CN115404202A

CN115404202A - Method for obtaining spinal cord motor neurons by using human pluripotent stem cell induction and application of method in mitochondria visualization

Info

Publication number: CN115404202A
Application number: CN202210943465.5A
Authority: CN
Inventors: 陈红; 罗红梅
Original assignee: Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Wuhan Hongchen Innovation Biotechnology Co ltd
Priority date: 2022-08-08
Filing date: 2022-08-08
Publication date: 2022-11-29

Abstract

The invention discloses a method for obtaining spinal cord motor neurons by using human pluripotent stem cell induction and mitochondrion visualization application thereof, and relates to the technical field of differentiation induction of pluripotent stem cells. Mitochondrial responses to different drug treatments were detected by mitochondrial staining of mature spinal motor neurons, live cell imaging, and. Can be used for researching related action mechanism of mitochondria in motor neuron diseases and establishing a drug screening platform; is beneficial to producing various medicines for treating neurodegenerative diseases and provides new diagnostic tools and treatment strategies for the neurodegenerative diseases.

Description

Method for obtaining spinal cord motor neurons by using human pluripotent stem cell induction and application of method in mitochondria visualization

Technical Field

The invention relates to the technical field of differentiation induction of pluripotent stem cells, in particular to a method for obtaining spinal motor neurons by using human pluripotent stem cells and application thereof.

Background

Motor Neurons (MN) are a special type of neurons in the central nervous system, mainly located in the cerebral cortex, brainstem and spinal cord, and are the basic units that control the movement of the body. Motor Neuron Disease (MND), a group of chronic degenerative diseases whose etiology has not been clear selectively invading the anterior horn cells of the spinal cord, the motor neurons of the brain stem, the cortical vertebral body cells and the vertebral bundles; its pathology is characterized by the degeneration, necrosis and apoptosis of the progressive upper and lower motor neurons. Different types of motor neuron diseases are formed due to different combinations of symptoms and signs, and comprise Amyotrophic Lateral Sclerosis (ALS), spinal Muscular Atrophy (SMA), primary Lateral Sclerosis (PLS), progressive Bulbar Palsy (PBP) and the like, wherein the ALS is the most common type of chronic motor neuron diseases, is commonly called as 'progressive freezing human disease', is one of five major absolute diseases recognized globally, is a fatal delayed neurodegenerative disease, selectively attacks upper and lower motor neurons of brain and spinal cord, and is clinically manifested by gradually-aggravated muscular atrophy and muscle weakness, even can cause difficulty in speaking, swallowing and breathing, and finally endangers life, and at present, no method for curing the ALS exists. ALS is divided into familial amyotrophic lateral sclerosis (familial ALS, faals) and sporadic amyotrophic lateral sclerosis (sporadic ALS, sALS). To date, more than 30 genes have been associated with fALS, including the common genes SOD1, C9orf72, FUS and TDP43 mutations accounting for 50% -70% of fALS, with SOD1 gene mutations accounting for 20% of fALS. The SOD1 gene is the ALS related gene which is discovered for the first time in 1993, and SOD1 protein is mainly positioned in the membrane gap of cytoplasm and mitochondria and is involved in regulating and controlling the dynamic processes of mitochondrial fusion and cleavage, movement, energy metabolism and the like, which means that the damage of mitochondrial dynamics can cause the degeneration of mitochondria due to the morphological and functional disorder of the mitochondria and the subsequent generation of neurons.

It is reported in literature that the mutant SOD1 protein is unstable in conformation and is easy to be misfolded, so that the mutant SOD1 protein is aggregated, and ALS motor neurons are degraded. In addition, misfolded SOD1 protein can impair mitochondrial function, leading to altered mitochondrial morphology and swelling, vacuoles and swelling in the mitochondrial matrix. Thread pelletThe body plays a key role in the survival and metabolism of cells, and is the site of oxidative phosphorylation of cells to produce Adenosine Triphosphate (ATP) which provides energy. Neurons are highly polarized cells with high metabolic demand, with the brain accounting for only 2% of the body mass, but consuming 20% of the body's ATP at rest. Neurons meet their high metabolic needs primarily by synthesizing ATP through oxidative phosphorylation, which makes neurons more prone to mitochondrial dysfunction. Such abnormalities can lead to dysfunction of motor neuron cells, eventually leading to apoptosis. The ALS rats and ALS patients have reports on the change of ultrastructural morphology of mitochondria such as motor neurons, muscles, brain tissues, lymphocytes, livers and the like and dysfunction of an electron transfer system. The mutated SOD1 results in a decrease in respiratory chain complex I, IV activity and a decrease in ATP production. Thus, abnormalities in ATP and ROS production, energy and calcium homeostasis, induction of apoptosis, and mitochondrial transport in axons were found in ALS transgenic mice and patients. ALS patients with SOD1 mutations have Ca deficiency in motor neurons ²⁺ Binding protein expression, resulting in cellular pair Ca ²⁺ Decreased uptake and increased neuronal excitability. Neurons, especially the synaptic terminal regions, need to rely on massive synthesis of ATP within the mitochondria and calcium homeostasis to maintain their function. Therefore, in these areas of high ATP demand and calcium homeostasis, mitochondrial transport is of paramount importance, and defects in mitochondrial axonal transport lead to abnormal neuronal metabolism.

An increasing number of literature studies have demonstrated that mitochondrial dysfunction drives or mediates the pathological processes of neurodegenerative diseases including ALS. In rats with SOD 1G 93A mutation, mutant SOD1 causes oxidative stress, protein aggregation, and neuronal excitotoxicity, eventually leading to motor neuron degeneration. Human pluripotent stem cells (hpscs) are pluripotent cells with self-renewal and self-replication capabilities, which can be differentiated into other somatic cells and mimic the pathogenesis of diseases, so the hPSC differentiation technology provides a good platform for studying ALS pathogenesis. In particular, hpscs can study mitochondrial dysfunction in the early pathological processes of ALS by providing valuable models of human motor neurons through directed differentiation.

In the prior art, the literature "Temporal expression profiles of lncRNA and mRNA in human embryonic stem cell-derived motor neurons degradation differentiation" (Xuejiao Sun, ming-Xing Li, etc., 2020 Nov.13) discloses Temporal expression profiles of lncRNA and mRNA during differentiation of human embryonic stem cell-derived motor neurons, and motor neurons with a purity of more than 90% are harvested from hESCs. However, the SHH agonist (Purmorphamine) used in this document is highly toxic, and therefore, we chose to use another SHH Agonist (SAG).

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for obtaining spinal motor neurons by using human pluripotent stem cell induction, which comprises the steps of firstly utilizing an induced differentiation culture medium to induce and differentiate human pluripotent stem cells (hPSC) to obtain neuroepithelial cells (NEP), then utilizing a patterned culture medium to induce and differentiate NEP to obtain spinal motor neuron progenitor cells, and then utilizing a maintenance patterned culture medium to induce and differentiate the spinal motor neuron progenitor cells to obtain HB9 ⁺ Spinal motor neurons, eventually undergoing neurotrophic maturation to obtain CHAT ⁺ Spinal motor neurons. The spinal cord motor neuron obtained by the method provided by the invention can be used for comprehensively and deeply researching the cell dysfunction of the motor neuron disease, researching the action mechanism related to mitochondria in the motor neuron disease by the mitochondria visualization application of the motor neuron, and establishing a drug screening platform. The method is helpful for screening clinical drugs, producing and preparing various drugs for treating neurodegenerative diseases, providing a new diagnosis tool and a new treatment strategy for the neurodegenerative diseases, and preventing and treating various neurodegenerative diseases, and is realized by the following technology.

A method for inducing and obtaining spinal cord motor neurons by using human pluripotent stem cells comprises the following steps:

s1, adding human pluripotent stem cells into an induced differentiation culture medium containing a first small molecule composition, and inducing and differentiating to obtain neuroepithelial cells;

the first small molecule composition comprises a GSK3 inhibitor, a TGF-beta/Smad inhibitor and a BMP inhibitor;

s2, adding the neuroepithelial cells obtained in the step S1 into a patterned culture medium containing a second small molecule composition, and inducing and differentiating to obtain spinal cord motor neuron progenitor cells;

the second small molecule composition comprises a GSK3 inhibitor, a TGF- β/Smad inhibitor, a BMP inhibitor, a RAR nuclear receptor activator, and a SHH agonist (Sonic hedgehog/Smoothened signaling pathway agonist);

in the actual operation of the step S2, all the products of the step S1 may be directly added to the maintenance patterned culture medium for direct culture, or the neuroepithelial cells NEP may be separated and purified by a certain technique, and then added to the maintenance patterned culture medium for induced differentiation to obtain the spinal motoneuron cells;

s3, adding the spinal cord motor neuron progenitor cells obtained in the step S2 into a maintenance patterned culture medium containing a third small molecule composition for suspension culture, and inducing differentiation to obtain HB9 ⁺ Spinal motor neurons;

the third small molecule composition comprises a RAR nuclear receptor activator and a SHH agonist;

step S3, in actual operation, all products obtained in step S2 can be directly added into a maintenance patterned culture medium, or spinal cord motor neuron progenitor cells can be separated and purified by a certain technology and then added into the maintenance patterned culture medium;

s4, taking the HB9 obtained in the step S3 ⁺ Culturing spinal cord motor neuron by adherence, performing neurotrophic maturation to obtain CHAT ⁺ Spinal motor neurons.

In the actual operation of step S4, all the products obtained in step S3 may be added directly to the neurotrophic medium, or HB9 may be isolated and purified first by a technique ⁺ Spinal cord motor neurons, which are then added to the neurotrophic medium.

Preferably, in step S1, the differentiation-inducing medium comprises DMEM/F12 and Neurobasal in a volume ratio of 1:1, and further comprises GlutaMax with a volume fraction of 1%, B27 with a volume fraction of 1%, N-2 with a volume fraction of 0.5%, AA with a volume fraction of 0.1mM, GSK3 inhibitor with a volume fraction of 3 μ M, TGF- β/Smad inhibitor with a volume fraction of 2 μ M, BMP inhibitor with a volume fraction of 2 μ M.

Preferably, in step S2, the patterned medium comprises DMEM/F12 and Neurobasal in a volume ratio of 1.

More preferably, in step S2, the second small molecule composition comprises CHIR99021 (GSK 3 inhibitor), DMH-1 (BMP inhibitor), SB431542 (TGF-. Beta./Smad inhibitor), RA (RAR nuclear receptor activator), and SAG (SHH agonist).

Preferably, in step S3, the maintenance patterning medium comprises DMEM/F12 and Neurobasal in a volume ratio of 1.

Preferably, step S4 is specifically: after inducing differentiation for 5 days by the method of step S3, the sterile round slide was placed in a well plate, and PLO solution was dropped on the slide at 37 ℃ and 5% CO ₂ Incubating overnight under the environment; absorbing and removing PLO the following day, washing, adding Laminin solution dropwise onto the slide, or using precooled Matrigel alone, and making the content of CO 5% at 37 ℃% ₂ Continuously incubating for more than 1h under the environment; finally, digesting the large neurosphere into a single spinal cord motor neuron or a small neurosphere by using cell digestive juice Accutase; inoculating the suspension of spinal motor neurons or small neurospheres on a slide treated by PLO/Lamin or Matrigel for adherent culture for 6-10 days, carrying out neurotrophic maturation to obtain the expression CHAT ⁺ Spinal motor neurons.

More preferably, in step S4, the components of the medium used for neurotrophic, including Neurobasal, further include GlutaMax at a volume fraction of 1%, N-2 at a volume fraction of 0.5%, B27 at a volume fraction of 1%, AA at 0.1mM, SAG at 0.1. Mu.M, RA at 0.01. Mu.M, IGF-1 at 10ng/mL, CNTF at 0.1. Mu.M, compound E at 10ng/mL, BDNF at 10ng/mL and GDNF at 10 ng/mL.

In the above step S4, HB9 ⁺ Spinal motor neurons may or may not be subjected to Accutase digestion for 7-10min optimal to yield a suspension of neurospheres containing on average about 50 cells per neurosphere.

It is noted that the first small molecule combinations provided by the present invention are selected from GSK3 inhibitors, TGF- β/Smad inhibitors and BMP inhibitors, i.e. selected from substances capable of inhibiting the GSK3, TGF- β/Smad and BMP signaling pathways, respectively. For example, although CHIR99021, DMH-1, SB431542 and their derivatives are preferable in the present invention, the above three pathways can be inhibited to achieve the corresponding culture purpose, and the concentrations of the three are not particularly limited; for example, it may be 3. Mu.M, 2. Mu.M and 2. Mu.M, respectively, but is not limited thereto.

The second small molecule composition is selected from a GSK3 inhibitor, a TGF-beta/Smad inhibitor, a BMP inhibitor, an RAR nuclear receptor activator and an SHH agonist, for example, CHIR99021, SB431542, RA and SAG can be preferable in the present invention, and the concentration thereof is not particularly limited as long as the purpose of inhibiting GSK3, TGF-beta/Smad and BMP signaling pathways, activating RAR nuclear receptors and inhibiting SHH signaling pathways can be achieved; for example, 1. Mu.M GSK3 inhibitor, 2. Mu.M BMP inhibitor, 2. Mu.M TGF-. Beta./Smad inhibitor, 0.1. Mu.M RAR nuclear receptor activator, and 1. Mu.M SHH agonist can be selected, but not limited thereto.

The third small molecule composition may be selected from an activator of RAR nuclear receptor and an agonist of SHH signaling pathway, for example, the present invention may preferably be RA, SAG and their derivatives, and the concentration thereof is not particularly limited as long as it can activate RAR nuclear receptor and SHH signaling pathway, and for example, 0.01. Mu.M of RAR nuclear receptor activator and 0.1. Mu.M of SHH signaling pathway agonist may be optimally selected, but not limited thereto.

The invention also provides a spinal cord motor neuron obtained by any one of the methods.

A composition or kit for obtaining spinal cord motor neurons by inducing human pluripotent stem cells, the composition or kit comprising the first small molecule composition, the second small molecule composition and the third small molecule composition provided in the method.

The spinal cord motor neuron is prepared by any one of the methods and used for researching the function change of mitochondria by dynamically observing the density, the motion state, the membrane potential and the intuitive change of the calcium ion concentration of the mitochondria of the spinal cord motor neuron, thereby evaluating the action of a medicament and screening clinical medicaments; for example, mito-tracker/TMRM/Rhod-2-AM and other dyes can be used for staining living cell mitochondria of spinal motor neurons, the density of the mitochondria of the spinal motor neurons can be dynamically observed in real time under a confocal microscope by applying or withdrawing drugs, and the change of functions of the mitochondria can be reflected by the intuitive change of the motion along axons, membrane potential and calcium ion concentration, so that the drug effect can be evaluated, and clinical drugs can be screened; or for preparing a medicament for treating spinal cord motor neuron loss or imbalance and dyskinesia and/or sensory disturbance caused by nervous system diseases or injuries; or used for pathological mechanism research and drug screening of the diseases.

Compared with the prior art, the invention has the advantages that: the invention provides a method for obtaining spinal cord motor neurons by a series of induced differentiation by utilizing human pluripotent stem cells. Compared with the method disclosed at present, the method provided by the invention has the advantages that the purity and yield of the finally obtained spinal cord motor neurons are higher by changing the raw material components of the culture medium at each stage. The spinal cord motor neuron and the mitochondrial visualization of the motor neuron can be widely applied to the research of pathological mechanisms of motor disorder and/or sensory disorder diseases caused by motor neuron diseases, neurodegenerative diseases and nervous system diseases or injuries and the research and the development of related medicaments.

Drawings

FIG. 1 is a flow chart of differentiation of hPSCs into spinal motor neurons;

FIG. 2 is a photograph showing immunofluorescence staining of cells obtained by inducing differentiation at each stage; wherein, FIG. 2-A is an immunofluorescence staining chart of NEP cell positive marker SOX1 (red) and nucleus marker Hochest (blue); FIG. 2-B is an immunofluorescence staining pattern of MNP cell positive marker Olig2 (green), nuclear marker Hochest (blue); FIG. 2-C is an immunofluorescence staining pattern for the spinal cord motor neuron positive marker HB9 (green), the neurofilament heavy chain marker NF200 (red); FIG. 2-D is a graph showing immunofluorescent staining of the positive marker CHAT (green) and the nuclear marker (Hochest) of mature motor neurons;

FIG. 3 is a graph of the distribution and size of mitochondria of D90D and D90A motor neurons on axons;

FIG. 4 is a graph of mitochondrial movement of D90D and D90A motor neurons;

FIG. 5 shows mitochondrial membrane potential changes in SOD1D 90A motoneurons;

FIG. 6 shows Ca placement into D90D and D90A motor neurons ²⁺ The change of concentration;

FIG. 7 is qRT-PCR of BCL-2, BCL-XL, BCL and BAX in spinal cord motor neurons; * P < 0.01.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.

The method for obtaining the glutamatergic interneurons of the spinal cord by inducing using the human pluripotent stem cells provided in the following embodiments mainly comprises four steps: (1) Utilizing primary hPSCs (human pluripotent stem cells) to induce and differentiate to obtain neuroepithelial NEP; (2) Inducing differentiation to obtain spinal motor neuron progenitor cells; (3) Inducing differentiation to obtain HB9 ⁺ Spinal motor neurons; (4) Culturing and maturing under adherent conditions to obtain CHAT ⁺ Spinal motor neurons.

1. Materials, instruments and apparatus

(1) Mice pregnant for 11.5 days: CF1 strain, purchased from shanghai slek laboratory animals llc.

(2) Human embryonic stem cell lines: h9 line (cell passage number 23-50).

(3) Inducible pluripotent stem cell lines: SOD1D 90A (cell passage number 20-50), SOD1D90D (cell passage number 20-50), cell line all derive from Zhang Su Chun professor laboratory friend situation of Wisconsin stem cell research center of USA and provide.

(3) Reagent and consumable

The reagent materials and consumables purchased are shown in table 1 below.

TABLE 1 procurement of reagent materials and consumables

The instruments, devices and apparatus used are shown in table 2 below.

TABLE 2 instruments, devices and instruments used

Name of instrument	Manufacturer(s) of
		Confocal microscope	Olympus Corp
Fluorescence inversion/righting microscope	Olympus Corp
		Optical microscope	Thermo of Nikon corporation
CO ₂ Cell culture box	Thermo of Fisher corporation
		Biological safety cabinet	Fisher Co Ltd
Electric liquid transfer device	Eppendorf Co Ltd
		Manual liquid transfer device	Eppendorf/Thermo Fisher Co
1.5ml freezing tube	Corning Ltd
		0.22 mu m sterile filter	Millipore Corp
Ultra-low temperature refrigerator at minus 80 DEG C	Panasonic corporation
		Culture dish (35/60/100 mm)	Corning Ltd
6 orifice plate/12 orifice plate/24 orifice plate	Corning Corp Ltd
		T25/T75 culture bottle	Eppendorf Co Ltd
Pipet (5/10/25 ml)	Thermo Fisher Co Ltd
		Disposable syringe (5/25 ml)	Corning Ltd
Water bath pot	Juzhou megasai laboratory devices manufacturing company

The antibodies used are shown in table 3 below.

Antibodies used in Table 3

Name of antibody	Company and goods number	Species and dilution ratio
			Sheep anti-SOX1 antibody	R&Company D	1:1000
Rabbit anti-HOXA3	Sigma Co Ltd	1:1000
			Rabbit anti-OLIG2	Millipore Corp	1:500
Mouse anti-HB9	Sigma Co Ltd	1:500
			Mouse anti-NF200	CST Corp Ltd	1:400
Sheep anti-CHAT	Millipore Corp	1:500
			Donkey anti-sheep Alexa Fluor 488	Invitrogen corporation	1:2000
Donkey anti-rabbit Alexa Fluor 488	Invitrogen corporation	1:2000
			Donkey anti-mouse Alexa Fluor 488	Invitrogen corporation	1:2000
Donkey anti-rabbit Alexa Fluor 594	Invitrogen corporation	1:2000
			Donkey anti-mouse Alexa Fluor 594	Invitrogen corporation	1:2000
Hoechst 33342	Invitrogen corporation	1:1000
			Mito-Tracker Green	Thermo Fisher Co Ltd
TMRM	Thermo Fisher Co Ltd
			Rhod-2-AM	Thermo Fisher Co Ltd
Fluo-4-AM	Thermo Fisher Co Ltd

The self-prepared reagents, culture medium materials are shown in table 4 below.

TABLE 4 self-prepared reagents and media

2. Test method

FIG. 1 is a flow chart of the cell differentiation process provided by the present invention. Each stage is 6 days, and HB9 is obtained after 18 days ⁺ Spinal motor neurons.

FIG. 2 is a photograph showing immunofluorescent staining of cells obtained by induced differentiation at each stage; wherein FIG. 2-A is an immunofluorescence staining pattern of NEP cell positive marker SOX1 (red), nuclear marker Hochest (blue); FIG. 2-B is an immunofluorescence staining pattern of MNP cell positive marker Olig2 (green) and nuclear marker Hochest (blue); FIG. 2-C is an immunofluorescence staining pattern for the spinal cord motor neuron positive marker HB9 (green), the neurofilament heavy chain marker NF200 (red); FIG. 2-D is a graph showing immunofluorescent staining of positive markers CHAT (green) and nuclear markers (hochests) of mature motor neurons.

(1) Primary culture of Mouse Embryonic Fibroblasts (MEFs)

(1) A Mouse Embryo Fibroblast (MEF) basal medium prepared in advance according to the formulation of table 4 above;

(2) killing mice pregnant for 13.5 days by a neck breaking method, exposing and taking out a beaded uterus, placing the beaded uterus in a 10cm culture dish containing PBS, and washing off bloodstain, fat connective tissue and the like;

(3) cutting the uterine wall, taking out embryos one by one, stripping fetal membrane tissues, taking out fetal rats, and sequentially placing the fetal rats in a 6cm culture dish containing PBS (phosphate buffer solution);

(4) cutting off the head, limbs, tail and internal organs of a fetal rat, and then placing the trunk of the fetal rat in a 6cm culture dish containing PBS;

(5) cutting fetal mouse trunk to 1mm ³ Adding 0.25% trypsin, sterilizing in an incubator at 37 ℃ for about 20min, periodically blowing with a gun head during the digestion to make the tissue block as small as possible, adding a prepared MEF basal medium with the same volume to stop digestion, and centrifuging at 1000rpm for 5min;

(6) discarding the supernatant, and inoculating the digested cells into a culture dish of 10 cm; typically, one embryo is inoculated into one petri dish.

(2) MEF cell subculture

(1) When the density of MEF cells is observed to reach about 90%, passage can be carried out;

(2) discard old culture medium and rinse 2 times with PBS medium, then add 0.05% trypsin 2.5mL, at 37 deg.C, 5% CO ₂ Digesting in the incubator for 3-5min;

(3) observing under a microscope, when the cells become round and float, immediately adding an equal volume of MEF basal medium to stop digestion, and blowing to form a single cell suspension;

(4) transferring MEF cells into a 15ml centrifuge tube, centrifuging at 1000rpm for 5min, and removing supernatant;

(5) add MEF basal medium to resuspend, inoculate to a new dish at a ratio of 1 ₂ And (5) culturing in a cell culture box.

(3) MEF cell irradiation

(1) When the third generation of cells overgrow to 90%, digesting MEF cells with 0.05% trypsin, adding an equal volume of MEF basal medium for neutralization, and then transferring into a 50mL centrifuge tube;

(2) irradiating MEF cells by gamma rays, wherein the total dose of the gamma rays reaches 40Gy;

(3) centrifuging the irradiated MEF cell suspension at 1000rpm for 6min;

(4) removing supernatant, resuspending cells with prepared cell freezing solution, placing into freezing box, and placing into-80 deg.C refrigerator overnight;

(5) the next day, the cryopreserved cells were placed in liquid nitrogen for long term cryopreservation.

(4) Cell culture and passage of human pluripotent stem cell hPSC

(1) Preparing a primary maintenance medium of human pluripotent stem cells (hPSCs) and a 0.1% gelatin solution in advance according to the formula of the above table 4;

(2) uniformly spreading 1mL per well of a six-well plate with 0.1% gelatin solution, placing at 37 deg.C, 5% CO ₂ Incubating in an incubator for at least 30min;

(3) sucking off gelatin solution, adding MEF basal medium, recovering MEF cells irradiated with radiation at a rate of 2.5 × 10 per well ⁵ Inoculating each cell in a six-well plate, mixing, placing at 37 deg.C, 5% CO ₂ Culturing in an incubator overnight;

(4) the next day, the MEF basal medium was aspirated off, the hpscs were subsequently seeded onto MEF cells, and 2ml per well of hPSC primary maintenance medium was added to culture the expanded hpscs;

(5) the culture medium is changed every day, the hPSCs are digested by dispase enzyme solution about 5-7 days, and then are subcultured on MEF cells according to the following passage ratio of 1.

(5) Differentiation of hPSCs into neuroepithelial cells (NEPs)

(1) Preparing an induced differentiation medium of NEP in advance according to the formula of the table 4;

(2) on the next day after the hPSC subculture, the hPSC medium was changed to an induced differentiation medium, and differentiation at the neuroepithelial cell stage was started, which was designated as NEP differentiation day 0;

(3) place the cells at 37 ℃ 5% CO ₂ In the cell culture box, the liquid is changed every other day, and the NEP cells can be differentiated after 6 days of culture.

(6) NEP differentiation into spinal cord motor neuron progenitor cells (MNP)

(1) MEF cells were plated on six-well plates one day in advance; preparing a patterned culture medium in advance according to the formula in the table 4 above;

(2) on day 6 of NEP differentiation, NEP cells were digested with dispase enzyme solution, incubated at 37 ℃ and 5% CO ₂ Digesting in the incubator for 3min, then bringing NEP cloning and crimping, washing out the enzyme solution, and then washing twice with DMEM/F-12 solution;

(3) using a patterned culture medium, inoculating the MEF cells according to the ratio of 1;

(4) place the cells at 37 ℃ 5% CO ₂ And in the cell culture box, changing the liquid every other day, and culturing for 6 days to differentiate into the MNP.

(7) MNP to HB9 ⁺ Spinal motor neuron differentiation

(1) Preparing a maintenance modeling culture medium in advance according to the formula of the table 4;

(2) on day 6 of MNP differentiation, resuspending the MNP by using MN culture medium, inoculating the MNP into a T25 culture flask for suspension culture, and changing the culture medium every other day, wherein the MNP stage is counted as day 0;

(3) placing the cells in a CO2 cell culture box at 37 deg.C, 5%;

(4) day 5 of differentiation, sterile round slides were removed, placed in 24-well plates, 50-100. Mu.L of 0.1mg/ml PLO solution was added to the slides, placed at 37 ℃,5% CO ₂ An incubator for overnight incubation;

(5) on the 6 th day of differentiation, the PLO is aspirated off, washed 3 times with sterile purified water, followed by adding 50-100. Mu.L of Lamin solution to the slide glass and placing at 37 ℃ 5% ₂ Incubating for more than 1h in an incubator;

(6) (ii) digesting day 6 neurospheres into single or very small neurospheres with Accutase, inoculating onto Laminin coated slides and placing at 37 ℃,5% ₂ Culturing in a cell culture box, changing liquid every other day, and dyeing after nerve fibers grow out.

(8)CHAT ⁺ Culture and preparation of spinal Motor Neuron (MN)

(1) Preparing a mature culture medium in advance according to the formula of the table 4;

(2) will lay HB9 well ⁺ Observing the 24-well plate of the spinal cord motor neuron under a microscope from an incubator, and finding that the nerve axons grow out of cell bodies;

(3) the maintenance patterned medium was aspirated, 500. Mu.L of maturation medium per well was slowly added along the walls, incubated at 37 ℃ in a 5% CO2 incubator;

(4) changing the whole or half of the culture solution every other day, and culturing for 6 days to obtain CHAT ⁺ Spinal cord motor neurons, nerve filaments winding the edge of the slide in a circle can be observed under a microscope.

3. Cellular immunofluorescence staining

(1) Before cellular immunofluorescence staining, a confining liquid (100 μ L) was prepared in advance according to the following ratio:

89.8. Mu.l PBS medium;

0.2μl Triton-X；

mu.l donkey serum;

primary antibody diluent (100 mu L) is prepared in advance according to the following proportion:

94.8 μ L PBS medium;

0.2. Mu.L of Triton-X solution;

50 μ L donkey serum;

prepare the secondary antibody diluent (100 μ L) in advance according to the following ratio:

95 μ L PBS medium;

50 μ L donkey serum;

(2) After passage, the cells at each stage are inoculated on a glass slide and cultured for 1 to 3 days;

(3) Absorbing the culture medium, washing with PBS culture medium for 2 times, adding 4% paraformaldehyde, and fixing at room temperature for 30min;

(4) Absorbing paraformaldehyde, washing for 3 times with PBS (phosphate buffer solution) culture medium, adding confining liquid, and incubating at room temperature for 1h;

(5) Sucking off the confining liquid, adding primary antibody solution, and incubating overnight at 4 ℃;

(6) The next day, the primary antibody solution was aspirated and washed 3 times with PBS medium for 10min each time;

(7) Adding a secondary antibody solution, adding Hoechest at the same time, and incubating for 45min at room temperature in a dark place;

(8) Removing the secondary antibody solution by suction, washing for 10min with PBS culture medium for 3 times, and then sealing with a sealing agent;

(9) The images were observed with a normal fluorescence microscope or photographed under a confocal microscope.

As shown in fig. 2, wherein fig. 2-a is an immunofluorescence staining pattern of NEP cell positive marker SOX1 (red), nuclear marker Hochest (blue); FIG. 2-B is an immunofluorescence staining pattern of MNP cell positive marker Olig2 (green), nuclear marker Hochest (blue); FIG. 2-C is an immunofluorescence staining pattern for the spinal cord motor neuron positive marker HB9 (green), the neurofilament heavy chain marker NF200 (red); FIG. 2-D is an immunofluorescence staining pattern for the positive marker CHAT (green) and the nuclear marker (Hochest) of mature motor neurons.

4. Mitochondrial fluorescent dye labeling

Mito-Tracker Green is a Green fluorescent probe that can be used for mitochondrial-specific fluorescent staining of living cells. The method comprises the following specific steps:

(1) The neurospheres were first inoculated into PLO and Laminin coated glass dishes and induced to differentiate further.

(2) The fluorescent dye Mito-Tracker Green was diluted with DMEM/F-12 medium to a final concentration of 300nM.

(3) The cells were washed 2 times with DMEM/F-12 medium, the DMEM/F-12 medium was aspirated,adding the prepared fluorescent dye and at 37 deg.C, 5% ₂ Incubate 30min.

(4) The dye was aspirated, washed 2 times with DMEM/F-12 medium, replaced with motor neuron medium, immediately observed under a confocal microscope and photographed. As shown in fig. 3.

5. Measurement of mitochondrial length and distribution index

According to the method, after mitochondrial fluorescent dye is marked and photographed, the length and the number of mitochondria in a neuron axon are measured through the function of a curve ROI of ImageJ; the mitochondria were classified and counted according to length of 2, 4, 6 μm. As shown in fig. 3.

Figure 3 shows the distribution and size of mitochondria on axons for D90D and D90A motor neurons, respectively. FIG. 3-A is mitochondria in neurons labeled by Mito-Tracker Green; FIG. 3-B is a statistical result of the distribution of the density of mitochondria on axons; FIG. 3-C is a statistics of the number of mitochondria on axons; FIG. 3-D is a statistical result of mitochondrial size length; FIGS. 3-E are statistics of the proportion of different length mitochondria. In fig. 3, P < 0.05; * P is less than 0.01; * P < 0.001; * P < 0.0001; NS, no design.

6. Measurement of mitochondrial movement

Confocal microscopy delayed real-time live cell imaging was used to detect axonal transport. According to the method, after mitochondrial fluorescent dye is marked and photographed, the marked motor neurons are placed under a confocal microscope for observation, and a video of mitochondrial movement is photographed for 10min, one per 4 seconds. Mitochondrial motion is analyzed by using a kymograph plug-in Image J, and the motion state and the motion direction of mitochondria are counted.

Mitochondria are dynamically transported in neurons to meet their sufficient energy supply and modification of mitochondria, and thus normal transport of mitochondria is an indicator of normal mitochondrial function. Mitochondria were labeled with the fluorescent dye TMRM, mitochondrial motion was photographed under laser confocal, and mitochondrial motion was analyzed by installing kymograph plug-in Image J software, with the results shown in fig. 4A. As shown in FIG. 4B, the ratio of motile mitochondria to total mitochondria was significantly reduced in D90A motor neurons compared to D90D motor neurons, 0.173 + -0.019 in the D90A group, 0.295 + -0.016 in the D90D group, and P < 0.0001. As shown in fig. 4C, the present application also analyzed the direction of mitochondrial movement, and the results showed that the reverse transport ratio of mitochondria of D90A motor nerve was significantly decreased, 0.074 ± 0.012 in D90A group, 0.160 ± 0.020 in D90D group, p =0.0014 in D90D group; the anterograde transport ratio also decreased, but there was no statistical difference between D90D group 0.135 + -0.016 and D90A group 0.099 + -0.014, with P > 0.05. The above results indicate that mitochondria are essentially in a quiescent state in SOD1D 90A ALS motor neurons and retrograde transport is impaired, with no significant change in anterograde transport, indicating that ALS spinomotor neuron retrograde transport is impaired prior to anterograde transport.

7. Mitochondrial potential detection

Mitochondrial potential detection is carried out by using TMRM fluorescent dye, and the specific method is as follows:

(1) Inoculating the motor neurospheres into a glass dish coated by PLO and Laminin, and inducing differentiation through a neural differentiation culture medium for subsequent dye dyeing for later use;

(2) Diluting TMRM dye to 200nM in DMEM/F-12 medium, washing motor neurons 2 times in DMEM/F-12 medium, adding diluted TMRM dye, and diluting at 37 deg.C with 5% CO ₂ Incubating in incubator for 30min;

(3) Washing off the dye, washing with DMEM/F-12 culture medium for 2 times, changing into motor neuron culture medium containing 40nM TMRM dye, and observing and taking pictures under confocal microscope;

mitochondrial membrane potential is the fundamental condition for mitochondria to undergo oxidative phosphorylation and constantly produce ATP to maintain cellular function, and a decrease in mitochondrial membrane potential is considered to be the earliest indicator of the onset of apoptosis. D90D and D90A motor neuron mitochondrial potentials were detected by using TMRM fluorescent probes, and the mean fluorescence intensities were counted.

As shown in FIG. 5-B, the mitochondrial potential of D90A motor neurons was significantly lower than that of D90D motor neurons, 29.74 + -2.176 in the D90A group, 45 + -2.248 in the D90D group, and P < 0.001. This indicates mitochondrial dysfunction in D90A motor neurons. The above results demonstrate that the SOD1D 90A mutation affects the membrane potential of motor neurons.

8. Mitochondrial and intracytoplasmic Ca ²⁺ Measurement of concentration

Rhod-2-AM is a calcium ion fluorescent probe for measuring mitochondrial Ca2+ concentration, while Fluo-4-AM is a fluorescent probe for detecting cytoplasmic Ca2+ concentration. The specific method comprises the following steps:

(1) Inoculating a motor neurosphere into a glass dish coated by PLO and Laminin, and inducing differentiation by using a motor neuron culture medium;

(2) Washing the motor neurons seeded on the glass plate with DMEM/F-12 medium for 2 times, replacing with serum-free medium, adding Rhod-2-AM and Fluo-4-AM dyes to a final concentration of 1. Mu.M, and then incubating at 37 ℃ for 5% CO ₂ Incubating in an incubator for 30min;

(3) The medium was aspirated, washed twice with DMEM/F-12 medium, then replaced with motoneuron medium, observed under a confocal microscope and photographed.

Mitochondrial Ca ²⁺ The concentration is closely related to the function of ALS motor neurons, and the shape and function of mitochondria are closely related to abnormal calcium ion homeostasis. Mitochondrial calcium overload can even lead to neuronal degeneration, and mitochondrial calcium signaling to cell death depends on increased mitochondrial calcium levels and stimulation of apoptosis.

The test first detects the intracytoplasmic and intracytoplasmic calcium ion concentration of D90D and D90A motor neurons by staining with Rhod-2 and Fluo-4, and as a result, it was found that, as shown in FIG. 6-A, the average intensity of Rhod-2 of D90D neurons should be significantly lower than that of D90A neurons, 12.84 + -0.431 for D90D group, 16.91 + -0.859 for D90A group, and P < 0.0001; however, as shown in FIG. 5-B, the mean fluorescence intensity of Fluo-4 did not change significantly.

9. Real-time fluorescent quantitative Real-time PCR reaction

(1) The reaction system is as follows: the reaction solution was prepared on ice

Reagent	Amount of the use
		SYBR Premix Ex TaqⅡ	10μl
PCR Forward Primer	0.8μl
		PCR Reverse Primer	0.8μl
ROX Reference Dye	0.8μl
		DNA template	0.8μl
Sterilized water	0.8μl
		Total	0.8μl

(2) Reaction conditions

Temperature of	Time	Number of cycles
			95℃	1min	1
95℃	15s					40
			60℃	30s

The final quantitative PCR result is calculated by using a formula of 2-delta-ct.

Statistical analysis was performed using SPSS 22.0 software, statistical mapping was performed using Graphpad Prism 7.0 software, fluorescence intensity was quantified using Image J software, and each experiment was repeated at least three times. Data are expressed as Mean ± SEM, continuous variable between two groups using student-t test, analysis of two sample rates using chi-square test. All results in this study were defined as statistically different as P < 0.05.

As shown in FIG. 7, since the mitochondrial membrane potential of SOD1D 90A motor neurons is reduced and the membrane potential reduction is an early indicator of mitochondrial apoptosis, the anti-apoptosis genes BCL-2 and BCL-XL and the pro-apoptosis genes BAX and BAK are detected by fluorescence quantitative PCR, and the BCL-XL gene is significantly reduced in D90A motor neurons, while the expression levels of BCL-2, BAX and BAK in D90D and D90A motor neurons are not significantly different statistically.

The practice of the present invention has been described in detail in the foregoing detailed description, however, the present invention is not limited to the specific details in the foregoing embodiment. Within the scope of the claims and the technical idea of the invention, a number of simple modifications and changes can be made to the technical solution of the invention, and these simple modifications are within the scope of protection of the invention.

Claims

1. A method for inducing and obtaining spinal cord motor neurons by using human pluripotent stem cells is characterized by comprising the following steps:

the second small molecule composition comprises a GSK3 inhibitor, a TGF-beta/Smad inhibitor, a BMP inhibitor, a RAR nuclear receptor activator, and a SHH agonist;

s4, taking HB9 obtained in the step S3 ⁺ Performing adherent culture of spinal motor neurons, performing neurotrophic maturation to obtain CHAT ⁺ Spinal motor neurons.

2. The method for obtaining spinal motor neurons by induction using human pluripotent stem cells according to claim 1, wherein in step S1, the differentiation-inducing medium comprises DMEM/F12 and Neurobasal in a volume ratio of 1, and further comprises GlutaMax in a volume fraction of 1%, B27 in a volume fraction of 1%, N-2 in a volume fraction of 0.5%, AA in a volume fraction of 0.1mM, a GSK3 inhibitor in a volume fraction of 3 μ M, a TGF- β/Smad inhibitor in a volume fraction of 2 μ M, and a BMP inhibitor in a volume fraction of 2 μ M.

3. The method for obtaining spinal motor neurons by induction using human pluripotent stem cells according to claim 1, wherein in step S2, the patterned medium comprises DMEM/F12 and Neurobasal in a volume ratio of 1.

4. The method for inducing acquisition of spinal motor neurons using human pluripotent stem cells according to claim 3, wherein in step S2, the second small molecule composition comprises CHIR99021, DMH-1, SB431542, RA and SAG.

5. The method for obtaining spinal motor neurons by induction using human pluripotent stem cells according to claim 1, wherein in step S3, the maintenance patterned medium comprises DMEM/F12 and Neurobasal in a volume ratio of 1, and further comprises GlutaMax in a volume fraction of 1%, B27 in a volume fraction of 1%, N-2 in a volume fraction of 0.5%, AA in a volume fraction of 0.1mM, an RAR nuclear receptor activator in a volume fraction of 0.01 μ M, and an SHH agonist in a volume fraction of 0.1 μ M.

6. The method for inducing and obtaining spinal motor neurons using human pluripotent stem cells according to claim 1, wherein the step S4 comprises: after inducing differentiation for 5 days by the method of step S3, the sterile round slide was placed in a well plate, and PLO solution was dropped on the slide at 37 ℃ and 5% CO ₂ Incubating overnight under the environment; absorbing and removing PLO the following day, washing, adding Laminin solution dropwise onto the slide, or using precooled Matrigel alone, and making the content of CO 5% at 37 ℃% ₂ Continuously incubating for more than 1h under the environment; finally, the large neurospheres are digested into single spinal cord motor neurons or small neurospheres by using cell digestive juice Accutase(ii) a Inoculating the suspension of spinal motor neurons or small neurospheres on a slide treated by PLO/Lamin or Matrigel for adherent culture for 6-10 days, carrying out neurotrophic maturation to obtain the expression CHAT ⁺ Spinal motor neurons.

7. The method for obtaining spinal cord motor neurons using induction of human pluripotent stem cells as claimed in claim 6, wherein in step S4, the composition of the medium for neurotrophic comprises Neurobasal, and further comprises GlutaMax at a volume fraction of 1%, N-2 at a volume fraction of 0.5%, B27 at a volume fraction of 1%, AA at 0.1mM, SAG at 0.1. Mu.M, RA at 0.01. Mu.M, IGF-1 at 10ng/mL, CNTF at 10ng/mL, compound E at 0.1. Mu.M, BDNF at 10ng/mL and GDNF at 10 ng/mL.

8. A spinal cord motor neuron obtained using the method of any one of claims 1-7.

9. A composition or kit for obtaining spinal cord motor neurons by induction of human pluripotent stem cells, comprising the first small molecule composition, the second small molecule composition, and the third small molecule composition according to any one of claims 1-6.

10. The use of spinal cord motor neurons prepared by the method of any one of claims 1 to 6 for studying mitochondrial function changes by dynamically observing the visualized changes of the density, motor state, membrane potential and calcium ion concentration of spinal cord motor neurons mitochondria, thereby evaluating drug effects and screening clinical drugs; or for preparing a medicament for treating spinal motor neuron loss or imbalance and dyskinesia and/or sensory disorders caused by nervous system diseases or injuries; or used for the pathological mechanism research and drug screening of the diseases.