A method for differentiating induced pluripotent stem cells into neural stem cells and application thereof

Abstract

The application discloses a method for differentiating induced pluripotent stem cells into neural stem cells and application thereof, and comprises the following steps: adding a small molecule inhibitor CHIR99021 and icariin into a culture medium to obtain an induction culture medium, and inducing differentiation of the induced pluripotent stem cells into the neural stem cells by using the induction culture medium. The present application only needs to combine the small molecule inhibitor with the icariin to complete the induction, and the method has the advantages of low cost, high differentiation efficiency, short induction period, clear culture medium components, simple operation process and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method and application for differentiating induced pluripotent stem cells into neural stem cells. Background Technology

[0002] Neural stem cells (NSCs) possess the ability to proliferate, self-renew, and generate a large number of functional progeny cells that can differentiate into neurons, astrocytes, and oligodendrocytes. Transdifferentiation technology allows for the large-scale proliferation, self-renewal, and multi-lineage differentiation of NSCs in vitro. After transplantation into the human body, NSCs can differentiate into corresponding nerve cells under the influence of the in vivo microenvironment, thus repairing the function of damaged nerves. Furthermore, implanted NSCs can migrate and focus towards the site of neurological lesions, promoting the recovery of damaged functions of the host nervous system. Neural stem cells can be used to treat neurodegenerative diseases and central nervous system injuries, therefore, they are becoming increasingly important in the treatment and mechanistic research of nervous system diseases.

[0003] Neural stem cells (NSCs) can be obtained through various methods, including embryonic stem cell transdifferentiation and in vitro differentiation of induced pluripotent stem cells (iPSCs). Among these, iPSCs are widely available, have no ethical constraints, and are considered an ideal method for obtaining NSCs in vitro. Induced pluripotent stem cells (iPSCs) are pluripotent cells created by introducing transcription factors (Oct4, Sox2, Klf4, and c-Myc) into animal somatic cells via gene transfection technology, causing the somatic cells to reprogram and become multipotent. iPSCs have been successfully cultured and differentiated into various somatic cells and tissues, including cardiac, neural, pancreatic, and bone cells. Early methods primarily used stromal cell co-culture to induce iPSC differentiation into neural stem cells; however, this method has the following drawbacks: the presence of stromal cells makes the culture conditions unclear, the induction and differentiation time is long, differentiation is uneven, and the differentiation efficiency is low. In addition, researchers have been working to optimize combinations of small molecule inhibitors, such as GSK-3 inhibitors CHIR99021, SB216763, and CHIR98014, BMP inhibitors DMH1, DOR, and LDN212854, and ALK inhibitors SB431542 and Gul788388, in order to obtain neural stem cells with short induction time and high differentiation efficiency. However, the cost of small molecule inhibitor combinations is high, and the purity of the differentiated neural stem cells is still relatively low. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide a method for efficiently differentiating induced pluripotent stem cells into neural stem cells by using a combination of small molecule inhibitors and icariin, so as to solve the technical problems of high cost, long cycle and instability of neural stem cell induction and differentiation in the prior art.

[0005] The present invention includes the following main contents:

[0006] A method for differentiating induced pluripotent stem cells into neural stem cells includes the following steps:

[0007] The small molecule inhibitor CHIR99021 was added to the culture medium in combination with icariin to obtain an induction medium. The induced pluripotent stem cells were induced to differentiate into neural stem cells using the induction medium.

[0008] The small molecule inhibitor CHIR99021 described in this invention, CAS number 1797989-42-4, has the molecular formula C 22 H 19 C l3 N8, a commercially available product.

[0009] The icariin (icariin) described in this invention, CAS No. 489-32-7, has the molecular formula C 33 H 40 O 15 Commercially available products.

[0010] As a preferred embodiment, the induced pluripotent stem cells are derived from fibroblasts.

[0011] As a preferred embodiment, the fibroblasts are human foreskin fibroblasts.

[0012] Currently, various somatic cells can be reprogrammed into iPS cells, such as fibroblasts, hepatocytes, umbilical cord blood cells, and keratinocytes. Reprogrammed iPS cells carry some genetic characteristics of the primary cells and possess the "epigenetic memory" of their source cells. Therefore, iPS cells from different sources exhibit differences in differentiation potential and efficiency. This invention has a particular advantage in inducing the differentiation of iPS cells derived from human foreskin fibroblasts into neural stem cells, with significant induction effects.

[0013] Induced pluripotent stem cells (iPSCs) can be generated through retroviruses, lentiviruses, Sendai virus, electroporation, and small molecule compounds. This invention utilizes the Sendai virus method for reprogramming to obtain iPS cells. This method does not induce the integration of exogenous genes, is relatively safe, and has high induction efficiency.

[0014] As a preferred embodiment, the mass concentration of the small molecule inhibitor CHIR99021 in the induction medium is 2 μmol / L to 6 μmol / L, and the mass concentration of icariin in the induction medium is 40 μg / mL to 80 μg / mL.

[0015] As a preferred embodiment, the induction medium contains the following components: glutamine, N2 additive, B27 additive, ascorbic acid, CHIR99021, icariin, DMEM / F12 medium, and nerve cell basal medium.

[0016] The N2 additive (i.e., N2 Supplement) and B27 additive described in this invention are serum-free additives and are commercially available products.

[0017] As a preferred embodiment, the induction medium contains the following components at the following mass concentrations: glutamine 0.5–1.5%, N2 additive 0.5–1.5%, B27 additive 1–2%, ascorbic acid 0.05–0.15 mmol / L, CHIR99021 2–6 μmol / L, icariin 40–80 μg / mL, and the remainder is DMEM / F12 medium and nerve cell basal medium.

[0018] As a more preferred embodiment, the induction medium contains the following components at mass concentrations: 1% glutamine, 1% N2 additive, 2% B27 additive, 0.1 mmol / L ascorbic acid, 3 μmol / L CHIR99021, 50 μg / mL icariin, and the remainder is DMEM / F12 medium and nerve cell basal medium in a volume ratio of 1:1.

[0019] As a preferred embodiment, the induction culture medium further contains 15–35 μg / mL of physalisin. Physalisin can accelerate the differentiation of pluripotent stem cells into neural stem cells.

[0020] The wild cherry glycoside described in this invention, CAS number 99-18-3, molecular formula: C 14 H 17 NO6, molecular weight: 295.29.

[0021] As a preferred option, the induction medium is used to induce differentiation when the induced pluripotent stem cell confluence reaches 20-30%.

[0022] On the other hand, the present invention also relates to the application of the method in the preparation of neural stem cell preparations.

[0023] The beneficial effects achieved by this invention are as follows:

[0024] This invention presents a novel method for inducing pluripotent stem cells to differentiate into neural stem cells. By adding the aforementioned induction culture medium, a large number of neural stem cells can be obtained within 7 days of induction, with an induction efficiency exceeding 90%. This induction method represents a relatively novel approach, eliminating the need for multiple inhibitor combinations. Induction can be achieved using only a small molecule inhibitor and icariin, simplifying the material selection.

[0025] The induction efficiency of CHIR99021 combined with icariin can reach over 90%, while the efficiency of CHIR99021 and icariin alone is lower than that of the combination.

[0026] The present invention also found that the addition of physalisin to the induction culture medium can accelerate the differentiation of pluripotent stem cells into neural stem cells. Attached Figure Description

[0027] Figures 1-3 Image showing the Nestin immunofluorescence staining results of neural stem cells induced to differentiate to day 6 in Experiment 1 (magnification: 100×). Figure 1 Nestin staining diagram; Figure 2 This is a DAPI nucleochromatogram. Figure 3 This is a diagram of Merge co-chromatic assemblages.

[0028] Figures 4-6 Image showing the SOX1 immunofluorescence staining results of neural stem cells induced to differentiate to day 6 in Experiment 1 (magnification: 100×). Figure 4 SOX1 staining diagram; Figure 5 This is a DAPI nucleochromatogram. Figure 6 This is a diagram of Merge co-chromatic assemblages.

[0029] Figures 7-8 The image shows the Nestin immunofluorescence staining results of neural stem cells induced to differentiate to day 6 in Experiment 2 (magnification: 100×), using an induction medium containing only small molecule inhibitors. Figure 7 Nestin staining diagram; Figure 8 This is a DAPI nucleochromatogram.

[0030] Figures 9-11 The image shows the Nestin immunofluorescence staining results of neural stem cells induced to differentiate to day 6 in Experiment 2 (magnification: 100×), using an induction medium containing only icariin. Figure 9 Nestin staining diagram; Figure 10 This is a DAPI nucleochromatogram. Figure 11 For white scenes. Detailed Implementation

[0031] To facilitate understanding of the technical content of this invention by those skilled in the art, the invention will be further described in detail below with reference to specific embodiments.

[0032] Example 1

[0033] Induction medium: 0.5% glutamine, 0.5% N2 additive, 1% B27 additive, 0.05 mmol / L ascorbic acid, 2 μmol / L CHIR99021, 40 μg / mL icariin, with the remainder being a 1:1 mixture of DMEM / F12 (1:1) and neurobasal medium. Prepare before use and store at 4°C for 14 days.

[0034] Example 2

[0035] Induction medium: 1.5% glutamine, 1.5% N2 additive, 2% B27 additive, 0.15 mmol / L ascorbic acid, 6 μmol / L CHIR99021, 80 μg / mL icariin, with the remainder being a 1:1 mixture of DMEM / F12 (1:1) and neurobasal medium. Prepare before use and store at 4°C for 14 days.

[0036] Example 3

[0037] Induction medium: 1% glutamine, 1% N2 additive, 1.5% B27 additive, 0.1 mmol / L ascorbic acid, 3 μmol / L CHIR99021, 60 μg / mL icariin, with the remainder being a 1:1 mixture of DMEM / F12 (1:1) and neurobasal medium. Prepare before use and store at 4°C for 14 days.

[0038] Experiment 1: The combined action of CHIR99021 and icariin induced the differentiation of induced pluripotent stem cells into neural stem cells.

[0039] 1) Obtaining induced pluripotent stem cells:

[0040] Four transcription factors (Oct4, Sox2, Klf4, and c-Myc) were introduced into human foreskin fibroblasts using Sendai virus to reprogram them and culture them to obtain iPS cells. The iPS cells were alkaline phosphatase positive, expressed stem cell-specific markers, had normal karyotypes, and possessed multi-lineage differentiation potential.

[0041] 2) Lamination of induced pluripotent stem cells:

[0042] a. Matrigel coating of 12-well plates: Thaw the dispensed Matrigel in advance on an ice box at 4°C, dilute with the appropriate amount of pre-cooled DMEM / F12, and spread 1000 μL / well onto a 12-well plate. Coat at 37°C for at least 1 hour before use.

[0043] b. Before plating, the induced pluripotent stem cells were in good condition, with cells closely arranged and few differentiated cells.

[0044] c. Discard the old culture medium, wash the cells once with 2 mL / well of DPBS (Dubor's phosphate buffer), then add 1 mL / well of accupase enzyme, and incubate the cells at 37°C for 3-4 min. When the intercellular spaces widen and the cells become translucent, carefully discard the accupase enzyme and add 1 mL of mTeSR to each well. TM After gently pipetting the Plus medium into a single-cell suspension, collect the cell suspension into a 15mL centrifuge tube and centrifuge at 800-1000rpm / min for 5min.

[0045] d. Discard the supernatant, resuspend in mTeSRTMPlus medium, count, and measure at 1×10⁻⁶. 5 / 12-hole plate.

[0046] e. Discard Matrigel before plating, and add mTeSR first. TM Add Plus+Y27632 (10 μg / mL) culture medium, then add cell suspension (1×10⁻⁶). 5 / well), the total amount of culture medium per well is set at 1mL / well.

[0047] f. Shake well to ensure even distribution of cells, then incubate in a 37°C CO2 incubator. Induce differentiation within 24 hours after plating.

[0048] 2) Induced differentiation of pluripotent stem cells

[0049] a. Before differentiation, the cell confluence is 20%-30%, and the cells are short spindle-shaped or irregularly shaped and adhere to the wall, with tentacles. When the cell confluence reaches 20-30%, the medium is replaced with induction medium to induce differentiation, and the induction medium is changed daily thereafter.

[0050] The induction medium formula consisted of 1% glutamine, 1% N2 additive, 2% B27 additive, 0.1 mmol / L ascorbic acid, 3 μmol / L CHIR99021, 50 μg / mL icariin, and the remainder was DMEM / F12 (1:1) and neurobasal medium in a 1:1 volume ratio.

[0051] b. During induction, cells proliferate continuously, mainly growing in clusters without significant shedding. The cells become tightly packed together. By the third day of induction, cells show bulging growth, and by the sixth day of induction, cell fusion reaches 95%-100%.

[0052] c. Induced differentiation to day 6, the obtained neural stem cells are P0 generation. They are then identified by Nestin and SOX1 immunofluorescence staining, or the induced cells are digested with neural stem cell digestion solution, passaged and expanded, and then identified accordingly.

[0053] 3) Experimental Results

[0054] See results Figures 1-6 The results showed that the obtained cells could express neural stem cell markers Sox1 and Nestin.

[0055] Further research showed that the proportion of cells positive for the marker Nestin was 97%, and the proportion of cells positive for the marker Sox1 was 94%.

[0056] Experiment 2: Small molecule inhibitors and icariin, acting alone, induced the differentiation of pluripotent stem cells into neural stem cells.

[0057] Group 1

[0058] An induction medium containing only a small molecule inhibitor (CHIR99021) in the optimal ratio was prepared. The induction medium consisted of 1% glutamine, 1% N2 additive, 2% B27 additive, 0.1 mmol / L ascorbic acid, 3 μmol / L CHIR99021, and the remainder was DMEM / F12 (1:1) and neurobasal medium in a 1:1 volume ratio.

[0059] Group 2

[0060] An induction medium containing only icariin in the optimal ratio was prepared. The induction medium consisted of 1% glutamine, 1% N2 additive, 2% B27 additive, 0.1 mmol / L ascorbic acid, 50 μg / mL icariin, and the remainder was DMEM / F12 (1:1) and neurobasal medium at a volume ratio of 1:1.

[0061] The differentiation induction procedure is the same as in Experiment 1.

[0062] The experimental results are shown in Figures 7-11 The results showed that although the obtained cells could express the neural stem cell marker Nestin, the positive rate was significantly lower than that of Experiment 1 (P < 0.01).

[0063] Example 4

[0064] Induction medium: 1% glutamine, 1% N2 additive, 2% B27 additive, 0.1 mmol / L ascorbic acid, 3 μmol / L CHIR99021, 50 μg / mL icariin, 20 μg / mL safflower glycoside, and the remainder was DMEM / F12 (1:1) and neurobasal medium at a volume ratio of 1:1.

[0065] Following the method described in Experiment 1 above, analysis revealed that after further addition of physalisin to the induction culture medium, cell confluence reached 95%-100% by day 5 of differentiation induction, with Nestin-positive cells accounting for over 97% and Sox1-positive cells accounting for over 94%. These results indicate that physalisin in the culture medium formulation can accelerate the differentiation of pluripotent stem cells into neural stem cells.

[0066] In this embodiment, the amount of physalisin added was 20 μg / mL. However, this amount is not fixed. Through experiments, the amount added in the culture medium can be 15 to 35 μg / mL.

[0067] The above description is only a partial preferred embodiment of the present invention and is not intended to limit the present invention. The scope of protection of the present invention is not limited to the above content. All equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of protection of the present invention.

Claims

1. A method for differentiating induced pluripotent stem cells into neural stem cells, characterized in that, Includes the following steps: The small molecule inhibitor CHIR99021 and icariin were added to a culture medium to obtain an induction medium. The induced pluripotent stem cells were induced to differentiate into neural stem cells using the induction medium. The concentration of the small molecule inhibitor CHIR99021 in the induction medium was 2 μmol / L to 6 μmol / L, and the concentration of icariin in the induction medium was 40 μg / mL to 80 μg / mL. The induction medium also contained 15 to 35 μg / mL of arugula glycoside.

2. The method for differentiating induced pluripotent stem cells into neural stem cells according to claim 1, characterized in that, The induced pluripotent stem cells are derived from fibroblasts.

3. The method for differentiating induced pluripotent stem cells into neural stem cells according to claim 2, characterized in that, The fibroblasts are human foreskin fibroblasts.

4. The method for differentiating induced pluripotent stem cells into neural stem cells according to claim 1, characterized in that, The induction medium contains the following components: glutamine, N2 additive, B27 additive, ascorbic acid, CHIR99021, icariin, DMEM / F12 medium, and nerve cell basal medium.

5. The method for differentiating induced pluripotent stem cells into neural stem cells according to claim 1, characterized in that, The induction medium contains the following components at the following mass concentrations: glutamine 0.5-1.5%, N2 additive 0.5-1.5%, B27 additive 1-2%, ascorbic acid 0.05-0.15 mmol / L, CHIR99021 2-6 μmol / L, icariin 40-80 μg / mL, and the remainder is DMEM / F12 medium and nerve cell basal medium.

6. The method for differentiating induced pluripotent stem cells into neural stem cells according to claim 1, characterized in that, The induction medium contains the following components at the following mass concentrations: glutamine 1%, N2 additive 1%, B27 additive 2%, ascorbic acid 0.1 mmol / L, CHIR99021 3 μmol / L, icariin 50 μg / mL, and the remainder is DMEM / F12 medium and nerve cell basal medium in a volume ratio of 1:

1.

7. The method for differentiating induced pluripotent stem cells into neural stem cells according to claim 1, characterized in that, Induction of differentiation was initiated using the aforementioned induction medium when the induced pluripotent stem cell confluence reached 20-30%.

8. The use of the method according to any one of claims 1 to 7 in the preparation of neural stem cell preparations.

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