Methods for differentiation of stem cells into caudal serotonergic neurons, culture media kits and uses thereof

Abstract

The application discloses a method for differentiating stem cells into caudal serotonergic neurons, a complete culture medium and application. Specifically, a new culture medium is formed by adding small molecule compounds, so that human pluripotent stem cells are gradually induced to obtain hindbrain neural stem cells, ventral hindbrain caudal neural stem cells, serotonergic precursor cells and caudal serotonergic neurons of the hindbrain. The culture method is simple, and can efficiently obtain mature serotonergic neurons of specific regions, thereby providing an effective cell model for research on diseases related to the serotonergic system.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a method for differentiating stem cells into caudal serotonin neurons, a complete set of culture media, and their applications. Background Technology

[0002] Serotonin neurons are distributed in the raphe nuclei of the hindbrain and can synthesize and secrete the neurotransmitter serotonin, which regulates human respiratory rhythm, sleep patterns, and emotions (Deneris, E.S. and S.C.Wyler, Serotonergic transcriptional networks and potential importance to mental health [J]. Nat Neurosci, 2012, 15(4): p. 519-27.). Studies on rodent embryonic development show that serotonin neurons originate from the most ventral side of the hindbrain and are distributed along the hindbrain. They are divided into two clusters by r4: r1 to r3 form the rostral cluster, and r5 to r8 form the caudal cluster. In adult rodents, serotonin neurons in the rostral cluster are distributed in the dorsal and medial raphe nuclei. Serotonin neurons in the dorsal raphe nuclei originate from r1 and project to the cortex, olfactory bulb, and paraventricular thalamic nucleus. Serotonin neurons in the medial raphe nucleus mainly originate from r2 and r3 and project to the olfactory bulb, hippocampus, and suprachiasmatic nucleus of the hypothalamus.

[0003] Serotonin neurons in the caudal cluster project downwards to regions such as the brainstem and spinal cord, playing roles in thermoregulation and respiratory regulation (Deneris, E. and P. Gaspar, Serotonin neuron development: shaping molecular and structural identities [J]. Wiley Interdiscip Rev Dev Biol, 2018.7). Sudden infant death syndrome (SIDS) is associated with abnormalities in the posterior caudal serotonin system (Kinney, HC, et al., The brainstem and serotonin in the sudden infant death syndrome [J]. Annu Rev Pathol, 2009.4: p.517-50.). However, a corresponding cell model is lacking to study the pathological mechanism, and the pathogenesis of this disease remains unclear. Human pluripotent stem cells possess the ability to differentiate into multiple lineages, and in vitro induction of differentiation can yield specific neuronal types. In 2016, Lu et al., through precise regulation of the WNT, SHH and FGF4 signaling pathways, for the first time directed the in vitro differentiation of human pluripotent stem cells into high-purity serotonin neurons in the r2-r3 region of the rostral side of the hindbrain (Lu, J., et al., Generation of serotonin neurons from human pluripotent stem cells[J].Nat Biotechnol,2016.34(1):p. 89-94.).

[0004] In 2021, Valilahi et al. obtained serotonin neurons in the caudal hindbrain by adding high concentrations of RA and purmorphamine in the early stage of differentiation. However, the high concentration of purmorphamine added in the early stage of differentiation resulted in a large number of floor plate cells in the final differentiation system, which affected the purity of serotonin neurons (Valiulahi P, Vidyawan V, Puspita L, et al. Generation of caudal-type serotonin neurons and hindbrain-fate organoids from hPSCs[J]. Stem Cell Reports, 2021.).

[0005] In summary, precise regulation of key signaling pathways in development is needed to direct the in vitro differentiation of pluripotent stem cells into high-purity hindbrain serotonin neurons, thereby providing an effective cell model for mechanistic research and drug screening of hindbrain serotonin system-related diseases such as sudden infant death syndrome. Summary of the Invention

[0006] The present invention aims to establish a method for the directed differentiation of human pluripotent stem cells into high-purity serotonin neurons in the hindbrain caudal region, providing a reliable cell model for the study of the mechanisms and phenotypes of nervous system-related diseases and for drug screening.

[0007] To achieve this objective, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a method for differentiating stem cells into caudal serotonin neurons, comprising the following steps:

[0009] S1. Culture pluripotent stem cells in E8 medium until the pluripotent stem cell confluence reaches 60-80%. Pass them to a new culture plate at a ratio of 1:3 and continue to culture in E8 medium.

[0010] S2. When the fusion rate of pluripotent stem cells reaches about 80%, replace E8 with the first culture medium and culture for 6-8 days.

[0011] S3. Digest cells with TrypLE cell digestion solution and passage them into Matrigel-coated well plates at a ratio of 1:5. Replace the medium with the second medium the next day and culture for 6-8 days.

[0012] S4. Digest cells with TrypLE cell digestion solution and passage them into Matrigel-coated well plates at a ratio of 1:3. Replace the medium with the third medium the next day and culture for 6-8 days.

[0013] S5. Digest cells using TrypLE cell digestion solution and at a concentration of 1.5–4.5 x 10⁻⁶. 4 / cm 2 Passed onto PO-Laminin-coated slides, the culture medium was changed to the fourth medium the next day. After culturing for 3-4 days, the culture medium was changed to neuronal differentiation medium. After culturing for another 2-4 weeks, mature serotonin neurons in the hindbrain caudal region could be obtained.

[0014] in,

[0015] The first culture medium includes: neural induction medium, SB431542, DMH1, and CHIR99021;

[0016] The second culture medium includes: neural induction medium, SB431542, DMH1, CHIR99021, purmorphamine, and RA;

[0017] The third culture medium includes: neural induction medium, SB431542, DMH1, CHIR99021, purmorphamine, RA, and FGF4;

[0018] The fourth culture medium includes: neural induction medium, SB431542, DMH1, CHIR99021, RA, and FGF4.

[0019] In this invention, SB431542 is a small molecule inhibitor of activin receptor-like kinase (ALK);

[0020] In this invention, DMH1 is a small molecule inhibitor of bone morphogenetic protein receptor (BMP);

[0021] In this invention, CHIR99021 is a small molecule inhibitor of glycogen synthase kinase 3 (GSK3);

[0022] In this invention, purmorphamine is a small molecule agonist of the Smo receptor;

[0023] In this invention, RA refers to all-trans retinoic acid;

[0024] In this invention, FGF4 refers to fibroblast growth factor 4.

[0025] Preferably, the neural induction medium includes DMEM / F12 medium, Neurobasal medium, 1×N2, 1×B27, 1×NEAA, and 1×GlutaMax.

[0026] Preferably, the neuronal differentiation culture medium includes Neurobasal medium, 1×N2, 1×B27, 1×NEAA, vitamin C, DAPT, GDNF, BDNF, TGFβ3, and IGF1.

[0027] Preferably, the volume ratio of DMEM / F12 medium to Neurobasal medium in the neural induction medium is 1:1.

[0028] Preferably, the molar concentration (μM) ratio of purmophamine and RA in the second culture medium is (0.4-1):(0.1-1).

[0029] Preferably, the molar concentration (μM) ratio of purmophamine and RA in the third culture medium is (1-2):(0.1-1).

[0030] Preferably, the molar concentration (μM) ratio of SB431542, DMH1, and CHIR99021 in the first culture medium is 2:2:(1-3).

[0031] Preferably, the second culture medium comprises SB431542, DMH1, CHIR99021, purmorphamine, and RA in a molar concentration (μM) ratio of 2:2:(1-3):(0.4-1):(0.1-1).

[0032] Preferably, the third culture medium comprises: SB431542, DMH1, CHIR99021, purmorphamine, and RA in a molar concentration (μM) ratio of 2:2:(1-3):(1-2):(0.1-1), and the concentration of FGF4 is 10 ng / ml.

[0033] Preferably, the fourth culture medium comprises: a molar concentration (μM) ratio (SB431542, DMH1, CHIR99021, RA) of 2:2:(1-3):(0.1-1), and an FGF4 concentration of 10 ng / ml.

[0034] A second aspect of the present invention provides a complete culture medium for the differentiation of stem cells into caudal serotonin neurons, comprising a first culture medium, a second culture medium, a third culture medium, and a fourth culture medium.

[0035] The first culture medium includes: neural induction medium, SB431542, DMH1, and CHIR99021;

[0036] The second culture medium includes: neural induction medium, SB431542, DMH1, CHIR99021, purmorphamine, and RA;

[0037] The third culture medium includes: neural induction medium, SB431542, DMH1, CHIR99021, purmorphamine, RA, and FGF4;

[0038] The fourth culture medium includes: neural induction medium, SB431542, DMH1, CHIR99021, RA, and FGF4.

[0039] Preferably, the molar concentration (μM) ratio of purmophamine and RA in the second culture medium is (0.4-1):(0.1-1).

[0040] Preferably, the molar concentration (μM) ratio of purmophamine and RA in the third culture medium is (1-2):(0.1-1).

[0041] Preferably, the neural induction medium includes DMEM / F12 medium, Neurobasal medium, 1×N2, 1×B27, 1×NEAA, and 1×GlutaMax.

[0042] Preferably, the molar concentration (μM) ratio of SB431542, DMH1, and CHIR99021 in the first culture medium is 2:2:(1-3).

[0043] Preferably, the second culture medium comprises SB431542, DMH1, CHIR99021, purmorphamine, and RA in a molar concentration (μM) ratio of 2:2:(1-3):(0.4-1):(0.1-1).

[0044] Preferably, the third culture medium comprises: SB431542, DMH1, CHIR99021, purmorphamine, and RA in a molar concentration (μM) ratio of 2:2:(1-3):(1-2):(0.1-1), and the concentration of FGF4 is 10 ng / ml.

[0045] Preferably, the fourth culture medium comprises: a molar concentration (μM) ratio (SB431542, DMH1, CHIR99021, RA) of 2:2:(1-3):(0.1-1), and an FGF4 concentration of 10 ng / ml.

[0046] A third aspect of the present invention provides the application of caudal serotonin neurons prepared by the method provided by the present invention in cell models for mechanistic studies and drug screening of diseases related to the caudal serotonin system in the hindbrain.

[0047] Preferably, the method includes the following steps:

[0048] S1. Culture pluripotent stem cells in E8 medium until the pluripotent stem cell confluence reaches 60-80%. Pass them to a new culture plate at a ratio of 1:3 and continue to culture in E8 medium.

[0049] S2. When the fusion rate of pluripotent stem cells reaches about 80%, replace E8 with the first culture medium and culture for 6-8 days.

[0050] S3. Digest cells with TrypLE cell digestion solution and passage them into Matrigel-coated well plates at a ratio of 1:5. Replace the medium with the second medium the next day and culture for 6-8 days.

[0051] S4. Digest cells with TrypLE cell digestion solution and passage them into Matrigel-coated well plates at a ratio of 1:3. Replace the medium with the third medium the next day and culture for 6-8 days.

[0052] S5. Digest cells using TrypLE cell digestion solution and at a concentration of 1.5–4.5 x 10⁻⁶. 4 / cm 2 Passed onto PO-Laminin-coated slides, the culture medium was changed to the fourth medium the next day. After culturing for 3-4 days, the culture medium was changed to neuronal differentiation medium. After culturing for another 2-4 weeks, mature serotonin neurons in the hindbrain caudal region could be obtained.

[0053] in,

[0054] The first culture medium includes: neural induction medium, SB431542, DMH1, and CHIR99021;

[0055] The second culture medium includes: neural induction medium, SB431542, DMH1, CHIR99021, purmorphamine, and RA;

[0056] The third culture medium includes: neural induction medium, SB431542, DMH1, CHIR99021, purmorphamine, RA, and FGF4;

[0057] The fourth culture medium includes: neural induction medium, SB431542, DMH1, CHIR99021, RA, and FGF4.

[0058] Compared with the prior art, the beneficial effects and significant progress of the present invention are as follows: The method, complete culture medium and application of stem cell differentiation into caudal serotonin neurons provided by the present invention are simple and efficient, and can obtain a large number of high-purity, functionally mature serotonin neurons in specific regions, providing an effective cell model for the study of related disease mechanisms as well as drug evaluation and screening. Attached Figure Description

[0059] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the embodiments of the present invention will be briefly introduced below.

[0060] Obviously, the accompanying drawings described below are only some of the drawings of the embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort, but these other drawings are also within the scope of the drawings required for the embodiments of the present invention.

[0061] Figure 1 The cell morphology of the human embryonic stem cell line H9 in the undifferentiated state in Example 1 of the present invention;

[0062] Figure 2 This is a flowchart illustrating the directed differentiation of human pluripotent stem cells into serotonin neurons in the hindbrain caudal region, as shown in Embodiment 2 of the present invention.

[0063] Figure 3 This is a morphological diagram of the different stages of serotonin neuron differentiation in the caudal hindbrain of Embodiment 2 of the present invention.

[0064] Figure 4 This is a cell immunofluorescence image of a marker for early differentiation of serotonin neurons in the caudal hindbrain of Example 3 of the present invention.

[0065] Figure 5 This is a cell immunofluorescence image of late-stage markers of hindbrain caudal neuronal differentiation in Example 3 of the present invention;

[0066] Figure 6 This is a cell immunofluorescence image of serotonin neuronal markers in the caudal hindbrain of Example 3 of the present invention;

[0067] Figure 7 This refers to the sodium-potassium current of the serotonin neurons in the posterior caudal lobes of the brain under voltage stimulation, as described in Embodiment 4 of the present invention.

[0068] Figure 8 These are the action potentials and spontaneous action potentials induced by electrical stimulation in the serotonin neurons on the caudal side of the hindbrain in Embodiment 4 of the present invention.

[0069] Figure 9 This is a diagram showing the serotonin neurotransmitter levels secreted by serotonin neurons in the caudal posterior part of the brain in Embodiment 5 of the present invention. Detailed Implementation

[0070] To make the objectives, technical solutions, beneficial effects, and significant advancements of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0071] Obviously, all the embodiments described are only some embodiments of the present invention, and not all embodiments; based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0072] What needs to be understood is:

[0073] The term "differentiation" refers to the process by which cells from the same origin gradually develop into cell groups with different morphological structures and functional characteristics. The result is spatial differences between cells and temporal differences between the same cell and its previous state. The essence of cell differentiation is the selective expression of the genome in time and space, ultimately producing marker proteins through the switching on or off of different gene expression.

[0074] The term "culture medium" refers to artificially prepared nutrients for the growth and maintenance of microorganisms, plant tissues, and animal tissues. It generally contains carbohydrates, nitrogenous substances, inorganic salts (including trace elements), vitamins, and water.

[0075] Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0076] It should also be noted that the following specific embodiments can be combined with each other, and the same or similar concepts or processes may not be described again in some embodiments.

[0077] The technical solution of the present invention will now be described in detail with reference to specific embodiments.

[0078] Example 1: Culture of human embryonic stem cells

[0079] Use E8 medium (TeSR) TM -E8 TM Human embryonic stem cell line H9 (WA09, WiCell) was cultured using Stem Cell Technology (catalog number 05990). The cells adhered to Matrigel (Corning, catalog number 354277) coated six-well plates. The culture medium was changed daily, and the cells were cultured for 4-6 days. When the cell confluence reached about 80%, the cells were passaged.

[0080] The specific steps for cell passage are as follows:

[0081] 1. Matrigel coating of well plates: Dilute Matrigel to pre-cooled high-sugar DMEM (Gibco, catalog number 11995065) at 4°C according to the dilution ratio in the instructions, then add it to the well plates, incubate at 37°C for 30 minutes, and remove it before passage.

[0082] 2. Aspirate the cell culture medium, wash once with DPBS (SIGMA), and add ReLeSR. TM Cell digestion solution (STEMCELL Technologies, catalog number 05872) was aspirated within 1 minute and left at room temperature for 7 minutes;

[0083] 3. After digestion, add E8 to resuspend the cells.

[0084] 4. Pass the cells at a 1:3 ratio and seed them into pre-coated Substrate gel-coated six-well plates. Culture in E8 medium, changing the medium daily and observing cell status. Cell morphology should be as follows: Figure 1 As shown.

[0085] Example 2: Directed Differentiation of Human Embryonic Stem Cells into Serotonin Neurons in the Caudal Side of the Back of the Brain

[0086] The steps of directed differentiation of human embryonic stem cells into serotonin neurons in the posterior tail of the brain are as follows: Figure 2 As shown, the specific steps are as follows:

[0087] Step 1: Differentiation of human embryonic stem cells into hindbrain neuroepithelial cells

[0088] When the fusion rate of the embryonic stem cells in Example 1 reaches about 80%, the medium is replaced with a neural induction medium (first medium) containing compound component 1, cultured for 6-8 days, and then passaged using TrypLE digestive enzyme.

[0089] The basal medium for neural induction contained DMEM / F12 (Gibco, catalog number 11330-032): Neurobasal medium (Gibco, catalog number 21103049) (volume ratio 1:1), 1×N2 (Gibco, catalog number 17502048), 1×B27 (Gibco, catalog number 12587010), 1×NEAA (Gibco, catalog number 11140050), and 1×GlutaMax (Gibco, catalog number 35050061). Compound component 1 contained: 2 μM SB431542, 2 μM DMH1, and 1.8 μM CHIR99021 (all small molecule compounds were purchased from Taoshu Biotechnology).

[0090] The specific digestion steps are as follows:

[0091] 1. Matrigel coating of well plates: Dilute Matrigel to pre-cooled high-sugar DMEM at 4°C according to the dilution ratio in the instructions, then add it to the well plates, incubate at 37°C for 30 minutes, and remove it before passage;

[0092] 2. After 6-8 days of neural induction culture, aspirate the culture medium, wash once with DPBS, add TrypLE (Gibco, catalog number 12604021) and wash once, aspirate the digestion solution, and incubate the cells in an incubator at 37°C for 4 minutes.

[0093] 3. Add neural induction medium containing compound combination 1 and add 10 μM Y27632 (Taoshu Biotechnology), resuspend the cells and passage them into well plates coated with matrix gel at a ratio of 1:5.

[0094] Step 2: Ventral and caudal transformation of hindbrain neuroepithelial cells

[0095] After obtaining the hindbrain neuroepithelial cells, on the second day of passage (step 1), the medium was replaced with a neural induction medium (second medium) containing compound component 2. The cells were cultured for 6-8 days and then passaged using TrypLE digestive enzyme.

[0096] The basal medium for neural induction contained DMEM / F12:Neurobasal medium (volume ratio 1:1), 1×N2, 1×B27, 1×NEAA, and 1×GlutaMax. Compound component 2 contained: 2 μM SB431542, 2 μM MMH1, 1.8 μM CHIR990210, 0.5 μM purmorphamine, and 100 nM RA.

[0097] The specific digestion steps are as follows:

[0098] 1. Matrigel coating of well plates: Dilute Matrigel to pre-cooled high-sugar DMEM at 4°C according to the dilution ratio in the instructions, then add it to the well plates, incubate at 37°C for 30 minutes, and remove it before passage;

[0099] 2. After 6-8 days of neural induction culture, aspirate the culture medium, wash once with DPBS, rinse once with TrypLE, aspirate the digestion solution, and incubate the cells at 37°C for 4 minutes.

[0100] 3. Add neural induction medium containing compound combination 2 and add 10 μM Y27632. After resuspending the cells, passage them into well plates coated with matrix gel at a ratio of 1:3.

[0101] Step 3: Differentiation of serotonin-producing neural progenitor cells in the caudal hindbrain

[0102] After obtaining the neuroepithelial cells from the ventral caudal side of the hindbrain (step 2), on the second day of passage, the medium was replaced with a neural induction medium (third medium) containing compound component 3. The cells were cultured for 6-8 days and then passaged using TrypLE digestive enzyme.

[0103] The basal medium for neural induction contains DMEM / F12:Neurobasal medium (volume ratio 1:1), 1×N2, 1×B27, 1×NEAA, and 1×GlutaMax.

[0104] Compound component 3 contains: 2 μM SB431542, 2 μM DMH1, 1.8 μM CHIR990210, 2 μM purmorphamine, 100 nM RA, and 10 ng / ml FGF4 (PeproTech, catalog number 100-31).

[0105] The specific digestion steps are as follows:

[0106] 1. PO-Laminin coated slides: Dilute polyornithine (PO, SIGMA) 200 times with sterile water, add 50 μL to each 12 mm diameter slide, coat overnight at room temperature, aspirate PO the next day, wash twice with sterile water, air dry, add laminin (Thermo Fisher, catalog number 23017015) diluted 50 times with DMEM / F12, coat at 37℃ for 3 hours, aspirate before passage;

[0107] 2. After 6-8 days of neural induction culture, aspirate the culture medium, wash once with DPBS, rinse once with TrypLE, aspirate the digestion solution, and incubate the cells at 37°C for 4 minutes.

[0108] 3. Add neural induction medium containing compound combination 3 and 10 μM Y27632, resuspend the cells, and then place them on 1.5–4.5 x 10⁻¹² glass slides (12 mm in diameter). 4 / cm 2 Cell seeding.

[0109] 4. Differentiation and maturation of serotonin neurons in the posterior caudal region of the brain: On the second day of passage in step 3), the medium was replaced with a neuroinduction medium (fourth medium) containing compound component 4. After culturing for 3-4 days, the medium was replaced with a neuronal differentiation medium and cultured for 2-4 weeks.

[0110] The neuronal differentiation medium contains the following components: Neurobasal medium, 1×N2, 1×B27, 1×NEAA, 0.2mM vitamin C (Sigma-Ardrich, catalog number A4403), 2.5μM DAPT (Taoshu Biotechnology, catalog number T6202), 10ng / ml GDNF (PeproTech, catalog number 450-10), 10ng / ml BDNF (PeproTech, catalog number 450-02), 1ng / ml TGFβ3 (PeproTech, catalog number 100-36E), and 10ng / ml IGF1 (PeproTech, catalog number 100-11).

[0111] Compound 4 includes: 2 μM SB431542, 2 μM DMH1, 1-3 μM CHIR99021, 0.1-1 μM RA, and 10 ng / ml FGF4.

[0112] The morphological diagrams of the different stages of serotonin neuron differentiation in the hindbrain and caudal lobes of this embodiment are shown below. Figure 3 As shown.

[0113] Example 3: Cell immunofluorescence assay to identify marker protein expression at different stages of differentiation

[0114] Cell immunofluorescence identification was performed on slides from the second day of digestion and passage in step 3 of the induction process in Example 2, as well as cell slides from 3 days and 2-3 weeks of culture in step 4. The specific steps are as follows:

[0115] 3.1. Aspirate the culture medium, wash once with DPBS, and then fix with 4% paraformaldehyde at room temperature for half an hour;

[0116] 3.2. Remove paraformaldehyde and wash three times with DPBS;

[0117] 3.3. Remove DPBS and add blocking solution. The blocking solution consists of DPBS containing 10% (v / v) donkey serum and 0.2% (v / v) Triton X-100. Incubate at room temperature for half an hour.

[0118] 3.4 Dilute the primary antibody with DPBS containing 5% (v / v) donkey serum and 0.2% (v / v) Triton X-100. See Table 1 for details.

[0119] 3.5. Remove the blocking solution, add the diluted primary antibody, and incubate overnight at 4°C.

[0120] 3.6. Remove the primary antibody, add DPBS and wash three times, 5 minutes each time.

[0121] 3.7 Dilute the secondary antibody and DAPI with DPBS containing 5% (v / v) donkey serum.

[0122] 3.8. Remove DPBS, add diluted secondary antibody and DAPI, and incubate at room temperature in the dark for 45 minutes.

[0123] 3.9. Remove the secondary antibody, add DPBS and wash three times, 5 minutes each time.

[0124] 3.10. Use anti-quenching sealing medium to seal the film.

[0125] 3.11. Observe and photograph using a fluorescence microscope.

[0126] Cell immunofluorescence identification image as shown Figure 4 , Figure 5 As shown. Figure 4 The markers are HOXB4, OLIG2, and NKX2.2, which are precursor cells of neurons in the posterior lateral hindbrain on day 21 of differentiation. HOXB4 is a marker of the posterior hindbrain caudal hindbrain, NKX2.2 is a marker of neuronal precursor cells in the ventral hindbrain, and OLIG2 is a marker of motor neurons. Figure 5 The results showed that the serotonin precursor cell markers in the hindbrain caudal lateral cerebral region were positive for HOXB4 and NKX2.2, but negative for OLIG2. Figure 6The presence of 5-HT and Tuj1 markers during the 3-week culture period of neurons indicates the formation of mature serotonin neurons in the hindbrain caudal region.

[0127] Table 1. Antibody information for cell immunofluorescence experiments.

[0128]

[0129] Example 4: Whole-cell patch-clamp assay to detect the electrophysiological function of serotonin neurons in the hindbrain derived from human embryonic stem cell differentiation.

[0130] After the electrode is drawn, it is filled with electrode fluid with a resistance of 6-8 MΩ. Before the electrode is immersed in the fluid, a weak positive pressure is applied inside through a conduit connected to the electrode holder. Once the tip begins to contact the cell and prepare for sealing, the conduit is immediately opened and a constant negative pressure is applied through it. After the sealing impedance reaches the GΩ level, the membrane potential is maintained at -60 mV. The adherent cells are then moved below the drug delivery tube of the rapid drug delivery perfusion system for the next experimental step. Whole-cell electrode fluid: 20 mM KCl, 10 mM Na + -HEPES, 121mM K + -gluconate, 10mM BAPTA, 4mM Mg 2+ -ATP (adjusted to pH 7.2, stored at 0℃). Whole-cell electrode extracellular solution: 127mM NaCl, 1.9mM KCl, 2.2mM CaCl2, 1.2mM KH2PO4, 26mM NaHCO3, 1.4mM MgSO4, 10mM glucose (adjusted to pH 7.3). Experiments were conducted at room temperature (23-25℃), with a filtering frequency of 1kHz. Clamp voltage commands and current recording were controlled using Clampex 10.3 (Molecular Devices) software via a DigiData-1440A converter interface.

[0131] Whole-cell sodium-potassium current recording

[0132] Under voltage clamping, with an injection voltage of -40 to 30 mV, and stimulation in 5 mV increments, the inward sodium current and outward potassium current were recorded, as follows: Figure 7 As shown, this indicates that serotonin neurons derived from differentiation possess mature sodium and potassium ion channels.

[0133] Action potential recording

[0134] Under current clamping, with a clamping current of 0 pA, and an injection of -40 to 100 pA, action potentials are generated under a 10 pA step stimulus, such as... Figure 8 As shown, this demonstrates that serotonergic neurons derived from differentiation possess mature electrophysiological characteristics.

[0135] Example 5: Enzyme-linked immunosorbent assay (ELISA) to detect the neurotransmitter secretion function of serotonin neurons in the hindbrain derived from human embryonic stem cell differentiation.

[0136] Cell culture media from serotonin-producing neurons at week 5 of differentiation and from non-serotonin-producing neurons that had been incubated overnight were collected. The serotonin content in the collected culture media was detected using an enzyme-linked immunosorbent assay (ELISA) kit (IBL, RE59141). Figure 9 As shown, extracellular serotonin levels were undetectable in non-serotonin neurons (NC, negative control) (Notdecteble, ND), while the levels of serotonin from differentiation-derived neurons (cSNs) were significantly higher than those from non-serotonin neurons. This indicates that serotonin neurons from the caudal hindbrain derived from differentiation possess normal neurotransmitter synthesis and secretion functions.

[0137] Comparative Example 1

[0138] 1. Obtain hindbrain neuroepithelial cells according to step 1 of Example 2;

[0139] 2. In compound component 2 of step 2 of Example 2, purmophamine and RA were added on the 7th day of differentiation to promote hindbrain caudal and ventral differentiation. We tried to induce differentiation for one week under 0 / 0.25 / 0.5 / 1 / 2μM purmophamine and 100nM RA conditions.

[0140] 3. Cell immunofluorescence assay to detect the proportion of ventral neural progenitor cell marker NKX2.2 and basal plate cell marker FOXA2 positive cells in cells differentiated for 14 days.

[0141] The results showed that the proportion of NKX2.2, a marker of ventral neural progenitor cells, was low in the 0.25 μM purmophamine group, while the proportion of FOXA2, a marker of basal plate cells, was high in the 2 μM purmophamine group. The 0.5 and 1 μM groups showed the best cell differentiation effects.

[0142] Comparative Example 2

[0143] 1. Obtain hindbrain neuroepithelial cells according to step 1 of Example 2.

[0144] 2. Obtain ventral hindbrain neural stem cells according to step 2 of Example 2.

[0145] 3. In compound component 3 of step 3 of Example 2, purmophamine, RA and FGF4 were added on day 14 of differentiation to further promote the differentiation of serotonin precursor. We tried to induce differentiation for one week with 0.5 / 2 μM purmophamine.

[0146] 4. Cell immunofluorescence assay to detect the proportion of ventral neural progenitor cell marker NKX2.2 and basal plate cell marker FOXA2 positive cells in cells differentiated for 21 days.

[0147] The results showed that the proportion of NKX2.2, a marker of ventral neural progenitor cells, was lower in the 0.5 μM purmophamine group, while the proportion of NKX2.2 was higher in the 2 μM purmophamine group, and the level of FOXA2, a marker of basal plate cells, remained low. Increasing the concentration of purmophamine from 0.5 μM to 2 μM in the later stage of differentiation promoted ventral differentiation without affecting differentiation into basal plate cells.

[0148] Comparative Example 3

[0149] 1. Obtain hindbrain neuroepithelial cells according to step 1 of Example 2.

[0150] 2. Obtain ventral hindbrain neural stem cells according to step 2 of Example 2.

[0151] 3. Obtain neuronal precursor cells from the ventral side of the hindbrain according to step 3 of Example 2.

[0152] 4. After digesting the precursor cells from step 3 in Example 2, seed them onto a glass slide, and then directly replace the neuron differentiation culture medium the next day.

[0153] 5. The proportion of serotonin neurons was detected by cellular immunofluorescence assay 45 days after neural differentiation. The results showed that the proportion of 5-HT-positive serotonin neurons was lower than that of serotonin neurons obtained in Example 2.

[0154] In the description process of the above instruction manual:

[0155] The terms "this embodiment," "an embodiment of the present invention," "as shown," "further," and "further improved technical solutions," etc., indicate that the specific features, structures, materials, or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms are not necessarily directed at the same embodiment or example, and the specific features, structures, materials, or characteristics described can be combined or combined in any suitable manner in one or more embodiments or examples. Furthermore, without causing contradiction, those skilled in the art can combine or combine the different embodiments or examples described in this specification and the features of the different embodiments or examples.

[0156] Finally, it should be noted that:

[0157] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them;

[0158] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Non-essential improvements, adjustments or substitutions made by those skilled in the art based on the content of this specification are all within the scope of protection claimed by the present invention.

Claims

1. A method for differentiating stem cells into caudal serotonin neurons, characterized in that, Includes the following steps: S1. Culture human embryonic stem cells in E8 medium until the fusion rate of human embryonic stem cells reaches 60-80%. Pass them to a new culture plate at a ratio of 1:3 and continue to culture in E8 medium. S2. When the fusion rate of human embryonic stem cells reaches about 80%, replace E8 with the first culture medium and culture for 6-7 days. The first culture medium is neural induction medium, SB431542, DMH1 and CHIR99021, wherein the molar concentration ratio of SB431542, DMH1 and CHIR99021 is 2:2: (1-3). S3. Digest cells using TrypLE cell digestion solution and passage them at a 1:5 ratio into Matrigel-coated wells. Replace the medium with a second culture medium the next day and culture for 6-8 days. The second culture medium consists of neural induction medium, SB431542, DMH1, CHIR99021, purmorphamine, and RA. The molar ratio of SB431542, DMH1, CHIR99021, purmorphamine, and RA is 2:2: (1-3): (0.4-1): (0.1-1). S4. Digest cells using TrypLE cell digestion solution and passage them in Matrigel-coated wells at a ratio of 1:

3. Replace the medium with a third culture medium the next day and culture for 6-8 days. The third culture medium consists of neural induction medium, SB431542, DMH1, CHIR99021, purmorphamine, RA, and FGF4. The molar ratio of SB431542, DMH1, CHIR99021, purmorphamine, and RA is 2:2:(1-3):(1-2):(0.1-1), and the concentration of FGF4 is 10 ng / ml. S5. Digest cells using TrypLE cell digestion solution and at a concentration of 1.5–4.5 x 10⁻⁶. 4 / cm 2 The culture medium was passaged onto PO-Laminin-coated slides, and the medium was replaced with a fourth culture medium the next day. After culturing for 3-4 days, the medium was replaced with neuronal differentiation medium, and cultured for another 2-4 weeks to obtain mature serotonin neurons in the hindbrain caudal region. The fourth culture medium consisted of neuroinduction medium, SB431542, DMH1, CHIR99021, RA, and FGF4. The molar ratio of SB431542, DMH1, CHIR99021, and RA was 2:2:(1-3):(0.1-1), and the concentration of FGF4 was 10 ng / ml. The neural induction medium is DMEM / F12 medium, Neurobasal medium, 1×N2, 1×B27, 1×NEAA, and 1×GlutaMax, wherein the volume ratio of DMEM / F12 medium to Neurobasal medium is 1:

1. The neuronal differentiation culture medium consisted of Neurobasal medium, 1×N2, 1×B27, 1×NEAA, vitamin C, DAPT, GDNF, BDNF, TGFβ3, and IGF1.

2. The method as described in claim 1, characterized in that, The caudal serotonin neurons prepared by the method are used in cell models for in vitro mechanistic studies and drug screening of nervous system-related diseases.

3. A complete culture medium for differentiating stem cells into caudal serotonin neurons, characterized in that, It consists of a first culture medium, a second culture medium, a third culture medium, a fourth culture medium, and a neuron differentiation culture medium. The first culture medium is: neural induction medium, SB431542, DMH1, CHIR99021, wherein the molar ratio of SB431542, DMH1, CHIR99021 is 2:2: (1-3); The second culture medium consisted of: neural induction medium, SB431542, DMH1, CHIR99021, purmorphamine, and RA, wherein the molar ratio of SB431542, DMH1, CHIR99021, purmorphamine, and RA was 2:2: (1-3): (0.4-1): (0.1-1). The third culture medium consists of: neural induction medium, SB431542, DMH1, CHIR99021, purmorphamine, RA, and FGF4, wherein the molar ratio of SB431542, DMH1, CHIR99021, purmorphamine, and RA is 2:2:(1-3):(1-2):(0.1-1), and the concentration of FGF4 is 10 ng / ml. The fourth culture medium is: neural induction medium, SB431542, DMH1, CHIR99021, RA, and FGF4, wherein the molar concentration ratio of SB431542, DMH1, CHIR99021, and RA is 2:2: (1-3): (0.1-1), and the concentration of FGF4 is 10 ng / ml; The neural induction medium is DMEM / F12 medium, Neurobasal medium, 1×N2, 1×B27, 1×NEAA, and 1×GlutaMax, wherein the volume ratio of DMEM / F12 medium to Neurobasal medium is 1:

1. The neuronal differentiation culture medium consisted of Neurobasal medium, 1×N2, 1×B27, 1×NEAA, vitamin C, DAPT, GDNF, BDNF, TGFβ3, and IGF1.

Images