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WORLD I>rrELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCX 

INTERNATIONAL APPUCATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification ^ : 

A61K 48/00, 35730, C12N 5/00, 5/06, 5/08 



Al 



(11) International Publication Number: WO 99/01159 

(43) IntemaUonal Publication Date: 14 January 1999 (14.01.99) 



(21) International Application Number: PCT/US98/ 13875 

(22) international Filing Date: 3 July 1998 (03.07.98) 



(30) Priority Data: 

08/909,435 
09/109.858 



4 July 1997 (04.07.97) US 
2 July 1998 (02.07,98) US 



(71) Applicant: UNIVERSITY OF UTAH RESEARCH FOUNDA- 

TION [US/US]; 210 Park Building, Salt Lake City. UT 
84112 (US). 

(72) Inventors: RAO. Mahendra, S.; 4590 S. Parkhill Drive, Salt 

Lake City. UT 84124 (US). MAYER-PROSCHEL, Margot; 
12169 S. Hidden Valley Road. Sandy. UT 84092 (US). 
KALYANI, Anjali, J.; 207 South 600 East. Salt Lake City, 
UT 84102 (US). 

(74) Agents: HOWARTH, Alan, J. et al.; Thorpe, North & Western. 
LLP, P.O. Box 1219, Sandy, UT 84091-1219 (US). 



(81) Designated States: AL, AM, AT, AU. AZ, BA, BE, BG. BR. 
BY, CA, CH, CN, CU, CZ, DE, DK. EE, ES, H, GB. GE. 
GH, GM. GW, HR. HU. ID. IL. IS. JP, KE, KG, KP, KR. 
KZ. LC. LK, LR. LS. LT. LU, LV, MD. MG, MK, MN. 
MW, MX. NO. NZ. PL. PT. RO, RU, SD, SE, SG, SI, SK. 
SL, TJ, TM, TR. TT, UA. UG, UZ. VN. YU. ZW. ARIPOrin 
patent (GH. GM, KE, LS. MW, SD. SZ, UG. ZW). Eurasian? K 
patent (AM, AZ, BY, KG. KZ, MD. RU, TJ, TM), European^ 



patent (AT, BE, CH, CY, DE, DK, ES, H. FR, GB, GR. 
IE. IT. LU. MC, NL, PT, SE), OAPI patent (BF, BJ, CF,' 
CG, CI, CM, GA, GN. ML, MR, NE. SN. TD, TG). 



21 



Published 

With international search report. 
Before the expiration of the time limit for amending 
claims and to be republished in the event of the receipt oX, 3 
amendments, 

m 

a 
o 

< 



(54) Title: LINEAGE-RESTRICTTED NEURONAL PRECURSORS 



(57) Abstract 



A self-renewing restricted stem cell population has been identified in developing (embryonic day 13.5) spinal cords that can 
differentiate into multiple neuronal phcnotypes, but cannot differentiate into glial phenotypes. This neuronal-restricted precursor (NRP) 
expresses highly polysialated or embryonic neural cell adhesion molecule (E-NCAM) and is moiphologically distinct from neuroepithelial 
sttm cells (NEP cells) and spinal glial progenitors derived from embryonic day 10.5 spinal cord. NRP cells self renew over multiple 
passages in the presence of fibroblast growth factor (FGF) and neurotrophin 3 (NT-3) and express a characteristic subset of neuronal 
epitopes. When cultured in the presence of RA and the absence of FGF. NRP cells differentiate into GABAergic, glutaminergic, and 
cholinergic immunoreactive neurons. NRP cells can also be generated from multipotent NEP cells cultured from embryonic day 10.5 
neural tubes. Clonal analysis shows that E-NCAM immunoreactive NRP cells arise from an NEP progenitor cell that generates other 
restricted CNS precursors. The NEP-derived E-NCAM immunoreactive cells undergo self renewal in defined medium and differentiate 
into multiple neuronal phenotypes in mass and clonal culture. Thus, a direct lineal relationship exists between multipotential NEP cells and 
more restricted neuronal precursor cells present in vivo at embryonic day 13.5 in the spinal cord. Methods for treating neurological diseases 
are also disclosed. 



FOR THE PURPOSES OF INFORMATION ONLY 
Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCX. 



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Annenia 


n 


iMnland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


hV 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


SZ 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


G£ 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madi^ascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


TTie former Yugoslav 


TM 


Turkmenistan 


BF 


Buricina Faso 


GR 


Greece 




Republic of Macedonia 


re 


Turkey 


BG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


VG 


Uganda 


BV 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mraico 


UZ 


Uzbekistan 


CF 


Central African Republic 


JP 


Japan 


NE 


Niger 


VN 


Vict Nam 


CG 


Congo 


KE 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgyzstan 


NO 


Norway 


ZW 


Znnbabwe 


CI 


C6te d'lvoire 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Ponugal 






CU 


Cuba 


KZ 


Kazakstan 


RO 


Romania 






cz 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






D£ 


Gennany 


LI 


Liechtenstein 


sn 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


S£ 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







159 



PCT/US98/1387S 



LINEAGE-RESTRICTED NEURONAL PRECURSORS 

CROSS-REFERENCE TO RELATED APPLICATIONS 
This application is a continuation-in-part of 
application Serial No. 08/909,435, filed July 4, 1997, 

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH 

OR DEVELOPMENT 
This invention was made with government support under 
a FIRST award and a Multidisciplinary Basic Cancer 
Research Training Grant Graduate Fellowship from the 
National Institutes of Health. The government has certain 
rights in the invention, 

BACKGROUND OF THE INVENTION 
This invention relates to lineage-restricted 
intermediate precursor cells and methods of making and 
using thereof. More particularly, the invention relates 
to neuronal-restricted precursors (NRP's) isolated from 
mammalian embryos, neuroepithelial stem (NEP) cells, or 
embryonic stem (ES) cells. These neuronal-restricted 
precursors are capable of self-renewal and differentiation 
into neurons, but not into glia, i.e. astrocytes and 
oligodendrocytes. Methods of generating, isolating, 
culturing, transfecting, and transplanting such neuronal- 
restricted precursor cells are also described. 

Multipotent cells with the characteristics of stem 
cells have been identified in several regions of the 
central nervous system and at several developmental 
stages. F.H. Gage et al., Isolation, Characterization and 
Use of Stem Cells from the CNS, 18 Ann. Rev, Neurosci. 
159^92 (1995); M. Marvin & R. McKay, Multipotential Stem 
Cells in the Vertebrate CNS, 3 Semin. Cell. Biol. 401-11 
(1992); R.P. Skoff, The Lineages of Neuroglial Cells, 2 
. The Neuroscientist 335-44 (1996) . These cells, often 
referred to as neuroepithelial stem oells <NEP cells) , 
have the capacity to undergo self renewal and to 
differentiate into neurons, oligodendrocytes, and 
astrocytes, thus representing multipotent stem cells. 



wo 99/01159 



PCT/iJS98/13875 



2 

A. A, Davis & S. Temple, A Self-Renewing Multipotential 
Stem Cell in Embryonic Rat Cerebral Cortex, 362 Nature 
363-72 (1994); A.G. Gritti et al., Multipotential Stem 
Cells from the Adult Mouse Brain Proliferate and Self- 
5 Renew in Response to Basic Fibroblast Growth Factor, 16 J, 

Neurosci. 1091-1100 (1996); B.A. Reynolds et al., A 
Multipotent EGF-Responsive Striatal Embryonic Progenitor 
Cell Produces Neurons and Astrocytes, 12 J. Neurosci, 
4565-74 (1992); B.A. Reynolds & S. Weiss, Clonal and 
10 Population Analyses Demonstrate that an EGF-Responsive 

Mammalian Embryonic CNS Precursor is a Stem Cell, 175 
Developmental Biol. 1-13 (1996); B,P. Williams et al., The 
Generation of Neurons and Oligodendrocytes from a Common 
Precursor Cell, 7 Neuron 685-93 (1991) . 
15 The nervous system also contains precursor cells with 

restricted differentiation potentials. T.J. Kilpatrick & 
P.F. Bartlett, Cloned Multipotential Precursors from the 
Mouse Cerebrum Require FGF-2, Whereas Glial Restricted 
Precursors are Stimulated with Either FGF-2 or EGF, 15 J. 
20 Neurosci. 3653-61 (1995); J. Price et al.. Lineage 

Analysis in the Vertebrate Nervous System by Retrovirus- 
Mediated Gene Transfer, 84 Developmental Biol. 156-60 
(1987); B.A. Reynolds et al., supra; B.A. Reynolds & S. 
Weiss, supra; B, Williams, Precursor Cell Types in the 
25 Germinal Zone of the Cerebral Cortex, 17 BioEssays 391-93 

(1995); B.P. Williams et al., supra. The relationship 
between multipotent stem cells and lineage restricted 
precursor cells is still unclear. In principal, lineage 
restricted cells could be derived from multipotent cells, 
30 but this is still a hypothetical possibility in the 

nervous system with no direct experimental ^evidence. 
Further, no method of purifying such precursors from 
multipotent cells has been described. 

As has been shown in copending U.S. Patent 
35 Application Serial No. 08/852,744, entitled ^'Generation, 

Characterization, and Isolation of Neuroepithelial Stem 
Cells and Lineage Restricted Intermediate Precursor," 
filed May 7, 1997, hereby incorporated by reference in its 



wo 99/01159 



PCT/US98/13875 



3 

entirety, NEP cells grow on fibronectin and require 
fibroblast growth factor (FGF) and an as yet 
uncharacterized component present in chick embryo extract 
(CEE) to proliferate and maintain an undifferentiated 
5 phenotype in culture. The growth requirements of NEP 

cells are different from neurospheres isolated from E14.5 
cortical ventricular zone cells. B.A. Reynolds et al., 
supra; B.A. Reynolds & S. Weiss, supra; WO 9615226; WO 
9615224; WO 9609543; WO 9513364; WO 9416718; WO 9410292; 
10 WO 9409119. Neurospheres grow in suspension culture and 

do not require CEE or FGF, but are dependent on epidermal 
growth factor (EGF) for survival. FGF itself is not 
sufficient for long term growth of neurospheres, though 
FGF may support their growth transiently. NEP cells, 
15 however, grow in adherent culture, are FGF dependent, do 

not express detectable levels of EGF receptors, and are 
isolated at a stage of embryonic development prior to 
which it has been possible to isolate neurospheres. Thus, 
NEP cells may represent a multipotent precursor 
20 characteristic of the brain stem and spinal cord, while 

neurospheres may represent a stem cell more characteristic 
of the cortex. Nonetheless, NEP cells provide a model 
system for studying the principles of lineage restriction 
from multipotent stem cells or precursor cells of the 
25 central nervous system. The principles elucidated from 

the study of NEP cells are expected to be broadly 
applicable to all CNS precursor cells sufficiently 
multipotent to generate both neurons and glia. Thus, the 
present application is intended to be applicable to any 
30 CNS precursor cells regardless of their site of derivation 

as long as they are able to differentiate to both neurons 
and glial cells. 

U.S. Patent No. 5,589,376, to D.J. Anderson and D.L. 
Stemple, discloses mammalian neural crest stem cells and 
35 methods of isolation and clonal propagation thereof, but 

fails to disclose cultured NEP cells, cultured lineage 
restricted precursor cells, and methods of generating, 
isolating, and culturing thereof. Neural crest cells 



wo 99/01 1S9 



PCT/US98/I3875 



differentiate into neurons and glia of the peripheral 
nervous system (PNS), whereas the neuroepithelial stem 
cells differentiate into neurons and glia of the central 
nervous system (CNS) . 
5 U.S. Serial No. 08/909,435, filed July 4, 1997, for 

"Isolation of Lineage Restricted Neuronal Precursors," 
hereby incorporated by reference in its entirety, 
describes neuronal restricted precursor (NRP) cells that 
are capable of differentiating into neurons, but not into 
10 glial cells. It was shown that NRP cells can be isolated 

from NEP cells, as well as directly from embryonic spinal 
cords . 

U.S. Serial No. 08/980,850, filed November 29, 1997, 
for "Lineage Restricted Glial Precursors from the Central 
15 Nervous System," hereby incorporated by reference in its 

entirety, describes glial restricted precursor (GRP) cells 
that are capable of differentiating into oligodendrocytes, 
A2B5* process-bearing astrocytes, and A2B5" f ibroblast-like 
astrocytes, but not into neurons. GRP cells can be 
20 isolated from differentiating NEP cells, as well as CNS 

tissue, and differ from oligodendrocyte-type-2 astrocyte 
(0-2A) progenitor cells in growth factor requirements, 
morphology, and progeny. 

In U.S. Patent Application Serial No. 09/073,881, 
25 filed May 6, 1998, for "Common Neural Progenitor for CNS 

and PNS," hereby incorporated by reference in its 
entirety, it was shown that NEP cells can be induced to 
differentiate into neural crest cells as well as other 
cells of the CNS and PNS. 
30 The neuron-restricted precursor cells described 

herein are distinct from the NEP cells, GRP cells, 
neurospheres, and neural crest stem cells that have -been 
described elsewhere. NEP cells are capable of 
differentiating into neurons or glia whereas NRPs can 
35 differentiate into neurons, but not glia, and NEP cells 

and NRPs display distinct cell markers. GRP cells can 
differentiate into glia, but not neurons. As mentioned 
above, neurospheres grow in suspension culture and do not 



wo 99/01159 PCT/US98/13875 

5 

require CEE or FGF, but are dependent on EGF for survival, 
whereas NRP cells grow in adherent culture and do not 
express detectable levels of EGF receptors. Further, 
neural crest cells differentiate into neurons and glia of 
5 the peripheral nervous system (PNS), whereas NRP cells 

differentiate into neurons of the central nervous system 
(CNS) . NRP cells express polysialated or embryonic neural 
cell adhesion molecule (E-NCAM) , but NEP cells, 
neurospheres, GRP cells, and neural crest cells do not. 
10 Therefore, NRP cells are different in their proliferative 

potential, expression of cell markers, and nutritional 
requirements from these other cell types. 

The ability to isolate and grow mammalian neuronal- 
restricted precursor cells in vitro allows for of using 
15 pure populations of neurons for transplantation, discovery 

of genes specific to selected stages of development, 
generation of cell-specific antibodies for therapeutic and 
diagnostic uses such as for targeted gene therapy, and the 
like. Further, NRP cells can be used to generate 
20 subpopulations of neurons with specific properties, i.e. 

motoneurons and other neuronal cells for analyzing 
neurotransmitter functions and small molecules in high 
throughput assays. Moreover, the methods of obtaining NRP 
cells from NEP cells or embryonic stem (ES) cells provides 
25 for a ready source of a large number of post-mitotic 

neurons. Post-mitotic cells obtained from a tumor cell 
line are already being commercially marketed (e.g., 
Clontech, Palo Alto, CA) . The present invention is also 
necessary to understand how multipotent neuroepithelial 
30 stem cells become restricted to the various 

neuroepithelial derivatives. In particular, culture 
conditions that allow the growth and self-renewal of 
mammalian neuronal-restricted precursor cells are 
desirable so that the particulars of the development of 
35 these mammalian stem cells can be ascertained. This is 

desirable because a number of tumors of neuroepithelial 
derivatives exist in mammals, particularly humans. 
Knowledge of mammalian neuroepithelial stem cell 



wo 99/01159 



PCT/US98/13875 



6 

development is therefore needed to understand these 
disorders in hiomans. 

In view of the foregoing, it will be appreciated that 
isolated populations of mammalian lineage restricted 
5 neuronal precursor cells and methods of generating, 

isolating, culturing, transfecting, and transplanting such 
cells would be significant advancements in the art. 

BRIEF SUMMARY OF THE INVENTION 
10 It is an object of the present invention to provide 

isolated (pure) populations of mammalian neuronal- 
restricted precursor cells and their progeny. 

It is another object of the invention to provide 
methods of generating, isolating, culturing, and 
15 regenerating of mammalian lineage-restricted neuronal 

precursor cells and their progeny. 

It is yet another object of the invention to provide 
a method for the generation of lineage-restricted neuronal 
precursor cells from a CNS multipotent precursor cell able 
20 to generate both neurons and glia. 

It is a still further object of the invention to 
provide pure differentiated populations of neuronal cells 
derived from lineage-restricted neuronal precursor cells. 
It is still another object of the invention to 
25 provide methods of transfecting and transplanting such 

neuronal restricted precursor cells. 

These and other objects can be achieved by providing 
an isolated, pure population of mammalian CNS neuron- 
restricted precursor cells. Preferably, such neuron- 
30 restricted precursor cells are capable of self-renewal, 

differentiation to CNS neuronal cells but not to CNS glial 
cells, and expressing embryonic neural cell adhesion 
molecule (E-NCAM) , but not expressing a ganglioside 
recognized by A2B5 antibody. These neuron-restricted 
35 precursor cells may or may not express nestin or 3-III 

tubulin. Thus, embryonic neural cell adhesion mol-ecule 
(E-NCAM) is a defining antigen for these cells. The NRP 
cells are able to differentiate into neurons that are 



wo 99/01159 



PCT/US98/13875 



7 

capable of releasing and responding to neurotransmitters. 
These neurons can demonstrate receptors for these 
neurotransmitters, and such cells are capable of 
expressing neurotransmitter-synthesizing enzymes. The NRP 
5 cells are also capable of differentiating into neurons 

that can form functional synapses and/or develop 
electrical activity. The NRP cells are also capable of 
stably expressing at least one material selected from the 
group consisting of growth factors for such cells, 

10 differentiation factors for such cells, maturation factors 

for such cells, and combinations of any of these. 
Further, the present neuron-restricted precursor cells may 
be selected, chosen, and isolated from human primates, 
non-human primates, equines, canines, felines, bovines, 

15 porcines, ovines, lagomorphs, and rodents. 

A method of isolating a pure population of mammalian 
CNS neuron-restricted precursor cells comprises the steps 
of: 

(a) isolating a population of mammalian multipotent 
20 CNS stem cells capable of generating both neurons and 

glia; 

(b) incubating the multipotent CNS stem cells in a 
mediiam configured for inducing such cells to begin 
differentiating ; 

25 (c) purifying from the -differentiating cells a 

subpopulation of cells expressing a selected antigen 
defining neuron-restricted precursor cells; and 

(d) incubating the purified subpopulation of cells 
in a medium configured for supporting adherent growth 

30 thereof. 

A preferred selected antigen defining neuron- 
restricted precursor cells is embryonic neural cell 
adhesion molecule. Preferably, the step of purifying the 
NRP cells comprises a procedure selected from the group 

35 consisting of specific antibody capture, fluorescence 

activated cell sorting, and magnetic bead capture. 
Specific antibody capture is especially preferred. In a 
preferred embodiment, the mammalian multipotent CNS stem 



wo 99/01159 



PCTAJS98/13875 



8 

cells are neuroepithelial stem cells. A preferred 
procedure for isolating a population of CNS 
neuroepithelial stem cells comprises: 

(a) removing a CNS tissue from a mammalian embryo 
5 at a stage of embryonic development after closure of the 

neural tube but prior to differentiation of cells in the 
neural tube; 

(b) dissociating cells comprising the neural tube 
removed from the mammalian embryo; 

10 (c) plating the dissociated cells in feeder-cell- 

independent culture on a substratum and in a medium 
configured for supporting adherent growth of the 
neuroepithelial stem cells comprising effective amounts of 
fibroblast growth factor and chick embryo extract; and 

15 (d) incubating the plated cells at a temperature 

and in an atmosphere conducive to growth of the 
neuroepithelial stem cells. 

Preferably, the mammalian embryo is selected from the 
group consisting of human and non-human primates, equines, 

20 canines, felines, bovines, porcines, ovines, lagomorphs, 

and rodents. It is also preferred that the sxibstratum is 
selected from the group consisting of fibronectin, 
vitronectin, laminin, and RGD peptides. In a preferred 
embodiment, the medium comprises effective amounts of 

25 fibroblast growth factor and neurotrophin 3 (NT-3) . 

A method of isolating a pure population of mammalian 
CNS neuron-restricted precursor cells comprises the steps 
of: 

(a) removing a sample of CNS tissue from a 

30 mammalian embryo at a stage of embryonic development after 

closure of the neural tube but prior to differentiation of 
glial and neuronal cells in the neural tube; 

(b) dissociating cells comprising the sample of CNS 
tissue removed from the mammalian embryo; 

35 (c) purifying from the dissociated cells a 

subpopulation expressing a selected antigen defining 
neuron-restricted precursor cells; 



wo 99/01159 



PCTAJS98/1387S 



9 

(d) plating the purified subpopulation of cells in 
feeder-cell-independent culture on a substratum and in a 
medium configured for supporting adherent growth of the 
neuron-restricted precursor cells; and 
5 (e) incubating the plated cells at a temperature 

and in an atmosphere conducive to growth of the neuron- 
restricted precursor cells. 

Preferably, the selected antigen defining neuron- 
restricted precursor cells is embryonic neural cell 

10 adhesion molecule. It is also preferred that the step of 

purifying comprises a procedure selected from the group 
consisting of specific antibody capture, fluorescence 
activated cells sorting, and magnetic bead capture. 
Specific antibody capture is especially preferred. It is 

15 further preferred that the mammalian embryo is selected 

from the group consisting of human and non-human primates, 
equines, canines, felines, bovines, porcines, ovines, 
lagomorphs, and rodents. 

A method of obtaining postmitotic neurons comprises: 

20 (a) providing neuron-restricted precursor cells and 

culturing the neuron-restricted precursor cells in 
proliferating conditions; and 

(b) changing the culture conditions of the neuron- 
restricted precursor cells from proliferating conditions 

25 to differentiating condition, thereby causing the neuron- 

restricted precursor cells to differentiate into 
postmitotic neurons . 

The changing of the culture conditions preferably 
comprises adding retinoic acid to basal medium or 

30 withdrawing a mitotic factor from basal medium. Such a 

mitotic factor is fibroblast growth factor. Changing the 
culture conditions can also comprise adding a neuronal 
maturation factor to basal medium. Preferred neuronal 
maturation factors are selected from the group consisting 

35 of sonic hedgehog, BMP-2, BMP-4, NT-3, NT-4, CNTF, LIF, 

retinoic acid, brain-derived neurotrophic factor <BDNF) , 
and combinations of any of the above. 



99/01 159 PCT/US98/I3875 

10 

Another preferred embodiment of the invention 
comprises an isolated cellular composition comprising the 
mammalian CNS neuron-restricted cells described herein. 
Another preferred embodiment of the invention comprises a 
pharmaceutical composition comprising a therapeutically 
effective amount of such composition and a 
pharmaceutically acceptable carrier. 

A method for treating a neuronal disorder in a mammal 
comprises administering to such mammal a therapeutically 
effective amount of the isolated cellular composition 
comprising the mammalian CNS neuron-restricted cells 
described herein. Another method for treating a neuronal 
disorder in a mammal comprising administering to said 
mammal a therapeutically effective amount of such 
pharmaceutical composition and a pharmaceutically 
acceptable carrier. Such composition can be administered 
by a route selected from the group consisting of 
intramuscular administration, intrathecal administration, 
intraperitoneal administration, intravenous 
administration, and combinations of any of the above. 
This method can also include the administration of a 
member selected from the group consisting of 
differentiation factors, growth factors, cell maturation 
factors and combinations of any of the above. Such 
differentiation factors are preferably selected from the 
group consisting of retinoic acid, BMP-2, BMP-4, and 
combinations of any of the above. 

A method for treating neurodegenerative symptoms in a 
mammal comprises the steps of: 

(a) providing a pure population of neuronal 
restricted precursor cells; 

(b) genetically transforming such neuronal 
restricted precursor cells with a gene encoding a growth 
factor, neurotransmitter, neurotransmitter synthesizing 
enzyme, neuropeptide, neuropeptide synthesizing enzyme, or 
substance that provides protection against free-radical 
mediated damage thereby resulting in a transfored 
population of glial restricted precursor cells that 



99/01159 



PCTAJS98/13875 



11 

express such growth factor, neurotransmitter/ 
neurotransmitter synthesizing enzyme, neuropeptide, 
neuropeptide synthesizing enzyme, or substance that 
provides protection against free-radical mediated damage; 
and 

(c) administering an effective amount of said 
transformed population of neuronal restricted precursor 
cells to such mammal. 

A method or screening compounds for neurological 
activity comprising the steps of: 

(a) providing a pure population of neuronal 
restricted precursor cells or derivatives thereof or 
mixtures thereof cultured in vitro; 

(b) exposing such cells or derivatives thereof or 
mixtures thereof to a selected compound at varying 
dosages; and 

(c) monitoring the reaction of such cells or 
derivatives thereof or mixtures thereof to said selected 
compound for selected time periods. 

A method for treating a neurological or 
neurodegenerative disease comprises administering to a 
mammal in need of such treatment an effective amount of 
neuronal restricted precursor cells or derivatives thereof 
or mixtures thereof. Such neuronal restricted precursor 
cells or derivatives thereof or mixtures thereof can be 
from either a heterologous donor or an autologous donor. 
The donor can be a fetus, juvenile, or adult. 

A method of isolating a pure population of mammalian 
CNS neuron-restricted precursor cells comprises the steps 
of: 

(a) providing a sample of mammalian embryonic stem 

cells : 

(b) purifying from the mammalian embryonic stem 
cells a subpopulation expressing a selected antigen 
defining neuron-restricted precursor cells; 

(c) plating the purified subpopulation of cells in 
feeder-cell-independent culture on a substratum and in a 



wo 99/01159 



PCT/US98/13875 



12 

medium configured for supporting adherent growth of the 
neuron-restricted precursor cells; and 

(d) incubating the plated cells at a temperature 
and in an atmosphere conducive to growth of the neuron- 
5 restricted precursor cells. 

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
FIG. 1 shows a siimmary of the immunoreactivities of 
NEP cells and their progeny, including NRP cells. 
10 FIG. 2 shows results of RT-PCR amplification of total 

RNA isolated from rat E-NCAM"" cells for determining 
expression of choline acetyl transferase (ChAT) , p75, 
islet^l (Isl-1), calbindin, glutamic acid decarboxylase 
(GAD), glutaminase, and cyclophilin (a housekeeping gene). 
15 FIG. 3 shows a bar graph of the number of cells 

responding to neurotransmitters on acutely dissociated 
(unshaded) and differentiated (shaded) E-NCAM* cells as 
measured by fura-2 calcium ion imaging: GABA (y-amino 
butyric acid), Gly (glycine), DA {dopamine), Glu 
20 (glutamate), Ach (acetyl choline), RR (rat ringers 

solution), 50 raw K RR (rat ringers solution modified by 
replacing Na'' with K'') . 

FIG. 4 shows an illustrative plot of the ratio 
(l34o/l3Bo) of Ca^* responses over time from an acutely 
25 dissociated E-NCAM" cell. 

FIG. 5 shows an illustrative plot of the ratio 
(l34o/l3eo) of Ca^'' responses over time from a differentiated 
E-NCAM* cell. 

FI<;. 6 shows the results of PGR analysis of a single 
30 E-NCAM'' clone for expression of markers of mature neurons. 

FIG. 7 shows a bar graph of the percentage of cells 
from four E-NCAM* clones that responded to 
neurotransmitters as measured by fura-2 calcium ion 
imaging: GABA (y-amino butyric acid), <^ly (glycine), DA 
35 (dopamine), Glu (glutamate), Ach (acetyl choline), RR (rat 

ringers solution), 50 mM K RR (rat ringers solution 
modified by replacing Na"" with K") . 



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13 

FIGS. 8 and 9 show illustrative traces of the ratio 
{I340/I380) of Ca^^ responses from two cells recorded from 
one E-NCAM* clone. 

FIG, 10 shows the effect of bone morphogenetic 
5 protein 2 (BMP-2) on cell division of E-NCAM^ cells as 

measured by BRDU incorporation. 

FIG. 11 shows the effect of sonic hedgehog (Shh) on 
cell division of E-NCAM^ cells as measured by BRDU 
incorporation. 

10 FIG. 12 shows results of RT-PCR amplification of 

total RNA isolated from mouse E-NCAM"^ cells for 
determining expression of (from left to right after the 
molecular weight markers at the far left) p75, Isl-1, 
ChAT, calbindin, GAD, and glutaminase. 

15 FIG. 13 shows results of RT-PCR amplification of 

total RNA isolated from differentiated mouse ES cells for 
determining expression of (from left to right) nestin, N- 
CAM, neurof ilament-M (NF-M) , microtubule associated 
protein 2 (Map-2), GFAP, DM-20/PLP. 

20 FIG. 14 shows results of RT-PCR amplification of 

total RNA isolated from differentiated mouse ES cells for 
determining expression of (from left to right) ChAT, p75, 
islet-1, calbindin, GAD, and glutaminase. 

25 DETAILED DESCRIPTION 

Before the present neuronal-restricted precursor 
cells and methods of making and methods of use thereof are 
disclosed and described, it is to be understood that this 
invention is not limited to the particular configurations, 

30 process steps, and materials disclosed herein as such 

configurations, process steps, and materials may vary 
somewhat. It is also to be understood that the 
terminology employed herein is used for the purpose of 
describing particular embodiments only and is not intended 

35 to be limiting since the scope of the present invention 

will be limited only by the appended claims and 
equivalents thereof. 



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It must be noted that, as used in this specification 
and the appended claims, the singular forms "a," "an," and 
"the" include plural referents unless the context clearly 
dictates otherwise. Thus, for example, reference to "an 
5 embryo" includes reference to two or more embryos, 

reference to "a mitogen" includes reference to a mixture 
of two or more mitogens, and reference to "a factor" 
includes reference to a mixture of two or more factors. 

In describing and claiming the present invention, the 
10 following terminology will be used in accordance with the 

definitions set out below. 

As used herein, "self renewal" refers, for example, 
to the capability of a neuroepithelial stem cell to divide 
to produce two daughter cells, at least one of which is a 
15 multipotent neuroepithelial stem cell, or to the 

capability of a neuronal-restricted precursor cell to 
divide to produce two daughter cells, at least one of 
which is a neuronal-restricted precursor cell. 

As used herein, "clonal density" and similar terms 
20 mean a density sufficiently low enough to result in the 

isolation of single, non-impinging cells when plated in a 
selected culture dish. An illustrative example of such a 
clonal density is about 225 cells/100 mm culture dish. 
As used herein, "feeder-cell-independent adherent 
25 culture" and similar terms mean the growth of cells in 

vitro in the absence of a layer of different cells that 
generally are first plated on a culture dish to which the 
cells from the tissue of interest are then added. In 
feeder cell cultures, the feeder cells provide a 
30 substratxam for the attachment of cells from the tissue of 

interest and additionally serve as a source of mitogens 
and survival factors. The feeder-cell-independent 
adherent cultures herein use a chemically defined 
substratum, for example fibronectin, and mitogens or 
35 survival factors are provided by supplementation of the 

liquid culture medium with either purified factors or 
crude extracts from other cells or tissues. Therefore, in 
feeder-cell-independent cultures, the cells in the culture 



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15 

dish are primarily cells derived from the tissue of 
interest and do not contain other cell types required to 
support the growth of cells derived from the tissue of 
interest. 

5 As used herein, "effective amount" means an amount of 

a growth factor or survival factor or other factor that is 
nontoxic but sufficient to provide the desired effect and 
performance. For example, an effective amount of FGF as 
used herein means an amount selected so as to support self 

10 renewal and proliferation of NEP cells when used in 

combination with other essential nutrients, factors, and 
the like. An effective amount of NRP cells or derivatives 
thereof or mixtures thereof for transplantation refers to 
an amount or number of cells sufficient to obtain the 

15 selected effect. NRP cells will generally be administered 

at concentrations of about 5-50,000 cells/microliter . 
Transplantation will generally occur in voliames up to 
about 15 microliters per injection site. However, 
transplantation subsequent to surgery on the central 

20 nervous system can involve volumes many times this size. 

Thus, the number of cells used for transplantation is 
limited only by utility, and such numbers can be 
determined by a person skilled in the art without undue 
experimentation . 

25 As used herein, ^'derivative" of an NRP cell means a 

cell derived from an NRP cell in vitro by genetic 
transduction, differentiation, or similar processes. 

As used herein, "administering an NRP cell to a 
mammal means transplanting or implanting such NRP cell 

30 into CNS tissue or adjacent to such CNS tissue of a 

recipient. Such administration can be carried out by any 
method known in the art, such as surgery, with an infusion 
cannula, needle, and the like. 

As used herein, ''heterologous" refers to individuals, 

35 tissues, or cells different from a transplant recipient. 

The transplant donor could be from the same species or a 
different species as the transplant recipient. For 
example, a heterologous donor of NRP cells for 



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16 

transplantation could be from a different species as the 
transplant recipient. 

As used herein, "autologous" refers to self-generated 
or originating within the body. Thus, for example, an 
5 autologous donor of tissue or cells for transplantation is 

the same individual that receives the transplant. By way 
of further example, autologous cells are cells arising, 
transferred, or transplanted within an individual. In 
vitro manipulation may take place between harvesting of 

10 the cells and transplanting such cells or derivatives 

thereof, but is not required prior to transplantation. 

As used herein, "transforming," "transducing," 
"transfection, " and similar terms mean insertion or 
transfer of a gene or genes into NRP cells regardless of 

15 the method of insertion r transfer- Thus, transformation 

can be accomplished by calcium phosphate trans fection, 
DEAE-dextran transfection, polybrene transfection, 
electroporation, lipofection, infection of viruses, and 
the like and any other methods known in the art. 

20 The present invention is illustrated using neuron- 

restricted precursor cells isolated from rats, mice, and 
humans. The invention, however, encompasses all mammalian 
neuronal-restricted precursor cells and is not limited to 
neuronal-restricted precursor cells from rats, mice, and 

25 humans. Mammalian neuron-restricted precursor .cells can 

be isolated from human and non-human primates, equines, 
canines, felines, bovines, porcines, ovines, lagomorphs, 
and the like. 

Pluripotent stem cells in the central nervous system 
30 may generate differentiated neurons and glia either 

directly or through the generation of lineage-restricted 
intermediate precursors. In the developing retina, it 
appears that multipotent retinal precursors can generate 
any combination of differentiated cells even at their 
35 final division, indicating that intermediate precursors do 

not exist. In other regions of the central nervous 
system, in contrast, retroviral labeling studies have 
suggested the existence of lineage-restricted precursors 



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17 

that generate only one type of cell or a limited number of 
cell types. Intermediate stage precursors such as the 
bipotential oligodendrocyte-type-2-astrocyte precursor (O- 
2A) and a neuronal precursor have also been described in 
5 tissue culture studies. Yet, the generation of 

intermediate lineage-restricted precursors from 
pluripotent embryonic or adult stem cells . or other stem 
cells capable of differentiating into neurons and glia had 
not been observed until recently, i.e. U.S. Patent 

10 Application Serial No. 08/909,435, filed July 4, 1997. 

Thus, the lineal relationship between pluripotent stem 
cells identified in culture and the committed precursors 
identified in vivo and in vitro had heretofore been 
unknown. Possible models of development have included (1) 

15 pluripotent and more committed stem cells representing 

lineally related cells or (2) such cells representing 
independent pathways of differentiation. 

The developing rat spinal cord represents an ideal 
model for studying this differentiation. At embryonic day 

20 10.5 (ElO-5), the caudal neural tube appears as a 

homogeneous population of nestin-immxinoreactive dividing 
cells in vivo and in vitro. These initially homogeneous 
cells are patterned over several days to generate neurons, 
oligodendrocytes, and astrocytes in a characteristic 

25 spatial and temporal profile. Neurogenesis occurs first 

on a ventro-dorsal gradient, with the earliest neurons 
becoming postmitotic on E13.5 in rats. Neurogenesis 
continues over an additional two days followed by 
differentiation of oligodendrocyte precursors and the 

30 subsequent differentiation of astrocytes. 

Methods for growing neuroepithelial stem (NEP) cells 
isolated from E10.5 rat embryos as undifferentiated cells 
for extended periods in vitro have been described in 
Serial No. 08/852,744., and it has been shown further that 

35 these populations were able to generate the three major 

cells types in the CNS. Thus, NEP cells represent a 
dividing multipotent stem cell that may differentiate into 
neurons either via an intermediate neuroblast or directly 



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18 

as a part of its terminal differentiation. To determine 
whether neurons differentiated from NEP cells via 
intermediate, more-restricted precursors, a variety of 
immunologically defined populations from differentiating 
5 cultures of NEP cells were isolated and characterized. It 

is shown herein that cells morphologically and 
. phenotypically identical to NRP' s can be isolated from NEP 
cell cultures. Clonal analysis shows that individual NEP 
cells generate neurons via the generation of neuronal 

10 precursors and that individual NEP cells can generate 

neuron-restricted and glial-restricted precursors. It is 
further shown that E-NCAM'' (embryonic neural cell adhesion 
molecule positive) cells are present in E13.5 neural tube 
cultures and that these cells are mitotic, self renewing 

15 stem cells that can generate multiple neuronal phenotypes, 

but not astrocytes or oligodendrocytes. Thus, neuron 
restricted precursors (NRPs) are an identifiable stage in 
the In vivo differentiation of neurons. Moreover, it is 
shown that NRPs can be isolated and cultured from mouse 

20 embryos, mouse embryonic stem (ES) cells, and from human 

embryonic spinal cords. These data provide a 
demonstration of a direct lineal relationship between 
multipotent and neuron-restricted stem cells and suggest 
that neural differentiation involves progressive 

25 restriction in developmental fate. 

FIG. 1 presents a model for spinal cord 
differentiation. This model is similar to that proposed 
for hematopoiesis and for differentiation of neural crest 
(see review by D.J. Anderson, The Neural Crest Lineage 

30 Problem: Neuropoiesis?, 3 Neuron 1-12 (1989)). According 

to this model, NEP cells 10 represent a homogeneous 
population of cells in the caudal neural tube that express 
nestin (i.e. nestin *) but no other lineage marker din"). 
These cells divide and self renew in culture and generate 

35 differentiated phenotypes. Previous data have suggested 

intermediate dividing precursors with a more restricted 
potential. Such precursors include glial restricted 
precursors 14 that generate oligodendrocytes 18 and 



159 



PCT/US98/1387S 



19 

astrocytes 22, as well as neuronal progenitors 26 that 
generate several kinds of neurons 30, 34. The model also 
shows that neural crest stem cells 38, which can 
differentiate into PNS neurons 42, Schwann cells 46, and 
smooth muscle cells 50, also descend from NEP cells. The 
model therefore suggests that the multipotent precursors 
(NEP cells) generate differentiated cells (i.e., 
oligodendrocytes, type 2 astrocytes, type 1 astrocytes, 
neurons, motoneurons, PNS neurons, Schwann cells, and 
smooth muscle cells) through intermediate precursors. 
Consistent with this model are the results presented 
herein showing the existence of cells with a neuron- 
restricted proliferative potential, 

NEP cell cultures provide a large source of transient 
cells that can be sorted to obtain differentiated cell 
types. The results described herein provide direct 
evidence to support a model describing initially 
multipotent cells undergoing progressive restriction in 
developmental potential under extrinsic influence to 
generate the different phenotypes within the CNS. 
Evidence is provided that initially multipotent NEP cells 
generate neuron-restricted precursors in vitro and that 
such neuron-restricted precursors are also present iTi 
vivo. It is also shown that NRPs fulfill criteria of 
blast cells and that a direct lineal relationship between 
multipotent stem cells and more restricted NEP cells 
exists . 

The results presented herein support that E-NCAM- 
immunoreactive cells are restricted in their developmental 
potential. E-NCAM* cells failed to differentiate into 
oligodendrocytes or astrocytes under any culture 
conditions tested. In contrast, NEP cells differentiated 
into neurons, astrocytes, and oligodendrocytes, and A2B5- 
immunoreactive cells differentiate into oligodendrocytes 
under identical conditions. For these reasons, E-NCAM- 
immunoreactive cells are described herein as neuron- 
restricted precursors or NRPs. 



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PCt/US98/13875 



20 

Iminunopanning and double^labeling data demonstrate 
that E-NCAM can be used to identify a specific neuronal 
sublineage that is generated from multipotential NEP 
cells. Like markers for intermediate precursors in the 
5 hematopoietic system and neural crest, however, E-NCAM, 

and the A2B5 glial precursor marker as well, is not unique 
to intermediate precursors. E-NCAM has been shown to 
label some astrocytes. Similarly, A2B5 has been shown to 
recognize neurons in some species and is transiently 

10 expressed by astrocytes in some culture conditions. 

Nevertheless, under the specific culture conditions 
defined herein these markers can be used to select 
intermediate precursors and therefore represent the first 
cell surface epitopes that are co-expressed in concordance 

15 with a restriction in developmental potential. 

The basal medixam (NEP medium) used in the experiments 
described herein comprises DMEM-F12 (GIBCO/BRL, 
Gaithersburg, MD) supplemented with 100 Mg/ml transferrin 
(Calbiochem, San Diego, CA) , 5 Mg/ml insulin (Sigma 

20 Chemical Co., St. Louis, MO), 16 ^g/ml putrescine (Sigma), 

20 nM progesterone (Sigma), 30 nM selenious acid (Sigma), 
1 mg/ml bovine serum albumin (GIBCO/BRL), plus B27 
additives (GIBCO/BRL) , 20 ng/ml basic fibroblast growth 
factor (bFGF) , and 10% chick embryo extract (CEE) . In 

25 general, these additives were stored as lOOX concentrates 

at -20**C until use. Normally, 200 ml of NEP medium was 
prepared with all additives except CEE and used within two 
weeks of preparation. CEE was added to the NEP medium at 
the time of feeding cultured cells. 

30 FGF and CEE were prepared as described in D.L. 

Stemple & D.J. Anderson, supra; M.S. Rao & D.J. Anderson, 
supra; L. Sommers et al.. Cellular Function of the bHLH 
Transcription Factor MASHl in Mammalian Neurogenesis, 15 
Neuron 1245-58 (1995), hereby incorporated by reference, 

35 FGF is also available commercially (UBI) . 

Briefly, CEE was prepared as follows. Chick eggs 
were incubated for 11 days at 38*'C in a humidified 
atmosphere. Eggs were washed and the embryos were removed 



wo 99/01159 



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21 

and placed in a petri dish containing sterile Minimal 
Essential Medium (MEM with glutamine and Earle's salts) 
(GIBCO/BRL) at 4°C. Approximately 10 embryos each were 
macerated by passage through a 30-ml syringe into a 50-ml 
5 test tube. This procedure typically produced about 25 ml 

of medium. To each 25 ml was added 25 ml of MEM. The 
tubes were rocked at 4'*C for 1 hour. Sterile 
hyaluronidase (1 mg/25 g of embryo) (Sigma) was added, and 
the mixture was centrifuged for 6 hours at 30,000 g. The 
10 supernate was collected, passed through a 0.45 /um filter 

and then through a 0,22 m filter, and stored at -SO^'C 
until use. 

Laminin (Biomedical Technologies Inc.) was dissolved 
in distilled water to a concentration of 20 mg/ml and 

15 applied to tissue culture plates (Falcon) . Fibronectin 

(Sigma) was resuspended to a stock concentration of 10 
mg/ml and stored at -80*'C and then diluted to a 
concentration of 250 /ig/ml in D-PBS (GIBCO/BRL) . The 
fibronectin solution was applied to tissue culture dishes 

20 and immediately withdrawn. Subsequently, the laminin 

solution was applied and plates were incubated for 5 
hours. Excess laminin was withdrawn, and the plates were 
allowed to air dry. Coated plates were then rinsed with 
water and allowed to -dry again. Fibronectin was chosen as 

25 a growth substrate for NEP cells because NEP cells did not 

adhere to collagen or poly-L-lysine (PLL) and adhered 
poorly to laminin. Thus, all subsequent experiments to 
maintain NEP cells in culture were performed on 
f ibronectin-coated dishes. Laminin-coated dishes were 

30 used, however, to promote differentiation of NEP stem 

cells. 

For clonal analysis, cells harvested by 
trypsinization were plated at a density of 50-100 cells 
per 35 mm dish. Individual cells were identified and 
35 located on the dish by marking the position with a grease 

pencil. Cells were grown in DMEM/F12 with additives, as 
described above, for a period ranging from 10-15 days. 



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22 

The cells of the present invention may be used in the 
preparation of compositions, including pharmaceutical 
compositions, which may be appropriately formulated and 
administered to treat and correct deficiencies, 
5 debilitations, and other dysfunctions that may result from 

injury, disease, or other degeneration of relevant neural 
tissue. By way of non-limiting examples, suitable cells 
prepared in accordance with the present invention may be 
administered, e.g., by implantation as a means of 

10 effecting cell-replacement therapy, to treat instances 

where cell injury or debilitation has taken place. Thus, 
for example, the cells may be prepared in appropriate 
growth mediim such as one for promotion of growth and 
differentiation. Suitable medium may include, for 

15 example, growth or differentiation factors, e.g., retinoic 

acid, BMP-2, BMP-4, or one or more members of the 
neurotrophins such as NT-3, NT-4, CNTF, BDNF and the like. 
Cells thus suitably prepared in such medium would be 
introduced either intrathecally, I. v., I. P., or wherever 

20 or by any means by which introduction of the cell 

preparation to the target site is best accomplished. The 
particulars of administration of this type may vary and 
would be within the skill set of the physician or 
practitioner, 

25 The cells of the present invention are. likewise 

useful in a variety of diagnostic applications and may, 
for example, be prepared for use in a screening assay, 
e.g., for identification of neuronal markers and other 
binding partners or ligands, modulators or other factors 

30 that may function as modulators of cell growth and/or 

differentiation. The cells of the present invention may 
also be used, e.g., as a positive control in an assay to 
identify deficiencies in cell growth and differentiation, 
and the factors that may be the cause thereof. 

35 The cells of the present invention may be utilized in 

a variety of therapeutic applications, including in the 
preparation of pharmaceutical compositions and appropriate 
carriers, for administration to individuals in need of 



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PCT/IIS98/13875 



23 

such therapy, to treat various cellular debilitation, 
dysfunctions or other irregularities or abnormalities 
associated with injury, disease or genetically caused 
neuronal deficits. Maladies or conditions contemplated 
5 herein include Parkinson's disease, Huntington's disease, 

Alzheimer's disease, dysfunctions resulting from injury or 
trauma, amyotrophic lateral sclerosis (ALS or Lou Gehrig's 
Disease), and anencephaly. 

10 Example 1 

To determine if a dividing neuron-restricted 
precursor is normally present in vivo, sections of E13.5 
rat spinal cords were analyzed with a panel of early 
neuronal markers. Sections were cut of embryos fresh 

15 frozen at 13.5 days gestation and then were labeled by 

immunocytochemistry . Staining procedures were carried out 
according to methods well known in the art. Cells were 
double-labeled with antibodies against E-NCAM 
(Developmental Studies Hybridoma Bank, Iowa) and 

20 tubulin (Sigma Chemical Co., St. Louis, Missouri) or were 

stained with E-NCAM and counters tained with DAPI, a 
. nuclear marker for identifying all cells. All secondary 
monoclonal antibodies were from Southern Biotechnology. 

Polysialated or embryonic N-C7^ (E-NCAM) appeared to 

25 be a likely marker for neuronal precursors. E-NCAM 

immunoreactivity was first detected at E13.5. E-NC7U>1 
immunoreactive cells could be seen in the margins of the 
neural tube, but not in the proliferating ventricular 
zone. Double-labeling with &-III tubulin indicated that 

30 most E-NCAM-immunoreactive cells co-expressed this 

neuronal marker, A small proportion of cells present more 
medially were E-NCAM", but did not express tubulin 
immunoreactivity, suggesting that E-NCAM may be an early 
and specific marker of differentiation into neuronal 

35 precursors that is expressed prior to P-III tubulin. 



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24 

Example 2 

To characterize E-NCAM-inununoreactive cells, E13.5 
spinal cords were dissociated and E-NCAM-immunoreactive 
cells were stained with a panel of antibodies (Table 1). 
5 Sprague-Dawley rat embryos were removed at embryonic day 

13.5 and placed in a petri dish containing Hanks balanced 
salt solutions (HBSS, Gibco) . The trunk segments of the 
embryos were dissected using tungsten needles, rinsed, and 
then transferred to fresh HBSS. Spinal cords were 

10 mechanically dissected free from the surrounding 

connective tissue using sharpened No. 5 forceps. 
Isolated spinal cords were incubated in 0.05% trypsin/EDTA 
solution for 20 minutes. The trypsin solution was 
replaced with fresh HBSS containing 10% fetal bovine serum 

15 (FBS) . The segments were gently triturated with a Pasteur 

pipette to dissociate cells. Cells dissociated by 
trituration were plated in PLL/laminin-coated 35 mm dishes 
(Nunc) at high density and stained after 24 hours. 

Staining for the cell surface markers, such as A2B5 

20 and a-GalC, was carried out with cultures of living cells - 

To stain cells with antibodies against internal antigens 
such as GFAP, which specifically recognizes astrocytes (A. 
Bignami et al.. Localization of the Glial Fibrillary 
Acidic Protein in Astrocytes, by Immunofluorescence, 43 

25 Brain Res. 429-35 (1972)), P-III tubulin (DARO) and RT-97, 

which stain neurons (E. Geisert & A. Frankfurter, The 
Neuronal Response to Injury as Visualized by 
Immunostaining of Class P-tubulin in the Rat, 102 
Neurosci, Lett. 137-41 (1989), nestin, which is a -marker 

30 for undifferentiated stem cells (U. Lendahl et al., CNS 

Stem Cells Express a New Class of Intermediate Filament 
Protein, 60 Cell 585-95 (1990)), or 5-bromodeoxyuridine 
(BrdU, Sigma), which is a marker for determining the 
niamber of dividing cells, cultures were fixed in ice-cold 

35 methanol. Double- or triple-labeling experiments were 

performed by simultaneously incubating cells in 
appropriate combinations of primary antibodies followed by 
non-cross-reactive secondary antibodies, , e.g. M. Mayer 



wo 99/01159 



PCT/US98/13875 



25 

et al., Ciliary Neurotrophic Factor and Leukemia 
Inhibitory Factor Promote the Generation, Maturation, and 
Survival of Oligodendrocytes, 120 Development 142-53 
(1994), hereby incorporated by reference. In triple-label 
5 experiments, cultures were incubated with the primary 

antibody in blocking buffer for a period of 1 hour, rinsed 
with PBS, and incubated with a species-specific secondary 
antibody in blocking buffer for 1 hour. Cultures were 
rinsed three times with PBS and examined under a 

10 fluorescence microscope. For labeling with 4 antibodies 

simultaneously, live cells were first incubated with the 
surface antibodies A2B5 and a-GalC without the secondary 
layers. Cells were then fixed in ice-cold methanol for 
ten minutes and stained with a-P-III tubulin and the 

15 appropriate secondary antibody. After scoring the results 

of this staining, which was usually negative, clones were 
stained with GFAP and the secondary layer for the first 
set of surface antibodies. Finally, the secondary 
antibody for GFAP was added. This procedure allowed 

20 staining with four antibodies using only three 

fluorescent-color conjugated secondary antibodies. 

E-NCAM-immunoreactive cells constituted 60%±3% of all 
cells present in dissociated culture 24 hours after 
plating. The majority of the remaining cells were A2B5*. 

25 It has been shown in U.S. Patent Application Serial No. 

08/852,744 that at this stage of development, A2B5- 
immunoreactive cells are glial precursor cells. 
Consistent with these results, 3-III tubulin or E-NCAM- 
immunoreactive cells did not co-express A2B5. The vast 

30 majority of cultured E-NCAM-immunoreactive cells (85%±8%) 

co-expressed P-III tubulin immunoreactivity as well as 
nestin immunoreactivity, but not markers characteristic of 
glial precursor immunoreactivity. Approximately 20% of 
the E-NCAM* cells divided in a 24-hour period. Most of 

35 the dividing E-NCAM* cells did not co-express p-III 

t\ibulin, indicating that this population of -cells could 
represent a dividing neuroblast. It is not yet known 



wo 99/01159 



26 



PCT/US98/13875 



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wo 99/01159 



PCT/US98/13875 



27. 

whether a higher percentage of the cells would be observed 
to divide under these conditions with longer labeling 
periods. However, even if this population were to include 
5 a subset of cells sufficiently committed to neuronal 

differentiation as to no longer engage in division, these 
committed neurons would be eliminated from the population 
with expansion and division in tissue culture. Table 2 
siammarizes results of the antigenic profile of the cells, 
10 showing the percentages of E-NCAM* cells from E13.5 

embryos that express various other antigens. These 
results show that E-NCAM* cells from E13.5 spinal cord 
express neuronal, but not glial, markers. 



15 



Table 2 | 


Antigen 


% Expression | 


a-Nestin 


98% 


a-3-III tubulin 


50% 


RT-97 


95% 


a-NF M 


100% 


a-MAP kinase 


100% 


A2B5 


0% 


a-GFAP 


0% 


1 a-NF 60 


0% 


1 a-GalC 


0% 


1 a-Peripherin 


0% 



Example 3 

To determine the differentiation potential of E-NCAM- 
30 immunoreactive cells, E-NCAM* cells were purified by 

immunopanning and plated at clonal density in gridded 
dishes. E13,5 cells were prepared according to the 
procedure of Example 2. An E-NCAM"" cell -population was 
purified from these E13.5 cells using a specific antibody- 
35 capture technique according to the procedure of L. Wysocki 

& V. Sato, '"Panning" for Lymphocytes: A Method for Cell 



wo 99/01159 



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28 

Selection, 75 Proc. Nat'l Acad. Sci. USA 2844-48 (1978); 
M. Mayer et al., supra, hereby incorporated by reference. 
In brief, cells were trypsinized and the resulting cell 
suspension was plated on an A2B5-antibody-coated dish to 
5 allow binding of all A2B5* cells to the plate. The 

supernate was removed, and the plate was washed with DMEM 
supplemented with additives described by J. Bottenstein 
and G. Sato, Growth of a Rat Neuroblastoma Cell Line in 
Seriam-free Supplemented Medium, 76 Proc. Nat'l Acad. Sci. 

10 USA 514-17 (1979), hereby incorporated by reference, 

(DMEM-BS) • The supernate was then plated on an E-NCAM- 
antibody-coated dish to allow binding of the E-NCAM- 
immunoreactive cells. The bound cells were scraped from 
the plate and replated on f ibronectin/laminin-coated glass 

15 coverslips in 300 ml DMEM-BS ± growth factors at 5000 

cells/well. 

The A2B5 and E-NCAM antibodies for coating the plates 
were used at concentrations of 5 Mg/ml. Cells were 
allowed to bind to the plate for 20-30 minutes in a 37**C 
20 incubator. Growth factors were added every other day at a 

concentration of 10 ng/ml. Recombinant bFGF and 
neurotrophin 3 (NT-3) were purchased from PeproTech, and 
retinoic acid (RA) was obtained from Sigma. 

After 24 hours, some immunopanned E-NCT^* cells were 
25 assayed by immunocytochemistry according to the procedure 

of Example 2. Greater than 95% of the cells were E-NCAM* 
at that time. Purified and stained cells were plated on 
gridded clonal dishes, and individual E-NCAM'' -cells were 
identified and followed over time by immunocytochemistry 
30 according to the procedure of Example 2. 

Of all the cytokines tested, optimum growth was 
observed when cells were cultured in FGF (10 ng/ml) and 
NT-3 (10 ng/ml). In the presence of FGF and NT-3, single 
E-NCAM* cells divided in culture to generate colonies 
35 ranging from one to several hundred cells. By day 5, most 

colonies contained between 20 and 50 daughter cells that 
continued to express E-NCAM immunor-eactivity . Daughter 
cells appeared phase bright and had short processes. At 



159 



PCTAJS98/I3875 



29 

this stage, most E-NCAM*positive cells did not express 3^ 
III tubulin or neurofilament -M iramunoreactivity . 

To promote differentiation of E-NCAM* clones, the 
FGF- and NT-3-containing medium was replaced with medium 
containing retinoic acid (RA) and from which the mitogen, 
bFGF, was withheld. In this differentiation medium, E- 
NCAM* cells stopped dividing and elaborated extensive 
processes and started to express neuronal markers. 
Quadruple-labeling of clones with neuronal and glial 
markers and DAPI histochemistry, to identify all cells, 
showed that all clones contained P-III tubulin- 
immunoreactive cells and neurof ilament-M (NF-M) 
immunoreactive cells and that none of the E-NCAM* clones 
differentiated into oligodendrocytes or astrocytes. 

Table 3 summarizes the results obtained by quadruple 
labeling of 124 E-NCAM* clones with DAPI, a-P-III tubulin, 
A2B5, and a-GFAP. 



Table 3 | 


Antigen Expressed 


% of Clones 1 


a-3-III tubulin 


100% 


A2B5 


0% 


a-GFAP 


0% 1 



Example 4 

In this example, immunopanned A2B5'' cells derived 
from dissociated E13.5 spinal cords according to the 
procedure of Example 2 were cultured in neuron-promoting 
medium, i.e. basal medium plus FGF and NT-3. Cultures 
were grown for 5 days and then switched to RA-containing 
medium as described in Example 3 , and sister plates were 
stained for either E-NCAM or A2B5 immunoreactivity . 

No A2B5 immunopanned cell expressed E-NCAM 
immunoreactivity when grown under conditions that promote 
growth of neuronal cells. All A2B5 immunopanned cells, 
however, continued to express A2B5 immunoreactivity, 
indicating that neuron-promoting conditions do not affect 



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the survival and proliferation of glial precursor cells. 
Thus, the inability to detect oligodendrocyte and 
astrocyte differentiation in Example 3 was unlikely to be 
due to the death in neuronal cultures of oligodendrocytes 
5 and astrocytes that might have differentiated from E-NCAM'' 

precursors since A2B5 glial precursor cells purified and 
grown in parallel in the presence of FGF and NT-3 
continued to express A2B5 without apparent cell death and 
generated healthy oligodendrocytes and astrocytes after 10 

10 days in culture. In addition, cells never generated 

neurons in the presence of FGF and NT-3 and showed no 
expression of E-NCAM at any time tested. Thus, E-NCAM 
immunoreactive cells, unlike A2B5-immunoreactive glial 
restricted precursors, could not differentiate into 

15 oligodendrocytes and appeared limited to neuronal 

differentiation when compared to multipotential E10.5 
neuroepithelial cells. 



Example 5 

20 While it has been clearly shown in the present system 

that E-NCAM identifies neuronally restricted precursor 
cells, it has been reported that certain glial precursors 
at later stages of development can also express E-NCAM 
immunoreactivity. This observation raises the possibility 

25 that some E-NCAM"" cells identified by the presently 

described methods may be bi-potential . To test this 
possibility, E-NCAM" cells were plated clonally in either 
neuron-promoting medium (F-GF + NT-3) or in glial-promoting 
medium (FGF +10% fetal calf serum) and compared for their 

30 development. Medium containing FGF with 10% fetal calf 

serum was chosen for glial differentiation since this 
medium promotes astrocyte differentiation of both -spinal 
cord NEP cells as well as A2B5 immunoreactive A2B5 glial 
precursor cells, as shown in U.S. Patent Application 

35 Serial No. 08/ 852,744. All E-NCAM* clones (24/24) that 

were grown in neuron-promoting medium contained only 3-III 
tubulin* cells after 8 days, while the clones grown in 
seriim-containing medium did not generate astrocytes or 



wo 99/01 159 PCT/US98/13875 

31 

proliferate. From a total of 97 E-NCAM"" cells grown in 
glial-promoting conditions, 90 clones (92%) consisted of a 
single dead cell after 24 hours, while the remaining 7 
clones (8%) contained 1 or 2 dead cells after 48 hours. 
5 Thus, E-NCAM iramunoreactive cells, in contrast with glial 

precursor cells, fail to proliferate or differentiate in 
astrocyte-promoting conditions - 

Example 6 

10 To determine whether the restriction of E-NCAM" cells 

to generation of neurons also includes a restriction to 
generation of certain subtypes of neurons, E-NCAM* clones 
grown in RA and NT-3 in the absence of FGF were examined 
for the expression of different neurotransmitters. The 

15 antibodies used in this example are described in Table 4. 



Table 4 


Antibody/Kind 


Source 


Antigen 
Recognized 


Cell Type 
Recognized 


Anti-ChAT/goat 
IgG 


Chemic 
on 


Choline acetyl 
transferase 


Motoneurons 


Anti- 
Glut amate / rabb 
it IgG 


Chemic 
on 


Glutamate 


Excitatory 
neurons 


Anti- 
GABA/ rabbit 
IgG 


Chemic 
on 


Gamma amino 
butyric acid 


Inhibitory 
neurons 



These results indicate that individual clones could 
generate GABA-ergic, glutaminergic, and cholinergic 
30 neurons. Of ten clones tested, all contained 

glutaminergic, GABAergic, and cholinergic neurons. Thus, 
E-NCAM-immunoreactive cells, while limited to 
differentiating neurons, are capable of generating 
excitatory, inhibitory, and cholinergic neurons. 

35 

Example 7 

Primary clones of E-NCAM* cells grown in FGF and NT-3 
according to the procedure of Example 5 grew to large 



159 



PCT/US98/13875 



32 

sizes of several hundred cells after 7 to 10 days in 
culture, indicating some degree of self renewal. To 
demonstrate prolonged self renewal of the E-NCAM" 
population, selected clones were followed by secondary and 
tertiary subcloning. Individual E-NCAM* cells from E13-5 
spinal cord were plated in f ibronectin/laminin and 
expanded for 7 days in the presence of FGF and NT-3. Five 
individual clones were randomly selected and replated at 
clonal density using the same expansion conditions. The 
nxamber of secondary clones was counted, and large clones 
were selected and replated. The number of tertiary clones 
obtained was counted, and clones were then induced to 
differentiate into postmitotic neurons by replacing FGF 
and RA. 

All clones examined generated niomerous daughter 
clones that subsequently generated tertiary clones. Small 
clones and very large clones showed similar self renewal 
potential. When tertiary clones were switched to a medium 
containing RA and lacking FGF, the majority of cells in a 
clone differentiated into post-mitotic neurons expressing 

tubulin. Thus, E-NCAM* cells are capable of 
prolonged self renewal and can generate multiple daughter 
cells capable of generating neurons. 

These results suggest that E-NCAM immunoreactivity 
identifies a neuroblast cell that can differentiate into 
multiple neuronal phenotypes in culture, even after 
multiple passages. NT-3 and FGF are required to maintain 
the blast cell in a proliferative state, while RA promotes 
differentiation. 

Example 8 

It has been shown previously that individual NEP 
cells derived from E10.5 spinal cord are an E-NCAM- 
immunonegative, multipotent, self renewing population of 
cells that <:an generate neurons, astrocytes, and 
oligodendrocytes (U.S. Patent Application Serial No. 
08/852,744). To determine if neuronal differentiation 
from NEP precursors involved the generation of an E-NCAM'' 



wo 99/01159 



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33 

intermediate neuronal precursor cell, NEP cell cultures 
that were induced to differentiate in vitro were examined 
for the presence of E-NCTVM"^ immunoreactive cells. 

NEP cells were prepared according to the method 
5 described in Serial No. 08/852,744. Briefly, Sprague 

Dawley rat embryos were removed at E10.5 (13-22 somites) 
and placed in a petri dish containing Ca/Mg-free Hanks 
balanced salt solution (HBSS, GIBCO/BRL) . The trunk 
segments of the embryos (last 10 somites) were dissected 

10 using tungsten needles, rinsed, and then transferred to 

fresh HBSS. Trunk segments were incubated at 4**C in 1% 
trypsin solution (GIBCO/BRL) for a period of ten to twelve 
minutes. The trypsin solution was replaced with fresh 
HBSS containing 10% fetal bovine serum (FBS, GIBCO/BRL) . 

15 The segments were gently triturated with a Pasteur pipette 

to release neural tubes free from surrounding somites and 
connective tissue. Isolated neural tuJ^es were transferred 
to a 0.05% trypsin/EDTA solution (GIBCO/BRL) for an 
additional period of ten minutes. Cells were dissociated 

20 by trituration and plated at high density in 35 ram 

f ibronectin-coated dishes in NEP medium. Cells were 
maintained at 37**C in 5% C02/95% air. Cells were replated 
at low density, i.e. ^5000 cells per 35 mm plate, one to 
three days after plating. Cells from several dishes were 

25 then harvested by trypsinization (0.05% trypsin/EDTA 

solution for two minutes). Cells were then pelleted, 
resuspended in a small volume, and counted. About 5000 
cells were plated in a 35 ram dish (Corning or Nunc) . 

NEP cells derived from E10.5 embryos were expanded in 

30 the presence of FGF and CEE for 5 days and differentiated 

by replating on laminin in the presence of CEE. 
Dif ferentiating NEP cells were triple-labeled with 
antibodies to E-NCAM, GFAP, and GalC. This showed that E- 
NCAM-immunoreactive cells that differentiated from NEP 

35 cells did not express astrocytic «3FAP) or 

oligodendrocytic (GalC) markers. A sister plate was 
double-labeled with antibodies to E-NCAM and nest in. This 
showed that E-NCAM immunoreactive cells that 



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34 

differentiated from NEP cells co-express nestin. 
Differentiating NEP cells were incubated for 24 hours with 
BrdU and subsequently double-labeled with an antibody 
against BrdU and E-NCAM. This showed that most E-NCAM- 
5 immunoreactive cells divided in 24 hours. This higher 

labeling rate may reflect differences in the isolate 
procedure as compared to the previous example. Table 5 
summarizes the antigenic profile of E-NCAK" cells derived 
from E10.5 NEP cells. Note that NEP-derived E-NCAM* cells 
10 are antigenically similar to E13.5 E-NCAM* cells and, like 

E13.5 E-NCAM*, do not express any of the glial markers 
examined. 



Table 5 



1 Antigen 


Expression 1 


a-Nestin 




a-3-III tubulin* 




A2B5 




a-GFAP 




a-Galc 





A siibset of cells express this marker. 



Thus, induced NEP cultures comprise multiple 
phenotypes, including E-NCAM* cells. Like the E13.5 E- 

25 NCAM* cells, NEP-derived E-NCAM* cells did not express 

glial markers, but co-expressed p-III tubulin (20-30%) and 
nestin (70-80%) iramunoreactivity . Ninety percent of 
panned E-NCAM* cells incorporated BrdU in culture and 
generated neurons after addition of RA or NT-3 and thus 

30 appeared similar to the E13.5 E-NCAM-immunoreactive cells. 

Example 9 

To determine whether single NEP-derived E-NCAM* cells 
were also restricted to neurons in their -differentiation 
35 potential, cells were studied in clonal culture, NEP 

cells were induced to differentiate by replating on 
laminin and withdrawal of CEE, as described in U.S. Patent 



wo 99/01159 



PCTAJS98/13875 



35 

Application Serial No. 08/852,744. NEP cells derived from 
E10.5 embryos were expanded in the presence of FGF and CEE 
for 5 days and differentiated by replating on laminin in 
the absence of CEE. Immunopanned E-NCAM-immunoreactive 
5 cells were then plated on clonal-grid dishes (Greiner 

Labortechnik) coated with f ibronectin/laminin, and single 
cells were followed in culture. After 5 days, clones were 
switched to RA and FGF was withdrawn. Clones were allowed 
to grow for an additional 3 days, fixed with 

10 paraformaldehyde, and triple-labeled with A2B5 and 

antibodies against GFAP and tubulin. In addition, 

cells were counterstained with DAPI to show individual 
cell nuclei. Table 6 summarizes the results of the 
staining of all 47 clones studied (8 of 47 clones did not 

15 survive replating) . Note that no clone contained 

astrocytes {GFAP"") cells or glial precursor cells (A2B5*) . 



Table 6 || 


Antigen Expressed 


% of Clones | 


a-3-III tubulin 


100% 1 


A2B5 


0% I 


1 a-GFAP 


0% 1 



25 Forty-eight hours after cells were induced to 

differentiate, 10-30% of the cells had begun to express E- 
NCAM immunoreactivity. NEP-cell-derived E-NCAM* cells 
were selected by immunopanning according to the procedure 
of Example 3, and individual E-NCAM"" cells were plated in 

30 medium containing FGF and NT-3 and clones were analyzed 

after 10 days. 

All clones contained only E-NCAM'^/P-III-tubulin"' 
cells, but not GFAP or A2B5 immunoreactive cells. In 
addition, individual E-NCAM"" cells failed to differentiate 

35 into oligodendrocytes or astrocytes under culture 

conditions that promoted astrocytic and oligodendroglial 
differentiation from the parent NEP cell population. E- 
NCAM'' cells could be maintained as dividing precursor 



wo 99/01159 



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36 

cells in defined medium in the presence of high 
concentrations of FGF (10 ng/ml) and NT-3 (10 ng/ml) . E- 
NCAM"" cells maintained for up to three months could 
readily differentiate into tubulin* mature neurons 

5 that expressed a variety of neurotransmitter phenotypes 

when exposed to RA grown on laminin. ThuSr E-NC7\M'' cells 
are similar to E13.5 neuronal precursors in their 
differentiation potential, antigenic profile, and in the 
conditions optimal for extended growth as a dividing 
10 precursor cell population. 

Example 10 

Differentiation of the E-NCAM"" population from an 
apparently homogeneous NestinVE-NCAM" NEP cell population 

15 suggests a progressive restriction in developmental fate. 

It was thought possible, but unlikely, that individual NEP 
cells could be pre-committed to generating neuroblasts or 
glioblasts. To rule out this possibility, individual NEP 
clones were examined for their ability to generate E-NCAM- 

20 imraxanoreactive cells and A2B5-immunoreactive cells. A2B5 

and E-NCAM were chosen since it had previously been shown 
that A2B5 immunoreactivity is unique to oligodendrocyte- 
astrocyte precursors at this stage of development. NEP 
cells derived from E10.5 embryos were expanded in the 

25 presence of FGF and CEE for 5 days, harvested by 

trypsinization, and replated at clonal density in gridded 
clonal dishes. After 7 days in culture, individual clones 
were double-labeled with antibodies against E-NCAM and 
A2B5 according to the procedure of Example 2. Of 112 NEP 

30 clones that were followed in culture, 83% generated both 

A2B5 and E-NCAM immune reactive cells. Five percent of the 
clones consisted of only A2B5 immunoreactive cells, and 
12% of the clones showed no convincing staining for either 
A2B5 or E-NCAM immunoreactivity. In all clones tested, E- 

35 NCAM and A2B5 were expressed in non-overlapping 

populations. That is, no cell co-expressed both markers. 
Table 1 summarizes the results obtained with 112 clones. 



wo 99/01159 



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37 



1 Table 7 


Antigen Expressed 


% of Clones 


Niamber of Clones 


E-NCAMVA2B5* 


83% 


93 


A2B5* alone 


5% 


6 


E-NCAM"/A2B5" 


12% 





Thus, the majority of NEP cells appear to be capable 
of generating precursors for glial restricted cells as 
well as neuronal restricted precursors. 

10 

Example 11 

To test if most neurons were generated via an E-NCAM* 
intermediate neuroblast, complement-mediated cell lysis 
was utilized to selectively kill E-NCAM"" cells. Twenty- 

15 four hours after replating NEP cells in differentiating 

conditions, E-NCAM-immunoreactive cells were killed using 
an IgM antibody to E-NCAM and guinea pig complement. In 
sister plates, glial precursors were killed using an anti- 
A2B5 IgM antibody and complement. At this stage in 

20 development, most E-NCAM+ cells do not express P-III 

tubulin- Treated plates were allowed to differentiate for 
an additional three days, and the development of neurons 
was monitored. E-NCAM-mediated lysis significantly 
reduced the niamber of P-III tubulin-immunoreactive cells 

25 that developed when compared to cultures treated with A2B5 

(219 ± 35 versus 879 ± 63, respectively) suggesting that 
neuronal differentiation from NEP cells in vitro requir-es 
a transition through an E-NCAM immunoreactive state. 

30 Example 12 

Diffg;:gntj-ated S-NCAM' Cells Can Pj.st4nffuished from 
Acutely Dissociated NRP Cells 

ENCAM"" cells were isolated by immunopanning according 
35 to the procedure of Example 3, plated in 35 mm dishes, and 

allowed to grow for 24 hours (acutely dissociated) or It) 
days (differentiated) . Cultured cells were then analyzed 
for cell division by BRDU incorporation, E-NCAM 



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38 

expression, NF-M expression, and synaptophysin expression 
according to the procedure of Example 2. TUDOut 70% of 
acutely dissociated E-NCAM'' cells incorporated BRDU, 
showing that such cells were dividing in culture, whereas 
5 after 10 days in differentiation promoting medium few or 

no cells incorporated BRDU, and had therefore stopped 
dividing. Double-labeling for E-NCAM and NF-M 
immunoreactivity showed that very few acutely dissociated 
cells expressed NF-M, whereas nearly all differentiated 

10 cells expressed this protein. Similarly, synaptophysin, a 

protein specifically associated with synaptic vesicles and 
functional synapses, see T.C. Sudhof, The Synaptic Vesicle 
Cycle: A Cascade of Protein-Protein Interactions, 375 
Nature 645-653 (1995), was expressed by differentiated but 

15 not acutely dissociated ENCAM* cells. Although 

synaptophysin protein expression was associated with 
synaptic vesicles, early expression could also be detected 
in the cell bodies and throughout the lengths of the 
processes where it was initially expressed during 

20 neurogenesis. M. Fujita et al.. Developmental Profiles of 

Synaptophysin in Granule Cells of Rat Cerebellum: An 
Immunocytochemical Study, 45 J. Electron Microsc, Tokyo 
185-194 (1996); D. Grabs et al.. Differential Expression 
of Synaptophysin and Synaptoporin during Pre- and 

25 Postnatal Development of the Rat Hippocampal Network, 6 

Eur. J. Neurosci. 1765-1771 (1994) . These results show 
that acutely dissociated E-NCAM* cells are immature, 
dividing cells that mature in culture. These results 
suggest that if NRP cells are induced to differentiate by 

30 RA and the removal of mitogen, they acquire many 

morphological and immunological properties of mature 
neurons . 



Example 13 

35 Numerous Neuronal .Phenotypes Can Re Dete<:ted in 

Differentiated but Not Acutely Dissociated E-NCAM^ Cells 

It was shown above that NRP cells can differentiate 
into postmitotic neurons, but not into oligodendrocytes or 



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39 

astrocytes. To determine if NRPs can differentiate into 
all of the major neuronal phenotypes present in the spinal 
cord, or whether they are more limited in their 
differentiation potential, the expression of 
5 neurotransmitter synthesizing enzymes and cell type 

specific markers for mature neurons was examined after 
inducing NRPs to differentiate- In addition, the 
expression of p75, Q, Yan & E.J. Johnson, An 
Immunocytochemical Study of the Nerve Growth Factor 

10 Receptor in Developing Rats, 8 J, Neurosci. 3481-3498 

(1988), and Islet-1, T. Tsuchida et al.. Topographic 
Organization of Embryonic Motor Neurons Defined by 
Expression of LIM Homeobox Genes, 79 Cell 957-970 (1994), 
which are characteristic of motoneurons in the spinal 

15 cord, and calbindin, which is often co-expressed with 

GABA, C. Batini, Cerebellar Localization and 
Colocalization of GABA and Calcium Binding Protein-D28K, 
128 Arch. Ital, Biol. 127-149 (1990), were examined. 
E-NCAM^ cells from E13.5 rat neural tube were 

20 isolated by immunopanning according to the procedure of 

Example 3, plated in 35 mm dishes, and cultured in 
differentiation-promoting medium. After 10 days in 
culture, total RNA was isolated from these cells and the 
ability to synthesize the neurotransmitters acetylcholine 

25 (Ach) , GABA, and glutamate was assessed by the expression 

of their synthesizing enzymes by RT-PCR. Total RNA was 
isolated from cells or whole tissues by a modification of 
the guanidine isothiocyanate-phenol-chloroform extraction 
method (TRIZOL, Gibco/BRL) . For cDNA synthesis, 1-5 of 

30 total RNA was used in a 20 //I reaction using SUPERSCRIPT 

II (Gibco/BRL) , a modified Maloney murine leukemia virus 
reverse transcriptase (RT) , and oligo (dT) 12-18 primers 
according to the Gibco/BRL protocol. 

For PCR amplification of the cDNA, aliquots of cDNA, 

35 equivalent to 1/20 of the reverse transcriptase reaction, 

were used in a 50 ^1 reaction vol\ime. PCR amplification 
was performed using ELONGASE polymerase (^ibco/BRL) . 
Primer sequences and cycling temperatures used for PCR 



wo 99/01 159 PCT/US98/13875 

40 

amplification of receptors are shown in Table 8. The 
reactions were run for 35 cycles, and a 10-minute 
incubation at 72**C was added at the end to ensure complete 
extension. The PGR products were purified using the 
5 ADVANTAGE PCR-PURE kit (Clontech, Palo Alto, CA) and 

sequenced to confirm their identities. 



Table 8 


Gene 


Product Size (bp) 


Primers (sense. 


p75 


329 


SEQ ID NOS:l and 


ChAT 


377 


SEQ ID N0S:3 and 
4 


Isl-1 


350 


SEQ ID NOS:5 and 
6 


GADes 


327 


SEQ ID N0S:7 and 
8 


calbindin28 


276 


SEQ ID N0S:9 and 
10 


glutaminase 


560 


SEQ ID N0S:11 and 
12 


1 cyclophilin 


302 


SEQ ID N0S:13 and 
14 



As shown in FIG. 2, all of these were present in 
differentiated cells (labeled ''D") . In contrast, none of 

20 these markers of neurotransmitter phenotypes could be 

detected from cells that were examined within 24 hours of 
isolation (teirmed "acutely dissociated;" labeled as "AD" 
in FIG. 2), even though expression of the housekeeping 
gene, cyclophilin, could be readily detected from both 

25 cell populations. These data show that NRP cells mature 

in culture and that NCAM expression and neuronal fate 
determination precede neurotransmitter synthesis. 



wo 99/01159 



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41 

The expression of neurotransmitter synthesizing 
enzymes was also examined by immunocytochemistry to 
determine whether all cells, or only a subset of 
differentiated cells, express these markers. Cells were 
5 grown in culture for 10 days and allowed to differentiate, 

fixed, and processed by immunocytochemistry according to 
the procedure of Example 2 to detect expression of choline 
acetyltransferase (ChAT) , glutamic acid decarboxylase 
(GAD), tyrosine hydroxylase (TH), glycine, and glutamate. 

10 Antibodies to ChAT, TH, and -GAD were obtained from 

Chemicon; antibodies to glutamate and glycine were from 
Signature Immunologicals. Virtually 100% of the 
differentiated cells expressed detectable glutamate 
levels, A much smaller percentage expressed glycine and 

15 GAD. Exact percentages varied between experiments from 

10-50%. The percentage of ChAT and TH'' cells were even 
smaller and ranged between 1-5%. However, substantially 
larger niombers could be seen by altering culture 
conditions. Since virtually 100% of the cells synthesized 

20 glutamate, it is likely that at least some cells 

synthesized more than one neurotransmitter. Nevertheless, 
these results clearly show that upon differentiation, E- 
NCAM^ cells are capable of maturing into a heterogeneous 
population with respect to their neurotransmitter 

25 phenotype. 

In contrast to the results obtained with 
differentiated cells, neither ChAT, -GAD, TH, nor glycine 
could be detected in acutely dissociated cells. <31utamate 
was detected in a small subset of such cells (less than 

30 10%) . Glutaminase, however, could not be detected in 

these cells by RT-PCR (FIG. 2), which suggests that 
glutamate was being taken up by these cells from the 
medivun. 



35 



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42 

Example 14 

The Neurotransmitter Receptor Profile of E-NCAM^ Cells 
Changes with Maturation 

5 Another important characteristic of mature neurons is 

their ability to respond to multiple neurotransmitters by 
expressing appropriate neurotransmitter receptors on their 
surfaces • To examine the ability of differentiated E- 
NOAM" cells to respond to glutamate, glycine, dopamine, 

10 and acetylcholine, fura-2 Ca^"" imaging techniques were 

used. £13. 5 E-NCAM* cells were grown in culture for 10 
days and allowed to differentiate. They were then loaded 
with fura-2, and the depolarizing response to 
neurotransmitter application was monitored. 

15 Cells were loaded with 5 Fura-2/AM, D. Grynkiewicz 

et al., A New Generation of Calciiim Indicators with 
Greatly Improved Fluorescence Properties, 260 J. Biol. 
Ghem. 3440-3450 (1985), hereby incorporated by reference, 
plus PLURONIC F127 (80 Mg/ml) in rat ringers (RR) at 23°C 

20 in the dark for 20 minutes followed by 3 washes in RR and 

a 30-minute desterif ication. Relative changes in 
intracellular calcium concentration were measured from the 
background-corrected ratio of fluorescence intensity by 
excitation at 340/380 nm. Response was defined as a 

25 minimiam rise of 10% of the ratioed baseline value, A 

Zeiss-Attof luor imaging system and software (Atto 
Instruments Inc., Rockville, MD) were used to acquire and 
analyze the data. Data points were sampled at 1 Hz. 
Neurotransmitters were made in RR and delivered by bath 

30 exchange using a small volume loop injector (200 Ml) . RR 

contained 140 mM NaCl, 3 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 
mM HEPES, and 10 mM glucose. In addition, 500 MM ascorbic 
acid was added to dopamine solutions to prevent oxidation. 
Control application of 500 MM ascorbic acid had no effect. 

35 The pH of all solutions was adjusted to 7.4 with NaOH. 

Further, 50 mM K* RR was made by substituting equimolar K"" 
for Na"" in the normal RR. 

FIG. 3 shows a bar graph of the nuitd^er of cells 
responding to application of the indicated 



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43 

neurotransmitter on acutely dissociated and differentiated 
cells. In general, the number of cells responding to 
neurotransmitters and the amplitude of the 
neurotransmitter-induced Ca^* responses increased in the 
5 differentiated cells. The most striking example was 

dopamine, where only 10% of the acutely dissociated cells 
responded to 500 dopamine with increases in internal 
Ca^" compared to 76% of differentiated cells, a net 
increase of 66%. Similar, but less striking, changes in 

10 the number of cells responding were seen for other 

excitatory neurotransmitters. The exceptions to this 
trend were the Ca^"" responses to GABA and glycine. 
Interestingly, 46% of the acutely dissociated cells 
responded to GABA compared to only 8% of the 

15 differentiated cells. Similarly, Ca^^ flux in response to 

glycine decreased from 20% in the acutely dissociated 
cells to 0% in the differentiated cells. This change in 
the inhibitory neurotransmitter profile probably reflects 
the decrease in internal chloride ion concentration with 

20 maturation that accounts for the shift from depolarizing 

to hyperpolarizing GABA and glycine responses. W. Wu et 
al.. Early Development of Glycine and GABA-Mediated 
Synapses in Rat Spinal Cor-d, 12 J. Neurosci. 3935-3945 
(1992). The possibility cannot be excluded, however, that. 

25 chloride ion levels remain elevated and fewer €ABA and 

glycine receptors are expressed in the differentiated 
cells. Representative plots of the ratio of {l3<o/l3Bo) Ca^^ 
responses over time from an acutely dissociated and 
differentiated cell are shown in FIG. 4 and FIG. 5, 

30 respectively. The acutely dissociated cell responded to 

GABA and glutamate, whereas the differentiated cell from 
the same embryo responded to dopamine, glutamate, and 
acetylcholine, but not to GABA or glycine. Comparison of 
Ca^* responses to the various transmitters in adjacent 

35 cells revealed that there is heterogeneity in the response 

profiles among cells, indicating that not only are the 
NCAM* cells heterogeneous in their ability to synthesize 
neurotransmitters, they are also sel-ected in terms of 



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transmitter receptor expression. In addition to 
neurotransmitters, elevated in rat ringers (50 mM K"" RR) 
was applied to depolarize the cells and allow Ca^"" entry 
through voltage-gated channels. In acutely dissociated 
5 cells, 4 9% responded to 50 mM RR compared to 85% of 

differentiated cells, suggesting that more of the 
differentiated cells were electrically competent than were 
the acutely dissociated cells. 

Thus, the contrast between the various properties of 

10 acutely dissociated E-NCAM* cells and fully differentiated 

E-NCAM"" cells, which are summarized in Table 9, is 
striking. Immature cells are mitotically active, but 
differentiated cells are not. Immature cells do not 
express any mature neuronal proteins such as NF-M, 

15 synaptophysin, or neurotransmitter synthetic enzymes, 

whereas all of these can be detected in differentiated 
cells. Moreover, acutely dissociated cells are overall 
less responsive than differentiated cells to 
neurotransmitter-induced Ca^"^ responses. 



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Table 9 






Property 


Acutely 
Dissociated 


Differentiated 




Mitotic Status 


Mitotic 


Postmitotic 


5 


Cell Size 

■ 


Comoa ra t i ve 1 v 
smaller 


Comparatively 
larger 




Process Outgrowth 


Little or none 


Extensive 




Neuronal Markers 


NCAM, PIII- 
tubulin, MAP-2 
kinase, nestin 


NCAM, PIII- 
tubulin, MAP-2 
kinase, NF-M, 
synaptophysin, 
peripherin 


10 


Neurotransmitters 

neurotransmitter 
synthetic 
enzymes, or other 
phenotypic 

1 ^riPf*! "fir* TTlAT'lcPT'^ 
1 OUCW^J«J.W illO X. JVC A. o 


None , except for 
a small amount of 
glutamate 
immunoreactivity . 


Glutamate, 
glycine, 

glutaminase, GAD, 
ChAT, Isl-1, p75, 
calbindin 


15 


Response to 
neurotransmitters 


Weak, in a small 
subset of cells. 


Robust and in 
virtually all 
cells . 




Depolarizing 
response to GABA 
and glycine 


All responses 
measured were 
depolarizing. 


Few or no 
depolarizing 
responses were 
detected. 



20 

Example 15 

Individual E-NCAM"^ Cells Can <Senerate Multiple 
Neurotransmitter Phenotvoes 

Mass culture experiments described above showed that 



25 the E-NCAM"" population can generate multiple 

neurotransmitter phenotypes. There existed the 
possibility, however, that individual cells could be pr«- 
committed to generating specific neuronal phenotypes. To 
determine whether the differentiation potential of NRPs i-n 

30 mass culture reflected the potential of an individual NRP, 

clonal analysis of E-NCAM^ cells was performed. E-NCAM"" 
cells were immunoselected according to the procedure of 
Example 3, plated at clonal density, and grown in FGF and 
NT-3, conditions that promote proliferation. Clones grew 



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to sizes of several hundred cells after 10 days in 
culture, after which their differentiation was promoted by 
withdrawal of FGF and addition of RA in the medium. 

Three different techniques were used to determine 
5 whether clones generated from individual NRP cells were 

composed of heterogeneous populations of neurons: RT-PCR 
according to the procedure of Example 13, 
immunocytochemistry according to the procedure of Example 
2, and calciiim imaging according to the procedure of 

10 Example 14. Six clones were examined by RT-PRC analysis. 

Five of the six clones expressed multiple neurotransmitter 
phenotypes: one clone expressed all six markers tested, 3 
clones expressed four markers, and 1 clone expressed three 
markers. Therefore, all but one clone were composed of 

15 heterogeneous populations of cells. One clone expressed 

detectable levels of only p75 and Isl-1, but not ChAT. 
This likely represented an immature clone that had not 
fully differentiated. FIG, 6 shows results from a 
representative clone that expressed all neurotransmitter 

20 markers tested. These results demonstrate that individual 

clones express multiple neurotransmitter synthetic enzymes 
or other phenotypic markers, and that most clones were 
composed of a heterogeneous population. 

To confirm the PGR results and to show heterogeneity 

25 at the protein level, clones were analyzed for expression 

of p75- No clone (0/17) consisted of exclusively p75 
immunoreactive cells, but all clones (17/17) contained p75 
iramunoreactive cells as well as other neurons. Similarly, 
staining for either glutamate or glycine immunor^activity 

30 showed that each transmitter was expressed by only a 

subset of cells in the same clonal population, indicating 
that clones are a heterogeneous population. 

Heterogeneity was demonstrated not only by the 
synthesis of different neurotransmitters, hut also hy 

35 heterogeneity in the receptors expressed by the cells. 

The response profiles of differentiated clonal cells to 
application of -GABA, glycine, dopamine, glutamate, 
acetylcholine, and 50 mM K+ RR, as evidence by increased 



wo 99/01 159 PCT/US98/13875 

47 

intracellular calcixom concentrations, were examined. Ca"^^ 
measurements were taken from as many as 113 cells from 4 
different clones. All clones examined (4/4) displayed 
heterogeneity in their response profiles, which varied 
5 somewhat between individual clones, FIG. 7 shows a bar 

graph of the percentage of cells from all 4 clones that 
responded to each of the applied neurotransmitters. 

As with the mass cultures of differentiated E-NCAM* 
cells, high percentages of clonal cells responded to 

10 glutamate (93%), acetylcholine (96%), 50 mM K+ RR (70%), 

and dopamine (50%), whereas few cells responded to GABA 
(27%) and glycine (1%) . FIGS. 8 and 9 show representative 
traces of the ratio (I340/I3B0) of Ca^" responses from two 
cells recorded from one clone. This heterogeneous 

15 expression of receptors also suggested a raultipotential 

characteristic of individual NRP cells. Thus, the 
maturation of clonal populations of cells closely 
resembled the maturation of cells in mass culture. 

By multiple independent methods, this clonal analysis 

20 demonstrates the multipotential characteristic of 

individual NRP cells. This analysis confirms the mass 
culture results that clearly define the developmental 
potential of the NRP cell. Although committed to 
generating neurons, the particular phenotypes of its 

25 progeny are dictated at some later stage in their 

development. Thus, the existence has been established of 
a neuronal precursor c^ll that can be purified and 
subsequently manipulated to define the transition between 
lineage restricted neuronal precursor and differentiated 

30 neuronal progeny. 



Example 16 

Extracellular Signa ls Influence the Fate of NRP Cells 
The results disclosed herein show that neuronal 
35 precursors can develop in vitro into mature neurons of 

multiple phenotypes in both mass and clonal cultures and 
that either application of RA or removal of FGF can 
promote differentiation into multiple phenotypes. In 



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normal development, however, differentiation is spatially 
and temporally regulated, with motoneurons being generated 
ventrally and sensory neurons being generated dorsally, 
suggesting that specific environmental signals may bias 
5 differentiation of neuronal precursors. In this example, 

the effects of two potentially regulatory molecules that 
are expressed in the spinal cord at the time of 
neurogenesis and have been shown to bias cells to either 
dorsal (BMP-2/4; J.M. Graff, Embryonic Patterning: To BMP 

10 or Not to BMP, That is the Question, 89 Cell 171-174 

(1997)) or ventral (Shh; M.J. Fietz et al.. The Hedgehog 
Gene Family in Drosophila and Vertebrate Development, 
Development (Suppl.) 43-51 (1994)) phenotypes. 

When BMP-2 was added to cultures of E-NCAM* cells, a 

15 dramatic reduction in cell division was seen. The effect 

of BMP-2 overrode the effect of the mitogen, FGF, and even 
in the presence of FGF, caused a 60% reduction in cell 
division (FIG. 10) . Identical effects were seen with BMP- 
4. BMP-2 was not a survival factor, since cells grown in 

20 BMP-2 alone did not survive. The decrease in mitosis was 

accompanied by the appearance of differentiated cells. 
Cell size increased and cells put out extensive processes. 
Cells grown in BMP-2 for 48 hours were also examined for 
neurotransmitter expression. €lutamatergic, GABAergic, 

25 dopaminergic, and cholinergic neurons were detected. The 

number of cholinergic neurons was signficantly larger than 
in untreated controls (5-10% v. 0-1%), however, there 
appeared to be no bias towards ventral phenotypes since 
the promotion of all other phenotypes was also 

30 significantly larger. Thus, BMP-2 acted as an antimitotic 

agent and promoted differentiation of E-NCAM* NRP cells, 
but did not appear to inhibit ventral fates. 

In contrast to the antimitotic and differentiation 
promoting effect of BMP, Shh appeared to be a mitogen. 

35 The mitotic effect of Shh at 100 ng/ml (the maximal 

response) was three-fold higher than controls, but was 
less than the effect of FGF at 10 ng/ml (FIG, 11). 
Experiments with Shh were done in the presence of NT-3, 



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which acts as a survival agent, Y.A. Barde, Neurotrophins : 
A Family of Proteins Supporting the Survival of Neurons, 
390 Prog. Clin. Biol. Res. 45-56 (1994), and not a 
mitogen, because Shh itself did not appear to be a 
5 survival factor for E-NCTVM* cells, i.e. E-NCAM^ cells grown 

in Shh alone did not survive. The effect of Shh on 
mitosis was only apparent after two days of exposure and 
was maintained over 5 days of the assay. No difference in 
cell division was seen during the first 24 hours. 

10 Shh did not appear to promote motoneuron 

differentiation over the 5 days of the assay. Cells 
continued to proliferate and no p75 or ChAT immunoreactive 
neurons could be detected. The failure to see cholinergic 
neurons was not due to an inability of the E-NCAM* cells 

15 to differentiate into p75 or ChAT positive cells, as 

sister cultures readily differentiated into ChAT and p7 5 
immunoreactive cells when treated with a differentiation 
agent such as BMP-2 or RA, Thus, E-NCAM* cells respond to 
Shh by proliferating. Shh unexpectedly did not promote 

20 motoneuron differentiation, at least over the time period 

tested. 

These results indicate that extracellular signaling 
molecules Shh and BMP modulate the phenotypic 
differentiation of E-NCAM" cells. BMP-2 inhibits cell 
25 proliferation and promotes differentiation and does not 

inhibit the differentiation of ventral phenotypes. In 
contrast, Shh promotes proliferation and inhibits the 
differentiation of any neuronal phenotypes, including p75 
and ChAT immunoreactive neurons. 

30 

Example 17 

Mouse Neural Tubes Contain E-NCAM Immunoreactive Neural 
Precursors 

35 To determine whether NRPs are present in mouse neural 

tubes, Ell mouse spinal cords were dissociated and 
examined for properties of E-NCAM immunoreactive cells, 
according to the procedures of Example 2. 



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50 

A large niomber of E-NCAM immunoreactive cells were 
found at Ell, and these cells comprised about 60% of the 
total population of cells. E-NCAM-positive cells appeared 
morphologically similar to neurons with extensive 
5 processes. At this stage of development, no co-expression 

of E-NCAM with either Gal-C or GFAP was observed in 
double-labeling experiments, suggesting that E-NCAM 
immunoreactivity may identify neuronal precursors. 

To determine if mouse E-NCAM-positive cells, like 

10 their rat counterparts, underwent cell division, cells 

were pulse labeled with BRDU and then double-labeled to 
detect cells that co-expressed BRDU and E-NCAM 
immunoreactivity. Results showed that E-NCAM-positive 
cells divided for at least three days in culture. E-NCAM 

15 -positive cells, thus, appeared similar to the NRPs 

previously described in rats. To confirm that E-NCAM- 
positive cells could generate multiple neuronal 
phenotypes, immunoselected E-NCAM cells prepared according 
to the procedure of Example 3 were allowed to 

20 differentiate in culture for 10 days. Plates were then 

harvested, and cDNA was prepared according to the 
procedure of Example 13 to assess neurotransmitter 
synthesis. As can be seen in FIG. 12, expression of p75, 
islet-1, ChAT, calbindin, GAD, and glutaminase were 

25 readily detected in differentiated populations. Thus, 

mouse E-NCAM immunoreactive cells can generate neurons 
that express cholinergic, excitatory, and inhibitory 
phenotypes . 



30 Example 18 

E-NCAM Imm unoreactive Neuroblasts Can Be Generated from ES 

In Example 17 it was shown that mouse spinal cords 
35 contain E-NCAM immunoreactive NRPs that are similar to rat 

NRP cells. To determine if similar lineage restricted 
precursors could be generated from ES cells, mouse ES 
cells were obtained from the Developmental Studies 
Hybridoma Bank (DSHB; University of Iowa, Iowa City, Iowa) 



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51 

and were then grown in culture and examined for the 
expression of E-NCAM, A2B5, and other neuroglial markers. 
As has been previously described, undifferentiated ES 
cells did not express detectable immunoreactivity for any 
5 of the markers tested. In contrast, when ES cells were 

plated in neural differentiation conditions ES cells 
altered their morphology and began to express multiple 
neuronal and glial markers (FIG. 13) . Differentiated ES 
cells were harvested and total RNA prepared for RT-PCR 

10 according to the procedure of Example 13. Of particular 

importance is the early expression of E-NCAM (early 
neuronal marker) and PLP/DM20 genes (known to be expressed 
by embryonic glial precursors) . Consistent with the 
detection of early neuronal and glial markers by PGR, high 

15 polysialiated NCAM expressing cells represented a small 

percentage of the total cells. Less than 5% of cells in 
culture expressed E-NCAM immunoreactivity after 5 days in 
culture. The percentage of A2B5 immunoreactive cells was 
significantly higher; about 10% of differentiated cells 

20 expressed this marker. 

To determine if E-NCAM immunoreactive cells 
represented neuronal precursors, the co-expression of 
neuronal and glial markers was examined. E-NCAM 
immunoreactive -cells co-expressed MAP-2 and tubulin 

25 immunoreactivity, but did not co-express GFAP and nestin 

immunoreactivity. E-NCAM-positive cells did not express 
Gal-C or other oligodendrocytic markers. Thus, E-NCAM 
immunoreactive cells that were derived from mouse ES cells 
appeared similar to spinal-cord-derived E-NCAM-positive 

30 NRPs. 

To confirm that ES-cell-derived neuronal precursors 
could generate multiple kinds of neurons, E-NCAM 
immunoreactive cells were immunosel-ected according to the 
procedure of Example 3 and such purified cells were 
35 allowed to differentiate for 10 days. Cells were then 

harvested and analyzed by immunocytochemistry and RT-PCR 
for the expression of phenotypic markers. FIG. 14 shows 
the results of an illustrative PGR experiment wherein 



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ChAT, p75, islet-1, calbindin, GAD, and glutaminase 
expression was readily detected in differentiated 
populations. Thus, ES-cell-derived E-NCAM iininunoreactive 
cells differentiated into postmitotic neurons that 
5 expressed multiple neurotransmitters, including 

cholinergic, excitatory, and inhibitory phenotypes. 
Therefore, ES cells can be used as a source of lineage 
restricted NRPs. 

10 Example 19 

NRPs in Human Neural Tubes 

To determine if NRPs are present in human neural 
tubes, human embryonic spinal cords were dissociated and 
the phenotypes of cells when grown in DMEM/F12 in a high 

15 concentration of FGF were examined according to the 

procedure of Example 2. 

Human spinal cord cells (HSCs) initially appeared 
morphologically similar to rat and mouse spinal cords, but 
rapidly differentiated into fibroblastic appearing cells 

20 with a significant proportion of cells having a neuronal 

morphology. HSCs continued to divide rapidly and most 
cells (95%) were nestin immunoreactive. At this stage, 
cultures did not contain astrocytes, oligodendrocytes, or 
their precursors as detected by the expression of GFAP or 

25 04/Gal-C immunoreactive cells. A substantial number of E- 

NCAM immunoreactive cells were present, however, and 
constituted about 40% of the total population. E-NCAM 
immunoreactive cells appeared morphologically similar to 
neurons, although some flat E-NCAM immunoreactive cells 

30 were also present. Both populations of E-NCAM-positive 

cells were MAP2K immunoreactive and also expressed a 
variety of other early neuronal markers, as summarized in 
Table 10. 



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53 



Table 10 


Antigen 


% E-NCAM" Cells 


Nestin 


100 


MAP2 


100 


Neurofilament H 


80 


Neurofilament -M 


Occasional Cell 


tubulin 


100 


Gal-C/04 


0 


GFAP 


0 



At this stage of development, no co-expression of E- 
NCAM with either Gal-C or GFAP was observed in double- 
labeling experiments, suggesting that E-NCAM 

15 immunoreactivity identifies neuronal precursors. That is, 

E-NCAM immunoreactive human spinal cord cells expressed 
neuronal but not non-neuronal antigens . To determine if 
human E-NCAM* cells, like their rat counterparts, underwent 
cell division, mixed cultures of HSCs were pulse labeled 

20 with BRDU and then double labeled to detect cells that co- 

expressed BRDU and E-NCAM immunoreactivity. The results of 
this experiment showed that E-NCAM-positive cells divided 
for at least three days in culture. Consistent with the 
results of Table 10 that E-NCAM immunoreactive cells also 

25 express NF-H, BRDU-incorporating cells also co-expressed 

neurofilament-H. Thus, as in fetal rodent spinal cord 
cultures, dividing nestin immunoreactive precursor cells 
from humans are present and E-NCAM immunoreactive cells 
represent a significant fraction of total precursor 

30 population at this age. E-NCAM"" cells appear similar to the 

NRPs previously described for rats and mice. 



35 



Transplanted cells can be administered to any animal, 
including humans, with abnormal neurological or 
neurodegenerative symptoms obtained in any manner. 



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54 

including as a result of chemical electrolytic lesions, 
experimental destruction of neural areas, or aging 
processes. Transplantation can be bilateral, or, for 
example in patients suffering from Parkinson's Disease, 
5 can be contralateral to the most-affected side. Surgery 

is preferably performed such that particular brain regions 
are located, such as in relation to skull sutures, and 
surgery performed with stereotactic techniques. 
Alternatively, cells can be implanted in the absence of 

10 stereotactic surgery. Cells can be delivered to any 

affected neural areas using any method of cell injection 
or transplantation known in the art. 

In another embodiment of the invention, NRP cells are 
transplanted into a host, and induced to proliferate 

15 and/or differentiate in that host by (1) proliferation 

and/or differentiation in vitro prior to being 
administered, or (2) differentiation in vitro prior to 
being administered and proliferation and differentiation 
in vivo after being administered, or (3) proliferation in 

20 vitro prior to being administered and then differentiation 

in vivo without further proliferation after being 
administered, or (4) proliferation and differentiation in 
vivo after being injected directly after being freshly 
isolated. 

25 NRP cells oan also be used for delivery of 

therapeutic or other compounds. Methods for bypassing the 
blood-brain barrier for purposes of delivery of 
therapeutic compounds include implanting cells in an 
encapsulation devioe according to methods known in the art 

30 or directly implanting genetically engineered cells such 

that the cells themselves produce the therapeutic 
compound- Such compounds may be small molecules, 
peptides, proteins, or viral particles- Cells can be 
genetically transduced by any means known in the art, 

35 including calcium phosphate transf ection, DEAE-dextran 

transfection, polybrene transfection, ^lactroporation, 
lipof ection, inf-ection of viruses, and the like. Cells 
are first genetically manipulated to express a therapeutic 



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55 

substance and then transplanted either as free cells able 
to diffuse and incorporate within the CNS parenchyma or 
are contained within an encapsulation device. R.P- Lanza 
& W.L. Chick, Encapsulated Cell Therapy, Sci. Amer.: Sci. 
5 & Med<, July/Aug, 16-25 (1995); P.M. Galletti, 

Bioartificial Organs, 16 Artificial Organs 55-60 (1992); 
A.S. Hoffman, Molecular Engineering of Biomaterials in the 
1990s and Beyond: A Growing Liaison of Polymers with 
Molecular Biology, 16 Artificial Organs 43-49 (1992); B.D, 
10 Ratner, New Ideas in Biomaterials Science - A Path to 

Engineered Biomaterials, 27 J. Biomed. Mat. Res. 837-850 

(1993) ; M.J. Lysaght et al., Recent Progress in 
Immunoisolated Cell Therapy, 56 J. Cell Biochem. 196-203 

(1994) , hereby incorporated by reference. 

15 Transplanted cells can be identified by prior 

incorporation of tracer dyes such as rhodamine or 
f luorescein-labeled microspheres, fast blue, bis- 
benzamide, or genetic markers incorporated by any genetic 
transduction procedure known in the art to allow 

20 expression of such enzymatic markers as P-galactosidase or 

alkaline phosphatase. 

Any expression system known in the art can be used to 
express the therapeutic compound, so long as it has a 
promoter that is active in the cell, and appropriate 

25 internal signals for initiation, termination, and 

polyadenylation. Examples of suitable expression vectors 
include recombinant vaccinia virus vectors including 
pSCll, or vectors derived from viruses such as simian 
virus 40 (SV40), Rous Sarcoma Virus (RSV) , mouse mammary 

30 tmor virus (MMTV) , adenovirus, herpes simplex virus 

(HSV) , bovine papilloma virus, Epstein-Barr virus, 
lentiviruses, or any other eukaryotic expression vector 
known in the art. Many of such expression vectors are 
commercially available. 

35 Cells can also be transduced to expr^ess any gene 

coding for a neurotransmitter, neuropepti<ie, 
neurotransmitter-synthesizing enzyme or neuropeptide 



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56 

synthesizing enzyme for which expression in the host is 
desired. 

NRP cells and/or their derivatives cultured in vitro 
can be used for the screening of potentially 
5 neurologically therapeutic compositions. These 

compositions can be applied to cells in culture at varying 
dosages, and the response of the cells monitored for 
various time periods. The induction of expression of new 
or increased levels of proteins such as enzymes, 

10 receptors, and other cell surface molecules, or of 

neurotransmitters, amino acids, neuropeptides, and 
biogenic amines can be analyzed with any technique known 
in the art that can identify the alteration of the level 
of such molecules, including protein assays, enzymatic 

15 assays, receptor binding assays, enzyme-linked 

immunosorbent assays, electrophoretic analysis, analysis 
with high performance liquid chromatography. Western 
blots, and radioimmune assays. Nucleic acid analysis, 
such as Northern blots, can be used to examine the levels 

20 of mRNA coding for these molecules, or for the enzymes 

that synthesize these molecules. Alternatively, cells 
treated with these pharmaceutical compositions can be 
transplanted into an animal and their survival, ability to 
form neurons, and to express any of the functions of these 

25 cell types can be analyzed by any procedure available in 

the art. 

NRP cells can be cryopreserved by any method known in 
the art. 

30 Example 20 

Use of NRP Cells a ndVor Their Derivatives for Treatment of 

Abnormal Nevrolggi^al or t^gurodgg^ngr-^tiv^ gymptoffig 

NRP cells are isolated by the methods of Examples 2, 
35 3, 8, 18, or 19. Cells are obtained from human embryonic 

or adult CNS or from xenographic sources from which 
immunorejection of cells is not a clinical problem, such 
as pigs genetically engineered so as not to present a 
foreign stimulus to the human immune system. Cells 



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57 

collected from embryos are obtained by dissection of CNS 
tissue following routine abortion procedures and tissue 
collection in a sterile collection apparatus. Cells from 
the postnatal CNS are obtained by digestion of tissue 
5 following routine autopsy. Tissue is prepared, cells are 

immunopurif ied, and the resulting purified cells are 
cultured as in Example 2. 

Cells can be transplanted directly or can first be 
expanded in vitro prior to transplantation. Populations 

10 expanded in vitro can further be expanded in conditions 

that enhance the generation of neurons or cells committed 
to the generation of neurons. 

Transplantation is routinely carried out at cell 
suspensions of 5-50,000 cells/Ml in physiological salt 

15 solutions, such as PBS. Cells can be transplanted into or 

near any CNS regions affected by the disease or condition. 
Transplantation procedures, with appropriate modifications 
for use in human patients, are in their essence similar to 
procedures well known to those skilled in the art of 

20 transplantation of 0-2A progenitor cells, e.g., A.K. 

Groves et al.. Repair of Demyelinated Lesions by 
Transplantation of Purified 0-2A Progenitor Cells, 362 
Nature 453-455 (1993), hereby incorporated by reference. 

More specifically, transplantation is performed using 

25 a computed tomographic stereotaxic guide. The patient is 

operated on using any of the procedures known in the art. 
In cases where precisely localized transplantation is 
desirable, the patient undergoes CT scanning to establish 
the coordinates of the region to receive the transplant. 

30 The injection cannula can be in any configuration used by 

those skilled in the relevant arts. The cannula is then 
inserted into the brain to the correct coordinates, then 
removed and replaced with a 19-gauge infusion cannula that 
has been preloaded with cell suspension in a small 

35 selected volume. The cells are then slowly infused, at 

rates generally of 1-10 ml per minute as the cannula is 
withdrawn. For some diseases in which it is desirable to 
spread cells over the largest possible area, multiple 



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58 

stereotactic needle passes may be made throughout the 
area. Patients are examined post-operatively for 
hemorrhage or edema. Neurological evaluations are 
performed at various post-operative intervals, as well as 
5 PET scans if these can be used to determine the metabolic 

activity of the implanted cells. These and similar 
procedures can be used for any implantation of NRP cells 
for any of the purposes indicated in this invention. 
Success of the procedure is detemined by non- 
10 invasive analysis with, for example, nuclear magnetic 

resonance image scanners, and/or by analysis of functional 
recovery according to methods well known in the art. 



Example 21 

15 Use of Genetically Engineered NRP Cells and/or Their 

Derivatives for Transplantation 

In this example, NRP cells are genetically modified 
ex vivo before introduction into or near regions of 

20 disease to express gene products that will make the 

transplanted cells resistant to destruction in vivo and/or 
to express gene products that provide trophic support to 
the host cells and/or to express gene products that limit 
destructive processes occurring in the host, -Genetic 

25 modification is carried out by any of the techniques known 

to those skilled in the art, including but not limited to 
calcium phosphate transf ection, DEAE-dextran transf ection, 
polybrene transf ection, electroporation, lipofection, 
infection of viruses, and the like. Gene products that 

30 would make cells resistant to destruction in vivo and/or 

to express gene products that provide trophic support to 
host cells and/or to express gene products that limit 
destructive processes occurring in the host include but 
are not limited to insulin-like growth factor-I, decay 

35 accelerating factor, catalase, superoxide dismutase, 

members of the neurotrophin family, glial-derived 
neurotrophic factor, ciliary neurotrophic factor, leukemia 
inhibitory factor, fas ligand, cytokines that inhibit 
inflammatory processes, receptor fragments that inhibit 



wo 99/01159 



PCTAJS98/I3875 



59 

inflammatory processes, antibodies that inhibit 
inflammatory processes, and so forth. 

Example 22 

5 Use of NRP Cells and/or Their Derivatives for the 

Screening of Potentially Neurologicallv Therapeutic 
Compositions 

NRP cells or derivatives thereof or mixtures thereof 

10 cultured in vitro can be exposed to compositions of 

interest at varying dosages, and the response of the cells 
monitored for various time periods. The induction of 
expression of new or increased levels of proteins such as 
enzymes, receptors, and other cell surface molecules or of 

15 neurotransmitters, amino acids, neuropeptides, and 

biogenic amines can be analyzed with any technique known 
in the art that can identify the alteration of the level 
of such molecules, including protein assays, enzymatic 
assays, receptor binding assays, enzyme-linked 

20 immunosorbent assays, electrophoretic analysis, analysis 

with high performance liquid chromatography. Western 
blots, and radioimmune assays. Nucleic acid analysis, 
such as Northern hybridization can be used to examine the 
levels of mRNA coding for these molecules, or for the 

25 enzymes that synthesize these molecules. Cells can also 

be used to screen for compounds able to promote the 
division of NRP cells and/or their derivatives by 
determining the ability of compounds to cause increases in 
NRP cell nimber or to promote DNA synthesis, as measured 

30 by, e.g. incorporation of bromodeoxyuridine or tritiated 

thymidine. Cells can also be used to screen for compounds 
that promote survival of NRP cells and/or their 
derivatives by applying compounds to cells in conditions 
where they would be expected to die (e.g., exposure to 

35 neurotoxic agents, withdrawal of all trophic factors) and 

examining cell survival using any of the techniques well 
known to practitioners of the art. Cells can also be u-sed 
to screen for compounds that specifically inhibit binding 
to particular receptors, by looking at the ability of said 



wo 99/01159 



PCT/US98/13875 



60 

blocking compounds to block the response elicited by 
binding of agonist to said receptors. Cells can also be 
used to screen for compounds able to activate particular 
receptors using ligand binding assays well known to 
5 practitioners of the art, or by looking at such 

physiological alterations as are associated with 
activation of the receptor, such as fluxes in calcixam 
levels, or other alterations well known to practitioners 
of the art. Alternatively, cells treated with these 
10 pharmaceutical compositions can be transplanted into an 

animal and their survival, ability to form neurons and to 
express any of the functions of these cells types can be 
analyzed by any procedures available in the art. 

Example 23 

In this example, cells were harvested from E13.5 rat 
spinal cords, and E-NCAM immunoreactive neuronal 
restricted precursor cells were isolated by immunopanning 
according to the procedure of Example 3. These cells were 
then labeled with a cell tracker and were transplanted to 
different cortical regions using a glass raicroelectrode . 
Animals were sacrificed after 3.5, 10 or 21 days, and the 
brain was sectioned according to methods well known in the 
art. Such transplanted cells were shown to survive and 
differentiate at all three times. 

Example 24 

In this example, cells were harvested, isolated, and 
plated in a 35 mm dish as described in Example 3. Cells 
30 were then incubated with a retroviral construct containing 

a green fluorescent protein (GFP) reporter gene under a 
cytomegalovirus (CMV) prompter. Cells were allowed to 
recover for 8 hours and then were analyzed for <3FP 
expression. GF? expression was detected as early as 24 
35 hours after infection, and <5FP expression persisted for up 

to two weeks, at which time the experiment was <:oncluded. 
These results show that ectopic genes can be expressed in 
NRPs under a heterologous promoter, and that infected 



15 



20 



25 



wo 99/01 159 PCT/US98/1 3875 

61 

cells continue to stably express the ectopic protein for 
several weeks. 



wo 99/01159 



PCTAJS98/13875 



62 
CLAIMS 

We claim: 

1. An isolated, pure population of mammalian CNS 
neuron-restricted precursor cells. 

5 

2. The population of claim 1 wherein said neuron- 
restricted precursor cells are capable of self -renewal . 

3. The population of claim 1 wherein said neuron- 
ic restricted precursor cells are capable of differentiation 

to CNS neuronal cells but not to CNS glial cells. 

4. The population of claim 1 wherein said neuron- 
restricted precursor cells express embryonic neural cell 

15 adhesion molecule. 

5. The population of claim 4 wherein said neuron- 
restricted precursor cells do not express a ganglioside 
recognized by A2B5 antibody. 

20 

6. The population of claim 4 wherein said neuron- 
restricted precursor cells do not express nestin. 

7. The population of claim 1 wherein said neuron- 
25 restricted precursor cells are selected from a mammalian 

embryo selected from the group consisting of human and 
non-hiaman primates, equines, canines, felines, bovines, 
porcines, ovines, lagomorphs, and the order Rodent ia. 

30 8. The population of claim 1 wherein said cells 

are able to differentiate into neurons that are capable of 
releasing and responding to neurotransmitters. 

9. The population of claim 8 wherein said neurons 
35 demonstrate receptors for said neurotransmitters, and said 

cells are capable of expressing neurotransmitter- 
synthesizing enzymes. 



wo 99/0nS9 



PCT/US98/13875 



63 

10. The population of Claim 1 wherein said cells 
are capable of differentiating into neurons which can form 
functional synapses and/or develop electrical activity. 

5 - 11. The population of claim 1 wherein said cells 

are capable of stably expressing at least one material 
selected from the group consisting of growth factors for 
said cells, differentiation factors for said cells, 
maturation factors for said cells, and combinations of any 
10 of these. 

12. A method of- isolating a pure population of 
mammalian CNS neuron-restricted precursor cells comprising 
the steps of: 

(a) isolating a population of mammalian multipotent 
CNS' stem cells capable of generating both neurons and 
glia; 

(b) incubating the multipotent CNS stem cells in a 
medium configured for inducing said cells to begin 
differentiating; 

(c) purifying from the differentiating cells a 
subpopulation of cells expressing a selected antigen 
defining neuron-restricted precursor cells; and 

(d) incubating the purified subpopulation of cells 
in a medium configured for supporting adherent growth 
thereof. 

13, The method of claim 12 wherein said selected 
antigen defining neuron-restricted precursor cells is 

30 embryonic neural cell adhesion molecule. 



15 



20 



25 



14. The method of claim 12 wherein said purifying 
comprises a procedure selected from the group consisting 
of specific antibody capture, fluorescence activated cell 

35 sorting, and magnetic bead capture. 

15. The method of claim 14 wherein said procedure 
is specific antibody capture. 



wo 99/01159 



PCTAJS98/13875 



64 

16. The method of claim 12 wherein said mammalian 
multipotent CNS stem cells are neuroepithelial stem cells. 

17. The method of claim 16 wherein said isolating 
5 a population of CNS neuroepithelial stem cells comprises: 

(a) removing a CNS tissue from a mammalian embryo 
at a stage of embryonic development after closure of the 
neural tube but prior to differentiation of cells in the 
neural tube; 

10 (b) dissociating cells comprising the neural tube 

removed from the mammalian embryo; 

(c) plating the dissociated cells in feeder-cell- 
independent culture on a substratum and in a medium 
configured for supporting adherent growth of the 

15 neuroepithelial stem cells comprising effective amounts of 

fibroblast growth factor and chick embryo extract; and 

(d) incubating the plated cells at a temperature 
and in an atmosphere conducive to growth of the 
neuroepithelial stem cells. 



20 



25 



18. The method of claim 17 wherein said mammalian 
embryo is selected from the group consisting of human and 
non-human primates, equines, canines, felines, bovines, 
porcines, ovines, lagomorphs, and the order Rodent ia. 

19. The method of claim 17 wherein said substratum 
is selected from the group consisting of fibronectin, 
vitronectin, laminin, and RGD peptides. 



30 20. The method of claim 12 wherein said medium 

comprises effective amounts of fibroblast growth factor 
and neurotrophin 3. 

21. A method of isolating a pure population of 
35 mammalian CNS neuron-restricted precursor cells comprising 

the steps of: 

(a) removing a sample of CNS tissue from a 
mammalian embryo at a stage of embryonic development after 



wo 99/01159 



PCTAJS98/I3875 



65 

closure of the neural tube but prior to differentiation of 
glial and neuronal cells in the neural tube; 

(b) dissociating cells comprising the sample of CNS 
tissue removed from the mammalian embryo; 
5 (c) purifying from the dissociated cells a 

subpopulation expressing a selected antigen defining 
neuron-restricted precursor cells; 

(d) plating the purified subpopulation of cells in 
feeder-cell-independent culture on a substratum and in a 

10 medium configured for supporting adherent growth of the 

neuron-restricted precursor cells; and 

(e) incubating the plated cells at a temperature 
and in an atmosphere conducive to growth of the neuron- 
restricted precursor cells. 

15 

22. The method of claim 21 wherein said selected 
antigen defining neuron-restricted precursor cells is 
embryonic neural cell adhesion molecule. 

20 23. The method of claim 21 wherein said purifying 

comprises a procedure selected from the group consisting 
of specific antibody capture, fluorescence activated cells 
sorting, and magnetic bead capture. 

25 24. The method of claim 23 wherein said procedure 

is specific antibody capture. 

25. The method of claim 21 wherein said mammalian 
embryo is selected from the group consisting of human and 
30 non-human primates, equines, canines, felines, bovines, 

porcines, ovines, lagomorphs, and the order Rodent ia. 



35 



26. A pure population of mammalian CNS neuron- 
restricted precursor cells isolated by the method of claim 
12. 



wo 99/01159 



PCT/US98/I3875 



66 

27. A pure population of mammalian CNS neuron- 
restricted precursor cells isolated by the method of claim 
21. 

5 28. A method of obtaining postmitotic neurons 

comprising: 

(a) providing neuron-restricted precursor cells and 
culturing the neuron-restricted precursor cells in 
proliferating conditions; and 
10 (b) changing the culture conditions of the neuron- 

restricted precursor cells from proliferating conditions 
to differentiating condition, thereby causing the neuron- 
restricted precursor cells to differentiate into 
postmitotic neurons. 

15 

29. The method of claim 28 wherein said changing 
the culture conditions comprises adding retinoic acid to 
basal medium. 

20 30. The method of claim 28 wherein said changing 

the culture conditions comprises withdrawing a mitotic 
factor from basal medium. 

31. The method of claim 30 wherein said mitotic 
25 factor is fibroblast growth factor. 

32. The method of claim 28 wherein said changing 
the culture conditions comprises adding a neuronal 
maturation factor to basal medium. 

30 

33. The method of claim 32 wherein said neuronal 
maturation factor is a member selected from the group 
consisting of sonic hedgehog, BMP-2, BMP-4, NT-3, NT-4, 
CNTF, LIF, retinoic acid, brain-derived neurotrophic 

35 factor {BDNF) , and combinations of any of the above. 



wo 99/01 159 PCT/US98/1 3875 

67 

34. An isolated cellular composition comprising the 
mammalian CNS neuron-restricted cells of any of claims 1-7. 

35. A pharmaceutical composition comprising a 

5 therapeutically effective amount of the composition of 

Claim 34 and a pharmaceutically acceptable carrier. 

36. A method for treating a neuronal disorder in a 
mammal comprising administering to said mammal a 

10 therapeutically effective amount of the composition of 

Claim 34. 

37. A method for treating a neuronal disorder in a 
mammal comprising administering to said mammal a 

15 therapeutically effective amount of the pharmaceutical 

composition of Claim 35. 

38. The method of Claim 34 wherein said composition 
is administered by a route selected from the group 

20 consisting of intramuscular administration, intrathecal 

administration, intraperitoneal administration, 
intravenous administration, and combinations of any of the 
above . 

25 39. The method of Claim 34 wherein said method also 

includes the administration of a member selected from the 
group consisting of differentiation factors, growth 
factors, cell maturation factors and combinations of any 
of the above. 



30 



35 



40. The method of Claim 39 wherein said 
differentiation factors are selected from the group 
consisting of retinoic acid, BMP-2, BMP-4, and 
combinations of any of the above. 

41. The composition of Claim 34 for use as a 
delivery vehicle for the delivery to glial cells of an 
agent selected from the group consisting of cell growth 



wo 99/01159 



PCT/US98/13875 



68 

factors, cell maturation factors, cell differentiation 
agents, and any combination of the above. 

42. The composition of Claim 34 for use as a 

5 delivery vehicle for the delivery of trophic factors to 

neurons . 

43. A method for treating neurodegenerative 
symptoms in a mammal comprising the steps of: 

10 (a) providing a pure population of neuronal 

restricted precursor cells; 

(b) genetically transforming said neuronal 
restricted precursor cells with a gene encoding a growth 
factor, neurotransmitter, neurotransmitter synthesizing 

15 enzyme, neuropeptide, neuropeptide synthesizing enzyme, or 

substance that provides protection against free-radical 
mediated damage thereby resulting in a transfored 
population of glial restricted precursor cells that 
express said growth factor, neurotransmitter, 

20 neurotransmitter synthesizing enzyme, neuropeptide, 

neuropeptide synthesizing enzyme, or substance that 
provides protection against free-radical mediated damage; 
and 

(c) administering an effective amount of said 

25 transformed population of neuronal restricted precursor 

cells to said mammal. 

44. A method or screening compounds for 
neurological activity comprising the steps of: 

30 (a) providing a pure population of neuronal 

restricted precursor cells or derivatives thereof or 
mixtures thereof cultured In vitro; 

(b) exposing said cells or derivatives thereof or 
mixtures thereof to a selected compound at varying 

35 dosages; and 

(c) monitoring the reaction of said cells -or 

* derivatives thereof or mixtures thereof to said selected 
compound for selected time periods. 



99/01 159 PCT/US98/13875 

69 

45. A method for treating a neurological or 
neurodegenerative disease comprising administering to a 
mammal in need of such treatment an effective amount of 
neuronal restricted precursor cells or derivatives thereof 
or mixtures thereof. 

46. The method of claim 4 5 wherein said neuronal 
restricted precursor cells or derivatives thereof or 
mixtures thereof are caused to proliferate and 
differentiate in vitro prior to being administered. 

47. The method of claim 45 wherein said neuronal 
restricted precursor cells or derivatives thereof or 
mixtures thereof are caused to proliferate in vitro prior 
to being administered, and then are caused to further 
proliferate and differentiate in vivo after being 
administered. 

48. The method of claim 45 wherein said neuronal 
restricted precursor cells or derivatives thereof or 
mixtures thereof are caused to proliferate in vitro prior 
to being administered, and then are caused to 
differentiate in vivo after being administered. 

49. The method of claim 45 wherein said neuronal 
restricted precursor cells or derivatives thereof or 
mixtures thereof are from a heterologous donor. 

50. The method of claim 4 9 wherein said donor is a 
fetus. 

51. The method of claim 49 wherein said donor is a 
juvenile. 



52. The method of claim 49 wherein said donor is an 
adult. 



wo 99/01159 



PCT/US98/13875 



10 



70 

53. The method of claim 45 wherein said neuronal 
restricted precursor cells or derivatives thereof or 
mixtures thereof are from an autologous donor. 

54. The method of claim 53 wherein said donor is a 
fetus. 

55. The method of claim 53 wherein said donor is a 
juvenile . 

56. The method of claim 53 wherein said donor is an 
adult . 



57. The method of claim 45 wherein said derivatives 
15 thereof are obtained by differentiation of neuronal 

restricted precursor cells in vitro, 

58. The method of claim 45 wherein said derivatives 
thereof are obtained by genetic transduction of neuronal 

20 restricted precursor cells. 

59. A method of isolating a pure population of 
mammalian CNS neuron-restricted precursor cells comprises 
the steps of: 

25 (a) providing a sample of mammalian embryonic stem 

cells : 

(b) purifying from the mammalian embryonic stem 
cells a subpopulation expressing a selected anti-gen 
defining neuron-restricted precursor cells; 

30 (c) plating the purified subpopulation of cells in 

feeder-cell-independent culture on a substratum and in a 
medium configured for supporting adherent growth of the 
neuron-restricted precursor cells; and 

(d) incubating the plated cells at a temperature 

35 and in an atmosphere conducive to growth of the neuron- 

restricted precursor cells. 



wo 99/01 159 



PCTAJS98/13875 



1/7 



NEP CELLS 



UN- 
NESTIN + 



SELF RENEWAL 

PNS PRECURSOR 
SELF RENEWAL 




UN- 
NESTIN+ 
p7S^ 



SELF RENEWAL 



38 



NEURAL CREST (NCSC) 



B-11U 
P7S 



P7S-¥ 
GFAP 



P7S- 
SMA+ 



NEURON 



42 



46 





??? CNS PRECURSOR 

UN- 
NESTIN-¥ 
P75- 




42S5-I- 

04- 
NCAM- 



NCAM + 
A2B5- 
P7S- 
B-111+ 



SCHWANN SMOOTH 
CELL MUSCLE 



OUGODENDROCYTE 

ASTROCYTE 




NEURON ti^uRON 



Fig. 1 



SUBSTITUTE SHEET (RULE 26) 



wo 99/01159 PCTAJS98/1387S 

2/7 



SOObp 




ChAT p75 /sM calbindin GAD glutam cyclophilin 

inase 



Fig. 2 



SUeSTITUTE SHEET <nULE 26) 



wo 99/01159 



PCT/US98/1387S 



tOO-r 



I 

8 40 



20- 



3/7 

n Acutely Dissociated 
ED Differentiated 

100 



49 



49 



100\~\ 49 
\l00 



E2 



100 

r 



99 

I 

y 43 
LA 



55 



55 

1 

49V 

I 



GAB>» G(K DA Glu ACh RRSOmMKRFt 
Substance 

Fig. 3 



0,4 ^ Acutely Dissociated 



0.3 iGABA 
Gly 



0.1 



Fig. 4 



SOmM KRR 




ACh RR 




SUBSTITUTE SHEET (RULE 26) 



WO99/0J159 PCT/US98/13875 



417 




GAD calbindin gluiam- p7S /s/-1 ChAT 

inase 



Fig, 6 



SUBSTITUTE SHEET (RULE 26) 



wo 99/01159 



PCT/US98/I3875 



5/7 



lOO-T 



n^102 



n^lll 



20-{ 
0 



n=111 



n=10S 



GABA Gly DA Glu ACh RR SOmMKRR 
Substance 

Fig. 7 




flff SOmMKRR 



Fig. 8 



SOmM KRR 




0.5 [ I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 r 

0 5 10 IS 20 25 30 
Time (min) 

Fig. 9 

SUBSTITUTE SHEET (RULE 26) 



wo 99/01159 



PCT/US98/13875 



6/7 

100 n 



"5 75- 




• + 



Fig, 11 

SUBSTITUTE SHEET (RULE 26) 



wo 99/01159 



PCT/US98/1387S 



7/7 



Fig. 12 



SOObp 
220bp 



Fig. 13 



SOObp 
220bp 



Fig. 14 



SUBSTITUTE SHEET <RULE 26) 



INTERNATIONAL SEARCH REPORT 



loteroational applicatioo No. 
PCTAJS98/13875 



A. CLASSinCATlON OF SUBJECT MATTER 

1PC(6) 'Ji6\K 48/00. 35/30; C12N S/00. 5A)6, 5/08 

US CL :Please See Extra Sheet 
Accordiog to loteroational Patent Classification (IPC) or to both national classification and IPC 

a FIELDS SEARCHED 

Minimum documentation searched (classification system followed by classificatioQ symbols) 

U.S. : 424/93.1, 932, 93^1. 93.7, 570; 435/4, 325, 368, 377. 378. 384 



Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched 



Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) 
Please See Extra Sheet 



DOCUMENTS CONSIDERED TO BE RELEVANT 



Category* 



Citation of document, with indication, where appropriate, of the relevant passages 



Relevant to claim No. 



X 
Y 



BLASS-KAMPMANN. S. et al. In Vitro Differentiation of E-N- 
CAM Expressing Rat Neural Precursor Cells Isolated by FACS 
During Prenatal Development. Journal of Neuroscience Researdi. 
1994. Vol 37. pages 3S9-373, see entire document. 



WO 93/01275 A (WEISS et al) 21 January 1993, see entire 
document. 



1, 2, 4. 7, 12-14, 
16, 21-23, 25-27, 
34 

3, 5, 6, 8-11, 15, 
17-20, 24, 28-33, 
35-59 

3, 5, 6, 8-11, 15, 
17-20, 24, 28-33, 
35-59 



\ xj Futtbcr docuneatt aie Gited ia Ifcc cooliauatiaa of Box C. | | Sec pataot faoiily aaacx. 



4osuB«Bt dafming IIm gaaml ■(■to of Am «rt which m not coniidtrwl 
to b« of partiottlar nimwmm 

•ariwr doouMafil fwAKrfiod on or mtUa ttM iotonMliaBal Tiling date 

docuaont whidk mwy Arc* doubti oo pnorify cUiB(a) or whsoh m 
citid to ••triilBfa ttM pubbntioa ditt of inoihof ottttoo or otfMr 
ipMiol r iiop (m tpoafiod) 

dociKaDt rvfcrrmg to an oral diaolaaiira, mo, cxhibitiofi or otb«r 
doouBCDt pubUabod prior lo fhm intMiiatioiwl fUing date but bftar t 



hm docuatnt pi^Uihod oflor tfw intomotkioal filins or priority 
data and not in oooflict «ilh Hm appUoatiao but cited to uodantKid 
ttia prioeipb or tfaaory imdarfyint ^ ■ wwi b ua 

docuBiOTit of paiftiouiir nlavanoa^ ttic dainad nvaotioti caiHMt ba 
ooniidOTad Doval or caniwn ba noiiiidarad to invohra an inv antiva Map 



dowt apt of p4il»j ^ntaT ralavaooa^ tba chunod ioTootioD cannot ba 
ooBiidarad to imohra aa iBTamiva atip wb«n tba doctmaDt i* 
cambinad wttfa ooa or mora otfatr tuofa doctnnonH, tuch oooibinatioo 
baing obvioin to a penoD ikiUad in lha at 



doowBaot nanbar of (ba i 



fanUjr 



Date of die actual completion of the international search 
02 DECEMBER 1998 


Date of mailing o^c international search report 


Name and mailing address of the ISAAJS 
Commissiooer of Patents tod Traderatfks 
Box PCT 

WathinglOB, D.C 30231 
Facsimile No. (703) 305-3230 


Teleph^eNoV (703)308-0196 y 



Form PCT/lSA/210 (second tbcetXJuly 1992)« 



INTERNATIONAL SEARCH REPORT 



IntcrnationAl applicatioD Mo. 
PCT/US98/13875 



C (Contiiiuation). DOCUMENTS CONSIDERED TO BE RELEVANT 


Category* 


Citation of document, with indication, where appropriate, of the relevant passages 


Relevant to claim No. 


Y 


HATTEN. M.E. et al. Embryonic Cerebellar Neurons Accumulate 
[3H]-gamma*Aminobutyric Acid:Visualization of Developing 
gamma-Aminobutyric Acid-Udlizing Neurons In Vitro and In 
Vivo. Journal of Neuroscience. May 1984. Vol 4. No. 5. pages 
1343-1353, especially pages 1345-1347. 


8-10 


Y 


MA. W. et al. Neuroepithelial Cells in the Rat Spinal Cord Express 
Glutamate Decarboxylase Immunoreactivity In Vivo and In Vitro. 
Journal of Comparative Neurology. 1992. Vol 325. pages 257-270, 
especially pages 262-266. 


8-10 


Y 


KIRSCHENBAUM. B. et at. In Vitro Neuronal Production and 
Differentiation by Precursor Cells Derived from the Adult Human 
Forebrain. Cerebral Cortex. December 1994. Vol 6. pages 576-589, 
e^ecially pages 582-583. 


8-10 


Y 


US 5.175,103 A (LEE et al) 29 December 1992, col. 4, lines 39-68 
through col. 14. 


3. 5. 6, 8-11, 15. 
17-20, 24. 28-33 


Y 


US 5,411,883 A (BOSS et al) 02 May 1995, col. 18, lines 30-68 
through col. 20. 


3, 5. 6. 8-11, 15. 
17-20. 24. 28-33 


X,P 

J 


MAYER-PROSCHEL. M. et al. Isolation of Lineage-Restricted 
Neuronal Precursors from Multipoint Neuroepidielial Stem Cells. 
Neuron. October 1997. Vol 19. pages 773-785, see entire 
document 


1-34 



Fonn K:T/1SA/2]0 (coalinuAtiaa of aecoDd sheetXJuly 1992)* 



INTERNATIONAL SEARCH REPORT 



International appltcatioo No. 
PCT/US98/13875 



A. CLASSIFICATION OF SUBJECT MATTER: 
USCL : 

424/93.1, 93.2, 93.21. 93.7, 570; 435/4, 325. 36g. 377. 378. 384 

B. FIELDS SEARCHED 

Electronic data bases consulted (Name of data base and where pvacticable terms used): 
APS. MEDLINE, EMBASE. BIOSIS. WPIDS. CAPLUS 

search terms: neural, neuronal, neuron, embryonic, cam. gtia, neural tube, postmitotic, growth factor, sonic hedgehog, 
neurotrophm, bmp, rctinoic acid, ncuroepithelia, c-ncam. neuron-restricted, embiyonic neural cell adhesion molecule, 
fibroblast growth factor, lif, brain-derived neurotrophic factor, lagomorpfa, ovme. porcine, bovine, primate, equine, 
canine, feline, progenitor, precursor, synapse, neurotransmitter 



Fonn PCT/ISA/210 (extra sheetXJuly 1992)* 



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