Note: Descriptions are shown in the official language in which they were submitted.
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1
GENERATION OF HUMAN NEURAL CREST STEM CELL LINE AND ITS
UTILIZATION IN HUMAN TRANSPLANTATION
PROVISIONAL PATENT APPLICATION
A Provisional Patent Application enabling and descriptive of the present
invention was filed with the U.S. Patent and Trademark Office as Application
No.
60!155,011 on September 21, 1999.
RESEARCH SUPPORT
Work for this invention was supported by grants from the Canadian Myelin
Research Initiative.
FIELD OF THE INVENTION
The present invention is concerned with the development of differentiated
cells and tissues deriving from neural crest cells; and is particularly
directed to the
isolation, expansion, and maintenance as stable clones of human neural crest
stem
cells and their direct daughter progeny cells.
BACKGROUND OF THE INVENTION
The neural crest of the vertebrate embryo is the main source of the cells of
the
peripheral nervous system (PNS) and autonomic nervous system (ANS). The neural
3 0 crest is a transitory embryonic structure arising from the lateral ridges
of the neural
primordium when they join mediodorsally during the closure of the neural tube.
This
structure has one striking developmental feature. Its cells have the capacity
to
f'.~~~
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2
undergo migration at precise periods of development along apparently definite
pathways, and to settle finally in particular locations where they
differentiate into a
variety of cell types. At the trunk level, the PNS arises entirely from the
neural crest,
while in the head, ectodermal placodes (that is, thickenings of the
superficial ectoderm
which differentiate at variable distances from the main neural primordium, the
neural
plate) also participate in the formation of the sensory ganglia of certain
cranial nerves.
Besides the PNS, the neural crest gives rise to a wide variety of structures.
All
the melanocytes of the body except those of the retinal pigmented epithelium
and
certain endocrine and paracrine cells (the adrenal medulla, the calcitonin-
producing
cells, the type I cells of the carotid bone) are of neural crest origin.
Moreover, the
cephalic neural crest yields mesenchymal cells that differentiate into the
brain
meninges, the entire facial and visceral arch skeleton and dermis, the musculo-
connective wall of the large arterial trunks arising from the aortic arches,
and the
connective tissues of the buccal and pharyngeal region, including that of the
salivary,
thyroid, parathyroid, and thymus glands. Neural crest development thus
involves the
establishment of different cell lineages and a patterning of crest cell
migration and
diversification is established during embryogenesis.
In the generation of the cells of the vertebrate nervous system, and in the
control of their phenotypic choices, the two best understood experimental
systems are
2 0 arguably the rat sympathoadrenal lineage that yields neurons and endocrine
cells of
the rat autonomic nervous system; and the avian lineage of neural crest cells
in-vivo.
Each experimental system has provided some useful information and insight.
The sympathoadrenal lineage is derived from the neural crest and
experimentally gives rise primarily to chromaffin cells of the adrenal gland
and
2 5 sympathetic neurons. These two cell types can be distinguished at a number
of levels.
Chromaffin cells are small cells, without significant processes, and they
secrete
primarily the catecholamine epinephrine into the circulation. Sympathetic
neurons are
much larger cells, with dendritic and axonal processes that receive and send
synaptic
connections, respectively. These neurons secrete primarily norepinephrine, a
3 0 catecholamine compound. While both cell types store their
neurotransmitters and
neuropeptides in vesicles, the chromaffin vesicles are about 150-350 nm in
diameter,
while neuronal synaptic vesicles are only 50 nm in diameter. A third,
apparently
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minor, population of cells in this lineage, known as small intensely
fluorescent (SIF)
cells, is somewhat intermediate in character between neurons and chromaffin
cells and
has vesicles of 75-120 nm in diameter.
In addition to these chemical and morphological characteristics, chromaffin
cells and sympathetic neurons can be distinguished by numerous molecular
markers,
such as genes encoding specific cytoskeletal, vesicle and surface proteins;
and
neurotransmitter-synthesizing enzymes such as phenyl N-methyl transferase
(which is
chromaffin cell specific). These markers have proven useful both in the
identification
of intermediate stages of differentiation and in the identification and
isolation of a
sympathoadrenal progenitor cell.
The step at which a multipotential neural crest cell becomes committed to the
sympathoadrenal lineage is not well understood. Catecholamine-producing cells
that
resemble neurons or chromaffin cells have been observed in cultures of neural
crest
cells but these possible sympathoadrenal precursors have not as yet been
isolated for
further study. Committed sympathoadrenal progenitors that are at least
bipotential
(giving rise to either chromaffin cells or sympathetic neurons) have, however,
been
isolated from embryonic adrenal medullae as well as embryonic and neonatal rat
sympathetic ganglia. Embryonic progenitors can be isolated by fluorescence-
activated
cell sorting (FACS), using several surface membrane antigens. In cell culture,
these
2 0 progenitors can be induced to become neurons or chromaffin cells that
express cell
type-specific antigens or genes, as well as the appropriate morphology and
ultra-
structure.
For more detailed information, empirical evidence, and reviews of the
sympathoadrenal lineage system, the reader is directed to the following list
of relevant
and representative publications: Patterson et al., Cell 62: 1035-1038 (1990);
Stemple,
D.L. and D.J. Anderson, Cell 71: 973-985 ( 1992); Anderson, D.J., Ann. Rev.
Neurosci. 16: 129-158 (1993); Le Douarin, N.M., Science 231: 1515-1522 (1986);
Kim et al., J. Neurosci. Res. 22: 50-59 (1989); Lo et al., Dev. Biol. 145: 139-
153
(1991); Jessen et al., Neuron 12: 509-527 (1994); Anderson, D.L., Curr. Opin.
3 0 Neurobiol. 3: 8-13 (1993); and the references cited within each of these
publications.
The avian embryo has also provided considerable information via in-vivo and
in-vitro experimental studies of the patterning of neural crest derivatives.
Much of the
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present knowledge of the developmental potential and fate of neural crest
cells comes
from research studies in avian systems. Fate maps have been established in
aves and
provide evidence that several different crest cell derivatives may originate
from the
same position along the neural tube. Schwann cells, melanocytes and sensory
and
sympathetic neurons can all derive from the truncal region of the avian neural
tube.
On the other hand, some derivatives were also found to originate from specific
regions
of the neural crest, e.g., enteric ganglia from the vagal and sacral regions.
These
studies revealed that the developmental potential of the neural crest
population at a
given location along the neural tube is greater than its developmental fate.
This also
suggests that a new environment encountered by the migrating neural crest
cells
influences their developmental fate.
Moreover, single-cell lineage analysis in-vivo, as well as clonal analysis in-
vitro, have reportedly shown that early avian neural crest cells are
multipotential
during or shortly after their detachment and migration from the neural tube.
In avian
systems, certain clones derived from single neural crest cells in culture were
found to
contain both catecholaminergic and pigmented cells; and that avian neural
crest cells
from the cephalic region could generate clones which gave rise to highly
heterogeneous progeny when grown on growth-arrested fibroblast feeder cell
layers.
An in-vivo demonstration of the multipotency of early neural crest cells has
2 0 been conducted in chickens. Individual neural crest cells, prior to their
migration
from the neural tube, were injected with a fluorescent dye. After 48 hours,
the clonal
progeny of injected cells were found to reside in many or all of the locations
to which
neural crest cells migrate, including sensory and sympathetic ganglia,
peripheral motor
nerves and the skin. Phenotypic analysis of the labeled cells revealed that at
least
2 5 some neural crest cells are multipotent in-vivo.
Furthermore, following migration from the neural tube, these early multipotent
neural crest cells become segregated into different sublineages, which
generate
restricted subsets of differentiated derivatives. The mechanisms whereby
neural crest
cells become restricted to the various sublineages continue to be poorly
understood.
3 0 The fate of avian neural crest derivatives is known to be controlled in
some way by
the embryonic location in which their precursors come to reside. Yet the
mechanism
of specification for neural crest cells derivatives remains unknown to date.
In culture
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studies described above, investigators reported that clones derived from
primary
neural crest cells exhibited a mixture of phenotypes. Some clones contained
only one
differentiated cell type whereas other clones contained many or all of the
assayable
crest phenotypes.
5 In short, the respective contributions of the avian "in embryo" and in-vitro
approaches to understanding of the neural crest cell differentiating
potentialities are
quite limited. It is pointed out that the search for survival and
proliferation factors
acting locally on neural crest derivatives when they are wandering and/or
settling in
various embryonic locations constitutes the recurring challenge for further
understanding their complex patterning and the highly diversified variety of
their
phenotypes.
For more detailed information, empirical data, and reviews of the avian
lineage
analyses for neural crest cells, the reader is directed to the following list
of
representative publications: Le Douarin et al., Dev. Biol. 159: 24-49 (1993);
N.M. Le
Douarin, Nature 286: 663-669 (1980); N.M. Le Douarin, The Neural Crest,
Cambridge University Press, Cambridge, U.K., 1982; Sieber-Blum et al., Dev.
Biol.
80: 96-106 (1980); Baroffio et al., Proc. Natl. Acad. Sci. USA 85: 5325-5329
(1988);
Bronner-Fraser et al., Neuron 3: 755-766 (1989); Cohen et al., Dev. Biol. 46:
262-280
(1975); Dupin et al., Proc. Natl. Acad. Sci. USA 87: 1119-1123 (1990); Duff et
al.,
Dev. Biol. 147: 451-459 (1991); Bronner-Frasier et al., Nature 355: 161-164
(1988).
Another approach and line of research inquiry in this technical area has been
the search to seek out, isolate and portray a "stem cell" for neurons and glia
derived
from the mammalian neural crest. Most illustrative of this approach are the
scientific
publications and issued U.S. patents of D.J. Anderson and his colleagues. In
the main,
2 5 these investigators have identified mammalian neural crest cells - chiefly
rat and
mouse cells - using antibodies to cell surface antigens; and have used sub-
cloning to
examine the developmental potential of these cells and their clonal progeny.
Three
main findings emerge from their analyses. First, some single mammalian neural
crest
cells are multipotent. Second, some multipotent neural crest cells generate
3 0 multipotent progeny, indicating that they are capable of self renewal and
therefore are
stem cells. Third, some multipotent neural crest cells can also generate some
clonal
progeny that form only neurons or glia, implying that the stem cells may
eventually
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produce committed neuroblasts and glioblasts. Their experiments collectively
suggest
that in-vivo neural crest cells may maintain their multipotency as they
migrate and
proliferate; and that initial lineage decisions may occur within developing
ganglia via
the generation of committed blast cells. Their data also demonstrate the
existence and
clonal propagation of a mammalian stem cell for neurons and glia via
experimental
manipulation of such cells and their environment.
Some of the relevant scientific publications of Anderson and his colleagues
include: D.J. Anderson, Neuron 3: 1-12 (1989); D.J. Anderson, Annu. Rev.
Neurosci.
16: 129-158 (1993); Stemple, D.L. and D.J. Anderson, Cell 71: 973-985 (1992);
Anderson et al., J. Neurosci. 11: 3507-3519 (1991); Anderson, D.L. and R.
Axel, Cell
42: 649-662 (1985); Anderson, D.L, and R. Axel, Cell 47: 1079-1090 (1986);
Shah et
al., Cell 85: 331-343 (1996); Lo et al., Dev. Biol. 145: 139-153 (1991).
The reader is also directed to the following issued U.S. Patents for further
information: U.S. Patent Nos. 6,001,654; 5,928,947; 5,849,553; 5,693,482;
5,824,489; 5,654,183; and 5,589,376. The texts of all these issued U.S.
Patents,
individually and collectively, are expressly incorporated by reference herein.
1t will be noted and appreciated, however, that despite this large body of
reported research inquiries, printed scientific publications, and issued
patents, no one
has yet isolated, or expanded, or characterized a human neural crest stem cell
as such;
2 0 and no person has to date succeeded in creating stable cultures or clones
of human
neural crest stem cells which proliferate and can be maintained indefinitely
under in-
vitro conditions; and no person, in so far as is presently known, has
demonstrated that
a human neural crest stem cell implanted in-vivo can develop into several
alternative
and different kinds of differentiated cells in-situ, including neurons,
Schwann cells,
2 5 adrenal chromaffin cells, and/or skeletal muscle cells.
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Clearly, were such an innovation created, it would be generally seen and
acknowledged as a major advance and unforeseen development in this technical
field.
SUMMARY OF THE INVENTION
The present invention has multiple aspects. A first major aspect provides a
primordial human neural crest stem cell and its descendant progeny cells which
are
suitable for implantation in-vivo into a living host subject, said primordial
human
neural crest stem cell comprising:
a pluripotent and self renewing neural crest stem cell of human origin which
(i) carries native human genomic DNA which has not been
genetically modified by human intervention means;
(ii) remains uncommitted and undifferentiated while passaged in-
vitro using as a mitotic cell line;
(iii) is implantable in-vivo as an uncommitted cell;
(iv) optionally migrates in-vivo after implantation from the
implantation site to another anatomic site for in-vivo integration within the
living host
subj ect;
(v) integrates in-situ after implantation into the body of the living
2 0 host subject at a local anatomic site; and
(vi) differentiates in-situ after integration into at least one
recognized type of differentiated cell of neural crest origin.
A second major aspect provides a genetically modified human neural crest
2 5 stem cell and its descendant progeny cells maintained as a stable cell
line in-vitro and
suitable for on-demand implantation in-vivo into a living host subject, said
genetically
modified human neural crest stem cell comprising:
a primordial neural crest stem cell of human origin which
(i) remains uncommitted and undifferentiated while passaged in-
3 0 vitro as a mitotic, self renewing cell line;
(ii) is implantable in-vivo as an uncommitted cell;
(iii) optionally migrates in-vivo after implantation from the
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implantation site to another anatomic site for integration within the body of
the living
host subject;
(iv) integrates in-situ after implantation into the body of the living
host subject at a local anatomic site; and
(v) differentiates in-situ after integration into a recognized type of
differentiated cell of neural crest origin; and
human genomic DNA which has been genetically modified to include a viral
vector carrying at least one DNA segment comprised of an exogenous gene coding
for
a specific protein product.
BRIEF DESCRIPTION OF THE FIGURES
The present invention may be more completely and easily understood when
taken in conjunction with the accompanying drawing, in which:
Figs. lA-1C illustrate the isolation, expansion and maintenance as a cultured
cell line of human neural crest stem cells;
Figs. 2A-2D are photographs illustrating attributes of human neural crest stem
cells;
Figs. 3A-3F are photographs illustrating HNC10 human neural crest stem cells
2 0 after differentiating into alternative and different functional cells;
Fig. 4 is a photograph showing the reverse transcriptase-polymerase chain
reaction analysis of genes expressed by the HNC 10 human neural crest stem
cell line;
Figs. SA-SC are graphs illustrating the differentiation of HNC10 human neural
crest stem cells into alternative types of differentiated cells after
stimulation by
2 5 various neurotropic factors;
Figs. 6A-6H illustrate the in-vivo changes caused by lacZ-expressing HNC 10
human neural crest stem cells which were implanted into newborn mouse brains
using
an intra-ventricular implantation technique and determined readily by their
blue color
reaction; and
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Figs. 7A-7D illustrate the results of HNC 10 human neural crest stem cells
which were implanted into myelin mutant shaker rat brains four weeks after
implantation in-vivo.
DETAILED DESCRIPTION OF THE INVENTION
The present invention constitutes the isolation, expansion, and maintenance of
human neural crest stem cells and their direct descendent progeny cells as a
pure
cloned culture; and the use of such cloned human neural crest cells as
therapeutic
implants when placed at a prechosen anatomic site in a living human or animal
host.
The generation and in-vitro culture of purified clones constituted of human
neural
crest stem cells and their direct daughter progeny cells is unique in this
field. These
stem cells and their direct progeny are medically suitable and biologically
compatible
for use as cellular implants in humans - particularly those afflicted with
debilitating
neurological disorders and diseases.
Among the many benefits and major advantages provided by the present
invention are the following:
1. When human neural crest stem cells are implanted into pathological myelin-
2 0 deficient areas of the CNS, their differential potential is strongly
influenced by the
environmental signals at the site of implantation. Thus, the implanted cells
are useful
for treating a variety of diseases characterized by profuse white matter
degeneration -
that would benefit by the replacement of myelin-forming cells. When neural
crest
stem cells are implanted into different areas of the developing nervous
system, they
2 5 generate progeny that become myelin-forming cells in that area.
2. Cell therapy has become a most promising strategy for the treatment of many
human diseases including neurological disorders. The objective of cell therapy
is to
replace lost cells and restore the function of damaged cells and tissues.
3 0 Transplantation of renewable, homogenous and well-characterized human
neural crest
stem cells into the damaged target tissue or organ will replace lost cells and
should
restore damaged function. These stem cells and their direct progeny are ideal
cells to
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l~
serve as donor cells for cell therapy in various neurological diseases
including motor
and sensory neuropathy, multiple sclerosis, Parkinson disease, Huntington
disease,
amyotrophic lateral sclerosis (ALS), spinal cord injury, stroke, Duchenne
muscular
dystrophy and pain control.
3. The stable immortalized human neural crest stem cell line described here
can
be expanded readily and provide a renewable and homogeneous population of
glial,
neuronal and muscle cells. These cells are most valuable for future research
studies of
fundamental questions in developmental neurobiology, cell and gene therapies,
and
development of new therapeutic drugs.
A considerable number of conventionally known abbreviations and minimal
designations are used by practitioners in this art as jargon. To aid the
reader in
understanding the detailed information and description presented hereinafter,
a
summary of such terms is given below.
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11
Abbreviation Proper title/name
or Desi,_ ng anon and nomenclature
A2B5 A clone of monoclonal antibody specifically
react to
GT1 ganglioside.
p75NGFR Low affinity nerve growth factor receptor.
FORSE-1 A cell surface molecule specific in earliest
stage of
neural tube formation and a marker for
neural stem
cells.
PSA-NCAM Polysialic acid - neural cell adhesion
molecule, a cell
type marker for neural progenitor cells.
NG2 A monoclonal antibody recognizes glycoprotein;
similar
to PSA-NCAM.
NF-L; -M; -H Neurofilament triplet proteins; low (L),
medium (M)
and high (H) molecular weight protein.
O1; 04 Clones of monoclonal antibodies specifically
react to
surface antigens of oligodendrocytes.
GFAP Glial fibrillary acidic protein.
trk A/B/C Cellular receptors specific for neurotrophins,
NGF,
2 BDNF, NT-3 and NT-4/5.
0
MBP Myelin basic protein.
B7-2 Co-stimulatory factor specific for antigen
presenting
cells (APC) and a marker for microglia.
PO Protein zero, a myelin protein specifically
found in the
2 PNS, and a marker for Schwann cells.
5
S 100 Protein found in Schwann cells.
FGF-2 Fibroblast growth factor-2, also called
basic fibroblast
growth factor.
NGF Nerve growth factor.
3 BDGF Brain derived growth factor.
0
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Abbreviation Proper title/name
or Desi ng ation and nomenclature
GGF Neuregulin/glial growth factor.
NT-3 Neurotrophin-3.
PDGF BB Platelet derived growth factor BB isoform.
TGF-b 1 Transforming growth factor-b 1.
BMP2 Bone morphogenic protein-2.
CNPase Cyclicnucleotide phosphodiesterase, a cell-type
specific
marker for oligodendrocytes.
TH Tyrosine hydroxylase.
DRG Dorsal root ganglia.
NCSC Neural crest stem cell(s).
PNS Peripheral nervous system.
ANS Autonomic nervous system.
CNS Central nervous system.
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The disclosure and detailed description of the present invention is presented
as
an incongruous approach and contrasting position to the conventional opinions
and
ordinary expectations of practitioners working in this technical field. The
present
invention often contravenes and stands opposite to conventional views and
positions;
and provides many striking examples of differences and distinctions of cell
qualities
and cell attributes not previously recognized or appreciated. For these
reasons, among
others, it is deemed both valuable and useful to provide the reader first with
the novel
neural crest cell paradigm and organized system of neural crest cellular
development
which underlies and supports the mode and manner by which the newly cloned
human
stem cells yield functional and completely differentiated cells.
I. The Neural Crest Cell Model And System Of Cellular Development
The substantive value and real significance of the present invention can only
be properly recognized and truly appreciated in the context of the model and
system of
cellular development that these unique cells evidence and embody. Much
confusion,
misleading views, ambiguity and inconsistency has been reported and
unfortunately is
the overall result to date of the different investigative attempts to
elucidate and specify
the stages of developmental and the various differentiated outcomes
originating with
and from the primordial neural crest stem cell. As evidenced and illustrated
by the
2 0 experiments and empirical data described herein, the present invention -
for the first
time - is able to present an organized system of developmental stages and
different
cell lineages and forms which originate with a single human neural crest stem
cell and
continue until a fully differentiated, phenotype cell is yielded. The model
system of
neural crest cellular origins and development is given by Flow Scheme A below.
Image
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As shown in Flow Scheme A, the original embryonic source of all these cells
is the neural crest stem cell. Such stem cells are: (i) uncommitted and
undifferentiated
cells; (ii) pluripotent cells having an unlimited proliferation capacity; and
(iii) are able
to self renew and self maintain their existence when replicating by producing
two
5 daughter progeny cells, one of which becomes a self renewed stem cell and
the other
becoming a direct and true descendent cell, now designated a "pre-progenitor
cell".
The daughter self renewed stem cell is identical to and indistinguishable from
its
parent cell. However, the daughter pre-progenitor cell is markedly different
from the
parent stem cell.
10 In comparison to its parent, the pre-progenitor cell has a large, but
limited
proliferation capacity. This direct descendent daughter cell is itself a
multipotent cell
which is and remains uncommitted and undifferentiated as such. However, the
rate of
proliferation for the daughter pre-progenitor cells is much greater than its
stem cell
parent. Stem cells are not found in abundance in any tissue, embryonic or
adult. The
15 pre-progenitor cell, however, proliferates rapidly during its limited
number of
reproductive cycles. Also the progeny of the single pre-progenitor cell
becomes
altered and thus these limited number of daughter cells are now termed
"primary
progenitor cells".
The dominant characteristic property and attribute of the "primary progenitor
2 0 cells" is their ability to become committed to a single cell lineage and
line of
development. It is therefore at this third stage of neural crest cell
descendancy that the
multipotency aspect and capacity of the ancestor cells becomes lost forever;
and that
the primary progenitor cell is the stage which becomes influenced by external
stimuli
and chemical signals in the local environment such that an irreversible
commitment is
2 5 made to one category of cell lineage and cell embodiment type. As shown by
Flow
Scheme A, the model system shows that not less than four (4) separate and
individual
cell pathway lineages exist. Each lineage provides for its own family tree and
pedigree; and each pathway provides at least one outcome and typically several
different possible formats of differentiation. The primary progenitor cell is
also able
3 0 to reproduce itself, presumably both before and after commitment to a
separate cell
lineage; but its proliferation capacity is believed to be markedly restricted
and limited
in comparison to its ancestors.
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16
Once cell lineage commitment occurs in the primary progenitor cell, the
development of the cells continues within carefully controlled and pre-
selected
pathway limits. Also, cell differentiation as such occurs only after a prior
commitment to a set cell lineage has been made. At these latter stages of
development, the particular form and phenotypic properties of the cell are
decided;
and a completely differentiated cell subsequently emerges as the full and
final
embodiment in the progression of events from early, to middle, to late stages
of cell
differentiation - as these have been classically identified and reviewed in
the scientific
literature. The nature of signals and molecules which determine cell lineage
or cell
fate are currently virtually unknown.
The human neural crest stem cells and their progeny cells - the pre-progenitor
cells and the primary progenitor cells - in this model system of cellular
development
are the cells constituting and comprising the present invention. As disclosed
and
described in full hereinafter, these cells are unique in their pluripotent
properties; are
uncommitted and undifferentiated cells; and are demonstrably able to be
implanted in-
vivo and yet provide a range of fully differentiated cells which differ in
function, in
morphology, and in phenotypic cell properties.
II. Isolation, Expansion, And Immortalization
2 0 Of Human Neural Crest Stem Cells
Isolation
Dissociated cell cultures were established from human embryonic dorsal root
ganglia (DRG) of 15 week gestation by trypsin treatment as described
previously by
Kim et al., J. Neurosci. Res. 22: 50-59 (1989). DRG cultures grown for 1-2
weeks
2 5 were consisted of small (10 um in diameter) or larger ( 15 um in diameter)
nerve cells
in singles or clusters, more numerous spindle shaped (15-20 um in length)
Schwann
cells, flat polyclonal fibroblasts and a small number of neural crest stem
cells [see Fig.
1A]. DRG cultures with 80% confluency were subjected to retrovirally mediated
transduction of vmyc oncogene and subsequent cloning.
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Retrovirus-Mediated Gene Transfer
Two different xenotropic, replication-incompetent retroviral vectors were used
to infect human NCSCs. The retroviral vector used for transducing NCSCs with
vmyc oncogene was an amphotropic replication-incompetent retroviral vector
encoding vmyc (transcribed from the LTR plus neo transcribed from an internal
SV40
early promoter) which permitted the propagation of human NCSC clones by
genetic
means. And also confirmation of the monoclonal origin of all progeny. This
amphotropic vector was generated using the ecotropic retroviral vector
encoding v_ myc
to infect the GP+envAMl2 amphotropic packaging lines [Snyder et al., Cell 68:
1-20
(1992); Ryder et al., J. Neurobiol. ?1: 356-375 (1990); Markowitz et al.,
Virology
167: 400-406 (1988)]. The second retroviral vector encoding lacZ transcribed
from
the viral long terminal repeat (LTR) plus neo transcribed from an internal
SV40 early
promoter similar to the BAG vector [Snyder et al., Cell 68: 1-20 (1992)]. This
vector
provided a stable, histochemically- and immuno-detectable genetic marker for
transplantation experiments. Successful infectants were selected and expanded.
Supernatants from these new producer cells contained replication-incompetent
retroviral particles bearing an amphotropic envelope at a titer of 4x105 CFUs
which
efficiently infected the human neural cells as indicated by 6418-resistance.
No helper
or replication-competent recombinant viral particles were produced.
Clones of Immortalized Human Neural Crest Stem Cells
After 7-14 days of 6418 selection, ten 6418-resistant clones, HNC10/A2, Bl,
B3, C2, D3, D4, E2, F5, F1, and G6 were isolated and expanded. The cloned
NCSCs
were tripolar or multipolar in morphology with 10-15 um in size [see Fig. 1B].
2 5 HNC 10/C2, one of these expanded clones, was subjected to elaborate study.
In the
proliferative growth condition, the clone HNC 10/C2 exhibited a doubling time
of
about 24 hr [see Fig. 1C].
Characteristics of the HNC 10 Clone of Immortalized Cells
3 0 The HNC 10/C2 cell line grows in culture in serum-free, chemically-defined
medium as single cells or clusters that can be subcultured and passaged
weekly. The
cells are typically grown on coverslips and can be processed for
immunocytochemical
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1g
staining to reveal cell type specific markers.
A summary of the cell-type specific markers and histochemical characteristics
for this stable clone of human neural crest stem cells is given by Table 1
below. A
more complete description of the experiments and empirical data supporting the
information provided by Table 1 is presented hereinafter. The content and
formulation of the serum-free, chemically-defined medium in which these cells
were
grown and maintained is given by Table 1A.
CA 02409713 2002-11-04
19
Table 1: Immortalizeduman Neural CrestCharacteristics
H Stem Cell
Presence Meaning/Value
(+)
Marker or of Marker Substance
T a Absence Indicator Detected
-
human mitochondria~~~(+) human cell origincell organelle
pan-myc oncoprotein~za(+) contains vm c cellular oncoprotein
nestin~3~ (+) NCSC cytoskeletal
protein
p75NGFR~~ (+) NCSC surface receptor
antigen
vimentin~s~ (+) NCSC cytoskeletal
protein
A2B5~6~ (+) NCSC surface antigen
Musashi'~ (+) neural progenitortranscription
2 cells factor
0
FORSE-1~8~ (+) neural progenitorsurface antigen
cells
2 PSA-NCAM~9~ (+) neural progenitorsurface antigen
5
cells
NG2 proteoglycan~~~(+) progenitor cells/surface antigen
glial precursor
cells
30
NF-L~"~ (+) early stage neuronspecific neuron
marker
NF-M~"~ (+) middle stage neuronspecific neuron
marker
3 NF-H~~ (+) differentiated specific neuron
5 neuron marker
~ tubulin~'~~ (-) differentiated specific neuron
neuron marker
isotype III
4 peripherin~~ (-) mature peripheralPNS specific
0 neuron
nervous system marker
neuron
trkA~~ (-) neuron neurotrophin
(NGF)
receptor protein
45
O1~~ (-) oligodendrocyte specific oligodendrocyte
marker
04~~1~ (-) oligodendrocyte specific oligodendrocyte
5 marker
0
GFAP~~~~ (-) astrocyte cell specific astrocyte
marker
CA 02409713 2002-11-04
20
Table 1: Immortalized racteristics
Human Neural (continued)
Crest Stem
Cell Cha
Presence Meaning/Value
(+)
Marker or of Marker Substance
T a Absence (-) Indicator Detected
MBP~~ (-) oligodendrocyte specific oligodendrite
marker
B7-2~~ (-) microglia specific marker
PO~~ (-) Schwann cell specific Schwann
cell marker
trkB/trkC~~ (-) special classes neurotrophin
of receptor
neurons protein
S 100~I~1~ (-) glial cell mature Schwann
cell marker
chromogranin~w~(-) adrenal chromaffin cell
chromaffin cell specific marker
desminw~ (-) skeletal muscle cytoskeletal
protein
2 cell
5
myosinw~ (-) skeletal muscle cytoskeletal
protein
cell
Table 1 References:
1. Flax et al., Nature Biotech 16: 1033-1039 (1998).
2. Spotts, R. and B. Hann, Mol. Cell Biol. 10: 3952-3956 (1990).
3. Lendahl et al., Cell 60: 585-595 (1990); Stemple, D.L. and D.J. Anderson,
Cell 71: 973-985
(1992); Friedman et al., J. Comp. Neurol. 295: 43-51 (1990); Hockfield et al.,
J. Neurosci. 5:
3310-3328 (1985); Reynolds et al., Science 255: 1707-1710 (1992).
4. Chao, M.V., Neuron 9: 583-593 (1992); Morrison et al., Cell 96: 737-749
(1999).
4 0 5. Houle, J. and S. Fedoroff, Dev. Brain Res. 9: 189-195 (1983).
6. Eisenbarth et al., Proc. Nat. Acad. Sci. USA 46: 4913-4917 (1979).
7. Sakakibara, S. and H. Okano, J. Neurosci. 17: 8300-8312 (1997).
8. Tole, S. and P.H. Patterson, J. Neurosci. 15: 970-980 (1995).
9. Grinspan, J.B. and B. Franceschino, J. Neurosci. Res. 41: 540-551 (1995).
4 5 10. Stallcup, W.B. and L. Beasley, J. Neurosci. 7: 2737-2744 (1987).
II. See Table 2 references.
III. See Table 3 references.
IV. See Table 4 references.
V. See Table S references.
CA 02409713 2002-11-04
21
Table 1A:
UBC 1 Serum-Free Chemically-Defined Medium
[mg/liter] [mg/liter]
albumin 1000 - 4000 aluminum chlorideØ001 - 0.01
6H20
D-biotin 0.015- 0.7 ammonium metavanadate0.0006 - 0.0012
ethanolamine 10 - 50 barium chloride 0.001 - 0.003
galactose 800 - 1000 cobalt chloride. 0.001 - 0.003
6H20
linoleic acid' 0.04 - 0.12 chromic potassium0.001 - 0.003
sulfate
oleic acid' 0.04 - 0.12 cupric sulfate. 0.0012 - 0.005
5Hz0
putrescine. 0.08 - 0.32 ferrous sulfate. 0.42 - 0.84
2H20 7H20
pyruvatez 55 - 220 germanium dioxide0.005 - 0.01
retinal acetate'0.01 - 0.05 lithium chloride 0.01 - 0.02
vitamin B 12 0.34 - 1.36 molybdic acid. 0.0001 - 0.0002
2Hz0
ascorbic acid 20 - 100 nickel nitrate. 0.0001 - 0.0002
6Hz0
catalase 20 - 100 rubidium chloride0.00001- 0.00002
glutathone, 0.05 - 2.0 silver chloride 0.0000044
reduced
2 superoxide dismutase20 - 100 sodium selenite 0.03 - 0.3
0
tocopherol acetate0.025- 0.5 stannous chloride0.0001 - 0.0003
HEPES 2900 - 6600 titanium oxide 0.001 - 0.003
zinc sulfate. 0.43 - 1.29
7H20
insulin/human3 5 - 10
2 transferrin/human5 - 10
5
hydrocortisone 0.2 - 0.4
progesterone 0.013- 0.026
triiodothyronine0.02 - 0.06
1. Water-soluble forms of linoleic/oleic acids and retinal acetate should be
used.
2. Pyruvate should be omitted when DMEM is used as a basal medium.
3. Insulin/ 10 mg should be dissolved in 1 ml of 0.1 N HCl and then add 1 ml
HzO.
CA 02409713 2002-11-04
22
As summarized by Table 1 above, the immortalized clone of HNC 10 cells
were immunoreaction-positive with an antibody against human mitochondria and
also
for pan-myc oncoprotein indicating that HNC10 cells are uniquely of human
origin
and also contain a copy or multiple copies of vmyc-encoding retrovirus (as
determined
relative to positive and negative controls run in parallel). Almost all HNC 10
cells
tested were immunoreaction-positive for nestin and p75NGFR indicating that HNC
10
cell line is indeed constituted of neural crest stem cells. Other cell-type
markers for
neural crest stem cells, vimentin and A2B5, were also demonstrated in HNC10
cells.
In addition, the HNC10 cells were also immunoreaction-positive for Musashi, a
transcription factor determining differentiation of neural progenitor cells;
FORSE-1, a
surface antigen marker for neural progenitor cells; PSA-NCAM, a surface
antigen
marker for neural progenitors and also for oligodendrocyte-type 2 astrocyte (O-
2A)
pre-progenitor cells; and NG2 proteoglycan, a surface antigen marker for
neural
progenitor cells.
These data, therefore, have established for the first time that the HNC 10
cell
lines (immortalized via retrovirus-mediated vmyc transfer and initially
derived from
embryonic DRG) are truly and properly the self renewing and multipotent human
neural crest stem cells. Never before, insofar as is presently known, has a
human
neural crest stem cell been isolated, expanded and maintained as a stable cell
line;
2 0 and, moreover, no immortalized neural crest stem cell clone has been
developed and
characterized in depth to demonstrate and prove its human origins and stem
cell
properties.
II. Lineage Commitment Of Human Neural Crest Stem Cells
2 5 Human neural crest cells and their progeny cells can commit to at least
four
different cell lineages when cultured in-vitro using media containing 5% fetal
bovine
serum and 5% horse serum. A variety of different immunological and
histochemical
markers have been identified in the earlier published scientific literature
which offers
reliable test assay [surface antigen phenotypes] procedures by which to
separate and
3 0 distinguish different cell types among the possible choices of cell
lineage
development. The experiments and empirical data unequivocally demonstrating a
commitment to a specific cell lineage and the undifferentiated nature of such
CA 02409713 2002-11-04
23
committed cells are described in detail hereinafter. However, the sum and
substance
of these differences is given in summary form by Tables 2-5 respectively
below.
CA 02409713 2002-11-04
24
Table 2: Neuronal Cell Lineage Characteristics
Absence Meaning or
(-)
Marker or Value of Substance
T a Presence of Indicator Detected
(+)
NF-L~~~ (+) early stage neuronneurofilament
protein-
specific neuron
marker
NF-M (+) middle stage neuronneurofilament
protein-
specific neuron
marker
NF-H (+) mature neuron neurofilament
protein-
specific neuron
marker
b tubulin~2~(+) mature neuron specific neuron
marker
isotype
III
2 peripherin~3~(+) mature peripheral specific neuron
0 marker
nervous system
neuron
trkA~4~ (+) sensory and receptor protein
specific
sympathetic neuronsfor NGF
trkB/trkC~s~(-) special classes receptor protein
of specific
neurons for BDNF (trkB)
or
NT-3 (trkC)
Table 2 References:
1. Julien et al., Biochem. Bionhys. Acta. 909: 10-20 (1987); Lee et al., EMBO
J.
7: 1947-1955 (1988); Myers et al., EMBO J. 6: 1617-1626 (1987).
2. Ferreira, A. and A. Caceres, J. Neurosci. Res. 32: 516-529 (1992).
3. Parysek, L.M. and R.D. Goldman, J. Neurosci. 8: 555-563 (1988); Gorham et
al., Dev. Brain Res. 57: 235-248 (1990).
4. Chao, M.V., Neuron 9: 583-587 (1992).
4 0 5. Obermeyer et al., J. Biol. Chem. 268: 22963-6? (1993).
CA 02409713 2002-11-04
Table 3: Glial Cell Lineage Characteristics
Absence Meaning or
(-)
5 Marker or Value of Substance
T a Presence Indicator Detected
(+)
glial fibrillary~l~(+) astrocyte and cellular protein
of
acidic protein Schwann cell astrocyte and Schwann
cells
10 (GFAP)
S100~2~ (+) Schwann cells Schwann cell specific
marker
15 O 1 ~3~ (-) oligodendrocyteoligodendrocyte specific
marker
04~~~ (-) oligodendrocyteoligodendrocyte specific
marker
20
MBP~S~ (-) oligodendrocyteoligodendrocyte specific
marker
p0~6~ (+) Schwann cell Schwann cell specific
2 marker
5
B7-2~~~ (-) mieroglia microglia specific
marker
Table 3 References
1. Jessen et al., Neuron 12: 509-527 (1994).
2. Jessen et al., Development 109: 91-103 (1990).
3. Sommer, L. and M. Schachner, Dev. Biol. 83: 311-327 (1981).
4. Schachner et al., Dev. Biol. 83: 328-338 (1981).
5. Sternberger et al., Proc. Nat. Acad. Sci. USA 75: 2521-2524 (1978).
6. Jessen et al., Neuron 12: 509-527 (1994).
7. Satoh et al., Brain Res. 704: 92-96 (1995).
CA 02409713 2002-11-04
26
Table 4: Lineage Characteristics of Adrenal Chromaffin Cells
Absence Meaning or
(-)
Marker or Value of Substance
T a Presence Indicator Detected
(-)
chromogranin~~~ (+) adrenal chromaffm cell surface
cell marker
tyrosine hydroxylase~2~(+) sympathetic neuronsspecific
of the autonomic enzyme
nervous system
PNMT~3~ adrenal chromaffm cell marker
cell
Table 4 References
1. Wilson, B.S. and R.V. Lloyd, Am. J. Pathol. 115: 458-468 (1984).
2. Pickel et al., Proc. Nat. Acad. Sci. USA 72: 659-663 (1975).
3. Connett, R. and N. Kirschner, J. Biol. Chem. 245: 329-334 (1970).
CA 02409713 2002-11-04
27
Table 5: Lineage Characteristics of Skeletal Muscle Cells
Absence (+) Meaning or
Marker or Value of Substance
T a Presence (-) Indicator Detected
morphologically flat, (+) myoblasts physical
elon ated, multinucleated appearance
~
cells of cells
~~
desmin~2~ (+) skeletal cellular
muscle cell filament
myosin~3? (+) skeletal cellular
muscle cell filament
Table S References
1. Le Douarin et al., Dev. Biol. 159: 24-49 (1993).
2. Naumann, K. and D. Pette, Differentiation 55: 203-211 (1994).
2 5 3. Ibid.
CA 02409713 2002-11-04
28
Thus, as summarized by Tables 2-5 respectively, neurons differentiated from
HNC 10 cells expressed neurofilament proteins, NF-L, NF-M and NF-H; and the
vast
majority of neurofilament-positive cells co-expressed ~ tubulin isotype III, a
cell type
specific marker for neurons as well as peripherin, a marker of mature
peripheral
nervous system neurons. These phenotypes are specifically and exclusively
expressed
by the human cells indicating that HNC10 cells can and do commit to and
differentiate into nerve cells under the serum-containing culture condition.
HNC 10 cells also differentiated into glial cells when grown in serum-
containing medium, and expressed glial fibrillary acidic protein (GFAP), a
cell type-
specific marker for Schwann cells. These lineage cells also were
immunoreaction-
positive for S 100, and PO cell type-specific markers for mature Schwann
cells. These
lineage cells were, however, immunoreaction-negative for O1 and 04, both cell
type
specific markers for oligodendrocytes.
In addition, HNC 10 cells grown in serum-containing medium for 2 weeks
yielded lineage cells which were immunoreaction-positive for chromogranin and
PNMT, two specific markers for adrenal chromaffin cells and were reaction
positive
for tyrosine hydroxylase, a marker for sympathetic neurons of the autonomic
nervous
system lineage. These results indicate that HNC 10 cells are sympathoadrenal
stem
cells which are capable of differentiation into sympathetic neuroblasts and
adrenal
2 0 chromaffin cells.
Lastly, after 1-2 weeks in serum-containing medium, HNC 10 stem cells gave
rise to large, flat, elongated, multinucleated cells, and many of these
multinucleated
cells expressed desmin- and myosin-immunoreaction. These results indicate that
HNC10 cells, human NCSCs, can commit to and differentiate into skeletal muscle
2 5 cells.
CA 02409713 2002-11-04
29
III. Promotion and Avoidance of Specific Cell Lineages by Individual
Growth Factors
In the embryonic stage of human development, NCSCs migrate from the
dorsal aspect of the neural tube and subsequently differentiate into a variety
of cell
types found in different embryonic locations. A variety of transplantation and
cell
culture studies support the view that the fate of pluripotent neural crest
stem cells is
determined by the local environment signals exerted in-vivo; and that at least
some of
such local environmental signals are created and provided by a range of growth
factors
such as fibroblast growth factor (FGF), transforming growth factor (TGF) and
neuregulin/glial growth factor (GGF).
The human neural crest stem cells and their direct descendent progeny cells
(the precursor progenitor cells and the primary progenitor cells) - both as
non-
transformed cells and stable immortalized cells maintained in culture - are
demonstrably influenced in different ways by a diverse range of different
growth
factors. A representative listing of empirically evaluated growth factors and
their
actions is given by Table 6 below.
CA 02409713 2002-11-04
Table 6: Environmental Factors and Signals
Promotes Blocks/Avoids
5 Growth Differentiation Differentiation
Factors Of Of
FGF-2 (bFGF) neurons; Schwann cells;--
10 skeletal muscle cells
NGF Schwann cells; --
( 100 ng/ml) skeletal muscle cells
15 BDNF Schwann cells; --
(10 ng/ml) skeletal muscle cells
NT-3 Schwann cells; --
(10 ng/ml) skeletal muscle cells
20
PDGF-BB Schwann cells skeletal muscle
cells
( 10 ng/ml)
neuregulin a Schwann cells; neuronal cells
1
2 ( 10 ng/ml) skeletal muscle cells
5
neuregulin b2 Schwann cells; neuronal cells
(10 ng/ml) skeletal muscle cells
3 TGF-b 1 Schwann cells; neuronal cells
0
( 10 ng/ml) skeletal muscle cells
CA 02409713 2002-11-04
31
These findings indicate that the choice of each of several alternative fates
available to NCSCs can be instructively promoted by different environmental
signals
including those growth factors previously reported by various investigators.
In the
present study, it was found that FGF-2 promotes differentiation of HNC 10
cells into
three cell types, i.e., neurons, Schwann cells and skeletal muscle cells.
Neuregulin
and TGFb 1 induced HNC 10 cells to differentiate into Schwann cells and
skeletal
muscle cells and those growth factors blocked neuronal differentiation of HNC
10
cells. Members of the neurotrophin family, NGF, BDNF and NT3 also favored
differentiation of HNC 10 cells into glial and skeletal muscle cells. These
findings
also showed that trophic effect was not specific for a particular
neurotrophin; and that
the mechanism actually in effect involves activation of the non-selective
neurotrophin
receptor, p75NGFR, by one of these neurotrophins. The results further indicate
that
neurotrophins and other pertinent growth factors affect differentiation of HNC
10 cells
at multiple levels for each and different lineage of cells. Moreover, these
results
reveal that concerted action of combinations of growth factors is important in
determining fate of the NCSCs rather than action of a single factor.
IV. In-Vivo Capabilities And Properties Of Human
Neural Crest Stem Cells And Their Progeny
The isolated human neural crest stem cells maintained in culture as a cloned
colony and their progeny cells - collectively, the precursor progenitor cells
and
primary progenitor cells - remain uncommitted and undifferentiated cells while
passaged in-vitro. This collection of cells, both stem cells and progeny
cells, are
2 5 suitable for on-demand implantation in-vivo, preferably at a prechosen
anatomic site,
into a living host subject, human or animal.
It will be noted and appreciated that at the time of in-vivo implantation,
these
cells are uncommitted to a specific cell lineage of development and remain
undifferentiated as to particular cell form or phenotype. All such cell
lineage
3 0 commitment and differentiation into cell form and phenotype occurs only
after the
cells are implanted in-vivo.
Moreover, after implantation into a living host at a known anatomic site, the
CA 02409713 2002-11-04
32
neural crest stem cells and their progenitor progeny cells optionally can
migrate to one
or more other anatomic sites. Such optional migration capabilities are
empirically
proven by the experimental data provided hereinafter.
Subsequently, whether at the original implantation site or another anatomic
location post-migration, the implanted cells will integrate with the
particular cells and
tissues then present and existing at that particular location. Such tissue
integration
brings the neural crest stem cells and their progenitor progeny cells into
effective
contact with and influence by the local environment - including such local
ligands,
activated cells, stimulating agents, immunlogically reactive substances, and
enzymatically active compositions as are present in or may be conveyed to that
local
environment. Among such chemical entities are various growth factors,
cytokines,
circulating antibodies and transitory cells, as well as a range of fluids
carrying
nutrients, minerals, co-factors and other reactive compositions, compounds and
ligands which typically serve as local environmental signals, stimulating
agents,
and/or modulating moieties.
As a consequence of integration at an identifiable anatomic site and the
influence and effects of signals and interactions, caused or provided by the
milieu and
setting of the local environment, the now integrated neural crest stem cells
and
progenitor progeny cells become committed to one particular cell lineage and
form of
2 0 cellular development in-situ; and become differentiated cells in-situ
consistent with a
particular cell lineage. Thus, the local environment will control and dictate
what
differentiated cell form and phenotype will appear in functional form in the
local
anatomic area. This is clearly demonstrated and evidenced by the experiments
and
empirical data described hereinafter by Experimental Series II.
2 5 Accordingly, the implanted and integrated neural crest stem cells and
their
progenitor progeny cells become committed and differentiated in-situ as
Schwann
cells, peripheral nerve cells, skeletal muscle cells, adrenal chromaffin
cells, as well as
the other differentiated cell forms and phenotypes shown by Flow Scheme A
previously herein. Each of these individual and different cell forms and
phenotypes
3 0 lies within the potential capacities and properties of the HNC 10 series
of cloned cells;
and each clone of cells after implantation in-vivo will become integrated at
and
differentiated by the environmental and development signals, ligands, and
cells
CA 02409713 2002-11-04
33
presented at or conveyed to the local anatomic site.
V. Experiments and Empirical Data
The following is a summation presenting the sum and substance of many
different in-vitro and in-vivo experiments. For ease of understanding and
presentation
clarity of information purposes, a lengthy recitation of the pertinent methods
and
materials background is given. It will be recognized that many of the
individual in-
vitro test and assay techniques and protocols are conventionally described in
the
scientific and medical/clinical published literature. The empirical studies
are recited
in substantive detail.
CA 02409713 2002-11-04
34
Materials and Methods:
UBC 1 (a serum-free, chemically-defined medium):
[mg/liter] [mg/liter]
albumin 1000 - 4000 aluminum chlorideØ001 - 0.01
6H20
D-biotin 0.015- 0.7 ammonium metavanadate0.0006 - 0.0012
ethanolamine 10 - 50 barium chloride 0.001 - 0.003
galactose 800 - 1000 cobalt chloride. 0.001 - 0.003
6H20
linoleic acids 0.04 - 0.12 chromic potassium 0.001 - 0.003
sulfate
oleic acid' 0.04 - 0.12 cupric sulfate. 0.0012 - 0.005
5Hz0
putrescine. 0.08 - 0.32 ferrous sulfate. 0.42 - 0.84
2Hz0 7H20
pyruvatez 55 - 220 germanium dioxide 0.005 - 0.01
retinal acetates0.01 - 0.05 lithium chloride 0.01 - 0.02
vitamin B 12 0.34 - 1.36 molybdic acid. 0.0001 - 0.0002
2Hz0
ascorbic acid 20 - 100 nickel nitrate. 0.0001 - 0.0002
6H20
catalase 20 - 100 rubidium chloride 0.00001- 0.00002
glutathone, 0.05 - 2.0 silver chloride 0.0000044
reduced
2 superoxide dismutase20 - 100 sodium selenite 0.03 - 0.3
0
tocopherol acetate0.025- 0.5 stannous chloride 0.0001 - 0.0003
HEPES 2900 - 6600 titanium oxide 0.001 - 0,003
zinc sulfate. 7Hz00.43 - 1.29
insulin/human3 5 - 10
2 transferrin/human5 - 10
5
hydrocortisone 0.2 - 0.4
progesterone 0.013- 0.026
triiodothyronine0.02 - 0.06
1. Water-soluble forms of linoleic/oleic acids and retinal acetate should be
used.
2. Pyruvate should be omitted when DMEM is used as a basal medium.
3. Insulin/ 10 mg should be dissolved in 1 ml of 0.1 N HCI and then add 1 ml
HZO.
CA 02409713 2002-11-04
Primary DRG cell culture
Thirty pairs of spinal dorsal root ganglia (DRG) were isolated from a 15 week
gestational human embryo and dissociated into single cells by incubating in
phosphate
buffer saline (PBS) containing 0.25% collagenase (Type CLS, Wothington
5 Biochemical, Lakewood, NJ) arid 40 ugh/ml DNase I (Sigma) for 1 hour at
37°C [Kim
et al., J. Neurosci. Sci. 22: 50-59 (1989)]. Dissociated cells (5X105
cells/ml)
suspended in culture medium consisted of Dullbecco's modified Eagle medium
(DMEM) containing 5% fetal bovine serum, 5% horse serum, 0.5% glucose, 20
ug/ml
gentamicin and 2.5 ug/ml amphotericin B, were plated on poly-L-lysine (10
ug/ml)-
10 coated 6 well dishes (Falcon). Two days later, medium was switched to serum-
free
culture medium consisted of DMEM containing UBC 1 supplements (contain human
insulin 5 ug/ml, human transferrin 10 ug/ml, sodium selenite 40 nM,
hydrocortisone
nM, triiodothyronine 3 nM and other nutrients and anti-oxidants), 0.5%
glucose, 20
ug/ml gentamicin, 2.5 ug/ml amphotericin B and FGF-2 (10 ng/ml, Peproteeh)
[Kim
15 et al., Brain Res. 275: 79-86 (1983)]. Culture medium was changed twice a
week.
DRG cultures grown for 1-3 weeks were consisted of DRG neurons, Schwann cells
and fibroblasts and were used for gene transfer experiments. The permission to
use
embryonic tissue was granted by the Clinical Research Screening Committee
involving Human Subjects of the University of British Columbia.
Retrovirus-mediated gene transfer into human DRG cells
An amphotropic replication-incompetent retroviral vector encoding vmvc
(transcribed from the retrovirus LTR plus neo transcribed from an internal
SV40 early
promotor) not only permitted the propagation of human NCSC clones by genetic
2 5 means, but also enabled confirmation of the monoclonal origin of all
progeny. This
amphotropic vector was generated using the ecotropic retroviral vector
encoding
vmyc, as described earlier for generating murine NSC clone C17-2 [Snyder et
al., Cell
68: 33-51 ( 1992)] to infect the GP+envAM 12 amphotropic packaging line
[Markowitz et al., Virolo~y 167: 400-406 (1988)]. Successful infectants were
3 0 selected and expanded. Supernatants from these new producer cells
contained
replication-incompetent retroviral particles which efficiently infected the
human
neural cells as indicated by 6418 resistance. Infection of human DRG cells in
6 well
CA 02409713 2002-11-04
36
plates was performed three times by the following procedures: 2 ml of
supernatant
(4X105 CFUs) from the packaging cell line and 8 ug/ml polybrene
(Aldrich/Sigma)
was added to target cells in 6 well plates and incubated for 4 hr at
37°C; the medium
was then replaced with fresh growth medium; infection was repeated 24 hr and
48 hr
later. Seventy two hr after the third infection, infected cells were selected
with 6418
(250 ug/ml, Sigma) in growth medium for 7-14 days and large clusters of clonal
cells
were individually isolated and grown in PL-coated 6 well plates. Individual
clones
were generated by limited dilution and propagated further. At this phase of
isolation,
individual clones were designated as human NCSC lines, HNC10. One of the HNC10
clones, HNC 10/C2, was propagated further and investigated for its in-vitro
characteristics. HNC 10/C2 clone is referred to simply as an HNC 10 line. One
of the
HNC 10 clones was subsequently transduced with a retroviral vector encoding
lacZ
gene and puromycin-resistant gene, enabling transfected cells to produce b-
galactosidase (b-gal) constitutively.
Determination of doubling time
HNC 10 cells at the concentration of 5 X 104 cells were plated in 30 mm dishes
with 2.5 ml of medium. After 12-72 hr of growth, cells were exposed to 0.1 %
trypsin
in PBS for 5 min at 37°C, collected by centrifugation at 1200 rpm for 8
min, and
2 0 resuspended in 0.5 ml PBS. Using a hemocytometer, cell number was counted
12, 24,
36, 48, 60 and 72 hr after plating under a Nikon inverted microscope.
Immunochemical characterization of human NCSCs
Immunochemical determination of cell type specific markers in HNC 10 cells
2 5 was performed as following: HNC 10 cells were grown on PL-coated Aclar
plastic
coverslips (9 mm in diameter) with serum-free medium for 3-7 days, fixed in 4%
paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for 3 min at room
temperature (RT), washed twice with PBS, and incubated with antibodies
specific for
A2B5 (specific for GT1 ganglioside; mouse IgM monoclonal, American Type
Culture
3 0 Collection/ATCC, Rockville, MD), human p75NGFR (mouse IgG monoclonal,
ATCC), FORSE-1 (mouse IgG monoclonal, ATCC), NG2 (specific for
transmembrane chondroitin sulfate proteoglycan; rabbit polyclonal, Chemicon)
and
CA 02409713 2002-11-04
37
PSA-NCAM (specific for embryonic polysialylated form of neural cell adhesion
molecule; mouse IgM monoclonal, Pharmingen). For cytoplasmic antigen staining,
coverslips bearing cells were fixed in cold acid alcohol (5% acetic acid/95%
ethanol)
for 15 min at -20°C, incubated with heat-inactivated goat serum (1:10)
for 30 min at
room temperature before the primary antibodies were applied, in order to block
any
potential interaction between Fc receptors and Fc fragments. The cells were
incubated
in antibodies specific for nestin (rabbit polyclonal), and vimentin (mouse IgG
monoclonal, Sigma) for 2 days at 4°C. Cells incubated with primary
antibodies were
followed by biotinylated secondary antibodies and avidin-biotin hydrogen
peroxidase
(ABC) immunochemical processing (Vector) and visualized with AEC (Sigma)
chromogen development.
For immunochemical characterization of differentiated cell types the following
antibodies were utilized: For neurons: neurofilament protein-L (NF-L, mouse
IgG
monoclonal, Sigma), NF-M (mouse IgG monoclonal, Sigma), NF-H (mouse IgG
monoclonal, Sigma), microtuble associated protein-2 (MAP2, mouse IgG
monoclonal,
Sigma), neuron specific enolase (NSE, rabbit polyclonal, DAKO), a tubulin
isotype
III (mouse IgG monoclonal, Sigma) and peripherin (rabbit polyclonal,
Chemicon).
For Schwann cells: S-100 A/B (rabbit polyclonal, DAKO). For oligodendrocytes:
CNPase (mouse IgG monoclonal, Sigma), galactocerebroside (GaIC, mouse IgG
2 0 monoclonal produced in our laboratory) and MBP (rabbit polyclonal, DAKO).
For
myoblast/ myotube: desmin (rabbit polyclonal, DAKO) and skeletal muscle myosin
(rabbit polyclonal, Chemicon). For sympathicoadrenal lineage cells: tyrosine
hydroxylase (rabbit polyclonal, pel-freeze), PNMT (rabbit polyclonal,
Chemicon), and
chromogranin (rabbit polyclonal, Dr. R. Angeletti).
2 5 For double-immunofluorescence of cell-surface and cytoplasmic antigens,
the
cells were fixed in 4% paraformaldehyde in 0.1 M PB for 3 min at RT, incubated
with
heat-inactivated normal goat serum (1:10) for 30 min at RT before the primary
antibodies were applied as described above. Cells incubated with primary
antibodies
were followed by fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgM,
IgG
3 0 or anti-rabbit IgG (Cappel, West Chester, PA). After several washes in
PBS, cells
were fixed in cold acid-alcohol for 1 S min. Cells were washed in PBS and then
incubated in second primary antibodies followed by rhodamine-conjugated second
CA 02409713 2002-11-04
38
antibodies as described above. After several washes, coverslips were mounted
on
slides with gelvatol and examined under a Zeiss Universal microscope equipped
with
phase contrast, fluorescein and rhodamin optics.
Cellular differentiation of human NCSCs
To differentiate the immortalized cells, the cells were plated onto PL coated
Aclar plastic coverslips (9 mm diameter) and then grown in serum-free medium
(DMEM containing UBC 1 supplements) for 4 days and then switched to
differentiation medium. The differentiation medium consisted of DMEM
containing
10% fetal bovine serum, 20 ug/ml gentamicin and 2.5 ug/ml amphoterine B. In
order
to study the effect of growth factors on differentiation, the following serum-
free
medium was utilized: DMEM containing UBC 1 supplements, 20 ug/ml gentamicin,
2.5 ug/ml amphotericin B, and one of the following factors: NGF (100 ng/ml),
BDNF
(10 ng/ml), NT-3 (10 ng/ml), PDGF- BB (10 ng/ml), neuregulin al (10 ng/ml),
neuregulin a2 ( 10 ng/ml) and TGF-b 1 ( 10 ng/ml).
RT-PCR analysis
Total RNA preparation was performed according to the acid guanidinium/
phenol/chloroform (AGPC) method previously described. Five ml of total RNA
from
2 0 each sample was subjected to DNase treatment and then processed for the
first strand
cDNA synthesis using Moloney marine leukemia virus (M-MLV) reverse
transcriptase (GIBCO-BRL). Five ml of each eDNA products was amplified by PCR
using the specific sense and antisense primers designed from the cDNA sequence
for
each marker gene for human CNS cells. The eDNA from HNC 10 cells was amplified
2 5 for human nestin and human p75NGFR as a marker gene for stem cells, human
NF-L,
-M, -H as marker genes for neurons, human MBP as a marker gene for
oligodendrocytes, human GFAP as a marker for astrocytes, human PO as a marker
gene for a peripheral myelin protein [Lee et al., Mol. Cell Neurosci. 8: 336-
350
( 1997)], human B7-2 as a marker for microglia, and human trkA, B, C as
markers for
3 0 subtypes of neurotrophin receptors. The following primers were used with
the
expected PCR product length in base pair (bp):
nestin sense: 5'-LCTCTGACCTG TCAGAAGAAT-3'
CA 02409713 2002-11-04
39
antisense: 5'-ACGCTGACACTTACAGAT-3' (316 bp)
first p75NGFR sense: 5'-LCTCACACCGGGGATGTG-3'
antisense: 5'-LGTGGGCCTTGTGGCCTAC-3'
nested primers for the second p75NGFR sense: 5'-TGTGGCCTACATAGCCTTC-3'
antisense: ATGTGGCAG- TGGACTCACT-3' (476 bp)
NF-L sense: 5'-TCCTACTACACCAGCCATGT-3'
antisense: 5'-TCCCCAGCACCTTCAACTTT-3' (284 bp)
NF-M sense: 5'-TGGG- AAATGGCTCGTCATTT-3'
antisense: 5'-CTTCATGGAAGCGGCCAATT-3' (333 bp)
NF-H sense: 5'-CTGGACGCTGAGCTGAGGAA-3'
antisense: 5'-CAGTCACTTCTTCAGTCACT-3' (316 bp)
MBP sense: 5'-ACACGGGCATCCTTGACTCCATCGG-3'
antisense: 5'-TCCGGAACCAGGTGGGTTTTCAGCG-3' (510 bp)
GFAP sense: 5'-GCAGAGATGATGGAGCTCAATGACC-3'
antisense: 5',GTTTCATCCTGGAGCTTCTGCCTCA-3' (266 bp)
PO sense: 5'-TTCTGGTCCAGTGAGTGGGTCTCAG-3'
antisense: 5- TCACTGTAGTCTAGGTTGTGTATGA-3' (209 bp)
B7-2 sense: CTC- TTTGTGATGGCCTTCCTG-3'
antisense: 5'-CTTAGGTTCTGGGTAACCGTG-3' (464 bp)
2 0 first TrkA sense: 5'-CCA- TCGTGAAGAGTGGTCTC-3
antisense: 5'-GGTGACATTGGCCAGGGTCA-3'
nested primers for the second TrkA sense: 5'-CCGTTTCGTGGCGCCAGATG-3'
antisense: 5'-GCCCCAGGGATGGCAGACCC-3' (438 bp)
first TrkB sense: 5'-AAGACCCTGAAGGATGCCAG-3'
2 5 antisense: 5'-AGTAGTCAGTGCTGTACACG-3'
nested primers for the second TrkB sense: 5'-CAATGCACGCAAGGACTTCC-3'
antisense: 5'-TCCCGGGACATCCCAAAGTC-3' (364 bp)
first TrkC sense: 5'-CTACAACCTCAGCCCGACCA-3'
antisense: 5'-GCTGTAG-ACATCTCTGGACA-3'
3 0 nested primers for the second TrkC sense: 5'-AGGACAAGATGCTTGTGGCT-3'
antisense: 5'-TGCCGAAGTCCCCAATCTTC-3' (308 bp)
CA 02409713 2002-11-04
PCR was carried out in a 50 u1 of reaction mixture containing Taq DNA
polymerase
buffer (20 mM Tris-HCI, pH 8.4, 50 mM KCI, 200 M d NTP, 2.5 mM MgClz., 1 mM
of each primer) and 2.5 U Taq DNA polymerase (GIBCO-BRL). The main
amplification program consisted of a denaturation step at 94°C for 1
min followed by
an annealing step at 55-57°C for 1 min, and a synthesis step at
74°C for 1 min, for
35-40 cycles. For the initial amplification, cDNA samples were denatured at
94°C for
5 min, annealed at 55-57°C for 1 min, and extended at 74°C for 3
min.
Brain transplantation study
10 Two different sets of these experiments (newborn mice or shaker rats) were
performed. First sets of experiments, heads of cryoanaesthetized postnatal day
0 CD 1
mice (n = 24) were transilluminated with a fiber optics and 2 u1 volume
containing
5X104 cells/ml plus 0.05% w/v trypan blue in PBS were gently expelled into
each
lateral ventricle through a finely drawn glass micropipette. The distribution
of trypan
15 blue confirmed filling of the ventricular system from as far rostral as the
olfactory
bulbs to as far caudal as the IVth ventricle and the rostral central spinal
canal [Snyder
et al., Nature 374: 367-370 (1995)]. All of these animals were sacrificed at
various
intervals between one and 4 weeks after transplantation. For second sets of
experiments, Long Evans shaker rats (4 weeks old, n = 32) were anesthetized
with
2 0 ketamine hydrochloride (MTC Pharmaceuticals, 35 mglkg) and xylazine (Bayer
Canada, 5 mg/kg) and held in a stereotaxic apparatus with ear bars. A finely
drawn
glass micropipette containing five u1 PBS with 5X104 HNC10/lacZ cells/ml were
slowly injected into the right caudate-putamen at 1.0 mm anterior, 3.0 mm
lateral
from bregma and 4.0 mm below the brain surface. Left side striatal tissues of
2 5 implanted animals served as built-in negative controls. All implanted
shaker rats
received daily cyclosporin A (Novartis Canada, 10 mg/kg) given
intraperitoneally.
All of these animals were sacrificed between one and 4 weeks after
transplantation.
The operated mice or shaker rats were sacrificed at various intervals after
transplantation. Under deep ketamine hydrochloride (35 mg/kg) and xylazine (5
3 0 mg/kg) anesthesia, each animal was perfused through the left cardiac
ventricle with
PBS followed by a fixative consisted of 0.1 M PB containing 4%
paraformaldehyde,
0.1 % glutaraldehyde and 0.2% picric acid. The brains were removed from the
skull
CA 02409713 2002-11-04
41
and postfixed in the same fixative for 24 hours at 4°C. After
cyroprotection with 0.1
M PB containing 15% sucrose, 20 um sections were cut on a freezing microtome
and
collected in 0.1 M PBS and 0.1% sodium azide. Sections were stored in the same
solution until staining was conducted as free-floating sections. Engrafted
cells were
then detected by X-gal (5-bromo-4-chloro-3-indolyl-D-galactoside)
histochemistry
and/or an anti-~-gal antibody (mouse monoclonal IgG, Cappel) to demonstrate
previously implanted ~-gal-positive HNC 10 (HNC 10/lacZ) cells. For X-gal
histochemistry cyrosections were stained by immersion in PBS containing 5 mM
K3Fe
(CN)6, 5 mM KaFe(CN)6, 2 mM MgCl2, 0.01 % sodium deoxycholate, 0.02% Nonidet
P-40, and 2 mg/ml X-gal at 37°C overnight. Sections were washed with
PBS and
placed onto gelatin-coated slides. Both X-gal and ~-gal staining methods
detect lac-Z
positive HNC 10/lac-Z cells. Cell type identity of grafted HNC 10 cells was
also
established by staining with following cell type specific markers: for
neurons: MAP-2;
for oligodendrocytes: CNPase and MBP; for astrocytes: GFAP.
Immunohistochemistry was performed on coronal, free-floating sections. The
sections were first incubated with 0.1 M PBS containing 5% normal goat serum
and
0.3% Triton X-100. After washing, the sections were treated for 30 min at RT
with
0.5% hydrogen peroxide to block endogenous peroxidase, and then rinsed three
times
for 10 min each in PBS. Afterwards, the sections were incubated either with
the anti-
2 0 MAP2, anti-MBP, anti-CNPase, or anti-GFAP antibodies for 3 days at
4°C. Sections
were then incubated with biotinylated secondary antibody (Vector Laboratories,
Burlingame, CA), diluted 1:1000 for 1 hour at RT, followed by incubation with
ABC
solution (Vector) diluted 1:1000 for 1 hour at RT. The peroxidase labeling was
visualized by incubation for 5 min with a mixture containing 0.02%
2 5 3,3'diaminobenzidine tetrahydrochloride (DAB), 0.0045% hydrogen peroxide
and
0.6% nickel ammonium sulfate in 0.05 M Tris-HCl buffer, pH 7.6. Sections were
mounted on glass slides, dehydrated and coverslipped with Permount. Sections
stained for anti-MAP2, MBP, CNPase and GFAP were counterstained with cresyl
violet.
Experimental Series I
A series of experiments were undertaken to isolate, characterize, and
CA 02409713 2002-11-04
42
selectively differentiate in-vitro human neural crest stem cells and their
descendent
multipotent progeny cells. The isolated individual clones of neural crest stem
cells are
stable cell lines; maintained in culture as immortalized cell lines; and are
suitable for
subsequent in-vivo use in a variety of different medical/clinical
applications.
Experiment 1: Isolation and expansion of human immortalized neural
crest stem cells (NCSCs)
Dissociated cell cultures were established from human embryonic dorsal root
ganglia (DRG) of 15 week gestation by collagenase treatment as described
previously
by Kim et al. [J. Neurosci. Res. 22: 50-59 (1989)]. DRG cultures were grown
for 1-3
weeks and consisted of small (10 um in diameter) or larger (15 um in diameter)
nerve
cells in singles or clusters, more numerous spindle shaped (15-20 um in
length)
Schwann cells, flat polygonal fibroblasts and a small number of NCSCs. The
result is
shown by Fig. 1A.
As seen in Fig. 1 A, a dissociated cell culture of human dorsal root ganglia
isolated from 15 week gestation embryo was grown in-vitro for 7 days. The
culture
contains a large number of nerve cells, Schwann cells and a small number of
neural
crest stem cells; and represents a starting material for the generation of
human neural
crest stem cell (NCSC) line. The bar indicates 20 um.
2 0 Subsequently, DRG cultures with 80% confluency were subjected to
retrovirally mediated transduction of vmyc oncogene and subsequent cloning.
After
7-14 days of 6418 selection, ten 6418-resistant clones were isolated and
expanded.
One of these clones, HNC 10/C2, was utilized for further study. The cloned
NCSC
was tripolar or multipolar in morphology with 10-15 um in size. This is shown
by
2 5 Fig. 1 B.
CA 02409713 2002-11-04
43
Fig. 1 B shows that a human neural crest stem cell line, HNC 10, can be grown
in UBC 1 serum-free medium which contains human insulin, human transferrin,
sodium selenite and other nutrients and FGF-2. HNC 10 cells can also be grown
in
serum-containing medium (10% fetal bovine serum) and differentiate into nerve
cells,
Schwann cells, adrenal chromaffin cells and skeletal muscle cells. The bar
indicates
20 um.
Afterwards, HNC10/C2, one of these clones, was subjected to further study. In
the proliferative growth condition, the clone HNC 10/C2 exhibited a doubling
time of
24 hr as shown by Fig. 1 C. When calculated by a population analysis the
doubling
time for HNC10 cells was determined to be 23.67 hr.
Experiment 2: Characterization of HNC 10 cells
HNC10/C2 cell line grew in culture as single cells or clusters that could be
subcultured and passaged weekly for 6 months. The cells grown on coverslips
were
processed for immunocytochemical staining of cell type specific markers.
As shown in Figs. 2A and 2B, HNC10 cells carry two cell-type-specific
markers for neural crest stem cells: nestin, a class of intermediate
cytoskeletal protein
only found in neural crest stem cells; and p75 neurotrophin receptor protein
(p75NGFR), another unique cell type-specific marker for neural crest stem
cells.
2 0 HNC 10 neural crest stem cells are shown to express a very strong nestin
and
p75NGFR immunoreactivity. The bar indicates 20 um.
HNC 10 cells were immunoreaction-positive with an antibody against human
mitochondria and also for pan-myc oncoprotein, as shown by Figs. 2C and 2D,
indicating that HNC 10 cells are uniquely human origin and contain a copy or
copies
2 5 of v-myc-encoding retrovirus, as determined relative to positive and
negative controls
run in parallel. Fig. 2C demonstrates that HNC 10 cells were of human donor
origin in
which HNC 10 cells were reacted with a monoclonal antibody specific for human
mitochondria antigen. Note that cytoplasmic mitochondria are intensely
positive for
the reaction. The bar indicates 20 um. Also, since human
CA 02409713 2002-11-04
44
dorsal root ganglia cells were transfected by the vmyc oncogene and
transformed to
become HNC 10 cell lines, presence of myc antigen was determined. This is
shown by
Fig. 2D. Immunoreactivity was found in nuclei of HNC 10 cells, indicating that
these
cells are indeed transformed by myc oncogene. The bar indicates 20 um.
Another cell-type marker for neural crest stem cells, vimentin, was also
demonstrated in HNC10 cells (results not shown). In addition, HNC10 cells were
also
immunoreaction-positive for Musashi, a transcription factor uniquely
detectable in
neural progenitor cells [see for example, Sakakibara et al., J. Neurosci. 17:
8300-8312
(1997)]; FORSE-1, a surface antigen marker for neural progenitor cells [see
for
example, Tole S. and P.H. Patterson, J. Neurosci. 15: 970-980 (1995)]; PSA-
NCAM,
a surface antigen marker for oligodendrocyte-type 2 astrocyte (O-2A) pre-
progenitor
cells and neural progenitor cells [see for example, Grinspan, J.B. and B.
Franceschini,
J. Neurosci. Res. 41: 540-551 (1995)]; and NG2 proteoglycan, a surface antigen
marker for glial progenitor cells and neural progenitor cells [see for
example, Stallcup,
W.B. and L. Beasley, J. Neurosci. 7: 2737-2744 (1987)].
Experiment 3: Neuronal and glial in-vitro differentiation of HNC10 cells
To determine if HNC 10 cells could differentiate into neurons and glial cells
in
the presence of serum, cultures grown under serum-containing medium with 10%
fetal
2 0 bovine serum were examined. The results are shown by Figs. 3A-3F.
When HNC 10 cells are grown in serum-containing medium, many of these
cells differentiate into nerve cells as shown by Fig. 3A. Note the highly-
branched,
well-differentiated live neurons in such conditions and the long and stout
dendritic
processes of nerve cells after 2 weeks in-vitro. The bar indicates 20 um.
2 5 Neurons differentiated from HNC 10 cells also expressed neurofilament
proteins, NF-
L (10.5 b' 3.5%), NF-M (13.2 d 4.1%) and NF-H (13.8 b' 3.6%) as shown by Fig.
3B;
and the vast majority of neurofilament-positive cells co-expressed b tubulin
isotype
III, a cell type specific marker for neurons [see for example, Ferreira, A.
and A.
Caceres, J. Neurosci. Res. 32: 516-529 (1992)]; and peripherin, a marker of
mature
3 0 peripheral nervous system (PNS) neurons [see for example, Parysek, L.M.
and R.D.
Goldman, J. Neurosci. 8: 555-563 (1988)]. These phenotypes are specifically
and
exclusively expressed by mammalian nerve cells including human nerve cells -
CA 02409713 2002-11-04
indicating that HNC 10 cells can dif=ferentiate into nerve cells under the
serum-
containing culture condition.
Moreover, as shown by Fig. 3C, HNC10 cells also differentiated into glial
cells when grown in serum-containing medium, and expressed S 100, a cell type-
s specific marker for mature Schwann cells [see for example, Jessen et al.,
Neuron 12:
509-527 (1994)]; but also immunoreaction positive for glial fibrillary acidic
protein
(GFAP), a marker for mature Schwann cells [see for example, Jessen et al.,
Development 109: 91-103 (1990)]. HNC10 cells were, however, immunoreaction-
negative for O1 and 04, both cell type specific markers for oligodendrocytes
[see for
10 example, Summer I, and M. Schachner, Dev. Biol. 83: 311-327 (1981); and
Schachner
et al., Dev. Biol. 83: 328-338 (1981)].
In addition, as shown by Fig. 3D, HNC10 cells grown in serum-containing
medium for 2 weeks were also immunoreaction-positive for chromogranin, a
specific
cell type marker for adrenal chromaffin cells [see for example, Wilson, B.S.
and R.V.
15 Lloyd, Am. J. Pathol. 115: 458-468 (1984)]; and tyrosine hydroxylase, a
marker for
sympathetic neurons of the autonomic nervous system lineage (data not shown).
These results indicate that HNC 10 cells are capable of differentiation into
not only
PNS sensory neurons and Schwann cells but also to sympathetic neurons and to
adrenal gland primordia where they differentiate into adrenal chromaffin cells
[see for
20 example, Anderson, D.J., Curr. Opin. Neurobiol. 3: 8-13 (1993)].
Equally important, as shown by Figs. 3E and 3F respectively, after 1-2 weeks
in serum-containing medium, HNC 10 cells gave rise to large flat, elongated,
multinucleated cells, and many of these multinucleated cells expressed desmin-
and
myosin-immunoreaction [see for example, Naumann, K. and D. Pette,
Differentiation
25 SS: 203-211 (1994)]. Note that Fig. 3E reveals that when HNC10 human neural
crest
stem cells were grown in culture in serum containing medium, the cells were
seen to
differentiate into myoblasts and myotubes indicating HNC 10 cells' ability to
develop
into skeletal muscle cells (living/phase contrast microscopy). The bar
indicates 20
um. Fig. 3F supplements Fig. 3E and shows that HNC10 cells differentiate into
3 0 myoblasts/myotubes and express immunoreactivity of human myosin. The bar
indicates 20 um. These results indicate that HNC10 cells, human NCSCs, can and
do
differentiate into skeletal muscle as reported earlier [see for example, N.M.
Le
CA 02409713 2002-11-04
46
Douarin, The Neural Crest, Cambridge University Press, Cambridge, U.K., 1982;
Le
Douarin et al., Dev. Biol. 159: 24-49 (1993)].
Experiment 4: Reverse transcriptase-polymerise chain reaction (RT-PCR)
analysis
Reverse transcriptase-polymerise chain reaction (RT-PCR) analysis of genes
expressed by the HNC 10 cell line were undertaken. Fig. 4 shows RNA
transcripts for
nestin and p75NGFR (both cell type markers for neural crest stem cells) were
demonstrated in HNC 10 cell line growth in serum-free medium. Cell type
markers
for neurons (neurofilament/NF-L, NF-M and NF-H), astrocytes/ Schwann cells
(GFAP), oligodendrocytes (MBP), Schwann cells (PO) or microglia (B7-2) were
not
detected in HNC 10 human neural crest stem cells grown in serum-free medium.
Fig.
4 reveals: Lane M: Molecular standards/bp; lane 1: nestin; lane 2: p75NGFR;
lane 3:
NF-L; lane 4: NF-M; lane 5: NF-H; lane 6: GFAP; lane 7: MBP; lane 8: PO (P-
zero);
lane 9: B7-2.
Results of RT-PCR analysis of cell type-specific markers for the HNC 10 cell
line grown in serum-free medium show that transcripts for nestin and p75NGFT,
cell
type-specific markers for NCSCs were clearly demonstrated. In addition
transcripts
for trkA, a subtype of neurotrophin receptors, were expressed by HNC 10 cells
(data
not shown); but trkB and trkC, other neurotrophin receptors, were not
detected.
2 0 Transcripts for NF-L, NF-M and NF-H, cell type-specific markers for early
and
differentiated neurons, GFAP (a marker for astrocytes), MBP (a marker for
oligodendrocytes), PO (a marker for Schwann cells) or B7-2 (a marker for
microglia)
were not demonstrated by the RT-PCR analysis.
This RT-PCR study of HNC 10 cells indicates that the cells express transcripts
2 5 for specific antigen markers of neural crest stem cells. It is evident
from the RT-PCR
results that HNC10, human NCSCs, grown in serum-free medium, an environment in
which NCSCs appear uncommitted as stem cells, expressed cell type-specific
phenotypes of NCSCs, nestin and p75NGFR.
CA 02409713 2002-11-04
47
Experiment 5: Effects of different growth factors in-vitro upon HNC 10 cells
HNC 10 cells were grown in serum-free medium (DMEM containing UBC 1
supplements) for 4 days and then supplemented with one of the following growth
factors (differentiation medium): FGF-2, NGF, BDNF, NT-3, PDGF-BB, and
neuregulin alpha and beta.
Differentiation of HNC 10 cells, human neural crest stem cells, into neuronal
and glial cells was determined by immunocytochemistry following stimulation by
various neurotropic factors including FGF-2 ( 10 ng/ml), NGF (50 ng/ml), BDNF
(50
ng/ml), NT-3 (50 ng/ml), PDGF-BB (20 ng/ml), neuregulin a 1 (20 ng/ml),
neuregulin
a2 (20 ng/ml) and TGFb 1 (20 ng/ml). HNC 10 cells were grown in serum-free
medium supplemented with one of the growth factors described above for 5-7
days
and then processed for immunostaining. The results are shown by Figs. 5A, SB
and
SC respectively.
Fig. 5A shows the differentiation of HNC 10 cells into neurons as identified
by
immunostaining with neurofilament protein 150 (NF-M). Neuregulins, TGFb 1 and
NT-3 are inhibitory to neuronogenesis, while others did not affect the
neuronal
induction of the HNC 10 cells. Fig. 5B shows glial cells/Schwann cells which
were
identified by GFAP immunoreaction. All the growth factors except neuregulin ~2
were effective in glial cell induction. It is noted that TGF~1 is most
effective as a
2 0 glial cell inducer. Finally, Fig. SC demonstrates the differentiation of
HNC 10 cells
into myoblasts/myotubes by immunostaining with skeletal muscle myosin. All the
growth factors except PDGF-BB are highly effective in myoblast/myotube
induction
of neural crest stem cells.
Overall therefore, there was a considerable degree of inhibition in
2 5 neuronogenesis of HNC 10 cells grown in neuregulins, TGF a 1, or NT-3,
while NGF,
BDNF or PDGF showed much less inhibitory effect on neuronal differentiation
(Fig.
5A). FGF-2 (bFGF) showed a small degree of neuron-inductive effect, although
it
was not statistically significant. TGFb 1 induced a greatly enhanced induction
of
Schwann cells (GFAP, 45.7 b' S.8%; 5100, 59.1 'd 9.2%). Similarly in HNC10
cells
3 0 treated with FGF-2, NGF, BDNF, NT-3, PDGF or neuregulin for 5-7 days,
there were
large numbers of GFAP- or S 100-immunoreactive cells indicating these growth
factors favor differentiation of NCSCs towards glial lineage (Fig. 5B). All
the growth
CA 02409713 2002-11-04
48
factors examined, FGF-2, NGF, BDNF, NT-3, neuregulins and TGF ~ 1, except
PDGF,
induced phenotypes of skeletal muscle such as skeletal muscle myosin and
desmin in
HNC 10 cells (Fig. SC).
Experiment 6: Cryostorage of HNC 10 cells
HNC 10 cells could be effectively cyropreserved with minimal adverse effects
on cell viability and no discernible effects on proliferation or
differentiation were
noted upon thawing and culturing. Cell viability after 6 months of storage in
a
nitrogen tank was invariably ?0-80% levels.
Experimental Series II
Another series of experiments were undertaken to reveal and demonstrate the
in-vivo effects of human neural crest stem cells and their pluripotent progeny
cells
after implantation into the brain of living animal hosts. These in-vivo animal
trials
clearly and convincingly evidence the valuable and desirable medical uses and
clinical
applications for these isolated human NCSC lines.
Experiment 7: Transplantation of HNC 10 cells into the brain of living animals
2 0 These experiments were designed to examine the ability of lacZ-expressing
(blue colored) HNC10 cells to survive, migrate and differentiate into neurons
or glial
cells in the brain of newborn mouse CNS. LacZ expressing HNC10 cells were
injected into lateral ventricles of newborn mice. Mouse brains were examined
at
various intervals between l and 8 weeks after implantation by X-gal
histochemistry or
2 5 by ~-gal immunostaining to demonstrate LacZ-expressing HNC 10 cells. The
HNC 10
cells integrated well within the subventricular zone of the ventricles and an
extensive
migration of X-gal-positive HNC10 cells from the site of implantation to
neighboring
anatomical sites was found. These outcomes and results are shown by Figs. 6A-
6F
respectively.
3 0 As shown herein, Fig. 6A presents a schematic map of mouse brain as shown
in Figs. 6B and 6C. Fig. 6B shows the brain two weeks post-operation. X-gal
reaction positive blue cells migrated from the lumen of the ventricule into
the
CA 02409713 2002-11-04
49
neighboring paraenchymal region of the brain (the bar indicates 0.3 mm). Fig.
6C
presents a higher magnification of Fig. 6B (the bar indicates 50 um). Fig. 6D
provides a schematic map of mouse brain as shown in Figs. 6E and 6F. Then,
Fig. 6E
shows a large number of X-gal-positive blue cells which are seen to migrate
from the
ventricular lumen into hippocampal formation (the bar indicates 0.2 um). Fig.
6F
provides a higher magnification of Fig. 6E (the bar indicates 50 um).
The results evidenced by Figs. 6A-6F respectively show that LacZ-expressing
HNC 10 cells migrated throughout the mouse brain; and the pattern of spread
indicates
that they migrated a considerable distance into the brain parenchyma along the
ventricular walls.
In several cases, also, a widespread presence of X-gal-positive cells along
the
neuronal layers of hippocampal fomtion was noted. This is shown by Figs. 6G
and
6H. Fig. 6G shows the result two weeks post-operation. Beta-gal-positive HNC
10
cells were found in brain parenchyma are indicated. The bar indicates 20 um.
Fig.
6H presents the same field as Fig. 6C. Beta-gal-positive HNC 10 cells are
doubly
immunoreactive to galactocerebroside antibody, indicating that these cells
differentiated into myelin-forming cells of Schwann cell lineage in response
to a
signal generated in the local environment (the bar indicates 20 um).
The empirical evidence and data thus show that these X-gal/-gal positive
2 0 cells expressed galactocerebroside (O 1 antibody) immunoreaction, a
specific cell type
marker for myelination, 2 weeks post-transplantation in the brain parenchyma.
This
indicates that HNC 10 cells can and had differentiated into myelin forming
glial cells,
probably Schwann cells (Figs. 6G and 6H). These results also indicate that
when
HNC 10 cells are implanted into the brains of the developing nervous systems,
they
2 5 generate progeny cells which become myelin-forming glial cells by
responding to
developmental signals originated in that area at the time the cells are
grafted.
Experiment 8: Evaluation of the myelinating capacity of HNC 10 cells in-vivo
The next objective was to evaluate the myelinating capacity of HNC 10 cells
3 0 and to determine whether transplanted HNC 10 cells could persist in the
myelin-
deficient mutant environment. HNC 10 cells were therefore grafted into the
unilateral
neostriatum of 4 week-old myelin mutant shaker rats. After 1-4 weeks post-
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transplantation, these shaker rats were sacrificed and processed for
immunohistochemical investigation of CNPase (oligodendrocytes/Schwann
cells/CNS
and PNS myelin), GFAP (astrocytes/Schwann cells) and MAP2 (neurons). The
results are illustrated by Figs. 7A-7D respectively.
Specifically, Fig. 7 provides a schematic map of myelin mutant shaker rat
brain was made where HNC 10 human neural crest stem cells were grafted
earlier.
Frontal sections through neostriatal region of shaker rat brain grafted with
HNC 10
cells. Four weeks post-implantation, CNPase (a specific marker for myelin)
immunostaining was performed. Fig. 7A shows the control side of the brain
where
the presence of CNPase-positive myelin is minimal (the bar indicates 0.3 mm).
Fig.
7B shows a large number of CNP-immunoreaction positive patches representing
myelinated axons are visible throughout the field. A brown colored strip
indicates the
track of HNC 10 cell implantation (the bar indicates 0.3 mm). Fig. 7C provides
a
higher magnification of Fig. 7A (the bar indicates 0.1 mm). Fig. 7D presents a
higher
magnification of Fig. 7B. Along the path of injection track, there are good
numbers of
myelin patches which represent remyelinated axons produced by Schwann cells
differentiated from HNC 10 human neural crest stem cells (the bar indicates
0.1 mm).
Thus, as shown by Figs. 7A-7D, a widespread remyelination of previously
unmyelinated axons is demonstrated as shown by an intense staining of
myelinated
2 0 axons by CNPase antibody, while CNPase staining was minimal in the control
areas.
Astrogliosis was not induced in the implanted sites as no GFAP-positive focus
was
found.
Conclusions Drawn From Experiments:
1. For the first time, immortalized cell lines of human NCSCs were generated.
These were created via retrovirus-mediated vmyc transfer into cells initially
obtained
from human embryonic DRG. HNC 10 cells, immortalized NCSCs, are self renewing
and multipotent in-vitro; and, in the proper setting, rise to four different
lineages - i.e.,
3 0 neurons, Schwann cells, adrenal chromaffin cells and skeletal muscle
cells. HNC 10
cells express phenotypes characteristic for neural crest stem cells, i.e.,
nestin and
p75NGFR, thereby indicating that HNC 10 cells without any reservation are of
neural
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S1
crest stem cell origin.
2. Isolated as stable cell lines, human NCSCs can be distinguished from CNS
stem cells by their morphology; by expression of low-affinity NGF receptor; by
the
multipotent progeny that they generate; and by their inability to generate CNS
derivatives. NCSCs can differentiate into sensory, sympathetic, and enteric
neurons;
or Schwann cells; or adrenal chromaffin cells; as well as into non-neural
derivatives
such as melanocytes, cartilage, bone, and smooth/skeletal muscle. HNC 10 cells
grown in serum-free medium expressed immunoreaction-positive for antibodies
specific for A2B5, PSA-NCAM, NG2, Musashi and FORSE-1, cell type-specific
markers known for neuronal and glial progenitors. When HNC 10 cells were grown
in
serum-containing medium, a large number of these cells expressed
immunoreaction
positive for neurofilaments NF-L, NF-M, NF-H and MAP-2, tubulin bIII and
neuron
specific enolase, cell type-specific markers for neurons; GFAP and S-100,
markers for
Schwann cells; chromagranin and PNMT, markers for adrenal chromaffin cells;
and
tyrosine hydrolase (TH), a marker for synthetic neurons. In addition, HNC 10
cells
could differentiate into skeletal muscle cells as shown by myosin staining.
3. Since the myelin basic protein (MBP) gene defect in mutant shaker rats
results
2 0 in a severe dysmyelinating in white matter, shaker rats serve as an
excellent animal
model for human demylinating diseases (such as multiple sclerosis). HNC 10
cells
were implanted into neostriatal region of 4 week old shaker rats, and the
brains were
examined 1-4 weeks post-operation by immunocytochemical staining for MBP and
CNPase, specific markers for myelin-forming glial cells and myelin. Implanted
2 5 HNC 10 cells survived well and differentiated into myelinating glial cells
(Schwann
cells) to remyelinate previously unmyelinated axons; and also migrated to
other
anatomical sites remote from the original grafted site. These results indicate
that
HNC 10 cells, human immortalized NCSCs, are capable of survival, migration and
remyelination of host axons in myelin deficient mutant brain tissue; and are
the cells
3 0 of choice for cell replacement and repair of brain with
demyelination/dysmyelination
pathological lesions.
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4. Mature NCSC-derived cells express phenotypic markers that can be used to
distinguish them from related cells in the CNS. For example, Schwann cells can
be
distinguished from astrocytes and oligodendrocytes by the co-expression of
GFAP and
myelination antigens such as galactocerebroside and 04. Peripherin expression
is
characteristic of peripheral neurons such as DRG neurons, derivatives of
NCSCs, but
is generally seen in only a limited population of CNS derivatives. Although
NCSCs
are multipotent stem cells of the PNS, they seem incapable of generating CNS
derivatives and transplantation of neural crest stem cells into the CNS
results in
primarily Schwann cell differentiation. Thus NSCs of the CNS and NCSCs
represent
two quite independent stem cells of two very different systems.
5. The choice of each of several alternative fates available to NCSCs can be
instructively promoted by different environmental signals including those
growth
factors previously reported in the scientific literature. In the present
study, it was
found that FGF-2 promotes differentiation of HNC10 cells into three cell
types, i.e.,
neurons, Schwann cells and skeletal muscle cells. Neuregulin and TGFb-1
induced
HNC10 cells to differentiate into Schwann cells and skeletal muscle cells and
these
growth factors blocked neuronal differentiation of HNC 10 cells. Members of
neurotrophin family, NGF, BDNF and NT3 also favored differentiation of HNC 10
2 0 cells into glial and skeletal muscle cells. These findings showed that
trophic effect
was not specific for a particular neurotrophin; and that the differentiation
mechanism
involves activation of the non-selective neurotrophin receptor, p75NGFR, by
one of
these neurotrophins. These results indicate that neurotrophins and other
pertinent
growth factors affect differentiation of HNC 10 cells at multiple levels for
each and
2 5 different cell lineage. Moreover, these results suggest that concerted
action of
combinations of growth factors is at least as important in determining the
fate of
NCSCs as the action of a single growth factor alone.
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6. HNC 10 cells possess a capability for myelin formation. As the empirical
results show, when HNC 10 cells were implanted into the neostriatum of 4-week
old
myelin mutant shaker rat brains, an extensive migration of HNC10 cells from
the site
of implantation to neighboring anatomical sites was found and a widespread
remyelination of previously unmyelinated axons was demonstrated at 1-5 weeks
post-
implantation. These results clearly demonstrate that when HNC 10 cells are
implanted
into pathological myelin-deficient areas of the CNS, their differential
potential is
strongly influenced by the environment signals at the site of implantation as
they form
myelin.
7. The present study shows that HNC10 cells, NCSCs, are useful for a variety
of
diseases characterized by profuse neural degeneration and cell death that
might benefit
from the replacement of lost neurons. Accordingly, when neural crest stem
cells are
implanted into different areas of the developing nervous system, they will
provide
progeny that would normally be generated in that area at the time the cells
are grafted.
Thus the same precursor cells can generate Purkinje cells when implanted into
the
cerebellum and hippocampal neurons when introduced into the hippocampus at the
time that these cells are being generated from endogenous precursors. The
present
studies show that LacZ expressing HNC 10 cells which were grafted into the
cerebral
2 0 ventricle of newborn mice migrated a great distance and became a part of
hippocampal pyramidal cell neurons 2-3 weeks post-operationally. Thus, when
multipotent neural crest stem cells are implanted into pathological areas of
the CNS,
their differentiation potential is influenced by the environment into which
they are
placed; and these become CNS neurons which are integrated into the
cytoarchitecture
2 5 of the host CNS.
8. The NCSC and their multipotent progeny are suitable for cell therapy
purposes. Cell therapy has become a most promising strategy for the treatment
of
many human diseases including neurological disorders; and the objective of
cell
3 0 therapy is to replace lost cells and restore the function of damaged cells
and tissues.
Transplantation of renewable, homogenous and well-characterized cells into the
damaged target tissue or organ should replace lost cells and restore damaged
function.
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HNC 10 cells, a human NCSC, can fulfill these criteria and serve as an ideal
cell type
as donor cells for cell therapy in various neurological diseases including
motor and
sensory neuropathy, multiple sclerosis, Parkinson disease, Huntington disease,
spinal
cord injury, stroke, Duchenne muscular dystrophy and pain control. In
addition,
human NCSCs can serve excellently as a vehicle to carry human genes by which
to
cure genetically incurred neurological diseases in an ever-expanding field of
gene
therapy. The stable immortalized human NCSC line described and characterized
here
can be expanded readily to provide a renewable and homogeneous population of
glial,
neuronal and muscle cells; and they will be most valuable for future research
studies
of fundamental questions in developmental neurobiology, cell and gene
therapies, as
well as the research and development of new drugs.
The present invention is not restricted in form nor limited in scope, except
by
the claims appended hereto.
