NZ712598B2 - 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 - Google Patents

Abstract

Disclosed is a method of in vitro culturing and maintaining an expandable neural cell wherein the cell is a progenitor cell or stem cell and wherein the cell maintains its capability to differentiate into neurons, astrocytes, and oligodendrocytes and its ability to differentiate into oligodendrocyte-lineage cells efficiently, the method comprising: a) providing at least one cell which has been isolated and dissociated from a human fetal neural tissue; b) 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 ?M 1-thioglycerol; c) passaging the cell from b) to obtain the expandable human neural cell. Also disclosed is an in vitro culture comprising an enriched population of neural cells which have been isolated from a mammalian central nervous system wherein the isolated cell is submerged in chemically defined serum-free culture medium comprising: - at least 5 ng/ml PDGF-AA, - at least 5 ng/ml bFGF, and - at least 10 ?M 1-thioglycerol. -lineage cells efficiently, the method comprising: a) providing at least one cell which has been isolated and dissociated from a human fetal neural tissue; b) 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 ?M 1-thioglycerol; c) passaging the cell from b) to obtain the expandable human neural cell. Also disclosed is an in vitro culture comprising an enriched population of neural cells which have been isolated from a mammalian central nervous system wherein the isolated cell is submerged in chemically defined serum-free culture medium comprising: - at least 5 ng/ml PDGF-AA, - at least 5 ng/ml bFGF, and - at least 10 ?M 1-thioglycerol.

Description

CULTURE METHOD TO OBTAIN AND MAINTAIN A PURE OR ENRICHED POPULATION OF MAMMALIAN NEURAL STEM CELLS AND/OR NEURAL ITOR CELLS THAT ARE PRONE TO DIFFERENTIATE INTO- OLIGODENDROCYTE-LINEAGE CELLS IN VITRO FIELD OF THE INVENTION This invention relates generally to the field of cell biology of neural stem cells and neural progenitor cells. more specificaIly, this invention provides a pure or enriched population of mammalian neural stem cells and/or neural progenitor cells that are prone to entiate into oIigodendrocyte—Iineage cells in vitro, suitable for use in biological research, drug screening and human therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to US. provisional patent application number 61/431,944 filed on January 12, 2011 and 61/558,527 fiIed on November 11, 2011.

STATEMENT REGARDING LLY SPONSORED CH OR DEVELOPMENT Not Applicable.

BACKGROUND OF THE INVENTION 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 s cell types that e the adult brain. The adult central nervous system mainly consists of neurons and giial cells, which include astrocytes and oligodendrocytes. The progenitor cells for s, astrocytes and oligodendrocytes originate sequentially from neural stem cells in the developing brain (see Figure 1).

WO 95730 PCT/1B2012/000030 Neuronai progenitor cells form first and differentiate into many types of neurons. Astrocytes p second and function to support neuron al.

Finally, oligodendrocyte progenitor cells start to appear and migrate throughout the central nervous system. They then differentiate into mature oligodendrocytes, which produce myelin necessary for proper neuronal Since oligodendrocytes play an important role in supporting the central nervous system, a pure or enriched tion 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 demyeiinating diseases (for example, Krabbe disease or Pelizaeus—Merzbacher disease), spinal cord injury and other conditions that result from s 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 sclerosis and phrenia. many neurological disorders such as multiple 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 ch 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 m involves obtaining neural stem cells and/or neural progenitor cells from tissue, expanding the cells in culture to obtain a ently 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 tion 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 humansvor non-human primates — 2012/000030 n the resulting expanded cell tion is primarily comprised of cells that retain the ability to differentiate into endrocytes.

Several scientists have reported obtaining oligodendrocyte Raff et al, progenitor cells from rats ((Raff et al, J. Neurosci, 311289, 1983; Proc. Natl. Acad. Sci.

Nature, 303:390, 1983; Espinosa de los Monteros et al, U. S. A., 90:50, 1993). These proliferative oligodendrocyte progenitors are differentiate in vitro into known as O-2A progenitors because of their ability to either oligodendrocytes or type 2 astrocytes. Other scientistshave identified rat culture ((Gallo, Armstrong or mouse oligodendrocyte pre-progenitors in y Neurosci. Res, RC, J. Neurosci., 151394, 1995; Grinspan, Franceschini B, J. 412540, 1995; Decker et al, Mol. Cell. ci, 161422, 2000). These cells and are expected are thought to be precursors of oligodendrocyte progenitors to be more beneficial in cell therapy because of their superior migration capacity as ed 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 02A progenitors from rat factor optic nerve or spinal cord using B104 conditioned medium or growth ations such as (i) platelet derived growth factor—AA (PDGF-AA) with basic fibroblast growth factor (bFGF or basic FGF) and neurotrophin—3 (NT—3), NT—3. or (ii) PDGF—AA with ciliary neurotrphic factor (CNTF) and However, no cell types from primate tissue one has succeeded in mass expansion of these using these growth factors.

Thus, it remains very difficult to obtain and expand a pure or enriched tion 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 es. 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 invitro.

BRIEF SUMMARY OF THE lNVENTlON human The present invention s to an isolated expandable cell neural cell wherein the cell is a progenitor cells or stem cell, wherein the maintains its capability to differentiate into neurons, astrocytes, .oligodendrocytes, wherein the cell maintains its ability to differentiate into oligodendrocyte lineage cells efficiently throughout subsequent passages, CD133 and CD140cr. wherein the cell expresses at least cell surface ns of in vitro culturing The present invention also relates to a method the cell is a progenitor cell or stem cell an expandable neural cell wherein wherein said cell maintains isolated from a mammalian central nervous system and oligodendrocytes and its capability to differentiate into neurons, astrocytes, cells ntly, wherein its y to differentiate into endrocyte—lineage least one cell from a human the method comprises isolating and dissociating at of 37°C, in an atmosphere fetal neural tissue; ing the cell at a temperature defined serum—free comprising 120% d 5% 002, and in a chemically at least 5 ng/ml PDGF-AA, at e medium, n the medium comprises and passaging the cell least 0.5 ng/ml bFGF, and at least 10 [JM 1—thioglycerol; to obtain the expandable human neural cell. ' of treating a The t invention further relates to a method condition caused by a loss of myelin or a loss of oligodendrocytes comprising of a composition administering to a subject a therapeutically effective amount is able to maintain comprising an isolated expandable human neural cell which and oligodendrocytes, its capability to differentiate into s, astrocytes, wherein the cell maintains its ability to differentiate into oligodendrocyte lineage and wherein the cell cells efficiently throughout subsequent passages, CD133 and CD140or. expresses at least cell surface antigens The present invention also relates to an in vitro culture comprising from a mammalian central nervous at least one isolated neural cell obtained system wherein the cell is submerged in chemically defined free culture PCT/1B2012/000030 medium which has at least 5 ng/ml PDGF—AA, - at least 5 ng/ml bFGF, and at least 10 prl 1—thioglycerol. neural The present invention moreover relates to a pharmaceutical human neural cell. stem cell composition comprising an isolated expandable to the use of a The present invention additionally relates to treat a pharmaceutical neural stem cell composition in a medicament condition. of in vitro culturing The present invention also relates to a method cells isolated from a and expanding neural stem cells and/or neural progenitor ed and expanded cells mammalian central nervous system wherein said cells. The maintain their ability to differentiate into oligodendrocyte~lineage culture of cells in the present invention is an adhesion culture. enriched The present invention further relates to an isolated pure or and/or neural progenitor population of ed mammalian neural stem cells cells (ie. 04— cells that are prone to entiate into endrocyte—lineage in Figure 7 and Figure 15) positive cells with spider web morphology as shown in vitro. mammalian The present invention moreover relates to differentiation from endrocyte-lineage cells via in vitro expansion and isolated from mammalian neural stem cells and/or neural itor cells central nervous system.

BRIEF DESCRIPTION OF THE DRAWlNGS and neural Figure 1 s the development of neural stem cells the brain- neurons, astrocytes progenitor cells into the three main cell types in and endrocytes; ' of various Figure 2 shows the comparison of marker expression CNS cells; Fetal Stem Cells (clone . Figure 3 depicts contrast images of Human #2b) in slides A—F; HFSC cells (clone #2b) in Figure 4 illustrates the expansion rate of the presence of different combinations of growth factors; of PDGF~AA (100 ng/ml) Figure 5 illustrates the effects of high dose cells (clone #Zb); and 1—thioglycerol on the proliferation of HFSC Figure 6 illustrates a growth curve of HFSC cells (clone #2b); HFSC cells in Figure 7 depicts spontaneous differentiation of serum—free medium in slides A—F; inverted Figure 8 is a phase contrast images taken with an at various microscope showing the morphology of HFSC cells (clone #3) Figure 9 illustrates a growth curve of HFSC cells (clone #3); inverted Figure 10 is a phase contrast images taken with an 4A and 48) at microscope showing the morphology of HFSC cells (clone various passages in slides A—F; Figure 11 illustrates a growth curve of HFSC cells (clone #4A and #48); Figure 12 illustrates the immuno-phenotype.of undifferentiated HFSC cells in slides A—S; Figure 13, in slides A-H, shows flow cytometry data illustrating proportion of undifferentiated HFSC cells (clone #2b, passage 13); HFSC ] Figure 14 illustrates the immuno-phenotype of differentiated cells (clone #3, e 15); and cells (clone Figure 15 illustrates the entiation potential of HFSC #2b, passage 15) in slides A—E.

ED DESCRIPTION OF THE DRAWINGS Figure 1 depicts thedevelopment of neural stem cells and neural progenitor cells into the three main cell types in the brain- neurons, ytes and endrocytes. Solid arrowed lines indicated the progression of one cell HFSC cell to neuronal—restricted type to another. The dashed line from PCT/182012/000030 the HFSC cell have a lower tendency to precursors (NRP) indicates that line from the HFSC cell to the 02A become neuronal fate. The heavy bolded to produce cells indicates that the HFSC cell has a greater tendency endrocyte—lineage cells. The multi—potentiality of HFSC cell to shown in Example differentiate into neuron, astrocyte and oligodendrocyte was I cell to differentiate into 7 (see Figure 14). The tendency of HFSC oligodendrocyte—tineage cells was shown in Example 2 (see Figure 7) and Example 8 (see Figure 15). of various CNS Figure 2 shows a comparison of marker expression in Example 7 (see cells. This invention disclosed the phenotype of HFSC cell in this figure. As Figure 12 and Figure 13) and summarizes the same and has discussed later, a HFSC cell is not the same as any other cell type both features of neural stem cell and oligodendrocyte Type—2 Astrocyte progenitor (02A).

Cells (clone Figure 3 depicts contrast images of Human Fetal Stem taken with an #2b) in slides A—F. Figure 3, slide A is a phase contrast image in DMEM/F12 inverted microscope showing HFSC cells (clone #2b) cultured 827 supplement containing glutamine and HEPES and supplemented with rogenTM), non-essential amino acids (NEAA) (lnvitrogenTM), 1.5 mM 1 mM N— pyruvate rogenTM), 55 pM B-mercaptoethanol (lnvitrogenTM),tand acetyl-L—cysteine (Sigma), (in combination referred to as “HFSCM1 incubator hereinafter) with 20 ng/ml A and 10 ng/ml bFGF in an from maintained at 37°C, 5% Oz, and 5% C02 incubator. The cells shown are formed spheres and these spheres were plated passage 0, day 7- The cells without dissociating Spheres onto a rnithine coated culture plate directly at passage 1. taken with an Figure 3, slide B & slide C, are phase st images cultured as described in inverted microscope showing HFSC cells (clone #2b) The cells shown are from the description of Figure 3, slide A hereinabove. passage 1, day ‘l and e 2, day 14, respectively. The cells plated directly and spread out from spheres onto a poly-ornithine coated culture plate attached ‘ PCT/18201 2/000030 (Figure 3, slide 8). The passaged cells could expand successfully in this slide C). culture condition in subsequent paSsage (Figure 3, with an Figure 3, slides D—F are phase contrast images taken inverted microscope showing HFSC cells (clone #2b) after multiple passages and cultured in HFSCM1 medium with 100 nglml PDGF-AA, 1O nglml bFGF, maintained at 37°C, 5% ng/ml lGF—1 and 50 pM 1—thioglycerol in an incubator Most HFSC cells were phase dark cells clustering - 02, and 5% 002 incubator. from the clusters with surrounding cells. The scattered cells that ted cells (so- tended to differentiate spontaneously into process-bearing multipolar called "spider’s ke" morphology) that is characteristic to pro- arrowheads oligodendroblasts or immature oligodendrocyte (indicated by white less in Figure 3, slides D & E) but their frequency of appearance was y F are from e 8, than 1%. The cells shown in Figure 3, slides D, E, and day 8, passage 11, day 11 and e 19, day 11, respectively. ' in Figure 4 illustrates the ion rate of HFSC cells (clone #2b) nglml PDGF~ the presence of different combinations of growth factors: (1): AA +10 nglml bFGF; (2): 20 nglml PDGF—AA + 10 nglml bFGF + 5 nglml NT—B; nglml (3): 20 nglml PDGF—AA + 10 nglml bFGF + 10 nglml lGF-1; (4): PDGF—AA + 10 nglml bFGF + 5 nglml NT—3 + 10 nglml lGF-1. The cells were in each harvested at day 11 of passage 3 (P3D11) and number of live cells condition that condition was counted. Then, they were passaged in the same The cells were harvested at was used at passage 3 at the same cell density. each condition was day 8 of passage 4 (P4D8) and number of live cells in counted (these cells were harvested before they became sub—confluent e they started forming spheres). Condition (4) was most effective at Condition (3) was effective at both passages. passage 3 but not at passage 4.

Figure 5 illustrates the s of high dose of PDGF-AA (100 nglml) and 1-thioglycerol on the proliferation of HFSC cells (clone #Zb); cultured in of growth HFSCM1 medium in the presence of the ing combinations factors: (1): 20 ng/mi PDGF—AA + 10 ng/ml bFGF + 10 nglml [GP—1; (2): 20 50 MA 1—thioglycerol; (3): ng/ml PDGF-AA +10 nglml bFGF + 10 ng/ml lGF-1 + PCT/IBZOIZ/00003O 100 nglml PDGF-AA +10 ng/ml bFGF +10 nglml lGF—l; (4): 100 nglml PDGF- AA +10 nglml bFGF + 10 ng/ml lGF—1 + 50 pM l-thioglycerol; (5): 100 nglml cells were harvested PDGF—AA +10 nglml bFGF + 50 prl l—thioglycerol. The before they became sub— at day 7 of passage 5 (these cells were harvested confluent because they started forming spheres as they did at e 4) of 20 number of live cells in each condition was counted. The combination cell nglml PDGF—AA + 10 nglml bFGF was also tested, but the recoVered number was too low to te (less than 1 x 10‘1 cells which was under the countable range) and its .data was eliminated from this figure. The condition (1) . The could expand cells at passage 3 but ’t expand cells at passage addition of 50 MA 1—thioglycerol [condition (2)] or the increase of PDGF-AA concentration to 100 nglml [condition (3)] had a very little positive effect on expansion rate. When the addition of 50 uM 1—thioglycerol and the increase of PDGF—AA concentration to 100 nglml were combined [condition (4)], the cell expansion rate improved dramatically and the cells could be ed sfully. When lGF-‘l was eliminated from this condition [condition (5)], the expansion rate decreased to <1, indicating that lGF—1 also ed HFSC cell proliferation and/or survival.

Figure 6 shows a growth curve for a human HFSC cells (Clone #Zb) in the (open circle with black line). HFSC cells (Clone #2b) were initially cultured bFGF. 10 nglml lGF—l was added from presence of 10 nglml PDGF-AA and 1D nglml sed from 20 nglml to 100 nglml passage 3. The concentration of PDGF-AA was from passage 6. However, they have very little or no effects on the expansion rate of the cells. 50 [1M 1—thioglycerol was added from passage 7. HFSC ceils (Clone #Zb) d to grow rapidly in the presence of 100 ng/ml PDGF-AA, 1O nglml bFGF, nglml IGF—1 and 50 pM 1-thioglycerol. 1'0 nglml NT—3 was added at e 17. NT—3 enhanced the cell growth a little bit but its effects disappeared after a passage, This panel also includes growth curves for human HFSC cells (Clone #2b) frozen at day of passage 10 (P10 Day 6: open circle with dashed line) and day 11 of passage (P11 Day 11: open circle with dot line). The frozen cells could be expanded at the similar speed after thawing, of HFSC cells in Figure 7 depicts spontaneous differentiation contrast images serum-free medium in slides A-F. Slides A and B are phase of HFSC taken with an inverted microscope Showing the morphological change slides A & B) after being cells (clone #Zb) at passage 12, day 9 (Figure 7, ng/mi ed in HFSClVH medium supplemented with 20 ng/ml PDGF—AA, slide A) or without 1- bFGF, 10 ng/ml lGF—1 and 50 pM 1-thioglycerol (Figure 7. ycerol (Figure 7, slide 8). HFSC'cells were differentiated spontaneously Even in the same without replenishing bFGF between ng medium. ion, HFSC cells didn't differentiate if bFGF was replenished ay and seemed to grow slowly. In on, HFSC cells were blocked to differentiate used. and formed clusters when 40 ng/ml or higher PDGF~AA was By and without decreasing the PDGFvAA concentration from 100 ng/ml to 20 ng/ml into replenishing bFGF, the cells could be differentiated neously process—bearing multipolar cells with spider3 weblike morphology, which expressed the O4 antigen (Figure 7, slides C & D) and/or GalC n (Figure ‘ cells [i.e., 7, slides E & F), a defining characteristic of oligodendrocyte—lineage . immature pro—oligodendroblast (O4—positive and GalC—negative), oligodendrocyte (O4—positive and GalC- positive)].

Figure 8 is a phase contrast images taken with an inverted at various microscope showing the morphology of HFSC cells (clone #3) stem cells were initially expanded in the passages. The conventional neural 8, slide A at Day 15 of presence of bFGF and EGF for 15 days (see Figure with 20 Passage 0). After that, the cells were cultured in the HFSClVl‘l medium ng/ml PDGF-AA and 10 ng/ml bFGF in an incubator maintained at 37°C, increased to 100 Oz, and 5% C302 incubator. PDGF—AA concentration was 50 pM ng/ml and 10 ng/ml of lGF—1 was added from e 4 (see Figure 9). 14thioglycerol was added from passage 8 (see Figure 9). Their morphology became almost identical with clone #Zb after several es.

Figure 9 shows a growth curve for a human HFSC cells (Clone #3) (open cultured in the presence of circle with black line). HFSC cells (Clone #3) were initially neural stem cells. Then, ng/ml EGF and 10 ng/ml bFGF to expand conventional PCT/IBZOIZ/000030 of A and 10 ng/ml the growth factor combination was d to 20 nglml bFGF from passage 1. 1O nglml IGF-1 and 10 nglml NT~3 were added from passage 2. Based on the data shown in Figure 4, NT-3 was removed form this culture and the concentration of PDGF‘AA was increased from 20 nglml to 100 nglml from passage of the cells as shown 'However, they have very little or no effects on the expansion rate in clone #2b. 50 pM 1-thioglycerol was added from passage 5, and then HFSC cells nglml (Ctone #3) started to grow rapidly in the presence of 100 ng/mt PDGF—AA, This bFGF, 10 nglml lGF-1 and 50 pM 1~thioglycerol as HFSC cells (Clone #2b). 7 of panel‘also inciudes growth curves for human HFSC cells (Clone #3) frozen at day dashed line). The frozen cells could be passage 10 (P10 Day 7: open circle with expanded at the simiiar speed after thawing.

Figure 10 is a phase contrast images taken with an inverted microscope showing the morphology of HFSC cells (clone 4A and 48) at s passages in slides A—F.. The cells were cultured in the HFSCM1 medium and 50 uM 1—thioglycerol with 20 nglml A, 20 nglml bFGF and ng/ml of lGF—1 (clone #4A) or 100 nglml PDGF-AA, 20 nglml bFGF and nglml of lGF-1 (clone #48) in an incubator maintained at 37°C, 5% 02, and 5% C02 incubator. Clone #4A grew slower than clone #48 lly but started to clone #48 from passage 2. They became grow at around the same speed as clone almost homogeneous and their morphology became almost identical with #2b or #3 after 3 passages, ] Figure 11 shows a growth curve for a human HFSC cells (Clone #4A: #48: open circle with black line). HFSC cells (Clone open square with dashed line and #4A) were cultured in the presence of 20 nglml of PDGF-AA, 20 nglml bFGF, cultured in the nglml, lGF—1 and 50 pM glycerol. HFSC cells (Clone #48) were ng/ml bFGF and 20 nglml lGF—1 and 50 [1M 1— presence of 100 nglml of PDGF—AA, thioglycerol. The cell number of both clones was decreased dramatically at first this decline was more prominent in clone #4A. After this initial decline in cell number, they started to expand rapidly This panel also includes a growth curve for human with HFSC cells (Clone #48) frozen at day 601‘ passage 5 (P5 Day 6: open circle dashed line).

Figure 12 iliustrates the immuno~phenotype of undifferentiated HFSC .cells in slides A—S; (clone #2b, passage 12—16) cultured in the presence PCT/IBZOIZ/OOOOISO ng/ml lGF—1, and 50 ulVl 1- of 100 ng/ml PDGF—AA, 10 ng/ml bFGF, 80x2, Nestin, Olig2, PDGF~ thioglycerol, and staining positive for either CD133, "Ra, NGZ, A285, PSA—NCAM, GFAP or Vimentin. DAPl was used to 90% of the cells were counterstain cell nuclei. These figures showed at least NGZ, A285 and vimentin positive for CD133, 80x2, Nestin,.Olig2, PDGF-Rd, The staining of PSA—NCAM was a little but there were no GFAP—positive cells. looked positive for AM. bit weak but still more than 90% of cells Figure 13, in slides A—H, shows flow cytometry data rating 13). Black line proportion of undifferentiated HFSC cells (clone #2b, passage and the gray—filled histograms histograms represented the iso—type l most of HFSC cells represented each tested antigen.‘ This data showed that 13 slide 8), were CD133—positive (Figure 13, slide A), (DDS-positive e slide D), A285 CD140a-positive (Figure 13, slide C), NG2-positive (Figure 13, slide F), and PSA—NCAM— positive (Figure 13, slide E), O4—positive (Figure 13, (data not positive (Figure 13, slide G). As tested by an immunocytochemistry shown), CD44 was ve (Figure 13, slide H). differentiated HFSC Figure 14 rates the immuno—phenotype of medium for 31 days cells (clone #3, passage 15) cultured in serum—containing Neurofilament—L, and stained with antibodies that recognize neuron (Blll Tublin, ed by a and MAP2), oligodendrocyte.(04, MBP) and astrocyte (GFAP) rstain cell nuclei. fluorescent secondary antibody. DAPl was used to marker. To evaluate HFSC cell could differentiate into cells positive for each co—stained with anti-Blll co—localization of neuronal axon and myelin, cells were slides A—C), anti- Tublin antibody and anti—MBP antibody (Figure 14, slides D-F), anti- Neurofilament-L dy and anti—MBP antibody (Figure 14, slides G-l).

IVIAP2 antibody and anti—MBP antibody (Figure 14, Only a few To evaluate co- al axon and myelin seemed to be co—localized. co-stained with 04 antibody and localization of O4 antigen and MBP, cells were BP antibody (Figure 14, slides J—L). Most signals for MBP were co— could also differentiate into localized with signal for O4 antigen. HFSC cells 14. slides MO). in astrocyte that was positive for GFAP antigen (Figure in many addition, many cells were positive for vimentin which was expressed epithelial cells including astrocyte and mesenchymal cells. Furthermore, undifferentiated cells were also positive for vimentin (data not shown). cells (clone Figure 15 illustrates the differentiation potential of HFSC #Zb, passage 15) in slides A-E. The cells were differentiated in DMEM/F12 containing gl'utamine and HEPES and supplemented with B27 supplement, 100 ng/ml IGF— supplement and 50 pM 1—thioglycerol with 10 ng/ml PDGF—AA, 1, 10 ng/ml BDNF, and 100 ull/l pCPT—cAMP. The cells were stained with anti- after GD3 dy and 04 antibody following fluorescent secondary antibody 4-day differentiation. Undifferentiated cells expressed GD3 er than oligodendrocyte progenitor cells and O4 vice versa (arrowhead of Figure 15, cell slide A, B and C shows undifferentiated . The most of differentiated showed a multipolar morphology with weak GD3 signal and strong 04 signal, indicating they were oligodendrocyte progenitor cells or pro-oligodendroblast.

Other cell types (e.g. astrocyte and neuron) were defined as a population GDS—nesgative and O4—negative cells. Figure 15, slide D shows the ratio of More each cell population from 10 ent imagesof the differentiated cells. than 70% of total cells differentiated into oligodendrocyte progenitor cells (75.8% i 2.09%). an 20% of total cells were still undifferentiated cells (23.5% :1: . The other cell type were less than 1 % of total cells (0.7% i: 0.41%). The ratio of oligodendrocyte progenitor cells and other cell types to differentiated cells (oligodendrocyte progenitor cells plus other cell types) were calculated and shown in Figure 15, slide E. endrocyte progenitor cell other cell types were 0.9% i- was 99.1% i‘0.56% of entiated cells whereas 0.56% of differentiated cells.

] DETAILED DESCRlPTION OF THE PREFERRED EMBODIMENTS OF THE lNVENTION The present invention provides a method for culturing and expanding neural stem cells or neural progenitor cells isolated from a - PCT/IBZOIZ/000030 mammalian central s system to produce a pure or enriched population neural stem cells or neural progenitor cells that have the ability to differentiate Neural stem into oligodendrocytes or oligodendrocyte—lineage cells in vitro. neuronal cells and neural progenitor cells both te progeny that are either cells (such as neuronal progenitors or mature neurons) or glial cells including self-renewable astrooytes and oligodendrocytes. While neural stem cells are but are not (i.e., able to proliferate indefinitely), neural progenitor cells may be, necessarily, capable of self renewal. The culture s of the present invention can produce an expanded cell population that can be entiated 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 neural stem cells to the pure or enriched population of expanded mammalian and/or neural progenitor cells that are described in this invention. ' [00056] The term “HFSCM1 ” refers to DMEM/F12 containing glutamine and HEPES and supplemented with 827 supplement (lnvitrogenTM), non-essential amino acids (NEAA) (InvitrogenTM), 1.5 mM pyruvate rogenTM), 55 pM B-mercaptoethanol (lnvitrogenm), and 1 mM N-acetyl—L- cysteine (Sigma) in combination.

The term “glial cells” refers to non~neuronal cells of the central nervous system and encompasses mature oligodendrocytes, astrocytes committed progenitor cells for either or both of these cell types.

The term “muttipotent progenitor cells” refers to neural progenitor limited cells that have the potential to give rise to cells from le, but a number of, lineages. potency” (derived from the Latin ”plurimus" or "very many“ and tia” 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, PCT/IBZOIZ/000030 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 itor cell that is committed, This is in contrast to a or ed, to become a specific type of mature cell. multipotent or a pluripotent progenitor cell, which has the potential to become cells which one of two or more types of mature cells (such as O—ZA progenitor on timing can become either oligodendrocytes or type-2 astrocytes, depending and environmental factors).

An “oligodendrocyte" is a type of gliatcell whose main function is to insulate nerve cell axons in the l nervous system of some rates.

The term “oligodendrocyte—lineage cells” refers to oligodendrocytes, pro-oligodendroblast and oligodendrocyte progenitors (e.g.02A progenitor).

This term does not include glial-restricted precursors or neural stem cells: The terms dendrocyte progenitor cells" and “oligodendrocyte progenitors" are used interchangeably throughout this ation and refer to cells that are committed to forming more itor cells and/or'progeny that are oligodendrocytes in preference to s or urological 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 Figure 1).

“Oligodendrocyte pre—progenitors" are predecessor cells of oligodendrocyte progenitors. ligodendroblasts” are predecessor cells to post-mitotic oligodendrocytes.

“Expanding" cells in culture means to increase cell number in the which stimulate cell presence of culture medium containing supplements 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 neural stem cells that are derived herein refers to neural progenitor cells or neural stem cells that have proliferated from ed neural progenitor cells or in vitro, producing the expanded cell population.

] "Passaging" cells (also known as lturing” or ting" cells) alive and growing under refers to a technique that enables cells to be kept of time by dissociating cells laboratory culture conditions for extended periods and then from one another (with enzymes like trypsin or collagenase vessel. Cells can be transferring a small number of cells into a new culture cultured for a longer time if they are passaged at regular intervals, as it avoids cell density. the premature senescence associated with ged high A “growth environment” is an environment in which the cells vitro. Features of the st will proliferate, differentiate and/or mature in environment include the medium in which the cells are Cultured, any growth and a supporting factors or differentiation—inducing factors that may be t, 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 Bonifacino et et al., eds, Wiley & Sons); Current Protocols in Cell Biology (J.S. et al. al., Wiley 8: Sons) and Current Protocols in lmmunology (J.E. Colligan eds., Wiley & Sons). Cell culture methods are described generally in the Basic Technique (R.l. current edition of Culture of Animal Cells: A Manual 'of Harrison Freshney etl, Wily & Sons); General ques of Cell Culture (MA. & l.F. Rae, Cambridge Univ. Press). Tissue Culture es and reagents are available from cial vendors such as Chemicon®. Millipore®, R&D Systems®, lnvitrogenTM, Nalgene—NuncTM lnternational, Sigma—AldrichTM, and ScienCell.

Specialized reference books relevant to this disclosure include McGraw—Hill 2000; Principles of Neuroscience, 4"1 edition, Kandel et al. eds, M. H.

CNS Regeneration: Basic e and Clinical es, Tuszynski & I’CT/132012/000030 Neuron: Cell and Molecular J.H Kordower, eds, Academic Press, 1999; The y, 3 rd edition, l. B. Levitan and L.K. Kaczmarek, Oxford U. Press, 2001; et al. eds, Cambridge U. Press Glial Cells: Role in Behavior, PR. Laming Matsas & 1998; The onal Roles of Glial Cells in Health and Disease, Jessen & Tsacopoulos eds, Plenum Pub. Corp, 1999: Glial Cell Development, Adrian dson eds, Oxford U. Press, 2001; and Man of Steel, Hill, 1996. in the context of cell ontogeny, the adjective “differentiated" is a further along the relative term. A differentiated cell is a cell that has ssed developmental pathway than the cell to which it is being compared. Thus, neural stem cells can differentiate to lineage-restricted progenitors. These, _ to end-stage turn, can differentiate into cells further along the pathway or differentiated cells, such as mature neurons or oligodendrocytes.

Differentiated cells of this invention can be characterized according of oligodendrocytes. to whether they express phenotypic markers teristic Classic immunocytochemical markers for these cells that may be t depending on the ty of the cell population are the following: 80x2: 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~RG)I the a chain of the platelet— derived growth factor receptor. A marker for endrocytes and their progenitors.

CD140a: the same as PDGF—Ror. CD140a antibody recognizes an extracellular domain of PDGF—Rd. A cell surface marker for oligodendrocytes and their progenitors.

CD9: a cell surface glycoprotein that is know to complex with cell surface integrins and other transmembrane 4 superfamily proteins. A marker for germline stem cell, neural stem cell, oligodendrocyte, mesenchymal stem cell.

PSA—NCAM: polysiaiylated-neural cell adhesion molecules. A cell neuronal progenitor, surface marker for neuronal-restricted precursor (NRP), — ~PCT/1132012/000030 u u v u This marker is negative in neurobiast and oligodendrocyte pre—progenitor. giial——restricted precursor (GRP) —restricted precursor (GRP) ] A2851a cell surface marker for glial— cell (OPC) and type 2 glial progenitor cells and oligodendrocyte itor neuronal—restricted precursor astrocytes. This cell surface marker is negative in (NRP). ‘ A cell surface marker for N62: a chondroitin sulfate proteoglycan. cells. macrophages and oligodendrocyte progenitor GD3: Ganglioside GD3. A marker for oligodendrocyte pre- progenitor and oligodendrocyte progenitors 04: a marker for oligodendrocytes and their progenitors.

Galactocerebroside C (GaIC): a marker for immature endrocytes.

Myelin basic protein (MBP): a marker for mature endrocyte. in cell—cell interactions, CD44: a cell-surface glycoprotein involved for hyaiuronic acid. A cell surface cell adhesion and migration and a receptor cells. marker for some epithelial cells and astrocyte lineage for astrocytes.

Glial fibrillary acidic protein (GFAP): a marker and neurons. ] [3]“ Tublin: a marker for neuronal progenitors Neurofilament—L: a marker for mature neurons.

Microtubule—associated protein 2 (MAPZ): a marker for mature neurons.

Tissue specific markers can be detected using any for cell surface immunological technique, such as flow immunocytochemistry of fixed cells or tissue s, or immunohistochemistry (for example, detailed method for flow sections) for ellular or cell—surface s. A Blood, 9611740, 2000. cytometry is is provided in Galiacher et ai, if a significantly Expression of a cell—surface antigen is defined as positive in a standard detectable amount of antibody will bind to the antigen after fixation of the lmmunocytochemistry or flow cytometry assay, optionally or other conjugate to cells, and optionally using a labeled secondary antibody PCT/132012/000030 WO 95730 it is often beneficial to amplify ng. To facilitate use in research or therapy, the characteristics maximize the proportion of cells in the population that have 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 form on, it is often desirable to minimize the ability of the cell tion to and cells of non— other cell types, particularly undifferentiated stem cells, ectodermal lineage. Depending on the application, it may also be neuronal lineage and advantageous to minimize the tion of cells of the and their committed their committed progenitors or cells of the astrocyte lineage invention progenitors. The contamination of the populations according to this these other types of have less than 30%, 20%, 10% or 5% contamination with cells.

The methods of the present invention cannot result in the development of an entire human organism.

The method'of the present ion involves culturing isolated central neural stem cells and/or neural progenitor cells from a mammalian the expansion of the cells nervous system in a defined medium that s method of the present through multiple passages. The cells cultured using the ion 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 ed over into 1,000 times while retaining their, ability to subsequently differentiate endrocyte—lineage cells. In some embodiments, the expanded cell population resulting from the culture method of the present invention comprises of cells having at least 30%, 50%, 70% or or can differentiate into a population serum-free culture 80% oligodendrocyte—lineage cells of differentiated cells in condition. ln a preferred embodiment, the expanded cell culture population ion comprises at least resulting from the culture method of the present PCT/IBZOIZ/OOOO30 WO 95730 cells. In preferred 90% oligodendrocyte—lineage cells of entiated In some embodiments the embodiments, the ed cells are otent. into oligodendrocyte— majority of expanded cells are capable of differentiating lineage cells upon culturing in decreased PDGF—AA medium (i. e., 20 ng/ml 50 uM 1-thioglycerol PDGF~AA, preferably with 10 ng/ml bFGF with or without bFGF between and optionally with at least 10 ng/ml lGF—t) without replenishing changing medium. ln some embodiments the majority of expanded cells are in10 capable of differentiating into oligodendrocyte—lineage cells upon culturing and 10 ng/ml BDNF ng/ml of PDGF-AA, 100 ng/ml lGF—1, 100 MA pCPT-CAMP and supplemented with B27 in DMEM/F12 containing glutamine and HEPES supplement, N2 ment and 50 pM 1—thioglycerol.

The isolated mammalian neural stem cells and/0r neural progenitor the central nervous cells for use in the present invention may be obtained from but not limited to, a system of a mammalian, preferably a e such as, human. Oligodendrocyte progenitors and pre~progenitors are known to exist in from white matter of the central s system As such suitable sources not limited which to isolate cells for use in the present invention include, but are cord. In on, isolated stem to, the optic nerve, corpus callosum and spinal such cells may be derived from a mammalian fetus, preferably a primate fetus, methods known in the art. In some as but not limited to a human fetus, using fetal spinal embodiments, the isolated stem cells are prepared from human cord tissue obtained from a human fetal spinal column. In a preferred obtained from 8» embcdiment, isolated cells for use in the present invention are human fetal 24 weeks ional age, preferably 12—18 weeks gestational age for example, spinal cord. Human fetal spinal columns can be obtained, commercially through companies such as Advanced Bioscience Resources, ed t lnc. (Alameda, CA, USA) with the lRB permission and an with from a donor. Spinal cord tissue can be dissected from the spinal column, The tissue then can be the meninges and peripheral nerves removed. dissociated, washed and placed in a culture vessel containing a growth medium that permits cell proliferation. limited to, culture ] Suitable culture vessels may e, but are not combination of poly-amino acids vessels with a culture surface having one or a tissue culture plastic and surfaces (e.g., poly—lysine and/or poly—ornithine), Cells generally may be plated at treated with laminin, vitronectin or fibronectin. to 105 cmz, preferably at a density of a y ranging from 10“ approximately 3 x 104 to 5 x 104 cells/cm? Poly~ornithine or poly—lysine may be et al, J. Neurosci., used to coat culture vessels as reported previously (Raff 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 2 to 20 pglml, more preferably 5 to 15 to 40 pg/ml of poly——ornithine preferably pg/ml. The strength of cell attachment can vary depending on , surface vessels. The l modification, format and c lot of culture for each source of culture concentration of coating materials can be determined vessels using methods known in the art. In some embodiments a two—hour is performed to coat incubation with 10 pg/ml of poly—ornithine or poly-lysine In some vessels, for example, from BD Faicon (Sparks, MD, USA). embodiments a 30—minute incubation with 5 pg/ml of poly-ornithine or poly— from Nalgen Nunc lysine can be performed to coat vessels, for example, International (Rochester, NY USA) cells The isolated neural stem cells and/or neural progenitor in a serum- ed from a mammalian central nervous system are cultured cell expansion without , free chemically defined culture medium that permits promoting differentiation of the cells (for example, into neurons, astrocytes such as, but endrocytes). The culture medium comprises a base medium Medium: Nutrient e F-12, 1:1 not limited to, Dulbecco’s Modified Eagle Medium, RPMI- (DMEM/F12) (Invitrogen®) (e.g., lscove’s Modified Dulbecco’s be supplemented with various 1640 and Neurobasal). The base medium may Such components may components to support cell‘health and survival. non-esSential amino acids [NEAA include, but are not limited to, at least 0.25% (Invitrogen®)— a 1% solution contains 100 pM of L—Alanine, L—Asparagine H20, and L—Serine], at least 1.0 L-Aspartic Acid, L-Glutamic Acid, e, L~Proline, PCT/IBZOIZ/00003O W0 2012/095730 1% 827 supplement mM glutamine, at least 0.5 mM pyruvate, at least (lnvitrogen®),‘at least 0.1 mM N-acetyl—cysteine and/or at least 10 uiVl B- mercaptoethanol. In some embodiments the base medium may comprise N821 (as sed in Y. Chen et al., J. Neurosci. Methods, 171:239, 2008) in place of 827 supplement. 827 supplement contains bovine serum albumin, retinol transferrin, insulin, progesterone, corticosterone, triiodo-l—thyronine, Linoleic acid, Linolenic acetate, DL tocopherol, DL tocopherol acetate, Biotin, d, catalase, acid, ethanolamine, Na Selenite, L—carnitine, glutathione superoxide ase, Degalactose and putrescine. lnvitrogen has disclosed its ingredients but hasn’t disclosed their concentration. However, concentration of each ingredients of their al formulation, B18 supplement, based on this information and the was sed. N821 was developed concentration of each gradient was disclosed. lt worked in neuronal culture as good as 827 supplement. In addition, N821 could be used to culture neural from human embryonic stem cells and oligodendrocyte progenitors derived stem cells. Therefore, this supplement is thought to be a good ate to replace B27 supplement. In a preferred embodiment, the culture medium comprises DMEM/F12 supplemented With 1-4 mM, more preferably 2.5 glutamine; 10—25 mM, more preferably 15 mM HEPES; 0 mM, more preferably 1 mM pyruvate; 1 to 4%, more preferably 2% 827 supplement; 0.25— 3%, more preferably 1% NEAA; 1-200 pM, more preferably 50 0M -100 thioglycerol; 0.1-3 mM, more preferably 1 mM N~acetyl~cysteine; and/or pM, more preferably 55 0M B—mercaptoethanol.

Furthermore, oxygen may facilitate entiation of neural itor cells. Therefore, to reduce cell differentiation, the cells may be ed in a 1—20% 02 growth environment. In a preferred embodiment, the cells are cultured in a culture flask in an incubator providing a 37°C, 1—10%, environment. After establishing HFSC more preferably 5% Oz, 5% CO; growth there was no cells, the effect of oxygen concentration was assessed but to 5% Oz se of differentiated celis in 20% Oz condition compared of HFSC condition in the culture condition to expand HFSC cells. The growth PCT/11320 12/000030 cells in 5% 0; condition was a little bit faster than that in 20% 0; condition. of p53 in Oxygen is a cause of oxidative stress and known to induce mutation rodent cells. To reduce a risk of mutation, we kept culturing HFSC cells in 5% 02 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 nglml platelet—derived growth -AA (PDGF—AA), at least 2.5, 5 or 10 nglml basic FGF (bFGF), and/or at least 10, 25 or 50 mm 1—thioglycerol to isolate the cells. In some embodiments the culture medium further comprises at least 1, 5 PDGF— or 10 nglml insulin—like growth factor—1 (lGF—1). in some embodiments AA may be replaced with PDGF-BB, PDGF—AB, PDGF—CC, or PDGF—DD. ln some embodiments bFGF may be replaced with other member of fibroblast growth factors (e.g. FGF-4 or FGF—9). In some embodiments lGF—1 may be replaced with lGF—Z. In a red embodiment, the e medium comprises 40-60 uM 1—thioglycerol, 40—200 nglml PDGF—AA, 5—100 nglml bFGF, and 5- 100 nglml lGF—1 to isolate the cells.

The ed neural stem cells and/or neural progenitor cells are cultured in culture medium further sing growth factors to ate proliferation after isolating the cells. The culture medium may contain at least 1, 2, or 5 nglml platelet—derived growth factor-AA (PDGE—AA), at least 0.5, 1 or ng/ml basic FGF (bFGF), and/or at least 10, 25 or 50 uM 1-thioglycer0l to expand the cells. In some embodiments the culture medium further comprises at least 1, 2 or 5 nglml insulin-Iikegrowth factor—1 (lGF—1). In a preferred embodiment, the culture medium comprises 40—60 uM 1-thioglycerol, 5—100 nglml PDGF—AA, 1—50 nglml bFGF, and 5-100 nglml lGF—‘l to expand the cells after the isolation of HFSC cells.

The isolated neural stem cells and/or neural itor cells grown under culture conditions of the present invention exhibit a doubling time of 50- 120 hours. In red embodiments, the ng time is about 60 to 100 hours. The cells can continue this proliferation rate through at least 8, 11, 14 or ' PCT/IBZOIZ/OOOOSO 17 passages. The cells may be ed at least 100, 250. or preferably at least 500 times per month. ln preferred embodiments, the cells cultured using the method of the present invention exhibits an expansion rate >1 for more than 18 passages.

Examples Example 1 base -— HFSC cell e and expansion; Identification of medium and growth ‘factcrs 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, CA, USA) with an ed consent of a donor. The cells were cultured in —12 (1:1) (DMEM/F12) (lnvitrogenTM, Carlsbad, CA, USA) supplemented with 2 mM glutamine llnvitrogenTM), 1 mM pyruvate (lnvitrogenTM) and 2% 827 supplement (lnvitrogenTM) initially. s growth factors were examined whether they stimulate growth of HFSC cells. Most effective growth s 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% 827 supplement could decrease vacuoles and increase cell number slightly (data not . Other supplements ing 1 mM N- acetyl—cysteine (Sigma—AldrichTM, St. Louis, MO, USA) and 55 pM [3- mercaptoethanol (lnvitrogenTM) seemed to improve the cells’ status but the improvements were not as prominent as with NEAA (data not . Thus, DMEM/F12 supplemented with 2 mM glutamine, 1 mM pyruvate, 2% 827 supplement, 1% NEAA, 1mM yl—cysteine and 55 pM B—mercaptoethanol to culture was identified as optimal base medium and was used thereafter HFSC cells.

Human 15-week fetal spinal cord was dissected from the spinal column, with the meninges and peripheral nerves removed. The tissue then PCT/IBZOIZ/OOOOSO and washed and cells ed from human was dissociated with Accutase vessel containing the following growth fetal spinal cord were placed in a culture and ’l5 mM HEPES, 2% medium: DMEM/F12 containing 2.5 mM glutamine 1.5 mM pyruvate (lnvitrogenTM), 55 827 supplement rogenTM), 1% NEAA, i lel N-acetyl—L~cysteine (Sigma- uM aptoethanol (lnvitrogenTM).

AldrichTM TM), 20 ng/ml PDGF-AA (R&D Systems, Inc., Minneapolis, MN, USA) then placed in an incubator and 10 ng/ml bFGF (R&D s). Cells were added daily to maintained at 37°C, 5% Oz, and 5% (302. bFGF (10 ng/ml) was 2-3 days during passage. the culture medium. Medium was Changed every cells were not so many in the Based on the preliminary experiments, g and bFGF and many cells stopped erating or died presence of A PDGF—Ro within a few weeks. This result seemed to be able because To remove cells that are not expressing cells are usually less than 5% of cells. responsive to PDGF-AA and bFGF, cells were cultured in an ultra-low adhesion The cells that are responsive to culture plate (Corning lnc., Corning, NY, USA). while cells that are PDGF-AA and bFGF made spheres (see Figure 3, slide A) not responsive to them did not form spheres. Spheres were collected at lower 1,000 rpm for speed of centrifugation (300 rpm for collecting s vs.

After 9 days, cells collecting single cells) for medium change and passaging. onto poly—ornithine—coated culture vessels. After were ted and passaged The cells exhibited a this period, cells were passaged every 7-14 days.

Figure 3, slides B and C) heterogeneous morphology at these early stages (see within a month. The proliferation and many of the cells stopped proliferating cells eventually did not expand. ln rate became slower over time and the vacuoles in their addition, the cells began to appear unhealthy, forming cytoplasm. condition for HSCF cell Example 2 —— Identification of optimal growth expansion of PDGF—AA and bFGF in the As mentioned in Example 1, inclusion of the HFSC cells initially, but the culture medium supported adequate growth NT—3 (R&D Systems) and lGF-1 protiferation rate slowed after 2 passages.

PCT/IBZOIZ/000030 (Sigma-AldrichTM) were tested to determine ifthey could enhance the proliferation of HFSC cells. The presence of 20 nglml PDGF—AA with 10 nglml bFGF was insufficient to stimulate the proliferation rate of the cells (the expansion rate was <1) (see Figure 4). Subsequent addition of 5 nglml NT-3 and/or10 nglml lGF-1 resulted in an enhanced proliferation rate (the expansion rate became >1). Combination of NT—3 and lGF—1 was most effective at further passaged to passage 3. The cells ed from each condition were confirm their effects. The cells were harvested earlier because the cells started forming spheres. At passage 4, the recovery of eration rate by combination of NT—3 and IGF—1 was decreased and single addition of lGF-1 became most effective at passage 4. Combination of NT—3 and lGF—1 might cause differentiation of cells or form more spheres and lost while changing medium. Thus, the addition of lGF—‘l was identified as a most effective survival factor and was used thereafter to culture HFSC cells. However, the proliferation rate of the HFSC cells began to slow again at the end of passage 4 and the cell number was decreased comparing to the seeded cell . ore, another supplement, 1-thioglycerol, was examined whether it could enhance the proliferation of HFSC cells at passage 5.

Figure 5 shows the effect of 1-thioglycerol on the eration of HFSC cells. The initial growth factor ation (20 nglml PDGF-AA +. 10 nglml bFGF) ‘t expand cells at all (data was not shown in this figure e it was under the countable range). The growth factor combination used from passage 3 (20 nglml PDGF~AA + 10 nglml bFGF + 10 nglml lGF—1) + 10 nglml was more effective than this combination (20 nglml A bFGF) but unable to stimulate HFSC cell proliferation well after passage 4 (Figure 4). Many cofactors related to the biosynthesis and degradation of fatty acids and related long-chain hydrocarbons feature thiols. 1—thioglycerol was next tested on the HFSC cells since it is one of thiol—based antioxidants and has been reported to ate the proliferation of some cells (i.e. mouse embryonic cortical and hippocampal neurons, mouse bone marrow mast cells, and human B cell lines) in culture. In addition, higher dose of PDGF—AA (100 PCT/IBZOIZ/000030 ng/ml) was also tested whether it could complement the decrease of reactivity to growth factors.

The addition of 50 pM 1—thioglycerol in the presence of 20 nglml PDGF~AA, 10 ng/ml bFGF and 10 ng/ml tGF-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-thiogiyceroi when the PDGF-AA concentration was increased to 100 ng/ml in the presence of 10 nglml bFGF + ng/ml'lGF—1. However, when both 50 pM 1—thioglycerol and sed PDGF-AA (100 nglml) were ed in the culture medium (along with 10 nglml bFGF and 10 nglml lGF—1), the two components appeared to work synergisticaliy, significantly increasing the cell number (expansion rate >1).

When lGF-1 was ated from this suppiement cocktail (i.e. total supplementation was 100 nglml A + 10 ng/mi bFGF + 50 11M thioglycerol), the expansion rate decreased to <1, indicating that lGF-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 pM 1—thioglycerol and the increase of PDGF—AA concentration to 100 ng/ml in addition to 10 nglml bFGF + 10 nglml lGF—1 were important to expand the cells. rmore, addition of lGF-1 might not be mandatory but was still effective even in the presence of 50 uM 1~thioglycerol and 100 nglml PDGF—AA to increase the ion rate. The combination of 50 pM 1—thiogiycerol and 100 nglml PDGF—AA in addition to 10 nglml bFGF + 10 nglml lGF-1 was used thereafter to e HFSC cells.

The HFSC cells were further expanded in the ce of 100 nglml PDGF-AA, 10 nglml bFGF, 10 nglml iGF—1 and 50 pM 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 The HFSC cells were able to was approximately 60—100 hours in this condition. maintain this proliferative state even after passage 8 (see Figure 3, slide D), and passage 19 (see Figure 3, slide F). passage 11 (see Figure 3, slide E) Thus, defined medium sing 100 ng/ml PDGF—AA, 10 nglml bFGF, 10 the optimal e ng/ml lGF-1 and 50 ulVl 1—thioglycerol was identified as cells and/or neural condition for long term expansion of the neural stem progenitor cells. of HSCF cell Example 3 — neous differentiation technique While testing various culture conditions, it was observed that when HFSC cells were cultured with the removal of bFGF from the medium, many cells (indicative of the cells seemed to differentiate into bipolar or multipolar oligodendrocyte) and died soon. To avoid these cell death, a spontaneous entiation que 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 entiation, medium was changed every of PDGF-AA 3 or4 days for this experiment. Basic FGF and high concentration cells but they were also important were thought to block differentiation of HFSC for HFSC cells to survive. Therefore, PDGF—AA concentration was decreased lGF—1 with from 100 to 20 ng/ml in the presence of 10 ng/ml bFGF, 10 ng/ml cells couldn’t survive well 50pM 1-thioglycerol or t 1—thioglycerol. HFSC if bFGF was removed. Usually, bFGF were replenished ay to keep HFSC cells in proliferative state. When replenishing bFGF was stopped, many HFSC cells separated from their clusters and formed complex web—like and/or immature processes ative of pro—oligodendroblasts I oligbdendrocytes) and survived well as shown in Figure 7, slidesA and B.

These process—bearing cells With s web—like morphology as shown in Figure 7, slides were positive for O4 antigen and/or GalC antigen C—F, thereforerthey were thought to be pro~oligodendroblast (O4—positive GalC~negative) or immature oligodendrocyte (O4—positive and GalC— positive). be differentiated but Even in the presence of 1—thioglycerol, HFSC cells could in the complexity of process looked simpler when 1-thioglycerol was present In addition, other cell types e medium as shown in Figure 7, slides A & B.

HFSC cells were thought (eg, astrocyte 'and neuron) were rarely observed and PCT/IBZOIZ/000030 cells. This to be prone to differentiate into only oligodendrocyte-lineage cells in a technique enabled to observe differentiated oligodendrocyte-lineage healthy state.

These data further indicate that the culture conditions of the t stem cells and/or invention are particularly useful for expanding isolated neural neural progenitor cells that are prone to entiate into oligodendrocytes, characteristics since most of the differentiated cells exhibited oligodendr’ocyte and resemble oligodendrocytes or igodendrocytes.’ cell Example 4- Induction of HFSC cell from conventional neural stem As shown in Figure 1, conventional neural stem cell is thought to be ' this relationship, it was examined a predecessor of HFSC cell. To confirm neural stem cell that whether HFSC cell could be induced from conventional from a second tissue sample of human fetal was reported previously. The cells of 10 spinal cord (11 weeks ion) were initially cultured in the presence mM glutamine, 15 nglml EGF and 10 nglml bFGF in DMEM/F12 containing 2.5 mM-HEPES, 2% 827 supplement, 1% NEAA, 1.5 mM pyruvate, 55 uM [3— mercaptoethanol, and ’l lel N—acetyl—bcysteine for 15 days to expand conventional neural stem cells. Many different cell types were present initially the end of the primary but some expandable cell population was obtained at A at Day 15 of e culture. These results are rated in Figure 8, slide ion as clone 0. After the first passage the cells were cultured in the same nglml lGF-1, 100 nglml #Zb (i.e. HFSCM1 medium with 10 nglml bFGF, PDGF-AA and 50 11M 1—thioglycerol) through 15 passages.

The morphology of the cells from the expandable cell population at around obtained at the end of the primary culture became homogenous to form clusters and s, similar to those passage 5 and the cells tended In addition, this clone that formed with clone #213 (see Figure 8, slides B~F). cells spontaneously (data not could differentiate into oligodendrocyte—lineage #2b (data not shown) shown), showed the same immunevphenotype as clone flow cytometry as described in and the same marker expression pattern by WO 95730 PCT/lBZOlZ/(lOQOLEO for future testing (e.g.

Example 7. The cells from this clone were frozen down Figure 12). This data suggests that HFSC cell could be d from is thought conventional neural stem cell and that conventional neural stem cell to be a essor of HFSC cell. {000117} PDGF-AA was thoUghtto work through PDGF receptor a. This members (PDGF-AA, receptor is known to be stimulated by all PDGF family used to PDGF-BB, PDGF—AB. PDGF—CC and PDGF—DD). PDGF-BB was HFSC cell clone examine whether PDGF-BB could replace PDGF—AA using condition #2b and clone #3. PDGF—BB could expand (both clones in the same except for PDGF—AA and it was proved that PDGF—AA could be successfully replaced with other PDGF family members.

Example 5— Confirmation of l cell cuiture components While the inventor tested various culture conditions after HFSC cell for each (clone #2b) was established, the inventor noticed that dose response growth factor has been changed. To isolate HFSC cell (clone #2b), higher concentration of PDGF—AA (100 ng/ml) was necessary in addition to 50 pM thioglycerol. After this clone became proliferate constantly, higher this concentration of PDGF—AA (100 ng/ml) was no more required to expand clone. Expansion rate was saturated at around 10-20 ng/ml of PDGF-AA and had no additional effects on their higher concentration of A (100 ng/ml) growth. This may be because of a long-term culture or a continuous usage 1—thioglycerol. To establish clone #2b and clone #3 of HFSC cells, 1— of higher thioglycerol was used after several passages. To e the effect of 1— concentration of PDGF—AA, new ciones were ished in the presence ycerol from-an initial e. In addition, the response to bFGF and lGF—1 When higher seemed to be ted at 20 ng/ml and 40 ng/ml, respectively. cells could concentration of lGF—1 (50600 ng/ml) was used, more differentiated be seen (it looked like around 1%). Therefore. 20 ng/ml lGF—1 was considered The usage of 20 ng/mi bFGF and 20 to be preferable to expand HFSC cells. 540%. compared to the usage of 10 ng/ml lGF—1 improved the expansion rate ng/ml bFGF and 10 ng/ml lGF-1. Therefore, 20 ng/ml bFGF and 20 ng/ml lGF- 1 were used for this experiment.

HFSC cells from a third sample (12 weeks gestation) were cultured in the identified optimal to see it higher tration of PDGF-AA is required culture medium components and if they would provide similar growth in the same characteristics in another batch of cells. The cells were cultured medium and 50 pM 1— culture medium as clone #2b and #3 (Le. HFSCM1 A in thioglycerol) with 20 ng/ml (clone #4A) or 100 ng/ml (clone #48) of the first passage, addition to 20 ng/ml bFGF and 20 ng/ml lGF—1.; At the end that of clone #48 (see cell number of clone.#4A was less than one third of Figure 11, slide A and slide B). However, clone #4A d to erate at the similar speed as clone #48 after passage 1 even with lower PDGF-AA concentration than clone #48. The logy of the cells became form clusters and spheres, homogenous at passage 3 and the cells tended to slides C—F). In r to those. that formed with clone #2b or #3 (see Figure 10, cells addition, these cells could differentiate into oligodendrocyte—lineage spontaneously as clone #2b and #3 did. This result suggested that higher isolate HFSC concentration of PDGF-AA was not mandatory but preferable to not necessary once HFSC cells and that higher concentration of PDGF-AA was cells were established. Furthermore, this culture method could provide similar growth characteristics in another batch of cells and it was confirmed that this The cells from this clone were frozen down for process could be repeatable. future testing. and grow after Example 6— Ability of expanded HFSC cells to recover freeze-thaw cycle of 8% The cells of clone #2b were cryopreserved in the presence frozen at DMSO at e 10 11 and 12 The HFSC cells (clone #2b) passage 11 have been deposited at ATCC (accession number PTA—12291) the presence of 8% DMSO at The cells of clone #3 were also cryopreservedin of clone #4A and #48 grew faster than clone #2b passage 9 and 10. The cells VVO 2012/095730 or #3, therefore they were cryopreserved in the presence of 8% DMSO at 4 and 5 (clone #48) in the same passage 4 and 5 (clone #4A) or passage 3, condition. The same medium to culture the HFSC cell (HFSCM1 medium and 50 pM 1—thiogiycerol) was used for freezing the HFSC cells. The cells were later thawed and cultured in the above described free HFSCM1 medium and 50 uM 1—thioglycerol with 10 ng/ml bFGF, 10 ng/ml lGF—1, 100 ng/ml PDGF-AA or 20 ng/mi bFGF, 20 ng/mi lGF—1, 20 ng/ml A. These cells were observed to proliferate at a similar rate as before freezing (see Figure 6, Figure 9 and Figure 11). The frozen cells were used for later experiments .shown in Figures 12—15.

Example 7 —— Characterization of ed and undifferentiated HFSC cells 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 lGF-1 and 50 pM 1- thiogiycerol, they grew in clusters and/or spheres as shown in Figure 3, slides D-F. The scattered cells that separated from the clusters tended to differentiate into oligodendrocytes as shown in Figures 3, slides D and E. Even when the . cells were passaged in a single cell state, they eventually began to gather and form clusters again. Their shape in this proliferative state resembled oligodendrocyte. pre-progenitor cells (see Ben—Hur et al, J. Neurosci., 1825777, 1998; Gago et al, Mol. Cell. Neurosci, 22:162. 2003), but not like O-2A progenitors which grow in a bipolar morphology without forming any clusters.

Even rat oligodendrocyte pre-progenitor celis were reported a long time ago, the human counterpart has not been reported. However, it was ssary to fy the precise stage of oligodendrocyte predecessor cell in the expanded cell culture since they retained their y to differentiate into oligodendrocytes (as shown below) regardless.

In order to characterize the -phenotype of the undifferentiated HFSC cells, the cells at passage 11-15 were dissociated into a single cell state with Accutase (lnnovative Cell Technologies, San Diego, CA, . USA) and grown in poiy-ornithine—coated l culture piates and cultured PCT/IBZOIZ/OOOOZSO ng/ml bFGF, 10 ng/ml 3-7 days in the presence of 100 ng/ml PDGF-AA, with 4% lGF— 1 and 50 uM 1 -thioglycerol The cells were then fixed paraformaldehyde and then washed with PBS. For staining surface antigens GalC and PSA-NCAM, cells were like CD133 PDGF-Roi, NG2 A285, 04, O1 and stained with blocked with PBS containing 3% normal gout serum (NGS) antibodies. For staining ellular antigens like 80x2, nestin, Olig2, myelin Blll Tublin, Neurofilament-L and MAP2, basic protein (MBP), Vimentin, GFAP, X—100 in ice-cold PBS for 10 min cells were permeabilized with 0.1% Triton before blocking. CD133/1 mouse lgG1 monoclonal antibody (Clone A0133, (Upstate), anti—N62 Miltenyi Biotec), anti—PDGF—‘Ro rabbit polyclonal antibody mouse lgM monoclonal rabbit polyclonal antibody (Millipore), anti—PSA—NCAM antibody pore), A285 mouse lgM onal antibody (Millipore), 01 mouse lgM onal mouse lgM monoclonal dy (R&D Systems), anti-Nestin antibody (Millipore), anti—80x2 rabbit polyclonal antibody (Millipore), mouse monoclonal mouse lgG1 monoclonal antibody (Millipore), anti-OligZ BP rat lgG2a antibody, anti-GalC monoclonal lgGB antibody pore), monoclonal monoclonal lgG2a antibody (Millipore), anti-Vimentin mouse lgG1 antibody pore), anti- antibody (Santa Cruz), anti-GFAP rabbit polyclonal anti—Neurofilament-L lSlll Tublin monoclonal mouse lgG1 antibody; (Millipore), anti-MAP2 rabbit polyclonal mouse monoclonal lgG1 antibody (Cell Signaling), antibody (Miilipore) and anti—MAPZ mouse lgG1 monoclonal antibody 1:600 (N62), (Millipore) were used at 1:300'(CD133/1), 1:300 (PDGF—Ro), 1:1000 (01), 121000 (30x2), 121000 (PSA—NCAM), 121000 , 121000 (04), 1:50 (MBP), 1.2500 (Vimentin), 1:200 (Nestin), 1:200 (Olig2), 1:100 (GalC), 1:1000 (GFAP), 1:100 (l3lll Tublin), 1:200 filament-L), 121000 (MAPZ, PBS containing 3% NGS. After polyclonal) or 1:200 (MAPZ, monoclonal) in with 3 s of PBS overnight incubation at 4°C, wells were washed cell staining for some cell surface containing 3% N68. ln some case, a live used to reduce non—specific antigen (N62, A285, 04, O1 and GD3) was each primary dy before signals. In such case, cells were stained with fixation without blocking. Anti-N62 rabbit poiyclonal antibody, A285 mouse WO 95730 IglVl monoclonal antibody, 04 mouse lglVl monoclonal antibody, 01 mouse lglVl monoclonal antibody, and anti—Disialoganglioside GD3 mouse monoclonal lgGE antibody (Millipore) were used at 1:150 (N62), 1:100 (A285), 1:200 (04), 1:100‘ (01) and 1:200 ((303) 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 lgG (Fcy nt specific), DyLight 488—conjugated Pure Goat anti—mouse lgG (Fcy Fragment specific), DyLight 488— conjugated AffiniPure Goat anti—rabbit lgG (H+L), DyLight 488—conjugated AffiniPure Goat anti—rat lgG (H+L), DyLight 594-conjugated AffiniPure Goat use lgG (H+L), and/or DyLight 594-conjugated Pure Goat anti— mouse lgM (u chain specific) (all ary antibodies were purchased from Jackson lmmunoResearch tories, lnc.) were used at dilution of 1:500 for 1 hour at room temperature. The cells were then washed with 2 s PBS. DAPl was used to counterstain cell nuclei. The cells were then observed using an Olympus 1X81 equipped for epifluorescence.

Most of the HFSC cells were CD133—positive (see Figure 12, slide A & B),.Sox2—positive (see Figure 12, slides C & D) and nestin-positive (see Figure 12, slide E & F), indicative of neural stem cells. Most of the HFSC cells cells for motor neuron and were OligZ—positive (often indicative of progenitor oligodendrocyte) (see Figure 12, slides G & H), PDGF-Ra-positive (often indicative of oligodendrocyte~lineage cells) (see Figure 12, slides I & J), A285- positive (A285 is y absent from neural stem cells and found on glial progenitor cells, oligodendrocyte progenitor cells and type—2 astrocytes) (see cells. Most of Figure 12, slides M & N), indicative of oligodendrocyte progenitor slides O 8: P) but the HFSC cells were PSA~NCAM—positive (see Figure 12, absent from there were variety in their expression levels (PSA—NCAM is usually neural stem cells and found on neuronal progenitor cells and oligodendrocyte ogenitor cells). No GFAP~positive cells could be seen while most of them At least 90% of HFSC cell were vimentin—positive (see Figure 12, slide 0-8). seemed to be positive for above markers except for GFAP but the precise counting was very difficult because HFSC cell tended to make clusters and to be detached from e vessels very easily during on and staining. To quantify their purity, a flow cytometry analysis was done and shown in Figure ln addition, erentiated HFSC cells were weakly stained with 04 antibody that is a marker for pro—oligodendroblast (O4-positive, GalC— negative) and immature oligodendrocyte (Oil—positive, GalC—positive) by immunocytochemistry but it was difficult to distinguish with weak staining and non-specific staining. Some pro—oligodendroblast Which shows strong 04— 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 s and neuronal morphology (see Figure 14, slide H). Such strong signal of MAP2 with neuronal morphology was not identified in undifferentiated HFSC cells. This weak signal disappeared when the cells were differentiated, so that this staining seemed to be ic and undifferentiated HFSC cells might not be non—specific staining.

Overall, HFSC cell expressed specific markers for neural stem cell , 80x2, and ) and specific markers for oligodendrocyte-lineage cells (Olig2, NGZ, A285, and 04). 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 endrocyte pre-progenitor cell.

Polysiaiic acid (PSA) of PSA—NCAM is a long, negatively charged, cell—surface glycan with an enourmous hydrated volume that serves to modulate the distance between cells. PSA is involved in a number of plasticity— related ses in the adult CNS, including changes in circadian and hormonal patterns, tions to pain and stress, and s of learning and WQ 2012/095730 PCT/IBZOIZ/000030 One of the roles of PSA memory (RutishauserNat. Rev. Neurosci., 9:26, 2008). in the neonatal nervous System is in the migration of oligodendrocyte progenitors. When PSA is d from migrating 02A progenitors, migration of 02A progenitor was inhibited in wound model (Barrel-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 cells correlates with the onset of precursors, and its down regulation on these myelination. PSA is also related plasticity-associated ses 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 e. 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 the same effect on treating an endogenous PSAexpressing cell and will have the trauma like brain injury or spinal cord injury.

To further characterize the HFSC cells (clone #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 passage13 were then subjected to flow cytometry using the ing antibodies: PE-conjugated D133/1 mouse lgG1 monoclonal antibody (Clone A0133, Miltenyi ); PE-conjugated CD140a mouse lgGZa monoclonal antibody (Clone 0R1, BD Pharmingen); PE—conjugated CD9 mouse lgG1 monoclonal antibody (Clone M—L13, BD ngen); PE—conjugated CD44 mouse lgGZb monoclonal antibody (Clone 644-25, BD ngen); PE—conjugated anti—PSA—NCAM mouse lgM monoclonal antibody (2—28, Miltenyi-Biotec); PE—conjugated A285 mouse lglVl monoclonal antibody (Clone 105H829, Miltenyi ); jugated O4 mouse lgM monoclonal dy (Clone O4, Miltenyi Biotec); and PE—conjugated anti—N62 mouse lgGi monoclonal antibody (R&D Systems). Briefly, after dissociation with Accutase, with 2 mM EDTA and the cells were washed and resuspended in ice—cold PBS number was 0.5% BSA and kept on ice. After cell number was counted and cell PCT/182012/000030 ed to 1 x 107ceils/ml using ice-cold PBS with 2 mM EDTA and 0.5% BSA. pl of cell suspension (250,000 cells) was erred 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 ended in ld PBS with 2 mM EDTA and 0.5% BSA. scence of celis was measured using FACS Canto ll (BD Bioscience) and each data was analyzed using Gatelogic software (lnivai Technologies Pty Ltd.) As show in Figure 13, all or most of the HFSC ceils (clone #2b) were CD133-positive (100% of cells), CD9-positive (100% of cells), CD140a— positive (98.8% of cells), of cells). AZBS-positive (99.9% , NGZ—positive (89.8% of cells), O4-positive (94.6% of cells), AM—positive (68.9% of , and CUM—negative (0.4% of cells were positive). By cytochemistry, the 04 signal could be hardly distinguished from non-specific staining and was much weaker than the 04 signal of pro-oligodendroblasts or oiigodendrocytes. From the result of flow cytometry, the 04 signal of HFSC cells (clone #Zb) could be separated from isotype control and HFSC cells (clone #2b) appeared weakly positive for O4 antigen. HFSC cells (clone #3) showed almOst the same phenotype by flow cytometry. They were CD133—positive (98.4% of cells), ODS-positive (99.4% of cells), CD140o—positive (91.5% of celis), sitive (63.4% of cells), A285~positive (99.8% of cells), O4—positive (71.8% of cells), PSA-NCAM-positive (74.7% of ceils), and CD44-negative (0.3% of cells were positive).

Example 8 - Differentiation potential of expanded HFSC cells in serum— containing medium To test the differentiation potential of the expanded cells, the HFSC cells (clone #3) were passaged to separate/single cell stage and cultured in WO 95730 ZOlZ/00003O serum-containing medium [Oligodendrocyte Precursor Cell Differentiation Medium (OPCDM)] (ScienCellTM ch Laboratories) to stimulate differentiation. The cells were then stained with antibodies that recognize and anti—MAP2 neurons (anti—Blll Tublin antibody, anti-Neurofiiament-L antibody antibody), oligodendrocyte itor cells and oligodendrocytes [04 antibody, 01 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 Research).

DAPI was used to rstain cell nuclei. 1] 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, Bill Tublin-positive cells, Neurofilament-L-positive cells, and MAP—Z—positive cells were detected (indicative of neurons). There were many Bill Tublin-positive cells (Figure 14, slide B) whereas small number of cells was positive for Neurofilament—L (Figure 14; slide E) or MAPZ (Figure 14, slide H). This result indicated many of them into MBP—positive are re neurons. HFSC cells could also differentiate cells. MBP is a major component of myelin and sed only in mature oligodendrocyte. This data indicates that HFSC cells have an ability to differentiate into mature oligodendrocytes. (Io—localization of MBP and above al markers was ted but only a few co-localization could be seen (Figure 14, slides C, F, I). This might be due to immaturity of neurons because myelin will not be wrapped on axon of immature neuron. Co—localization of MBP and O4 antigen was also ted (Figure 14, slides J—L). Most signal of MBP co-localized with signal of O4 antigen but only half of signal of O4 antigen co—localized with signal of MBP. This result was reasonable because 04 antigen is expressed in oligodendrocyte progenitors, immature oligodendrocytes and mature oligodendrocytes while MBP is expressed only that is mature oligodendrocyte. These cells were also positive for GalC antigen not shown). a marker for immature oligodendrocytes (data GFAP-positive cells (astrocytes) and vimentin-positive cells were also detected as shown in Figure PCT/IBZOIZ/OOOOSO 14, slides M-O. These data te that the expanded HFSC cells are multipotent and that, therefore, they are likely neural stem cells, since they are capable of giving rise to the three major central‘nervous system cell phenotypes depending on the environment. The HFSC cell (clone #2b) was also tested in the same condition and showed the same multipotency as the HFSC cell did (clone #3).

Example 9 —‘ Differentiation potential of expanded HFSC cells in serum-free medium HFSC cells showed good differentiation potency into oligodendrocyte by reducing PDGF-AA concentration without replenishing bFGF as shown in Figure 5. However, the differentiated cells were less than half of total cells. If HFSC cells were seeded at very low density (< 0.5 x 104 cells/cmz) without bFGF with 10 ng/ml of PDGF-AA and 100 ng/ml lGF-1, most cells seemed to differentiate but they were lost within a day. To examine their potency to differentiate into oligodendrocyte further, an ion of differentiation and a long-term cell survival were t to be very important Cyclic AMP (CAMP) is known to induce differentiation of many cell types. ' pCPT-cAMP which is a cell-permeable analog of CAMP was tested whether it could induce entiation in HFSC cells. 100 pM pCPT—cAMP could induce differentiation of HFSC cells at high density (3 x 104 cells/cmz) but cell couldn't survive well even in the presence of 100 ng/ml lGF-i. BDNF is reported to enhance differentiation of oligodendrocyte itors and support cell survival of entiated cells. When HFSC cells were differentiated in the presence of ng/ml of PDGF—AA, 100 ng/ml IGF—1, 100 pM AMP and 10 ng/ml BDNF in DMEM/FlZ containing glutamine and HEPES and supplemented with 827 supplement, N2 supplement and 50 uM 1-thioglycerol, at least more than half of HFSC cells were differentiated into process—bearing cells. Because HFSC cell ses several oligodendrocyte markers like 04 or N62, the new method to distinguish HFSC cell and dendrocyte-lineage cell was required.

Based on several trials, the inventor noticed that undifferentiated cells express ~ - - GD3 stronger than oligodendrocyte itor cells and O4 vice versa head of Figure 11, slide A, slide B and slide C). When the cells were stained with GD3 and O4, oligodendrocytes could be distinguished using their staining pattern and their logy. In addition, other cell types like neuron or yte also could be identified because they don‘t express GD3 or 04 antigen. Figure 15, slide D shows the ratio of each cell type. Undifferentiated cells were 23.5% i 2.0% of total cells. Oligodendrocyte progenitor cells were 75.8% i 2.1% of total cells. The other cell types were only 0.9% :t 0.6%. As mentioned above, the ratio of oligodendrocyte progenitor cells to differentiated cells (oligodendrocyte itor cells plus other cell types) were 99.1% i 0.56% of differentiated cells whereas other cell types-were 0.9% i 0.56% of entiated cells. This data indicates that HFSC cell has the high potential to differentiate into oligodendrocyte-lineage cells.

Example 10- HFSC cell is enriched or selected by a fluorescent activated cell sorting (FACS) method or a magnetic sorting method The present invention disclosed the phenotype of HFSC cell that is CD133-positive, CD140a-positive, CDQ-positive, CD44—negative, PSA—NCAM— positive, A285-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. (309 is also used as a marker for neural stem cell but some oligodendrocytes are known to express CD9. PSA-NCAM and A285 are used to detect neuronal-restricted precursor or restricted precursor. Most neural precursors and progenitors are thought to s PSA-NCAM and A285 or either PSA—NCAM or A285, their usage cannot enrich HFSC cell so well, especially in the first and second trimester. CD140a, NG2, A285 and 04 are used as markers for oligodendrocyte precursor cell, pro-oligodendroglia and oligodendrocyte. The sion level of CD140a and NGZ were higher in HFSC cell than oligodendrocyte precursor cell, pro—oligodendroglia or oligodendrocyte, whereas the expression level of A285 and 04 were lower in HFSC cell than oligodendrocyte sor cell, pro-oligodendroglia or oligodendrocyte. The to be more appropriate to enrich HFSC usage of CD140a and NG2 are thought cell. Based on above information and the data bed in this invention, the effectiveness of each marker to enrich HFSC cell will be CD140a > NG2 > CD9 > CD133 > A285 > 04, 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 009) and one of oligodendrocyte—lineage markers (CD140a, NG2) will be very effective to select HFSC cells. Based on above dge, the most efficient combinations of s to select the HFSC cells will be CD133 and CD140a among these combinations but other combinations should be also more ive than ion 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 their gestation week. Most very low (less than 1% of total cells) depending on 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.

Claims (21)

1. A method of in vitro culturing and maintaining an expandable neural cell wherein the cell is a itor cell or stem cell and wherein said cell maintains its capability to differentiate into neurons, ytes, and oligodendrocytes and its ability to entiate into oligodendrocyte-lineage cells efficiently, said method comprising: a) providing at least one cell which has been isolated and dissociated from a human fetal neural tissue; b) 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 ses: -at least 5 ng/ml PDGF-AA, -at least 0.5 ng/ml bFGF, and -at least 10 µM 1-thioglycerol; c) passaging the cell from b) to obtain said expandable human neural cell.

2. The method of claim 1, wherein said culture medium further comprises at least 1.0 ng/ml IGF-1.

3. The method of claim 1, wherein said neural tissue is selected from the group consisting of spinal cord, cerebral cortex, hippocampus, striatum, basal forebrain, ventral mesencephalon, locus ceruleus, hypothalamus, cerebellum, corpus callosum and optic nerve.

4. The method of claim 1, wherein in step a), said neural tissue is from human fetal spinal cord at 8-24 weeks gestation.

5. The method of claim 1, wherein in step a), said isolating was performed by an isolating method which uses at least one of fluorescence activated cell sorting (FACS) or immunopanning.

6. A method of in vitro isolating an expandable neural cell wherein the cell is a itor cell or stem cell and wherein said cell maintains its capability to entiate into neurons, astrocytes, and oligodendrocytes and its ability to differentiate into oligodendrocyte-lineage cells efficiently, said method comprising: a) providing at least one cell which has been isolated and dissociated from a human fetal neural tissue; b) 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: 200 ng/ml PDGF-AA, 100 ng/ml bFGF, 100 µM 1-thioglycerol, and - 5-100 ng/ml IGF-1. c) passaging the cell from b) to obtain said expandable human neural cell.

7. The method of claim 6 n said culture medium comprises: - about 100 ng/ml A, - about 20 ng/ml bFGF, - about 50 µM 1-thioglycerol, and - about 20 ng/ml IGF-1.

8. The method of claim 1 further comprising IGF-1, wherein said culture medium ses: - 5-100 ng/ml PDGF-AA, - 1-50 ng/ml bFGF, - 10-100 µM 1-thioglycerol, and - 5-100 ng/ml IGF-1.

9. The method of claim 8 wherein said culture medium comprises: - about 20 ng/ml PDGF-AA, -about 20 ng/ml bFGF, -about 50 µM 1-thioglycerol, and -about 20 ng/ml IGF-1.

10. The method of claim 2 wherein said culturing step takes place in a culture vessel coated with rnithine or poly-lysine.

11. The method of claim 2 wherein the chemically defined serum-free culture medium comprises DMEM/F12 mented with non-essential amino acids, ine, pyruvate, B27, N-acetyl-cysteine and -mercaptoethanol.

12. Use of an expandable neural cell in the preparation of a ment for treating a loss of myelin or a loss of oligodendrocytes in a patient, wherein the cell is a progenitor cell or stem cell and wherein said cell maintains its capability to differentiate into neurons, astrocytes, and oligodendrocytes and its ability to differentiate into oligodendrocyte-lineage cells efficiently, and n the cell is ed and maintained by a method comprising: a) providing at least one cell which has been isolated and dissociated from a human fetal neural tissue; b) 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; c) passaging the cell from b) to obtain said able human neural cell. .

13. The use of claim 12, wherein said condition is a demyelinating disease or a neurodegenerative disease.

14. The use of claim 13, wherein said demyelinating disease is selected from the group consisting of spinal cord injury (SCI), multiple sclerosis (MS), hereditary leukodystrophy, transverse athy/myelitis, progressive multiple focal leukoencephalopathy and other congenital demyelinating diseases.

15. The use of claim 13, n said neurodegenerative disease is selected from the group consisting of Alzheimer’s disease, senile dementia of Alzheimer type (SDAT), Parkinson’s disease, Huntington’s disease, Amyotrophic Lateral Sclerosis (ALS), ischemia, blindness and a egenerative disease caused by injury to myelinated neurons.

16. An in vitro culture comprising an enriched tion of neural cells which have been isolated from a mammalian l nervous system wherein the isolated cell is submerged in chemically defined free culture medium comprising: - at least 5 ng/ml PDGF-AA, - at least 5 ng/ml bFGF, and - at least 10 µM 1-thioglycerol.

17. The in vitro e of claim 16, further comprising at least 1.0 ng/ml IGF-1.

18. The in vitro culture of claim 17 wherein said cell has been obtained from human fetal spinal cord.

19. The in vitro culture of claim 18 wherein the in vitro culture is in a culture vessel coated with poly-ornithine or poly-lysine.

20. The in vitro culture of claim 18 wherein the chemically defined serum-free culture medium further comprises DMEM/F12 supplemented with nonessential amino acids, glutamine, pyruvate, B27, N-acetyl-cysteine and - mercaptoethanol.

21. The in vitro e of claim 16 wherein said enriched population of neural cells is CD133-positive, CD140a-positive, A2B5-positive, and PSA-NCAM- positive.