CA2104697A1 - Nestin expression as an indicator of neuroepithelial tumors - Google Patents

Abstract

A gene encoding a protein, nestin, whose expression distinguishes neural multipotential stem cells and brain tumor cells from the more differentiated neural cell types (e.g., neuronal, glial and muscle cells). Methods of detecting the expression of nestin as a means of diagnosing and treating brain tumors are also described.

Description

WO92/1~21 PCT/US92/01375 ~ NESTIN EXPRESSION AS AN INDICATOR OF
________________ NEUROEPITHELIAL TUMORS
:, Descri~ion Back~round 05 Braln tumors are a leadlng cause of cAncer teaths in people under tho age of 35. An important concopt in our understanding of brain tumors is that the neoplAstic cells may arise from undifferentiated neuroectodermal cells (Rubinstein, L.J. J. Neurosur~ 62: 795-805 ~1985)).
At the gaserula stage of central nervous system (CNS) development, neuroectoderm, formin~ the neural plate is derived from ectoderm which has been induced by und-rlyin~ mcsoderm. ~Spemann, H. Yale University Press 1938)). The neural plate then folds, 5n the process callot n-urulation, to form the noural tube. The cells of the neural tube are the precursors for the ma~or differentiated cell types in the central neirvous system:
, astrocytes, oligodendrocytos, and the ~arious types of nouron (Sauer, F.C. J. Com~ Neurol. 62: 377-405 (1935)). The cells of the neural tube may also be the precursors to neoplastic cells which form brain tumors.
~ Recent success in "tagging~ individual CNS pre-;, cursors and their daughter cells, either through in-fection by a markor-bearing retrovirus or by in;ection of 1 25 stable, specific dyes and enzymes, has shown that neurons i,, and non-nouronal cells (i.e., ~lia cells) are often `~ derived from a common preoursor. In retina a common l precursor for neurons and the Mueller glial cell exists :. I .
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very late in de~elopment (Holt, C.E., T.W. Bertsch et al., Neuron 1: 15-26 (1988); Turner, D.L. and C.L. Cepko, Nature 238: 131-136 tl987); Wetts, R. and S.E. Fraser, Science 239: 1142-1145 (1988); a common precursor for 05 different neuronal and glial cell types has also been demonstrated in cortex and tectum (Luskin, M.B., A.L.
Pearl~an et al., Neuron 1: 635-647 (1988); Price, J.
and L. Thurlow, Develo~ment 104: 473-482 (1988); Walsh, C. and C. Cepko, Science 241: 1342-1345 (1988)).
Tissue culture systems have provided additional information about the differentiat$on potential of CNS
stem cells. Single-cell microculture of embryonic day 14 rat forebrain has produced clones containing both neurons and glia (Temple, S. Nature 340: 471-473 (1989)).
Analysis of a later precursor in the glial different-iation pathway has established specific factors influ-, ancing difierentiation to oligodendrocytes or type II
~ astrocytes (Raff, M Science 243: 1450-1455 1989)). Cell ~i lines established by immortalization of CNS seem cells ;~20 also reflect features of the stem cell and can different-iate along both the neuronal and glial pathways (Cepko, C.L. Ann. Rev. Neurosci. 12: 47-65 (1989); Frederiksen, ~K. et al., supra (1988)).
',Antibodies have also proven useful for analyzing ~'25 stem cells. For example, antibodies A2B5 (Raff, M.C.
supra 1989) Dl.l (Levine, J.M., L. Beasley et al., J.
Neurosci 4: 820-831 ~1984), Rat 401 (Hockfield, S. and R.
McKay, J. Neurosci 5. 3310-3328 (1985~, and antisera against the intermediate filament vimentin (Bignami, A., 30 T. Raju et al Dev. Biol 91: 286-295 (1982); Federoff, S.
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~3-` Molecular Bases of Neural Develo~ment (1985); Tapscott, - S.J., G.S. Bennett et Bl., Dev. Biol. 86: 40-54 (1981) bind to ~ntigens enriched in the proliferative zone of the neural tube. However, A2B5 and Dl.l recogni~e 05 glycol~p~d epitopes And are less useful for analysis of gene expression. Vimentin, which Appears transiently in brain de~elopment, is an smbiguous marker largely because of its promiscuous expression in many cultured cells as well as in a variety of developing and differentiated tissues (Traub, N. Intermetiate filaments a review.
Berlin, SpringerVerlag (1985)).
The correlation between Rat 401 and proliferating cells in the developing nervous system has been examined ; in detail. Rat 401 was found to recognize a transient population of embryonic columnar epithelial cells and radial glial cells in many regions of the CNS (Hockfield, ;~; S. and McKay J. Neurosci. 5: 3310-3328 (1985)). The number of Rat 401 positive cells, their proliferative rate, and the developmental kinetics of Rst 401 expres- -sion relative to neuronal differentiation revealed that the immediate precursors to neurons are also Rat 401 positive (~rederiksen ~nd McKay,J. J. Neurosci 8: 1144-,~ 1151 (1988)). Neither neurons nor glia in the adult brain express the epitope recognized by the Rat 401 antibody.
Although "taggingr, tissue culture systems and antibodies have proven useful in analyzing neural pre-cursor cells, there is still an incomplete understanding of the lineage of tumor development. For example, medulloblastoma, a common brain tumor of children, is associated predominantly with the cerebellum and brain "
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W092/1~21 PCT/US92/013~5 b ^~ 7 stem. Medulloblasto~a tumors contain cell types with differentiated charscteristics of neurons, glia and muscle (Rub~nstsin, supra 1985; Coakham H.B., et al., J.
Clin Patkol. 38: 165-173 (1985); Velasco, N.E. et al., OS Surg. Neurol. 23: 177-182 (1985); Hayashi, K. e~ al., Acta Patholo~ica Ja~onlca 37: 85-96 (1987); Cras, P. et al., Acta. Ne~ro~athol 75: 377-384 (1938)).
Because of the presence of muleiple differentiated cell types found in these tumors, Bailey and Cushing (Bailey, P. and H. Cushing, Arch. Neurol. Psychiatry 14:
192-224 (1925) proposed that rather than being multiple colncident tumors, they were derlved from a multi-potential stem cell, which they called a "medulloblast".
, According to this model, a medulloblastoma tumor would result from uncontrolled proliferation and different-iation of the medulloblast. However, the hypothetical medulloblast has not been identified.
Due to the incomplete understanding of brain tumor i development, there is currently no adequate assay for detecting brain tumors at an early stage in their . development. In addition, there is no specific noninvasive method of treating brain tumors. Methods of detecting and treating brain tumors would be very useful.
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, Summary of the Invention The present invention relates to a gene encoding a protein whose expression distinguishes neural multi-~ potential stem cells and brain tumor cells from the more ;, differentiated neural cell types (e.g. neuronal, glial ' and muscle cells) of the mammalian brain.

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W092/l4821 'lUi~v 7 PCT/US92/01375 The present invention relates to the nestln gene, particularly a nestin gene of mammalian origin, and the encoded nestin protein. As described herein, tw~ nestin genes of mammalian origin, the rat nestin gene and the 05 human nestin gene, have been isolated and sequenced. The coding sequence of the rat nestin gene transcript is 5415 bp, which corresponds to an expeceed molecular weight of approximately 200 kD ~or the nestin protein. The relative molecular weight of the nestin protein as - 10 determined by SDS polyacrylamide gels is ~pproximately 240 kD. The predicted amino acid sequence of the rat nestin gene product shows that nestin deflnes a distinct sixth class of intermediate filament protein.
The present inventlon also relates to the human ~5 nestin gene and the encoded human nestin protein The coding sequence of the human nestin gene transcript is 4854 bp. The predicted amino acid sequence is 1618 amino acids in length, and shows significant sequence homology with the rat nestin sequenee.
The present invention further relates to methods of detecting the expression of nestin as a means of diagnosing a predisposition to the development of a brain tumor or the presence of a braln tumor in an adult individual. In one embodiment, DNA present in a sample from the brain of an adult individual is hybridized to a DNA probe whic'n is complementary to all or a portion of the nestin gene. As used herein, the term the nestin gene includes the human nestin gene whose sequence is represented herein, the rat nestin gene whose sequence is al50 represeneed herein and equivalent genes fror other ,' : .
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species, such as those which are substar,tially homologous to the human or the rat gene sequence. Detection of ; hybridization is an indication of a predisposition to the development of a brain tumor or the presence of a brain ; 05 tumor. In another embodiment, cerebral spinal fluid or a serum sample from the brain of Rn ~dult individual can be stained with anti-nestin antibodies. Detection of stained cells is an indication of a predisposition to the development of a brain tumor or the presence of a brain lO tumor. - -., .
Brief Descri~tion of the Drawin~s Figure l is the nucleotide and deduced amino acid sequences of the nes~in transcript derived from the central nervous system of rat embryos. The three intron positions (912, 1038 and llll) are indicated. The canonical poly A addition signal (5924-5929) is under-lined.
:: ' ~ Figure 2 is the nucleotide sequence of human nestin.
,~ Pigure 3 is the deduced amino acid sequence of human ~ 20 nestin.
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DetMiled Descri~tion of the lnventio_ The present invention relates to a gene of mammalian ^ origin encoding a protein whose expression distinguishes neural multipotential stem cells and brain tumor cells from the more differentiated neural cell types (e.g., neuronal, glial and muscle cells of the adult brain).
The present invention more particularly relates to the nestin gene and the encoded proteins and their use in , ': '.
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diagnosing tu~ors of the brain, such as medulloblsstomas, gliablastomas and oligodendroglioma. As described her~in, the hu~an nestin gene and the rat nestin gene have been isolatet snd sequenced. In addition, expres-OS sion of the nestin gene and its structural features aredescribed. The intracellular distribution of the nestin gene product, both in an embryonic rat brRin cell line ~ST15A3 and human brain tissue, including tumor cells, using immunocytochemical and immunohistochemical tech-niques is also described. Finally, nestin expression inthe developing human central nervous system and brain tumor samples is also described herein.
, CLONING AND CHARACTERIZATION OF THE RAT AND HUMAN NESTIN
GENE

The Rat Nestin Gene 1, A cDNA library in the expression vector lambda gtll ~' was construct~d from poly(A)+ RNA from CNS of embryonic ~: day 15 (E15) rat embryos as described in detail in : Example 1. The library was screened with the monoclonal ~0 antibody Rat 401 sfter induction of protein expression and ~mmunopositive plaques were identified. The cDNA
insert from the most immunopositi~e clone, gtll.401.19, was used as probe to isolate additional clones, gtlO.401:16, gtlO.401:18 and gtlO.401:9, in a lambda gtlO
library from rat E15 CNS constructed in parallel with the gtll library. A number of hybridizing clones were . identified and sequenced and together represent 5333 base pair (bp) of ~ranscribed sequence, including 4C4 bp of 3' ' ., :
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W092~1~21 PCT/US92/Ot375 -~lU~b"7 , untranslated sequence and a consensus polyA addition site.
The remaining portion of the nestin gene was ob-tainet from genomic sequences. Genomic Southern blots, 05 even when hybridized at low stringency, revealed a pattern consistent wi~h a single rat gene. A genomic rat library in the vector Charon 4A was screened with the nestin cDNA clones. Hybritizing clones were organized by restriction mapping and the region upstrcam of the cDNA
clone gtlO.401:9 was sequenced.
An open reading frame continues for an additional 600 bp from the 5' end of the most upstream cDNA clone.
This region from the genomlc clone Cha.401:14 was sub-divided into three different fragments with the restric-' 15 tion enzyme BamHl. These three fragments all hybridized to an mRNA of the correct size, 6.2 kb, when used as probes on Northern blots with rat E15 CNS RNA.
;i The exact location of the 5' end was determined by a comb~nat1on of Sl nuclease mapp~ng and primer extension.
-~ 20 A probe (bp -121 to +194 rela~ive to +1 - the sub-~, sequently defined transcription start) was generated overlapping the end of the genomic open reading frame by . polymerase chain reaction between two specific primers.
~ This PCR product protected a 194 bp fragment in Sl :` 25 nuclease analysis. Primer extension from the downstream primer used to generate the PCR fragment als~ produced a 194 bp fragment. The fact that these two experiments gave the same fragment suggests that the transcription start site has been defined. ~-Figure 1 shows the nucleotide sequence of the rat : ~;
:~ nestin gene. The total length of the nestin gene trans-cript is 5945 bp, excluding the poly A tail.
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WO92/1~21 PCTtUS92/01375 ~V'LbY7 There are two potential inltiation codons fi~ positions +127 and +160. Assuming the first methionine is the initiation codon, the coding sequence is 5415 bp. This corresponds to an expected ~oleGular weight of approxi- I
05 mately 200 kD; the relative molecular weight as deter-; mined by migration on SDS polyacrylamide gels is ap-proximately 240 kD (Hockfield and McKay, supra (1985)).
Sequence comparison of cDNA and genomic clones revesls three introns at positions 912, 1038, and 1111 in the transcribed region.
To confirm thst the cloned gene indeed represents the gene encoding the Rat401 antigen, a synthetic peptide from the C-terminal 20 amino acids was made. The extreme ; C-terminus was chosen because it is not included in the ; 15 clone recognized by the original monoclonal antibody, and therefore represents a distinct epitope. A polyclonal antiaeru~ against the peptide was raised in rabbits and identifies a band in Western blots and a pattern in immunostaining experiments which are indistinguishable from that recognized 'oy the ~onoclonal antibody Rat 401.

Structural Features of the Rat Nestin Gene .~ The sequence of the entire transcribed region of ~he rat nestin gene was compared to the Genbank and EMBL
databases. The only significant similarities found are to the five classes of intermediate filaments: acidic , keratins (class I); basic keratins (class II); desmin, `` glial fibrillary acidic protein, peripherin, and vimentin (class III); the neurofilament triplet (class IV); and ~ nuclear lamins (class V) (Steinert and Roop, 1588)).
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WO92/1~21 PCT/US92/01375 , ,; ri b ~ o -The similarity between the nestin gene and the genes of the other five classes of intermediate filaments ranges from 16 to 29 ~ at the amino ~cid level in a 307 amino acid long region starting close to the N-terminus 05 of the nestin gene, corresponding to the conserved alpha-helical rod or ~core" domain of the intermediate filaments. This region of the predicted nestin amino ~cid sequence also contains a repeated hydrophobic heptsd motif characteristic of ~ntermedia~e filaments. The degreH of amino acid similarity is comparable to that found between different classes of intermediate filament, both in degrse and in location, i.e., conserved heptad-conta~ning alpha-helical stretches w~thin the core domain are separated by less well conserved, non-heptad spacers (Stelnert, P.N.ant D.R. Roop, Ann. Rev. Biochem. 57:
593-625 (1988)). The locations of the nestin gene's three introns are not conserved with rQspect to those of ~,~ other cla8ses of intermediate filaments.
In the regions outside the conserved rod do~a~n, no strong similarities to other characterized genes were found. The N-terminAl domain is only ll amino acid residues long, shorter than in other intermediate fila-ments. The large C-terminal domain loosely resembles those of neurofila~ents in that it is highly charged, bears'glutamate-rich regions, and features a repeat, in this case the ll amino acid motif S/P-L-E-E/K-E-X-Q-E-S/L-L-R (underlined residues are strongly conserved).
There are approximately 35 of these repeats in the region , between amino scids 512 and 1050.
;1 30 The deduced amino acid sequence of nestin suggests ~` that it is a member of the intermediate filament protein :;
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: :-' W092~1~2l PCT/US92/01375 family. However, its degree of sequence homology to other intermediate filaQents in the core domain, its dissimilarity in the head and tail domains, and its tifferent splicing pattern suggest that lt defines A new 05 class of intermediate filament protein.

The Human Nestin Gene ~,As described in detail in Example 5, the human nestin ~ene was isolated using low stringency DNA hybri-~;tization of a human genomic bacteriophage lambda library with a rat nestin probe.
Figure 2 shows the nucleotide sequence of the human nestin gene. The total length of the human nestin gene sequence is 4854 bp. Figure 3 shows the deduced amino acid sequence encoded by the human nestin gene which is 1618 amino acid residues in length. The alignment of the deduced amino acid sequences of human nestin with the deduced amino acid sequence of rat nestin showed greater than 75~ sequence similarity between the two sequences, wich greater than 60~ sequence identity.

DEVELOPMENTAL EXPRESSION OF THE RAT AND HUMAN NESTIN GENE
; AND ENCODED PRODUCT
,, De~elo~mental Ex~ression of the Rat Nestin Qene During development, CNS stem cells differentiate i~to neurons and glia on a stereotyped schedule;
different brain regions become post-mitotic at different times. The nestin protein was originally identified by the Rat 401 antibody, which transiently stains radial ' ' ., ~
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WO92/1~21 PCT/US92/01375 ~ 7 -12-glial cells of the neural tube in cross sections of rat embryos (Fredericksen, ~. and R. McKay, J. Ne~rosci 5:
3310-3328 (1988); Hockfield and McKay, supra (1985)).
The distribution of staining in $he neural tube at 05 different developmental stages, combined with quanti-tstive double-label experiments using tr~tiated thymidine autoradiography and ~ACS cell cycle analysis, showed that expression of the Rat 401 antigen was localized to proliferating CNS stem cells during embryogenesls (Frederiksen and McKay, supra (1988)) and U.S. Patent Applications Serial Nos. 180,548; 201,762 and 603,803 herein incorporated by reference. These experiments have beon extended by analyzing the developmentsl expression of the nestin gene.
Northern blots of RNA from E15 rat CNS or from the Rat 401 positive cell line STlSA were probed with nestin ' cDNA inserts, a single 6.2 kb mRNA species was ~-identified. To investigate the temporal profile of nestin RNA expression in the developing CNS, representatives of early (cerebrum) and late (cerebellum) developing regions of the brain were compared.
In cerebrum, nestin expression was found to decline from a maxi~um on day E16, such that only extremely low levels of RNA could be detected at day P9 and P12 and none at all in the adult cerebral cortex, even after long exposures. The 109s of nestin mRNA preciselv parallels the decline of Rat 401 positive stem cells in cerebrum during neurogenesis (Frederiksen and McKay, supra ~1988)). The postnatal cerebellum expresses the nestin gene with a maximum on day P5; no expression can be :
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_ Nestin is not detected by Northern blot analysis in 05 adult tissue. The loss of nsstin expression coincides with terminal differentiation of these early multi-potential cells. There are two important implications of these results. First, nestin ~ppears to be a general marker for the CNS stem cell from the newly closed Ell neural tube through the postnatally developing cerebellum. Second, nestin expression is a consequence of transcriptional regulation of the gene.
The only expression of nestin observed outside developing CNS is traces of nestin messa~e from day P4 developing skeletal muscle. This finding is consistent with previous observations from cross sections of rat embryos, where Rat 401 staining in the developing somites was observed (Hockfield and McKay, supra tl985)).
Developing nervous tissue and developing skeletal muscle may share unexpected kinship with regard to their potential fates.

Intracellular Distribution of the Nestin Gene Product in the STl5A Cell Line ' The associations of nestin with different components `' 25 of the cytoskeleton were analyzed by a series of double-label immunocytochemistry experiments. These experiments were carried out in the immortalized CNS precursor cell . line STl5A which expresses, in addition to components of ;' microtubules and microfilaments, nestin and the inter-. . . ~
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WO92/1~21 PCT/US92/01375 mediate fil~ment vimentin. ST15A produces a nestin mRNA
indistinguishable by Northern blot analysis from that found in E15 CNS.
In the first set of doubls-lsbel experi~ents the 05 intracellular distributlon of the nestin gene product, detected by the monoclonal ~t.401 antibody and an appropriate second antibody, was compared to the distri-bution of actin, tubulin and vimentin. The latter two proteins were detected by polyclonal antibodies with matching second antibodies while the actin pattern in microfilaments was visualized by rhodamine-conjugated phalloidin.
The intracellular distributions of nestin, micro-tubules (tubulin), and microfilaments (actin) are quite different. MLcrotubules are more evenly distributed in the cell with no particular aggregation around the nucl~us, whereas nestin is found in a fiber-like pattern, with a pronouncet perinuclear flccumulation. Ue also observe distinct differences when microfilaments ~nd nestin are stained in the same cell: microfilaments form stra~ght and parallel fibers, in contrast to the gently curved fibers of nestin.
Differences ln nestin and vimentin staining in the same ST15A cells are much less clear. Vimentin staining is stronger than nestin in the cell periphery, and there ;~ are regions where intensity varies between the two proteins, but common features are also evident. The overall organization of the two networks is quite similar, with filamene arrays radiating out from a perinuclear center. Moreover, there are regions in cells where it seems likely that nestin and vimentin colo-calize.

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W092/1~21 PCT/US9210~375 l 5 ~ b ~ 7 In another sst of experiments, microfilaments and microtubules were disrupted by cytochalasin B anid col-chicine, respectively, and the effects on nestin distri-bution analyzet. When STlSA cells were treated with 10 OS ug/ml cytochalasin B for one hour the microfilaments becams highly disorganized, while the overall morphology of the nestin network was preserved. Microtubules also remained intact Aftcr the cytochalasin B treat~ent.
Colchicine treatment (25 ug/ml) for 24 hours caused a collapse of the microtubules. The nestin network could still be identified, although the perinuclear distri-; bution became much more prominent. A similsr pattern was observed for vimentin ~s has been previously reported (Hynes, RØ and A.T.Destres, Cell 13:151 103 (1978):
Monteiro, M.J. and D.W. Cleveland, J. Cell Biol.:108:579-593 (1989). Microfilaments were essentially un-affected by the colchicine treatment. The inferred relationship of nestin to intermediate filaments based on its deduced amino acid sequence is thus supported by the intracellular distribution of the nestin gene product under various conditions.

ST15A Cells have ~eatures Ex~ected of a Neural Precurso_ to Brain Tumors _______________ .
"The prevailing name 'medulloblastoma' given by Bailey and Cushing (1925), is unfortunate because there is no embryonal cell that has been identified as a medulloblast." This sentence begins the discussion of ; medulloblastoma in a prominent text on neural tumors , (Russell, D. and L.J.Rubinstein, Pathologiy of Tumors of :, .
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the Nervous System, Baltimore, Williams and Uilkins (1989)). The following pr~8ents evidence that 8 cell l~ne derived from rat cerebellum, STlSA, h~s festuras expected of a medulloblast.
05 ST15A wag derlved from postnatAl day 4 rat cere-~ bellum. In the rat, as in other mammals, much of cere-bellar development occurs ~ter birth~ so that there are a lArge number of diviting precursox cells present at the time from which this line was der$Yed. ~hus, STlSA is derived from the ~ppropriate tissue and stage of develop-ment. It has previously been ~hown that ST15A cells can differentiate along neuron~l and glial pathways. The following experiments, which are described in det~il in Example 3. show that ST15A cells can also dif~erentiate ~nto muscle. This result su~gests that a sin~le neuro-ectodermal cell can g~ve rise to the different cell types iound in brain tumors.
~ he cerebellar cell line ST15A was one of several cell lines obtained by infecting pri~ary rat cerebellar cells with a recombinant retrovirus carrying SV40 T-antigen. A tempersture 9enSitive allele of T-antigen was used to establish ~hese cell lines. At the permissive temperature (i.e., when the T-antigen protein is active) the cells proliferate and express the intermediate filament protein nestin.
At the non-permissive te~perature the T-anti~en protein is rapidly degraded and the cells differentiate into either neurons or glial cell~ depending on the conditions. The differentiation into neuron~ snd glial ~' 30 cells $s best accomplished by growing ST15A cells in <,:"
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WO 92/14821 ~ J 7 PCltUS92/01375 co-:uleure vieh p~i~ary oerebellar cells. Pareial differentlation has also been observed when ST15A cells are cultured alone; serum-free medium promotes neuronal differentiation and medium containing fetal calf serum OS promotes glial differentiation.
ST15A cells also can differentiate into muscle cells. This fate was first noted when spontaneously contracting cells were observ2d in long term cultures of ST15A at 39C, the non-permissi~e temperature for T-antigen. Horse serum is often used to support thedifferentiation of primary myoblasts. When ST15A cells are grown in horse serum at 39C, they reproducibly differentiate into multi-nucleate cells which express muscle specific proteins, regenerative action potentials and spontaneously contract.
' ST15A cells grown for two days at 33C in 10~ fetal calf serum (FCS) in DMEM grew in a disorganized manner;
in contrast ST15A cells grown at 33C in 104 horse serum at 39C became aligned. The monoclonal antibody Rat 401 recognizes the 220 kD intermediate filament protein nestin. ST15A cells express nestin at both 33C and 39C. A second monoclonal ant~body which recognizes a skeletal and cardiac muscle isotype of troponin T was reacted with the cells (Lin, J. Biol. Chem. 263: 7309 (1988)). Immunofluorescence shows that troponin T
expression is induced when ST15A cells are cultured at 39C in horse serum.
; Immunoblots of proteins extracted from ST15A cells i verify that anti-troponin T antibody recognizes a strong band of 38 Kd, the appropriate molecular weight, in the ,, .
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W092/1~82l PCT/US92/01375 ' lU~b~7 -18-cells grown at 39C in horse serum; at 33C no band is present. The troponin band in STlSA co-migrates with troponin extracted from rat postnatal day 4 muscle. The double band seen in both muscle cells and differentiated 05 ST15A cells may represent the adult and embryonic iso-forms of troponin T which differ by 3,000 daltons (Lin, J. Biol. Chem. 263: 7309 (19B8)). These two isoforms are derived from the same primary transcript by differ-ential splicing. Lanes carrying the same amount of protein were also probed for nestin with the monoclonal antibody Rat 401. Nestin is present at both temperatures but at elevated levels at the higher temperature. The lower bands are proteolytic fragments of nestin which are often present in protein extracts even in the presence of protease inhibitors. Nestin is also present in postnatal rat muscle.
A family of DNA binding factors has been shown to play a critical role in the differentiation of muscle.
These proteins, myoDl, myogenin, myfS and herculin/MRF4, contain DNA binding motifs of the helix-loop-helix type in a region of homology to c-myc (Wright, W.E., et al., Cell. 56: 607 (1989); Miner, J.~. and B.Wold, Proc. Nat.
Acad. Sci. 87: 1089-1093 (l990); Davis, R.L. et al., Cell. Sl: 987-lO00 ~1987); Braun, T et al., EMBO J 8:
: 25 701-709 (1989); Rhodes S.J.and S.F.Konieczny, ~enes and dev. 3: 2050-2061 (1989). The expression of these factors at the mRNA level in STlSA cells as they differ-entiate into muscle was examined.
Northern blot analysis of total RNA from differ-entiating STlSA cells probed with the indicated sequences was performed. Each probe hybridized only to the bands :

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W092/l~2l PCT/US92/01375 'lU4~7 of the expected mobilities. ~yoD1 is e~pressed in passaging ST15A cells at the permissi~e temperature for the T antigen and continues to be expre~sed when the cells are shifted to 39C. Myogenin ~s not expressed 05 under normal growth conditions but is expressed by 2 days after confluent cell~ are shi~ted to 39C in horse serum ~HS); if confluent cells are placed in HS ~nd held at 33 C, the inductlon of myogenin expression is dramatic-ally inhibitet.
Herculin~MRF4 was found in adult skeletal muscle but was not detected in ST15A. Myf5 gave a very weak signal in STl5A cells compared to the myoblast cell line C2C12.
Nestin mRNA accumulates with time at 39C, as does the protein. ~-actin serves as a control for the amount of RNA at 33C and in the short time points at 39 C ; the decrease in ~-actin signal at 3 and 7 days at 39C may be due to the changin~ composition of the cytoskeleton accompanying differentiation, as levels of myoDl mRNA
appear constant throughout the experiment. These results indicate the STl5A cells express myogenic transcription factors and that the induction of myogenin correlates with the differentiation into the muscle fate.
As expected of muscle cells, STl5A cells become j multinucleate as they differentiate. The most striking : 25 similarity to muscle, which initiated this series of ;, experiments, was that STl5A cells grown to confluence in - FCS at 33C and cultured for up to 15 days at 39 in lO~
horse serum can contract spontaneously. The initial action potentials were generated by a series of hyper-,30 polarizing pulses. The final hyper-polarizing pulse was :~followed by a train of spontaneous action potentials.
Spontaneous action potentials were seen after 12 days at 39C in 10% horse serum.

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W092/1~2~ PCT/US92/01375 ~ l VL~ 20-Coincident with the appearance of contracting cells, the resting membrane potential falls snd it becomes easier to stimulate trains of action potentials with hyperpolarizing current pulses. ST15A cells also 05 twitched in response to stimuli which elicited action potentials. These results Qhow that ST15A cells differ-entiate into muscle, expressing myogenic transcription factors, muscle-specific cytoskeletal components and electrical excitability.
An interesting question raised by the properties of ST15A cells is whether primary cerebellar cells can differentiate into the muscle fate. Dissociated cells from postnatal day 5 rat cerebellum were cultured for 6 days under conditions which promote muscle different-15 iation of ST15A cells (39 C in the presence of 10% horse serum) and stained with monoclonal antibcdy against troponin T. These culture conditions promote extensive differentiation of neurons and glia. Troponin T positive ¦ cells with an elongated, multinucleate morphology of muscle cells were also seen, but these cells were very ~ rare, occurring less than one per hundred thousand cells.

; Develo~mental ex~ression of Human Nestin Gene . To characterize human Destin and determine the timing of its induction in the normal human developing ~ 25 CNS, studies were carried out, to examine this class VI
'~ ~F protein in the normal human developing central nervous system (CNS), human brain tumor derived cell lines, and tissue samples of human CNS tumors.
, Human nestin exhibited biochemical and immuno-chemical properties similar to those of rat nestin.
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W09~/l~2l PCT/US92/01375 ~lV,~ ~ !; 7 Fureher, in the hum~n, nestin was detected immuno-histochemically in several different types of immature human CNS cells, i.e. germinal matrix cells, neuro-epithelial cells lining the central canal, radial glia 05 and endothelial cells. Nestin appeared in the~e cells at the earliest gestation~l a&e (l.e., 6 weeks) examined here ~nd then it declined in all but the endothelial cells at later embryonic-stages. Nestin ~lso was detected by immunocytochemlstry in 6 of 7 primitive neuroectodermal tumor cell lines and in both of the malignant glioma cell lines examined. In these cell lines, nestin co-localized incompletely with bundles of IFs containing other IF proteins (i.e. vimentin, glial filament, nuerofilament). Nestin was ubiquitous in a ~i 15 wide variety of brain tumors, but was most prominent in gliom~s. These studies document the existence of a human counterpart of rat nestin.

Distribution of the Nestin Gene Product in the Human ~ Developin~ CNS
; 20 Cervical le~els of six developing human spinal cords with gestational ages (GAs) of 6 to 40 weeks were immuno-:. histochemically stained with anti-nestin 129. The spinal cord of the 6 weeks GA human fetus was co~posed of three distinct layers, i.e. a layer of primiti~e neuro-epithelial cells lining the centrsl canal, a mantle layer and sn outer marginal layer. Anti-nestin 129 stained most cells in the primitive neuroepithelial layer, i.e.
the presumptive miltipotential stem cells that give rise to CNS neurons and glia. In addition, thin elongated radial glial fibers extending from the primitive neuro-. . .~
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WO92/14821 PCTtUS92/01375 ~ ~ U~b~ 7 -22-epithelial layer to the subpial region wers stained by this antiserum. ~hese neuroepithelial stem cells and radial glial fibers also expressed vimentin but not GFAP.
Neuroblasts in the mantle layer (identified by their 05 morphology and NF protein posltively) did not express nestin.
At 11 weeks GA, cells in the ependymal l~uer were positively stained with anti-nestin 129, as were radial ~lial fibers. These radial glial fibers were also stained with the anti-vimentin MAb. Nestin immuno-reactivity decreased at 17 weeks GA and disappeared at 20 weeks GA. From this gestational sge to maturity, GFAP
positive glial cells and NF positive neurons were seen in the spinal cord, but no elements other than endothelial cells were nestin positive.
The telencephalic germinal matrix of two cases with GAs of 17 and 20 weeks were examined with anti-nestin 129. At both time points, many presumptive neuroe-pithelial stem cells in the germinmal matirx cells were nestin positive. These neuroepithelial precursor cells also were labeled by ehe anti-vimentin MAb, but no germinal matrix cells were stained by the MAbs to GFAP or ' NF proteins. At 40 weeks GA, a well defined germinal - matrix was not evident, but clusters of morphologically immature cells (presumably resitual, multipotential precursor cells) were seen in the subependymal region.
Nestin immunoreactivity was recognized in only a few of these cells.
To determine if neuroepithelial precursors in the i 30 cerebellum also expressed nestin, four developing human cerebelli at GAs of 17 to 40 weeks were studied. Late in , , , : .

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:, WO92/148~1 PCTIUS92/01375 i 7 development, the cerebellar cortex has four distinct layers, l.e. the internal and extern~l granular layers, the Purkin~e cell layer and the molecular layer. How-ever, these layers wcre not yet evident at 17 weeks GA
05 and Purkin~e cells were not recognizable by ~orphological ~ criteria. Nevertheless, immature NF positive cells were ;~ observed a~ 17 weeks GA in the superficial internal granular layer, and ~hese cells probably correspond to , nascent Purkin~e cells. Nestin immunoreactivity was lO detected at this time in radial glial cells in the internal granular layer and in radial glial processes that extended to the external granular layer. These radial glial cells, which mature into the Bergmann glia of the adult cerebellum, also were stained by the anti-lS vimentin NAb (data not shown), but not by the anti-GFAP
MAb. However, the anti-GFAP MAb did stain ependymal cells lining the fourth ventricle. At 20 weeks GA, nestin immunoreactivity in radial glia was markedly diminished and by 40 weeks GA, it was weakly present in 20 only a few radial glia fibers, while the blood vessel y endothelial cells at 40 weeks GA were clearly nestin I positive. In contrast, radial glial fibers expressed - both GFAP and vimentin (data not shown) at these time points.

-~ 25 Nesti__E_pressio__i_ Brain Tumor derived Cell Lines Indirect immunofluorescence studies of 9 well ~, characterized human brain tumor derived cell lines showed variable nestin immunoreactivity in 6 or 7 primitive ~ ;
neuroectodermal tumor (PNET) cell lines, and very intense 30 nestin staining in 2 glioma cell lines. Table l .:~
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summarizes the data on the expression of nestin and oth~r IF proteins in these cell lines.

IMMUNOFLUORESCENCE ASSAY OF BRAIN
TUMOR-DERIVED CELL LINES
CELL LINES ~ESTIN VIMENTIN GFAP NF L NF M NF H
Daoy _ +
D283 Med + + - + ~ +
D341 Ned + ~ - - + +
D384 ~ed ~ + - + + +
D425 Med ~ + - ~ ~ +
D458 Med + + ~ +
; CHOP707m + ~ ~ +
U251 MG + + I -~
`~ 15 Summary of the data obtained from the indirect immunofluorescence studies of each cell line. The pAtterDS of stainin~ are described as - - no staining, I - positive staining. NF-L, NF-~ and NF-H are ~he low, ~iddle and high molecular weight neurofilament subun~ts, respectively. The followin~
antibodies w0re used to obtain these data, nestin (rabbit anti-nestin antiserum 129), vimentin (V9) GFAP (2.2B10), NE-L (NR-4, RMS12), NF-M (RMdO20 R~0254, H014), NF-H (DPl, TA51, RN024).
__ _______ Except for CHOP707m, which was deri~ed from a cerebral neuroblastoma (Baker, D.L. et al., Am. Neurol.
28:136 (1990)), the other 6 PNET cell lines were obtsined from cerebellar medulloblastomas (see Hockfield, S. et ~ .
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:,- ' : . , , W092~1~21 PCT/US92/01375 ~21 U 1 ~ ~ 7 al., J. Neuro~ci 5:3310 (l985)) ant citations therein).
Daoy does not exhibit any evidence of glial or n~uron~l differentintion and it was nestin negative, while the other 6 PNET cell lines resembled th&t of embryonic 05 neur~blasts (He, ~. et al., J. Neuro~athol. Ex~. Neurol.
48:48 (1989)). Double immunofluorescent stain~ng oi D283 Med, which i9 the most differentlated PNET cell line, showed incomplete co-localization of nestin and NF
proteins in bundles of IFs in the same cells. Similar observations were made using nestin and vimentin anti-bodies. CHOP707m expressed extensive nestin immunore-activity and double immunofluorescence of nestin and vimentin in this cell line co-localized both proteins in the same cells, but the nes~in positive filament bundles were located mainly in the perinuclear area while:
vimentin positive filamene bundles were present through-out these cells. The Daoy line expresses vimentin, but not NF proteins, GFAP or other molecular markers of neurons or glia (He, X. et al., J. Neuro~athol. Ex~.
:! 20 Neurol. 48:48 (1989)). Hence, Daoy is the least differ-, entiated PNET cell line, and it did not express nestin.
The foregoing co-localization stuties also indicsted that the anti-nestin 129 did not cross react with vimentin or NF proteins.
Since nestin is expressed primar~ly in CNS stem cells and is eliminated during the progressive maturation of the progeny of these stem cells, the Daoy cell line may resemble a CNS precursor that subsequently develops into a vimentin and nestin positive CNS stem cell like :~ 30 those observed here in the human spinal cord at a GA of 6 .~ weeks, Al~ern=tive1y, t==or cell li=es contain genetic ~.

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mutations and their phenotype ~ay not fully re~licate that of normal cells. However, the 2 glioma cell lines (V251 MG and U373 MG) resembled immature spinal cord radial glia (i.e. at ll weeks GA~ since these cells 05 co-expressed nestin, GFAP and vimentin in the triple fluorescence studics.
Finally, Western blo~ studies were performed which showed that anti-nestin 129 labeled a sin~le band in cytoskeletal extrscts of U251 MG cells, and this lmmuno-band had a Mr similar to that of the bacterial TrpE-nestin fusion protein and lower than that of rat nestin expressed in P6 rat cerebellum. The significance of the difference in the Mr of rat and human nestin is unclear and will require further studies. Since these extracts contained vimentin, GFAP and other cytoskeletal p:roteins, these data further demonstrate the specificity of the anti-nestin antiserum for human nestin.
' ', THE EXPRESSION QF NESTIN IN BRAIN TUMQR TISSUE

Brain Tumors Co-t~in-N~sti---ositive-cells STl5A cells express properties expected of the precursor to brain tumor cells as described in Exampie 3.
Therefore, brain tumor cells were tested to determine whether they express fea~ures in common with STl5A, such as expression of nestin. As described in detail in Example 6, tissue from five medulloblastomas was obtained. Three of these samples came from recurrent tumors occurrin~ in a 16 year old male, a 26 year old male and a 5 year old female. The remaining tumors were .. ,', ' .
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-WO 92/14~21 PCI/VS92tO1375 ''lU'it)~'7 newly diagnosed in an 11 year old male and a 10 year old female. The primary tumor in the 11 year old male was also used as a source of cultured cells.
Immunohi6tochemistry on sections of tissue from each S of the five tumors studied showed regions with large numbers cf nestin immunoreactive cells. The express~on of nestin in ~edulloblasto~a tissue was not uniform and in some cases parts of the tumor tissue showed a few nestin-positive cells among nestin-negative cells. The pre-immune serum showed no reaction with either human embryonic or tumor tis~ue. The nestin positive cells were small with a cytoplasmic distribution of the antigen. In one tumor, there were groups of sligned nestin positive cells which were very similar in their morphology to the embryonic neuroepithelium.
A cell line which was generated by growing cells out of the medulloblastoma from the 11 year old male was also ~: tested for expression of nestin. Immunocytochemistry 1 with polyclonal anti-nestin antiserum shows that these cells are uniformly positive for nestin expression and reveals the filamentous pattern characteristic of this . ~ntermediate filament protein. ~estern blotting using , anti-nestin serum shows that the antigen detected in ~,, these cells comigrates with rat nestin.
Sections of nor~al fetal and adult human cerebellum :` were also stained with polyclonal antiserum against nestin. The fetal tissue showed many positive cells in the proliferative, external granular layer of the developing cerebellum and in the outer part of the internal granular layer. In contrast, ~here was no .. ', , , :, ) ,. ~ .

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', . : : ' W092~14821 PCT/US92/Ot3~5 ~ I U~b'~7 -28-inmunoreactivity in cerebellum from a 2.5 year old.
These results are consistent with the stem cell specific expression of nestln seen in developing mouse and rat brain.

05 Utility The adult cell types of the brain express distinct intermediate filament proteins: neurofilaments are expressed by neurons, peripherin by a subset of neurons (Leonard, Gorham, et al., ~l988)), and glial fibrillary acidic protein by astrocytes. The expression of nestin defines an earlier stage in the pathway of intermediate filament gene e~pression. Early embryonic cells are nestin negative but exprcss certain cytokeratins (Jackson, B.W., C. Grund et al., Differentiation 20:
203-216 (1980)). During neurulation neurectodermal cells become nestin positive. Upon terminal different-iation, CNS cells stop producing nestin and express class III or IV intermetiate filaments typical of their differ-entiated cell type.
Nestin positive neurectodermal cells have been shown to be capable of differentisting into neuronal, glial and muscle cells (i.e., the different cell types found in tumors). Nestin expression has also been directly detec~ed in tumor tissue and in a medulloblastoma derived 2S cell line. Nestin positive cells derived from tumors are likely to be renewin& stem cells which ~ive rise to the ; differentiated cells in the tumor.
The detection of nestin expression in brain cells , obtained from an adul~ using known methods is indicative of the presence of a brain tumor or of neural precursor ', '''~
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WO92/1~21 ~ 1U ~ PCT/US92/01375 cells capable of developing into a brain tumor. For the purposes of the subJect invention, an "adult" is an indi~idual whose central nervous sygtem is developed and, therefore, contains differentiated cells. For example, 05 the central nervous system of human neonates is in continual development up until the a~e of about one year.
Therefore, a human adult, for ~he purposes of the subJect invention, is an individual who is older than about one year.
lONestin mRNA expression in a brain tissue sample cBn be detected ~sing a DNA probe; this is indicative of expression of nestin, whlch is, in t~rn a~ l~dication of a predisposition to the develop~ent of a brain tumor or ' of the presence of a brain tumor in the individual. For " 15 example, a sample of brain tissue from of an indi~idual ^, can be hybridized to a DNA probe which is complementary ;¦ to all or a portion of the nestin gene. Detection of ;~;; hybridization is an indication of a predisposition to the development of a brain tumor or the presence of a brain ` 20 tumor.
Alternatively, expression of the nestin protein can ' be detected using polyclonal antibodies, (e.~., anti- .
nest~n antiserum 129) or monoclonal antibodies (e.g., Rat 401). For example, cerebral spinal fluid or a serum 25 sample from the brain of an individual can be stained ~, with anti-nestin antibodies. Detection of stained cells is an indication of a predisposition to the development :'~ of a brain tumor or the presence of a brain tumor.
;, The findin~ that neural precursors to tumors express 30 nestin also has important therapeutic implications. For example, the nestin marker enables further investigation :.
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WO92/1~21 PCT/US92/01375 ~ b~7 -30 into the proliferation and differentiation of the stem cells of a brain tumor, so that existing chemotherapies can be lmplemented at an earlier stage in the tumor development and 50 that new methods of inhibiting tumor 05 development can be developed.
The present lnvention will now be illustrated by the following examples, which are not to be seen as limiting in any way.

EXEMPLI~ICATION

EXAMPLE 1 Clonin~ and Characterization of the Rat Nestin Gene Construction and Screenin~ of Libraries ~ gtlO and ~gtll cDNA libraries were constructed from oligo-d~-primed poly(A)+ RNA from embryonic day 15 dissected rat CNS according to procedures which were slightly modified from those described (Young, R.A, and R.W.Davis, Proc. Natl. Acad. Sci. USA. 80: 1194-1198 ~1983)). The second strand cDNA was synthesized using the RNase H modification of the Okayama-Berg method 20 (Gubler, U. and R. Hoffman, Gene 25: 263-269 (1983)).
After induced protein expression, the ~gtll library was screened with the monoclonal antibody Rat 401 (undiluted hybridoma supernatant) and positive clones were visual-ized with an anti-mouse second antibody con~ugated to alkaline phosphatase (Promega Protoblot). Immunopositive clones were selected and the cDNA insert from the clone giving the strongest hybridization signal, ~gt~1.401:16, ..

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W092/1~21 PCT/US92/01375 J l ~ 7 was 2P-labelled tFeinberg, A.P.and B. Vogelstein, Anal.
Bioche~. 137: 266-267 (1983) and used as probe to screen the ~ gtlO library (Benton U.D.and R.W. Davis, Science.
196: 180-182 (1977); Thomas, P.S. Proc. Natl. Acad.
05 Sci__USA. 77: 5201-5~05 (1980)). Several clones were identified. A commercial rat genomic DNA library (Clontech) in the vector Charon 4A w~s screened with 2P-labelled nestin cDNA inserts as sbove. A number of hybridizing clones were obtained, and a genomic map comprising 20 kb of the nestin gene region was established by restriction mapping of the genomic clones, cross-hybridizations, and sequencing of selected genomic ; subclones.

DNA Sequencin~ and Com~uter Qnalysis cDNA inserts and genomic subclones were sequenced after subcloning into M13mpl8, M13mpl9, or Bluescript KS+
(Stratagene). M13 phage particles and Bluescript were grown and circular DNA prepared according to standard procedures, Both single stranded (M13) and double stranded (Bluescript) sequencing was performed using a modified T7 DNA polymerase (Sequenase, USB), as suggested ! by the manufacturer. Approximately 90~ of the gene, including all exons and cDNA inserts, was sequenced on both strands. Ambiguous regions were further resolved ` 25 using deoxyinosine in the standard Sequenase protocol.
~- Computer analysis was performed using the Vniversity of Wisconsin Genetics Computer Group program package, including the FASTP and TFASTA algorithms (Devereu~, , Haeberli ee al., 1984)). The complete sequence, :l 30 including introns, will be stored in the EMBL Database.
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Isolation of RNA and Northern Blott~n~
Tissue was rapidly dissected and quickly fsozen in liquid nitrogen. After homogenization in a quanidinium thiocyanate containing buf~er, total RNA was lsolated by 05 centrifugation through a CsCl cushion (Chirgwin, Przybyla et al., 1979)). Poly(A)+ RNA was isolated by an oligo-dT
chromatography column ~Aviv and Leder, 1972) or by a poly(A)+ RNA extraction kit (Fast Trak, Invitrogen). For Northern blot anslysis RNA was denatured and electro-phoresed in 14 agarose gels containing 2.2 M formaldehydein lX MOPS buffer (Maniatis, Fritsch et al., 1982) at ~4-C for 8 hours. The RNA was then transferred to nitrocellulose filters (GeneScreen Plus, New England Nuclear) (Thomas, 1980)). The filters were prehybridized and hybridized (50~ formamide, 0.75M NaCl, 100 mM Tris pH
7.8, 5 mM EDTA, 1% sodium dodecyl sulphate at 42C for 16 hours) with 2 x 106 cpm/ml of the 32P-labeled nestin cDNA
insert from ~ gtlO.401.16 (specific activity 3 x 108 cpm~ug). After hybridization, the filters were washed (final wash - 2 x 30 minutes at 65C in 30 mM NaCl, 4 mM
Tris pH 7.8, 0.2 mM EDTA and 0.25% SDS) and exposed to x-ray film with intensifying screens. Quantity and integrity of the RNA were monitored by rehybridizing the blots with a cDNA probe from the human beta-tublin gene under identic~l conditions. The size of the hybridizing RNA was determined by running RNA molecules of known size (HMW RNA ladder, BRL) in parallel.
~:' Sl Nuclease Ma~in~_and Prlmer Extension To generate a probe for Sl nuclease protection 30 experiments, we amplified a region of a genomic clone ' :

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; between two synthetic primers using the polymerase chain reaction (kit from Perkin-Elmer Cetus). The resulting fragment was 315 bp lon3 and located between bp -121 and +194 (bp +l indicates the subsequently defined initiation 05 of transcription). After 32p end-labelling, 300 fmole of : the fragment was hybridized to S0 ug of El5 CNS rat total RNA or 50 ug of yeast total RNA according to standard procedures, and the Sl resistant hybrids separated on ; denaturing polyacrylamite gels (Manistis, T., E.F.
Fritsch et al., Molecular clonin~ A laboratorY manual (Cold S~rin~ Harbor, New York- (1982)). 32p end-labeled restriction digested plasmids and sequencing reactions of known DNAs were used as size standards. The dried gels were exposed to x-ray film. Primer extensions were . 15 performed by hybridizing 125 fmole of one of the oligo-, nucleotides used in the polymerase chain reaction (pos ,1 +170 - +194) to lO ug of poly(A)+ RNA from rat El5 CNS
according to standard procedures. The reaction product was sub;ected to gel electrophoresis as in the Sl ~s 20 nuclease experiments.
., .
Immunocytochemistry For all experiments cells from the immortalized cell line STl5A were ~rown for 2 days (to 404 confluence) in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum on sterile, polyornithine coated glass cover slips, as described in U.S. Patent Applicaticns Serial Nos. 07/603,803; 07/201,762 and 180,548. All antibody incubations lasted 60 minutes at room eemper-aeure and were followe~ by ~hree rinses in PbS (0.14M) . , ~ .
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: ~ .: , WO92/1~21 PCT/US92/01375 ' lU~b~7 34 NaCl, 2.7 m~ KCl, 8 mM Na2HP04 and 8 mM KH2P04 pH 7.2).
In the double-lable experiments with nestin and tubulin antibodies the STl5A cells were briefly rinsed ~n Pss~
preso~ked ~n microtubule stabilizing buffer tMTSB - 2M
05 glycerol, 0.1 M PIPES pH 6.9, 1 mM MgS04, 2 mM EDTA) for 2 minutes and then fixed for 5 minutes in 4~ para-formsldehyde, 0.5% NP-40 iD NTSB for 5 minutes. After two brief rinsos in PBS and 5 minutes incubation in blocking solution (0.754 Triton X-100, 500 ug/ml bovine serum albumin, 1% horse seru~ and 0.05~ sodium azide), ; the cells were incubated with a polyclonal rabbit-anti-tubulin antiserum (kind gift of Dr. Frank Solomon) diluted 1:10000 in PBS. This was followed by an incu-bation with a fluorescein-conjugsted goat-anti-rabbit IgG
15 antiserum (Cappel, No. 1612-0081) diluted 1:100 in PBS.
The cells were then postfixed in 95~ ethanol/5% acetic acid for 5 seconds and rinsed three times in PBS. This was followed by an incubation with undiluted hybridoma supernatant from the monoclonal mouse-anti-nestin anti-body (Rat 401). Finally, the cells were incubated with a - rhodamine-con~ugated polyclonal goat-anti-mouse IgG
antibody (1:100 dilution, Cappel No. 2611-0231). After two brief rinses in water, the cells were covered with Immumount (Shandon? and invert~d onto clean glass micro-scope slides. As control experiments for potential cross-hybridization and spillover between the fluorescein and rhodamine spectra in the fluorescence microscope, the first antibodies (rabbit-anti-tubulin and mouse-anti-nestin) were omitted in two separate experiments under otherwise identical conditions. No cross-hybridization , . .- I .
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or significant spillover was observed. In the colchicine experiments the Stl5A cells were grown as above and treated with colchicine at 25 ug/ml for 24 hours followed by fixation and staining as described here.
05 In the actin/nestin double-label experiments STlSA
cells were ~rown ~s gbove and fixed in 4~ parafor~alde-hyde, 0.14 Triton X-100 in PBS for 5 minutes. After blocking as described above, the cells were incubated with 0.16 uMi rhodamine-conjugated phalloidin, as suggestQd by the manufacturer (Molecular Probes).
Rinses, postfixation and staining with the nestin mono-clonal antibody were performed as described above, with the sxception that a fluorescein-con~ugated sheep-anti-mouse IgG second antibody (diluted 1:00, Cappel No.
1606-3152) was used. No cross-hybridization or spillover was observed after omitting the rhodamin-conjugated - phalloidin or the nestin antibody. In the cytoch~lasin B
. , .
experinents the ST15A cells were treated with cyto-! chalasin B at a final concentration of 10 ug/ml for 60 i 20 minutes, otherwise all im~unostaining procedures were identical.
1 In the ~imentin/nestin double-label experiments .,! ST15A cells were fixed in 4~ paraformaldehyde, 0.1~
Triton X-100 in PBS for S minutes. After blocking, the cells were incubated with a polyclonal goat-anti-vimentin antiserum (dilution 1:20, MILES 65-794) followed by a ' fluorescein-conju~ated rabbit-anti-goat IgG second ~ antibody (dilution 1:100, ICN 65-176). Incubation times i and rinsing procedures were as described above. Post-fixation and incubation with the anti-nestin antibody . .
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WO92/1~21 PCTtUS92~0137S

~ l U ~ 7 were performed as in the tubulin/nestin experiments. No cross-hybridization or spillover was detected after omitt~ng the anti-vimentin and anti-nestin sntibodies, respectively. In all experiments the mounted cells were 05 photographed on Kodak Ectachrome 400 ASA using a 63X
object~ve on a Zeiss fluorescence microscope.

EXAMPLE 2 Production of the Rat 401 Monoclonal Antibody Developing monoclonsl antibodies were generated to fixed spinal cord from embryonic day 15 (El5) rats.
Timed pregnant female rats were obtained from Taconic Animal Supply Co. and housed until the appropriate gestation age. Uteri were dissected from pregnant ; animals into ice-cold phosphate buffer ~pH 7.4), indi-vidual enbryos were removed and the spinal cord was dissected free of other tissue into fixative (4~ para-formaldehyde in O.l M phosphate buffer).
The immunizat$on and fusion protocol has been described previously (McKay and Hockfield, Proc. Natl.
Acad. Sci. USA 79: 6747-6751 (1982)). Briefly, BALB/c mice received two intraperltoneal immunizat~ons with fixed tissue homogenized in saline and suspended in an equal volume of Freud's complete adjuvant and a final intravenous boost of unfixed tissue with adjuvant.
Spleen cells from immunized mice were fused with NSl ;.'J~ 25 myeloma cells and resulting hybrid cell lines were screened immunohistochemically on l00 ~m-thick Vibratome :
sections of 4% paraformaldehyde-fixed El5 rats. Lines ~, producing antibodies of interest were cloned in soft agar or by limiting dilution and antibody subclass was deter-mined by the Ouchteriony method (Ouchteriony and Nilsson, ' :, ' .
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WO 92/1~1 PCT/US92~01375 ~ i u i ~ ~ 7 Immunodiffusion and Im~unoelectrophorssis. In Handbook of Ex~erimental Immunolo~y D.M. Weir, ed., Blackwell ;~ Scientific Publications, Oxford (1978).
. _ ~- Immunohistochemistry 05 Tissue for immunohistochemistry was fixed either by intravascular perfusion or by immersion. All postnatal material was fixed by intravascular perfusion (under ether anesthesia) with 44 paraformaldehyde at pH 7.4 for light microscopy or with either 4% paraformaldehyde at pH
10.0 or 4% paraformaldehyde, 0.1% glutaraldehyde at pH
7.4 for electron microscopy. The CNS was dissected out and stored in 0.1 m phosphate buffer (pH 7.4) with 0.1%
~i sodium azide. Por prenatal material, pregnant females were ether anesthetized, uteri were dissected into cold ` 15 0.1 m phosphate buffer and the females were killed by cervical dislocation. Embryos were dissected indi-:~ vidually into Sylgard-coated Petri dishes and p~nned in place. Intravascular perfusion was performed by opening the skin of the thoracic cavity, nicking the right atrium with a fine forceps and placing a 25 gauge needle into , the left ventricle. One to 5 ml of fixative (as above) :l were in;ected into the heart. The embryos were placed in a vial containing the same fixa~ive for 4 hr and then ~; stored in phosphate buffer. E12 to term embryos were fixed by perfusion (using a 30 gauge needle for E12 embryos); E10 and Ell embryos were fixed by immersion in fixative. E13 and older animals were sectioned at 50 to ~ 100 ~m on a Vibratome without additional support. E10 to j E12 embryos were embedded in 15~ gelatin before seoeioni=g.

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wo 92/14821 PCr/lJS92~01375 f~ l U ~ 3 8 -Immunohistochemic~l resctions were performed on free-floating sections by sequential incubstions in monoclonal ant~body as full-strength supern~tant (12 to 20 hr); horseradish peroxidase (HRP)-conjugated goat 05 anti-mouse antibody at a dilution of l:100 in tissue culture medium with 10~ serum; and 3.3'-diaminobenzidine (DAB; 0.025~) with H202 (0.002~). Peroxidase-conjugated lectins (Sigma Chemical Co.) were used at serial dilutions in the same manner with the omission of the peroxidase-conjugated goat anti-mouse antibody. For light microscopy, 2~ Triton X-100 was included in both antibody solutions. For electron microscopy sections carried through this procsdure (without Triton X-100) were post-fixed in 2% 24 embedded in Epon/Araldite between plastic coverslips, and cut at l ~m and 0.12 ~m for correlative light and electron microscopy. For high resolution light microscopy, sections (Triton-treated) ' were embedded in plastic as for electron microscopy and sectioned at 2 ~m.
.
I_mu_oblots The apparent molecular weights of antigens recog-. nized by antibodies were determined using the method of ; Towbin et al., Proc. Natl Acad. Sci. USA 76: 4350-4354 (1979)). Proteins were extracted from unfixed tissue with sodium dodecyl sulfate (SDS) and ~-mercaptoethanol and run on an SDS-polyacrylamide gel. The proteins were . electroblotted onto nitrocellulose paper which was then exposed to primary (l hr) and secondary (l hr) anti-bodies. hRP w}Y vis~alLYed with 4-chloronaphthol.

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EXAMPLE 3 Chacterization of Rat Nestin from the ST15A Cell Line ST15A cells were derived from r~t postnatal day 2 cerebellum after iniection of a primary culture with a 05 retroviruS transducing the temperature sensitive, tsA58 allele of SV40 T antigen (Frederiksen et ~1., 1988~).
ST15A cells were plated on tissue cultur¢ plastic coated with polyornithine ~nd psssaged at the permissive te~per-ature, 33C, in Dulbecco's modlfied Eagle's medium (DNE) supplemented with 2 m~ L-glutamine, l mM sodium pyruvate and 15 mM HEPES, pH 7.2, containing lO~ fetal calf serum (FCS). For the experiment in Figure 8, confluent cells (labelled 'day 0') were transferred into DME/lO~ horse serum (HS) and cultured at 33C or 39C as marked for the indicated time. 39C is the non-permissive temperature ~-j for tsA58.
Primary cultures were prepared from P4 rat cere-~- bellum as previously described (McKay, R.D.G. et al., _ ld SPrin~ Ha_bor Symp. ~uant. Biol. (l990). Briefly, tissue was dissociated by trypsinization followed by ~, trituration. Cells were plated on polyornithine-coated coverslips in DME/lO~ FCS/10% HS and grown at 39 C for 24 ~J hours. The medium was changed to DNE/104 HS and culture continued for 6 days.
The medulloblastoma cell line was deri~ed by growing ~ cells from dispersed, fresh medulloblastoma tissue. The -~ tissue was remo~ed from a previously untreated tumor in a lO year old female. The cell line has not been cloned.
Cells were passaged in Ham's Fl2 supplemented with 20mm L-glutamine ~nd 10~ FCS.

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~ 97 -40-Im_unoCytoche-istrx Cells for immunocytochemistry were fixed in 4~
paraformaldehyde buffered with 50 mM sodium phosph~te, pH
7.2, for troponin st~ining or buffered with 50mM sodium 05 borate, pH 9.5, for nestin staining. The antibodies used were mouse monoclonal Rat 401 (Hockfield and McKay, supra (1985), mouse monoclo~al antibody agalnst erOpOnin T
(Sigma T-6277), and r~bbit polyclonal serum raised against the C-termlnal region of nestin expressed in bacteria. Fixed cells were lncubated in primary antibody for one hour, rinsed 3 times in PBS, incubated for one hour with flourescein-con~ugated goat anti-rabbit serum (Organon-Tetnika) and mounted in Immumount (Shandon, Inc.) with 2~ DABC0. Medulloblastoma tissue and normal human cerebellum were frozen and subsequently fixèd in formalin. 50 um sections were cut and stained with rabbit polyclonal serum raised against the C-terminal region of nestin.

Isolation of_RNA and Northern blottin Cultured cells were scraped into 5~ guanidine thiocyanate/25 mM citrate, pH 6.8/ 0.54 N-lauryl sarcosine/0.l ~-mercaptoethanol and RNA pelleted through a CsCl step gradient (Chir~win et al., 1979). 20 ug of total RNA was electrophoresed through a 1% agarose/2.2M
formaldehyde gel and transferred to nitrocel'ulose.
Prehybridization and hybridization were in 50%
formamide/SxSSC/5X Denhardt's/0.l% SDS/0.l mg/ml salmon sperm DNA at 42C. Filters were washed in 2xSSC/0.l~ SDS
. at room temperature and in 0.lxSSC/0.1% SDS at 55 C and -:
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WO92J1~21 ~lU~ 7 PCT/US92/~1375 exposed to X-ray film with an intensifying screen.
Filters were stripped by washing 6 times for 3 minutes each in boiling 0.05xSSC/0.1~ SDS/20 mM EDTA, pH8, and rehybridized. Probes were prepared for hybridization by 05 rsndom priming on fragments isolated on low melting temperature agarose gels (Felnberg and Vogelstein, supra (1984). The following plasmids were used to generate probes: mouse myoDl cDNA pEMClls (Davis, R.L. et al., Cell_ 51: 987-1000 (1987); mouse myogenin cDNA p65 10 (Wright et al., supra (1989); rat nestin cDNA p401-16;
humAn B-act~n cDNA (Gunning, P. et al., Mol. Cell. Biol.

3: 787-795 (1983)).

Western Blottin~
` Proteins were extracted irom cultured cells or rat postnatal day 5 limb muscle (Sprague-Dawley; Taconic, Inc.) in the presence of aprotinin and PMSF in 2% SDS, 5 mM EDTA and 15% glycerol in 0.1 M Tris, pH 6.8. 50 ug of total protein was run in each lane of an SDS reducing gel according to Laemmli (Laemmli 1970); proteins were .; 20 transferred to Immobilon P membranes (Millipore) using a Hoeffer transfer appartus at 50 volts for 4 hours.
Filters were blocked in 5% BLOTTO (Johnson, D.A. et al., Gene Anal. Tech. 1: 3-8 (1984)) overnight. Rat 401 hybridoma supernatant was used undiluted; troponin T
antibody was diluted 1:500; polyclonal anti-nestin antiserum was diluted 1:1000; HRP conjugated secondary antibodies were purchased from BioRad.
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W092/1~21 PCT~USg2/01375 ~ 42-EXAMPLE 4 Characterization of Human Nesein _________ ________ _______________________ To characterize human nestin, a new antiserum, designated anti-nestin 129, was raised to recombinant rat nestin expressed in E. coli since the rat-401 m~b failed 05 to recognize human nestin in ~mmunohistochemical and immunochemical assays.

Production of Anti nestin Antiserum i The insert from the clone ~gtlO 401:16 (Lendahl, U.
et al., Cell 60: 585 (1990)) was isolated and li~ated into the pATHl vector (Tzagaloff, A. et al., J. Biol.
Chem. 262: 17163 (1986)), producing a clone in which the bacterial TrpE protein (37.2 kD) was fused at its C
terminus with the last 1197 amino acids of nestin. The fusion protein was induced in E. coli strain HB101 with ` 1~ 20 ~g/ml indoleacrylic ncid (Sigma I1625) for 4 hours as described in (T~pscott, S. et al., Science 242: 405 ~, (1988)). The TrpE-nestin fusion protein migrates at ~reater than 200 kD in 64 SDS PAGE gels, making it the larsest protein in the bacterial lysate. The fusion ' 20 protein was purified by cutting the top band from the gel. Anti-nestin antiserum 129 was produced by in;ecting : the fusion protein subcutaneously into a female NZW
rabbit. The characteriz~tion of anti-nestin 129 and its ~ specificity for human nestin is described here.
; 25 First, this antiserum was compared to the Rat-401 MAb in ~mmunochemical and immunohistochemical studies of : postnatal day 6 (P6) rat cerebellum. Then, anti-nestin 129 was used to probe human CNS tissues, tumors and tumor derived cell lines. To determine if nestin co-localized ;j 30 with other IF proteins, double and triple immuno-.,`

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W092/l~82i PCT/US92/01375 ~ 1 u~ ~ y 7 fluorescence experimcnts 81so were performed using anti-nestin 129 and antibodies to vimentin, glial fibrillsry acidic protein (G~AP) snd neurofila~ent (NF) triplet proteins. Finally, Western blots were performed 05 on cytosksletal ex~racts of gome of the tumor derived cell lines to identify human nestin and to compare it with rat nestin as well as with the cl~ss III and IV IF
proteins expressed by CNS cells.

Specificity of Anti Nestin 129_i__t_e__at The rabbit anti-nestin antiseru~ produced as des-cribed herein, i.e. anit-nestin 129, yielded results itentical to those produced by the Rat-401 MAb whe~ both were us~d to probe P6 and adult rat CNS tissues by immunoblots and immunohistochemistry. On immunoblots of cytoskeletal extracts from P6 rat cerebellum separated by SDS-PAGE, anti-nestin 129 identified a band with an apparent Mr of 240 kD. This band was identical to the band labeled by Rat-401. Further, both antibodies labeled similar bands in lysates of the induced bacteria producing the TrpE-nestin fusion protein.
In immunohistochemical studies of the P6 rat cere-bellum, anti-nestin 129 labelled radial gli21 cells very intens$vely, The processes of these cells radiated from the ~nternal granular layer to the external granular layer. These radial glia also were labeled with Rat-401 and the anti-vi~entin MAb. Neither of the anti-nestin antibodies stained immature Purkinje cells or other neurons. In contrast to the P6 rat cerebellum, the anti-nestin 129 and Rat-401 antibodies only stained blood 30 vessel endothelial cells in the adult rat cerebellu~, :`

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W092/l482l PCT/US92/01375 -~l~L~ 44-while the anti-vimentin MAb (V9) labelled blood vessels, whi~e matter astrocytes and radlal glial fibers.
Furthermore, nestin immunoreactivity was demonstrated by both anti-nestin antibodies in immature skeletal muscle 05 of the P6 rat as well as in Schwann cells of ~dult rats (data not shown).
These studies dcmonstrate that this new anti-nestin antiserum yields immunohistochemical results in the human developing spinal cord, cerebru~ and cerebellum that were nearly identical to those produced by Rat-401 and the anti-nestin antiserum in the rat.

EXANPLE 5 Cloni ~ of the Human Nestin Gene The human nestin gene was isolated using low-stringency DNA hybridization of a human genomic bacterio-phage lambda library with a rat nestin probe.
A genomic library in the vector EMBL 3 made frompartially Sau3A-digested human genomic DNA (Clontech) was screened using a P-labelled probe derived from the rat nestin gene starting at 202 nucleotides upstream of the ` 20 translation start and ending at nucleotide 483 after the translation start (Lendahl, et al., 1990) under the following conditions: prehybridization in 6X SSC, 5X
Denbardt's solution, 0.5~ SDS and 100 micrograms/ml salmon sperm DNA at 65C for 1 hour; hybridization in the ' 25 same conditions except for using 50 micrograms/ml salmon sper~ DNA and hybridizing for 16 hours. The lilters were then washed for 2 X 30 minutes at 48C in 2X SSC and 0.25~ SDS.
One positive clone out of 1.5 X 106 placques ~0 screened contained the entire human nestin gene and was ~'', '~' : .~
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restriction map was established and EcoRI fragments were subcloned in the plasmid vector Bluescript KS I
05 (Stratagene).

EXAMPLE 6 Nestin Ex~ression in Brain Tu~or Sam~les Immunoh'stochemlcal data on the expression of nestin, other IF proteins and synaptophysin (SYP) by a diverse group of 34 CNS tumors are summarized in Table 2.

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WO 92tl4821 PCI`/US92/01375 ~lU'~7 Le~end to Table 2 Summary of the data obtained from the immun-operoxidsse studles of each of the brain tumor samples.
Results from frozen (~) and paraffin (P) material are combined in the cases in which both kinds of material were studied. The patterns of staining are described as - - no staining, + - positive in less than 5~ of tumor cells, +~ - positive in between 5 - 50~ of tumor cel].s, 1l+ - positive in between 50 - 95~ of tumor cells ++++ -positive in more than ~54 of tumor cells, E - positive cells equivocal, i.e. reactive astrocytes of tumor cells P - interstitial filamentous staining in the neuropil.
The following antibodies were used to obtain these data;
nestin (rabbit ~nti-nestin antiserum 129), vimentin (V9) GFAP (2.2B10), NF (RMS12, RMdO20, RM0254, H014, DPl, TA51, R~024). NA - not available due to limited material; PNET+N,G - PNET with neuronal and glial differ-entation; PNET+N - PNET with neuronal differentiation;
PNET + G - PNET with glial differentiation; PNET-NOS -PNE~, not otherwise specified. areccur~nt tumor of case 7, autopsy case, beneighn astrocytoma genigh~
strocytoma ~ angioma, anaplastic astrocytoma glih-blastoma multiforme, igmixet ependymo-astrocytoma, ana- 7 plastic enpendymoma, choroid plexus carcinoma.
--_______ . 25 The results from frozen and ethanol-fixed paraffin-embedded samples from the same biopsy are combined in the 10 cases from which these paired samples were a~ailable.
.;
Nctably, the immunohistochemical results obtained from each of these paired samples were identical, although the primary antibodies were used at slightly higher dilutions '7 in the frozen material compared with the paraffin ;~ material.
PNETs The anti-nestin antiserum stained tumor cells , in 12 of the 15 PNETS examined here. Nestin immunore-activiSy was found in the cytoplasm of individual neo-I plastic cells. Reactivity was also observed in the . coarse processes of large stellate cells, as well as in ! .

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WO 92/1~1 PCT/US92/01375 blood vessel endothelial cells. In the PNETs with an insular architecture, nestin reactive cells were found both within and outside the islands. Since PNETs express SYP and other neuroendocrine msrkers as well as all 05 classes of IF proteins (Gould, V.E. et al., Human Pathol.
21:2~5 (1990))~ the PNETs were probed for SYP. These studies demonstr~ted SYP in 14 of 15 PNETs. Vimentin was detected in all the PNETs. Ten of the 15 PNETs expressed GFAP and 10 expressed 1 or more NF proteins. Double immunofluorescence studies of nestin and NF proteins, as well as nestin and GFAP on frozen sections showed their co-localization in the same cells (data not shown). The immunohistochemical date on GFAP and NF proteins suggested that 15 PNETs could be classified into 4 subtypes, i.e. 3 cases of PNET-NOS (not otherwise specified), 2 cases of PNET+G (PNET with glial tiffer-entiation), 2 cases of PNET+N (PNET with neuronal differ-entiation) and 8 cases of PNET+G,N (PNET with glial and neuronal differentiation). All 8 cases of PNET+G,N
showed nestin immunoreactivity and 2 cases of PNET+G
expressed nestin. On the other hand, 2 cases of PNET+N
were negative for nestin. These results may indicate thàt PNETs with neuronal differentiatioD tend to lack nestin expression in contrast to PNETs with glial differ-: 25 entiation. This phenomenon paralleled normal differ-entiation in that postmitotic neuroblasts did not express ` nestin whereas radial glial cells did, even at relatively `; late developmental stages. Two of 3 cases of PNET-NOS
showed nestin positive tumor cells. This immunohisto-: 30 chemical phenotype might represent primitive neuro-; epithelial cells or immature radial glial cells in the : .
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WO9~/1~21 PCT/US92J01375 ~1~4~7 developing CNS. The other PNET-NOS case was negative for nestin and i e might correspond to extremely immature CNS
precursor cells Just like the most undifferentiated PNET
cell line (i.e~ Daoy).
05 Astrocyt~c tumors Five of 7 astrocytic tumors .
dlsplayed pcsitivity for nestin in the perinuclear cytoplasm of the tumor cells as well as in the cyto-plasmic processes of these cells. Additionally blood vessel walls were nestin positive. Two of 4 benign astrocytomas were negative for nestin. One anaplastic - astrocytoma and 2 glioblastomas showed strong nestin positivity. In the glioblastomas, mul~inuclear giant - cells were stained strongly.
E~endymomas Six tumors were histologically diagnosed as ependymomas and all showed nestin positivity. Immunoreactivity for nestin was found in the tumer cells which formed perlvascular pseudo-rosetts.
One ependymoma (with marked nuclear pleomorphism necrosis and a few mitoses) was diagnosed as an ana-plastic ependymoma and it demonstrated the strongestnestin im~unoreactivity among the 6 ependymomas.
Expression of nestin described here indicates that nestin is the dominant IF protein of early CNS precursor cells.
However, nestin is rapidly extinguished in neurons, glia and other CNS cell types derived irom these precursors.
he mechanisms responsible for tbese rapid changes in IF
protein expression are unknown.
In contrast to the restricted expression of nestin in normal developing CNS cells, this IF protein was ~ ¦
30 ubiquitously present in a wide variety of CNS neoplasms.
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Claims (20)

1. Isolated DNA encoding a protein of mammalian origin whose expression distinguishes neural multipotential seem cells from neuronal, glial and muscle cells.

2. Isolated DNA of Claim 1 wherein the protein is nestin.

3. Isolated DNA of Claim 2 having the nucleotide sequence of all or a portion of the nucleotide sequence of Figure 1 or Figure 2.

4. Isolated DNA having a nucleotide sequence corres-ponding to all or a portion of the nucleotide sequence of Figure 1.

5. Isolated DNA having a nucleotide sequence corres-ponding to all or a portion of the nucleotide sequence of Figure 2.

6. Isolated DNA which hybridizes to all or a portion of the DNA sequence represented in Figure 1.

7. Isolated DNA which hybridizes to all or a portion of the DNA sequence represented in Figure 2.

8. Isolated DNA having a nucleotide sequence corres-ponding to all or a portion of the nucleotide sequence of Figure 1 which encodes a protein of mammalian origin whose expression distinguishes neural multipotential stem cells from neuronal, glial and muscle cells.

9. Isolated DNA having a nucleotide sequence corres-ponding to all or a portion of the nucleotide sequence of Figure 2 which encodes a protein of mammalian origin whose expression distinguishes neural multipotential stem cells from neuronal, glial and muscle cells.

10. A DNA probe for the detection of a brain tumor, the probe comprising DNA which hybridizes to all or a portion of the DNA sequence represented in Figure 1 or to all or a portion of a DNA sequence sufficient-ly homologous to the DNA sequence of Figure 1 that it encodes a protein which is indicative of a predisposition to the development of a brain tumor in an individual.

11. A DNA probe for the detection of a brain tumor, the probe comprising DNA which hybridizes to all or a portion of the DNA sequence represented in Figure 2 or to all or a portion of a DNA sequence sufficient-ly homologous to the DNA sequence of Figure 2 that it encodes a protein which is indicative of a predisposition to the development of a brain tumor in an individual.

12. A method of diagnosing a predisposition to the development of a brain tumor or the presence of a brain tumor in an individual, comprising:
a) obtaining a sample from the brain of an individual;
b) treating the sample in a manner that renders DNA present in the sample available for hybridization with a complementary DNA sequence thereby producing A treated sample;
c) contacting the treated sample with at least one probe which is a DNA sequence which hybridizes with all or a portion of a DNA sequence repre-sented in Figure 1 or Figure 2; and d) detecting the hybridization of DNA from the sample with the DNA probe; wherein hybridi-zation is an indication of a predisposition to the development of a brain tumor or the presence of a brain tumor in the individual.

13. A method of diagnosing a predisposition to the development of a brain tumor or the presence of a brain tumor in an individual, comprising:
a) obtaining a sample from the brain of an individual;
b) treating the sample in a manner that renders the cells available for staining with a nestin-specific antibody thereby producing a treated sample;
c) staining the cells of the treated sample with labelled anti-nestin antibodies; and d) detecting stained cells as an indication of a predisposition to the development of brain tumors or the presence of a brain tumor in an individual.

14. A method of Claim 13 wherein the antibodies are labelled with a label selected from the group consisting of fluorescein, rhodamine and peroxidase.

15. A method of Claim 13 wherein the nestin-specific antibody is a polyclonal antibody.

16. A method of Claim 15 wherein the polyclonal antibody is anti-nestin 129.

17. A kit for diagnosing a brain tumor, comprising at least one nucleic acid probe, which hybridizes with a DNA sequence represented in Figure 1 or Figure 2.

18. A kit for diagnosing a brain tumor, comprising a first antibody which is nestin-specific and a second antibody which binds to the first antibody and is labelled.

19. A nestin-specific antibody or a pharmaceutical composition comprising a nestin-specific antibody formulated in a solution for parenteral adminis-tration for use in in vivo diagnosis, e.g., tumor imaging.

20. Use of a nestin-specific antibody for the manu-facture of a diagnostic agent for in vivo tumor imaging.