Neural Stem Cells (NSCs) and Proteomics
2015; Elsevier BV; Volume: 15; Issue: 2 Linguagem: Inglês
10.1074/mcp.o115.052704
ISSN1535-9484
AutoresLorelei D. Shoemaker, Harley I. Kornblum,
Tópico(s)Alzheimer's disease research and treatments
ResumoNeural stem cells (NSCs) can self-renew and give rise to the major cell types of the CNS. Studies of NSCs include the investigation of primary, CNS-derived cells as well as animal and human embryonic stem cell (ESC)-derived and induced pluripotent stem cell (iPSC)-derived sources. NSCs provide a means with which to study normal neural development, neurodegeneration, and neurological disease and are clinically relevant sources for cellular repair to the damaged and diseased CNS. Proteomics studies of NSCs have the potential to delineate molecules and pathways critical for NSC biology and the means by which NSCs can participate in neural repair. In this review, we provide a background to NSC biology, including the means to obtain them and the caveats to these processes. We then focus on advances in the proteomic interrogation of NSCs. This includes the analysis of posttranslational modifications (PTMs); approaches to analyzing different proteomic compartments, such the secretome; as well as approaches to analyzing temporal differences in the proteome to elucidate mechanisms of differentiation. We also discuss some of the methods that will undoubtedly be useful in the investigation of NSCs but which have not yet been applied to the field. While many proteomics studies of NSCs have largely catalogued the proteome or posttranslational modifications of specific cellular states, without delving into specific functions, some have led to understandings of functional processes or identified markers that could not have been identified via other means. Many challenges remain in the field, including the precise identification and standardization of NSCs used for proteomic analyses, as well as how to translate fundamental proteomics studies to functional biology. The next level of investigation will require interdisciplinary approaches, combining the skills of those interested in the biochemistry of proteomics with those interested in modulating NSC function. Neural stem cells (NSCs) can self-renew and give rise to the major cell types of the CNS. Studies of NSCs include the investigation of primary, CNS-derived cells as well as animal and human embryonic stem cell (ESC)-derived and induced pluripotent stem cell (iPSC)-derived sources. NSCs provide a means with which to study normal neural development, neurodegeneration, and neurological disease and are clinically relevant sources for cellular repair to the damaged and diseased CNS. Proteomics studies of NSCs have the potential to delineate molecules and pathways critical for NSC biology and the means by which NSCs can participate in neural repair. In this review, we provide a background to NSC biology, including the means to obtain them and the caveats to these processes. We then focus on advances in the proteomic interrogation of NSCs. This includes the analysis of posttranslational modifications (PTMs); approaches to analyzing different proteomic compartments, such the secretome; as well as approaches to analyzing temporal differences in the proteome to elucidate mechanisms of differentiation. We also discuss some of the methods that will undoubtedly be useful in the investigation of NSCs but which have not yet been applied to the field. While many proteomics studies of NSCs have largely catalogued the proteome or posttranslational modifications of specific cellular states, without delving into specific functions, some have led to understandings of functional processes or identified markers that could not have been identified via other means. Many challenges remain in the field, including the precise identification and standardization of NSCs used for proteomic analyses, as well as how to translate fundamental proteomics studies to functional biology. The next level of investigation will require interdisciplinary approaches, combining the skills of those interested in the biochemistry of proteomics with those interested in modulating NSC function. Neural stem cells, which are present both during development and in the adult, are most commonly defined by the ability to self-renew and the capacity to generate the major cell types in the central nervous system (CNS) 1The abbreviations used are:NSCsneural stem cellsCNScentral nervous systemESCsembryonic stem cellsiPSCsinduced pluripotent stem cellsNSPCsneural stem/progenitor cellsMNmotoneuronsFACSfluorescent activated cell sortingRAretinoic acidFGFfibroblast growth factorEGFepidermal growth factorCpG-C-phosphate-G-ChIRP-MS comprehensive identification of RNA-binding proteins by mass spectrometrylncRNAlong noncoding RNAChIP-MSchromatin precipitation mass spectrometry., including oligodendrocytes, astrocytes, and neurons. Within this seemingly simple definition, however, the diversity of what is termed "neural stem cells" is quite large. There is a broad spectrum of NSCs with varying degrees of potency from multi- to more limited progenitors, each with unique lineages, fates, and spatial and temporal molecular signatures, that ultimately give rise to the vast numbers of mature CNS cell types (1.Molyneaux B.J. Arlotta P. Menezes J.R. Macklis J.D. Neuronal subtype specification in the cerebral cortex.Nat. Rev. Neurosci. 2007; 8: 427-437Crossref PubMed Scopus (1015) Google Scholar, 2.Zuchero J.B. Barres B.A. Intrinsic and extrinsic control of oligodendrocyte development.Curr. Opin. Neurobiol. 2013; 23: 914-920Crossref PubMed Scopus (92) Google Scholar, 3.Shoemaker L.D. Arlotta P. Untangling the cortex: advances in understanding specification and differentiation of corticospinal motor neurons.BioEssays. 2010; 32: 197-206Crossref PubMed Scopus (0) Google Scholar, 4.Guérout N. Li X. Barnabé-Heider F. Cell fate control in the developing central nervous system.Exp. 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While the first evidence of proliferating cells within the human brain was found in the 1800s, the age of NSC studies began in earnest in the 1990s with the development of advanced techniques, including approaches to isolation and purification, in vitro models, lineage tracing, and molecular profiling (historical review (6.Breunig J.J. Haydar T.F. Rakic P. Neural stem cells: Historical perspective and future prospects.Neuron. 2011; 70: 614-625Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar)). As illustrated in Fig. 1, there are currently three primary means of obtaining NSCs: (1) direct isolation from the developing or adult CNS using a variety of markers; (2) amplification of isolated cells in vitro; and (3) directed differentiation from pluripotent cells consisting of either embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Each of these approaches to isolation or enrichment comes with its own drawbacks. Ideally, all studies would use bona fide NSCs purified from in vivo sources. However, despite a great deal of effort, there are no protein markers that absolutely purify even one type of NSCs, perhaps a reasonable finding given NSC diversity. On the other hand, any tissue culture method being used will introduce both heterogeneity as well as tissue culture artifacts. The first neural stem cells identified were in vitro models isolated from embryonic rat forebrain (7.Temple S. Division and differentiation of isolated CNS blast cells in microculture.Nature. 1989; 340: 471-473Crossref PubMed Google Scholar) and adult mouse brain (8.Reynolds B.A. Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.Science. 1992; 255: 1707-1710Crossref PubMed Google Scholar). 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Embryonic stem cell lines derived from human blastocysts.Science. 1998; 282: 1145-1147Crossref PubMed Google Scholar) ushered in a new era for the field and has led to novel approaches to drive ESCs to a NSC fate (Fig. 1A) (15.Reubinoff B.E. Itsykson P. Turetsky T. Pera M.F. Reinhartz E. Itzik A. Ben-Hur T. Neural progenitors from human embryonic stem cells.Nat. Biotechnol. 2001; 19: 1134-1140Crossref PubMed Scopus (886) Google Scholar, 16.Zhang S.-C. Wernig M. Duncan I.D. Brustle O. Thomson J.A. In vitro differentiation of transplantable neural precursors from human embryonic stem cells.Nat. Biotechnol. 2001; 19: 1129-1133Crossref PubMed Scopus (1424) Google Scholar, 17.Bain G. Kitchens D. Yao M. Huettner J.E. Gottlieb D.I. Embryonic stem cells express neuronal properties in vitro.Dev. Biol. 1995; 168: 342-357Crossref PubMed Scopus (971) Google Scholar). Advances in understanding the maintenance of ESC pluripotency led directly to the manipulation of key molecules associated with ESC pluripotency, in particular octamer-binding transcription factor 4 (OCT4), (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (KFL4), and the transcription factor, c-MYC, to generate pluripotent iPSCs from fully differentiated somatic cells (18.Gurdon J.B. The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles.J. Embryol. Exp. Morphol. 1962; 10: 622-640PubMed Google Scholar, 19.Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (16415) Google Scholar). Approaches to generating iPSCs have evolved since the initial description and now include many innovations and much diversity in methodologies, including various cocktails of transcription factors, transcription factor delivery options, xeno-free cultures, and nonintegrating approaches (20.Yu J. Hu K. Smuga-Otto K. Tian S. Stewart R. Slukvin I.I. Thomson J.A. Human induced pluripotent stem cells free of vector and transgene sequences.Science. 2009; 324: 797-801Crossref PubMed Scopus (1608) Google Scholar) (reviewed in (21.Lowry W.E. Plath K. The many ways to make an iPS cell.Nat. Biotechnol. 2008; 26: 1246-1248Crossref PubMed Scopus (37) Google Scholar)). The generation of human iPSCs has led to technological advances in the study of NSCs (Fig. 1B) (22.Park I.-H. Zhao R. West J.A. Yabuuchi A. Huo H. Ince T.A. Lerou P.H. Lensch M.W. Daley G.Q. 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With the lofty goal of neural repair, there is clear potential for clinical uses of NSCs, either through transplantation of NSCs, of more committed cell types, of autologous iPSC-derived cells, or of manipulation of endogenous stem cells in vivo, for diseases ranging from ALS, Parkinson's disease, spinal cord injury, and stroke (reviewed in (25.Aboody K. Capela A. Niazi N. Stern J.H. Temple S. Translating stem cell studies to the clinic for CNS repair: Current state of the art and the need for a Rosetta stone.Neuron. 2011; 70: 597-613Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar)). In the initial approaches of NSCs for use in neural repair, investigators were primarily focused on discovering the means to direct cell fate in a general fashion: that is, to guide cells to become either neurons, astrocytes or oligodendrocytes, or, at best, to guide cells to synthesize a specific neurotransmitter, such as dopamine for Parkinson's disease. Despite decades of successful research in the field (26.Greig L.C. Woodworth M.B. Galazo M.J. Padmanabhan H. Macklis J.D. Molecular logic of neocortical projection neuron specification, development and diversity.Nat. Rev. Neurosci. 2013; 14: 755-769Crossref PubMed Scopus (382) Google Scholar, 27.Franco S.J. Müller U. Shaping our minds: stem and progenitor cell diversity in the mammalian neocortex.Neuron. 2013; 77: 19-34Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), many questions remain, including what defines NSCs molecularly, how this signature may be manipulated to produce certain cell types, how similar NSCs are when derived from different sources, what are the primary signaling pathways, master transcriptional controls, key protein posttranslational modifications and epigenetic changes, and what drives cell specification and differentiation in vitro and in vivo? However, as the knowledge of developmental biology and the technology of reprogramming have grown, simplistic approaches have given way to attempts to direct general cell fate in a more specific fashion. For instance, although clinical studies of differentiated iPSCs are in their infancy, a recent report detailed the transplantation of cortically fated neuronal progenitors derived from human iPSCs, resulting in mature, functional cortical neurons and functional recovery in a rodent stroke model (28.Tornero D. Wattananit S. Grønning Madsen M. Koch P. Wood J. Tatarishvili J. Mine Y. Ge R. Monni E. Devaraju K. Heverner R.F. Brüstle O. Lindvall O. Kokaia Z. Human induced pluripotent stem cell-derived cortical neurons integrate in stroke-injured cortex and improve functional recovery.Brain. 2013; 136: 3561-3577Crossref PubMed Scopus (154) Google Scholar). By being more sophisticated in (re)programming specific progenitors or cell types and by providing a finer molecular definition of those cells, we may be in a better position to affect and direct clinical outcomes, to improve efficacy and safety for future transplantation. A major challenge lies in the understanding of the mechanisms of action of NSC transplants. It is unclear how NSC treatments might exert their benefit—will they be capable of generating complex circuitry, or will their effects be to provide general support for endogenous repair mechanisms? The existing theories for potential benefit of NSCs include the delivery of trophic support to the injured tissue, increased host cell survival, provision of immunomodulation, contributions to angiogenesis, and integration into the host tissue to provide cellular scaffolding and re-establishing synapses and neural circuits. To understand the in vivo consequences of NSC transplants, technological advances are necessary to address for instance the difficult task of distinguishing host versus donor tissue. In recent elegant work, Kumamaru et al. describe the in vivo isolation and RNA-seq profile of mouse spinal cord derived neural stem/progenitor cells (NSPCs) from host tissue following transplantation into a spinal cord injury model (29.Kumamaru H. Ohkawa Y. Saiwai H. Yamada H. Kubota K. Kobayakawa K. Akashi K. Okano H. Iwamoto Y. Okada S. Direct isolation and RNA-seq reveal environment-dependent properties of engrafted neural stem/progenitor cells.Nat. Commun. 2012; 3: 1140Crossref PubMed Scopus (43) Google Scholar). This type of study has yet to be accomplished at the level of the proteome with current proteomics approaches, and remains a technological challenge. Importantly, NSC research was previously dominated primarily by rodent-derived stem cells, but human ESC and in particular human iPSC technology has led to an increase in human-derived stem cell studies. iPSC technologies are increasingly being utilized to develop "disease-in-dish" approaches using patient-derived cells, offering a significantly more "human," albeit in vitro, environment and have been used to study diseases as diverse as epilepsy, Huntington's disease, schizophrenia, and Parkinson's disease (reviewed in (30.Han S.S. Williams L.A. Eggan K.C. Constructing and deconstructing stem cell models of neurological disease.Neuron. 2011; 70: 626-644Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) and covered in greater detail in this special issue of MCP). Although a detailed review of NSC biology is beyond the scope of this review, we will briefly cover some basics to illustrate the diversity of NSCs present throughout development. The purpose here is to familiarize the nonexpert reader, who may be interested in interrogating NSC with proteomics, with the basics of the field. During embryonic and fetal development, NSCs are found throughout the neuraxis in specialized pseudostratified columnar neuroepthelium lining the surfaces of the developing ventricular system and central canal. During these stages, NSCs express the intermediate filament, nestin (31.Lendahl U. Zimmerman L.B. McKary R.D. CNS stem cells express a new class of intermediate filament protein.Cell. 1990; 60: 585-595Abstract Full Text PDF PubMed Scopus (2658) Google Scholar), and the extracellular protein, Lex/SSEA1/CD15 (32.Capela A. Temple S. LeX is expressed by principle progenitor cells in the embryonic nervous system, is secreted into their environment and binds Wnt-1.Dev. Biol. 2006; 291: 300-313Crossref PubMed Scopus (135) Google Scholar) and have a high proliferative and neuronogenic capacity. These cells are, in fact, radial glia (33.Noctor S.C. Flint A.C. Weissman T.A. Dammerman R.S. Kriegstein A.R. Neurons derived from radial glial cells establish radial units in neocortex.Nature. 2001; 409: 714-720Crossref PubMed Scopus (1427) Google Scholar), long thought to be mere scaffolds for neuronal migration, and have attachments extending from the ventricular to the pial (or outer) surfaces. As development proceeds, in most CNS regions, the ventricular zone thins. However, in the forebrain, some NSCs detach from the ventricular surface to form the subventricular zone, which then gives rise to an additional, even more superficial zone termed the outer radial glia zone (34.Hansen D.V. Lui J.H. Parker P.R. Kriegstein A.R. Neurogenic radial glia in the outer subventricular zone of human neocortex.Nature. 2010; 464: 554-561Crossref PubMed Scopus (734) Google Scholar), which also contains NSCs. Over the course of development within most brain regions, NSCs are first predominantly neuronogenic, generating primarily neurons, and later become largely gliogenic, a pattern that holds to some extent in vitro, with earlier derived NSCs giving rise mainly to neurons and later ones primarily to glia (35.Qian X. Shen Q. Goderie S.K. He W. Capela A. Davis A.A. Temple S. Timing of CNS cell generation: A programmed sequence of neuron and glial cell production from isolated murine cortical stem cells.Neuron. 2000; 28: 69-80Abstract Full Text Full Text PDF PubMed Google Scholar). During this time, NSCs generally switch their intermediate filament expression from nestin to glial fibrillary acidic protein (36.Doetsch F. Caillé I. Lim D.A. García-Verdugo J.M. Alvarez-Buylla A. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain.Cell. 1999; 97: 703-716Abstract Full Text Full Text PDF PubMed Scopus (3048) Google Scholar, 37.Garcia A.D. Doan N.B. Imura T. Bush T.G. 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GFAP-expressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain.Nat. Neurosci. 2004; 7: 1233-1241Crossref PubMed Scopus (706) Google Scholar). In the forebrain subventricular zone lining the lateral ventricle, NSCs give rise to olfactory bulb neurons that may participate in certain forms of olfactory learning and memory (39.Gregorian C. Nakashima J. Le, Belle J. Ohab J. Kim R. Liu A. Smith K.B. Groszer M. Garcia A.D. Sofroniew M.V. Carmichael S.T. Kornblum H.I. Liu X. Wu H. Pten deletion in adult neural stem/progenitor cells enhances constitutive neurogenesis.J. Neurosci. 2009; 29: 1874-1886Crossref PubMed Scopus (164) Google Scholar), although it is not clear whether this is the case in humans, as at least some subventricular zone NSCs participate in the genesis of neurons in the adjacent caudate nucleus (40.Ernst A. Alkass K. Bernard S. Salehpour M. Perl S. Tisdale J. Possnert G. Druid H. Frisén J. Neurogenesis in the striatum of the adult human brain.Cell. 2014; 156: 1072-1083Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar). In addition to these well-known NSC locations and sources, there are other, more poorly understood or less well-known regions harboring neural stem cells. Though controversial, there is a growing body of evidence suggesting that in some brain regions, ependymal cells (specialized ciliated cells lining the ventricular surfaces) also function as NSCs (41.Carlén M. Meletis K. Göritz C. Darsalia V. Evergren E. Tanigaki K. Amendola M. Barnabé-Heider F. Yeung M.S. Naldini L. Honjo T. Kokaia Z. Shupliakov O. Cassidy R.M. Lindvall O. Frisén J. Forebrain ependymal cells are Notch-dependent and generate neuroblasts and astrocytes after stroke.Nat. Neurosci. 2009; 12: 259-267Crossref PubMed Scopus (315) Google Scholar, 42.Luo Y. Coskun V. Liang A. Yu J. Cheng L. Ge W. Shi Z. Zhang K. Li C. Cui Y. Lin H. Luo D. Wang J. Lin C. Dai Z. Zhu H. Zhang J. 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Kessaris N. Richardson W.D. Götz M. Hedin-Pereira C. The marginal zone/layer I as a novel niche for neurogenesis and gliogenesis in developing cerebral cortex.J. Neurosci. 2007; 27: 11376-11388Crossref PubMed Scopus (0) Google Scholar). NSCs from different brain regions and developmental stages possess distinct characteristics, as illustrated in Figs 1C-1E. This includes intrinsic neuronogenic and gliogenic capacity but also dorsal-ventral patterning signatures and many other qualities. Furthermore, within a distinct brain region, there may be multiple types of cells with the characteristics of NSC self-renewal and multipotency. Indeed, the act of placing cells in culture may obscure some of these differences. For example, oligodendrocytes are normally specified from select brain regions during development, but in vitro, all NSCs are capable of giving rise to oligodendrocytes. To further complicate the situation, cells that behave only as short-term progenitors in vivo may behave as long-, self-renewing NSCs in vitro(45.Doetsch F. Petreanu L. Caille I. Garcia-Verdugo J.M. Alvarez-Buylla A. EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells.Neuron. 2002; 36: 1021-1034Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar). There has been a large effort to develop reliable systems to purify neural stem cells from living tissue for biochemical analysis. Early research focused on standard FACS methodologies using combinations of cell size and protein expression (46.Rietze R.L. Valcanis H. Brooker G.F. Thomas T. Voss A.K. Bartlett P.F. Purification of a pluripotent neural stem cell from the adult mouse brain.Nature. 2001; 412: 736-739Crossref PubMed Scopus (574) Google Scholar) as well as dye exclusion based on expression of ABC transporters (47.Kim M. Morshead C.M. 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High levels of Id1 expression define B1 type adult neural stem cells.Cell Stem Cell. 2009; 5: 515-526Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 53.Mich J.K. Signer R.A. Nakada D. Pineda A. Burgess R.J. Vue T.Y. Johnson J.E. Morrison S.J. Prospective identification of functionally distinct stem cells and neurosphere-initiating cells in adult mouse forebrain.eLife. 2014; 3: e02669Crossref PubMed Scopus (82) Google Scholar). Even given these improvements, obtaining sufficient
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