JP2004254682A - Side population cell derived from human amnion and use of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a stem cell capable of being stably supplied, without causing a problem of compatibility, when transplanted. <P>SOLUTION: A side population (SP) cell is separated from a human amniotic mesenchymal cell layer, is recognized that it expresses an Oct-4 gene, an Sox-2 gene, an FGF-4 gene, and an Rex-1 gene in RT-PCR, and is positive to vimentin and CK 19 in staining of an immunocyte. It is discovered that the SP cell exists in the human amniotic mesenchymal cell layer and a human amniotic epithelial cell layer, and the SP cell is separated at first, and further it is confirmed at first that the SP cell comprises the stem cell. Because the SP cell is at least differentiated into a nerve cell, the SP cell is useful as a supply source for a substance which the nerve cell produces, and further is used as a drug-delivery system, etc., for the substance which the nerve cell produces, by transplanting the SP cell to the brain of a patient suffering from refractory neuropath, such as Parkinson's disease and metabolic neuropath. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to side population cells isolated from human amniotic membrane. The cell of the present invention is useful as a source of a substance produced by a nerve cell, and is also a substance produced by a nerve cell by transplanting it into the brain of a patient with intractable neuropathy such as Parkinson's disease or metabolic neurological disease. It is useful as a drug delivery system. Moreover, since the cell of the present invention produces a specific enzyme, it is useful as a cell for treating metabolic diseases such as lysosomal disease.

  Pluripotent stem cells are undifferentiated cells that can be differentiated to form various tissues, and are important in the fields of regenerative medicine and tissue engineering. Conventionally known stem cells include bone marrow stem cells obtained from bone marrow and umbilical cord blood stem cells, but these are difficult to supply stably. It was also announced that a large amount of pluripotent stem cells can be collected from human placenta. However, since the placenta is derived from the mother's body, when transplanting cells differentiated from placenta-derived stem cells, it is necessary to examine the suitability in order to prevent rejection, and for incompatible patients There is a problem that it cannot be transplanted.

  On the other hand, a method using a fluorescent dye called Hoechst33342 has been reported as a method for purifying stem cells (Goodell, M.A. et al., J. Exp. Med., 183: 1797-1806, 1996). Hoechst33342 is a fluorescent dye that is incorporated into the AT base sequence of DNA, has a strong cell membrane permeability, and can stain cells alive. For this reason, Hoechst33342 has been used to study the cell cycle in a living state. Hoechst33342 emits two types of fluorescence having a wavelength of 450 nm and a wavelength of 675 nm when irradiated with ultraviolet rays. Goodell et al. Stained mouse bone marrow cells with Hoechst33342 and applied to a fluorescence activated cell sorter (FACS), taking the fluorescence intensity at a wavelength of 675 nm on the horizontal axis and the fluorescence intensity at a wavelength of 450 nm on the vertical axis. As a result of dimensional analysis, it was found that a cell population exists in the lower left part of the graph, that is, in a region where the fluorescence intensities at both wavelengths are both weak, and this is referred to as side population cells (hereinafter referred to as “SP cells”). ”). Furthermore, it was clarified that hematopoietic stem cells are frequently contained in SP cells. Subsequent studies revealed that SP cells are present in humans other than mice, monkeys, pigs, dogs, zebrafish, etc., and that SP cells are also contained in livers and muscles other than bone marrow. (Experimental Medicine, Vol.19, No.15 (extra number) 2001, pp.68-73).

  SP cells are thought to be less stained by Hoechst33342 due to the high efflux of Hoechst33342. It is thought that Hoechst33342 is excreted out of the cell by the molecule. In stem cells, Hoechst33342 is thought to be excreted extracellularly due to active MDR expression, and it is suggested that the excretion ability of Hoechst33342 is a property common to stem cells (Experimental Medicine, supra) . This SP cell fraction disappears completely after the addition of verapamil, which is a function inhibitor of MDR molecules (117th Annual Meeting of the Japanese Society for Medical Science, pp.67-74 (August 2000)).

  On the other hand, lysosomal diseases in which enzymes in various lysosomes are deficient are known. In lysosome disease, the nervous system such as the brain is easily damaged, causing progressive intelligence decline, growth delay, convulsions, and the like. As a treatment for lysosomal disease, a treatment method for replenishing a deficient enzyme is adopted, but it is necessary to continue replenishment of the enzyme, and there is no fundamental treatment method.

Goodell, M.A. et al., J. Exp. Med., 183: 1797-1806, 1996 Experimental Medicine, Vol.19, No.15 (Special Issue) 2001, pp.68-73

  An object of the present invention is to provide a stem cell that can be stably supplied and does not cause compatibility in transplantation. Another object of the present invention is to provide cells useful for the treatment of metabolic diseases such as lysosomal disease.

  As a result of diligent research, the present inventors have found that SP cells are present in the human amniotic mesenchymal cell layer and the human amniotic epithelial cell layer, and express several stem cell markers, thereby completing the present invention. did. In addition, the present inventors have found that SP cells obtained from a human amniotic mesenchymal cell layer produce a large amount of enzymes such as β-galactosidase and β-glucosidase, and can be transplanted to the brain. It has been found that SP cells are useful as cells for transplantation or cells for treating cerebral metabolic diseases.

  That is, the present invention is isolated from the human amnion mesenchymal cell layer, the expression of Oct-4 gene, Sox-2 gene, FGF-4 gene, Rex-1 gene is observed in RT-PCR, and vimentin in immune cell staining Side population cells that are positive and CK19 positive are provided. The present invention also provides a side population cell isolated from a human amniotic epithelial cell layer. Furthermore, the present invention relates to a side population that is isolated from a human amnion mesenchymal cell layer, is positive for vimentin and positive for CK19, and negative for major histocompatibility antigen class II. A population of erythroid cells. Furthermore, the present invention provides a transplant cell comprising a side population cell isolated from a human amnion mesenchymal cell layer. Furthermore, the present invention provides a cell for treating cerebral metabolic diseases containing a side population cell isolated from a human amnion mesenchymal cell layer as an active ingredient. Furthermore, the present invention provides a cell for treating β-galactosidase or β-glucosidase-deficient disease, which contains a side population cell isolated from a human amnion mesenchymal cell layer as an active ingredient.

  According to the present invention, it was found that SP cells exist in the human amniotic mesenchymal cell layer and the human amniotic epithelial cell layer, and the SP cells were isolated for the first time, and it was confirmed for the first time that they were stem cells. Since the HAMC layer-derived SP cells of the present invention can be differentiated into at least neuronal cells, they are useful as a source of substances produced by the neuronal cells, and are useful for patients with intractable neurological diseases such as Parkinson's disease and metabolic neurological diseases. By transplanting into the brain, it can be used as a drug delivery system for substances produced by nerve cells. Moreover, the HAMC layer-derived SP cells of the present invention that are negative for HLA Class II and negative for HLA Class I are cells that do not cause rejection upon transplantation immunization.

  Moreover, the side population cells isolated from the human amnion mesenchymal cell layer can be transplanted into the brain and produce various lysosomal enzymes as specifically described in the following examples. Accordingly, transplantation of SP cells of the present invention into the brain has made it possible to treat cerebral metabolic diseases such as lysosomal disease.

  The SP cells of the present invention can be obtained from a human amniotic mesenchymal cell layer or human amniotic epithelial cell layer by applying a known SP cell separation method. That is, as specifically described in the following examples, a human amniotic mesenchymal cell layer or a human amniotic epithelial cell layer is collected, separated into cell units, stained with Hoechst33342, subjected to FACS under ultraviolet irradiation. Call. Each cell is plotted with the fluorescence intensity at a wavelength of 675 nm on the horizontal axis and the fluorescence intensity at a wavelength of 450 nm on the vertical axis. In this graph, SP cells are cells present in the lower left portion, that is, in the region where both of the fluorescence intensities are weak, that is, in the region protruding like a corner from the position of the main cell population. FIG. 1 shows the above graph for cells separated from the human amniotic mesenchymal cell (HAMC) layer and the human amniotic epithelial cell (HAEC) layer. The cells in the regions surrounded by the solid solid lines are shown in FIG. SP cells. Such SP cells can be collected by a normal method using a FACS apparatus. In the present specification, the “SP cell” includes not only the entire cell population in the region surrounded by the rectangular solid line in FIG. 1, but also a partial cell population in the entire cell population, Furthermore, it means one cell included in the cell population, and further, a progeny cell population obtained by subculturing these cell populations or one cell, and exhibiting the properties of SP cells. It also means things. However, the term “cell population” is sometimes used particularly when referring to a population of cells in a limited manner.

  The HAMC layer-derived SP cells of the present invention express the Oct-4 gene (POU5 gene) in RT-PCR. The Oct-4 gene is known to be a master gene (differentiation suppressor gene) that works to maintain an undifferentiated state expressed only in ES cells, EC cells, and EG cells, which are undifferentiated cell lines that can be cultured in vitro ( Literature: Nichols, J et al .: Cell, 95, 379-391 (1998)). Furthermore, the HAMC layer-derived SP cells of the present invention express the Sox-2 gene, FGF-4 gene, and Rex-1 gene. These genes are downstream genes of the Oct-4 gene. Sox-2 and Rex-1 are undifferentiated state-maintaining molecular models that are activated in cooperation with FGF-4 and Rox-1 (cofactor), respectively. (Literature name: Niwa, H et al .: Nature Genetics, 24, 372-376 (2000). Therefore, in the HAMC layer-derived SP cells of the present invention, the above genes work to maintain an undifferentiated state. Therefore, the HAMC layer-derived SP cells of the present invention are pluripotent stem cells, and are positive for vimentin, a mesenchymal cell marker antigen, and positive for CK19, an epithelial cell marker antigen, in immune cell staining. It is.

  The SP cells of the present invention that have been separated from the HAMC layer have negative major histocompatibility antigen (HLA) class II and a mixture of negative and positive class I. In order to use for cell transplantation, it is preferable that class I is negative. Therefore, it is preferable to collect and use only class I negative cells. The present invention also provides an SP cell that is included in the HAMC layer-derived SP cell population of the present invention and is negative for HLA-Class I. Class I-negative cells can be easily obtained by collecting cells that were not stained with the HLA-Class I antibody in the immune cell staining described below. This recovery can be performed by a conventional method using commercially available flow cytometry, or can be recovered by negative selection using magnetic beads.

  The SP cells of the present invention can be subcultured by culturing under the following conditions. Culture conditions: 10% fetal bovine serum (FBS) -containing DMEM / F-12 (1: 1) culture medium (human leukemia inhibitory factor (hLIF, manufactured by alomone labo, Israel) 10 ng / ml, and 0.2 mM 2- Mercaptoethanol (2-ME, manufactured by Sigma) was added and cultured in a collagen-coated culture dish at 37 ° C. in a 5% CO 2 incubator. Subsequent cells also have the ability to proliferate as stem cells because they maintain the properties of SP cells and the expression of the markers described above.

  Since the HAMC layer-derived SP cells of the present invention express at least a neural stem cell marker, they can be used as at least neural stem cells. That is, the cells of the present invention are useful at least as a source of substances produced by nerve cells, and are transplanted into the brains of patients with intractable neurological diseases such as Parkinson's disease and metabolic neurological diseases. It is useful as a drug delivery system for substances to be produced.

  Furthermore, SP cells isolated from the human amnion mesenchymal cell layer are considered to be a cell population in which a plurality of types of cells are mixed, and these include expression of the above genes, immunostaining, and expression of tumor tissue compatible antigens. Therefore, it can be transplanted as it is without further selection, and since it produces an enzyme in lysosome, it is useful as a cell for treating metabolic diseases such as lysosomal disease. Accordingly, the present invention also provides a side population cell population that is isolated from a human amnion mesenchymal cell layer and has a negative major histocompatibility antigen class II and a mixture of negative and positive class I. This SP cell population can also be cultured under the same conditions as the SP cells of the present invention.

  As specifically described in the Examples below, it was confirmed that the side population cells isolated from the human amnion mesenchymal cell layer can be transplanted into the brain. Accordingly, the present invention also provides a cell for transplantation consisting of side population cells isolated from a human amnion mesenchymal cell layer. In addition, as specifically described in the Examples below, SP cells isolated from a human amnion mesenchymal cell layer produce a large amount of lysosomal enzymes such as β-galactosidase and β-glucosidase. Lysosome disease is common in the brain and is one of the typical cerebral metabolic diseases. Therefore, the present invention also provides a cell for treating cerebral metabolic diseases containing, as an active ingredient, a side population cell isolated from a human amnion mesenchymal cell layer. In addition, SP cells isolated from human amnion mesenchymal cell layers produce β-galactosidase and β-glucosidase particularly in comparison to other cells, so that among other metabolic diseases, especially for β-galactosidase or β-glucosidase deficient diseases. It can be used as a therapeutic cell.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Example 1 Separation of SP cells Isolation and primary culture of amniotic cells
(1) After performing informed consent, the amniotic epithelial cell layer and mesenchymal cell layer were separated from the villi layer and separated from the placenta of the scheduled cesarean delivery.
(2) Treated with 0.25% trypsin solution / 1.3 mM EDTA at 37 ° C. for 15 minutes. This was repeated 4 times, and the trypsin solution fraction was centrifuged to collect the cells, which were then washed 3 times with phosphate buffer (PBS) (HAEC).
(3) After washing the undigested cell fraction with phosphate buffer, shake with mixed enzyme (0.01% papain, 1 mg / ml collagenase, 0.01% DNase, 0.1% neutral protease) at 37 ° C for 1 hour. It was.
(4) Centrifugation was performed at 2000 rpm for 10 minutes, and the sediment was washed three times with PBS, and passed through a 40 μm filter to obtain a mixed enzyme-treated fraction (HAMC).
(5) Both fractions (HAEC and HAMC) in 10% fetal bovine serum (FBS) -containing DMEM / F-12 (1: 1) culture solution (human leukemia inhibitory factor (hLIF, manufactured by alomone labo, Israel)) Primary culturing was performed at 37 ° C. in a 5% CO 2 incubator on a collagen-coated culture dish with 10 ng / ml and 0.2 mM 2-mercaptoethanol (2-ME, manufactured by Sigma).

2. SP cell detection
(1) The primary cultured cells were treated with 0.125% trypsin solution / 1.3 mM EDTA at 37 ° C. for 10 minutes, centrifuged at 2000 rpm for 5 minutes, and the sediment was washed twice with PBS.
(2) Suspended in HBSS + (Hanks Balanced Salt Solution, Gibco 2% FCS, 10 mM HEPES buffer, Gibco), and nucleated cells were counted.
(3) The cells are centrifuged and suspended in DMEM + (DMEM, Gibco 2% FCS, 10 mM HEPES buffer, Gibco) at a concentration of 1 × 10 6 cells / mL. Mixed.
(4) Hoechst 33342 dye final concentration of 5 μg / mL was added.
(5) The cells were mixed and incubated in a 37 ° C. water bath for 120 minutes.
(6) After incubation, the mixture was centrifuged while passing through a 70 μm filter, and resuspended in cold HBSS + (2% FCS, 10 mM HEPES buffer, Gibco manufactured by Hanks Balanced Salt Solution, Gibco).
(7) The cells were kept at 4 ° C., and PI (propidium iodide) was added to a final concentration of 2 μg / mL.
(8) Hoechst33342 dye (final concentration 5 μg / mL) and 50 μM Verapamil were added to the cells prepared in (1) to (3), and operations (4) to (5) were performed to obtain a target sample.
(9) The Hoechst 33342 dye was excited with a UV laser of 350 nm using EPICS ALTRA HyPerSort (Beckman Coulter, Inc.), and Hoechst Blue / 350 nm and Hoechst Red / 675 nm were detected.

3. SP cell isolation
(1) 10,000 SP region cells were sorted and separated.
(2) The isolated cells were 10 ng / ml of human leukemia inhibitory factor (hLIF, manufactured by alomone labo, Israel) in a DMEM / F-12 (1: 1) culture medium containing 10% fetal bovine serum (FBS), and 0.2 mM 2-mercaptoethanol (2-ME, manufactured by Sigma) was added and cultured on a collagen-coated culture dish at 37 ° C. in a 5% CO 2 incubator.
(3) Cultured cells were seeded at an average of 1.2 × 10 5 to 2.8 × 10 5 cells / cm 2 and subcultured every 3 or 4 days.

  The result of FACS is shown in FIG. As described above, the part surrounded by the rectangular solid line is the SP cell region. About 0.6% of HAMC and about 1.5% of HAEC were SP cells. This SP cell fraction disappeared completely when verapamil, which is a function inhibitor of MDR molecules, was added (indicated as “+ Ver” in FIG. 1). Therefore, it was confirmed that the cell population contained in both SP areas was SP cells.

Example 2 Gene expression analysis by RT-PCR
(1) Total RNA was extracted from 10 passage culture cells using High Pure RNA Isolation Kit (Roche).
(2) cDNA synthesis was performed from the above total RNA using M-MuLV Reverse Transscriptase (Roche).

5 x Incubation buffer 4 μl
10 mM dNTP mix. 2 μl
0.1 M DTT 2 μl
Random primer 1 μl
(or Oligo dT (18) primer) 1 μl
RNase inhibitor 0.5μl
DEPC treated water 5 μl
Reverse Transcriptase 0.5μl
RNA 5 μl
Total 20 μl

The PCR reaction was performed under the following conditions.

10x reaction buffer 5 μl
2.5 mM dNTP mix.5 μl
50 μM forward primer 1 μl
50 μM reverse primer 1 μl
Distilled water 32.5μl
Taq DNA polymerase 0.5μl
cDNA 5 μl
Total 50 μl

(Primer)
OCT-4 (annealing temperature: 62 ℃)
5'-ctt gct gca gaa gtg ggt gga gga a-3 '
5'-ctg cag tgt ggg ttt cgg gca-3 '

Nestin (annealing temperature: 58 ℃)
5'-gag agg gag gac aaa gtc cc-3 '
5'-tcc ctc aga gac tag cgc at-3 '

Musashi-1 (annealing temperature: 60 ° C)
5'-gaa tgg acg cct tca tgc tg-3 '
5'-cgc tga tgt aac tgc tga cc-3 '

Sox-2 (annealing temperature: 60 ℃)
5'-ccc ccg gcg gca ata gca-3 '
5'-tcg gcg ccg ggg aga tac at-3 '

FGF-4 (annealing temperature: 55 ° C)
5'-cta caa cgc cta cga gtc cta ca-3 '
5'-gtt gca cca gaa aag tca gag ttg-3 '

Rex-1 (annealing temperature: 56 ℃)
5'-gcg tac gca aat taa agt cca ga-3 '
5'-cag cat cct aaa cag ctc gca gaa t-3 '

  As a result of RT-PCR, the SP cells derived from the HAMC layer were Oct-4 positive, nestin positive, Sox-2 positive, FGF-4 positive, Rex-1 positive (Musashi-1 was the sixth passage cell) Is negative and positive in the 11th passage cell). Oct-4 is a master gene that works to maintain an undifferentiated state expressed only in ES cells, EC cells, and EG cells, which are undifferentiated cell lines that can be cultured in vitro. Therefore, the HAMC layer-derived SP cells of the present invention are pluripotent. It became clear that it was a sex stem cell. Further, since nestin is a neural stem cell marker, it has been revealed that the cells of the present invention are at least neural stem cells, that is, can be differentiated into nerve cells. In addition, it has been reported that among the downstream genes of the Oct-4 gene, Rex-1 is activated in cooperation with Sox-2 and Rox-1 (cofactor). Therefore, it became clear that the above results worked to maintain the undifferentiated state.

Example 3 Immunostaining
(1) The cultured cells were fixed with 4% paraformaldehyde for 1 minute and incubated with the primary antibody for 2 hours at room temperature.
(2) The mixture was incubated for 2 hours with a secondary antibody diluted with 0.3% Triton X100 (trade name).
(3) The immunoblotted cells were observed with an Olympus IX10 fluorescence microscope and analyzed by confocal images using an Olympus Fluoview laser scanning microscope.
(4) Each cell marker includes, as primary antibodies, anti-human nestin polyclonal antibody (provided by Dr. Okano), anti-human Musashi-1 monoclonal antibody (provided by Dr. Okano), CK19 (manufactured by Santa Cruz), vimentin (manufactured by PROGEN) ), CD4 (manufactured by IMMUNOTECH), CD8 (manufactured by IMMUNOTECH), CD13 (manufactured by IMMUNOTECH), CD29 (manufactured by IMMUNOTECH), CD34 (manufactured by IMMUNOTECH), CD38 (manufactured by IMMUNOTECH) , CD43 (IMMUNOTECH), CD44 (IMMUNOTECH), CD45 (IMMUNOTECH), CD49b (IMMUNOTECH), CD50 (IMMUNOTECH), CD56 (IMMUNOTECH), Thy-1 (IMMUNOTECH) ), CD106 (made by IMMUNOTECH), c-kit (made by IMMUNOTECH), HLA-DR (made by Ancell), HLA Class I (made by company), Flt-1 (made by SANT CRUZ), AFP (DAKO) As a secondary antibody, anti-rabbit IgG rhodamine (1: 100 Chemicon) and anti-rabbit IgG FITC (ZYMED) were used in accordance with a conventional method.

  As a result, the HAMC layer-derived SP cells of the present invention are vimentin positive, CK19 positive, CD29, CD44, Flt-1, and nestin weak positive. HLA Class II was negative, but HLA Class I positive cells and negative cells were mixed, and the others were negative.

Example 4 Intracerebral transplantation of SP cells Informed consent was performed, the amniotic membrane was detached from the placental tissue provided, and the SP cell population was separated in the same manner as in Example 1. The resulting SP cell population was stained and labeled with PKH26 dye. Specifically, this was performed as follows. That is, 500 μl of buffer solution was added to PKH26 dye and left at room temperature for 5 minutes. Thereafter, 500 μl of serum was added, centrifuged, and washed with phosphate buffer. Subsequently, about 5 × 10 ⁴ to 2 × 10 ⁵ cells after staining were transplanted into the brain (hippocampus) of a rat (Wistar Rat 8W male). Specifically, this was performed as follows. Rats were anesthetized by Nembutal injection, a small hole was formed in the skull on one side, and cells were injected into the brain. One week after cell transplantation, rats were anesthetized with ether. After recirculation fixation with 4% paraformaldehyde, the brain was taken out, the brain was fixed in tissue, and a sliced specimen was prepared. Sliced specimens were observed with a fluorescence microscope.

Results SP cells transplanted into the rat brain migrated along the corpus callosum and observed extensive migration to the opposite brain. Furthermore, some of the SP cells engrafted in the dentate gyrus had changed from an original round shape to a neuron-like morphology that extended the process to the surrounding tissue. Thus, since the transplanted SP cells engraft in the brain and change its form like a nerve cell, it was confirmed that the SP cells of the present invention can be transplanted into the brain.

Example 5 Production of enzyme in lysosome by SP cells Informed consent was performed, the amniotic membrane was detached from the provided placental tissue, and the SP cell population was separated in the same manner as in Example 1. Production of 17 types of enzymes shown in Table 1 below by the obtained SP cells was examined as follows.

(1) Preparation of enzyme sample Purified water was added to a cryopreserved cultured cell pellet to a protein concentration of 0.5 to 1 mg / mL, and sonicated with an ultrasonic homogenizer (Branson model 250) to obtain an enzyme sample.

(2) Protein concentration measurement Total protein in the enzyme solution was quantified by the pyrogallol red method (Micro TP Test Wako (Wako Pure Chemical Industries)).

(3) Measurement of enzyme activity (Enzyme 1)
Substrate was added to the enzyme sample and incubated at 37 ° C. for 1 hour, followed by addition of stop solution.
[Enzyme 2]
Substrate was added to the enzyme sample and incubated at 37 ° C for 4 hours, followed by addition of LEBT, further incubation at 37 ° C for 24 hours, and stop solution.
[Enzyme 3]
Substrate was added to the enzyme sample and incubated at 37 ° C for 17 hours, then α-glucosidase was added, further incubated at 37 ° C for 24 hours, and a stop solution was added.
[Enzyme 5]
Substrate was added to the enzyme sample and incubated at 37 ° C for 17 hours, followed by addition of stop solution.
[Enzyme 6]
Substrate was added to the enzyme sample and incubated at 37 ° C for 17 hours, then β-glucosidase was added, further incubated at 37 ° C for 24 hours, and a stop solution was added.
For enzymes 1 to 3, 5, and 6, after adding a stop solution, fluorescence intensity at EX: 365 nm and EM: 450 nm was measured with a plate reader (Molecular Device Spectra max).
[Enzyme 8-16]
Substrate was added to the enzyme sample and incubated at 37 ° C. for 1 hour, followed by addition of stop solution. Thereafter, the fluorescence intensity at EX: 365 nm and EM: 450 nm was measured with a fluorescence analysis system (JASCO).
[Enzyme 7, 17]
Substrate was added to the enzyme sample and incubated at 37 ° C. for 30 minutes before adding stop solution. Enzyme 7 was incubated at 37 ° C. for 30 minutes and 90 minutes, and then a stop solution was added. Thereafter, the absorbance at 515 nm was measured with a spectrophotometer (Hitachi U3300).
[Enzyme 4]
Substrate was added to the enzyme sample and incubated at 37 ° C. for 18 hours, then stop solution was added. Thereafter, the absorbance at 400 nm was measured with a plate reader (Molecular Device Spectra max).
(The numbers after “enzyme” indicate the numbers given to each enzyme shown in Table 1)

  The results are shown in Table 2 below. As shown in Table 2, seven types of enzymes (iduronate-2-sulfatase, heparan-N-sulfatase, β-glucosidase, β-galactosidase, α-fucosidase, and allylsulfatase A) are used for enzyme activity in leukocytes or fibroblasts. Exceeded twice. In particular, β-galactosidase and β-glucosidase were very active, 4.2 times and 25 times, respectively. Therefore, the SP cells of the present invention are useful as cells for treating diseases deficient in these enzymes.

In an Example, it is a figure which shows the result of FACS about the cell isolate | separated from the human amniotic mesenchymal cell (HAMC) layer and the human amniotic epithelial cell (HAEC) layer.

Claims (8)

  Isolated from human amnion mesenchymal cell layer, RT-PCR shows expression of Oct-4 gene, Sox-2 gene, FGF-4 gene, Rex-1 gene, immunocytostaining positive for vimentin and CK19 Side population cells.   The side population cell according to claim 1, wherein major histocompatibility antigen class II is negative and class I is negative.   Side population cells isolated from human amnion epithelial cell layer.   A side population cell population isolated from a human amnion mesenchymal cell layer and containing major histocompatibility antigen class II negative and class I negative and positive mixed.   Transplanted cells consisting of side population cells isolated from human amnion mesenchymal cell layers.   The cell for transplantation according to claim 5, which is for transplantation into the brain.   A therapeutic cell for cerebral metabolic diseases comprising a side population cell isolated from a human amnion mesenchymal cell layer as an active ingredient. A cell for treating β-galactosidase or β-glucosidase-deficient disease, comprising as an active ingredient a side population cell isolated from a human amnion mesenchymal cell layer.

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