CN115584343A - Method for stem cell differentiation of caudal serotonin neurons, complete culture medium and application - Google Patents

Method for stem cell differentiation of caudal serotonin neurons, complete culture medium and application Download PDF

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CN115584343A
CN115584343A CN202211190987.9A CN202211190987A CN115584343A CN 115584343 A CN115584343 A CN 115584343A CN 202211190987 A CN202211190987 A CN 202211190987A CN 115584343 A CN115584343 A CN 115584343A
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陆建峰
许婷
曹立宁
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Tongji University
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Abstract

The invention discloses a method for differentiating caudal serotonin neurons by stem cells, a complete set of culture medium and application, and particularly relates to a novel culture medium formed by adding a small molecular compound, so that human pluripotent stem cells are gradually induced to obtain hindbrain neural stem cells, ventral hindbrain caudal neural stem cells, serotonin precursor cells and hindbrain caudal serotonin neurons. The culture method is simple and convenient, can efficiently obtain mature serotonin neurons in a specific region, and provides an effective cell model for the research of related diseases of a serotonin system.

Description

Method for stem cell differentiation of caudal serotonin neurons, complete culture medium and application
Technical Field
The invention relates to the field of biological medicines, in particular to a method for differentiating caudal serotonin neurons by stem cells, a complete set of culture medium and application.
Background
Serotonin neurons are distributed in the posterior suture nucleus, and are capable of synthesizing and secreting serotonin neurotransmitters, regulating the respiratory rhythm, sleep patterns, mood, etc. in humans (Deneris, e.s. and s.c. wyler, sensorial transport networks and social interaction to mental health J. Nat Neurosci,2012.15 (4): p.519-27). Embryonic development studies in rodents have shown that serotonin neurons originate most ventrally in the hindbrain, distributed along the hindbrain. The two clusters are separated by r4, the r1 to r3 are beak clusters, and the r5 to r8 are tail clusters. In adult rodents, the rostral cluster of serotonin neurons is distributed in the dorsal and ventral raphe nuclei, with the serotonin neurons of the dorsal raphe nuclei originating from r1, projecting into the cortex, olfactory bulb and paraventricular thalamic nuclei. Serotonin neurons of the raphe nucleus originate mainly from r2 and r3, project to the olfactory bulb and hippocampus and the suprachiasmatic nucleus of the hypothalamus.
The serotonergic neurons of The tail cluster project down to areas such as The brainstem and spinal cord, and have functions such as thermoregulation and respiratory regulation (Deneris, e.and p. Gaspar, serotonin neuron depth: mapping molecular and structural identities [ J ]. Wiley interniscip Rev Biol, 2018.7), and sudden infant death syndrome is associated with abnormalities in The hindbrain caudal serotonergic system (Kinney, h.c., et al., the brazinten and Serotonin in The summer amino acid loss syndrome [ J ]. Annu Rev pathway, 2009.4 p.517-50.. However, the pathogenesis of the disease is not clear, and no corresponding cell model is available for studying the pathogenesis. The human pluripotent stem cells have the capacity of multidirectional differentiation, and specific neuron types can be obtained by in vitro induced differentiation. 2016 Lu et al, by precisely regulating WNT, SHH and FGF4 signaling pathways, first directed differentiation of human pluripotent stem cells into high purity serotonin neurons in the r2-r3 region on the rostral posterior brain beak side in vitro (Lu, J., et al, generation of serotonin neurons from human pluripotent cells [ J ]. Nat Biotechnol,2016.34 (1): p. 89-94.) was performed in vitro.
Valilahii et al obtained hindbrain-caudal serotonin neurons by adding high concentrations of RA and purmorphamine early in differentiation, but this method added high concentrations of purmorphamine early in differentiation, resulting in the presence of large numbers of Floor plate cells in the final differentiation system, affecting the purity of serotonin neurons (Valilahii P, vidyawan V, puspita L, et al. Generation of cad-type serotonin neurons and hindbrain-tissue oligonucleotides from hPSCs [ J ]. Stem Cell Reports, 2021.).
In conclusion, a key signal path for precisely regulating and controlling development is needed to directionally differentiate pluripotent stem cells into high-purity hindbrain caudal serotonin neurons in vitro, so that an effective cell model is provided for mechanism research and drug screening of diseases related to the hindbrain caudal serotonin system such as sudden infant death syndrome and the like.
Disclosure of Invention
The invention aims to establish a method for directionally differentiating human pluripotent stem cells into high-purity hindbrain caudal serotonin neurons, and provides a reliable cell model for mechanism and phenotype research of nervous system related diseases and drug screening.
To achieve the purpose, the invention provides the following technical scheme:
in a first aspect of the present invention, there is provided a method for differentiating caudal serotonin neurons from stem cells, comprising the steps of:
s1, culturing the pluripotent stem cells by using an E8 culture medium, wherein the fusion degree of the pluripotent stem cells reaches 60-80%, carrying out passage to a new culture plate according to 1:3, and continuously culturing by using E8;
s2, when the fusion degree of the pluripotent stem cells reaches about 80%, replacing the E8 with a first culture medium, and culturing for 6-8 days;
s3, digesting the cells by using trypLE cell digestive juice, carrying out passage to a Matrigel coated pore plate according to 1:5, replacing the cell digestive juice with a second culture medium the next day, and culturing for 6-8 days;
s4, digesting the cells by using trypLE cell digestive juice, carrying out passage to a Matrigel coated pore plate according to 1:3, replacing the cell digestive juice with a third culture medium on the next day, and culturing for 6-8 days;
s5, digesting the cells by using trypLE cell digestive juice and adding 1.5-4.5 x10 4 /cm 2 Subculturing on PO-Laminin coated glass slide, changing to fourth culture medium the next day, culturing for 3-4 days, changing to neuron differentiation culture medium, and continuously culturing for 2-4 weeks to obtain mature hindbrain caudal serotonin neurons;
wherein,
the first medium comprises: a nerve induction medium, SB431542, DMH1, CHIR99021;
the second medium comprises: a nerve induction medium, SB431542, DMH1, CHIR99021, purmorphamine, RA;
the third medium comprises: a nerve induction culture medium, SB431542, DMH1, CHIR99021, purmorphamine, RA and FGF4;
the fourth medium comprises: nerve induction medium, SB431542, DMH1, CHIR99021, RA and FGF4.
In the present invention, SB431542 is a small molecule inhibitor of activin receptor-like kinase (ALK);
in the present invention, DMH1 is a small molecule inhibitor of the bone morphogenetic protein receptor (BMP);
in the present invention, CHIR99021 is a small molecule inhibitor of glycogen synthase kinase 3 (GSK 3);
in the present invention, purmorphamine is a small molecule agonist of the Smo receptor;
in the present invention, RA means all-trans retinoic acid;
in the present invention, FGF4 refers to fibroblast growth factor 4.
Preferably, the neural induction medium includes DMEM/F12 medium, neurobasal medium, 1 xn 2,1 xb 27,1 xneaa, 1 xgutamax.
Preferably, the neuron differentiation medium includes Neurobasal medium, 1 xn 2,1 xb 27,1 xneaa, vitamin C, DAPT, GDNF, BDNF, TGF β 3, IGF1.
Preferably, the volume ratio of DMEM/F12 medium to Neurobasal medium in the nerve induction medium is 1:1.
Preferably, the molar concentration (μ M) ratio of purmophamine to RA in the second medium is: (0.4-1): (0.1-1).
Preferably, the molar concentration (μ M) ratio of purmophamine to RA in the third medium is: (1-2): (0.1-1).
Preferably, in the first culture medium, the molar concentration (μ M) ratio of SB431542, DMH1, CHIR99021 is 2: (1-3).
Preferably, the second medium comprises: the molar concentration (μ M) ratio of SB431542, DMH1, CHIR99021, purmorphamine, RA was 2:2: (1-3): (0.4-1): (0.1-1).
Preferably, the third medium comprises: the molar concentration (μ M) ratio of SB431542, DMH1, CHIR99021, purmorphamine, RA was 2:2: (1-3): (1-2): (0.1-1), the concentration of FGF4 is 10ng/ml.
Preferably, the fourth medium comprises: the molar concentration (μ M) ratio of the neural induction medium, SB431542, DMH1, CHIR99021, RA was 2:2: (1-3) (0.1-1), the concentration of FGF4 is 10ng/ml.
In a second aspect of the invention, a kit for differentiation of caudal serotonin neurons from stem cells is provided, which comprises a first medium, a second medium, a third medium and a fourth medium,
the first medium comprises: nerve induction medium, SB431542, DMH1, CHIR99021;
the second medium comprises: a nerve induction medium, SB431542, DMH1, CHIR99021, purmorphamine, RA;
the third medium comprises: a nerve induction culture medium, SB431542, DMH1, CHIR99021, purmorphamine, RA and FGF4;
the fourth medium comprises: a nerve induction culture medium, SB431542, DMH1, CHIR99021, RA and FGF4.
Preferably, the molar concentration (μ M) ratio of the purmophamine to the RA in the second medium is: (0.4-1): (0.1-1).
Preferably, the molar concentration (μ M) ratio of purmophamine to RA in the third medium is: (1-2): (0.1-1).
Preferably, the neural induction medium includes DMEM/F12 medium, neurobasal medium, 1 xn 2,1 xb 27,1 xneaa, 1 xgutamax.
Preferably, in the first culture medium, the molar concentration (μ M) ratio of SB431542, DMH1, CHIR99021 is 2: (1-3).
Preferably, the second medium comprises: the molar concentration (μ M) ratio of SB431542, DMH1, CHIR99021, purmorphamine, RA was 2:2: (1-3): (0.4-1): (0.1-1).
Preferably, the third medium comprises: the molar concentration (μ M) ratio of SB431542, DMH1, CHIR99021, purmorphamine, RA was 2:2: (1-3): (1-2): (0.1-1), the concentration of FGF4 is 10ng/ml.
Preferably, the fourth medium comprises: the molar concentration (μ M) ratio of the neural induction medium, SB431542, DMH1, CHIR99021, RA was 2:2: (1-3) (0.1-1), wherein the concentration of FGF4 is 10ng/ml.
In a third aspect, the invention provides an application of the caudal serotonin neuron prepared by the method provided by the invention in a cell model for in vitro mechanism research and drug screening of diseases related to the caudal serotonin system of the hindbrain.
Preferably, the method comprises the steps of:
s1, culturing the pluripotent stem cells by using an E8 culture medium, wherein the fusion degree of the pluripotent stem cells reaches 60-80%, carrying out passage to a new culture plate according to 1:3, and continuously culturing by using E8;
s2, when the fusion degree of the pluripotent stem cells reaches about 80%, replacing the E8 with a first culture medium, and culturing for 6-8 days;
s3, digesting the cells by using trypLE cell digestive juice, carrying out passage to a Matrigel coated pore plate according to 1:5, replacing the cell digestive juice with a second culture medium the next day, and culturing for 6-8 days;
s4, digesting the cells by using trypLE cell digestive juice, carrying out passage to a Matrigel coated pore plate according to 1:3, replacing the cell digestive juice with a third culture medium on the next day, and culturing for 6-8 days;
s5, digesting the cells by using trypLE cell digestive juice and adding 1.5-4.5 x10 4 /cm 2 Passage on PO-Laminin coated glass slide, changing to fourth culture medium the next day, culturing for 3-4 days, changing to neuron differentiation culture medium, and continuously culturing for 2-4 weeks to obtain mature hindbrain caudal serotonin neurons;
wherein,
the first medium comprises: nerve induction medium, SB431542, DMH1, CHIR99021;
the second medium comprises: a nerve induction medium, SB431542, DMH1, CHIR99021, purmorphamine, RA;
the third medium comprises: a nerve induction culture medium, SB431542, DMH1, CHIR99021, purmorphamine, RA and FGF4;
the fourth medium comprises: nerve induction medium, SB431542, DMH1, CHIR99021, RA and FGF4.
Compared with the prior art, the invention has the beneficial effects and remarkable progress that: the method for differentiating the caudal serotonin neurons by the stem cells, the complete culture medium and the application can be simple, convenient and efficient, can obtain a large amount of high-purity serotonin neurons with mature functions in a specific region, and provides an effective cell model for the mechanism research of related diseases and the evaluation and screening of drugs.
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To more clearly illustrate the technical solution of the present invention, the drawings required for the embodiment of the present invention will be briefly described below.
It should be apparent that the drawings in the following description are only drawings of some embodiments of the invention, and that other drawings can be obtained by those skilled in the art without inventive exercise, and the other drawings also belong to the drawings required by the embodiments of the invention.
FIG. 1 is a diagram showing the cell morphology of the human embryonic stem cell line H9 in an undifferentiated state, according to example 1 of the present invention;
FIG. 2 is a flow chart of the differentiation process of the directed differentiation of human pluripotent stem cells into retrocerebro caudal serotonin neurons according to example 2 of the present invention;
FIG. 3 is a morphological diagram of the various stages of the differentiation of the hindbrain caudal serotonin neurons in example 2 of the present invention;
FIG. 4 is a cellular immunofluorescence plot of markers for the early stage of hindbrain caudal serotonin neuron differentiation of example 3 of the present invention;
FIG. 5 is a cellular immunofluorescence map of a late differentiation marker of a hindbrain caudal neuron in accordance with example 3 of the present invention;
FIG. 6 is a cellular immunofluorescence plot of a hindbrain caudal serotonin neuron marker of example 3 of the present invention;
FIG. 7 is the sodium potassium current of the hindbrain caudal serotonin neurons of example 4 of the invention under voltage stimulation;
FIG. 8 shows the action potential and spontaneous action potential induced by the caudal-brain serotonin neurons of example 4 of the present invention under current stimulation;
FIG. 9 is a graph of secreted serotonin neurotransmitter levels of the hindbrain caudal serotonin neurons of example 5 of the present invention.
Detailed Description
In order to make the objects, technical solutions, beneficial effects and significant progress 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 with reference to the embodiments of the present invention.
It is to be understood that all of the described embodiments are only some, and not all, of the present invention; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It is to be understood that:
the term "differentiation" refers to the process by which cells of the same origin gradually develop populations of cells with different morphological, structural and functional characteristics, resulting in spatial differentiation of the cells, and temporal differentiation of the same cell from its previous state. The essence of cell differentiation is the selective expression of the genome in time and space, with the expression of different genes turned on or off, ultimately producing marker proteins.
The term "culture medium" refers to an artificially prepared nutrient for the growth and maintenance of microorganisms, plant tissues, and animal tissues, and generally contains carbohydrates, nitrogen-containing substances, inorganic salts (including trace elements), vitamins, water, and the like.
The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
It should be further noted that the following embodiments may be combined with each other, and the same or similar concepts or processes may not be repeated in some embodiments.
The technical means of the present invention will be described in detail with reference to specific examples.
EXAMPLE 1 culture of human embryonic Stem cells
Use of E8 Medium (TeSR) TM -E8 TM ,Stem Cell technology, cat No. 05990) cultured human embryonic stem cell line H9 (WA 09, wiCell), cells were attached to Matrigel (Corning, cat No. 354277) coated six-well plates, cells were replaced with fresh medium every day, cultured for 4-6 days, and passaged with cell fusion of about 80%.
The specific steps of cell passage are as follows:
1. coating a pore plate with matrigel: according to the dilution ratio of the specification, the Matrigel is diluted to 4 ℃ precooled high-glucose DMEM (Gibco, cat No. 11995065), then added into a pore plate, incubated at 37 ℃ for 30 minutes, and sucked away before passage;
2. sucking off cell culture solution, adding DPBS (SIGMA) for washing, adding ReLeSR TM Cell digests (STEMCELL Technologies, cat # 05872) were aspirated within 1 minute and left at room temperature for 7 minutes;
3. after digestion, E8 resuspended cells were added
4. The cells were passaged as 1:3, seeded in six-well plates previously coated with matrigel, cultured using E8, and the cells were changed and observed for cell state every day, and the cell morphology is shown in fig. 1.
Example 2 directed differentiation of human embryonic Stem cells into caudate-caudal serotonin neurons
The steps of the directional differentiation of human embryonic stem cells into caudate-caudal serotonin neurons are shown in fig. 2, and the specific steps are as follows:
step 1, differentiation of human embryonic stem cells into retroencephalic neuroepithelial cells
When the confluency of the embryonic stem cells in example 1 reached about 80%, the cells were cultured for 6 to 8 days in a neural induction medium (first medium) containing compound component 1, and then subjected to digestion passaging using TrypLE digestive enzyme.
Wherein the basal medium of the nerve induction medium comprises DMEM/F12 (Gibco, cat # 11330-032), neurobasal medium (Gibco, cat # 21103049) (volume ratio 1:1), 1 xN 2 (Gibco, cat # 17502048), 1 xB 27 (Gibco, cat # 12587010), 1 xNEAA (Gibco, cat # 11140050), 1 xGlutaMax (Gibco, cat # 35050061). Compound component 1 comprises: 2 μ M SB431542, 2 μ M DMH1, 1.8 μ M CHIR99021 (small molecule compounds are all purchased from Chiozotan organisms).
The specific digestion steps are as follows:
1. coating a pore plate with matrigel: according to the dilution ratio of the specification, diluting the Matrigel to 4 ℃ precooled high-sugar DMEM, adding a pore plate, incubating at 37 ℃ for 30 minutes, and sucking away before passage;
2. performing nerve induction culture for 6-8 days, sucking culture solution, washing with DPBS, adding trypLE (Gibco, cat # 12604021) and washing, sucking digestive juice, placing cells in incubator, incubating at 37 deg.C for 4 min
3. Neural induction medium containing compound combination 1 was added and 10 μ M Y27632 (Tao Shu organism) was added, and after cell resuspension, the cells were passaged to matrigel coated well plates at a rate of 1:5.
Step 2, ventral and caudal organization of the hindbrain neuroepithelial cells
After obtaining the hindbrain neuroepithelial cells, namely the next day of passage in the step 1), the cells are replaced by a nerve induction medium (second medium) containing the compound component 2, cultured for 6 to 8 days, and digested and passaged by using TrypLE digestive enzyme.
Wherein the basic culture medium of the nerve induction culture medium contains DMEM/F12: neurobasal culture medium (the volume ratio is 1:1), 1 xN 2,1 xB 27,1 xNEAA and 1 xGlutaMax. Compound component 2 comprises: 2 μ M SB431542, 2 μ M DMH1, 1.8 μ M CHIR990210, 0.5 μ M purmorphamine, 100nM RA.
The specific digestion steps are as follows:
1. coating a pore plate with matrigel: according to the dilution ratio of the specification, diluting the Matrigel to 4 ℃ precooled high-sugar DMEM, adding a pore plate, incubating at 37 ℃ for 30 minutes, and sucking away before passage;
2. performing nerve induction culture for 6-8 days, sucking culture solution, washing with DPBS, moistening with TrypLE, sucking digestive juice, placing cells in incubator, incubating at 37 deg.C for 4 min
3. Neural induction medium containing compound combination 2 was added and 10 μ M Y27632 was added, after resuspending the cells, passaged to matrigel coated well plates at 1:3.
Step 3, differentiation of the hindbrain caudal serotonin neural precursor cells
After obtaining the neuroepithelial cells on the caudal ventral side of the hindbrain, i.e., the next day of the passage in step 2), the cells were replaced with a neural induction medium (third medium) containing compound component 3, cultured for 6 to 8 days, and subjected to digestion passage using TrypLE digestive enzyme.
Wherein the basic culture medium of the nerve induction culture medium contains DMEM/F12: neurobasal culture medium (the volume ratio is 1:1), 1 xN 2,1 xB 27,1 xNEAA and 1 xGlutaMax.
Compound component 3 comprises: 2 μ M SB431542, 2 μ M DMH1, 1.8 μ M CHIR990210, 2 μ M purmorphamine, 100nM RA, 10ng/ml FGF4 (PeproTech, cat # 100-31).
The specific digestion steps are as follows:
1. PO-Laminin coated slide: diluting polyornithine (PO, SIGMA) 200 times with sterile water, adding 50 μ L per slide with diameter of 12mm, coating at room temperature overnight, sucking PO off the next day, washing with sterile water twice, air drying, adding lamin (Thermo fisher, cat No. 23017015) diluted 50 times with DMEM/F12, coating at 37 deg.C for 3 hr, and sucking off before passage;
2. performing nerve induction culture for 6-8 days, sucking culture solution, washing with DPBS, moistening with TrypLE, sucking digestive juice, placing cells in incubator, incubating at 37 deg.C for 4 min
3. Adding the nerve induction culture medium containing the compound combination 3 and adding 10 mu M Y27632, after cell resuspension, 1.5-4.5 x10 of each slide with the diameter of 12mm 4 /cm 2 And (4) inoculating the cells.
4. Differentiation and maturation of hindbrain caudad serotonin neurons: the next day of passage in step 3), the culture was changed to a neural induction medium (fourth medium) containing compound component 4, and the culture was continued for 3 to 4 days, and then to a neuronal differentiation medium, and the culture was continued for 2 to 4 weeks.
Wherein the neuron differentiation medium comprises the following components: neurobasal medium, 1 XN 2,1 XB 27,1 XNEAA, 0.2mM vitamin C (sigma-Ardrich, cat # A4403), 2.5. Mu.M DAPT (Tao Shu organism, cat # T6202), 10ng/ml GDNF (PeproTech, cat # 450-10), 10ng/ml BDNF (PeproTech, cat # 450-02), 1ng/ml TGF beta 3 (PeproTech, cat # 100-36E), 10ng/ml IGF1 (PeproTech, cat # 100-11).
Compound 4 includes: 2 μ M SB431542, 2 μ M DMH1, 1-3 μ M CHIR99021, 0.1-1 μ M RA, 10ng/ml FGF4.
The morphological pattern of the various stages of differentiation of the caudal-cranial serotonin neurons of this example is shown in FIG. 3.
Example 3 identification of marker protein expression at various stages of differentiation by cellular immunofluorescence assay
The slides which were digested the next day of passaging in step 3 of the induction process of example 2 and cell slides which were cultured for 3 days and 2-3 weeks in step 4 were taken for cellular immunofluorescence identification, with the following specific steps:
3.1, sucking away the culture solution, adding DPBS (dimethyl-benzene-sulfonate) for washing once, and then adding 4% paraformaldehyde for fixing at room temperature for half an hour;
3.2, absorbing paraformaldehyde, and adding DPBS for washing for 3 times;
3.3, absorbing the DPBS and adding a sealing liquid, wherein the sealing liquid comprises the following components: DPBS containing 10% (v/v) donkey serum, 0.2% (v/v) Triton X-100 is incubated for half an hour at room temperature;
3.4 dilution of primary antibody with DPBS containing 5% (v/v) donkey serum and 0.2% (v/v) Triton X-100, for information see Table 1;
3.5, sucking off the confining liquid, adding the diluted primary antibody, and incubating overnight at 4 DEG C
3.6, removing primary antibody by suction, adding DPBS to wash for 3 times, 5 minutes each time
3.7 dilution of secondary antibody with DPBS containing 5% (v/v) donkey serum and DAPI
3.8, aspirate DPBS, add diluted secondary antibody and DAPI, incubate 45 minutes at room temperature in the dark
3.9, suck the secondary antibody, add DPBS to wash 3 times, each for 5 minutes
3.10 mounting using anti-quenching mounting agent
3.11 Observation and photography with a fluorescence microscope
The cellular immunofluorescence identification map is shown in fig. 4 and fig. 5. Fig. 4 is the hindbrain caudal neuronal precursor cell markers HOXB4, OLIG2, NKX2.2 for day 21 of differentiation, where HOXB4 is a postbrain caudal marker, NKX2.2 is a postbrain ventral neuronal precursor cell marker, and OLIG2 is a motor neuron marker. Figure 5 is a posterocaudal serotonin neuron precursor cell marker assay at day 24 of differentiation, in which HOXB4 is positive, NKX2.2 is positive, and OLIG2 is negative. FIG. 6 is a graph of markers 5-HT and Tuj1 at the neuronal differentiation stage cultured for 3 weeks, indicating that mature retrocerebro-caudal serotonin neurons were obtained.
TABLE 1 antibody information from cellular immunofluorescence experiments
Figure BDA0003869359800000101
Example 4 Whole cell patch Clamp assay for electrophysiological function of human embryonic stem cell differentiation-derived hindbrain caudal serotonin neurons
And filling the electrode solution after the electrode is drawn, wherein the resistance value is 6-8M omega. Before the electrode invades the liquid surface, a weak positive pressure is given to the interior of the electrode holder through a conduit connected with the electrode holder, and when the tip begins to contact the cell to prepare for forming a seal (seal), the conduit is opened by screwing and a constant negative pressure is applied through the conduit. After the sealing impedance reaches G omega level, the membrane potential is kept at-60 mV, and then the adherent cells are lifted to the lower part of the dosing tube of the rapid drug delivery perfusion system for the next experiment. Whole-cell electrode internal solution: 20mM KCl,10mM Na + -HEPES，121mM K + -gluconate，10mM BAPTA，4mM Mg 2+ ATP (adjusted pH =7.2, stored at 0 ℃). Whole-cell electrode external liquid: 127mM NaCl,1.9mM KCl,2.2mM CaCl 2 ，1.2mM KH 2 PO 4 ， 26mM NaHCO 3 ，1.4mM MgSO 4 10mM glucose (pH = 7.3). The experiment control is carried out at the room temperature of 23-25 ℃, the filtering frequency is 1kHz, and the recording of the clamping voltage command and the current is controlled by clamp 10.3 (Molecular devices) software through a DigiData-1440A conversion interface.
Whole cell sodium potassium current recording
Under voltage clamping, a voltage of-40-30 mV was injected, and inward sodium and outward potassium currents were recorded under a 5mV one step stimulation, as shown in FIG. 7, indicating that the differentiating-derived serotonin neurons have mature sodium potassium channels.
Action potential recording
Under current clamp, the clamp current is 0pA, and the action potential is delivered under-40-100pA and 10pA one-step stimulation, as shown in FIG. 8, which shows that the serotonergic neuron from differentiation source has mature electrophysiological characteristics.
Example 5 detection of neurotransmitter secretion function of human embryonic Stem cell differentiation derived hindbrain caudal serotonin neurons by enzyme-linked immunosorbent assay
Cell culture fluid of the fifth week of the differentiation stage of the serotonin neurons and overnight incubation of non-serotonin neurons is collected, and the content of serotonin in the collected culture fluid is detected by using an enzyme linked immunosorbent assay kit (IBL, RE 59141). As shown in fig. 9, the extracellular serotonin content of non-serotonin neurons (NC, negative control) was Not detectable (Not deciteleble, n.d.), while the differentiation-derived serotonin (cSNs) content was significantly higher than the extracellular level of non-serotonin. Indicating that the differentiation-derived hindbrain caudal serotonin neurons have normal neurotransmitter synthesis and secretion functions.
Comparative example 1
1. Hindbrain neuroepithelial cells were obtained according to example 2, step 1;
2. example 2 in compound component 2 of step 2, on day 7 of differentiation, purmophamine and RA were added to promote posterocaudal and ventral differentiation, and we tried 0/0.25/0.5/1/2 μ M purmopamine to induce differentiation under 100nM RA for one week;
3. the cell immunofluorescence detects the proportion of ventral nerve precursor cell marker NKX2.2 and basal plate cell marker FOXA2 positive cells in cells differentiated for 14 days to total cells.
The results showed that the ratio of the ventral nerve precursor cell marker NKX2.2 in the 0.25. Mu.M puerhamine group was low, while the ratio of the basal plate cell marker FOXA2 in the 2. Mu.M puerhamine group was high, and the differentiation of the cells in the 0.5 and 1. Mu.M groups was the best.
Comparative example 2
1. Hindbrain neuroepithelial cells obtained according to step 1 of example 2
2. Hindbrain ventral neural stem cells obtained according to step 2 of example 2
3. Example 2 in compound fraction 3 of step 3, purmophamine was added on day 14 of differentiation, RA and FGF4 further promoted serotonin precursor differentiation, and we tried 0.5/2 μ M purmophamine to induce differentiation for one week.
4. The cell immunofluorescence detects the proportion of ventral nerve precursor cell marker NKX2.2 and basal plate cell marker FOXA2 positive cells in cells differentiated for 21 days to the total cells.
The results showed that the ratio of the ventral nerve precursor cell marker NKX2.2 was low in the 0.5. Mu.M pueramine group, while the ratio of NKX2.2 was high in the 2. Mu.M pueramine group and the basal plate cell marker FOXA2 remained low, and that increasing the concentration of the pueramine from 0.5. Mu.M to 2. Mu.M in the late stage of differentiation promoted ventral localization without affecting differentiation into basal plate cells
Comparative example 3
1. Hindbrain neuroepithelial cells obtained according to step 1 of example 2
2. Hindbrain ventral neural stem cells obtained according to step 2 of example 2
3. Hindbrain ventral neuronal precursor cells were obtained according to step 3 of example 2,
4. the precursor cells of step 3 in example 2 were digested and plated on glass slides, and the neuronal differentiation medium was changed directly the next day.
5. Cell immunofluorescence for 45 days of neural differentiation detects the ratio of serotonin neurons, and the result shows that the ratio of the final 5-HT positive serotonin neurons is lower than that of the serotonin neurons obtained in example 2.
During the description of the above description:
the description of the terms "this embodiment," "an embodiment of the invention," "as shown in … …," "further improved technical solution," etc., means that a particular feature, structure, material, or characteristic described in this embodiment or example is included in at least one embodiment or example of the invention; in this specification, the terminology used above is not necessarily intended to refer to the same embodiment or example, and the particular features, structures, materials, or characteristics described, etc., may be combined or coupled in any suitable manner in any one or more embodiments or examples; furthermore, those of ordinary skill in the art may combine or combine features of different embodiments or examples and features of different embodiments or examples described in this specification without undue conflict.
Finally, it should be noted that:
the above embodiments are only used for illustrating the technical solution of the present invention, and not for limiting the same;
although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that various changes and modifications may be made, and equivalents may be substituted for elements thereof without departing from the scope of the embodiments of the present invention.

Claims (10)

1. A method for differentiating caudad serotonin neurons from stem cells, comprising the steps of:
s1, culturing the pluripotent stem cells by using an E8 culture medium, wherein the fusion degree of the pluripotent stem cells reaches 60-80%, carrying out passage to a new culture plate according to 1:3, and continuously culturing by using E8;
s2, when the fusion degree of the pluripotent stem cells reaches about 80%, replacing the E8 with a first culture medium, and culturing for 6-7 days;
s3, digesting the cells by using trypLE cell digestive juice, carrying out passage to a Matrigel coated pore plate according to 1:5, replacing the cell digestive juice with a second culture medium the next day, and culturing for 6-8 days;
s4, digesting the cells by using trypLE cell digestive juice, transferring the cells to a Matrigel coated pore plate according to 1:3, changing the cell digestive juice to a third culture medium on the next day, and culturing for 6-8 days;
s5, digesting the cells by using trypLE cell digestive juice and adding 1.5-4.5 x10 4 /cm 2 Subculturing on PO-Laminin coated glass slide, changing to fourth culture medium the next day, culturing for 3-4 days, changing to neuron differentiation culture medium, and continuously culturing for 2-4 weeks to obtain mature hindbrain caudal serotonin neurons;
wherein,
the first medium comprises: nerve induction medium, SB431542, DMH1, CHIR99021;
the second medium comprises: a nerve induction medium, SB431542, DMH1, CHIR99021, purmorphamine, RA;
the third medium comprises: a nerve induction culture medium, SB431542, DMH1, CHIR99021, purmorphamine, RA and FGF4;
the fourth medium comprises: nerve induction medium, SB431542, DMH1, CHIR99021, RA and FGF4.
2. The method of differentiating caudal serotonin neurons according to claim 1, wherein said neural induction medium comprises DMEM/F12 medium, neurobasal medium, 1 xn 2,1 xb 27,1 xneaa, 1 xgutamax.
3. The method of claim 1, wherein the neuronal differentiation medium comprises Neurobasal medium, 1 xn 2,1 xb 27,1 xneaa, vitamin C, DAPT, GDNF, BDNF, TGF β 3, IGF1.
4. The method for differentiating caudad serotonin neurons from stem cells according to claim 2, wherein the volume ratio of DMEM/F12 medium to Neurobasal medium in the neural inducing medium is 1:1.
5. The method of differentiating caudal serotonin neurons from stem cells according to claim 1, wherein said second medium comprises the molar concentration (μ M) ratio between purmophamine and RA: (0.4-1): (0.1-1).
6. The method of differentiating caudal serotonin neurons from stem cells according to claim 1, wherein said third medium comprises the molar concentration (μ M) ratio between purmophamine and RA: (1-2): (0.1-1).
7. A complete set of culture medium for differentiating caudal serotonin neurons by stem cells is characterized by comprising a first culture medium, a second culture medium, a third culture medium and a fourth culture medium,
the first medium comprises: nerve induction medium, SB431542, DMH1, CHIR99021;
the second medium comprises: a nerve induction medium, SB431542, DMH1, CHIR99021, purmorphamine, RA;
the third medium comprises: a nerve induction culture medium, SB431542, DMH1, CHIR99021, purmorphamine, RA and FGF4;
the fourth medium comprises: nerve induction medium, SB431542, DMH1, CHIR99021, RA and FGF4.
8. The set of stem cell differentiation caudal serotonin neurons according to claim 7, wherein said second medium comprises purmophamine and RA in molar concentration (μ M) ratio: (0.4-1): (0.1-1).
9. The set of media of claim 7, wherein the ratio of the molar concentrations (μ M) of purmophamine and RA in the third medium is: (1-2): (0.1-1).
10. Use of caudal serotonin neurons prepared according to the method of any one of claims 1 to 6 in cell models for in vitro mechanistic studies and drug screening of diseases associated with the nervous system.
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