U.S. patent application number 16/410516 was filed with the patent office on 2019-08-29 for method of obtaining terminally differentiated neuronal lineages and uses thereof.
This patent application is currently assigned to Hadasit Medical Research Services & Development Limited. The applicant listed for this patent is Hadasit Medical Research Services & Development Limited. Invention is credited to Dimitrios KARUSSIS, Ibrahim KASSIS.
Application Number | 20190264172 16/410516 |
Document ID | / |
Family ID | 52811169 |
Filed Date | 2019-08-29 |
United States Patent
Application |
20190264172 |
Kind Code |
A1 |
KARUSSIS; Dimitrios ; et
al. |
August 29, 2019 |
METHOD OF OBTAINING TERMINALLY DIFFERENTIATED NEURONAL LINEAGES AND
USES THEREOF
Abstract
Provided is a method of inducing transdifferentiation of
mesenchymal stem cells (MSC), the method including (a) culturing
MSC in a first culture medium including a growth factor selected
for allowing formation of neuralized MSC (NMSC); (b) allowing the
NMSC to proliferate for a sufficient time during which said culture
medium is renewed at least once; and (c) culturing the NMSC of (b)
in a second culture media including cerebrospinal fluid (CSF) for a
time sufficient for the NMSC to differentiate into a population of
cells including terminally differentiated neurons, astrocytes and
oligodendrocytes. Also provided by the present invention is the use
of MSC or NMSC for providing a composition including said
population and to kits including MSC or NMSC and instructions for
use of same.
Inventors: |
KARUSSIS; Dimitrios;
(Jerusalem, IL) ; KASSIS; Ibrahim; (Abu Gush,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hadasit Medical Research Services & Development
Limited |
Jerusalem |
|
IL |
|
|
Assignee: |
Hadasit Medical Research Services
& Development Limited
Jerusalem
IL
|
Family ID: |
52811169 |
Appl. No.: |
16/410516 |
Filed: |
May 13, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15029127 |
Apr 13, 2016 |
10336985 |
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PCT/IL2014/050884 |
Oct 7, 2014 |
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16410516 |
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61890481 |
Oct 14, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2506/1353 20130101;
C12N 2501/11 20130101; C12N 2500/84 20130101; A61K 35/28 20130101;
C12N 5/0618 20130101; C12N 5/0622 20130101; C12N 5/0619 20130101;
A61P 25/00 20180101; A61P 25/28 20180101 |
International
Class: |
C12N 5/079 20060101
C12N005/079; A61K 35/28 20060101 A61K035/28; C12N 5/0793 20060101
C12N005/0793 |
Claims
1.-24. (canceled)
25. A composition comprising a population of cells comprising
terminally differentiated neurons, astrocytes and oligodendrocytes,
wherein said population of cells is characterized by at least 10%
expression GFAP and of Tubulin-beta-III, at least 10% expression of
MAP2 and of S100 and at least a 10% expression of GalC and of
CNPase.
26. The composition of claim 25, wherein said population is
characterized by at least 65% expression GFAP and of
Tubulin-beta-III, at least a 35% expression of MAP2 and of S100, at
least 10% expression of GalC and of CNPase.
27. The composition of claim 25, wherein said population is
characterized by expression of MAP2 in an amount of 75-85%,
Tubulin-beta-III in an amount of 68-77%, GFAP in an amount of
45-55%, S100 in an amount of 35-45%, GalC in an amount of 15-20%
and CNPase in an amount of 10-18%.
28. The composition of claim 25, wherein said population comprises
about 80% neurons, about 15% astrocytes, and about 5%
oligodendrocytes.
29. The composition of claim 25, wherein said population of cells
is characterized by secretion of NGF or BDNF to a level greater
than level of expression of same by naive mesenchymal stem
cells.
30. The composition of claim 25, wherein said population of cells
comprises mesenchymal stem cells (MSC)-derived cells.
31. The method of claim 25, wherein said population of cells is
derived from neutralized MSC (NMSC).
32. The composition of claim 25, wherein said population of cells
is obtainable by a method comprising culturing NMSC cells in a
culture media comprising cerebrospinal fluid (CSF) for a time
sufficient for the NMSC to differentiate into a population of cells
comprising terminally differentiated neurons, astrocytes and
oligodendrocytes.
33. A method of treatment comprising administering to a subject in
need of treatment a composition comprising a population of cells
comprising terminally differentiated neurons, astrocytes and
oligodendrocytes, wherein said population of cells is characterized
by at least 10% expression GFAP and of Tubulin-beta-III, at least
10% expression of MAP2 and of S100 and at least a 10% expression of
GalC and of CNPase.
34. The method of claim 33, wherein said population is
characterized by at least 65% expression GFAP and of
Tubulin-beta-III, at least a 35% expression of MAP2 and of S100, at
least 10% expression of GalC and of CNPase.
35. The method of claim 33, for treatment of a pathological
condition of the nervous system.
36. The method of claim 34, for treatment of a pathological
condition of the nervous system.
37. The method of claim 35, wherein the pathological condition of
the nervous system is a neurodegenerative disease or a spinal cord
injury.
38. The method of claim 36, wherein the pathological condition of
the nervous system is a neurodegenerative disease or a spinal cord
injury.
39. The method of claim 35, wherein the pathological condition of
the nervous system is multiple sclerosis.
40. The method of claim 36, wherein the pathological condition of
the nervous system is multiple sclerosis.
Description
TECHNOLOGICAL FIELD
[0001] The present disclosure concerns stem cells manipulation and
in particular the manipulation of mesenchymal stem cells into
neuronal lineages.
PRIOR ART
[0002] References considered to be relevant as background to the
presently disclosed subject matter are listed below: [0003]
Pittenger M F. Mackay A M. Beck S C et al. "Multilineage potential
of adult human mesenchymal stem cells". Science, 284(5411), 143-147
(1999). [0004] Gonzales C, Vio K, Munoz RI and Rodriguez EM "The
CSF of normal H-Tx rats promotes neuronal differentiation from
neurospheres but CSF of hydrocephalic H-Tx rats does not"
Cerebrospinal Fluid Res. 3(Suppl 1): S10. (2006) [0005] Judith
Buddensiek, Alexander Dressel, Michael Kowalski, Uwe Runge, Henry
Schroeder, Andreas Hermann, Matthias Kirsch, Alexander Storch,
Michael Sabolek Cerebrospinal fluid promotes survival and
astroglial differentiation of adult human neural progenitor cells
but inhibits proliferation and neuronal differentiation
Neuroscience 11:48 (2010) [0006] Harris V K. Yan Q J. Vyshkina T.
Sahabi S. Liu X. Sadiq S A. Clinical and pathological effects of
intrathecal injection of mesenchymal stem cell-derived neural
progenitors in an experimental model of multiple sclerosis. J
Neurol Sci, 313(1-2), 167-177 (2012) #1. [0007] Harris V K, Faroqui
R, Vyshkina T, Sadiq S A. Characterization of autologous
mesenchymal stem cell-derived neural progenitors as a feasible
source of stem cells for central nervous system applications in
multiple sclerosis. Stem Cells Dev. 1(7), 536-547 (2012) #2. [0008]
Ying Ye, Yin-Ming Zeng, Mei Rong Wan, Xian Fu Lu "Induction Of
Human Bone Marrow Mesenchymal Stem Cells Differentiation Into
Neural-Like Cells Using Cerebrospinal Fluid" Cell Biochem biophys
59:179-184 (2011). [0009] Radtke et al. "Peripheral glial cell
differentiation from neurospheres derived from adipose mesenchymal
stem cells", Int. J. Devl Neuroscience, 27:817-823 (2009).
[0010] Acknowledgement of the above references herein is not to be
inferred as meaning that these are in any way relevant to the
patentability of the presently disclosed subject matter.
BACKGROUND
[0011] MSCs are an important member of the bone marrow stem cell
repertoire. These cells are described as nonhematopoietic stromal
cells and their classical role is to support the process of
hematopoiesis and HSC engraftement and to give rise to cells of
mesodermal origin, such as osteoblasts, adipocytes and chondrocytes
[Pittenger M F et al. 1999].
[0012] Various studies have depicted roles of MSCs, among others,
their ability to transdifferentiate into cells of endodermal and
ectodermal origin, including possible neural transdifferentiation
and broad immunomodulating properties. In one publication it was
shown that cerebrospinal fluid (CSF) of normal H-Tx rats promotes
neuronal and glial differentiation from neurospheres and that the
CSF from hydrocephalic H-Tx rats interferes with neuronal
differentiation (Gonzales et. al. 2006). Another publication
reported that CSF can promote survival and astroglial
differentiation of adult human neural progenitor cells but inhibits
proliferation and neuronal differentiation (Buddensiek et al.
2010)
[0013] There are recent reports that multiple intrathecal
injections of mouse derived MSC neural progenitors (MSC-NPs) in an
experimental model of multiple sclerosis (Harris V K et al. (2012)
#1) induced a strong beneficial clinical effect on EAE. In another
recent study by the same group, neurosphere-like cells were
generated from multiple sclerosis patients and healthy donors
(Harris V K et al. (2012) #2]. The investigators reported that
multiple injections of MSC-NPs are advantageous as compared to a
single injection and they improve the clinical and pathological
parameters of EAE, and promote endogenous repair mechanisms.
[0014] The publications WO 2004/046348, WO 2006/134602, WO
2007/066338, WO 2009/144718 describe methods of neuronal
differentiation.
[0015] Radtke et al (2009) describes the formation of neurospheres
from adipose-derived stem cells and their differentiation in
culture to peripheral glial-like cells.
[0016] In yet another study it was reported that CSF from healthy
human donors can induce human bone marrow MSC to differentiate into
neural-like cells (Ying Ye et al. (2011)).
GENERAL DESCRIPTION
[0017] The present disclosure provides a method of inducing
transdifferentiation of mesenchymal stem cells (MSCs), the method
comprising: (a) culturing MSCs in a first culture medium comprising
a growth factor selected to allow formation of neuralized MSCs
(NMSC); (b) allowing the NMSC to proliferate for a sufficient time
during which the culture medium is renewed at least once; and (c)
culturing the NMSC in a second culture media comprising
cerebrospinal fluid (CSF) for a time sufficient for the NMSC to
differentiate into a population of cells comprising terminally
differentiated neurons, astrocytes and oligodendrocytes.
[0018] The present disclosure also provides the use of MSC for the
preparation of a composition comprising a population of cells
comprising terminally differentiated neurons, astrocytes and
oligodendrocytes.
[0019] Within this aspect, the present disclosure also provides the
use of neutralized mesenchymal stem cells (NMSC) for the
preparation of a population of cells comprising terminally
differentiated neurons, astrocytes and oligodendrocytes.
[0020] Also provided by the present disclosure is a kit comprising
a composition comprising mesenchymal stem cells (MSC) and
instructions for use of the MSC in a method of preparing a
population of cells comprising terminally differentiated neurons,
astrocytes and oligodendrocytes, the method being as defined
herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] In order to better understand the subject matter that is
disclosed herein and to exemplify how it may be carried out in
practice, embodiments will now be described, by way of non-limiting
example only, with reference to the accompanying drawings, in
which:
[0022] FIGS. 1A-1C are images showing generation of neuralized
mesenchymal stem cells-neurospheres (NMSC) from MSC; FIG. 1A show
example of human MSC isolated from MS patients (passage 1) having
fibroblast-like spindle shape morphology, FIGS. 1B and 1C show that
after culturing MSC with neurosphere generation media for 5 days,
floating NMSC having neurosphere-like structures emerged in culture
(FIG. 1B--big window=X20 magnification, bar=100 .mu.m; small
window=X40 magnification of the spheres, bar=50 .mu.m); FIG. 1C
shows staining for the nuclei of the cells forming the spheres is
demonstrated using the DAPI staining (bar 50 .mu.m).
[0023] FIGS. 2A to 2D show characterization of NMSC, FIGS. 2A and
2B are image showing that NMSC generated from hMSC were positively
stained for the marker Nestin (FIG. 2A) and PS-NCAM (FIG. 2B); FIG.
2C is a merged micrograph of the two markers measured in FIGS. 2A
and 2B, FIG. 2D shows a representative FACS analysis of hMSC and
NMSC showing that hMSC stained positively for the mesodermal
markers CD90 and CD105 while being negative for the hematopoietic
markers CD34 and CD45, whereas NMSC were stained positively for
Nestin and PSNCAM while showing low to negative staining for the
mesenchymal and hematopoietic markers CD34, CD45, CD90 and
CD105.
[0024] FIG. 3 is a graph showing survival and proliferation
kinetics of NMSC, the best culture conditions for NMSC were found
to be with the combination of the two growth factors EGF and FGF as
a supplement for the culture media; loss of one of these growth
factors resulted in halting the proliferation rate whereas loss of
both of the growth factors resulted eventually in death and
dissociation of the NMSC.
[0025] FIGS. 4A to 4J are images of neuronal cells; FIGS. 4A-4D
show neuronal differentiation of NMSC; NMSC seeded on Poly-L-Lysine
coated culture wells (from the second week of culture) were
cultured with 0.2% allogenic CSF (of MS patient), morphological
changes were observed within the culture resembling neural and
glial-like cells, a positive staining of the differentiated cells
was observed for the neuronal marker MAP2 (FIG. 4A), a neuronal
marker Class III .beta.-tubulin (FIG. 4B), the astrocytic marker
GFAP (FIG. 4C) and for the oligodendrocytic marker CNPase (FIG.
4D), bar=100 .mu.m. FIG. 4E is an image of an astrocytes isolated
from the brain (prior art); FIGS. 4F-4J show MSC cultured with
allogenic CSF, the cells were grown and treated by the method
described in Ye et al. FIGS. 4F-4G show immunohistochemistry
staining with GFAP (FIG. 4F), Class III tubulin (FIG. 4G); FIGS.
4H-4J show immunofluorescence staining with GFAP (FIG. 4H), Class
III tubulin (FIG. 4I), and the merging of the two markers (FIG.
4J).
[0026] FIG. 5 is a bar graph showing that NMSC suppress lymphocytes
proliferation; a significant dose dependent suppression of the
proliferation of lymphocytes obtained from peripheral blood of
healthy donor by NMSC was observed using a 3H-Thymidine
incorporation assay (* p<0.05, **p<0.001).
[0027] FIG. 6 is a graph showing the clinical score of chronic EAE
in treated or non-treated EAE induced mice.
DETAILED DESCRIPTION OF EMBODIMENTS
[0028] Neurodegenerative disorders such as multiple sclerosis,
alzheimer's disease, parkinson's disease and huntington's disease
involve the death of neurons in the brain. The same is true also
for spinal cord injuries. Treatment of such diseases is currently
limited and thus, there is a need for alternative therapies. One
approach involves the providence of transplantable cells produced
for example from stem cells which may be used to replace the
inactive neurons.
[0029] As shown herein the inventors have developed a novel method
for generating of terminally differentiated neuronal cells from
human bone marrow-derived mesenchymal stem cells (MSCs) via the
establishment of intermediate neurospheres as discussed below.
[0030] Specifically, the inventors have found that neurospheres
obtained from culturing MSC in the presence of growth factors
resemble (in terms of same morphology and markers) neurospheres
generated from adult neural stem cells (these neurospheres are thus
being regarded as neuralized mesenchymal stem cells (NMSC)). These
NMSC were found to be stable, i.e. maintained their morphology and
expressed markers through subsequent proliferation stages.
[0031] Further, it was shown by the inventors that treatment of
these stable NMSC with cerebrospinal fluid (CSF) allowed the NMSC
to differentiate into all three cell types of the ectoderm lineage,
namely, astrocytes, neurons and oligodendrocytes, as exhibited by
their specific markers (see Table 2 below).
[0032] The differentiation induced by CSF was cell specific and the
NMSC lost their potential to differentiate into cells of a
mesodermal lineage.
[0033] Thus, in accordance with its first aspect, the present
disclosure provides a method of inducing transdifferentiation of
mesenchymal stem cells (MSCs), the method comprising: [0034] (a)
culturing MSCs in a first culture medium comprising a growth factor
selected for allowing formation of neutralized MSCs (NMSC); [0035]
(b) allowing the NMSC to proliferate for a sufficient time during
which said culture medium is renewed at least once; [0036] (c)
culturing the NMSC in a second culture media comprising
cerebrospinal fluid (CSF) for a time sufficient for the NMSC to
differentiate into a population of cells comprising terminally
differentiated neurons, astrocytes and oligodendrocytes.
[0037] The above steps are conducted under commonly acceptable
incubator conditions that maintains optimal temperature (e.g.
approximately 37.degree. C.), humidity (typically >95%) and
other conditions such as the carbon dioxide (CO.sub.2, typically
5%) and oxygen content of the atmosphere inside the incubator with
periodical refreshing (replace and replenish) of the various
culture media.
[0038] As used herein, the term "differentiation" refers to a
process by which a less specialized cell becomes a more specialized
cell type. Differentiation is a common process where, for example,
stem cells divide and create partially or fully differentiated
daughter cells, e.g. during tissue repair and during normal cell
turnover. Differentiation dramatically changes a cell's size,
shape, membrane potential, metabolic activity, and responsiveness
to signals.
[0039] Further, as used herein, the term "transdifferentiation"
denotes a process that takes place when a non-stem cell transforms
into a different type of cell, or when an already differentiated
stem cell creates cells outside its already established
differentiation path. In accordance with the present disclosure,
the mesenchymal stem stells (the neutralized ones)
transdifferentiated into neurons, astrocytes and oligodendrocytes,
each being obtained at statistically significant amounts.
[0040] As used herein, the term "mesenchymal stem cells" (MSC)
denotes multipotent stromal stem cells that have the ability to
differentiate into cells of the mesodermal lineage, such as
adipocytes (fat cells), osteoblasts (bone cells) and chondrocytes
(cartilage cells).
[0041] The term "multipotent" refers to stem cells which are
capable of giving rise to many number of cell types.
[0042] The source of the MSC according to the present disclosure is
not limited and may be derived from any appropriate biological
source, for example from bone marrow, adipose tissue, umbilical
cord tissue, umbilical cord blood and peripheral blood. The source
may be human or non-human. In one embodiment, the MSC are human
MSC.
[0043] In some embodiments, the MSC are obtained from bone marrow.
The "bone marrow" (BM) refers to the flexible tissue found in the
interior of bones.
[0044] In some embodiments, the MSC are not obtained from an
adipose tissue.
[0045] In some further embodiments, the NMSC are not formed from
adipose-derived stem cells.
[0046] The MSCs may be obtained from the BM by conventional methods
such as aspiration or biopsy or any other method for providing
MSCs. In BM aspiration a semi-liquid is obtained which may be
further diluted with peripheral blood.
[0047] In some embodiments, the BM is harvested and the BM sample
is treated to select the mesenchymal cells (also referred to as
stromal cells). Selecting for BM mesanchymal/stromal cells may be
in a number of ways. For example, stromal cells may be
disaggregated and cultured inside a plastic container and then
separated by their survival in specific media and adherence to the
plastic.
[0048] The BM sample obtained from a subject may be stored under
appropriate conditions prior to use for example the sample can be
stored in liquid nitrogen after the separation of BM in mononuclear
cells using, for example, Sepax separation method or may be used
immediately after removal.
[0049] In some embodiments the BM may be obtained from a healthy
donor. Alternatively, the BM may be obtained from a subject
diagnosed with a disease, including, without being limited thereto,
neurodegenerative diseases or inflammatory disorders, including
autoimmune disorders. In some embodiments, the BM is obtained from
a subject diagnosed with multiple sclerosis (MS).
[0050] As noted herein, the BM is a source of MSCs (stromal cells).
The MSC may be identified by using five markers by FACS analysis
for identification of the cells.
[0051] For example, the cells may be characterized by at least of
the being negative for CD34 and CD45 and positive for CD73, CD105
and CD90. The term negative is used to note that no intensity or an
intensity that is like the control intensity is observed in the
FACS analysis. The term positive is used to note that a higher
intensity than control is observed in the FACS analysis.
[0052] In accordance with the present disclosure, the MSCs are
cultured in a culture medium that supports formation of MSC-derived
NPs like structures from MSC which are referred to herein as
"neutralized MSC".
[0053] As used herein the terms "neuralized MSC" or "NMSC" which
are used herein interchangeably, refer to non-adherent
(free-floating) spherical clusters of stem cells and progeny
therefrom. The NMSC have a neurosphere-like structures
characterized by the non-limiting markers, Nestin and PS-NCAM,
which are characteristic antigens for neurospheres; the NMSC are
further characterized by the capability to induce a dose-dependent
suppression of lymphocytes proliferation. As such, while having a
neurosphere structure, they are distinguished from classical
neurospheres generated from adult neural stem cells (at least for
the reason that they are produced from a different cell
source).
[0054] The culture medium that supports formation of NMSC from MSCs
at minimum comprises at least one growth factor. In addition, the
culture medium comprises a serum free medium supplemented with a
serum substitute. In some embodiments, the culture medium comprises
at least two growth factors. This culture medium is referred to
herein as the "first culture medium".
[0055] The first culture medium that supports formation of NMSC
from MSC may include a variety of combinations of serum free media,
serum substitutes and growth factors, as known in the art. For
example and without being limited thereto, the first culture medium
that supports the directed formation of NMSC comprises a basic
serum free medium selected from the group consisting of
Neurobasal.TM. (Gibco, Invitrogen cell culture, USA Cat. No.
21103-049 1998/1999), DMEM-F12 (Gibco, Invitrogen cell culture, USA
Cat. No. 11320-033), Cellgro Stem Cell Growth Medium (Cat No. 2001
CellGenix Germany 2005), KO-DMEM (Cat. No, 10829-018 Gibco
1998/1999) and X-Vivo 10 (Cat. No. 04-380Q Lonza Switzerland
2007).
[0056] In some embodiments, the serum free medium is DMEM-F12. The
serum free medium is DMEM-F12 typically comprises the following
ingredients:
TABLE-US-00001 COMPONENTS Concentration (.+-.5%) (mg/L) INORGANIC
SALTS Calcium chloride (CaCl2) 116.70 Cupric sulfate (CuSO4--5H2O)
0.0013 Ferric nitrate (Fe(NO3)3--9H20) 0.05 Ferrous sulfate
(FeSO4--7H2O) 0.417 Potassium chloride (KCl) 311.80 Magnesium
chloride (MgCl2) 28.64 Magnesium sulfate (MgSO4) 48.84 Sodium
chloride (NaCl) 6995.50 Sodium bicarbonate (NaHCO3) 1200 Sodium
phosphate, mono. 62.50 (NaH2PO4--H20) Sodium phosphate, dibas
(Na2HPO4) 71.02 Zinc sulfate (ZnSO4--7H2O) 0.432 OTHER COMPOUNDS
D-Glucose 3151.00 Hypoxanthine 2.05 Linolcic Acid 0.042 Lipoic Acid
0.105 Phenol red 8.10 Putrescine-2HCl 0.081 Sodium Pyruvate 55.00
HEPES 3575.00 Thymidine 0.365 AMINO ACIDS L-Alanine 4.45 L-Arginine
hydrochloride 147.50 L-Asparagine-H2O 7.50 L-Aspartic acid 6.65
L-Cysteine-HCl--H2O 17.56 L-Cystine 24.00 L-Glutamic acid 7.35
Glycine 18.75 L-Histidine-HCl--H2O 31.48 L-Isoleucine 54.47
L-Leucine 59.05 L-Lysine hydrochloride 91.25 L-Methionine 17.24
L-Phenylalanine 35.48 L-Proline 17.25 L-Serine 26.25 L-Threonine
53.45 L-Tryptophan 9.02 L-Tyrosine 38.70 L-Valine 52.85 VITAMINS
Biotin 0.0035 D-Calcium pantothenate 2.24 Choline chloride 8.98
Folic acid 2.65 i-Inositol 12.60 Niacinamide 2.02 Pyridoxal
hydrochloride 2.00 Pyridoxine hydrochloride 0.031 Riboflavin 0.219
Thiamine hydrochloride 2.17 Vitamin B12 0.68
[0057] The culture medium may be further supplemented by components
known to be used in culture, such as serum free supplement. In some
other embodiments, the serum free supplement is B27.
[0058] The B27 components are provided below:
Components
[0059] Biotin [0060] DL Alpha Tocopherol Acetate [0061] DL
Alpha-Tocopherol [0062] Vitamin A (acetate) [0063] BSA, fatty acid
free Fraction V [0064] Catalase [0065] Human Recombinant Insulin
[0066] Human Transferrin [0067] Superoxide Dismutase [0068]
Corticosterone [0069] D-Galactose [0070] Ethanolamine HCl [0071]
Glutathione (reduced) [0072] L-Carnitine HCl [0073] Linoleic Acid
[0074] Linolenic Acid [0075] Progesterone [0076] Putrescine 2HCl
[0077] Sodium Selenite [0078] T3 (triodo-I-thyronine)
[0079] The first culture medium also included at least one growth
factor, preferably, at least two growth factors, the growth
factor(s) being capable of stimulating at least growth, and
possible also proliferation and differentiation of the cells in the
medium. In some embodiments, the growth factor is selected from the
group consisting of Epidermal growth factor (EGF), Fibroblast
growth factor (FGF), such as FGF-1 (bFGF), Platelet-derived growth
factor (PDGF), Transforming growth factor alpha (TGF-.alpha.),
Transforming growth factor beta (TGF-.beta.). In some embodiments,
at least two growth factors are used. When using two growth
factors, a preferred embodiment comprises the use of EGF and
bFGF.
[0080] As shown in FIG. 3, the presence of growth factors enhances
the formation of NMSC. Specifically, the NMSC proliferation and
viability requires the presence of at least one of EGF and bFGF in
the culture media. Absence of one of these particular factors
results in decline of the proliferation and viability of the NMSC.
Absence of both growth factors eventually resulted in the
dissociation and death of the NMSC.
[0081] Accordingly, in some embodiments, the growth factor is at
least one of EGF and bFGF. In some other embodiments, the growth
factor is the combination of EGF and bFGF.
[0082] In some embodiments, EGF is human EGF, this includes,
without being limited thereto, the 6045 Da EGF protein known in the
art to promote cell growth.
[0083] In accordance with some embodiment, the formation of NP like
structures is on plastic dishes (flasks). In some further
embodiments, the cells are grown in flasks with minimal cell
adhesion. This may be achieved by using ultra low-Adherence.TM.
flasks as known in the art. The use of such non-adherent growth
environment allows the free floatation of the cells and formation
of the non-adherent floating spheres.
[0084] The first culture media is preferably refreshed at least
once and after several days. In one embodiment, the first culture
media is replaced every 2-4 days and in yet some embodiments, at
least once a week and even twice a week.
[0085] In accordance with some embodiments, the culture in the
first culture media is for a time sufficient to allow the
establishment/formation of the NMSC (exhibited by at least one of
the markers characteristic of NMSC, e.g. Nestin and PS-NCAM and
disappearance of the markers of their origin, i.e. the markers of
MSC, as further discussed below).
[0086] As appreciated, the minimal time for the formation of NMSC
from MSC will dependent, inter alia, on the conditions of culture
but also on the original MSC culture and the density of the MSC. A
person versed in the art will be able to determine the time
sufficient for formation of NMSC, for example, by trial and
error.
[0087] In some embodiments, formation of NMSC is observed after at
least 24-48 hours. Notwithstanding this fact, at times, the NMSC
are maintained in culture with the first culture media for more
than 48 so as to maximize density of the NMSC in the culture and
their stability. To this end, and in accordance with some
embodiments, the NMSC are cultured in the first culture media for
at least 2-10 days, preferably 4-7 days (with periodical
replenishing) to obtain the desired quality and density of the
NMSC. Accordingly, in some embodiments, the time sufficient for
culturing in a first culture medial is between 2-10 days, at times,
between 2 to 9 days, 3-8 days, 3-7 days and preferably between 4-7
days. Under these conditions, the culture is predominantly composed
of a low but significant proportion (95%) of NMSC (less than 5% of
MSC).
[0088] Once the modified culture (i.e. that containing
predominantly NMSC) is obtained, namely there is evidence for the
formation of neurosphere-like structures in the culture, the first
culture medium is replaced at least once with a fresh amount of
either the same first culture medium or modified version thereof
which contains at least one growth factor and/or at least a serum
free medium (this being regarded as the "renew culture medium"). In
some embodiments, this culture medium being renewed comprises the
same first culture medium, possibly serum free medium and growth
factors and is lacking the serum substitute (e.g. B-27 supplement).
In one embodiment, for proliferation and maintenance of the NMSC
the medium comprises at least DMEM-F12, EGE and bFGF.
[0089] The inventors have found that in contrast to cultured MSC
which express on their surface CD90 and CD105, more than 90% of the
cells within a single neurosphere like structure (namely, within
the NMSC) express Nestin and PS-NCAM, which are characteristic
antigens for neutralized MSC.
[0090] The NMSC formation was thus characterized by low expression
or lack of (negative, in scientific terms) a MSC marker selected
from the group consisting of CD90, and CD105 and positive
expression of a neurosphere marker selected from the group
consisting of nestin and PSNCAM.
[0091] In addition, the inventors have found that the NMSC have
immunomodulatory properties similar to the immunomodulatory
properties of naive MSCs as well of neural stem cells and were
shown to induce a dose-dependent suppression of the proliferation
of lymphocytes.
[0092] The NMSC are allowed to proliferation in the replaced
(renew) growth medium. It should be noted that the time period for
proliferation in the renewed medium is dependent inter alia on
several factors for example the density of the NMSC, number of NMSC
and viability of the NMSC.
[0093] In some embodiments, the time sufficient for further
proliferation (e.g. in the renewed media) is between 1 day to 7
days, at times between 2 days to 6 days, at times even between 3
days to 4 days. After a suitable period of time of about 3 days or
4 days the NMSC can be further used.
[0094] Once desired NMSC are formed ("desired" meaning expressing
at least the aforementioned markers and after the first culture
medium is at least once replaced with a new volume of first culture
medium), the culture medium may be replaced with a second culture
medium comprising at least a serum free medium as defined above and
cerebrospinal fluid (CSF). In some embodiments, the serum free
medium comprises at least DMEM-F12 together with CSF.
[0095] When referring to CSF it is to be understood as meaning the
clear colorless fluid produced in the brain and which may be
collected from the spinal cord of either the subject that needs
treatment, as further discussed below, or from a healthy donor, by
any known technique, including, without being limited thereto,
lumbar puncture. The amount of CSF may vary in the second culture
medium and in some embodiments, the volume is between about 0.1%
(v/v) to about 1% (v/v) CSF out of the total volume of the
medium.
[0096] As noted above, the CSF may be "allogeneic or "autologous".
In some embodiments, the CSF is an allogeneic CSF.
[0097] When referring to allogeneic CSF it is noted that the CSF
provided to a recipient from a genetically non-identical donor.
Namely, the CSF is obtained from a donor/patient and provided to a
different person in need of the resulting neuronal cells.
[0098] The CSF may be collected from donors who are either patients
for example in the emergency room or at a treatment department or
from donors per se.
[0099] When referring to autologous CSF it is noted that the CSF is
obtained from and provided to the same patient.
[0100] The second culture medium induces differentiation of the
NMSC. Specifically, visual inspection and immunehistochemistry of
specific markers, showed that the NMSC differentiate into cells of
an ectoderm lineage including terminally differentiated astrocyte,
neurons and oligodendrocyte. Specifically, as shown in FIG. 4A-4D,
the inventors have found that differentiation induced by CSF
resulted in the formation of Microtubule-associated protein 2
(MAP2)- and Class III .beta.-tubulin (.beta.III-tubulin or
.beta.-tubulin III), positive cells with neuronal morphology (FIGS.
4A and 4B, respectively). Glial fibrillary acidic protein
(GFAP)-positive cells with astrocyte morphology (FIG. 4C), and
2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNPase)-positive
cells with oligodendrocyte morphology (FIG. 4D).
[0101] In general, astrocytes are "star-shaped" cells as shown in
FIG. 4E. When referring to "astrocytes" for example in ex-vivo
tissues or in-vitro isolated, it should be understood to refer to
cells having both the matured astrocytes morphology (i.e. the star
shape) and being positive for staining for GFAP. Cells showing only
positive for staining for GFAP are not considered astrocytes.
[0102] In addition, it is known that naive mesenchymal stem cells
may hold a positive expression for GFAP (Foudah et al 2012,
Blondheim et al 2006) even not in their differentiated state. Thus,
morphological similarity for "star shaped" astrocytes is essential
for defining astrocytes.
[0103] In some embodiments, the astrocytes according to the
invention have a "star-shaped" like structures indicating their
resemblance to astrocytes.
[0104] As shown in FIG. 4C, the cells show a clear "star-shaped"
like structures and were positively stained with GFAP similarly to
real astrocytes isolated from the brain (FIG. 4E, prior art).
[0105] Thus, in some further embodiments, the differentiated NMSC
have both the star shape and are positive for GFAP. This is
supported, inter alia, by the following findings:
[0106] To compare with the cells of Ye et al., MSC were treated
with CSF (as described in Ye et al. ibid.) to obtain "neuron like
cells". FIGS. 4F to 4J, show that treatment of MSC with CSF
resulted in cells which showed positive staining for GFAP and Class
III .beta.-tubulin, respectively. FIGS. 4F and 4G show
immunohistochemistry staining of GFAP and Class III D-tubulin
respectively and FIGS. 4H and 4I show immunofluorescence staining
of GFAP and Class III f-tubulin respectively.
[0107] However, the morphology of these cells did not resemble real
neurons as no "star-shaped" like structures was observed. These
cells conserved their elongated fibroblast-like shape that is
identified with naive MSC.
[0108] Table 2 shows that cells obtained by the method disclosed
herein (i.e. after treatment of NMSC (and not MSC) with CSF)
express MAP2, Tubulin-beta-III, GFAP, S100, GalC and CNPase as
determined from staining of these markers. The cells obtained by
the method disclosed herein showed at least a 10% expression of
these markers.
[0109] For example, the cells obtained by the method disclosed
herein showed at least a 10% expression of GFAP and
Tubulin-beta-III, at times at least 20% expression, at times at
least 30% expression, at times at least 40% expression, at times at
least 50% expression, at times at least 60% expression, at times
even at least 65% expression.
[0110] For example, the cells obtained by the method disclosed
herein showed at least a 10% expression of MAP2 and S100, at times
at least 20% expression, at times at least 30% expression, at times
even at least 35% expression.
[0111] For example, the cells obtained by the method disclosed
herein showed at least a 10% expression of GalC and CNPase, at
times even at least 15% expression.
[0112] In connection with the present disclosure expression is
determined from the ratio of the positively stained cells
determined as detailed herein below. Staining may be obtained from
any method known in the art such as but not limited to FACS,
immunofluorescence.
[0113] In contrast, MSC treated directly with CSF (according to the
method of Ye et al) showed reduced expression as compared to the
cells obtained by the method disclosed herein (i.e. after treatment
of NMSC (and not MSC) with CSF).
[0114] As shown in Table 2 the expression as determined from
staining of the markers MAP2. Tubulin-beta-III. GFAP. S100. GalC
and CNPas is increased in the cells obtained by the method
disclosed herein (i.e. after treatment of NMSC (and not MSC) with
CSF).
[0115] Specifically, positive staining of the "neuron-like cells"
with GFAP and S100 was observed in the cells obtained by the method
of Ye et al. However, staining of .beta.-tubulin or MAP2 markers
was hardly observed and no staining was detected for the
oligodendrocytic markers GalC and CNPase.
[0116] Without being bound by theory, these results suggest that no
oligodendrocytes are obtained by the method described by Ye et
al.
[0117] Taken together, these results suggested that treatment of
MSC directly with CSF and not via the a priori formation on NMSC,
did not result in formation of neurons that need to exhibit an
arsenal of features characterizing neurons and did not have the
activity neurons.
[0118] On the other hand, the neurons derived from NMSC in
accordance with the present disclosure shows the required features
for defining the resulting cells as neurons. Specifically, the
resulting NMSC derived cells showed staining of antigens and cell
morphology which was identical to the staining and morphology
observed in differentiated neurospheres derived from adult neural
stem cells.
[0119] The resulting population of cells includes predominantly the
mixture of astrocytes, neural cells and oligodendrocytes.
[0120] The term "comprising predominantly" is used to denote a
population wherein at least 50%, at times 60%, at times even 90%,
at times 95%, at time even 99% or even 100% of the cells exhibit
characteristics of the above three cell types. The ratio between
the cell types may vary.
[0121] In some embodiments, the ratio between the three cell types
is 16:3:1 for neurons:astrocytes:oligodendrocytes. In some other
embodiments, the population comprises about 80% neurons, about 15%
astrocytes and about 5% oligodendrocytes.
[0122] In addition, if was found that the NMSC potential to
differentiate into cells of a mesodermal lineage was lost including
their ability to differentiate into adipocytes, osteoblasts, or
chondrocytes (data not shown).
[0123] Further, it was found that the neuronal cells that
differentiated from MSC not only express neuronal markers but
importantly were also functional.
[0124] Specifically, as shown in Table 1 in the Examples the effect
of differentiated NMSC on neurite length in N2A cells was superior
over the non-differentiated NMSC or MSC and as good as the effect
of the positive control used.
[0125] In addition, as shown in Table 3 neurite length in N2A cells
at the cells obtained by the method of Ye et al, namely MSC treated
with CSF, was lower compared to the cells obtained by the method
developed herein.
[0126] In general, when cells differentiate in culture towards
cells from the neuronal lineage, they secrete neurotrophic factors
such as: Brain-derived neurotrophic factor (BDNF), Nerve growth
factor (NGF), Ciliary neuronotrophic factor (CNTF) etc. Secretion
of these factors is highly important in terms of neuroprotection
and possible neuro-regeneration. As shown in Table 4, the
population of cells obtained in accordance with the present
disclosure secreted at least 5 times, 6 times, 7 times, 8 times, 9
times and even 10 times the amount of NGF as compared to naive MSC,
and at least 1.5 times, between 1.5 and 2 times or even essentially
twice the amount of BDNF as compared to naive MSC. In comparison,
treatment of MSC with CSF (in accordance with the method of Ye et
al.) resulted in secretion of much lower amounts of these growth
factors, as shown in Table 4 (column "MSC induced with CSF").
[0127] Thus, in some embodiments, the population of cells secreted
at least the above neurotrophic factors. In some other embodiments,
the population of cells secreted at least BDNF and NFG.
[0128] It was thus suggested that the population of cells
comprising terminally differentiated neurons, astrocytes and
oligodendrocytes obtained as disclosed herein have neurotrophic
effects and as such can promote protection and repair of
neurodegenerative diseases.
[0129] In other words, it was concluded that culturing stable NMSC
for at least 48 hours with CSF leads to a controlled path of
differentiation favoring ectoderm lineage including terminally
differentiated astrocyte, neurons and oligodendrocyte over
mesodermal lineage.
[0130] Without being bound by theory, it was suggested by the
inventors that formation of NMSC is a crucial step in affecting the
differentiation route towards ectoderm lineage to thereby obtain
the terminally differentiated astrocyte, neurons and
oligodendrocyte are obtained.
[0131] Taken together, the results presented herein provide a cell
population comprising terminally differentiated astrocyte, neurons
and oligodendrocyte that is valuable for neuronal survival,
neuronal growth and differentiation.
[0132] Once the population of cells is formed, they may used for
various applications, both in research and in medicine. When
referring to a population of cells it is to be understood as
meaning a population comprising terminally differentiated
astrocyte, neurons and oligodendrocyte.
[0133] As shown in FIG. 6 in the Examples the effect of
differentiated NMSC on clinical score of cEAE and animals mortality
was superior over the non-differentiated NMSC or MSC.
[0134] In some aspects, the present disclosure provides use of MSC
for the preparation of a composition comprising a population of
cells comprising terminally differentiated neurons, astrocytes and
oligodendrocytes.
[0135] In some other aspects, the present disclosure provides use
of neutralized mesanchymal stem cells (NMSC) for the preparation of
a population of cells comprising terminally differentiated neurons,
astrocytes and oligodendrocytes.
[0136] In some embodiments, the NMSC according with the present
disclosure may be characterized by low expression or lack of an MSC
marker selected from the group consisting of CD90, and CD105 and
expression of a neurosphere marker selected from the group
consisting of nestin and PSNCAM. In some other embodiments, the
population of cells may be characterized by expression of human
microtubule-associated protein 2 (MAP-2) being characteristic of
neurons, expression of Glial fibrillary acidic protein (GFAP) being
characteristic of astrocytes, and expression of 2'3'-cyclic
nucleotide 3'-phosphodiesterase (CNPase) being characteristic of
oligodendrocytes. The population of cells may be characterized by
secretion of neurotrophic growth factors at a level greater than
their level from naive mesenchymal stem cells. For example, the
population of cells may be characterized by secretion of NGF or
BDNF at a level greater than their level from naive mesenchymal
stem cells
[0137] The composition described herein may be a pharmaceutical
composition. The pharmaceutical composition optionally further
comprise at least one pharmaceutically acceptable excipient or
carrier. As used herein "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings and the
like. The use of such media and agents for pharmaceutical active
substances is well known in the art.
[0138] In some embodiments, the pharmaceutical composition is for
treating a pathological condition of the nervous system. In some
other embodiments, the pharmaceutical composition is for treating a
neurodegenerative disease or spinal cord injury. In yet some other
embodiments, the use and pharmaceutical composition is for treating
conditions that involve death of neurons and the treatment by the
population of cells in accordance with the invention involves
regeneration of neurons.
[0139] In the context of the present disclosure the term
"Neurodegenerative diseases" is used to denote any condition that
is characterized by progressive nervous system dysfunction and/or
neuron cell death. There are more than 600 disorders afflict the
nervous system. Neurodegenerative diseases may be associated with
cognition, movement, strength, coordination, or myelin impairment,
which are associated with the peripheral nervous system (PNS) or
the autonomous nervous system (ANS).
[0140] In some embodiments the neurodegenerative disease may be one
of but not limited to Parkinson's Disease (PD), Alzheimer's Disease
(AD) and other dementias, Degenerative Nerve Diseases,
Encephalitis, Epilepsy, Genetic Brain Disorders, Head and Brain
Malformations, Hydrocephalus, Multiple Sclerosis, Amyotrophic
Lateral Sclerosis (ALS or Lou Gehrig's Disease), Huntington's
Disease (HD), Prion Diseases, Frontotemporal dementia, Dementia
with Lewy bodies, Progressive supranuclear palsy, Corticobasal
degeneration, Multiple system atrophy, Hereditary spastic
paraparesis, Spinocerebellar atrophies, Amyloidoses, Motor neuron
diseases (MND), Spinocerebellar ataxia (SCA), stroke and Spinal
muscular atrophy (SMA).
[0141] In some embodiments, the neurodegenerative disease is
multiple sclerosis (MS). Multiple Sclerosis, also known as
disseminated sclerosis or encephalomyelitis disseminata, refers to
an inflammatory disease in which myelin sheaths around axons of the
brain and spinal cord are damaged, leading to loss of myelin and
scarring.
[0142] In the context of this aspect, when referring to treating it
may include inhibiting, preventing, ameliorating or delaying onset
it is to be understood as meaning improvement of at least one
characteristic of the disease such as: increase in disease free
periods, decrease in acute disease periods or decrease in severity
of the disease in the subject exhibiting at least the same disease
characteristics.
[0143] The population of cells to the subject in need thereof may
be self-administration as well as administration to the subject by
another person.
[0144] The composition may comprise an amount of the population of
cells that results in a medically statistically improvement of the
subject's condition based on criteria acceptable for the particular
condition being treated.
[0145] In some embodiments, the method involves administration by
transplantation of the cell population into a subject's brain or
cerebrospinal fluid.
[0146] The present disclosure provides in accordance with some
further aspects, a kit comprising: (i) a composition comprising
mesanchymal stem cells (MSC) (ii) instructions for use of the MSC
in a method of preparing a population of cells comprising
terminally differentiated neurons, astrocytes and oligodendrocytes,
the method comprising: (a) culturing MSC in a first culture medium
comprising a growth factor selected for allowing formation of
neuralized MSC (NMSC); (b) allowing the NMSC to proliferate for a
sufficient time during which said culture medium is renewed at
least once; (c) culturing the NMSC of (b) in a second culture media
comprising cerebrospinal fluid (CSF) for a time sufficient for the
NMSC to differentiate into a population of cells comprising
terminally differentiated neurons, astrocytes and
oligodendrocytes.
[0147] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification.
[0148] As used in the specification and claims, the forms "a", "an"
and "the" include singular as well as plural references unless the
context clearly dictates otherwise. For example, the term "a stem
cell" includes one or more stem cells and the term "stem cells"
includes one stem cell as well as more than one stem cell.
[0149] As used herein, the term "or" means one or a combination of
two or more of the listed choices.
[0150] Further, as used herein, the term "comprising" is intended
to mean that the methods and culture systems includes the recited
elements, but does not exclude others. Similarly, "consisting
essentially of" is used to define methods and systems that include
the recited elements but exclude other elements that may have an
essential significance on the functionality of the culture systems
of the inventions. For example, a culture system consisting
essentially of a basic medium and medium supplements will not
include or will include only insignificant amounts (amounts that
will have an insignificant effect on the propagation of cells in
the culture system) of other substances that have an effect on
cells in a culture. Also, a system consisting essentially of the
elements as defined herein would not exclude trace contaminants.
"Consisting of" shall mean excluding more than trace amounts of
other elements. Embodiments defined by each of these transition
terms are within the scope of this invention.
[0151] Further, all numerical values, e.g., concentration or dose
or ranges thereof, are approximations which are varied (+) or (-)
by up to 20%, at times by up to 10%, from the stated values. It is
to be understood, even if not always explicitly stated that all
numerical designations are preceded by the term "about". It also is
to be understood, although not always explicitly stated, that the
reagents described herein are merely exemplary and that equivalents
of such are known in the art.
NON-LIMITING EXAMPLES
Materials and Methods:
Bone Marrow Aspiration
[0152] Bone marrow aspiration was performed under short general
anesthesia with puncture from the posterior superior iliac crest
while the patient was lying in a left or a right lateral position.
Approximately 200 mL of bone marrow inocula is usually obtained
from each patient.
MSC Preparation and Culture
[0153] A culture of purified MSCs was prepared under aseptic
conditions (positively pressurized clean rooms) using filtered
sterilized Dulbecco modified Eagle medium with low glucose levels
(Qiagen, Valencia, Calif.) supplemented with 10% fetal bovine
serum, 1% L-glutamine, and 1% penicillin-streptomycin-nystatin
solution (all from Biological Industries, Kibbutz Beit-Haemek,
Israel).
[0154] Mesenchymal cells were then cultured for 40 to 60 days,
until they reached confluency, and were then harvested and
cryo-preserved in 10% dimethyl sulfoxide-containing medium in
liquid nitrogen (-196.degree. C.). At 2 weeks, a sample was taken
for sterility testing and quality control.
Generation and Propagation of Neutralized MSC (NMSC) from hMSC
[0155] For the generation of NMSC, hMSC were cultured in ultra
low-Adherence.TM. flasks (Corning, Mexico) in DMEM-F12 serum free
media (Biological Industries, Israel) containing 2% B-27 supplement
(Gibco, USA), 20 ng/ml basic fibroblast growth factor (bFGF,
Peprotech, Israel), 25 ng/ml epidermal growth factor (EGF,
Peprotech, Israel), 1% non-essential amino acids (Biological
Industries, Israel).
[0156] The cells were cultured for 14 days with medium exchange
twice a week. Floating neurosphere-like structures (NMSC) were
visible after 48 hours. The suspension was then washed gently by
centrifugation and cells pellet was re-suspended in DMEM-F12 serum
free media supplemented with 20 ng/ml bFGF, 25 ng/ml EGF, 5
.mu.g/ml heparin, 1% non-essential amino acids and 1% MEM-alpha
vitamins and seeded in ultra low-Adherence.TM. flasks for
propagation.
NMSC Differentiation Using Cerebrospinal Fluid (CSF)
[0157] To terminally differentiate NMSC into neural cell lineages,
intact NMSC from passage 2 were used (namely, after the NMSC
propagated at least once in the renew medium, i.e. the medium was
at least once replenished). The NMSC from passage 2 were cultured
in DMEM-F12/GlutaMax.TM. serum free medium (Invitrogen)
supplemented with 1% non-essential vitamins (biological industries)
and 0.2% autologous/allogenic CSF seeded in regular attachment
tissue vessels (NUNC, USA).
Immunostaining of NMSC and Naive MSC
[0158] The medium was aspirated and the NMSC were washed gently
with 0.03% Tween 20 (Sigma-Aldrich, Rehovot, Israel) diluted in
DPBS and then fixed with fresh paraformaldehyde, 4%, for 20 minutes
at room temperature. To stain the intracellular components, the
cells were permeabilized with Triton X-100 (Sigma-Aldrich, Rehovot,
Israel), 0.1%, for 10 minutes. For blocking nonspecific binding,
the cells were rinsed with 2.5% bovine serum albumin in DPBS for 45
minutes at room temperature on a slowly rotating plate. Then, the
cells were washed 3 times with 0.03% Tween 20 diluted in PBS, and
incubated for 120 minutes with the following primary antibodies for
mouse: anti human CD34, anti human CD45, anti human CD90, anti
human CD105 anti human Nestin, anti human PS-NCAM, anti human MAP2,
anti human GFAP, anti human MAP2 and anti human CNPase (all from
Abcam, UK) diluted to the required concentrations with DPBS buffer
containing 1% bovine serum albumin. After washing, the cells were
incubated with goat anti mouse fluorescein
isothiocyanate-conjugated and goat anti rabbit tetramethylrhodamine
isothiocyanate-conjugated secondary antibodies diluted in bovine
serum albumin buffer, 1%, on a slowly rotating plate for 45 minutes
in the dark at room temperature. The cells were mounted on slides
with DAPI-mounting solution (Vectashield, CA, USA) and examined
under fluorescence and light microscopy. The number of
immunoreactive cells was determined under a fluorescence microscope
(Nikon, Japan) in relation to the nuclei stained with DAPI.
Proliferation Assay for NMSC (Immunological Potential Test)
[0159] NMSC growth was measured using the XTT based assay
(Biological Industries, Israel). NMSC were seeded into 96 well
plates at a density of 2000 NMSC/well and cultured for 3 days in
the presence or absence of EGF (20 ng/ml, Peprotech, Israel), FGF-2
(25 ng/ml, Peprotech, Israel). After 3 days, 50 .mu.l of MTT
reaction solution was added to each well and plates were incubated
at 37.degree. C. for 2 hours. The absorbance of the samples against
background ground control as blank was measured with ELISA reader
(Beckman Coulter, USA) at a wavelength of 450-500 nm. In order to
measure reference absorbance (for non-specific binding), a
wavelength of 630-690 nm was used and subtracted from the 450-500
nm measurements. Within each experiment, absorbance at 590 nm
values was averaged across 3 replicate wells.
Differentiation of N2A Cells (Neurotrophic Effects Model)
[0160] N2A cell were cultured in 24-wells plates (200 cells/well)
in culture medium containing DMEM (high glucose formulation), 5%
FBS and 1% penicillin-streptomycin (all from Biological Industries,
Kibbutz Beit-Haemek, Israel). Differentiation was initiated in
cultures confluent at a degree of 25-50%. Conditioned medium from
naive-MSC. NMSC and terminally differentiated cells were added to
the cultured N2A cells for 3 days. To detect neurite outgrowth,
cultured cells were immunostained with the neuronal marker MAP2
(Abcam.RTM., Cambridge, UK). To measure neurites after
differentiation the computerized analysis software ImageJ was used
and the neurite length was calculated in pixels.
MSCs Treatment by Cerebrospinal Fluid (CSF)
[0161] The following protocol was adopted from Ye et al for
comparative study. Briefly, for differentiation into neuronal-like
cells, bone marrow stromal cells from two healthy volunteers were,
respectively, plated at 2.times.10.sup.6 cell/well (0.1 ml) on
poly-1-lysine coated (100 mg/ml Sigma) coverslips in six-well
plate. When cells grew to 70% confluence, cells were cultured with
10 .mu.l of auto-CSF supplemented to the culture medium every day
for 7 days.
Characterization of Cells after Treatment by Cerebrospinal Fluid
(CSF)
[0162] The CSF treated NMSCs were further analyzed by studying
additional parameters such as: Staining for the markers MAP2
(neuronal), S100 (Astrocytic) and GalC and CNPase (Oligodenrocyic),
Computerized evaluation of the neurites length and Growth factors
secretion by the differentiated cells. The results were compared to
the result obtained by the method of the present invention.
In Vivo Studies
[0163] Naive MSC were isolated from bone marrow of multiple
sclerosis patients as described above. These cells were induced to
form floating neruosphere-like structure (NMSC). Following NMSC
generation (as described above) the cells were exposed to neuronal
differentiation protocol using allogenic CSF to obtain
differentiated NMSC as described above. Chronic Experimental
Autoimmune Encephalomyelitis (cEAE) was induced with MOG.sub.35-55
peptide in female C57BL/6 mice. On day 8 after EAE induction, the
NMSC or the differentiated NMSC were injected intraventricularly
into the cEAE induced mice. The mice were scored daily for
neurological symptoms according to the EAE clinical severity scale
(0=asymptomatic; 1=partial loss of tail tonicity; 2=tail paralysis;
3=hind limb weakness; 4=hind limb paralysis; 5=4-limb paralysis;
6=death).
[0164] In a further study with MOG-induced EAE in C57bl/6 mice, are
intracerebroventriculary (ICV) injected on day 8 (following
induction) in accordance with one of the following treatment
groups: [0165] (1) EAE non-treated animals (n=8) [0166] (2) EAE
injected with naive MSC (n=8) [0167] (3) EAE injected with MSC
exposed to CSF (n=10) [0168] (4) EAE injected with NMSC (n=8)
[0169] (5) EAE injected NMSC exposed to CSF (n=10)
[0170] During the 30 days from induction, the animals are evaluated
by EAE clinical scores and histopathological parameters
(inflammation and axonal loss).
Results
[0171] Derivation of NMSC from BM-MSC
[0172] To generate NMSC, MSCs (of passage 2-3, FIG. 1A) were
trypsinized, collected, and cultured NMSC induction media
containing DMEM-F12 serum free media supplemented with B27
supplement, epidermal growth factor (EGF) and basic fibroblast
growth factor (basic FGF). Under these culture conditions cells
started to form spheres (the NMSC) after 2 day of culture (FIGS. 1B
and 1C).
Characterization of NMSC
[0173] To determine if the NMSC derived from MSCs resembled
neurospheres, it was studied whether these spheres expressed two
characteristic antigens for neurospheres, Nestin and PS-NCAM.
[0174] Immunocytochemistry revealed that most of the cells
(>90%) within a single sphere express Nestin and PS-NCAM (FIGS.
2A and 2B respectively, and FIG. 2C). In addition, it was
investigated whether these MSC-NPs express antigens that are
characteristic of MSCs. FACS analysis showed that these MSC-derived
spheres did not express CD34, CD45 but did weakly express
CD105(.about.5%), and CD90(.about.10%) (FIG. 2D).
Proliferation and Expansion of NMSC
[0175] In order to evaluate the proliferation rates and factors
affect the growth kinetics in vitro the XTT-based assay was used.
In these experiments, the essentiality of the epidermal and basic
growth factors was evaluated. It was found that MSC-NPs
proliferation and viability requires the presence of both growth
factors EGF and bFGF in the culture media (FIG. 3). Absence of one
of the factors results in decline of the proliferation and
viability of the NMSC. Absence of both growth factors eventually
resulted in the dissociation and death of the NMSC.
Neural Differentiation of NMSC
[0176] NMSC were cultured for differentiation as described in
methods. Fluorescence immunocytochemistry for neuronal (MAP2) and
glial cell antigens (GFAP and CNPase) was performed for MSC-NPs
cultured for 7 days under differentiation conditions. MSC-NPs were
able to differentiate into GFAP-positive cells with astrocyte
morphology, MAP2-positive cells with neuronal morphology, and
CNPase-positive cells with oligodendrocyte morphology (FIGS. 4A to
4D). When analyzed for their potential to differentiate into cells
of a mesodermal lineage, it was found that these MSC-NPs have lost
their ability to differentiate into adipocytes, osteoblasts, or
chondrocytes (data not shown).
NMSC Suppress Lymphocytes Proliferation In Vitro
[0177] To explore the capacity of NMSC to inhibit the proliferation
of lymphocytes isolated from peripheral blood donation, naive NMSC
at different doses (50, 100 and 250 spheres) were co-cultured with
lymphocytes at the presence of the mitogen PHA. The proliferation
was measured using the .sup.3H-incorporation assay after 3 days of
co-culture. As shown in FIG. 5. The NMSC were found to inhibit the
lymphocytes proliferations in a dose-dependent manner, similarly to
the inhibitory effect observed by naive MSC (Kassis et al., 2008)
and NSC (Einstein et al., 2007). Further, the NMSC were shown to
have greater similarity to classical neurospheres derived from
adult NSC.
[0178] In addition, as shown in Table 4, the cells treated with CSF
secreted neurotrophic factors such as: BDNF, NGF. These factors are
known to be of high importance in terms of neuroprotection and
possible neuro-regeneration and suggest the clinical use of the
cells obtained by the method of the present invention.
NMSC and Terminally Differentiated Cells have Neurotrophic Effects
In Vitro
[0179] Evaluation of the neurotrophic effects of NMSC and cells
differentiated from the NMSC was studied in the N2A cell
system.
[0180] In normal conditions, N2A cells undergo neural
differentiation after exposure to retinoic acid or serum
withdrawal. N2A cells were cultured with conditioned media from
NMSC and cells differentiated from NMSC (collected after 72 hours
of culture). The effects of the supernatant on neurite outgrowth of
the N2A were visualized by immunostaining with the neuronal marker
MAP2 and neurites length was calculated by the ImageJ Software.
[0181] Positive staining for the neuronal marker MAP2 was detected
after culturing for 72 hours with the supernatant of cells from all
cell types (not shown). As shown in Table 1, the length of the
generated neurites was 85.+-.10.7 and 127.+-.20.43 pixels/neurite
(calculating 20 cells, 150 neurites) following culture with
supernatant from NMSC and the differentiated NMSC respectively,
(p>0.1).
TABLE-US-00002 TABLE 1 Neurite outgrowth Diff MSC-NPs (induced with
Control MSC MSC-NPs CSF) 198 .+-. 32.2 57 .+-. 12.3 85 .+-. 10.7
127 .+-. 20.43
[0182] As a positive control, N2A were cultured with 20 .mu.M
retinoic acid. The differentiated cells showed positive staining
for the neuronal marker MAP2. The neurite length measurement of the
retinoic acid differentiated cells was 198.+-.32.2 pixels/neurite
meaning that the differentiated MSC-NP are as almost good as the
positive control indicating that NMSC hold a very powerful
neurotrophic effect.
Comparative Study:
[0183] The following describes the differences in the morphology of
the cells obtained in the method according to the present invention
vs. the cells obtained by the method described by Ye et al. In
addition, characterization of the cells obtained by the method
described by Ye et al is presented.
[0184] As shown below, the results show that the cells obtained by
the method of Ye et al do not differentiate to form
oligodendrocytes and are not active as the cells according to the
present invention.
[0185] MSC by grown using the protocol used by Ye et al and treated
with CSF also showed a positive staining Class III .beta.-tubulin.
However, in contrast to the above, MSC by grown using the protocol
used by Ye et al and treated with CSF, does not resemble the
morphology of astrocytes isolated from the brain (FIGS. 4F-4I).
This is in contrast to the cells obtained by the method described
herein, which have a "star-shaped" like structures and astrocytes
isolated from the brain (FIG. 4C).
[0186] The cells obtained by the method of Ye et al were compared
to the cells obtained by the method of the present invention. The
results are summarized in Table 2.
TABLE-US-00003 TABLE 2 Ratio of the positive stained cells MSC
induced Cell Type with CSF NMSC induced Marker (marker) (Ye et al)*
with CSF* MAP2 Neurons 0-5% 75-85% Tubulin-beta-III Neurons 0-3%
68-77% GFAP Astrocytes 5-10% 45-55% S100 Astrocytes 5-8% 35-45%
GalC Oligodendrocytes 0% 15-20% CNPase Oligodendrocytes 0% 10-18%
*% positive cells, replicates of 5 wells.
[0187] As can be seen in Table 2, in the cells obtained by the
method of Ye et al, low positive staining of neuron-like cells with
the markers Tub-beta-III or MAP2 was observed. Some of the cells
indeed expressed positive staining for the astrocytic markers GFAP
and S100.
[0188] In addition, no positive staining was detected for the
oligodendrocytic markers GalC and CNPase. Further, to the
morphological results showing that the CSF-treated cells conserved
their elongated fibroblast-like shape that is identified with naive
MSC, the results suggest that treatment of MSC with CSF is not
sufficient to induce differentiation as obtained by the method of
the present invention.
[0189] To detect neurite outgrowth, cultured cells were
immunostained with the neuronal marker MAP2. To measure neurites
after differentiation the computerized analysis software ImageJ was
used and the neurite length was calculated in pixels. The results
are shown in Table 3:
TABLE-US-00004 TABLE 3 Neurite outgrowth MSC induced with CSF* NMSC
induced with CSF* 37 .+-. 8 pixels/neurite 118 .+-. 12
pixels/neurite *pixels .+-. SD, calculation of 150 neurites
TABLE-US-00005 TABLE 4 Growth factor secretion Factor MSC induced
(0.D.sub.405 nm) Naive MSC with CSF NMSC induced with CSF NGF 0.2
.+-. 0.35* 0.8 .+-. 0.09* 2.7 .+-. 1.3* BDNF 1.4 .+-. 0.7* 1.7 .+-.
0.4* 3.3 .+-. 0.9* *Triplicates
[0190] As shown in Table 4, the cells obtained by the method of Ye
et al secrete low amounts of neurotrophic factors (BDNF, NGF)
compared to the cells obtained by the method described herein.
NMSC Attenuated Chronic Experimental Autoimmune Encephalomyelitis
(cEAE) In Vivo
[0191] As shown in FIG. 6, while NMSC attenuated disease severity
after transplantation, differentiated NMSC provided significantly
higher clinical scores. Specifically, the clinical course of cEAE
was improved in NMSC treated animals (n=8), with 0% mortality and
mean maximal EAE score 1.75 vs. 33% mortality and 3.33 mean maximal
score in non-treated animals (n=10). Moreover, using differentiated
NMSC (n=7) the effect was more superior with 0% mortality and mean
maximal EAE score of 0.6 vs. 1.75 of NMSC and 3.33 of untreated
animals.
* * * * *