Method for differentiating human pluripotent stem cells into dopaminergic neurons

Abstract

The invention relates to a method for differentiating human pluripotent stem cells into dopaminergic neurons. The method mainly comprises the following steps: preparing and culturing cells, inducing ventral neural progenitor cells of a midbrain, and performing terminal differentiation on the dopaminergic neurons. Compared with the existing differentiation technology, the method disclosed by the invention has the characteristic of short differentiation period, serum and animal-derived components are eliminated in the differentiation process, and a serum-free differentiation system conforming to clinical standards is established; meanwhile, non-target differentiated cells are inhibited by accurately regulating and controlling a signal channel, functional maturation of dopaminergic neurons is promoted, the release capacity of dopamine is ensured to be equivalent to that of in-vivo neurons, and good application prospects are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical fields of cell biology and regenerative medicine, and particularly relates to a method for differentiating human pluripotent stem cells into dopaminergic neurons. Background Art

[0002] Dopaminergic Neurons (DNs) are key cell types in the central nervous system, mainly distributed in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) of the midbrain. Their dysfunction is closely related to Parkinson's Disease (PD). Current PD treatment methods (such as levodopa, deep brain stimulation) can only relieve symptoms and cannot reverse neuronal degeneration. Cell replacement therapy based on human pluripotent stem cells (hPSCs) is regarded as a potential radical cure.

[0003] The existing differentiation technologies generally have the following problems: 1. Long differentiation cycle: Traditional protocols require more than 50 days, with low efficiency and high costs. 2. Dependence on serum and animal components: The use of fetal bovine serum (FBS) or feeder cells (such as MEF) leads to batch differences and clinical translation risks (such as immune rejection, pathogen contamination). 3. Immature function: The dopamine release ability of the differentiated neurons is weak, and there is a lack of high expression of mature markers (such as TH, Nurr1, Pitx3). 4. Insufficient signal pathway regulation: There is a lack of fine design for the synergistic effects of key pathways such as Wnt, SHH, and TGF-β, resulting in a high proportion of non-neural or ectopic differentiation.

[0004] Therefore, developing an efficient, stable, and reproducible method for differentiating dopaminergic neurons is of great significance for the basic research, cell therapy, and drug screening of Parkinson's disease. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for differentiating human pluripotent stem cells into dopaminergic neurons, which solves the problems of long differentiation cycle, dependence on serum and animal components, and insufficient signal pathway regulation in the existing differentiation technologies.

[0006] The present invention provides a method for differentiating human pluripotent stem cells into dopaminergic neurons, comprising the following steps:

[0007] (1) Cell preparation and culture: Resuscitate human pluripotent stem cells and culture them in a serum-free medium;

[0008] (2) Induction of midbrain progenitor cells: Prepare an induction medium to perform the first-stage differentiation of the above cells, wherein the induction medium contains a nutritional additive and a signal pathway regulator A; after culturing for 5 - 7 days, adjust the composition of the induction medium and perform the second-stage differentiation;

[0009] (3) Dopaminergic neuron terminal differentiation: The cells after step (2) are subjected to terminal differentiation in a terminal differentiation medium, and the terminal differentiation medium is further optimized at the functional maturation stage to obtain dopaminergic neurons; wherein the terminal differentiation medium contains a nutrient factor and a signaling pathway regulator B.

[0010] Preferably, the human pluripotent stem cells in step (1) include human embryonic stem cells (such as H1, H9) or human induced pluripotent stem cells (hiPSC).

[0011] Preferably, the serum-free medium in step (1) is mTeSR TM Plus.

[0012] Preferably, the basal medium of the induction medium in step (2) is DMEM / F12 and Neurobasal meida at a volume ratio of 1:1.

[0013] Preferably, the signaling pathway regulator A in step (2) includes one or more of a WNT signaling pathway activator, a SHH signaling pathway activator, a BMP signaling pathway inhibitor, and a TGF-β signaling pathway inhibitor A.

[0014] More preferably, the WNT signaling pathway activator includes CHIR99021 at a concentration of 1 - 5 μM; the SHH signaling pathway activator includes Purmorphamine and C25II at concentrations of 1 - 5 μM and 200 ng / ml - 1 μg / ml respectively; the BMP signaling pathway inhibitor includes LDN193189 at a concentration of 100 - 500 nM; the TGF-β signaling pathway inhibitor A includes SB431542 at a concentration of 10 - 20 μM.

[0015] Preferably, adjusting the components of the induction medium in step (2) refers to adjusting the concentrations of the WNT signaling pathway activator and the SHH signaling pathway activator.

[0016] Preferably, the nutrient additives in step (2) include one or more of N2 (1x) and non-essential amino acids (NEAA, 1x).

[0017] Preferably, the nutrient factors in step (3) include one or more of N2, B27, BDNF, GDNF, and Ascorbic Acid.

[0018] More preferably, the concentrations of N2 and B27 are both 1x, providing nutritional support and a differentiation induction environment.

[0019] More preferably, the concentrations of BDNF and GDNF are 10 - 50 ng / mL, supporting neuron survival and synapse formation.

[0020] More preferably, the concentration of Ascorbic Acid is 100 - 150 μM.

[0021] Preferably, the signal pathway regulator B in step (3) includes one or more of a Notch signal inhibitor, a cAMP signal pathway activator, and a TGF-β signal pathway activator B.

[0022] More preferably, the Notch signal inhibitor includes DAPT ((3,5-difluorophenylacetyl)-L-alanyl-L-2-phenylglycine tert-butyl ester), with a concentration of 10 - 15 μM, accelerating maturation.

[0023] More preferably, the cAMP signal pathway activator includes cAMP, with a concentration of 0.1 - 0.5 mM, promoting TH expression.

[0024] More preferably, the TGF-β signal pathway activator B includes TGF-β3, with a concentration of 1 - 3 ng / mL, stabilizing the dopaminergic phenotype.

[0025] Preferably, the functional maturation stage in step (3) refers to the 21st - 25th day of induced culture after cell thawing.

[0026] Preferably, the further optimization of the terminal differentiation medium in step (3) refers to adding a signal pathway regulator C and / or an antioxidant to the terminal differentiation medium.

[0027] More preferably, the signal pathway regulator C includes an NF-κB activator. Further preferably, the NF-κB activator includes IL-1β. The concentration of IL-1β is 5 - 10 ng / mL, simulating a neuroinflammatory microenvironment and enhancing functional adaptability.

[0028] More preferably, the antioxidant includes N-acetylcysteine. The concentration of N-acetylcysteine is 1 - 5 mM, reducing oxidative stress damage.

[0029] Beneficial effects

[0030] The method of the present invention has the characteristic of a short differentiation cycle compared with the existing differentiation techniques. During the differentiation process, serum and animal-derived components are eliminated, and a serum-free differentiation system that meets clinical standards is established. At the same time, by precisely regulating the signal pathway, non-target differentiation is inhibited, the functional maturation of neurons is promoted, and the dopamine release ability is ensured to be equivalent to that of in vivo neurons, showing good application prospects. Brief Description of the Drawings

[0031] Figure 1Representative immunofluorescence staining of human induced pluripotent stem cells (scale bar: 100 μm).

[0032] Figure 2 Representative immunofluorescence staining of ventral midbrain progenitor cells (scale bar: 100 μm).

[0033] Figure 3 Representative immunofluorescence of dopaminergic neurons (scale bar: 100 μm). Detailed implementation manners

[0034] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0035] Example 1

[0036] 1. iPSC cell culture

[0037] 1.1 Coat a 6-well cell culture plate with Laminin-521 (10 μg / mL) and pre-incubate at 37 °C for ≥ 2 hours;

[0038] 1.2 Rapidly thaw human induced pluripotent stem cells (iPSC) in a 37 °C water bath, and after centrifugation, resuspend them in mTeSRPlus medium containing 10 μM Y-27632 (ROCK inhibitor);

[0039] 1.3 Discard the Laminin-521 coating solution and seed at 0.5×10 6 cells / well into the coated culture plate;

[0040] 1.4 Change the medium daily until the cell density reaches 70%-80%.

[0041] 2. Induction of ventral midbrain neural progenitor cells (days 1-12)

[0042] 2.1 First-stage differentiation (days 1-7)

[0043] 2.1.1 Coat a 6-well cell culture plate with Laminin-521 (10 μg / mL) and pre-incubate at 37 °C for ≥ 2 hours;

[0044] 2.1.2 Digest iPSC into single cells with Accutase and incubate at 37 °C for 5-10 minutes;

[0045] 2.1.3 Observe under the microscope that the cell dissociation is complete, and add mTeSR TMPlus medium, centrifuge at 200 x g for 5 min;

[0046] 2.1.4 Resuspend in mTeSR Plus medium containing 10 μM Y-27632 and seed into a 6-well plate at a density of 2 x 10 6 cells / well. After swirling gently, incubate at 37 °C and 5% CO2 for 24 hours;

[0047] 2.1.5 On the next day, when the cell density is >90% under the microscope, change the mTeSR TM Plus to DA induction basal medium (DMEM / F12: Neurobasal meida = 1:1), and add the following components: N2 (1x), NEAA (1x), CHIR99021 (3 μM), C25II (500 ng / mL), Purmorphamine (1 μM), LDN193189 (200 nM), SB431542 (10 μM).

[0048] Culture conditions: 37 °C, 5% CO2. Change the medium daily for 7 days.

[0049] 2.2 Second-stage differentiation (days 8 - 12)

[0050] Small molecule adjustment of DA induction basal medium: CHIR99021 (2.5 μM), C25II (200 ng / mL), Purmorphamine (0.5 μM). Detection: On day 12, detect the FOXA2 + / LMX1A + ratio (>85%) by flow cytometry / immunofluorescence staining.

[0051] 2.3 Dopaminergic neuron terminal differentiation (days 13 - 25)

[0052] 2.3.1 Terminal differentiation stage (days 13 - 17)

[0053] DA maturation medium (DMEM / F12: Neurobasal meida = 1:1), add: N2 (1x), B27 (1x), BDNF (20 ng / mL), GDNF (20 ng / mL), Ascorbic Acid (100 μM), cAMP (0.5 mM), TGF-β3 (1 ng / mL). Culture conditions: 37 °C, 5% CO2. Change the medium every 2 days for 5 days.

[0054] 2.3.2 Terminal differentiation stage (days 18 - 20)

[0055] DA maturation medium (DMEM / F12: Neurobasal meida = 1:1), supplemented with: N2 (1x), B27 (1x), BDNF (20 ng / mL), GDNF (20 ng / mL), Ascorbic Acid (100 μM), cAMP (0.5 mM), TGF-β3 (1 ng / mL), DAPT (10 μM).

[0056] Culture conditions: 37 °C, 5% CO2, change the medium every 2 days.

[0057] 2.3.3 Functional maturation stage (days 21 - 25)

[0058] DA maturation medium (DMEM / F12: Neurobasal meida = 1:1), supplemented with: N2 (1x), B27 (1x), BDNF (20 ng / mL), GDNF (20 ng / mL), Ascorbic Acid (100 μM), cAMP (0.5 mM), TGF-β3 (1 ng / mL), DAPT (10 μM), IL-1β (5 ng / mL), N-acetylcysteine (NAC, 1 mM).

[0059] * Detection: Immunofluorescence detection of TH on day 25 + / Nurr1 + Double positive cells (>40%).

[0060] 3. Cryopreservation and recovery

[0061] 3.1 Cryopreservation (optimal on days 13 - 19)

[0062] 3.1.1 Digest the cells into small aggregates (5 - 20 cells / aggregate) with Accutase and incubate at 37 °C for 3 - 5 minutes;

[0063] 3.1.2 Observe the degree of cell dissociation under the microscope, add mTeSR Plus medium equal in volume to the digestive solution, centrifuge at 200 xg for 5 min;

[0064] 3.1.3 Resuspend in a cryopreservation solution containing 20% DMSO and 60% KnockOut serum (3×10 6 cells / mL);

[0065] 3.1.4 Transfer to liquid nitrogen for storage after programmed cooling.

[0066] 3.2 Recovery and re-culture

[0067] 3.2.1 Coat a 6-well cell culture plate with Laminin-521 (10 μg / mL) and pre-incubate at 37 °C for ≥2 hours;

[0068] 3.2.2 Rapidly thaw the ventral midbrain neural progenitor cells in a 37 °C water bath, resuspend them in DA maturation medium containing 10 μM Y-27632 (ROCK inhibitor) after centrifugation, and add: N2 (1x), B27 (1x), BDNF (20 ng / mL), GDNF (20 ng / mL), Ascorbic Acid (100 μM), cAMP (0.5 mM), TGF-β3 (1 ng / mL), DAPT (10 μM);

[0069] 3.2.3 Discard the Laminin-521 coating solution and seed the cells at 2.5×10 6 cells / well into the coated culture plate;

[0070] 3.2.4 Replace the DA medium supplemented with small molecules on the second day, and then replace the medium every two days.

[0071] The method of the present invention has the following characteristics:

[0072] ① High efficiency and rapidity: The differentiation cycle is shortened to 28 days, and the efficiency is increased to 50%+;

[0073] ② Serum-free system: Completely avoid animal components, meet cGMP standards, and reduce clinical risks;

[0074] ③ Signal synergy optimization: Through the combination of CHIR99021 / LDN193189 / SB431542, the non-target differentiation rate is <5%;

[0075] ④ High functional maturity: The dopamine release amount reaches 0.8 ng / 10^6 cells / hour (detected by ELISA), which is comparable to that of adult substantia nigra neurons.

[0076] It can be seen from Figure 1 that the human induced pluripotent stem cell markers Oct4 and Sox1 are well expressed.

[0077] It can be seen from Figure 2 that on the 11th day of differentiation, the immunofluorescence of the ventral midbrain neural progenitor cell marker LMX1 is well expressed.

[0078] It can be seen from Figure 3 that on the 19th day of differentiation, the dopaminergic neuron marker TH can be highly expressed.

Claims

1. A method for differentiating human pluripotent stem cells into dopaminergic neurons, comprising the following steps: (1) Cell preparation and culture: Resuscitate human pluripotent stem cells and culture them in a serum-free medium; (2) Induction of ventral midbrain neural progenitor cells: Prepare an induction medium to perform the first-stage differentiation on the above cells, wherein the induction medium contains a nutritional additive and a signaling pathway regulator A; after culturing for 5-7 days, adjust the components of the induction medium to perform the second-stage differentiation; (3) Terminal differentiation of dopaminergic neurons: Perform terminal differentiation on the cells after step (2) in a terminal differentiation medium, and optimize the terminal differentiation medium during the functional maturation stage to obtain dopaminergic neurons; wherein the terminal differentiation medium contains a nutritional factor and a signaling pathway regulator B.

2. The method according to claim 1, wherein: The human pluripotent stem cells in step (1) include human embryonic stem cells or human induced pluripotent stem cells.

3. The method according to claim 1, characterized in that: The signaling pathway regulator A in step (2) includes one or more of a WNT signaling pathway activator, a SHH signaling pathway activator, a BMP signaling pathway inhibitor, and a TGF-β signaling pathway inhibitor A.

4. The method according to claim 3, characterized in that: The WNT signaling pathway activator includes CHIR99021 at a concentration of 1-5 μM; the SHH signaling pathway activator includes Purmorphamine and C25II at concentrations of 1-5 μM and 200 ng / ml - 1 μg / ml, respectively; the BMP signaling pathway inhibitor includes LDN193189 at a concentration of 100-500 nM; the TGF-β signaling pathway inhibitor A includes SB431542 at a concentration of 10-20 μM.

5. The method according to claim 3, characterized in that: Adjusting the components of the induction medium in step (2) refers to adjusting the concentrations of the WNT signaling pathway activator and the SHH signaling pathway activator.

6. The method according to claim 1, characterized in that: The nutritional additive in step (2) includes one or more of N2 and non-essential amino acids.

7. The method according to claim 1, characterized in that: The nutritional factor in step (3) includes one or more of N2, B27, BDNF, GDNF, and Ascorbic Acid.

8. The method according to claim 1, characterized in that: The signaling pathway regulator B in step (3) includes one or more of a Notch signaling inhibitor, a cAMP signaling pathway activator, and a TGF-β signaling pathway activator B.

9. The method according to claim 1, characterized in that: The functional maturation stage in step (3) refers to the 21st - 25th day after thawing and induction culture of the cells.

10. The method according to claim 1, wherein: Re-optimizing the terminal differentiation medium in step (3) refers to adding a signaling pathway regulator C and / or an antioxidant to the terminal differentiation medium.

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