USES OF INDUCED NEURAL STEM CELLS DERIVED FROM PERIPHERAL BLOOD MONONUCLEAR CELLS

Abstract

A method of treating neurodegenerative diseases or disorders, especially Parkinson's disease and a method of inducing neural stem cells from peripheral blood mononuclear cells. The induced neural stem cells can express neural stem cell-related genes and differentiate into neurons, astrocytes and oligodendrocytes. The dopaminergic precursors derived from the induced neural stem cells are transplanted into the striatum of the PD mouse models without any sign of tumorigenesis, thereby improving the behaviors of the PD mouse models and slowing down the progression of Parkinson's disease.

Claims

1. A method of treating Parkinson's disease in a subject, comprising: a) inducing neural stem cells (NSCs); b) selecting and differentiating the induced neural stem cells (iNSCs) into dopaminergic precursors; and c) administering the differentiated dopaminergic precursors into striatum of the subject thereby treating the Parkinson's disease.

2. The method of claim 1, wherein step a comprises: i) extracting mononuclear cells from peripheral blood to obtain peripheral blood mononuclear cells (PBMNCs) and expanding the PBMNCs in a medium; ii) transducing the expanded PBMNCs with a Sendai viral vector carrying OCT4, SOX2, c-MYC and KLF-4 genes; iii) seeding the transduced PBMNCs onto a Matrigel-coated plate followed by culturing in a medium until neural stem cell clones appear; and iv) transferring the neural stem cell clones for neural stem cell expansion in which the neural stem cells are subjected to a high-temperature culture to obtain Sendai virus-inactivated neural stem cells.

3. The method of claim 2, wherein step b comprises: 1) culturing the neural stem cells obtained in step iv) in a medium comprising a basic medium consisting of DMEM/F12, 1?N2, 1?B27, 1% of GlutaMAX and 1% of NEAA; SAG1 and FGF8 for 10 days; and optionally, 2) after 10 days of culture in step 1), transferring the neural stem cells to a medium comprising a basic medium consisting of DMEM/F12, 1?N2, 1?B27, 1% of GlutaMAX and 1% of NEAA; BDNF, GDNF, AA, DAPT, cAMP and TGF-? III and then culturing the neural stem cells for two weeks to obtain the dopaminergic precursors.

4. The method of claim 2, wherein in step i), by using Ficoll density gradient centrifugation, an intermediate cloudlike layer comprising CD34+ hematopoietic stem cells and the PBMNCs comprising lymphocytes and monocytes is obtained.

5. The method of claim 2, wherein in step iv), the high-temperature culture is carried out at 38.5-39.5? C. for one week to one month until Sendai virus is inactivated.

6. The method of claim 2, wherein in step i), the medium for expanding the PBMNCs comprises a basic medium consisting of 48.96-48.97% of Iscove's modified Dulbecco's medium (IMDM), 48% of Ham's F-12, 1% of insulin-transferrin-selenium, 1% of chemically defined concentration concentrate, 1% of L-glutamine, 0.05 mg/ml of L-vitamin C, 5 mg/ml of bovine serum albumin (BSA) and 0.018 ?L/mL of thioglycerol; 100 ng/mL of recombinant human stem cell factor; 10 ng/mL of recombinant human interleukin 3; 2 U/mL of erythropoietin; 40 ng/mL of insulin-like growth factor IGF-1; 1 ?M of dexamethasone, and 100 ?g/mL of human transferrin.

7. The method of claim 2, wherein in step iii), the medium for culturing comprises a basic medium consisting of DMEM/F12 and 1?N2, neurobasal and 1?B27, 1% of GlutaMAX and 1% of NEAA; 10 ng/mL of recombinant human leukemia inhibitory factor (rhLIF), CHIR99021 and SB431542.

8. The method of claim 3, wherein the differentiated neural stem cells in step 1) or in step 2) are administered into the striatum of the subject with Parkinson's disease.

9. The method of claim 2, wherein the expanded PBMNCs is predominantly erythroid progenitor cells.

10. A method of treating a neurodegenerative disease or disorder in a subject, comprising: a) inducing neural stem cells (NSCs); b) selecting and differentiating the induced neural stem cells (iNSCs) into a neuronal precursor corresponding to the neurodegenerative disease or disorder; and c) administering the neuronal precursor to the subject thereby treating the neurodegenerative disease; wherein the neurodegenerative disease or disorder is selected from the group consisting of Parkinson's disease, stroke, injury of cranial nerves, spinal cord injury and amyotrophic lateral sclerosis (ALS).

11. The method of claim 10, wherein step a comprises: i) extracting mononuclear cells from peripheral blood to obtain peripheral blood mononuclear cells (PBMNCs) and expanding the PBMNCs in a medium; ii) transducing the expanded PBMNCs with a Sendai viral vector carrying OCT4, SOX2, c-MYC and KLF-4 genes; iii) seeding the transduced PBMNCs onto a Matrigel-coated plate followed by culturing in a medium until neural stem cell clones appear; and iv) transferring the neural stem cell clones for neural stem cell expansion in which the neural stem cells are subjected to a high-temperature culture to obtain Sendai virus-inactivated neural stem cells.

12. The method of claim 11, the induced neural stem cells is capable of expressing neural stem cell-related genes, and differentiating into different kinds of neurons comprising dopaminergic neurons, and different kinds of glial cells.

13. A method of producing dopaminergic precursors, comprising: a) inducing neural stem cells (NSCs) by a method comprising: i) extracting mononuclear cells from peripheral blood to obtain peripheral blood mononuclear cells (PBMNCs) and expanding the PBMNCs in a medium; ii) transducing the expanded PBMNCs with a Sendai viral vector carrying OCT4, SOX2, c-MYC and KLF-4 genes; iii) seeding the transduced PBMNCs onto a Matrigel-coated plate followed by culturing in a medium until neural stem cell clones appear; and iv) transferring the neural stem cell clones for neural stem cell expansion in which the neural stem cells are subjected to a high-temperature culture to obtain Sendai virus-inactivated neural stem cells; and b) selecting and differentiating the induced neural stem cells (iNSCs) into dopaminergic precursors by a method comprising: 1) culturing the neural stem cells obtained in step iv) in a medium comprising a basic medium consisting of DMEM/F12, 1?N2, 1?B27, 1% of GlutaMAX and 1% of NEAA; SAG1 and FGF8 for 10 days; and optionally 2) after 10 days of culture in step 1), transferring the neural stem cells to a medium comprising a basic medium consisting of DMEM/F12, 1?N2, 1?B27, 1% of GlutaMAX and 1% of NEAA; BDNF, GDNF, AA, DAPT, cAMP and TGF-? III and then culturing the neural stem cells for 2 weeks to produce the dopaminergic precursors.

14. The method of claim 13, wherein in step iv), the high-temperature culture is carried out at 38.5-39.5? C. for one week to one month until Sendai virus is inactivated.

15. The method of claim 13, wherein in step i), the medium for expanding the PBMNCs comprises a basic medium consisting of 48.96-48.97% of Iscove's modified Dulbecco's medium (IMDM), 48% of Ham's F-12, 1% of insulin-transferrin-selenium, 1% of chemically defined concentration concentrate, 1% of L-glutamine, 0.05 mg/ml of L-vitamin C, 5 mg/ml of bovine serum albumin (BSA) and 0.018 ?L/mL of thioglycerol; 100 ng/mL of recombinant human stem cell factor; 10 ng/mL of recombinant human interleukin 3; 2 U/mL of erythropoietin; 40 ng/mL of insulin-like growth factor IGF-1; 1 ?M of dexamethasone, and 100 ?g/mL of human transferrin.

16. The method of claim 13, wherein in step iii), the medium for culturing comprises a basic medium consisting of DMEM/F12 and 1?N2, neurobasal and 1?B27, 1% of GlutaMAX and 1% of NEAA; 10 ng/mL of recombinant human leukemia inhibitory factor (rhLIF), CHIR99021 and SB431542.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG. 1 shows an intermediate cloudlike layer after centrifugation of peripheral blood by using Ficoll density gradient centrifugation, where the arrow indicates the cloudlike mononuclear cell layer.

[0058] FIG. 2 shows morphology of peripheral blood mononuclear cells after 14 days of in vitro expansion.

[0059] FIG. 3 shows the emergence of neural stem cell clones after peripheral blood mononuclear cells are expanded in vitro for 2 weeks and then transducted with Sendai viral vectors for 10 days.

[0060] FIG. 4 shows morphology of neural stem cells during adherent culture, which is similar to that of neuroepithelial cells.

[0061] FIG. 5 shows identification of the expression of neural stem cell marker Nestin protein by induced neural stem cells using immunocytochemical staining, where green indicates Nestin protein and blue indicates DAPI cell nucleus.

[0062] FIG. 6 shows identification of the expression of neural stem cell marker Pax6 protein by induced neural stem cells using immunocytochemical staining, where red indicates Pax6 protein and blue indicates DAPI cell nucleus.

[0063] FIG. 7 shows identification of the expression of neural stem cell marker Sox1 protein by induced neural stem cells using immunocytochemical staining, where red indicates Sox1 protein and blue indicates DAPI cell nucleus.

[0064] FIG. 8 shows the induced neural stem cells having a normal male karyotype (i.e., 46XY).

[0065] FIG. 9 shows identification of in vitro differentiation of induced neural stem cells into mature neurons using immunocytochemical staining, where green indicates TUJ1 protein and blue indicates DAPI cell nucleus.

[0066] FIG. 10 shows identification of in vitro differentiation of induced neural stem cells into oligodendrocytes using immunocytochemical staining, where red indicates 04 protein and blue indicates DAPI cell nucleus.

[0067] FIG. 11 shows identification results using immunohistochemical staining in which the induced neural stem cells in immunodeficient mice is capable of differentiation without tumorigenicity, where green indicates Nuclei protein; red indicates NeuN protein; and blue indicates DAPI cell nucleus.

[0068] FIG. 12 shows identification of in vitro differentiation of neural stem cells into dopaminergic neurons using immunohistochemical staining, where red indicates TH protein and blue indicates DAPI cell nucleus.

[0069] FIG. 13 is an HPLC curve for dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) secreted by dopaminergic neurons.

[0070] FIG. 14A is a flow chart showing PD immunodeficient mice modeling, cell transplantation and behavioral test. FIG. 14B shows the behavioral test results of PD mice, ***p<0.001.

[0071] FIGS. 15A-B show histological staining results and statistical analysis for PD mouse cell transplantation group. (A): histological staining results for mice after cell transplantation. (B): statistical results of histological staining for the mouse cell transplantation group. It can be seen that the cells can differentiate into mature midbrain dopaminergic neurons, where TH+ cells account for 13.84%; a ratio of FOXA2 to TH is 86.78%; a ratio of NURR1 to TH is 91.72%; and a ratio of GIRK2 to TH is 98.77%.

[0072] FIG. 16 shows expression ratio of different dopaminergic neuron markers on day 10, 18 and 24 of in vitro differentiation of induced neural stem cells. On day 24, the proportion of TH+ dopaminergic neurons reaches 57%, and the proportion of GIRK2+ cells is 28%, where GIRK2 is an A9 region-specific mature dopaminergic neuron marker.

DETAILED DESCRIPTION OF EMBODIMENTS

[0073] The present application will be further illustrated below with reference to the embodiments. It should be understood that these embodiments are merely illustrative of the invention and are not intended to limit the scope thereof.

EXAMPLE 1

Induction of Neural Stem Cells from Peripheral Blood Mononuclear Cells (PBMNCs)

[0074] Step 1: Isolation of PBMNCs

[0075] (1) At room temperature, 6 mL of blood was collected from a peripheral vein of an adult to store in a heparin anticoagulant tube which was mixed upside down 5 times.

[0076] (2) Mononuclear cells were harvested by using Ficoll density gradient centrifugation. The peripheral blood was diluted with PBS in a ratio of 1:2, and stored in a 50 mL centrifuge tube at room temperature. If the volume of the diluted blood was less than 35 mL, PBS supplement was added.

[0077] (3) Another 50 mL centrifuge tube containing 15 mL of Ficoll-Paque Premium was tilted 45 degrees allowing for slow inflow of the diluted blood.

[0078] (4) The centrifuge tube was then centrifuged at 750?g and at 25? C. for 30 min.

[0079] (5) After centrifugation, an upper layer in the centrifuge tube was plasmas, and an intermediate cloudlike layer was mononuclear cells of interest, as shown in FIG. 1.

[0080] (6) The upper layer was pipetted, and the cloudlike layer was transferred to a 50 mL centrifuge tube.

[0081] (7) The cloudlike layer was added with 30 mL of PBS and centrifuged at 350?g and at 4? C. for 10 min, during which the centrifuge was opened for braking.

[0082] (8) The supernatant was pipetted, and the cells were resuspended with 25 mL of PBS followed by centrifugation at 300?g and at 4? C. for 10 min.

[0083] (9) The supernatant was pipetted, and the cells were resuspended with 25 mL of PBS followed by centrifugation at 300?g and at 4? C. for 10 min (that is, step (8) was repeated).

[0084] (10) The supernatant was removed, and the cells were resuspended with 5 mL of PBS for counting.

[0085] Step 2: Expansion of PBMNCs

[0086] (1) On day 14, the PBMNCs obtained by centrifugation were resuspended in a medium at 2?10.sup.6?3?10.sup.6 cell s/mL and incubated at 37? C. and 5% CO.sub.2 for 2 days.

[0087] (2) On day 11, the cells were collected and centrifuged at 200?g. The supernatant was discarded, and the cells were then resuspended in a medium at 1?10.sup.6 cells/mL and incubated in an incubator for 3 days.

[0088] (3) On day 8, the cells were collected and centrifuged at 200?g. The supernatant was discarded, and the cells were then resuspended in a medium at 1?10.sup.6 cells/mL and incubated in an incubator for 4 days.

[0089] (4) On day 4, the cells were collected and centrifuged at 200?g. The supernatant was discarded, and the cells were then resuspended in a medium at 1?10.sup.6 cells/mL and incubated in an incubator for 4 days.

[0090] Step 3: Transduction

[0091] (1) On day 0, the cells were collected, counted and centrifuged. The supernatant was discarded and 2?10.sup.6 PBMNCs were resuspended with 5 mL of PBS. The resuspension was centrifuged at 200?g and room temperature for 5 min and the supernatant was discarded. The cell morphology was shown in FIG. 2.

[0092] (2) The obtained cells were resuspended in 3 mL of a medium containing viruses for transduction.

[0093] (3) The incubation was performed at 37? C. and at 5% CO.sub.2.

[0094] Step 4: Culture of NSCs

[0095] (1) Preparation: 1 mL of Matrigel diluted with a medium in a ratio of 1:100 was placed in a 6-well plate and incubated overnight for future use.

[0096] (2) The PBMNCs was centrifuged 2 days after transduction, and the supernatant was discarded. The cells were resuspended in a medium and seeded into the 6-well plate at (2-4)?10.sup.5/well. The medium was replaced every other day. On day 10 or so, stem cell clones appeared, and the medium was replaced to allow for continuous expansion of the cells.

[0097] (3) On day 20 to day 30, the clones expanded and the cells were blown down using a pipette and transferred to a 96-well plate coated with PDL-Laminin for later expansion. As shown in FIG. 4, the induced neural stem cells had a morphology similar to that of the neural stem cells under light microscope.

[0098] (4) After one month of passage, the iNSCs were incubated at 39? C. for a week. Then PCR assay was then used to identify the iNSCs.

EXAMPLE 2

Expression of Neural Stem Cell Marker Protein by the Induced Neural Stem Cells

[0099] 5?10.sup.4 iNSCs were plated onto a 12 mm slide coated with poly-D-lysine and laminin, and stained after cultured in a medium for 48 h. The specific steps were described as follows.

[0100] (1) The medium was pipetted, and the cells were washed twice with PBS and added with 4% paraformaldehyde for immobilization for 10 minutes.

[0101] (2) The paraformaldehyde was removed and the cells were added with 1 mL of 0.3% PBST, which is repeated twice at an interval of 5 minutes.

[0102] (3) The cells were blocked by 3% donkey serum at room temperature for 1 h. After that, the cells were added with primary antibodies (prepared by adding antibodies such as Nestin (in a dilution of 1:500, mouse, BD bioscience) and Sox1 (in a dilution of 1:200, goat, BD bioscience) to 1% donkey serum in a certain ratio) and incubated at 4? C. overnight.

[0103] (4) The primary antibodies were pipetted, and the cells were added with corresponding secondary antibodies and placed at room temperature in the dark for 2 h. FITC-conjugated donkey anti-mouse corresponding to Nestin was prepared in a dilution of 1:200, and Cy3-conjugated donkey anti-goat corresponding to Sox1 and Sox2 was prepared in a dilution of 1:400.

[0104] (5) The secondary antibodies were pipetted, and the cells were washed with PBS three times and then added with DAPI (Sigma-Aldrich) prepared in a dilution of 1:1000 for incubation for 10 minutes.

[0105] (6) The slide was mounted and then photographed by laser scanning confocal microscope. As shown in FIGS. 5, 6 and 7, the stem cells were found to express Nestin, Sox1, and Sox2 proteins. The iNSCs in the proliferative phase were lysed for karyotype analysis, and the results showed that the iNSCs had a normal karyotype (shown in FIG. 8).

EXAMPLE 3

Differentiation of Neural Stem Cells into Mature Neurons

[0106] 2?10.sup.4 iNSCs were plated onto a 12 mm slide coated with poly-D-lysine and laminin, and cultured in a medium for 24 h. After that, the medium was replaced with a medium for neuronal differentiation comprising DMEM: F12, 1% N2, 1% B27, 1% glutamine and 1% non-essential amino acid (NEAA, Life Technologies). The medium was replaced every other day, and the cells were immobilized after 6 weeks for immunocytochemical staining of MAP2 (in a dilution of 1:200, mouse, Sigma) and Neun (in a dilution of 1:400, rabbit, Millpore). The cells expressed mature neuronal proteins Map 2 and Neun, as shown in FIG. 9.

EXAMPLE 4

Differentiation of Neural Stem Cells into Dopaminergic Neurons

[0107] 2?10.sup.4 iNSCs were plated onto a 12 mm slide coated with poly-D-lysine and laminin, and cultured in a medium for 24 h. After that, the medium was replaced with a basal medium for neuronal differentiation comprising DMEM: F12, 1% N2, 1% B27, 1% glutamine and 1% non-essential amino acid (NEAA). At the first stage, the basal medium was supplemented with chemical small molecules: 1 ?M SAG1 (Enzo) and 100 ng/m1 FGF8 (PeproTech). After 10 days of culture, the medium was replaced with a medium (prepared by adding BDNF, GDNF, AA, DAPT, cAMP and TGF?III to the basal medium) for second stage. Then cellular immunochemical staining of TH (in a dilution of 1:500, Sheep, Mllpore) in the primary antibodies was performed after 2 weeks of culture. As shown in FIG. 10, the iNSCs were able to differentiate into TH+ dopaminergic neurons, where 42.51% of the TH+ cells also expressed A9 region-specific dopaminergic neuron marker GIRK2, indicating that the midbrain substantia nigra-specific dopaminergic neurons may be obtained in vitro in a fast and efficient manner. Statistical results of positive cells at different time points during differentiation were shown in FIG. 16. It can be seen that the method of the application can efficiently induce neural stem cells to differentiate into TH+ and midbrain substantia nigra-specific dopaminergic neurons. Determination of physiological secretion of dopaminergic neurons by HPLC was shown in FIG. 13.

EXAMPLE 5

Differentiation of Neural Stem Cells into Oligodendrocytes

[0108] 2?10.sup.4 iNSCs were plated onto a 12 mm slide coated with poly-D-lysine and laminin, and cultured in a medium for 24 h. After that, the medium was replaced with a basal medium for neuronal differentiation to which the following small molecules were added, including 1 ?M trans-retinoic acid RA (Sigma-Aldrich), 20 ng PDGF-AB (PeproTech), 10 ng/mL bFGF (PeproTech) and SAG1 (Enzo). Two weeks later, the medium was replaced with another basal medium for neuronal differentiation to which the following small molecules were added, including 20 ng PDGF-AB (PeproTech), SAG1 (Enzo), 60 ng/mL thyroxine T3 (Sigma-Aldrich), 1 mM cyclic adenosine monophosphate (Signa-Aldrich), 10 ng/mL insulin-like growth factor IGF-1 (PeproTech) and 10 ng/mL neurotrophic factor 3 NT3 (PeproTech). The cells were then subjected to cytochemical staining of O1 (in a dilution of 1:300, mouse, eBioscience) as the primary antibody. As shown in FIG. 11, the iNSCs differentiate into oligodendrocytes with O1 as a marker protein, scale bars: 50 ?m.

EXAMPLE 6

In Vivo Differentiation of Neural Stem Cells without Tumorigenesis

[0109] Two neural stem cell lines derived from the same parental PBMNCs were selected for whole-genome sequencing to analyze the mutation between two daughter cells and parental cells by comparison. No mutation of the oncogene (that is, no tumorigenic risk) was found in the two daughter iNSCs.

[0110] The iNSCs were resuspended in 5% glucose solution at 1?10.sup.5/?L. After the immunodeficient mice were anesthetized, the cell suspension was injected into the unilateral corpus striatum in the mouse by microsyringe under a stereotaxic condition. Two months later, the mice were perfused at general anesthesia, and the brain tissue was frozen-sliced and stained. It can be seen in FIG. 12 that the transplanted iNSCs can differentiate into Tuj-1 positive cells in mice.

EXAMPLE 7

Transplantation of DA Precursors Differentiated from iNSCs into PD Mice with Significant Behavioral Difference

[0111] 5?10.sup.3 iNSCs were plated onto a 6-well plate coated with poly-D-lysine and laminin, and cultured in a medium for 24 h. After that, the medium was replaced with a medium I for neuronal differentiation comprising DMEM: F12, 1% N2, 1% B27, 1% glutamine, 1% non-essential amino acid (NEAA), SAG1 and FGF8. After 10 days of culture, the medium I was replaced with a medium II to which BDNF, GDNF, cAMP, AA (ascorbic acid), TGF-?III and DAPT were supplemented for further 2 weeks of culture. The mice (SCID-beige) were lesioned unilaterally on the right side of the striatum by injecting 6-OHDA to generate PD models. 2?10.sup.5 DA precursors including cells from day 10 and day 13 differentiation at a ratio of 1:7 were transplanted into the lesioned sites and the behavioral tests were performed 2, 4, 6, 8 and 12 weeks respectively after transplantation. Tumor development was not observed in the brain of PD mice in the engrafted group which showed significantly improved behavioral performance that is different from that of the buffer group (***p<0.001). As shown in FIG. 14, a flow chart including SCID-beige PD mouse modeling, cell transplantation and behavior testing was illustrated. The histological staining results indicated the differentiation of iNSCs into mature midbrain dopaminergic neurons, as shown in FIG. 15.

[0112] Unless otherwise specified, reagents used herein are commercially available.

[0113] Obviously, these embodiments are merely illustrative of the invention, and are not intended to limit the scope of the invention. Various forms of variations and modifications that are not described in detail here, may be made by those skilled in the art based on the above description, and obvious variations and modifications derived therefrom should fall within the scope of the invention.