WO2018120437A1 - 成体多功能嗅干细胞及其分离方法以及其应用 - Google Patents

成体多功能嗅干细胞及其分离方法以及其应用 Download PDF

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WO2018120437A1
WO2018120437A1 PCT/CN2017/076703 CN2017076703W WO2018120437A1 WO 2018120437 A1 WO2018120437 A1 WO 2018120437A1 CN 2017076703 W CN2017076703 W CN 2017076703W WO 2018120437 A1 WO2018120437 A1 WO 2018120437A1
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stem cells
olfactory
adult
olfactory stem
multifunctional
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English (en)
French (fr)
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徐伟成
许重義
周德阳
林振寰
李玮
陈三元
郑隆宾
蔡长海
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中国医药大学
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Priority to JP2019509486A priority Critical patent/JP6990932B2/ja
Priority to EP17889464.8A priority patent/EP3536777A4/en
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the present invention relates to an undifferentiated animal cell, and more particularly to an adult tissue stem cell.
  • Stem Cell is a non-differentiated primary cell in an organism that has the ability to continuously replicate, renew, and differentiate into mature cells with specific patterns and functions.
  • Stem cells can be mainly divided into embryonic stem cells (ESCs) and adult stem cells.
  • the embryonic stem cells are taken from the inner cell mass in the blastocyst, and the adult stem cells are derived from various tissues. .
  • Stem cells can be divided into three major categories according to their differentiation ability. One is totipotent stem cells, which have complete ability to differentiate into intact embryos or organisms.
  • the other is pluripotent stem cells. ), which has the ability to differentiate into three germ layers, but can form all cells of a certain tissue or organ, but cannot develop into a complete embryo or organism; three are multipotent stem cells, including specific tissues.
  • Stem cells such as neural stem cells, blood cell stem cells, liver stem cells, skin stem cells, and the like.
  • pluripotent stem cells can differentiate into different cell lines, they can be used to treat a variety of degenerative or hereditary diseases.
  • embryonic stem cells are considered to have the above functions.
  • moral doubts have hampered the application of human embryonic stem cells in research and therapy, while embryonic stem cells, which are not embryos, can circumvent this obstacle.
  • Such non-embryonic pluripotent stem cells include adult bone marrow mesenchymal stem cells or stromal stem cells and cord blood stem cells.
  • the clinical application of these cells is limited, and therefore, another pluripotent cell is required.
  • Adult tissue stem cells refer to undifferentiated cells present in an already differentiated tissue and are present in various tissues and organs of the body. Traditionally, adult adult stem cells are considered to be only proliferating and differentiated into defined cell types within the tissue. However, in recent years, the results of the research have questioned this traditional concept, pointing out that the adult tissue contains a population of stem cells with multiple differentiation capabilities.
  • Adult pluripotent stem cells are good news for medical applications because they can replace human embryos with moral controversy. Stem cells, therefore, the search for adult tissue stem cells with self-renewal and pluripotent differentiation from adult tissues has become one of the main topics in the research and development of stem cell medical related technologies.
  • the object of the present invention is to provide an adult multifunctional olfactory stem cell and an isolated method thereof, and an application thereof, wherein the isolated adult multifunctional olfactory stem cells have self-renewal ability and pluripotent differentiation ability.
  • the pluripotency markers Oct-4, Sox-2, Nanog and SSEA-4 are more prominent.
  • the isolated adult multifunctional olfactory stem cells of the present invention exhibit B cell-specific Moloney murine leukemia virus insertion site 1 on the cell surface, which has the characteristics of self-renewal and pluripotent differentiation.
  • Another object according to the invention is to provide a method of isolating pluripotent adult olfactory stem cells comprising the following steps.
  • a mixture of cells from olfactory tissue of a mammal is provided.
  • the separation step is performed by isolating cells positive for Oct-4, Sox-2, Nanog, and SSEA-4 from the cell mixture to obtain pluripotent adult olfactory stem cells.
  • the mammal can be human or murine.
  • the separating step further comprises a B-lymphoma Moloney murine leukemia virus insertion region-1 (B-lymphoma Moloney murine leukemia virus insertion region-1, Bmi-1) positive cells.
  • the method for isolating pluripotent adult olfactory stem cells of the present invention can screen pluripotency for Oct-4, Sox-2, Nanog and SSEA-4 from a mixture of cells derived from mammalian olfactory tissue.
  • the cells which are positive for labeling can be rescreened for cells of the B cell-specific Moloney murine leukemia virus insertion site 1.
  • the selected cells are adult olfactory stem cells with pluripotent differentiation ability, so that the cells can be rapidly And specific purification of pluripotent adult sniffing Cell.
  • Still another object according to the present invention is to provide a use of isolated adult multifunctional olfactory stem cells for the preparation of a medicament for treating brain tissue damage in an individual.
  • the brain tissue damage may be caused by a cerebral ischemic disease or may be caused by a neurodegenerative disease.
  • the cerebral ischemic disease may be a stroke
  • the neurodegenerative disease may be Alzheimer's disease, Parkinson's disease or epilepsy.
  • the isolated adult multifunctional olfactory stem cells of the present invention are used for cell therapy to improve the nerve function of the brain tissue damaged individual, and the implanted adult multifunctional olfactory stem cells can migrate to the damaged part of the brain, thereby repairing and receiving The nerve cells in the damaged part can be used to treat individuals with brain tissue damage.
  • FIGS. 1A to 1C are diagrams showing the results of pluripotency labeling of adult multifunctional olfactory stem cells isolated from human olfactory mucosa;
  • Figure 1D is an amplification index diagram of adult multifunctional olfactory stem cells isolated from human olfactory mucosa
  • Figure 1E is a graph showing the measurement of the proliferation index of adult multifunctional olfactory stem cells isolated from human olfactory mucosa;
  • 1F and 1G are graphs showing the results of pluripotency marker expression of adult multifunctional olfactory stem cells isolated from murine olfactory tissue;
  • 2A is a graph showing the results of pluripotency labeling of adult multifunctional olfactory stem cells isolated from human olfactory mucosa after three-dimensional culture;
  • Figure 2B shows the pluripotency marker of adult olfactory stem cells isolated from murine olfactory tissue after three-dimensional culture. Result chart
  • FIG. 3A to FIG. 3E are photomicrographs of tissue cells of the present invention in which adult multi-functional olfactory stem cells are induced to differentiate into different germ layers;
  • FIG. 3A to FIG. 3E are photomicrographs of tissue cells of the present invention in which adult multi-functional olfactory stem cells are induced to differentiate into different germ layers;
  • FIG. 3F is a photomicrograph of a tissue cell of the present invention in which the adult multifunctional olfactory stem cells spontaneously differentiate into different germ layers;
  • FIG. 3G is a diagram showing the results of analysis of different germ layer markers after spontaneous differentiation of adult multifunctional olfactory stem cells according to the present invention.
  • 4A to 4E are photomicrographs of tissue cells of adult multifunctional olfactory stem cells isolated from human olfactory mucosa tissues differentiated into different germ layers in mice;
  • 5A to 5D are photomicrographs of tissue cells of adult multi-functional olfactory stem cells isolated from mouse olfactory tissue differentiated into different germ layers in mice;
  • Figure 6A is a diagram showing the distribution results of adult multifunctional olfactory stem cells isolated from human olfactory mucosa
  • Figure 6B is a diagram showing the distribution results of adult multifunctional olfactory stem cells isolated from mouse olfactory tissue in vivo;
  • Figure 6C is a graph showing the results of analysis of basal cell marker expression of adult multi-functional olfactory stem cells cultured in vitro;
  • Figure 7A is a graph showing the results of analysis of Bmi-1 expression of adult multifunctional olfactory stem cells cultured in vitro;
  • Figure 7B is a graph showing the results of analysis of Bmi-1 expression of adult multifunctional olfactory stem cells isolated from human olfactory mucosa;
  • Figure 7C is a graph showing the results of analysis of Bmi-1 expression of adult multifunctional olfactory stem cells isolated from murine olfactory tissue;
  • Figure 8A is a graph showing the effect of Bmi-1 on adult multifunctional olfactory stem cells
  • Figure 8B is a graph showing the results of aging-related galactosidase analysis
  • Figure 8C is a quantitative result of reverse transcription PCR of p16 Ink4a of Bmi-1 +/+ olfactory tissue and Bmi-1 -/- olfactory tissue;
  • Figure 8D is an amplification index diagram of Bmi-1 +/+ adult multifunctional olfactory stem cells and Bmi-1 ⁇ / ⁇ adult multifunctional olfactory stem cells;
  • Figure 8E is an amplification index diagram of human adult multifunctional olfactory stem cells into which shRNA is introduced;
  • Figure 9A is a diagram showing the volume of brain tissue infarction of a stroke mouse transplanted with an adult multifunctional olfactory stem cell
  • FIG. 9B is a graph of the statistical result of FIG. 9A.
  • Figure 9C is a graph showing the results of a vertical activity test of a stroke mouse
  • Figure 9D is a graph showing the results of the vertical movement test of stroke mice.
  • Figure 9E is a graph showing the results of a vertical activity time test of a stroke mouse
  • Figure 9F is a graph showing the results of CD31 expression in brain tissue of a mouse with a transplanted murine adult olfactory stem cells
  • Figure 10A is a nuclear magnetic resonance angiogram of a subject
  • Figure 10B is a nuclear magnetic resonance diffusion tensor image of the subject
  • Figure 10C is a correlation diagram of Bmi-1 relative performance and Fog-Mel assessment score.
  • the present specification provides an isolated adult mammalian olfactory stem cell that expresses a specific cell surface receptor and has self-renewal ability and pluripotent differentiation ability.
  • the present disclosure further provides a method of isolating pluripotent adult olfactory stem cells that can be rapidly and specifically screened for pluripotent adult olfactory stem cells from a mixture of cells from olfactory tissue of a mammal.
  • the present invention provides an isolated adult mammalian olfactory stem cell which exhibits a B cell-specific Moloney murine leukemia virus insertion region-1 (B-lymphoma Moloney murine leukemia virus insertion region-1).
  • Bmi-1 which may be derived from a human cell or a murine cell, preferably, which exhibits the pluripotency markers Oct-4, Sox-2, Nanog and SSEA-4.
  • the method for isolating pluripotent adult olfactory stem cells of the present invention is to screen cells positive for Oct-4, Sox-2, Nanog and SSEA-4 from a mixture of cells derived from olfactory mucosa of mammals.
  • the cells that are positive for Bmi-1 can be rescreened, that is, the adult olfactory stem cells with pluripotency can be isolated from the cell mixture, wherein the mammals can be human or murine.
  • the adult multifunctional olfactory stem cells of the isolated mammals of the present invention can be applied to the preparation of a medicament for treating brain tissue damage in an individual. Furthermore, the isolated adult multifunctional olfactory stem cells of the present invention are used for cell therapy to improve the neurological function of individuals with brain tissue damage, and the implanted adult multifunctional olfactory stem cells can migrate to damaged parts of the brain, thereby further Repairing damaged nerve cells, so it can be used to treat brain tissue damage.
  • Brain tissue damage can be cerebral ischemic disease or neurodegenerative disease, while cerebral ischemic disease can be stroke.
  • Neurodegenerative diseases can be Zhaimer, Parkinson's or epilepsy.
  • the mammalian tissue used in this test case is human olfactory mucosa tissue or murine olfactory tissue.
  • the human olfactory mucosa (5mm 3 , 0.5g weight) was taken from the nasal septum under general anesthesia. The sampling process was approved by the Institutional Review Board of the China Medical University Hospital and the patients were also obtained. Written consent.
  • the murine olfactory tissue was taken from 8 or 11 weeks old mice, sacrificed after anesthesia, and the olfactory tissue was isolated from the upper turbinate bone under a dissecting microscope.
  • the rodent used contained SD (Sprague- Dawley) Rat, C57BL/6JNarl mouse, GFP transgenic mouse, Bmi-1 +/+ mouse, and Bmi-1 knockout mouse (Bmi-1 -/- ).
  • the collected human olfactory mucosa tissue was placed in a sterilized box containing Hank's salt balanced solution (Gibco/BRL), and primary culture was performed within 24 hours by explant culture method. Said. Each olfactory tissue was cut into small pieces under a dissecting microscope, placed in phosphate buffer at room temperature, and centrifuged at 600 x g for 10 minutes to collect tissue explants, and contained 2 ⁇ g/mL heparin.
  • Hank's salt balanced solution Gibco/BRL
  • heparin heparin; Sigma
  • FGF2 fibroblast growth factor
  • EGF epidermal growth factor
  • penicillin/streptomycin 100 U/ml
  • the medium was resuspended in the culture medium, and the tissue explants were placed in a 25 cm 2 cell culture dish, and cultured for 5 to 7 days in a 5% CO 2 , 37 ° C environment, and the cells were allowed to grow from the explants.
  • the extracellular growth, while the primary adherent cells are adult multifunctional olfactory stem cells isolated from humans.
  • the adult multifunctional olfactory stem cells isolated from the mouse were prepared in the same manner as above, except that 20 ng/mL of epidermal growth factor (Invitrogen) was additionally added to the DMEM/F12 medium in addition to the above-mentioned supplement.
  • epidermal growth factor Invitrogen
  • the pluripotency marker expression of the above-mentioned primary cultured adult multifunctional olfactory stem cells was analyzed by immunofluorescence staining, reverse transcription PCR and flow cytometry.
  • the analyzed pluripotency marker contains the key transcription factors necessary for the developmental embryo sac and the cell surface glycosphingolipids (glycosphingolipids) present in undifferentiated human embryonic stem cells, wherein the key transcription factors include Nanog, Sox-2 and Oct-4.
  • the glycosphingolipid is SSEA-4 (stage-specific embryonic antigen 4).
  • FIG. 1A to FIG. 1C are the results of pluripotency labeling of adult multifunctional olfactory stem cells isolated from human olfactory mucosa tissue
  • FIG. 1A is a result of immunofluorescence staining, and the cell nucleus is labeled with DAPI
  • the fluorescence of the 1) to (4) portions are signals of Nanog, Oct-4, Sox-2, and SSEA-4, respectively.
  • Fig. 1B is a graph showing the results of reverse transcription PCR in which a control group is a group to which no template is added, APOSC is an adult multifunctional stem cell, and hES is a human embryonic stem cell as a positive control group.
  • Figure 1C is a flow cytometry analysis showing the analysis of adult multifunctional olfactory stem cells isolated from 6 different donors with different algebras (p2-p14).
  • FIG. 1A show that adult multifunctional olfactory stem cells isolated from human olfactory mucosa do exhibit pluripotency markers such as Nanog, Oct-4, Sox-2, and SSEA-4, especially adult morphological stem cells on the cell surface.
  • the expression of SSEA-4 showed that it was a more primitive stage of the cell, and nuclear expression of Nanog and Oct-4 in adult multi-functional olfactory stem cells isolated from 6 different donors was also confirmed by immunofluorescence staining (data not shown).
  • FIG. 1B show that adult olfactory stem cells isolated from human olfactory mucosa can express pluripotency markers such as Nanog, Oct-4 and Sox-2, and can express pluripotent stem cells (induced pluripotent).
  • Stem cells, iPS) produced c-Myc and KLF-4.
  • the above data demonstrates that adult multifunctional olfactory stem cells isolated from different donors and subsequent passages of different generations consistently exhibit pluripotency markers associated with embryonic stem cells.
  • FIG. 1D is an amplification index diagram of the isolated adult multifunctional olfactory stem cells of the present invention.
  • the results of Figure 1D show that adult multifunctional olfactory stem cells proliferate exponentially for 48 days (17 generations in total) and grow slowly after 83 days (25th generation).
  • the doubling time of adult multifunctional olfactory stem cells was 20.9 ⁇ 3.9 hours (donor 1, mean of passages 3 to 17) or 25.3 ⁇ 6.7 hours (average of donor 2, 5th to 19th generation).
  • adult multi-functional olfactory stem cells were calibrated with 10 ⁇ M CFSE (carboxyfluorescein diacetate succinimidyl ester; Molecular Probes), and adult morphological stem cells were cultured for 4 days, followed by flow cytometry (Becton). Dickinson) detected the distribution of CFSE in adult multi-functional olfactory stem cells, and further calculated the proliferation index (PI) of adult multi-functional olfactory stem cells using MODFIT software (Verity Software House, ME). The higher the calculated proliferation index was. , indicating that the cell proliferation ability is more it is good.
  • CFSE carboxyfluorescein diacetate succinimidyl ester
  • ME proliferation index
  • FIG. 1E is a graph showing the measurement results of the proliferation index of the isolated adult multifunctional olfactory stem cells of the present invention.
  • the results in Figure 1E show that during the 96-hour follow-up period, 50.9% of the adult multifunctional olfactory stem cells completed 3 cell divisions, and 32.7% of the adult multifunctional olfactory stem cells completed 2 cell divisions, showing adult multifunctional olfactory stem cells.
  • the cell cycle dynamics are identical to human embryonic stem cells and induced pluripotent stem cells (doubling time between 24 hours and 48 hours) and superior to some adult pluripotent stem cells (doubling time ranging from 30 hours to 72 hours) .
  • FIG. 1F the results of pluripotency labeling of adult multifunctional olfactory stem cells isolated from murine olfactory tissue are shown in FIG. 1F as a result of immunofluorescence staining, and the nucleus is indicated by DAPI.
  • the fluorescence of the parts (1) to (3) is the signals of Nanog, Oct-4 and Sox-2, respectively.
  • Fig. 1G is a graph showing the results of reverse transcription PCR in which the control group is a group to which no template is added, APOSC is an adult multifunctional stem cell, and mES is a mouse embryonic stem cell as a positive control group.
  • stem cells One of the unique characteristics of stem cells is the ability to form cell spheres in a three-dimensional and simulated natural environment. Therefore, in addition to the pluripotency marker of the adult multifunctional olfactory stem cells of the present invention by attaching growth, adult multi-functional olfactory stem cells are cultured under three-dimensional culture conditions in this experiment to examine whether the adult multifunctional olfactory stem cells of the present invention retain the forming cells. The stemness of the ball.
  • the three-dimensional culture steps are as follows.
  • the adult multifunctional olfactory stem cells which have occupied the growth plane space are trypsinized, resuspended in suspension culture medium, and the adult multifunctional olfactory stem cells are adjusted to 7 ⁇ 10 4 cells/mL, wherein
  • the suspension culture medium was containing 2% B27supplement (Gibco), 20 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor and 1% penicillin/streptomycin (100 U/ml).
  • DMEM/F12 medium was containing 2% B27supplement (Gibco), 20 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor and 1% penicillin/streptomycin (100 U/ml).
  • the cell sphere formed in the first generation is called the first adult multifunctional olfactory stem cell sphere.
  • adult multi-functional olfactory stem cell spheres were further cultured in suspension culture medium containing Bromodeoxyuridine (BrdU) to calibrate DNA.
  • FIG. 2A is a graph showing the results of pluripotency labeling of adult multifunctional olfactory stem cells isolated from human olfactory mucosa after three-dimensional culture, wherein (1) is a photomicrograph in the bright field, (2) is Fluorescence photomicrograph of BrdU after calibration.
  • the results of Figure 2A show that human adult multifunctional olfactory stem cells can form tight cell spheres via three-dimensional culture.
  • the human adult multi-functional olfactory stem cell sphere embedded in BrdU can be proved to continue into the S phase of the cell cycle.
  • the cell proliferation marker Ki67 was also abundantly expressed in the first adult multifunctional olfactory stem cell sphere (data not shown).
  • the number of cell spheres formed can represent stem cell self-renewal activity, and the size of the formed sphere can indicate the proliferation ability of stem cells.
  • the first adult multi-functional olfactory stem cell sphere cultured for 3 days was digested into single cells by trypsin digestion, and the number of cells was counted, and then re-inoculated with suspension culture solution. After three-dimensional culture, the cell sphere formed by the three-dimensional culture is called a second adult multifunctional olfactory stem cell sphere.
  • the diameter of the second adult multifunctional olfactory stem cell sphere was measured on days 2, 5 and 9 of the culture. Two days after dissociation and reculture, 50% of the adult multifunctional olfactory stem cells survived, and a second adult multifunctional olfactory stem cell sphere was observed in the three-dimensional culture. The second adult multifunctional stem cell spheres cultured for more than 9 days increased the average diameter from 59 ⁇ m to 81 ⁇ m.
  • FIG. 2A Please refer to FIG. 2A again, wherein (3) is a result of detecting Alkaline phosphatase (ALP) activity by Vector Red Alkaline phosphatase Substrate Kit I, and (4) to (6) are respectively stained by immunofluorescence.
  • ALP Alkaline phosphatase
  • FIG. 2A shows that adult multi-function olfactory stem cells exhibited Nanog, Oct-4, and SSEA-4, which were similar to embryonic stem cells.
  • alkaline phosphatase activity can be detected in adult multifunctional olfactory stem cell spheres.
  • FIG. 2B is a graph showing the results of pluripotency labeling of adult multifunctional olfactory stem cells isolated from mouse olfactory tissue after three-dimensional culture, wherein (1) is a fluorescence micrograph of ki67 after calibration, ( 2) Results of alkaline phosphatase activity, and (3) and (4) are immunofluorescence staining results of Oct-4 and Nanog.
  • the results in Figure 2B show that ki67, alkaline phosphatase, Oct-4 and Nanog were also observed in murine multi-function olfactory stem cell spheres.
  • the above results show that the pluripotency marker, self-renewal and alkaline phosphatase activity exhibited by the adult multifunctional olfactory stem cell sphere of the present invention show that it has similar characteristics to embryonic stem cells.
  • This test case further investigates whether the adult multifunctional olfactory stem cells of the present invention have pluripotent differentiation ability to differentiate into three germ layer cells by in vitro differentiation assay.
  • adult multi-functional olfactory stem cells were cultured in differentiation medium containing different growth factors, and they were induced to differentiate into ectoderm (neuronal cells) and mesoderm (adipocytes, osteoblasts, chondrocytes, and Cells of endothelial cells and endoderm (hepatocytes) were observed under a microscope, and the cell types after differentiation were further confirmed by staining.
  • 3A is a photomicrograph of the neural cells of the adult multifunctional olfactory stem cells differentiated into ectoderm. In this test case, it is confirmed by immunofluorescence staining whether the differentiated cells exhibit mature neural markers, mature neural markers. It includes Tuj-1 (Neuron-specific class III beta-tubulin), GFAP (glial fibrillary acidic protein) and MAP-2 (microtubule-associated protein 2). The results in Fig.
  • 3A show that the three mature neural markers of the cells cultured in the differentiation medium are all expressed, and they are shown to exhibit neuronal morphology, including the multipolar morphology and branches of part (1) (arrows Point)), in part (2), similar to the long bipolar linear morphology of the Olfactory receptor neuron (ORN) (points indicated by the arrows), the bead axis of part (3) The protruding structure (where the arrow points) and the mesh axon-like structure of part (4) (where the arrow points). It was demonstrated that the differentiated cells were indeed nerve cells.
  • 3B is a photomicrograph of hepatocytes in which the adult multifunctional olfactory stem cells differentiate into endoderm, wherein (1) to (3) are confirmed by immunofluorescence staining whether the differentiated cells are expressed by the liver.
  • ⁇ -FP alpha-fetoprotein
  • Fig. 3B show that cells differentiated and cultured by hepatocytes are hepatocyte-specific gene-positive cells which can express albumin, ⁇ 1-antitrypsin and ⁇ -FP.
  • this test further measures the sugars in the cells by PAS staining (Periodic Acid-Schiffstain) to examine whether the cytoplasm of the differentiated adult multifunctional olfactory stem cells has stored glycogen ( Glycogen).
  • 3C is a photomicrograph of the adult multifunctional olfactory stem cells stained with PAS, wherein U represents undifferentiated adult multifunctional olfactory stem cells, and D represents differentiated adult multifunctional olfactory stem cells.
  • Fig. 3C show that no stored glycogen was found in (1) undifferentiated adult multi-functional olfactory stem cells, but was visible in both (2) and (3) differentiated adult multi-functional olfactory stem cells.
  • the above results demonstrate that adult multifunctional olfactory stem cells can indeed be induced to differentiate into functional hepatocytes.
  • FIG. 3D is a photomicrograph of the adult multifunctional olfactory stem cells differentiated into endothelial cells by angiogenesis assay.
  • adult multi-functional olfactory stem cells are treated with vascular endothelial growth factor (VEGF), bFGF and heparin for angiogenesis assay, and cell morphology is observed under bright field.
  • VEGF vascular endothelial growth factor
  • bFGF vascular endothelial growth factor
  • heparin heparin
  • FIG. 3D shows that In the angiogenesis assay, adult multifunctional olfactory stem cells migrate toward each other within 2-4 hours and form a capillary-like tubular structure with a maturity of about 6 hours. After 21 hours, the formed tubular structure will separate from the matrix. It can be seen that the angiogenesis kinetics of adult multifunctional olfactory stem cells showed the same performance as endothelial cells.
  • FIG. 3E is a photomicrograph of the adipocytes, osteoblasts, chondrocytes and endothelial cells of the adult multi-functional olfactory stem cells differentiated into mesoderm, and the oil red staining (oil red) is used in FIG. 3E.
  • O stain is used as an indicator of intracellular lipid accumulation to confirm that the differentiated cells are fat cells.
  • the status of calcium mineralization was revealed by Alizarin red S stain to confirm that the differentiated cells were osteoblasts.
  • Alican blue stain was used as an indicator of proteoglycans synthesis to confirm that the differentiated cells were chondrocytes.
  • adult multifunctional olfactory stem cells were not induced by vascular endothelial growth factor and their cell type was observed under bright field.
  • the results of Fig. 3E show that adult multifunctional olfactory stem cells can differentiate into adipocytes, osteoblasts, chondrocytes and tubular structures with capillary-like structure in the culture medium of differentiation, although adult multifunctional olfactory stem cells spontaneously differentiate to form tubular
  • the structure started from 48 hours, but it shows that it can not only have tissue-specific growth factors provided by exogenous, but also has the potential to spontaneously differentiate into three germ layer cells like embryonic stem cells.
  • FIG. 3F is a photomicrograph of the tissue cells of the present invention for spontaneously differentiating into different germ layers
  • FIG. 3G is a different germ layer marker after spontaneous differentiation of the adult multifunctional olfactory stem cells of the present invention.
  • Fig. 3F(1) show that neuron-like cells can be observed in the spontaneously differentiated population, and the results of (2) show that they can express the ectoderm marker Tuj-1.
  • the results of (3) show that it can express the mesodermal marker ⁇ -smooth muscle actin ( ⁇ -SMA), and the results of (4) and (5) show that it can express the marker of endoderm. Protein and alpha 1-antitrypsin. Further, the results of (6) also showed that hepatocytes having glycogen deposition in the cells were confirmed to be endothelial layers by PAS staining measurement.
  • the stroke mouse model was subjected to an ischemia-reperfusion model to simulate the symptoms of transient local cerebral ischemia in mice, and the test animals used were 25-30 g of C57BL/6 mice.
  • the mice were anesthetized by intraperitoneal injection of chloral hydrate (0.4 g/kg).
  • the model of cerebral ischemia/reperfusion in mice was to block the common carotid arteries (CCAs) and right brain of the mice.
  • CCAs common carotid arteries
  • MCA middle cerebral artery
  • CBF cortical blood flow
  • the core temperature of the mice was monitored with a thermistor temperature probe and the body temperature of the mice was maintained at 37 °C with a heating pad. After recovery from anesthesia, the body temperature of the mice was also maintained at 37 ° C with a heat lamp.
  • hAPOSC-Luc Human adult multi-function olfactory stem cells calibrated with Luciferase were transfected with a lentivirus encoding the Luc gene prior to human adult multi-function olfactory stem cell transplantation. Then the next three 3.5mm cortical regions 106 hAPOSC-Luc injected into mice to stroke stereotaxic dura. Four weeks later, the mice were sacrificed, and the brain tissue was taken for immunofluorescence staining to label the neuronal marker MAP2, the glial cell marker GFAP, the endothelial cell marker vWF (Von Willebrand factor), and laminin (laminin), and confirmed the transplanted hAPOSC. Whether -Luc differentiates into neurons, glial cells or endothelial cells.
  • FIG. 4A to FIG. 4D wherein FIG. 4A and FIG. 4B are photomicrographs of human adult multifunctional olfactory stem cells differentiated into neurons and glial cells in mice, and FIG. 4C and FIG. 4D are diagrams.
  • the results of Figures 4A and 4B show that hAPOSC-Luc migrates to the penumbra of the ischemic hemisphere, the lateral ventricle (LV), and the hippocampal dentate gyrus (DG), and is immunofluorescent.
  • LV lateral ventricle
  • DG hippocampal dentate gyrus
  • hAPOSC-Lucs were further percutaneously injected into the liver of newborn (day 2) mice, and transplanted. The mice were sacrificed in the last six weeks, and liver tissues were taken for immunofluorescence staining to label albumin synthesized by hepatocytes, and it was confirmed whether or not the transplanted hAPOSC-Luc differentiated into hepatocytes.
  • Fig. 4E a photomicrograph of human adult multifunctional olfactory stem cells differentiated into hepatocytes in a mouse is shown.
  • hepatocytes co-expressing luciferase and albumin can be detected on mouse liver tissue.
  • the above results show that hAPOSC-Luc transplanted into mouse brain can indeed differentiate into ectodermal cells (neurons and glial cells), mesoderm cells (endothelial cells) and endoderm cells (hepatocytes).
  • FIG. 5A to FIG. 5D photomicrographs of tissue cells of adult multi-functional olfactory stem cells isolated from mouse olfactory tissue differentiated into different germ layers in mice, murine adult multifunctional olfactory stem cells (mAPOSC-GFP) Is isolated from GFP transgenic mice, so it is calibrated with fluorescence, and other test procedures are not repeated here.
  • mAPOSC-GFP murine adult multifunctional olfactory stem cells
  • the results of Figures 5A to 5D show that the transplanted mAPOSC-GFP also migrates to the penumbra of stroke mice and differentiates into neurons (MAP2+ and Nestin+) or endothelial cells (vWF+ and laminin+).
  • the adult multifunctional olfactory stem cells of the present invention express a plurality of specific pluripotency markers, and therefore, the specific pluripotency markers will be used in this test case to further investigate the distribution of adult multifunctional olfactory stem cells in vivo, and Endogenous adult multifunctional olfactory stem cell niche in olfactory mucosa (OM).
  • OM olfactory mucosa
  • Fig. 6A is a schematic diagram of the cell type of human olfactory mucosa, wherein OE stands for olfactory mucosa, BM stands for basal membrane, LP stands for lamina basement, GBC Represents globose basal cells, and HBC stands for horizontal basal cells.
  • horizontal basal cells and spheroidal basal cells remained in the biopsies of human olfactory mucosa, but other cell types were not completely retained in the biopsy.
  • Horizontal basal cells are present adjacent to the basement membrane (where indicated by the dashed line) and the laminalitis is below the olfactory mucosa.
  • Fig. 6A (2) to (7) for the distribution of adult multi-functional olfactory stem cells isolated from human olfactory mucosa in vivo.
  • Immunofluorescence staining is used to calibrate human adult multifunctional olfactory stem cells.
  • the markers of performance are Nanog, Sox-2, Oct-4 and SSEA-4, and the basal cell marker K14 (cytokeratin 14).
  • both horizontal basal cells and squamous basal cells exhibit K14 and have round cell bodies. This is the only horizontal basal cell in mouse olfactory tissue that exhibits K14 and exhibits a flat/horizontal pattern. different.
  • the murine adult multi-function olfactory stem cells were further identified in this test. The state of distribution in the body. Please refer to the part of Fig.
  • OE stands for olfactory mucosa
  • BM stands for basement membrane
  • LP lamina propria
  • GBC spheroid basal cells
  • HBC horizontal basal cells
  • ORN Represents olfactory receptor neurons and Sus represents sustentacular cells.
  • Fig. 6B (2) to (4) for the distribution of adult olfactory stem cells isolated from rat olfactory tissue in vivo. The mouse is divided into adult olfactory stem cells by immunofluorescence staining. The markers Nang, Sox-2 and Oct-4, as well as the basal cell marker K14, were expressed.
  • FIG. 6C Please refer to FIG. 6C again for the analysis results of the basal cell marker expression of the adult multi-functional olfactory stem cells cultured in vitro, and the analyzed cells include adult multi-functional olfactory stem cells and adult multifunctional olfactory stem cell spheres, and analyzed basal cell markers.
  • the results of Fig. 6C (1) to (3) show that in vitro cultured adult multi-functional olfactory stem cells and adult multi-functional olfactory stem cell spheres all exhibit K14 or ICAM-1.
  • FIG. 7A is a graph showing the results of Bmi-1 expression of adult multifunctional olfactory stem cells cultured in vitro
  • FIG. 7B is a Bmi-1 expression of adult multifunctional olfactory stem cells isolated from human olfactory mucosa tissues.
  • the results of the analysis are shown in Fig. 7C as a graph showing the results of analysis of Bmi-1 expression of adult multifunctional olfactory stem cells isolated from murine olfactory tissue.
  • Fig. 7C shows the results of analysis of Bmi-1 expression of adult multifunctional olfactory stem cells isolated from murine olfactory tissue.
  • Bmi-1 and Ki67 are not detected in the olfactory mucosa of Bmi-1 -/- olfactory tissue, but the expression of Bmi-1 and Ki67 can be detected by Bmi-1 +/+ olfactory tissue.
  • the basal cells of Bmi-1 have proliferative activity.
  • Bmi-1 affects the self-renewal of adult multifunctional olfactory stem cells located in the natural location.
  • the olfactory neuroepithelial cells of Bmi-1 +/+ mice and Bmi-1 -/- mice were induced to induce damage, and whether the expression of Bmi-1 would affect the self-renewal of adult multifunctional stem cells.
  • the induced injury test was performed by intraperitoneal injection of 50 ⁇ g/g methimazole (Sigma) into mice, and after 3 days, the olfactory tissue was fixed for immunofluorescence staining to detect proliferative marker Ki67 and basal cell marker K14 and neuronal marker.
  • Tuj1 and NeuroD1 are manifested by the proliferation of basal cells as an indicator of self-renewal of adult multifunctional olfactory stem cells.
  • (2) is an olfactory tissue treated without an induced damage test
  • (3) is an olfactory tissue treated by an induced damage test.
  • (4) The results of double staining of Tuj1 and K14, and the location of the nucleus is indicated by DAPI.
  • DAPI The staining result of NeuroD1, and the position of the nucleus is indicated by DAPI.
  • (6) is a statistical chart of the staining results of (3) to (5), where * represents P ⁇ 0.05. From the results of Fig.
  • Bmi-1 maintains the pluripotency of adult tissue stem cells by inhibiting early cell senescence and abnormal cell death, so this study will further explore whether Bmi-1 plays the same role in adult multifunctional olfactory stem cells. Detecting senescence markers in Bmi-1 +/+ olfactory tissue and Bmi-1 -/- olfactory tissue by senescence-associated (SA) ⁇ -galactosidase ( ⁇ -Gal) activity assay The activity of ⁇ -galactosidase at pH 6. Referring to Fig.
  • FIG. 8C there is a quantitative result plot of reverse transcription PCR of p16 Ink4a of Bmi-1 +/+ olfactory tissue and Bmi-1 -/- olfactory tissue, where * represents P ⁇ 0.05.
  • the p16 Ink4a gene is a gene that determines the normal function of the cell cycle and cell proliferation, differentiation, and apoptosis, and induces cellular senescence when the expression of p16 Ink4a is increased.
  • the results in Figure 8C show that the expression of the p16 Ink4a gene is significantly positively regulated in Bmi-1 -/- olfactory tissue.
  • This test case further detects the phenotype of adult multifunctional olfactory stem cells isolated from Bmi-1 +/+ olfactory tissue and Bmi-1 ⁇ / ⁇ olfactory tissue.
  • Adult multi-functional olfactory stem cells were prepared by Bmi-1 +/+ olfactory tissue and Bmi-1 -/- olfactory tissue, and Bmi-1 +/+ adult multi-functional olfactory stem cells were also detected by SA- ⁇ -Gal staining.
  • FIG 8B (3) is a photomicrograph of the adult multifunctional olfactory stem cells in the bright field.
  • Figure 8B (4) is an adult multifunctional olfactory stem cell stained with SA- ⁇ -Gal.
  • FIG 8D is a Bmi-1 + / + adult stem cells and olfactory multifunctional Bmi-1 - / - adult olfactory dry multifunctional of the amplification cell of FIG.
  • the results of Fig. 8B (3) and (4) and Fig. 8D show that the Bmi-1 -/- adult multi-functional olfactory stem cells cultured in vitro stopped dividing after 21 days, and had flat and enlarged cell types, indicating that it was early.
  • Bmi-1 +/+ adult olfactory stem cells remain dividing for more than 32 days, most cells remain spindle-shaped, and only 16% ⁇ 1% of Bmi-1 +/+ adult olfactory stem cells Showing beta-galactosidase.
  • a shRNA targeting Bmi-1 was introduced by lentivirus (LV-Bmi-1-sh, sc-29815-V, Santa Cruz Biotechnology).
  • lentivirus lentivirus
  • LV-Bmi-1-sh-hAPOSC human adult multi-function olfactory stem cells
  • FIG. 8E is an amplification index diagram of human adult multifunctional olfactory stem cells into which shRNA is introduced.
  • the results of Figure 8E show that when the Bmi-1 expression in human adult multifunctional olfactory stem cells is inhibited by shRNA, the long-term expansion ability of human adult multifunctional olfactory stem cells is significantly reduced.
  • no increase in apoptosis in Bmi-1 -/- olfactory mucosa was detected by TUNEL assay (data shown at the end).
  • Bmi-1 affects the self-renewal ability of adult multi-functional olfactory stem cells, and that Bmi-1 regulates the self-renewal ability of adult multi-functional olfactory stem cells by inhibiting premature cellular senescence rather than by apoptosis.
  • the adult multifunctional olfactory stem cells of the present invention are used for treating brain tissue damage
  • the experimental examples of this part will discuss the adult multifunctional olfactory stem cells of the present invention. It is used to treat the effects and potential of individuals with brain tissue damage.
  • FIG. 9A is a brain tissue infarction volume diagram of a stroke mouse transplanted with an adult multifunctional olfactory stem cell
  • FIG. 9B is a statistical result diagram of FIG. 9A.
  • the results of Figures 9A and 9B show that the infarct volume of brain tissue was significantly reduced in stroke mice transplanted with mAPOSC-GFP and control stroke mice (untransplanted mAPOSC-GFP).
  • the time point of detection of neurobehavioral was from day 6 to day 28 after cerebral ischemia/reperfusion.
  • the neurological deficit model is to assess the ability of mice to move.
  • the mobility of the mice was detected by the VersaMax Animal Activity Monitoring System (Accuscan Instruments) for 2 hours.
  • the VersaMax animal activity monitoring system consists of a 16-level infrared sensor and 8 vertical infrared sensors. The vertical sensors are located 10 cm above the bottom of the chamber.
  • the rats' mobility is interrupted by the movement of the rats in the chamber.
  • the number of beams is quantified.
  • the three vertical motion parameters measured are: vertical activity, vertical activity time, and number of vertical motions.
  • FIG. 9C is a result diagram of a vertical activity test of a stroke mouse
  • FIG. 9D is a result diagram of a vertical movement quantity test of a stroke mouse
  • FIG. 9E is a result diagram of a vertical activity time test of a stroke mouse.
  • the results of Figures 9C to 9E show that compared with the control group of stroke mice, the movement activities of stroke mice receiving mAPOSC-GFP transplantation from day 6 to day 28 after cerebral ischemia include vertical activity, vertical movement Both the number and the vertical movement time are significantly increased.
  • FIG. 9F is a graph showing the results of CD31 expression in brain tissue of a stroke mouse transplanted with mAPOSC-GFP.
  • the results of Figure 9F show that the number of newly generated blood vessels in the penumbra is significantly increased in stroke mice transplanted with mAPOSC-GFP compared to control mice.
  • transplantation of the adult multifunctional olfactory stem cells of the present invention can significantly improve the neurological function of stroke mice, and the implanted adult multifunctional olfactory stem cells migrate significantly into the brain of stroke mice, and stem cells migrate to the edge of the stroke. Phenomenon, and then repair the nerve cells in the damaged area.
  • Table 1 shows the clinical characteristics of the subject and the conditions for transplantation of the adult multifunctional olfactory stem cells of the present invention
  • FIG. 10A is a nuclear magnetic resonance angiogram of the subject.
  • subjects selected patients with old-fashioned stroke who developed from 35 to 70 years of age, and MRI showed vascular distribution (M1 and M2 positions) in the cerebral artery region.
  • vascular distribution M1 and M2 positions
  • NIHSS NIH stroke scale
  • the transplanted cells are adult multifunctional olfactory stem cells isolated from the autologous olfactory mucosa of the subject, and the amount of cells transplanted is 2 ⁇ 10 6 cells, and the follow-up condition of the subject after implantation is tracked every 1 to 3 months, and the follow-up is performed. The period is 12 months.
  • the results of the trial were monitored by an independent safety committee whose monitoring program included the frequency of adverse reactions.
  • the clinical scores of the Fuger-Meyer Assessment at 6 months and 12 months after implantation of adult multifunctional olfactory stem cells were the primary end points.
  • none of the 6 subjects had systemic or local adverse reactions, providing prima facie evidence of the safety and feasibility of transplantation of the adult multifunctional olfactory stem cell treatment regimen of the present invention.
  • Subject 1 2 3 4 5 6 Symptoms and signs Right limb hemiplegia Right limb hemiplegia Right limb hemiplegia Hemiplegia of the left limb Hemiplegia of the left limb Hemiplegia of the left limb Stroke history (month) 51 11 12 16 20 29 Injection volume 2x 10 6 2x 10 6 2x 10 6 2x 10 6 2x 10 6 6
  • a nuclear magnetic resonance diffusion tensor image of one of the subjects is to observe the diffusion of water molecules in the brain tissue, and to evaluate the brain tissue structure and nerve fiber direction by the diffusion direction of the water molecules.
  • Water molecules diffuse freely in three dimensions in the body, and the direction in which water molecules diffuse is affected by the permeability of surrounding tissues, the direction of axons, and even the microtubule depolymerization of intracellular microtubules. Therefore, by observing the anisotropy of water molecules (that is, whether water molecules diffuse in a single direction), it can be inferred whether there is a change in the microstructure of the white matter.
  • the fractional anisotropy (FA) can be used as the tensor of water molecules and the directionality of water molecules.
  • the value of FA is between 0 and 1. The larger the value, the more water molecules are. Diffusion in a single direction.
  • the results of Fig. 10B show that the subject had a FA of 0.47 before transplantation of the adult multifunctional olfactory stem cells of the present invention (BT), and the FA of the adult multifunctional olfactory stem cells of the present invention after 6 months (6AT) was 0.39.
  • the FA was 0.33, which showed that the transplantation of the adult multifunctional olfactory stem cells of the present invention significantly increased the number of corticospinal tract fibers in the subject.
  • FIG. 10C is a correlation diagram of the Bmi-1 relative performance and the Fog-Meel assessment (FMT) score improvement percentage.
  • FMT Fog-Meel assessment
  • the isolated adult multifunctional olfactory stem cell line of the present invention exhibits Bmi-1 and pluripotency markers Oct-4, Sox-2, Nanog and SSEA-4 on the cell surface, which have self-renewal and pluripotency.
  • the characteristics of differentiation can be screened for pluripotency markers such as Oct-4, Sox-2, Nanog and SSEA-4 from a mixture of cells derived from mammalian olfactory tissue.
  • the cells can be rescreened for Bmi-1 cells, and the selected cells are adult olfactory stem cells with pluripotent differentiation ability, so that pluripotent adult olfactory stem cells can be rapidly and exclusively purified.
  • the isolated adult multifunctional olfactory stem cells of the present invention are used for cell therapy, they can be used for treating brain tissue damage of an individual, and further, in the part of treating brain tissue damage of an individual, the individualization of the present invention
  • the mesenchymal stem cells can improve the nerve function of individuals with brain tissue damage.
  • the implanted adult multifunctional olfactory stem cells can migrate to the damaged parts of the brain, repair the nerve cells in the damaged area, and reduce the infarct volume of the stroke. Therefore, it can be used to treat individuals with brain tissue damage.

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Abstract

提供了一种成体多能嗅干细胞及其分离方法以及其应用，经分离的成体多能嗅干细胞，其表达B细胞特异性莫洛尼鼠白血病病毒插入位点1。

Description

成体多功能嗅干细胞及其分离方法以及其应用 技术领域
本发明是关于一种未分化的动物细胞，特别是关于一种成体组织干细胞。
背景技术
干细胞(Stem Cell)是为生物体内尚未分化的原生细胞，其具有可以长时间地不断复制、更新，并分化衍生成具有特殊型态和功能的成熟细胞的能力。干细胞依其来源主要可分成胚胎干细胞(embryonic stem cells，ESCs)及成体干细胞(adult stem cell)两种，胚胎干细胞取自囊胚里的内细胞团，而成体干细胞则来自各式各样的组织。干细胞又依其分化能力，主要可分成三大类，一为全能性干细胞(totipotent stem cells)，其具有完全能力，可分化发育成为完整胚胎或生物体；二为多能性干细胞(pluripotent stem cells)，其具有分化为三个胚层的能力，而可形成某种组织或器官的所有细胞，但无法发育成为完整胚胎或生物体；三为复效性干细胞(multipotent stem cells)，包括特定组织的干细胞，如神经干细胞、血球干细胞、肝脏干细胞、皮肤干细胞等。
由于多能性干细胞可分化成不同的细胞系，故可用于治疗多种退化性或遗传性疾病。在各种多能干细胞之中，胚胎干细胞被认为具有上述的功能。然而，道德上的疑虑一直阻碍着人类胚胎干细胞在研究及治疗上的应用，而非胚胎的胚胎干细胞则能够规避此一障碍。此类非胚胎的多能干细胞包括成人骨髓间叶干细胞或基质干细胞及脐带血干细胞。然而，由于体外扩增的需求及人类白血球抗原配对的条件，限制了这些细胞在临床上的应用，因此，需要有另一种多能细胞。
成体组织干细胞是指存在于一种已经分化组织中的未分化细胞，存在于机体的各种组织器官中，传统上成体组织干细胞被认为只能增生分化成为所在组织内限定细胞种类。然而近年研究结果纷纷质疑这传统概念，指出成体组织内蕴藏具有多重分化能力的干细胞族群，成体多功能干细胞对医疗应用方面是项喜讯，因为能替代具有道德争议性的人类胚胎 干细胞，因此自成体组织中找寻具有自我更新和多能性分化能力的成体组织干细胞，已成为干细胞医疗相关技术研发的主要课题之一。
发明内容
本发明的目的在于提供一种成体多功能嗅干细胞及其分离方法以及其应用，其经单离的成体多功能嗅干细胞具有自我更新能力和多能性分化的能力。
依据本发明的一目的是在提供一种经单离的成体多功能嗅干细胞，其表现B细胞特异性莫洛尼鼠白血病病毒插入位点1(B-lymphoma Moloney murine leukemia virus insertion region-1，Bmi-1)。
依据前述经单离的成体多功能嗅干细胞，其更表现多能性标记Oct-4、Sox-2、Nanog和SSEA-4。
依据前述经单离的成体多功能嗅干细胞，其具有碱性磷酸酶(Alkaline phosphatase，ALP)活性。
借此，本发明的经单离的成体多功能嗅干细胞是在细胞表面表现B细胞特异性莫洛尼鼠白血病病毒插入位点1，其具有自我更新和多能性(pluripotent)分化的特性。
依据本发明的另一目的是在提供一种分离具多能性的成体嗅干细胞的方法，包含下述步骤。提供来自哺乳类动物的嗅觉组织的细胞混合物。进行分离步骤，是自细胞混合物单离对于Oct-4、Sox-2、Nanog和SSEA-4呈阳性的细胞，以获得具多能性的成体嗅干细胞。
依据前述的分离具多能性的成体嗅干细胞的方法，其中哺乳类动物可为人类或鼠类。
依据前述的分离具多能性的成体嗅干细胞的方法，其中分离步骤还包含单离对B细胞特异性莫洛尼鼠白血病病毒插入位点1(B-lymphoma Moloney murine leukemia virus insertion region-1，Bmi-1)呈阳性的细胞。
借此，本发明的分离具多能性的成体嗅干细胞的方法，可自哺乳动物的嗅觉组织来源的细胞混合物中，筛选对于Oct-4、Sox-2、Nanog和SSEA-4等多能性标记呈阳性的细胞，优选地，可再筛选对于B细胞特异性莫洛尼鼠白血病病毒插入位点1的细胞，筛选出的细胞即为具多能性分化能力的成体嗅干细胞，故可快速且专一的纯化多能性的成体嗅干细 胞。
依据本发明的再一目的是在提供一种经单离的成体多功能嗅干细胞的用途，其是用于制备治疗个体脑组织损伤的药物。
依据前述的经单离的成体多功能嗅干细胞的用途，其中脑组织损伤可由脑缺血疾病造成或可由神经退化性疾病造成。而脑缺血疾病可为中风，神经退化性疾病可为阿兹海默症、巴金森氏症或癫痫症。
借此，本发明的经单离的成体多功能嗅干细胞用于细胞治疗时，改善脑组织损伤个体的神经功能，植入的成体多功能嗅干细胞可迁移至脑中受损部位，进而修补受损部位的神经细胞，故可用以治疗脑组织损伤的个体。
上述发明内容旨在提供本揭示内容的简化摘要，以使阅读者对本揭示内容具备基本的理解。此发明内容并非本揭示内容的完整概述，且其用意并非在指出本发明实施例的重要/关键元件或界定本发明的范围。
附图说明
为让本发明的上述和其他目的、特征、优点与实施例能更明显易懂，结合附图说明如下：
图1A至图1C为分离自人类嗅黏膜组织的成体多功能嗅干细胞的多能性标记表现的结果图；
图1D为分离自人类嗅黏膜组织的成体多功能嗅干细胞的扩增指数图；
图1E为分离自人类嗅黏膜组织的成体多功能嗅干细胞的增生指数测量结果图；
图1F和图1G为分离自鼠类嗅觉组织的成体多功能嗅干细胞的多能性标记表现的结果图；
图2A为分离自人类嗅黏膜组织的成体多功能嗅干细胞经三维培养后多能性标记表现的结果图；
图2B为分离自鼠类嗅觉组织的成体多功能嗅干细胞经三维培养后多能性标记表现的 结果图；
图3A至图3E为本发的明的成体多功能嗅干细胞诱导分化为不同胚层的组织细胞的显微照片图；
图3F为本发的明的成体多功能嗅干细胞自发分化为不同胚层的组织细胞的显微照片图；
图3G为本发的明的成体多功能嗅干细胞自发分化后不同胚层标记表现的分析结果图；
图4A至图4E为分离自人类嗅黏膜组织的成体多功能嗅干细胞于小鼠体内分化为不同胚层的组织细胞的显微照片图；
图5A至图5D为分离自鼠类嗅觉组织的成体多功能嗅干细胞于小鼠体内分化为不同胚层的组织细胞的显微照片图；
图6A为分离自人类嗅黏膜组织的成体多功能嗅干细胞于体内的分布结果图；
图6B为分离自鼠类嗅觉组织的成体多功能嗅干细胞于体内的分布结果图；
图6C为体外培养的成体多功能嗅干细胞的基底细胞标记表现的分析结果图；
图7A为体外培养的成体多功能嗅干细胞的Bmi-1表现的分析结果图；
图7B为分离自人类嗅黏膜组织的成体多功能嗅干细胞的Bmi-1表现的分析结果图；
图7C为分离自鼠类嗅觉组织的成体多功能嗅干细胞的Bmi-1表现的分析结果图；
图8A为表现Bmi-1对于成体多功能嗅干细胞影响的结果图；
图8B为老化相关半乳糖苷酶分析的结果图；
图8C为Bmi-1+/+嗅觉组织和Bmi-1-/-嗅觉组织的p16Ink4a的反转录PCR的定量结果图；
图8D为Bmi-1+/+成体多功能嗅干细胞和Bmi-1-/-成体多功能嗅干细胞的扩增指数图；
图8E为导入shRNA的人类成体多功能嗅干细胞的扩增指数图；
图9A为移植成体多功能嗅干细胞的中风小鼠的脑组织梗塞体积图；
图9B为图9A的统计结果图；
图9C为中风小鼠垂直活动测试的结果图；
图9D为中风小鼠垂直运动数量测试的结果图；
图9E为中风小鼠垂直活动时间测试的结果图；
图9F为移植鼠类成体多功能嗅干细胞的中风小鼠脑组织中CD31表现的结果图；
图10A为受试者的核磁共振造影图；
图10B为受试者的核磁共振扩散张量影像图；
图10C为Bmi-1相对表现量和傅格-梅尔评估量分数的相关图。
具体实施方式
本说明书公开内容提出一种经单离的哺乳类动物的成体多功能嗅干细胞，其表达特定的细胞表面受体，且具自我更新能力和多能性(pluripotent)分化能力。本说明书公开内容另提供一种分离具多能性的成体嗅干细胞的方法，其可由来自哺乳类动物的嗅觉组织的细胞混合物中，快速且专一性的筛选出具多能性的成体嗅干细胞。
更进一步说，本发明提供一种单离的哺乳类动物的成体多功能嗅干细胞，其表现B细胞特异性莫洛尼鼠白血病病毒插入位点1(B-lymphoma Moloney murine leukemia virus insertion region-1，Bmi-1)，其来源可为人类细胞或鼠类细胞，优选地，其表现多能性标记Oct-4、Sox-2、Nanog和SSEA-4。而本发明的分离具多能性的成体嗅干细胞的方法，是自哺乳类动物的嗅黏膜组织来源的细胞混合物中，筛选对于Oct-4、Sox-2、Nanog和SSEA-4呈阳性的细胞，优选地，可再筛选对于Bmi-1呈阳性的细胞，即可自细胞混合物中分离出具多能性的成体嗅干细胞，其中哺乳类动物可为人类或鼠类。
本发明的经单离的哺乳类动物的成体多功能嗅干细胞可应用于制备治疗个体脑组织损伤之药物。更进一步地说，本发明的经单离的成体多功能嗅干细胞用于细胞治疗时，改善脑组织损伤个体的神经功能，植入的成体多功能嗅干细胞可迁移至脑中受损部位，进而修补受损部位的神经细胞，故可用以治疗脑组织损伤的个体，其中脑组织损伤可为脑缺血疾病或神经退化性疾病，而脑缺血疾病可为中风，神经退化性疾病可为阿兹海默症、巴金森氏症或癫痫症。
兹以下列具体试验例进一步示范说明本发明，用以有利于本发明所属技术领域的技术人员，可在不需过度解读的情形下完整利用并实践本发明，而不应将这些试验例视为对本发明范围的限制，但用于说明如何实施本发明的材料及方法。
<试验例>
一、本发明的成体多功能嗅干细胞
1.1.成体多功能嗅干细胞的制备
为制备成体多功能嗅干细胞，在本试验例中所使用的哺乳动物组织为人类的嗅黏膜组织或鼠类的嗅觉组织。人类的嗅黏膜组织(5mm3，0.5克重)是在全身麻醉下取自鼻中隔附近，取样的流程经过中国医药大学附设医院的人体试验委员会(Institutional Review Board)的认可，亦已取得受试患者的书面同意。而鼠类的嗅觉组织则取自8周或11周龄的鼠类，将鼠类麻醉后牺牲，并在解剖显微镜下自上鼻甲骨分离嗅觉组织，其中所使用的鼠类包含SD(Sprague-Dawley)大鼠、C57BL/6JNarl小鼠、GFP转基因小鼠、Bmi-1+/+小鼠和Bmi-1基因剔除小鼠(Bmi-1-/-)。
将所收集的人类嗅黏膜组织置于含有Hank’s盐类平衡溶液(Gibco/BRL)的已灭菌盒中，并于24小时内以外植体培养法(explant culture method)进行初代培养，其步骤如下述。在解剖显微镜下将每个嗅觉组织分切成小片，在室温下将其置于磷酸盐缓冲液中，再以600×g的离心力离心10分钟收集组织外植体，并以含有2μg/mL肝素(heparin；Sigma)、20ng/mL成纤维细胞生长因子(FGF2；R&D Systems)、20ng/mL表皮生长因子(EGF；R&D Systems)和1％青霉素/链霉素(100U/ml)的DMEM/F12培养基再悬浮组织沉淀物，再将组织外植体置于25cm2的细胞培养皿中，在5％CO2、37℃的环境下进行培养5至7天后，让细胞自外植体中向外生长，而初代贴壁的细胞即为分离自人类的成体多功能嗅干细胞。分离自鼠类的成体多功能嗅干细胞其制备步骤同上述，但在DMEM/F12培养基除了添加上述的补充物外，再额外添加20ng/mL表皮生长因子(Invitrogen)。
再以免疫萤光染色、反转录PCR和流式细胞仪分析前述初代培养的成体多功能嗅干细胞的多能性标记表现。分析的多能性标记包含对发育中胚囊必要的关键转录因子和存在于未分化人类胚胎干细胞的细胞表面鞘糖脂(glycosphingolipids)，其中关键转录因子包含Nanog、Sox-2和Oct-4，鞘糖脂为SSEA-4(stage-specific embryonic antigen 4)。
请参照图lA至图1C，为分离自人类嗅黏膜组织的成体多功能嗅干细胞的多能性标记表现的结果图，其中图lA为免疫萤光染色的结果图，细胞核是以DAPI标示，(1)至(4)部分的萤光分别为Nanog、Oct-4、Sox-2和SSEA-4的信号。图1B为反转录PCR的结果图，其中对照组为未加入模板的组别，APOSC表示成体多功能嗅干细胞，hES表示作为正对照组的人类胚胎干细胞。图1C为流式细胞仪分析结果图，其分析的成体多功能嗅干细胞是分离自6个不同的供体，且具有不同的代数(p2-p14)。
图lA的结果显示，分离自人类嗅黏膜组织的成体多功能嗅干细胞确实能表现Nanog、Oct-4、Sox-2和SSEA-4等多能性标记，尤其是成体多功能嗅干细胞于细胞表面表现SSEA-4显示其为更原始阶段的细胞，经由免疫萤光染色也证实Nanog和Oct-4在分离自6个不同供体的成体多功能嗅干细胞中的核表达(数据未显示)。而图1B的结果显示，分离自人类嗅黏膜组织的成体多功能嗅干细胞能表达Nanog、Oct-4和Sox-2等多能性标记，且能表达能助于诱导多潜能性干细胞(induced pluripotent stem cells，iPS)产生的c-Myc和KLF-4。图1C也有一致的结果，显示分离自人类嗅黏膜组织的成体多功能嗅干细胞确实能表达Nanog、Oct-4、Sox-2和SSEA-4等多能性标记，且有52.9±19.3％(n＝6)的成体多功能嗅干细胞对SSEA-4呈阳性，甚至0.1％至5％的成体多功能嗅干细胞(n＝6)可检测到更原始阶段的标记-SSEA-3(数据未显示)。上述的数据可证实分离自不同供体和后续继代不同代数后的成体多功能嗅干细胞皆一致地表现与胚胎干细胞相关的多能性标记。
为了分析体外培养的成体多功能嗅干细胞的增殖能力，将成体多功能嗅干细胞进行长期扩增，并分析成体多功能嗅干细胞的生长动力学。请参照图1D，为本发明的经单离的成体多功能嗅干细胞的扩增指数图。图lD的结果显示，成体多功能嗅干细胞指数地增殖48天(共17代)，并且在83天后(第25代)缓慢生长。而成体多功能嗅干细胞的倍增时间为20.9±3.9小时(供体1，第3至17代的平均值)或25.3±6.7小时(供体2，第5至第19代的平均值)。
为了监测成体多功能嗅干细胞的增殖，试验上以10μM的CFSE(carboxyfluorescein diacetate succinimidyl ester；Molecular Probes)标定成体多功能嗅干细胞，并续培养成体多功能嗅干细胞4天后，以流式细胞仪(Becton Dickinson)检测CFSE在成体多功能嗅干细胞中的分布状况，并进一步以MODFIT软体(Verity Software House，ME)计算成体多功能嗅干细胞的增生指数(proliferation index，PI)，所计算的增生指数越高，表示细胞增生能力越 好。CFSE萤光在每个连续的细胞分裂中会精准地减半，因此可通过CFSE的标定可追踪细胞的分裂，进一步证实成体多功能嗅干细胞的活性生长表型。请参照图1E，为本发明的经单离的成体多功能嗅干细胞之增生指数测量结果图。图1E的结果显示，在96小时的追踪期内，50.9％的成体多功能嗅干细胞完成了3次细胞分裂，32.7％的成体多功能嗅干细胞完成了2次细胞分裂，显示成体多功能嗅干细胞的细胞周期动力学与人类胚胎干细胞和诱导多潜能性干细胞相同(倍增时间介于24小时至48小时之间)，并优于一些成体多能干细胞(倍增时间从30小时至72小时不等)。
请再参照图1F和图1G，为分离自鼠类嗅觉组织的成体多功能嗅干细胞的多能性标记表现的结果图，其中图1F为免疫萤光染色的结果图，并以DAPI标示细胞核的位置，(1)至(3)部分的萤光分别为Nanog、Oct-4和Sox-2之信号。图1G为反转录PCR的结果图，其中对照组为未加入模板的组别，APOSC表示成体多功能嗅干细胞，mES表示作为正对照组的鼠类胚胎干细胞。由图1F和图1G的结果显示，分离自鼠类嗅觉组织的成体多功能嗅干细胞也会在核中表现Nanog、Oct-4和Sox-2，以及表现KLF-4和c-Myc。而分离自鼠类嗅觉组织的成体多功能嗅干细胞的倍增时间为24.6±2.0小时。
1.2.成体多功能嗅干细胞的三维培养
干细胞的独特特征之一为在三维和模拟天然的环境下具有形成细胞球的能力。因此除了以贴附生长检视本发明的成体多功能嗅干细胞的多能性标记，本试验中利用三维培养条件下培养成体多功能嗅干细胞，以检视本发明的成体多功能嗅干细胞是否保留形成细胞球的干细胞性(stemness)。
三维培养的步骤如下，将已占满生长平面空间的成体多功能嗅干细胞以胰蛋白酶消化后，以悬浮培养液再悬浮，并将成体多功能嗅干细胞调整为7×104cells/mL，其中悬浮培养液为含有2％的B27supplement(Gibco)、20ng/mL的碱性成纤维细胞生长因子(bFGF)、20ng/mL的表皮生长因子和1％的青霉素/链霉素(100U/ml)的DMEM/F12培养基。在接种前述细胞悬浮液之前，先将15mg/ml的聚甲基丙烯酸羟乙基酯(poly HEMA，Sigma，P3932)涂覆在细胞培养皿上，以避免细胞贴附于细胞培养皿的底部。初代形成的细胞球称为第一成体多功能嗅干细胞球。为了检测成体多功能嗅干细胞球的增值潜力，进一步将成体多功能嗅干细胞球培养于含有尿嘧啶类似物(Bromodeoxyuridine，BrdU)的悬浮培养液中标定DNA。
请参照图2A，为分离自人类嗅黏膜组织的成体多功能嗅干细胞经三维培养后多能性标记表现的结果图，其中(1)为明视野下的显微照片图，(2)为以BrdU标定后的萤光显微照片图。图2A的结果显示，人类成体多功能嗅干细胞经由三维培养可形成紧密的细胞球。而嵌入BrdU的人类成体多功能嗅干细胞球则可证明其持续进入细胞周期中的S期。此外，第一成体多功能嗅干细胞球中也大量表现细胞增殖标记物Ki67(数据未显示)。
干细胞经过数代三维培养后，所形成的细胞球数量可表示干细胞自我更新活性，而所形成的球体大小则可表示干细胞的增殖能力。为了评估成体多功能嗅干细胞球的自我更新能力，将经过3天培养的第一成体多功能嗅干细胞球以胰蛋白酶消化解离为单细胞，计数细胞数后再以悬浮培养液重新接种再次进行三维培养，尔后所形成的细胞球称为第二成体多功能嗅干细胞球。并在培养的第2、5和9天测量第二成体多功能嗅干细胞球的直径。经解离再培养的2天后，50％的成体多功能嗅干细胞存活，并可在三维培养中观察到第二成体多功能嗅干细胞球。而培养9天以上的第二成体多功能嗅干细胞球，其平均直径从59μm增加到81μm。
请再次参照图2A，其中(3)为以Vector Red Alkaline phosphatase Substrate Kit I侦测碱性磷酸酶(Alkaline phosphatase，ALP)活性的结果图，(4)至(6)分别为以免疫萤光染色标定Nanog、Oct-4和SSEA-4的结果图，由第2A图的结果显示，成体多功能嗅干细胞球表现Nanog、Oct-4和SSEA-4，和胚胎干细胞的表现相似。此外，可在成体多功能嗅干细胞球中检测到碱性磷酸酶的活性。
请再参照图2B，为分离自鼠类嗅觉组织的成体多功能嗅干细胞经三维培养后多能性标记表现的结果图，其中(1)为以ki67标定后的萤光显微照片图，(2)为碱性磷酸酶活性的结果图，(3)和(4)为Oct-4和Nanog的免疫萤光染色结果图。图2B的结果显示，在鼠类多功能嗅干细胞球中也观察到其表现ki67、碱性磷酸酶、Oct-4和Nanog。上述结果显示，本发明的成体多功能嗅干细胞球所表现的多能标志物、自我更新和碱性磷酸酶活性显示其具有与胚胎干细胞相似的特征。
1.3.成体多功能嗅干细胞的体外多能分化能力
本试验例以体外分化试验进一步探讨本发明的成体多功能嗅干细胞是否具有分化为三胚层细胞的多能分化能力。实验上分别以含有不同生长因子的分化培养基培养成体多功能嗅干细胞，引导其分化为外胚层(神经细胞)、中胚层(脂肪细胞、成骨细胞、软骨细胞和 内皮细胞)和内胚层(肝细胞)的细胞，并在显微镜下观察细胞型态，以及进一步以染色法确认分化后的细胞种类。
请参照图3A，为本发明的成体多功能嗅干细胞分化为外胚层的神经细胞的显微照片图，在本试验例以免疫萤光染色确认分化后的细胞是否表现成熟神经标记，成熟神经标记包含Tuj-1(Neuron-specific class III beta-tubulin)、GFAP(glial fibrillary acidic protein)和MAP-2(microtubule-associated protein 2)。第3A图的结果显示，以分化培养基培养后的细胞3种成熟神经标记皆有表现，且可见其表现出神经元形态，包含第(1)部份的多极形态学和分支(箭头所指之处)，第(2)部份中类似发展中嗅神经细胞(Olfactory receptor neuron，ORN)的长双极线状形态(箭头所指之处)，第(3)部份的珠状轴突样结构(箭头所指之处)和第(4)部份的网状轴突样结构(箭头所指之处)。证明分化后的细胞确实为神经细胞。
请参照图3B，为本发明的成体多功能嗅干细胞分化为内胚层之肝细胞的显微照片图，其中(1)至(3)为利用免疫萤光染色确认分化后的细胞是否表现由肝细胞合成的白蛋白(albumin)、α1-抗胰蛋白酶(α1-antitrypsin)和甲型胎儿蛋白(α-fetoprotein，α-FP)。(4)在利用反转录PCR确认分化后的成体多功能嗅干细胞的白蛋白表现的结果图，其中人类肝癌细胞株HepG2为正对照组，U表示未分化的成体多功能嗅干细胞，D表示分化后的成体多功能嗅干细胞。图3B的结果显示，经由肝细胞诱导分化培养后的细胞为肝细胞特异性基因阳性的细胞，其可表现白蛋白、α1-抗胰蛋白酶和α-FP。为了测试分化后的肝细胞代谢功能，本试验例进一步以PAS染色(Periodic Acid-Schiffstain)检测细胞中的醣类，以检视分化后的成体多功能嗅干细胞的细胞质中是否具有储存的糖原(glycogen)。请参照图3C，为本发明的成体多功能嗅干细胞PAS染色后的显微照片图，其中U表示未分化的成体多功能嗅干细胞，D表示分化后的成体多功能嗅干细胞。图3C的结果显示，在(1)未分化的成体多功能嗅干细胞中未见有储存的糖原，但在(2)和(3)经分化后的成体多功能嗅干细胞中皆可见的储存的糖原，其中(2)和(3)的放大倍率分别为40x和400x。上述结果证明成体多功能嗅干细胞确实可被诱导分化成具功能性的肝细胞。
请参照图3D，为本发明的成体多功能嗅干细胞以血管形成测定分化为内皮细胞的显微照片图。为了证明成体多功能嗅干细胞可以被诱导分化为内皮细胞，将成体多功能嗅干细胞以血管内皮生成因子(VEGF)、bFGF和肝素诱导处理以进行血管形成测定，并以明视野下观察细胞型态以确认成体多功能嗅干细胞是否会分化为内皮细胞。图3D的结果显示， 在血管形成测定中，成体多功能嗅干细胞在2-4小时内会朝向彼此迁移并形成毛细管样的管状结构，其成熟时间约6小时。而21小时后，所形成的管状结构会从基质分离。可见成体多功能嗅干细胞的血管形成动力学显示其与内皮细胞的表现相同。
请再参照图3E，为本发明的成体多功能嗅干细胞分化为中胚层之脂肪细胞、成骨细胞、软骨细胞和内皮细胞的显微照片图，第3E图中利用油红组织染色(oil red O stain)作为细胞内脂质累积的指示剂，以确认分化后的细胞为脂肪细胞。利用茜素红染色(Alizarin red S stain)显示钙矿化的状况，以确认分化后的细胞为成骨细胞。利用阿新蓝染色(Alican blue stain)作为蛋白聚糖(proteoglycans)合成的指示，以确认分化后的细胞为软骨细胞。而在图3E中，成体多功能嗅干细胞未先以血管内皮生成因子诱导处理，并于明视野下观察其细胞型态。图3E的结果显示，成体多功能嗅干细胞在分化培养基的培养下，可以分化为脂肪细胞、成骨细胞、软骨细胞以及具有毛细管样的管状结构，虽然成体多功能嗅干细胞自发分化以形成管状结构是从48小时才开始，但显示其不仅可以通过外源提供的组织特异性生长因子，也可能像胚胎干细胞一样，具有自发分化为三个胚层细胞的潜力。
因此，在本试验例中进一步测试本发明的成体多功能嗅干细胞是否可以在未添加任何生长因子诱导而自发分化成三个胚层细胞。将成体多功能嗅干细胞或第一成体多功能嗅干细胞球接种到涂覆明胶的细胞培养皿上培养15天，再以染色法和反转录PCR检视其是否分化为三个胚层的细胞。请参照图3F和图3G，图3F为本发明的成体多功能嗅干细胞自发分化为不同胚层的组织细胞之显微照片图，图3G为本发明的成体多功能嗅干细胞自发分化后不同胚层标记表现的分析结果图。图3F(1)的结果显示，在自发分化的群体中可观察到神经元样细胞，(2)的结果显示其可表现外胚层标记物Tuj-1。(3)的结果显示其可表现中胚层标记物α-平滑肌肌动蛋白(Alpha-smooth muscle actin，α-SMA)，(4)和(5)的结果显示其可表现内胚层的标记物白蛋白和α1-抗胰蛋白酶。此外，(6)的结果也显示经由PAS染色测定显示细胞中的具有糖原沉积，证明其为内皮层的肝细胞。图3G的反转录PCR结果显示，在明胶上生长的成体多功能嗅干细胞或第一成体多功能嗅干细胞球会表现外胚层的标记物MAP2和PAX6、中胚层标志物brachyury和Nkx2.5，以及内皮层标记物GATA6，α-FP和FOXA2。再次证明本发明的成体多功能嗅干细胞可以自发分化为三个胚层细胞。
1.4.成体多功能嗅干细胞的体内多能分化能力
为了证实本发明的成体多功能嗅干细胞具有体内(in vivo)多能分化能力，在本试验例中 将人类成体多功能嗅干细胞移植到中风小鼠体内，再以免疫萤光染色确定移植入的人类成体多功能嗅干细胞是否能于中风小鼠的脑缺血部位分化为神经元、神经胶质细胞或内皮细胞。
中风小鼠模型采取脑缺血/再灌注模型(ischemia-reperfusion model)来模拟小鼠中暂时性局部脑缺血的症状，所使用的试验动物为体重25-30克C57BL/6小鼠。将小鼠先腹腔注射水合氯醛(0.4g/kg)进行麻醉，小鼠的脑缺血/再灌注模型是以阻塞小鼠的双侧颈总动脉(common carotid arteries；CCAs)及右侧大脑中动脉(middle cerebral artery；MCA)诱发局部性脑缺血，阻塞期间的皮层血流量(cortical blood flow，CBF)以激光多普勒流量计(PF-5010，Periflux system)测量。阻塞120分钟后解除以进行再灌注。在麻醉期间，小鼠的核心温度以热敏电阻温度探测器监测，并以加热垫使小鼠的体温维持在37℃。从麻醉中恢复后，小鼠体温也以热灯保持在37℃。
在人类成体多功能嗅干细胞移植之前，先以编码Luc基因的慢病毒进行转染以得到萤光素酶(luciferase)标定的人类成体多功能嗅干细胞(hAPOSC-Luc)。再将106个hAPOSC-Luc以立体定位注射至中风小鼠的硬脑膜下3.5mm的三个皮层区域。四周后牺牲小鼠，取脑组织进行免疫萤光染色标定神经元标记MAP2、神经胶质细胞标记GFAP、内皮细胞标记vWF(Von Willebrand factor)和laminin(层黏连蛋白)，确认移植入的hAPOSC-Luc是否分化为神经元、神经胶质细胞或内皮细胞。
请参照图4A至图4D，其中图4A和图4B为本发明的人类成体多功能嗅干细胞于小鼠体内分化为神经元和神经胶质细胞的显微照片图，图4C和图4D为本发明的人类成体多功能嗅干细胞在小鼠体内分化为内皮细胞的显微照片图。图4A和图4B的结果显示，hAPOSC-Luc会迁移到缺血半球的半影区、侧脑室(lateral ventricle，LV)和海马齿状回(hippocampal dentate gyrus，DG)中，且由免疫萤光染色共定位结果显示，部份hAPOSC-Luc在DG共表达MAP2，在半影区共表达GFAP。而图4C和图4D的结果显示，血管腔周围的细胞共表达萤光素酶和内皮细胞标记物vWF或laminin。
为了进一步证实本发明的成体多功能嗅干细胞可在体内分化为内胚层的细胞，在本试验例中进一步将106个hAPOSC-Luc经皮注射到新生(第2天)小鼠肝脏中，移植后六周牺牲小鼠，取肝组织进行免疫萤光染色标定由肝细胞合成的白蛋白，确认移植入的hAPOSC-Luc是否分化为肝细胞。请参照图4E，为本发明的人类成体多功能嗅干细胞在小 鼠体内分化为肝细胞的显微照片图。图4E的结果显示，可在小鼠肝组织上检测到共表达萤光素酶和白蛋白的肝细胞。上述结果显示，移植入小鼠脑内的hAPOSC-Luc确实能分化为外胚层细胞(神经元和神经胶质细胞)、中胚层细胞(内皮细胞)和内胚层细胞(肝细胞)。
请再参照图5A至图5D，为分离自鼠类嗅觉组织的成体多功能嗅干细胞于小鼠体内分化为不同胚层之组织细胞的显微照片图，鼠类成体多功能嗅干细胞(mAPOSC-GFP)是分离自GFP转基因小鼠，因此其标定有萤光，其他试验步骤同上述在此不再赘述。图5A至图5D的结果显示，移植的mAPOSC-GFP也会迁移到中风小鼠的半影区，并分化成神经元(MAP2+和Nestin+)或内皮细胞(vWF+和laminin+)。
1.5.成体多功能嗅干细胞源自成人和鼠嗅上皮的基底层
由前述的试验例可知本发明的成体多功能嗅干细胞表达多个特殊的多能性标记，因此本试验例将利用特殊的多能性标记来进一步探讨成体多功能嗅干细胞在体内的分布，以及嗅粘膜组织(olfactory mucosa，OM)中的内源性成体多功能嗅干细胞区位(niche)。
请参照图6A(1)的部分，为人类嗅黏膜组织的细胞型态示意图，其中OE代表嗅黏膜(olfactory mucosa)，BM代表基底膜(basal membrane)，LP代表固有层(lamina propria)，GBC代表球状基底细胞(globose basal cell)，以及HBC代表水平基底细胞(horizontal basal cell)。由图6A的结果显示，水平基底细胞和球状基底细胞仍保留在人类嗅黏膜组织的活组织切片中，但其他细胞类型未能完整保留在活组织切片中。水平基底细胞存在于邻近基底膜之处(虚线所指之处)，固有层在嗅黏膜之下。请再参照图6A(2)至(7)的部分，为分离自人类嗅黏膜组织的成体多功能嗅干细胞于体内的分布结果图，试验上以免疫萤光染色标定人类成体多功能嗅干细胞所表现之标记Nanog、Sox-2、Oct-4和SSEA-4，以及基底细胞标记K14(cytokeratin 14)。图6A(2)至(5)的结果显示，在人类嗅粘膜组织中，Nanog(细胞核中的萤光)、Sox-2(细胞核中的萤光)、Oct-4(核仁和细胞质中的萤光)和SSEA-4(细胞膜上的萤光)皆与K14(细胞质中的萤光)共表达，显示表现Nanog、Sox-2、Oct-4和SSEA-4的细胞分布于嗅黏膜的基底层。此外，图6A(6)和(7)的结果显示，分布于基底层的细胞共表达SSEA-4和Nanog，以及共表达Sox-2和Nanog。
而在人类嗅黏膜组织中，水平基底细胞和球状基底细胞皆会表现K14，并且具有圆形细胞体，此与鼠类嗅觉组织中仅有水平基底细胞会表现K14，并呈现平坦/水平型态不同。为了精确鉴定成体多功能嗅干细胞区位，本试验例中进一步鉴定鼠类成体多功能嗅干细胞 在体内的分布状态。请参照图6B(1)的部分，为鼠类嗅觉组织的细胞型态示意图，其中OE代表嗅黏膜，BM代表基底膜，LP代表固有层，GBC代表球状基底细胞、HBC代表水平基底细胞、ORN代表嗅觉神经元(olfactory receptor neurons)和Sus代表支持细胞(sustentacular cells)。请再参照图6B(2)至(4)的部分，为分离自鼠类嗅觉组织的成体多功能嗅干细胞于体内的分布结果图，试验上以免疫萤光染色标定鼠类成体多功能嗅干细胞所表现的标记Nanog、Sox-2和Oct-4，以及基底细胞标记K14。图6B(2)至(4)的结果显示，在鼠类嗅觉组织中，Nanog、Sox-2和Oct-4皆与K14共表达，显示表现Nanog、Sox-2和Oct-4的细胞分布于嗅黏膜的基底层。
请再参照图6C，为体外培养的成体多功能嗅干细胞之基底细胞标记表现的分析结果图，分析的细胞包含体外培养的成体多功能嗅干细胞和成体多功能嗅干细胞球，分析的基底细胞标记为K14和ICAM-1(Intercellular Adhesion Molecule 1)，由图6C(1)至(3)的结果显示，体外培养的体外培养的成体多功能嗅干细胞和成体多功能嗅干细胞球皆会表现K14或ICAM-1。此外由图6C(4)的结果显示，成体多功能嗅干细胞一旦诱导分化为神经元，其表现神经元标记Tuj-1但不表现基底细胞标记K14。上述结果显示，不论是人类成体多功能嗅干细胞或鼠类成体多功能嗅干细胞皆存在于嗅黏膜的基底层。
1.6.Bmi-1对成体多功能嗅干细胞自我更新能力的必要性
本试验例进一步探讨Bmi-1维持成体多功能嗅干细胞自我更新能力的作用机制。请参照图7A至图7C，图7A为体外培养的成体多功能嗅干细胞的Bmi-1表现的分析结果图，图7B为分离自人类嗅黏膜组织的成体多功能嗅干细胞的Bmi-1表现的分析结果图，图7C为分离自鼠类嗅觉组织的成体多功能嗅干细胞的Bmi-1表现的分析结果图。在图7A中，(1)为贴附培养的成体多功能嗅干细胞，(2)为经三维培养所形成的成体多功能嗅干细胞球，其中萤光为Bmi-1的信号，并以DAPI标示细胞核，可见体外培养的成体多功能嗅干细胞和成体多功能嗅干细胞球皆会表现Bmi-1。而图7B和图7C的结果显示，在人类的嗅黏膜组织和鼠类的嗅觉组织中，Bmi-1明显表现于成体多功能嗅干细胞的细胞核中，且与多能性标记Nanog、Sox-2和Oct-4以及基底细胞标记K14共表达。此外，图7C(1)的结果显示，大量的Bmi-1累积在嗅觉神经元中作为多梳家族体，此结果与先前研究中指出成熟脑和眼组织中有丝分裂后的神经元会表现Bmi-1一致。图7B和图7C的结果亦显示，除了水平基底细胞和嗅觉神经元，在球状基底细胞和支持细胞中仅观察到少量的Bmi-1。上述结果显 示，本发明的成体多功能嗅干细胞不仅表现胚胎干细胞的多能性标记，其也表现成体干细胞的Bmi-1基因。
本试验例进一步探讨Bmi-1的表现对成体多功能嗅干细胞的影响。试验上分别取Bmi-1+/+小鼠和Bmi-1-/-小鼠的嗅觉组织，再以免疫萤光染色检视Bmi-1的表现和增生标记Ki67的表现。请先参照图8A(1)，为Bmi-1+/+嗅觉组织和Bmi-1-/-嗅觉组织的Bmi-1和Ki67的表现。可见在Bmi-1-/-嗅觉组织中的嗅黏膜侦测不到Bmi-1和Ki67的表现，但Bmi-1+/+嗅觉组织则可侦测到Bmi-1和Ki67的表现，显示表现Bmi-1的基底细胞具有增殖活性。
因此本试验例中将进一步探讨Bmi-1的表现是否影响位于自然区位的成体多功能嗅干细胞的自我更新。试验上分别对Bmi-1+/+小鼠和Bmi-1-/-小鼠的嗅神经上皮进行诱导损伤，再观察Bmi-1的表现是否会影响成体多功能嗅干细胞的自我更新。诱导损伤试验是以腹腔注射50μg/g的甲硫咪唑(methimazole，Sigma)至小鼠体内，并在3天后固定嗅觉组织进行免疫萤光染色侦测增生标记Ki67和基底细胞标记K14和神经元标记Tuj1和NeuroD1的表现的表现，以基底细胞的增生作为成体多功能嗅干细胞自我更新的指示。请参照图8A(2)至(6)的部分，其中(2)为未经诱导损伤试验处理的嗅觉组织，(3)为经诱导损伤试验处理的嗅觉组织。(4)为Tuj1和K14双染的结果，并以DAPI标示细胞核的位置。(5)为NeuroD1的染色结果，并以DAPI标示细胞核的位置。(6)为(3)至(5)染色结果的统计图，其中*代表P＜0.05。由图8A(2)的结果显示，仅有少部分的基底细胞(K14+)自发性增生，而图8A(3)至(6)的结果显示，在诱导损伤试验的再生研究下，化学损伤(如甲硫咪唑)引起嗅觉神经元破坏会刺激基底细胞的增殖和分化以代替神经元损失。和Bmi-1+/+嗅觉组织相比，Bmi-1-/-嗅觉组织经诱导损伤试验处理后，其中增生的成体多功能嗅干细胞数量显著减少。计算Ki67+K14+细胞数量的统计结果显示，在诱导损伤后3天，79％的Bmi-1+/+基底细胞被刺激增生，但仅32％的Bmi-1-/-基底细胞可以被刺激增殖。此外在Bmi-1-/-嗅觉组织中可见未成熟感觉神经元标记Tuj-1和神经元前体标记NeuroD1的表现显著地减少以及型态上的改变。
先前研究中指出Bmi-1通过抑制早期细胞衰老和异常细胞死亡来维持成体组织干细胞的多能性，因此本试验例将进一步探讨Bmi-1是否在成体多功能嗅干细胞中扮演相同的角色。试验上以老化相关半乳糖苷酶分析(senescence-associated(SA)β-galactosidase(β-Gal)activity assay)侦测Bmi-1+/+嗅觉组织和Bmi-1-/-嗅觉组织中衰老标记β-半乳糖苷酶在pH 6下的活性。请参照图8B(1)至(3)，为老化相关半乳糖苷酶分析的结果图，其中(1)为经苏木 素-伊红染色(HE Stain)的嗅觉组织。(2)为经SA-β-Gal染色的成年小鼠(8周大)嗅觉组织，其中被染色的部分代表β-半乳糖苷酶活性，虚线代表基底膜位置。图8B的结果显示，Bmi-1-/-嗅觉组织中成体多功能嗅干细胞所在的基底膜和嗅觉神经元可见被染色的细胞，显示其为衰老表型(phenotype)的细胞，而在Bmi-1+/+嗅觉组织中则较少被染色的细胞。请再参照图8C，为Bmi-1+/+嗅觉组织和Bmi-1-/-嗅觉组织的p16Ink4a的反转录PCR的定量结果图，其中*代表P＜0.05。p16Ink4a基因为决定细胞周期的正常运转和细胞增殖、分化及凋亡的基因，当p16Ink4a表现增加时会诱导细胞衰老。图8C的结果显示，在Bmi-1-/-嗅觉组织中p16Ink4a基因的表现显著地正向调控。
本试验例进一步检测分离自Bmi-1+/+嗅觉组织和Bmi-1-/-嗅觉组织的成体多功能嗅干细胞的表型。试验上以Bmi-1+/+嗅觉组织和Bmi-1-/-嗅觉组织制备成体多功能嗅干细胞，并同样以SA-β-Gal染色侦测Bmi-1+/+成体多功能嗅干细胞和Bmi-1-/-成体多功能嗅干细胞中衰老标记β-半乳糖苷酶在pH 6下的活性。请参照图8B(3)和(4)以及图8D，第8B图(3)为成体多功能嗅干细胞于明视野下的显微照片图。第8B图(4)为经SA-β-Gal染色的成体多功能嗅干细胞。图8D为Bmi-1+/+成体多功能嗅干细胞和Bmi-1-/-成体多功能嗅干细胞之扩增指数图。图8B(3)和(4)以及图8D的结果显示，体外培养的Bmi-1-/-成体多功能嗅干细胞在21天后即停止分裂，且具有扁平和扩大的细胞型态，显示其提早衰老，且有40％±5％的Bmi-1-/-成体多功能嗅干细胞表现大量与衰老相关的β-半乳糖苷酶。相较之下，Bmi-1+/+成体多功能嗅干细胞保持分裂超过32天，大部分细胞保持纺锤状型态，且仅16％±1％的Bmi-1+/+成体多功能嗅干细胞表现β-半乳糖苷酶。
为了再次证明Bmi-1的表现会影响成体多功能嗅干细胞的衰老，试验上再以慢病毒导入靶向Bmi-1的shRNA(LV-Bmi-1-sh，sc-29815-V，Santa Cruz Biotechnology)至分离自人类成体多功能嗅干细胞中(LV-Bmi-1-sh-hAPOSC)，以降低成体多功能嗅干细胞中Bmi-1的表现，实验上也使用慢病毒分别导入控制组的shRNA(LV-control-sh，sc-108080，Santa Cruz Biotechnology)至人类成体多功能嗅干细胞中(LV-control-sh-hAPOSC)做为实验对照组。请参照图8E，为导入shRNA的人类成体多功能嗅干细胞之扩增指数图。图8E的结果显示，当人类成体多功能嗅干细胞中的Bmi-1表现被shRNA抑制时，人类成体多功能嗅干细胞的长期扩增能力显著降低。此外，以TUNEL测定未侦测到Bmi-1-/-嗅粘膜组织中细胞凋亡的增加(数据末显示)。以上结果显示Bmi-1的表现会影响成体多功能嗅干细胞的自我更新 能力，且Bmi-1系通过抑制过早的细胞衰老而非通过凋亡来调节成体多功能嗅干细胞的自我更新能力。
二、本发明的成体多功能嗅干细胞用于治疗脑组织损伤
根据前述已知本发明的成体多功能嗅干细胞具有自我更新能力及多能分化能力，且其显示神经源性和血管生成能力，此部份的试验例将探讨将本发明的成体多功能嗅干细胞用于治疗脑组织损伤个体的效果和潜力。
2.1.成体多功能嗅干细胞的移植可减少中风大鼠的梗塞体积大小和改善中风大鼠模型的神经行为
试验上在中风小鼠在脑缺血后一周，将106个mAPOSC-GFP以立体定位注射至中风小鼠脑中，四周后检查中风小鼠脑缺血后的梗塞体积，并透过神经功能缺损模式检测中风前后的神经行为，以评估神经功能是否恢复。
试验上牺牲移植mAPOSC-GFP后28天的中风小鼠并取其脑组织，将脑组织切片进行HE染色。为了测量右侧皮质中的梗塞体积，试验上自左半球的总皮层区域减去右皮质中的非梗塞区域，并以分析软体(NIH Image J)计算梗塞体积。请参照图9A和图9B，图9A为移植成体多功能嗅干细胞的中风小鼠的脑组织梗塞体积图，图9B为图9A的统计结果图。图9A和图9B的结果显示，移植mAPOSC-GFP的中风小鼠和对照组中风小鼠(未移植mAPOSC-GFP)相比，其脑组织的梗塞体积显著地减少。
神经行为的检测时间点为脑缺血/再灌注后第6天至第28天。神经功能缺损模式为评估小鼠的活动能力。小鼠的活动能力是使用VersaMax动物活动监测系统(Accuscan Instruments)侦测大鼠2小时。VersaMax动物活动监测系统包含16水平红外线感测器和8个垂直红外感测器，垂直感测器分别位于腔室底板上方10公分处，大鼠的活动能力运动由腔室内大鼠因运动打断光束的次数进行定量。测量的3个垂直运动参数为：垂直活动、垂直活动时间以及垂直运动数量。
请参照图9C至图9E，图9C为中风小鼠垂直活动测试的结果图，图9D为中风小鼠垂直运动数量测试的结果图，图9E为中风小鼠垂直活动时间测试的结果图。图9C至图9E的结果显示，和对照组中风小鼠相比，接受mAPOSC-GFP移植的中风小鼠在脑缺血后第6天至第28天之间的运动活动包括垂直活动，垂直运动数量和垂直运动时间皆显著地增加。
为了确定移植mAPOSC-GFP是否能诱导中风小鼠脑中的血管生成，试验上以免疫萤光染色法定量CD31的表现量以测量血管密度。请参照图9F，为移植mAPOSC-GFP的中风小鼠脑组织中CD31表现的结果图。图9F的结果显示，移植mAPOSC-GFP的中风小鼠和对照组中风小鼠相比，其半影区中的新生成的血管数量显著地增加。
上述结果显示，移植本发明的成体多功能嗅干细胞可以明显改善中风小鼠的神经功能，此外植入的成体多功能嗅干细胞明显地迁移至中风小鼠脑中，并且有干细胞移行至中风边缘的现象，进而修补受损部位的神经细胞。
2.2.追踪植入成体多功能嗅干细胞的中风患者
为了确认移植自体的成体多功能嗅干细胞以改善中风诱发的神经功能障碍在临床治疗的安全性和可行性，在本试验进行人体试验，其是由中国医药大学附设医院的人体试验委员会(Institutional Review Board)的批准，受试者共6位。
请参照下表一和图10A，表一为受试者的临床特征和移植本发明的成体多功能嗅干细胞的条件，图10A为受试者的核磁共振造影图。在本次人体试验，受试者选择病发于35岁至70岁的成年人的陈旧性脑中风患者，且核磁共振造影显示大脑动脉区域的血管分布(M1和M2位置)梗塞者为主(排除出血型脑中风)，并且其NIHSS(NIH stroke scale)定位在5至15分的范围内。移植的细胞为分离自受试者自体嗅黏膜组织的成体多功能嗅干细胞，移植的细胞量为2x106个细胞，并后续每1至3个月追踪受试者植入细胞后的状况，追踪期为12个月。此外由独立的安全委员会监测试验的结果，其监测项目包括不良反应的频率。而本试验以傅格-梅尔评估量表(Fugl-Meyer Assessment)在植入成体多功能嗅干细胞后6个月和12月的临床分数为主要疗效的评估指标(primary end points)。在12个月追踪期，6位受试者皆无全身或局部不良反应，提供了移植本发明的成体多功能嗅干细胞治疗方案的安全性和可行性的初步证据。
表一、受试者的临床特征和移植成体多功能嗅干细胞的条件
受试者 1 2 3 4 5 6
症状和迹象 右肢偏瘫 右肢偏瘫 右肢偏瘫 左肢偏瘫 左肢偏瘫 左肢偏瘫
中风病史(月) 51 11 12 16 20 29
注射细胞量 2x 106 2x 106 2x 106 2x 106 2x 106 2x 106
P75+细胞纯度 91.5％ 90.4％ 95.5％ 91.7％ 91％ 91％
IC轨迹数量 3 3 3 3 3 3
请再参照图10B，为其中一位受试者的核磁共振扩散张量影像图，是观察大脑组织中水分子的扩散情形，并通过水分子的扩散方向来评估大脑的组织结构与神经纤维走向。水分子在体内以三维方向自由扩散，水分子扩散的方向会受周围组织的渗透性，神经轴突方向，甚至细胞内微小管的解聚(microtubule depolymerization)影响。故由观察水分子的非等向性(anisotropy，即水分子是否以单一方向扩散)可以此推测神经白质的微结构是否有变化。非等向性指标(fractional anisotropy，FA)可作为代表水分子扩散张量的大小及水分子扩散的方向性，FA的数值介于0到1之间，数值越大越表此部分水分子越以单一方向扩散。第10B图的结果显示，受试者在未移植本发明的成体多功能嗅干细胞前(BT)其FA为0.47，移植本发明的成体多功能嗅干细胞6个月后(6AT)其FA为0.39，移植本发明的成体多功能嗅干细胞12个月后(12AT)其FA为0.33，显示移植本发明的成体多功能嗅干细胞后明显增加受试者的皮质脊髓束纤维数量。
再利用线性相关分析观察受试者临床改善的状况和成体多功能嗅干细胞中Bmi-1表现的关系。请参照图10C，为Bmi-1相对表现量和傅格-梅尔评估量(FMT)分数改善百分比的相关图。结果显示，在植入成体多功能嗅干细胞后12个月所测量的FMT分数改善百分比与Bmi-1的相对表现量具有高度相关性(相关系数：r＝0.97；p＜0.001)。上述结果显示，具有高Bmi-1表现的自体成体多功能嗅干细胞的受试者，比低Bmi-1表现的自体成体多功能嗅干细胞的受试者有更显著良好的临床结果。
综合上述，本发明的经单离的成体多功能嗅干细胞系于细胞表面表现Bmi-1以及多能性标记Oct-4、Sox-2、Nanog和SSEA-4，其具有自我更新和多能性分化的特性。本发明的分离具多能性的成体嗅干细胞的方法，可自哺乳动物的嗅觉组织来源的细胞混合物中，筛选对于Oct-4、Sox-2、Nanog和SSEA-4等多能性标记呈阳性的细胞，优选地，可再筛选对于Bmi-1的细胞，筛选出的细胞即为具多能性分化能力的成体嗅干细胞，故可快速且专一的纯化多能性的成体嗅干细胞。将本发明的经单离的成体多功能嗅干细胞用于细胞治疗时，其可用于治疗个体的脑组织损伤，进一步地说，在治疗个体的脑组织损伤的部份，本发明的经单离的间质干细胞可以改善脑组织损伤个体的神经功能，植入的成体多功能嗅干细胞可迁移至脑中受损部位，进而修补受损部位的神经细胞，并可降低中风的梗塞体积， 故可用以治疗脑组织损伤的个体。
然本发明已以实施方式公开如上，然其并非用以限定本发明，任何所属领域的一般技术人员，在不脱离本发明的精神和范围内，当可作各种的更动与润饰，因此本发明的保护范围当视权利要求所界定的为准。

Claims (11)

  1. 一种经单离的成体多功能嗅干细胞，其特征在于，表现B细胞特异性莫洛尼鼠白血病病毒插入位点1。
  2. 如权利要求1所述的经单离的成体多功能嗅干细胞，其特征在于，所述经单离的成体多功能嗅干细胞还表现多能性标记Oct-4、Sox-2、Nanog和SSEA-4。
  3. 如权利要求1所述的经单离的成体多功能嗅干细胞，其特征在于，所述成体多功能嗅干细胞具有碱性磷酸酶活性。
  4. 一种分离具多能性的成体嗅干细胞的方法，其特征在于，包含：
    提供来自哺乳类动物的嗅觉组织的细胞混合物；以及
    进行分离步骤，所述分离步骤是自所述细胞混合物单离对于Oct-4、Sox-2、Nanog和SSEA-4呈阳性的细胞，以获得所述具多能性的成体嗅干细胞。
  5. 如权利要求4所述的分离具多能性的成体嗅干细胞的方法，其特征在于，所述哺乳类动物为人类或鼠类。
  6. 如权利要求4所述的分离具多能性的成体嗅干细胞的方法，其特征在于，所述分离步骤还包含单离对B细胞特异性莫洛尼鼠白血病病毒插入位点1呈阳性的细胞。
  7. 一种如权利要求1所述的经单离的成体多功能嗅干细胞的用途，其特征在于，是用于制备治疗个体脑组织损伤的药物。
  8. 如权利要求7所述的经单离的成体多功能嗅干细胞的用途，其特征在于，所述脑组织损伤是由脑缺血疾病造成。
  9. 如权利要求8所述的经单离的成体多功能嗅干细胞的用途，其特征在于，所述脑缺血疾病为中风。
  10. 如权利要求7所述的经单离的成体多功能嗅干细胞的用途，其特征在于，所述脑组织损伤是由神经退化性疾病造成。
  11. 如权利要求10所述的经单离的成体多功能嗅干细胞的用途，其特征在于，所述神经退化性疾病为阿兹海默症、巴金森氏症或癫痫症。
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