Wanner IB, Mahoney J, Jessen KR, Wood PM, Bates M, Bunge MB (2006) Invariant mantling of growth cones by Schwann cell precursors characterize growing peripheral nerve fronts. Glia 54(5):424–438. https://doi.org/10.1002/glia.20389
Vadivelu RK, Ooi CH, Yao RQ, Tello Velasquez J, Pastrana E, Diaz-Nido J, Lim F, Ekberg JA et al (2015) Generation of three-dimensional multiple spheroid model of olfactory ensheathing cells using floating liquid marbles. Sci Rep 5:15083. https://doi.org/10.1038/srep15083
Levi AD, Guenard V, Aebischer P, Bunge RP (1994) The functional characteristics of Schwann cells cultured from human peripheral nerve after transplantation into a gap within the rat sciatic nerve. J Neurosci 14(3 Pt 1):1309–1319
Stratton JA, Kumar R, Sinha S, Shah P, Stykel M, Shapira Y, Midha R, Biernaskie J (2017) Purification and characterization of Schwann cells from adult human skin and nerve. eNeuro 4(3):ENEURO.0307–ENEU16.2017. https://doi.org/10.1523/ENEURO.0307-16.2017
Emery E, Li X, Brunschwig JP, Olson L, Levi AD (1999) Assessment of the malignant potential of mitogen stimulated human Schwann cells. J Peripher Nerv Syst 4(2):107–116
Casella GT, Wieser R, Bunge RP, Margitich IS, Katz J, Olson L, Wood PM (2000) Density dependent regulation of human Schwann cell proliferation. Glia 30(2):165–177. https://doi.org/10.1002/(SICI)1098-1136(200004)30:2<165::AID-GLIA6>3.0.CO;2-L
Mathon NF, Malcolm DS, Harrisingh MC, Cheng L, Lloyd AC (2001) Lack of replicative senescence in normal rodent glia. Science 291(5505):872–875. https://doi.org/10.1126/science.1056782
Wewetzer K, Radtke C, Kocsis J, Baumgartner W (2011) Species-specific control of cellular proliferation and the impact of large animal models for the use of olfactory ensheathing cells and Schwann cells in spinal cord repair. Exp Neurol 229(1):80–87. https://doi.org/10.1016/j.expneurol.2010.08.029
Brushart TM, Aspalter M, Griffin JW, Redett R, Hameed H, Zhou C, Wright M, Vyas A et al (2013) Schwann cell phenotype is regulated by axon modality and central-peripheral location, and persists in vitro. Exp Neurol 247:272–281. https://doi.org/10.1016/j.expneurol.2013.05.007
Acknowledgements
We thank the technical assistance provided by Ketty Bacallao, Michael McGrath, Talia Robinson, Kristine Ravelo, and Natalia Andersen with cell culture and Derek Van Booven with RNA-seq. We acknowledge the human SC manufacturing team of The Miami Project to Cure Paralysis for evaluating the purity and viability of cell stocks and Yan Shi for assisting in automated image analysis. We greatly appreciate the contribution of Patrick Wood for communicating unpublished data from many years of research and facilitating access to cryopreserved stocks of human SCs, hybridoma cell lines, and antibodies. We thank the important contribution made by Mary Bunge and Margaret Bates on TEM studies. This work was supported by the National Institutes of Health Grants 1R21NS084326 (to P.V.M.), 1R01NS089525 (to G.W.), The Craig H. Neilsen Foundation (339576 to P.V.M.), The Miami Project to Cure Paralysis, and The Buoniconti Fund.
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Monje, P.V., Sant, D. & Wang, G. Phenotypic and Functional Characteristics of Human Schwann Cells as Revealed by Cell-Based Assays and RNA-SEQ. Mol Neurobiol 55, 6637–6660 (2018). https://doi.org/10.1007/s12035-017-0837-3
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DOI: https://doi.org/10.1007/s12035-017-0837-3