U.S. patent application number 14/371701 was filed with the patent office on 2015-01-08 for mammalian neural plate border stem cells capable of forming neural tube and neural crest cell lineages including central and peripheral neurons.
The applicant listed for this patent is Max- Planck-Gesellschaft zur Forderung der Wissenschaften E.V.. Invention is credited to Michael Glatza, Peter Reinhardt, Hans R. Schoeler, Jared L. Sterneckert.
Application Number | 20150010515 14/371701 |
Document ID | / |
Family ID | 47594687 |
Filed Date | 2015-01-08 |
United States Patent
Application |
20150010515 |
Kind Code |
A1 |
Schoeler; Hans R. ; et
al. |
January 8, 2015 |
MAMMALIAN NEURAL PLATE BORDER STEM CELLS CAPABLE OF FORMING NEURAL
TUBE AND NEURAL CREST CELL LINEAGES INCLUDING CENTRAL AND
PERIPHERAL NEURONS
Abstract
The present invention relates to a method for producing
mammalian neural plate border stem cells (NPBSCs), comprising: (a)
differentiation of mammalian pluripotent stem cells by (a-i)
culturing mammalian pluripotent stem cells in pluripotent stem cell
medium for about 24 to about 96 hours, wherein the pluripotent stem
cell medium comprises: (i) an inhibitor of the activin/TGF-.beta.
signalling pathway; (ii) an inhibitor of the BMP signalling
pathway; (iii) an activator of the canonical WNT signalling
pathway; and (iv) an activator of the Hedgehog signalling pathway;
subsequently (a-ii) culturing the cells obtained in step (a-i) for
about 24 to about 96 hours in a neural medium, wherein the neural
medium comprises: (i) an inhibitor of the Activin/TGF-.beta.
signalling pathway; (ii) an inhibitor of the BMP signalling
pathway; (iii) an activator of the canonical WNT signalling
pathway; and (iv) an activator of the Hedgehog signalling pathway;
subsequently (a-iii) culturing the cells obtained in step (a-ii)
for about 24 to about 96 hours in a neural medium, wherein the
neural medium comprises: (i) an activator of the canonical WNT
signalling pathway; (ii) an activator of the Hedgehog signalling
pathway; and (iii) an inhibitor of oxidation; and (b) plating the
obtained differentiated mammalian pluripotent stem cells in NPBSCs
expansion medium, wherein the NPBSCs expansion medium comprises (i)
an activator of the canonical WNT signalling pathway; (ii) an
activator of the Hedgehog signalling pathway; and (iii) an
inhibitor of oxidation; and expanding the cells in the NPBSCs
expansion medium for about 24 to about 96 hours; (c) splitting the
cells obtained in (b) and further expanding the cells in the NPBSCs
expansion medium; and (d) repeating step (c) at least two times.
The present invention further relates to neural plate border stem
cells obtainable by the method of the invention and the use of the
cells of the invention in medicine.
Inventors: |
Schoeler; Hans R.;
(Muenster, DE) ; Sterneckert; Jared L.; (Muenster,
DE) ; Glatza; Michael; (Hamm, DE) ; Reinhardt;
Peter; (Muenster, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Max- Planck-Gesellschaft zur Forderung der Wissenschaften
E.V. |
Muenchen |
|
DE |
|
|
Family ID: |
47594687 |
Appl. No.: |
14/371701 |
Filed: |
January 11, 2013 |
PCT Filed: |
January 11, 2013 |
PCT NO: |
PCT/EP2013/050482 |
371 Date: |
July 10, 2014 |
Current U.S.
Class: |
424/93.7 ;
435/377 |
Current CPC
Class: |
C12N 2501/148 20130101;
C12N 5/0622 20130101; C12N 2501/13 20130101; C12N 5/0623 20130101;
C12N 2501/385 20130101; C12N 5/0619 20130101; C12N 2501/16
20130101; C12N 2506/45 20130101; C12N 5/0647 20130101; A61K 35/30
20130101; C12N 2501/119 20130101; C12N 2501/155 20130101; C12N
2500/38 20130101; C12N 2501/15 20130101; C12N 2501/41 20130101;
C12N 2501/999 20130101; C12N 2533/90 20130101; C12N 2501/415
20130101; C12N 2501/01 20130101; C12N 2506/02 20130101; C12N 5/062
20130101; C12N 2501/70 20130101 |
Class at
Publication: |
424/93.7 ;
435/377 |
International
Class: |
C12N 5/0797 20060101
C12N005/0797; C12N 5/079 20060101 C12N005/079; C12N 5/0793 20060101
C12N005/0793 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 11, 2012 |
EP |
12000143.3 |
Claims
1. A method for producing mammalian neural plate border stem cells
(NPBSCs), comprising: (a) differentiation of mammalian pluripotent
stem cells by (a-i) culturing mammalian pluripotent stem cells in
pluripotent stem cell medium for about 24 to about 96 hours,
wherein the pluripotent stem cell medium comprises: (i) an
inhibitor of the activin/TGF-.beta. signalling pathway; (ii) an
inhibitor of the BMP signalling pathway; (iii) an activator of the
canonical WNT signalling pathway; and (iv) an activator of the
Hedgehog signalling pathway; subsequently (a-ii) culturing the
cells obtained in step (a-i) for about 24 to about 96 hours in a
neural medium, wherein the neural medium comprises: (i) an
inhibitor of the activin/TGF-.beta. signalling pathway; (ii) an
inhibitor of the BMP signalling pathway; (iii) an activator of the
canonical WNT signalling pathway; and (iv) an activator of the
Hedgehog signalling pathway; subsequently (a-iii) culturing the
cells obtained in step (a-ii) for about 24 to about 96 hours in a
neural medium, wherein the neural medium comprises: (i) an
activator of the canonical WNT signalling pathway; (ii) an
activator of the Hedgehog signalling pathway; and (iii) an
inhibitor of oxidation; (b) plating the obtained differentiated
mammalian pluripotent stem cells in NPBSCs expansion medium,
wherein the NPBSCs expansion medium comprises (i) an activator of
the canonical WNT signalling pathway; (ii) an activator of the
Hedgehog signalling pathway; and (iii) an inhibitor of oxidation
and expanding the cells in the NPBSCs expansion medium for about 24
to about 96 hours; (c) splitting the cells obtained in (b) and
further expanding the cells in the NPBSCs expansion medium; and (d)
repeating step (c) at least two times.
2. The method of claim 1, wherein the differentiated mammalian
pluripotent stem cells are plated in step (b) at a density of about
1000 to 100,000 per cm.sup.2.
3. The method of claim 1, wherein the NPBSCs are characterised by
the expression of at least three markers selected from the group
consisting of FORSE1, MSX1, PHOX2B, PAX3, PAX6, SOX1, SOX2, NESTIN,
IRX3, HOXA2, HOXB2, HES5, DACH1, PLZF, LMO3, EVI1 and ASCL1.
4. The method of claim 1, wherein the NPBSCs are characterised by a
lack of expression of at least one of the markers OCT4, NANOG, AFP,
T, SOX17, EOMES, GSH2, OLIG2, CK8, CK18, NKX2.2, NKX6.1, HOXB8,
HOXA5, FOXA2 and VCAM-1.
5. The method of claim 1, wherein the NPBSCs are characterised by
(i) the expression of SOX1, MSX1 and PHOX2B and the lack of
expression of NKX6.1 and VCAM-1; or (ii) the expression of SOX2,
IRX3, and MSX1 and the lack of expression of HOXB8, HOXA5 and
VCAM-1.
6. The method of claim 1, further comprising differentiating the
NPBSCs obtained in step (d) into: (i) peripheral nervous system
neurons; (ii) central nervous system neurons; (iii) midbrain
dopaminergic neurons; (iv) motor neurons; (v) neural crest-derived
mesenchymal cells; (vi) astrocytes; or (vii) neural rosettes.
7. The method of claim 6, wherein the differentiation of NPBSCs
into peripheral nervous system neurons comprises: (i) culturing the
NPBSCs obtained in step (d) in a neural medium comprising an
activator of the canonical WNT signalling pathway for about 48 to
72 hours; (ii) adding an activator of the BMP pathway to the
culture of step (i) for about 192 hours; and (iii) culturing the
cells obtained in step (ii) for about 336 hours in a neural medium
containing at least two different neurotrophins and an inhibitor of
oxidation; thereby differentiating the NPBSCs into peripheral
nervous system neurons.
8. The method of claim 6, wherein the differentiation of NPBSCs
into central nervous system neurons comprises: (i) culturing the
NPBSCs obtained in step (d) in a neural medium; thereby
differentiating the NPBSCs into central nervous system neurons.
9. The method of claim 6, wherein the differentiation of NPBSCs
into midbrain dopaminergic neurons comprises: (i) culturing the
NPBSCs obtained in step (d) in a neural medium comprising (a) an
activator of the FGF signaling pathway, (b) an activator of the
hedgehog signaling pathway and (c) an inhibitor of oxidation, for
about 168 to about 192 hours; (ii) changing the medium to a neural
medium comprising (a) at least two different neurotrophins, (b) an
inhibitor of oxidation; and culturing the cells for about 24 to
about 96 hours; and (iii) further culturing the cells in a neural
medium comprising (a) at least two different neurotrophins; and (b)
an inhibitor of oxidation; thereby differentiating the NPBSCs into
midbrain dopaminergic neurons.
10. The method of claim 6, wherein the differentiation of NPBSCs
into motor neurons comprises: (i) culturing the NPBSCs obtained in
step (d) in a neural medium comprising an activator of the hedgehog
signaling pathway for about 24 to about 48 hours; (ii) adding
retinoic acid to the culture of step (i) for about 168 to about 192
hours; and (iii) further culturing the cells in a neural medium
comprising at least two different neurotrophins, thereby
differentiating the NPBSCs into motor neurons.
11. The method of claim 6, wherein the differentiation of NPBSCs
into neural crest-derived mesenchymal cells comprises: (i)
culturing the NPBSCs obtained in step (d) in a neural medium
comprising an activator of the canonical WNT signalling pathway for
about 48 to 72 hours; and (ii) culturing the cells obtained in step
(i) in a cell culture medium comprising serum, thereby
differentiating the NPBSCs into neural crest-derived mesenchymal
cells.
12. The method of claim 6, wherein the differentiation of NPBSCs
into astrocytes comprises: (i) culturing the NPBSCs obtained in
step (d) in a cell culture medium comprising an activator of FGF
signalling for about 12 to 96 hours; and (ii) culturing the cells
obtained in step (i) in a cell culture medium comprising fetal calf
serum, fetal bovine serum and/or CNTF for about 14 to 60 days;
thereby differentiating the NPBSCs into astrocytes.
13. The method of claim 6, wherein the differentiation of NPBSCs
into neural rosette cells comprises: (i) culturing the NPBSCs
obtained in step (d) in a neural medium comprising an activator of
FGF signalling for about 12 to 96 hours; thereby differentiating
the NPBSCs into neural rosette cells.
14. The method of claim 1, wherein the cells obtained are free or
substantially free of pathogens.
15. Neural plate border stem cells obtained by the method of claim
1.
16. (canceled)
17. (canceled)
18. A method for treatment of a disease selected from the group
consisting of Parkinson's disease, amyotrophic lateral sclerosis,
spinal muscular atrophy, peripheral neuropathy, Hirschsprung's
disease, DiGeorge syndrome, familial dysautonomia, congenital
insensitivity to pain with anhididrosis and Charcot-Marie-Tooth
disease, comprising administering to a subject presenting with said
disease neural plate border stem cells obtained by the method of
claim 1.
Description
[0001] The present invention relates to a method for producing
mammalian neural plate border stem cells (NPBSCs), comprising: (a)
differentiation of mammalian pluripotent stem cells by (a-i)
culturing mammalian pluripotent stem cells in pluripotent stem cell
medium for about 24 to about 96 hours, wherein the pluripotent stem
cell medium comprises: (i) an inhibitor of the activin/TGF-.beta.
signalling pathway; (ii) an inhibitor of the BMP signalling
pathway; (iii) an activator of the canonical WNT signalling
pathway; and (iv) an activator of the Hedgehog signalling pathway;
subsequently (a-ii) culturing the cells obtained in step (a-i) for
about 24 to about 96 hours in a neural medium, wherein the neural
medium comprises: (i) an inhibitor of the Activin/TGF-.beta.
signalling pathway; (ii) an inhibitor of the BMP signalling
pathway; (iii) an activator of the canonical WNT signalling
pathway; and (iv) an activator of the Hedgehog signalling pathway;
subsequently (a-iii) culturing the cells obtained in step (a-ii)
for about 24 to about 96 hours in a neural medium, wherein the
neural medium comprises: (i) an activator of the canonical WNT
signalling pathway; (ii) an activator of the Hedgehog signalling
pathway; and (iii) an inhibitor of oxidation; and (b) plating the
obtained differentiated mammalian pluripotent stem cells in NPBSCs
expansion medium, wherein the NPBSCs expansion medium comprises (i)
an activator of the canonical WNT signalling pathway; (ii) an
activator of the Hedgehog signalling pathway; and (iii) an
inhibitor of oxidation; and expanding the cells in the NPBSCs
expansion medium for about 24 to about 96 hours; (c) splitting the
cells obtained in (b) and further expanding the cells in the NPBSCs
expansion medium; and (d) repeating step (c) at least two times.
The present invention further relates to neural plate border stem
cells obtainable by the method of the invention and the use of the
cells of the invention in medicine.
[0002] In this specification, a number of documents including
patent applications and manufacturer's manuals are cited. The
disclosure of these documents, while not considered relevant for
the patentability of this invention, is herewith incorporated by
reference in its entirety. More specifically, all referenced
documents are incorporated by reference to the same extent as if
each individual document was specifically and individually
indicated to be incorporated by reference.
[0003] The neural tube and neural crest cell lineages are specified
during gastrulation from the neural plate through a combination of
WNT and bone morphogenetic protein (BMP) signals. WNT signals
specify caudal identity, and BMPs direct differentiation into the
neural plate border fate, which will go on to form neural crest
cell derivatives including peripheral nervous system (PNS) and
mesenchymal cells. Sonic hedgehog (SHH) signaling antagonizes the
specification of neural crest cells by BMPs and WNT by inducing
ventral neural tube progenitors.
[0004] Sensory information is conveyed through neurons of the
peripheral nervous system (PNS) to the central nervous system
(CNS), which processes it and responds with output signals through
motor neurons. These two systems, PNS and CNS, are specified during
gastrulation from the neural plate. The neural plate is a
thickening of medial ectoderm and marks the onset of neurulation.
The border region of the neural plate will form neural crest cells,
but the medial neural plate will invaginate and form the neural
tube, which will differentiate into the central nervous system
(CNS).
[0005] The caudal neural plate border is specified during
gastrulation by a combination of bone morphogenetic protein (BMP)
and WNT signals (Patthey et al., 2009; Patthey et al., 2008).
During gastrulation, WNT signals form a morphogenic gradient with
WNT proteins expressed in the caudal portion of an embryo and
inhibitors of WNT signals being produced rostrally (Kiecker and
Niehrs, 2001; Nordstrom et al., 2002). This graded WNT signal
induces caudal character in the neural plate in a dose dependent
manner (Kiecker and Niehrs, 2001; Nordstrom et al., 2002). In
addition, exogenous WNT signals induce anterior neural plate cells
to differentiate into caudal neural plate border cells, which will
form the neural crest (Patthey et al., 2008). In contrast,
inhibition of early WNT signals prevents neural plate border
specification and results in CNS specification (Patthey et al.,
2009).
[0006] BMP signaling also plays an important role in neural plate
border specification. Neural border identity is induced when caudal
plate cells are exposed to BMP protein (Patthey et al., 2008). In
addition, although WNT is sufficient to induce caudal neural border
character in anterior cells, inhibition of BMP inhibits the
specification by WNT and results in CNS identity (Patthey et al.,
2009). Patthey et alia demonstrated that a temporal expression
pattern of WNT and BMP is responsible for induction of neural
border identity (Patthey et al., 2009). Early WNT signaling
specifies caudal identity, whereas BMP signaling instructs neural
plate border differentiation (Patthey et al., 2009).
[0007] Sonic hedgehog (SHH) forms an morphogenic gradient that
opposes both the BMP and WNT gradients during the developing neural
tube (Ulloa and Briscoe, 2007). Both BMPs and WNTs induce dorsal
neural progenitor identity, which expresses many of the same
markers as the neural border region including MSX1 and PAX3 (Lee
and Jessell, 1999). SHH protein, expressed by the notochord and
floor plate, antagonizes BMP and WNT signals and specifies ventral
neural identity (Jessell, 2000). As such, SHH represses the dorsal
markers MSX1 and PAX3 and induces ventral markers such as OLIG2,
NKX2.2, and FOXA2 (Jessell, 2000; Ulloa and Briscoe, 2007). It has
been shown that SHH likewise represses neural crest cell
differentiation, which are formed from the neural plate border
(Selleck et al., 1998).
[0008] Alvarez-Medina et al. 2009 describe the co-ordinated
regulation of proliferation mediated by Wnt and Shh activities. The
authors found that these two pathways appear to interact to control
progression of the G1 phase of the cell cycle. However, the work by
Alvarez-Medina and co-workers relates to progenitors that are
already committed to the central nervous system (e.g. the neural
tube) and, thus, represents a strictly proliferative effect on CNS
progenitors. Developmentally earlier precursor cell types with the
capacity to differentiate into both neural tube and neural crest
cell lineages are not discussed in this publication.
[0009] Despite the fact that a lot of effort has been invested into
methods to direct the differentiation of pluripotent stem cells
into precursor cells of neurons from either the CNS or the PNS, no
method exists so far to induce pluripotent stem cells into even
earlier precursor cells, i.e. precursor cells that are capable of
self-renewal in culture with the ability to differentiate into both
CNS and PNS neurons. Accordingly, there is still a need to provide
such early precursor cells that provide a useful source of CNS and
PNS neurons for example for disease modelling and drug
discovery.
[0010] This need is addressed by the provision of the embodiments
characterized in the claims.
[0011] Accordingly, the present invention relates to a method for
producing mammalian neural plate border stem cells (NPBSCs),
comprising: [0012] (a) differentiation of mammalian pluripotent
stem cells by [0013] (a-i) culturing mammalian pluripotent stem
cells in pluripotent stem cell medium for about 24 to about 96
hours, wherein the pluripotent stem cell medium comprises: [0014]
(i) an inhibitor of the activin/TGF-.beta. signalling pathway;
[0015] (ii) an inhibitor of the BMP signalling pathway; [0016]
(iii) an activator of the canonical WNT signalling pathway; and
[0017] (iv) an activator of the Hedgehog signalling pathway;
subsequently [0018] (a-ii) culturing the cells obtained in step
(a-i) for about 24 to about 96 hours in a neural medium, wherein
the neural medium comprises: [0019] (i) an inhibitor of the
activin/TGF-.beta. signalling pathway; [0020] (ii) an inhibitor of
the BMP signalling pathway; [0021] (iii) an activator of the
canonical WNT signalling pathway; and [0022] (iv) an activator of
the Hedgehog signalling pathway; subsequently [0023] (a-iii)
culturing the cells obtained in step (a-ii) for about 24 to about
96 hours in a neural medium, wherein the neural medium comprises:
[0024] (i) an activator of the canonical WNT signalling pathway;
[0025] (ii) an activator of the Hedgehog signalling pathway; and
[0026] (iii) an inhibitor of oxidation; and [0027] (b) plating the
obtained differentiated mammalian pluripotent stem cells in NPBSCs
expansion medium, wherein the NPBSCs expansion medium comprises
[0028] (i) an activator of the canonical WNT signalling pathway;
[0029] (ii) an activator of the Hedgehog signalling pathway; and
[0030] (iii) an inhibitor of oxidation; [0031] and expanding the
cells in the NPBSCs expansion medium for about 24 to about 96
hours; [0032] (c) splitting the cells obtained in (b) and further
expanding the cells in the NPBSCs expansion medium; and [0033] (d)
repeating step (c) at least two times.
[0034] In accordance with the present invention, the term "neural
plate border stem cells", which are also abbreviated herein as
NPBSCs, relates to a novel precursor cell type capable of
differentiating into either neural tube or neural crest cell
lineages, which form CNS and PNS neurons, respectively. The NPBSCs
of the present invention are characterized by the expression of at
least three markers selected from the group consisting of MSX1,
PHOX2B, PAX3, PAX6, SOX1, SOX2, NESTIN, IRX3, HOXA2, HOXB2, HES5,
DACH1, PLZF, LMO3, EVI1 and ASCL1, as defined herein below.
[0035] The term "mammalian" is taxonomically well known in the
art.
[0036] Preferably, the mammalian cells are derived from a mammal
selected from the group consisting of e.g. human, mouse, rat,
hamster, cow, cat, pig, dog, horse, rabbit or monkey. More
preferably, the mammalian NPBSCs are derived from human or mouse,
most preferably the mammalian neural plate border stem cells are
human neural plate border stem cells.
[0037] The term "pluripotent stem cells", in accordance with the
present invention, relates to a cell type having the capacity for
self-renewal, an ability to go through numerous cycles of cell
division while maintaining the undifferentiated state, and the
potential of differentiation, i.e. the capacity to differentiate
into specialized cell types. Pluripotent stem cells are the
descendants of totipotent cells and can differentiate into nearly
all cells, i.e. cells derived from any of the three primary germ
layers: ectoderm, endoderm, and mesoderm. The term pluripotent stem
cells also encompasses stem cells derived from the inner cell mass
of an early stage embryo known as a blastocyst. Recent advances in
embryonic stem cell research have led to the possibility of
creating new embryonic stem cell lines without destroying embryos,
for example by using a single-cell biopsy similar to that used in
preimplantation genetic diagnosis (PGD), which does not interfere
with the embryo's developmental potential (Klimanskaya et al.,
2006). Furthermore, a large number of established embryonic stem
cell lines are available in the art (according to the U.S. National
Institutes of Health, 21 lines are currently available for
distribution to researchers), thus making it possible to work with
embryonic stem cells without the necessity to destroy an
embryo.
[0038] In a preferred embodiment, the pluripotent stem cells are
not embryonic stem cells obtained via the destruction of a human
embryo.
[0039] In accordance with the present invention, the pluripotent
stem cells can be induced pluripotent stem cells. The term "induced
pluripotent stem (iPS) cells", as used herein, refers to
pluripotent stem cells derived from a non-pluripotent cell,
typically an adult somatic cell, by inducing a "forced" expression
of certain genes. Induced pluripotent stem cells are identical to
natural pluripotent stem cells, such as e.g. embryonic stem cells,
in many respects including, for example, unlimited self-renewal in
vitro, a normal karyotype, the expression of certain stem cell
genes and proteins such as for example Oct3/4, Sox2, Nanog,
alkaline phosphatase (ALP) as well as stem cell-specific antigen 3
and 4 (SSEA3/4), chromatin methylation patterns, doubling time,
embryoid body formation, teratoma formation, viable chimera
formation, and potency and differentiability (Takahashi and
Yamanaka 2006, Cell 126: 663-676; Hanna, J., et al. (2007). Science
318(5858): 1920-3; Meissner, A., et al. (2007). Nat Biotechnol
25(10): 1177-81; Nakagawa, M., et al. (2008). Nat Biotechnol
26(1):101-106; Okita, K., et al. (2007). Nature 448(7151): 313-7;
Takahashi, K., et al. (2007 Cell 131(5): 861-72; Wernig, M., et al.
(2007). Nature 448(7151): 318-24; Yu, J., et al. (2007). Science
318(5858): 1917-20; Park, I. H., et al. (2008). Nature 451(7175):
141-6). Induced pluripotent stem cells are an important advancement
in stem cell research, as they allow researchers to obtain
pluripotent stem cells without the use of embryos (Nishikawa et
al., 2008). The induced pluripotent stem cells may be obtained from
any adult somatic cell, preferably from fibroblasts, such as for
example from skin tissue biopsies. The pluripotency of iPS cells
can tested, e.g., by in vitro differentiation into neural, glia and
cardiac cells and the production of germline chimaeric animals
through blastocyst injection. Human iPS cells lines can be analysed
through in vitro differentiation into neural, glia and cardiac
cells and their in vivo differentiation capacity can be tested by
injection into immuno-deficient SCID mice and the characterisation
of resulting tumours as teratomas.
[0040] Methods for the generation of human induced pluripotent stem
cells are well known to the skilled person. For example, induced
pluripotent stem cells can be generated from human skin tissue
biopsies (Park and Daley, 2009; Park et al., 2008). Fibroblasts can
be grown in MEM-medium containing chemically defined and
recombinant serum components. For reprogramming, the human
fibroblasts are preferably retrovirally transduced with OCT4, SOX2,
c-MYC and NANOG genes. For this, genes are usually cloned into a
retroviral vector and transgene-expressing viral particles are
produced in the HEK293FT cell line. Human skin fibroblasts can be
co-transduced with all four vectors. The obtained iPS cells are
preferably cultured according to protocols established for human
embryonic stem cells in DMEM-medium containing serum replacement
factors and recombinant growth factors. The iPS cells can then be
analyzed for normal morphology and normal karyotype and can be
studied by fingerprinting analysis and immunostaining for OCT3/4,
NANOG, SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81. Gene transcripts for
OCT4, SOX2, NANOG, KLF4, c-MYC, REX1, GDF3 and hTERT are analyzed
by real-time RT-PCR. Furthermore, multi-lineage differentiation of
iPS cells can be confirmed by embryoid body, teratoma formation and
differentiation into adult cell types (Choi et al., 2009; Zhang et
al., 2009). As another example, human iPS cells can also be
obtained from embryonic fibroblasts without viral integration using
adenoviral vectors expressing c-Myc, Klf4, Oct4, and Sox2 (Zhou and
Freed, 2009). Further methods are described e.g. in WO2009115295,
WO2009144008 or EP2218778.
[0041] In accordance with the present invention, any medium
suitable as a culture medium for pluripotent stem cells may be
employed as the "pluripotent stem cell medium". Such media are well
established in the art. For example, the pluripotent stem cell
medium can be a basal cell culture medium, for example knock-out
DMEM comprising: (i) knockout serum replacement; (ii)
.beta.-mercaptoethanol; (iii) non-essential amino acids; and (iv)
penicillin/streptomycin/glutamine.
[0042] DMEM is well known in the art and refers to Dulbecco's
Modified Eagle Medium. Knock-out DMEM is a basal medium optimized
for growth of undifferentiated embryonic and induced pluripotent
stem cells and can be commercially obtained, for example from
Invitrogen. Also knockout serum replacement, .beta.-mercaptoethanol
and non-essential amino-acids (NEAA) can be commercially obtained,
for example from Invitrogen and penicillin, streptomycin and
glutamine may for example be commercially obtained from PAA.
[0043] The skilled person is aware of suitable amounts of these
compounds to be employed in a cell culture medium, such as the
pluripotent stem cell medium of the present invention. Preferably,
the knockout serum replacement is added in an amount of at least
5%, such as e.g. at least 10%, at least 15% and most preferably at
least 20%. Preferably, the .beta.-mercaptoethanol is added in an
amount of at least 0.01 mM, such as e.g. at least 0.05 mM, at least
0.07 mM, at least 0.1 mM and most preferably at least 0.11 mM.
Mixtures of non-essential amino acids preferably comprise glycine,
L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid,
L-proline and L-serine at 10 mM and preferably, at least 0.5%, such
as e.g. at least 1% of this mixtures is added to the pluripotent
stem cell medium. Penicillin, streptomycin and glutamine mixtures
typically consist of 200 mM L-glutamine, 10,000 Units/ml Penicillin
and 10 mg/ml streptomycin and are preferably added to the
pluripotent stem cell medium in an amount of at least 0.5%, more
preferably at least 1%.
[0044] In a preferred embodiment, the pluripotent stem cell medium
is knock-out DMEM comprising 20% knockout serum replacement, 0.11
mM .beta.-mercaptoethanol, 1% non-essential amino acids and 1% of a
penicillin, streptomycin and glutamine mixture.
[0045] Preferably, the pluripotent stem cell medium is free of
FGF2. The term "FGF2" refers to the basic fibroblast growth factor,
a member of the fibroblast growth factor family, that is also
referred to as bFGF or FGF-.beta. in the art (Kurokawa et al.,
1987). The omission of FGF2 is preferred, as this growth factor may
slow down the differentiation of the pluripotent stem cells.
[0046] In accordance with the present invention, the pluripotent
stem cell medium further comprises [0047] (i) an inhibitor of the
activin/TGF-3 signalling pathway; [0048] (ii) an inhibitor of the
BMP signalling pathway; [0049] (iii) an activator of the canonical
WNT signalling pathway; and [0050] (iv) an activator of the
Hedgehog signalling pathway.
[0051] The term "inhibitor", as used herein, refers to a compound
that reduces or abolishes the biological function or activity of
the recited signalling pathway, by interfering with a specific
target protein that is part of this signalling pathway or by
interfering with the interaction between two or more target
proteins. An inhibitor may perform any one or more of the following
effects in order to reduce or abolish the biological function or
activity of the protein to be inhibited: (i) the transcription of
the gene encoding the protein to be inhibited is lowered, i.e. the
level of mRNA is lowered, (ii) the translation of the mRNA encoding
the protein to be inhibited is lowered, (iii) the protein performs
its biochemical function with lowered efficiency in the presence of
the inhibitor, and (iv) the protein performs its cellular function
with lowered efficiency in the presence of the inhibitor.
[0052] Compounds suitable to achieve the effect described in (i)
include compounds interfering with the transcriptional machinery
and/or its interaction with the promoter of said gene and/or with
expression control elements remote from the promoter such as
enhancers. Compounds suitable to achieve the effect described in
(ii) comprise antisense constructs and constructs for performing
RNA interference (e.g. siRNA, shRNA, miRNA) well known in the art
(see, e.g. Zamore (2001) Nat. Struct. Biol. 8(9), 746; Tuschl
(2001) Chembiochem. 2(4), 239). Compounds suitable to achieve the
effect described in (iii) interfere with molecular functions of the
protein to be inhibited. Compounds suitable to achieve the effect
described in (iv) include compounds which do not necessarily bind
directly to the target protein, but still interfere with their
activity, for example by binding to and/or inhibiting the function
or expression of members of a pathway which comprises the target
protein. These members may be either upstream or downstream of the
protein to be inhibited within said pathway.
[0053] Such compounds further include, without being limiting,
small molecules, antibodies, aptamers and ribozymes. Suitable
compounds further include but are not limited to, peptides such as
soluble peptides, including Ig-tailed fusion peptides and members
of random peptide libraries (see, e.g., Lam et al. (1991) Nature
354: 82-84; Houghten et al. (1991) Nature 354: 84-86) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids or phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al. (1993) Cell 72: 767-778).
[0054] A "small molecule" according to the present invention may
be, for example, an organic molecule. Organic molecules relate or
belong to the class of chemical compounds having a carbon basis,
the carbon atoms linked together by carbon-carbon bonds. The
original definition of the term organic related to the source of
chemical compounds, with organic compounds being those
carbon-containing compounds obtained from plant or animal or
microbial sources, whereas inorganic compounds were obtained from
mineral sources. Organic compounds can be natural or synthetic.
Alternatively, the "small molecule" in accordance with the present
invention may be an inorganic compound. Inorganic compounds are
derived from mineral sources and include all compounds without
carbon atoms (except carbon dioxide, carbon monoxide and
carbonates). Preferably, the small molecule has a molecular weight
of less than about 2000 amu, or less than about 1000 amu such as
less than about 500 amu, and even more preferably less than about
250 amu. The size of a small molecule can be determined by methods
well-known in the art, e.g., mass spectrometry. The small molecules
may be designed, for example, based on the crystal structure of the
target molecule, where sites presumably responsible for the
biological activity, can be identified and verified in in vivo
assays such as in vivo high-throughput screening (HTS) assays. Such
small molecules may be particularly suitable to inhibit
protein-protein-interaction by blocking specific bindings sites of
the target molecule. Suitable small molecules currently employed in
the inhibition of the recited signalling pathways include, without
being limiting:
4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-
-benzamide hydrate (SB431542) for the inhibition of the
activin/TGF-.beta. signalling pathway;
6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyri-
midine dihydrochloride (dorsomorphin) for the inhibition of the BMP
signalling pathway, and
(5R)-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxyfuran-2(5H)-one
(ascorbic acid) for the inhibition of oxidation.
[0055] The term "antisense nucleic acid molecule" is known in the
art and refers to a nucleic acid which is complementary to a target
nucleic acid, i.e. a nucleic acid encoding the target protein. An
antisense molecule in accordance with the invention is capable of
interacting with the target nucleic acid, more specifically it is
capable of hybridising with the target nucleic acid. Due to the
formation of the hybrid, transcription of the target gene(s) and/or
translation of the target mRNA is reduced or blocked. Standard
methods relating to antisense technology have been described (see,
e.g., Melani et al., Cancer Res. (1991) 51:2897-2901).
[0056] In accordance with the present invention, the term "small
interfering RNA (siRNA)", also known as short interfering RNA or
silencing RNA, refers to a class of 18 to 30, preferably 19 to 25,
most preferred 21 to 23 or even more preferably 21 nucleotide-long
double-stranded RNA molecules that play a variety of roles in
biology. Most notably, siRNA is involved in the RNA interference
(RNAi) pathway where the siRNA interferes with the expression of a
specific gene. In addition to their role in the RNAi pathway,
siRNAs also act in RNAi-related pathways, e.g. as an antiviral
mechanism or in shaping the chromatin structure of a genome.
[0057] siRNAs naturally found in nature have a well defined
structure: a short double-strand of RNA (dsRNA) with 2-nt 3'
overhangs on either end. Each strand has a 5' phosphate group and a
3' hydroxyl (--OH) group. This structure is the result of
processing by dicer, an enzyme that converts either long dsRNAs or
small hairpin RNAs into siRNAs. siRNAs can also be exogenously
(artificially) introduced into cells to bring about the specific
knockdown of a gene of interest. Essentially any gene of which the
sequence is known can thus be targeted based on sequence
complementarity with an appropriately tailored siRNA. The
double-stranded RNA molecule or a metabolic processing product
thereof is capable of mediating target-specific nucleic acid
modifications, particularly RNA interference and/or DNA
methylation. Exogenously introduced siRNAs may be devoid of
overhangs at their 3' and 5' ends, however, it is preferred that at
least one RNA strand has a 5'- and/or 3'-overhang. Preferably, one
end of the double-strand has a 3'-overhang from 1-5 nucleotides,
more preferably from 1-3 nucleotides and most preferably 2
nucleotides. The other end may be blunt-ended or has up to 6
nucleotides 3'-overhang. In general, any RNA molecule suitable to
act as siRNA is envisioned in the present invention. The most
efficient silencing was so far obtained with siRNA duplexes
composed of 21-nt sense and 21-nt antisense strands, paired in a
manner to have 2-nt 3' overhangs on either end. The sequence of the
2-nt 3' overhang makes a small contribution to the specificity of
target recognition restricted to the unpaired nucleotide adjacent
to the first base pair (Elbashir et al. 2001). 2'-deoxynucleotides
in the 3' overhangs are as efficient as ribonucleotides, but are
often cheaper to synthesize and probably more nuclease resistant.
Delivery of siRNA may be accomplished using any of the methods
known in the art, for example by combining the siRNA with saline
and administering the combination intravenously or intranasally or
by formulating siRNA in glucose (such as for example 5% glucose) or
cationic lipids and polymers can be used for siRNA delivery in vivo
through systemic routes either intravenously (IV) or
intraperitoneally (IP) (Fougerolles et al. (2008), Current Opinion
in Pharmacology, 8:280-285; Lu et al. (2008), Methods in Molecular
Biology, vol. 437: Drug Delivery Systems--Chapter 3: Delivering
Small Interfering RNA for Novel Therapeutics).
[0058] A short hairpin RNA (shRNA) is a sequence of RNA that makes
a tight hairpin turn that can be used to typically silence gene
expression via RNA interference. shRNA can for example use a vector
introduced into cells, in which case preferably the U6 promoter is
utilized to ensure that the shRNA is always expressed. This vector
is usually passed on to daughter cells, allowing the gene silencing
to be inherited. The shRNA hairpin structure is cleaved by the
cellular machinery into siRNA, which is then bound to the
RNA-induced silencing complex (RISC). This complex binds to and
cleaves mRNAs which match the siRNA that is bound to it.
[0059] Preferably, si/shRNAs to be used in the present invention
are chemically synthesized using conventional methods that, for
example, appropriately protected ribonucleoside phosphoramidites
and a conventional DNA/RNA synthesizer. Suppliers of RNA synthesis
reagents are Proligo (Hamburg, Germany), Dharmacon Research
(Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science,
Rockford, Ill., USA), Glen Research (Sterling, Va., USA), ChemGenes
(Ashland, Mass., USA), and Cruachem (Glasgow, UK). Most
conveniently, siRNAs or shRNAs are obtained from commercial RNA
oligo synthesis suppliers, which sell RNA-synthesis products of
different quality and costs. In general, the RNAs applicable in the
present invention are conventionally synthesized and are readily
provided in a quality suitable for RNAi.
[0060] Further molecules effecting RNAi include, for example,
microRNAs (miRNA). Said RNA species are single-stranded RNA
molecules which, as endogenous RNA molecules, regulate gene
expression. Binding to a complementary mRNA transcript triggers the
degradation of said mRNA transcript through a process similar to
RNA interference. Accordingly, miRNA may be employed as an
inhibitor of the signalling pathways in accordance with the present
invention.
[0061] The term "antibody" as used in accordance with the present
invention comprises polyclonal and monoclonal antibodies, as well
as derivatives or fragments thereof, which still retain the binding
specificity. Antibody fragments or derivatives comprise, inter
alia, Fab or Fab' fragments as well as Fd, F(ab').sub.2, Fv or scFv
fragments; see, for example Harlow and Lane "Antibodies, A
Laboratory Manual", Cold Spring Harbor Laboratory Press, 1988 and
Harlow and Lane "Using Antibodies: A Laboratory Manual" Cold Spring
Harbor Laboratory Press, 1999. The term "antibody" also includes
embodiments such as chimeric (human constant domain, non-human
variable domain), single chain and humanised (human antibody with
the exception of non-human CDRs) antibodies.
[0062] Various techniques for the production of antibodies are well
known in the art and described, e.g. in Harlow and Lane (1988) and
(1999), loc. cit. Thus, the antibodies can be produced as
peptidomimetics. Further, techniques described for the production
of single chain antibodies (see, inter alia, U.S. Pat. No.
4,946,778) can be adapted to produce single chain antibodies
specific for the target of this invention. Also, transgenic animals
or plants (see, e.g., U.S. Pat. No. 6,080,560) may be used to
express (humanized) antibodies specific for the target of this
invention. Most preferably, the antibody is a monoclonal antibody,
such as a human or humanized antibody. For the preparation of
monoclonal antibodies, any technique which provides antibodies
produced by continuous cell line cultures can be used. Examples for
such techniques are described, e.g. in Harlow and Lane (1988) and
(1999), loc. cit. and include the hybridoma technique originally
developed by Kohler and Milstein Nature 256 (1975), 495-497, the
trioma technique, the human B-cell hybridoma technique (Kozbor,
Immunology Today 4 (1983), 72) and the EBV-hybridoma technique to
produce human monoclonal antibodies (Cole et al., Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96).
Surface plasmon resonance as employed in the BIAcore system can be
used to increase the efficiency of phage antibodies which bind to
an epitope of STIM2 or an epitope of a STIM2-regulated plasma
membrane calcium channel (Schier, Human Antibodies Hybridomas 7
(1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). It
is also envisaged in the context of this invention that the term
"antibody" comprises antibody constructs which may be expressed in
cells, e.g. antibody constructs which may be transfected and/or
transduced via, inter alia, viruses or plasmid vectors.
[0063] Aptamers are nucleic acid molecules or peptide molecules
that bind a specific target molecule. Aptamers are usually created
by selecting them from a large random sequence pool, but natural
aptamers also exist in riboswitches. Aptamers can be used as
macromolecular drugs. Aptamers can be combined with ribozymes to
self-cleave in the presence of their target molecule. These
compound molecules have additional research, industrial and
clinical applications (Osborne et. al. (1997), Current Opinion in
Chemical Biology, 1:5-9; Stull & Szoka (1995), Pharmaceutical
Research, 12, 4:465-483).
[0064] More specifically, aptamers can be classified as nucleic
acid aptamers, such as DNA or RNA aptamers, or peptide aptamers.
Whereas the former normally consist of (usually short) strands of
oligonucleotides, the latter preferably consist of a short variable
peptide domain, attached at both ends to a protein scaffold.
[0065] The term "peptide" as used herein describes a group of
molecules consisting of up to 30 amino acids, whereas the term
"protein" as used herein describes a group of molecules consisting
of more than 30 amino acids. Peptides and proteins may further form
dimers, trimers and higher oligomers, i.e. consisting of more than
one molecule which may be identical or non-identical. The
corresponding higher order structures are, consequently, termed
homo- or heterodimers, homo- or heterotrimers etc. The terms
"peptide" and "protein" (wherein "protein" is interchangeably used
with "polypeptide") also refer to naturally modified
peptides/proteins wherein the modification is effected e.g. by
glycosylation, acetylation, phosphorylation and the like. Such
modifications are well-known in the art.
[0066] A ribozyme (from ribonucleic acid enzyme, also called RNA
enzyme or catalytic RNA) is an RNA molecule that catalyzes a
chemical reaction. Many natural ribozymes catalyze either their own
cleavage or the cleavage of other RNAs, but they have also been
found to catalyze the aminotransferase activity of the ribosome.
Non-limiting examples of well-characterized small self-cleaving
RNAs are the hammerhead, hairpin, hepatitis delta virus, and in
vitro-selected lead-dependent ribozymes, whereas the group I intron
is an example for larger ribozymes. The principle of catalytic
self-cleavage has become well established in the last 10 years. The
hammerhead ribozymes are characterized best among the RNA molecules
with ribozyme activity. Since it was shown that hammerhead
structures can be integrated into heterologous RNA sequences and
that ribozyme activity can thereby be transferred to these
molecules, it appears that catalytic antisense sequences for almost
any target sequence can be created, provided the target sequence
contains a potential matching cleavage site. The basic principle of
constructing hammerhead ribozymes is as follows: An interesting
region of the RNA, which contains the GUC (or CUC) triplet, is
selected. Two oligonucleotide strands, each usually with 6 to 8
nucleotides, are taken and the catalytic hammerhead sequence is
inserted between them. Molecules of this type were synthesized for
numerous target sequences. They showed catalytic activity in vitro
and in some cases also in vivo. The best results are usually
obtained with short ribozymes and target sequences.
[0067] A recent development, also useful in accordance with the
present invention, is the combination of an aptamer recognizing a
small compound with a hammerhead ribozyme. The conformational
change induced in the aptamer upon binding the target molecule is
supposed to regulate the catalytic function of the ribozyme.
[0068] Also encompassed herein are modified versions of these
inhibitory compounds.
[0069] The term "modified versions of these inhibitory compounds"
in accordance with the present invention refers to versions of the
compounds that are modified to achieve i) modified spectrum of
activity, organ specificity, and/or ii) improved potency, and/or
iii) decreased toxicity (improved therapeutic index), and/or iv)
decreased side effects, and/or v) modified onset of therapeutic
action, duration of effect, and/or vi) modified pharmacokinetic
parameters (resorption, distribution, metabolism and excretion),
and/or vii) modified physico-chemical parameters (solubility,
hygroscopicity, color, taste, odor, stability, state), and/or viii)
improved general specificity, organ/tissue specificity, and/or ix)
optimised application form and route by, for example, (a)
esterification of carboxyl groups, or (b) esterification of
hydroxyl groups with carboxylic acids, or (c) esterification of
hydroxyl groups to, e.g. phosphates, pyrophosphates or sulfates or
hemi-succinates, or (d) formation of pharmaceutically acceptable
salts, or (e) formation of pharmaceutically acceptable complexes,
or (f) synthesis of pharmacologically active polymers, or (g)
introduction of hydrophilic moieties, or (h) introduction/exchange
of substituents on aromates or side chains, change of substituent
pattern, or (i) modification by introduction of isosteric or
bioisosteric moieties, or (j) synthesis of homologous compounds, or
(k) introduction of branched side chains, or (k) conversion of
alkyl substituents to cyclic analogues, or (l) derivatisation of
hydroxyl groups to ketales, acetales, or (m) N-acetylation to
amides, phenylcarbamates, or (n) synthesis of Mannich bases,
imines, or (o) transformation of ketones or aldehydes to Schiff's
bases, oximes, acetales, ketales, enolesters, oxazolidines,
thiazolidines; or combinations thereof.
[0070] The various steps recited above are generally known in the
art. They include or rely on quantitative structure-action
relationship (QSAR) analyses (Kubinyi, "Hausch-Analysis and Related
Approaches", VCH Verlag, Weinheim, 1992), combinatorial
biochemistry, classical chemistry and others (see, for example,
Holzgrabe and Bechtold, Deutsche Apotheker Zeitung 140(8), 813-823,
2000).
[0071] In a preferred embodiment, the activity of the target
protein of the respective signalling pathway is inhibited such that
it has less than 90%, more preferred less than 80%, less than 70%,
less than 60% or less than 50% of the activity as compared to the
activity it has in the absence of any inhibition. Even more
preferred its activity is reduced such that it is less than 25%,
more preferred less than 10%, less than 5%, or less than 1% of the
activity as compared to the activity it has in the absence of any
inhibition. Most preferably, the activity of the target protein is
fully inhibited, i.e. no expression or activity is detectable.
[0072] The efficiency of an inhibitor can be quantified by
comparing the level of expression and/or activity in the presence
of an inhibitor to that in the absence of the inhibitor. For
example, as a measure may be used: the change in amount of mRNA
formed, the change in amount of protein formed, the change in
biological activity of the target proteins as described herein
below, and/or the change in the cellular phenotype or in the
phenotype of an organism. In other words, the efficiency of an
inhibitor can be quantified by comparing e.g. the amount of target
protein in the presence of an inhibitor to that in the absence of
the inhibitor or by determining the biological activity of the
target protein present prior to and after administration of the
inhibitor, wherein a reduction in the amount or biological activity
of the target protein in the presence of or after administration of
the inhibitor as compared to in the absence of or prior to said
administration is indicative of a successful inhibition of the
target protein. Means and methods to determine the amount of mRNA
or proteins in a sample or for determining biological activities
are well known in the art and include, without being limiting, the
following methods.
[0073] In cases where an inhibitor acts by affecting the expression
level of the target protein, the determination of the expression
level of the protein can, for example, be carried out on the
nucleic acid level or on the amino acid level.
[0074] Methods for determining the expression of a protein on the
nucleic acid level include, but are not limited to, northern
blotting, PCR, RT-PCR or real RT-PCR. Methods for the determination
of the expression of a protein on the amino acid level include but
are not limited to western blotting or polyacrylamide gel
electrophoresis in conjunction with protein staining techniques
such as Coomassie Brilliant blue or silver-staining. Also of use in
protein quantification is the Agilent Bioanalyzer technique. These
methods are well known in the art.
[0075] The determination of binding of potential inhibitors can be
effected in, for example, any binding assay, preferably biophysical
binding assay, which may be used to identify binding test molecules
prior to performing the functional/activity assay with the
inhibitor. Suitable biophysical binding assays are known in the art
and comprise fluorescence polarisation (FP) assay, fluorescence
resonance energy transfer (FRET) assay and surface plasmon
resonance (SPR) assay. For example, a modulator acting via binding
to an enzyme, and thereby modulating the activity of said enzyme,
may be tested by FRET by labelling either the modulator or the
enzyme with a donor chromophore and the other molecule with an
acceptor chromophore. These chromophore-labelled molecules are then
mixed with each other. When they are dissociated, donor emission
can be detected upon donor excitation at the appropriate
wavelength. However, when the donor and acceptor are in proximity
(1-10 nm) due to the interaction of the modulator with the enzyme,
the acceptor emission is predominantly observed because of the
intermolecular FRET from the donor to the acceptor.
[0076] The function of an inhibitor suitable for the method of the
present invention may be identified and/or verified by using high
throughput screening assays (HTS). High-throughput assays,
independently of being biochemical, cellular or other assays,
generally may be performed in wells of microtiter plates, wherein
each plate may contain, for example 96, 384 or 1536 wells. Handling
of the plates, including incubation at temperatures other than
ambient temperature, and bringing into contact of test compounds
with the assay mixture is preferably effected by one or more
computer-controlled robotic systems including pipetting devices.
Where large libraries of test compounds are to be screened and/or
screening is to be effected within short time, mixtures of, for
example 10, 20, 30, 40, 50 or 100 test compounds may be added to
each well. In case a well exhibits biological activity, said
mixture of test compounds may be de-convoluted to identify the one
or more test compounds in said mixture giving rise to the observed
biological activity.
[0077] The "activin/TGF-.beta. signalling pathway" is well known in
the art and has been described, for example, in Keiji Miyazawa et
al. (Genes to Cells (2002), 7:1191-1204); Joan Massague and David
Wotton (EMBO J. (2000) 19:1745-1754), and Xin-Hua Feng and Rik
Derynck (Annu. Rev. Cell Dev. (2005) 21:659-693). Receptor ligands,
including, for example, TGFB1, TGFB2, TGFB3, ACTIVIN A, ACTIVIN B,
ACTIVIN AB, and/or NODAL, bind to a heterotetrameric receptor
complex consisting of two type I receptor kinases, including, for
example, TGFBR2, ACVR2A, and/or ACVR2B, and two type II receptor
kinases, including, for example, TGFBR1, ACVR1B, and/or ACVR1C.
This binding triggers phosphorylation and activation of a
heteromeric complex consisting of an R-smad, including, for
example, SMAD2, and/or SMAD3, and a Co-smad, including, for
example, SMAD4.
[0078] Accordingly, the term "inhibitor of the activin/TGF-.beta.
signalling pathway" refers to an inhibitor of any one of the above
recited molecules that form part of this signalling pathway.
[0079] Preferably, the inhibitor of the activin/TGF-.beta.
signalling pathway is selected from the group consisting of
4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide
hydrate (SB431542; Laping et al. 2002), Lefty1 (Gene symbol:
LEFTY1; Chen and Shen, Curr Biol (2004) 14:618-624), Lefty2 (Gene
symbol: LEFTY2; Meno et al., Molecular Cell (2000) 4:287-298),
Follistatin (Gene symbol: FST; Massague and Chen, Genes Dev (2000)
14:627-644), and Cerberus (Gene symbol: CER1; Piccolo et al.,
Nature (1999) 397:707-710). Even more preferably, the inhibitor of
the activin/TGF-.beta. signalling pathway is SB431542.
[0080] Preferred amounts of SB431542 to be employed are between
about 0.1 and about 100 .mu.M, more preferably between about 1 and
about 50 .mu.M, such as for example between about 5 and about 20
.mu.M and most preferably the amount is about 10 .mu.M. Preferred
amounts of Lefty2 to be employed are between about 10 and about
10000 ng/ml, more preferably between about 100 and about 1000
ng/ml, such as for example between about 200 and about 500 ng/ml
and most preferably the amount is about 500 ng/ml. Preferred
amounts of Follistatin to be employed are between about 10 and
about 10000 ng/ml, more preferably between about 100 and about 1000
ng/ml, such as for example between about 200 and about 5000 ng/ml
and most preferably the amount is about 500 ng/ml. Preferred
amounts of Cerberus to be employed are between about 10 and about
10000 ng/ml, more preferably between about 100 and about 1000
ng/ml, such as for example between about 200 and about 500 ng/ml
and most preferably the amount is about 500 ng/ml.
[0081] The term "about", as used herein, encompasses the explicitly
recited amounts as well as deviations therefrom of .+-.15%. More
preferably, a deviation of .+-.10%, and most preferably of .+-.5%
is encompassed by the term "about".
[0082] The "BMP signalling pathway" is well known in the art and
has been described, for example, in Keiji Miyazawa et al. (Genes to
Cells (2002), 7:1191-1204), Joan Massague and David Wotton (EMBO J.
(2000) 19:1745-1754), Xin-Hua Feng and Rik Derynck (Annu. Rev. Cell
Dev. (2005) 21:659-693), and Mazerbourg et al. (J Biol Chem (2005)
280:32122-32132). Receptor ligands, including, for example, BMP2,
BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMP9, BMP10, BMP15, GDF2,
GDF5, GDF6, GDF7, and/or MSTN, bind to a heterotetrameric receptor
complex consisting of two type I receptor kinases, including, for
example, BMPR1A, BMPR1B, and/or MISR2, and two type II receptor
kinases, including, for example, BMPR2, BMPR2B, and/or ALK2. This
binding triggers phosphorylation and activation of a heteromeric
complex consisting of an R-smad, including, for example, SMAD1,
SMAD5, and/or SMAD8, and a Co-smad, including, for example,
SMAD4.
[0083] Accordingly, the term "inhibitor of the BMP signalling
pathway" refers to an inhibitor of any one of the above recited
molecules that form part of this signalling pathway.
[0084] Preferably, the inhibitor of the BMP signalling pathway is
selected from the group consisting of
6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyri-
midine dihydrochloride (dorsomorphin; Yu et al. 2008), noggin (gene
symbol: NOG) and/or chordin (gene symbol: CHRD) (Massague and Chen,
Genes Dev (2000) 14:627-644). Most preferably, the inhibitor of the
BMP signalling pathway is dorsomorphin.
[0085] Preferred amounts of dorsomorphin to be employed are between
about 0.1 and about 10 .mu.M, more preferably between about 0.5 and
about 5 .mu.M, and most preferably the amount is about 1 .mu.M.
Preferred amounts of Noggin to be employed are between about 10 and
about 10000 ng/ml, more preferably between about 100 and about 1000
ng/ml, and most preferably the amount is between about 200 and
about 500 ng/ml. Preferred amounts of chordin to be employed are
between about 10 and about 10000 ng/ml, more preferably between
about 100 and about 1000 ng/ml, such as for example between about
200 and about 500 ng/ml and most preferably the amount is about 500
ng/ml.
[0086] The term "activator", as used herein, is defined as a
compound enhancing the activity of a target molecule, preferably by
performing one or more of the following effects: (i) the
transcription of the gene encoding the protein to be activated is
enhanced, (ii) the translation of the mRNA encoding the protein to
be activated is enhanced, (iii) the protein performs its
biochemical function with enhanced efficiency in the presence of
the activator, and (iv) the protein performs its cellular function
with enhanced efficiency in the presence of the activator.
Accordingly, the term "activator" encompasses both molecules that
have a directly activating effect on the specific pathway but also
molecules that are indirectly activating, e.g. by interacting for
example with molecules that negatively regulate (e.g. suppress)
said pathway.
[0087] The above recited definitions for such compounds as well as
methods of testing them apply mutatis mutandis to the definition of
an activator. Preferably, the activator is a small molecule or
protein/polypeptide.
[0088] Preferably, the level of activity is 10% more than the
activity in absence of the activator, more preferred, the level of
activity is 25%, such as 50% more than the activity in absence of
the activator. Yet more preferred are activators enhancing the
level of activity to 75%, 80%, 90% or 100% more than the activity
in absence of the activator.
[0089] The term "canonical WNT signalling pathway" is well known in
the art and has been described, for example, in Logan and Nusse
(Annu. Rev. Cell Dev. Biol. (2004) 20:781-810). Wnt ligands,
including, for example Wnt1, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt7a,
Wnt7b, and/or Wnt11, bind to a heteromeric receptor complex,
including, for example, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD8,
FZD9, FZD10, LRP5, and/or LRP6, causing a signal to be transduced
to proteins including, for example, dishevelled, axin, adenomatous
polyposis coli, glycogen synthase kinase 3-beta, and beta-catenin.
This results in beta-catenin forming a complex with transcription
factors including, for example, lymphoid enhancer-binding factor 1,
and T cell-specific transcription factor, which then modulates
transcription.
[0090] Accordingly, the term "activator of the canonical WNT
signalling pathway" refers to an activator of any one of the above
recited molecules that form part of this signalling pathway.
[0091] Preferably, the activator of the canonical WNT signalling
pathway is selected from the group consisting of
6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidiny-
l]amino]ethyl]amino]-3-pyridinecarbonitrile (CHIR 99021; Ring et
al., Diabetes (2003) 52:588-595),
3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione
(SB 216763; Coghlan et al., Chemistry & Biology (2000)
7:793-803),
3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]-phenol
(TWS119; Ding et al., Proc. Natl. Acad. Sci. USA (2003):
7632-7637),
3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dion-
e (SB 415286; Coghlan et al., Chemistry & Biology (2000)
7:793-803),
6-bromo-3-[(3E)-1,3-dihydro-3-(hydroxyimino)-2H-indol-2-ylidene]-1,3-dihy-
dro-(3Z)-2H-indol-2-one (BIO; Sato et al. Nat Med (2004) 10:55-63),
Wnt1 (Gene Symbol: WNT1; Logan and Nusse (Annu. Rev. Cell Dev.
Biol. (2004) 20:781-810) and Wnt3a (Gene Symbol: WNT3A; Logan and
Nusse (Annu. Rev. Cell Dev. Biol. (2004) 20:781-810), and Wnt3
(Gene Symbol: WNT3; Logan and Nusse (Annu. Rev. Cell Dev. Biol.
(2004) 20:781-810). Most preferably, the activator of the canonical
WNT signalling pathway is CHIR 99021.
[0092] Preferred amounts of CHIR 99021 to be employed are between
about 0.05 and about 4 .mu.M, more preferably between about 1.5 and
about 3.5 .mu.M, and most preferably the amount is about 3 .mu.M.
Preferred amounts of SB 216763 to be employed are between about
0.05 and about 30 .mu.M, more preferably between about 1.5 and
about 3.5 .mu.M, and most preferably the amount is about 3 .mu.M.
Preferred amounts of TWS119 to be employed are between about 0.005
and about 10 .mu.M. Preferred amounts of SB 415286 to be employed
are between about 0.01 and about 10 .mu.M. Preferred amounts of BIO
to be employed are between about 0.01 and about 30 .mu.M. Preferred
amounts of Wnt1 to be employed are between about 1 and about 500
ng/ml. Preferred amounts of Wnt3a to be employed are between about
1 and about 500 ng/ml. Preferred amounts of Wnt3 to be employed are
between about 1 and about 500 ng/ml.
[0093] The "Hedgehog signalling pathway" is well known in the art
and has been described, for example, in Rubin and de Sauvage
(Nature Reviews Drug Discovery (2006) 5:1026-1033). Hedgehog
ligands, including, for example, Sonic hedgehog, Indian hedgehog,
and/or Desert hedgehog, bind to a receptor, including, for example,
Patched1, causing a signal to be transduced to proteins including,
for example, Smoothened, and/or SUFU, GLI1, GLI2, GLI3, which then
modulates transcription.
[0094] Accordingly, the term "activator of the Hedgehog signalling
pathway" refers to an activator of any one of the above recited
molecules that form part of this signalling pathway.
[0095] Preferably, the activator of the Hedgehog signalling pathway
is selected from the group consisting of
9-cyclohexyl-N-[4-(morpholinyl)phenyl]-2-(1-naphthalenyloxy)-9H-purin-6-a-
mine (purmorphamine (PMA); Sinha and Chen, Nat. Chem. Biol. (2006)
2:29-30), SHH (Gene Symbol: SHH) and SHH C2411 (Taylor et al.,
Biochemistry (2001) 40:4359-4371),
N-Methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-
-1,4-diaminocyclohexane (smoothened agonist SAG; Chen et al., Proc.
Natl. Acad. Sci. USA (2002) 99:14071-14076), and
3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylam-
ino)cyclohexyl)benzo[b]thiophene-2-carboxamide (Hh-Ag1.5;
Frank-Kamenetsky et al. journal of Biology (2002) 1:10.2-10.19).
Most preferably, the activator of the Hedgehog signalling pathway
is PMA.
[0096] Preferred amounts of PMA to be employed are between about
0.25 and about 1 .mu.M, more preferably between about 0.4 and about
0.8 .mu.M, and most preferably the amount is about 0.5 .mu.M.
Preferred amounts of SHH to be employed are between about 50 and
about 1000 ng/ml. Preferred amounts of SHH C2411 to be employed are
between about 10 and about 500 ng/ml. Preferred amounts of
smoothened agonist SAG to be employed are between about 1 and about
100 nM. Preferred amounts of Hh-Ag1.5 to be employed are between
about 1 and about 50 nM.
[0097] The term "oxidation" is well known in the art and has been
described, for example, in Jomova et al. (Basic Neurochemistry:
Molecular, Cellular, and Midcal Aspects (6.sup.th Edition; 1998)
pages 711-730). Free Radicals are molecules or fragments of
molecules containing unpaired electrons, including, for example,
superoxide, nitric oxide, peroxynitrite, and hydroxyl radicals.
Free radicals cause cellular damage by processes including, for
example, protein oxidation, DNA oxidation, DNA fragmentation, lipid
oxidation, carbonyl compound formation, and/or protein
cross-linking. Proteins including, for example, superoxide
dismutase 1, superoxide dismutase 2, superoxide dismutase 3,
catalase, and/or glutathione can detoxify free radicals.
[0098] Accordingly, the term "inhibitor of oxidation" refers to an
inhibitor of any one of the above recited molecules involved in
cellular oxidative processes.
[0099] Preferably, the inhibitor of oxidation is selected from the
group consisting of
(5R)-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxyfuran-2(5'-1)-one
(ascorbic acid; Jomova et al. Molecular and Cellular Biochemistry
(2010) 345:91-104); superoxide dismutase 1 (Gene Symbol SOD1),
superoxide dismutase 2 (Gene Symbol: SOD2) and superoxide dismutase
3 (Gene Symbol: SOD3) (Zeiko et al., Free Radical Biology and
Medicine (2002) 33:337-349);
(2S)-2-amino-5-[[(1R)-1-[[(2R)-2-[[(4S)-4-amino-5-hydroxy-1,5-dioxopentyl-
]amino]-3-(carboxymethylamino)-3-oxopropyl]disulfanylmethyl]-2-(carboxymet-
hylamino)-2-oxoethyl]amino]-5-oxopentanoic acid (glutathione;
Jomova et al. Molecular and Cellular Biochemistry (2010)
345:91-104), (R)-5-(1,2-dithiolan-3-yl)pentanoic acid (lipoic acid;
Jomova et al. Molecular and Cellular Biochemistry (2010)
345:91-104), [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)
chroman-3-yl]3,4,5-trihydroxybenzoate (epigallocatechin gallate;
Jomova et al. Molecular and Cellular Biochemistry (2010)
345:91-104),
(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione
(curcumin; Jomova et al. Molecular and Cellular Biochemistry (2010)
345:91-104), N-[2-(5-methoxy-1H-indol-3-yl)ethyl]acetamide
(melatonin; Hardeland, Endocrine (2005) 27:119-130),
4-(2-Hydroxyethyl)-1,2-benzenediol (hydroxytyrosol; Fabiani et al.,
J. Nutr. (2008) 138:1411-1416), and 2-[(2E,6E,
10E,14E,18E,22E,26E,30E,34E)-3,7,11,15,19,23,27,31,35,39-decamethyltetrac-
onta-2,6,10,14,18,22,26,30,34,38-decaenyl]-5,6-dimethoxy-3-methylcyclohexa-
-2,5-diene-1,4-dione (ubiquinone; Cabrini et al., Free Radic. Res.
Commun. (1986) 2:85-92), catalase, vitamin E (Bucioli et al. BMC
Complement Altern Med. 2011 Dec. 20; 11(1):133). Most preferably,
the inhibitor of oxidation is ascorbic acid.
[0100] Preferred amounts of ascorbic acid to be employed are
between about 50 .mu.M and about 1 mM, more preferably between
about 100 and about 500 .mu.M, and most preferably the amount is
about 150 .mu.M. Preferred amounts of superoxide dismutase 1, 2 or
3 to be employed are between about 10 and about 500 units/ml.
Preferred amounts of glutathione to be employed are between about 1
and about 10 ng/.mu.l. Preferred amounts of lipoic acid to be
employed are between about 200 and about 1000 .mu.M. Preferred
amounts of epigallocatechin gallate to be employed are between
about 10 and about 100 .mu.g/ml. Preferred amounts of curcumin to
be employed are between about 10 and about 100 .mu.M. Preferred
amounts of melatonin to be employed are between about 10 and about
200 .mu.M. Preferred amounts of hydroxytyrosol to be employed are
between about 10 and about 100 .mu.M. Preferred amounts of
ubiquinone to be employed are between about 10 and about 50 .mu.M.
Preferred amounts of catalase to be employed are between about 10
and about 500 units/ml. Preferred amounts of vitamin E to be
employed are between about 100 and about 1000 .mu.M.
[0101] All of the above recited compounds and proteins are well
known in the art and are commercially available to the skilled
person. For example, SB-431542 may be obtained from Ascent
Scientific, dorsomorphin may be obtained from Tocris, CHIR 99021
may be obtained from Axon Medchem, PMA may be obtained from Alexis
and ascorbic acid may be obtained from Sigma.
[0102] In accordance with the present invention, the term "neural
medium" refers to a medium comprising about 90 to about 9.5% of a
basal cell culture medium and about 0.5 to about 10% of serum-free
supplements. Non-limiting examples of serum-free supplements are N2
supplement and/or B27 supplement. Preferred amounts of e.g. N2
supplement are between about 0.5 and about 5% and preferred amounts
of B27 supplement are up to about 5%. Further additives such as
e.g. antibiotics or amino acid can be included in the medium. The
skilled person is aware of such additional compounds and how to use
them.
[0103] In a preferred embodiment, the neural medium is N2B27 medium
comprising about 50% DMEM-F12 (e.g. from Invitrogen)/about 50%
Neurobasal (e.g. from Invitrogen)/about 1:200 N2 supplement (e.g.
from Invitrogen)/about 1:100 B27 supplement lacking vitamin A (e.g.
from Invitrogen) and 1% Penicillin/Streptomycin/Glutamine (e.g.
from PAA).
[0104] Depending on the respective embodiment, the neural medium
may further comprise the compounds (inhibitors and/or activators)
specifically recited in the respective embodiments.
[0105] Employing the above described culture conditions,
differentiation of mammalian pluripotent stem cells is achieved in
step (a). Differentiation may be carried out in suspension culture
or on a solid support. When using suspension culture, cells are
grown under conditions in which they do not adhere to a matrix or
the bottom of the dish. When grown on a solid support, cells are
grown under conditions that enable the adherence of cells to a
surface. These cell culture conditions and methods are well known
in the art and the skilled person is capable of choosing the
conditions and methods most suitable. Most preferably, the
mammalian pluripotent stem cells are are cultured such that
embryoid bodies are formed. Even more preferably, the formation of
embryoid bodies is achieved by culture in suspension culture.
[0106] The term "embryoid bodies" as used herein refers to
aggregates of cells derived from pluripotent stem cells. Embryoid
bodies (embryoid body; EB) are generally comprised of a large
variety of differentiated cell types. Cell aggregation can for
example be imposed by hanging drop or other methods that prevent
cells from adhering to a surface, thus allowing the embryoid bodies
to form their typical colony growth. Upon aggregation,
differentiation is typically initiated and the cells begin to a
limited extent to recapitulate embryonic development.
[0107] In the subsequent step, these differentiated mammalian
pluripotent stem cells are plated onto a solid support, such as
e.g. a suitable cell culture dish. The differentiated mammalian
pluripotent stem cells may be plated as they are (i.e. without
disaggregation). In those cases where the differentiated mammalian
pluripotent stem cells form embryoid bodies, the embryoid bodies
may be plated without disaggregation or, alternatively, may be
mechanically disaggregated into fragments. For example, using a 1
ml pipette and pipetting the solution comprising the embryoid
bodies up and down is sufficient for disaggregation. Most
preferably, the fragments of the embryoid bodies comprise about 500
cells. Most preferably, when using embryoid bodies, then the
embryoid bodies are disaggregated prior to plating.
[0108] The term "solid support", as used herein, refers to a
surface enabling the adherence of cells thereto. Said surface may
be, for example, the wall or bottom of a culture vessel, a plastic
or glass slide such as for example a microscope slide or (a)
bead(s) offering a surface for adherence. Conditions suitable to
allow attachment of the cells are well known to the skilled person
and have been described, for example, in Schmitz, 2009 (Schmitz, S.
(2009). Der Experimentator: Zellkultur. Spektrum Akademischer
Verlag, 2. Aufl.). Preferably, said conditions are achieved by
coating the solid support with an agent that enhances attachment of
cells to the solid support. Such coating agents as well as methods
of using them are also well known in the art and include, without
being limiting, matrigel as for example described in the examples
below, but also gelatine, fibronectin, poly-L-lysin,
poly-L-ornithin, collagen, tenascin, perlecan, phosphocan,
brevican, neurocan, thrombospondin, laminin, or defined mixtures of
these attachment molecules, such as CellStart (Invitrogen). Most
preferably, the solid support is coated with matrigel. Matrigel is
well known in the art and is commercially available, for example
from BD Biosciences.
[0109] After plating, the differentiated mammalian pluripotent stem
cells are cultured in NPBSC expansion medium.
[0110] In accordance with the present invention, the NPBSC
expansion medium is based on the above defined neural medium,
further comprising an activator of the canonical WNT signalling
pathway, an activator of the Hedgehog signalling pathway and an
inhibitor of oxidation. As used herein, the term "NPBSC expansion
medium" always refers to a medium comprising these three
specifically recited activators and inhibitors. Preferred compounds
and amounts thereof have been defined herein above.
[0111] The term "expanding", in accordance with the present
invention, refers to a multiplication of cells, thus resulting in
an increase in the total number of cells. Preferably, cells are
expanded to at least twice their original number, more preferably
to at least 10 times their original number, such as for example at
least 100 times, such as at least 1,000 times their original number
and most preferably to at least 10,000 times, such as at least
100,000 times their original number.
[0112] Expansion of the cells may be achieved by known methods,
e.g. by culturing the cells under appropriate conditions to high
density or confluence and subsequent splitting (or passaging) of
the cells, wherein the cells are re-plated at a diluted
concentration into an increased number of culture dishes or onto
solid supports. With increasing passage number, the amount of cells
obtained therefore increases due to cell division. The skilled
person is aware of means and methods for splitting cells and can
determine the appropriate time point and dilution for splitting
cells. Preferably, cells are split between 1:5 and 1:10 every five
to seven days.
[0113] The splitting and expansion step defined in (c) is repeated
at least two times. After two repeats, the cells obtained are the
neural plate border stem cells of the invention and the cultures
are (substantially) free of contaminating non-NPBSCs. As defined
elsewhere herein, the term "at least" refers to a minimum
requirement of repeats and includes two or more repeats, such as
e.g. (at least) three repeats, (at least) four repeats, (at least)
five repeats, (at least) six repeats, (at least) seven repeats, (at
least) eight repeats, (at least) nine repeats, (at least) ten
repeats, (at least) twenty repeats, (at least) fivty repeats and so
on. It will be understood that the number of repeats defined in
step (d) refers to the total number of times step (c) is to be
carried out. In other words, when step (d) requires "at least two
times", then the splitting and expansion of step (c) is carried out
twice.
[0114] In accordance with the present invention, a method is
provided for the generation of a novel type of neuronal precursor
cells from pluripotent stem cells, such as e.g. induced pluripotent
stem cells. These neural plate border stem cells can be clonally
expanded under chemically defined conditions (i.e. as characterised
in step (d) of the method of the invention), thus demonstrating
that these cells are stem cells. Moreover, the cells obtained by
the method of the present invention can differentiate into neural
tube and neural crest cells. As such, NPSCs can differentiate into
both PNS neurons and CNS neurons, including midbrain dopaminergic
and motor neurons. Thus, they provide a useful source of CNS and
PNS neurons for disease modelling and drug discovery.
[0115] Surprisingly, it was found that the combination of
inhibition of the activin/TGF-.beta. and the BMP signalling
pathways with an activation of both the Hedgehog signalling pathway
and the canonical WNT signalling pathway enables the production of
a new precursor cell type, namely neural plate border stem cells.
Canonical WNT and Hedgehog signalling--in the presence of
inhibition of cellular oxidation processes--specifies self-renewal
of NPBSCs by maintaining a neural plate border-like identity. The
morphogens employed in accordance with the present invention have a
well-defined role in development. During gastrulation, the neural
plate is specified into the medial or border areas, which then
differentiate to form neurons in the CNS and PNS, respectively. WNT
and BMP signaling control this specification. WNT proteins induce
caudal neural plate border specification in neural plate. BMP
signalling is necessary for instruction into the neural plate
border identity, and SHH has been shown to antagonize BMP induced
specification.
[0116] Based on the knowledge in the prior art, the skilled person
was aware that Hedgehog and WNT signalling oppose each other during
patterning of cells of the central nervous system and the
peripheral nervous system. Accordingly, when aiming at providing
cells capable of differentiating into both the central nervous
system and the peripheral nervous system, the dual usage of
Hedgehog and WNT signalling is counter-intuitive, as it would be
expected that the dual signal would disrupt the normal patterning
signals, thus resulting in a cell without clear differentiation
signals. Surprisingly, however, it was found in accordance with the
present invention that the combination of Hedgehog and WNT
signalling leads to the preservation of the ability to
differentiate into both the central nervous system and the
peripheral nervous system. The cells maintain themselves in this
undifferentiated state and expand robustly while maintaining this
differentiation competence to both central and peripheral nervous
system cell types.
[0117] Therefore, through a combination of opposing morphogens, WNT
and Hedgehog, an environment was created that induces the
self-renewal of NPBSCs and inhibits their commitment to either CNS
or PNS neuronal cell types. Furthermore, as is shown in example 2,
the NPBSCs obtained by the method of the present invention are
developmentally upstream of neural rosette-derived cells previously
described in the art (Zhang et al., Nat Biotechnol 19, 1129-1133;
Koch et al., Proc Natl Acad Sci USA 106, 3225-3230).
[0118] The neural plate border stem cells of the present invention
are a valuable source of material for disease modelling studies.
NPBSCs can be readily derived from mammalian (e.g. human)
pluripotent stem cells, including patient-specific iPS cells,
without the need of manual selections steps. However, unlike other
neural cell types, NPBSCs can be cultured under chemically defined
conditions in which proteins can be substituted for small molecules
and have a consistently high splitting ratio (usually 1:10 every
five to seven days). This considerably reduces both the
batch-to-batch variability inherent to purified proteins as well as
the expense. The ability for efficient expansion as well as
competence for efficient differentiation into both PNS and CNS
neurons enables the large-scale production of disease models for
applications such as high-throughput drug discovery screens.
[0119] In a further preferred embodiment of the method of the
invention, the differentiated mammalian pluripotent stem cells are
plated in step (b) at a density of about 1000 to 100,000 per
cm.sup.2.
[0120] In another preferred embodiment of the method of the
invention, the NPBSCs are characterized by the expression of at
least three markers selected from the group consisting of FORSE1,
MSX1, PHOX2B, PAX3, PAX6, SOX1, SOX2, NESTIN, IRX3, HOXA2, HOXB2,
HES5, DACH1, PLZF, LMO3, EVI1 and ASCL1.
[0121] All of the marker proteins referred to herein are defined in
accordance with the pertinent prior art.
[0122] In accordance with the present invention, "FORSE1" refers to
the surface epitope recognized by the antibody FORSE1 and has been
described in the art, for example in Tole et al., J Neurosci (1995)
15:957-969. FORSE1 is a carbohydrate epitope expressed on the cell
surface expressed in specific populations during neuronal
development and has been described in the art, for example in
Allendoerfer et al., Mol. Cell. Neurosci. (1995) 6:381-395.
[0123] In accordance with the present invention, "MSX1" refers to
Msh homeobox 1, a protein that in humans is encoded by the MSX1
gene. MSX1 is a transcriptional repressor during embryogenesis
through interactions with components of the core transcription
complex and other homeoproteins. Human MSX1 is represented by the
NCBI reference NP.sub.--002439.2 and has been described in the art,
for example in Davidson, Trends in Genetics (1995) 11:405-411.
[0124] "PHOX2B", as used throughout the present invention, refers
to Paired-like homeobox 2b, a protein that in humans is encoded by
the PHOX2B gene. PHOX2B is a transcriptional regulator. Human
PHOX2B is represented by the NCBI reference NP.sub.--003915.2 and
has been described in the art, for example in Samad et al.,
Development (2004) 131:4071-4083.
[0125] In accordance with the present invention, "PAX3" and "PAX6"
refer to Paired box 3 and Paired box 6, respectively, which are
proteins that in humans are encoded by the PAX3 and PAX6 genes,
respectively. PAX3 and PAX6 are transcriptional regulators. Human
PAX3 is represented by the NCBI references NP.sub.--000429.2,
NP.sub.--001120838.1, NP.sub.--039230.1, NP.sub.--852122.1,
NP.sub.--852123.1, NP.sub.--852124.1, NP.sub.--852125.1 and
NP.sub.--852126.1; and PAX6 is represented by the NCBI references
NP.sub.--000271.1, NP.sub.--001121084.1, NP.sub.--001595.2. PAX3
and PAX6 have been described in the art, for example in Strachan
and Read (Curr. Opin. Genet. Dev. (1994) 4:427-438).
[0126] In accordance with the present invention, "SOX1" and "SOX2"
refer to Sex determining region Y-box 1 and Sex determining region
Y-box 2, which are proteins that in humans are encoded by the SOX1
and SOX2 genes, respectively. SOX1 and SOX2 are transcriptional
regulators. Human SOX1 is represented by the NCBI reference
NP.sub.--005977.2, and SOX2 is represented by the NCBI reference
NP.sub.--003097.1. SOX1 and SOX2 have been described in the art,
for example in Uchikawa et al., (Mech. Dev. (1999) 84:103-120).
[0127] "NESTIN", as used throughout the present invention, refers
to NESTIN, a protein that in humans is encoded by the NES gene.
NESTIN is an intermediate filament. Human NESTIN is represented by
the NCBI reference NP.sub.--006608.1 and has been described in the
art, for example in Michalcyzk and Ziman (Histol. Histopathol.
(2005) 20:665-671).
[0128] In accordance with the present invention, "IRX3" refers to
Iroquois homeobox 3, a protein that in humans is encoded by the
IRX3 gene. IRX3 is a transcriptional regulator. Human IRX3 is
represented by the NCBI reference NP.sub.--077312.2 and has been
described in the art, for example in Briscoe and Ericson (Current
Opinion in Neurobiology (2001) 11:43-49).
[0129] In accordance with the present invention, "HOXA2" and
"HOXB2" refer to Homeobox A2 and Homeobox B2, which are proteins
that in humans are encoded by the HOXA2 and HOXB2 genes,
respectively. HOXA2 and HOXB2 are transcriptional regulators. Human
HOXA2 is represented by the NCBI reference NP.sub.--006726.1, and
HOXB2 is represented by the NCBI reference NP.sub.--002136.1. HOXA2
and HOXB2 have been described in the art, for example in Davenne et
al. (Neuron (1999) 22:677-691).
[0130] "HES5", as used throughout the present invention, refers to
Hairy and enhancer of split 5, a protein that in humans is encoded
by the HES5 gene. HES5 is a transcriptional repressor, which is
activated downstream of the Notch pathway. Human HES5 is
represented by the NCBI reference NP.sub.--001010926.1 and has been
described in the art, for example in Ohtsuka et al. (EMBO J. (1999)
18:2196-2207).
[0131] In accordance with the present invention, "DACH1" refers to
Dachshund homolog 1, a protein that in humans is encoded by the
DACH1 gene. DACH1 is a chromatin-associated protein that interacts
with transcription factors to regulate gene expression. Human DACH1
is represented by the NCBI references NP.sub.--004383.3,
NP.sub.--542937.2, NP.sub.--542938.2 and has been described in the
art, for example in Watanabe et al. (Proc. Natl. Acad. Sci. USA
(2011) 108:12384-12389).
[0132] "PLZF", as used throughout the present invention, refers to
Promyelotic leukemia zinc finger, a protein that in humans is
encoded by the ZBTB16 gene. PLZF is a transcriptional regulator.
Human PLZF is represented by the NCBI references
NP.sub.--001018011.1 and NP.sub.--005997.2, and has been described
in the art, for example in Raberger et al. (Proc. Natl. Acad. Sci.
(2008) 105:17919-17924).
[0133] In accordance with the present invention, "LMO3" refers to
LIM domain only 3, a protein that in humans is encoded by the LMO3
gene. LMO3 is a transcriptional regulator. Human LMO3 is
represented by the NCBI reference NP.sub.--001001395.1,
NP.sub.--001230538.1, NP.sub.--001230539.1, NP.sub.--001230540.1,
NP.sub.--001230541.1, NP.sub.--001230542.1, and NP.sub.--061110.2
and has been described in the art, for example in Isogai et al.
(PloS ONE (2011) 6:e19297).
[0134] In accordance with the present invention, "EVI1" refers to
Ectopic viral integration site 1, a protein that in humans is
encoded by the MECOM gene. EVI1a transcriptional regulator. Human
EVI1 is represented by the NCBI reference NP.sub.--001098547.3,
NP.sub.--001098548.2, NP.sub.--001157471.1, NP.sub.--001157472.1,
NP.sub.--001192123.1, NP.sub.--004982.2, and NP.sub.--005232.2 and
has been described in the art, for example in Wieser (Gene (2007)
396:346-357).
[0135] "ASCL1", as used throughout the present invention, refers to
achaete-scute complex homolog 1, a protein that in humans is
encoded by the ASCL1 gene. ASCL1 is a transcriptional regulator.
Human ASCL1 is represented by the NCBI reference NP.sub.--004307.2
and has been described in the art, for example in Kageyama et al.
(Int J Biochem Cell Biol (1997) 29:1389-1399).
[0136] The term "at least three", as used herein, encompasses also
at least four, at least five, at least six, at least seven, at
least eight, at least nine, at least ten different amino acids or
more, such as at least eleven, at least 12, at least 13, at least
14, at least 15 or all 16 of the recited markers. It will be
appreciated by the skilled person that this term further
encompasses exactly three, exactly four, exactly five, exactly six,
exactly seven, exactly eight, exactly nine, exactly ten, exactly
eleven, exactly 12, exactly 13, exactly 14, exactly 15 or exactly
16 markers from the recited list of markers. Preferably, one of
said at least three markers is PHOX2B. For example, the at least
three markers may comprise a combination of PHOX2B with one or more
markers selected of MSX1, IRX3 or PAX3 and one or more markers
selected of PAX6, SOX1, SOX2 or NES, e.g. the at least three
markers may include: MSX1, PAX6 and PHOX2B; IRX3, SOX1 and PHOX2B;
MSX1, SOX1 and PHOX2B; MSX1, SOX2 and PHOX2B; IRX3, SOX2 and
PHOX2B; IRX3, PAX6 and PHOX2B; MSX1, NES and PHOX2B; IRX3, NES and
PHOX2B; PAX3, PAX6 and PHOX2B; PAX3, SOX2 and PHOX2B; PAX3, SOX1
and PHOX2B; and/or PAX3, NES and PHOX2B. Alternatively, the cells
are characterized by the combined expression of the markers SOX2,
IRX3, and MSX1 or of the markers SOX1, SOX2, NES, and PAX6.
[0137] Preferably, the at least three markers are human marker
proteins.
[0138] Preferably, the NPBSCs are a population of cells comprising
at least 70% of cells expressing three or more of the above defined
markers, more preferably at least 80%, such as at least 85%, such
as at least 90%, such as at least 95%, more preferably at least
98%, even more preferably at least 99% and most preferably
100%.
[0139] The skilled person is aware of suitable methods of
determining whether three or more of the above recited markers are
expressed by the cells. Such methods include, without being
limiting, determining the expression of a marker on the amino acid
level as well as on the nucleic acid level.
[0140] Methods for the determination of expression levels of a
marker on the amino acid level include but are not limited to
immunohistochemical methods as described in the appended examples
but also e.g. western blotting or polyacrylamide gel
electrophoresis in conjunction with protein staining techniques
such as Coomassie Brilliant blue or silver-staining. Also of use in
protein quantification is the Agilent Bioanalyzer technique.
Further methods of determination include, without being limiting,
cell sorting approaches such as magnetic activated cell sorting
(MACS) or flow cytometry activated cell sorting (FACS) or panning
approaches using immobilised antibodiesm as described for example
in Dainiak et al. (Adv Biochem Eng Biotechnol. 2007; 106:1-18).
Methods for determining the expression of a protein on the nucleic
acid level include, but are not limited to, northern blotting, PCR,
RT-PCR or real time PCR as well as techniques employing
microarrays. All these methods are well known in the art and have
been described in part in the appended examples.
[0141] In a further preferred embodiment of the method of the
invention, the NPBSCs are characterized by a lack of expression of
at least one of the markers OCT4, NANOG, AFP, T, SOX17, EOMES,
GSH2, OLIG2, CK8, CK18, NKX2.2, NKX6.1, HOXB8, HOXA5, FOXA2 and
VCAM-1.
[0142] In accordance with the present invention, "OCT4" refers to
Octamer binding protein 4, a protein that in humans is encoded by
the POU5f1 gene. OCT4 is a transcriptional regulator. Human OCT4 is
represented by the NCBI references NP.sub.--001167002.1,
NP.sub.--001167002.1, and NP.sub.--976034.4 and has been described
in the art, for example in Wang and Dai (Stem Cells (2010)
28:885-893).
[0143] In accordance with the present invention, "NANOG" refers to
Nanog homeobox, a protein that in humans is encoded by the NANOG
gene. NANOG a transcriptional regulator. Human NANOG is represented
by the NCBI reference NP.sub.--079141.2 and has been described in
the art, for example in Chambers et al. (Cell (2003)
113:643-655).
[0144] In accordance with the present invention, "AFP" refers to
Alpha-fetoprotein, a protein that in humans is encoded by the AFP
gene. AFP is a plasma protein produced by the yolc sak and liver
during fetal life. Human OCT4 is represented by the NCBI reference
NP.sub.--001125.1 and has been described in the art, for example in
Marubashi et al. (Ann Surg Oncol (2011) 18:2200-2209).
[0145] In accordance with the present invention, "T" refers to T,
brachyury homolog, a protein that in humans is encoded by the T
gene. T is a transcriptional regulator. Human T is represented by
the NCBI reference NP.sub.--003172.1 and has been described in the
art, for example in Fernando et al. (J Clin Invest (2010)
120:533-544).
[0146] In accordance with the present invention, "SOX17" refers to
Sex determining region Y-box 17, a protein that in humans is
encoded by the SOX17 gene. SOX17 is a transcriptional regulator.
Human SOX17 is represented by the NCBI reference NP.sub.--071899.1
and has been described in the art, for example in Chew et al. (J
Neurosci (2011) 31:13921-13935).
[0147] In accordance with the present invention, "EOMES" refers to
Eomesodermin, a protein that in humans is encoded by the EOMES
gene. EOMES is a transcriptional regulator. Human OCT4 is
represented by the NCBI reference NP.sub.--005433.2 and has been
described in the art, for example in Teo et al. (Genes Dev (2011)
25:238-250).
[0148] In accordance with the present invention, "GSH2" refers to
GS homeobox 2, a protein that in humans is encoded by the GSX2
gene. GSH2 is a transcriptional regulator. Human GSH2 is
represented by the NCBI reference NP.sub.--573574.1 and has been
described in the art, for example in Waclaw et al. (Neuron (2009)
63:451-465).
[0149] In accordance with the present invention, "OLIG2" refers to
Oligodendrocyte lineage transcription factor 2, a protein that in
humans is encoded by the OLIG2 gene. OLIG2 is a transcriptional
regulator. Human OLIG2 is represented by the NCBI reference
NP.sub.--005797.1 and has been described in the art, for example in
Ahn et al. (PloS ONE (2008) 3:e3917).
[0150] In accordance with the present invention, "CK8" refers to
Cytokeratin 8, a protein that in humans is encoded by the KRT8
gene. CK8 forms intermediate filaments through dimerization with
other keratin family proteins. Human CK8 is represented by the NCBI
reference NP.sub.--002264.1 and has been described in the art, for
example in Merjava et al. (Invest Opthalmol V is Sci (2011)
52:787-794).
[0151] In accordance with the present invention, "CK18" refers to
Cytokeratin 18, a protein that in humans is encoded by the KRT18
gene. CK18 forms intermediate filaments through dimerization with
other keratin family proteins. Human CK18 is represented by the
NCBI references NP.sub.--000215.1 and NP.sub.--954657.1 and has
been described in the art, for example in Stanke et al. (BMC Med
Genet (2011) 12:62).
[0152] In accordance with the present invention, "NKX2.2" refers to
NK transcription factor related locus 2, a protein that in humans
is encoded by the NKX2-2 gene. NKX2.2 is a transcriptional
regulator. Human NKX2.2 is represented by the NCBI reference
NP.sub.--002500.1 and has been described in the art, for example in
Smith et al. (Cancer Cell (2006) 9:405-416).
[0153] In accordance with the present invention, "NKX6.1" refers to
NK6 homeobox 1 a protein that in humans is encoded by the NKX6-1
gene. NKX6.1 is a transcriptional regulator. Human NKX6.1 is
represented by the NCBI reference NP.sub.--006159.2 and has been
described in the art, for example in Donelan et al. (J Biol Chem
(2010) 285:12181-12189).
[0154] In accordance with the present invention, "HOXB8" refers to
Homeobox B8, a protein that in humans is encoded by the HOXB8 gene.
HOXB8 is a transcriptional regulator. Human HOXB8 is represented by
the NCBI reference NP.sub.--076921.1 and has been described in the
art, for example in Knoepfler et al. (Oncogene (2001)
20:5440-5448).
[0155] In accordance with the present invention, "HOXA5" refers to
Homeobox A5, a protein that in humans is encoded by the HOXA5 gene.
HOXA5 is a transcriptional regulator. Human HOXA5 is represented by
the NCBI reference NP.sub.--061975.2 and has been described in the
art, for example in Gray et al. (JOP (2011) 12:216-219).
[0156] In accordance with the present invention, "FOXA2" refers to
Forkhead box A2, a protein that in humans is encoded by the FOXA2
gene. FOXA2 is a transcriptional regulator. Human FOXA2 is
represented by the NCBI references NP.sub.--068556.2 and
NP.sub.--710141.1 and has been described in the art, for example in
Popovic et al. (Biochem Biophys Acta (2010) 1799:411).
[0157] In accordance with the present invention, "VCAM-1" refers to
Vascular cell adhesion molecule 1, a protein that in humans is
encoded by the VCAM-1 gene. VCAM-1 is a surface sialoglycoprotein
mediating leukocyte-endothelial cell adhesion and signal
transduction. Human VCAM-1 is represented by the NCBI references
NP.sub.--001069.1, NP.sub.--001186763.1, and NP.sub.--542413.1 and
has been described in the art, for example in Nishihira et al.
(Cell Biol Int (2011) 35:475-81).
[0158] All of the definitions provided herein above for markers
expressed by the cells in accordance with the invention, in
particular the methods referred for determining the presence or
absence of marker expression, apply mutatis mutandis to these
markers that are lacking expression in the cells in accordance with
the invention. Moreover, it is preferred that the NPBSCs are a
population of cells comprising less than 30% of cells expressing
one or more of the markers defined in this embodiment, more
preferably less than 20%, such as less than 15%, such as less than
10%, such as less than 5%, more preferably less than 3%, even more
preferably less than 1% and most preferably 0%. Preferably, the
markers to be absent are human marker proteins.
[0159] In a particularly preferred embodiment of the method of the
invention, the NPBSCs are characterised by (i) the expression of
SOX1, MSX1, and PHOX2B and the lack of expression of NKX6.1 and
VCAM-1; or (ii) the expression of SOX2, IRX3, and MSX1 and the lack
of expression of HOXB8, HOXA5 and VCAM-1.
[0160] In a preferred embodiment, the method of the present
invention further comprises differentiating the NPBSCs obtained in
step (d) into: (i) peripheral nervous system neurons; (ii) central
nervous system neurons; (iii) midbrain dopaminergic neurons; (iv)
motor neurons; (v) neural crest-derived mesenchymal cells; (vi)
astrocytes; or (vii) neural rosettes.
[0161] Methods of differentiating known precursor cells into the
above recited cell types are known in the art and the skilled
person is capable of testing and employing these conditions for
achieving the desired differentiation also when using the NPBSCs of
the present invention as the starting point. Preferred methods of
differentiating the NPBSCs of the present invention are described
below.
[0162] Accordingly, in one preferred embodiment, the method of the
present invention further comprises (i) culturing the NPBSCs
obtained in step (d) in a neural medium comprising an activator of
the canonical WNT signalling pathway for about 48 to 72 hours; (ii)
adding an activator of the BMP pathway to the culture of step (i)
for about 192 hours and (iii) maturing the culture of step (ii) for
about 336 hours in a neural medium containing at least two
different neurotrophins and an inhibitor of oxidation; thereby
differentiating the NPBSCs into peripheral nervous system
neurons.
[0163] The "BMP signalling pathway" or "BMP pathway" is well known
in the art and has been described herein above. Accordingly, the
term "activator of the BMP pathway" refers to an activator of any
one of the above recited molecules that form part of this
signalling pathway.
[0164] Preferably, the activator of the BMP signalling pathway is
selected from the group consisting of BMP4 BMP2, BMP5, BMP6, BMP7,
BMP8A, and BMP8B (Gene Symbols: BMP4, BMP2, BMP5, BMP6, BMP7,
BMP8A, and BMP8B, respectively; Mazerbourg et al. (J Biol Chem
(2005) 280:32122-32132. Most preferably, the activator of the BMP
signalling pathway is BMP4.
[0165] Preferred amounts of BMP4 to be employed are between about 2
and about 100 ng/ml, more preferably between about 5 and about 20
ng/ml, and most preferably the amount is about 10 ng/ml. Preferred
amounts of BMP2, BMP5, BMP6, BMP7, BMP8A, and BMP8B to be employed
are also between about 2 and about 100 ng/ml, more preferably
between about 5 and about 20 ng/ml, and most preferably the amount
is about 10 ng/ml.
[0166] The term "neurotrophins", as used herein, relates to a
family of proteins that regulate the survival, development, and
function of neurons. Family-members include for example nerve
growth factor (NGF), brain-derived neurotrophic factor (BDNF),
neurotrophin-3 (NT-3), neurotrophin-4 (NT-4) as well as the GDNF
family of ligands and ciliary neurotrophic factor (CNTF).
[0167] Accordingly, the term "at least two different neurotrophins"
refers to two or more of the above recited molecules. Preferably,
the at least two different neurotrophins are BDNF and GDNF (Gene
Symbols: BDNF and GDNF, respectively; Jiang et al. (Chin Med J
(Engl) (2011) 124:1540-1544); Glavaski-Joksimovic et al. (J
Neurosci res (2010) 88:2669-2681)).
[0168] Preferred amounts of BDNF and GDNF to be employed are
between about 5 and about 50 ng/ml each, more preferably between
about 7.5 and about 25 ng/ml each, and most preferably the amount
is about 10 ng/ml each. BDNF and GDNF may for example be obtained
from Peprotech.
[0169] All other definitions and preferred embodiments are as
provided elsewhere herein.
[0170] In accordance with this embodiment of the invention,
peripheral nervous system neurons are obtained. Such cells are
particularly suitable to model, without being limiting,
Hirschsprung's Disease, DiGeorge syndrome, Waardenburg syndrome,
Charcot-Marie-tooth disease, familial disautonomia, congenital
insensitivity to pain with anhidrosis and pediatric cancers, such
as neuroblastoma, the treatment of such diseases by transplantion
of the cells obtained by the method of this embodiment as well as
their use for drug screening.
[0171] In another preferred embodiment of the present invention,
the method further comprises culturing the NPBSCs obtained in step
(d) in a neural medium, thereby differentiating the NPBSCs into
central nervous system neurons.
[0172] As is shown in the appended examples, changing NPBSC
expansion medium to a neural medium, such as e.g. N2B27 medium
without additional supplements, results in differentiation of the
NPBSC into central nervous system neurons of hindbrain character.
Such cells find numerous applications, such as in drug discovery
screens, for transplantations in order to treat diseases correlated
with hindbrain pathology (e.g. Parkinson's disease) as well as for
modelling such diseases.
[0173] In another preferred embodiment of the method of the
invention, the method further comprises: [0174] (i) culturing the
NPBSCs obtained in step (d) in a neural medium comprising [0175]
(a) an activator of the FGF signaling pathway, [0176] (b) an
activator of the hedgehog signaling pathway and [0177] (c) an
inhibitor of oxidation, [0178] for about 168 to about 192 hours;
[0179] (ii) changing the medium to a neural medium comprising
[0180] (a) at least two different neurotrophins, [0181] (b) an
inhibitor of oxidation; [0182] and culturing the cells for about 24
to about 96 hours; and [0183] (iii) further culturing the cells in
a neural medium comprising [0184] (a) at least two different
neurotrophins; and [0185] (b) an inhibitor of oxidation; thereby
differentiating the NPBSCs into midbrain dopaminergic neurons.
[0186] The "FGF signaling pathway" is well known in the art and has
been described, for example, in Dorey and Amaya (Development (2010)
137:3731-3742) and Omitz and Otoh (Genome Biol (2001)
2:REVIEWS3005). FGF ligands, including, for example, FGF1, FGF2,
FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12,
FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22,
and/or FGF23, induce dimerization upon binding to a receptor,
including, for example, FGFR1, FGFR2, FGFR3, and/or FGFR4, causing
a signal to be transduced to proteins including, for example, SRC,
FRS2, GRB2, SHP2, SOS, RAS, cRAF, MEK, ERK, GAB1, GAB2, PI3K, PKB,
PLCgamma, PKC, and/or CAMKII.
[0187] Accordingly, the term "activator of the FGF signaling
pathway" refers to an activator of any one of the above recited
molecules that form part of this signalling pathway.
[0188] Preferably, the activator of the FGF signaling pathway is
selected from the group consisting of FGF2, FGF1, FGF4, FGF8, FGF17
and FGF18 (Gene Symbols: FGF2, FGF1, FGF4, FGF8, FGF17 and FGF18,
respectively; Omitz and Otoh (Genome Biol (2001) 2:REVIEWS3005)).
Most preferably, the activator of the FGF signalling pathway is
FGF8.
[0189] Preferred amounts of FGF2, FGF1, FGF4, FGF8, FGF17, or FGF18
to be employed are between about 1 and about 500 ng/ml, more
preferably between about 10 and about 250 ng/ml, and most
preferably the amount is about 100 ng/ml.
[0190] All other definitions and preferred embodiments are as
provided elsewhere herein.
[0191] In accordance with this embodiment of the invention,
midbrain dopaminergic neurons are obtained. Such cells are
particularly suitable to model Parkinson's disease, conduct drug
discovery screens, or treat Parkinson's disease.
[0192] In a more preferred embodiment of this method of the
invention of differentiating the NPBSCs into midbrain dopaminergic
neurons, the medium in step (ii) further comprises: [0193] (c) an
activator of the activin/TGF-.beta. signalling pathway; and/or
[0194] (d) an activator of adenylate cyclase or a cAMP analogue;
and/or [0195] (e) an activator of the hedgehog signaling
pathway.
[0196] The "activin/TGF-.beta. signalling pathway" is well known in
the art and has been described herein above.
[0197] Accordingly, the term "activator of activin/TGF-.beta.
signalling pathway" refers to an activator of any one of the above
recited molecules that form part of this signalling pathway.
[0198] Preferably, the activator of activin/TGF-.beta. signalling
pathway is selected from the group consisting of TGFB1, TGFB2,
TGFB3, ACTIVIN A, ACTIVIN B, ACTIVIN AB, and/or NODAL (Gene
Symbols: TGFB1, TGFB2, TGFB3, ACTIVIN A, ACTIVIN B, ACTIVIN AB, and
NODAL, respectively; Xin-Hua Feng and Rik Derynck (Annu. Rev. Cell
Dev. (2005) 21:659-693)). Most preferably, the activator of the
activator of activin/TGF-.beta. signalling pathway is activator of
TGFB3.
[0199] Preferred amounts of TGFB1, TGFB2, TGFB3, ACTIVIN A, ACTIVIN
B, ACTIVIN AB, and/or NODAL to be employed are between about 0.5
and about 100 ng/ml, more preferably between about 0.75 and about
10 ng/ml, and most preferably the amount is about 1 ng/ml.
[0200] Preferred examples of an "activator of adenylate cyclase or
a cAMP analogue" include, without being limiting,
8-Piperidinoadenosine 3',5'-monophosphate (Skalhegg, B. S., et al.,
J. Biol. Chem. 267, 15707-15714, (1992)),
8-(6-Aminohexyl)aminoadenosine 3':5'-cyclic monophosphate
(Whitehouse, B. J. and Abayasekara, D. R.; J. Mol. Endocrinol. 12,
195-202, (1994)), Sp-Adenosine 3',5'-cyclic monophosphorothioate
(Scholubbers, et al. Eur. J. Biochem. 138, 101-109, (1984),
N.sup.6-Benzoyladenosine-3',5'-cyclic monophosphate (Christensen,
A. E., et al. J. Biol. Chem. 278, 35394-35402, (2003)),
8-Chloroadenosine-3',5'-cyclic monophosphorothioate (Yokozaki, H.
Cancer Res. 52, 2504, (1992)), Rp-isomer, 8-Bromoadenosine
3',5'-cyclic monophosphate (Meyer, R. B. Jr., and Miller, J. P.,
Life Sci. 14, 1019-1040, (1974)),
8-(6-Aminohexyl)aminoadenosine-3',5'-cyclic monophosphate
(Skalhegg, B. S., et al. J. Biol. Chem. 267, 15707-15714, (1992)),
8-Chloroadenosine 3',5'-cyclic-monophosphate (Juranic, et al., J.
Exp. Clin. Cancer Res 17, 269-275, (1998)),
8-(4-Chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic
monophosphate (Enserink, J. M., et al., Nat. Cell Biol. 4, 901-906,
(2002)), N.sup.6,2'-O-Dibutyryladenosine 3',5'-cyclic monophosphate
(dbcAMP; Hei, Y. J., et al., Mol. Pharmacol. 39, 233, (1991)),
(3R,4aR,5S,6S,6aS,10S,10aR,
10bS)-6,10,10b-trihydroxy-3,4-a,7,7,10a-pentamethyl-1-oxo-3-vinyldodecahy-
dro-1H-benzo[f]chromen-5-yl acetate (forskolin; Seamon et al., Proc
Natl Acad Sci USA (1981) 78:3363-3367)). Most preferably, the cAMP
analogue is dbcAMP.
[0201] Preferred amounts of 8-Piperidinoadenosine
3',5'-monophosphate, 8-(6-Aminohexyl)aminoadenosine 3':5'-cyclic
monophosphate, Sp-Adenosine 3',5'-cyclic monophosphorothioate,
N.sup.6-Benzoyladenosine-3',5'-cyclic monophosphate,
8-Chloroadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer,
8-Bromoadenosine 3',5'-cyclic monophosphate,
8-(6-Aminohexyl)aminoadenosine-3',5'-cyclic monophosphate,
8-Chloroadenosine 3',5'-cyclic-monophosphate,
8-(4-Chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic
monophosphate, dbcAMP to be employed are between about 1 and about
1000 .mu.M, more preferably between about 100 and about 750 .mu.M,
and most preferably the amount is about 500 .mu.M. Preferred
amounts of forskolin to be employed are between about 0.5 and about
100 .mu.M, more preferably between about 1 and about 20 .mu.M, and
most preferably the amount is about 10 .mu.M.
[0202] Most preferably, all three compounds recited in (c) to (e)
are additionally present in the medium in step (ii).
[0203] In a further more preferred embodiment of this method of the
invention of differentiating the NPBSCs into midbrain dopaminergic
neurons, the medium in step (iii) further comprises: [0204] (c) an
activator of the activin/TGF-.beta. signalling pathway; and/or
[0205] (d) an activator of adenylate cyclase or a cAMP
analogue.
[0206] Most preferably, both compounds recited in (c) and (d) are
additionally present in the medium in step (iii). Even more
preferably, all three compounds recited in (c) to (e) above are
additionally present in the medium in step (ii) and both compounds
recited in (c) and (d) are additionally present in the medium in
step (iii).
[0207] In another preferred embodiment of the method of the
invention, the method further comprises: [0208] (i) culturing the
NPBSCs obtained in step (d) in a neural medium comprising an
activator of the hedgehog signaling pathway for about 24 to about
48 hours; [0209] (ii) adding retinoic acid to the culture of step
(i) for about 168 to about 192 hours; and [0210] (iii) further
culturing the cells in a neural medium comprising at least two
different neurotrophins, thereby differentiating the NPBSCs into
motor neurons.
[0211] The term "retinoic acid" as used herein, refers to
(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-
-tetraenoic acid, which is also known as all-trans retinoic acid,
and is well known in the art and has been described, for example,
in Tang and Gudas, Annu. Rev. Pathol. Mech. Dis. (2011)
6:345-364.
[0212] Preferred amounts of retinoic acid to be employed are
between about 0.1 to about 20 .mu.M, more preferably between about
0.5 and about 5 .mu.M, and most preferably the amount is about 1
.mu.M. Retinoic acid may be obtained from e.g. Sigma.
[0213] All other definitions and preferred embodiments are as
provided elsewhere herein.
[0214] In accordance with this embodiment of the invention, motor
neurons are obtained. Such cells find numerous applications, for
example in the generation of disease models of motor neuron
disorders, the treatment of such diseases by transplantion of the
cells obtained by the method of this embodiment as well as their
use for drug screening.
[0215] In a more preferred embodiment of this method of the
invention of differentiating the NPBSCs into motor neurons, the
medium in step (iii) further comprises: [0216] (c) an activator of
adenylate cyclase or a cAMP analogue.
[0217] In another preferred embodiment of the method of the
invention, the method further comprises: [0218] (i) culturing the
NPBSCs obtained in step (d) in a neural medium comprising an
activator of the canonical WNT signalling pathway for about 48 to
72 hours; and [0219] (ii) culturing the cells obtained in step (i)
in cell culture medium comprising serum, thereby differentiating
the NPBSCs into neural crest-derived mesenchymal cells.
[0220] The term "cell culture medium", as used in accordance with
this embodiment, refers to any commonly used cell culture medium
known in the art, preferably a medium selected from the group
consisting of DMEM, knock-out DMEM, F-10, F-12, RPMI medium 1640,
MEM, IMEM, GMEM and DMEM/F12. Preferably, the serum is selected
from fetal calf serum (FCS) and fetal bovine serum (FBS).
[0221] Preferred amounts of serum to be employed are between about
1% and about 50%, more preferably between about 5 and about 25%,
and most preferably the amount is about 10%. Serum such as for
example FCS may be obtained from e.g. PAA.
[0222] In accordance with this embodiment of the invention, neural
crest-derived mesenchymal cells are obtained. Neural crest-derived
mesenchymal cells emerge during development in the area between the
neural and non-neural ectoderm. These cells migrate extensively and
give rise to a variety of different cell types, such as peripheral
neurons, glia, melanocytes, and endocrine cells, being able to from
smooth muscle, bone and cartilage, without being limiting. Neural
crest-derived mesenchymal cells are characterized by the expression
of specific markers, including, without being limiting, SNAI2,
PAX7, SOX9 and HNK-1. Neural crest-derived mesenchymal cells can be
used for drug screening as well as modelling or therapeutic
treatment of neural crest-associated diseases. Well-investigated
examples of these diseases comprise, without being limiting,
Hirschsprung's Disease, DiGeorge syndrome, Waardenburg syndrome,
Charcot-Marie-tooth disease, familial disautonomia, congenital
insensitivity to pain with anhidrosis and pediatric cancers, such
as neuroblastoma, as described herein below. These applications and
further characterizations of these cells are well-described in the
art, such as in Lee and others, Nat. Protocols. (2010)
5:688-701.
[0223] In accordance with the present invention, "SNAI2" refers to
the zinc-finger protein snail homolog 2, also known as human SLUG,
a protein that in humans is encoded by the SNAI2 gene. SNAI2 is a
transcriptional regulator. Human SNAI2 is represented by the NCBI
reference NP.sub.--003059.1 and has been described in the art, for
example in Cohen et al. (1998). Genomics 51 (3): 468-71.
[0224] In accordance with the present invention, "PAX7" refers to
Paired box protein 7, a protein that in humans is encoded by the
PAX7 gene. PAX7 is a transcriptional regulator. Human PAX7 is
represented by the NCBI reference NP.sub.--002575 and has been
described in the art, for example in Stapleton et al. (1995). Nat
Genet. 3 (4): 292-298.
[0225] In accordance with the present invention, "SOX9" refers to
SRY (sex determining region Y)-box 9, a protein that in humans is
encoded by the SOX9 gene. SOX9 is a transcriptional regulator.
Human SOX9 is represented by the NCBI reference NP.sub.--000337.1
and has been described in the art, for example in Tommerup et al.
(1993). Nat Genet. 4 (2): 170-4.
[0226] In accordance with the present invention, "HNK-1" refers to
an epitope detected by a monoclonal antibody designated HNK-1 and
has been described in the art, for example in Lipinski et al.
(1983), J Exp Med 158: 1775-1780.
[0227] In another preferred embodiment, the method of the present
invention further comprises [0228] (i) culturing the NPBSCs
obtained in step (d) in a neural medium comprising an activator of
FGF signalling for about 12 to 96 hours; and [0229] (ii) culturing
the cells obtained in step (i) in cell culture medium comprising
fetal calf serum, fetal bovine serum and/or CNTF for about 14 to 60
days; thereby differentiating the NPBSCs into astrocytes.
[0230] The term "CNTF" refers to "ciliary neurotrophic factor",
which is a protein that in humans is encoded by the CNTF gene (Gene
Symbol: CNTF; Protein sequence: NP.sub.--000605.1) and has been
described in the art, e.g. in Dutt et al., In Vitro Cell Dev Biol
Anim (2010) 46:635-646.
[0231] Preferred amounts of CNTF to be employed are between about 1
and about 500 ng/ml, more preferably between about 5 and about 50
ng/ml, and most preferably the amount is about 10 ng/ml.
[0232] All other definitions and preferred embodiments are as
provided elsewhere herein.
[0233] In accordance with this embodiment of the invention,
astrocytes are obtained. Such cells find numerous applications, for
example in the generation of disease models of neuralogical
disorders, the treatment of such diseases by transplantion of the
cells obtained by the method of this embodiment as well as their
use for drug screening.
[0234] In another preferred embodiment, the method of the present
invention further comprises culturing the NPBSCs obtained in step
(d) in a neural medium comprising an activator of FGF signalling
for about 12 to 96 hours; thereby differentiating the NPBSCs into
neural rosette cells.
[0235] All definitions and preferred embodiments are as provided
elsewhere herein.
[0236] In accordance with this embodiment of the invention, neural
rosette cells are obtained. Such cells are well known in the art
and have been described, e.g. in Zhang et al., Nat Biotechnol 19,
1129-1133 and Koch et al., Proc Natl Acad Sci USA 106, 3225-3230.
Such cells find numerous applications, for example in the
generation of disease models of neurological disorders, the
treatment of such diseases by transplantion of the cells obtained
by the method of this embodiment as well as their use for drug
screening.
[0237] In another preferred embodiment of the method of the present
invention, the cells obtained are free or substantially free of
pathogens.
[0238] Pathogens to be avoided are well known to the skilled person
and include, without being limiting, viruses such as for example
Hepatitis virus A, B, C, Epstein-Barr-Virus or HIV-Virus and
bacteria such as for example mycoplasm or chlamydia.
[0239] The present invention also relates to neural plate border
stem cells obtained or obtainable by the method of the
invention.
[0240] To the inventors' best knowledge, such cells do not occur
naturally at any point in development. As shown in the example
sections, the NPBSCs derived by the method of the present invention
express the markers PAX6 and SOX1, which occur in vivo only after
neural tube closure and the onset of somitogenesis. Accordingly,
the NPBSCs have features of neural tube stage progenitors. In
addition, NPBSCs express PHOX2B, HOXA2, and HOXB2. This profile is
indicative of motor neuron progenitors in the hindbrain
(approximately at the level of rhombomere 4). As is shown in the
appended examples, motor neurons form efficiently from the NPBSCs
of the invention. In addition, NPBSCs also express MSX1 and IRX3,
which are not expressed by motor neuron progenitors. Instead, MSX1
and IRX3 are markers of neural crest and dorsal progenitors,
respectively. In agreement with this, the present inventors have
shown that NPBSCs also efficiently form peripheral neurons, which
are neural crest derivatives. In summary, NPBSCs express a
combination of markers that is not found during development. This
unique combination of markers is reflected by the unique
developmental potential of NPBSCs, which is not comparable to any
cells of the neural tube.
[0241] The present invention further relates to the neural plate
border stem cells of the invention for use in medicine or
medical/pharmaceutical research.
[0242] The cells of the invention as well as a composition
comprising the neural plate border stem cells of the can be used in
a variety of experimental as well as therapeutic scenarios. The
cells of the invention have no transgenic expression elements and
are more differentiated than pluripotent or totipotent stem cells.
Accordingly, there is an overall reduced risk of these cells
developing into cancerous cells, which renders them particularly
beneficial in gene therapy, regenerative medicine, cell therapy or
drug screening.
[0243] Gene therapy, which is based on introducing therapeutic DNA
constructs for correcting a genetic defect into germ line cells by
ex vivo or in vivo techniques, is one of the most important
applications of gene transfer. Suitable vectors and methods for in
vitro or in vivo gene therapy are described in the literature and
are known to the person skilled in the art (Davis P B, Cooper M J.,
AAPS J. (2007), 19; 9(1):E11-7; Li S, Ma Z., Curr Gene Ther.
(2001),1(2):201-26). In accordance with the invention, pluripotent
stem cells obtained from a patient could, for example, be
genetically corrected by methods known in the art and subsequently
be reprogrammed into neural plate border stem cells having the
ability to differentiate into neural tube or neural crest cell
derivatives, including, for example, CNS or PNS neurons,
respectively. This evidences the applicability of the NPBSCs in
gene therapy and/or cell therapy.
[0244] Regenerative medicine can be used to potentially cure any
disease that results from malfunctioning, damaged or failing tissue
by either regenerating the damaged tissues in vivo or by growing
the tissues and organs in vitro and subsequently implanting them
into the patient. The NPBSCs of the invention being capable of
differentiating into neural tube or neural crest cell derivatives,
including, for example, CNS or PNS neurons, respectively, that can
be used in neurobiological aspects of regenerative medicine and
hence drastically reduce the need for ES cells.
[0245] The neural plate border stem cells of the invention can also
be used to identify drug targets and test potential therapeutics
hence reducing the need for ES cells and in vivo studies.
Experimental setups and methods to identify and/or assess effects
of a potential drug including, for example, target-site and
-specificity, toxicity or bioavailability are well-known to the
person skilled in the art. Further, the neural plate border stem
cells may be used to study the prevention and treatment of birth
defects or study cell differentiation. Finally, the neural plate
border stem cells of the invention may also be useful in
experimental settings--besides therapeutic applications--to study a
variety of aspects related to neuronal differentiation. The neural
plate border stem cells can further be subject to studies relating
to, e.g., gene therapy, gene targeting, differentiation studies,
tests for safety and efficacy of drugs, transplantation of
autologous or allogeneic regenerated tissue, tissue repair,
diseases like, e.g., Parkinson's disease, amyotrophic lateral
sclerosis, spinal muscular atrophy, peripheral neuropathy and
Charcot-Marie-Tooth disease, embryonal gene expression, genetic
manipulation of embryonal genes, early embryology and fetal
development, identification of embryonic cell markers, cell
migration or apoptosis.
[0246] In a more preferred embodiment, the neural plate border stem
cells of the invention are for use in the treatment of a disease
selected from the group consisting of Parkinson's disease,
amyotrophic lateral sclerosis, spinal muscular atrophy, peripheral
neuropathy, Hirschsprung's disease, DiGeorge syndrome, familial
dysautonomia, congenital insensitivity to pain with anhididrosis
and Charcot-Marie-Tooth disease. These diseases are well known in
the art and have been described, e.g. in Davie, Br Med Bull (2008)
86:109-127 (Parkinson's disease), Ilieva et al., J Cell Biol (2009)
187:761-772 (amyotrophic lateral sclerosis), Burghes et al., Nat
Rev Neuroscience (2009) 10:597-609 (spinal muscular atrophy),
Hughes, BMJ (2002) 342:466-469 (peripheral neuropathy), Guiguis,
Can Fam Physician. (1986) 32: 1521-1523 (Hirschsprung's disease),
Greenberg, J Med. Genet. (1993) 30: 803-806 (DiGeorge syndrome),
Gold-von Simson et al., J Pediatr (2009) 155:934-936 (familial
dysautonomia), Mardy et al., Am J Hum Genet (1999) 64:1570-1579
(congenital insensitivity to pain with anhididrosis) and Pareyson
and Marchesi The Lancet Neurology (2009) 8:654-667
(Charcot-Marie-Tooth disease).
[0247] All the steps recited in the embodiments of the present
invention are carried out in the order listed and subsequently to
each other, unless defined otherwise.
[0248] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. In case
of conflict, the patent specification, including definitions, will
prevail.
[0249] The figures show:
[0250] FIG. 1. Early temporal window of competence for hedgehog
induced specification of ventral neural tube progenitors. qRT-PCR
analysis for the ventral markers NKX6.1 (A) and OLIG2 (B) on
differentiating EBs treated with PMA beginning on the indicated
day. Error bars are the variation of the mean from one human ESC
line and one human iPSC line.
[0251] FIG. 2. Derivation of NPBSCs. (A) Plated EBs differentiated
in the presence of both PMA and CHIR for 6 days. (B) Phase contrast
images of NPBSCs on the indicated days after splitting. (C)
Immunostaining of hESC-derived NPBSCs with antibodies raised
against the indicated neural progenitor markers. Nuclei are
counterstained with Hoechst. Scale bars are 100 .mu.m.
[0252] FIG. 3. NPBSCs have a stable diploid karyotype. DAPI stained
metaphase spread analysis for 3 independent NPBSC lines derived
from either human ESCs or iPSCs as indicated, analyzed at passage
25 and showing a diploid karyotype of 46 chromosomes.
[0253] FIG. 4. EB formation enables NPBSC derivation efficiency.
(A) EBs at day 6 of NPBSC derivation show massive outgrowth of
neuroepithelial cells. (B) Disaggregated EBs plated on
matrigel-coated dishes prior to first splitting. (C) NPBSC colonies
1 day after the first splitting already showing that most cells
present in the cultures are neural cells. (D) Confluent NPBSC
culture 6 days after first splitting. (E) Four passages already
yield very homogenous NPBSC cultures. (F) When treated as a
monolayer with NPBSC derivation conditions, no epithelial outgrowth
was observed at day five of differentiation. (G) When treated with
PMA only, plated EBs already show intensive neurite formation,
marked by arrowheads, more clearly visible after the first split,
shown in (H). (I) Derivation of NPBSC with only CHIR produced many
non-neural cells that overgrew the culture (J) and that were
negative for the neural markers SOX1 and NESTIN (K). (L) qRT-PCR
analysis for the indicated markers of non-neural differentiation on
EBs treated with the indicated small molecules. Only the
combination of CHIR and PMA together resulted in efficient
formation of homogenous NPBSC cultures and inhibited formation of
non-neural cells. Scale bars are 100 .mu.m.
[0254] FIG. 5. Microarray analysis of NPBSC. Expression levels for
the indicated genes derived from microarray analysis of two NPBSC
lines and their parental human pluripotent cell lines. NPBSCs
consistently expressed neural progenitor and rosette markers, but
not markers of pluripotency or mesendodermal differentiation.
[0255] FIG. 6. NPBSCs adopt a stable identity of regionally
specified neural progenitors after four passages.
[0256] qRT-PCR analysis of 2 indicated NPBSC line at the indicated
passage number for the indicated gene. OCT4, SOX2=pluripotency
markers. SOX2, SOX1, PAX6=neural progenitor markers. PAX3=neural
plate marker. FOXG1=anterior neural progenitor marker. AFP,
SOX17=endodermal markers. T, CK8, CK18=mesodermal marker.
[0257] FIG. 7. NPBSC show a stable growth rate over extended
passaging. (A) Doubling time of NPBSCs derived from hESCs is stable
over multiple passages. (B) Doubing time of NPBSCs from different
human pluripotent cell lines have comparable doubling times. (C)
0.5 .mu.M is the preferred PMA concentration for NPBSC growth. When
grown with 0.25 .mu.M or 1 .mu.M PMA concentrations, the doubling
time was higher.
[0258] FIG. 8. NPBSCs are capable of forming neural rosettes. (A)
Phase contrast image of NPBSCs before and after treatment with FGF2
for 2 days. (B) Immunostaining for ZO-1 on NPBSCs before and after
treatment with FGF2. Arrowheads indicate ZO-1 expression in the
center of neural rosettes, in contrast to the diffuse expression in
NPBSCs. Scale bars are 100 .mu.m.
[0259] FIG. 9. NPBSCs can be respecified along both the
dorsoventral and rostrocaudal axes. (A) qRT-PCR analysis for the
indicated marker on NPBSCs cultured under the indicated conditions
for 6 days. Error bars represent the standard deviation from three
independent cultures. (B) qRT-PCR analysis for the indicated
rostrocaudal marker on NPBSCs cultured with 1 .mu.M RA for 8 days.
Error bars represent the variation from two independent
cultures.
[0260] FIG. 10. Differentiation of PNS neurons and mesenchymal
cells from NPBSCs. (A) Immunostaining for PERIPHERIN and TUBBIII of
hESC-derived NPBSCs differentiated in the presence of BMP4. Nuclei
are stained with Hoechst. (B) qRT-PCR demonstrating upregulation of
PERIPHERIN and BRN3A in NPBSCs differentiated for 8 days in the
presence of BMP4, but not PMA. (C) Confocal imaging demonstrating
BRN3A and PERIPHERIN double positive peripheral neurons. (D) More
than 40% of cells are double positive for PERIPHERIN and TUBBIII
after patterning with BMP4. Error bars represent variation from two
independent cultures. (E) Immunostaining of VIMENTIN, CD9 and SMA
positive mesenchymal cells differentiated from NPBSCs treated with
fetal calf serum. Scale bars are 100 .mu.m.
[0261] FIG. 11. Directed differentiation of NPBSCs into mDA
neurons. (A) hESC-derived NPBSCs were differentiated into mDA
neurons and immunostained for TH and FOXA2 and counterstained for
nuclei with Hoechst. (B) Immunostaining of NPBSC-derived mDA
neurons for TH and TUBIII and counterstained for nuclei with
Hoechst. (C) qRT-PCR analysis of NPBSC-derived cultures for the
indicated markers of mDA neuron specification at day 21. Error bars
show tandard deviation from three different experiments. (D)
Efficiency of mDA neuron formation for three independent NPBSC
lines. Error bars represent the variation between two independent
cultures. Scale bars are 100 .mu.m.
[0262] FIG. 12. Directed differentiation of NPBSCs into motor
neurons. (A) Immunostaining of NPBSC-derived motor neuron
progenitors at day 8 of differentiation for OLIG2, and
counterstained for nuclei with Hoechst. (B) qRT-PCR analysis of
NPBSC-derived cultures for the indicated markers of mDA neuron
specification at day 21 of differentiation. Error bars show
standard deviation of three independent experiments. (C)
hESC-derived NPBSCS were differentiated into motor neurons and
immunostained for ISLET1, CHAT, and TUBIII and counterstained for
nuclei with Hoechst. (D) Immunostaining of NPBSC-derived motor
neurons showing colocalization of HB9 and TUBIII. (E) Motor neuron
differentiation efficiency from NPBSCs was approximately 50% as
determined by TUBBIII and HB9 colocalization. Error bars represent
variation from three independent cultures. Scale bars are 100
.mu.m.
[0263] FIG. 13. Glial differentiation of NPBSCs. (A) NPBSCs were
differentiated by withdrawal of CHIR and PMA. After three weeks,
TUBBIII positive neurons and GFAP positive astrocytes are
generated. (B) Mostly GFAP-positive astrocytes were observed when
cultures were differentiated with 10% fetal calf serum and split as
single cells once confluent. Scale bars are 100 .mu.m.
[0264] FIG. 14. NPBSC-derived neurons acquire excitable properties
of neuronal cells. (A) The net of transmembrane currents, elicited
by the voltage steps from holding potential -70 mV to +20 mV with
10 mV increment (the above panel shows stimulation paradigm). (B)
Current-voltage relationship of inward and outward currents,
measured on the peak and normalized to cell capacitance (n=8). (C)
Cells demonstrate spontaneous firing of APs likewise neurons. Right
panel shows more detailed view on the unitary APs.
[0265] FIG. 15. Miniature spontaneous activity in NPBSC-derived
neurons. (A-E) Amplitude and kinetic parameters (n=7 cells) and an
exemplary recording of minis (F) performed at holding potential -70
mV.
[0266] FIG. 16: Marker gene expression analysis after cell culture
with different concentrations of compounds employed. The numbering
refers to: 1=human embryonic stem cells--replicate 1; 2=human
embryonic stem cells--replicate 1; 3=5 .mu.M SB431542--replicate 1;
4=5 .mu.M SB431542-replicate 2; 5=20 .mu.M SB431542--replicate 1;
6=20 .mu.M SB431542--replicate 2; 7=0.5 .mu.M
dorsomorphin--replicate 1; 8=0.5 .mu.M dorsomorphin--replicate 2;
9=5 .mu.M dorsomorphin--replicate 1; 10=5 .mu.M
dorsomorphin--replicate 2; 11=2 .mu.M CHIR 99021-replicate 1; 12=2
.mu.M CHIR 99021-replicate 2; 13=4 .mu.M CHIR 99021-replicate 1;
14=4 .mu.M CHIR 99021-replicate 2; 15=0.25 .mu.M PMA--replicate 1;
16=0.25 .mu.M PMA--replicate 2; 17=1 .mu.M PMA--replicate 1; 18=1
.mu.M PMA--replicate 2; 19=0 .mu.M ascobric acid--replicate 1; 20=0
.mu.M ascobric acid--replicate 2; 21=500 .mu.M ascobric
acid--replicate 1; 22=500 .mu.M ascobric acid--replicate 2;
23=original conditions--replicate 1 and 24=original
conditions--replicate 2.
[0267] FIG. 17: Marker gene expression analysis after cell culture
with different time periods of treatment employed. The numbering
refers to: 1=human embryonic stem cells--replicate 1; 2=human
embryonic stem cells--replicate 2; 3=each step for 24 hours--cells
at passage 2; 4=each step for 24 hours--cells at
passage3--replicate 1, 5=each step for 24 hours--cells at
passage3-replicate 2; 6=each step for 96 hours--cells at
passage2--replicate 1; 7=each step for 96 hours--cells at
passage2--replicate 2; 8=original conditions--cells at passage 17
9=original conditions--replicate 1; 10=original
conditions--replicate 2.
[0268] FIG. 18: Marker gene expression analysis after cell culture
with varying compounds. The numbering refers to: 1=human embryonic
stem cells; 2=100 ng/mL Noggin (instead of dorsomoprhin)--replicate
1; 3=100 ng/mL Noggin (instead of dorsomoprhin)--replicate 2; 4=2
.mu.M SB525334 (instead of SB431542)--replicate 1; 5=2 .mu.M
SB525334 (instead of SB431542)--replicate 2; 6=original conditions;
7=original conditions; 8=10 .mu.M SB216763 (instead of CHIR99021);
9=0.5 .mu.M SAG (instead of PMA)--replicate 1; 10=0.5 .mu.M SAG
(instead of PMA)--replicate 2; 11=original conditions--passage
17.
[0269] FIG. 19: Heatmap demonstrating that NPBSCs express markers
that are unique to them and distinguish NPBSCs from the other cell
types previously published. P10=passage 10, P20=passage 20,
HUES6=human embryonic stem cells, IPSCs=induced pluripotent stem
cells, pNSCs=primitive neural stem cells, hNPCs=human neural
progenitor cells.
[0270] The examples illustrate the invention:
EXAMPLE 1
Methods and Materials
[0271] Pluripotent Stem Cell Culture
[0272] Human ESCs and iPSCs were cultured on a layer of mitotically
inactivated with mitomycin c (Tocris) mouse embryo fibroblasts
(MEFs) in hESC medium. hESC medium consisted of Knockout DMEM
(Invitrogen) with 20% Knockout Serum Replacement (Invitrogen), 0.11
mM beta-mercaptoethanol (Invitrogen), 1% non-essential amino-acids
(NEAA, Invitrogen), 1% Penicillin/Streptomycin/Glutamine (PAA),
freshly supplemented with 5 ng/ml FGF2 (Peprotech). Pluripotent
stem cells were split 1:5 to 1:8 every five to seven days. Colonies
were mechanically disaggregated with 1 mg/ml collagenase IV
(Invitrogen). 10 .mu.M ROCK Inhibitor (Ascent Scientific) was added
for 24 hours after splitting.
[0273] NPBSC Derivation
[0274] For generation of NPBSCs from pluripotent stem cells,
colonies were detached from the MEFs three to four days after
splitting, using 2 mg/ml collagenase IV (Invitrogen). Colony pieces
were collected by sedimentation and resuspended in hESC medium
(without FGF2) supplemented with 10 .mu.M SB-431542 (Ascent
Scientific), 1 .mu.M dorsomorphin (Tocris) for neural induction, as
well as 3 .mu.M CHIR 99021 (Axon Medchem), 0.5 .mu.M PMA (Alexis)
and cultured in petri dishes. Medium was replaced on day two by
N2B27 supplemented with the same small molecule supplements. N2B27
consisted of DMEM-F12 (Invitrogen)/Neurobasal (Invitrogen) 50:50
with 1:200 N2 supplement (Invitrogen), 1:100 B27 supplement lacking
vitamin A (Invitrogen) with 1% Penicillin/Streptomycin/Glutamine
(PAA). On day four, SB-431542 and dorsomorphin were withdrawn and
150 .mu.M Ascorbic Acid (AA; Sigma) was added to the medium. On day
six, the EBs were triturated with a 1000 .mu.l pipette into smaller
pieces and plated on Matrigel-coated (Matrigel, Growth factor
reduced, high concentration; BD Biosciences) 12well plates at a
density of about ten to fifteen per well in NPBSC expansion medium
(N2B27 with CHIR, PMA and AA). For coating, Matrigel was diluted to
a final dilution of 1:100 in Knockout DMEM (Invitrogen) prior to
coat 500 .mu.l per well of a 12well plate over night. Coated plates
were wrapped with Parafilm and kept in the fridge for up to one
month. The first split was performed at a 1:5 to 1:10 ratio on day
2 to 4 after plating. All the remaining splitting ratios were 1:10.
Note that higher splitting ratios selected better for NPBSC
colonies and led to a high purity with fewer splits. After a
maximum of five splits, cultures were virtually free of
contaminating non-NPBSC cells.
[0275] NPBSC Culture
[0276] NPBSC were cultured on Matrigel-coated 12well plates (Nunc)
cell-culture plates. NPBSC expansion medium consisted of N2B27
freshly supplemented with CHIR, PMA and AA, with a medium change
every other day. Typically, cells were split 1:10 every five or six
days. For splitting, cells were digested to single cells for about
15 minutes at 37.degree. C. with prewarmed Accutase (PAA). Cells
were diluted in DMEM (PAA) for centrifugation at 200.times.g for 5
minutes. The cell pellet was resuspended in fresh NPBSC expansion
medium and plated on Matrigel-coated cell culture dishes.
[0277] Differentiation of NPBSCs
[0278] For general differentiation, it is sufficient to change
NPBSC expansion medium to N2B27 medium without supplements. Once
cultures became too confluent, they were split at a 1:2 to 1:3
ratio by digesting with 1 mg/ml Collagenase IV for 5 minutes at
37.degree. C., detachment with a cell spatula and replating on
fresh Matrigel-coated plates.
[0279] For generation of more ventral CNS neurons, including
midbrain dopaminergic neurons (mDA), NPBSC expansion medium was
changed two days after splitting to N2B27 medium with 100 ng/ml
FGF8 (Peprotech), 1 .mu.M PMA and 200 .mu.M AA. After eight days in
this medium, maturation medium--N2B27 with 10 ng/ml BDNF
(Peprotech), 10 ng/ml GDNF (Peprotech), 1 ng/ml TGF-b3 (Peprotech),
200 .mu.M AA and 500 .mu.M dbcAMP (Sigma Aldrich)--was used for the
maturation of neurons. 0.5 .mu.M PMA was added to this medium for
two more days. One day after changing to maturation medium, the
cultures were split at a 1:3 ratio as small clumps, using
collagenase IV (Invitrogen). Cultures were analyzed after two weeks
in maturation conditions unless otherwise indicated.
[0280] For induction of posterior cells, including motor neurons,
NPBSC expansion medium was changed to N2B27 with 1 .mu.M PMA three
days after splitting. Two days later, 1 .mu.M retinoic acid (RA,
Sigma) and 1 .mu.M PMA were added for eight days. Following one day
in maturation medium (N2B27 with BDNF, GDNF and dbcAMP), cultures
were also split as clumps at a ratio of 1:2 to 1:3. Cells were
cultured in maturation medium for two weeks.
[0281] For generation of PNS neurons, NPBSCs two days after
splitting were switched to N2B27 with only CHIR for two days.
Afterward, 10 ng/ml BMP4 (R&D Systems) was added for eight
days. Splitting and maturation was performed as described for the
generation of motor neurons. For astrocyte and mesenchymal neural
crest differentiation, NPBSCs were cultured with DMEM (PAA) with
10% fetal calf serum (PAA) and 1% Pen/Strep/Glutamin (PAA),
beginning two days after splitting. Cultures were split twice at a
1:3 ratio when confluent using trypsin (Invitrogen).
[0282] Immunocytochemistry
[0283] For confocal microscopy, cells were plated on
Matrigel-coated glass coverslips. Cultures were fixed for 20
minutes with 4% paraformaldehyde (Electron Microscopy Sciences) in
PBS (Invitrogen) and washed twice with PBS. Permeabilization and
blocking was done in one step using 0.1% Triton X-100 (Sigma
Aldrich), 10% fetal calf serum (PAA) and 1% BSA in PBS for 45
minutes. Plates or coverslips were washed once with 0.1% BSA in PBS
and the primary antibodies applied overnight at 4.degree. C. in 1%
BSA in PBS. The next day, following one washing step with 0.1% BSA
in PBS, secondary antibodies were applied for one hour at room
temperature in 1% BSA in PBS. Finally, cells were washed three
times with 0.1% BSA in PBS-T (0.05% Tween 20), including a Hoechst
counterstaining for nuclei in the second washing step. Cells were
mounted in Vectashield mounting medium (Vector Labs) and imaged on
a Zeiss PALM/Axiovert fluorescence microscope or a Zeiss LSM700
confocal microscope. If necessary, images were merged using ImageJ
and Adobe Photoshop.
[0284] The primary antibodies used in this study are mouse anti
NESTIN (1:150, R&D), goat anti SOX1 (1:150, R&D), rabbit
anti PAX6 (1:300, Millipore), goat anti SOX2 (1:200, Santa Cruz),
mouse anti FOXA2 (1:100, Santa Cruz), rabbit anti TH (1:500, Pel
Freez), mouse anti TUBBIII (1:1000, Covance), rabbit anti OLIG2
(1:200, Sigma Aldrich), rabbit anti TUBBIII (1:2000, Covance),
rabbit anti ISLET1 (1:500, Abcam), goat anti CHAT (1:100,
Millipore), mouse anti BRN3A (1:500, Santa Cruz) and rabbit anti
PERIPHERIN (1:200, Millipore). All secondary antibodies were
obtained from Invitrogen and were conjugated to AlexaFluor
fluorochromes.
[0285] Quantitative RT-PCR (qRT-PCR)
[0286] Total RNA was isolated from cultured cells using RNeasy
columns (Qiagen), according to manufacturer instructions, including
an on-column DNase digest. Isolated RNA was reverse-transcribed
using M-MLV Reverse Transcriptase (USB) with oligo-dT.sub.16
primers (Metabion) for 1 h at 42.degree. C. qRT-PCR was performed
on an Applied Biosystems 7500 Real-Time PCR system with SYBR green
PCR master mix (ABI) and 56 ng of original RNA equivalents per 200
PCR reaction. Cycling conditions were 40 cycles of 15 s, 95.degree.
C./60 s 60.degree. C. Relative expression levels were calculated
using the 2.sup.-2.DELTA. method, normalized to biological
reference samples and using GAPDH and ACTB as housekeeping
genes.
[0287] Whole Genome Expression Analysis
[0288] DNA-free total RNA samples (500 ng) to be hybridized on
Illumina human-12 V3 expression BeadChips were processed using a
linear amplification kit (Ambion) generating biotin-labeled cRNA
(IVT duration: 14 h). This was quality-checked on a 2100
Bioanalyzer (Agilent) and hybridized as recommended and using
materials/reagents provided by the manufacturer. In BeadStudio, raw
data were background-subtracted and normalized using the "cubic
spline" algorithm. Differential gene expression was assessed on the
basis of thresholds for both expression ratios and statistical
significance employing the "Illumina custom" algorithm considering
standard deviations from replicate beads within each array. Signal
intensities below ca. 50% of the detection threshold were
arbitrarily trimmed to the value corresponding to 50% of detection.
This procedure underestimates expression changes for genes
undetectable in the reference sample (or vice versa) but avoids
nonsense ratios, such as negative or unrealistically high
values.
[0289] Karyotype Analysis
[0290] NPBSCs at passage 25 were cultured until confluent. 0.2
.mu.g/ml colcimid (Invitrogen) was added and the cells incubated at
37.degree. C. After 45 minutes, the colcimid containing medium was
removed, the cells washed with PBS and digested to a single cell
suspension with prewarmed Accutase, diluted in DMEM and collected
by centrifugation. The cell pellet was resuspended in 37.degree. C.
prewarmed 75 mM KCl solution and incubated at room temperature for
ten minutes. Cells were collected by 5 minutes centrifugation at
250.times.g, once again resuspended in prewarmed KCl solution and
immediately collected by centrifugation. The pellet was resuspended
in 5000 KCl solution and ice-cold fixation solution (3:1
methanol/acetic acid) was added drop wise while carefully shaking
the cell suspension. Once fixed, the cells were collected by
centrifugation and carefully resuspended in fresh fixative and
again pelleted. This procedure was repeated until the supernatant
after centrifugation remained clear. Cells were spread by dropping
different dilutions in fixative on glass slides (Menzel Glaser,
Thermo Scientific). One day later, cells were mounted in
Vectashield with DAPI (Vector Labs) and metaphase spreads were
analyzed on a Zeiss AxioVision Fluorescence microscope at 63.times.
magnification with oil immersion. At least 10-15 countable spreads
were recorded and counted for each line.
[0291] Generation of Single-Cell Clonal Lines
[0292] For the generation of single cell clones, NPBSCs were
infected with a pLenti CMV-SV40-Blasticidine construct based on the
pLenti6/V5 expression system (Invitrogen), which includes a
blasticidin resistance cassette. Virus production was performed in
293T cells using the ViraPower packaging mix (Invitrogen). One 6 cm
plate 293T cells were transfected using FuGENE 6 (Roche) according
to the manufacturer's instructions with 2 .mu.g packaging mix and 1
.mu.g expression construct. One day after transfection, medium was
changed against N2B27 medium. The following day, the medium
supernatant was filtered to remove 293T cells, supplemented with 6
.mu.g/ml protamine sulfate (Sigma), 3 .mu.M CHIR 99021, 0.5 .mu.M
PMA, 150 .mu.M AA and directly used for infection of freshly plated
NPBSC. The next day, infected NPBSC were washed four times with PBS
and fed with fresh NPBSC expansion medium. Selection with 5
.mu.g/ml blasticidine (PAA) in NPBSC expansion medium started two
days later and was maintained for two more weeks.
[0293] Blasticidin resistant NPBSC were digested and triturated to
single cells using Accutase for 30 minutes and filtered using a 40
.mu.m cell strainer (BD Biosciences) to remove remaining cell
aggregates. Single cells were counted and seeded at a density of 50
cells per well on a Matrigel-coated well of a 6well plate, together
with approximately 200,000 uninfected NPBSCs in expansion medium.
Four days later, cells were again selected with 5 .mu.g/ml
blasticidin, until only resistant, single colonies remained on the
plate that were spotted and marked. Selection was maintained for
one more week, single colonies picked, replated on 4well-plates and
expanded under standard NPBSC conditions, blasticidin resistance
was continued for one more week to exclude surviving non-resistant
cells. Once sufficiently expanded, single cell-derived clones were
differentiated as described above.
[0294] Evaluation of Electrophysiological Function
[0295] The transmembrane currents and spontaneous activity were
recorded from NPBSC-derived neurons, differentiated for 3 weeks,
using the whole-cell configuration of the patch-clamp technique
(Hamill et al., 1981). The patch pipettes were fabricated from
borosilicate glass on a PIP-6 pipette puller (HEKA Elektronik,
Lambrecht, Germany). When filled with pipette solution they had tip
resistances of 5-7 MO. Recordings were done using a HEKA EPC-9
amplifier (HEKA Elektronik, Lambrecht, Germany) and Pulse 8.61
Aqusition Software (HEKA Elektronik, Lambrecht, Germany). Series
resistance and pipette and whole-cell capacitance were cancelled
electronically. Cells were perfused with a bath solution containing
(mM): NaCl-140, KCl 2.4, MgCl2 1.3, CaCl2 2.5, HEPES 10, D-glucose
10, pH 7.4. The pipette solution contained (mM): K-gluconate 125,
NaCl 10, EGTA 1, MgATP 4, HEPES 10, D-glucose 10, pH 7.4. All
experiments were performed at room temperature. Recordings of
current-voltage relationship ("I-V curves") or miniature
spontaneous activity ("minis") were done in voltage-clamp mode at
holding potential -70 mV. Recordings of spontaneous firing of
action potentials ("AP") were performed in current-clamp mode at 0
pA holding current i.e. at own cell's membrane potential.
[0296] Data were analyzed using Patcher's Power Tool routine
(developed by Dr. F. Mendez and F. Wurriehausen, MPI BPC,
Gottingen, Germany) for Igor Pro (WaveMetrics, Lake Oswego, Oreg.,
USA) and Origin 7.5 (Origin Lab Corp., Northampton, Mass., USA).
Minis were analyzed with Mini Analysis 6.0 software (Synaptosoft
Inc., Fort Lee, N.J., USA).
EXAMPLE 2
Derivation and Characterization of Human NPBSCs
[0297] Differentiation of human pluripotent stem cells via embryoid
bodies (hEBs) was used to model human embryogenesis. To ensure
reproducibility, all experiments were conducted with both human
embryonic stem cells (hESCs; human ES cell line HUES6 from Chad A.
Cowan, et al. N Engl J Med 2004; 350:1353-1356) and human induced
pluripotent stem cells (hiPSCs). Neural induction was initiated
through the use of dual inhibition of SMAD signaling (Chambers et
al., 2009). First, we sought to determine the window of competence
to respond to patterning by SHH signaling for differentiating human
cells. Previously, Fasano et alia demonstrated that floor plate
differentiation was most efficiently induced when SHH was added
from day 1 of the differentiation of hESCs (Fasano et al., 2010).
Since floor plate is most ventral portion of the neural tube, we
reasoned that hESCs might have an early window of competence for
the efficient specification of ventral neural tube progenitor
lineages by SHH signaling. To test this, differentiating hEBs were
exposed to purmorphamine (PMA), which is a small molecule agonist
of the SHH receptor SMO, starting on different days (FIG. 1).
Consistent with the results of Fasano et alia, quantitative RT-PCR
(qRT-PCR) analysis demonstrated that the ventral neural progenitor
markers NKX6.1 and OLIG2 were most efficiently upregulated when PMA
was applied as early a day 2 during differentiation (FIG. 1)
(Fasano et al., 2010). A significant decrease in the efficiency of
ventral neural tube fate specification as marked by NKX6.1 and
OLIG2 expression was observed when PMA was delayed as little as two
more days (FIG. 1).
[0298] Because the temporal window for competence to respond to SHH
is so narrow, it would be impossible to significantly expand the
number of cells in culture while maintaining their ability to be
efficiently specified by SHH signaling into ventral neural tube
lineages. As such, we sought to identify culture conditions that
enable the expansion of cells in vitro that retain the ability to
be patterned by hedgehog signaling, which is normally lost within 4
days of the initiation of differentiation. WNT proteins are potent
mitogens, and WNT signaling is known to oppose SHH signaling. We
speculated that WNT signaling might facilitate the expansion of the
window of competence for differentiating hESCs to remain responsive
to SHH signaling. Therefore, we tested the effects of adding both
WNT and SHH signals to cultures of differentiating hESCs. The small
molecule CHIR99021 (CHIR), which is a GSK3B inhibitor, was added to
stimulate the canonical WNT signaling pathway. Differentiating hEBs
exposed to CHIR and PMA were marked by the formation and expansion
of an epithelium morphologically resembling neural plate about day
6 (FIG. 2A). When disaggregated and plated on Matrigel, homogeneous
colonies of epithelial cells were formed (FIG. 2B). These cells,
which we named NPBSCs, could be expanded as cell lines for more
than 70 population doublings and maintained a diploid karyotype
(FIG. 3). Interestingly, attempts to derive NPBSCs from monolayer
differentiation cultures were unsuccessful, which suggest that
factors produced or the 3-dimensional environment of cells within
EBs are necessary for NPBSC derivation (FIG. 4). Immunostaining
demonstrated that NPBSC colonies uniformly expressed the neural
progenitor markers SOX1, SOX2, NES, and PAX6 (FIG. 2C). Therefore,
NPBSCs express characteristic markers of neural progenitors.
[0299] To further characterize NPBSCs, we performed microarray
expression analysis. As expected, NPBSCs showed no significant
expression of the pluripotent markers OCT4 and NANOG, nor
mesendodermal markers AFP, T, and SOX17, nor of the trophoblast
marker EOMES (FIG. 5). In contrast, NPBSCs showed high expression
of neural markers including SOX2, PAX6, HES5, and ASCL1 (FIG. 5).
qRT-PCR analysis confirmed that NPBSCs express markers of neural
progenitors, including PAX6, SOX2, SOX1, and PAX3, which were
stably expressed beginning at about passage 4 (FIG. 6). qRT-PCR
also confirmed that non neural markers including OCT4, AFP, SOX17,
CK8, CK18, and T were not expressed by NPBSCs (FIG. 5). An analysis
of doubling time indicated that NPBSCs divided approximately once
per day, which was stable over multiple passages and also multiple
cell lines (FIG. 7). These results demonstrate that NPBSCs are a
pure population of neural progenitors and maintain a stable
expression pattern beginning at passage 4.
[0300] Interestingly, although NPBSCs do not morphologically
resemble neural rosettes, microarray analysis demonstrated the
expression of (pre-) neural rosette genetic markers DACH1, PLZF,
and LMO3 (FIG. 5). This suggested that NPBSCs retain the ability to
form neural rosettes. We tested this by culturing NPBSC colonies in
the presence of FGF2, which has previously been reported to induce
neural rosette formation by hEBs that have been plated (Zhang et
al., 2001). After 2 days of culturing NPBSC colonies with FGF2,
numerous neural rosettes were formed (FIG. 8A). For further
characterization, we immunostained for ZO-1, which is expressed by
neural rosettes but spatially localized to the apical surface
(Elkabetz et al., 2008). Although ZO-1 expression was readily
detected in colonies of NPBSCs, it demonstrated no preferential
spatial localization within the colonies (FIG. 8B). In contrast,
after FGF2 treatment, ZO-1 expression had re-oriented to the apical
surface of the rosettes (FIG. 8B). Therefore, we conclude that
NPBSCs express rosette markers and are capable of forming neural
rosettes when cultured under the appropriate conditions, which
suggests that they are developmentally upstream of neural
rosettes.
EXAMPLE 3
NPBSCs Resemble Caudal Neural Plate Border Cells
[0301] Since both WNT and SHH signaling are potent developmental
morphogens, we sought to identify the regional identity of NPBSCs.
As shown above, SHH signaling is a potent signal for
ventralization. For this reason, we first used microarray data to
determine the dorsoventral character of NPBSCs (FIG. 2A).
Interestingly, NPBSCs expressed high amounts of IRX3. PAX6 and MSX1
were readily detectable, and PAX3 was present, but in smaller
quantities (FIG. 2A). However, the most dorsal neural progenitor
marker, GSH2, was not detectable. In addition, ventral markers such
as NKX6.1, OLIG2, NKX2.2, and FOXA2 were not expressed (FIG. 2A).
These results indicate that NPBSCs have a moderately dorsal
character, which is consistent with the known opposing roles of WNT
and SHH in specifying dorsoventral identity. Microarray data for
rostrocaudal markers also demonstrated that only the genes HOXA2
and HOX82, which mark anterior hindbrain identity, were
significantly expressed (FIG. 2B). This result is consistent with
the known role of WNT signaling in specifying caudal identity
(Kiecker and Niehrs, 2001). Therefore, we conclude that NPBSCs are
neural progenitors with a moderately dorsal, hindbrain
character.
[0302] Next, we sought to determine if NPBSCs remain competent for
WNT and SHH mediated patterning of neural fate commitment. NPBSCs
were cultured with different concentrations of CHIR and PMA alone
and in combination for 6 days. qRT-PCR analysis demonstrated that
dorsal neural progenitor markers MSX1 and PAX3 were upregulated by
cells cultured with CHIR in a dose-dependent manner (FIG. 9A). SHH
signaling opposes dorsal neural fates and specifies ventral fates
(Ulloa and Briscoe, 2007). In keeping with this, NPBSCs exposed to
both PMA and CHIR together expressed significantly less MSX1 and
PAX3 than NPBSCs exposed to CHIR alone (FIG. 9A). In contrast,
increasing the PMA 1 .mu.M in combination with CHIR or PMA alone
(without CHIR) induced upregulation of ventral neural markers
NKX6-1, NKX2-1, OLIG2, and FOXA2 in a dose dependent manner (FIG.
9A). Immunostaining for FOXA2 confirmed the dose dependent
specification of floor plate by PMA (FIG. 9A). GLI2 is a mediator
of SHH signaling and was likewise upregulated by PMA (Bai et al.,
2002). Therefore, we conclude that NPBSCs are receptive to WNT and
SHH induced dorsoventral patterning of neural fate
specification.
[0303] WNT signaling has been shown to induce caudal neural plate
border fate in anterior neural plate, and it is significant to note
that dorsal neural progenitor markers MSX1 and PAX3 are also
markers of neural plate border and neural crest cells (Goulding et
al., 1991; Patthey et al., 2009; Tribulo et al., 2003). As such,
the upregulation of these markers by NPBSCs could also be because
of assuming a neural plate border and/or neural crest identity. To
test this possibility, we assessed the responsiveness of the CNS
progenitor marker SOX1 to SHH and WNT signaling. We found that SOX1
expression was induced by PMA and inhibited by CHIR (FIG. 9A). The
inhibition by CHIR is the same that induced MSX1 and PAX3
upregulation. These results suggest that NPBSCs are not only
capable of forming different dorsoventral neural tube lineages of
the CNS, but may also be capable of forming neural plate border and
neural crest lineages, which include PNS neurons.
[0304] We also assessed the responsiveness of NPBSCs to
repatterning signals along the rostrocaudal axis. Retinoids are
produced in vivo by somites and specify spinal cord fate, which can
be mimicked in vitro with all-trans retinoic acid (RA) (Novitch et
al., 2003). NPBSCs treated with RA for 8 days downregulated HOXA2,
and upregulated HOXA4, HOX84, but not HOXB9 (FIG. 9B). This
demonstrates that NPBSCs can be repatterned into posterior fates,
including spinal cord lineages. However, despite repeated attempts,
no conditions were found to be able to induce forebrain markers
such as BF1. Therefore, we conclude that NPBSCs can be respecified
along the rostrocaudal axis, but are unable to for forebrain
lineages. Taken together, these data demonstrate that NPBSCs most
closely resemble caudal neural plate border cells.
EXAMPLE 4
Directed Differentiation of NPBSCs into PNS Neurons
[0305] Since PNS neurons are derived from the neural plate border
region, we tested the capacity of NPBSCs to differentiate into PNS
neurons. In vivo, neural plate border cells are specified by BMP
proteins (Patthey et al., 2009). Since SHH, which is used to expand
NPBSCs, is known to antagonize BMP induced patterning, we cultured
NPBSCs in the presence of BMP4 for 8 days. To rule out possible
heterogeneity within the cultures as an explanation for
differentiation results, we repeated the experiments with clonal
NPBSC lines derived from single NPBSCs for all subsequent
experiments. Interestingly, BMP4 did not inhibit neurogenesis (FIG.
10A). Instead, immunostaining for PERIPHERIN, a marker of PNS
neurons, demonstrated that the majority of the neurons were
PERIPHERIN-positive, indicative of PNS neurons (FIG. 10A). Cultures
of PERIPHERIN-positive neurons also stained positive for BRN3A,
which is a marker of PNS sensory neurons (FIG. 10C). qRT-PCR
analyses confirmed that PERIPHERIN and BRN3A were upregulated by
BMP4 (FIG. 10B). As expected, 8 days of treatment with PMA
essentially abolished expression of these markers (FIG. 10B).
Overall, the efficiency of directing differentiation into
PERIPHERIN and TUBBIII double positive cells was about 40 to 50%
when three independent NPBSC lines were treated with BMP4 (FIG.
10D). Therefore, we conclude that NPBSCs are capable of forming PNS
neurons, including sensory neurons.
[0306] Since PNS neurons are derived from neural crest cells, this
suggests that NPBSCs are capable of forming other neural
crest-derived cell types including non-neural cells. To test this,
NPBSCs were differentiated using serum containing medium for 21
days after two days of treatment with CHIR alone. NPBSCs formed
VIMENTIN, CD9 and SMA positive cells, which were distinctly
mesenchymal in morphology (FIG. 10E). From these data, we conclude
that NPBSCs are capable of forming PNS neurons as well as
mesenchymal neural crest cell derivatives.
EXAMPLE 5
Directed Differentiation of NPBSCs into CNS Lineages
[0307] Having established the ability to differentiate into PNS
neurons, we next assessed the ability of NPBSCs to differentiate
into CNS neuronal lineages. First, we exposed NPBSCs to PMA and
FGF8 for 8 days, which are the patterning factors for midbrain
dopaminergic (mDA) neurons (Gale and Li, 2008). After maturation
for 2 weeks, immunostaining demonstrated that NPBSCs had
differentiated into TH, FOXA2, and TUBBIII positive neurons, which
specifically marks mDA neurons (FIGS. 11A and B). Real-time RT-PCR
showed upregulation of markers of mDA differentiation, including
EN-1, LMX1A, LMX1B, NURR1, FOXA2, and AADC (FIG. 11C). The overall
efficiency of differentiation of mDA neurons was consistent between
3 different NPBSC lines (FIG. 11D). Taken together, these results
demonstrate the formation of mDA neurons by NPBSCs using
developmentally appropriate patterning signals. SHH and RA in
combination specify the formation of motor neurons (Wichterle et
al., 2002). Since we had previously observed NPBSCs respond to
these signals individually, we next tested the ability of SHH and
RA together to direct differentiation of NPBSCs into the motor
neuron lineage. Immunostaining of NPBSCs treated for 8 days with
PMA and RA showed a large number of nuclei expressing OLIG2, which
is a marker motor neuron progenitors (FIG. 12A). After 8 days
patterning with double PMA and 1 .mu.M RA, and maturation for 2
weeks, qRT-PCR demonstrated that markers of motor neuron
differentiation including HB9, ISLET1, CHAT and HOXB4 were
significantly upregulated compared to undifferentiated NPBSCs (FIG.
12B). Immunostaining showed that most TUBBIII-positive neurons were
also ISLET1 and CHAT double positive, which indicates a high
frequency of motor neuron formation (FIG. 12C). Immunostaining also
demonstrated the presence of HB9 and TUBIII double positive cells,
which is consistent with a motor neuron identity (FIG. 12D).
Immunostaining of single-cell plated cells demonstrated that NPBSCs
formed motor neurons with an efficiency of approximately 50% (FIG.
12E). We also tested NPBSC capacity to form astroglial cells by
exposing NPBSCs to fetal calf serum for 2 weeks. Immunostaining
demonstrated the abundant formation of GFAP-positive astrocytes,
indicative of glial differentiation potential (FIG. 13). Therefore,
we conclude that NPBSCs have the developmental potential to form
CNS lineages, including mDA and motor neurons as well as glia, and
PNS neurons using developmentally appropriate specification
signals.
[0308] Next, we sought to determine if the ability to form both CNS
and PNS neurons is retained within a single NPBSC, or if it is due
to the presence of mixed heterogeneous cultures. To answer this
question, we generated three clonal NPBSC lines from single
hESC-derived NPBSCs. These clonal lines expressed the neural
progenitor makers NES, SOX2, SOX1, and PAX6. Finally, each of these
three lines could be efficiently directed to differentiate into mDA
neurons, motor neurons, and PNS neurons. Therefore, we conclude
that NPBSCs are clonally competent to form both CNS and PNS
neurons.
EXAMPLE 6
Neurons Formed from NPBSCs are Electro-Physiologically
Functional
[0309] Our final objective was to evaluate the electrophysiological
function of NPBSC-derived neurons using patch clamping. On average,
the recorded membrane potential from NPBSC-derived neurons was
-35.+-.2 mV (n=12) and the cell membrane capacitance was
31.88.+-.4.36 pF (n=12). These values are consistent with
previously published results of neurons differentiated from human
stem cells (Coyne et al., 2011; Moe et al., 2005; Westerlund et
al., 2003). Stepping the membrane holding potential from -70 to +20
mV with 10 mV increment elicited a fast-activating,
fast-inactivating inward current followed by a slower activating,
slowly deactivating outward current (FIG. 14A). The I-V curves of
both currents are typical for sodium inward current through
voltage-gated sodium channels and potassium outward current through
voltage gated potassium channels (FIG. 14B) described in neurons
(Cummins et al., 1994; Simard et al., 1993). Current-clamp
recordings demonstrated the presence of neurons that spontaneously
fired action potentials (APs) with frequencies of up to 2.1 Hz
(mean 1.00, 0.28 Hz, n=12; FIG. 14C)--a feature common to excitable
cells like neurons or muscles.
[0310] Next, we sought to determine if NPBSC-derived neurons could
form functional synaptic connections using spontaneous miniature
events, which has been proposed to represent the postsynaptic the
postsynaptic response, evoked by releasing of neurotransmitter from
a single synaptic vesicle (Del Castillo and Katz, 1954).
Spontaneous activity was measured using patch-clamp method in
voltage clamp whole-cell configuration at holding potential -70 mV
and appeared with the frequency 0.35.+-.0.11 Hz. The average
amplitude of miniature spontaneous postsynaptic currents was
21.18.+-.2.47 pA (peak value; n=7 cells, 360 events analyzed).
Representative trace and offline analysis results are shown in FIG.
15. The offline analysis revealed that recorded minis have the
amplitude or kinetic parameters comparable to those of neurons
(Edwards et al., 1990; Inenaga et al., 1998; Wyllie et al., 1994),
suggesting that NPBSC-derived neurons have not only acquired the
electrical properties of excitable neurons, but have even developed
synaptic contacts between neurons.
EXAMPLE 7
Variations of the Standard Cell Culture Conditions
[0311] In order to test whether variations in the cell culture
conditions affect the protocol for deriving NPBSC, several
modifications of the cell culture conditions were tested. As a
control, NPBSC were derived using standard conditions (each step
following the other) as follows: [0312] 1. Culturing pluripotent
stem cells as embryoid bodies (EBs) in pluripotent stem cell medium
containing 10 .mu.M SB-431542, 1 .mu.M dorsomorphin, 0.5 .mu.M
purmorphamine, 3 .mu.M CHIR-99021 for 48 hours. [0313] 2. Culturing
EBs in neural medium containing 10 .mu.M SB-431542, 1 .mu.M
dorsomorphin, 0.5 .mu.M purmorphamine, 3 .mu.M CHIR-99021 for 48
hours. [0314] 3. Culturing EBs in neural medium (containing B27,
which contains an antioxidant) containing 0.5 .mu.M purmorphamine,
3 .mu.M CHIR-99021, and 150 .mu.M ascorbic acid for 48 hours.
[0315] 4. EBs were triturated, plated, and cultured in neural
medium (containing B27, which contains an antioxidant) containing
0.5 .mu.M purmorphamine, 3 .mu.M CHIR-99021. [0316] 5. RNA samples
were taken at passage 3 and expression levels were normalized to
the originating pluripotent stem cells.
[0317] Different Concentrations.
[0318] In addition to the standard conditions, the effects of
changing the concentration of individual factors were tested, as
shown in FIG. 16. Specifically, the following alternatives to the
standard concentrations were tested: [0319] SB-431542 at 5 .mu.M
and 20 .mu.M [0320] dorsomorphin at 0.5 .mu.M and 5 .mu.M, [0321]
purmorphamine at 0.25 .mu.M and 1 .mu.M, [0322] CHIR-99021 at 2
.mu.M and 4 .mu.M, [0323] ascorbic acid at 0 .mu.M and 500
.mu.M.
[0324] All data are compared to undifferentiated human embryonic
stem cells.
[0325] Different Timing.
[0326] Further, the effect of changing the duration of treatments
was analysed, as shown in FIG. 17. In particular, the effects of
reducing each step to 24 hours and the effects of using 96 hours
for each step were tested. Also depicted are NPBSC after prolonged
passaging (passage 17).
[0327] All data are compared to undifferentiated human embryonic
stem cells.
[0328] Different Factors.
[0329] In addition to the standard compounds, the effects of
replacing several factors (see FIG. 18) were tested as follows:
[0330] SB-525334 at 2 .mu.M was used instead of SB-431542, [0331]
Noggin at 100 ng/ml was used instead of dorsomorphin [0332]
SB-216763 at 10 .mu.M was used instead of CHIR-99021 [0333]
Smoothened Agonist (SAG) at 0.5 .mu.M was used instead of
purmorphamine.
[0334] The derivation experiments were done in duplicates, and RNA
samples taken at passage 3. Also depicted are NPBSCs after
prolonged passaging (passage 17).
[0335] All data are compared to undifferentiated human embryonic
stem cells.
[0336] The data provided in FIGS. 16 to 18 show that the various
alterations of the standard conditions tested herein with regard to
the nature of the factors, their concentrations, and the timing of
their addition consistently results in the derivation of NPBSCs.
Adjusting the conditions to the most advantageous combination of
conditions in a particular laboratory setting is thus within the
skill of the skilled person.
EXAMPLE 8
NPBSCs have a Unique Expression Signature
[0337] The Illumina microarray platform was used to profile the
global gene expression of NPBSCs at passage 10 (P10) and passage 20
(P20), human embryonic stem cells (HUES6), induced pluripotent stem
cells (IPSCs) as well as primitive neural stem cells (pNSCs) and
human neural progenitor cells (hNPCs), which were derived as
described by Li et alia 2011 and Koch et alia, 2009, respectively.
The heatmap demonstrates that NPBSCs express markers that are
unique to these cells and distinguish NPBSCs from the other cell
types previously published.
REFERENCES
[0338] Alvarez-Medina, R., Le Dreau, G., Ros, M. and Marti, E.
(2009). Hedgehog activation is required upstream of Wnt signalling
to control neural progenitor proliferation. Development 136,
3301-3309. [0339] Bai, C. B., Auerbach, W., Lee, J. S., Stephen,
D., and Joyner, A. L. (2002). Gli2, but not Gli1, is required for
initial Shh signaling and ectopic activation of the Shh pathway.
Development 129, 4753-4761. [0340] Chambers, S. M., Fasano, C. A.,
Papapetrou, E. P., Tomishima, M., Sadelain, M., and Studer, L.
(2009). Highly efficient neural conversion of human ES and iPS
cells by dual inhibition of SMAD signaling. Nat Biotechnol 27,
275-280. [0341] Coyne, L., Shan, M., Przyborski, S. A., Hirakawa,
R., and Halliwell, R. F. (2011). Neuropharmacological properties of
neurons derived from human stem cells. Neurochem Int 59, 404-412.
[0342] Cummins, T. R., Xia, Y., and Haddad, G. G. (1994).
Functional properties of rat and human neocortical
voltage-sensitive sodium currents. J Neurophysiol 71, 1052-1064.
[0343] Del Castillo, J., and Katz, B. (1954). Quantal components of
the end-plate potential. J Physiol 124, 560-573. [0344] Edwards, F.
A., Konnerth, A., and Sakmann, B. (1990). Quantal analysis of
inhibitory synaptic transmission in the dentate gyrus of rat
hippocampal slices: a patch-clamp study. J Physiol 430, 213-249.
[0345] Elkabetz, Y., Panagiotakos, G., Al Shamy, G., Socci, N. D.,
Tabar, V., and Studer, L. (2008). Human ES cell-derived neural
rosettes reveal a functionally distinct early neural stem cell
stage. Genes Dev 22, 152-165. [0346] Fasano, C. A., Chambers, S.
M., Lee, G., Tomishima, M. J., and Studer, L. (2010). Efficient
derivation of functional floor plate tissue from human embryonic
stem cells. Cell Stem Cell 6, 336-347. [0347] Gale, E., and Li, M.
(2008). Midbrain dopaminergic neuron fate specification: Of mice
and embryonic stem cells. Mol Brain 1, 8. [0348] Goulding, M. D.,
Chalepakis, G., Deutsch, U., Erselius, J. R., and Gruss, P. (1991).
Pax-3, a novel murine DNA binding protein expressed during early
neurogenesis. EMBO J. 10, 1135-1147. [0349] Hamill, O. P., Marty,
A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981). Improved
patch-clamp techniques for high-resolution current recording from
cells and cell-free membrane patches. Pflugers Arch 391, 85-100.
[0350] Inenaga, K., Honda, E., Hirakawa, T., Nakamura, S., and
Yamashita, H. (1998). Glutamatergic synaptic inputs to mouse
supraoptic neurons in calcium-free medium in vitro. J
Neuroendocrinol 10, 1-7. [0351] Jessell, T. M. (2000). Neuronal
specification in the spinal cord: inductive signals and
transcriptional codes. Nat Rev Genet. 1, 20-29. [0352] Kiecker, C.,
and Niehrs, C. (2001). A morphogen gradient of Wnt/beta-catenin
signalling regulates anteroposterior neural patterning in Xenopus.
Development 128, 4189-4201. [0353] Koch P, Opitz T, Steinbeck J A,
Ladewig J, Brustle 0 (2009) A rosette-type, self-renewing human ES
cell-derived neural stem cell with potential for in vitro
instruction and synaptic integration. Proc Natl Acad Sci USA 106:
3225-3230. [0354] Krencik, R., Weick, J. P., Liu, Y., Zhang, Z. J.,
and Zhang, S. C. (2011). Specification of transplantable astroglial
subtypes from human pluripotent stem cells. Nat Biotechnol 29,
528-534. [0355] Lee, K. J., and Jessell, T. M. (1999). The
specification of dorsal cell fates in the vertebrate central
nervous system. Annu Rev Neurosci 22, 261-294. [0356] Li W, Sun W,
Zhang Y, Wei W, Ambasudhan R, et al. (2011) Rapid induction and
long-term self-renewal of primitive neural precursors from human
embryonic stem cells by small molecule inhibitors. Proc Natl Acad
Sci USA 108: 8299-8304. [0357] Moe, M. C., Varghese, M., Danilov,
A. I., Westerlund, U., Ramm-Pettersen, J., Brundin, L., Svensson,
M., Berg-Johnsen, J., and Langmoen, I. A. (2005). Multipotent
progenitor cells from the adult human brain: neurophysiological
differentiation to mature neurons. Brain 128, 2189-2199. [0358]
Nordstrom, U., Jessell, T. M., and Edlund, T. (2002). Progressive
induction of caudal neural character by graded Wnt signaling. Nat
Neurosci 5, 525-532. [0359] Novitch, B. G., Wichterle, H., Jessell,
T. M., and Sockanathan, S. (2003). A requirement for retinoic
acid-mediated transcriptional activation in ventral neural
patterning and motor neuron specification. Neuron 40, 81-95. [0360]
Patthey, C., Edlund, T., and Gunhaga, L. (2009). Wnt-regulated
temporal control of BMP exposure directs the choice between neural
plate border and epidermal fate. Development 136, 73-83. [0361]
Patthey, C., Gunhaga, L., and Edlund, T. (2008). Early development
of the central and peripheral nervous systems is coordinated by Wnt
and BMP signals. PLoS One 3, e1625. [0362] Selleck, M. A.,
Garcia-Castro, M. I., Artinger, K. B., and Bronner-Fraser, M.
(1998). Effects of Shh and Noggin on neural crest formation
demonstrate that BMP is required in the neural tube but not
ectoderm. Development 125, 4919-4930. [0363] Simard, J. M., Song,
Y., Tewari, K., Dunn, S., Werrbach-Perez, K., Perez-Polo, J. R.,
and Eisenberg, H. M. (1993). Ionic channel currents in cultured
neurons from human cortex. J Neurosci Res 34, 170-178. [0364]
Tribulo, C., Aybar, M. J., Nguyen, V. H., Mullins, M. C., and
Mayor, R. (2003). Regulation of Msx genes by a Bmp gradient is
essential for neural crest specification. Development 130,
6441-6452. [0365] Ulloa, F., and Briscoe, J. (2007). Morphogens and
the control of cell proliferation and patterning in the spinal
cord. Cell Cycle 6, 2640-2649. [0366] Westerlund, U., Moe, M. C.,
Varghese, M., Berg-Johnsen, J., Ohlsson, M., Langmoen, I. A., and
Svensson, M. (2003). Stem cells from the adult human brain develop
into functional neurons in culture. Exp Cell Res 289, 378-383.
[0367] Wichterle, H., Lieberam, I., Porter, J. A., and Jessell, T.
M. (2002). Directed differentiation of embryonic stem cells into
motor neurons. Cell 110, 385-397. [0368] Wyllie, D. J., Manabe, T.,
and Nicoll, R. A. (1994). A rise in postsynaptic Ca2+ potentiates
miniature excitatory postsynaptic currents and AMPA responses in
hippocampal neurons. Neuron 12, 127-138. [0369] Zhang, S. C.,
Wernig, M., Duncan, I.D., Brustle, O., and Thomson, J. A. (2001).
In vitro differentiation of transplantable neural precursors from
human embryonic stem cells. Nat Biotechnol 19, 1129-1133.
* * * * *