CN112852715A - Method for directionally differentiating induced pluripotent stem cells into inner ear hair cell-like cells - Google Patents
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Abstract
The invention provides a method for directionally differentiating induced pluripotent stem cells into inner ear hair cell-like cells. The method mainly comprises the steps of utilizing a culture medium with definite chemical components, timely adding a small molecular compound to induce and differentiate iPSC into inner ear progenitor cells, and co-culturing chick embryo oocyst cells and the inner ear progenitor cells to obtain inner ear hair cell-like cells. The method can effectively directionally differentiate the induced pluripotent stem cells into the inner ear hair cell-like cells, and the inner ear hair cell-like cells obtained by differentiation have cilium hair bundle structures, express specific proteins of the inner ear hair cells and have the electrophysiological functions of the hair cells.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a method for directionally differentiating induced pluripotent stem cells into inner ear hair cell-like cells.
Background
Hearing impairment is a major public health problem worldwide, and estimated by the world health organization in 2019, about 4.6 million people worldwide have hearing impairment (http:// www.who.int/en /). The inner ear hair cells are important sensory epithelial cells in China in the human auditory system, convert sound waves sensed by the inner ear into electric stimulation in a mechanical-electric conversion mode, and conduct the electric stimulation to the brain along the auditory nerve to form the auditory sense. It is generally accepted that mammalian inner ear hair cells are difficult to regenerate following injury, and thus inner ear hair cell injury is a significant cause of hearing loss in humans. Recent studies suggest that mammalian inner ear hair cells can achieve partial regeneration under certain experimental conditions. In 2013, Edge team studies found that noise-traumatized mice produced new inner ear hair cells and hearing was partially restored after using Notch pathway inhibitors (Mizutari K, Fujioka M, Hosoya M, Bramhall N, Okano HJ, Okano H, Edge AS. Notch inhibition cochlear hair cell regeneration and recovery of hearing after environmental tract. neuron.2013; 77(1): 58-69.). In 2019, Chen team found that activation of Myc and Notch1 could effectively achieve proliferation of adult mouse cochlear sensory epithelial cells, and that the supporting cells could transdifferentiate into hair-cell-like cells in response to the transcription factor Atoh1 (Shu Y, Li W, Huang M, Quan YZ, Scheffer D, Tian C, Tao Y, Liu X, Hochedlinger K, Indazyhkulian AA, Wang Z, Li H, Chen ZY. Renewwed promotion in adult mouse cochlea and regeneration of hair cells. Nat Commun.2019; 10(1): 5530.). However, there remain significant challenges in the efficiency and safety of inner ear hair cell regeneration under in vivo conditions.
Yamanaka and Takahashi obtained Induced Pluripotent Stem Cells (iPSCs) by successfully utilizing mouse cells in 2006, the iPSCs having similar cellular characteristics as Embryonic Stem Cells (ESCs), such as expression of sternness marker genes, having a potential for differentiation into three germ layers, and the like (Takahashi K, Yamanaka S.indication of Pluripotent Stem cells from motor tissue and adult tissue cells defined factors. cell.2006; 126): 663-76.). Through directional differentiation of iPSC into various tissue cells, research can be carried out in the fields of cell fate determination, organ development, regeneration and the like from the perspective of tissue specificity, and the iPSC has a considerable clinical application prospect. Currently, scientists worldwide have successfully reprogrammed embryonic fibroblasts, dermal fibroblasts, blood cells, urine cells, and the like, into iPSCs in a variety of models of animals and humans (Theunissen TW, Jaenisch R. molecular control of induced pluralityotency. cell Stem cell 2014; 14(6): 720-34.). Therefore, the iPSC is directionally differentiated into the inner ear hair cell-like cells in vitro, an important cell source is provided for the research in the fields of inner ear hair cell fate determining molecular mechanism, inner ear hair cell regenerative drug screening, cell regenerative therapy and the like, and the iPSC has important research and application values.
Disclosure of Invention
The invention aims to provide a method for directionally differentiating induced pluripotent stem cells into inner ear hair cell-like cells, which is realized by the following steps:
(1) inoculating iPSC to a Laminin coated culture plate, and culturing for 10-12 days by using an inner ear progenitor cell culture medium;
(2) then inoculating the cells to a gelatin-coated culture plate containing inactivated chick embryo oval bursa cells, and culturing for 14-28 days by using an inner ear hair cell culture medium to obtain mature inner ear hair cell-like cells.
The iPSC can be induced pluripotent stem cells obtained by reprogramming through methods of retrovirus, lentivirus, Sendai virus, episome plasmid, protein, RNA or small molecules and the like.
The plate was coated with the described lamin by adding lamin diluted with PBS to the plate at a concentration of 5. mu.g/cm 2, allowing to stand at 37 ℃ for 2 hours, and then discarding the solution.
The cell culture environment is 37 ℃ and 5% CO2Constant temperature incubator in humid environment
The progenitor cell culture medium is prepared by adding 1% of N2supplement, 2% of B27 supplement, 0.2% of Normocin (500X), 1mmol/L sodium pyruvate, 50ng/mL FGF3 and 50ng/mL FGF10 into DMEM/F12(1:1) culture medium by volume percent.
Preferably, the medium added on days 0-2 in step (1) may have a final concentration of 10. mu. mol/L of Y-27632.
Preferably, the culture medium of the inner ear progenitor cells should be used for alternate days during the culture process in the step (1).
Preferably, the expression level of the inner ear progenitor cell marker gene is detected in the culture process in the step (1) by means of reverse transcription PCR, immunofluorescence staining and the like.
The inner ear progenitor cell marker gene comprises a plurality of combinations of PAX2, PAX8, SIX1, DLX5, GATA3, EYA1, NESTIN and the like.
The inner ear hair cell culture medium is prepared by adding 1 volume percent of N2supplement, 2 volume percent of B27 supplement, 0.2 volume percent of Normocin (500 x), 1mmol/L of sodium pyruvate, 1 mu mol/L of all-trans retinoic acid and 20ng/mL of EGF into DMEM/F12(1:1) culture medium.
Preferably, the medium added on days 0-2 of step (2) may have a final concentration of 10. mu. mol/L of Y-27632.
Preferably, the inner ear hair cell culture medium should be used for changing liquid every other day during the culture process in the step (2).
Preferably, the expression level of the inner ear hair cell marker gene is detected in the culture process in the step (2) by means of reverse transcription PCR, immunofluorescence staining and the like.
The inner ear hair cell marker genes comprise a plurality of combinations of ATOH1, MYO7A, POU4F3, USH1C, ESPN and the like.
The method for coating the culture plate by gelatin comprises the following steps: adding gelatin with mass volume fraction of 0.2% into PBS solution, autoclaving, adding appropriate volume into culture plate to cover the bottom surface of culture, standing at 37 deg.C for 0.5 hr, and discarding solution.
The preparation method of the chick embryo oval bursa cell comprises the following steps: taking the eggs fertilized for 18.5 days, taking out the chicken embryos in a sterile environment, separating out oval sacs of the chicken embryos by a body type lens dissection mode, digesting the oval sacs for 30min by using a thermolysin solution, digesting for 5min by using 0.25% pancreatin after the digestion is stopped, stopping the digestion again, removing redundant tissues from the mixed solution by using a 200-mesh screen, centrifuging for 5min by using 300g, re-suspending the cells by using a growth culture medium, inoculating a culture dish, and recording as P0 generation cells.
The preparation method of the inactivated chick embryo oval bursa cell comprises the following steps: and (3) culturing the P0 generation chick embryo oval sac cells until the confluence reaches 90%, carrying out passage to obtain P1 generation cells, repeatedly culturing and carrying out passage until P3 generation cells are obtained, culturing the chick embryo oval sac cells for 3 hours by using a DMEM medium added with mitomycin C with the final concentration of 2 mug/mL, and rinsing the cells by PBS to obtain the inactivated chick embryo oval sac cells.
The thermophilic protease solution is obtained by adding 0.5mg/mL of thermophilic protease into a DMEM/F12(1:1) culture medium.
The reagents of the invention are all commercially available.
The invention has the beneficial effects that: the method can effectively directionally differentiate the induced pluripotent stem cells into the inner ear hair cell-like cells, and the inner ear hair cell-like cells obtained by differentiation have cilium hair bundle structures, express specific proteins of the inner ear hair cells and have the electrophysiological functions of the hair cells.
Drawings
FIG. 1 shows the morphological changes of cells observed under an optical microscope at the 2 nd, 6 th and 10 th days of the directed differentiation of iPSCs into inner ear progenitor cells.
FIG. 2 differentiation of iPSC into inner ear progenitor cells on day 12, the expression of inner ear progenitor cell marker proteins NESTIN, PAX2, PAX8 was identified as positive by immunofluorescence staining.
FIG. 3 directed differentiation of iPSC into inner ear progenitor cells on day 12, expression of inner ear progenitor marker genes PAX2, PAX8, SIX1, DLX5, GATA3, EYA1 was identified as positive by reverse transcription PCR.
FIG. 4 shows that the expression of inner ear hair cell marker proteins POU4F3, MYO7A and ATOH1 was identified as positive by immunofluorescence staining when the inner ear progenitor cells were directionally differentiated into inner ear hair cells for 3 weeks.
FIG. 5 shows the cilia structure on the surface of inner ear hair cell-like cells observed by scanning electron microscopy when the progenitor cells of the inner ear are directionally differentiated into inner ear hair cells for 3 weeks.
FIG. 6 depicts the identification of inner ear hair cell-like cells as having outward potassium current, inward potassium current and calcium current by patch clamp techniques when inner ear progenitor cells are committed to differentiate into inner ear hair cells for 3 weeks.
Detailed Description
The invention is further described with reference to the following description and embodiments in conjunction with the accompanying drawings.
Example 1 directed differentiation of induced pluripotent Stem cells into inner ear progenitor cells
(1) Normally, induced pluripotent stem cells with good growth are cultured, digested into single cells by using Accutase, diluted by a DMEM/F12(1:1) culture medium, centrifuged at 300g for 5min, and resuspended and counted. According to 5. mu.g/cm2Adding Lamin diluted with PBS into a 12-well plate, standing at 37 ℃ for 2 hours, and then discarding the solution. Laminin coated 12-well plates, per wellInoculating into 1X 104The cells were cultured for 2 days in progenitor cell medium supplemented with Y-27632 at a final concentration of 10. mu. mol/L, followed by 12 days of medium change every other day. The progenitor cell culture medium is DMEM/F12(1:1) culture medium added with 1% by volume of N2supplement, 2% by volume of B27 supplement, 0.2% by volume of Normocin (500X), 1mmol/L sodium pyruvate at final concentration, 50ng/mL FGF3 and 50ng/mL FGF 10.
(2) The total RNA of the progenitor cells of the inner ear, which are directionally differentiated for 12 days, is extracted by a TRIzol method, the total RNA is reversely transcribed into cDNA, the primers of marker genes PAX2, PAX8, SIX1, DLX5, GATA3 and EYA1 of the progenitor cells of the inner ear are used for carrying out PCR, and the products are analyzed for the expression condition by agarose gel electrophoresis.
(3) Fixing the inner ear progenitor cells which are directionally differentiated for 12 days by paraformaldehyde, rinsing by PBS, adding PAX2, PAX8 and NESTIN antibodies into each hole after closing, incubating overnight at 4 ℃, adding the corresponding species of fluorescent secondary antibody after rinsing by PBS, incubating for 2 hours at room temperature, dyeing by DAPI, rinsing by PBS, and observing and photographing under a fluorescent microscope.
EXAMPLE 2 Targeted differentiation of inner ear progenitor cells into inner ear hair cell-like cells
(1) Taking the eggs fertilized for 18.5 days, taking out the chicken embryos in a sterile environment, separating out oval sacs of the chicken embryos by a body type lens dissection mode, digesting the oval sacs for 30min by using a thermolysin solution, digesting for 5min by using 0.25% pancreatin after the digestion is stopped, stopping the digestion again, removing redundant tissues from the mixed solution by using a 200-mesh screen, centrifuging for 5min by using 300g, re-suspending the cells by using a growth culture medium, inoculating a culture dish, and recording as P0 generation cells. And (3) culturing the P0 generation chick embryo oval sac cells until the confluence reaches 90%, carrying out passage to obtain P1 generation cells, repeatedly culturing and carrying out passage until P3 generation cells are obtained, culturing the chick embryo oval sac cells for 3 hours by using a DMEM medium added with mitomycin C with the final concentration of 2 mug/mL, and rinsing the cells by PBS to obtain the inactivated chick embryo oval sac cells.
(2) Adding gelatin with mass volume fraction of 0.2% into PBS solution, autoclaving, adding appropriate volume into culture plate to cover the bottom surface of culture, standing at 37 deg.C for 0.5 hr, and discarding solution. The cells were seeded at 1X 10 per well in 12-well plates5InactivatedChick embryo oocyst cells were cultured overnight.
(3) Digesting the directional differentiation for 12 days with 0.05% pancreatin, stopping digestion, counting the weight of the suspension, inoculating 1 × 10 per well according to 12-well plate5Culturing inner ear progenitor cells in a progenitor cell culture medium with the addition of Y-27632 with the final concentration of 10 mu mol/L for 2 days, and culturing in an inner ear hair cell culture medium for 12-26 days to obtain mature inner ear hair cell-like cells. The inner ear hair cell culture medium is prepared by adding 1% by volume of N2supplement, 2% by volume of B27 supplement, 0.2% by volume of Normocin (500X), 1mmol/L by volume of sodium pyruvate, 1. mu. mol/L by volume of all-trans retinoic acid, and 20ng/mL by volume of EGF to DMEM/F12(1: 1).
(4) Extracting total RNA of inner ear hair cell-like cells which are directionally differentiated for 14-28 days by a TRIzol method, carrying out reverse transcription on the total RNA to obtain cDNA, carrying out PCR by using primers of inner ear hair cell-like cell marker genes ATOH1, MYO7A, POU4F3, USH1C and ESPN, and analyzing the expression condition of products by agarose gel electrophoresis.
(5) Fixing inner ear hair cell-like cells which are directionally differentiated for 14-28 days by paraformaldehyde, rinsing by PBS, sealing, adding antibodies of ATOH1, MYO7A and POU4F3 into each hole respectively, incubating overnight at 4 ℃, rinsing by PBS, adding a corresponding species fluorescent secondary antibody, incubating for 2 hours at room temperature, dyeing by DAPI, rinsing by PBS, and observing and photographing under a fluorescent microscope.
Claims (6)
1. A method for the directed differentiation of induced pluripotent stem cells into inner ear hair cell-like cells, comprising the steps of:
(1) inoculating iPSC to a Laminin coated culture plate, and culturing for 10-12 days by using an inner ear progenitor cell culture medium;
(2) then inoculating the cells to a gelatin-coated culture plate containing inactivated chick embryo oval bursa cells, and culturing for 14-28 days by using an inner ear hair cell culture medium to obtain mature inner ear hair cell-like cells.
2. The method for committed differentiation of induced pluripotent stem cells into inner ear hair cell-like cells according to claim 1, wherein the progenitor cell culture medium in step (1) is a culture medium prepared by adding 1% by volume of N2supplement, 2% by volume of B27 supplement, 0.2% by volume of Normocin (500X), 1mmol/L final concentration of sodium pyruvate, 50ng/mL of FGF3 and 50ng/mL of FGF10 to DMEM/F12 medium.
3. The method for the directed differentiation of induced pluripotent stem cells into inner ear hair cell-like cells according to claim 1, wherein the culture conditions in step (1) are all 37 ℃ and 5% CO2A constant temperature incubator in a humid environment.
4. The method for directed differentiation of induced pluripotent stem cells into inner ear hair cell-like cells according to claim 1, wherein the inner ear hair cell culture medium in step (2) is a culture medium prepared by adding 1 vol% of N2supplement, 2 vol% of B27 supplement, 0.2 vol% of Normocin (500X), 1mmol/L of sodium pyruvate, 1. mu. mol/L of all-trans retinoic acid, and 20ng/mL of EGF to DMEM/F12 medium.
5. The method for directed differentiation of induced pluripotent stem cells into inner ear hair cell-like cells according to claim 1, wherein the gelatin-coated culture plate of step (2) is prepared by: adding gelatin with mass volume fraction of 0.2% into PBS solution, autoclaving, adding appropriate volume into culture plate to cover the bottom surface of culture, standing at 37 deg.C for 0.5 hr, and discarding solution.
6. The method for directed differentiation of induced pluripotent stem cells into inner ear hair cell-like cells according to claim 1, wherein the inactivation method of chick embryo oocyst in step (2) comprises culturing chick embryo oocyst in DMEM medium supplemented with mitomycin C at a final concentration of 2 μ g/mL for 3 hours, rinsing the cells with PBS, and obtaining inactivated chick embryo oocyst cells.
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