CN112852715B - Method for directionally differentiating induced pluripotent stem cells into inner ear hair cell-like cells - Google Patents

Method for directionally differentiating induced pluripotent stem cells into inner ear hair cell-like cells Download PDF

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CN112852715B
CN112852715B CN202110251574.6A CN202110251574A CN112852715B CN 112852715 B CN112852715 B CN 112852715B CN 202110251574 A CN202110251574 A CN 202110251574A CN 112852715 B CN112852715 B CN 112852715B
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管敏鑫
陈潮
冀延春
孟飞龙
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Zhejiang University ZJU
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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

Method for directionally differentiating induced pluripotent stem cells into inner ear hair cell-like cells
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 as estimated by the world health organization in 2019, about 4.6 million people worldwide have hearing impairment (http:// www.who.int/en /). Inner ear hair cells are important sensory epithelial cells in China in the human auditory system, sound waves sensed by the inner ear are converted into electric stimulation in a mechanical-electric conversion mode and are conducted to the brain along the auditory nerve to form 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, the Edge team research found that noise-traumatized mice produced new inner ear hair cells and hearing was partially restored by 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. Renewed 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 tri-germ layer differentiation, and the like (Takahashi K, yamanaka S.indication of Pluripotent Stem cells from motor tissue and adult tissue cells by defined factors. Cell.2006;126 (4): 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 various types of cells, such as embryonic fibroblasts, skin fibroblasts, blood cells, urine cells, etc., to iPSCs in various model animals and humans (Theunissen TW, jaenisch R. Molecular control of induced pluripotency. Cell Stem cell.2014;14 (6): 720-34.). Therefore, the iPSC is directionally differentiated into inner ear hair cell-like cells in vitro, provides an important cell source for the research in the fields of inner ear hair cell fate determination molecular mechanism, inner ear hair cell regeneration drug screening, cell regeneration treatment and the like, and 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 Lamin by adding Lamin diluted with PBS to the plate at a concentration of 5. Mu.g/cm 2, and allowing the plate to stand at 37 ℃ for 2 hours and then discarding the solution.
The cell culture environment is 37 ℃,5% CO 2 Constant temperature incubator in humid environment
The progenitor 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 of sodium pyruvate, 50ng/mL of FGF3 and 50ng/mL of FGF10 to DMEM/F12 (1.
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 for the inner ear progenitor cells is used in the culture process of step (1) at intervals.
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.
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 oocyst cells 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 oocyst 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 oocyst 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 oocyst cells.
The thermolysin solution was obtained by adding 0.5mg/mL of thermolysin to a DMEM/F12 (1.
The reagents of the invention are 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 cilia hair bundle structures, express specific proteins of the inner ear hair cells and have the electrophysiological functions of the hair cells.
Drawings
FIG. 1 directed differentiation of iPSCs into inner ear progenitor cells on days 2, 6, and 10, the morphological changes of the cells were observed under an optical microscope.
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, the expression of inner ear progenitor cell 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 is identified as positive by immunofluorescence staining when the inner ear progenitor cells are 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) The well-grown induced pluripotent stem cells were cultured normally, digested into single cells by Accutase, diluted by DMEM/F12 (1) medium, centrifuged at 300g for 5min, and counted by resuspension. According to 5. Mu.g/cm 2 Adding 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 were inoculated with 1X 10 seeds per well 4 The 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.
(2) Extracting total RNA of the inner ear progenitor cells subjected to directional differentiation for 12 days by a TRIzol method, performing reverse transcription on the total RNA to form cDNA, performing PCR by using primers of inner ear progenitor cell marker genes PAX2, PAX8, SIX1, DLX5, GATA3 and EYA1, and analyzing the expression condition of the products 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 for 4 ℃ incubation overnight, adding the fluorescent secondary antibody of the corresponding species 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 oocyst cells until the confluence reaches 90%, carrying out passage to obtain P1 generation cells, repeatedly culturing and carrying out passage to obtain P3 generation cells, culturing the chick embryo oocyst 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 inactivated chick embryo oocyst 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 plates 5 Inactivated chick embryo oocyst cells were cultured overnight.
(3) Digesting the cells with 0.05% pancreatin for 12 days after directional differentiation, counting the number of the cells after termination of digestion, inoculating the cells into 12-well plates at a rate of 1X 10/well 5 Culturing 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.
(4) Extracting total RNA of inner ear hair cell-like cells which are directionally differentiated for 14-28 days by a TRIzol method, performing reverse transcription on the total RNA to cDNA, performing 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, respectively adding ATOH1, MYO7A and POU4F3 antibodies into each hole after closing, incubating overnight at 4 ℃, rinsing by PBS, adding corresponding species of 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 (2)

1. The application of the method for inducing the directional differentiation of the pluripotent stem cells in obtaining the inner ear hair cell-like cells is characterized by comprising the following steps:
(1) iPSC was inoculated to a Laminin-coated plate and cultured for 10-12 days using an inner ear progenitor cell culture medium prepared by adding 1% by volume of N2supplement,2% by volume of B27 supplement, 500X of 0.2% by volume of Normocin, 1mmol/L of sodium pyruvate at a final concentration, 50ng/mL of FGF3, and 50ng/mL of FGF10 to DMEM/F12 medium under 37 ℃ and 5% CO 2 A constant temperature incubator in a humid environment;
(2) Inoculating the cells to a gelatin-coated culture plate containing inactivated chick embryo oocyst 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, wherein the inner ear hair cell culture medium is a culture medium prepared by adding 1 volume percent of N2supplement,2 volume percent of B27 supplement,500 multiplied by 0.2 percent of Normocin, 1mmol/L final concentration of sodium pyruvate, 1 mu mol/L all-trans retinoic acid and 20ng/mL EGF into a DMEM/F12 culture medium; 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.
2. The use according to claim 1, wherein the inactivation method of chicken embryo oocyst cells in step (2) comprises culturing chicken embryo oocyst cells in DMEM medium supplemented with mitomycin C at a final concentration of 2 μ g/mL for 3 hours, and rinsing the cells with PBS to obtain inactivated chicken embryo oocyst cells.
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