Human multipotent neural stem cells could effectively be used for the treatment of a variety of neurological disorders. However, a defining signature of neural stem cell lines that would be expandable, non-tumorigenic, and differentiate into desirable neuronal/glial phenotype after in vivo grafting is not yet defined. Employing a mass spectrometry approach, based on selected reaction monitoring, we tested a panel of well-described culture conditions, and measured levels of protein markers routinely used to probe neural differentiation, i.e. POU5F1 (OCT4), SOX2, NES, DCX, TUBB3, MAP2, S100B, GFAP, GALC, and OLIG1. Our multiplexed assay enabled us to simultaneously identify the presence of pluripotent, multipotent, and lineage-committed neural cells, thus representing a powerful tool to optimize novel and highly specific propagation and differentiation protocols. The multiplexing capacity of this method permits the addition of other newly identified cell type-specific markers to further increase the specificity and quantitative accuracy in detecting targeted cell populations. Such an expandable assay may gain the advantage over traditional antibody-based assays, and represents a method of choice for quality control of neural stem cell lines intended for clinical use.

Department of Cell Biology Faculty of Science Charles University Albertov 6 Prague CZ 12843 Czech Republic

Department of Histology and Embryology Faculty of Medicine Masaryk University Kamenice 753 5 Brno CZ 62500 Czech Republic

Department of Quantitative Biomedicine University of Zurich Winterthurerstrasse 190 Zürich CH 8057 Switzerland

Laboratory of Applied Proteome Analyses and Research Center PIGMOD Institute of Animal Physiology and Genetics of The Czech Academy of Sciences Rumburska 89 Libechov CZ 27721 Czech Republic

Laboratory of Biochemistry and Molecular Biology of Germ Cells Institute of Animal Physiology and Genetics of The Czech Academy of Sciences Rumburska 89 Libechov CZ 27721 Czech Republic

Laboratory of Cell Regeneration and Plasticity and Research Center PIGMOD Institute of Animal Physiology and Genetics of The Czech Academy of Sciences Rumburska 89 Libechov CZ 27721 Czech Republic

Neuroregeneration Laboratory Sanford Consortium for Regenerative Medicine Department of Anesthesiology University of California San Diego 2880 Torrey Pines Scenic Dr La Jolla CA 92037 USA

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Aebersold, R. and Mann, M. (2003). Mass spectrometry-based proteomics. Nature 422, 198-207. 10.1038/nature01511 PubMed DOI

Barker, R. A. and de Beaufort, I. (2013). Scientific and ethical issues related to stem cell research and interventions in neurodegenerative disorders of the brain. Prog. Neurobiol. 110, 63-73. 10.1016/j.pneurobio.2013.04.003 PubMed DOI

Bjarkam, C. R., Glud, A. N., Margolin, L., Reinhart, K., Franklin, R., Deding, D., Ettrup, K. S., Fitting, L. M., Nielsen, M. S., Sørensen, J.-C. H.et al. (2010). Safety and function of a new clinical intracerebral microinjection instrument for stem cells and therapeutics examined in the göttingen minipig. Stereotact. Funct. Neurosurg. 88, 56-63. 10.1159/000268743 PubMed DOI

Bodenmiller, B. (2016). Multiplexed epitope-based tissue imaging for discovery and healthcare applications. Cell Syst 2, 225-238. 10.1016/j.cels.2016.03.008 PubMed DOI

Bohaciakova, D., Hruska-Plochan, M., Tsunemoto, R., Gifford, W. D., Driscoll, S. P., Glenn, T. D., Wu, S., Marsala, S., Navarro, M., Tadokoro, T.et al. (2019). A scalable solution for isolating human multipotent clinical-grade neural stem cells from ES precursors. Stem Cell Res. Ther. 10, 83. 10.1186/s13287-019-1163-7 PubMed DOI PMC

Boulis, N. M. (2010). Floating spinal cannula and method of use. U.S. Patent US7833217B2 filed July 1, 2009, and issued November 16, 2010.

Carpenter, M. K., Cui, X., Hu, Z., Jackson, J., Sherman, S., Seiger, Å. and Wahlberg, L. U. (1999). In Vitro expansion of a multipotent population of human neural progenitor cells. Exp. Neurol. 158, 265-278. 10.1006/exnr.1999.7098 PubMed DOI

Cattaneo, E. and McKay, R. (1991). Identifying and manipulating neuronal stem cells. Trends Neurosci. 14, 338-340. 10.1016/0166-2236(91)90158-Q PubMed DOI

Červenka, J., Tylečková, J., Kupcová Skalníková, H., Vodičková Kepková, K., Poliakh, I., Valeková, I., Pfeiferová, L., Kolář, M., Vaškovičová, M., Pánková, T.et al. (2021). Proteomic characterization of human neural stem cells and their secretome during in vitro differentiation. Front. Cell. Neurosci. 14, 612560. 10.3389/fncel.2020.612560 PubMed DOI PMC

Chang, C.-Y., Picotti, P., Hüttenhain, R., Heinzelmann-Schwarz, V., Jovanovic, M., Aebersold, R. and Vitek, O. (2012). Protein significance analysis in selected reaction monitoring (SRM) measurements. Mol. Cell. Proteomics 11, M111.014662. 10.1074/mcp.M111.014662 PubMed DOI PMC

Choi, M., Chang, C.-Y., Clough, T., Broudy, D., Killeen, T., MacLean, B. and Vitek, O. (2014). MSstats: an R package for statistical analysis of quantitative mass spectrometry-based proteomic experiments. Bioinformatics 30, 2524-2526. 10.1093/bioinformatics/btu305 PubMed DOI

Cizkova, D., Kakinohana, O., Kucharova, K., Marsala, S., Johe, K., Hazel, T., Hefferan, M. P. and Marsala, M. (2007). Functional recovery in rats with ischemic paraplegia after spinal grafting of human spinal stem cells. Neuroscience 147, 546-560. 10.1016/j.neuroscience.2007.02.065 PubMed DOI PMC

Conti, L. and Cattaneo, E. (2010). Neural stem cell systems: physiological players or in vitro entities? Nat. Rev. Neurosci. 11, 176-187. 10.1038/nrn2761 PubMed DOI

Cunningham, M. G. (1998). Systems and methods for delivering therapeutic agents to selected sites in a subject. U.S. Patent US5792110A filed June 26, 1996, and issued August 11, 1998.

Doetsch, F., Caillé, I., Lim, D. A., García-Verdugo, J. M. and Alvarez-Buylla, A. (1999). Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97, 703-716. 10.1016/S0092-8674(00)80783-7 PubMed DOI

Donega, V., Burm, S. M., van Strien, M. E., van Bodegraven, E. J., Paliukhovich, I., Geut, H., van de Berg, W. D. J., Li, K. W., Smit, A. B., Basak, O.et al. (2019). Transcriptome and proteome profiling of neural stem cells from the human subventricular zone in Parkinson's disease. Acta Neuropathol Commun. 7, 84. 10.1186/s40478-019-0736-0 PubMed DOI PMC

Dunkley, T., Costa, V., Friedlein, A., Lugert, S., Aigner, S., Ebeling, M., Miller, M. T., Patsch, C., Piraino, P., Cutler, P.et al. (2015). Characterization of a human pluripotent stem cell-derived model of neuronal development using multiplexed targeted proteomics. Proteomics Clin. Appl. 9, 684-694. 10.1002/prca.201400150 PubMed DOI

Eriksson, P. S., Perfilieva, E., Björk-Eriksson, T., Alborn, A.-M., Nordborg, C., Peterson, D. A. and Gage, F. H. (1998). Neurogenesis in the adult human hippocampus. Nat. Med. 4, 1313-1317. 10.1038/3305 PubMed DOI

Ernst, A., Alkass, K., Bernard, S., Salehpour, M., Perl, S., Tisdale, J., Possnert, G., Druid, H. and Frisén, J. (2014). Neurogenesis in the striatum of the adult human brain. Cell 156, 1072-1083. 10.1016/j.cell.2014.01.044 PubMed DOI

Flax, J. D., Aurora, S., Yang, C., Simonin, C., Wills, A. M., Billinghurst, L. L., Jendoubi, M., Sidman, R. L., Wolfe, J. H., Kim, S. U.et al. (1998). Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes. Nat. Biotechnol. 16, 1033-1039. 10.1038/3473 PubMed DOI

Hefferan, M. P., Galik, J., Kakinohana, O., Sekerkova, G., Santucci, C., Marsala, S., Navarro, R., Hruska-Plochan, M., Johe, K., Feldman, E.et al. (2012). Human neural stem cell replacement therapy for amyotrophic lateral sclerosis by spinal transplantation. PLoS ONE 7, e42614. 10.1371/journal.pone.0042614 PubMed DOI PMC

Jensen, M. B., Yan, H., Krishnaney-Davison, R., Al Sawaf, A. and Zhang, S.-C. (2013). Survival and differentiation of transplanted neural stem cells derived from human induced pluripotent stem cells in a rat stroke Model. J. Stroke Cerebrovasc. Dis. 22, 304-308. 10.1016/j.jstrokecerebrovasdis.2011.09.008 PubMed DOI PMC

Johansson, C. B., Momma, S., Clarke, D. L., Risling, M., Lendahl, U. and Frisén, J. (1999). Identification of a neural stem cell in the adult mammalian central nervous system. Cell 96, 25-34. 10.1016/S0092-8674(00)80956-3 PubMed DOI

Kelly, S., Bliss, T. M., Shah, A. K., Sun, G. H., Ma, M., Foo, W. C., Masel, J., Yenari, M. A., Weissman, I. L., Uchida, N.et al. (2004). Transplanted human fetal neural stem cells survive, migrate, and differentiate in ischemic rat cerebral cortex. Proc. Natl Acad. Sci. USA 101, 11839-11844. 10.1073/pnas.0404474101 PubMed DOI PMC

Klein, S. M., Behrstock, S., McHugh, J., Hoffmann, K., Wallace, K., Suzuki, M., Aebischer, P. and Svendsen, C. N. (2005). GDNF delivery using human neural progenitor cells in a rat model of ALS. Hum. Gene. Ther. 16, 509-521. 10.1089/hum.2005.16.509 PubMed DOI

Kobayashi, Y., Okada, Y., Itakura, G., Iwai, H., Nishimura, S., Yasuda, A., Nori, S., Hikishima, K., Konomi, T., Fujiyoshi, K.et al. (2012). Pre-evaluated safe human iPSC-derived neural stem cells promote functional recovery after spinal cord injury in common marmoset without tumorigenicity. PLoS ONE 7, e52787. 10.1371/journal.pone.0052787 PubMed DOI PMC

Kupcova Skalnikova, H., Cizkova, J., Cervenka, J. and Vodicka, P. (2017). Advances in proteomic techniques for cytokine analysis: focus on melanoma research. Int. J. Mol. Sci. 18, 2697. 10.3390/ijms18122697 PubMed DOI PMC

Lam, M., Sanosaka, T., Lundin, A., Imaizumi, K., Etal, D., Karlsson, F. H., Clausen, M., Cairns, J., Hicks, R., Kohyama, J.et al. (2019). Single-cell study of neural stem cells derived from human iPSCs reveals distinct progenitor populations with neurogenic and gliogenic potential. Genes Cells 24, 836-847. 10.1111/gtc.12731 PubMed DOI PMC

Lange, V., Picotti, P., Domon, B. and Aebersold, R. (2008). Selected reaction monitoring for quantitative proteomics: a tutorial. Mol. Syst. Biol. 4, 222. 10.1038/msb.2008.61 PubMed DOI PMC

Lee, G., Kim, H., Elkabetz, Y., Al Shamy, G., Panagiotakos, G., Barberi, T., Tabar, V. and Studer, L. (2007). Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nat. Biotechnol. 25, 1468-1475. 10.1038/nbt1365 PubMed DOI

Lu, P., Wang, Y., Graham, L., McHale, K., Gao, M., Wu, D., Brock, J., Blesch, A., Rosenzweig, E. S., Havton, L. A.et al. (2012). Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell 150, 1264-1273. 10.1016/j.cell.2012.08.020 PubMed DOI PMC

Lu, P., Woodruff, G., Wang, Y., Graham, L., Hunt, M., Wu, D., Boehle, E., Ahmad, R., Poplawski, G., Brock, J.et al. (2014). Long-distance axonal growth from human induced pluripotent stem cells after spinal cord injury. Neuron 83, 789-796. 10.1016/j.neuron.2014.07.014 PubMed DOI PMC

MacLean, B., Tomazela, D. M., Shulman, N., Chambers, M., Finney, G. L., Frewen, B., Kern, R., Tabb, D. L., Liebler, D. C. and MacCoss, M. J. (2010). Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26, 966-968. 10.1093/bioinformatics/btq054 PubMed DOI PMC

Marsala, M. (2014). Spinal cord pulsation-cancelation injection system. World Intellectual Property Organization Patent WO2014047540A1 filed September 23, 2013, and issued March 27, 2014.

Melo-Braga, M. N., Schulz, M., Liu, Q., Swistowski, A., Palmisano, G., Engholm-Keller, K., Jakobsen, L., Zeng, X. and Larsen, M. R. (2014). Comprehensive quantitative comparison of the membrane proteome, phosphoproteome, and sialiome of human embryonic and neural stem cells. Mol. Cell. Proteomics 13, 311-328. 10.1074/mcp.M112.026898 PubMed DOI PMC

Melo-Braga, M. N., Meyer, M., Zeng, X. and Larsen, M. R. (2015). Characterization of human neural differentiation from pluripotent stem cells using proteomics/PTMomics—Current state-of-the-art and challenges. Proteomics 15, 656-674. 10.1002/pmic.201400388 PubMed DOI

Meyer, K., Ferraiuolo, L., Miranda, C. J., Likhite, S., McElroy, S., Renusch, S., Ditsworth, D., Lagier-Tourenne, C., Smith, R. A., Ravits, J.et al. (2014). Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS. Proc. Natl Acad. Sci. USA 111, 829-832. 10.1073/pnas.1314085111 PubMed DOI PMC

Nagato, M., Heike, T., Kato, T., Yamanaka, Y., Yoshimoto, M., Shimazaki, T., Okano, H. and Nakahata, T. (2005). Prospective characterization of neural stem cells by flow cytometry analysis using a combination of surface markers. J. Neurosci. Res. 80, 456-466. 10.1002/jnr.20442 PubMed DOI

Patro, N., Naik, A. and Patro, I. K. (2015). Differential temporal expression of S100β in developing rat brain. Front. Cell. Neurosci. 9, 87. 10.3389/fncel.2015.00087 PubMed DOI PMC

R Core Team (2020). R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing.

Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671-675. 10.1038/nmeth.2089 PubMed DOI PMC

Shoemaker, L. D. and Kornblum, H. I. (2016). Neural stem cells (NSCs) and proteomics. Mol. Cell. Proteomics 15, 344-354. 10.1074/mcp.O115.052704 PubMed DOI PMC

Song, Y., Subramanian, K., Berberich, M. J., Rodriguez, S., Latorre, I. J., Luria, C. M., Everley, R., Albers, M. W., Mitchison, T. J. and Sorger, P. K. (2019). A dynamic view of the proteomic landscape during differentiation of ReNcell VM cells, an immortalized human neural progenitor line. Scientific Data 6, 190016. 10.1038/sdata.2019.16 PubMed DOI PMC

Soste, M., Hrabakova, R., Wanka, S., Melnik, A., Boersema, P., Maiolica, A., Wernas, T., Tognetti, M., von Mering, C. and Picotti, P. (2014). A sentinel protein assay for simultaneously quantifying cellular processes. Nat. Methods 11, 1045-1048. 10.1038/nmeth.3101 PubMed DOI

Svendsen, C. N., Caldwell, M. A., Shen, J., ter Borg, M. G., Rosser, A. E., Tyers, P., Karmiol, S. and Dunnett, S. B. (1997). Long-term survival of human central nervous system progenitor cells transplanted into a rat model of Parkinson's disease. Exp. Neurol. 148, 135-146. 10.1006/exnr.1997.6634 PubMed DOI

The International Stem Cell Initiative* (2007). Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat. Biotechnol. 25, 803-816. 10.1038/nbt1318 PubMed DOI

Tyleckova, J., Valekova, I., Zizkova, M., Rakocyova, M., Marsala, S., Marsala, M., Gadher, S. J. and Kovarova, H. (2016). Surface N-glycoproteome patterns reveal key proteins of neuronal differentiation. J. Proteomics 132, 13-20. 10.1016/j.jprot.2015.11.008 PubMed DOI

United Nations. Nearly 1 in 6 of world's population suffer from neurological disorders – UN report (2007). UN News.

Usvald, D., Vodicka, P., Hlucilova, J., Prochazka, R., Motlik, J., Kuchorova, K., Johe, K., Marsala, S., Scadeng, M., Kakinohana, O.et al. (2010). Analysis of dosing regimen and reproducibility of intraspinal grafting of human spinal stem cells in immunosuppressed minipigs. Cell Transplant. 19, 1103-1122. 10.3727/096368910X503406 PubMed DOI

van Gorp, S., Leerink, M., Kakinohana, O., Platoshyn, O., Santucci, C., Galik, J., Joosten, E. A., Hruska-Plochan, M., Goldberg, D., Marsala, S.et al. (2013). Amelioration of motor/sensory dysfunction and spasticity in a rat model of acute lumbar spinal cord injury by human neural stem cell transplantation. Stem Cell Res. Ther. 4, 57. 10.1186/scrt209 PubMed DOI PMC

Vescovi, A. L., Parati, E. A., Gritti, A., Poulin, P., Ferrario, M., Wanke, E., Frölichsthal-Schoeller, P., Cova, L., Arcellana-Panlilio, M., Colombo, A.et al. (1999). Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation. Exp. Neurol. 156, 71-83. 10.1006/exnr.1998.6998 PubMed DOI

Vinci, L., Ravarino, A., Fanos, V., Naccarato, A. G., Senes, G., Gerosa, C., Bevilacqua, G., Faa, G. and Ambu, R. (2016). Immunohistochemical markers of neural progenitor cells in the early embryonic human cerebral cortex. Eur. J. Histochem. 60, 13-19. 10.4081/ejh.2016.2563 PubMed DOI PMC

Wittchen, H. U., Jacobi, F., Rehm, J., Gustavsson, A., Svensson, M., Jönsson, B., Olesen, J., Allgulander, C., Alonso, J., Faravelli, C.et al. (2011). The size and burden of mental disorders and other disorders of the brain in Europe 2010. Eur. Neuropsychopharmacol. 21, 655-679. 10.1016/j.euroneuro.2011.07.018 PubMed DOI

World Health Organization. Neurological disorders: public health challenges (2006). World Health Organization.

Yabut, O. R. and Pleasure, S. J. (2016). The crossroads of neural stem cell development and tumorigenesis. Opera Med. Physiol. 2, 181-187. 10.20388/omp2016.003.0040 PubMed PMC

Yocum, A. K., Gratsch, T. E., Leff, N., Strahler, J. R., Hunter, C. L., Walker, A. K., Michailidis, G., Omenn, G. S., O'Shea, K. S. and Andrews, P. C. (2008). Coupled global and targeted proteomics of human embryonic stem cells during induced differentiation. Mol. Cell. Proteomics 7, 750-767. 10.1074/mcp.M700399-MCP200 PubMed DOI PMC

Yuan, S. H., Martin, J., Elia, J., Flippin, J., Paramban, R. I., Hefferan, M. P., Vidal, J. G., Mu, Y., Killian, R. L., Israel, M. A.et al. (2011). Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS ONE 6, e17540. 10.1371/journal.pone.0017540 PubMed DOI PMC

Yuan, T., Liao, W., Feng, N.-H., Lou, Y.-L., Niu, X., Zhang, A.-J., Wang, Y. and Deng, Z.-F. (2013). Human induced pluripotent stem cell-derived neural stem cells survive, migrate, differentiate, and improve neurologic function in a rat model of middle cerebral artery occlusion. Stem Cell Res. Ther. 4, 73. 10.1186/scrt224 PubMed DOI PMC

Zhang, S.-C., Wernig, M., Duncan, I. D., Brüstle, O. and Thomson, J. A. (2001). In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotechnol. 19, 1129-1133. 10.1038/nbt1201-1129 PubMed DOI

Zizkova, M., Sucha, R., Tyleckova, J., Jarkovska, K., Mairychova, K., Kotrcova, E., Marsala, M., Gadher, S. J. and Kovarova, H. (2015). Proteome-wide analysis of neural stem cell differentiation to facilitate transition to cell replacement therapies. Expert Rev. Proteomics 12, 83-95. 10.1586/14789450.2015.977381 PubMed DOI

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