CN117025531A

CN117025531A - Method for inducing multifunctional stem cells or embryonic stem cells to form astrocytes by rapid and efficient small molecule method

Info

Publication number: CN117025531A
Application number: CN202310687763.7A
Authority: CN
Inventors: 陈纯海; 龚明月; 马秦龙; 林敏�; 张宽; 杨志奇; 余争平
Original assignee: Third Military Medical University TMMU
Current assignee: Third Military Medical University TMMU
Priority date: 2023-06-09
Filing date: 2023-06-09
Publication date: 2023-11-10

Abstract

The application relates to the technical field of cell induction culture, in particular to a method for inducing human induced multifunctional stem cells or human embryonic stem cells to form astrocytes by a rapid and efficient small-molecule method. The scheme uses a culture medium containing retinoic acid and valproic acid to culture human embryonic stem cells to obtain neuroepithelial cells; next, culturing the neural epithelial cells by using a mixed culture medium and then using an NI culture medium to obtain neural stem cells; the mixed culture medium consists of 0-100% of KSR culture medium and 0-100% of NI culture medium: the neural stem cells were cultured using an As medium to obtain astrocytes. By adopting the scheme, as can be obtained within 30 days, the conversion efficiency reaches 94.89%, the technical problems of complicated process, long required induction culture time and low cell conversion rate of the existing method are solved, and an important platform is provided for basic research, drug screening and regenerative medicine research.

Description

Method for inducing multifunctional stem cells or embryonic stem cells to form astrocytes by rapid and efficient small molecule method

Technical Field

The application relates to the technical field of cell induction culture, in particular to a method for inducing human induced multifunctional stem cells or human embryonic stem cells to form astrocytes by a rapid and efficient small-molecule method.

Background

Human astrocytes (As) are obtained in vitro by stem cell technology, and the method has important significance for drug screening and treating brain diseases. Currently, the main method of in vitro directed induction of human induced multifunctional stem cells (human induced plutipotent stem cell, hiPSC) or human embryonic stem cells (human embryonic stem cell, hESC) to differentiate into astrocytes is to induce the hiPSC/hESC cells to differentiate into embryoid bodies containing three germ layers, then to cut the ectodermal cells to differentiate into neural stem cells or precursor cells, and finally to induce the differentiation of the neural stem cells or precursor cells into terminal cells such as astrocytes.

The currently reported methods for directed differentiation of hiPSC/hESC into astrocytes essentially go through 4 stages:

(1) Suspension culturing to form Embryoid Body (EB);

(2) EB further differentiated into rosette-like structures (rosette structure);

(3) The rosette structure is mechanically separated and amplified to form a neurosphere (neurosphere);

(4) The neurospheres are inoculated into a culture plate coated with a matrix, and a culture medium and an induction factor for differentiating astrocytes are added to obtain target cells.

Although astrocytes satisfying the conditions can be obtained, the whole process has a long induction time (about 180 days are required), the transformation efficiency is low, the induction process is complicated, and the culture medium and the induction reagent are required to be used very much. Meanwhile, when the final target cells are formed, only about 20% of the cells can be effectively converted into the target cells, while the remaining about 80% of the cells cannot be clearly identified in type, so that the clinical application effect of the cell population containing a large amount of mixed cells is difficult to ensure. Therefore, the astrocyte culture method of the prior art is unfavorable for large-scale development and has low efficiency, and a rapid, simple and efficient induction method is urgently needed.

Disclosure of Invention

The application aims to provide a method for inducing human-induced multifunctional stem cells or human embryonic stem cells to form astrocytes by a rapid and efficient small-molecule method, so as to solve the technical problems of complicated process, long required induction culture time and low cell conversion rate of the astrocyte induction culture method in the prior art.

In order to achieve the above purpose, the application adopts the following technical scheme:

a method for inducing human induced multifunctional stem cells or human embryonic stem cells to form astrocytes by a rapid and efficient small molecule method comprises the following steps in sequence:

s1: culturing human induced multifunctional stem cells or human embryonic stem cells by using a complete mTESR1 culture medium containing retinoic acid and valproic acid to obtain neuroepithelial cells;

s2: firstly, culturing nerve epithelial cells by using a mixed culture medium and then using an NI culture medium to obtain nerve stem cells; the mixed culture medium consists of 0-100% of KSR culture medium and 0-100% of NI culture medium; the ratio of the NI medium in the mixed medium increases with the passage of incubation time;

s3: the neural stem cells were cultured using an As medium to obtain astrocytes.

The technical principle of the technical scheme is as follows:

the induction differentiation stage of human induced pluripotent stem cells or human embryonic stem cells (hiPSC/hESC) into neuroepithelial cells (NESC) was induced using a medium containing retinoic acid and valproic acid. Through the induction of retinoic acid and valproic acid, the efficiency of the transformation from hiPSC/hESC cells to NESC cells is improved, and conditions are created for the subsequent induction of astrocytes.

In the stage of inducing differentiation of NESC cells into neural stem cells (NSC/NPC), the cells are cultured by adopting a method of mixing KSR and NI and changing liquid and increasing the proportion of NI every day, and finally the culture is completely changed into NI culture, and finally a large number of neural stem cells (NSC/NPC) are obtained. KSR culture can result in the accumulation of cell debris around the final neurosphere, which is generated by the fact that cells which have not been transformed into the next stage cannot survive in a medium unsuitable for culture, and eventually undergo death, and these cell debris can adhere around the neurosphere and release apoptosis-related growth factors, so that the neurosphere which has been successfully transformed also undergoes apoptosis; and KSR and NI are adopted for mixed culture, so that a buffer process of the cells which are not differentiated is provided, the generation of the situation is reduced, and finally, the cell transformation efficiency is improved.

Further, in S1, the final concentrations of retinoic acid and valproic acid were 10. Mu.M and 0.5mM, respectively.

Further, at the beginning of the culturing process of S1, human induced pluripotent stem cells or human embryonic stem cells were cultured using mTeSR1 complete medium containing Rock inhibitor, retinoic acid and valproic acid; the Rock inhibitor is then removed.

Further, in S2, the process of culturing the neuroepithelial cells using the mixed medium is continued for five days, and a suspension culture manner is adopted; the mixed culture mediums used each day are respectively as follows: a mixed medium containing 100% KSR medium, a mixed medium containing 75% KSR medium and 25% NI medium, a mixed medium containing 67% KSR medium and 33% NI medium, a mixed medium containing 50% KSR medium and 50% NI medium, a mixed medium containing 100% NI medium.

Further, in S2, the process of culturing the neuroepithelial cells using the NI medium includes a suspension culture process and an adherent culture process, which are sequentially performed.

Further, the NI medium is a DMEM/F-12 medium containing 1% N-2 additive, 2% B-27 additive, 10ng/ml basic fibroblast growth factor, 10ng/ml epidermal cell growth factor, 1% diabody, 1% MEM nonessential amino acid solution, 1% L-alanyl-L-glutamine.

Further, the KSR medium was a DMEM/F-12 medium containing 20% KSR serum replacement, 1% MEM non-essential amino acid solution, 1% L-alanyl-L-glutamine, 50uM beta-mercaptoethanol, 1% diabody.

Further, in S3, the method of culturing neural stem cells using As medium is: the neural stem cells were cultured using an As first medium, an As second medium, and an As third medium in this order.

Further, the As first medium is a DMEM/F-12 medium containing 1% of B-27 additive, 30% of fetal bovine serum, 10ng/mL of human ciliary neurotrophic factor, and 1% of diabody;

as second medium is DMEM/F-12 medium containing 0.5% B-27 additive, 15% fetal bovine serum, 10ng/mL human ciliary neurotrophic factor, 1% diabody;

the As third medium is DMEM/F-12 high sugar medium containing 10% of fetal bovine serum and 1% of diabody.

Further, prior to S1, human induced pluripotent stem cells or human embryonic stem cells were cultured and passaged using mTeSR1 complete medium containing Rock inhibitors.

To sum up, the beneficial effects of this technical scheme lie in:

in the application, we develop a method for rapidly and efficiently transforming human induced multifunctional stem cells or human embryonic stem cells (hiPSC/hESC) into astrocytes (As), the conversion efficiency of the hiPSC/hESC into As can reach 94.89% in 30 days, meanwhile, the As markers GFAP and S100b are highly expressed, and in-vitro calcium imaging experiments show that the As induced by the method can be in network communication with other glial cells, and has the functional characteristics of primary astrocytes. In summary, this rapid and efficient method of inducing hiPSC/hESC-derived As can provide an important platform for basic research, drug screening and regenerative medicine research.

The prior art generally employs the culture of hiPSC/hESC into Embryoid Bodies (EB), followed by induction of neural stem cells (NSC/NPC), and finally, the induction of As cells. The above process is complicated to operate, requires a long culture time, and has low induction efficiency. In order to overcome the above problems, the inventors have made extensive studies on a method of inducing As cells. The inventors first tried a way to induce hiPSC/hESC cells directly using retinoic acid, but Retinoic Acid (RA) alone resulted in the difficulty of inducing hiPSC/hESC cells to form neural stem cells. Although the prior art reports that RA is associated with As cells, practical experimental attempts have not found this approach viable. The inventors have found that hiPSC/hESC cells can be efficiently induced into neuroepithelial cells (NESC) if hiPSC/hESC cells are treated with Retinoic Acid (RA) and valproic acid (VPA) simultaneously, and the transformation efficiency of hiPSC/hESC cells into neural stem cells is greatly improved in the subsequent induction process of forming the neural stem cells.

In addition to the NESC induction phase being very important for improving the induction transformation efficiency and shortening the culture time, the inventor has also discovered a method for effectively improving the culture effect in the induction process of NESC cells to neural stem cells. The proportion of the KSR culture medium and the NI culture medium is gradually regulated in the initial stage of NESC induction, and the dosage of the NI culture medium is gradually increased, so that the induction conversion efficiency is greatly improved and the culture time is shortened. The inventors further analyzed that the reason for the occurrence of the above phenomenon was: cell differentiation is a linear process, and at a specific time point, cells are not 100% completely transformed into differentiated cells of the next stage, so that the technical scheme selects to gradually increase the culture medium of differentiated cells of the next stage in the transformation process, and a buffer process is carried out on the cells which are not differentiated, thereby improving the transformation efficiency of the final cells.

Drawings

FIG. 1 is a flow chart of the induction culture of example 1.

FIG. 2 is a cell micrograph (10X) of example 1 at various stages of inducing the differentiation of human embryonic stem cells into astrocytes.

FIG. 3 is a cell micrograph (10X) of comparative example 1 at various stages of EB induced differentiation of human embryonic stem cells into astrocytes.

FIG. 4 is a cell micrograph (10X) of comparative example 2 at various stages of RA induced differentiation of human embryonic stem cells into astrocytes.

FIG. 5 is the astrocyte differentiation efficiency statistics of example 1, comparative example 1 and comparative example 2.

FIG. 6 is an immunofluorescence micrograph of neural stem cells of example 1 (PAX 6 and Ki67; 40X).

FIG. 7 is an immunofluorescence micrograph of neural stem cells of comparative example 1 (PAX 6 and Ki67; 40X).

FIG. 8 is a statistical graph of differentiation efficiency of neural stem cells of example 1 and comparative example 1.

FIG. 9 is an immunofluorescence micrograph of astrocytes of example 1 (primary astrocyte markers: GFAP and S-100deg.C; 40X).

FIG. 10 shows the results of in vitro calcium imaging analysis of astrocytes of example 1 (ATP, thapsigargin induces Ca in human embryonic stem cell-derived astrocytes ²⁺ Activity).

FIG. 11 is a microscopic image (10X) of cells of comparative example 3 using KSR medium for neural stem cell induction.

FIG. 12 is a microscopic image (10X) of cells of comparative example 3 using a fixed ratio of KSR and NI mixed medium for neural stem cell induction.

Detailed Description

The present application will be described in further detail with reference to examples, but embodiments of the present application are not limited thereto. Unless otherwise indicated, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used are all commercially available.

Example 1:

total procedure for human-induced pluripotent stem cells or human embryonic stem cell (hiPSC/hESC) -derived astrocytes (As) -induced culture

In order to improve the conversion efficiency of hiPSC/hESC-derived astrocytes and promote the clinical application thereof, the inventor improves the differentiation method of hiPSC/hESC, and generates a rapid and efficient method for inducing the direct conversion of hiPSC/hESC into astrocytes, which is specifically as follows:

(1) Passage and culture of hiPSC/hESC cells

(1.1) hiPSC/hESC medium formulation: the hiPSC/hESC cells are cultured and passaged by using a conventional culture medium mTESR1, wherein the mTESR1 complete culture medium is a common human embryonic stem cell/pluripotent stem cell culture medium and can be purchased through commercial means. The complete mTeSR1 medium included a volume ratio of 4:1 (mTeSR 1 Basal Medium) and mTeSR1 additive (mTeSR 1 Supplement).

Culture of hiPSC/hESC cells requires coating of commercially available Matrigel in culture flasks. The preparation of Matrigel is carried out by the following general procedures according to the specification: mixing Matrigel original glue and conventional DMEM-F12 culture solution according to description, adding Matrigel into culture container (culture flask, culture plate, etc.), and incubating at 37deg.C for 1 hr or overnight at 4deg.C to complete coating Matrigel. The Matrigel adhesive specification is described as follows: an aliquot of Corning Matrigel hESC-acceptable substrate (original gum) was added to 25mL DMEM/F-12 to coat four 6-well plates (1 mL/well) or three 100mm dishes (8 mL/dish). At room temperature (15-25 ℃) for at least 1 hour, the remaining liquid is aspirated from the dish before use.

(1.2) passage and culture: the cells were passaged to about 80% and, at passaging, the original medium was discarded, washed 2 times with PBS, reLeSR dispersion was added until the edge of each clone was rolled up to the center, digestion was stopped by adding DMEM-F12, centrifugation at 800rpm for 4min, and cells were resuspended using complete medium of mTESR1 with Rock inhibitor at a final concentration of 10. Mu.M. Next, the cells were inoculated onto a culture vessel previously coated with Matrigel and cultured using mTESR1 complete medium containing Rock inhibitor at a final concentration of 10. Mu.M at 37℃in 5% CO ₂ And (3) in a saturated humidity incubator, observing and changing the liquid every day.

(2) Induced differentiation of hiPSC/hESC into neural Stem cells (NSC/NPC)

(2.1) the medium used included neuro-induction medium (Neural induction medium; NI medium), KSR medium (KSR medium), mTESR1 complete medium containing retinoic acid and valproic acid.

The NI culture medium is prepared on the basis of DMEM/F-12 culture mediumWherein the composition comprises 1% of N-2 additive (N-2 Supplement), 2% of B-27 additive (B-27 Supplement), 10ng/ml of basic fibroblast growth factor (bFGF), 10ng/ml of Epidermal Growth Factor (EGF), 1% of diabody, 1% of MEM nonessential amino acid solution (MEM-NEAA), 1% of Glutamax ^TM -I (L-alanyl-L-glutamine). All of the above ingredients were purchased from suppliers, "%" is percent by volume.

The KSR medium was also formulated on the basis of DMEM/F-12 medium, containing 20% KSR serum replacement (Knockout serum replacement), 1% MEM-NEAA, 1% Glutamax ^TM -I, 50uM beta-mercaptoethanol (beta-ME), 1% diabody. All of the above ingredients were purchased from suppliers, "%" is percent by volume.

The complete medium of mTESR1 containing Retinoic acid and Valproic acid is prepared by adding 10 μm Retinoic Acid (RA) and 0.5mM Valproic acid (VPA) into the complete medium of mTESR 1.

A complete mTESR1 culture medium containing Rock inhibitor, retinoic acid and Valproic acid is prepared by adding Rock inhibitor with a final concentration of 10 μm, retinoic Acid (RA) with a final concentration of 10 μm and Valproic acid (VPA) with a final concentration of 0.5mM to the complete mTESR1 culture medium.

The reagents for preparing the above media are all commercially available.

(2.2) Induction of differentiation Process

Induced differentiation can be performed when hiPSC/hESC cells reached 60% -70% confluency, reference to (1.2) passaging step, 8×10 at final inoculation ³ Individual/cm ² Density inoculation is performed on a culture plate coated with Matrigel in advance, and mTESR1 complete medium containing Rock inhibitor at a final concentration of 10. Mu.M, retinoic Acid (RA) at a final concentration of 10. Mu.M, and Valproic acid (VPA) at a final concentration of 0.5mM is added, time-stamped as day 0. After 24h, rock inhibitor was removed from the medium, RA and VPA concentrations were unchanged, and the fluid was changed daily until day7 cells were plated to the bottom of the plate, forming neuroepithelial cells that were considered the most primitive neural stem cell state during embryonic development (neuroepithelial stem cell,NESC). Repeating the step (1.2) to isolate NESC, and inoculating at the end at 8×10 ⁴ Individual/cm ² The medium was changed to KSR medium in a 10cm dish, time-stamped day7. Changing liquid by standing method every day, mixing KSR culture medium with NI culture medium, day7-day11, increasing NI proportion every day, day7 being 100% KSR, day8 being 75% KSR and 25% NI, day9 being 67% KSR and 33% NI, day10 being 50% KSR and 50% NI, day11 being 100% NI; when day11 was completely changed to NI medium, the suspension culture was continued for 7 days (suspension culture using ultra-low suspension dishes/plates, which were specially treated so that the cells did not tend to grow on the wall and were not coated with Matrigel), and the neurospheres were further grown on the wall for 7 days (using ordinary dishes/plates and coated with Matrigel) until the plate bottom was completely filled with cells, and the induction of hiPSC/hESC-derived neural stem cells was completed, thus obtaining neural stem cells.

(3) hiPSC/hESC-derived astrocytes (As) induced differentiation

The culture medium used in (3.1) comprises an As first culture medium, an As second culture medium and an As third culture medium, and the specific preparation method is As follows:

as first medium was formulated on the basis of DMEM/F-12 medium containing 1% B-27Supplement (B-27 Supplement), 30% Fetal Bovine Serum (FBS), 10ng/mL human ciliary neurotrophic factor (CNTF), 1% diabody. All of the above ingredients were purchased from suppliers, "%" is percent by volume.

As second medium was formulated on the basis of DMEM/F-12 medium, containing 0.5% B-27Supplement (B-27 Supplement), 15% Fetal Bovine Serum (FBS), 10ng/mL human ciliary neurotrophic factor (CNTF), 1% diabody. All of the above ingredients were purchased from suppliers, "%" is percent by volume.

As third medium was prepared on the basis of DMEM/F-12 high glucose medium (DMEM/F-12-high glucose) containing 10% Fetal Bovine Serum (FBS) and 1% diabody. All of the above ingredients were purchased from suppliers, "%" is percent by volume. All of the above ingredients were purchased from suppliers, "%" is percent by volume.

(3.2) hiPSC/hESC-derived As-induced differentiation

When the fusion rate of the hiPSC/hESC-derived neural stem cells reaches 90%, the cells are digested into single cells by using Ackutase digestive juice, counted and expressed at the ratio of 4 multiplied by 10 ⁵ Density of individual/flask (T25 flask) was inoculated onto flask previously coated with Matrigel matrix, as first medium was added, and the flask was placed at 37℃with 5% CO ₂ Culturing in a saturated humidity incubator, changing the As second culture medium after 2 days, and changing the As third culture medium after 2 days. On day30, the hiPSC/hESC-derived As induction ended. If the cells reach 90% confluency during the culture process, the cells must be passaged.

The above-described induction of As by hiPSC/hESC can be seen in the flow chart shown in FIG. 1. The first stage is to conduct conventional subculture on the hESC cells, the second stage is to induce the hESC cells to form NESC cells, the third stage is to induce the NESC cells to form neural stem cells, and the fourth stage is to induce the neural stem cells to form As cells.

Specific examples of (two) hiPSC/hESC-derived astrocyte-induced culture

The following describes the induced transformation culture process of hiPSC/hESC-derived astrocytes using hESC cells (human embryonic stem cells, human embryonic stem cell, hESC) as an example. Both human induced pluripotent stem cells (Human induced plutipotent stem cell, hiPSC) and human embryonic stem cells (Human embryonic stem cell, hESC) can be used as primary cells in the actual induction of astrocytes.

(1) Culturing of hESC: resuscitates 1 hESC cell (human embryonic stem cell, human embryonic stem cell, hESC), inoculates into matrigel coated T25 flasks, cultures with mTESR1 medium containing Rock inhibitor at final concentration of 10. Mu.M, and places at 37deg.C, 5% CO ₂ Culturing in a saturated humidity incubator for 2-3 days, observing and changing liquid every day, and carrying out passage when the cell fusion degree reaches 80%.

(2) Induction of hescs into neural stem cells:

hESC cells were seeded on plates coated with Matrigel prior to seeding,inoculation density of 8X 10 ³ Individual/cm ² . A complete medium of mTESR1 was added, time-stamped day0, containing Rock inhibitor at a final concentration of 10. Mu.M, retinoic Acid (RA) at a final concentration of 10. Mu.M, and Valproic acid (VPA) at a final concentration of 0.5mM. After 24h, rock inhibitors were removed from the medium, RA and VPA concentrations were unchanged, the cell morphology was changed daily, and if the cell fusion reached 90% cells were observed daily, conventional passaging treatments were required until day7, forming neuroepithelial cells (neuroepithelial stemcell, NESC) which were considered the most primitive neural stem cell state during embryonic development.

After induction of NESC cells, the next induction is continued. Cells were washed twice with 1 XPBS and then with 2ml ReLeSR ^TM After 1min of the dispersion, the cells were aspirated, digested at 37℃for 5min, and after all cells were observed under a microscope to round, digestion was stopped using DMEM-F12 containing 1% FBS, the bottom of the bottle was gently blown with a Pasteur tube, the cells were detached, and the cells were gently transferred into a 15mL centrifuge tube without blowing. 400rpm,4min centrifugation, and 100% KSR medium was added for resuspension. The cells were grown at 8X 10 ⁴ Individual/cm ² The density was seeded in ultra low suspension dishes/plates. During day7-day11, cells were cultured using KSR medium and/or NI medium, and the medium was changed daily using a static method. More specifically: day7 was 100% KSR medium, day8 was 75% KSR medium and 25% NI medium, day9 was 67% KSR medium and 33% NI medium, day10 was 50% KSR medium and 50% NI medium, and day11 was 100% NI medium. Then, the culture was continued for seven days in suspension until day18, and during the culture, the medium was changed every 1 day, and 100% NI medium was used. At day18, cells were inoculated into matrigel coated common six well plates, and adherent culture was performed with liquid changes every 1 day until day25, to obtain mature neural stem cells.

(3) Induction of Astrocytes (AS) by neural stem cells

At day25, cells were washed with 1 XPBS, 2-3ml Accutase was added, digested at 37℃for 3-5min, and after all cells were observed to round under the microscope, digestion was stopped with DMEM-F12 containing 1% FBS. Cells were added to a centrifuge tube, centrifuged at 800rpm for 4min, the supernatant discarded, and the cells resuspended in As first medium. Cells were separated into plates pre-plated with Matrigel gel and cultured using As first medium. After two days of culture, i.e. at day27, the medium was replaced with As second medium. After two days of culture, i.e. at day29, the medium was replaced with As third medium. At day30, neural stem cells are substantially converted to As, which are inoculated into a cell slide for immunofluorescence detection and in vitro calcium imaging assay detection.

By using the present protocol for As induction, the cell status at different time points can be seen in FIG. 2.Day0 is the state of the cells prior to inoculation. By day7, cells form a fusiform or triangle, have a strong stereoscopic impression, and have a tendency to aggregate into neurospheres at multiple locations, at which time NESC can be determined to have formed. Demonstrating conversion of hESC cells to NESC cells by seven days of induction of retinoic acid and valproic acid; microscopic images of day7 showed that cells in the field proliferated in large numbers and exhibited the characteristics of NESC cells. The scheme adopts a mode of mixed induction of KSR culture medium and NI culture medium, and on the 21 st day, a large number of cells already show the characteristics of neural stem cells: the cell surface is smooth, the edge of the aggregated cells climbs out of fusiform or triangular cells, and the cell morphology is full. The obtained neural stem cells are further induced, a large number of As cells can be obtained on the 30 th day, and the under-lens cells have the characteristics of good cell state, smooth cell surface, obvious axons and dendrites, high proliferation speed, close contact between cells, no impurities such As organelles and the like.

The cell area statistics was performed on the As cells obtained in this example, and the ratio of the total area of the As cells to the total area of all cells was calculated to show the astrocyte differentiation efficiency, and the experimental results are shown in fig. 5. In the statistics, 10-fold mirror fields were selected from the two experimental groups, and the percentage of As cell area to total cell area under the 10-fold mirror fields was counted, and then the average was taken. The statistical time point was day 30. Comparing this example with EB and RA methods, the efficiency of induced formation of As cells in this protocol was far higher than other methods, giving unexpected technical results.

Immunofluorescence study was performed on the neural stem cells obtained in this example, and the levels of PAX6 and Ki67 (neural stem cell markers) in the cells were detected, and the experimental results are shown in fig. 6 (data acquisition time is day25 of culture). The ratio of the number of PAX6/Ki67 positive cells to the number of living cells was counted to show the efficiency of hESC differentiation into neural stem cells, and the experimental results are shown in FIG. 8 (data acquisition time is 25 days of culture). In the statistics, 40-fold mirror fields were selected from the two experimental groups, and the percentage of PAX6/Ki67 positive cells in the living cells under the 40-fold mirror fields was counted, and then the average value was obtained. Compared with the EB method, the nerve stem cell transformation efficiency of the scheme is quite ideal and is obviously higher than that of the EB method.

Immunofluorescence studies were performed on As cells obtained in this example to detect GFAP and S-100. Beta. Levels (As cell markers) in the cells, and the experimental results are shown in FIG. 9. The results of the in vitro calcium imaging analysis of hiPSC/hESC-derived As are shown in FIG. 10. The identification of As cells is realized through immunofluorescence and in vitro calcium wave experiments. In FIG. 10, (A) confocal microscope Ca was performed on GCaMP6f (green) labeled hESC-derived astrocytes ²⁺ Imaging, imaging results at 4 time points after addition of 200. Mu. Mol/L ATP (dashed circles are selected cells); (B): 200. Mu. Mol/L Ca of ATP-induced cells ²⁺ 4 time points in C corresponding to signal activity; (C): comparison of Ca in astrocytes induced by ATP-free (control group) and ATP-free ²⁺ Signal amplitude (Δf/f) (each group comprised 78 cells, z= -10.728, p=4.175E-27; × P < 0.001, double sided Wilcoxon symbol rank test); (D): confocal microscopy of GCaMP6f (Green) labeled hESC-derived astrocytes Ca ²⁺ Imaging, imaging results at 4 time points after addition of 10. Mu. Mol/L thapsigargin; (E): ca in 10. Mu. Mol/L thapsigargin-induced cells ²⁺ 4 time points in F corresponding to signal activity; (F): comparison of Ca in astrocytes induced by non-thapsigargin (control group) and thapsigargin ²⁺ Signal amplitude (Δf/f) (78 cells per group, z= -10.728, p= 4.176E-27; × P < 0.001, double sided Wilcoxon rank sum test). All data in the figures are arithmetic mean ± s.e.m.

Comparative example 1: induction of As cells was carried out using conventional methods (i.e., EB method, robert 2011 document description method)

EB method refers to the literature "Robert Krencik, su-Chun Zhang. Directed differentiation of functional astroglial subtypes from human pluripotent stem cells, nat Protoc.2011Oct 13;6 (11): 1710-7.Doi:10.1038/nprot.2011.405. The general procedure is as follows:

inducing differentiation when hESC cells reach 60% -70% fusion rate, transferring the digested hESC colonies into a culture dish, blowing the culture medium into small cell clusters by adding mTESR1 complete culture medium containing Rock inhibitor with the final concentration of 10 mu M, inoculating the culture medium into an ultralow suspension plate for culture (namely suspension culture), changing liquid every day until day10, and forming embryoid bodies; culturing in NI culture medium until day21, climbing out spindle-shaped or triangle cell, i.e. nerve stem cell, performing immunofluorescence identification, adding human ciliary neurotrophic factor (CNTF), and performing AS induction. According to the technical scheme, when the neural stem cells are induced, embryoid bodies are formed firstly, then the neural stem cells are induced to form, and finally AS cells are induced to form. With this method, the time is long and the induction efficiency is limited.

Induction of As was performed using this comparative protocol, and the cell status at various time points can be seen in fig. 3.EB method since retinoic acid and valproic acid were not used, the cells remained Embryoid Bodies (EB) after 10 days of culture (day 10). Only a small number of cells were transformed into neural stem cells on day21 (day 21). Cells were cultured for a long period (e.g., day 180) and only a small number of As cells appeared under the mirror.

The cell area of the As cells obtained in this comparative example was counted, and the ratio of the total area of the As cells to the total area of all cells was calculated to represent the astrocyte differentiation efficiency, and the experimental results are shown in FIG. 5. In the statistics, 10-fold mirror fields were selected from the two experimental groups, and the percentage of As cell area to total cell area under the 10-fold mirror fields was counted, and then the average was taken. The statistical time point was day 180.

Immunofluorescence study was performed on the neural stem cells obtained in this example, and the levels of PAX6 and Ki67 (neural stem cell markers) in the cells were detected, and the experimental results are shown in fig. 7 (data acquisition time is day 90 of culture). The ratio of PAX6/Ki67 positive cell number to living cell number was counted to show the efficiency of hESC differentiation into neural stem cells, and the experimental results are shown in FIG. 8 (data acquisition time is day 90 of culture). In the statistics, 40-fold mirror fields were selected from the two experimental groups, and the percentage of PAX6/Ki67 positive cells in the living cells under the 40-fold mirror fields was counted, and then the average value was obtained.

Comparative example 2: induction of neural stem cells by RA method

RA method reference is made to "Zhou JM, chu JX, chen XJ.an improved protocol that induces human embryonic stem cells to differentiate into neural cells in vitro. Cell Biol W t.2008, 32 (1): 80-85", "Baharvand H, mehrjardi N Z, hatami M, et al.Neurol differentiation from human embryonic stem cells in a defined adherent culture condition. Int J Dev Bio1.2007,51 (5): 371-378". The general procedure for induction culture by RA method is as follows: the digested hESC colonies were transferred to a petri dish, and the medium was blown into small cell clusters with the addition of complete medium containing Rock inhibitor at a final concentration of 10. Mu.M and Retinoic Acid (RA) at a final concentration of 10. Mu.M, inoculated into ultra-low suspension plates for cultivation, and changed every day until day5.

Induction of As was performed using this comparative protocol, and the cell status at various time points can be seen in fig. 4. As can be seen from the cell microscopic image of fig. 4, the efficiency of transformation of hESC cells into neural stem cells is very low, very few neural stem cells are observed in the visual field, and As cells are more difficult to form without forming a large number of neural stem cells. Therefore, the induction experiment was terminated at about 5 days of culture in this comparative example due to the too low transformation efficiency at the neural stem cell stage. Therefore, in the process of inducing the hESC cells to form As, the induction needs to be carried out by simultaneously using retinoic acid and valproic acid, otherwise, the hESC cells are difficult to effectively form the neural stem cells, and the induction efficiency of the As cells is greatly reduced.

The cell area of the As cells obtained in this comparative example was counted, and the ratio of the total area of the As cells to the total area of all cells was calculated to represent the astrocyte differentiation efficiency, and the experimental results are shown in FIG. 5. In the statistics, 10-fold mirror fields were selected from the two experimental groups, and the percentage of As cell area to total cell area under the 10-fold mirror fields was counted, and then the average was taken. The statistical time point is day5.

Comparative example 3: method for inducing formation of neural stem cells from NESC cells

The procedure of example 1 was followed to obtain NESC in the same manner as in example 1, and the procedure of example 1 was followed to examine the induction of neural stem cells from NESC cells. The specific process is as follows:

attempt 1: NESC was isolated and then inoculated for neural stem cell induction, and the graded mixed medium of example 1 was changed to KSR medium, and cultured in suspension from day7 to day11, at which time the cells were microscopically observed, see FIG. 11.KSR culture can result in the accumulation of cell debris around the final neurosphere that has not been transformed into a subsequent stage of cell death in media unsuitable for its culture, and eventually results from the attachment of these cell debris around the neurosphere, release of apoptosis-related growth factors, and the tendency of the transformed neurosphere to apoptosis.

Attempt 2: unlike trial 1, where the KSR medium was changed to 50% KSR medium and 50% NI medium, day11 was used for microscopic observation of cells, see FIG. 12. Instead of gradually adjusting the ratio of KSR medium to NI medium as in example 1, the experiment directly uses a fixed ratio of KSR medium to induce culture of NESC cells. The culture method also causes the NESC cells to be insufficiently differentiated, the phenomenon of cell aggregation is generated, cell fragments are also collected around neurospheres, and the conversion rate of hESC cells to As cells is greatly and negatively affected. The inventors further analyzed that the reason for the occurrence of the above phenomenon was that: cell differentiation is a linear process, and at a specific time point, cells are not 100% completely transformed into differentiated cells of the next stage, so that the technical scheme selects to gradually increase the culture medium of differentiated cells of the next stage in the transformation process, and a buffer process is carried out on the cells which are not differentiated, thereby improving the transformation efficiency of the final cells.

The foregoing is merely exemplary of the present application, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, and these should also be regarded as the protection scope of the present application, which does not affect the effect of the implementation of the present application and the practical applicability of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.

Claims

1. A method for inducing human induced multifunctional stem cells or human embryonic stem cells to form astrocytes by a rapid and efficient small molecule method is characterized in that: the method comprises the following steps of:

2. The method for inducing human induced pluripotent stem cells or human embryonic stem cells to form astrocytes by a rapid and efficient small molecule method according to claim 1, wherein the method comprises the steps of: in S1, the final concentrations of retinoic acid and valproic acid were 10. Mu.M and 0.5mM, respectively.

3. The method for inducing human induced pluripotent stem cells or human embryonic stem cells to form astrocytes by a rapid and efficient small molecule method according to claim 2, wherein the method comprises the steps of: at the beginning of the culturing process of S1, human induced pluripotent stem cells or human embryonic stem cells are cultured using mTeSR1 complete medium containing Rock inhibitor, retinoic acid and valproic acid; the Rock inhibitor is then removed.

4. The method for inducing human induced pluripotent stem cells or human embryonic stem cells to form astrocytes by a rapid and efficient small molecule method according to claim 1, wherein the method comprises the steps of: in S2, the process of culturing the neuroepithelial cells by using the mixed culture medium lasts for five days, and a suspension culture mode is adopted; the mixed culture mediums used each day are respectively as follows: a mixed medium containing 100% KSR medium, a mixed medium containing 75% KSR medium and 25% NI medium, a mixed medium containing 67% KSR medium and 33% NI medium, a mixed medium containing 50% KSR medium and 50% NI medium, a mixed medium containing 100% NI medium.

5. A method for inducing human induced pluripotent stem cells or human embryonic stem cells to form astrocytes by a rapid and efficient small molecule method according to claim 4, wherein the method comprises the steps of: in S2, the process of culturing the neuroepithelial cells using the NI medium includes a suspension culture process and an adherent culture process, which are sequentially performed.

6. The method for inducing human induced pluripotent stem cells or human embryonic stem cells to form astrocytes by a rapid and efficient small molecule method according to claim 5, wherein the method comprises the steps of: the NI culture medium is DMEM/F-12 culture medium containing 1% of N-2 additive, 2% of B-27 additive, 10ng/ml of basic fibroblast growth factor, 10ng/ml of epidermal growth factor, 1% of diabody, 1% of MEM nonessential amino acid solution and 1% of L-alanyl-L-glutamine.

7. The method for inducing human induced pluripotent stem cells or human embryonic stem cells to form astrocytes by a rapid and efficient small molecule method according to claim 6, wherein the method comprises the steps of: the KSR medium was DMEM/F-12 medium containing 20% KSR serum replacement, 1% MEM nonessential amino acid solution, 1% L-alanyl-L-glutamine, 50uM beta-mercaptoethanol, 1% diabody.

8. The method for inducing human induced pluripotent stem cells or human embryonic stem cells to form astrocytes by a rapid and efficient small molecule method according to claim 1, wherein the method comprises the steps of: in S3, the method of culturing neural stem cells using As medium is: the neural stem cells were cultured using an As first medium, an As second medium, and an As third medium in this order.

9. The method for inducing human induced pluripotent stem cells or human embryonic stem cells to form astrocytes by a rapid and efficient small molecule method according to claim 8, wherein the method comprises the steps of: as first culture medium is DMEM/F-12 culture medium containing 1% of B-27 additive, 30% of fetal calf serum, 10ng/mL of human ciliary neurotrophic factor and 1% of diabody;

10. The method for inducing human induced pluripotent stem cells or human embryonic stem cells to form astrocytes by a rapid and efficient small molecule method according to claim 1, wherein the method comprises the steps of: prior to S1, human induced pluripotent stem cells or human embryonic stem cells were cultured and passaged using mTESR1 complete medium containing Rock inhibitor.