CN116836927A - Method for inducing iPSCs into neural stem cells - Google Patents
Method for inducing iPSCs into neural stem cells Download PDFInfo
- Publication number
- CN116836927A CN116836927A CN202311126745.8A CN202311126745A CN116836927A CN 116836927 A CN116836927 A CN 116836927A CN 202311126745 A CN202311126745 A CN 202311126745A CN 116836927 A CN116836927 A CN 116836927A
- Authority
- CN
- China
- Prior art keywords
- induction
- neural stem
- stem cells
- ipscs
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000001178 neural stem cell Anatomy 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 28
- 230000001939 inductive effect Effects 0.000 title claims abstract description 16
- 230000006698 induction Effects 0.000 claims abstract description 70
- 210000004027 cell Anatomy 0.000 claims abstract description 49
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 20
- 102000045246 noggin Human genes 0.000 claims description 10
- 108700007229 noggin Proteins 0.000 claims description 10
- AQGNHMOJWBZFQQ-UHFFFAOYSA-N CT 99021 Chemical compound CC1=CNC(C=2C(=NC(NCCNC=3N=CC(=CC=3)C#N)=NC=2)C=2C(=CC(Cl)=CC=2)Cl)=N1 AQGNHMOJWBZFQQ-UHFFFAOYSA-N 0.000 claims description 7
- 239000002609 medium Substances 0.000 abstract description 40
- 239000001963 growth medium Substances 0.000 abstract description 10
- 238000012258 culturing Methods 0.000 abstract description 7
- 230000004069 differentiation Effects 0.000 abstract description 5
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 30
- 101150092640 HES1 gene Proteins 0.000 description 9
- 101000687905 Homo sapiens Transcription factor SOX-2 Proteins 0.000 description 9
- 102000008730 Nestin Human genes 0.000 description 9
- 108010088225 Nestin Proteins 0.000 description 9
- 102100024270 Transcription factor SOX-2 Human genes 0.000 description 9
- 210000005055 nestin Anatomy 0.000 description 9
- 239000011248 coating agent Substances 0.000 description 7
- 238000000576 coating method Methods 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 7
- 239000007788 liquid Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000000872 buffer Substances 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000012744 immunostaining Methods 0.000 description 4
- 208000014674 injury Diseases 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 229920002714 polyornithine Polymers 0.000 description 3
- 108010055896 polyornithine Proteins 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 208000024827 Alzheimer disease Diseases 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 description 2
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 208000018737 Parkinson disease Diseases 0.000 description 2
- 206010039966 Senile dementia Diseases 0.000 description 2
- LZAXPYOBKSJSEX-UHFFFAOYSA-N blebbistatin Chemical compound C1CC2(O)C(=O)C3=CC(C)=CC=C3N=C2N1C1=CC=CC=C1 LZAXPYOBKSJSEX-UHFFFAOYSA-N 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 208000010877 cognitive disease Diseases 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 210000004498 neuroglial cell Anatomy 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 210000000278 spinal cord Anatomy 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 101000803709 Homo sapiens Vitronectin Proteins 0.000 description 1
- 208000020358 Learning disease Diseases 0.000 description 1
- 208000026139 Memory disease Diseases 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 238000010806 PrimeScriptTM RT Reagent kit Methods 0.000 description 1
- 206010071390 Resting tremor Diseases 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 102100035140 Vitronectin Human genes 0.000 description 1
- 108010031318 Vitronectin Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000003920 cognitive function Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000408 embryogenic effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000002518 glial effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 201000003723 learning disability Diseases 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000008450 motivation Effects 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000033667 organ regeneration Effects 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
- 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
-
- 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
- C12N2501/999—Small molecules not provided for elsewhere
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Neurology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Neurosurgery (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for inducing iPSCs into neural stem cells, which comprises the steps of inducing and culturing the digested iPSCs in an induction medium I for 2 days to form initial embryoid cells, then inducing and culturing the initial embryoid cells in an induction medium II for 2 days to form later embryoid cells, and finally inducing and culturing the initial embryoid cells in an induction medium III for 3 days to form the neural stem cells; the invention adopts a specific induction culture medium to induce the iPSCs into the neural stem cells, the induction time is short, and the induction time is only 7 days; the method for inducing the iPSCs into the neural stem cells has high induction rate and uniform induction and differentiation.
Description
Technical Field
The invention relates to a method for inducing iPSCs into neural stem cells, belonging to the technical field of genetic engineering.
Background
Senile dementia and parkinson's disease are two of the most common chronic progressive neurodegenerative diseases, which affect more than 1% of the population over 65 years old. The main symptoms of senile dementia include gradually serious cognitive disorder (memory disorder, learning disorder, attention disorder, spatial cognitive function, disorder of problem solving ability) and gradually inability to adapt to society. The main symptoms of parkinson's disease include resting tremor, stiffness, slow motion, and unstable posture. Late stage symptoms also include signs of not being conscious, sensory, mental and cognitive disturbances.
Currently, the field of regenerative medicine is striving to restore the structure and function of damaged tissues and organs, finding a way to treat full or partial injuries and diseases due to disease, trauma and aging. Whereas for any regeneration strategy, stem cells are considered to be an important vital control center. It is a challenge and motivation to excavate a pluripotent stem cell with tissue and organ regeneration capability and transport such stem cell to the site of injury.
The discovery of Induced Pluripotent Stem Cells (iPSCs) and their related culture protocols both eliminates ethical issues and the possibility of being autologous therapy significantly reduces the risk of immune rejection. CN116083362a adopts adipose-derived stem cells to induce and differentiate into neural stem cells, the induction time is 12 days, and the number of added factors is up to more than 10. CN115125210a is the induction of iPSCs into lumbosacral spinal cord neural stem cells, mainly against injuries at specific sites of the spinal cord.
In recent years, the application of iPSCs technology to induce neural stem cells becomes a research hot spot, and the most commonly used N2/B27 culture medium is used to induce the neural stem cells at present, but the induction culture time is longer, the induction rate is low, the differentiation is nonuniform, and meanwhile, the culture medium is provided with a plurality of small molecular compounds.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a method for inducing iPSCs into neural stem cells, which realizes the following aims: short induction time, high induction rate and uniform induction and differentiation.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for inducing iPSCs into neural stem cells, wherein the method comprises the steps of inducing and culturing the digested iPSCs in an induction medium I for 2 days to form initial embryoid cells, then inducing and culturing the initial embryoid cells in an induction medium II for 2 days to form later embryoid cells, and finally inducing and culturing the initial embryoid cells in an induction medium III for 3 days to form the neural stem cells;
the induction medium I is a Neurobasal medium added with 0.5 XN 2, 0.5 XB 27, 10 mu M of Y-27632, 10 mu M of SB431542, 100ng/mL of Noggin, 2mM of L-Glu and 1% of Vol of double antibody;
the induction medium II is a Neurobasal medium added with 1 XN 2, 1 XB 27, 10 mu M Y-27632, 10 mu M SB431542, 100ng/mL Noggin, 2mM L-Glu, 1% Vol double antibody, 1 mu M CHIR99021 and 50ng/mL SHH;
the induction medium III is a Neurobasal medium added with 0.5XN2, 0.5XB27, 10 mu M Y-27632, 10 mu M SB431542, 100ng/mL Noggin, 2mM L-Glu, 1% Vol double antibody, 1 mu M CHIR99021 and 50ng/mL SHH.
The initial cell density of the iPSCs in the induction medium I is 1 multiplied by 10 6 Individual cells/mL.
The late embryoid cells and the induction medium III are subjected to induction culture in double-coated T75 culture flasks.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention adopts a specific induction culture medium to induce the iPSCs into the neural stem cells, the induction time is short, and the induction time is only 7 days;
(2) The method for inducing the iPSCs into the neural stem cells has high induction rate and uniform induction and differentiation, and three markers SOX2, nestin and Hes1 of the neural stem cells induced by the method are all dyed, wherein the positive cell proportion of Nestin is 98.32%, the positive cell proportion of SOX2 is 95.68%, and the positive cell proportion of Hes1 is 97.35%.
Drawings
FIG. 1 is a microscopic view of iPSCs of the present invention;
FIG. 2 is a microscopic view of the neural stem cells induced by the present invention;
FIG. 3 is an immunostaining fluorescence plot of the identification of neural stem cell markers induced by the present invention and comparative examples;
wherein FIG. 3A is an immunostained fluorescent image of the identification of the neural stem cell markers induced by the present invention; FIG. 3B is an immunostaining fluorescence plot of comparative induced neural stem cell marker identification;
FIG. 4 is a bar graph showing the relative expression levels of neural stem cell markers induced by the present invention and the comparative example;
FIG. 5 is an immunostaining fluorescence of glial cells and neuronal markers of example 5.
Detailed Description
EXAMPLE 1 resuscitating and passaging of iPSCs
iPSCs, vitronectin (human vitronectin), ROCK inhibitor Blebbistatin, EDTA digest, nuwacell bulk ncEpic hPSC Medium were all purchased from the company of the flourishing biotechnology limited in the Anhui.
(1) T75 bottle coating
Thawing the coated protein Vitronin at room temperature, subpackaging into 120 mu L/tube, adding 9mL of DMEM/F12 medium into 120 mu L of Vitronin, gently mixing, diluting, adding into a T75 bottle, gently shaking, and mixing to obtain a T75 bottle coated with Vitronin coating solution. After standing at room temperature for 2 hours, the coating liquid is sucked up by a pipette when the coating liquid is used.
(2) Cell resuscitation
Preheating the water bath to 40 ℃, taking out 1 piece of frozen iPSCs (1 mL), placing the iPSCs into the water bath at 40 ℃, slightly shaking the iPSCs by hand, thawing the iPSCs within 1 min, and taking out the iPSCs after ice crystals in the cell suspension are about to disappear completely by naked eyes to obtain the cell suspension. Wiping the surface of the freezing and storing tube with 75% alcohol dust-free paper, and transferring into an ultra-clean bench; the cell suspension was transferred to a 50mL centrifuge tube prepared in advance, and then 10mL of DMEM/F12 medium was added dropwise, the cells were gently mixed during the process, and the cells were centrifuged at 850rpm for 5 min. The supernatant was aspirated, 10mL of ncEpic complete medium (pre-warmed to room temperature) containing Blebbistatin (final concentration 2.5 μm) was added, the cells were mixed, blowing was avoided as much as possible, and a cell culture solution was obtained.
Absorbing and discarding the T75 bottle coated in the step (1) and the Vitronidin coating liquid, inoculating the uniformly mixed cell culture liquid into the T75 bottle according to 10 mL/bottle, shaking horizontally for three times, and placing at 37 ℃ and 5% CO 2 The culture was carried out in a concentration incubator for 24 hours, and then replaced with new ncEpic complete medium (10 mL was added per bottle), followed by daily replacement of ncEpic complete medium.
(3) Passage of cells
When the cell confluency reached about 85% (as in FIG. 1), passaging was performed according to 1:5 passaging.
Cells passed to passage 2 were used for the following experiments when cell confluency was 80% or more.
Example 2 Induction of iPSCs into neural Stem cells
(1) Taking out iPSCs in incubator, digesting with EDTA for 7min, observing cell digestion under microscope, sucking out digestive juice after most cells become round to obtain digested iPSCs, blowing off cells with induction medium, inoculating into T75 bottle, and inoculating with cell density of 1×10 6 The amount of the first induction medium added per bottle was 10mL per cell/mL, and was designated day0.
(2) The resulting primary embryogenic cells were collected at day2, centrifuged at 100g for 5min, resuspended in 10mL induction medium and inoculated into a new T75 flask.
(3) The resulting post-embryonic somatic cells were collected at day4, 100g, centrifuged for 5min, resuspended in 10mL induction medium three and inoculated into double-coated T75 flasks.
(4) Day7 collection of induced neural stem cells, which together were 7.5X10 6 Individual cells/flasks (fig. 2).
The preparation method of the double-coated T75 culture flask comprises the following steps:
(1) Preparing polyornithine at room temperature to obtain 15mg/mL mother liquor, adding 10mL of 10 mu L of mother liquor into 10mL of PBS (purchased from Soy pal), gently mixing and diluting, adding into a T75 bottle, gently shaking and mixing to obtain a T75 bottle coated with polyornithine, and placing into a 37 ℃ incubator.
(2) Standing for 2 days, removing polyornithine coating liquid in T75 bottle, cleaning with PBS, adding 10mL PBS containing laminin with final concentration of 5 μg/mL into each bottle, performing secondary coating, and standing in a 37 ℃ incubator for use.
(3) After further 2 days of standing, the coated T75 bottle was washed with PBS and then dried for use.
Wherein the first induction culture medium is: neurobasal medium of double antibody of 0.5XN2 (Vol), 0.5XB27 (Vol), 10. Mu.M Y-27632, 10. Mu.M SB431542, 100ng/mL Noggin, 2mM L-Glu, 1% Vol was added.
The second induction culture medium is: 1 XN 2 (Vol), 1 XB 27 (Vol), 10. Mu.M Y-27632, 10. Mu.M SB431542, 100ng/mL Noggin, 2mM L-Glu, 1% Vol diabody, 1. Mu.M CHIR99021, 50ng/mL SHH Neurobasal medium were added.
The induction medium III is as follows: 0.5 XN 2 (Vol), 0.5 XB 27 (Vol), 10. Mu.M Y-27632, 10. Mu.M SB431542, 100ng/mL Noggin, 2mM L-Glu, 1% Vol diabody, 1. Mu.M CHIR99021, 50ng/mL SHH Neurobasal medium was added.
N2, B27, L-Glu and diabodies were purchased from Gibco, Y-27632, SB431542, noggin, CHIR99021, SHH from MCE.
Comparative example
The induction of neural stem cells was performed using the digested iPSCs of example 2 of the present invention, and simultaneously using the induction medium and induction time of example 1 of CN115125210a, the specific induction method was as follows:
(1) Induction culture with first induction medium on day 1
Blowing the digested iPSCs with a first induction medium, inoculating into a T75 flask, and the cell density after inoculation is 1×10 6 The amount of first induction medium added per bottle was 10mL per cell/mL.
(2) Induction culture with the second induction medium on days 2-3
The first induction medium was removed and cells were resuspended in 10mL of second induction medium per flask and inoculated into new T75 flasks.
(3) Induction culture with third induction medium on day4
The second induction medium was removed and cells were resuspended in 10mL of third induction medium per flask and inoculated into new T75 flasks.
(4) Induction culture with fourth induction medium on day 5-10
Removing the third induction culture medium, re-suspending the cells with 10mL fourth induction culture medium, inoculating into new double-coated T75 culture flask, inducing for 10 days, collecting the induced neural stem cells together with 6×10 6 Individual cells/bottles.
Double-coated T75 flasks were coated in the same manner as in example 2 of the present invention.
Example 3 identification of neural stem cells Using immunofluorescent staining
The immunofluorescence staining method is used for identifying the markers SOX2, nestin and Hes1 of the neural stem cells, the neural stem cells induced by the method of the invention are induced for 7 days, and the neural stem cells induced by the method of CN115125210A are induced for 10 days. Three of these antibodies were purchased from Abcam and were mainly prepared as follows:
(1) Inoculating the cells to be detected on a cell slide (purchased from Jingan organism, specification phi 14 mm), 37 ℃ and 5% CO 2 Culturing in an incubator for 1 day.
(2) The cell slide was removed and washed 3 times with PBS buffer (available from Soy Corp., cat# P1020-500 ml) for 1 minute/time.
(3) 200. Mu.L of 4wt% paraformaldehyde was added and the mixture was fixed for 15min, the paraformaldehyde was sucked off, dried in air for 5min, and washed 3 times with PBS buffer for 2 min/time.
(4) 200. Mu.L of DPBS (available from Soy Corp., product number: D1040-500 ml) containing 0.5vol% Triton X-100 was added, and left at room temperature for 20 minutes; the PBS buffer solution is washed for 2 times and 2 minutes/time, and the action is not easy to be violent in the washing process.
(5) 200. Mu.L of serum blocking solution was added, left at room temperature for 20min, and the excess solution was decanted.
(6) The primary antibodies were diluted in a ratio of 1:20 (volume ratio), added to the slide respectively, placed in a wet box, incubated at 37℃for 1 hour, washed 3 times with PBS buffer, 2 minutes/time.
(7) Adding a secondary antibody, and incubating for 30min at 37 ℃; the PBS buffer was washed 3 times, 2 min/time.
(8) Drying at room temperature for 5min, sealing with neutral resin, and observing under fluorescent microscope.
As shown in FIG. 3A, three markers SOX2, nestin and Hes1 of the neural stem cells induced by the method of the present invention were stained, wherein the positive cell proportion of Nestin was 98.32%, the positive cell proportion of SOX2 was 95.68% and the positive cell proportion of Hes1 was 97.35%. As shown in fig. 3B, the marker expression rate of neural stem cells induced by CN115125210a was lower than that of the present invention, the positive cell proportion of Nestin was 96.98%, the positive cell proportion of SOX2 was 86.78%, and the positive cell proportion of Hes1 was 84.32%; the method has the advantages that the induction rate of the neural stem cells induced by the method is higher, and the differentiation is more uniform.
Example 4 detection of induced neural Stem cells Using RT-QPCR
The RNA of Induced Pluripotent Stem Cells (iPSCs), of the invention iPSCs-induced Neural Stem Cells (NSCs) and of the comparative example-induced Neural Stem Cells (NSCs) was first extracted using RNeasy Plant Mini Kit (from Qiagen, cat# 74904) and the extraction method was as described in the kit instructions. RNA from the three were reverse transcribed to cDNA using a PrimeScript RT reagent Kit with gDNA Eraser kit (available from TaKaRa, cat# RR 047A) and the procedure was as described in the kit. The three cDNAs are used as templates, fluorescent quantitative PCR reagents are purchased from Saenox corporation, and detected genes comprise SOX2, nestin and Hes1, and beta-attin genes are used as reference genes. The quantitative PCR primer sequences are shown in Table 1. Each PCR reaction was repeated 3 times. The procedure for RT-PCR was set as follows: fluorescence was collected during annealing at 94℃for 3min, (94℃for 15s,58℃for 30s,72℃for 30 s). Times.35 cycles.
TABLE 1 fluorescence quantitative Gene and primer sequences
As shown in figure 4, the mRNA expression levels of the neural stem cell markers SOX2, nestin and Hes1 induced by the invention are obviously improved compared with the original iPSCs, and the mRNA expression levels have obvious differences, so that the invention is proved to successfully induce the iPSCs into the neural stem cells. The expression level of Nestin of the neural stem cells induced by the invention is basically consistent with that of the neural stem cells induced by the comparative example, and the expression levels of SOX2 and Hes1 of the neural stem cells induced by the invention are obviously higher than those of the neural stem cells induced by the comparative example, which is probably caused by inconsistent types of final neural stem cells induced by the two.
EXAMPLE 5 identification of the Multi-differentiation of neural Stem cells
The neural stem cells induced in example 2 were inoculated into F12 medium containing 10% Vol of FBS (purchased from Gibco), 20ng/mL of EGF (purchased from MCE), 20ng/mL of bFGF (purchased from MCE) and differentiated for 7 days, and identification of glial markers GFAP and neuronal markers TH was performed. Expression of GFAP and TH was identified using fluorescent immunostaining, both antibodies were purchased from Abcam. As shown in fig. 5, both stained, indicating the potential of neural stem cells to differentiate into glial cells and neurons.
At the time of inoculation, 10mL of culture medium is added into each bottle, and 6×10 culture medium is added into each bottle 6 And (3) neural stem cells.
Claims (3)
1. A method of inducing neural stem cells from iPSCs, comprising: the method comprises the steps of performing induction culture on digested iPSCs in an induction medium I for 2 days to form initial embryoid cells, performing induction culture in an induction medium II for 2 days to form final embryoid cells, and performing induction culture in an induction medium III for 3 days to form neural stem cells;
the induction medium I is a Neurobasal medium added with 0.5 XN 2, 0.5 XB 27, 10 mu M of Y-27632, 10 mu M of SB431542, 100ng/mL of Noggin, 2mM of L-Glu and 1% of Vol of double antibody;
the induction medium II is a Neurobasal medium added with 1 XN 2, 1 XB 27, 10 mu M Y-27632, 10 mu M SB431542, 100ng/mL Noggin, 2mM L-Glu, 1% Vol double antibody, 1 mu M CHIR99021 and 50ng/mL SHH;
the induction medium III is a Neurobasal medium added with 0.5XN2, 0.5XB27, 10 mu M Y-27632, 10 mu M SB431542, 100ng/mL Noggin, 2mM L-Glu, 1% Vol double antibody, 1 mu M CHIR99021 and 50ng/mL SHH.
2. The method of claim 1, wherein the method is performed by iPSCs induced into neural stem cells, and the method comprises the steps of: the initial cell density of the iPSCs in the induction medium I is 1 multiplied by 10 6 Individual cells/mL.
3. The method of claim 1, wherein the method is performed by iPSCs induced into neural stem cells, and the method comprises the steps of: the late embryoid cells and the induction medium III are subjected to induction culture in double-coated T75 culture flasks.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311126745.8A CN116836927B (en) | 2023-09-04 | 2023-09-04 | Method for inducing iPSCs into neural stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311126745.8A CN116836927B (en) | 2023-09-04 | 2023-09-04 | Method for inducing iPSCs into neural stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116836927A true CN116836927A (en) | 2023-10-03 |
| CN116836927B CN116836927B (en) | 2023-12-01 |
Family
ID=88162087
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311126745.8A Active CN116836927B (en) | 2023-09-04 | 2023-09-04 | Method for inducing iPSCs into neural stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116836927B (en) |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050153446A1 (en) * | 2003-11-07 | 2005-07-14 | Steindler Dennis A. | Culturing and differentiating neural precursor cells |
| JP2014236701A (en) * | 2013-06-07 | 2014-12-18 | 大日本印刷株式会社 | Cell culture substrate which promotes differentiation inducing to nerve cell |
| CN109294991A (en) * | 2018-11-07 | 2019-02-01 | 北京赛贝生物技术有限公司 | The method of differentiation of neural stem cells culture medium and differentiation of neural stem cells |
| WO2019023793A1 (en) * | 2017-08-04 | 2019-02-07 | University Health Network | Generation of oligodendrogenic neural progenitor cells |
| CN109385404A (en) * | 2018-10-19 | 2019-02-26 | 国典(北京)医药科技有限公司 | A kind of differentiation of stem cells is the method and neuron and application of neuron |
| CN110760476A (en) * | 2019-09-03 | 2020-02-07 | 广州瑞臻再生医学科技有限公司 | Preparation method of cerebral cortex neural stem cells and glutamatergic neurons |
| CN111440768A (en) * | 2020-04-23 | 2020-07-24 | 青岛海尔生物科技有限公司 | Application of recombinant human Notch1 protein in preparation of neural stem cells and cortical neurons |
| CN112626022A (en) * | 2021-01-12 | 2021-04-09 | 中国人民解放军海军军医大学 | Forebrain neural stem cell in-vitro induction and long-term culture system, induction culture method and application |
| CN115125210A (en) * | 2022-08-31 | 2022-09-30 | 华科星河(北京)生物科技有限公司 | Culture medium and method for lumbosacral segment spinal cord neural stem cells induced from iPSC |
| CN115851597A (en) * | 2022-11-25 | 2023-03-28 | 海南苏生生物科技有限公司 | Method for differentiating induced pluripotent stem cells into neural stem cells and application |
| CN115916240A (en) * | 2020-08-17 | 2023-04-04 | 中国科学院动物研究所 | Amplification culture medium and culture method of neural cells |
| CN115975914A (en) * | 2023-03-14 | 2023-04-18 | 山东兴瑞生物科技有限公司 | Method for inducing pluripotent stem cells by reprogramming of chemical small molecule drugs |
-
2023
- 2023-09-04 CN CN202311126745.8A patent/CN116836927B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050153446A1 (en) * | 2003-11-07 | 2005-07-14 | Steindler Dennis A. | Culturing and differentiating neural precursor cells |
| JP2014236701A (en) * | 2013-06-07 | 2014-12-18 | 大日本印刷株式会社 | Cell culture substrate which promotes differentiation inducing to nerve cell |
| WO2019023793A1 (en) * | 2017-08-04 | 2019-02-07 | University Health Network | Generation of oligodendrogenic neural progenitor cells |
| CN109385404A (en) * | 2018-10-19 | 2019-02-26 | 国典(北京)医药科技有限公司 | A kind of differentiation of stem cells is the method and neuron and application of neuron |
| CN109294991A (en) * | 2018-11-07 | 2019-02-01 | 北京赛贝生物技术有限公司 | The method of differentiation of neural stem cells culture medium and differentiation of neural stem cells |
| CN110760476A (en) * | 2019-09-03 | 2020-02-07 | 广州瑞臻再生医学科技有限公司 | Preparation method of cerebral cortex neural stem cells and glutamatergic neurons |
| CN111440768A (en) * | 2020-04-23 | 2020-07-24 | 青岛海尔生物科技有限公司 | Application of recombinant human Notch1 protein in preparation of neural stem cells and cortical neurons |
| CN115916240A (en) * | 2020-08-17 | 2023-04-04 | 中国科学院动物研究所 | Amplification culture medium and culture method of neural cells |
| CN112626022A (en) * | 2021-01-12 | 2021-04-09 | 中国人民解放军海军军医大学 | Forebrain neural stem cell in-vitro induction and long-term culture system, induction culture method and application |
| CN115125210A (en) * | 2022-08-31 | 2022-09-30 | 华科星河(北京)生物科技有限公司 | Culture medium and method for lumbosacral segment spinal cord neural stem cells induced from iPSC |
| CN115851597A (en) * | 2022-11-25 | 2023-03-28 | 海南苏生生物科技有限公司 | Method for differentiating induced pluripotent stem cells into neural stem cells and application |
| CN115975914A (en) * | 2023-03-14 | 2023-04-18 | 山东兴瑞生物科技有限公司 | Method for inducing pluripotent stem cells by reprogramming of chemical small molecule drugs |
Non-Patent Citations (3)
| Title |
|---|
| RUI BAI等: "Ascorbic acid can promote the generation and expansion of neuroepithelial-like stem cells derived from hiPS/ES cells under chemically defined conditions through promoting collagen synthesis", STEM CELL RESEARCH & THERAPY, vol. 12, pages 48 * |
| 王乐等: "人类诱导多能干细胞体外定向分化为神经干细胞的方法", 郑州大学学报(医学版), vol. 56, no. 5, pages 619 - 622 * |
| 陈丽: ""尾侧化"与"腹侧化"的分化模式对人胚胎干细胞定向诱导为中脑多巴胺能神经元的影响研究", 中国优秀硕士学位论文全文数据库 基础科学辑, no. 2022, pages 006 - 31 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116836927B (en) | 2023-12-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2013127293A1 (en) | Culture medium for preparing neural stem cells and use thereof | |
| CN112626023B (en) | Spinal cord olig2+ nerve progenitor cell induction and self-renewal culture system, induction method and application | |
| CN109674809B (en) | Composition containing miR-124-3P and application thereof in medicine for inducing neuron formation | |
| CN115975914B (en) | Method for inducing pluripotent stem cells by reprogramming chemical small molecule drugs | |
| CN114214279A (en) | Human spinal cord neural stem cell induced differentiation culture system and method | |
| CN112143708A (en) | Umbilical cord mesenchymal stem cells, stem cell essence factor and application of umbilical cord mesenchymal stem cells and stem cell essence factor in aspect of resisting skin aging | |
| CN112795541B (en) | Method for inducing differentiation of neural stem cells into neurons and astrocytes by umbilical cord mesenchymal stem cells | |
| CN113462639B (en) | Method for separating fibroblasts from human induced pluripotent stem cells and application of method | |
| CN116836927B (en) | Method for inducing iPSCs into neural stem cells | |
| CN114250199A (en) | Method and medium for culturing spine-spinal cord organoid | |
| Aleksandrova et al. | Characteristics of human neural stem cells in vitro and after transplantation into rat brain | |
| US11898164B2 (en) | Method for producing pluripotent stem cell-derived spherical neural mass having suppressed teratoma formation | |
| CN112626022B (en) | Forebrain neural stem cell in-vitro induction and long-term culture system, induction culture method and application | |
| CN110396496B (en) | Culture method and application of duck small intestine epithelial cells | |
| CN113528576A (en) | Recurrent grapevine patient specific induced pluripotent stem cell line containing NLRP7 pure and mutant and construction method thereof | |
| CN103215226A (en) | Method for co-culturing with nerve stem cell to inducing rASCs to be DA Neuron by Lmx1a mediation | |
| CN101935635A (en) | In vitro amplification method of human marrow pluripotent stem cells and method for directionally differentiating same into dopaminergic neuron | |
| CN114540282B (en) | Method for obtaining mesenchymal stem cells by induced differentiation of pluripotent stem cells | |
| CN111235110A (en) | In-vitro culture method of neural stem cells | |
| CN111849900A (en) | Intelligent culture method for autologous fat MSCs (mesenchymal stem cells) derived precursor cells | |
| CN112852735A (en) | Method for inducing differentiation of induced pluripotent stem cells into neural precursor cells | |
| CN116536255B (en) | Culture medium and method for efficiently inducing muscle stem cells | |
| CN109182269B (en) | Culture system and method for efficiently differentiating spermatogonial stem cells to nerve cells | |
| CN113717935B (en) | Application of Nigroain-E1 polypeptide in maintaining adipose-derived stem cell (Stem) stem property | |
| CN108085316B (en) | Method for promoting in-vitro amplification of mouse retina precursor cells by inhibiting micro ribonucleic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |