Embryonic StemCDerived Versus Somatic Neural Stem Cells: A Comparative Analysis - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:08 |只看该作者 |倒序浏览 |打印

作者：Elena Colomboa, Serena G. Giannellia, Rossella Gallia, Enrico Tagliaficob, Chiara Foronia, Elena Tenedinib, Sergio Ferrarib, Stefano Ferrarib, Giorgio Cortec,d, Angelo Vescovia, Giulio Cossua, Vania Broccolia作者单位：a Stem Cell Research Department, San Raffaele Scientific Institute, Milan, Italy;b Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy;c National Institute for Cancer Research, Genoa, Italy;d Department of Oncology, Biology and Genetics, Genova University Medical . l3 t2 j) y# y
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      【摘要】, F2 b5 H3 ?. o& x& T3 u1 Y
   Reliable procedures to induce neural commitment of totipotent undifferentiated embryonic stem (ES) cells have provided new tools for investigating the molecular mechanisms underlying cell fate choices. We extensively characterized the developmental potential of ES-induced neural cells obtained using an adaptation of the multistep induction protocol. We provided evidence that ES-derived neural proliferating cells are endowed with stem cell properties such as extensive self-renewal capacity and single-cell multipotency. In differentiating conditions, cells matured exclusively into neurons, astrocytes, and oligodendrocytes. All these features have been previously described in only somatic neural stem cells (NSCs). Therefore, we consider it more appropriate to rename our cells ES-derived NSCs. These similarities between the two NSC populations induced us to carefully compare their proliferation ability and differentiation potential. Although they were very similar in overall behavior, we scored specific differences. For instance, ES-derived NSCs proliferated at higher rate and consistently generated a higher number of neurons compared with somatic NSCs. To further investigate their relationships, we carried out a molecular analysis comparing their transcriptional profiles during proliferation. We observed a large fraction of shared expressed transcripts, including genes previously described to be critical in defining somatic NSC traits. Among the genes differently expressed, candidate genes possibly responsible for divergences between the two cell types were selected and further investigated. In particular, we showed that an enhanced MAPK (mitogen-activated protein kinase) signaling is acting in ES-induced NSCs, probably triggered by insulin-like growth factor¨CII. This may contribute to the high proliferation rate exhibited by these cells in culture.
      【关键词】 Neural stem cell Embryonic stem cell Neural differentiation Self-renewal Multipotency Transcriptional profile% N' S! q: @+ j% D. H  N
   INTRODUCTION0 C% C; X* b9 ]4 }7 G

Stem cells are defined by three fundamental features: the ability to self-renew, to give rise to a differentiated progeny, and to maintain these features over a long period of time . However, the nature of the in vitro¨Cderived neural progenitors is still elusive, and a better characterization of their self-renewal features and neural potential over time is desirable.' Q6 M# ~/ a6 J, V/ ?% \

Somatic neural stem cells (NSCs) can be retrieved from the embryonic neural tissue or from the neurogenetic regions of the adult brain (subependymal layer and dentate gyrus) and can self-renew in vitro for a long period of time and differentiate into neurons, astrocytes, and oligodendrocytes . ES cell¨Cderived neural progenitors, on the other hand, have not been studied as well, and their clonogenic potentials are still unknown. Moreover, a thorough comparison between ES cell¨Cderived neural progenitors and somatic NSCs in terms of stable growth, differentiation potential, and overall gene expression is still missing. To fill this gap, we studied a population of ES-derived cells that showed all the cardinal properties of stem cells and thus defined ES-derived NSCs. Moreover, we extensively compared them with NSCs isolated from embryonic neural tissue, in terms of proliferation ability, neural differentiation potential, and overall gene expression.
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MATERIALS AND METHODS. H% M, ^6 c9 D' T5 e
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Materials and methods are available as supplemental online data.* \: ^* n- G" i: \1 K' w& H/ g
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RESULTS

Derivation of Homogenous and Stably Proliferating Neural Progenitors from ES Cells

To differentiate ES cells toward a neural phenotype, we employed a protocol based on a three-step procedure established by Okabe et al. . Furthermore, matrigel-coated dishes allowed a better cell attachment and spreading than did gelatin, used in previous studies.
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Within 5 days, a homogenous population of cells expressing the neural precursor markers Nestin and Vimentin had grown  (Fig. 1C¨C1E). The overall procedure required 8 days, thus shortening the period of time needed to obtain neural commitment as compared with the protocol of Okabe et al. After the first selection phase, we were able to maintain stable cell growth of undifferentiated neural progenitors in NSC culture medium for several months.* @# ~4 ]7 t$ ~3 u8 |' ]  z
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Figure 1. Establishment of homogenous cultures of ES cell¨Cderived NPs. (A): Outline of the overall protocol based on three major steps: I, embryoid body formation; II, plating in matrigel-coated dishes and cultured with NSC medium; and III, disaggregation after amplification. (B): Determination of the cell growth in a window of 1 month. (C): Phase-bright microphotograph showing the general morphology of the ES-derived NSCs in their undifferentiated state. Immunofluorescence for nestin (D) and vimentin (E) on proliferating ES-derived cells. (F): Normal karyotype (2n = 40) isolated from P17 cell cultures. (G): Reverse transcription¨Cpolymerase chain reaction analysis of a series of molecular markers of naïve ES cells (Oct4) and forebrain neural stem cells (Sox2, Emx2, Pax6, Lhx2, and Foxg1). Abbreviations: dNP, differentiated embryonic stem cell¨Cderived neural progenitors; EGF, epidermal growth factor; ES, embryonic stem; FGF, fibroblast growth factor; KSR, knockout serum replacement; LIF, leukemia inhibitory factor; NP, neural progenitor; NSC, neural stem cell.
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To monitor neural induction in our cells, we tested a number of neural-specific genes by reverse transcription¨Cpolymerase chain reactions (RT-PCRs). Interestingly, the expression of molecular markers typical of totipotent ES cells such as Oct4, Cripto, and Nanog was not detected in the neural progenitor cultures (Fig. 1G; data not shown), whereas genes specific to somatic NSCs, such as Sox2, and forebrain-specific genes such as Emx2, Pax6, Lhx2, and Foxg1 were activated and detectable at similar levels (Fig. 1G) . Cultures of ES cell¨Cderived neural progenitors were stable for months without undergoing either senescence or growth factor¨Cindependent proliferation. They showed a stable doubling time (approximately 24 hours) and a normal caryotype (2n = 40) as tested up to 17 passages, spanning 1 month of in vitro culture (Fig. 1B, 1F).

ES cell¨Cderived neural progenitors were differentiated by sequential removal of FGF-2 and EGF and finally switched to a hippocampal culture medium (see Materials and Methods) .
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Figure 2. Differentiation protocol and phenotypes of embryonic stem (ES)-derived neural cells. (A, B): Low magnification of differentiated cell cultures stained with ß-III-tubulin (neurons) and GFAP (astrocytes). (C): Quantitative analysis of neurons, glia, and oligodendroglia in the differentiated cells. (D, E): Morphological characterization of the ES cell¨Cderived neurons stained with NF160 and MAP2. (F): Quantitative analysis of the different types of neurons according to their neurotransmitter content. (G, G'): Mature oligodendrocyte highlighted in the ES cell¨Cderived cultures by GalC and O4 antibody stainings, respectively. (H, J, K, L): Immunocytochemistry for neuron subtype¨Cspecific markers in the differentiated cell cultures using antibodies against GABA (H), glutamate (J), TH (K), and ChAT (L). (I): Double staining for synaptophysin (green) and ß-III-tubulin (red) to assess the formation of synaptic buttons in neurites of ES cell¨Cderived neurons. Abbreviations: ßIIItub, ß-III-tubulin; ChAT, choline acetyltransferase; GABA, -aminobutyric acid; GalC, galactocerebroside; GFAP, glial fibrillary acidic protein; MAP2, microtubule associated protein 2; TH, tyrosine hydroxylase.! u* X0 h+ m4 `! Q0 X
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To evaluate the in vivo differentiation potential of ES cell¨Cderived precursors, we took advantage of the YC5 ES cell line that constitutively expresses enhanced green fluorescent protein (eGFP) . Neural precursors derived from this line showed stable growth and multilineage differentiation in vitro. We transplanted the cells into the lateral ventricles of embryonic day 14.5 (E14.5) mouse embryos, and results were analyzed between P1 and P5. A diffuse integration of eGFP cells was normally observed in the neural parenchyma of different regions, depending on the area of graft integration (supplemental online Fig. 2). Generally, the lateral cortex, the olfactory bulbs, and the ventral forebrain showed many integrated eGFP cells. In particular, in the lateral cortex, many GFP cells were detected in all the layers and spread along the dorsal-ventral axis (supplemental online Fig. 2). eGFP integrated cells expressed markers of differentiated neurons and astrocytes as assessed by ß-III-tubulin or NeuN and GFAP immunofluorescence, respectively (supplemental online Fig. 2). In all the experiments analyzed (n = 3), neurons, astrocytes, and oligodendrocytes were scored, but the ratio among the different cell types was highly variable, depending on the injection site.7 E* u9 a, \! h) u  v5 A' o5 F
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ES Cell¨CDerived Neural Progenitors Are Clonogenic and Multipotent5 z! u: a! @! H: Q2 O7 c! y. t
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Clonal analysis is a stringent paradigm for testing multipotency at the single-cell level. Although it has been extensively used in the NSC field, it has rarely been applied to ES cell¨Cderived neural cultures. Individual ES-derived cells from different passages (P3, P7, and P11) were transferred into four-well chamber slides (1 cell/1 well) pre-coated with matrigel. In the presence of FGF-II/EGF, approximately 5% of these single cells proliferated, giving rise to a clone in approximately 3 weeks (Fig. 3A¨C3D). Upon differentiation, cells were tested for ß-III-tubulin, GFAP, and GalC immunofluorescence (Fig. 3E¨C3H). In all cases (n = 12), immunostainings revealed neural tri-lineage commitment, confirming the multipotent nature of the clone founder cells. The percentages of mature cell progenies (neurons, astrocytes, and oligodendrocytes) over the whole differentiated population derived from either a single clone or the sister uncloned culture were very similar (Fig. 3I). This indicated that each individual clonal culture maintained its differentiation potentialities with no alteration. Finally, we tested the growth rate of the three cultures derived by single-cell expansion (no. 35 from P3, no. 102 from P7, and no. 155 from passage P11) in comparison with their sister bulk cultures. The four cultures exhibited a similar growth rate when measured for a period of 4 weeks (Fig. 3J). These results suggest that the ES cell¨Cderived progenitors exhibit self-renewal features as tested with stringent criteria.
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Figure 3. Clonogenic assays and analysis of bulk and cloned cell cultures. (A¨CD): Bright-field pictures of a growing clone. (E¨CH): Immunoreactivity for markers of neuronal (ß-III-tubulin ) fate of a clone derived by single-cell expansion. (I): Characterization of the mature phenotypes observed in both bulk and cloned cell cultures (nos. 35, 102, and 135). (J): Compared analysis of the growth rate of the bulk and three cloned cell cultures over a period of 3 weeks. Abbreviations: ßIIITub, ß-III-tubulin; GalC, galactocerebroside; GFAP, glial fibrillary acidic protein.- }8 B; S0 }/ t: ]( i# T" X
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Taken together, these data demonstrate that ES cell¨Cderived neural progenitors are self-renewing, multipotent, and clonogenic and maintain these potentialities for a long period of time in vitro. In conclusion, neural precursors derived from ES cells are endowed with all the cardinal features of stem cell lines and thus represent bona fide NSCs. We thus renamed them ES-derived NSCs. This finding cleared the way for a comparison of the cellular and molecular phenotypes of these two NSC lines in similar experimental conditions.2 ^; d) t3 U! }8 i
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ES-Derived NSCs and Somatic NSCs Compared with In Vitro Phenotypes

We isolated somatic NSCs from forebrain regions of E14.5 mouse embryos to compare in vitro growth and differentiation potential of somatic and ES-derived NSCs. Therefore, we maintained both cell lines in the same culture medium (see Materials and Methods) and compared cells at the same passages (between P8 and P14).
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The two cell lines grew in different ways. Whereas somatic NSCs proliferated better as clusters of cells floating freely in the medium, generally called neurospheres, the ES-derived NSCs grew optimally when adhering to a substrate (supplemental online Fig. 3). In fact, ES-derived NSCs were unable to form growing "spheres," and their growth was impaired when unable to attach to a substrate (data not shown). Under optimal conditions for each of the cultures and using the same culture medium, cells showed a different proliferation rate at similar passage numbers. In fact, ES-derived NSCs showed a two-time log 10 increase with respect to embryonic somatic NSCs after 6 passages, spanning less that 1 month of in vitro culture (supplemental online Fig. 3). Then, we analyzed their differentiation potential. Both cell lines were plated on laminin-coated dishes and exposed to similar differentiation conditions (see Materials and Methods). After 8 days, cells were processed for immunofluorescence to detect neurons (ß-III-tubulin), astroglia (GFAP), and oligodendroglia (GalC). In all cases analyzed (n = 18), the proportion of neuronal and glial cells was different between the two cell lines. In fact, differentiated progenies derived from either ES-derived or somatic NSCs contained 54% ¡À 4% and 40% ¡À 3% of neurons, 36% ¡À 2% and 52% ¡À 3% of astrocytes, and 10% ¡À 3% and 8% ¡À 3% of oligodendrocytes, respectively (200 cells, n = 5) (Fig. 4). Furthermore, we assessed the differentiation potential of NSCs derived from adult brains in similar conditions and we observed a further general decrease in neuronal cell production (28% ¡À 4%) and a corresponding enrichment in astrocytes (64% ¡À 5%) (200 cells, n = 5) (Fig. 4C, 4F, 4H). Overall, ES-derived NSCs displayed a higher efficiency in neuronal differentiation compared with somatic NSCs. We then assessed the neuronal subtype composition of the mature neurons in embryonic NSCs and ES-derived NSCs. In both cases, glutamatergic neurons were the most abundant phenotype (69% ¡À 6% and 53% ¡À 4%) (200 cells, n = 5), whereas GABAergic neurons represented 29% ¡À 3% and 37% ¡À 3% of the entire differentiated cell population, respectively (n = 5) (supplemental online Fig. 4). However, only in the mature progeny derived by ES-derived NSCs was it possible to observe a portion of dopaminergic (6% ¡À 4%) and cholinergic (4% ¡À 3%) neurons (200 cells, n = 5) (Fig. 2I; supplemental online Fig. 4).

Figure 4. Composition and morphology of differentiated cell cultures derived from embryonic somatic NSCs (A, D), adult somatic NSCs (B, E), and ES-derived NSCs (C, F, G). (A¨CC): Double stainings for ß-III-tubulin and GFAP to visualize the ratio of neurons/astrocytes in the overall differentiated cultures. (D¨CG): Neural morphology visualized by ß-III-tubulin immunostaining of neurons derived from either somatic embryonic (D) and adult (E) NSCs or ES-derived NSCs (F, G) with nuclei labeled with 4',6-diamidino-2-phenylindole. (H): Quantitative analysis of the neural, glial, and oligodendroglial phenotypes obtained by differentiation of somatic and ES-derived cell cultures.*, p

ES-Derived NSCs and Somatic NSCs Compared with Transcription Profiles

To test the molecular heterogeneity between ES-derived NSCs and somatic NSCs and to score genes differently expressed in the two cell populations, we performed a compared global transcriptional profile by means of gene chip analysis comparing cells between passages P9 and P10. Briefly, fluorescent-labeled cRNAs derived from ESC-derived NSCs and E14.5 embryonic striatal NSCs (between P8 and P9) were hybridized to the Affymetrix Mouse Expression 430 set (Santa Clara, CA, http://www.affymetrix.com). This platform consists of two GeneChip probe arrays (MOE430A and MOE430B) containing more than 45,000 probe sets representing more than 39,000 transcripts and variants, including more than 34,000 well substantiated mouse genes. As shown in the scatter plot in Figure 5A, both NSC lines expressed a large number of common sequences. In this category, 15,031 sequences were not changed in the two cell populations. Although 3,158 and 2,482 showed an increased expression in ES-derived NSCs and somatic NSCs, respectively, as identified by Affymetrix MAS 5.0 absolute and comparative analyses, the number of sequences, which can be considered specifically expressed in either cell population, on the basis of a ratio equal to or higher than threefold (i.e., a signal-log ratio grater than or equal to 1.5 or less than or equal to ¨C1.5), was 276 in ES-derived NSCs (among them, 185 present only ES-derived NSCs) and 372 in somatic NSCs (among them, 201 present only in somatic NSCs). Thus, less than 2% of the studied transcripts were found significantly changed, indicating a substantial transcriptional homogeneity of the two cell populations.! R0 {) b0 l8 h0 Q9 i2 g

Figure 5. Compared global transcriptional profile of somatic and ES-derived NSCs. (A): Scatter plot showing the distribution of hybridization signals of somatic (E14-NSCs) on the y-axis and ES-derived (ES-NSCs) NSCs on the x-axis. Transcripts more abundant in E14-NSCs are shown in red, transcripts more abundant in ES-NSCs are shown in green, and unchanged transcripts are shown in yellow. (B): Probe sets considered as positive controls for the microarray experiment: Xist, H2-K, H2-D1, and H13. (C): Microarray data of selected genes chosen for their proven or suggested role played in somatic NSCs. (D¨CF): Signaling pathways whose main components show equal expression in both ES-derived and somatic NSCs. (E): Wnt-ß-catenin schematic transduction pathway and Western blotting showing Wnt5A and ß-catenin protein levels in both NSC cell lines. (C, D): Signals are shown as color-coded cells (black for "absent" or "marginal" transcripts and blue to red for "present" transcripts: blue for signals 1,001). The fold change is also shown as SLR. (F): Notch transduction pathway and protein level analysis of Notch1 and Hes1. (G): Hh schematic molecular pathway and analysis of Dhh and Smo protein levels in both stem cell lines. Abbreviations: Bmpr1a, bone morphogenetic protein receptor 1a; Bmpr2, bone morphogenetic protein receptor 1; Ccnb1, cyclinb1; Ccnnd1, cyclind1; Ccnd2, cyclind2; Dhh, desert hedgehog; Disp1, Dispatched1; Dll1, Delta1; Dnclc1, dynein light chain 1; Dvl1, Dishevelled 1; E14, embryonic day 14; Egfr1, epidermal growth factor 1; ES-NSC, embryonic stem¨Cneural stem cell; Fgfr1, fibroblast growth factor receptor 1; Fz2/9, Frizzled receptor 2, 9; Fu, fused; Hdac, histone deacetylase; Hdgf, hepathoma-derived growth factor; Hh, Hedgehog; Jag1, Jagged1; Nes, nestin; Nic, nicastrin, NISD, notch intracellular domain; Nr3c1, nuclear receptor 3c1; Pdgfa, platelet-derived growth factor A; PS1, presenilin1; Ptch1, Patched1; SLR, signal log 2 ratio; Smo, Smoothened; Stmn1, Stathmin1; ThrA, thyroid receptor A; Vegfb, vascular endothelial growth factor B; Vim, vimentin.! O% D7 O' h& V7 @9 X' X
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Importantly, the Xist gene resulted as one of the most widely differentially expressed (Fig. 5B). This gene is exclusively expressed in the female lineage from the blastocyst stage onward because it produces a main molecular switch that inactivates one of the two X chromosomes. In fact, the ES cell lines used for the transcriptional profile were all male (XY), and thus the Xist gene was not expressed. Conversely, the NSCs were derived by a pool of both male and female E14.5 embryos, and thus Xist gene expression was clearly detectable in the screening. Another expected difference between the two cell lines was represented by the low expression of some genes coding for major histocompatibility complex (MHC) class I proteins in ES-derived NSCs (H2-K, H2-D1, and H13) (Fig. 5C), as already stated in previous reports describing the very low level of MHC class I gene expression in mouse and human ES cells with respect to many somatic cell lines .- g; G$ l, Z( j1 t+ T( G, K# z. ^

Among the 15,031 genes equally expressed in the two cell lines, several genes encode for a variety of proteins already known to play a critical role in somatic NSCs, such as the cytoskeletal components nestin  were highly expressed in both cell lines, as expected with their function in modulating cell cycle progression in a variety of cells (Fig. 5D).3 r8 J5 _6 e1 ?  c( S
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We scored a similar presence not only of single genes but also of entire transduction pathways implicated in regulating somatic NSC behavior. For instance, three transduction pathways highly related to proliferation and self-renewal activity of somatic NSCs, such as the Wnt¨Cß-catenin (ß-cat), Notch, and Hedgehog (Hh) pathways, were similarly expressed in ES-derived NSCs . Both the Hh receptor Patched1 (Ptch1) and its coreceptor Smoothened (Smo) as well as the downstream effectors Gli2-Gli3 were found expressed in both cell lines (Fig. 5G). Interestingly, we found only Desert Hedgehog (Dhh), out of the whole Hh ligand family, to be expressed in both cell types. Indeed, we confirmed these results by Western blot analysis, which indicated the presence of both Dhh and Ptch1 proteins (Fig. 5G). Taken together, these data show that somatic and ES-derived NSCs share a great part of their molecular phenotype, including important molecular components and specific signal transduction pathways, suggesting the similar nature and potentials of the two cell lines.
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Furthermore, this study identified 245 sequences specifically enriched in either of the two NSC lines (supplemental online Table 1). We identified three groups of genes among them that may unravel the molecular pathways at the basis of the differences of the two cell lines. Some of the genes most differently expressed coded for proteins of the extracellular matrix (ECM) such as collagen proteins such as Col1a1, Col3a1, and Col1a2 (Fig. 6A). Furthermore, other important components of the ECM, such as fibronectin (Fn) and fibrillins (Fbn-1 and -2), were upregulated in ES-derived NSCs . To independently confirm the reproducibility of the array data, RT-PCRs were performed. Semiquantitative amplification reactions for Col1a1, Fn1, and Fbn1 confirmed gene expression diversity in the two cell populations and showed even greater differences as in the case of Fbn1 (6.5-fold increase in ES-derived NSCs vs. a 3.5-fold increase in the gene-chip analysis) (Fig. 6A). The enhanced expression of extracellular components in ES-derived cells may explain why these cells show a higher ability to attach and grow over many types of surfaces in respect to somatic NSCs, which typically cannot grow on a naïve substrate.
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Figure 6. Classes of genes differentially expressed in ES-derived NSCs (ES-NSCs) versus somatic NSCs (E14-NSCs). (A): Microarray and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of ES-derived NSC¨Cenriched transcripts coding for extracellular matrix proteins. (B): Gene expression analysis by Gene-Chip and RT-PCR of transcripts coding for transcription factors contributing to caudal neural tube identity. (C): Microarray and RT-PCR study of genes coding for IGF signaling pathway. In tables, signals are shown as color-coded cells (black for "absent" transcripts and blue to red for "present" transcripts, as in Figure 5). The fold change is also shown as SLR. Abbreviations: aNSC, adult neural stem cell; Col1a1, collagen 1a1; Col3a1, collagen 3a1, Col1a2, collagen 1a2; Col6a3, collagen 6a3; E14-NSC, embryonic day 14¨Cneural stem cell; ES-NSC, embryonic stem¨Cneural stem cell; Fn1, fibronectin 1; Fbn1, fibrillin 1; Fbn2, fibrillin 2; Itga9, Integrin a9; SLR, signal log 2 ratio.
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Among the 75 sequences found enriched in the ES-derived NSCs and corresponding unambiguously to known genes, eight (Nkx2.2, Irx2, En1, Hoxa1, Hoxa2, Hoxa3, Hoxa4, and Hoxa5) were represented by genes coding for transcription factors mostly or exclusively active along the caudal neural tube. During embryonic development, a series of transcription factors regionalize the neural tube along the anterior-posterior (A-P) axis. Growing evidence supports the hypothesis that these molecules are producing a molecular identity that specifies the different regions of the neural tube such as forebrain, midbrain, and hindbrain. Many of these transcription factors were expressed in both cell lines; however, we found a general enrichment in ES-derived NSCs of mRNAs of transcription factors that act on the spinal cord. Nkx2.2, Irx2, and En1 are genes expressed starting from the mid-hindbrain region all along the ventral spinal cord, whereas the Hox genes are active in the most caudal regions of the body. We confirmed these findings by means of semiquantitative RT-PCRs (Fig. 6B) and widened this data set by analysis other Hox genes not included in the microarray platform, such as Hoxa9, Hoxa10, and Hoxa13 (Fig. 6B; data not shown). In all cases, these genes were expressed in ES-derived NSCs but were represented at a low or undetectable level in somatic NSCs. These data strongly suggested that ES-derived NSCs displayed the positional molecular information of the whole A-P axis of the developing neural tube. Conversely, somatic NSCs showed a general low expression or absence of members of the Hox gene family and other transcription factors of the Nkx and Irx classes, which are critical for the establishment and shaping of the spinal cord.  W+ W! I( q0 s' M- ^3 d5 E# h  G

Among the 75 known transcripts enriched in the ES-derived NSCs, insulin-like growth factor (IGF)-II is coding for the growth factor most expressed in a relative and absolute manner in these cells. IGF-II belongs to the IGF family and shares many pleiotropic activities in neural cells, such as supporting cell proliferation, migration, and survival, with the other members of the family, IGF-I and IGF-III , were expressed at comparable levels (Fig. 6C). These data prompted us to determine whether the IGF signaling was effectively upregulated in the ES-derived NSCs and to correlate this molecular diversity with a particular aspect of the in vitro behavior of these cells.$ K3 N4 j+ R& {* s, L! j
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The Enhanced IGF Signaling Is Responsible for the High Proliferation Rate of ES-Derived NSCs In Vitro

To verify whether the IGF-II enhanced expression resulted in the functional activation of the downstream targets, we evaluated the relative amounts of either phosphorylated MAPK (mitogen-activated protein kinase) and phophorylated Akt, two key molecules of the Ras-Raf-MAPK and PI3-Kinase (PI3K) pathways, respectively. Indeed, we found an increase in the phosphorylated forms of MAPK and Akt, in the ES-derived in respect to the somatic NSCs, without any clear increase of the total amount of these proteins (Fig. 7A). Thus, a sustained increase in IGF signaling was exhibited by ES-derived NSCs during their proliferating stage. Previous studies revealed an essential action of IGF-I in maintaining proliferation of somatic NSCs in cultures . In addition, a culture medium deprived of insulin, but supplemented with IGF-I or IGF-II (100 nM), sustained the growth of ES-derived NSCs. Interestingly, the effect of IGF-I on proliferation was higher than that of IGF-II, although the latter still had a significant effect.+ w0 a& b; H- C+ y
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Figure 7. Active IGF signaling is enhanced in ES-derived NSCs with respect to somatic NSCs. (A): Western blotting analysis of IGF-II and active forms of key components of the pathway. p42/p44 (Erk1/2) and Akt phosphorylated forms are enriched in ES-derived NSCs. Cell lysates were normalized with respect to their ß-actin protein levels. (B): IGF growth factors sustained ES cell¨Cderived neural proliferation in culture in the absence of insulin. (C): Functional arrest of IGFR-I function by a blocking antibody strongly decreased IGF-II¨Cdependent cell proliferation. Suramin is an inhibitor of both IGF and epidermal growth factor. (D): Cell proliferation elicited by IGF-II is mediated mostly by activation of the mitogen-activated protein kinase signal transduction pathway. Abbreviations: aNSC, adult neural stem cell; DIV, days in vitro; ES-NSC, embryonic stem¨Cneural stem cell; IGF, insulin-like growth factor; IGFR-I, insulin-like growth factor receptor I.

To identify the receptor through which IGF-II promoted cell proliferation, we added blocking antibodies for either IGF receptor I (IGF-IR) or IGF receptor II (IGF-IIR) in conditions in which cell growth was sustained by IGF-II (see Materials and Methods). Only cultures treated with anti¨CIGF-IR, but not anti-IGF-IIR, exhibited a dramatic decrease in cell proliferation (Fig. 7C). Accordingly, we found a reduction in MAPK phosporylation in cultures exposed to IGF-RI blocking antibody (Fig. 7C). These findings suggest that IGF-II elicits cell growth mainly by signaling via the IGF-IR. Finally, we investigated the intracellular pathway involved in mediating IGF action. Specific inhibitors of the MAPK or the PI3K pathways were used, in particular inhibitors of the ERK kinases such as PD098059, U0126, and LY294002, to block PI3-kinase . Addition of the MAPKK inhibitor PD098059 to IGF-II treated cultures abolished the effect of the growth factor on the increased proliferation in a dose-dependent manner (Fig. 7C). Conversely, a minor effect was noted on the IGF-II¨Cinduced proliferation upon the addition of the PI3-K inhibitor, LY294002 (Fig. 7C). These results suggest that IGF-II¨Cinduced proliferation is mediated mainly by the activation of the MAPK signaling and only marginally by the PI3K molecular component pathway.
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DISCUSSION( }( c7 c' v# u

ES Cell¨CDerived Neural Progenitors Exhibited Main Properties of Stem Cells

Several protocols suitable for ESC differentiation toward a neural cell lineage have been established over the years. In particular, the work originally described by Okabe et al. . A series of clonogenic assays allowed the authors to emphasize the stem cell nature of the induced ES cells. All these results indicate that different strategies may be applied to derive NSCs from more primitive totipotent cells. This suggests that further studies are required to understand the differences among various NSC-inducing systems and the developmental potential of derived cells.
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Cellular and Molecular Phenotypes of Somatic and ES-Derived NSCs
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The stem cell features of these ES cell¨Cderived populations prompted us to investigate their relationships with somatic NSCs. We used embryonic NSCs derived from the forebrain regions as a basis of comparison with the ES-derived NSCs, although in some circumstances adult NSCs isolated from the subependymal zone were added in the analysis.+ g* |, \1 l( m/ T% q. n

ES-derived NSCs showed an in vitro behavior completely different from that displayed by somatic NSCs. In fact, the former grew attached to a substrate, whereas the latter formed clonal aggregates known as neurospheres. Our large-scale gene transcription analysis, based on GeneChip technology, provided some possible explanations for these dissimilar behaviors. In fact, we found ES-derived NSCs to highly express the genes coding for Fibronectin, Fibrillin-1 and -2, and various forms of Collagens. All of them are important components of the extracellular matrix; Fibronectin, in particular, allows the cells to strongly adhere, linking the substrate to the cell membrane and to the intracellular cytoskeleton via -5-ß-1 integrin. Undifferentiated ES cells show a highly organized extracellular matrix, forming focal adhesions in specific circumstances . Thus, a similar function may be supposed in NSCs, where it shows a high level of expression.
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ES-Derived NSCs Exhibit a High Proliferative Index and Sustained IGF Signaling
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ES-derived NSCs displayed a higher proliferation rate in comparison with both embryonic and adult somatic NSCs. Seeking the molecular basis of this behavior, we used the microarray analysis to identify the enriched expression of IGF-II in the ES-derived cell population. IGFs are widely expressed molecules that regulate proliferation, survival, and differentiation. In particular, several studies have revealed their essential role in supporting proliferation of NSCs either in culture or during neural development . Thus, these and other data suggest that MAPK acts as a primary signaling in regulating proliferation in ES-derived and somatic NSCs, reinforcing the view of a general homogeneity between the two NSC cell populations. Taken together, these findings represent an initial step toward the molecular characterization of different types of NSCs, highlighting specific features for each cell line. Furthermore, these characterizations will reveal new molecules able to control cell differentiation and cell lineage choices.

ACKNOWLEDGMENTS9 h) b5 N; ]( f/ y  T0 E' M5 p
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We thank L. Cornaghi and T. Veneroso for expert technical assistance, Drs. A. Faedo and A. Bulfone for initial support in the microarray analysis, and Dr. A. Nagy for providing YC5-ES cells. This research was supported by Istituto Superiore di Sanit¨¢ grant CS-71 to V.B.% Y- X3 _' T0 t8 M
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DISCLOSURES( i' `7 V6 j  ]/ |. L3 ?  b
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The authors indicate no potential conflicts of interest.+ U. R4 c0 }4 M% h; }" L
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