Register or Login To Download This Patent As A PDF
|United States Patent Application
Carpenter, Melissa K.
January 24, 2002
Neural progenitor cell populations
This invention provides populations of neural progenitor cells,
differentiated neurons, glial cells, and astrocytes. The populations are
obtained by culturing stem cell populations (such as embryonic stem
cells) in a cocktail of growth conditions that initiates differentiation,
and establishes the neural progenitor population. The progenitors can be
further differentiated in culture into a variety of different neural
phenotypes, including dopaminergic neurons. The differentiated cell
populations or the neural progenitors can be generated in large
quantities for use in drug screening and the treatment of neurological
Carpenter, Melissa K.; (Castro Valley, CA)
230 CONSTITUTION DRIVE
May 16, 2001|
|Current U.S. Class:
||435/6; 424/93.21; 435/368 |
|Class at Publication:
||435/6; 424/93.21; 435/368 |
||A61K 048/00; C12Q 001/68; C12N 005/08|
What is claimed as the invention is:
1. A cell population that proliferates in an in vitro culture, obtained by
differentiating primate pluripotent stem (pPS) cells, wherein at least
.about.30% of the cells in the population are committed to form neuronal
cells, glial cells, or both.
2. A cell population that proliferates in an in vitro culture, obtained by
differentiating primate pluripotent stem (pPS) cells, comprising at least
.about.60% neural progenitor cells, wherein at least 10% of the cells can
differentiate into neuronal cells, and at least 10% of the cells can
differentiate into glial cells.
3. A cell population that proliferates in an in vitro culture, obtained by
differentiating primate pluripotent stem (pPS) cells, comprising at least
.about.60% neural progenitor cells, wherein at least 10% of the cells
express A2B5, and at least 10% of the cells express NCAM.
4. The cell population of claim 1, wherein the pPS cells are human
embryonic stem (hES) cells.
5. The cell population of claim 1, obtained by differentiating pPS cells
in a medium containing at least two ligands that bind growth factor
receptors, selected from the group consisting of EGF, bFGF, PDGF, IGF-1,
and antibodies to receptors for these ligands.
6. The cell population of claim 1, obtained by differentiating pPS cells
in a medium containing growth factors, sorting the differentiated cells
for expression of NCAM or A2B5, and then collecting the sorted cells.
7. The cell population of claim 1, which can be induced to produce a
population of cells of which at least 30% of the cells have morphological
features of mature neurons and are NCAM positive.
8. The cell population of claim 7, wherein the cells having morphological
features of mature neurons have at least three of the following
characteristics: a) at least 60% of the cells show calcium flux when
administered acetylcholine; b) at least 60% of the cells show calcium
flux when administered GABA; c) at least 10% of the cells show calcium
flux when administered norepinephrine; d) at least 60% of the cells show
calcium flux when subjected to an external potassium concentration of 50
mM; or e) at least 25% of the cells demonstrate action potentials when
subject to stimulation in a whole-cell patch clamp apparatus.
9. The cell population of claim 1, which can be induced to produce a
population of cells in which at least 1% of the cells stain positively
for tyrosine hydroxylase.
10. The cell population of claim 1, comprising cells genetically altered
to express telomerase reverse transcriptase.
11. A cell population comprising mature neurons, astrocytes,
oligodendrocytes, or any combination thereof, obtained by further
differentiating the cell population according to claim 1.
12. The cell population of claim 11, comprising a subpopulation of at
least 30% of the cells that have the morphological characteristics of
neurons and are NCAM positive, wherein the subpopulation has the
following properties: a) at least 60% show calcium flux when administered
acetylcholine; b) at least 60% show calcium flux when administered GABA;
c) at least 10% show calcium flux when administered norepinephrine; d) at
least 60% show calcium flux when subjected to an external potassium
concentration of 50 mM; or e) at least 25% demonstrate action potentials
when subject to stimulation in a whole-cell patch clamp apparatus.
13. The cell population of claim 11, in which at least 1% of the cells
stain positively for tyrosine hydroxylase.
14. The cell population of claim 11, obtained by culturing the cell
population of claim 1 in a medium containing an activator of cAMP, a
neurotrophic factor, or a combination thereof.
15. An isolated neural precursor cell, obtained by providing the cell
population of claim 1, and selecting therefrom a cell having
characteristics of a neural precursor cell.
16. An isolated mature neuron, astrocyte, or oligodendrocyte, obtained by
providing the cell population of claim 11, and selecting therefrom a cell
having characteristics of a neuron, astrocyte, or oligodendrocyte,
17. The isolated mature neuron of claim 16, which is a dopaminergic
18. The isolated mature neuron of claim 16, obtained by culturing the cell
of claim 1 in a medium containing an activator of cAMP, a neurotrophic
factor (such as nerve growth factor, neurotrophin 3, or brain-derived
neurotrophic factor), or a combination thereof.
19. A cell population comprising at least .about.60% neural progenitor
cells and/or mature neurons that have the same genome as an established
human embryonic stem (hES) cell line.
20. The cell population of claim 18, wherein the neural progenitor cells
and/or mature neurons express NCAM, A2B5, MAP-2, or Nestin.
21. A method for obtaining neural precursor cells capable of producing a
cell population comprising at least 1% tyrosine hydroxylase positive
cells, comprising differentiating human embryonic stem cells.
22. The method of claim 21, wherein the differentiating comprises
culturing in a medium containing at least two ligands that bind growth
factor receptors, selected from the group consisting of EGF, bFGF, PDGF,
IGF-1, and antibodies to receptors for these ligands.
23. A method for obtaining a cell population comprising at least 1%
tyrosine hydroxylase positive cells, comprising differentiating human
embryonic stem cells.
24. The method of claim 21, comprising obtaining neural precursor cells
according to claim 19, and then culturing the cells obtained thereby in a
medium containing an activator of cAMP, a neurotrophic factor (such as
nerve growth factor, neurotrophin 3, or brain-derived neurotrophic
factor), or a combination thereof.
25. The method of claim 21, further comprising genetically altering the
cells to express telomerase reverse transcriptase before or after
26. A method of screening a compound for neural cell toxicity or
modulation, comprising combining the compound with a cell population
according to claim 1; determining any phenotypic or metabolic changes in
the cell that result from contact with the compound; and correlating the
change with neural cell toxicity or modulation.
27. A method of screening a compound for neural cell toxicity or
modulation, comprising combining the compound with a cell population
according to claim 11; determining any phenotypic or metabolic changes in
the cell that result from contact with the compound; and correlating the
change with neural cell toxicity or modulation.
28. A method for obtaining a polynucleotide comprising a nucleotide
sequence contained in an mRNA more highly expressed in neural progenitor
cells, the method comprising: a) determining the level of expression of a
plurality of mRNAs in one or more cells in the cell population of claim
1, in comparison to the level of expression of the same mRNAs in more
mature neural cells; b) identifying an mRNA expressed at a higher level
in the cell(s) from the cell population, relative to that in the more
mature cells; and c) preparing a polynucleotide comprising a nucleotide
sequence of at least 30 consecutive nucleotides contained in the mRNA
selected in step b).
29. A method of reconstituting or supplementing central nervous system
(CNS) function in an individual, comprising administering to the
individual a cell population according to claim 1.
30. A method of reconstituting or supplementing CNS function in an
individual, comprising administering to the individual a cell population
according to claim 11.
REFERENCE TO RELATED APPLICATIONS
 This application claims priority to U.S. Provisional Patent
Applications Ser. No. 60/205,600, filed May 17, 2000; and No. 60/257,608,
filed Dec. 22, 2000. The priority applications are hereby incorporated
herein by reference in their entirety.
 This invention relates generally to the field of cell biology of
embryonic cells and neural progenitor cells. More specifically, this
invention relates to the directed differentiation of human pluripotent
stem cells to form cells of the neuronal and glial lineages, using
special culture conditions and selection techniques.
 Repairing the central nervous system is one of the frontiers that
medical science has yet to conquer. Conditions such as Alzheimer's
disease, Parkinson's disease, epilepsy, Huntington's disease, and stroke
can have devastating consequences for those who are afflicted. Traumatic
injury to the head or the spinal chord can instantly propel someone from
the bounds of everyday life into the ranks of the disabled.
 What makes afflictions of the nervous system so difficult to manage
is the irreversibility of the damage often sustained. A central hope for
these conditions is to develop cell populations that can reconstitute the
neural network, and bring the functions of the nervous system back in
 For this reason, there is a great deal of evolving interest in
neural progenitor cells. Up until the present time, it was generally
thought that multipotent neural progenitor cells commit early in the
differentiation pathway to either neural restricted cells or glial
restricted cells. These in turn are thought to give rise to mature
neurons, or to mature astrocytes and oligodendrocytes. Multipotent neural
progenitor cells in the neural crest also differentiate to neurons,
smooth muscle, and Schwann cells. It is hypothesized that various
lineage-restricted precursor cells renew themselves and reside in
selected sites of the central nervous system, such as the spinal chord.
Cell lineage in the developing neural tube has been reviewed in the
research literature by Kalyani et al. (Biochem. Cell Biol. 6:1051, 1998).
 Putative multipotent neuroepithelial cells (NEP cells) have been
identified in the developing spinal cord. Kalyani et al. (Dev. Biol.
186:202, 1997) reported NEP cells in the rat. Mujtaba et al. (Dev. Biol.
214:113, 1999) reported NEP cells in the mouse. Differentiation of NEP
cells is thought to result in formation of restricted precursor cells
having characteristic surface markers.
 Putative neural restricted precursors (NRP) were characterized by
Mayer-Proschel et al. (Neuron 19:773, 1997). These cells express
cell-surface PS-NCAM, a polysialylated isoform of the neural cell
adhesion molecule. They reportedly have the capacity to generate various
types of neurons, but do not form glial cells.
 Putative glial restricted precursors (GRPs) were identified by Rao
et al. (Dev. Biol. 188: 48, 1997). These cells apparently have the
capacity to form glial cells but not neurons.
 Ling et al. (Exp. Neurol. 149:411, 1998) isolated progenitor cells
from the germinal region of rat fetal mesencephalon. The cells were grown
in EGF, and plated on poly-lysine coated plates, whereupon they formed
neurons and glia, with occasional tyrosine hydroxylase positive
(dopaminergic) cells, enhanced by including IL-1, IL-11, LIF, and GDNF in
the culture medium.
 Wagner et al. (Nature Biotechnol. 17:653, 1999) reported cells with
a ventral mesencephalic dopaminergic phenotype induced from an
immortalized multipotent neural stem cell line. The cells were
transfected with a Nurr1 expression vector, and then cocultured with VM
type 1 astrocytes. Over 80% of the cells obtained were claimed to have a
phenotype resembling endogenous dopaminergic neurons.
 Mujtaba et al. (supra) reported isolation of NRP and GRP cells from
mouse embryonic stem (mES) cells. The NRPs were PS-NCAM immunoreactive,
underwent self-renewal in defined medium, and differentiated into
multiple neuronal phenotypes. They apparently did not form glial cells.
The GRPs were A2B5-immunoreactive, and reportedly differentiated into
astrocytes and oligodendrocytes, but not neurons.
 A number of recent discoveries have raised expectations that
embryonic cells may become a pluripotential source for cells and tissues
useful in human therapy. Pluripotent cells are believed to have the
capacity to differentiate into essentially all types of cells in the body
(R. A. Pedersen, Scientif. Am. 280(4): 68, 1999). Early work on embryonic
stem cells was done using inbred mouse strains as a model (reviewed in
Robertson, Meth. Cell Biol. 75:173,1997; and Pedersen, Reprod. Fertil.
Dev. 6:543, 1994).
 Compared with mouse ES cells, monkey and human pluripotent cells
have proven to be much more fragile, and do not respond to the same
culture conditions. Only recently have discoveries been made that allow
primate embryonic cells to be cultured ex vivo.
 Thomson et al. (Proc. Natl. Acad. Sci. USA 92:7844, 1995) were the
first to successfully culture embryonic stem cells from primates, using
rhesus monkeys and marmosets as a model. They subsequently derived human
embryonic stem (hES) cell lines from human blastocysts (Science 282:114,
1998). Gearhart and coworkers derived human embryonic germ (hEG) cell
lines from fetal gonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci.
USA 95:13726, 1998). Both hES and hEG cells have the long-sought
characteristics of human pluripotent stem (hPS) cells: they are capable
of ongoing proliferation in vitro without differentiating, they retain a
normal karyotype, and they retain the capacity to differentiate to
produce all adult cell types.
 Reubinoff et al. (Nature Biotechnol. 18:399, 2000) reported somatic
differentiation of human blastocysts. The cells differentiated
spontaneously in culture, with no consistent pattern of structural
organization. After culturing for 4-7 weeks to high density,
multicellular aggregates formed above the plane of the monolayer.
Different cells in the culture expressed a number of different
phenotypes, including expression of .beta.-actin, desmin, and NCAM.
 Spontaneous differentiation of pluripotent stem cells in culture or
in teratomas generates cell populations with a highly heterogeneous
mixture of phenotypes, representing a spectrum of different cell
lineages. For most therapeutic purposes, it is desirable for
differentiated cells to be relatively uniform--both in terms of the
phenotypes they express, and the types of progeny they can generate.
 Accordingly, there is a pressing need for technology to generate
more homogeneous differentiated cell populations from pluripotent cells
of human origin.
 This invention provides a system for efficient production of
primate cells that have differentiated from pluripotent cells into cells
of the neuronal or glial lineage. Populations of cells are described
which contain precursors for either lineage, which provide a source for
generating additional precursor cells, the mature cells of the central
nervous system: neurons, astrocytes, or oligodendrocytes. Certain
embodiments of the invention have the ability to generate cells of both
lineages. The precursor and mature cells of this invention can be used a
number of important applications, including drug testing and therapy to
restore nervous system function.
 One embodiment of this invention is a cell population that
proliferates in an in vitro culture, obtained by differentiating primate
pluripotent stem (pPS) cells, wherein at least about 30% of the cells in
the population are committed to form neuronal cells, glial cells, or
both. A second embodiment is a cell population that proliferates in an in
vitro culture, comprising at least about 60% neural progenitor cells,
wherein at least 10% of the cells can differentiate into neuronal cells,
and at least 10% of the cells can differentiate into glial cells. A third
embodiment is a cell population that proliferates in an in vitro culture,
comprising at least about 60% neural progenitor cells, wherein at least
10% of the cells express A2B5, and at least 10% of the cells express
 Certain cell populations of the invention are obtained by
differentiating primate pluripotent stem cells, such as human embryonic
stem cells. Some are obtained by differentiating stem cells in a medium
containing at least two or more ligands that bind growth factor
receptors. Some are obtained by differentiating pPS cells in a medium
containing growth factors, sorting the differentiated cells for
expression of NCAM or A2B5, and then 0 collecting the sorted cells.
Certain cell populations are enriched such that at least 70% of the cells
express NCAM or A2B5.
 Another embodiment of this invention is a cell population
comprising mature neurons, astrocytes, oligodendrocytes, or any
combination thereof, obtained by further differentiating a precursor cell
population of this invention. Some such populations are obtained by
culturing neural precursors in a medium containing an activator of cAMP,
a neurotrophic factor, or a combination of such factors. As described
below, neurons produced by such methods may be capable of exhibiting an
action potential, may show gated sodium and potassium channels, and may
show calcium flux when administered with neurotransmitters or their
equivalents. Included are populations of cells containing a substantial
proportion of dopaminergic neurons, detectable for example by staining
for tyrosine hydroxylase.
 Also embodied in the invention are isolated neural precursor cells,
neurons, astrocytes, and oligodendrocytes, obtained by selecting a cell
for the desired phenotype from one of the cell populations.
 Where derived from an established line of pPS cells, the cell
populations and isolated cells of this invention will typically have the
same genome as the line from which they are derived. This means that the
chromosomal DNA will be over 90% identical between the pPS cells and the
neural cells, which can be inferred if the neural cells are obtained from
the undifferentiated line through the course of normal mitotic division.
Neural cells that have been treated by recombinant methods to introduce a
transgene (such as TERT) or knock out an endogenous gene are still
considered to have the same genome as the line from which they are
derived, since all non-manipulated genetic elements are preserved.
 A further embodiment of the invention is a method of screening a
compound for neural cell toxicity or modulation, in which a culture is
prepared containing the compound and a neural cell or cell population of
this invention, and any phenotypic or metabolic change in the cell that
results from contact with the compound is determined.
 Yet another embodiment of the invention is a method for obtaining a
polynucleotide comprising a nucleotide sequence contained in an mRNA more
highly expressed in neural progenitor cells or differentiated cells, as
described and exemplified further on in this disclosure. The nucleotide
sequence can in turn be used to produce recombinant or synthetic
polynucleotides, proteins, and antibodies for gene products enriched or
suppressed in neural cells. Antibodies can also be obtained by using the
cells of this invention as an immunogen or an adsorbent to identify
markers enriched or suppressed in neural cells.
 A further embodiment of the invention is a method of reconstituting
or supplementing central nervous system (CNS) function in an individual,
in which the individual is administered with an isolated cell or cell
population of this invention. The isolated cells and cell populations can
be used in the preparation of a medicament for use in clinical and
veterinary treatment. Medicaments comprising the cells of this invention
can be formulated for use in such therapeutic applications.
 Other embodiments of the invention are methods for obtaining the
neural precursor cells and fully differentiated cells of this invention,
using the techniques outlined in this disclosure on a suitable stem cell
population. Included are methods for producing cell populations
containing dopaminergic cells at a frequency of 1%, 3% or 5%--and
populations of progenitor cells capable of generating dopaminergic cells
at this frequency--from primate embryonic stem cells. This is
particularly significant in view of the loss in dopamine neuron function
that occurs in Parkinson's disease. The compositions, methods, and
techniques described in this disclosure hold considerable promise for use
in diagnostic, drug screening, and therapeutic applications.
 These and other embodiments of the invention will be apparent from
the description that follows.
 FIG. 1 is a graph representing the growth of cells bearing neural
markers that were derived from human embryonic stem cells. The upper
panel shows growth of cells maintained in the presence of CNTF, bFGF, and
NT3, and then sorted for expression of NCAM. The lower panel shows growth
of cells maintained in the presence of EGF, bFGF, PDGF, and IGF-1, and
then sorted for expression of A2B5. Four different hES cell lines were
used: H1, H7, H9, and H13. The A2B5 selected population has been passaged
over 7 times, and can be further differentiated into both neuronal and
 FIG. 2 is a schematic diagram outlining an exemplary procedure for
obtaining A2B5-positive cells. Abbreviations used: MEF-CM=medium
conditioned by culturing with mouse embryonic fibroblasts; +/-SHH=with or
without sonic hedgehog; D/F12=DMEM/F12 medium; N2 and B27, culture
supplements (Gibco); EPFI=differentiation agents EGF, PDGF, bFGF, and
IGF-1; PLL=poly-L lysine substrate; PLL/FN=substrate of poly-L lysine and
 FIG. 3 is a half-tone reproduction of a fluorescence micrograph of
the brains from neonatal rats administered with cells that express green
fluorescent protein. Left panels: parental hES cell line. Middle panels:
embryoid body cells formed from the parental line. Right panels:
differentiated cells sorted for expression of NCAM. Undifferentiated hES
cells and embryoid body cells remain in the area of administration and
show evidence of necrosis. In contrast, the differentiated
NCAM.sup.+cells appear as single cells, and have migrated away from the
 FIG. 4 is a phot
ocopy reproduction of a fluorescence micrograph
showing a cell staining for tyrosine hydroxylase (TH), a marker for
dopaminergic cells. Embryoid bodies made from human ES cells were
maintained in 10 .mu.m retinoic acid for 4 days, plated into a
neural-supportive cocktail, and then passaged into medium containing 10
ng/mL NT-3 and 10 ng/mL BDNF. Certain populations of this invention
contain >1% TH-positive cells.
 FIG. 5 is a series of graphs showing response of the
neural-restricted precursors to various neurotransmitters. Panel A shows
the ratio of emission data from single cells on two different coverslips.
Both cells responded to GABA, elevated potassium, acetylcholine and ATP.
Panel B shows the frequency of cells tested that responded to specific
neurotransmitters. Panel C shows the combinations of neurotransmitter
 FIG. 6 is a series of graphs showing electrophysiology of
neural-restricted precursors. Panel A shows sodium and potassium currents
observed in two cells depolarized to test potentials between -80 and 80
mV from a holding potential of -100 mV. Panel B shows the inward
(Na.sup.+) and outward (K) peak current-voltage relationships observed.
Panel C shows action potentials generated by the same cells n response to
depolarizing stimuli. These measurements show that neural precursor cells
derived from human ES cells are capable of generating action potentials
characteristic of neurotransmission.
 This invention provides a system for preparing and characterizing
neural progenitor cells, suitable for use for therapeutic administration
and drug screening.
 It has been discovered that when pluripotent stem cells are
cultured in the presence of selected differentiating agents, a population
of cells is derived that has a remarkably high proportion of cells with
phenotypic characteristics of neural cells. Optionally, the proportion of
neural cells can be enhanced by sorting differentiated cells according to
cell-surface markers. Since certain types of pluripotent stem cells (such
as embryonic stem cells) can proliferate in culture for a year or more,
the invention described in this disclosure provides an almost limitless
supply of neural precursors. Certain cell populations of this invention
are capable of generating cells of the neuronal or glial lineage, and
themselves can be replicated through a large number of passages in
 FIG. 1 shows the growth curve of cells that have been cultured with
differentiating agents, and then selected according to whether they bear
polysialylated NCAM, or the A2B5 epitope. Either of these cell
populations can be proliferated through a large number of cell doublings.
 Differentiated cells positively selected for A2B5 expression
comprise cells that appear to express A2B5 without NCAM, and cells that
express A2B5 and NCAM simultaneously. In one of the experiments described
below, maturation of these cells produced 13% oligodendrocytes, and 38%
neurons. Since these cells proliferate in long-term culture without
losing their phenotype, the population can provide a reserve of
multipotential cells. Upon administration to a subject with CNS
dysfunction, the population would comprise cells that may repopulate both
the neuronal and glial cell lineage, as needed.
 If desired, the neural precursor cells can be further
differentiated ex vivo, either by culturing with a maturation factor,
such as a neurotrophic factor, or by withdrawing one or more factors that
sustain precursor cell renewal. Neurons, astrocytes, and oligodendrocytes
are mature differentiated cells of the neural lineage that can be
obtained by culturing the precursor cells in this fashion. The neurons
obtained by these methods have extended processes characteristic of this
cell type, show staining for neuron-specific markers like neurofilament
and MAP-2, and show evidence of synapse formation, as detected by
staining for synaptophysin. FIG. 5 shows that these cells respond to a
variety of neurotransmitter substances. FIG. 6 shows that these cells are
capable of action potentials as measured in a standard patch-clamp
system. In all these respects, the cells are apparently capable of full
 Of particular interest is the ability of this system to generate a
supply of dopaminergic neurons (FIG. 4). Cells of this type are
particularly desirable for the treatment of Parkinson's disease, for
which the best current modality is an allograft of fetal brain tissue.
The use of fetal tissue as a clinical therapy is fraught with supply and
procedural issues, but no other source described previously can supply
the right kind of cells with sufficient abundance. The neural precursor
cells of this invention are capable of generating differentiated cells in
which several percent of the neurons have a dopaminergic phenotype. This
is believed to be a sufficient proportion for cell replacement therapy in
Parkinson's disease, and warrants the development of the progenitor
populations of this invention for therapeutic use.
 Since pluripotent stem cells and some of the lineage-restricted
precursors of this invention proliferate extensively in culture, the
system described in this disclosure provides an unbounded supply of
neuronal and glial cells for use in research, pharmaceutical development,
and the therapeutic management of CNS abnormalities. The preparation and
utilization of the cells of this invention is illustrated further in the
description that follows.
 For the purposes of this disclosure, the terms "neural progenitor
cell" or "neural precursor cell" mean a cell that can generate progeny
that are either neuronal cells (such as neuronal precursors or mature
neurons) or glial cells (such as glial precursors, mature astrocytes, or
mature oligodendrocytes). Typically, the cells express some of the
phenotypic markers that are characteristic of the neural lineage.
Typically, they do not produce progeny of other embryonic germ layers
when cultured by themselves in vitro, unless dedifferentiated or
reprogrammed in some fashion.
 A "neuronal progenitor cell" or "neuronal precursor cell" is a cell
that can generate progeny that are mature neurons. These cells may or may
not also have the capability to generate glial cells.
 A "glial progenitor cell" or "glial precursor cell" is a cell that
can generate progeny that are mature astrocytes or mature
oligodendrocytes. These cells may or may not also have the capability to
generate neuronal cells.
 A "multipotent neural progenitor cell population" is a cell
population that has the capability to generate both progeny that are
neuronal cells (such as neuronal precursors or mature neurons), and
progeny that are glial cells (such as glial precursors, mature
astrocytes, or mature oligodendrocytes), and sometimes other types of
cells. The term does not require that individual cells within the
population have the capability of forming both types of progeny, although
individual cells that are multipotent neural progenitors may be present.
 In the context of cell ontogeny, the adjective "differentiated" is
a relative term. A "differentiated cell" is a cell that has progressed
further down the developmental pathway than the cell it is being compared
with. Thus, pluripotent embryonic stem cells can differentiate to
lineage-restricted precursor cells, such as hematopoetic cells, which are
pluripotent for blood cell types; hepatocyte progenitors, which are
pluripotent for hepatocytes; and various types of neural progenitors
listed above. These in turn can be differentiated further to other types
of precursor cells further down the pathway, or to an end-stage
differentiated cell, which plays a characteristic role in a certain
tissue type, and may or may not retain the capacity to proliferate
further. Neurons, astrocytes, and oligodendrocytes are all examples of
terminally differentiated cells.
 A "differentiation agent", as used in this disclosure, refers to
one of a collection of compounds that are used in culture systems of this
invention to produce differentiated cells of the neural lineage
(including precursor cells and terminally differentiated cells). No
limitation is intended as to the mode of action of the compound. For
example, the agent may assist the differentiation process by inducing or
assisting a change in phenotype, promoting growth of cells with a
particular phenotype or retarding the growth of others, or acting in
concert with other agents through unknown mechanisms.
 Unless explicitly indicated otherwise, the techniques of this
invention can be brought to bear without restriction on any type of
progenitor cell capable of differentiating into neuronal or glial cells.
 Prototype "primate Pluripotent Stem cells" (pPS cells) are
pluripotent cells derived from pre-embryonic, embryonic, or fetal tissue
at any time after fertilization, and have the characteristic of being
capable under appropriate conditions of producing progeny of several
different cell types that are derivatives of all of the three germinal
layers (endoderm, mesoderm, and ectoderm), according to a standard
art-accepted test, such as the ability to form a teratoma in 8-12 week
old SCID mice.
 Included in the definition of pPS cells are embryonic cells of
various types, exemplified by human embryonic stem (hES) cells, described
by Thomson et al. (Science 282:1145, 1998); embryonic stem cells from
other primates, such as Rhesus stem cells (Thomson et al., Proc. Nati.
Acad. Sci. USA 92:7844, 1995), marmoset stem cells (Thomson et al., Biol.
Reprod. 55:254, 1996) and human embryonic germ (hEG) cells (Shamblott et
al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Other types of
pluripotent cells are also included in the term. Any cells of primate
origin that are capable of producing progeny that are derivatives of all
three germinal layers are included, regardless of whether they were
derived from embryonic tissue, fetal tissue, or other sources. The pPS
cells are not derived from a malignant source. It is desirable (but not
always necessary) that the cells be karyotypically normal.
 pPS cell cultures are described as "undifferentiated" when a
substantial proportion of stem cells and their derivatives in the
population display morphological characteristics of undifferentiated
cells, clearly distinguishing them from differentiated cells of embryo or
adult origin. Undifferentiated pPS cells are easily recognized by those
skilled in the art, and typically appear in the two dimensions of a
microscopic view in colonies of cells with high nuclear/cytoplasmic
ratios and prominent nucleoli. It is understood that colonies of
undifferentiated cells within the population will often be surrounded by
neighboring cells that are differentiated. "Feeder cells" or "feeders"
are terms used to describe cells of one type that are co-cultured with
cells of another type, to provide an environment in which the cells of
the second type can grow. For example, certain types of pPS cells can be
supported by primary mouse embryonic fibroblasts, immortalized mouse
embryonic fibroblasts, or human fibroblast-like cells differentiated from
hES cell. pPS cell populations are said to be "essentially free" of
feeder cells if the cells have been grown through at least one round
after splitting in which fresh feeder cells are not added to support the
growth of the pPS.
 The term "embryoid bodies" is a term of art synonymous with
"aggregate bodies". The terms refer to aggregates of differentiated and
undifferentiated cells that appear when pPS cells overgrow in monolayer
cultures, or are maintained in suspension cultures. Embryoid bodies are a
mixture of different cell types, typically from several germ layers,
distinguishable by morphological criteria and cell markers detectable by
 A "growth environment" is an environment in which cells of interest
will proliferate, differentiate, or mature in vitro. Features of the
environment include the medium in which the cells are cultured, any
growth factors or differentiation-inducing factors that may be present,
and a supporting structure (such as a substrate on a solid surface) if
 A cell is said to be "genetically altered", "transfected", or
"genetically transformed" when a polynucleotide has been transferred into
the cell by any suitable means of artificial manipulation, or where the
cell is a progeny of the originally altered cell that has inherited the
polynucleotide. The polynucleotide will often comprise a transcribable
sequence encoding a protein of interest, which enables the cell to
express the protein at an elevated level. The genetic alteration is said
to be "inheritable" if progeny of the altered cell have the same
 The term "antibody" as used in this disclosure refers to both
polyclonal and monoclonal antibody. The ambit of the term deliberately
encompasses not only intact immunoglobulin molecules, but also such
fragments and derivatives of immunoglobulin molecules (such as single
chain Fv constructs, diabodies, and fusion constructs) as may be prepared
by techniques known in the art, and retaining a desired antibody binding
 General Techniques
 For further elaboration of general techniques useful in the
practice of this invention, the practitioner can refer to standard
textbooks and reviews in cell biology, tissue culture, and embryology.
Included are Teratocarcinomas and embryonic stem cells: A practical
approach (E. J. Robertson, ed., IRL Press Ltd. 1987); Guide to Techniques
in Mouse Development (P. M. Wasserman et al. eds., Academic Press 1993);
Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles, Meth. Enzymol.
225:900, 1993); Properties and uses of Embryonic Stem Cells: Prospects
for Application to Human Biology and Gene Therapy (P. D. Rathjen et al.,
Reprod. Fertil. Dev. 10:31, 1998).
 For elaboration of nervous system abnormalities, and the
characterization of various types of nerve cells, markers, and related
soluble factors, the reader is referred to CNS Regeneration: Basic
Science and Clinical Advances, M. H. Tuszynski & J. H. Kordower, eds.,
Academic Press, 1999.
 Methods in molecular genetics and genetic engineering are described
in Molecular Cloning: A Laboratory Manual, 2nd Ed. (Sambrook et al.,
1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell
Culture (R. I. Freshney, ed., 1987); the series Methods in Enzymology
(Academic Press); Gene Transfer Vectors for Mammalian Cells (J. M. Miller
& M. P. Calos, eds., 1987); Current Protocols in Molecular Biology and
Short Protocols in Molecular Biology, 3rd Edition (F. M. Ausubel et al.,
eds., 1987 & 1995); and Recombinant DNA Methodology II (R. Wu ed.,
Academic Press 1995). Reagents, cloning vectors, and kits for genetic
manipulation referred to in this disclosure are available from commercial
vendors such as BioRad, Stratagene, Invitrogen, and ClonTech.
 General techniques used in raising, purifying and modifying
antibodies, and the design and execution of immunoassays including
immunohistochemistry, the reader is referred to Handbook of Experimental
Immunology (D. M. Weir & C. C. Blackwell, eds.); Current Protocols in
Immunology (J. E. Coligan et al., eds., 1991); and R. Masseyeff, W. H.
Albert, and N. A. Staines, eds., Methods of Immunological Analysis
(Weinheim: VCH Verlags GmbH, 1993).
 Sources of Stem Cells
 This invention can be practiced using stem cells of various types,
which may include the following non- limiting examples.
 U.S. Pat. No. 5,851,832 reports multipotent neural stem cells
obtained from brain tissue. U.S. Pat. No. 5,766,948 reports producing
neuroblasts from newborn cerebral hemispheres. U.S. Pat. Nos. 5,654,183
and 5,849,553 report the use of mammalian neural crest stem cells. U.S.
Pat. No. 6,040,180 reports in vitro generation of differentiated neurons
from cultures of mammalian multipotential CNS stem cells. WO 98/50526 and
WO 99/01159 report generation and isolation of neuroepithelial stem
cells, oligodendrocyte-astrocyte precursors, and lineage-restricted
neuronal precursors. U.S. Pat. No. 5,968,829 reports neural stem cells
obtained from embryonic forebrain and cultured with a medium comprising
glucose, transferrin, insulin, selenium, progesterone, and several other
 Except where otherwise required, the invention can be practiced
using stem cells of any vertebrate species. Included are stem cells from
humans; as well as non-human primates, domestic animals, livestock, and
other non-human mammals.
 Amongst the stem cells suitable for use in this invention are
primate pluripotent stem (pPS) cells derived from tissue formed after
gestation, such as a blastocyst, or fetal or embryonic tissue taken any
time during gestation. Non-limiting examples are primary cultures or
established lines of embryonic stem cells or embryonic germ cells.
 Embryonic Stem Cells
 Embryonic stem cells can be isolated from blastocysts of members of
the primate species (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844,
1995). Human embryonic stem (hES) cells can be prepared from human
blastocyst cells using the techniques described by Thomson et al. (U.S.
Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133
ff., 1998) and Reubinoff et al, Nature Biotech. 18:399,2000.
 Briefly, human blastocysts are obtained from human in vivo
preimplantation embryos. Alternatively, in vitro fertilized (IVF) embryos
can be used, or one-cell human embryos can be expanded to the blastocyst
stage (Bongso et al., Hum Reprod 4: 706,1989). Embryos are cultured to
the blastocyst stage in G1.2 and G2.2 medium (Gardner et al., Fertil.
Steril. 69:84, 1998). The zona pellucida is removed from developed
blastocysts by brief exposure to pronase (Sigma). The inner cell masses
are isolated by immunosurgery, in which blastocysts are exposed to a 1:50
dilution of rabbit anti-human spleen cell antiserum for 30 min, then
washed for 5 min three times in DMEM, and exposed to a 1:5 dilution of
Guinea pig complement (Gibco) for 3 min (Solter et al., Proc. Natl. Acad.
Sci. USA 72:5099, 1975). After two further washes in DMEM, lysed
trophectoderm cells are removed from the intact inner cell mass (ICM) by
gentle pipetting, and the ICM plated on mEF feeder layers.
 After 9 to 15 days, inner cell mass-derived outgrowths are
dissociated into clumps, either by exposure to calcium and magnesium-free
phosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispase or
trypsin, or by mechanical dissociation with a micropipette; and then
replated on mEF in fresh medium. Growing colonies having undifferentiated
morphology are individually selected by micropipette, mechanically
dissociated into clumps, and replated. ES-like morphology is
characterized as compact colonies with apparently high nucleus to
cytoplasm ratio and prominent nucleoli. Resulting ES cells are then
routinely split every 1-2 weeks by brief trypsinization, exposure to
Dulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase
(.about.200 U/mL; Gibco) or by selection of individual colonies by
micropipette. Clump sizes of about 50 to 100 cells are optimal.
 Embryonic Germ Cells
 Human Embryonic Germ (hEG) cells can be prepared from primordial
germ cells present in human fetal material taken about 8-11 weeks after
the last menstrual period. Suitable preparation methods are described in
Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726,1998 and U.S. Pat.
 Briefly, genital ridges are rinsed with isotonic buffer, then
placed into 0.1 mL 0.05% trypsin/0.53 mM sodium EDTA solution (BRL) and
cut into <1 mm.sup.3 chunks. The tissue is then pipetted through a 100
.mu.L tip to further disaggregate the cells. It is incubated at
37.degree. C. for .about.5 min, then .about.3.5 mL EG growth medium is
added. EG growth medium is DMEM, 4500 mg/L D-glucose, 2200 mg/L mM
NaHCO.sub.3; 15% ES qualified fetal calf serum (BRL); 2 mM glutamine
(BRL); 1 mM sodium pyruvate (BRL); 1000-2000 U/mL human recombinant
leukemia inhibitory factor (LIF, Genzyme); 1-2 ng/ml human recombinant
bFGF (Genzyme); and 10 .mu.M forskolin (in 10% DMSO). In an alternative
approach, EG cells are isolated using hyaluronidase/collagenase/DNAse.
Gonadal anlagen or genital ridges with mesenteries are dissected from
fetal material, the genital ridges are rinsed in PBS, then placed in 0.1
ml HCD digestion solution (0.01% hyaluronidase type V, 0.002% DNAse I,
0.1% collagenase type IV, all from Sigma prepared in EG growth medium).
Tissue is minced, incubated 11 h or overnight at 37.degree. C.,
resuspended in 1-3 mL of EG growth medium, and plated onto a feeder
 Ninety-six well tissue culture plates are prepared with a
sub-confluent layer of feeder cells (e.g., STO cells, ATCC No. CRL 1503)
cultured for 3 days in modified EG growth medium free of LIF, bFGF or
forskolin, inactivated with 5000 rad .gamma.-irradiation. .about.0.2 mL
of primary germ cell (PGC) suspension is added to each of the wells. The
first passage is done after 7-10 days in EG growth medium, transferring
each well to one well of a 24-well culture dish previously prepared with
irradiated STO mouse fibroblasts. The cells are cultured with daily
replacement of medium until cell morphology consistent with EG cells is
observed, typically after 7-30 days or 1-4 passages.
 Propagation of pPS Cells in an Undifferentiated State
 pPS cells can be propagated continuously in culture, using culture
conditions that promote proliferation without promoting differentiation.
Exemplary serum-containing ES medium is made with 80% DMEM (such as
Knock-Out DMEM, Gibco), 20% of either defined fetal bovine serum (FBS,
Hyclone) or serum replacement (WO 98/30679), 1% non-essential amino
acids, 1 mM L-glutamine, and 0.1 mM .gamma.-mercaptoethanol. Just before
use, human bFGF is added to 4 ng/mL (WO 99/20741, Geron Corp.).
 Traditionally, ES cells are cultured on a layer of feeder cells,
typically fibroblasts derived from embryonic or fetal tissue. Embryos are
harvested from a CF1 mouse at 13 days of pregnancy, transferred to 2 mL
trypsin/EDTA, finely minced, and incubated 5 min at 37.degree. C. 10% FBS
is added, debris is allowed to settle, and the cells are propagated in
90% DMEM, 10% FBS, and 2 mM glutamine. To prepare a feeder cell layer,
cells are irradiated to inhibit proliferation but permit synthesis of
factors that support ES cells (.about.4000 rads .gamma.-irradiation).
Culture plates are coated with 0.5% gelatin overnight, plated with
375,000 irradiated mEFs per well, and used 5 h to 4 days after plating.
The medium is replaced with fresh hES medium just before seeding pPS
 Scientists at Geron have discovered that pPS cells can
alternatively be maintained in an undifferentiated state even without
feeder cells. The environment for feeder-free cultures includes a
suitable culture substrate, particularly an extracellular matrix such as
Matrigel.RTM. or laminin. The pPS cells are plated at >15,000 cells
cm.sup.-2 (optimally 90,000 cm.sup.-2 to 170,000 cm.sup.-2). Typically,
enzymatic digestion is halted before cells become completely dispersed
(say, .about.5 min with collagenase IV). Clumps of .about.10-2000 cells
are then plated directly onto the substrate without further dispersal.
 Feeder-free cultures are supported by a nutrient medium typically
conditioned by culturing irradiated primary mouse embryonic fibroblasts,
telomerized mouse fibroblasts, or fibroblast-like cells derived from pPS
cells. Medium can be conditioned by plating the feeders at a density of
.about.5.6.times.10.sup.4 cm.sup.-2 in a serum free medium such as KO
DMEM supplemented with 20% serum replacement and 4 ng/mL bFGF. Medium
that has been conditioned for 1-2 days is supplemented with further bFGF,
and used to support pPS cell culture for 1-2 days.
 Under the microscope, ES cells appear with high nuclear/cytoplasmic
ratios, prominent nucleoli, and compact colony formation with poorly
discernable cell junctions. Primate ES cells express stage-specific
embryonic antigens (SSEA) 3 and 4, and markers detectable using
antibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science
282:1145, 1998). Mouse ES cells can be used as a positive control for
SSEA-1, and as a negative control for SSEA-4, Tra-1-60, and Tra-1-81.
SSEA-4 is consistently present on human embryonal carcinoma (hEC) cells.
Differentiation of pPS cells in vitro results in the loss of SSEA-4,
Tra-1-60, and Tra-1-81 expression and increased expression of SSEA-1.
SSEA-1 is also found on hEG cells.
 Materials and Procedures for Preparing Neural Precursors and
Terminally Differentiated Cells
 Certain neural precursor cells of this invention are obtained by
culturing, differentiating, or reprogramming stem cells in a special
growth environment that enriches for cells with the desired phenotype
(either by outgrowth of the desired cells, or by inhibition or killing of
other cell types). These methods are applicable to many types of stem
cells, including primate pluripotent stem (pPS) cells described in the
 Typically, the differentiation takes place in a culture environment
comprising a suitable substrate, and a nutrient medium to which the
differentiation agents are added. Suitable substrates include solid
surfaces coated with a positive charge, such as a basic amino acid,
exemplified by poly-L-lysine and polyornithine. Substrates can be coated
with extracellular matrix components, exemplified by fibronectin. Other
permissive extracellular matrixes include Matrigel.RTM. (extracellular
matrix from Engelbreth-Holm-Swarm tumor cells) and laminin. Also suitable
are combination substrates, such as poly-L-lysine combined with
fibronectin, laminin, or both.
 Suitable differentiation agents include growth factors of various
kinds, such as epidermal growth factor (EGF), transforming growth factor
a (TGF-.alpha.), any type of fibroblast growth factor (exemplified by
FGF-4, FGF-8, and basic fibroblast growth factor=bFGF), platelet-derived
growth factor (PDGF), insulin-like growth factor (IGF-1 and others), high
concentrations of insulin, sonic hedgehog, members of the neurotrophin
family (such as nerve growth factor=NGF, neurotrophin 3=NT-3,
brain-derived neurotrophic factor=BDNF), bone morphogenic proteins
(especially BMP-2 & BMP-4), retinoic acid (RA) and ligands to receptors
that complex with gp 30 (such as LIF, CNTF, and IL-6). Also suitable are
alternative ligands and antibodies that bind to the respective
cell-surface receptors for the aforementioned factors. Typically, a
plurality of differentiation agents is used, which may comprise 2, 3, 4,
or more of the agents listed above or in the examples below. Exemplary is
a cocktail containing EGF, bFGF, PDGF, and IGF-1 (Examples 1 and 2).
 The factors are supplied to the cells in a nutrient medium, which
is any medium that supports the proliferation or survival of the desired
cell type. It is often desirable to use a defined medium that supplies
nutrients as free amino acids rather than serum. It is also beneficial to
supplement the medium with additives developed for sustained cultures of
neural cells. Exemplary are N2 and B27 additives, available commercially
 Where the stem cells are pPS cells, the cells (obtained from feeder
cell supported or feeder-free cultures) are differentiated by culturing
in the presence of a suitable cocktail of differentiation agents.
 In one method of affecting differentiation, pPS cells are plated
directly onto a suitable substrate, such as an adherent glass or plastic
surface, such as coverslips coated with a polyamine. The cells are then
cultured in a suitable nutrient medium that is adapted to promote
differentiation towards the desired cell lineage. This is referred to as
the "direct differentiation" method.
 In another method, pPS cells are first let differentiate into a
heterogeneous cell population. In an exemplary variation, embryoid bodies
are formed from the pPS cells by culturing them in suspension.
Optionally, one or more of the differentiation agents listed earlier
(such as retinoic acid) can be included in the medium to promote
differentiation within the embryoid body. After the embryoid bodies have
reached sufficient size (typically 3-4 days), they are plated onto the
substrate of the differentiation culture. The embryoid bodies can be
plated directly onto the substrate without dispersing the cells. This
allows neural cell precursors to migrate out of the embryoid bodies and
on to the extracellular matrix. Subsequent passaging of these cultures
into an appropriate medium helps select out the neural progenitor cells.
 Cells prepared according to these procedures have been found to be
capable of further proliferation (Example 1). As many as 30%, 50%, 75% or
more of the cells express either polysialylated NCAM or the A2B5 epitope,
or both. Typically, at least about 10%, 20%, 30% or 50% of the cells
express NCAM, and at least about 10%, 20%, 30% or 50% of the cells
express A2B5--which implies that they have the capacity to form cells of
the neuronal lineage, and the glial lineage, respectively.
 Optionally, the differentiated cells can be sorted based on
phenotypic features to enrich for certain populations. Typically, this
will involve contacting each cell with an antibody or ligand that binds
to a marker characteristic of neural cells, followed by separation of the
specifically recognized cells from other cells in the population. One
method is immunopanning, in which specific antibody is coupled to a solid
surface. The cells are contacted with the surface, and cells not
expressing the marker are washed away. The bound cells are then recovered
by more vigorous elution. Variations of this are affinity chromatography
and antibody-mediated magnetic cell sorting. In a typical sorting
procedure, the cells are contacted with a specific primary antibody, and
then captured with a secondary anti-immunoglobulin reagent bound to a
magnetic bead. The adherent cells are then recovered by collecting the
beads in a magnetic field.
 Another method is fluorescence-activated cell sorting, in which
cells expressing the marker are labeled with a specific antibody,
typically by way of a fluorescently labeled secondary
anti-immunoglobulin. The cells are then separated individually according
to the amount of bound label using a suitable sorting device. Any of
these methods permit recovery of a positively selected population of
cells that bear the marker of interest, and a negatively selected
population of cells that not bear the marker in sufficient density or
accessibility to be positively selected. Negative selection can also be
effected by incubating the cells successively with a specific antibody,
and a preparation of complement that will lyse cells to which the
antibody has bound. Sorting of the differentiated cell population can
occur at any time, but it has generally been found that sorting is best
effected shortly after initiating the differentiation process.
 It has been found that cells selected positively for polysialylated
NCAM can provide a population that is 60%, 70%, 80%, or even 90% NCAM
positive (Example 1). This implies that they are capable of forming some
type of neural cell, including neurons.
 It has also been found that cells selected positively for A2B5 can
provide a population that is 60%, 70%, 80%, or even 90% A2B5 positive
(Example 2). This implies that they are capable of forming some type of
neural cell, possibly including both neurons and glial cells. The A2B5
positive cells can be sorted again into two separate populations: one
that is A2B5 positive and NCAM negative, and one that is both A2B5
positive and NCAM positive.
 Differentiated or separated cells prepared according to this
procedure can be maintained or proliferated further in any suitable
culture medium. Typically, the medium will contain most of the
ingredients used initially to differentiate the cells.
 If desired, neural precursor cells prepared according to these
procedures can be further differentiated to mature neurons, astrocytes,
or oligodendrocytes. This can be effected by culturing the cells in a
maturation factor, such as forskolin or other compound that elevates
intracellular cAMP levels, such as cholera toxin, isobutylmethylxanthine,
dibutyladenosine cyclic monophosphate, or other factors such as c-kit
ligand, retinoic acid, or neurotrophins. Particularly effective are
neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF). Other
candidates are GDNF, BMP-2, and BMP-4. Alternatively or in addition,
maturation can be enhanced by withdrawing some or all of the factors that
promote neural precursor proliferation, such as EGF or FGF.
 For use in therapeutic and other applications, it is often
desirable that populations of precursor or mature neurological cells be
substantially free of undifferentiated pPS cells. One way of depleting
undifferentiated stem cells from the population is to transfect them with
a vector in which an effector gene under control of a promoter that
causes preferential expression in undifferentiated cells. Suitable
promoters include the TERT promoter and the OCT-4 promoter. The effector
gene may be directly lytic to the cell (encoding, for example, a toxin or
a mediator of apoptosis). Alternatively, the effector gene may render the
cell susceptible to toxic effects of an external agent, such as an
antibody or a prodrug. Exemplary is a herpes simplex thymidine kinase
(tk) gene, which causes cells in which it is expressed to be susceptible
to ganciclovir. Suitable pTERT-tk constructs are provided in
International Patent Publication WO 98/14593 (Morin et al.).
 Characteristics of neural precursors and terminally differentiated
 Cells can be characterized according to a number of phenotypic
criteria. The criteria include but are not limited to microscopic
observation of morphological features, detection or quantitation of
expressed cell markers, enzymatic activity, or neurotransmitters and
their receptors, and electrophysiological function.
 Certain cells embodied in this invention have morphological
features characteristic of neuronal cells or glial cells. The features
are readily appreciated by those skilled in evaluating the presence of
such cells. For example, characteristic of neurons are small cell bodies,
and multiple processes reminiscent of axons and dendrites. Cells of this
invention can also be characterized according to whether they express
phenotypic markers characteristic of neural cells of various kinds.
 Markers of interest include but are not limited to .beta.-tubulin
III, microtubule-associated protein 2 (MAP-2), or neurofilament,
characteristic of neurons; glial fibrillary acidic protein (GFAP),
present in astrocytes; galactocerebroside (GaIC) or myelin basic protein
(MBP), characteristic of oligodendrocytes; Oct-4, characteristic of
undifferentiated hES cells; Nestin, characteristic of neural precursors
and other cells; and both A2B5 and polysialylated NCAM, as already
described. While A2B5 and NCAM are instructive markers when studying
neural lineage cells, it should be appreciated that these markers can
sometimes be displayed on other cell types, such as liver or muscle
cells. .beta.-Tubulin III was previously thought to be specific for
neural cells, but it has been discovered that a subpopulation of hES
cells is also .beta.-tubulin III positive. MAP-2 is a more stringent
marker for fully differentiated neurons of various types.
 Tissue-specific markers listed in this disclosure and known in the
art can be detected using any suitable immunological technique--such as
flow immunocytochemistry for cell-surface markers, immunohistochemistry
(for example, of fixed cells or tissue sections) for intracellular or
cell-surface markers, Western blot analysis of cellular extracts, and
enzyme-linked immunoassay, for cellular extracts or products secreted
into the medium. Expression of an antigen by a cell is said to be
"antibody-detectable" if a significantly detectable amount of antibody
will bind to the antigen in a standard immunocytochemistry or flow
cytometry assay, optionally after fixation of the cells, and optionally
using a labeled secondary antibody or other conjugate (such as a
biotin-avidin conjugate) to amplify labeling.
 The expression of tissue-specific gene products can also be
detected at the mRNA level by Northern blot analysis, dot-blot
hybridization analysis, or by reverse transcriptase initiated polymerase
chain reaction (RT-PCR) using sequence-specific primers in standard
amplification methods. See U.S. Pat. No. 5,843,780 for further details.
Sequence data for the particular markers listed in this disclosure can be
obtained from public databases such as GenBank (URL www.ncbi.nlm.nih.gov:
80/entrez). Expression at the mRNA level is said to be "detectable"
according to one of the assays described in this disclosure if the
performance of the assay on cell samples according to standard procedures
in a typical controlled experiment results in clearly discernable
hybridization or amplification product. Expression of tissue-specific
markers as detected at the protein or mRNA level is considered positive
if the level is at least 2-fold, and preferably more than 10- or 50-fold
above that of a control cell, such as an undifferentiated pPS cell, a
fibroblast, or other unrelated cell type.
 Also characteristic of neural cells, particularly terminally
differentiated cells, are receptors and enzymes involved in the
biosynthesis, release, and reuptake of neurotransmitters, and ion
channels involved in the depolarization and repolarization events that
relate to synaptic transmission. Evidence of synapse formation can be
obtained by staining for synaptophysin. Evidence for receptivity to
certain neurotransmitters can be obtained by detecting receptors for
.gamma.-amino butyric acid (GABA), glutamate, dopamine,
3,4-dihydroxyphenylalanine (DOPA), noradrenaline, acetylcholine, and
 Differentiation of particular neural precursor cell populations of
this invention (for example, using NT-3 and BDNF) can generate cell
populations that are at least 20%, 30%, or 40% MAP-2 positive. A
substantial proportion, say 5%, 10%, 25%, or more of the NCAM or MAP-2
positive cells will be capable of synthesizing a neurotransmitter, such
as acetylcholine, glycine, glutamate, norepinephrine, serotonin, or GABA.
 Certain populations of the invention contain NCAM or MAP-2 positive
cells that are 0.1%, and possibly 1%, 3%, or 5% or more (on a cell count
basis) that are positive for tyrosine hydroxylase (TH), measured by
immunocytochemistry or mRNA expression. This generally considered in the
art to be a marker for dopamine synthesizing cells.
 To elucidate further mature neurons present in a differentiated
population, the cells can be tested according to functional criteria. For
example, calcium flux can be measured by any standard technique, in
response to a neurotransmitter, or other environmental condition known to
affect neurons in vivo. First, neuron-like cells in the population are
identified by morphological criteria, or by a marker such as NCAM. The
neurotransmitter or condition is then applied to the cell, and the
response is monitored (Example 6). The cells can also be subjected to
standard patch-clamp techniques, to determine whether there is evidence
for an action potential, and what the lag time is between applied
potential and response. Differentiation of neural precursor populations
of this invention can generate cultures that contain subpopulations that
have morphological characteristics of neurons, are NCAM or MAP-2
positive, and show responses with the following frequency: a response to
GABA, acetylcholine, ATP, and high sodium concentration in at least about
40%, 60% or 80% of the cells; a response to glutamate, glycine, ascorbic
acid, dopamine, or norepinephrine in at least about 5%, 10% or 20% of the
cells. A substantial proportion of the NCAM or MAP-2 positive cells (at
least about 25%, 50%, or 75%) can also show evidence of an action
potential in a patch-clamp system.
 Other desirable features consistent with functioning neurons,
oligodendrocytes, astrocytes, and their precursors can also be performed
according to standard methods to confirm the quality of a cell population
according to this invention, and optimize conditions for proliferation
and differentiation of the cells.
 Telomerization of neural precursors It is desirable that neural
precursors have the ability to replicate in certain drug screening and
therapeutic applications, and to provide a reservoir for the generation
of differentiated neuronal and glial cells. The cells of this invention
can optionally be telomerized to increase their replication potential,
either before or after they progress to restricted developmental lineage
cells or terminally differentiated cells. pPS cells that are telomerized
may be taken down the differentiation pathway described earlier; or
differentiated cells can be telomerized directly.
 Cells are telomerized by genetically altering them by transfection
or transduction with a suitable vector, homologous recombination, or
other appropriate technique, so that they express the telomerase
catalytic component (TERT), typically under a heterologous promoter that
increases telomerase expression beyond what occurs under the endogenous
promoter. Particularly suitable is the catalytic component of human
telomerase (hTERT), provided in International Patent Application WO
98/14592. For certain applications, species homologs like mouse TERT (WO
99/27113) can also be used. Transfection and expression of telomerase in
human cells is described in Bodnar et al., Science 279:349, 1998 and
Jiang et al., Nat. Genet. 21:111, 1999. In another example, hTERT clones
(WO 98/14592) are used as a source of hTERT encoding sequence, and
spliced into an EcoRI site of a PBBS212 vector under control of the MPSV
promoter, or into the EcoRi site of commercially available pBABE
retrovirus vector, under control of the LTR promoter.
 Differentiated or undifferentiated pPS cells are genetically
altered using vector containing supernatants over a 8-16 h period, and
then exchanged into growth medium for 1-2 days. Genetically altered cells
are selected using 0.5-2.5 .mu.g/mL puromycin, and recultured. They can
then be assessed for hTERT expression by RT-PCR, telomerase activity
(TRAP assay), immunocytochemical staining for hTERT, or replicative
capacity. The following assay kits are available commercially for
research purposes: TRAPeze.RTM. XL Telomerase Detection Kit (Cat. s7707;
Intergen Co., Purchase N.Y.); and TeloTAGGG Telomerase PCR ELISApIus
(Cat. 2,013,89; Roche Diagnostics, Indianapolis Ind.). TERT expression
can also be evaluated at the mRNA by RT-PCR. Available commercially for
research purposes is the LightCycler TeloTAGGG hTERT quantification kit
(Cat. 3,012,344; Roche Diagnostics). Continuously replicating colonies
will be enriched by further culturing under conditions that support
proliferation, and cells with desirable phenotypes can optionally be
cloned by limiting dilution.
 In certain embodiments of this invention, pPS cells are
differentiated into multipotent or committed neural precursors, and then
genetically altered to express TERT. In other embodiments of this
invention, pPS cells are genetically altered to express TERT, and then
differentiated into neural precursors or terminally differentiated cells.
Successful modification to increase TERT expression can be determined by
TRAP assay, or by determining whether the replicative capacity of the
cells has improved.
 Other methods of immortalizing cells are also contemplated, such as
transforming the cells with DNA encoding myc, the SV40 large T antigen,
or MOT-2 (U.S. Pat. No. 5,869,243, International Patent Applications WO
97/32972 and WO 01/23555). Transfection with oncogenes or oncovirus
products is less suitable when the cells are to be used for therapeutic
purposes. Telomerized cells are of particular interest in applications of
this invention where it is advantageous to have cells that can
proliferate and maintain their karyotype--for example, in pharmaceutical
screening, and in therapeutic protocols where differentiated cells are
administered to an individual in order to augment CNS function.
 Use of Neural Precursors and Terminally Differentiated Cells
 This invention provides a method to produce large numbers of neural
precursor cells and mature neuronal and glial cells. These cell
populations can be used for a number of important research, development,
and commercial purposes.
 The cells of this invention can be used to prepare a cDNA library
relatively uncontaminated with cDNA preferentially expressed in cells
from other lineages. For example, multipotent neural progenitor cells are
collected by centrifugation at 1000 rpm for 5 min, and then mRNA is
prepared from the pellet by standard techniques (Sambrook et al., supra).
After reverse transcribing into cDNA, the preparation can be subtracted
with cDNA from any or all of the following cell types: cells committed to
the neuronal or glial cell lineage, mature neurons, astrocytes,
oligodendrocytes, or other cells of undesired specificity. This produces
a select cDNA library, reflecting transcripts that are preferentially
expressed in neuronal precursors compared with terminally differentiated
cells. In a similar fashion, cDNA libraries can be made that represent
transcripts preferentially expressed in neuronal or glial precursors, or
mature neurons, astrocytes, and oligodendrocytes.
 The differentiated cells of this invention can also be used to
prepare antibodies that are specific for markers of multipotent neural
progenitors, cells committed to the neuronal or glial cell lineage, and
mature neurons, astrocytes, and oligodendrocytes. This invention provides
an improved way of raising such antibodies because cell populations are
enriched for particular cell types compared with pPS cell cultures, and
neuronal or glial cell cultures extracted directly from CNS tissue.
 Polyclonal antibodies can be prepared by injecting a vertebrate
animal with cells of this invention in an immunogenic form. Production of
monoclonal antibodies is described in such standard references as Harrow
& Lane (1988), U.S. Pat. Nos. 4,491,632, 4,472,500 and 4,444,887, and
Methods in Enzymology 73B: 3 (1981). Other methods of obtaining specific
antibody molecules (optimally in the form of single-chain variable
regions) involve contacting a library of immunocompetent cells or viral
particles with the target antigen, and growing out positively selected
clones. See Marks et al., New Eng. J. Med. 335:730, 1996, International
Patent Applications WO 94/13804, WO 92/01047, WO 90/02809, and McGuiness
et al., Nature BiotechnoL 14:1449, 1996. By positively selecting using
pPS of this invention, and negatively selecting using cells bearing more
broadly distributed antigens (such as differentiated embryonic cells) or
adult-derived stem cells, the desired specificity can be obtained. The
antibodies in turn can be used to identify or rescue neural cells of a
desired phenotype from a mixed cell population, for purposes such as
costaining during immunodiagnosis using tissue samples, and isolating
precursor cells from terminally differentiated neurons, glial cells, and
cells of other lineages.
 Gene Expression Analysis
 The cells of this invention are also of interest in identifying
expression patterns of transcripts and newly synthesized proteins that
are characteristic for neural precursor cells, and may assist in
directing the differentiation pathway or facilitating interaction between
cells. Expression patterns of the differentiated cells are obtained and
compared with control cell lines, such as undifferentiated pPS cells,
other types of committed precursor cells (such as pPS cells
differentiated towards other lineages, cells committed to the neuronal or
glial cell lineage), other types of putative neural stem cells such as
those obtained from neural crest, neurospheres, or spinal chord, or
terminally differentiated cells, such as mature neurons, astrocytes,
oligodendrocytes, smooth muscle cells, and Schwann cells.
 Suitable methods for comparing expression at the protein level
include the immunoassay or immunohistochemistry techniques described
above. Suitable methods for comparing expression at the level of
transcription include methods of differential display of mRNA (Liang,
Peng, et al., Cancer Res. 52:6966, 1992), whole-scale sequencing of cDNA
libraries, and matrix array expression systems.
 The use of microarray in analyzing gene expression is reviewed
generally by Fritz et al Science 288:316, 2000; " Microarray Biochip
Technology", L Shi, www.Gene-Chips.com. Systems and reagents for
performing microarray analysis are available commercially from companies
such as Affymetrix, Inc., Santa Clara CA; Gene Logic Inc., Columbia MD;
HySeq Inc., Sunnyvale CA; Molecular Dynamics Inc., Sunnyvale CA; Nanogen,
San Diego CA; and Synteni Inc., Fremont CA (acquired by Incyte Genomics,
Palo Alto CA).
 Solid-phase arrays are manufactured by attaching the probe at
specific sites either by synthesizing the probe at the desired position,
or by presynthesizing the probe fragment and then attaching it to the
solid support (U.S. Pat. Nos. 5,474,895 and 5,514,785). The probing assay
is typically conducted by contacting the array by a fluid potentially
containing the nucleotide sequences of interest under suitable conditions
for hybridization conditions, and then determining any hybrid formed.
 An exemplary method is conducted using a Genetic Microsystems array
generator, and an Axon GenePixTM Scanner. Microarrays are prepared by
first amplifying cDNA fragments encoding marker sequences to be analyzed,
and spotted directly onto glass slides To compare mRNA preparations from
two cells of interest, one preparation is converted into Cy3-labeled
cDNA, while the other is converted into Cy5-labeled cDNA. The two cDNA
preparations are hybridized simultaneously to the microarray slide, and
then washed to eliminate non- specific binding. he slide is then scanned
at wavelengths appropriate for each of the labels, the resulting
fluorescence is quantified, and the results are formatted to give an
indication of the relative abundance of mRNA for each marker on the
 Identifying expression products for use in characterizing and
affecting differentiated cells of this invention involves analyzing the
expression level of RNA, protein, or other gene product in a first cell
type, such as a pluripotent neuronal precursor cell of this invention, or
a cell capable of differentiating along the neuronal or glial pathway;
then analyzing the expression level of the same product in a control cell
type; comparing the relative expression level between the two cell types,
(typically normalized by total protein or RNA in the sample, or in
comparison with another gene product expected to be expressed at a
similar level in both cell types, such as a house-keeping gene); and then
identifying products of interest based on the comparative expression
 Drug Screening
 Neural precursor cells of this invention can be used to screen for
factors (such as solvents, small molecule drugs, peptides,
polynucleotides) or environmental conditions (such as culture conditions
or manipulation) that affect the characteristics of neural precursor
cells and their various progeny.
 In some applications, pPS cells (undifferentiated or
differentiated) are used to screen factors that promote maturation into
neural cells, or promote proliferation and maintenance of such cells in
long-term culture. For example, candidate maturation factors or growth
factors are tested by adding them to cells in different wells, and then
determining any phenotypic change that results, according to desirable
criteria for further culture and use of the cells.
 Other screening applications of this invention relate to the
testing of pharmaceutical compounds for their effect on neural tissue or
nerve transmission. Screening may be done either because the compound is
designed to have a pharmacological effect on neural cells, or because a
compound designed to have effects elsewhere may have unintended side
effects on the nervous system. The screening can be conducted using any
of the neural precursor cells or terminally differentiated cells of the
invention, such as dopaminergic, serotonergic, cholinergic, sensory, and
motor neurons, oligodendrocytes, and astrocytes.
 The reader is referred generally to the standard textbook "In vitro
Methods in Pharmaceutical Research", Academic Press, 1997, and U.S. Pat.
No. 5,030,015. Assessment of the activity of candidate pharmaceutical
compounds generally involves combining the differentiated cells of this
invention with the candidate compound, either alone or in combination
with other drugs. The investigator determines any change in the
morphology, marker phenotype, or functional activity of the cells that is
attributable to the compound (compared with untreated cells or cells
treated with an inert compound), and then correlates the effect of the
compound with the observed change.
 Cytotoxicity can be determined in the first instance by the effect
on cell viability, survival, morphology, and the expression of certain
markers and receptors. Effects of a drug on chromosomal DNA can be
determined by measuring DNA synthesis or repair. [.sup.3H]-thymidine or
BrdU incorporation, especially at unscheduled times in the cell cycle, or
above the level required for cell replication, is consistent with a drug
effect. Unwanted effects can also include unusual rates of sister
chromatid exchange, determined by metaphase spread. The reader is
referred to A. Vickers (pp 375-410 in "In vitro Methods in Pharmaceutical
Research," Academic Press, 1997) for further elaboration.
 Effect of cell function can be assessed using any standard assay to
observe phenotype or activity of neural cells, such as receptor binding,
neurotransmitter synthesis, release or uptake, electrophysiology, and the
growing of neuronal processes or myelin sheaths--either in cell culture
or in an appropriate model.
 Therapeutic Use
 This invention also provides for the use of neural precursor cells
to restore a degree of central nervous system (CNS) function to a subject
needing such therapy, perhaps due to an inborn error in function, the
effect of a disease condition, or the result of an injury.
 To determine the suitability of neural precursor cells for
therapeutic administration, the cells can first be tested in a suitable
animal model. At one level, cells are assessed for their ability to
survive and maintain their phenotype in vivo. Neural precursor cells are
administered to immunodeficient animals (such as nude mice, or animals
rendered immunodeficient chemically or by irradiation) at an observable
site, such as in the cerebral cavity or in the spinal chord. Tissues are
harvested after a period of a few days to several weeks or more, and
assessed as to whether pPS derived cells are still present.
 This can be performed by administering cells that express a
detectable label (such as green fluorescent protein, or
.beta.-galactosidase); that have been prelabeled (for example, with BrdU
or [3H]thymidine), or by subsequent detection of a constitutive cell
marker (for example, using human-specific antibody). Where neural
precursor cells are being tested in a rodent model, the presence and
phenotype of the administered cells can be assessed by
immunohistochemistry or ELISA using human-specific antibody, or by RT-PCR
analysis using primers and hybridization conditions that cause
amplification to be specific for human polynucleotide sequences. Suitable
markers for assessing gene expression at the mRNA or protein level are
provided elsewhere in this disclosure.
 Various animal models for testing restoration of nervous system
function are described in "CNS Regeneration: Basic Science and Clinical
Advances", M. H. Tuszynski & J. H. Kordower, eds., Academic Press, 1999.
 Differentiated cells of this invention can also be used for tissue
reconstitution or regeneration in a human patient in need thereof. The
cells are administered in a manner that permits them to graft or migrate
to the intended tissue site and reconstitute or regenerate the
functionally deficient area.
 Certain neural progenitor cells embodied in this invention are
designed for treatment of acute or chronic damage to the nervous system.
For example, excitotoxicity has been implicated in a variety of
conditions including epilepsy, stroke, ischemia, Huntington's disease,
Parkinson's disease and Alzheimer's disease. Certain differentiated cells
of this invention may also be appropriate for treating dysmyelinating
disorders, such as Pelizaeus-Merzbacher disease, multiple sclerosis,
leukodystrophies, neuritis and neuropathies. Appropriate for these
purposes are cell cultures enriched in oligodendrocytes or
oligodendrocyte precursors to promote remyelination.
 By way of illustration, neural stem cells are transplanted directly
into parenchymal or intrathecal sites of the central nervous system,
according to the disease being treated. Grafts are done using single cell
suspension or small aggregates at a density of 25,000-500,000 cells per
.mu.L (U.S. Pat. No. 5,968,829). The efficacy of transplants of motor
neurons or their precursors can be assessed in a rat model for acutely
injured spinal cord as described by McDonald et al . (Nat. Med. 5:1410,
1999). A successful transplant will show transplant-derived cells present
in the lesion 2-5 weeks later, differentiated into astrocytes,
oligodendrocytes, and/or neurons, and migrating along the cord from the
lesioned end, and an improvement in gate, coordination, and
 The neural progenitor cells and terminally differentiated cells
according to this invention can be supplied in the form of a
pharmaceutical composition, comprising an isotonic excipient prepared
under sufficiently sterile conditions for human administration. For
general principles in medicinal formulation, the reader is referred to
Cell Therapy. Stem Cell Transplantation, Gene Therapy, and Cellular
Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University
Press, 1996; and Hematopoletic Stem Cell Therapy, E. D. Ball, J. Lister &
P. Law, Churchill Livingstone, 2000.
 The composition may optionally be packaged in a suitable container
with written instructions for a desired purpose, such as the
reconstitution of CNS function to improve some neurological abnormality.
The Following Examples are Provided as Further Non-limiting Illustrations
of Particular Embodiments of the Invention.
 Experimental Procedures
 This section provides details of some of the techniques and
reagents used in the Examples below.
 hES cells are maintained either on primary mouse embryonic
fibroblasts, or in a feeder-free system. The hES cells are seeded as
small clusters either on irradiated mouse embryonic fibroblasts, or on
plates coated with Matrigel.RTM. (1:10 to 1:30 in culture medium). hES
cell cultures on feeder cells are maintained in a medium composed of 80%
KO DMEM (Gibco) and 20% Serum Replacement (Gibco), supplemented with 1%
non-essential amino acids, 1 mM glutamine, 0.1 mM .beta.-mercaptoethanol
and 4 ng/mL human bFGF (Gibco). Cultures free of feeder cells are
maintained in the same medium that has previously been conditioned by
culturing with embryonic fibroblasts, and resupplemented with 4 ng/mL
bFGF (replaced daily).
 Cells are expanded by serial passaging. The monolayer culture of ES
colonies is treated with 1 mg/mL collagenase for 5-20 minutes at
37.degree. C. The cultures are then gently scraped to remove the cells.
The clusters are gently dissociated, and replated as small clusters onto
fresh feeder cells.
 Embryoid bodies are produced as follows. Confluent monolayer
cultures of hES cells are harvested by incubating in 1 mg/mL collagenase
for 5-20 min, following which the cells are scraped from the plate. The
cells are then dissociated into clusters and plated in non-adherent cell
culture plates (Costar) in a medium composed of 80% KO ("knockout") DMEM
(Gibco) and 20% non-heat-inactivated FBS (Hyclone), supplemented with 1%
non-essential amino acids, 1 mM glutamine, 0.1 mM .beta.-mercaptoethanol.
The cells are seeded at a 1:1 or 1:2 ratio in 2 mL medium per well (6
well plate). The EBs are fed every other day by the addition of 2 mL of
medium per well.
 When the volume of medium exceeds 4 mL/well, the EBs are collected
and resuspended in fresh medium. After 4-8 days in suspension, the EBs
are plated onto a substrate and allowed to differentiate further, in the
presence of selected differentiation factors.
 Differentiating into neural precursors is typically performed on
wells coated with fibronectin (Sigma) at a final concentration of 20
.mu.g/mL in PBS. Using 1 mL/well (9.6 cm .sup.2), plates are incubated at
4.degree. C. overnight or at room temperature for 4 h. The fibronectin is
then removed, and the plates are washed with PBS or KO DMEM once before
 Immunocytochemistry for NCAM and A2B5 expression is conducted as
follows: Live cells are incubated in primary antibody diluted in culture
medium with 1% goat serum for 15 minutes at 37.degree. C., washed once
with medium, and then incubated with labeled secondary antibody for 15
min. After washing, the cells are then fixed for 15-20 min in 2%
paraformaldehyde. For other markers, cultures are fixed for 10-20 min
with 4% paraformaldehyde in PBS, washed 3 times with PBS, permeabilized
for 2 min in 100% ethanol, and washed with 0.1 M PBS. Cultures are then
incubated in a blocking solution of 0.1 M PBS with 5% NGS (normal goat
serum) for at least 1 hour at room temperature. Cultures are then
incubated in primary antibody diluted in 0.1M PBS containing 1% NGS for
at least 2 h at room temperature. They are then washed in PBS before a 30
min incubation with secondary antibody in the same buffer. Antibodies
used include those shown in Table 1.
Antibody for Neural Cell Phenotypic
Anti- Working Epitope
body Isotype Dilution
5A5 mouse IgM 1:1 Polysialylat-
ed NCAM studies hybridoma
A2B5 mouse IgM 1:1 ganglioside ATCC-CRL1520
.beta.-tub- IgG 1:1000 Sigma T-8660
polyclonal 1:500 DAKO 2-334
GalC mouse IgG3 1:25
 Bead immunosorting is conducted using the following reagents and
equipment: magnetic cell separator; Midi MACs.TM. column; buffer of PBS
CMF containing 0.5% BSA and 2 mM EDTA; primary antibody against NCAM or
A2B5; rat anti-mouse IgG (or IgM) microbeads; pre-separation filter; rat
anti-mouse kappa PE; and a FACScan device. Cells are harvested using
trypsinlEDTA (Gibco) and dissociated. After removing the trypsin, the
cells are resuspended in MACs.TM. buffer. Cells are then labeled with
primary antibody for 6-8 min at room temp., and washed 2 times in
MACs.TM. buffer by spinning cells at 300.times.g for 10 min and
aspirating the buffer. The cells are then resuspended in 80 .mu.I
(minimum vol.) per 10.sup.7 cells. 20 .mu.I (minimum vol.) MACs ram.TM.
IgG microbeads per 10.sup.7 cells are added for 15 min at 6-12.degree. C.
The sample is then washed 2 times in MACs.TM. buffer before magnetic
separation. With the column in the magnetic cell separator, the cell
suspension is applied to the column (LS+Midi) in .about.3-5 mL buffer.
Negative cells are passed through by washing 3 times with 3 mL of
MACs.TM. buffer. The column is then removed from the magnetic field, and
positive cells are eluted with 5 mL of MACs.TM. buffer.
 After separation, A2B5+ or NCAM+ cells are maintained on plates
coated with poly-lysine and laminin in DMEM/F12 (Biowhittaker)
supplemented with N2 (Gibco 17502-014), B27 (Gibco 17504-010) and the
factors indicated. Source of the factors is shown in Table 2.
Factors used for Neural Cell Culture
Growth Factor Source Concentration
human EGF R & D Systems 10 ng/mL
human bFGF Gibco 10-25 ng/mL
human CNTF R & D Systems 1-10 ng/mL
human PDGF R & D Systems
human IGF-I R & D Systems 1 ng/mL
 RT-PCR analysis of expression at the transcription level is
conducted as follows: RNA is extracted from the cells using RNAeasy
Kit.TM. (Qiagen) as per manufacturer's instructions. The final product is
then digested with DNAse to get rid of contaminating genomic DNA. The RNA
is incubated in RNA guard (Pharmacia Upjohn) and DNAse I (Pharmacia
Upjohn) in buffer containing 10 mM Tris ph 7.5, 10 mM MgCI.sub.2, and 5
mM DDT at 37.degree.C. for 30-45 min. To remove protein from the sample,
phenol chloroform extraction is performed, and the RNA precipitated with
3 M sodium acetate and 100% cold ethanol. The RNA is washed with 70%
ethanol, and the pellet is air-dried and resuspended in DEPC-treated
 For the reverse transcriptase (RT) reaction, 500 ng of total RNA is
combined with a final concentration of 1.times.First Strand Buffer
(Gibco), 20 mM DDT and 25,.mu.g/mL random hexamers (Pharmacia Upjohn).
The RNA is denatured for 10 min at 70.degree. C., followed by annealing
at room temperature for 10 min. dNTPs are added at a final concentration
of 1 mM along with 0.5.mu.L of Superscript II RT (Gibco), incubated at 42
.degree. C. for 50 minutes, and then heat-inactivated at 80 .degree. C.
for 10 min. Samples are then stored at -20 .degree. C. till they are
processed for PCR analysis. Standard polymerase chain reaction (PCR) is
performed using primers specific for the markers of interest in the
following reaction mixture: cDNA 1.0,.mu.L, 10.times.PCR buffer (Gibco)
2.5 .mu.L, 10.times.MgCI.sub.2 2.5 .mu.L, 2.5 mM dNTP 3.0 .mu.L, 5 .mu.M
3'-primer 1.0 .mu.L, 5 .mu.M 5'-primer, 1.0 .mu.L, Taq 0.4,.mu.L,
DEPC-water 13.6 .mu.L.
 This experiment focused on determining whether the human embryonic
stem cells (hES) could undergo directed differentiation to NCAM-positive
progenitor cells. The hES cells were harvested either from mEF-supported
cultures or feeder-free cultures, and then differentiated via embryoid
body (EB) formation in suspension culture using medium containing 20%
FBS. The EBs were then plated intact onto fibronectin in DMEM/F12 medium,
supplemented with N2 supplement (Gibco) and 25 ng/mL human bFGF. After
culturing for about 2-3 days, NCAM-positive cells and A2B5-positive cells
were identified by immunostaining.
 Magnetic bead sorting and immunopanning were both successful in
enriching NCAM-positive cells. The starting population of cells typically
contained 25-72% NCAM-positive cells. After immuno-isolation, the
NCAM-positive proportion was enriched to 43-72%. Results are shown in
Differentiation and Sorting Conditions for
NCAM positive Cells
Cells staining positively
used in for NCAM
hES Cell Line used Differentiation Positive
for Differentiation Culture Type of Sort Before sort sort
H13 p28 CFN bead sort 33 92 41
panning 25 n/a n/a
H9 p32 CFN panning 64 72 51
H1 p32 CFN
bead sort 27 77 9
H9 p19 CFN bead sort 58 76 32
545.184 CFN bead sort 50 91 67
H1 p40 545.185 CFN bead sort A 65
H1 p40 545.185 CFN bead sort B 63 81 33
545.187 CFN bead sort A 53 92 45
H7NG p28/4 545.187 CFN bead sort
A 72 87 50
H1p39 545.189 CFNIP bead sort 16 43 6
667.004 CFNIP bead sort 25 73 10
H1p43 667.010 CFNIP bead sort 47
H1p44 667.012 CFNIP bead sort 52 89 34
H1 p46 667.020
EPFI bead sort 60 23 8
H1 p47 667.031 EPFI--EPFI bead sort 53 91
H1 p47 667.033 CFN-F bead sort 41 76 24
H9 p40MG 667.038
EPFI bead sort 55 80 25
C-ciliary neurotrophic factor (CNTF)
F-basic fibroblast growth
N-neurotrophin 3 (NT3)
growth factor (IGF-1)
P-platelet-derived growth factor (PDGF)
T-thyroid hormone T.sub.3
 In the first 10 experiments shown, NCAM positive cells retrieved
from the sort were plated on poly-L-lysine/laminin in DMEM/F12 with N2
and B27 supplements and 2 mg/mL BSA, 10 ng/mL human CNTF, 10 ng/mL human
bFGF and 1 ng/mL human NT-3. In subsequent experiments, cells were
maintained in DMEM/F12 with N2 and B27 supplements and 10 ng/mL EGF, 10
ng/mL bFGF, 1 ng/mL PDGF, and 1 ng/mL IGF-1.
 FIG. 1 (Upper Panel) shows the growth curves for the NCAM positive
cells. The cells studied in this experiment were prepared by forming
embryoid bodies in 20% FBS for 4 days in suspension, then plating onto a
fibronectin matrix in DMEM/F12 with N2 and B27 supplements and 25 ng/mL
bFGF for 2-3 days. The cells were then positively sorted for NCAM
expression, and maintained in a medium containing CNTF, bFGF, and NT3.
The sorted cells did not show increased survival relative to the unsorted
population. It was found that some of the NCAM positive cells also
express .beta.-tubulin 111, indicating that these cells have the capacity
to form neurons. They also had morphology characteristic of neuronal
cells. There were also A2B5 positive cells within this population, which
may represent glial progenitor cells. However, very few cells were
positive for GFAP, a marker for astrocytes. Although this cell population
proliferated in culture, the proportion of NCAM positive cells (and the
capacity to form neurons) diminished after several passages.
 Cells in this experiment were immunoselected for the surface marker
A2B5. hES cells were induced to form EBs in 20% FBS. After 4 days in
suspension, the EBs were plated onto fibronectin in DMEM/F12 with N2 and
B27 supplemented with 10 ng/mL human EGF, 10 ng/mL human bFGF, 1 ng/mL
human IGF-l, and 1 ng/mL human PDGF-AA. After 2-3 days in these
conditions, 25-66% of the cells express A2B5. This population is enriched
by magnetic bead sorting to 48-93% purity (Table 4).
Differentiation and Sorting Conditions for
Cells staining positively
used in for NCAM
hES Cell Line used for Differentiation Positive
Differentiation Culture Type of Sort Before sort sort
H7 p32 667.004 CFNIP bead sort 25 77 10
667.010 CFNIP bead sort 62 n/a 50
H1 p44 667.012 CFNIP bead sort
56 89 32
H1 p46 667.020 EPFI bead sort 27 48 2
667.032 EPFI bead sort 57 93 30
H9 p40MG 667.038 EPFI bead sort 66
H9 p42 667.041 EPFI bead sort 27 70 6
C-ciliary neurotrophic factor (CNTF)
F-basic fibroblast growth factor (bFGF)
N-neurotrophin 3 (NT3)
I-insulin-like growth factor (IGF-1)
growth factor (PDGF)
T-thyroid hormone T.sub.3
 FIG. 2 shows an exemplary procedure for obtaining A2B5-positive
cells. Abbreviations used: MEF-CM=medium conditioned by culturing with
mouse embryonic fibroblasts; +/-SHH=with or without sonic hedgehog;
 D/F12=DMEM/F12 medium; N2 and B27, culture supplements (Gibco);
EPFI=growth factors EGF, PDGF, bFGF, and IGF-1; PLL=poly-L lysine
substrate; PLL/FN=substrate of poly-L lysine and fibronectin.
 FIG. 1 (Lower Panel) shows the growth curves for the sorted
A2B5-positive cells. The cells were maintained in the same media
formulation on poly-l-lysine coated plates. The cells proliferate when
Maturation of A2B5-Positive Cells
 A2B5-positive cells were induced to differentiate by the addition
of forskolin. These cells have been assessed through different culture
passages, as shown in Table 5.
Phenotypic Features of Mature Neural Cells
No. of passages Method of morphology Cells
Staining Positively for:
after A2B5 sort Maturation visible
.beta.-tubulin GFAP GalC A2B5 NCAM
1 PICNT + Fk yes 38
.+-. 9% 13 .+-. 7% 79 .+-. 3% 28 .+-. 6%
3 PICNT +
Fk yes +++ + +++++ ++
7 +/- EF yes + + ++ +++ -
insulin-like growth factor (IGF-1)
P -- platelet-derived growth
T -- thyroid hormone T.sub.3
Ra -- retinoic
Fk -- Forskolin
C -- ciliary neurotrophic factor
F -- basic fibroblast growth factor (bFGF)
neurotrophin 3 (NT3)
 Even though the cells were sorted for A2B5 expression, the
population demonstrated the capacity to generate not only
oligodendrocytes, and astrocytes, but also a large proportion of neurons.
This is surprising: it was previously thought that A2B5 expressing cells
were glial precursors, and would give rise to oligodendrocytes, and
astrocytes--while NCAM expressing cells were neuronal precursors, giving
rise to mature neurons. This experiment demonstrates that pPS cells can
be differentiated into a cell population that proliferates repeatedly in
culture, and is capable of generating neurons and glia.
Transplantation of Differentiated Cells into the Mammalian Brain
 Transplantation of neural precursor cells was done using cells
derived from two hES cell lines: the line designated H1, and a
genetically altered line designated H7NHG. The H7NHG cell line carries an
expression cassette that permits the cells to constitutively express
green fluorescent protein (GFP).
 Neonatal Sprague Dawley rats received unilateral intrastriatal
implants of one of the following cell populations:
 undifferentiated hES cells
 embryoid bodies derived from hES cells
 neural precursors sorted for NCAM expression (Example 1)
 neural precursors sorted for A2B5 expression (Example 2)
 Control animals received grafts of irradiated mouse embryonic
fibroblasts upon which the undifferentiated hES cells were maintained. To
determine if cell proliferation occurred after grafting, some animals
were pulsed with intraperitoneal injections of BrdU, commencing 48 h
prior to sacrifice. Fourteen days after transplantation, the rats were
transcardially perfused with 4% paraformaldehyde and the tissue was
processed for immunohistochemical analysis.
 FIG. 3 shows the fluorescence observed in sections from animals
administered cells expressing GFP.
 Surviving cells were detected in all transplanted groups. The
undifferentiated cells presented as large cell masses, suggesting
unregulated growth with areas of necrosis and vacuolation of surrounding
tissue (Left-side Panels). Immunostaining for AFP in an animal
transplanted with Hi cells showed that undifferentiated hES cells
transformed into visceral endoderm after transplantation. Embryoid bodies
remained in the graft core with little migration, and were also
surrounded by areas of necrosis. (Middle Panels). In contrast, sorted
NCAM-positive cells appeared as single cells and showed some degree of
migration distal to the site of implantation.
Differentiation to Mature Neurons
 To generate terminally differentiated neurons, the first stage of
differentiation was induced by forming embryoid bodies in FBS medium with
or without 10 .mu.M retinoic acid (RA). After 4 days in suspension,
embryoid bodies were plated onto fibronectin-coated plates in defined
medium supplemented with 10 ng/mL human EGF, 10 ng/mL human bFGF, 1 ng/mL
human PDGF-AA, and 1 ng/mL human IGF-1. The embryoid bodies adhered to
the plates, and cells began to migrate onto the plastic, forming a
 After 3 days, many cells with neuronal morphology were observed.
The neural precursors were identified as cells positive for BrdU
incorporation, nestin staining, and the absence of lineage specific
differentiation markers. Putative neuronal and glial progenitor cells
were identified as positive for polysialylated NCAM and A2B5. Forty one
to sixty percent of the cells expressed NCAM, and 20-66% expressed A2B5,
as measured by flow cytometry. A subpopulation of the NCAM-positive cells
was found to express .beta.-tubulin III and MAP-2. There was no
co-localization with glial markers such as GFAP or GaIC. The A2B5
positive cells appeared to generate both neurons and glia. A
subpopulation of the A2B5 cells expressed .beta.-tubulin III or MAP-2,
and a separate subpopulation expressed GFAP. Some of the cells with
neuronal morphology double-stained for both A2B5 and NCAM. Both the NCAM
positive and A2B5 positive populations contained far more neurons than
 The cell populations were further differentiated by replating the
cells in a medium containing none of the mitogens, but containing 10
ng/mL Neurotrophin-3 (NT-3) and 10 ng/mL brain-derived neurotrophic
factor (BDNF). Neurons with extensive processes were seen after about 7
days. Cultures derived from embryoid bodies maintained in retinoic acid
(RA) showed more MAP-2 positive cells (.about.26%) than those maintained
without RA (.about.5%). GFAP positive cells were seen in patches. GaIC
positive cells were identified, but the cells were large and flat rather
than having complex processes.
 A summary of cell types and markers expressed at different stages
of differentiation is provided in Table 6.
Phenotypic Markers (Immunocytochemistry)
Undifferentiated hES colonies NCAM-positive
progenitors A2B5 positive progenitors
Tra-1-60 + Nestin
subset Nestin subset
Tra-1-81 + A2B5 subset NCAM subset
SSEA-4 + .beta.-tubulin III subset .beta.-tubulin III subset
.beta.-tubulin III + + Map-2 subset Map-2 subset
Nestin - GFAP -
Map-2 - GalC - GalC -
Neurofilament (NF) - AFP -
GFAP - muscle-specific actin - muscle-specific actin -
muscle-specific actin -
Neurons Astrocytes Oligodendrocytes
.beta.-tubulin III + GFAP + GalC +
Neurofilament (NF) subset
 The presence of neurotransmitters was also assessed.
GABA-immunoreactive cells were identified that co-expressed
.beta.-tubulin IlIl or MAP2, and had morphology characteristic of
neuronal cells. Occasional GABA-positive cells were identified that did
not co-express neuronal markers, but had an astrocyte-like morphology.
Neuronal cells were identified that expressed both tyrosine hydroxylase
(TH) and MAP-2. Synapse formation was identified by staining with
 FIG. 4 shows TH staining in cultures differentiated from the H9
line of human ES cells. Embryoid bodies were maintained in 10 .mu.M
retinoic acid for 4 days, then plated onto fibronectin coated plates in
EGF, basic FGF, PDGF and IGF for 3 days. They were next passaged onto
laminin in N2 medium supplemented with 10 ng/mL NT-3 and 10 ng/mL BDNF,
and allowed to differentiate further for 14 days. The differentiated
cells were fixed with 2% formaldehyde for 20 min at room temp, and then
developed using antibody to TH, a marker for dopaminergic cells.
 Standard fura-2 imaging of calcium flux was used to investigate the
functional properties of the hES cell derived neurons. Neurotransmitters
studied included GABA, glutamate (E), glycine (G), elevated potassium (50
mM K.sup.+instead of 5 mM K.sup.+), ascorbic acid (control), dopamine,
acetylcholine (ACh) and norepinephrine. The solutions contained 0.5 mM of
the neurotransmitter (except ATP at 10 pM) in rat Ringers (RR) solution:
140 mM NaCI, 3 mM KCI, 1 mM MgCI.sub.2, 2 mM CaCI.sub.2, 10 mM HEPES
buffer, and 10 mM glucose. External solutions were set to pH 7.4 using
NaOH. Cells were perfused in the recording chamber at 1.2-1.8 mL/min, and
solutions were applied by bath application using a 0.2 mL loop injector
located .about.0.2 mL upstream of the bath import. Transient rises in
calcium were considered to be a response if the calcium levels rose above
10% of the baseline value within 60 sec of application, and returned to
baseline within 1-2 min.
 FIG. 5 shows the response of neural-restricted precursors to
various neurotransmitters. Panel A shows the ratio of emission data from
single cells on two different coverslips. Addition of the
neurotransmitters is indicated above by labeled triangles.
 Panel B shows the frequency of cells tested that responded to
specific neurotransmitters. Panel C shows the combinations of
neurotransmitter responses observed. Of the 53 cells tested, 26 responded
to GABA, acetylcholine, ATP and elevated potassium. Smaller subsets of
the population responded to other combinations of agonists. Only 2 of the
cells failed to respond to any of the agonists applied.
 Standard whole-cell patch-clamp technique was conducted on the hES
cell derived neurons, to record ionic currents generated in voltage-clamp
mode and the action potential generated in current-clamp mode. The
external bath solution was rat Ringers solution (Example 6). The internal
solution was 75 mM potassium-aspartate, 50 mM KF, 15 mM NaCI, 11 mM EGTA,
and 10 mM HEPES buffer, set to pH 7.2 using KOH.
 All 6 cells tested expressed sodium and potassium currents, and
fired action potentials. Passive membrane properties were determined with
voltage steps from -70 to -80 mV; and produced the following data:
average capacitance (C.sub.m)=8.97.+-.1.17 pF; membrane resistance
(R.sub.m)=487.8.+-.42.0 M.OMEGA.; access resistance
(R.sub.a)=23.4.+-.3.62 M.OMEGA.. Ionic currents were determined by
holding the cells at -100 mV, and stepping to test voltages between -80
and 80 mV in 10 mV increments, producing the following data: average
sodium current I.sub.Na=-531.8.+-.136.4 pA; average potassium current
I.sub.K=441.7.+-.113.1 pA; I.sub.Na(density)=-57.7.+-.7.78 pA/pF;
 FIG. 6 shows results from a typical experiment. Panel A shows
sodium and potassium currents observed in two cells depolarized to test
potentials between -80 and 80 mV from a holding potential of -100 mV.
Panel B shows the inward (Na.sup.+) and outward (K.sup.+) peak
current-voltage relationships observed. Sodium current activates between
-30 and 0 mV, reaching a peak at -10 or 0 mV. Potassium current activates
above -10 mV, becoming equal or larger in magnitude than the sodium
current at voltages between 20 and 40 mV. Panel C shows action potentials
generated by the same cells n response to depolarizing stimuli. Cell
membranes were held at voltages between -60 and -100 mV in -80 or -150 pA
of current, and depolarized for short durations
Dopaminergic Cells Derived From Neural Progenitor Ells
 Embryoid bodies were cultured in suspension with 10 .mu.M retinoic
acid for 4 days, then plated into defined medium supplemented with EGF,
bFGF, PDGF, and IGF-1 for 3-4 days. Cells were then separated by magnetic
bead sorting or immunopanning into A2B5-positive or NCAM-positive
 The immuno-selected cells were maintained in defined medium
supplemented with 10 ng/mL NT-3 and 10 ng/mL BDNF. After 14 days,
25.+-.4% of the NCAM-sorted cells were MAP-2 positive--of which
1.9.+-.0.8% were GABA-positive, and 3 .+-.1% were positive for tyrosine
hydroxylase (TH): the rate-limiting enzyme for dopamine synthesis,
generally considered to be representative of dopamine-synthesizing cells.
 In the cell population sorted for NCAM, the cells that were NCAM+ve
did not express glial markers, such as GFAP or GaIC. These data indicate
that a population comprising neuron restricted precursors can be isolated
directly from hES cell cultures, essentially uncontaminated with glial
 Cells sorted for A2B5, on the other hand, have the capacity to
generate both neurons and astrocytes. After the enrichment, the cells
were placed into defined media supplemented with NT-3 and BDNF and
allowed to differentiate for 14 days. Within the first 1-2 days after
plating, cells in the A2B5 enriched population began to extend processes.
After two weeks, cells took on the morphology of mature neurons, and
32.+-.3% of the cells were MAP-2 positive. Importantly, 3.+-.1% of the
MAP-2 cells were TH-positive, while only 0.6.+-.0.3% were GABA
immunoreactive. These data indicate that a population of cells can be
obtained from hES cells that comprise progenitors for both astrocytes and
neurons, including those that synthesize dopamine.
 Further elaboration of conditions for obtaining TH-expression
neurons was conduced as follows. Embryoid bodies were generated from
confluent hES cells of the H7 line at passage 32 by incubating in 1 mg/mL
collagenase (37.degree. C., 5-20 min), scraping the dish, and placing the
cells into non-adherent culture plates (Costar.RTM.). The resulting EBs
were cultured in suspension in media containing FBS and 10 .mu.M
all-trans retinoic acid. After four days, the aggregates were collected
and allowed to settle in a centrifuge tube. The supernatant was then
aspirated, and the aggregates were plated onto poly L-lysine and
fibronectin coated plates in proliferation medium (DMEM/F12 1:1
supplemented with N2, half-strength B27, 10 ng/mL EGF (R&D Systems), 10
ng/mL bFGF (Gibco), 1 ng/mL PDGF-AAA (R&D Systems), and 1 ng/mL IGF-1 (R
& D Systems).
 The EBs were allowed to attach and proliferate for three days; then
collected by trypsinizing .about.1 min (Sigma) and plated at
1.5.times.10.sup.5 cells/well onto poly l-lysine and laminin coated
4-well chamber slides in proliferation medium for one day. The medium was
then changed to Neural Basal medium supplemented with B27, and one of the
following growth cocktails:
 10 ng/mL bFGF (Gibco), 10 ng/mL BDNF, and 10 ng/mL NT-3
 10 ng/mL bFGF, 5000 ng/mL sonic hedgehog, and 100 ng/mL FGF8b
 10 ng/mL bFGF alone
 The cells were maintained in these conditions for 6 days, with
feeding every other day. On day 7, the medium was changed to Neural Basal
medium with B27, supplemented with one of the following cocktails:
 10 ng/mL BDNF, 10 ng/mL NT-3
 1 .mu.M cAMP, 200 .mu.M ascorbic acid
 1 .mu.M cAMP, 200 .mu.M ascorbic acid, 10 ng/mL BDNF, 10 ng/mL NT-3
 The cultures were fed every other day until day 12 when they were
fixed and labeled with anti-TH or MAP-2 for immunocytochemistry.
Expression of the markers was quantified by counting four fields in each
of three wells using a 40X objective lens.
 Results are shown in Table 7. Initial culturing in bFGF, BDNF and
NT-3 yielded the highest proportion of TH positive cells.
Conditions for Producing Dopaminergic
Culture conditions % MAP-2 % MAP-2 cells that are
days 1-6 days 6-12 positive TH positive
B, N, F B, N 26%
B, N, F CA, AA 35% 4.0%
B, N, F CA, AA, B, N 25% 8.7%
F, F8, S B, N 37% 3.7%
F, F8, S CA, AA 34% 3.9%
F8, S CA, AA, B, N 21% 5.8%
F B, N 28% 3.5%
F CA, AA 26%
F CA, AA, B, N 22% 5.7%
F-basic fibroblast growth factor (bFGF)
N-neurotrophin 3 (NT3)
B-brain-derived neurotrophic factor (BDNF)
 It is understood that certain adaptations of the invention
described in this disclosure are a matter of routine optimization for
those skilled in the art, and can be implemented without departing from
the spirit of the invention, or the scope of the appended claims.
* * * * *