MULTIPOTENT NEURAL STEM CELLS THAT EXPRESS PLATELET DERIVED GROWTH FACTOR (PDGF) RECEPTOR AND METHODS OF USE THEREOF
PRIORITY CLAIM
[0001] This application claims priority to and the benefit of US Provisional Application No. 62/422,389, filed November 15, 2016, the contents of which are incorporated herein by reference in their entirety.
FIELD
[0002] The disclosure provides multipotent neural stem cells that express platelet derived growth factor (PDGF)-alpha receptor and the use of such cells to produce oligodendrocytic cells.
BACKGROUND
[0003] Glial cells, which include oligodendrocytes and astrocytes, play a critical role in the function of the mammalian central nervous system (CNS). Among other functions, these cells provide the insulating myelin needed for the efficient propagation of impulses along nerve fibers, provide trophic support for neuronal cells, and remove toxins and excess neurotransmitters from the interstitial space. Transplantation of glial cells or glial progenitors into the CNS can have therapeutic benefits in a number of disease pathologies. However, generation of large quantities of glia, in particular oligodendrocytes, for transplantation is technically challenging. Furthermore, generation of glial progenitors from sources such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells poses potential safety risks due to the tumorigenic potential of undifferentiated cells.
[0004] Thus, there exists a need for compositions and methods for the generation and expansion of myelinating oligodendrocytes from tissue-restricted neural stem cells.
SUMMARY
[0005] The present disclosure relates to multipotent neural stem cells that express PDGF receptor (e.g., PDGF-alpha receptor) and the use of such cells to produce oligodendrocytic precursor cells. [0006] Also provided herein is a culture of human multipotent neural stem cells, including a culture of expanded human multipotent neural stem cells, that express PDGF receptor.
[0007] In some embodiments, the expanded multipotent neural stem cells that express PDGF receptor differentiate (e.g., upon withdrawal of growth factors such as insulin, PDGF, and/or basic fibroblast growth factor (bFGF)) to produce greater than 25 percent oligodendrocytic cells.
[0008] In some embodiments, the expanded multipotent neural stem cells that express PDGF receptor produce greater than 50 percent oligodendrocytic cells.
[0009] In some embodiments, the expanded multipotent neural stem cells that express PDGF receptor produce greater than 75 percent oligodendrocytic cells.
[0010] In some embodiments, the expanded multipotent neural stem cells express PDGF receptor alpha.
[0011] The present disclosure also provides a method for producing human oligodendrocytic cells from human multipotent neural stem cells comprising cultivating expanded human multipotent neural stem cells in a serum-free medium comprising insulin or IGF-1, and optionally PDGF and/or bFGF, wherein the cultivated expanded human multipotent neural stem cells produce human oligodendrocytic cells.
[0012] In some embodiments, PDGF is present at a concentration of at least 1 ng/ml.
[0013] In some embodiments, bFGF is present at a concentration of at least 1 ng/ml.
[0014] In some embodiments, insulin is present at a concentration of at least 1 ng/ml.
[0015] In some embodiments, insulin is present at a concentration of less than about 25 μg/ml.
[0016] In some embodiments, insulin is present at a concentration of about 5 μg/ml.
[0017] In some embodiments, IGF-1 is present at a concentration of at least 1 ng/ml.
[0018] In some embodiments, the PDGF receptor is PDGF receptor alpha.
[0019] In some embodiments, the oligodendrocytic cells produced from the human multipotent neural stem cells exceed about 25 percent of the differentiated cells.
[0020] In some embodiments, the oligodendrocytic cells produced from the human multipotent neural stem cells exceed about 50 percent of the differentiated cells. [0021] In some embodiments, the oligodendrocytic cells produced from the human multipotent neural stem cells exceed about 75 percent of the differentiated cells.
[0022] In some embodiments, greater than about 25 percent of the cells produced from the human multipotent neural stem cells are 01ig2+.
[0023] In some embodiments, greater than about 50 percent of the cells produced from the human multipotent neural stem cells are 01ig2+.
[0024] In some embodiments, greater than about 75 percent of the cells produced from the human multipotent neural stem cells are 01ig2+.
[0025] In some embodiments, the human multipotent neural stem cells are derived from embryonic stem cells.
[0026] In some embodiments, the human multipotent neural stem cells are derived from fetal CNS.
[0027] In some embodiments, the human multipotent neural stem cells are derived from induced pluripotent stem cells.
[0028] The present disclosure also provides an isolated human oligodendrocytic cell produced by any of the methods disclosed herein.
[0029] The present disclosure also provides a human neural stem or an oligodendrocytic cell that expresses one or more antigenic markers selected from the group consisting of CD44 (ECMRII), CD49c (Integrin a3), CD49e (Integrin <x5), CD54 (ICAM-1), CD56 (NCAM), CD57 (HNK1), CD58 (LFA-3), CD59 (H19, Protectin), CD63 (LIMP), CD71 (Transferrin receptor), CD73 (NT5E), CD81 (TAPAl), CD90 (Thy-1), CD98 (SLC3A2), CD99 (MIC2), CD99R (CD99 MAB restricted), CD 140a (PDGFa receptor), CD 142 (Thromboplastin), CD 146 (MCAM), CD147 (Basigin), CD151 (PETA-3), CD164 (MGC-24), CD165 (AD2), CD166 (ALCAM), CD171 (LI CAM), CD271 (NGFR, p75NTR), β2 microglobulin, EGFR, HLA-A, HLA-B, HLA- C, HLA-A2, HLA-DQ, HLA-DR, HLA-DP, GD2, and CD49f (integrin a6).
[0030] Also provided by the present disclosure is a serum-free composition for producing oligodendrocytic cells from an expanded culture of human multipotent neural stem cells that express PDGF receptor, the composition comprising insulin or IGF-1 ; and optionally bFGF and/or PDGF.
[0031] In some embodiments, bFGF is present at a concentration of about 10 ng/ml.
[0032] In some embodiments, PDGF is present at a concentration of about 20 ng/ml. [0033] In some embodiments, insulin is present at a concentration of less than about 25 μg/ml.
[0034] In some embodiments, insulin is present at a concentration of about 5 μg/ml.
[0035] In some embodiments, insulin is present at a concentration of greater than about 1 ng/ml.
[0036] In some embodiments, the serum-free composition is entirely lacking insulin-like growth factor 1 (IGF-1).
[0037] The present disclosure also provides a serum-free composition for producing differentiated oligodendrocytes from the cells of claim 6, comprising insulin but lacking bFGF and PDGF.
[0038] In some embodiments, insulin is present at a concentration of less than about 25 μg/ml.
[0039] In some embodiments, insulin is present at a concentration of about 5 μg/ml.
[0040] In some embodiments, insulin is present at a concentration of greater than about 1 ng/ml.
[0041] In some embodiments, the serum-free composition is entirely lacking IGF-1.
[0042] The present disclosure also provides a method for producing differentiated oligodendrocytes comprising the steps of culturing human multipotent neural stem cells in the serum free composition as disclosed herein and isolating differentiated cells including differentiated oligodendrocytes therefrom.
[0043] In some embodiments, the differentiated oligodendrocytes produced from the multipotent neural stem cells exceed about 30 percent of the differentiated cells.
[0044] In some embodiments, greater than about 30 percent of the differentiated cells produced from the multipotent neural stem cells express oligodendrocyte marker 4 (04).
[0045] In some embodiments, greater than about 30 percent of the differentiated cells produced from the multipotent neural stem cells express class III beta-tubulin (TuJl).
[0046] In some embodiments, greater than about 30 percent of the differentiated cells produced from the multipotent neural stem cells express galactosylceramidase (GalC).
[0047] The present disclosure also provides a method for treating a mammalian subject with demyelinated nerve tissue, comprising engrafting the subject with the multipotent neural stem cells as disclosed herein. [0048] In some embodiments, greater than about 70 percent of the engrafted multipotent neural stem cells differentiate into myelinating oligodendrocytes in vivo.
[0049] In some embodiments, greater than about 70 percent of the engrafted multipotent neural stem cells express myelin basic protein (MBP) in vivo after differentiation.
[0050] In some embodiments, between 80 and 97 percent of the engrafted multipotent neural stem cells differentiate into myelinating oligodendrocytes in vivo.
[0051] In some embodiments, between 80 and 97 percent of the engrafted multipotent neural stem cells express MBP in vivo after differentiation.
[0052] The present disclosure also provides compositions and methods for the generation of oligodendrocytic cells from multipotent neural stem cells, and/or the large-scale expansion of these multipotent neural stem cells while retaining their capacity to differentiate into oligodendrocytes. These methods can be used to generate large quantities of multipotent neural stem cells that can give rise to myelinating oligodendrocytes for the treatment of neurodegenerative diseases or trauma to the nervous system. They can also be used as components in a process for screening activity or toxicity of drugs or molecular entities in vitro. Therapeutic applications may include those that rely on the efficient generation of myelinating oligodendrocytes, including but not limited to multiple sclerosis, transverse myelitis, optic neuritis, traumatic brain injury, stroke, and spinal cord injury.
[0053] In an embodiment, the disclosure comprises a medium composition for expanding multipotent neural stem cells with improved capacity for generating oligodendrocytic cells.
[0054] In an embodiment, the disclosure comprises a medium composition promoting specific expansion of multipotent neural stem cells. In such embodiment, the disclosure comprises a serum free medium comprising insulin and optionally bFGF and/or PDGF. In one embodiment, bFGF is human bFGF and PDGF is human PDGF. In one embodiment, insulin is human insulin. In one embodiment, insulin is present at less than 25 μg/ml and more than 1 ng/ml, preferably about 5 μg/ml, and optionally bFGF is present at about 10 ng/ml and/or PDGF is present at about 20 ng/ml. In a related embodiment, the serum free medium is entirely lacking IGF-1.
[0055] In an embodiment, the disclosure comprises methods and compositions for producing myelinating oligodendrocytes from multipotent neural stem cells. In some embodiments, the compositions may comprise a serum free medium comprising insulin and entirely lacking bFGF and PDGF. In one embodiment, the insulin is present at less than 25 μg/ml and more than 1 ng/ml, preferably about 5 μg/ml. In a related embodiment, the serum free medium is entirely lacking IGF-1.
[0056] In one embodiment, the disclosure comprises a method for producing oligodendrocytic progenitor cells from multipotent neural stem cells wherein the multipotent neural stem cells are derived from fetal brain tissue or embryonic tissue. In another embodiment, the multipotent neural stem cells are derived from an induced pluripotent or an embryonic stem cell.
[0057] In one embodiment, the neural stem cells are obtained from a fetal tissue of about 8 weeks or older gestational age. In a further embodiment, the fetal tissue is from the forebrain, midbrain, hindbrain, or spinal cord.
[0058] In one embodiment, the disclosure comprises a method for producing from multipotent neural stem cells oligodendrocytes capable of producing myelin.
[0059] In an embodiment of the disclosure, multipotent neural stem cells are engrafted to repair demyelinated nervous tissue in a subject suffering from nerve damage or chronic conditions that are associated with loss of oligodendrocytes, nerve demyelination, or reduction or dysfunction of products made by oligodendrocytes. In a related embodiment, the disclosure comprises engrafting oligodendrocytic cells in a subject suffering from nerve damage or chronic conditions that are associated with oligodendrocytes or nerve demyelination.
[0060] In an embodiment, more than about 70 percent of engrafted multipotent neural stem cells differentiate into oligodendrocytes. In a preferred embodiment, 80 to 97 percent of the engrafted multipotent neural stem cells differentiate into oligodendrocytes.
[0061] In an embodiment of the disclosure, human oligodendrytic cells are treated with drug candidates to identify compounds that promote or inhibit growth, survival, or differentiation of such cells. In a related embodiment, differentiated oligodendrocytes are treated with drug candidates to identify compounds that promote or inhibit cell growth, survival, migration, or production of myelin. DESCRIPTION OF THE DRAWINGS
[0062] The foregoing summary as well as the following detailed description will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the disclosure, shown in the figures are embodiments that are presently preferred. It should be understood, however, that the disclosure is not limited to the precise arrangements, examples and instrumentalities shown.
[0063] Figure 1 shows the proportion of oligodendrocytes generated by human neural stem cell cultures derived from various gestational ages. Neural stem cells isolated from human hindbrain CNS tissue of various gestational ages were expanded in the presence of bFGF and PDGF, were forced to differentiate, and were evaluated for the presence of oligodendrocytes.
[0064] Figure 2 shows the expression of PDGF receptor alpha in NSI-777 neural stem cells, which are capable of generating high proportions of oligodendrocytes, and NSI-566 stem cells, which do not generate high proportions of oligodendrocytes.
[0065] Figure 3 shows differentiation properties of three representative neural stem cell lines in vitro and in vivo. Cells were expanded under the conditions described and allowed to differentiate in vitro or were grafted into MBP-deficient mice and evaluated 4 months later and analyzed for expression of phenotypic markers.
[0066] Figure 4 shows differentiation of neural stem cells into oligodendrocytes capable of myelination after transplantation into myelin-deficient mice. GFP-labeled human cells (green, left image) grafted into myelin-deficient mice were also positive for myelin basic protein (red, middle image). Combined image is shown on the right.
[0067] Figure 5 shows generation of myelin sheath around axons in the graft location in myelin-deficient mice that have received expanded NSI-777.
DETAILED DESCRIPTION
[0068] Provided herein are methods for the generation of oligodendrocytic cells from NSCs that express PDGF receptor grown under defined serum-free conditions, including the use of a survival factor such as insulin at concentrations ranging from 0.001-25 μg/L and optionally the use of a mitogen such as bFGF at approximately 10 ng/ml. Surprisingly, the inventors have discovered that NSCs (e.g., NSCs obtained from gestational age about 8-12 week hindbrain tissue) grown with a reduced concentration of insulin and optionally PDGF express PDGF-alpha receptor and preferentially differentiate upon removal of growth factors (e.g., insulin) into oligodendrocytic cells. Optionally, the cells may also be grown in the presence of bFGF. The resulting cell population can be expanded through many passages and generated 25-95 percent oligodendrocytes. Such cells may be used to repair demyelinated nervous tissue in a subject suffering from nerve damage or chronic conditions that are associated with nerve demyelination.
[0069] The disclosure provides methods for producing human oligodendrocytic cells from human multipotent neural stem cells, the methods comprising cultivating human multipotent neural stem cells in a serum-free medium comprising insulin or IGF-1, and optionally comprising PDGF and/or bFGF (e.g., the serum-free medium may comprise an initial concentration of insulin or IGF-1, and may optionally comprise an initial concentration of PDGF and/or bFGF), wherein the cultivated human multipotent neural stem cells differentiate into human oligodendrocytic cells including, for example, upon withdrawal of one or more growth factors (e.g., the serum-free medium comprises a second concentration of insulin or IGF-1, PDGF and/or bFGF that is zero or substantially zero). Notably, the cultivation of the human multipotent neural stem cells by the methods disclosed herein generates an expanded culture of human multipotent neural stem cells that express PDGF-alpha receptor (i.e., the human multipotent neural stem cells are programmed by the cultivation methods disclosed herein to express PDGF-alpha receptor).
[0070] Neural stem cells expressing PDGF receptors (e.g., PDGF-alpha receptors) were expanded by the described compositions and processes and subsequently differentiated such that greater than 25 percent, 30 percent, 35 percent, 40 percent, 45 percent, 50 percent, 55 percent, 60 percent, 65 percent, 70 percent, 75 percent, 80 percent, 90 percent or 95 percent or more of the differentiated progeny cells represent oligodendrocytes.
[0071] The presence of PDGF is responsible for determining whether NSCs can efficiently generate oligodendrocytes. NSCs expanded in the presence of PDGF and relatively low concentrations of insulin (approximately 1-5 μg/ml of medium) retain the ability to generate neurons, astrocytes, and oligodendrocytes. In particular, these cells give rise to at least 25 percent and as much as 95 percent oligodendrocytes upon differentiation, with the remaining cells divided between neurons and astrocytes. [0072] Undifferentiated NSCs expanded under these conditions express additional antigens (markers) that distinguish them from NSCs that do not express PDGF-alpha receptor. These include 01ig2 and A2B5, which are recognized markers for undifferentiated oligodendrocyte progenitors. The presence of 01ig2, A2B5, or PDGF-alpha receptor can be used to isolate the desired oligodendrytic progenitor NSCs, for example, by well-known affinity methods utilizing antibodies such as AB 15328 specific to 01ig2 (EMD Millipore, Billerica, MA), anti-A2B5 (LifeSpan Biosciences, Inc., Seattle, WA), and/or olaratumab specific to PDGF receptor alpha (Eli Lilly, Indianapolis, IN). Upon grafting into various animal models of demyelinating disorders, these cells are capable of migrating long distances from the graft site, integrating into the host tissue, and differentiating into oligodendrocytes with the ability to generate myelin and ensheath host neurons.
[0073] In addition to expressing 01ig2, A2B5, or PDGF-alpha receptor, neural stem cells produced by the methods described herein also uniquely express one or more of the markers listed in Table 1, and can be identified by use of combinations of the listed cognate antibodies. Neural stem cells expressing one or more of these markers produce much higher levels of differentiated oligodendrocytes under the conditions described herein than those derived from other methods and sources, while simultaneously minimizing the risk of turn orogenesis associated with differentiated stem cell transplants.
Table 1
Antibody Antigenic Marker
G44-26 CD44 (ECMRII)
C3 II.1 CD49c (Integrin a3)
VC5 CD49e (Integrin a5)
LB-2 CD54 (ICAM-1)
B159 CD56 (NCAM)
NK-1 CD57 (HNK1)
1C3 CD58 (LFA-3)
p282 (HI 9) CD59 (HI 9, Protectin)
H5C6 CD63 (LIMP)
M-A712 CD71 (Transferrin receptor)
AD2 CD73 (NT5E)
JS-81 CD81 (TAPAl)
5E10 CD90 (Thy-1)
UM7F8 CD98 (SLC3A2)
TU12 CD99 (MIC2) Antibody Antigenic Marker
HIT4 CD99R (CD99 MAB restricted)
alpha Rl CD 140a (PDGFa receptor)
HTF-1 CD 142 (Thromboplastin)
P1H12 CD146 (MCAM)
HIM6 CD 147 (Basigin)
14A2.H1 CD151 (PETA-3)
N6B6 CD 164 (MGC-24)
SN2 CD 165 (AD2)
3A6 CD 166 (ALCAM)
5G3 CD171 (LI CAM)
C40-1457 CD271 (NGFR, p75NTR)
TU99 β2 microglobulin
EGFR1 EGFR
G46-2.6 HLA-A, B, C
BB7.2 HLA-A2
TU169 HLA-DQ
G46-6 HLA-DR
TU39 HLA-DP, DQ, DR
14.G2A GD2
GoH3 CD49f (integrin <x6)
G44-26 CD44 (ECMRII)
C3 II.1 CD49c (Integrin a3)
[0074] The neural stem cells obtained by the methods provided herein may be used to treat a mammalian subject with demyelinated nerve tissue, comprising engrafting the subject with an amount (e.g., an effective amount) of the cells disclosed herein. Notably, greater than about 70 percent (including between 80 and 97 percent) of the engrafted cells differentiate into oligodendrocytes in vivo. The methods of the disclosure may be used to treat, including reverse, a neurodegenerative disease or disorder.
[0075] As used herein, a neurodegenerative condition can include any disease or disorder or symptoms or causes or effects thereof involving the damage or deterioration of neurons. Neurodegenerative conditions can include, but are not limited to, Alexander Disease, Alper's Disease, Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Ataxia Telangiectasia, Canavan Disease, Cockayne Syndrome, Corticobasal Degeneration, Creutzfeldt-Jakob Disease, Huntington Disease, Kennedy's Disease, Krabbe Disease, Lewy Body Dementia, Machado- Joseph Disease, Multiple Sclerosis, Parkinson's Disease, Pelizaeus-Merzbacher Disease, Niemann-Pick's Disease, Primary Lateral Sclerosis, Refsum's Disease, Sandhoff Disease, Schilder's Disease, Steele-Richardson-Olszewski Disease, Tabes Dorsalis or any other condition associated with damaged neurons. Other neurodegenerative conditions can include or be caused by traumatic spinal cord injury, ischemic spinal cord injury, stroke, traumatic brain injury, and hereditary conditions.
[0076] In some embodiments, "treating" or "treatment" of a disease, disorder, or condition includes at least partially (1) preventing the disease, disorder, or condition, i.e., causing the clinical symptoms of the disease, disorder, or condition not to develop in a mammal that is exposed to or predisposed to the disease, disorder, or condition but does not yet experience or display symptoms of the disease, disorder, or condition; (2) inhibiting the disease, disorder, or condition, i.e., arresting or reducing the development of the disease, disorder, or condition or its clinical symptoms; or (3) relieving the disease, disorder, or condition, i.e., causing regression of the disease, disorder, or condition or its clinical symptoms.
[0077] In some embodiments, "effective amount," as used herein, refers to the amount of an active composition that is required to confer a therapeutic effect on the subject. A "therapeutically effective amount," as used herein, refers to a sufficient amount of an agent or a compound being administered that will relieve to some extent one or more of the symptoms of the disease, disorder, or condition being treated. In some embodiments, the result is a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, in some embodiments, an "effective amount" for therapeutic uses is the amount of the composition including a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms without undue adverse side effects. In some embodiments, an appropriate "effective amount" in any individual case is determined using techniques, such as a dose escalation study. The term "therapeutically effective amount" includes, for example, a prophylactically effective amount. In other embodiments, an "effective amount" of a compound disclosed herein, such as a compound of Formula (A) or Formula (I), is an amount effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects. In other embodiments, it is understood that "an effective amount" or "a therapeutically effective amount" varies from subject to subject, due to variation in metabolism, age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.
[0078] This disclosure is further illustrated by the following examples, which are provided to facilitate the practice of the disclosed methods. These examples are not intended to limit the scope of the disclosure in any way.
Example 1
Isolation and propagation of NSCs.
[0079] NSCs were obtained from tissue isolated post-mortem from aborted human fetuses. Tissue donations were made with full consent of the donor following guidelines established and monitored by an independent review board. Multiple independent isolates of NSCs were successfully achieved using tissue from multiple regions and gestational ages, including cortex, hippocampus, thalamus, midbrain, cerebellum, and hindbrain; spinal cord; and dorsal root ganglia. Upon identification of the region of interest, tissue was minced in a solution of Hanks buffered saline solution (HBSS) using a tungsten needle, and cells were then manually dissociated using gentle trituration. Cells were then plated on tissue culture-treated dishes precoated with poly D-lysine and fibronectin and cultured in serum-free growth medium (consisting of DMEM/F12 base media with glucose, glutamine, sodium bicarbonate, HEPES, 25 μg/ml insulin, apo-transferrin, progesterone, putrescine, sodium selenite, and 10 ng/ml bFGF). Media was completely replaced every other day and bFGF was added fresh daily.
[0080] Cells were expanded under these conditions until they reached approximately 80 percent confluence, at which point they were passaged by washing twice with FIBSS and incubating briefly in the presence of 0.05 percent trypsin. Trypsin was inactivated by the addition of soybean trypsin inhibitor and cells were pelleted by centrifugation, then resuspended in growth medium and replated on precoated 15 cm dishes (surface area 175 cm2) at a density of 1.0-1.5xl06 cells per dish. This process was continued for multiple passages to generate sufficient numbers of cells for cryopreservation. Example 2
Expansion of neural stem cells.
[0081] One NSC line, NSI-777, was used to develop and optimize the procedures described herein. Early passage cells were grown under standard conditions (as described in Example 1) or were switched to conditions consisting of growth medium with the following modifications: reduced insulin (5 μg/ml) and the use of PDGF (20 ng/ml) as mitogen in addition to bFGF. We found that cells from human hindbrain tissue derived from gestational ages about 11-12 weeks responded optimally to this treatment by generating more oligodendrocytes upon differentiation than cells derived from earlier gestational age tissue (Figure 1). In contrast to the stem cell line NSI-566, NSI-777 is immunopositive for PDGF receptor alpha by immunocytochemistry (Figure 2).
Example 3
Prolonged expansion of glial precursor cell lines.
[0082] We discovered two additional NSC lines under the same growth conditions and evaluated their ability to expand and then generate oligodendrocytes upon withdrawal of growth factors. During a period of over three months in culture, cells from all 3 lines proliferated with consistent and predictable kinetics, showing no signs of crisis or changes in morphology (Figure 3). All cell lines evaluated in this way showed similar growth and differentiation properties throughout the evaluation. One line was carried well beyond this point, and showed no significant changes to expansion or proliferation.
[0083] Upon differentiation in vitro, all 3 lines gave rise to neurons, astrocytes, and oligodendrocytes, with the oligodendrocyte population typically comprising at least 30 percent of total cells (Figure 3).
Example 4
Transplantation of glial progenitors into animal models of demyelinating disease.
[0084] In order to demonstrate the capacity of NSI-777-derived oligodendrocytes to produce myelin, we transplanted the expanded cell population into shiverer mice that are deficient in the ability to produce myelin. NSI-777 survived and differentiated into oligodendrocytes that produce myelin (Figure 4). Electron microscopy showed the presence of myelin with classical appearance in intimate proximity with axons in the region of the graft (Figure 5).
[0085] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
[0086] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0087] The terms "a," "an," "the" and similar referents used in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed. No language in the specification should be construed as indicating any non- claimed element essential to the practice of the disclosure.
[0088] Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified, thus fulfilling the written description of all Markush groups used in the appended claims.
[0089] Certain embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.
[0090] Specific embodiments disclosed herein can be further limited in the claims using "consisting of and/or "consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term "consisting of excludes any element, step, or ingredient not specified in the claims. The transition term "consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the disclosure so claimed are inherently or expressly described and enabled herein.
[0091] It is to be understood that the embodiments of the disclosure disclosed herein are illustrative of the principles of the present disclosure. Other modifications that can be employed are within the scope of the disclosure. Thus, by way of example, but not of limitation, alternative configurations of the present disclosure can be utilized in accordance with the teachings herein. Accordingly, the present disclosure is not limited to that precisely as shown and described.
[0092] While the present disclosure has been described and illustrated herein by references to various specific materials, procedures and examples, it is understood that the disclosure is not restricted to the particular combinations of materials and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the disclosure being indicated by the following claims. All references, patents, and patent applications referred to in this application are herein incorporated by reference in their entirety.