CN113846060A

CN113846060A - Application of nano material in promoting differentiation of embryonic stem cells to neural precursor cells

Info

Publication number: CN113846060A
Application number: CN202111271780.XA
Authority: CN
Inventors: 朱融融; 程黎明; 王照洁
Original assignee: Shanghai Tongji Hospital
Current assignee: Shanghai Tongji Hospital
Priority date: 2021-10-29
Filing date: 2021-10-29
Publication date: 2021-12-28
Anticipated expiration: 2041-10-29
Also published as: CN113846060B

Abstract

The invention relates to an application of a nano material in promoting differentiation of embryonic stem cells to neural precursor cells. The method firstly adopts a coprecipitation method to synthesize the nano-layered double hydroxide consisting of different metal ions at normal temperature, a cell survival rate detection experiment proves that the nano-layered double hydroxide has low cytotoxicity on the embryonic stem cells, and by detecting the expression of a neural precursor cell marker gene or protein, the nano-layered double hydroxide added into a system for inducing the embryonic stem cells to differentiate into the neural precursor cells is found to remarkably promote the differentiation, and the adding time and concentration of the nano-layered double hydroxide are further optimized, so that the induced differentiation effect is remarkably improved. The invention provides a new strategy for promoting the differentiation of embryonic stem cells to neural precursor cells and creates conditions for treating related diseases of the nervous system.

Description

Application of nano material in promoting differentiation of embryonic stem cells to neural precursor cells

Technical Field

The invention relates to induced differentiation of stem cells, in particular to application of a nano material in promoting differentiation of embryonic stem cells to neural precursor cells.

Background

Stem cell transplantation brings new hopes for the treatment of central nervous system diseases, and the regulation and mechanism analysis of stem cell fate are leading research fronts and hot spots in the field. Embryonic Stem Cells (ESCs) with totipotency of differentiation can be differentiated into specific nerve cell types under a proper external signal induction condition, including Neural Precursor Cells (NPCs), dopaminergic neurons, motor neurons, oligodendrocyte precursor cells and the like, and provide a series of solutions for treatment after nerve injury. Among them, NPCs are not only capable of self-renewal and differentiation into all types of nerve cells, but also capable of migration and integration into damaged sites of the central nervous system, and thus are ideal therapeutic means for nervous system-related diseases. Therefore, finding a new method for regulating differentiation of ESCs into NPCs and revealing the mechanism of ESCs have important scientific significance.

The prior art discloses some differentiation methods of ESCs to NPCs, for example, patent document CN107254442A discloses a method for differentiating neural precursor cells from embryonic stem cells, which is characterized in that the embryonic stem cells are cultured in vitro in the form of colonies, and peripheral cells are more easily differentiated due to the compact cells in the middle of the colonies and strong interaction among the cells; journal paper (plum, chenhong wei, huixing, etc. directed differentiation of human embryonic stem cells into neural precursor cells [ J ] zoology research, 2007,28 (3)) discloses inducing the directional differentiation of human embryonic stem cells cultured in a homologous feeder layer under a serum-free condition by adopting a single-layer adherent differentiation method to obtain a high proportion of neural precursor cells; journal papers (huzhixing, gunny, liangming. hepatocyte growth factor promotes differentiation of human embryonic stem cells into neural precursor cells [ J ]. journal of chinese pathophysiology, 2010,26(4) ], disclose that a serum-free neural differentiation system containing Hepatocyte Growth Factor (HGF) and G5 can effectively induce neural differentiation of hESCs.

Since the size of the nano material is similar to that of biological molecules, the nano material is easy to interact with DNA or protein in organs, tissues or cells, and the research of regulating and controlling the fate of ESCs through the physicochemical properties of the nano material is an important research direction in the field. In recent years, more and more studies have been conducted to control the neural differentiation process of ESCs using nanomaterials with stable biochemical properties. The nanomaterial can induce differentiation of ESCs into motor neurons, dopaminergic neurons, and glial cells. However, no report related to the regulation of differentiation from ESCs to NPCs by nanomaterials is available at present.

Disclosure of Invention

The invention aims to provide a new application of nano-layered double hydroxide aiming at the defects in the prior art.

It is still another object of the present invention to provide a method for promoting differentiation of embryonic stem cells into neural precursor cells.

In order to achieve the first purpose, the invention adopts the technical scheme that:

the application of the nano layered double hydroxide in promoting the differentiation of embryonic stem cells to neural precursor cells.

As a preferred embodiment of the invention, the nano-layered double hydroxide has divalent metal cations selected from Mg²⁺、Zn²⁺And Ca²⁺。

As another preferred example of the present invention, the nano-layered double hydroxide has a trivalent metal cation selected from Al³⁺And Fe³⁺。

As another preferred example of the present invention, the nano-layered double hydroxide has an interlayer anion selected from Cl^-、NO₃ ^-And SO₄ ^2-。

In another preferred embodiment of the present invention, the molar ratio of the divalent metal ions to the trivalent metal ions of the nano-layered double hydroxide is (0.1-5): 1.

As another preferable example of the present invention, the nano-layered double hydroxide has a general formula of [ M²⁺ _(1-x)M³⁺ _x(OH)₂]_x+[A^n-]_x/n·zH₂O, wherein M²⁺Is a divalent metal cation, M³⁺Is a trivalent metal cation, A^n-Is an anion with the valence of n between layers, x is the molar ratio of trivalent cations to all cations, and z is the number of crystal water between each nano-layered double hydroxide molecule.

In order to achieve the second object, the invention adopts the technical scheme that:

a method for promoting differentiation of embryonic stem cells into neural precursor cells comprises the step of adding a nano layered double hydroxide into a system for differentiation of embryonic stem cells into neural precursor cells.

As a preferred example of the present invention, the nano-layered double hydroxide is added on days 3 to 5 of the induced differentiation culture.

As another preferable example of the present invention, the concentration of the nano-layered double hydroxide in the differentiation system is 1 to 40. mu.g/mL.

More preferably, the concentration of the nano-layered double hydroxide in the differentiation system is 10 μ g/mL.

The invention has the advantages that:

1. the method specifically induces the differentiation of the ESCs to the NPCs by adopting the nano-layered double hydroxide for the first time, defines the new function of the nano-layered double hydroxide in the regulation and control of the fate of the ESCs, and provides a strategy for optimizing a differentiation system of the ESCs to the NPCs.

2. According to the invention, the nanolayered double hydroxide and the ESCs are co-cultured, materials are added into a differentiation system of the ESCs according to concentration gradients and different time windows respectively, and the expression levels of the corresponding NPCs marker genes and proteins are detected, so that the appropriate concentration and the key time window for promoting the NPCs differentiation by the nanolayered double hydroxide are found, and the effect of induced differentiation is obviously improved.

3. The invention adopts the immunoblotting method to determine the effect of the nano-layered material in promoting the differentiation of the ESCs to the NPCs, and provides a new strategy for optimizing the neural differentiation system of the ESCs.

Drawings

FIG. 1: characterization of the nanomaterial LDH and effect on the cell proliferative capacity of mESCs. (A) TEM detects the structure of two LDHs; (B) detecting the Zeta potential of the surface of the material; (C) CCK8 measures cell viability at different concentrations of LDH-treated

mESCs

24h and 48 h.

FIG. 2: qPCR detects the influence on the expression level of NPCs marker genes Sox1, Pax6, N-cadherin and Map2 after MgFe-LDH and MgAl-LDH materials are added in the differentiation of ESCs to NPCs.

FIG. 3: screening the optimal time window for MgFe-LDH materials to promote the differentiation of mESCs to NPCs. (A) The qPCR detects the expression level of NPCs marker genes Sox1, Pax6, N-cadherin and Map2 after MgFe-LDH is added on the 1 st to 5 th days during the differentiation of ESCs to NPCs; (B) immunofluorescence detects the fluorescence signals of the marker proteins Sox1, Pax6 and N-cadherin.

FIG. 4: and (3) screening the optimal concentration of the MgFe-LDH material for promoting differentiation of ESCs to NPC. (A) qPCR detects the expression level of NPCs marker gene after materials with different concentrations are added in the 5 th day of the differentiation process of NPCs; (B) detecting the expression of the NPCs marker protein by Western blot; (C) and (5) carrying out quantitative statistics on the Western blot.

Detailed Description

The following detailed description of the present invention will be made with reference to the accompanying drawings.

Mouse Embryonic Stem Cells (ESCs) are derived from a blastocyst inner cell mass, are pluripotent stem cells with self-renewal and multidirectional differentiation potential, are induced into Neural Precursor Cells (NPCs) and have high survival rate and plasticity in nerve transplantation, and can reduce the risk of teratoma formation. Therefore, a complete set of induction culture system for NPCs differentiation can provide a foundation for future clinical cell transplantation treatment.

In the following examples, DMEM, fetal bovine serum, GMEM, serum analogs (KOSR), non-essential amino acids, glutamine and diabody, which are used as essential components of cell culture media for maintaining normal cell growth, are commercially available products from Gibco, USA, and other non-self-made reagents and materials are commercially available.

Example 1

1. Two LDH materials with different metal elements are successfully prepared, and characterization and biocompatibility detection are carried out on the LDH materials

(1) Preparation of magnesium-aluminum-magnesium-iron LDH (layered double hydroxide) nano material

Step one, preparing 60mL salt solution by using 6mmol of magnesium nitrate and 2mmol of aluminum nitrate or 2mmol of ferric nitrate, wherein Mg²⁺With Al³⁺Or Fe³⁺The molar ratio therebetween was 3: 1.

Step two, CO removal₂ddH of₂A0.016M NaOH solution (40 mL) was prepared using O (double distilled water) as a solvent.

Step three, continuously administering N₂Meanwhile, the mixed salt prepared in the step one60mL of the solution was added to 40mL of 0.016M NaOH solution obtained in step two under vigorous stirring (400 rpm) to obtain a first suspension.

And step four, transferring the first suspension to a hydrothermal synthesis kettle, and heating for 16h at 100 ℃ to obtain a second suspension.

Step five, centrifuging 20000g for 15min to collect the product and removing CO₂After washing twice with the double distilled water, the colloidal material was stored in a refrigerator at 4 ℃.

And step six, drying the second suspension in a vacuum drying oven to obtain magnesium-aluminum LDH (MgAl-LDH) or magnesium-iron LDH (MgFe-LDH).

And (3) detecting the structure of the material by using a Transmission Electron Microscope (TEM), preparing two LDHs into a suspension of 0.1mg/mL, and ultrasonically dispersing for 30min to enable the sample to be a uniform solution, wherein FIG. 1A is a transmission electron microscope morphology image of the two LDHs. As shown in FIG. 1A, MgAl-LDH and MgFe-LDH both have hexagonal layered structures, are distributed uniformly, and have particle sizes of about 80-120nm (FIG. 1A).

(2) Surface potential determination of nano LDH

The two LDHs are prepared into 1mg/mL suspension, the sample is made into a uniform solution by ultrasonic dispersion for 30min, particle size detection and surface Zeta potential detection are carried out by using a particle size analyzer, and the detection result is shown in figure 1B. FIG. 1B is the surface potential of both LDHs at a concentration of 0.5mg/mL, indicating that the surface potential of MgAl-LDH is about 24.4mV and the surface potential of MgFe-LDH is about 13.4mV, indicating that both LDH materials in this example have Zeta potentials that are positive, are stable, and facilitate contact with negatively charged cell membranes and entry into the cells.

(3) Cell viability assay for ESCs following Nano-LDH treatment

Step one, culturing the mouse embryonic stem cell ESCs. ESCs were plated on 96-well plates at 8000 plates/well in medium at 37 ℃ with 5% CO₂Culturing for 24h under the condition for subsequent treatment of the medicine or the material. The ESCs culture medium comprises the following raw materials: DMEM; FBS; (ii) glutamine; double antibody. In this experiment, the incubation time was 24 h.

And step two, preparing solutions with five concentrations, wherein the concentrations are 2.5 mu g/mL, 5 mu g/mL, 10 mu g/mL, 20 mu g/mL and 40 mu g/mL in sequence.

And step three, discarding the supernatant of the ESCs cells cultured in the step one, adding LDH solutions with different concentrations prepared in the step two into each hole, culturing for 24 hours, adding 10 mu L of CCK-8 solution with the concentration of 5mg/mL into each hole, placing for 2 hours in a dark place, vibrating for 10s in the dark place, and measuring the OD (optical density) value at the position of 455nm wavelength by using a microplate reader, wherein the detection result is shown in figure 1C. The cell proliferation capacity of the mESCs is detected after the two LDH materials are respectively treated for 7 days according to concentration gradients of 2.5 mu g/mL, 5 mu g/mL, 10 mu g/mL, 20 mu g/mL and 40 mu g/mL, and the result shows that the cell proliferation capacity is not influenced.

2. MgFe-LDH can better promote mESCs to differentiate into NPCs than MgAl-LDH material

Step one, culturing the mouse embryonic stem cell ESCs. ESCs were plated on 6cm suspension culture dishes at a plating density of 2X 10⁵Cell/well, cultured in Neural Precursor Cells (NPCs) medium at 37 deg.C in 5% CO₂Performing differentiation culture for 1-5 days under the condition for subsequent treatment of medicine or material. The NPCs culture medium comprises the following raw materials: GMEM; 8% KOSR; 1% glutamine; 1% double antibody. The media components are commercially available from Gibco.

And step two, respectively preparing MgFe-LDH and MgAl-LDH solutions with the concentrations of 2.5 mug/mL, 5 mug/mL and 10 mug/mL, adding two LDH materials with different concentrations for treating for 48 hours on the 5 th day of differentiation from the ESCs to the NPCs, collecting cells at each time point, cracking by Trizol, extracting total RNA, carrying out reverse transcription by using a cDNA reverse transcription kit to obtain cDNA, and detecting expression of NOC differentiation related genes by real-time quantitative PCR (Sox1, Pax6, N-cadherin and Map 2).

And step three, finding the NPCs marker gene expression by using qPCR detection, and finding that the NPCs treated by MgFe-LDH under various concentration conditions can better promote the expression of NPCs marker genes Sox1, Pax6, N-cadherin and Map2 compared with a control group and an MgAl-LDH group by using the result of figure 2, so that the material is adopted in subsequent experiments.

3. Optimum time point for MgFe-LDH nano material to promote differentiation of ESCs to NPCs

Step one, inoculating ESCs on a 6cm suspension culture dish, and paving the dish at a density of 2 multiplied by 10⁵And/well, considering that the differentiation of NPCs reaches higher maturity substantially at day 5, we added 10. mu.g/mL of MgFe-LDH material to the differentiation of ESCs to NPCs at days 1-5, changed the culture broth every 3 days and harvested the cells to examine the differentiation efficiency of NPCs at day 7 by qPCR, respectively, FIG. 3A is the effect on the differentiation marker genes Sox1, Pax6, N-cadherin and Map2 of NPCs after the addition of MgFe-LDH material at days 1, 2, 3, 4 and 5, respectively, and the results show that LDH can promote the expression of Sox1, Pax6, N-cadherin and Map2 to the differentiation thereof after the addition of the early (days 1-2) and late (days 3-5) stages of NPCs differentiation.

And step two, adding MgFe-LDH materials on the 1 st day, the 3 rd day and the 5 th day of the NPCs differentiation, collecting cells of each group on the 7 th day of the differentiation, and detecting differentiation markers of the NPCs after treatment of each group by immunofluorescence staining, wherein the differentiation markers comprise Sox1, Pax6 and N-cadherin. Fixing the cells with 4% paraformaldehyde for 10min, washing with PBS for three times, permeating with 0.25% Triton X-100 (polyethylene glycol octyl phenyl ether) for 10min, then blocking with 5% goat serum for 1h to inhibit nonspecific antibody binding, incubating overnight at 4 ℃ with Sox1, Pax6 and N-cadherin primary antibody (diluted 1: 500), washing with PBS for three times, incubating at room temperature with fluorescent secondary antibody (diluted 1: 200) for 1h, washing with PBS for three times, incubating DAPI (37 ℃, 15min) for cell nucleus staining, and observing staining condition with a fluorescence confocal microscope. The antibodies used therein were all commercially available from Abcam. The results shown in FIG. 3B show that the amounts of Sox1, Pax6 and N-cadherin in NPCs after addition of the materials on day 1 were lower than those of the control group (treated without addition of the materials), that the fluorescence intensities of Pax6 and N-cadherin after addition of the materials on day 3 were higher than those of the control group, and that the protein fluorescence intensities of Pax6 and N-cadherin after addition of the materials on day 5 were the highest. This indicates that the optimal time point for MgFe-LDH nanomaterials to promote differentiation of ESCs into NPCs was day 5 of differentiation.

4. Optimal concentration screening of nano LDH material for promoting differentiation of ESCs to NPC

Step one, adding 2.5, 5, 10, 20 and 40 mu g/mL of MgFe-LDH materials respectively on the 5 th day of the differentiation process of NPCs, and detecting the differentiation efficiency of the NPCs after MgFe-LDH treatment under different concentration conditions by utilizing qPCR (figure 4A) and WB (figure 4B) on the 7 th day, wherein the qPCR results in figure 4A show that under the concentration condition of 10 mu g/mL, the expression levels of marker genes Sox1, Pax6, N-cadherin and Map2 corresponding to the NPCs are higher than that under the treatment under other concentration conditions;

step two, respectively adding 2.5, 5, 10, 20 and 40 mu g/mL MgFe-LDH materials in the NPCs differentiation process on the 5 th day, detecting the influence on the NPCs marker proteins after the MgFe-LDH materials are treated under different concentration conditions by using a protein immunoprecipitation technology (WB) on the 7 th day, quantitatively detecting the total proteins extracted from each group of cells, loading the total proteins to the sample according to the same protein amount, performing polyacrylamide gel electrophoresis, performing membrane transfer, sealing and incubation corresponding to a primary antibody and a secondary antibody on the gel after the electrophoresis is finished, exposing corresponding protein bands by using ECL luminescence liquid, and comparing the expressions of the NPCs marker proteins Sox dh 1, Pax6 and N-calcin after each group of treatment. The WB level test results in FIG. 4B also demonstrated that the protein expression levels of N-cadherin and Pax6 were higher at a concentration of 10. mu.g/mL than in the other groups under the condition of consistent expression of the internal reference protein, and the quantitative statistics in FIG. 4C also demonstrated that the optimal concentration condition of MgFe-LDH for promoting the differentiation of mESCs into NPCs was 10. mu.g/mL.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims

1. The application of the nano layered double hydroxide in promoting the differentiation of embryonic stem cells to neural precursor cells.

2. Use according to claim 1, characterized in that the nano-layered double hydroxide has a divalent metal cation selected from Mg²⁺、Zn²⁺And Ca²⁺。

3. Use according to claim 1, characterized in that the nano-layered double hydroxide has its trivalent metal cation selected from Al³⁺And Fe³⁺。

4. Use according to claim 1, characterized in that the nano-layered double hydroxide has interlayer anions selected from the group consisting of Cl^-、NO₃ ^-And SO₄ ^2-。

5. The use according to claim 1, wherein the nano-layered double hydroxide has a molar ratio of divalent metal ions to trivalent metal ions of (0.1-5): 1.

6. Use according to claim 1, characterized in that the nano-layered double hydroxide has the general formula [ M [ ]²⁺ _(1-x)M³ ⁺ _x(OH)₂]_x+[A^n-]_x/n·zH₂O, wherein M²⁺Is a divalent metal cation, M³⁺Is a trivalent metal cation, A^n-Is an anion with the valence of n between layers, x is the molar ratio of trivalent cations to all cations, and z is the number of crystal water between each nano-layered double hydroxide molecule.

7. A method for promoting differentiation of embryonic stem cells into neural precursor cells, which comprises the step of adding a nano-layered double hydroxide into a system for differentiation of embryonic stem cells into neural precursor cells.

8. The method of claim 7, wherein the nano-layered double hydroxide is added on days 3-5 of the induced differentiation culture.

9. The method according to claim 7, wherein the concentration of the nano-layered double hydroxide in the differentiation system is 1 to 40 μ g/mL.

10. The method according to claim 9, wherein the concentration of the nano-layered double hydroxide in the differentiation system is 10 μ g/mL.