专利汇可以提供Culture method to obtain and maintain a pure or enriched population of mammalian neural stem cells and/or neural/progenitor cells that are prone to differentiate into oligodendrocyte-lineage cells in vitro专利检索,专利查询,专利分析的服务。并且An isolated expandable human neural stem or progenitor cell wherein the cell is a progenitor cells or stem cell, maintains its capability to differentiate into neurons, astrocytes, and oligodendrocytes, maintains its ability to differentiate into oligodendrocyte lineage cells efficiently throughout subsequent passages, and the cell expresses at least cell surface antigens CD133 and CD140α. Also provided is a method of in vitro culturing an expandable neural progenitor or stem cell isolated from a mammalian central nervous system, and the culture itself, wherein said cell maintains its capability to differentiate into neurons, astrocytes, and oligodendrocytes and its ability to differentiate into oligodendrocyte-lineage cells efficiently. In addition, a method of treating a condition caused by a loss of myelin or a loss of oligodendrocytes is provided as is a composition comprising an isolated expandable neural stem cell or one cultured by the methods of the invention.,下面是Culture method to obtain and maintain a pure or enriched population of mammalian neural stem cells and/or neural/progenitor cells that are prone to differentiate into oligodendrocyte-lineage cells in vitro专利的具体信息内容。
I claim:
This invention relates generally to the field of cell biology of neural stem cells and neural progenitor cells. More specifically, this invention provides a pure or enriched population of mammalian neural stem cells and/or neural progenitor cells that are prone to differentiate into oligodendrocyte-lineage cells in vitro, suitable for use in biological research, drug screening and human therapy.
This application is related to U.S. provisional patent application No. 61/431,944 filed on Jan. 12, 2011 and 61/558,527 filed on Nov. 11, 2011.
Not Applicable.
During development of the central nervous system, primitive, multipotent neural stem cells (NSC) proliferate, giving rise to transiently dividing progenitor cells that eventually differentiate into the various cell types that compose the adult brain. The adult central nervous system mainly consists of neurons and glial cells, which include astrocytes and oligodendrocytes. The progenitor cells for neurons, astrocytes and oligodendrocytes originate sequentially from neural stem cells in the developing brain (see
Since oligodendrocytes play an important role in supporting the central nervous system, a pure or enriched population of oligodendrocytes or their predecessor cells (i.e., oligodendrocyte pre-progenitor cells and/or oligodendrocyte progenitor cells) would be useful for cell therapies and regenerative medicine such as in the treatment of neurological disorders including congenital demyelinating diseases (for example, Krabbe disease or Pelizaeus-Merzbacher disease), spinal cord injury and other conditions that result from defects in the myelin sheath that insulates nerve cells. These cells also can be used for research and for identifying new drugs for the treatment of many neurological disorders such as multiple sclerosis and schizophrenia.
Mature oligodendrocytes do not proliferate and do not survive well in culture, and the ability to obtain oligodendrocytes directly from tissue samples in quantities sufficient for use in research or human therapy is extremely difficult. As a result, the use of oligodendrocytes for these purposes is hindered by the lack of availability of these cells.
One solution to this problem involves obtaining neural stem cells and/or neural progenitor cells from tissue, expanding the cells in culture to obtain a sufficiently large quantity of cells which can subsequently differentiate into oligodendrocytes. Differentiation can take place either in vitro or in vivo, such as in the case of transplantation. This would result in a large population of oligodendrocytes or their progenitors or pre-progenitors for use in research and human therapy.
However, scientists have struggled to identify culture conditions that permit long term culture and mass expansion of oligodendrocyte progenitors and/or pre-progenitors—particularly from humans or non-human primates—wherein the resulting expanded cell population is primarily comprised of cells that retain the ability to differentiate into oligodendrocytes.
Several scientists have reported obtaining oligodendrocyte progenitor cells from rats ((Raff et al, J. Neurosci., 3:1289, 1983; Raff et al, Nature., 303:390, 1983; Espinosa de los Monteros et al, Proc. Natl. Acad. Sci. U.S.A., 90:50, 1993). These proliferative oligodendrocyte progenitors are known as O-2A progenitors because of their ability to differentiate in vitro into either oligodendrocytes or type 2 astrocytes. Other scientists have identified rat or mouse oligodendrocyte pre-progenitors in primary culture ((Gallo, Armstrong R C, J. Neurosci., 15:394, 1995; Grinspan, Franceschini B, J. Neurosci. Res., 41:540, 1995; Decker et al, Mol. Cell. Neurosci., 16:422, 2000). These cells are thought to be precursors of oligodendrocyte progenitors and are expected to be more beneficial in cell therapy because of their superior migration capacity as compared to oligodendrocyte progenitors. Unfortunately, scientists have been unable to effectively expand these cells for long periods of time in vitro. In contrast, scientists have reported culturing O2A progenitors from rat optic nerve or spinal cord using B104 conditioned medium or growth factor combinations such as (i) platelet derived growth factor-AA (PDGF-AA) with basic fibroblast growth factor (bFGF or basic FGF) and neurotrophin-3 (NT-3), or (ii) PDGF-AA with ciliary neurotrophic factor (CNTF) and NT-3. However, no one has succeeded in mass expansion of these cell types from primate tissue using these growth factors.
Thus, it remains very difficult to obtain and expand a pure or enriched population of oligodendrocytes and/or their predecessor cells from mammals other than rat or mouse. It is particularly difficult to obtain and expand these cells from humans and non-human primates. Therefore, a great need exists for methods for generating pure or enriched populations of mammalian neural stem cells or progenitor cells which are prone to differentiate into oligodendrocyte-lineage cells in vitro.
The present invention relates to an isolated expandable human neural cell wherein the cell is a progenitor cells or stem cell, wherein the cell maintains its capability to differentiate into neurons, astrocytes, and oligodendrocytes, wherein the cell maintains its ability to differentiate into oligodendrocyte lineage cells efficiently throughout subsequent passages, and wherein the cell expresses at least cell surface antigens CD133 and CD140α.
The present invention also relates to a method of in vitro culturing an expandable neural cell wherein the cell is a progenitor cell or stem cell isolated from a mammalian central nervous system wherein said cell maintains its capability to differentiate into neurons, astrocytes, and oligodendrocytes and its ability to differentiate into oligodendrocyte-lineage cells efficiently, wherein the method comprises isolating and dissociating at least one cell from a human fetal neural tissue; culturing the cell at a temperature of 37° C., in an atmosphere comprising 1-20% O2, and 5% CO2, and in a chemically defined serum-free culture medium, wherein the medium comprises at least 5 ng/ml PDGF-AA, at least 0.5 ng/ml bFGF, and at least 10 μM 1-thioglycerol; and passaging the cell to obtain the expandable human neural cell.
The present invention further relates to a method of treating a condition caused by a loss of myelin or a loss of oligodendrocytes comprising administering to a subject a therapeutically effective amount of a composition comprising an isolated expandable human neural cell which is able to maintain its capability to differentiate into neurons, astrocytes, and oligodendrocytes, wherein the cell maintains its ability to differentiate into oligodendrocyte lineage cells efficiently throughout subsequent passages, and wherein the cell expresses at least cell surface antigens CD133 and CD140α.
The present invention also relates to an in vitro culture comprising at least one isolated neural cell obtained from a mammalian central nervous system wherein the cell is submerged in chemically defined serum-free culture medium which has at least 5 ng/ml PDGF-AA,—at least 5 ng/ml bFGF, and at least 10 μM 1-thioglycerol.
The present invention moreover relates to a pharmaceutical neural stem cell composition comprising an isolated expandable human neural cell.
The present invention additionally relates to the use of a pharmaceutical neural stem cell composition in a medicament to treat a condition.
The present invention also relates to a method of in vitro culturing and expanding neural stem cells and/or neural progenitor cells isolated from a mammalian central nervous system wherein said cultured and expanded cells maintain their ability to differentiate into oligodendrocyte-lineage cells. The culture of cells in the present invention is an adhesion culture.
The present invention further relates to an isolated pure or enriched population of expanded mammalian neural stem cells and/or neural progenitor cells that are prone to differentiate into oligodendrocyte-lineage cells (i.e. O4-positive cells with spider web morphology as shown in
The present invention moreover relates to mammalian oligodendrocyte-lineage cells via in vitro expansion and differentiation from neural stem cells and/or neural progenitor cells isolated from mammalian central nervous system.
The present invention provides a method for culturing and expanding neural stem cells or neural progenitor cells isolated from a mammalian central nervous system to produce a pure or enriched population of neural stem cells or neural progenitor cells that have the ability to differentiate into oligodendrocytes or oligodendrocyte-lineage cells in vitro. Neural stem cells and neural progenitor cells both generate progeny that are either neuronal cells (such as neuronal progenitors or mature neurons) or glial cells including astrocytes and oligodendrocytes. While neural stem cells are self-renewable (i.e., able to proliferate indefinitely), neural progenitor cells may be, but are not necessarily, capable of self renewal. The culture methods of the present invention can produce an expanded cell population that can be differentiated into at least 70%, 80%, 90% or 95% oligodendrocyte-lineage cells, which are oligodendrocyte progenitor cells and oligodendrocytes, of differentiated cells.
The following abbreviations and definitions are used throughout this application:
The term “HFSC cell” or human fetal spinal cord-derived cell, refers to the pure or enriched population of expanded mammalian neural stem cells and/or neural progenitor cells that are described in this invention.
The term “HFSCM1 medium” refers to DMEM/F12 containing glutamine and HEPES and supplemented with B27 supplement (Invitrogen™), non-essential amino acids (NEAA) (Invitrogen™), 1.5 mM pyruvate (Invitrogen™), 55 μM β-mercaptoethanol (Invitrogen™), and 1 mM N-acetyl-L-cysteine (SIGMA-ALDRICH™) in combination.
The term “glial cells” refers to non-neuronal cells of the central nervous system and encompasses mature oligodendrocytes, astrocytes and committed progenitor cells for either or both of these cell types.
The term “multipotent progenitor cells” refers to neural progenitor cells that have the potential to give rise to cells from multiple, but a limited number of, lineages.
“Pluripotency” (derived from the Latin “plurimus” or “very many” and “potentia” or “powered”) refers to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system). Neural stem cells are multipotent and not pluripotent. Embryonic stem cells are pluripotent and not multipotent.
A “committed progenitor cell” is a progenitor cell that is committed, or destined, to become a specific type of mature cell. This is in contrast to a multipotent or a pluripotent progenitor cell, which has the potential to become one of two or more types of mature cells (such as O-2A progenitor cells which can become either oligodendrocytes or type-2 astrocytes, depending on timing and environmental factors).
An “oligodendrocyte” is a type of glial cell whose main function is to insulate nerve cell axons in the central nervous system of some vertebrates.
The term “oligodendrocyte-lineage cells” refers to oligodendrocytes, pro-oligodendroblast and oligodendrocyte progenitors (e.g. O2A progenitor). This term does not include glial-restricted precursors or neural stem cells.
The terms “oligodendrocyte progenitor cells” and “oligodendrocyte progenitors” are used interchangeably throughout this application and refer to cells that are committed to forming more progenitor cells and/or progeny that are oligodendrocytes in preference to neurons or non-neurological tissue. Unless otherwise specified, they may, but do not necessarily, have the capability of making other types of glial cells (such as type-2 astrocytes). This term as used herein does not encompass oligodendrocyte pre-progenitors or glial-restricted precursors (see
“Oligodendrocyte pre-progenitors” are predecessor cells of oligodendrocyte progenitors.
“Pro-oligodendroblasts” are predecessor cells to post-mitotic oligodendrocytes.
“Expanding” cells in culture means to increase cell number in the presence of culture medium containing supplements which stimulate cell proliferation.
The cell “expansion rate” refers to the cell number on a particular date divided by the initial cell number at the start of culture.
“Expanded” neural progenitor cells or neural stem cells as used herein refers to neural progenitor cells or neural stem cells that are derived from isolated neural progenitor cells or neural stem cells that have proliferated in vitro, producing the expanded cell population.
“Passaging” cells (also known as “subculturing” or “splitting” cells) refers to a technique that enables cells to be kept alive and growing under laboratory culture conditions for extended periods of time by dissociating cells from one another (with enzymes like trypsin or collagenase and then transferring a small number of cells into a new culture vessel. Cells can be cultured for a longer time if they are passaged at regular intervals, as it avoids the premature senescence associated with prolonged high cell density.
A “growth environment” is an environment in which the cells of interest will proliferate, differentiate and/or mature in vitro. Features of the environment include the medium in which the cells are cultured, any growth factors or differentiation-inducing factors that may be present, and a supporting structure (such as a substrate on a solid surface) if present.
General Techniques
General methods in cell biology, protein chemistry, and antibody techniques can be found in Current Protocols in Protein Science (J. E. Colligan et al., eds., Wiley & Sons); Current Protocols in Cell Biology (J. S. Bonifacino et al., Wiley & Sons) and Current Protocols in Immunology (J. E. Colligan et al. eds., Wiley & Sons). Cell culture methods are described generally in the current edition of Culture of Animal Cells: A Manual of Basic Technique (R. I. Freshney etl, Wily & Sons); General Techniques of Cell Culture (M. A. Harrison & I. F. Rae, Cambridge Univ. Press). Tissue Culture supplies and reagents are available from commercial vendors such as Chemicon®, Millipore®, R&D Systems®, Invitrogen™, Nalgene-Nunc™ International, Sigma-Aldrich™, and SCIENCELL™ Research Laboratories.
Specialized reference books relevant to this disclosure include Principles of Neuroscience, 4th edition, Kandel et al. eds., McGraw-Hill 2000; CNS Regeneration: Basic Science and Clinical Advances, M. H. Tuszynski & J. H Kordower, eds., Academic Press, 1999; The Neuron: Cell and Molecular Biology, 3 rd edition, I. B. Levitan and L. K. Kaczmarek, Oxford U. Press, 2001; Glial Cells: Their Role in Behavior, P. R. Laming et al. eds., Cambridge U. Press 1998; The Functional Roles of Glial Cells in Health and Disease, Matsas & Tsacopoulos eds., Plenum Pub. Corp, 1999: Glial Cell Development, Jessen & Richardson eds., Oxford U. Press, 2001; and Man of Steel, Adrian Hill, 1996.
In the context of cell ontogeny, the adjective “differentiated” is a relative term. A differentiated cell is a cell that has progressed further along the developmental pathway than the cell to which it is being compared. Thus, neural stem cells can differentiate to lineage-restricted progenitors. These, in turn, can differentiate into cells further along the pathway or to end-stage differentiated cells, such as mature neurons or oligodendrocytes.
Differentiated cells of this invention can be characterized according to whether they express phenotypic markers characteristic of oligodendrocytes. Classic immunocytochemical markers for these cells that may be present depending on the maturity of the cell population are the following:
Sox2: a marker for pluripotent stem cells and neural stem cells.
Nestin: a marker for neural stem cells.
CD133: a cell surface marker for neural stem cells.
PDGF-Receptor alpha (PDGF-Rα): the α chain of the platelet-derived growth factor receptor. A marker for oligodendrocytes and their progenitors.
CD140a: the same as PDGF-Rα. CD140a antibody recognizes an extracellular domain of PDGF-Rα. A cell surface marker for oligodendrocytes and their progenitors.
CD9: a cell surface glycoprotein that is know to complex with integrins and other transmembrane 4 superfamily proteins. A cell surface marker for germline stem cell, neural stem cell, oligodendrocyte, and mesenchymal stem cell.
PSA-NCAM: polysialylated-neural cell adhesion molecules. A cell surface marker for neuronal-restricted precursor (NRP), neuronal progenitor, neuroblast, and oligodendrocyte pre-progenitor. This marker is negative in glial-restricted precursor (GRP).
A2B5: a cell surface marker for glial-restricted precursor (GRP), glial progenitor cells and oligodendrocyte progenitor cell (OPC) and type 2 astrocytes. This cell surface marker is negative in neuronal-restricted precursor (NRP).
NG2: a chondroitin sulfate proteoglycan. A cell surface marker for macrophages and oligodendrocyte progenitor cells.
GD3: Ganglioside GD3. A marker for oligodendrocyte pre-progenitor and oligodendrocyte progenitors
O4: a marker for oligodendrocytes and their progenitors.
Galactocerebroside C (GalC): a marker for immature oligodendrocytes.
Myelin basic protein (MBP): a marker for mature oligodendrocyte.
CD44: a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration and a receptor for hyaluronic acid. A cell surface marker for some epithelial cells and astrocyte lineage cells.
Glial fibrillary acidic protein (GFAP): a marker for astrocytes.
βIII Tublin: a marker for neuronal progenitors and neurons.
Neurofilament-L: a marker for mature neurons.
Microtubule-associated protein 2 (MAP2): a marker for mature neurons.
Tissue specific markers can be detected using any suitable immunological technique, such as flow immunocytochemistry for cell surface markers, or immunohistochemistry (for example, of fixed cells or tissue sections) for intracellular or cell-surface markers. A detailed method for flow cytometry analysis is provided in Gallacher et al, Blood., 96:1740, 2000. Expression of a cell-surface antigen is defined as positive if a significantly detectable amount of antibody will bind to the antigen in a standard immunocytochemistry or flow cytometry assay, optionally after fixation of the cells, and optionally using a labeled secondary antibody or other conjugate to amplify labeling. To facilitate use in research or therapy, it is often beneficial to maximize the proportion of cells in the population that have the characteristics of oligodendrocytes or their progenitors. It is possible to obtain populations of cells that are at least 50%, 60%, 70%, 90% or 95% specific lineage cells, identified as being positive for one or more of the phenotypic markers characteristic of such cells.
For therapeutic applications relating to reconstitution of neural function, it is often desirable to minimize the ability of the cell population to form other cell types, particularly undifferentiated stem cells, and cells of non-ectodermal lineage. Depending on the application, it may also be advantageous to minimize the proportion of cells of the neuronal lineage and their committed progenitors or cells of the astrocyte lineage and their committed progenitors. The contamination of the populations according to this invention have less than 30%, 20%, 10% or 5% contamination with these other types of cells.
The methods of the present invention cannot result in the development of an entire human organism.
The method of the present invention involves culturing isolated neural stem cells and/or neural progenitor cells from a mammalian central nervous system in a defined medium that permits the expansion of the cells through multiple passages. The cells cultured using the method of the present invention retain their ability, throughout expansion, to differentiate into oligodendrocyte-lineage cells. The cells cultured using the method of the present invention can be passaged more than 6 times and expanded over 1,000 times while retaining their ability to subsequently differentiate into oligodendrocyte-lineage cells. In some embodiments, the expanded cell population resulting from the culture method of the present invention comprises or can differentiate into a population of cells having at least 30%, 50%, 70% or 80% oligodendrocyte-lineage cells of differentiated cells in serum-free culture condition. In a preferred embodiment, the expanded cell culture population resulting from the culture method of the present invention comprises at least 90% oligodendrocyte-lineage cells of differentiated cells. In preferred embodiments, the expanded cells are multipotent. In some embodiments the majority of expanded cells are capable of differentiating into oligodendrocyte-lineage cells upon culturing in decreased PDGF-AA medium (i.e., 20 ng/ml PDGF-AA, preferably with 10 ng/ml bFGF with or without 50 μM 1-thioglycerol and optionally with at least 10 ng/ml IGF-1) without replenishing bFGF between changing medium. In some embodiments the majority of expanded cells are capable of differentiating into oligodendrocyte-lineage cells upon culturing in 10 ng/ml of PDGF-AA, 100 ng/ml IGF-1, 100 μM pCPT-cAMP and 10 ng/ml BDNF in DMEM/F12 containing glutamine and HEPES and supplemented with B27 supplement, N2 supplement and 50 μM 1-thioglycerol.
The isolated mammalian neural stem cells and/or neural progenitor cells for use in the present invention may be obtained from the central nervous system of a mammalian, preferably a primate such as, but not limited to, a human. Oligodendrocyte progenitors and pre-progenitors are known to exist in white matter of the central nervous system. As such, suitable sources from which to isolate cells for use in the present invention include, but are not limited to, the optic nerve, corpus callosum and spinal cord. In addition, isolated stem cells may be derived from a mammalian fetus, preferably a primate fetus, such as but not limited to a human fetus, using methods known in the art. In some embodiments, the isolated stem cells are prepared from human fetal spinal cord tissue obtained from a human fetal spinal column. In a preferred embodiment, isolated cells for use in the present invention are obtained from 8-24 weeks gestational age, preferably 12-18 weeks gestational age human fetal spinal cord. Human fetal spinal columns can be obtained, for example, commercially through companies such as Advanced Bioscience Resources, Inc. (Alameda, Calif., USA) with the IRB permission and an informed consent from a donor. Spinal cord tissue can be dissected from the spinal column, with the meninges and peripheral nerves removed. The tissue then can be dissociated, washed and placed in a culture vessel containing a growth medium that permits cell proliferation.
Suitable culture vessels may include, but are not limited to, culture vessels with a culture surface having one or a combination of poly-amino acids (e.g., poly-lysine and/or poly-ornithine), tissue culture plastic and surfaces treated with laminin, vitronectin or fibronectin. Cells generally may be plated at a density ranging from 104 to 105 cells/cm2, preferably at a density of approximately 3×104 to 5×104 cells/cm2. Poly-omithine or poly-lysine may be used to coat culture vessels as reported previously (Raff et al, J. Neurosci., 3:1289, 1983; Raff et al, Nature., 303:390, 1983; Protocols for Neural Cell Culture, 3rd edition, Humana Press, Inc.). Culture vessels may be coated with 1 to 40 μg/ml of poly-ornithine, preferably 2 to 20 μg/ml, more preferably 5 to 15 μg/ml. The strength of cell attachment can vary depending on vendor, surface modification, format and specific lot of culture vessels. The optimal concentration of coating materials can be determined for each source of culture vessels using methods known in the art. In some embodiments a two-hour incubation with 10 μg/ml of poly-ornithine or poly-lysine is performed to coat vessels, for example, from BD Falcon (Sparks, Md., USA). In some embodiments a 30-minute incubation with 5 μg/ml of poly-ornithine or poly-lysine can be performed to coat vessels, for example, from Nalgen Nunc International (Rochester, N.Y., USA).
The isolated neural stem cells and/or neural progenitor cells obtained from a mammalian central nervous system are cultured in a serum-free chemically defined culture medium that permits cell expansion without promoting differentiation of the cells (for example, into neurons, astrocytes or oligodendrocytes). The culture medium comprises a base medium such as, but not limited to, Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12. 1:1 (DMEM/F12) (Invitrogen®) (e.g., Iscove's Modified Dulbecco's Medium, RPMI-1640 and Neurobasal). The base medium may be supplemented with various components to support cell health and survival. Such components may include, but are not limited to, at least 0.25% non-essential amino acids [NEAA (Invitrogen®)-a 1% solution contains 100 μM of L-Alanine, L-Asparagine H2O, L-Aspartic Acid, L-Glutamic Acid, Glycine, L-Proline, and L-Serine], at least 1.0 mM glutamine, at least 0.5 mM pyruvate, at least 1% B27 supplement (Invitrogen®), at least 0.1 mM N-acetyl-cysteine and/or at least 10 μM β-mercaptoethanol. In some embodiments the base medium may comprise NS21 (as disclosed in Y. Chen et al., J. Neurosci. Methods., 171:239, 2008) in place of B27 supplement. B27 supplement contains bovine serum albumin, transferrin, insulin, progesterone, corticosterone, triiodo-l-thyronine, retinol acetate, DL tocopherol, DL tocopherol acetate, Biotin, Linoleic acid, Linolenic acid, ethanolamine, Na Selenite, L-carnitine, glutathione reduced, catalase, superoxide dismutase, D-galactose and putrescine. Invitrogen has disclosed its ingredients but hasn't disclosed their concentration. However, the concentration of each ingredients of their original formulation, B18 supplement, was disclosed. NS21 was developed based on this information and the concentration of each gradient was disclosed. It worked in neuronal culture as good as B27 supplement. In addition, NS21 could be used to culture neural stem cells and oligodendrocyte progenitors derived from human embryonic stem cells. Therefore, this supplement is thought to be a good candidate to replace B27 supplement. In a preferred embodiment, the culture medium comprises DMEM/F12 supplemented with 1-4 mM, more preferably 2.5 mM glutamine; 10-25 mM, more preferably 15 mM HEPES; 0.5-2.0 mM, more preferably 1 mM pyruvate; 1 to 4%, more preferably 2% B27 supplement; 0.25-3%, more preferably 1% NEAA; 1-200 μM, more preferably 50 μM 1-thioglycerol; 0.1-3 mM, more preferably 1 mM N-acetyl-cysteine; and/or 10-100 μM, more preferably 55 μM β-mercaptoethanol.
Furthermore, oxygen may facilitate differentiation of neural progenitor cells. Therefore, to reduce cell differentiation, the cells may be cultured in a 1-20% O2 growth environment. In a preferred embodiment, the cells are cultured in a culture flask in an incubator providing a 37° C., 1-10%, more preferably 5% O2, 5% CO2 growth environment. After establishing HFSC cells, the effect of oxygen concentration was assessed but there was no increase of differentiated cells in 20% O2 condition compared to 5% O2 condition in the culture condition to expand HFSC cells. The growth of HFSC cells in 5% O2 condition was a little bit faster than that in 20% O2 condition. Oxygen is a cause of oxidative stress and known to induce mutation of p53 in rodent cells. To reduce a risk of mutation, we kept culturing HFSC cells in 5% O2 condition.
The neural stem cells and/or neural progenitor cells are cultured in culture medium further comprising growth factors to stimulate proliferation for isolating the cells. The culture medium may contain at least 5, 10, 20 or 40 ng/ml platelet-derived growth factor-AA (PDGF-AA), at least 2.5, 5 or 10 ng/ml basic FGF (bFGF), and/or at least 10, 25 or 50 μM 1-thioglycerol to isolate the cells. In some embodiments the culture medium further comprises at least 1, 5 or 10 ng/ml insulin-like growth factor-1 (IGF-1). In some embodiments PDGF-AA may be replaced with PDGF-BB, PDGF-AB, PDGF-CC, or PDGF-DD. In some embodiments bFGF may be replaced with other member of fibroblast growth factors (e.g. FGF-4 or FGF-9). In some embodiments IGF-1 may be replaced with IGF-2. In a preferred embodiment, the culture medium comprises 40-60 μM 1-thioglycerol, 40-200 ng/ml PDGF-AA, 5-100 ng/ml bFGF, and 5-100 ng/ml IGF-1 to isolate the cells.
The isolated neural stem cells and/or neural progenitor cells are cultured in culture medium further comprising growth factors to stimulate proliferation after isolating the cells. The culture medium may contain at least 1, 2, or 5 ng/ml platelet-derived growth factor-AA (PDGF-AA), at least 0.5, 1 or 5 ng/ml basic FGF (bFGF), and/or at least 10, 25 or 50 μM 1-thioglycerol to expand the cells. In some embodiments the culture medium further comprises at least 1, 2 or 5 ng/ml insulin-like growth factor-1 (IGF-1). In a preferred embodiment, the culture medium comprises 40-60 μM 1-thioglycerol, 5-100 ng/ml PDGF-AA, 1-50 ng/ml bFGF, and 5-100 ng/ml IGF-1 to expand the cells after the isolation of HFSC cells.
The isolated neural stem cells and/or neural progenitor cells grown under culture conditions of the present invention exhibit a doubling time of 50-120 hours. In preferred embodiments, the doubling time is about 60 to 100 hours. The cells can continue this proliferation rate through at least 8, 11, 14 or 17 passages. The cells may be expanded at least 100, 250, or preferably at least 500 times per month. In preferred embodiments, the cells cultured using the method of the present invention exhibits an expansion rate >1 for more than 18 passages.
Several medium and supplements were tested in a preliminary experiment using HFSC cells derived from human 12-week fetal spinal cord, which was obtained from Advanced Bioscience Resources, Inc. (Alamada, Calif., USA) with an informed consent of a donor. The cells were cultured in DMEM:F-12 (1:1) (DMEM/F12) (Invitrogen™, Carlsbad, Calif., USA) supplemented with 2 mM glutamine (Invitrogen™), 1 mM pyruvate (Invitrogen™) and 2% B27 supplement (Invitrogen™) initially. Various growth factors were examined whether they stimulate growth of HFSC cells. Most effective growth factors were the combination of PDGF-AA and bFGF. The cells could grow in the presence of PDGF-AA and bFGF but showed vacuoles and looked unhealthy. Several supplements were tested and it was observed that DMEM/F12 supplemented with 1% NEAA in addition to 2 mM glutamine, 1 mM pyruvate and 2% B27 supplement could decrease vacuoles and increase cell number slightly (data not shown). Other supplements including 1 mM N-acetyl-cysteine (Sigma-Aldrich™, St. Louis, Mo., USA) and 55 μM β-mercaptoethanol (Invitrogen™) seemed to improve the cells' status but the improvements were not as prominent as with NEAA (data not shown). Thus, DMEM/F12 supplemented with 2 mM glutamine, 1 mM pyruvate, 2% B27 supplement, 1% NEAA, 1 mM N-acetyl-cysteine and 55 μM β-mercaptoethanol was identified as optimal base medium and was used thereafter to culture HFSC cells.
Human 15-week fetal spinal cord was dissected from the spinal column, with the meninges and peripheral nerves removed. The tissue then was dissociated with Accutase and washed and cells obtained from human fetal spinal cord were placed in a culture vessel containing the following growth medium: DMEM/F12 containing 2.5 mM glutamine and 15 mM HEPES, 2% B27 supplement (Invitrogen™), 1% NEAA, 1.5 mM pyruvate (Invitrogen™), 55 μM β-mercaptoethanol (Invitrogen™), 1 mM N-acetyl-L-cysteine (Sigma-Aldrich™), 20 ng/ml PDGF-AA (R&D SYSTEMS™, Inc., Minneapolis, Minn., USA) and 10 ng/ml bFGF (R&D SYSTEMS™). Cells were then placed in an incubator maintained at 37° C., 5% O2, and 5% CO2. bFGF (10 ng/ml) was added daily to the culture medium. Medium was changed every 2-3 days during passage. Based on the preliminary experiments, growing cells were not so many in the presence of PDGF-AA and bFGF and many cells stopped proliferating or died within a few weeks. This result seemed to be reasonable because PDGF-Rα expressing cells are usually less than 5% of cells. To remove cells that are not responsive to PDGF-AA and bFGF, cells were cultured in an ultra-low adhesion culture plate (OWENS CORNING™ Inc., Corning, N.Y., USA). The cells that are responsive to PDGF-AA and bFGF made spheres (see
As mentioned in Example 1, inclusion of PDGF-AA and bFGF in the culture medium supported adequate growth of the HFSC cells initially, but the proliferation rate slowed after 2 passages. NT-3 (R&D SYSTEMS™) and IGF-1 (Sigma-Aldrich™) were tested to determine if they could enhance the proliferation of HFSC cells. The presence of 20 ng/ml PDGF-AA with 10 ng/ml bFGF was insufficient to stimulate the proliferation rate of the cells (the expansion rate was <1) (see
The addition of 50 μM 1-thioglycerol in the presence of 20 ng/ml PDGF-AA, 10 ng/ml bFGF and 10 ng/ml IGF-1 stimulated proliferation slightly but the cell number was still decreased (expansion rate <1). This result was similar to that obtained in the absence of 1-thioglycerol when the PDGF-AA concentration was increased to 100 ng/ml in the presence of 10 ng/ml bFGF+10 ng/ml IGF-1. However, when both 50 μM 1-thioglycerol and increased PDGF-AA (100 ng/ml) were included in the culture medium (along with 10 ng/ml bFGF and 10 ng/ml IGF-1), the two components appeared to work synergistically, significantly increasing the cell number (expansion rate >1). When IGF-1 was eliminated from this supplement cocktail (i.e. total supplementation was 100 ng/ml PDGF-AA+10 ng/ml bFGF+50 μM 1-thioglycerol), the expansion rate decreased to <1, indicating that IGF-1 also promoted HFSC cell proliferation and/or survival. However, if HFSC cells were cultured in this condition longer, HFSC cells might be expanded even in this condition. This data indicated that the addition of 50 μM 1-thioglycerol and the increase of PDGF-AA concentration to 100 ng/ml in addition to 10 ng/ml bFGF+10 ng/ml IGF-1 were important to expand the cells. Furthermore, addition of IGF-1 might not be mandatory but was still effective even in the presence of 50 μM 1-thioglycerol and 100 ng/ml PDGF-AA to increase the expansion rate. The combination of 50 μM 1-thioglycerol and 100 ng/ml PDGF-AA in addition to 10 ng/ml bFGF+10 ng/ml IGF-1 was used thereafter to culture HFSC cells.
The HFSC cells were further expanded in the presence of 100 ng/ml PDGF-AA, 10 ng/ml bFGF, 10 ng/ml IGF-1 and 50 μM 1-thioglycerol after passage 6. The cells began to proliferate more rapidly under these conditions after passage and were expanded 3-4 times within a week. The doubling time was approximately 60-100 hours in this condition. The HFSC cells were able to maintain this proliferative state even after passage 8 (see
While testing various culture conditions, it was observed that when HFSC cells were cultured with the removal of bFGF from the medium, many of the cells seemed to differentiate into bipolar or multipolar cells (indicative of oligodendrocyte) and died soon. To avoid these cell death, a spontaneous differentiation technique was developed.
Culture medium was usually changed every 2 days for expanding HFSC cells and it was thought to be very important to keep HFSC cells in proliferative state. To enhance cell differentiation, medium was changed every 3 or 4 days for this experiment. Basic FGF and high concentration of PDGF-AA were thought to block differentiation of HFSC cells but they were also important for HFSC cells to survive. Therefore, PDGF-AA concentration was decreased from 100 to 20 ng/ml in the presence of 10 ng/ml bFGF, 10 ng/ml IGF-1 with 50 μM 1-thioglycerol or without 1-thioglycerol. HFSC cells couldn't survive well if bFGF was removed. Usually, bFGF were replenished everyday to keep HFSC cells in proliferative state. When replenishing bFGF was stopped, many HFSC cells separated from their clusters and formed complex web-like processes (indicative of pro-oligodendroblasts and/or immature oligodendrocytes) and survived well as shown in
These process-bearing cells with spider's web-like morphology were positive for O4 antigen and/or GalC antigen as shown in
These data further indicate that the culture conditions of the present invention are particularly useful for expanding isolated neural stem cells and/or neural progenitor cells that are prone to differentiate into oligodendrocytes, since most of the differentiated cells exhibited oligodendrocyte characteristics and resemble oligodendrocytes or pro-oligodendrocytes.
As shown in
The morphology of the cells from the expandable cell population obtained at the end of the primary culture became homogenous at around passage 5 and the cells tended to form clusters and spheres, similar to those that formed with cell line #2b (see
PDGF-AA was thought to work through PDGF receptor a. This receptor is known to be stimulated by all PDGF family members (PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and PDGF-DD). PDGF-BB was used to examine whether PDGF-BB could replace PDGF-AA using HFSC cell cell line #2b and cell line #3. PDGF-BB could expand both cell lines in the same condition except for PDGF-AA and it was proved that PDGF-AA could be successfully replaced with other PDGF family members.
While the inventor tested various culture conditions after HFSC cell (cell line #2b) was established, the inventor noticed that dose response for each growth factor has been changed. To isolate HFSC cell (cell line #2b), higher concentration of PDGF-AA (100 ng/ml) was necessary in addition to 50 μM 1-thioglycerol. After this cell line became proliferate constantly, higher concentration of PDGF-AA (100 ng/ml) was no more required to expand this cell line. Expansion rate was saturated at around 10-20 ng/ml of PDGF-AA and higher concentration of PDGF-AA (100 ng/ml) had no additional effects on their growth. This may be because of a long-term culture or a continuous usage of 1-thioglycerol. To establish cell line #2b and cell line #3 of HFSC cells, 1-thioglycerol was used after several passages. To examine the effect of higher concentration of PDGF-AA, new cell lines were established in the presence of 1-thioglycerol from an initial culture. In addition, the response to bFGF and IGF-1 seemed to be saturated at 20 ng/ml and 40 ng/ml, respectively. When higher concentration of IGF-1 (50-500 ng/ml) was used, more differentiated cells could be seen (it looked like around 1%). Therefore, 20 ng/ml IGF-1 was considered to be preferable to expand HFSC cells. The usage of 20 ng/ml bFGF and 20 ng/ml IGF-1 improved the expansion rate 5-10% compared to the usage of 10 ng/ml bFGF and 10 ng/ml IGF-1. Therefore, 20 ng/ml bFGF and 20 ng/ml IGF-1 were used for this experiment.
HFSC cells from a third sample (12 weeks gestation) were cultured to see if higher concentration of PDGF-AA is required in the identified optimal culture medium components and if they would provide similar growth characteristics in another batch of cells. The cells were cultured in the same culture medium as cell line #2b and #3 (i.e. HFSCM1 medium and 50 μM 1-thioglycerol) with 20 ng/ml (cell line #4A) or 100 ng/ml (cell line #4B) PDGF-AA in addition to 20 ng/ml bFGF and 20 ng/ml IGF-1. At the end of the first passage, cell number of cell line #4A was less than one third of that of cell line #4B (see
The cells of cell line #2b were cryopreserved in the presence of 8% DMSO at passage 10, 11 and 12. The HFSC cells (cell line #2b) frozen at passage 11 have been deposited at ATCC (accession number PTA-12291). The cells of cell line #3 were also cryopreserved in the presence of 8% DMSO at passage 9 and 10. The cells of cell line #4A and #4B grew faster than cell line #2b or #3, therefore they were cryopreserved in the presence of 8% DMSO at passage 4 and 5 (cell line #4A) or passage 3, 4 and 5 (cell line #4B) in the same condition. The same medium to culture the HFSC cell (HFSCM1 medium and 50 μM 1-thioglycerol) was used for freezing the HFSC cells. The cells were later thawed and cultured in the above described serum-free HFSCM1 medium and 50 μM 1-thioglycerol with 10 ng/ml bFGF, 10 ng/ml IGF-1, 100 ng/ml PDGF-AA or 20 ng/ml bFGF. 20 ng/ml IGF-1, 20 ng/ml PDGF-AA. These cells were observed to proliferate at a similar rate as before freezing (see
When the HFSC cells of Example 2 were cultured in the presence of 100 ng/ml PDGF-AA, 10 ng/ml bFGF, 10 ng/ml IGF-1 and 50 μM 1-thioglycerol, they grew in clusters and/or spheres as shown in
In order to characterize the immuno-phenotype of the undifferentiated HFSC cells, the cells at passage 11-15 were dissociated into a single cell state with Accutase (Innovative Cell Technologies, San Diego, Calif., USA) and grown in poly-ornithine-coated 24-well culture plates and cultured for 3-7 days in the presence of 100 ng/ml PDGF-AA, 10 ng/ml bFGF, 10 ng/ml IGF-1 and 50 μM 1-thioglycerol. The cells were then fixed with 4% paraformaldehyde and then washed with PBS. For staining surface antigens like CD133, PDGF-Rα, NG2, A2B5, O4, O1, GalC and PSA-NCAM, cells were blocked with PBS containing 3% normal gout serum (NGS) and stained with antibodies. For staining intracellular antigens like Sox2, nestin, Olig2, myelin basic protein (MBP), Vimentin, GFAP, βIII Tublin, Neurofilament-L and MAP2, cells were permeabilized with 0.1% Triton X-100 in ice-cold PBS for 10 min before blocking. CD133/1 mouse IgG1 monoclonal antibody (Clone AC133, Miltenyi Biotec), anti-PDGF-Rα rabbit polyclonal antibody (Upstate), anti-NG2 rabbit polyclonal antibody (Millipore®), anti-PSA-NCAM mouse IgM monoclonal antibody (Millipore®), A2B5 mouse IgM monoclonal antibody (Millipore®), O4 mouse IgM monoclonal antibody (R&D SYSTEMS™), O1 mouse IgM monoclonal antibody (Millipore®), anti-Sox2 rabbit polyclonal antibody (Millipore®), anti-Nestin mouse IgG1 monoclonal antibody (Millipore®), anti-Olig2 mouse monoclonal IgG2a antibody, anti-GalC monoclonal IgG3 antibody (Millipore®), anti-MBP rat monoclonal IgG2a antibody (Millipore®), anti-Vimentin mouse IgG1 monoclonal antibody (Santa Cruz), anti-GFAP rabbit polyclonal antibody (Millipore®), anti-βIII Tublin monoclonal mouse IgG1 antibody (Millipore®), anti-Neurofilament-L mouse monoclonal IgG1 antibody (CELL SIGNALING TECHNOLOGY®), anti-MAP2 rabbit polyclonal antibody (Millipore®) and anti-MAP2 mouse IgG1 monoclonal antibody (Millipore®) were used at 1:300 (CD133/1), 1:300 (PDGF-Rα), 1:600 (NG2), 1:1000 (PSA-NCAM), 1:1000 (A2B5), 1:1000 (O4), 1:1000 (O1). 1:1000 (Sox2), 1:200 (Nestin), 1:200 (Olig2), 1:100 (GalC), 1:50 (MBP), 1:500 (Vimentin), 1:1000 (GFAP), 1:100 (βIII Tublin), 1:200 (Neurofilament-L), 1:1000 (MAP2, polyclonal) or 1:200 (MAP2, monoclonal) in PBS containing 3% NGS. After overnight incubation at 4° C., wells were washed with 3 changes of PBS containing 3% NGS. In some case, a live cell staining for some cell surface antigen (NG2, A2B5, O4, O1 and GD3) was used to reduce non-specific signals. In such case, cells were stained with each primary antibody before fixation without blocking. Anti-NG2 rabbit polyclonal antibody. A2B5 mouse IgM monoclonal antibody, O4 mouse IgM monoclonal antibody, O1 mouse IgM monoclonal antibody, and anti-Disialoganglioside GD3 mouse monoclonal IgG3 antibody (Millipore®) were used at 1:150 (NG2), 1:100 (A2B5), 1:200 (O4), 1:100 (O1) and 1:200 (GD3) in PBS containing 0.5% BSA. After 30 minutes incubation at room temperature, wells were washed with 3 changes of PBS containing 0.5% BSA. The secondary antibodies, DYLIGHT™ 488-conjugated AffiniPure Goat anti-rabbit IgG (Fcγ Fragment specific), DYLIGHT™ 488-conjugated AffiniPure Goat anti-mouse IgG (Fcγ Fragment specific), DYLIGHT™ 488-conjugated AffiniPure Goat anti-rabbit IgG (H+L), DYLIGHT™ 488-conjugated AffiniPure Goat anti-rat IgG (H+L), DYLIGHT™ 594-conjugated AffiniPure Goat anti-mouse IgG (H+L), and/or DYLIGHT™ 594-conjugated AffiniPure Goat anti-mouse IgM (μ chain specific) (all secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc.) were used at dilution of 1:500 for 1 hour at room temperature. The cells were then washed with 2 changes of PBS. DAPI was used to counterstain cell nuclei. The cells were then observed using an Olympus IX81 equipped for epifluorescence.
Most of the HFSC cells were CD133-positive (see
In addition, undifferentiated HFSC cells were weakly stained with O4 antibody that is a marker for pro-oligodendroblast (O4-positive, GalC-negative) and immature oligodendrocyte (O4-positive, GalC-positive) by immunocytochemistry but it was difficult to distinguish with weak staining and non-specific staining. Some pro-oligodendroblast which shows strong O4-positive cells with multipolar morphology could be seen in this culture but their frequency of appearance was less than 1% of total cells.
In addition, undifferentiated HFSC cells were weakly stained with anti-MAP2 antibody but their morphology was not like neuron. When the antibody was used with the cells differentiated in the presence of serum, neuron was identified with strong signals and neuronal morphology (see
Overall, HFSC cell expressed specific markers for neural stem cell (CD133, Sox2, and Nestin) and specific markers for oligodendrocyte-lineage cells (Olig2, NG2, A2B5, and O4). These data suggested that HFSC cell might be an intermediate cell between neural stem cell and oligodendrocyte progenitor cell. Furthermore, HFSC cell expressed PSA-NCAM in addition above antigens, indicative to be the human counterpart of rat oligodendrocyte pre-progenitor cell.
Polysialic acid (PSA) of PSA-NCAM is a long, negatively charged, cell-surface glycan with an enormous hydrated volume that serves to modulate the distance between cells. PSA is involved in a number of plasticity-related responses in the adult CNS, including changes in circadian and hormonal patterns, adaptations to pain and stress, and aspects of learning and memory (Rutishauser Nat. Rev. Neurosci., 9:26, 2008). One of the roles of PSA in the neonatal nervous system is in the migration of oligodendrocyte progenitors. When PSA is removed from migrating O2A progenitors, migration of O2A progenitor was inhibited in wound model (Barral-Moran et al, J. Neurosci. Res., 72:679, 2003). Another role is controlling differentiation timing of cells. PSA is expressed on both developing axons and oligodendrocyte precursors, and its down regulation on these cells correlates with the onset of myelination. PSA is also related plasticity-associated responses of the adult CNS. Given the ability of PSA to regulate developmental and adult plasticity, it follows that PSA-expressing cell could have the therapeutic value in situation in which tissues have been damaged by injury or disease. Axonal regrowth in the PSA-expressing region (engineered PSA expression in the scar or on grafted Schwann cells) were observed through the scar in trauma model. HFSC cell is an endogenous PSA-expressing cell and will have the same effect on treating the trauma like brain injury or spinal cord injury.
To further characterize the HFSC cells (cell line #2b), cells that were frozen at passage 11 were thawed, cultured in the growing condition described above and passaged every 7-9 days. The cells cultured for 9 days at passage 13 were then subjected to flow cytometry using the following antibodies: PE-conjugated anti-CD133/1 mouse IgG1 monoclonal antibody (Clone AC133, Miltenyi Biotec); PE-conjugated CD140a mouse IgG2a monoclonal antibody (Clone αR1, BD Pharmingen); PE-conjugated CD9 mouse IgG1 monoclonal antibody (Clone M-L13, BD Pharmingen); PE-conjugated CD44 mouse IgG2b monoclonal antibody (Clone G44-26, BD Pharmingen); PE-conjugated anti-PSA-NCAM mouse IgM monoclonal antibody (2-2B, Miltenyi Biotec); PE-conjugated A2B5 mouse IgM monoclonal antibody (Clone 105HB29, Miltenyi Biotec); PE-conjugated O4 mouse IgM monoclonal antibody (Clone O4, Miltenyi Biotec); and PE-conjugated anti-NG2 mouse IgG1 monoclonal antibody (R&D SYSTEMS™). Briefly, after dissociation with Accutase, the cells were washed and resuspended in ice-cold PBS with 2 mM EDTA and 0.5% BSA and kept on ice. After cell number was counted and cell number was adjusted to 1×107 cells/ml using ice-cold PBS with 2 mM EDTA and 0.5% BSA. 25 μl of cell suspension (250,000 cells) was transferred into each 1.5-ml tube. Primary antibodies then were added into each tube following the manufacturer's recommendation. PE-conjugated isotype controls for each antibody were used to set appropriate gates. After 20-min incubation on ice, cells were washed with ice-cold PBS with 2 mM EDTA and 0.5% BSA and resuspended in fixation buffer (BD Bioscience). After 20 minutes fixation on ice, cells were washed and resuspended in ice-cold PBS with 2 mM EDTA and 0.5% BSA. Fluorescence of cells was measured using FACS Canto II (BD Bioscience) and each data was analyzed using Gatelogic software (Inivai Technologies Pty Ltd.).
As show in
To test the differentiation potential of the expanded cells, the HFSC cells (cell line #3) were passaged to separate/single cell stage and cultured in serum-containing medium [Oligodendrocyte Precursor Cell Differentiation Medium (OPCDM)](ScienCell™ Research Laboratories) to stimulate differentiation. The cells were then stained with antibodies that recognize neurons (anti-βIII Tublin antibody, anti-Neurofilament-L antibody and anti-MAP2 antibody), oligodendrocyte progenitor cells and oligodendrocytes [O4 antibody, O1 antibody, anti-GalC antibody and anti-myelin basic protein (MBP) antibody], and astrocytes (anti-GFAP antibody) followed by a fluorescent dye-conjugated secondary antibody (DYLIGHT™ 488 or DYLIGHT™ 594, Jackson ImmunoResearch). DAPI was used to counterstain cell nuclei.
All three major central nervous system (CNS) phenotypes were observed following treatments to stimulate differentiation of HFSC cells. When the HFSC cells were cultured in serum-containing medium, βIII Tublin-positive cells, Neurofilament-L-positive cells, and MAP-2-positive cells were detected (indicative of neurons). There were many βIII Tublin-positive cells (
HFSC cells showed good differentiation potency into oligodendrocyte by reducing PDGF-AA concentration without replenishing bFGF as shown in
The present invention disclosed the phenotype of HFSC cell that is CD133-positive, CD140a-positive, CD9-positive, CD44-negative, PSA-NCAM-positive, A2B5-positive, O4-positive, and NG2-positive. This information enables to select or enrich HFSC cell without culturing. CD133 is a marker for neural stem cell and not expressed in progenitor or precursor cells. CD9 is also used as a marker for neural stem cell but some oligodendrocytes are known to express CD9. PSA-NCAM and A2B5 are used to detect neuronal-restricted precursor or glial-restricted precursor. Most neural precursors and progenitors are thought to express PSA-NCAM and A2B5 or either PSA-NCAM or A2B5, their usage cannot enrich HFSC cell so well, especially in the first and second trimester. CD140a, NG2, A2B5 and O4 are used as markers for oligodendrocyte precursor cell, pro-oligodendroglia and oligodendrocyte. The expression level of CD140a and NG2 were higher in HFSC cell than oligodendrocyte precursor cell, pro-oligodendroglia or oligodendrocyte, whereas the expression level of A2B5 and O4 were lower in HFSC cell than oligodendrocyte precursor cell, pro-oligodendroglia or oligodendrocyte. The usage of CD140a and NG2 are thought to be more appropriate to enrich HFSC cell. Based on above information and the data described in this invention, the effectiveness of each marker to enrich HFSC cell will be CD140a>NG2>CD9>CD133>A2B5>O4, PSA-NCAM but this order will be vary depend on their gestation week.
However, a single marker will not be enough to select HFSC cells and combination of 2 markers can select HFSC cell more specifically. The combination of one of neural stem cell markers (CD133 or CD9) and one of oligodendrocyte-lineage markers (CD140a, NG2) will be very effective to select HFSC cells. Based on above knowledge, the most efficient combinations of markers to select the HFSC cells will be CD133 and CD140a among these combinations but other combinations should be also more effective than selection with a single marker.
The frequency of appearance of CD133 or CD140a is usually low (less than 5%) and the appearance of CD140a is later (expression starts from around 8-week and maximum at around 18-week of gestation week) than that of CD133. Therefore, the cells expressing both CD133 and CD140a will be very low (less than 1% of total cells) depending on their gestation week. Most of CD133-positive cells may not express CD140a if cells are derived from human fetal tissue at gestation week 15 or earlier. Because CD133-positive and CD140a-negative cell will express CD140a later, the HFSC cell can be obtained when the cells are cultured in the same condition for HFSC cells after the initial enrichment of CD133-positive cell.
The cell line #2b was deposited on Nov. 30, 2011, with Accession number PTA-12291 in the depository American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas Va. 20110 USA. The deposit is named Human neural stem cell: HFSC #2b.
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