WO2013071469A1 - Procédé, trousse et application pour l'application d'une différentiation inverse à des cellules somatiques humaines pour générer des cellules souches autologues de la rétine et des cellules autologues de la rétine - Google Patents

Procédé, trousse et application pour l'application d'une différentiation inverse à des cellules somatiques humaines pour générer des cellules souches autologues de la rétine et des cellules autologues de la rétine Download PDF

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WO2013071469A1
WO2013071469A1 PCT/CN2011/002065 CN2011002065W WO2013071469A1 WO 2013071469 A1 WO2013071469 A1 WO 2013071469A1 CN 2011002065 W CN2011002065 W CN 2011002065W WO 2013071469 A1 WO2013071469 A1 WO 2013071469A1
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cells
growth factor
autologous
retinal
loong
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PCT/CN2011/002065
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Chinese (zh)
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林雄斌
石海鹏
李贵铃
廖焕昭
任海英
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北京善尔创生生物科技有限公司
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    • 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
    • C12N5/062—Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • 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
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
    • C12N2506/115—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells from monocytes, from macrophages

Definitions

  • the book reverses the differentiation of human somatic cells to produce autologous retinal stem cells
  • the present invention relates to the field of biomedical technology, and in particular, to a cell culture method and application for the reverse differentiation of human somatic cells to produce autologous retinal stem cells and autologous retinal cells. Background technique
  • blind eye diseases such as infectious eye diseases and cataracts have been effectively diagnosed and treated.
  • the incidence and blindness rate of retinal degenerative diseases have increased.
  • One of the major blinding eye diseases worldwide, its current treatment is not only the hotspots and difficulties of ophthalmology but also neuroscience research.
  • iPS stem cells induced autologous pluripotent stem cells
  • Somatic cells are progeny cells that have been produced by the directional differentiation of stem cells and have specific functions.
  • the chromosome DM of the somatic cell and the chromosomal DNA of the stem cell do not differ in the number of gene structures and genes. The most important difference between somatic and stem cells may be that certain functional genes are in different active expression states. Differences in functional gene expression determine differences in specific functions and morphologies of cells. We speculate that after the initiation of the process of stem cell differentiation into somatic cells, the dry cells themselves The cell characteristic determines that the gene switch is turned off, and then the stem cells differentiate to form a somatic cell with a specific function; in other words, the retinal stem is still present in the somatic chromosome DM sequence.
  • the cell characteristic determining gene such as Pax6 , Rx, Mi tf, Chxl O, these genes are only closed or closed. Using certain substances to open the characteristics of the retinal stem cells in the somatic DM sequence determines the genes Pax6, Rx, Mi tf, ChxlO and other switches, these somatic cells may re-differentiate to form new retinal stem cells, and then differentiate into autologous retinal cells.
  • the first generation of autologous retinal stem cells can be produced in a few weeks, which not only provides a new treatment for various ophthalmic diseases.
  • the unique pluripotent stem cells provide a good basis for the industrial development of retinal tissue engineering and autologous retinal stem cell production.
  • the technical problem to be solved by the present invention is to develop a new human somatic cell reverse differentiation regulation technology, without changing any human somatic chromosome DM sequence, without inserting any foreign gene or DM film: segment, the application contains A series of formulations of certain plant extracts and proteins that reactivate the retinal stem cell characteristics in somatic DM sequences in vitro determine gene switches that re-differentiate these somatic cells to form new retinal stem cells and autologous retinal cells.
  • an object of the present invention to provide a method for reversely dividing human somatic cells to produce autologous retinal stem cells. Another object of the present invention is to provide a method of producing autologous retinal cells using the autologous retinal stem cells. Still another object of the present invention is to provide autologous retinal stem cells and autologous retinal cells obtained by the above method, and their use in the preparation of a medicament for treating various diseases. A further object of the present invention is to provide a plurality of cell culture fluids containing plant extracts and proteins for use in the above methods and the use of the culture fluids. Further, the present invention provides a corresponding kit for preparing autologous retinal stem cells and autologous retinal cells.
  • the invention provides a method of reverse-differentiating human somatic cells to produce autologous retinal stem cells, the method comprising:
  • the cell culture is carried out at 5% CO 2 and 37 °C.
  • the human somatic cells include, but are not limited to, human peripheral blood cells, cord blood cells, placental cells, skin cells, and fat cells.
  • the human somatic cells are cultured for 24-48 hours at a density of 2- 5 10 6 at 5% CO 2 and 37 ° C before being cultured in the cell culture medium D1.
  • the cells are first cultured for 72 hours in cell culture D1, which is DMEM, and contains hazelnut extract 50 mg/ml, chrysanthemum extract 50 mg/ml, Y-27632 10 ⁇ , interleukin.
  • D1 which is DMEM
  • hazelnut extract 50 mg/ml hazelnut extract 50 mg/ml
  • chrysanthemum extract 50 mg/ml chrysanthemum extract 50 mg/ml
  • Y-27632 10 ⁇ interleukin.
  • the cell culture solution D2 is composed of hazelnut extract 50 mg/ml, chrysanthemum extract 50 mg/ml, leech extract 50 mg/ml, ⁇ -27632 10 ⁇ , interleukin 3 (IL3) (IL3) 10 ng/m leukocyte 6 (IL6) 10ng/mK nerve growth forceps 10ng/mK Dkk-1 l Ong/mK Lef ty- A l Ong/ml DMEM; then switch to cell culture D3 at 5% C0 2 and 37 °C
  • the culture medium is cultured for 10-20 days, and the cell culture liquid D3 is 50 mg/ml containing hazelnut extract, 50 mg/ml of chrysanthemum
  • the invention provides two methods of producing autologous retinal cells, the method comprising: Manual cell culture was D4 culture plants and proteins induced obtained above autologous retinal stem cells, cultured 15--20 days formed rod cells, cone cells and pigment epithelium cells, the cell culture fluid D4 of DMEM, and comprising B27 2% ( ⁇ / ⁇ ), ⁇ -27632 5-30 ⁇ , nerve growth factor 1-lOOng/mK B-type transforming growth factor ( TGF- ⁇ ) 1-lOOng/m basic fibroblast growth factor ( bFGF ) 1 -lOOng/m insulin 1-lOOng/m vascular endothelial growth factor ; '1-lOOng/mK Dkk-1 1-lOOng/mK Lefty- A 1-lOOng/mK RA 1-lOOng/mK Taurine 5 -30 ⁇ , disulfoxide (DMSO) 5-30 ⁇ ;
  • Cell culture was carried out at C0 2 and 37 ° C;
  • the obtained plant and protein-induced autologous retinal stem cells are cultured in the cell culture medium D4 for 9-15 days to form rod cells, cone cells and pigment epithelial cells, and the cell culture medium D4 contains Y-27632 ⁇ .
  • the plant and protein-induced autologous retinal stem cells obtained above were cultured in a cell culture medium D5, and cultured for 15-20 days to form optic ganglion cells, the cell culture medium D5 was DMEM, and contained B27 2% ( ⁇ / ⁇ ), ⁇ -27632 5-30 ⁇ , growth factor 1-100ng/ml, type B transforming growth factor (TGF- ⁇ ) 1-100ng/ml, basic fibroblast growth factor (bFGF) 1-lOOng/mK insulin 1-lOOng/mK vascular endothelial growth factor 1-lOOng/mK Dkk-1 1-lOOng/mK Lefty-A 1-lOOng/mK RA 1-lOOng/mK taurine 5- 30 ⁇ , Heparin 1-100 g/ml, disulfoxide (DMSO) 5-30 ⁇ ;
  • DMSO disulfoxide
  • the cell culture is carried out at 5% C0 2 and 37 ° C;
  • the obtained plant and protein-induced autologous retinal stem cells are cultured in the cell culture medium D5 for 9-15 days to form optic ganglion cells
  • the cell culture solution D5 is DMEM, and contains B27 2% (v/v), Y-27632 10 ⁇ , nerve growth factor 10ng/ml, type B transforming growth factor (TGF- ⁇ ) 10ng/ml, basic fibroblast growth factor (bFGF) 10ng/ml, insulin lOng/mK vascular endothelial growth factor 10ng/ mK Dkk-1 10 ng/m Lefty-A lOng/mK RA 5 ng/mK Taurine 10 ⁇ , Heparin 2 ⁇ g/ml, diterpenoid (DMSO) 10 ⁇ .
  • TGF- ⁇ type B transforming growth factor
  • bFGF basic fibroblast growth factor
  • lOng/mK vascular endothelial growth factor 10ng/ mK Dkk-1 10 ng/m Lefty-A lOn
  • the invention provides autologous retinal stem cells and autologous retinal cells prepared by the above methods.
  • the present invention also provides the use of the autologous retinal stem cells and autologous retinal cells.
  • the present invention provides the use of the autologous retinal stem cells and autologous retinal cells for the preparation of a medicament for treating diseases involving organ regeneration and repair;
  • the disease is pathological damage, traumatic necrosis and various degenerative degeneration of cells, tissues and organs such as the retina and the nervous tissue system; further preferably, the disease is an ophthalmic disease, a retinal degenerative disease, an optic nerve disease; more preferably The disease is age-related macular degeneration, retinitis pigmentosa, and optic nerve damage atrophy.
  • the present invention provides a cell culture fluid for reversely differentiating human somatic cells, wherein the culture fluid is selected from the group consisting of cell culture fluids D1, D2, D3, D4 and! 5, the culture solution D1 is DMEM and contains hazelnut extract l-100mg/ml, chrysanthemum extract 1-100mg/ml, Y-27632 5- 30 ⁇ , interleukin 3 (IL3) 5-30ng/ml , interleukin 6 (IL6) 5-30ng/mK nerve growth factor l-100ng/ml, disulfoxide (DMS0) 5-30 ⁇ ; the culture solution D2 is DMEM, and contains hazelnut extract l-100mg /ml, chrysanthemum extract l-100mg/ml, leech extract l-100mg/ml, ⁇ -27632 5-30 ⁇ , interleukin 3 (IL3) 5-30ng/ra interleukin 6 ( IL6 ) 5-30ng /
  • the present invention provides the above cell culture fluids D1, D2 and D3 in culturing human somatic cells.
  • the book reversely differentiates to produce autologous retinal stem cells.
  • the present invention also provides the use of cell culture fluids D1, D2, D3, D4 and D5 for the reverse differentiation of cultured human somatic cells to produce autologous retinal stem cells, which are then differentiated to produce autologous retinal cells.
  • the present invention provides a kit for preparing autologous retinal stem cells, wherein the kit comprises the above cell culture fluids D1, D2 and D3; preferably, the kit further comprises human somatic cells; Somatic cells include, but are not limited to, peripheral blood cells, cord blood cells, placental cells, skin cells, and fat cells.
  • the present invention also provides a kit for preparing autologous retinal cells, wherein the kit comprises the above cell culture fluids D1, D2, D3, D4 and !
  • the kit further comprises human somatic cells; the somatic cells include, but are not limited to, peripheral blood cells, cord blood cells, placental cells, skin cells, and fat cells.
  • the invention also provides the use of the kit in the preparation of autologous retinal stem cells and autologous retinal cells and preparation of a medicament for treating diseases involving regeneration and repair of organs;
  • the disease is pathological damage, wound necrosis and various degenerative degeneration of cells, tissues and organs such as the retina and the nervous tissue system;
  • the disease is an ophthalmic disease, a retinal degenerative disease, an optic nerve disease; more preferably, the disease is age-related macular degeneration, retinitis pigmentosa, diabetic retinal degeneration, and optic nerve damage atrophy.
  • the disease is age-related macular degeneration, retinitis pigmentosa, diabetic retinal degeneration, and optic nerve damage atrophy.
  • the human body cells of the human body selected for the specification include, but are not limited to, human umbilical cord blood cells, placental cells, commercially available non-immortalized and immortalized human somatic cell lines, blood and white blood cell suspensions preserved in conventional blood banks, Freshly prepared human skin cells, blood nucleated cells, and fat cells may be peripheral blood cells, cord blood cells, placental cells, skin cells, and fat cells.
  • the culture of the selected raw body cells comprises: culturing at a density of 2-5 ⁇ 10 ⁇ with the corresponding cell culture medium at 5% CO 2 and 37 ° C for 24-48 hours;
  • the reverse differentiation of the raw material cells to produce human autologous retinal stem cells includes: using the corresponding raw somatic cells to culture with the corresponding cell culture medium at 5% C0 2 and 37 ° C for 24-48 hours, and then switching to the fine 'cell culture medium Dl (DMEM, hazelnut extract 50 mg/ml, chrysanthemum extract 50 mg/ml, Y-27632 10 ⁇ , interleukin-3 lOng/mK interleukin-610 ng/mK nerve growth factor 10 ng/m DMSO ⁇ ) continued at 5% C0 2 and Incubate for 72 hours under conditions of 37 , then switch to cell culture medium D2 (DMEM, hazelnut extract 50 mg/ml, chrysanthemum extract 50 mg/ml, leech extract 50 mg/ml, ⁇ -27632 ⁇ , interleukin 310 ng/ml, interleukin 610ng/ml, nerve growth factor lOng/mK Dkk-1 10ng/m
  • the person obtained autologous retinal stem cells can be detected by the following methods: using retinal progenitor cells and optic nerve progenitor cells specific cell phenotype (Rx, 3 ⁇ 4 ChxlO and Pax6-), the pigment-specific progenitor cells Cell phenotype (Mitf), specific cell phenotype (Crx, Nrl) of photoreceptor precursor cells, etc., as indicators of detection, on the 3rd, 6th, 9th and 12th days after the cell culture medium D3 was replaced.
  • RT-PCR and cellular immunofluorescence techniques were used to observe the formation rate, generation rate, production quantity and purity of various retinal stem cells (using Pax6, Rx, ChxlO, Mi t if, etc. as detection indicators and their detection, refer to Meyer JS Waiting
  • the differentiation of plant and protein-induced autologous retinal stem cells to produce rods, cones, and pigment epithelial cells includes: plant and protein-induced retinal stem cells cultured in cell culture medium D3, application Cell culture medium D4 (DMEM, B272% (v/v), Y-27632 piano, nerve growth factor lOng/mK B type transforming growth factor (TGF- ⁇ ) 10ng/ml, basic fibroblast growth factor (bFGF) 10 ng/ml, insulin 10 ng/ml, vascular endothelial growth factor 10 ng/mK Dkk-1 lOng/mK Lefty-A lOng/mK RA 5 ng/mK Taurine ⁇ , disulfoxide (DMSO) ⁇ ) Continue Cultured for 15-20 days to form autologous retinal rod cells, cone cells and pigment epithelial cells.
  • DMEM Cell culture medium D4
  • bFGF basic fibroblast growth factor
  • the differentiation of the plant-derived autologous retinal stem cells into the ganglion cells comprises: plant-derived and retinal stem cells cultured in cell culture medium D3, using cell culture medium D5 (DMEM, B27) 2% ( ⁇ / ⁇ ), ⁇ -27632 10 ⁇ , 'Nerve growth factor 10ng/ml, B-type transforming growth factor (TGF-(3) 10ng/ml, basic fibroblast growth factor (bFGF) 10ng/m insulin 10 ng/ml, vascular endothelial growth factor 10 ng/mK Dkk-1 lOng/mK Lefty-A lOng/mK RA 5 ng/mK taurine, 10 ⁇ , Heparin 2 ⁇ g/mK dimethyl sulfoxide (DMSO) ⁇ Continue to culture for 15-20 days to form autologous retinal ganglion cells.
  • DMEM cell culture medium D5
  • DMSO cell culture medium D5
  • various types of autologous retinal cells produced can be detected by: detecting retinal cells, such as rod cells (Rhodopsiru Recovering cones), using cellular immunofluorescence techniques and reverse transcription PCR techniques.
  • retinal cells such as rod cells (Rhodopsiru Recovering cones)
  • red opsin, Red/grennopsin ganglion cell (MAth5, Brn3b)
  • pigment epithelial (RPE-65, CRAL'BP) specific protein expression (refer to Meyer JS et al. "Modeling early retinal development with human embryonic and induced Pulur ipotent stem eel Is" PNAS 2009; 106, 16543-4. Osakada F et al. "Stepwise differentiation of pluripotent stem eel Is into retinal eel Is" Nat Protocoll 2009; 4(6): 811-24).
  • the plant and protein-induced autologous retinal stem cells obtained according to the present invention can be used as an excellent seed cell for organ regeneration and repair, for optic atrophy, injury, age-related macular degeneration, optic nerve trauma, retinopathy, choroidal laceration, diabetic retina Eye diseases such as lesions, retinal contusion, and retinal detachment have the potential for regeneration and repair, and have good therapeutic application prospects;
  • plant and protein-induced autologous retinal stem cells can also be used for in vitro tissue engineering of retina, producing autologous retinal stem cells and retinal cells.
  • the present invention also has the possibility of large-scale industrialization of autologous retinal stem cells and retinal cells. Compared with the prior art, the present invention has at least the following advantages:
  • the present invention differs from the existing use of genetic recombination technology to induce somatic cell reverse differentiation to produce 'stem cells (iP stem cells), and provides a novel technical method for producing autologous retinal stem cells and autologous retinal cells, using plant extracts and protein formulas.
  • the technology induces the stem cells produced by the reverse differentiation of somatic cells (Plants and Proteins induced plur i potent s etem 1, PPiPS stem cells), which makes the cells close to natural stem cells and has high safety.
  • the present invention employs a plant extract and a protein formulation which have been subjected to screening optimization design by adopting human conventional somatic cells without changing the somatic chromosome DM sequence, without inserting any foreign gene or DNA fragment.
  • Somatic cells re-differentiate to form autologous retinal stem cells, which in turn differentiate into autologous retinal cells.
  • the technology is unique and innovative, providing new ideas and ways to solve the current difficulties in the treatment of ophthalmic diseases, and solving the problem of difficulty in the source of human autologous retinal stem cells and retinal cells.
  • the method of the present invention can directly use somatic cells isolated from a patient to reversely differentiate the somatic cells to produce autologous retinal stem cells and autologous retinal cells, and the use of the autologous retinal stem cells and autologous retinal cells avoids the use of allogeneic retinal cells or allogeneic retinas.
  • the immune rejection caused by cell transplantation provides significant convenience for clinical applications.
  • Autologous retinal stem cells and retinal cells prepared by the method of the present invention have a normal diploid karyotype.
  • the mice were subcutaneously transplanted with lx 10 8 plant and protein-induced retinal stem cells or 8 human natural embryonic stem cells to observe whether there was tumor formation at the inoculation site. After 90 days of continuous observation, it was found.
  • the test group using the plant and protein-induced retinal stem cells of the present invention none of the 10 mice produced tumors, and the human natural embryonic stem cell test. All the mice of the test group produced tumors, and thus the plant and protein-induced retinal stem cells of the present invention. And retinal cells have a higher safety.
  • the invention is also a novel technical method capable of producing autologous retinal stem cells and retinal cells in human individuals of any age, and can be widely applied. Audiences with related diseases in different age groups.
  • the method of the present invention can produce tens of millions of levels of the first generation of autologous retinal stem cells and retinal cells in the blood of 200 ml in a period of 3 to 4 weeks, and the production speed is fast, and the yield reaches 1-1.
  • 1-A is a raw material cell, 1-B, (is a retinal stem cell;
  • Figure 2-A shows the specific mRM of Rx, Pax6, Mi tf, Chxl O, Six3, S ix6, Sox2, Lhx2 shown by plant and protein-induced retinal stem cells using reverse transcription PCR;
  • Figure 2-B uses immunization Fluorescence chemistry showed that plant and protein-induced retinal stem cells have specific phenotypes of Pax6, Rx, Mi tf and Chxl O;
  • Figure 3 shows the use of immunofluorescence chemistry to show that plant and protein-induced cones have a blue ops in, Red/grenn ops in specific phenotype
  • Figure 4 shows the use of immunofluorescence chemistry to show that plant and protein-induced rods have a specific phenotype of Rhodops in, Recover in;
  • Figure 5 shows specific mRNAs of Blue ops in, Red/grenn ops in ⁇ Rhodops in, Recover in and MAth5 which are shown to be plant and protein-induced cones, rods, and ganglion cells using reverse transcription PCR;
  • Figure 6 shows the RPE-65, CRALBP-specific phenotypes of plant and protein-induced pigment epithelial cells using immunofluorescence chemistry
  • Figure 7 shows the specific phenotype of MAth5, . Brn3b in plant and protein-induced ganglion cells using immunofluorescence chemistry. detailed description
  • the culture medium used is:
  • Cell culture medium D1 DMEM (purchased from GIC0 company), containing hazelnut extract (purchased from Henan Natural Plant Material Factory, Zhengzhou Yanuo Biotechnology Co., Ltd.) 50mg/ml, chrysanthemum extract (purchased from Henan Natural Plant Material Factory) , Zhengzhou Yanuo Biotechnology Co., Ltd.) 50mg/ml, Y-27632 (purchased from Sigma) 10 ⁇ , interleukin 3 (IL3) (purchased from R&D) 10ng/ml, interleukin-6 (IL6) Purchased from R&D Company) 10ng/ml, nerve growth factor (purchased from R&D Company) 10ng/ml Disulfoxide (DMS0) (purchased from Sigma) ⁇ ⁇ ;
  • Cell culture medium D2 DMEM (purchased from GIC0 company), containing hazelnut extract (purchased from Henan Natural Plant Material Factory, Zhengzhou Yinuo Biotechnology Co., Ltd.) 50mg/ml, chrysanthemum extract (purchasing 3 ⁇ 4 Henan Natural Plant Material Factory) , Zhengzhou Yunuo Biotechnology Co., Ltd.) 50mg/ml, leech extract (purchased from Zhengzhou Yinuo Biotechnology Co., Ltd.) 50mg/ml, Y-27632 (purchased from Sigma) 10 ⁇ , interleukin 3 UL3 ) ( Purchased from R&D) 10 ng/ml, interleukin 6 (IL6) (purchased from R&D) l Ong/ml, nerve growth factor (purchased from R&D) 10 ng/ml, Dk'k-l (purchased from Sigma) 10ng/mK Lef ty-A (purchased from Sigma) 10ng/ml
  • Cell culture medium D3 DMEM (purchased from GICO), containing hazelnut extract (purchased from Henan Natural Plant Material Factory, Zhengzhou Yanuo Biotechnology Co., Ltd.) 50mg/ml, chrysanthemum extract (purchased from the river : southern natural plant) Raw Material Plant, Zhengzhou Yinuo Biotechnology Co., Ltd.) 50mg/ml, Leech Extract (purchased from Zhengzhou Yanuo Biotechnology Co., Ltd.) 50mg/mK Y-27632 (purchased from Sigma) 10 ⁇ , ⁇ 27 (purchased from S igma Company) 2% (v/v), interleukin 3 (IL3) (purchased from R&D) 10 ng/ml, interleukin 6 (IL6) (purchased from R&D) 10 ng/ml, nerve growth factor (purchased from R&D) l Ong/m Dkk-1 (purchased from Sigma) 10ng/ml, Lef ty-A (purchased from Sigma
  • DMEM purchased from GICO
  • B 27 purchased from Sigma
  • Y-27632 purchasedd from Sigma
  • 10 ⁇ nerve growth factor (purchased from R&D) Company) 10 ng/mK B-type transforming growth factor (purchased from R&D) (TGF-(3) l Ong/mK basic fibroblast growth factor (purchased from R&D) (bFGF) l Ong/mK insulin (purchased from R&D Company) 10ng/mL, vascular endothelial growth factor (purchased from R&D) l Ong/ml, Dkk-l (purchased from S igma Specification company) lOng/m Lefty-A (purchased from Sigma) 10ng/mK RA (purchased from Sigma) 5ng/ml, taurine (purchased from Sigma) 10 ⁇ , disulfoxide (DMSO) (purchased from Sigma) ⁇ ;
  • DMSO disulfoxide
  • Cell culture medium D5 DMEM (purchased from GICO), ⁇ 27 (purchased from Sigma) 2% (v/v), Y-27632 (purchased from Sigma) 10 ⁇ , nerve growth factor (purchased from R&D) lOng/mK B-type transforming growth factor (TGF- ⁇ ) (purchased from R&D) lOng/mK basic ; fibroblast growth factor (bFGF) (purchased from R&D) 10ng/ml, insulin (purchased from R&D) 10ng/ml, vascular endothelial growth factor (purchased from R&D) 10ng/nil, Dkk-1 (purchased from Sigma) lOng/mK Lefty-A (purchased from Sigma) lOng/mK RA (purchased from Sigma) 5ng /ml, taurine (purchased from Sigm) 10 ⁇ , Heparin (purchased from Sigma) 2 g/ml, disulfoxide (DMSO) (purchased from Sigma) 10 ⁇ .
  • Peripheral venous blood and cord blood were collected aseptically. Before collecting peripheral venous blood/umbilical cord blood, the consent of the blood donor or immediate family member should be obtained, and the genetic and infectious disease history of the blood donor and family and all relevant virus test results of the hospital should be recorded.
  • Peripheral venous blood / cord blood routine anticoagulation, stored at 4 ° C. Send it to the stem cell production center within 24 hours. After the center needs to do the corresponding virus detection, enter the central computer registration process, and after recording the corresponding barcode number, the blood sample enters the sterile stem cell production workshop.
  • the culture medium of DMEM was cultured at a density of 2-5 X 10 6 at 5% CO 2 and 37 ° C for 24-48 hours.
  • the cell culture medium D1 was further cultured at % C02 and 37 ° C for 72 hours. Then switch to cell culture medium D2 and continue to culture for 9-15 days at 5% CO 2 and 37 ° C; then switch to cell culture medium D3 and continue to culture at 5% C02 and 37 ° C for 10-20 days to form plants.
  • protein-induced autologous retinal stem cells is performed using Ficoll standard separation technique (Modern Immunology, Chapter 12, page 288) to separate mononuclear cells and perform single nuclear cell counting.
  • the culture medium of DMEM was cultured at a density of 2-5 X 10 6 at 5% CO 2 and 37 ° C for 24-48 hours.
  • the cell culture medium D1 was further cultured at % C02 and 37 ° C for 72 hours. Then switch to cell culture medium D2 and continue to culture for 9-15 days at 5% CO 2
  • Peripheral venous blood and cord blood were collected aseptically.
  • the consent of the donor or immediate family member should be obtained before collecting the peripheral venous blood/umbilical cord blood, and the history of the genetic and infectious diseases and the relevant viral examination results of the hospital and the family should be recorded.
  • Peripheral venous blood/umbilical cord blood is routinely anticoagulated, and stored at 4 °C. Send it to the stem cell production center within 24 hours. After the center needs to do the corresponding virus detection, enter the central computer registration process, and after recording the corresponding barcode number, the blood sample enters the sterile stem cell production workshop.
  • DMEM medium was cultured at a density of 2-5 xl O 6 at 5% CO 2 and 37 ° C for 24-48 hours; replaced with cell culture medium D1 and continued to culture at 5% CO 2 and 37 ° C for 72 hours. Then, switch to cell culture medium D 2 and continue to culture for 9-15 days at 5% CO 2 and 37 ° C; then switch to cell culture medium D3 and continue to culture at 5% CO 2 and 37 ° C for 10-20 days. and form a plant protein induced autologous stem cells depending 3 ⁇ 4 retina.
  • Plant and protein-induced retinal stem cells cultured in cell culture medium D3 were cultured for 15-20 days using D5 cell culture medium to form autologous retinal ganglion cells.

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Abstract

L'invention concerne une solution de culture et un procédé pour la différentiation inverse de cellules somatiques pour générer des cellules souches autologues de la rétine, et l'application de ceux-ci. La solution de culture pour la différentiation inverse de cellules somatiques pour générer des cellules souches autologues de la rétine consiste en un mélange de multiples extraits végétaux et de multiples protéines, et peut permettre une différentiation induite inverse sur des cellules somatiques humaines, telles que des cellules mononuclées du sang périphérique humain, pour générer en quelques semaines des dizaines de milliards de cellules souches autologues de la rétine de première génération et des cellules autologues de la rétine de première génération. Le procédé possède les avantages que le nombre de cellules souches de la rétine produites est élevé et que la qualité des cellules souches peut être bien surveillée. Les cellules souches autologues humaines de la rétine induites par des plantes et des protéines ont des phénotypes cellulaires spécifiques similaires et une variété de capacités de différentiation cellulaire à ceux des cellules souches endogènes de la rétine, et peuvent subir une différentiation pour former des cellules photosensibles, les cellules épithéliales pigmentaires de la rétine, et les cellules ganglionnaires de la rétine, et sont non seulement applicables au traitement de diverses maladies oculaires, mais sont également applicables à l'établissement de la banque de cellules souches autologues de la rétine pour préserver des cellules souches autologues de la rétine d'une façon permanente, et, en tant que cellules primaires autologues et cellules pour la régénération et la réparation, ont des applications potentielles souhaitables dans le traitement de maladies oculaires, l'ingénierie de tissus et d'organes autologues de la rétine, et le domaine industriel de la production de cellules souches autologues de la rétine.
PCT/CN2011/002065 2011-01-20 2011-12-12 Procédé, trousse et application pour l'application d'une différentiation inverse à des cellules somatiques humaines pour générer des cellules souches autologues de la rétine et des cellules autologues de la rétine WO2013071469A1 (fr)

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CN201110022102XA CN102604886A (zh) 2011-01-20 2011-01-20 使人的体细胞逆向分化产生自体视网膜干细胞和自体视网膜细胞的方法、试剂盒及用途
CN201110022102.X 2011-11-20

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CN106148271A (zh) * 2016-05-31 2016-11-23 宁夏医科大学 一种枸杞菊花提取液培养晶状体上皮细胞的方法
CN106822182A (zh) * 2016-12-27 2017-06-13 广州姿生生物科技有限公司 一种细胞提取物及其用途
CN107326009B (zh) * 2017-05-02 2019-03-08 深圳百年干细胞技术研究院有限公司 使血液单个核细胞逆向分化产生人血源性自体视网膜干细胞的方法、试剂盒及用途

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