Proteomics of neural stem cells

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Proteomics of neural stem cells Expert Rev. Proteomics 5(2), 175–186 (2008)

Helena Skalnikova, Petr Vodicka, Suresh Jivan Gadher and Hana Kovarova† †

Author for correspondence Institute of Animal Physiology & Genetics, Academy of Sciences of the Czech Republic, Rumburska 89, 277 21 Libechov, Czech Republic; Joint Proteome Laboratory, Videnska 1083, 142 20 Prague, Czech Republic Tel.: +42 031 563 9582 Fax: +42 031 563 9510 [email protected]

The isolation of neural stem cells from fetal and adult mammalian CNS and the demonstration of functional neurogenesis in adult CNS have offered perspectives for treatment of many devastating hereditary and acquired neurological diseases. Due to this enormous potential, neural stem cells are a subject of extensive molecular profiling studies with a search for new markers and regulatory pathways governing their self-renewal as opposed to differentiation. Several in-depth proteomic studies have been conducted on primary or immortalized cultures of neural stem cells and neural progenitor cells, and yet more remains to be done. Additionally, neurons and glial cells have been obtained from embryonic stem cells and mesenchymal stem cells, and proteins associated with the differentiation process have been characterized to a certain degree with a view to further investigations. This review summarizes recent findings relevant to the proteomics of neural stem cells and discusses major proteins significantly regulated during neural stem cell differentiation with a view to their future use in cell-based regenerative and reparative therapy. KEYWORDS: cell-based regenerative and reparative therapy • conditioned media • differentiation • neural stem cell • neurodegenerative disease • proteomics

Origin of neural stem cells

For many decades, neurogenesis was believed to proceed only in the prenatal period of mammalian development, and adult brain was supposed to contain a fixed number of neurons. Experiments performed by Altman et al. in the 1960s documented incorporation of tritiated thymidine into dividing cells of rodent brains [1,2]. Although some of these cells were identified as neurons by morphological evidence, these findings were largely ignored until the mid-1990s. At the same time, new labeling techniques for dividing cells using injection of bromo-deoxyuridine or retroviral vectors carrying the LacZ gene finally demonstrated the generation of new neurons in the adult rodent CNS [3,4] and brought the first evidence of neurogenesis in primates [5], including neurogenesis in human hippocampus [6]. In 1992, undifferentiated cells from adult mouse brain were successfully isolated and expanded in vitro in the form of free-floating cell clusters (neurospheres) when stimulated with EGF [7]. Culture of these cells on poly-L-ornithine-coated cover slips induced differentiation into neurons and astrocytes [7]. Currently, such cells able to selfrenew or differentiate into the three major cell

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types of CNS: neurons, astrocytes and oligodendrocytes, correspond to the definition of multipotent neural stem cells (NSCs) (FIGURE 1). In contrast to these NSCs, neural progenitor cells have limited capacity to self-renew and are often unipotent [8]. Since 1992, neural stem cells and progenitor cells have been isolated not only from two major neurogenic areas of adult brain (subventricular zone and hippocampus), but also from various areas of the CNS including cortex, striatum, cerebellum or different levels of the spinal cord of mouse, rat or human (reviewed in [9]). Alternative sources of NSCs

As the sources of human NSCs available for large-scale analyses, including proteomics, and for their potential practical use in cell-based reparative and regenerative therapies are very limited, alternative sources of cells capable of neural differentiation are being investigated. During mammalian embryonic development, embryonic stem (ES) cells are formed in the inner cell mass of blastocyst. ES cells are capable of developing into cells of all tissues from embryo in vivo and they are also able to produce various cell types, including neural cells in vitro.

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50 µm

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Figure 1. Porcine neural stem cells. (A) Undifferentiated neural stem cells grown as neurospheres (14 days in vitro, relief contrast). (B) Differentiation of neural stem cells induced by all-trans retinoic acid (5 days in vitro, fluorescent microscopy). Differentiating neurons, labeled by anti-βIII-tubulin antibody (red, arrow) and astrocytes, labeled by anti-GFAP antibody (green, arrowheads) migrating out of neurosphere (lower left, asterisk). Nuclei counterstained by DAPI (blue).

Currently, neural cells can be in vitro derived from mouse ES cells using feeder-free and serum-free chemically defined medium [10]. Another way to produce unlimited source of NSCs is creation of immortalized cell lines. Cell immortalization is usually achieved by genetic modification, often by transduction of cells with different oncogenes. Such cell lines are thus not regarded as safe for use in clinical transplantations, but can be very useful for in vitro studies. Roy et al. created a cell line restricted to neuronal differentiation by transducing fetal human spinal progenitor cells with human telomerase [11]. Two NSC lines (from human ventral mesencephalon: ReNcell VM and from cortex: ReNcell CX) were prepared by Donato et al. by introduction of v-myc and c-myc oncogenes [12]. One of these cell lines, ReNcell VM, was used for extensive proteomic study of in vitro-induced neural cell differentiation [13]. The stem cells are directed to divide and differentiate by microenvironment (called ‘niche’) formed by complex effects of nutrients, growth factors, hormones, autocrine factors and interactions with other cells and the extracellular matrix. According to these interactions, the undifferentiated stem cell is usually committed to produce predefined cell types. Under specific experimental conditions, the stem cells of different origins can transit to NSCs phenotypes and differentiate to neural cells (neurons, astrocytes or oligodendrocytes). Transdifferentiation to NSCs/neural cells can be induced in vitro by specific treatments of cells, such as rat muscle pluripotent stem cells [14], epidermal neural crest cells [15], CD133+

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hematopoietic stem cells, both CD34+ and CD34- cord blood cells, bone marrow hematopoietic stem cells, and mesenchymal stem cells (MSCs) from umbilical blood, bone marrow, adipose tissue or scalp tissue (reviewed in [8]). Neural transdifferentiation of MSCs is of great interest because MSCs can be obtained in large quantities from patient’s own bone marrow, allowing for autologous transplantation. However, the extent to which transdifferentiated cells resemble genuine cells still remains controversial [16]. The fact that MSCs are readily available in large quantities and because of their great therapeutical potential, extensive proteomic study of induced MSCs neural differentiation is of great interest. Applications of NSCs & neural progenitor cells

The isolation of NSCs from the fetal and adult mammalian CNS, including human, and the demonstration of functional neurogenesis in adult CNS has offered perspectives for the development of new strategies for the treatment of many devastating acquired and hereditary diseases [17]. Approaches to cell transplantation differ among transplantations of undifferentiated NSCs [18], transplantations of restricted progenitors or in vitro-differentiated neural cells [11,19]. One particular example is Parkinson’s disease (PD), where a specific population of dopaminergic neurons in the striatum must be restored for therapeutic effect, which seems to be a good target for cellular transplantation or gene therapy [20]. Early clinical trials with transplantation of human fetal neural tissue into CNS of PD patients demonstrated some beneficial results [21]. The stimulation of endogenous neurogenesis by growth factors administration, either by direct infusion [22] or by gene delivery by viral vectors [23], also seems to be a very promising approach. Another potential target for NSCbased therapies is Huntington’s disease (HD) [24]. This autosomal dominant hereditary neurodegenerative disorder is characterized by degeneration of the striatal GABAergic medium spiny neurons, caused by accumulation of mutant huntingtin protein with polyglutamine tract expansion. The exact mechanism of mutant huntingtin toxicity is still unknown, although many studies suggest that neurons with huntingtin aggregates are selectively vulnerable to glutamate excitotoxicity [25]. Similar mechanisms including oxidative stress [26] or aberrant protein–protein interactions [27] may also be considered. Different neuroprotective or cell-replacement strategies using NSCs were tested as a therapeutic approach to HD [28,29]. Transplantation of human fetal neuroblasts into the striatum of several HD

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patients showed functional improvement in some of them [30], but long-term follow-up study showed such an improvement to be only temporary [31]. Stroke [32] and spinal cord injuries [33,34] are also good candidates for reparative stem cell therapy, which could benefit a large population. Molecular characterization of NSCs

Due to the enormous potential of using NSCs or precursors in cell-based therapies, these cells are subject of extensive molecular profiling studies with search for new markers and regulatory pathways governing their self-renewal versus differentiation. A number of transcriptomic and proteomic studies have been conducted on stem cells (reviewed in [35–37]). Large-scale transcriptomic studies, such as cDNA microarrays, are very powerful and provide information on the expression of many thousands of genes in one experiment. Due to amplification by PCR, expression of very low-abundance mRNAs can be monitored using a very small amount of biological sample. Although the changes in mRNA level are likely to impact subsequent protein level, there are many other dynamically regulated processes involved (e.g., stability of mRNA, rate of translation and stability of protein products), very often resulting in lack of correlation between gene expression and protein level [38]. A further complication that may contribute to the disparity between mRNA profiling and protein profiling, is the complexity arising from the situation that several protein forms after splice variants and post-translational modifications correspond to one gene, resulting in difficulties in combining data from genomic and proteomic databases [36]. More importantly, the final functional entities in cells are not mRNAs, but proteins that undergo many post-translational modifications controlling protein activity, localization and interactions with other proteins and molecules. It is the final protein that defines cellular phenotype and fate. As a demonstrative example in differentiation of NSCs, a lack of concordance between transcriptomic and proteomic data was observed in rat striatal progenitor cell line ST14A, where comparison of native and glial cell line-derived neurotrophic factor (GDNF)-transfected cells were examined [39]. In this study, 43 proteins were more than 1.5-fold up- or downregulated between native and GDNF-transfected cells in at least five of eight timepoints investigated. Twofold upregulation of only mRNA for one protein (acidic leucine-rich nuclear phosphoprotein 32 family member A [AN32A]) was also detected in transcriptional level using microarray analysis [40]. Proteomics of NSCs Proteomic tools applied to study NSCs

The lack of amplification methods for proteins (such as PCR for nucleic acids) makes the proteomic studies very demanding in terms of biological material. Typically, several millions of cells

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are required for complex analysis of proteins. Although many protocols for NSC isolation, expansion and differentiation have been established in the last few years, sources of NSCs remain very limited and only a narrow spectrum of proteomic methods has been applied to date (TABLE 1). Until now, the most often utilized method for separation of NSC proteins has been 2DE. This technique separates proteins according to their isoelectric points in the first dimension, followed by SDS-PAGE in the second dimension [41]. Separated proteins are then identified by mass spectrometry (MS). Recently, the 2DE/MS approach was used to profile proteins expressed in primary NSC cultures from rat hippocampus [42] and pig fetal brain [43], or cell lines derived from rat striatal progenitor cells [39] and human fetal midbrain stem cells [13]. In order to characterize the pluripotent potential of embryonic stem cells, including their transition to neural precursors, their protein profiles in mouse model have been characterized using either 2DE/MS [44] or a gel-free approach based on 2D liquid chromatography of peptides [45]. To address protein changes during differentiation of NSCs, 2DE and/or its modification with fluorescent labeling, differential in-gel electrophoresis (DIGE) [46], allowing improvements in reproducibility and quantitation were frequently used [13,39,43,47–51] (TABLE 1). Due to the limitation of 2DE to separate highly hydrophobic membrane proteins, high-resolution 1D SDS-PAGE was preferred for the analysis of these proteins in embryonic carcinoma cell lines differentiating into neurons [52]. An advanced proteomic technique based on 2D chromatographic separation of peptides digested from proteins and labeled by isobaric mass tags (iTRAQ™), thus allowing subsequent protein identification and quantification by tandem MS [53] was used by Salim et al. [54]. They compared protein expression during differentiation of mouse subventricular zone neural progenitor cells (TABLE 1). Such an approach revealed 55 proteins that demonstrated altered expression during differentiation, and only 14 of them were identified in another study on the same cells using subcellular fractionation and DIGE [50]. Whilst the gel-based approach characterized a total of 115 distinct proteins that underwent regulatory changes during neural progenitor cell differentiation, the power of iTRAQ technology was demonstrated by the identification of 41 additional differentially regulated proteins that were not found by DIGE. These proteins included growth factors, signaling molecules, cell cycle-related proteins, heat-shock proteins and other proteins involved in transcriptional and translational machinery, and metabolic regulation. Many of the proteins identified by iTRAQ were proteins that were not amenable to 2DE DIGE due to extremely high or low molecular sizes or isoelectric points. In this sense, the results of both technologies, such as gel-dependent DIGE and gel-free MS-centered iTRAQ, are complementary and helpful in increasing the coverage of proteome under study. Alternative proteomic methods were employed to analyze proteins secreted by NSCs into the culture medium. 1D SDS-PAGE was used to purify a 21-kDa protein

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Organism Sample type

Cells

Treatment

Mouse

Mouse

Rat

Rat

Rat

Rat

Pig

Human

Rat

Salim et al. (2006)

Salim et al. (2007)

Maurer et al. (2003)

Maurer et al. (2004)

Maurer et al. (2007)

Chatterjee et al. (2005)

Skalnikova et al. (2007)

Hoffrogge et al. (2006)

Hoffrogge et al. (2007)

Cell line

Cell line

Primary culture

Primary culture

Primary culture

Primary culture

Primary culture

Primary culture

Primary culture

Primary culture

Striatal progenitor cell line ST14A, transgenic with temperaturesensitive mutation, tsA58U19 in SV40 large T antigen

Fetal midbrain stem cell line (ReNcell VM), immortalized by retroviral transformation using the myc transcription factor

Fetal brain hemispheres

Cerebellar granule cells

Adult subventricular zone

Adult hippocampus

Adult hippocampus

Adult subventricular zone

Adult subventricular zone

Fetal cortex

Mouse

Embryonic stem cells

PKU ES cell line

0.5 µM RA

39°C (induces differentiation), transfection with GDNF gene

4 days

3, 6, 9, 12, 24, 48, 72 h

4 and 7 days

5 days

Differentiation by 1 µM all-trans RA Withdrawal of growth factors EGF and bFGF

1, 7 and 21 days

3 days

48 h

none

24 h

24 h

1h

Not mentioned

Inhibition of GSK3β (SB216763))

2% (v/v) bovine fetal serum and withdrawing mitogens (EGF, FGF)

None

Differentiation by EGF and FGF withdrawal

Differentiation by EGF and FGF withdrawal

1% serum

2DE

2DE

2DE

2DE

2DE

2DE

2DE

2DE

DIGE + subcellular fractionation

SCX + RPLC of iTRAQ™ labeled peptides

2DE

Duration of Protein separation treatment method

700–850

652

956

848 in SCs, 992 in differentiated cells

2200

410 in inhibited neurospheres, 353 in controls

1141 in SCs, 843 in differentiated cells

1141

2000

Not mentioned

[67]

[39]

[13]

[43]

[48]

[47]

[61]

[42]

[50]

[54]

[49]

Average number of Ref. protein spots detected on 2D gel

1DE: 1D electrophoresis; 2DE: 2D electrophoresis; AHP: Adult hippocampus-derived neural progenitor cell; BHA: Butylated hydroxyanisole; ES: Embryonic stem; FACS: Fluorescence-activated cell sorting; FCS: Fetal calf serum; FGF: Fibroblast growth factor; GDNF: Glial cell line-derived neurotrophic factor; IEF: Isoelectric focusing; MSC: Mesenchymal stem cell; RA: Retinoic acid; RPLC: Reversed-phase liquid chromatography; SCXLC: Strong cation-exchange liquid chromatography.

Guo et al. (2001)

Embryonic stem cells – neural differentiation

Human

Pearce et al. (1999)

Neural stem cells (isolated from brains or immortalized cultures)

Study

Table 1. Proteomic studies on neural stem cells and/or neural differentiation.

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Embryonic stem cells

Mouse

Mouse

Mouse

Mouse

Mouse

Rat

Rat

Human

Wang et al. (2005)

Battersby et al. (2007)

An et al. (2005)

Inberg et al. (2007)

Sakaguchi et al. (2006)

Taupin et al. (2000)

Dahl et al. (2003)

Choi et al. (2006)

AHPs

AHPs

Medium conditioned by OP9 stromal cell line

P19 cell line

P19 cell line (P19C6 subclone)

Embryonic stem cells E 14 cell line

E14 cells (ES cells)

ES cells derived, from 129 mice or the BALB/c mouse

Cells

45 min

10 days

Not mentioned

Pre-induction 20% FCS + 10 ng/ml 1-24 h FGF overnight, induction 2% DMSO, 200 µM BHA, 25 mM KCl, 2 mM valporic acid, 10 µM forskolin, 1 µM hydrocortisone and 5 µg/ml insulin

None

None

48 h

0-6 days

Sparse (1000 cells/mm2) or dense (4000 cells/mm2) cell culture (cells differentiate much faster in the dense culture) None

2-3 weeks

none

Serum-free medium N2

FACS purified cells expressing Oct4 and Nestin

600 spots (18-cm strip), 400 spots (11-cm strip)

3500

175 confirmed and matched spots

1200

2DE

1000

Liquid-phase IEF + SDS Not mentioned PAGE

Sepharose 4b column, strong cation exchange column + SDS PAGE

SELDI-TOF-MS

1DE of membrane proteins, 2DE of cytosolic proteins

2DE

DIGE

2DE

[16]

[56]

[55]

[57]

[52]

[63]

[51]

[65]

[83]

Average number of Ref. protein spots detected on 2D gel

2DE (phosphoprotein Not mentioned enrichment by FeIII agarose) + radioactive labeling

Duration of Protein separation treatment method

2 µM RA - differentiated to spinal 10 days progenitor cells and motor neurons or without retinoic acid - different to dopaminergic neurons; differentiated Ca2+ concentrations

Chemical ischemia induced by glucose-free low K+ solution supplemented with 1 mM KCN

Treatment

1DE: 1D electrophoresis; 2DE: 2D electrophoresis; AHP: Adult hippocampus-derived neural progenitor cell; BHA: Butylated hydroxyanisole; ES: Embryonic stem; FACS: Fluorescence-activated cell sorting; FCS: Fetal calf serum; FGF: Fibroblast growth factor; GDNF: Glial cell line-derived neurotrophic factor; IEF: Isoelectric focusing; MSC: Mesenchymal stem cell; RA: Retinoic acid; RPLC: Reversed-phase liquid chromatography; SCXLC: Strong cation-exchange liquid chromatography.

Mesenchymal Bone marrow MSCs, stem cells differentiated into true (MSCs) or pseudo neurons

Conditioned medium

Conditioned 2DE medium

Conditioned medium

Embryonic carcinoma cells

Embryonic carcinoma cells

Embryonic stem cells

Embryonic stem cells

Organism Sample type

Schrattenholz Mouse et al. (2005)

Others

Study

Table 1. Proteomic studies on neural stem cells and/or neural differentiation (cont.).

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that potentiates mitogenic activity of bFGF on NSCs. This factor was identified as a glycosylated form of cystatin C [55]. Dahl et al. used an alternative approach to 2DE by using liquid-phase isoelectric focusing in Rotophor unit followed by SDS-PAGE of each Rotophor fraction to characterize proteins secreted by adult rat hippocampal progenitor cells into the culture medium [56]. In another study, Sakaguchi et al. identified Galectin-1 as a candidate protein that stimulated in vitro proliferation of NSCs using protein chip technology [57]. This technique is based on the selective binding of proteins to different chemically defined surfaces, followed by direct monitoring of bound proteins by time-of-flight MS (SELDI) [58]. In summary, application of the aforementioned proteomic approaches reflects the initial stages of NSC proteomics. The 2D gel-based methods are only able to display the more abundant proteins, but with good potential to reveal protein posttranslational modifications. By contrast, MS-based methods allow access to proteins of extreme molecular sizes and/or isoelectric points and hydrophobic proteins. Unfortunately, this category of methods is most often coupled to protein digestion and peptide prefractionation and, consequently, there is loss of information about post-translational modifications of a given protein.

gels and also western blots with immunodetection of human fetal midbrain stem cell line ReNcell VM after 4 and 7 days of differentiation by growth factor withdrawal [13]. The level of PCNA was further analyzed during differentiation of rat striatal progenitor cell line ST14A and only a very slow decrease of PCNA was observed after 2 and 3 days of differentiation, which could be a hint for a later onset of downregulation of PCNA in differentiating ST14A cells [39]. Downregulation of PCNA is in concordance with the arrest of proliferation of neural stem/progenitor cells after induction of differentiation. Neuronal differentiation-related protein

A potential marker associated with undifferentiated NSCs may be neuronal differentiation-related protein (NDRP). NDRP gene was identified by Kato et al. in 2000 and its expression was observed in sensory neurons in olfactory epithelia and neural layer of retina during embryonic development [60]. In motor neurons, the expression of NDRP became detectable during regeneration after axotomy [60]. NDRP protein was detected by 2DE to be highly expressed in undifferentiated hippocampal NSCs compared with cells differentiated for 2 days by 2% fetal calf serum [61]. Stromal cell-derived factor receptor 1 isoform-α

Potential markers of NSCs & their differentiated counterparts identified in proteomic studies

Neural stem cells and progenitor cells of different origins are used in proteomic studies, and various stimuli are applied to induce differentiation. NSCs are capable of differentiating into a mixture of neurons, astrocytes and oligodendrocytes, and the proportion of each cell type is dependent on the origin of NSCs and the differentiation protocol used. Besides this, only a limited spectrum of proteomic methods (mentioned previously) with relatively low sensitivity have been applied so far, resulting mostly in the identification of more abundant proteins that are not neural cell specific. For example, neuronal receptors for certain neurotransmitters that characterize neuronal subtypes could not be detected in such studies. Selected proteins related to NSC differentiation and identified in recent studies are discussed below. Interestingly, the majority of proteins were upregulated and only two proteins were downregulated during neural differentiation. Proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) interacts with other protein partners involved mainly in DNA replication, and is a well-known marker of proliferating cells [59]. Decrease of PCNA level associated with the transition of proliferating NSCs to differentiated neural cells was reported in four studies. Downregulation of PCNA could be detected by 2DE, iTRAQ and western blot with immunodetection as early as 24 h upon removal of growth factor and induction of differentiation of mouse subventricular zone neural progenitor cells [50,54]. Significant downregulation of PCNA was further confirmed by 2DE

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Expression of the receptor for stromal cell-derived factor 1-α is significantly upregulated when fetal human NSCs are differentiated into neuronal precursors. When neurons mature, localization of receptor changes from the cell body in neuronal precursors to only axons and dendrites in mature neurons. Stromal cell-derived factor 1-α and its receptor may be important for regulation of neuronal migration and guidance during development and repair [62]. High expression of stromal cell-derived factor receptor 1 in neurons differentiated from mouse embryonic carcinoma cells was also observed by An et al. [63]. Cellular retinoic acid-binding protein II

Retinoic acid is used in embryonic stem cell culture to aggregate cells and induce neural differentiation. Stimulation of embryonic carcinoma cells by retinoic acid transiently induced expression of cellular retinoic acid-binding protein II. After withdrawal of retinoic acid and full differentiation of retinoic acid-committed cells to neurons, the level of cellular retinoic acid-binding protein II returned to its basal level [63]. Proteins regulating intracellular redox balance

Various systems, including peroxiredoxins, glutathione-Stransferases (GSTs) or thioredoxin peroxidases, are involved in the protection of cells against oxidative stress and reactive oxygen species. Upregulation of these proteins was observed, particularly in differentiated cells compared with NSCs or neural progenitor cells, thereby suggesting that their principal role was the protection of neurons. In a 2DE proteomic analysis of differentiation

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of human NSC line ReNcell VM, induced by removal of growth factors, Hoffrogge et al. found significantly increased expression of peroxiredoxin 1 after 4 and 7 days of differentiation and immunoblot analysis confirmed its upregulation [13]. However, in experiments with rat striatal progenitor cell line ST14A, the level of peroxiredoxin-1 was unchanged during the first 72 h of differentiation (TABLE 1) [39]. The different time of cell culture may contribute to observed discrepancy between these two observations. Nonetheless, additional peroxidase activity functioning independently might play a further role in neuronal differentiation. Significant increases in peroxiredoxin 1 levels have been found in patients with Alzheimer’s disease (AD) and Down syndrome [64]. In mouse ES cells and embryonic carcinoma cells differentiated to neurons, a fourfold increase in peroxiredoxin-4 and upregulation of peroxiredoxin-6 were observed by Wang and Gao [65] and An et al. [63], respectively. Oxidized peroxiredoxin 1 can be regenerated by GST-π isoform, thus indicating cross-talk among redox balancing proteins [66]. The GST-π isoform belongs to the family of glutathione transferases, which are involved through the glutathionylation of acceptor proteins in the detoxification of many chemical compounds and participate in adaptive responses to oxidative stress. The GST-π isoform was downregulated in neural cells differentiated from porcine fetal NSCs [43], and GST was down-regulated in neurons derived from mouse ES cells [65]. By contrast, in experiments performed by An et al. [63] and Salim et al., the level of GST as well as level of GST-µ 1 isoform, respectively, were higher in differentiated cells [54]. This disparity might be due to different treatment durations and/or different origins of cells. Additionally, expression levels of thioredoxin peroxidase-2 were significantly increased during differentiation of ES cells to neural cells [67]. Proteins involved in cellular organization

Annexins belong to family of Ca2+- and phospholipid-binding proteins that can influence a number of membranerelated events; for example, membrane trafficking and the organization of compartment membranes and the plasma membrane. Through their apparent ability to organize, perturb or integrate into the membranes with which they interact, annexins may have roles as effectors, regulators and mediators of Ca2+ signals. Despite many reports in which the expression of individual annexins was correlated with cell proliferation, differentiation or transformation, the exact role of annexin molecules in cellular differentiation remains unknown [68]. An increase in the level of annexin 5 in differentiated cells was confirmed in two proteomics studies on different cell lines. A twofold increase was detected by Hoffroge et al. by 2DE as well as western blot with immunodetection after differentiation of rat striatal progenitor cells (line ST14A) transfected with GDNF gene [39]. GDNF is one of the crucial factors controlling neuronal differentiation, axonal

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sprouting and promoting neuronal repair. The level of annexin 5 was further increased during differentiation of native ST14A cells, suggesting a function of annexin 5 in differentiation independent of GDNF overexpression. In retinoic acid-induced differentiation of mouse embryonal carcinoma cell line to neurons, protein expression in two cultures, sparse with 1000 cells/mm2 and dense with 4000 cells/mm2, was compared by Inberg et al. [52]. The dense cultures exhibited faster maturation and accelerated neurite outgrowth. The level of annexin 5 was more than four-times higher in faster maturating dense cultures compared with sparse culture. These findings corroborated the observations published by Hoffroge et al. [39]. Cytoskeletal proteins (e.g., actin, tubulin and vimentin), belong to the most abundant proteins in NSCs, and this aspect allows them to be detected often in proteomic studies. Although the change of cellular shape from spherical NSCs to differentiated cells with irregular shape and long elongations is accompanied by re-organization of cytoskeleton, the role of cytoskeleton proteins as markers of neural differentiation is debatable. An interesting member of cytoskeleton-associated proteins is cofilin. Increased levels of cofilin were detected in parallel in two proteomic studies after 4 days of induction of neural differentiation of mouse ES and embryonic carcinoma cells by retinoic acid [63,67]. The expression level of cofilin was also significantly increased in faster differentiating dense cultures (4000 cells/mm2) of mouse carcinoma cell lines that were induced to neurons as compared with sparse cultures (1000 cells/mm2) [52]. Cofilin is a member of actin-depolymerizing factor/cofilin (AC) family of actin-binding proteins. AC proteins destabilize polymeric F-actin and thus produce a source of monomeric actin that can be reused for building of F-actin filaments support for growing cell edges. AC proteins thus mediate several intracellular events that require rapid actin filament dynamics [69]. In neurons, AC proteins are involved in filopodia formation followed by neurites and axon formation, together with elongation [70]. Molecular chaperons

Most of the neurodegenerative diseases are characterized by accumulation of misfolded and/or aggregated proteins, such as α-synuclein and synphilin-1 in PD, amyloid-β and tau in AD, superoxide dismutase 1 in some forms of amyotrophic lateral sclerosis, polyglutamine protein in HD or prion protein in prion diseases [71]. During protein folding in the endoplasmic reticulum, protein disulphide isomerase (PDI) catalyses thiol/disulfide exchange, thus facilitating disulfide bond formation, rearrangement reactions and structural stability of proteins. Accumulation of misfolded or immature proteins in endoplasmic reticulum in many neurodegenerative disorders and also after cerebral ischemia can be balanced by upregulation of PDI, leading to protein refolding and neuronal cell protection [71,72] .

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In proteomic studies, induction of PDI was observed to accompany superoxide dismutase-1 aggregation in spinal cord in a mouse model for amyotrophic lateral sclerosis, and inhibition of PDI by bacitracin increased aggregate formation [73]. Three other proteomic studies confirmed upregulation of PDI isoforms after differentiation of stem cells to neural cells. Protein disulphide isomerase A3 was upregulated in neurons in comparison with mouse ES cells [65] and also in cells differentiated from mouse subventricular zone precursors [50]. Protein disulphide isomerase isoform A6 was upregulated in differentiated neural cells from porcine NSCs [43]. Heat-shock proteins are the major molecular chaperons that provide a line of defense against misfolded proteins and are among the most potent suppressors of neurodegeneration in animal models [74]. Among the small HSP family, human HSP 27 (analog to mouse hsp25) and αB-crystallin share similar function as molecular chaperons and similar tissue-specific expression patterns. Both proteins can be phosphorylated on three serine residues [75]. Besides its function as a chaperone, HSP27 interacts with actin cytoskeleton in early and also later stages of neurite growth, where inhibition of HSP27 phosphorylation results in aberrant neurite growth [76]. Mouse HSP25 was detected in three protein spots on 2DE gel, and all of the spots were downregulated during formation of neuronic embryoid bodies from ES cells [51]. In a proteomic study on differentiation of porcine NSCs, αB-crystallin was also detected in three protein spots and the intensity of the most acidic spot was significantly increased in differentiated cells [43]. Metabolic enzymes

Glucose is the main energy source in CNS, and triosephosphate isomerase is one of the key glycolytic enzymes. Inhibition of triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase by α-monochlorohydrine in primary cell cultures from mouse cortices leads to decreased ATP levels and progressive neuronal death [77]. Glycolytic enzymes are thus important in ensuring energy production in the CNS. In proteomic studies, triosephosphate isomerase levels were upregulated in differentiated cells, namely neurons differentiated from mouse ES cells [65] and cells differentiated from porcine NSCs [43].

Other proteins

Phosphatidylethanolamine-binding protein-1 (PEBP1), also known as Raf-kinase inhibitor protein, is a multifunctional protein. It modulates the MAPK signaling cascade and thus may play a role in cellular growth and development. In a study on immortalized H19-7 cells generated from rat embryonic hippocampal cells, PEBP inhibited Raf-1, but had no effect on Raf-B kinase [80]. In addition, PEBP protein is a precursor of hippocampal cholinergic neurostimulating peptide that possesses cholinergic neuronal stimulatory activity. A decrease in PEBP and hippocampal cholinergic neurostimulating peptide was observed in brains from patients with AD, and the decrease of PEBP level accompanied accumulation of amyloid-β and formation of amyloid plaques in mouse model of AD [81]. The cellular level of PEBP was increased in undifferentiated rat hippocampal NSCs [61] and was also upregulated in rat striatal progenitor cell lineST14A transfected by GDNF. However, the PEBP1 level did not change during differentiation of the GDNF-transfected cell line [39]. PEBP1 was further identified among proteins secreted by rat hippocampal adult NSCs into the culture medium [56]. Proteins secreted into the culture medium

Cystatin C (active as N-glycosylated form) is a factor secreted by NSCs into the culture medium. Cystatin C was identified in the search for autocrine/paracrine factors that in vitro potentiated mitogenic activity of FGF-2 on adult rat hippocampus-derived neural progenitor cells. Stimulation of neurogenesis in adult hippocampus was confirmed also by in vivo experiments with the combined delivery of FGF-2 and cystatin C to the dentate gyrus of hippocampus [55]. Presence of cystatin C in culture medium was identified also by Dahl et al. by proteomic analysis of medium conditioned with adult rat hippocampus-derived neural progenitor cells [56]. It was discovered later that the medium conditioned by neurospheres (primary culture of NSCs from mouse striatum) was sufficient for survival and generation of NSCs from mouse ES cells in the absence of fetal bovine serum and feeder cells in ES cell culture. One of the secreted molecules responsible for NSCs generation was again identified as cystatin C [82]. Expert commentary

Proteins involved in mRNA processing

Heterogeneous nuclear ribonucleoproteins (hnRNPs) play an important role in pre-mRNA splicing. Multiple mRNAs can be produced from a single gene transcript by alternative splicing. Alternative splicing is an important mechanism in the developmental and cell type-specific control of gene expression. In the CNS, hnRNP H is one of the important factors participating in alternative splicing and co-ordinates specific splicing of, for example, pre-mRNA encoding src protein or glutamate NMDA R1 receptor [78,79]. Increased levels of hnRNP H were detected on 2D gels of cells differentiated from mouse subventricular zone precursors [50] and porcine NSCs [43].

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Availability of protein separation techniques and advances in MS allowing sensitive protein identification together with the development of protocols for NSC isolation and cell culture triggered the first attempts to analyze proteins expressed in NSCs. Protein profiles of NSCs of different origins have been constructed and changes in the levels of various proteins during different treatments of NSCs have been reported. Among the proteins identified, many relatively high-abundance proteins prevail, and these results represent only initial steps towards understanding NSCs biology. For future studies, the combination of prefractionation steps with sensitive protein separations and quantitative MS techniques would allow identification of low-abundance regulatory

Expert Rev. Proteomics 5(2), (2008)

Proteomics of neural stem cells

and signaling molecules, including membrane-associated proteins. In addition, protein array methodology appears to be promising, namely antibody arrays. Such antibody array approaches may circumvent many of the limitations, such as sensitivity, quantitation and post-translational modifications. Furthermore, spatial distribution and intracellular localization in differentiated neural cells of significantly regulated proteins identified by proteomic studies would benefit from additional studies using microscopic techniques. Computer modeling of biological interaction networks using datasets of candidate protein targets could provide a comprehensive view of mechanisms involved in neurogenesis, identifying potential points of therapeutic intervention. Five-year view

Future proteomics studies of NSCs and their differentiation together with application of a novel spectrum of analytical methodologies would dig deeper in the quest for low-abundance proteins, including signaling proteins that are more strictly related to differentiation to neurons. In order to be able to detect these molecules, greater effort must be focused on protein separation and prefractionation techniques to reduce sample complexities. Decreasing sample complexities on a subcellular or protein level would be one of the future approaches in NSCs proteomics.

Review

In addition, studies of post-translational protein modifications and especially signaling molecules and key regulatory proteins using microproteomics would be specifically applied. Multiplexed protein/antibody microarrays will enable the monitoring of changes that accompany NSCs self-renewal and differentiation. Very sensitive detection protocols using antibodies may also help to overcome the problems caused by restricted sample availability. Most importantly, functional studies and in vivo experiments will be necessary to verify protein roles in NSC self-renewal and differentiation. The knowledge of regulatory pathways that control NSC fate and understanding of interactions among proteins in protein complexes and between proteins and other molecules, may subsequently lead to the development of successful and primarily safe protocols for stem cells transplantation in treatment of CNS disorders. Financial & competing interests disclosure

This study was supported by the Centre for Cell Therapy and Tissue Repair (1M0538) and Institutional Research Concept IAPG No. AV0Z50450515. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Key issues • Differentiated neural cells can be obtained by default differentiation process of neural stem cells (NSCs) from the CNS or alternatively by differentiation of embryonic stem cells or transdifferentiation of mesenchymal stem cells. • Different origin of stem cells and various treatments used for studying neural differentiation make the selection of potential markers of neural differentiation very demanding. • Proliferating cell nuclear antigen is a common marker of dividing cells, and high levels of this protein are also typical of proliferating NSCs. • An increase in peroxiredoxin levels during differentiation was reported in various types of NSCs. These proteins may play an important role in the protection of neurons from oxidative damage. • Differentiation of NSCs is associated with changes in levels of small heat-shock proteins. • Other potential markers of NSCs differentiation could be neuronal differentiation-related protein, stromal cell-derived factor receptor-1 or cofilin. • Cystatin C is a secreted protein produced by NSCs that stimulates proliferation and generation of neural precursors. • Verification of candidate markers and elucidation of their functional role in differentiation of NSCs deserve further investigations. • Information collected from transcriptomic, proteomic and transplantation experiments must be collated to develop protocols for the safe use of NSCs in cell-based therapies.

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Affiliations •

Helena Skalnikova, PhD Institute of Animal Physiology & Genetics, Academy of Sciences of the Czech Republic, Rumburska 89, 277 21 Libechov, Czech Republic; Joint Proteome Laboratory, Videnska 1083, 142 20 Prague, Czech Republic Tel.: +42 031 563 9580 Fax: +42 031 563 9510 [email protected]



Petr Vodicka, PhD Institute of Animal Physiology & Genetics, Academy of Sciences of the Czech Republic,, Rumburska 89, 277 21 Libechov, Czech Republic Tel.: +42 031 563 9566 Fax: +42 031 563 9510 [email protected]



Suresh Jivan Gadher Beckman Coulter International S.A., 1260 Nyon, Switzerland; Joint Proteome Laboratory, Videnska 1083, 142 20 Prague, Czech Republic Tel.: +41 223 653 745 Fax: +41 223 653 700 [email protected]



Hana Kovarova, PhD Institute of Animal Physiology & Genetics, Academy of Sciences of the Czech Republic, Rumburska 89, 277 21 Libechov, Czech Republic; Joint Proteome Laboratory, Videnska 1083, 142 20 Prague, Czech Republic Tel.: +42 031 563 9582 Fax: +42 031 563 9510 [email protected]

Expert Rev. Proteomics 5(2), (2008)

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