Patent Application
20230235282
The present invention belongs to biological field. Specifically, it relates to a method for inducing pluripotent stem cells into sensory neuron cells, an induction medium and uses thereof.
In 2006, Shinya Yamanaka's team invented a “cocktail” composed of four transcription factors (i.e., OCT4, SOX2, KLF4 and c-Myc), which could successfully reprogram terminally differentiated skin fibroblasts into stem cells with differentiation pluripotency. Such stem cells are called induced pluripotent stem cells (Cell, 2006, 124(4) pp. 663-676; Cell, 2007, 131(5) pp. 861-872). These stem cells have a similar differentiation potential to embryonic stem cells, and can form the three most basic germ layers in human development: ectoderm, mesoderm and endoderm, and can eventually form a variety of somatic cells. This invention breaks through the ethical limitation of using human embryonic stem cells in medicine, can solve the problem of immune rejection in cell transplantation therapy, and greatly expands the application potential of stem cell technology in clinical medicine.
Peripheral sensory neurons, known as nociceptors, can sense extreme temperatures and pressures, as well as detect damage-related chemicals; and can further convert these stimuli into electrical signals, which are transmitted to the central nervous system over long distances. The diversity of peripheral sensory neuron activation and information transmitted therefrom also contributes to the diversity of pain levels and types. Therefore, peripheral sensory neurons are the focus of pain mechanism research and development of analgesic medicines. Taking the analgesic medicines in nervous system medicines as an example, pain is essential to protect organs from serious injury or damage. Depending on the type, location, and intensity of pain generation, this information is transmitted to the spinal cord via sensory neurons in the DRG or trigeminal ganglia, and ultimately to the thalamus and cerebral cortex. The circuit mechanisms, biochemical responses and molecular interactions of pain are complex and highly variable. Alterations in nociceptor pathway can lead to allergic and chronic pain, which affects 20% of adults, including arthritis, neuralgia caused by infection, and cancer pain. Therefore, understanding the signaling and modulation of pain is important to develop better analgesic medicines. Non-human mammalian neurons (usually derived from rats) are generally used in traditional methods for medicine screening. However, these animal cell culture models are quite different from human physiology and cannot fully represent human physiological, pharmacological and pharmacodynamic responses. Therefore, many medicines in human clinical trials do not have similar effects to animal experiments, thereby leading to unnecessary risks and waste of early research and development.
Based on the great differentiation potential of induced pluripotent cells, human pluripotent stem cells are also widely used in medicine screening. By using sensory neurons differentiated from human pluripotent stem cells, it is possible to gain an in-depth understanding of the working principle of sensory neurons and how neurons interact with medicines to generate therapeutic mechanisms, thereby further discovering new therapeutic targets.
In the field of sensory nervous system, primary cell lines, immortalized cell lines, and trans-ion channel gene cell lines have been used for in vitro studies, but there are considerable limitations for the use of primary cells from patient/body donations, for example, the limited number of in vitro proliferation limits the use of primary cells in high-throughput screening; while immortalized cell lines and transgenic cell lines are quite different from primary cells in many indices, and cannot accurately represent the physiological function of natural cells. Therefore, human induced pluripotent stem cell (iPSC)-derived sensory neurons serve as a novel alternative that can be broadly applied to test new medicine candidates. From pluripotent stem cells to neural stem cells, there are currently two most widely used induction methods. The first is a two-step method: firstly, cells aggregate into clusters to form embryonic bodies, and then cells are further induced to become neurons. The disadvantage of this method is that the cell differentiation is not synchronized, and the purity of neuronal cells is not high; in addition, the use of animal-derived growth factors also restricts the clinical use of neuronal cells. The second method is Dual SMAD inhibition (Nat Biotechnol, 2009, 05; 27(5): 485). The neural stem cells obtained by this method can differentiate into other types of neuron cells. The mechanism of the method is to simulate the signaling pathway in early embryonic development by inhibiting the BMP and TGFB pathways, thereby inducing the generation of neural stem cells. In this way, sensory neurons can be induced in the presence of Knockout Serum Replacement (Nat Biotechnol., 2013, 30(7): 715-720). Feeder cells are not used in this method, but Knockout Serum Replacement is still needed, which may have a certain interference effect on medicine screening. In addition, some non-target cells are mixed in the induced cells, which may affect the purity of cells.
The present application discloses a serum-free and feeder cell-free directional induction method, which combines pure chemical factor induction and growth factor, simulates the signal pathway of early embryonic development, and can induce directional differentiation of human induced pluripotent stem cells from various sources into sensory neurons which express a variety of ion channel proteins, and have electrophysiological functions, thereby greatly expanding the use of induced nerve cells in medicine screening.
The present disclosure relates to molecules and culture medium for inducing into sensory neuron cells, and correspondingly induced and cultured sensory nerve precursor cells and sensory neuron cells.
Specifically, the present disclosure relates to the following aspects:
1. Use of LY2157299, RepSox, SB5253334 or TEW7179 in the induction of pluripotent stem cells to differentiate into sensory nerve precursor cells and sensory neuron cells.
2. The use of item 1, wherein the dosage of LY2157299, RepSox, SB5253334 or TEW7179 is 55 nM-25 μM, preferably 100 nM, 300 nM, 500 nM, 700 nM, 900 nM, 1 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, 17.5 μM, 20 μM or 22 μM.
3. Use of human-NT3 or CHIR98014 in culturing sensory neurons.
4. The use of item 3, wherein the dosage of human-NT3 is 0.1-49 ng/ml, or the amount of CHIR98014 is 0.5 nM-3 μM.
5. An induction medium, characterized in that a basal medium is formed by 50% (v/v) DMEM medium and 50% (v/v) neural basal medium, then the basal medium is supplemented with 0.5%-2% (v/v) N2 supplement (preferably 0.7%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7% (v/v)), 0.2%-3.1% (v/v) GS21 supplement or B27 supplement (preferably 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0% (v/v)), 0.1-15 mM HEPES buffer (preferably 1, 5, 10 mM), 5-22 mM glycine-glutamine monohydrate (preferably 10, 15, 20 mM), 12-180 nM LDN-193189 or LDN-193189 2HCl (preferably 20, 50, 70, 80, 90, 100, 110, 120, 150, 170 nM), and 55 nM-25 LY2157299, RepSox, SB5253334 or TEW7179 (preferably 100 nM, 300 nM, 500 nM, 700 nM, 900 nM, 1 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, 20 μM or 22 μM).
6. The induction medium of item 5, characterized in that a basal medium is formed by 50% (v/v) DMEM medium and 50% (v/v) neural basal medium, then the basal medium is supplemented with 1% (v/v) N2 supplement, 2% (v/v) GS21 supplement or B27 supplement, 0.5 mM HEPES buffer, 20 mM glycine-glutamine monohydrate, 100 nM LDN-193189 or LDN-193189 2HCl, and LY2157299, RepSox, SB5253334 or TEW7179 selected from 5 μM, 7.5 μM or 12.5 μM.
7. The induction medium of item 5 or 6, wherein the induction medium is formed as follows: a basal medium formed by 50% (v/v) DMEM medium and 50% (v/v) neural basal medium, then supplemented with 1% (v/v) N2 supplement, 2% (v/v) GS21 supplement or B27 supplement, 0.5 mM HEPES buffer, 20 mM glycine-glutamine monohydrate, 100 nM LDN-193189 or LDN-193189 2HCl, and LY2157299 selected from 5 μM, 7.5 μM or 12.5 μM; preferably, the sensory neuron cell induction medium is further supplemented with a serum replacement (such as serum albumin, transferrin, fatty acid), more preferably, the serum replacement is in purified form.
8. A sensory neuron cell medium, characterized in that the induction medium of any one of items 5-7, and is supplemented with 3-10 μM SU5402 (preferably 5 μM, 6 μM, 7 μM, 8 μM, 9 μM), 3-10 μM DAPT (4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM), 1-3 μM CHIR99021 (preferably 1.3 μM, 1.5 μM, 1.7 μM, 1.9 μM, 2.0 μM, 2.2 μM, 2.5 μM, 2.7 μM) or 0.5 nM-3 μM CHIR98014 (preferably 5 nM, 50 nM, 100 nM, 200 nM, 300 mM, 500 mM, 700 mM, 900 mM, 1 μM, 1.4 μM, 1.8 μM, 2.0 μM, 2.5 μM, 2.7 μM), and 0.1-49 ng/ml human-NT3 (preferably 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 ng/ml), preferably, the sensory neuron cell medium is further supplemented with a serum replacement (e.g., serum albumin, transferrin, fatty acid), more preferably, the serum replacement is in purified form.
9. A method for inducing pluripotent stem cells to differentiate into sensory neuron cells, characterized in that the pluripotent stem cells are cultured in the induction medium of any one of items 5-7 to obtain sensory nerve precursor cells, and then the sensory nerve precursor cells are cultured in the sensory neuron cell medium of item 8 to obtain sensory neuron cells, preferably, the culture is performed in the presence of a basal membrane, more preferably, the basal membrane is composed of one or more of Matrigel (STEMCELL Technologies), Laminin and Vitronectin.
10. Sensory nerve precursor cells, which are prepared by culturing pluripotent stem cells by using the induction medium of any one of items 5-7.
11. Sensory neuron cells, which are prepared by culturing the sensory nerve precursor cells of item 10 by using the sensory neuron cell medium of item 8.
12. A method for inducing sensory neuron cells comprising, culturing pluripotent stem cells by the induction medium of any one of items 5-7 to obtain sensory nerve precursor cells, and then culturing the sensory nerve precursor cells by the sensory neuron cell medium of item 8.
13. The method of item 9 or 12, wherein the pluripotent stem cells are prepared from somatic cells by using a reprogramming medium, preferably, wherein the reprogramming medium consists of a DMEM-F12 basal medium and supplementary components, the supplementary components include 60 μg/mL-180 μg/mL L-ascorbic acid, 5.3 μmol/L-74 μmol/L hydrocortisone, 3 ng/mL-89 ng/mL sodium selenite, 8 μmol/L-23 μmol/L Optiferrin, 0.5 μmol/L-7.4 μmol/L retinyl acetate, 40 ng/mL-60 ng/mL plant-derived recombinant human basic growth factor, 8 μg/mL-12 μg/mL IGF, 0.2 μmol/L-0.6 μg/mL A-83, 2 μmol/L-6 μmol/L CHIR99021, and 100 μmol/L-450 μmol/L sodium butyrate.
14. The method of item 13, characterized in that the supplementary components include 70 μg/mL-90 μg/mL L-ascorbic acid, 40 μmol/L-60 μmol/L hydrocortisone, 10 ng/mL-30 ng/mL sodium selenite, 8 μmol/L-12 Optiferrin, 3 μmol-6 μmol/L retinyl acetate, 45 ng/mL-55 ng/mL plant-derived recombinant human basic growth factor, 8 μg/mL-12 μg/mL IGF, 0.3 μmol/L-0.5 μg/mL A-83, 3 μmol/L-5 μmol/L CHIR99021 and 280 μmol/L-410 μmol/L sodium butyrate.
15. The method of item 13, characterized in that the supplementary components comprise 80 μg/mL L-ascorbic acid, 50 μmol/L hydrocortisone, 20 ng/mL sodium selenite, 10 μmol/L Optiferrin, 4 μmol/L retinyl acetate, 50 ng/mL plant-derived recombinant human basic growth factor, 10 μg/mL IGF, 0.4 μg/mL A-83, 4 μmol/L CHIR99021 and 400 μmol/L sodium butyrate.
16. The method of any one of items 12-15, wherein the culturing is performed in the presence of a basal membrane, preferably, the basal membrane is composed of one or more of Matrigel (STEMCELL Technologies), Laminin and Vitronectin.
17. The method of any one of items 13-15, wherein the somatic cells are human mesenchymal cells, human CD34+ cells or human fibroblasts (e.g. skin fibroblasts, lung fibroblasts, bladder fibroblasts, foreskin fibroblasts, uterine fibroblasts).
18. Use of the sensory neuron of item 11 in screening a medicine for treating or alleviating pain or preparing a model for screening a medicine for treating or alleviating pain.
In some embodiments, the preparation of human induced sensory neuron comprises the following steps:
Step 1: Human derived somatic cells are subjected to somatic reprogramming by a plasmid method to obtain induced pluripotent stem cells, which are then identified cytologically and biochemically.
Step 2: The induced pluripotent stem cells obtained in step 1 are cultured in an adherent monolayer in a serum-free sensory neuron induction medium that does not contain substances acting on BMP transduction pathway, but contains, for example, GS21 Cell culture supplements and amino acid hydrates; thereby obtaining neuron precursor cells. In a particular embodiment, the substances acting on BMP signal transduction pathway include one or more of the following proteins in any combination: BMP2, BMP4, BMP4, Smad1, Smad5, Smad8; wherein, the induced pluripotent stem cells are cultured adherently for 15 days by using the serum-free sensory neuron induction medium to obtain an adherent monolayer of sensory nerve precursor cells.
Step 3: The precursor cells in Step 2 are cultured adherently in a serum-free sensory neuron maturation medium that does not contain one or more substances acting on BMP transduction pathway, TGF transduction pathway, Notch transduction pathway or VEGFR pathways, but contains, for example, GS21 cell culture supplements, neurotrophic factors and amino acid hydrates; thereby obtaining mature sensory neuron cells expressing ion channel proteins and having electrophysiological activity. In a particular embodiment, the substances acting on BMP signal transduction pathway include one or more proteins independently selected from the group consisting of: BMP2, BMP4, BMP4, Smad1, Smad5, and Smad8; wherein the substances acting on TGF transduction pathway include one or more proteins independently selected from the group consisting of: Activin, TGF-beta, Nodal, Smad2, and Smad3; the substances acting on Notch transduction pathway include one or more proteins independently selected from the group consisting of: Notch, NICD, and r-Secretase; the substances acting on VEGFR transduction pathway include one or more proteins independently selected from the group consisting of: VEGFR2, FGFR1, and PDGF-Rβ. The neuron precursor cells are further cultured adherently for 15 days in a serum-free sensory neuron maturation medium to obtain an adherent monolayer of sensory neuron cells, which express ion channel proteins and have electrophysiological activities.
In a particular embodiment, the adherent culture in steps 1 to 3 is preferably performed in the presence of a basal membrane. The basal membrane of the present invention can form a thin film composed of extracellular matrix molecules on the surface of culture dishes, and can provide support similar to the in vivo environment for parameters such as cell morphology, growth and differentiation, and movement. In a particular embodiment, the basal membrane is composed of one or more of Matrigel (STEMCELL Technologies), Laminin and Vitronectin.
The serum-free medium in the present invention means that it does not contain serum directly separated from blood. Serum is the clear liquid portion of plasma that does not contain fibrinogen or blood cells and remains liquid after the blood clots. The serum-free medium may contain serum replacements. Examples of serum replacements include purified substances such as serum albumin, transferrin, fatty acids, etc., which are substances well known in the art that can substitute for serum.
In the present invention, the expressions “sensory neuron cell” and “sensory neuron” may be used interchangeably.
The present invention provides a new combination of chemical molecules, which can significantly improve the purity of induced sensory neurons in vitro and the expression intensity of functional ion channel proteins, and the formula of the present invention can support in vitro culture of induced peripheral neuron cells, thereby establishing a sensory neuron cell induction culture system without serum and animal-derived substances. Therefore, sensory neuron cells are obtained by reprogramming and directional differentiation of somatic cells from various sources, and they have various complete biological functions. The state is stable in multiple batches, and the reproducibility is good, which can solve the problem that sensory neuron cells are not easy to obtain in medicine screening and scientific research. The invention also solves the long-standing problems involving animal-derived culture methods and feeder cell contamination, and can be used for in vitro screening of medicines for neurological diseases, especially analgesic medicines, and has great economic and social effects.
The implementation process and beneficial effects of the present invention are described in detail below through specific examples, which are intended to help readers better understand the essence and characteristics of the present invention, and are not intended to limit the scope of the present application.
A 6-well culture plate was coated with Matrigel (STEMCELL Technologies), and then incubated in a 37° C. incubator for more than one hour. The following human somatic cells were reprogrammed to induced pluripotent stem cells by using a Neon Electrotransformation Kit (Thermo Fisher) when somatic cell reprogramming was performed using the Epi5 Reprogramming Kit (Invitrogen): Human Mesenchymal Cells (Lonza, PT-2501), human CD34+ cells (PromoCELL, c-12921), and human skin fibroblasts (PromoCELL, c-12302).
The steps were as follows: after centrifugation and washing, the human somatic cells were resuspended in a resuspension buffer provided in the electrotransformation kit, and the electrotransformation was performed according to the following procedures: 1650V pulse voltage, 10 ms pulse width, 3 pulses. After electrotransformation, the cells were removed into a 6 ml tube of reprogramming medium, mixed well, and added to each well of a 6-well plate. The conditions for cell culture were 37° C., 5% CO2 (Panasonic, MCO-18AC), and the cells were cultured by standing still. Thereafter, the medium was half quantity changed a day until the tenth day after electrotransformation, and the medium was changed every other day from the tenth day until the 28th day.
The medium and method used in the reprogramming process were implemented with reference to the patent “Reprogramming Medium and Culture Method for Reprogramming Induced Pluripotent Stem Cell” (ZL 201910050800.7). The reprogramming medium consisted of DMEM-F12 basal medium and supplementary components, wherein the supplementary components are 80 μg/ml ascorbic acid, 50 μmol/L hydrocortisone, 20 ng/ml sodium selenite, 10 μmol/L Optiferrin, 0.4 μg/ml IGF, 0.4 μg/ml A-83, 4 μmol/L CHIR9901 and 400 μmol/L sodium butyrate.
(2.1) The counting of induced pluripotent stem cell clones and identification of pluripotency were performed by using alkaline phosphatase staining (Cat #SCR004, Millipore). Firstly, the cell culture medium in the petri dish was aspirated, and the cells were washed with PBS, fixed for 1-2 min by adding 4% paraformaldehyde, then washed with TBST for 3 times, added with staining working solution provided in the kit (taking 24-well plate as an example, 0.5 ml per well), and placed at room temperature in the dark for 15-20 min; after staining, the staining solution was aspirated, and the cells were washed with phosphate buffer for 2-3 times, and the staining results were observed by a microscope (DMi8, Leica).
(2.2) Oct3/4 immunofluorescence identification of induced pluripotent stem cells by using immunofluorescence.
The induced pluripotent stem cells in Example 1 were respectively taken and identified by immunofluorescence staining as follows: cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes, washed twice with DPBS buffer; then treated with 0.1% Triton X-100 for 50 minutes, washed twice with DPBS buffer; then incubated overnight at 4° C. with DPBS buffer containing 10% horse serum and 0.1% Triton X-100; added with primary antibody diluted in DPBS buffer, incubated at 37° C. for 2 hours, washed three times with DPBS buffer; then added with corresponding secondary antibody diluted in DPBS buffer, incubated at 37° C. for 2 hours, washed three times with DPBS buffer; and then being subjected to nuclear staining with 0.1 μg/ml DAPI solution for 2 minutes, washed with DPBS buffer for three times, and then photographed (DMi8, Leica). The details for the antibodies used were shown in Table 1.
The results for alkaline phosphatase staining and immunofluorescence staining were shown in
(3.1) Induction of Sensory Nerve Precursor Cells
A T25 cell culture flask was coated with Matrigel (STEMCELL Technologies), and then incubated in a 37° C. incubator for more than one hour. Human induced pluripotent stem cells from various sources obtained in Example 1 were seeded in a T25 culture flask at 1×106 cells.
When the induced pluripotent stem cells reached 70% coverage, they were digested with 0.05% trypsin/EDTA for 5 min at 37° C., and the cell digestion was terminated by using DMEM. After the cells were washed and centrifuged, they were re-seeded in a T25 culture plate at 2×105 per flask, and cultured in a sensory neuron induction medium at 37° C., 5% CO2 (Panasonic, MCO-18AC) for 10 days until the formation of neural plate, which confirmed the formation of sensory nerve precursor cells.
The results of three different cells cultured in the sensory neuron induction medium were shown in
Formula for sensory neuron induction medium (N2GS212I-1) was as follows:
50% (v/v) DMEM medium and 50% (v/v) Neurobasal medium formed a basal medium, which was supplemented with 1% (v/v) N2 supplement, 2% (v/v) GS21 supplement, 0.5 mM HEPES buffer, 20 mM glycine-glutamine monohydrate, 100 nM LDN-193189, and 7.5 μM LY2157299.
(3.2) Induction of Sensory Nerve Precursor Cells by Using a Medium Containing Serum Components (Represented by CK, Also Referred to as “Serum Induction Method” in the Present Invention)
According to published method (Nat Biotechnol., 2013, 30(7): 715-720), three types of cells (fibroblasts, mesenchymal cells, and CD34+ cells) were induced to sensory nerve precursor cells by using a serum-containing media. The specific steps were as follows: The basal medium was prepared by 820 ml Knockout Duchenne's modified Eagle's medium (Knockout DMEM medium, Cat. No. 10829018, Thermo Fisher), 150 ml Knockout Serum Replacement (Cat. No. 10828028, Thermo Fisher), 1 mM L-Glutamine (Cat. No. 25030081, Thermo Fisher), 100 μM Minimum Essential Medium Non-Essential Amino Acids (Cat. No. 11140076, Thermo Fisher) and 0.1 mM 0-mercaptoethanol (Cat. No. 21985023, Thermo Fisher), and the basal medium was supplemented with 100 nM LDN-193189 and 10 μM SB431542 on Day 0 to Day 5. N2 medium was formed by adding 1% (v/v) N2 supplement (Cat. No. 17502045, Thermo Fisher) to neural basal medium (Neurobasal Medium, Cat. No. 21103049, Thermo Fisher). N2 medium was added every other day starting from day 4 in 25% increments (100% N2 on day 10). On day 2 to day 10, three inhibitors, 3 μM CHIR99021 (Selleck, Cat. No. S2924), 10 μM SU5402 (Tocris, Cat. No. 3300/1) and 10 μM DAPT (Selleck, Cat. No. S2215), were added to induce sensory nerve precursor cells-CK. The results were shown in
(3.3) Culture of Sensory Neuron Cells
After obtaining the above-mentioned various sensory nerve precursor cells, the medium was removed, and cells were washed with DPBS, and then treated with 0.05% trypsin/EDTA at 37° C. for 5 minutes, and then cell digestion was terminated with DMEM. After the cells were washed and centrifuged, the cells were re-seeded in a T25 culture flask at 1-5×105 per plate, and the sensory nerve precursor cells were induced to differentiate and expand into mature sensory neurons by using sensory neuron cell medium. The conditions for cell culture were 37° C., 5% CO2. The results were shown in
Sensory Neuron Cell Medium was prepared as follows: 10 μM SU5402 (Tocris, Cat. No. 3300/1), 10 μM DAPT (Selleck, Cat. No. S2714), 4.9 μM CHIR98014 (Selleck, Cat. No. S2745); 49 ng/ml human-NT3 (Novoprotein, Cat. No. C079) were added into Sensory Neuron Induction Medium N2GS212I-1.
(4.1) Identification of Molecular Markers Specific for Sensory Neurons
Induced sensory neurons were identified for marker expression by using immunofluorescence (immunofluorescence staining of multiple markers for different cell types). The various sensory neuron cells in Example 3 were respectively taken, and identified by immunofluorescence staining according to section (2.2) of Example 2. Details for antibodies used were shown in Table 2.
The immunofluorescence staining results of various sensory neuron cells obtained in Example 3 were shown in
a-l illustrate sensory neurons formed by using induced pluripotent stem cells from different sources, wherein the sensory neuron cells induced by the sensory neuron induction medium of the present invention had no significant difference in ion channel expression.
The sensory neurons produced by serum-induction of iPSCs from mesenchymal cells and fibroblasts were similar to those in
(4.2) Immunofluorescence Identification of the Expression and Localization of Various Ion Channel Proteins in Sensory Neuron Cells
Sensory neuron cells were identified for marker expression by using immunofluorescence. Taking the sensory neuron cells differentiated from iPSCs induced by CD34+ source of Example 3 as an example, immunofluorescence staining was carried out according to the description in section (2.2) of Example 2. The details for the antibodies used were shown in Table 3.
The results were shown in
The results showed that in present invention, a variety of somatic cells can be successfully induced into sensory neuron cells, expressing a series of sensory neuron-specific markers, and showing a higher level of marker expression compared with the serum induction method.
(4.3) Transcription changes of different marker genes during induction from pluripotent stem cells to sensory neurons were detected by Q-PCR.
Total RNA from different sensory neuron cells was extracted by RNeasy Mini or Micro Kit (QIAGEN), and cDNA was synthesized from 1 mg RNA by SuperScript III First-Strand Synthesis System (Invitrogen). SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa) were used for labeling and reaction of Quantitative PCR, and beta-Actin was used as internal control. All data were analyzed by delta-Ct method. Triplicates were used for each group of experiments, and statistics were performed with ANOVA. Primer sequences used to identify coding genes of different cellular markers were shown in Table 4.
Taking the sensory neuron cells obtained from CD34+ source by the method of Example 3 and the sensory neuron cells CK obtained by serum induction method as examples, the results were shown in Fig. Compared with the serum induction method, the sensory neuron cells obtained by the present invention had significant increase in the expressions of various important channel genes such as potassium ion channel genes (KV4.3, KCNIP2, KV7.2, KV7.3), calcium ion channel proteins (CaV2.1, CaV2.2, CaV3.2), potassium-calcium complex ion channels (KCa2.1, KCa2.2), sodium ion channels (SCN9A, SCN11A), aminobutyric acid (ABA) receptors (GABRA3), aspartate receptors (NR2A, NR2B), TRP ion channel family (TRPV1, TRPV4, TPRM8). The results showed that in present invention, a variety of somatic cells can be successfully induced into sensory neuron cells, expressing a series of sensory neuron-specific markers, and showing a higher level of marker expression compared with the sensory neuron cells obtained by serum induction method.
(4.4) Electrophysiological Activity Assay of Sensory Neuron Cells
(4.4.1) Spontaneous discharge signals of induced sensory neuron cells were detected by multi-channel electrodes. A 48-well or 96-well MEA system multi-channel electrode plate (AXION Biosystem, US) was coated with 100 ng/ml polylysine (Poly-L-lysine, Sigma-Aldrich, P4707) in an incubator (Panasonic, MCO-18AC) at 37° C., 5% CO2 for 12 hours; and then the poly-lysine-coated MEA multi-channel electrode plate was taken out, with the poly-lysine being removed, and then washed three times with sterile water. After that, a PBS solution containing 3 μg/ml gelatin (laminin 521) as a nerve cell coating matrix, was added to the MEA multi-channel electrode plate, and then the plated was coated in a cell incubator (Panasonic, MCO-18AC) at 37° C., 5% CO2 for 3 hours. After the MEA multi-channel electrode plate was completely coated, the induced sensory neuron precursor cells were seeded at 5×104 cells per well. The culture was performed by the sensory neuron cell medium of Example 3.3. The detection of spontaneous neuron electrical signals was performed after 14 days (i.e. the early stage of neuron maturation). The MEA multi-channel electrode plate for culturing induced sensory neuron cells was placed in a MEA chamber, and the cell culture conditions were adjusted in AxIS Navigator 2.0.2 software to 37° C., 5% carbon dioxide, and run for 10 minutes until the chamber environment was stable. Cell spontaneous discharge signals were recorded with AxIS Navigator 2.0.2 software (AXION Biosystem, US). The results were shown in
(4.4.2) The MEA plate and cells were prepared by the method of (4.4.1), and cultured for 20 days (i.e. the neuron mature period). 3 μM tetrodotoxin (TOCRIS, 1078/1) was used as non-specific sodium ion inhibitor to verify the electrophysiological activity of sodium ion channels on cells. The MEA plate was placed in a MEA chamber, and the cell culture condition was adjusted to 37° C., 5% carbon dioxide, running for 10 minutes until the chamber environment was stable, and then the spontaneous discharge of cells was detected. The cell electrode plate was taken out, and added with 3 μM tetrodotoxin per well for 10 minutes of reaction, and then the MEA plate was placed in the MEA chamber; the cell culture conditions were adjusted to 37° C., 5% carbon dioxide, running for 10 minutes until the chamber environment was stable; the spontaneous discharge of cells under the action of 3 μM tetrodotoxin was detected. Cell spontaneous discharge signals were recorded with AxIS Navigator 2.0.2 software (AXION Biosystem, US). Data analysis was performed by AxIS Metrictool and NruralMethics software (AXION Biosystem, US). The results were shown in
(4.4.3) The MEA plate and cells were prepared by the method of (4.4.2), and PF-05089771 (TOCRIS, 5931), a specific inhibitor of sodium ion channel 1.7, at a concentration of 1 μM was used to verify the electrophysiological activity of sodium ion channels 1.8 and 1.9 on cells. The MEA plate was placed in a MEA chamber, and the cell culture conditions were adjusted to 37° C., 5% carbon dioxide, running for 10 minutes until the chamber environment was stable, and then the spontaneous discharge of cells was detected. The cell electrode plate was taken out, and added with 1 μM PF-05089771 per well, and then the MEA plate was placed in the MEA chamber; the cell culture conditions were adjusted to 37° C., 5% carbon dioxide, running for 10 minutes until the chamber environment was stable; the spontaneous discharge of cells under the action of 1 μM PF-05089771 was detected. Cell spontaneous discharge signals were recorded with AxIS Navigator 2.0.2 software (AXION Biosystem, US). Data analysis was performed by AxIS Metrictool and NruralMethics software (AXION Biosystem, US). Taking the induction of fibroblasts as an example, the chemical small molecule induction method used in the present invention and the serum induction method were compared, and the results were shown in
(5.1) Morphology of Peripheral Sensory Cells Induced by Different LY2157299 Concentrations
The steps of Examples 3-4 were repeated by taking CD34+-derived cells as an example, and the effects of different concentrations of LY2157299 on sensory neuron cells were observed, including the effect on the induction of sensory nerve precursor cells, and the effect on the expression of specific markers of sensory neuron cells, and the effect on the electrophysiological activity.
The results were shown in
(5.2) Taking CD34+-derived cells as an example, different small molecules (5 μM RepSox, 5 μM SB525334 and 5 μM TEW-7197 molecules) were used to replace the LY2157299 in the sensory neuron induction medium N2GS212I-2 in Section (3.1) of Example 3; steps in Section (3.1) of Example 3 were repeated to observe the effects of different small molecules on the induced sensory nerve precursor cells. The results were shown in
(5.3) Taking CD34+-derived cells as an example, steps of Example 4 (4.3) were repeated by using the sensory nerve precursor cells obtained in (5.2), and the effects of different small molecules on the expression of neuronal precursor cell markers during the formation of sensory nerve precursor cells were observed. The results were shown in
(5.4) Effects of Different LDN Concentrations on the Formation of Induced Sensory Neuron Cells
The steps of Examples 3-4 were repeated by taking CD34+-derived cells as an example, and the effects of different concentrations of LDN (that is, low concentration (12 nM), medium concentration (50 nM) and high concentration (180 nM)) on sensory neuron cells were observed, including the effect on the induction of sensory nerve precursor cells, and the effect on the expression of specific markers of sensory neuron cells, and the effect on the electrophysiological activity. The results were shown in
(5.5) Effects of Different N2 Concentrations on the Formation of Induced Sensory Neuron Cells
The steps of Examples 3-4 were repeated by taking CD34+-derived cells as an example, and the effects of different concentrations of N2 (that is, low concentration (0.5%), medium concentration (0.5%) and high concentration (2%)) on sensory neuron cells were observed, including the effect on the induction of sensory nerve precursor cells, and the effect on the expression of specific markers of sensory neuron cells, and the effect on the electrophysiological activity. The results were shown in
(5.6) Effects of Different GS21 Concentrations on the Formation of Induced Sensory Neuron Cells
The steps of Examples 3-4 were repeated by taking CD34+-derived cells as an example, and the effects of different concentrations of GS21 (that is, low concentration (0.2%), medium concentration (2.0%) and high concentration (3.1%)) on sensory neuron cells were observed, including the effect on the induction of sensory nerve precursor cells, and the effect on the expression of specific markers of sensory neuron cells, and the effect on the electrophysiological activity. The results were shown in
(5.7) Effects of Different Concentrations of Glycine-Glutamine Monohydrate on the Formation of Induced Sensory Neuron Cells
The steps of Examples 3-4 were repeated by taking CD34+-derived cells as an example, and the effects of different concentrations of glycine-glutamine monohydrate (that is, low concentration (5 mM), medium concentration (12 mM) and high concentration (22 mM)) on sensory neuron cells were observed, including the effect on the induction of sensory nerve precursor cells, and the effect on the expression of specific markers of sensory neuron cells, and the effect on the electrophysiological activity. The results were shown in
In order to observe the effect of different concentrations of glycine-glutamine monohydrate on osmotic pressure, the effect of different concentrations of glycine-glutamine monohydrate on the osmotic pressure of culture system was detected by using an automatic freezing point osmometer (FM-8P, Shanghai Medical University Instrument Co., Ltd.), and specific operations were performed according to the product manual (FM-8P, Shanghai Medical University Instrument Co., Ltd.). The test results were shown in
(5.8) Effects of Different HEPES Buffer Concentrations on the Formation of Induced Sensory Neuron Cells
The steps of Examples 3-4 were repeated by taking CD34+-derived cells as an example, and the effects of different concentrations of HEPES buffer (that is, low concentration (5 mM), medium concentration (10 mM) and high concentration (15 mM)) on sensory neuron cells were observed, including the effect on the induction of sensory nerve precursor cells, and the effect on the expression of specific markers of sensory neuron cells, and the effect on the electrophysiological activity. The results were shown in
Steps in (5.7) were repeated to observe the effect of the medium with different HEPES buffer concentrations on the osmotic pressure. The results were shown in
(5.9) Effects of Different CHIR98014 Concentrations on the Formation of Peripheral Sensory Neuron Cells
The steps of Examples 3-4 were repeated by taking CD34+-derived cells as an example, and the effects of different concentrations of CHIR98014 (0.3 nM-5 μM) on sensory neuron cells were observed, including the effect on the induction of sensory nerve precursor cells, and the effect on the expression of specific markers of sensory neuron cells.
The results were shown in
(5.10) Effects of Different SU5402 Concentrations on the Formation of Peripheral Sensory Neuron Cells
The steps of Examples 3-4 were repeated by taking CD34+-derived cells as an example, and the effects of different concentrations of SU5402 (that is, low concentration (3 μM), medium concentration (6 μM) and high concentration (10 μM)) on sensory neuron cells were observed, including the effect on the induction of sensory nerve precursor cells, and the effect on the expression of specific markers of sensory neuron cells, and the effect on the electrophysiological activity. The results were shown in
a-c showed that cells could form axons at low concentration (3 μM), medium concentration (6 μM) and high concentration (10 μM) of SU5402;
(5.11) Effects of Different DAPT Concentrations on the Formation of Peripheral Sensory Neuron Cells
The steps of Examples 3-4 were repeated by taking CD34+-derived cells as an example, and the effects of different concentrations of DAPT (that is, low concentration (3 μM), medium concentration (6 μM) and high concentration (10 μM)) on sensory neuron cells were observed, including the effect on the induction of sensory nerve precursor cells, and the effect on the expression of specific markers of sensory neuron cells, and the effect on the electrophysiological activity. The results were shown in
a-c showed that cells were able to form axons at low (3 μM), medium (6 μM) and high (10 μM) concentrations of DAPT.
In conclusion, as compared with the known methods, in the absence of serum components, the combination of small molecules and concentrations thereof in the present invention significantly promoted the expressions of various functional ion channels in peripheral neuron cells.
The cell viability was quantitatively detected by Cyquant assay to compare the effects of different combinations of small molecule compounds on maintaining physiological states of cells during long-term culture of induced sensory neurons.
Cell plate coating was performed in a 96-well opaque cell culture plate according to the method in Example 4 (4.4.1). After the coating was completed, the induced nerve precursor cells were seeded at 5×104 cells per well, and three parallel replicates were set up (the average of the three groups was calculated as the data). The cells were cultured in the sensory neuron cell induction medium of Example 3 with different concentrations of NT3, and the culture conditions were 37° C., 5% carbon dioxide, and the medium was half quantity changed every three days. The medium in the known method was used as a control group. Samples were taken at day 1, day 3, day 5 and day 10, respectively, and the cell viability detection was carried out by using CyQuant Kit (Invitrogen, X12223) according to the instruction manual, and date were read by SpectraMax i3 Multi-Mode Microplate Reader (VWR, ID3-STD). Taking the induced sensory neurons derived from fibroblasts as an example, the results were shown in
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/091041 | 5/19/2020 | WO |
